AU2016231617B2 - Single-chain multivalent binding proteins with effector function - Google Patents

Abstract

Abstract The present application relates to maltivalent binding peptides, including bi-specific binding peptides having minnnoglobulin effector function, nucleic acids, vectors, host cells and compositions comprising them. The present application also describes uses of and methods of treating a wide variety of diseases such as cancers, auto immune diseases, inflaunation and viral infections etc. using the multivalent binding peptides. Methods of preparation of these peptides are also described. WO 2007/146968 PCT/US2007/071052 BDI EFD BD2 SUBSTITUTE SHEET (RULE 26)

Description

FIELD OF THE INVENTION

The invention relates generally to the field of multivalent binding 5 molecules and therapeutic applications thereof.

The sequence listing is being submitted as a text file and as a PDF file in compliance with applicable requirements for electronic filing. The sequence listing was created on June 12, 2007. The sequence listing is incorporated herein by reference in its entirety.

BACKGROUND

In a healthy mammal, the immune system protects the body from damage from foreign substances and pathogens. In some instances though, the immune system goes awry, producing traumatic insult and/or disease. For example, B-cells can produce antibodies that recognize self-proteins rather than foreign proteins, leading to the production of the autoantibodies characteristic of autoimmune diseases such as lupus erythematosus, rheumatoid arthritis, and the like. In other instances, the typically beneficial effect of the immune system in combating foreign materials is counterproductive, such as following organ transplantation. The power of the mammalian immune system, and in particular the human immune system, has been recognized and efforts have been made to control the system to avoid or ameliorate the deleterious consequences to health that result either from normal functioning of the immune system in an abnormal environment (e.g., organ transplantation) or from abnormal functioning of the immune system in an otherwise apparently normal environment (e.g., autoimmune disease progression). Additionally, efforts have been made to exploit the immune system to provide a number of target-specific diagnostic and therapeutic methodologies, relying on the capacity of antibodies to specifically recognize and bind antigenic targets with specificity,

One way in which the immune system protects the body is by production of specialized cells called B lymphocytes or B-cells. B-cells produce antibodies that bind to, and in some cases mediate destruction of, a foreign substance or pathogen. In some instances though, the human immune system, and specifically the B lymphocytes of the human immune system, go awry and disease results. There are

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2016231617 23 Sep 2016 numerous cancers that involve uncontrolled proliferation of B-cells. There are also numerous autoimmune diseases that involve B-cell production of antibodies that, instead of binding to foreign substances and pathogens, bind to parts of the body. In addition, there are numerous autoimmune and inflammatory diseases that involve B5 cells in their pathology, for example, through inappropriate B-cell antigen presentation to T-cells or through other pathways involving B-cells. For example, autoimmune-prone mice deficient in B-cells do not develop autoimmune kidney disease, vasculitis or autoantibodies. (Shlomchik et al., J Exp. Med. 1994, 180:1295306). Interestingly, these same autoimmune-prone mice which possess B-cells but are deficient in immunoglobulin production, do develop autoimmune diseases when induced experimentally (Chan et al., J Exp. Med. 1999, 189:1639-48), indicating that B-cells play an integral role in development of autoimmune disease.

B-cells can be identified by molecules on their cell surface. CD20 was the first human B-cell lineage-specific surface molecule identified by a monoclonal antibody. It is a non-glycosylated, hydrophobic 35 kDa B-cell transmembrane phosphoprotein that has both its amino and carboxy ends situated inside the cell. Einfeld et al., EMBO J. 1988,7:711-17. CD20 is expressed by all normal mature Bcells, but is not expressed by precursor B-cells or plasma cells. Natural ligands for CD20 have not been identified, and the function of CD20 in B-cell biology is still incompletely understood.

Another B-cell lineage-specific cell surface molecule is CD37. CD37 is a heavily glycosylated 40-52 kDa protein that belongs to the tetraspanin transmembrane family of cell surface antigens. It traverses the cell membrane four times forming two extracellular loops and exposing its amino and carboxy ends to the cytoplasm. CD37 is highly expressed on normal antibody-producing (s!g+)B-cells, but is not expressed on pre-B-cells or plasma cells. The expression of CD37 on resting and activated T cells, monocytes and granulocytes is low and there is no detectable CD37 expression on NK. cells, platelets or erythrocytes. See, Belov et al,, Cancer Res., 61(11 ):44834489 (2001); Schwartz-Albiez et al., J. Immunol., 140(3): 905-914 (1988); and Link et al,, J. Immunol., 137(9): 3013-3018 (1988). Besides normal B-cells, almost all malignancies of B-cell origin are positive for CD37 expression, including CLL, NHL, and hairy cell leukemia (Moore, et al. 1987; Merson and Brochier 1988; Faure, et al.

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1990). CD37 participates in regulation of B-cell function, since mice lacking CD37 were found to have low levels of serum IgGl and to be impaired in their humoral response to viral antigens and model antigens. It appears to act as a nonclassical costimulatory molecule or by directly influencing antigen presentation via complex formation with MHC class II molecules. See Knobeloch et al., Mol. Cell. Biol., 20(15):5363-5369 (2000).

Research and drug development has occurred based on the concept that B-cell lineage-specific cell surface molecules such as CD37 and CD20 can themselves be targets for antibodies that would bind to, and mediate destruction of, cancerous and autoimmune disease-causing B-cells that have CD37 and CD20 on their surfaces. Termed immunotherapy, antibodies made (or based on antibodies made) in a nonhuman animal that bind to CD37 or CD20 were given to a patient to deplete cancerous or autoimmune disease-causing B-cells.

Monoclonal antibody technology and genetic engineering methods have facilitated development of immunoglobulin molecules for diagnosis and treatment of human diseases. The domain structure of immunoglobulins is amenable to engineering, in that the antigen binding domains and the domains conferring effector functions may be exchanged between immunoglobulin classes and subclasses. Immunoglobulin structure and function are reviewed, for example, in Harlow et al.,

Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988). An extensive introduction as well as detailed information about all aspects of recombinant antibody technology can be found in the textbook “Recombinant Antibodies” (John Wiley & Sons, NY, 1999). A comprehensive collection of detailed antibody engineering lab Protocols can be found in R,

Kontermann and S. Dtibel (eds.), “The Antibody Engineering Lab Manual” (Springer Verlag, Heidelberg/New York, 2000).

An immunoglobulin molecule (abbreviated Ig), is a multimeric protein, typically composed of two identical light chain polypeptides and two identical heavy chain polypeptides (H2L2) that are joined into a macromolecular complex by interchain disulfide bonds, i.e., covalent bonds between the sulfhydryl groups of neighboring cysteine residues. Five human immunoglobulin classes are defined on the basis of their heavy chain composition, and are named IgG, IgM, IgA, IgE, and

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IgD. The IgG-class and IgA-class antibodies are further divided into subclasses, namely, IgGl, IgG2, lgG3, and IgG4, and IgAl and IgA2, respectively. Intrachain disulfide bonds join different areas of the same polypeptide chain, which results in the formation of loops that, along with adjacent amino acids, constitute the immunoglobulin domains. At the amino-terminal portion, each light chain and each heavy chain has a single variable region that shows considerable variation in amino acid composition from one antibody to another. The light chain variable region, Vt, has a single antigen-binding domain and associates with the variable region of a heavy chain, Vh (also containing a single antigen-binding domain), to form the antigen binding site of the immunoglobulin, the Fv.

In addition to variable regions, each of the full-length antibody chains has a constant region containing one or more domains. Light chains have a constant region containing a single domain. Thus, light chains have one variable domain and one constant domain. Heavy chains have a constant region containing several domains.

The heavy chains in IgG, IgA, and IgD antibodies have three domains, which are designated Chi, Ch2, and Ch3_; the heavy chains in IgM and IgE antibodies have four domains, Chi, Ch2, Ch3 and Chx Thus, heavy chains have one variable domain and three or four constant domains. Noteworthy is the invariant organization of these domains in all known species, with the constant regions, containing one or more domains, being located at or near the C-terminus of both the light and heavy chains of immunoglobulin molecules, with the variable domains located towards the N-termini of the light and heavy chains. Immunoglobulin structure and function are reviewed, for example, in Harlow et al., Eds., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (1988).

The heavy chains of immunoglobulins can also be divided into three functional regions: the Fd region (a fragment comprising Vh and Chi, ie., the two Nterminal domains of the heavy chain), the hinge region, and the Fc region (the fragment crystallizable region). The Fc region contains the domains that interact with immunoglobulin receptors on cells and with the initial elements of the complement cascade. Thus, the Fc region or fragment is generally considered responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent

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2016231617 23 Sep 2016 cytotoxicity) and complement fixation, binding to Fc receptors, greater half-life in vivo relative to a polypeptide lacking an Fc region, protein A binding, and perhaps even placental transfer. Capon et al,, Nature, 337: 525-531, (1989). Further, a polypeptide containing an Fc region allows for dimerization/multimerization of the polypeptide. These terms are also used for analogous regions of the other immunoglobulins.

Although all of the human immunoglobulin isotypes contain a recognizable structure in common, each isotype exhibits a distinct pattern of effector function.

IgG, by way of nonexhaustive example, neutralizes toxins and viruses, opsonizes, fixes complement (CDC) and participates in ADCC. IgM, in contrast, neutralizes blood-bome pathogens and participates in opsonization. IgA, when associated with its secretory piece, is secreted and provides a primary defense to microbial infection via the mucosa; it also neutralizes toxins and supports opsonization. IgE mediates inflammatory responses, being centrally involved in the recruitment of other cells needed to mount a full response. IgD is known to provide an immunoregulatory function, controlling the activation of B cells. These characterizations of isotype effector functions provide a non-comprehensive illustration of the differences that can be found among human isotypes.

The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG 1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgGl, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly-proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgGl hinge), containing 62 amino acids (including 21 pro lines and 11 cysteines), forming an

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2016231617 23 Sep 2016 inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgGl and its flexibility is intermediate between that of IgGl and IgG2. The flexibility of the hinge regions reportedly decreases in the order IgG3>IgGl>IgG4>IgG2. The four IgG subclasses also differ from each other with respect to their effector functions. This difference is related to differences in structure, including differences with respect to the interaction between the variable region, Fab fragments, and the constant Fc fragment.

According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of Chi to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the Ch2 domain and includes residues in Ch2·

Id. The core hinge region of human IgGl contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgAl contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.

Conformational changes permitted by the structure and flexibility of the immunoglobulin hinge region polypeptide sequence may also affect the effector functions of the Fc portion of the antibody. Three general categories of effector functions associated with the Fc region include (1) activation of the classical complement cascade, (2) interaction with effector cells, and (3) compartmentalization

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2016231617 23 Sep 2016 of immunoglobulins. The different human IgG subclasses vary in the relative efficacies with which they fix complement, or activate and amplify the steps of the complement cascade. See, e.g., Kirschfink, 2001 Immunol. Rev. 180:177;

Chakraborti et al., 2000 Cell Signal 12:607; Kohl etal., 1999 Mol. Immunol. 36:893;

Marsh et al., 1999 Curr. Opin. Nephrol. Hypertens. 8:557; Speth etal., 1999 Wien Klin. Wochenschr. 111:378.

Exceptions to the H2L2 structure of conventional antibodies occur in some isotypes of the immunoglobulins found in camelids (camels, dromedaries and llamas; Hamers-Casterman etal., 1993 Nature363:446; Nguyen etal., 19987. Mol. Biol

275:413), nurse sharks (Roux et al., 1998 Proc. Nat. Acad. Sci. USA 95:11804), and in the spotted ratfish (Nguyen, et al., 2002 Immunogenetics 54(1):39-47). These antibodies can apparently form antigen-binding regions using only heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (referred to as heavy-chain antibodies or HCAbs). Despite the advantages of antibody technology in disease diagnosis and treatment, there are some disadvantageous aspects of developing whole-antibody technologies as diagnostic and/or therapeutic reagents. Whole antibodies are large protein structures exemplified by the heterotetrameric structure of the IgG iso type, containing two light and two heavy chains. Such large molecules are sterically hindered in certain applications.

For example, in treatments of solid tumors, whole antibodies do not readily penetrate the interior of the tumor. Moreover, the relatively large size of whole antibodies presents a challenge to ensure that the in vivo administration of such molecules does not induce an immune response. Further, generation of active antibody molecules typically involves the culturing of recombinant eukaryotic cells capable of providing appropriate post-translational processing of the nascent antibody molecules, and such cells can be difficult to culture and difficult to induce in a manner that provides commercially useful yields of active antibody.

Recently, smaller immunoglobulin molecules have been constructed to overcome problems associated with whole immunoglobulin methodologies. A single30 chain variable antibody fragment (scFv) comprises an antibody heavy chain variable domain joined via a short peptide to an antibody light chain variable domain (Huston et al., Proc. Natl. Acad. Sci. USA, 1988, 85: 5879-83). Because of the small size of

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2016231617 23 Sep 2016 scFv molecules, they exhibit more effective penetration into tissues than whole immunoglobulin. An anti-tumor scFv showed more rapid tumor penetration and more even distribution through the tumor mass than the corresponding chimeric antibody (Yokota et al., Cancer Res. 1992, 52:3402-08).

Despite the advantages that scFv molecules bring to serotherapy, several drawbacks to this therapeutic approach exist. An scFv is rapidly cleared from the circulation, which may reduce toxic effects in normal cells, but such rapid clearance impedes delivery of a minimum effective dose to the target tissue. Manufacturing adequate amounts of scFv for administration to patients has been challenging due to difficulties in expression and isolation of scFv that adversely affect the yield. During expression, scFv molecules lack stability and often aggregate due to pairing of variable regions from different molecules. Furthermore, production levels of scFv molecules in mammalian expression systems are low, limiting the potential for efficient manufacturing of scFv molecules for therapy (Davis et al, J Biol. Chem.

1990,265:10410-18); Traunecker et al., EMBO J 1991, 10: 3655-59). Strategies for improving production have been explored, including addition of glycosylation sites to the variable regions (Jost, C. R. U.S. Pat. No. 5,888,773, Jost et al, J. Biol. Chem.

1994, 69: 26267-73).

Another disadvantage to using scFv for therapy is the lack of effector function.

An scFv without a cytolytic function, such as the antibody-dependent cell-mediated cytotoxicity (AJDCC) and complement dependent-cytotoxicity (CDC) associated with the constant region of an immunoglobulin, may be ineffective for treating disease. Even though development of scFv technology began over 12 years ago, currently no scFv products are approved for therapy.

Alternatively, it has been proposed that fusion of an scFv to another molecule, such as a toxin, could take advantage of the specific antigen-binding activity and the small size of an scFv to deliver the toxin to a target tissue. Chaudary et al., Nature 1989,339:394; Batra et al., Mol. Cell. Biol. 1991, 11:2200. Conjugation or fusion of toxins to scFvs has thus been offered as an alternative strategy to provide potent, antigen-specific molecules, but dosing with such conjugates or chimeras can be limited by excessive and/or non-specific toxicity due to the toxin moiety of such preparations. Toxic effects may include supraphysiological elevation of liver

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2016231617 23 Sep 2016 enzymes and vascular leak syndrome, and other undesired effects. In addition, immunotoxins are themselves highly immunogenic upon administration to a host, and host antibodies generated against the immunotoxin limit potential usefulness for repeated therapeutic treatments of an individual.

Nonsurgical cancer therapy, such as external irradiation and chemotherapy, can suffer from limited efficacy because of toxic effects on normal tissues and cells, due to the lack of specificity these treatments exhibit towards cancer cells. To overcome this limitation, targeted treatment methodologies have been developed to increase the specificity of the treatment for the cells and tissues in need thereof. An example of such a targeted methodology for in vivo use is the administration of antibody conjugates, with the antibody designed to specifically recognize a marker associated with a cell or tissue in need of treatment, and the antibody being conjugated to a therapeutic agent, such as a toxin in the case of cancer treatment. Antibodies, as systemic agents, circulate to sensitive and undesirable body compartments, such as the bone marrow. In acute radiation injury, destruction of lymphoid and hematopoietic compartments is a major factor in the development of septicemia and subsequent death. Moreover, antibodies are large, globular proteins that can exhibit poor penetration of tissues in need of treatment.

Human patients and non-human subjects suffering from a variety of end-stage disease processes frequently require organ transplantation. Organ transplantation, however, must contend with the untoward immune response of the recipient and guard against immunological rejection of the transplanted organ by depressing the recipient's cellular immune response to the foreign organ with cytotoxic agents which affect the lymphoid and other parts of the hematopoietic system. Graft acceptance is limited by the tolerance of the recipient to these cytotoxic chemicals, many of which are similar to the anticancer (antiproliferative) agents. Likewise, when using cytotoxic antimicrobial agents, particularly antiviral drugs, or when using cytotoxic drugs for autoimmune disease therapy, e.g., in treatment of systemic lupus erythematosis, a serious limitation is the toxic effects of the therapeutic agents on the bone marrow and the hematopoietic cells of the body.

Use of targeted therapies, such as targeted antibody conjugate therapy, is designed to localize a maximum quantity of the therapeutic agent at the site of desired

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WO 2007/146968 PCT/US2007/071052 action as possible, and the success of such therapies is revealed by the relatively high signal-to-background ratio of therapeutic agent. Examples of targeted antibodies include diagnostic or therapeutic agent conjugates of antibody or antibody fragments, cell-or tissue-specific peptides, and hormones and other receptor-binding molecules.

For example, antibodies against different determinants associated with pathological and normal cells, as well as associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. In these methods, the targeting antibody is directly conjugated to an appropriate detecting or therapeutic agent as described, for example, in Hansen et al.,

U.S. Pat. No. 3,927,193 and Goldenberg, U.S. Pat. Nos. 4,331,647,4,348,376, 4,361,544,4,468,457,4,444,744,4,460,459,4,460,561,4,624,846 and 4,818,709.

One problem encountered in direct targeting methods, i.e., in methods wherein the diagnostic or therapeutic agent (the active agent) is conjugated directly to the targeting moiety, is that a relatively small fraction of the conjugate actually binds to the target site, while the majority of conjugate remains in circulation and compromises in one way or another the function of the targeted conjugate. To ensure maximal localization of the active agent, an excess of the targeted conjugate is typically administered, ensuring that some conjugate will remain unbound and contribute to background levels of the active agent. A diagnostic conjugate, e.g., a radioimmunoscintigraphic or magnetic resonance imaging conjugate that does not bind its target can remain in circulation, thereby increasing background and decreasing resolution of the diagnostic technique. In the case of a therapeutic conjugate having a toxin as an active agent (e.g., a radioisotope, drug or toxic compound) attached to a long-circulating targeting moiety such as an antibody, circulating conjugate can result in unacceptable toxicity to the host, such as marrow toxicity or systemic side effects.

U.S, Pat. No. 4,782,840 discloses a method for reducing the effect of elevated background radiation levels during surgery. The method involves injection of a patient with antibodies specific for neoplastic tissue, with the antibodies labeled with radioisotopes having a suitably long half-life, such as Iodine-125. After injection of the radiolabeled antibody, the surgery is delayed at least 7-10 days, preferably 14-21

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2016231617 23 Sep 2016 days, to allow any unbound radiolabeled antibody to be cleared to a low background level.

U.S. Pat. No. 4,932,412 discloses methods for reducing or correcting for nonspecific background radiation during intraoperative detection. The methods include the administration to a patient who has received a radiolabeled primary antibody, of a contrast agent, subtraction agent or second antibody which binds the primary antibody.

Apart from producing the antibodies described above, the immune system includes a variety of cell types that have powerful biological effects. During hematopoiesis, bone marrow-derived stem cells differentiate into either mature cells of the immune system (“B” cells) or into precursors of cells that migrate out of the bone marrow to mature in the thymus (“T” cells).

B cells are central to the humoral component of an immune response. B cells are activated by an appropriate presentation of an antigen to become antibody15 secreting plasma cells; antigen presentation also results in clonal expansion of the activated B cell. B cells are primarily responsible for the humoral component of an immune response. A plasma cell typically exhibits about 10⁵ antibody molecules (IgD and IgM) on its surface.

T lymphocytes can be divided into two categories. The cytotoxic T cells, Tc lymphocytes or CTLs (CD8+ T cells), kill cells bearing foreign surface antigen in association with Class IMHC and can kill cells that are harboring intracellular parasites (either bacteria or viruses) as long as the infected cell is displaying a microbial antigen on its surface. Tc cells kill tumor cells and account for the rejection of transplanted cells. Tc cells recognize antigen-Class I MHC complexes on target cells, contact them, and release the contents of granules directly into the target cell membrane, which lyses the cell.

A second category of T cells is the helper T cell or Th lymphocyte (CD4+ T cells), which produces lymphokines that are “helper” factors in the maturation of B cells into antibody-secreting plasma cells. Th cells also produce certain lymphokines that stimulate the differentiation of effector T lymphocytes and the activity of macrophages. Thl cells recognize antigen on macrophages in association with Class

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II MHC and become activated (by IL-1) to produce lymphokines, including the IFN-γ that activates macrophages and NK cells. These cells mediate various aspects of the cell-mediated immunity response including delayed-type hypersensitivity reactions. Th2 cells recognize antigen in association with Class II MHC on an antigen presenting cell or APC (e.g., migratory macrophages and dendritic cells) and then produce interleukins and other substances that stimulate specific B-cell and T-cell proliferation and activity.

Beyond serving as APCs that initiate T cell interactions, development, and proliferation, macrophages are involved in expression of cell-mediated immunity because they become activated by IFN-γ produced in a cell-mediated immune response. Activated macrophages have increased phagocytic potential and release soluble substances that cause inflammation and destroy many bacteria and other cells. Natural Killer cells are cytotoxic cells that lyse cells bearing new antigen, regardless of their MHC type, and even lyse some cells that bear no MHC proteins. Natural

Killer T cells, or NK cells, are defined by their ability to kill cells displaying a foreign antigen (e.g., tumor cells), regardless of MHC type, and regardless of previous sensitization (exposure) to the antigen. NK cells can be activated by IL-2 and IFN-γ, and lyse cells in the same manner as cytotoxic T lymphocytes. Some NK cells have receptors for the Fc domain of the IgG antibody (e.g, CD 16 or FcyRIlI) and are thus able to bind to the Fc portion of IgG on the surface of a target cell and release cytolytic components that kill the target cell via antibody-dependent cell-mediated cytotoxicity.

Another group of cells is the granulocytes or polymorphonuclear leukocytes (PMNs). Neutrophils, one type of PMN, kill bacterial invaders and phagocytose the remains. Eosinophils are another type of PMN and contain granules that prove cytotoxic when released upon another cell, such as a foreign cell. Basophils, a third type of PMN, are significant mediators of powerful physiological responses (e.g., inflammation) that exert their effects by releasing a variety of biologically active compounds, such as histamine, serotonin, prostaglandins, and leukotrienes. Common to all of these cell types is the capacity to exert a physiological effect within an organism, frequently by killing, and optionally scavenging, deleterious compositions such as foreign cells.

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Although a variety of mammalian cells, including cells of the immune system, are capable of directly exerting a physiological effect (e.g., cell killing, typified by Tc, NK, some PMN, macrophage, and the like), other cells indirectly contribute to a physiological effect. For example, initial presentation of an antigen to a naive T cell of the immune system requires MHC presentation that mandates cell-cell contact.

Further, there often needs to be contact between an activated T cell and an antigenspecific B cell to obtain a particular immunogenic response. A third form of cell-cell contact often seen in immune responses is the contact between an activated B cell and follicular dendritic cells. Each of these cell-cell contact requirements complicates the targeting of a biologically active agent to a given target.

Complement-dependent cytotoxicity (CDC) is believed to be a significant mechanism for clearance of specific target cells such as tumor cells. CDC is a series of events that consists of a collection of enzymes that become activated by each other in a cascade fashion. Complement has an important role in clearing antigen, accomplished by its four major functions: (1) local vasodilation; (2) attraction of immune cells, especially phagocytes (chemotaxis); (3) tagging of foreign organisms for phagocytosis (opsonization); and (4) destruction of invading organisms by the membrane attack complex (MAC attack). The central molecule is the C3 protein. It is an enzyme that is split into two fragments by components of either the classical pathway or the alternative pathway. The classical pathway is induced by antibodies, especially IgG and IgM, while the alternative pathway is nonspecifically stimulated by bacterial products like lipopolysaccharide (LPS). Briefly, the products of the C3 split include a small peptide C3a which is chemotactic for phagocytic immune cells and results in local vasodilation by causing the release of C5a fragment from C5. The other part of C3, C3b, coats antigens on the surface of foreign organisms and acts to opsonize the organism for destruction. C3b also reacts with other components of the complement system to form an MAC consisting of C5b, C6, C7, C8 and C9.

There are problems associated with the use of antibodies in human therapy because the response of the immune system to any antigen, even the simplest, is polyclonal, i.e,, the system manufactures antibodies of a great range of structures both in their binding regions as well as in their effector regions.

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Two approaches have been used in an attempt to reduce the problem of immunogenic antibodies. The first is the production of chimeric antibodies in which the antigen-binding part (variable regions) of a mouse monoclonal antibody is fused to the effector part (constant region) of a human antibody. In a second approach, antibodies have been altered through a technique known as complementarity determining region (CDR) grafting or humanization. This process has been further improved to include changes referred to as “reshaping” (Verhoeyen, et al., 1988 Science 239:1534-1536; Riechmann, etal., 1988 Nature 332:323-337; Tempest, etal., Bio/Technol 1991 9:266-271), “hyperchimerization” (Queen, et al., 1989 Proc Natl

AcadSci USA 86:10029-10033; Co, etal., 1991 Proc Natl Acad Sci USA 88:2869 2873; Co, et al., 1992 J Immunol 148:1149-1154), and “veneering” (Mark, et al., In·. Metcalf BW, Dalton BJ, eds. Cellular adhesion: molecular definition to therapeutic potential. New York: Plenum Press, 1994:291-312).

An average of less than one therapeutic antibody per year has been introduced to the market beginning in 1986, eleven years after the publication of monoclonal antibodies. Five murine monoclonal antibodies were introduced into human medicine over a ten year period from 1986-1995, including “muromonab-CD3” (OrthoClone 0KT3®) for acute rejection of organ transplants; “edrecolomab” (Panorex®) for colorectal cancer; “odulimomab” (Antilfa®) for transplant rejection; and, “ibritumomab” (Zevalin® yiuxetan) for non-Hodgkin’s lymphoma. Additionally, a monoclonal Fab, “abciximab” (ReoPro®) has been marketed for preventing coronary artery reocclusion. Three chimeric monoclonal antibodies were also launched: “rituximab” (Rituxan®) for treating B cell lymphomas; “basiliximab” (Simulect®)for transplant rejection; and “infliximab” (Remicade®) for treatment of rheumatoid arthritis and Crohn’s disease. Additionally, “abciximab” (ReoPro®), a 47.6 kD Fab fragment of a chimeric human-murine monoclonal antibody is marketed as an adjunct to percutaneous coronary intervention for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention. Finally, seven “humanized” monoclonal antibodies have been launched. “Daclizumab” (Zenapax®) is used to prevent acute rejection of transplanted kidneys; “palivizumab” (Synagis®) for RSV; “trastuzumab” (Herceptin®) binds HER-2, a growth factor receptor found on breast cancers cells; “gemtuzumab” (Mylotarg®) for acute myelogenous leukemia (AML); and “alemtuzumab” (MabCampath®) for chronic

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2016231617 23 Sep 2016 lymphocytic leukemia; “adalimumab” (Humira® (D2E7)) for the treatment of rheumatoid arthritis; and, “omalizumab” (Xolair®), for the treatment of persistent asthma.

Thus, a variety of antibody technologies have received attention in the effort 5 to develop and market more effective therapeutics and palliatives. Unfortunately, problems continue to compromise the promise of each of these therapies. For example, the majority of cancer patients treated with rituximab relapse, generally within about 6-12 months, and fatal infusion reactions within 24 hours of rituximab infusion have been reported. Acute renal failure requiring dialysis with instances of fatal outcome has also been reported in treatments with rituximab, as have severe, occasionally fatal, mucocutaneous reactions. Additionally, high doses of rituximab are required for intravenous injection because the molecule is large, approximately 150 kDa, and diffusion into the lymphoid tissues, where many tumor cells may reside is limited.

Trastuzumab administration can result in the development of ventricular dysfunction, congestive heart failure, and severe hypersensitivity reactions (including anaphylaxis), infusion reactions, and pulmonary events. Daclizumab immunosuppressive therapy poses an increased risk for developing lymphoproliferative disorders and opportunistic infections. Death from liver failure, arising from severe hepatotoxicity, and from veno-occlusive disease (VOD), has been reported in patients who received gemtuzumab.

Hepatotoxicity was also reported in patients receiving alemtuzumab. Serious and, in some rare instances fatal, pancytopenia/marrow hypoplasia, autoimmune idiopathic thrombocytopenia, and autoimmune hemolytic anemia have occurred in patients receiving alemtuzumab therapy. Alemtuzumab can also result in serious infusion reactions as well as opportunistic infections. In patients treated with adalimumab, serious infections and sepsis, including fatalities, have been reported, as has the exacerbation of clinical symptoms and/or radiographic evidence of demyelinating disease, and patients treated with adalimumab in clinical trials had a higher incidence of lymphoma than the expected rate in the general population. Omalizumab reportedly induces malignancies and anaphylaxis.

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Cancer includes a broad range of diseases, affecting approximately one in four individuals worldwide. Rapid and unregulated proliferation of malignant cells is a hallmark of many types of cancer, including hematological malignancies. Although patients with a hematologic malignant condition have benefited from advances in cancer therapy in the past two decades, Multani etal., 1998 J. Clin. Oncology 16:3691-3710, and remission times have increased, most patients still relapse and succumb to their disease. Barriers to cure with cytotoxic drugs include, for example, tumor cell resistance and the high toxicity of chemotherapy, which prevents optimal dosing in many patients.

Treatment of patients with low grade or follicular B cell lymphoma using a chimeric CD20 monoclonal antibody has been reported to induce partial or complete responses in patients. McLaughlin et al., 1996 Blood 88:90a (abstract, suppl. 1); Maloney et al., 1997 Blood 90:2188-95. However, as noted above, tumor relapse commonly occurs within six months to one year. Further improvements in serotherapy are needed to induce more durable responses, for example, in low grade B cell lymphoma, and to allow effective treatment of high grade lymphoma and other B cell diseases.

Another approach has been to target radioisotopes to B cell lymphomas using monoclonal antibodies specific for CD20, While the effectiveness of therapy is reportedly increased, associated toxicity from the long in vivo half-life of the radioactive antibody increases, sometimes requiring that the patient undergo stem cell rescue. Press et al., 1993 N. Eng. J. Med. 329:1219-1224; Kaminski et al., 1993 N. Eng.J. Med. 329:459-65. Monoclonal antibodies to CD20 have also been cleaved with proteases to yield F(ab’)2 or Fab fragments prior to attachment of radioisotope.

This has been reported to improve penetration of the radioisotope conjugate into the tumor and to shorten the in vivo half-life, thus reducing the toxicity to normal tissues. However, these molecules lack effector functions, including complement fixation and/or ADCC.

Autoimmune diseases include autoimmune thyroid diseases, which include

Graves' disease and Hashimoto's thyroiditis. In the United States alone, there are about 20 million people who have some form of autoimmune thyroid disease. Autoimmune thyroid disease results from the production of autoantibodies that either

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2016231617 23 Sep 2016 stimulate the thyroid to cause hyperthyroidism (Graves' disease) or destroy the thyroid to cause hypothyroidism (Hashimoto's thyroiditis). Stimulation of the thyroid is caused by autoantibodies that bind and activate the thyroid stimulating hormone (TSH) receptor. Destruction of the thyroid is caused by autoantibodies that react with other thyroid antigens. Current therapy for Graves' disease includes surgery, radioactive iodine, or antithyroid drug therapy. Radioactive iodine is widely used, since antithyroid medications have significant side effects and disease recurrence is high. Surgery is reserved for patients with large goiters or where there is a need for very rapid normalization of thyroid function. There are no therapies that target the production of autoantibodies responsible for stimulating the TSH receptor. Current therapy for Hashimoto's thyroiditis is levothyroxine sodium, and lifetime therapy is expected because of the low likelihood of remission. Suppressive therapy has been shown to shrink goiters in Hashimoto's thyroiditis, but no therapies that reduce autoantibody production to target the disease mechanism are known.

Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints, leading to swelling, pain, and loss of function. RA affects an estimated 2.5 million people in the United States. RA is caused by a combination of events including an initial infection or injury, an abnormal immune response, and genetic factors. While autoreactive T cells and B cells are present in RA, the detection of high levels of antibodies that collect in the joints, called rheumatoid factor, is used in the diagnosis of RA. Current therapy for RA includes many medications for managing pain and slowing the progression of the disease. No therapy has been found that can cure the disease. Medications include nonsteroidal anti-inflammatory drugs (NSAIDS), and disease modifying anti-rheumatic drugs (DMARDS). NSAIDS are useful in benign disease, but fail to prevent the progression to joint destruction and debility in severe RA. Both NSAIDS and DMARDS are associated with significant side effects. Only one new DMARD, Leflunomide, has been approved in over 10 years. Leflunomide blocks production of autoantibodies, reduces inflammation, and slows progression of RA. However, this drug also causes severe side effects including nausea, diarrhea, hair loss, rash, and liver injury.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by recurrent injuries to blood vessels in multiple organs, including the kidney, skin, and

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WO 2007/146968 PCT/US2007/071052 joints. SLE is estimated to affect over 500,000 people in the United States. In patients with SLE, a faulty interaction between T cells and B cells results in the production of autoantibodies that attack the cell nucleus. These include anti-double stranded DNA and anti-Sm antibodies. Autoantibodies that bind phospholipids are also found in about half of SLE patients, and are responsible for blood vessel damage and low blood counts. Immune complexes accumulate in the kidneys, blood vessels, and joints of SLE patients, where they cause inflammation and tissue damage. No treatment for SLE has been found to cure the disease, NSAIDS and DMARDS are used for therapy depending upon the severity of the disease. Plasmapheresis with plasma exchange to remove autoantibodies can cause temporary improvement in SLE patients. There is general agreement that autoantibodies are responsible for SLE, so new therapies that deplete the B cell lineage, allowing the immune system to reset as new B cells are generated from precursors, would offer hope for long lasting benefit in SLE patients.

Sjogren's syndrome is an autoimmune disease characterized by destruction of the body’s moisture-producing glands. Sjogren's syndrome is one of the most prevalent autoimmune disorders, striking up to an estimated 4 million people in the United States. About half of the people stricken with Sjogren’s syndrome also have a connective tissue disease, such as RA, while the other half have primary Sjogren’s syndrome with no other concurrent autoimmune disease. Autoantibodies, including anti-nuclear antibodies, rheumatoid factor, anti-fodrin, and anti-muscarinic receptor are often present in patients with Sjogren's syndrome. Conventional therapy includes corticosteroids, and additional more effective therapies would be of benefit.

Immune thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to blood platelets and cause their destruction. Some cases of ITP are caused by drugs, and others are associated with infection, pregnancy, or autoimmune disease such as SLE. About half of all cases are classified as being of idiopathic origin. The treatment of ITP is determined by the severity of the symptoms. In some cases, no therapy is needed although in most cases immunosuppressive drugs, including corticosteroids or intravenous infusions of immune globulin to deplete T cells, are provided. Another treatment that usually results in an increased number of platelets is removal of the spleen, the organ that destroys antibody-coated platelets. More potent

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2016231617 23 Sep 2016 immunosuppressive drugs, including cyclosporine, cyclophosphamide, or azathioprine are used for patients with severe cases. Removal of autoantibodies by passage of patients’ plasma over a Protein A column is used as a second line treatment in patients with severe disease. Additional more effective therapies are needed.

Multiple sclerosis (MS) is also an autoimmune disease. It is characterized by inflammation of the central nervous system and destruction of myelin, which insulates nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are primary contributors to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebrospinal fluid of patients with MS, and some predict that the B cell response leading to antibody production is important for mediating the disease. No B cell depletion therapies have been studied in patients with MS, and there is no cure for MS. Current therapy is corticosteroids, which can reduce the duration and severity of attacks, but do not affect the course of MS over time. New biotechnology interferon (IFN) therapies for MS have recently been approved but additional more effective therapies are required.

Myasthenia Gravis (MG) is a chronic autoimmune neuromuscular disorder that is characterized by weakness of the voluntary muscle groups. MG affects about

40,000 people in the United States. MG is caused by autoantibodies that bind to acetylcholine receptors expressed at neuromuscular junctions. The autoantibodies reduce or block acetylcholine receptors, preventing the transmission of signals from nerves to muscles. There is no known cure for mg. Common treatments include immunosuppression with corticosteroids, cyclosporine, cyclophosphamide, or azathioprine. Surgical removal of the thymus is often used to blunt the autoimmune response. Plasmapheresis, used to reduce autoantibody levels in the blood, is effective in mg, but is short-lived because the production of autoantibodies continues. Plasmapheresis is usually reserved for severe muscle weakness prior to surgery. New and effective therapies would be of benefit.

Psoriasis affects approximately five million people, and is characterized by autoimmune inflammation in the skin. Psoriasis is also associated with arthritis in 30% (psoriatic arthritis). Many treatments, including steroids, uv light retinoids,

2016231617 23 Sep 2016 vitamin D derivatives, cyclosporine, and methotrexate have been used but it is also clear that psoriasis would benefit from new and effective therapies. Scleroderma is a chronic autoimmune disease of the connective tissue that is also known as systemic sclerosis. Scleroderma is characterized by an overproduction of collagen, resulting in a thickening of the skin, and approximately 300,000 people in the United States have scleroderma, which would also benefit from new and effective therapies.

Apparent from the foregoing discussion are needs for improved compositions and methods to treat, ameliorate or prevent a variety of diseases, disorders and conditions, including cancer and autoimmune diseases.

SUMMARY

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The invention satisfies at least one of the aforementioned needs in the art by providing proteins containing at least two specific binding domains, wherein those two domains are linked by a constant sub-region derived from an antibody molecule attached at its C-terminus to a linker herein referred to as a scorpion linker, and nucleic acids encoding such proteins, as well as production, diagnostic and therapeutic uses of such proteins and nucleic acids. The constant sub-region comprises a domain derived from an immunoglobulin Ch2 domain, and preferably a domain derived from an immunoglobulin Ch3 domain, but does not contain a domain or region derived from, or corresponding to, an immunoglobulin Chi domain. Previously, it had been thought that the placement of a constant region derived from an antibody in the interior of a protein would interfere with antibody function, such as effector function, by analogy to the conventional placement of constant regions of antibodies at the carboxy termini of antibody chains. In addition, placement of a scorpion linker, which may be an immunoglobulin hinge-like peptide, Cterminal to a constant sub-region is an organization that differs from the organization of naturally occurring immunoglobulins. Placement of a constant sub-region (with a scorpion linker attached C-terminal to the constant region) in the interior of a polypeptide or protein chain in accordance with the invention, however, resulted in proteins exhibiting effector

50β«27_1 (GHMatters) P79707.AU 1

2016231617 23 Sep 2016 function and multivalent (mono- or multi-specific) binding capacities relatively unencumbered by steric hindrances. As will be apparent to one of skill in the art upon consideration of this disclosure, such proteins are modular in design and may be constructed by selecting any of a variety of binding domains for binding domain 1 or binding domain 2 (or for any additional binding domains found in a particular protein according to the invention), by selecting a constant sub-region having effector function, and by selecting a scorpion linker, hinge-like or non-hinge like (e.g., type II C-lectin receptor stalk region peptides), with the protein exhibiting a general organization of Nbinding domain 1-constant sub-region-scorpion linker- binding domain 2-C. Those of skill will further appreciate that proteins of such structure, and the nucleic acids encoding those proteins, will find a wide variety of applications, including medical and veterinary applications.

One aspect of the invention provides a single-chain binding protein comprising from amino-terminus to carboxy-terminus:

(a) a first binding domain comprising variable regions from an immunoglobulin or immunoglobulin-like molecule;

(b) an immunoglobulin constant sub-region that comprises immunoglobulin CH2 and CH3 domains;

(c) a scorpion linker peptide, wherein said scorpion linker peptide comprises an amino acid sequence derived from a hinge of an immunoglobulin or a stalk region of a type II C-lectin protein; and (d) a second binding domain comprising variable regions from an immunoglobulin or immunoglobulin-like molecule.

One further aspect of the invention is drawn to a multivalent single-chain binding protein with effector function, or scorpion (the terms are used interchangeably), comprising a first binding domain derived from an immunoglobulin (e.g., an antibody) or an immunoglobulin-like molecule, a constant sub-region providing an effector function, the constant sub-region located C-terminal to the first binding domain; a scorpion linker located C-terminal to the constant sub-region; and a second binding domain derived from an immunoglobulin (such as an antibody) or immunoglobulin-like molecule, located Cterminal to the constant sub-region; thereby localizing the constant sub-region between the first binding domain and the second binding domain. The single-chain binding protein may be multispecific, e.g., bispecific in that it could bind two or more distinct targets, or it may be monospecific, with two binding sites for the same target. Moreover, all of the domains of the protein are found in a single chain, but the protein may form homo-multimers, e.g., by interchain disulfide bond formation. In some embodiments, the first binding domain and/or the second binding domain is/are derived from variable regions of light and heavy (<3ΗΜβ„·«> P79767.AU.1

21a

2016231617 23 Sep 2016 immunoglobulin chains from the same, or different, immunoglobulins (e.g., antibodies). The immunoglobulin(s) may be from any vertebrate, such as a mammal, including a human, and may be chimeric, humanized, fragments, variants or derivatives of naturally occurring immunoglobulins.

The invention contemplates proteins in which the first and second binding domains are derived from the same, or different immunoglobulins (e.g., antibodies), and wherein the first and second binding domains recognize the same, or different, molecular targets (e.g., cell surface markers, such as membrane-bound proteins). Further, the first and second binding domains may recognize the same, or different, $065527.1 (GHMellere) P797&7AU1

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2016231617 23 Sep 2016 epitopes. The first and second molecular targets may be associated with first and second target cells, viruses, carriers and/or objects. In preferred embodiments according to this aspect of the invention, each of the first binding domain, second binding domain, and constant sub-region is derived from a human immunoglobulin, such as an IgG antibody. In yet other embodiments, the multivalent binding protein with effector function has at least one of the first binding domain and the second binding domain that recognizes at least one cell-free molecular target, e.g., a protein unassociated with a cell, such as a deposited protein or a soluble protein. Cell-free molecular targets include, e.g., proteins that were never associated with a cell, e.g., administered compounds such as proteins, as well as proteins that are secreted, cleaved, present in exosomes, or otherwise discharged or separated from a cell.

The target molecules recognized by the first and second binding domains may be found on, or in association with, the same, or different, prokaryotic cells, eukaryotic cells, viruses (including bacteriophage), organic or inorganic target molecule carriers, and foreign objects. Moreover, those target molecules may be on physically distinct cells, viruses, carriers or objects of the same type (e.g., two distinct eukaryotic cells, prokaryotic cells, viruses or carriers) or those target molecules may be on cells, viruses, carriers, or objects that differ in type (e.g., a eukaryotic cell and a virus). Target cells are those cells associated with a target molecule recognized by a binding domain and includes endogenous or autologous cells as well as exogenous or foreign cells (e.g., infectious microbial cells, transplanted mammalian cells including transfused blood cells). The invention comprehends targets for the first and/or second binding domains that are found on the surface of a target cell(s) associated with a disease, disorder or condition of a mammal such as a human. Exemplary target cells include a cancer cell, a cell associated with an autoimmune disease or disorder, and an infectious cell (e.g., an infectious bacterium), A cell of an infectious organism, such as a mammalian parasite, is also contemplated as a target cell. In some embodiments, a protein of the invention is a multivalent (e.g., multispecific) binding protein with effector function wherein at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a

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Dendritic cell target, an NK cell target, a monocyte/macrophage cell target and an angiogenesis target.

In some embodiments of the above-described protein, the tumor antigen is selected from the group consisting of SQUAMOUS CELL CARCINOMA ANTIGEN

1 (SCCA-1), (PROTEIN T4-A), SQUAMOUS CELL CARCINOMA ANTIGEN 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B) (KJAA0049), MUCIN 1 (TUMOR-ASSOCIATED MUCIN), (CARCINOMA-ASSOCIATED MUCIN), (POLYMORPHIC EPITHELIAL MUCIN),(ΡΕΜ),(ΡΕΜΤ),(EPI SI ALIN), (TUMOR-ASSOCIATED EPITHELIAL MEMBRANE

ANTIGEN),(EMA),(H23AG), (PEANUT-REACTIVE URINARY MUCIN), (PUM), (BREAST CARCINOMA- ASSOCIATED ANTIGEN DF3), CTCL tumor antigen sel-1, CTCL tumor antigen sel4-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-l, CTCL tumor antigen se37-2, CTCL tumor antigen se57-l, CTCL tumor antigen se89-l, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi’s sarcoma-associated herpesvirus, MAGE-CI (cancer/testis antigen CT7), MAGE-B1 ANTIGEN (MAGEXP ANTIGEN) (DAM 10), MAGE-B2 ANTIGEN (DAM6), MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU-12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated brain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplastic neuronal antigen), Neuro-oncological ventral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma, X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NY-BR15, Serologically defined breast cancer antigenNY-BR-16, Chromogranin A; parathyroid secretory protein 1, DUP AN-2, CA 19-9, CA 72-4, CA 195 and L6.

Embodiments of the above-described method comprise a B cell target selected from the group consisting of CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37,

CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b,

CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDwl30, CD138 and CDwl50.

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In other embodiments of the above-described method, the TNF receptor superfamily member is selected from the group consisting of 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSFl IB,

BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11A,

CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B,

DcR3/TNFRSF6B, TNF RI/TNFRSF1 A, DcTRAIL R1/TNFRSF23, TNF RII/TNFRSF1B, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A, DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, EDAR, TRAIL R4/TNFRSF10D, Fas/TNFRSF6,

TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, Lymphotoxin beta R/TNFRSF3,4-1 BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin beta/TNFSF3, BAFF/TNFSF13C, 0X40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-alpha/TNFSFlA, CD40 Ligand/TNFSF5, TNF-beta/TNFSFIB, EDA-A2,

TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.

The above-described method also includes embodiments in which the Hedgehog family member is selected from the group consisting of Patched and Smoothened. In yet other embodiments, the proteoglycan-related molecule is selected from the group consisting of proteoglycans and regulators thereof.

Additional embodiments of the method are drawn to processes in which the receptor tyrosine kinase is selected from the group consisting of Axl, FGF R4, Clq R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF-I R, EGF R, IGF-II R, Eph, INSRR, EphAl, Insulin R/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4,

MSP R/Ron, EphA5, MuSK, EphA6, PDGF R alpha, EphA7, PDGF R beta, EphA8, Ret, EphBl, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, Tie2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF Rl, VEGF Rl/Flt-1, FGF R2, VEGF R2/Flk-1, FGF R3 and VEGF R3/Flt-4.

In other embodiments of the method, the Transforming Growth Factor (TGF)30 beta superfamily member is selected from the group consisting of Activin RIA/ALK2, GFR alpha-1, Activin RIB/ALK-4, GFR alpha-2, Activin RIIA, GFR alpha-3, Activin RUB, GFR alpha-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGFWO 2007/146968

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2016231617 23 Sep 2016 beta RI/ALK-5, BMPR-IB/ALK-6, TGF-beta RII, BMPR-II, TGF-beta Rllb, Endoglin/CD105 and TGF-beta RJII.

Yet other embodiments of the method comprise a Wnt-related molecule selected from the group consisting of Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9,

Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4,

Frizzled-7, MFRP, LRP 5, LRP 6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-lOb, Wnt-4, Wnt-11, Wnt-5a, Wnt-9a and Wnt-7a.

In other embodiments of the method, the receptor ligand is selected from the group consisting of 4-1BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13,

Lymphotoxin beta/TNFSF3, BAFF/TNFSF13C, 0X40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-alpha/TNFSFlA, CD40 Ligand/TNFSF5, TNF-beta/TNFSFIB, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12, LIGHT/TNFSF14, Amphiregulin, NRG1 isoform GGF2, Betacellulin, NRG1 Isoform

SMDF, EGF, NRG 1-alpha/HRGl-alpha, Epigen, NRGl-beta 1/HRGl-beta 1,

Epiregulin, TGF-alpha, HB-EGF, TMEFFl/Tomoregulin-1, Neuregulin-3, TMEFF2, IGF-I, IGF-II, Insulin, Activin A, Activin B, Activin AB, Activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5,

Decapentaplegic, BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6,

GDF-15, GDF-7, Artemin, Neurturin, GDNF, Persephin, TGF-beta, TGF-beta 2, TGF-beta 1, TGF-beta 3, LAP (TGF-beta 1), TGF-beta 5, Latent TGF-beta 1, Latent TGF-beta bpl, TGF-beta 1.2, Lefty, Nodal, MIS/AMH, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin-1, PIGF,

Neuropilin-2, P1GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGFC, PDGF-D, VEGF-D and PDGF-AB.

In still other embodiments, the T-cell target is selected from the group consisting of 2B4/SLAMF4, IL-2 R alpha, 4-1BB/TNFRSF9, IL-2 R beta, ALCAM, B7-1/CD80, IL-4 R, B7-H3, BLAME/SLAMF8, BTLA, IL-6 R, CCR3, IL-7 R alpha,

CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R alpha, CCR7, IL-10 R beta, CCR8, IL-12 R beta 1, CCR9, IL-12 R beta 2, CD2, IL-13 R alpha 1, IL-13, CD3, CD4, ILT2/CD85J, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, Integrin alpha 4/CD49d,

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CD5, Integrin alpha E/CD103, CD6, Integrin alpha M/CD1 lb, CD8, Integrin alpha X/CD 11c, Integrin beta 2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD3O/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 LigandZTNFSF5, LAG-3, CD43, LAIRI, CD45, LAIR2, CD83, Leukotriene B4 RI,

CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common gamma Chain/IL-2 R gamma, Osteopontin, CRACC/SLAMF7, PD-1, CRT AM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP beta 1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoietin,

EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas

Ligand/TNFSF6, TIM-4, Fc gamma RIII/CD16, TIM-6, GITR/TNFRSF18, TNF RI/TNFRSF1A, Granulysin, TNF RII/TNFRSF1B, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-gamma RI, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP,

IL-1 RI and TSLP R.

In other embodiments, the NK cell receptor is selected from the group consisting of 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, DNAM-1, LMIR5/CD300LB, Fc epsilon RII, LMIR6/CD300LE, Fc gamma

RI/CD64, MICA, Fc gamma RIIB/CD32b, MICB, Fc gamma RIIC/CD32c, MULT-1, Fc gamma RIIA/CD32a, Nectin-2/CDl 12, Fc gamma RIII/CD16, NKG2A, FcRHl/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTAl, NKp30, FcRH5/IRTA2, NKp44, Fc Receptor-like 3/CD16-2, NKp46/NCRl, NKp80/KLRFl, NTB-A/SLAMF6, Rae-1, Rae-1 alpha, Rae-1 beta, Rae-1 delta, H60, Rae-1 epsilon,

ILT2/CD85j, Rae-1 gamma, ILT3/CD85k, TREM-1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3.

In other embodiments, the monocyte/macrophage cell target is selected from the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common beta Chain, Integrin alpha 4/CD49d, BLAME/SLAMF8, Integrin alpha X/CD11 c, CCL6/CI0, Integrin beta 2/CD18, CD155/PVR, Integrin beta 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3, LeukotrieneB4 RI, CD40/TNFRSF5,

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LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD 163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc gamma RI/CD64, Osteopontin, Fc gamma RIIB/CD32b, PD-L2, Fc gamma

RIIC/CD32c, Siglec-3/CD33, Fc gamma RIIA/CD32a, SIGNR1/CD209, Fc gamma RIII/CD16, SLAM, GM-CSF R alpha, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFNgamma RI, TLR4, IFN-gamma R2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF4, IL-10 R alpha, ALCAM,

IL-10 R beta, Aminopeptidase N/ANPEP, ILT2/CD85j, Common beta Chain,

ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin alpha 4/CD49d, CCR5, Integrin alpha M/CD1 lb, CCR8, Integrin alpha X/CD1 lc, CD155/PVR, Integrin beta 2/CD18, CD14, Integrin beta 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotriene B4 RI, CD68, LIMPII/SR-B2,

CD84/SLAMF5, LMIRI/CD300A, CD97, LMIR2/CD300c, CD163,

LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, Fc gamma RI/CD64, PSGL-1, Fc gamma R1II/CD16, RP105, G-CSF R,

L-Selectin, GM-CSF R alpha, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL-6 R, TREM-2, CXCR1/IL-8 RA, TREM-3 and TREML1/TLT-1.

In yet other embodiments of the method, a Dendritic cell target is selected from the group consisting of CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163,

SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB Ligand/TNFSF9, IL-12/IL23 p40,4-Amino-l,8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8οχο-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin alpha 4/CD49d, Aag, Integrin beta 2/CD18, AMICA, Langerin, B7-2/CD86, Leukotriene B4 RI, B7-H3,

LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2FWO 2007/146968

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10/SLAMF9, Osteoactivin/GPNMB, Chem 23, PD-L2, CLEC-1, RP105, CLEC-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DCSIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, SigIec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209, DEP5 1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc gamma

RI/CD64, TLR3, Fc gamma RIIB/CD32b, TREM-1, Fc gamma RIIC/CD32c, TREM2, Fc gamma RIIA/CD32a, TREM-3, Fc gamma RIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid RE

In still other embodiments of the method, the angiogenesis target is selected 10 from the group consisting of Angiopoietin-1, Angiopoietin-like 2, Angiopoietin-2,

Angiopoietin-like 3, Angiopoietin-3, Angiopoietin-like 7/CDT6, Angiopoietin-4, Tie1, Angiopoietin-like 1, Tie-2, Angiogenin, iNOS, Coagulation Factor III/Tissue Factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr, EGVEGF/PK1, Proliferin, Endostatin, ROBO4, Erythropoietin, Thrombospondin-1,

Kininostatin, Thrombospondin-2, MFG-E8, Thrombospondin-4, Nitric Oxide, VG5Q, eNOS, EphAl, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphBl, EphB4, EphB2, EphB6, EphB3, Ephrin-Al, Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-Bl, Ephrin-B3, Ephrin-B2, FGF acidic, FGF-12, FGF basic, FGF13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF20 21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF R1, FGF R4, FGF R2, FGF

R5, FGF R3, Neuropilin-1, Neuropilin-2, Semaphorin 3A, Semaphorin 6B, Semaphorin 3C, Semaphorin 6C, Semaphorin 3E, Semaphorin 6D, Semaphorin 6A, Semaphorin 7A, MMP, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP-15, MMP-8, MMP-16/MT3-MMP, MMP-9, MMP25 24/MT5-MMP, MMP-10, MMP-25/MT6-MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4,

ACE, IL-13 R alpha 1, IL-13, Clq R1/CD93, Integrin alpha 4/CD49d, VE-Cadherin, Integrin beta 2/CD18, CD31/PECAM-1, KLF4, CD36/SR-B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Nectin-2/CDl 12, Coagulation Factor III/Tissue Factor, E-Selectin, D6, P-Selectin, DC-SIGNR/CD299, SLAM, EMMPR1N/CD147, Tie-2,

Endoglin/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1B, Erythropoietin R, TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF10B, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/FR-4, IL-1 Rl and VG5Q.

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Other embodiments of the method provide multivalent binding proteins wherein at least one of binding domain 1 and binding domain 2 specifically binds a target selected from the group consisting of Prostate-specific Membrane Antigen (Folate Hydrolase 1), Epidermal Growth Factor Receptor (EGFR), Receptor for

Advanced Glycation End products (RAGE, also known as Advanced Glycosylation End product Receptor or AGER), IL-17 A, IL-17 F, P19 (IL23A and IL12B), Dickkopf-1 (Dkkl), NOTCH1, NG2 (Chondroitin Sulfate ProteoGlycan 4 or CSPG4), IgE (IgHE or IgH2), IL-22R (IL22RA1), IL-21, Amyloid β oligomers (Ab oligomers), Amyloid β Precursor Protein (APP), NOGO Receptor (RTN4R), Low Density

LipoproteinReceptor-Related Protein 5 (LRP5), IL-4, Myostatin (GDF8), Very Late Antigen 4, an alpha 4, beta 1 integrin (VLA4 or ITGA4), an alpha 4, beta 7 integrin found on leukocytes, and IGF-1R. For example, a VLA4 target may be recognized by a multivalent binding protein in which at least one of binding domain 1 and binding domain 2 is a binding domain derived from Natalizumab (Antegren).

In some embodiments, the cancer cell is a transformed, or cancerous, hematopoietic cell. In certain of these embodiments, at least one of the first binding domain and the second binding domain recognizes a target selected from the group consisting of a B-cell target, a monocyte/macrophage target, a dendritic cell target, an NK-cell target and a T-cell target, each as herein defined. Further, at least one of the first binding domain and the second binding domain can recognize a myeloid targets, including but not limited to, CD5, CD 10, CDllb, CD1 lc, CD 13, CD 14, CD 15,

CDI8, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD29, CD30, CD31, CD33, CD34, CD35, CD38, CD43, CD45, CD64, CD66, CD68, CD70, CD80, CD86, CD87, CD88, CD89, CD98, CD100, CD103, CD111, CD112, CD114, CD115, CD116,

CD117, CD118, CD119, CD120a, CD120b, CDwl23, CDwl3l, CD141, CD162,

CD163, CD177, CD312,1RTA1, IRTA2, IRTA3, IRTA4, IRTA5, B-B2, B-B8 and Bcell antigen receptor.

Other embodiments of the invention are drawn to the multivalent binding protein, as described herein, comprising a sequence selected from the group consisting of SEQ ID NOS:2,4,6, 103, 105, 107, 109, 332,333, 334, and 345. Other embodiments are directed to the multivalent binding protein comprising a sequence

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WO 2007/146968 PCT/US2007/071052 selected from the group consisting of SEQ ID NOS:355, 356, 357, 358, 359, 360, 361, 362, 363,364 and 365.

In other embodiments, the multivalent and multispecific binding protein with effector function has a first binding domain and a second binding domain that recognize a target pair selected from the group consisting of EPHB4-KDR and TIETEK. In such embodiments, the protein has a first binding domain recognizing EPHB4 and a second binding domain recognizing KDR or a first binding domain recognizing KDR and a second binding domain recognizing EPHB4, Analogously, the protein may have a first binding domain recognizing TIE and a second binding domain recognizing TEK, or a first binding domain recognizing TEK and a second binding domain recognizing TIE.

In a related aspect, the invention provides a multivalent binding protein with effector function, wherein the constant sub-region recognizes an effector cell Fc receptor (e.g., FcyRI, FqyRII, FcyRIII, FcaR, and Fc£RI. In particular embodiments, the constant sub-region recognizes an effector cell surface protein selected from the group consisting of CD2, CD3, CD 16, CD28, CD32, CD40, CD56, CD64, CD89, FceRI, KIR, thrombospondin R, NKG2D, 2B4/NAIL and 4IBB. The constant subregion may comprise a Ch2 domain and a Ch3 domain derived from the same, or different, immunoglobulins, antibody isotypes, or allelic variants. In some embodiments, the Ch3 domain is truncated and comprises a C-terminal sequence selected from the group consisting of SEQ ID NOS: 366, 367, 368,369, 370 and 371. Preferably, the Chi domain and the scorpion linker are derived from the same class, or from the same sub-class, of immunoglobulin, when the linker is a hinge-like peptide derived from an immunoglobulin.

Some proteins according to the invention are also contemplated as further comprising a scorpion linker of at least about 5 amino acids attached to the constant sub-region and attached to the second binding domain, thereby localizing the scorpion linker between the constant sub-region and the second binding domain. Typically, the scorpion linker peptide length is between 5-45 amino acids. Scorpion linkers include hinge-like peptides derived from immunoglobulin hinge regions, such as IgGl, IgG2, IgG3, IgG4, IgA, and IgE hinge regions. Preferably, a hinge-like scorpion linker will retain at least one cysteine capable of forming an interchain disulfide bond under

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WO 2007/146968 PCT/US2007/071052 physiological conditions. Scorpion linkers derived from IgGl may have 1 cysteine or two cysteines, and will preferably retain the cysteine corresponding to an N-terminal hinge cysteine of IgGl. In some embodiments, the scorpion linker is extended relative to a cognate immunoglobulin hinge region and, in exemplary embodiments, comprises a sequence selected from the group consisting of SEQ ID NOS:351, 352, 353 and 354. Non-hinge-like peptides are also contemplated as scorpion linkers, provided that such peptides provide sufficient spacing and flexibility to provide a single-chain protein capable of forming two binding domains, one located towards each protein terminus (N and C) relative to a more centrally located constant sub10 region domain. Exemplary non-hinge-like scorpion linkers include peptides from the stalk region of type II C-lectins, such as the stalk regions of CD69, CD72, CD94, NKG2A and NKG2D. In some embodiments, the scorpion linker comprises a sequence selected from the group consisting of SEQ ID NOS:373, 374,375, 376 and 377.

The proteins may also comprise a linker of at least about 5 amino acids attached to the constant sub-region and attached to the first binding domain, thereby localizing the linker between the constant sub-region and the first binding domain. In some embodiments, linkers are found between the constant sub-region and each of the two binding domains, and those linkers may be of the same or different sequence, and of the same or different lengths.

The constant sub-region of the proteins according to the invention provides at least one effector function. Any effector function known in the art to be associated with an immunoglobulin (e.g., an antibody) is contemplated, such as an effector function selected from the group consisting of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), relatively extended in vivo half-life (relative to the same molecule lacking a constant sub-region), FcR binding, protein A binding, and the like. In some embodiments, the extended halflives of proteins of the invention are at least 28 hours in a human. Of course, proteins intended for administration to non-human subjects will exhibit relatively extended half-lives in those non-human subjects, and not necessarily in humans.

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In general, the proteins (including polypeptides and peptides) of the invention exhibit a binding affinity of less than IO⁹ M, or at least 10'⁶ M, for at least one of the first binding domain and the second binding domain.

Another aspect of the invention is drawn to a pharmaceutical composition 5 comprising a protein as described herein and a pharmaceutically acceptable adjuvant, carrier or excipient. Any adjuvant, carrier, or excipient known in the art is useful in the pharmaceutical compositions of the invention.

Yet another aspect of the invention provides a method of producing a protein as described above comprising introducing a nucleic acid encoding the protein into a host cell and incubating the host cell under conditions suitable for expression of the protein, thereby expressing the protein, preferably at a level of at least 1 mg/liter. In some embodiments, the method further comprises isolating the protein by separating it from at least one protein with which it is associated upon intracellular expression. Suitable host cells for expressing the nucleic acids to produce the proteins of the invention include, but are not limited to, a host cell selected from the group consisting of a VERO cell, a HeLa cell, a CHO cell, a COS cell, a W138 cell, a BHK cell, a HepG2 cell, a 3T3 cell, a RIN cell, an MDCK cell, an A549 cell, a PC 12 cell, a K.562 cell, a HEK293 cell, an N cell, a Spodoptera frugiperda cell, a Saccharomyces cerevisiae cell, a Pichia pastoris cell, any of a variety of fungal cells and any of a variety of bacterial cells (including, but not limited to, Escherichia coli, Bacillus subtilis, Salmonella typhimurium, and a Streptomycete).

The invention also provides a method of producing a nucleic acid encoding the protein, as described above, comprising covalently linking the 3’ end of a polynucleotide encoding a first binding domain derived from an immunoglobulin variable region to the 5 ’ end of a polynucleotide encoding a constant sub-region, covalently linking the 5’ end of a polynucleotide encoding a scorpion linker to the 3’ end of the polynucleotide encoding the constant sub-region, and covalently linking the 5’ end of a polynucleotide encoding a second binding domain derived from an immunoglobulin variable region to the 3’ end of the polynucleotide encoding the scorpion linker, thereby generating a nucleic acid encoding a multivalent binding protein with effector function. Each of these coding regions may be separated by a coding region for a linker or hinge-like peptide as part of a single-chain structure

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2016231617 23 Sep 2016 according to the invention. In some embodiments, the method produces a polynucleotide encoding a first binding domain that comprises a sequence selected from the group consisting of SEQ ID NO: 2 (anti-CD20 variable region, oriented VlVh), SEQ ID NO: 4 (anti-CD28 variable region, oriented Vl-Vh) and SEQ ID NO: 6 (anti-CD28 variable region, oriented Vh-Vl) in single-chain form, rather than requiring assembly from separately encoded polypeptides as must occur for heteromultimeric proteins, including natural antibodies. Exemplary polynucleotide sequences encoding first binding domains are polynucleotides comprising any of SEQ ID NOS: 1,3 or 5.

This aspect of the invention also provides methods for producing encoding nucleic acids that further comprise a linker polynucleotide inserted between the polynucleotide encoding a first binding domain and the polynucleotide encoding a constant sub-region, the linker polynucleotide encoding a peptide linker of at least 5 amino acids. Additionally, these methods produce nucleic acids that further comprise a linker polynucleotide inserted between the polynucleotide encoding a constant subregion and the polynucleotide encoding a second binding domain, the linker polynucleotide encoding a peptide linker of at least 5 amino acids. Preferably, the encoded peptide linkers are between 5 and 45 amino acids.

The identity of the linker regions present either between BD1 and EFD or

EFD and BD2 may be derived from other sequences identified from homologous -Ig superfamily members. In developing novel linkers derived from existing sequences present in homologous members of the -Ig superfamily, it may be preferable to avoid sequence stretches similar to those located between the end of a C-like domain and the transmembrane domain, since such sequences are often substrates for protease cleavage of surface receptors from the cell to create soluble forms. Sequence comparisons between different members of the -Ig superfamily and subfamilies can be compared for similarities between molecules in the linker sequences that join multiple V-like domains or between the V and C like domains. From this analysis, conserved, naturally occurring sequence patterns may emerge; these sequences when used as the linkers between subdomains of the multivalent fusion proteins should be more protease resistant, might facilitate proper folding between Ig loop regions, and

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2016231617 23 Sep 2016 would not be immunogenic since they occur in the extracellular domains of endogenous cell surface molecules.

The nucleic acids themselves comprise another aspect of the invention, Contemplated are nucleic acids encoding any of the proteins of the invention described herein, As such, the nucleic acids of the invention comprise, in 5’ to 3’ order, a coding region for a first binding domain, a constant sub-region sequence, and a coding region for a second binding domain. Also contemplated are nucleic acids that encode protein variants wherein the two binding domains and the constant subregion sequences are collectively at least 80%, and preferably at least 85%, 90%,

95%, or 99% identical in amino acid sequence to the combined sequences of a known immunoglobulin variable region sequence and a known constant sub-region sequence. Alternatively, the protein variants of the invention are encoded by nucleic acids that hybridize to a nucleic acid encoding a non-variant protein of the invention under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. Variant nucleic acids of the invention exhibit the capacity to hybridize under the conditions defined immediately above, or exhibit 90%, 95%,

99%, or 99.9% sequence identity to a nucleic acid encoding a non-variant protein according to the invention,

In related aspects, the invention provides a vector comprising a nucleic acid as described above, as well as host cells comprising a vector or a nucleic acid as described herein. Any vector known in the art may be used (e.g., plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, shuttle vectors and the like) and those of skill in the art will recognize which vectors are particularly suited for a given purpose. For example, in methods of producing a protein according to the invention, an expression vector operable in the host cell of choice is selected.

In like manner, any host cell capable of being genetically transformed with a nucleic acid or vector of the invention is contemplated. Preferred host cells are higher eukaryotic host cells, although lower eukaryotic (e.g., yeast) and prokaryotic (bacterial) host cells are contemplated.

Another aspect of the invention is drawn to a method of inducing damage to a target cell comprising contacting a target cell with a therapeutically effective amount

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2016231617 23 Sep 2016 of a protein as described herein. In some embodiments, the target cell is contacted in vivo by administration of the protein, or an encoding nucleic acid, to an organism in need. Contemplated within this aspect of the invention are methods wherein the multivalent single-chain binding protein induces an additive amount of damage to the target cell, which is that amount of damage expected from the sum of the damage attributable to separate antibodies comprising one or the other of the binding domains. Also contemplated are methods wherein the multivalent single-chain binding protein induces a synergistic amount of damage to the target cell compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. In some embodiments, the multivalent single-chain binding protein is multispecific and comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD19/CD20, CD20/CD21, CD20/CD22, CD20/CD40, CD20/CD79a, CD20/CD79b, CD20/CD81,

CD21/CD79b, CD37/CD79b, CD79b/CD81, CD19/CLII (i.e., MHC class II),

CD20/CLII, CD30/CLII, CD37/CLII, CD72/CLII, and CD79b/CL II.

This aspect of the invention also comprehends methods wherein the multispecific, multivalent single-chain binding protein induces an inhibited amount of damage to the target cell compared to the sum of the damage induced by a first antibody comprising the first binding domain but not the second binding domain and a second antibody comprising the second binding domain but not the first binding domain. Exemplary embodiments include methods wherein the multi-specific, multivalent single-chain binding protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20/CLII,

CD21/CD79L·, CD22/CD79b, CD40/CD79b, CD70/CD79b, CD72/CD79b,

CD79a/CD79b, CD79b/CD80, CD79b/CD86, CD21/CLII, CD22/CL II, CD23/CL II, CD40/CLII, CD70/CLII, CD80/CLII, CD86/CLII, CD19/CD22, CD20/CD22, CD21/CD22, CD22/CD23, CD22/CD30, CD22/CD37, CD22/CD40, CD22/CD70, CD22/CD72, CD22/79a, CD22/79b, CD22/CD80, CD22/CD86 and CD22/CLII.

In a related aspect, the invention provides a method of treating a cell proliferation disorder, e.g., cancer, comprising administering a therapeutically effective amount of a protein (as described herein), or an encoding nucleic acid, to an

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2016231617 23 Sep 2016 organism in need. Those of skill in the art, including medical and veterinary professionals, are proficient at identifying organisms in need of treatment. Disorders contemplated by the invention as amenable to treatment include a disorder selected from the group consisting of a cancer, an autoimmune disorder, Rous Sarcoma Virus infection and inflammation. In some embodiments, the protein is administered by in vivo expression of a nucleic acid encoding the protein as described herein. The invention also comprehends administering the protein by a route selected from the group consisting of intravenous injection, intraarterial injection, intramuscular injection, subcutaneous injection, intraperitoneal injection and direct tissue injection.

Another aspect of the invention is directed to a method of ameliorating a symptom associated with a cell proliferation disorder comprising administering a therapeutically effective amount of a protein, as described herein, to an organism in need. Those of skill in the art are also proficient at identifying those disorders, or diseases or conditions, exhibiting symptoms amenable to amelioration. In some embodiments, the symptom is selected from the group consisting of pain, heat, swelling and joint stiffness.

Yet another aspect of the invention is drawn to a method of treating an infection associated with an infectious agent comprising administering a therapeutically effective amount of a protein according to the invention to a patient in need, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. Infectious agents amenable to treatment according to this aspect of the invention include prokaryotic and eukaryotic cells, viruses (including bacteriophage), foreign objects, and infectious organisms such as parasites (e.g., mammalian parasites).

A related aspect of the invention is directed to a method of ameliorating a symptom of an infection associated with an infectious agent comprising administering an effective amount of a protein according to the invention to a patient in need, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. Those of skill in the medical and veterinary arts will be able to determine an effective amount of a protein on a case-by-case basis, using routine experimentation.

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Yet another related aspect of the invention is a method of reducing the risk of infection attributable to an infectious agent comprising administering a prophylactically effective amount of a protein according to the invention to a patient at risk of developing the infection, wherein the protein comprises a binding domain that specifically binds a target molecule of the infectious agent. Those of skill in the relevant arts will be able to determine a prophylactically effective amount of a protein on a case-by-case basis, using routine experimentation.

Another aspect of the invention is drawn to the above-described multivalent single-chain binding protein wherein at least one of the first binding domain and the second binding domain specifically binds an antigen selected from the group consisting of CD 19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide.

In certain embodiments, one of the first binding domain and the second binding domain specifically binds CD20, and in some of these embodiments, the other binding domain specifically binds an antigen selected from the group consisting of CD 19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. For example, in one embodiment, the first binding domain is capable of specifically binding CD20 while the second binding domain is capable of specifically binding, e.g., CD19. In another embodiment, the first binding domain binds CD19 while the second binding domain binds CD20. An embodiment in which both binding domains bind CD20 is also contemplated.

In certain other embodiments according to this aspect of the invention, one of the first binding domain and the second binding domain specifically binds CD79b, and in some of these embodiments, the other binding domain specifically binds an antigen selected from the group consisting of CD 19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. Exemplary embodiments include distinct multi-specific, multivalent single-chain binding proteins in which a first binding domain.second binding domain specifically binds CD79b:CD19 or CD19:CD79b. A

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2016231617 23 Sep 2016 multivalent binding protein having first and second binding domains recognizing CD79b is also comprehended.

In still other certain embodiments, one of the first binding domain and the second binding domain specifically binds a major histocompatibility complex class II peptide, and in some of these embodiments, the other binding domain specifically binds an antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. For example, in one embodiment, the first binding domain is capable of specifically binding a major histocompatibility complex class II peptide while the second binding domain is capable of specifically binding, e.g., CD 19. In another embodiment, the first binding domain binds CD 19 while the second binding domain binds a major histocompatibility complex class II peptide. An embodiment in which both binding domains bind a major histocompatibility complex class II peptide is also contemplated.

In yet other embodiments according to this aspect of the invention, one of the first binding domain and the second binding domain specifically binds CD22, and in some of these embodiments, the other binding domain specifically binds an antigen selected from the group consisting of CD 19, CD20, CD21, CD22, CD23, CD30,

CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and a major histocompatibility complex class II peptide. Exemplary embodiments include distinct multi-specific, multivalent single-chain binding proteins in which a first binding domain:second binding domain specifically binds CD22:CD19 or CD19:CD22. A multivalent binding protein having first and second binding domains recognizing

CD22 is also comprehended.

A related aspect of the invention is directed to the above-described multivalent single-chain binding protein wherein the protein has a synergistic effect on a target cell behavior relative to the sum of the effects of each of the binding domains. In some embodiments, the protein comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD19, CD20-CD21, CD20-CD22, CD20-CD40, CD20-CD79a, CD20-CD79b and CD20CD81.

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The invention further comprehends a multivalent single-chain binding protein as described above wherein the protein has an additive effect on a target cell behavior relative to the sum of the effects of each of the binding domains. Embodiments according to this aspect of the invention include multi-specific proteins comprising a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-CD23, CD20-CD30, CD20-CD37, CD20-CD70, CD20CD80, CD20-CD86, CD79b-CD37, CD79b-CD81, major histocompatibility complex class II peptide-CD30, and major histocompatibility complex class II peptide-CD72.

Yet another related aspect of the invention is a multivalent single-chain 10 binding protein as described above wherein the protein has an inhibitory effect on a target cell behavior relative to the sum of the effects of each of the binding domains,

In some embodiments, the protein is multispecific and comprises a binding domain pair specifically recognizing a pair of antigens selected from the group consisting of CD20-major histocompatibility complex class II peptide, CD79b-CD19, CD79b15 CD20, CD79b-CD21, CD79b-CD22, CD79b-CD23, CD79b-CD30, CD79b-CD40,

CD79b-CD70, CD79b-CD72, CD79b-CD79a, CD79b-CD80, CD79b-CD86, CD79bmajor histocompatibility complex class II peptide, major histocompatibility complex class II peptide-CD19, major histocompatibility complex class II peptide-CD20, major histocompatibility complex class II peptide-CD21, major histocompatibility complex class II peptide-CD22, major histocompatibility complex class II peptideCD23, major histocompatibility complex class II peptide-CD37, major histocompatibility complex class II peptide-CD40, major histocompatibility complex class II peptide-CD70, major histocompatibility complex class II peptide-CD79a, major histocompatibility complex class II peptide-CD79b, major histocompatibility complex class II peptide-CD80, major histocompatibility complex class II peptideCD81, major histocompatibility complex class II peptide-CD86, CD22-CD19, CD22CD40, CD22-CD79b, CD22-CD86 and CD22-major histocompatibility complex class II peptide.

Another aspect of the invention is a method of identifying at least one of the binding domains of the multivalent binding molecule, such as a multispecific binding molecule, described above comprising: (a) contacting an anti-isotypic antibody with an antibody specifically recognizing a first antigen and an antibody specifically

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2016231617 23 Sep 2016 recognizing a second antigen; (b) further contacting a target comprising at least one of said antigens with the composition of step (a); and (c) measuring an activity of the target, wherein the activity is used to identify at least one of the binding domains of the multivalent binding molecule. In some embodiments, the target is a diseased cell, such as a cancer cell (e.g., a cancerous B-cell) or an auto-antibody-producing B-cell.

In each of the foregoing methods of the invention, it is contemplated that the method may further comprise a plurality of multivalent single-chain binding proteins. In some embodiments, a binding domain ofa first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein induce a synergistic, additive, or inhibitory effect on a target cell, such as a synergistic, additive, or inhibitory amount of damage to the target cell. The synergistic, additive or inhibitory effects of a plurality of multivalent single-chain binding proteins is determined by comparing the effect of such a plurality of proteins to the combined effect of an antibody comprising one of the binding domains and an antibody comprising the other binding domain.

A related aspect of the invention is directed to a composition comprising a plurality of multivalent single-chain binding proteins as described above. In some embodiments, the composition comprises a plurality of multivalent single-chain binding proteins wherein a binding domain ofa first multivalent single-chain binding protein and a binding domain of a second multivalent single-chain binding protein are capable of inducing a synergistic, additive, or inhibitory effect on a target cell, such as a synergistic, additive or inhibitory amount of damage to the target cell.

The invention further extends to a pharmaceutical composition comprising the composition described above and a pharmaceutically acceptable carrier, diluent or excipient. In addition, the invention comprehends a kit comprising the composition as described herein and a set of instructions for administering said composition to exert an effect on a target cell, such as to damage the target cell.

Finally, the invention also comprehends a kit comprising the protein as described herein and a set of instructions for administering the protein to treat a cell proliferation disorder or to ameliorate a symptom of the cell proliferation disorder.

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Other features and advantages of the present invention will be better understood by reference to the following detailed description, including the examples.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows a schematic representation of the multivalent single-chain 5 molecules envisioned by the invention. Individual subdomains of the fusion protein expression cassette are indicated by separate shapes/blocks on the figure. BD1 refers to binding domain 1, linker 1 refers to any potential linker or hinge like peptide between BD1 and the “effector function domain”, indicated as EFD. This subdomain is usually an engineered form of the Fc domain of human IgGl, but may include other subdomains with one or more effector functions as defined herein. Linker 2 refers to the linker sequence, if any, present between the carboxy terminus of the EFD and the binding domain 2, BD2.

Figure 2 shows a Western blot of non-reduced proteins expressed in COS cells. Protein was secreted into the culture medium, and culture supernatants isolated after 48-72 hours from transiently transfected cells by centrifugation. Thirty microliters, 30 μΐ of crude supernatant were loaded into each well of the gel. Lane identifications: 1- molecular weight markers, with numerals indicating kilodaltons; 22H7-IgG-STDl-2E12 LH; 3- 2H7-IgG-STDl-2EI2 HL, 4- 2H7-IgG-STD2-2E12 LH; 5- 2H7-IgG-STD2-2E12 HL; 6- 2E12 LH SMIP; 7- 2E12 HL SMIP; 8- 2H7 SMIP.

“2H7” refers to a single-chain construct, where BD1 encodes the CD20 specific binding domain (2H7) in the VLVH orientation; “2E12” refers to a binding domain specific for CD28; -IgG-refers to a single-chain construct, with a hinge encoding a sequence where all C are mutated to S (sss), and the CH2 and CH3 domains of IgGl contain mutations which eliminate both ADCC and CDC effector functions (P238S and P331 S), “STD 1 refers to a 20-amino-acid linker (identified in Figure 7 as “STDl=20aa “) inserted adjacent to the BD2 in the VL-VH orientation, or 2EI2 (VlVh). “STD1- HL” refers to a similar construct as just described, but with the BD2 V regions in the VH-VL orientation as follows: 2H7-sssIgG (P238/33lS)-20-aminoacid linker-2EI2 (V_H-V_L). “STD2- LH” refers to 2H7-sssIgG (P238/33 lS)-38-amino30 acid linker-2E12 (V_L-V_H); “STD2-LH” refers to 2H7-sssIgG (P238/331 SS)-38amino-acid linker-2E12 (Vh-Vl); “SMIP” refers to small modular immunopharmaceutical; and “H” generally refers to Vh, while “L” generally refers to

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Vl. Unless otherwise indicated, all protein orientations are N-terminal to C-terminal orientations.

Figure 3 shows two columnar graphs illustrating the binding properties of the 2H7-sssIgG (P238S/P331S)-STDl-2el2 LH and HL derivatives expressed from COS cells. These experiments were performed with crude culture supernatants rather than purified proteins. Serial dilutions from undiluted to 16X of the culture supernatants were incubated with either CD20 expressing cells (WIL-2S) or CD28 expressing cells (CD28 CHO). Binding activity in the supernatants was compared to control samples testing binding of the relevant single specificity SMIP, such as TRU-015, or 2el2

VLVH, or 2el2VHVL SMIPs. Binding in each sample was detected using fluorescein isothyocyanate (FITC) conjugated goat anti-human IgG at a dilution of 1:100.

Figure 4 is a histogram showing the binding pattern of protein A purified versions of the proteins tested in Figure 3 to WIL2-S cells. “TRU015” is a SMIP specific for CD20. Two multispecific binding proteins with effector function were also analyzed: “2H7-2E12 LH” has binding domain 2, specific for CD28, in Vl-Vh orientation; “2H7-2E12 HL” has binding domain 2, specific for CD28, in Vh-Vl orientation. Each of the proteins was tested for binding at 5 pg/ml, and binding detected with FITC goat anti-human IgG at 1:100. See the description for Figure 2 above for more complete descriptions of the molecules tested.

Figure 5 shows two histograms illustrating the binding by protein A purified multispecific binding proteins with effector function to CHO cells expressing CD28. “2H7-2E12 LH” has binding domain 2, specific for CD28, in Vl-Vh orientation; “2H7-2E12 HL” has binding domain 2, specific for CD28, in Vh-Vl orientation.

Each of the proteins was tested for binding at 5 pg/ml, and binding was detected with FITC goat anti-human IgG at 1:100. See the descriptions in Figure 2 for a more complete description of the molecules tested.

Figure 6 A) shows a table which identifies the linkers joining the constant sub-region and binding domain 2. The linkers are identified by name, sequence, sequence identifier, sequence length, and the sequence of the fusion with binding domain 2. B) shows a table identifying a variety of constructs identifying elements of

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WO 2007/146968 PCT/US2007/071052 exemplified molecules according to the invention.. In addition to identifying the multivalent binding molecules by name, the elements of those molecules are disclosed in terms of binding domain 1 (BD1), the constant sub-region (hinge and effector domain or EFD), a linker (see Fig. 6A for additional information regarding the linkers), and binding domain 2 (BD2). The sequences of a number of exemplified multivalent binding proteins are provided, and are identified in the figure by a sequence identifier. Other multivalent binding proteins have altered elements, or element orders, with predictable alterations in sequence from the disclosed sequences.

Figure 7 shows a composite columnar graph illustrating the binding of 10 purified proteins at a single, fixed concentration to CD20 expressing WIL-2S cells and to CHO cells expressing CD28. “H1-H6” refers to the 2H7-sss-hIgG-Hx-2el2 molecules with the H1-H6 linkers and the 2el2 V regions in the orientation of Vh-Vl“L1-L6” refers to the 2H7-sss-hIgG-Lx-2el2 molecules with the L1-L6 linkers and the 2el2 V regions in the orientation of Vl-Vh. All the molecules were tested at a concentration of 0.72 pg/ml, and the binding detected using FITC conjugated goat anti-human IgG at 1:100. The mean fluorescence intensity for each sample was then plotted as paired bar graphs for the two target cell types tested versus each of the multivalent constructs being tested, L1-L6, or H1-H6.

Figure 8 shows photographs of Coomassie stained non-reducing and reducing

SDS-PAGE gels. These gels show the effect of the variant linker sequence/length on the 2H7-sss-hlgG-Hx-2el2 HL protein on the amounts of the two predominate protein bands visualized on the gel.

Figure 9 shows Western Blots of the [2H7-sss-hIgG-H6-2el2] fusion proteins and the relevant single specificity SMIPs probed with either (a) CD28mIgG or with (b) a Fab reactive with the 2H7 specificity. The results show that the presence of the

H6 linker results in the generation of cleaved forms of the multivalent constructs which are missing the CD28 binding specificity.

Figure 10 shows binding curves of the different linker variants for the [TRU015-sss-IgG-Hx-2el2 HL] H1-H6 linker forms. The first panel shows the binding curves for binding to CD20 expressing W1L-2S cells. The second panel shows the binding curves for binding of the different forms to CD28 CHO cells.

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These binding curves were generated with serial dilutions of protein A purified fusion protein, and binding detected using F1TC conjugated goat anti-human IgG at 1:100.

Figure 11 shows a table summarizing the results of SEC fractionation of 2H7sss-IgG-2el2 HL multispecific fusion proteins with variant linkers H1-H7. Each row in the table lists a different linker variant of the [2H7-sss-IgG-Hx-2el2-HL] fusion proteins. The retention time of the peak of interest (POI), and the percentage of the fusion protein present in POI, and the percentage of protein found in other forms is also tabulated. The cleavage of the molecules is also listed, with the degree of cleavage indicated in a qualitative way, with (Yes), Yes, and YES, or No being the four possible choices.

Figure 12 shows two graphs with binding curves for [2H7-sss-hIgG-Hx-2el2] multispecific fusion proteins with variant linkers H3, H6, and H7 linkers to cells expressing CD20 or CD28, Serial dilutions of the protein A purified fusion proteins from 10 pg/ml down to 0.005 pg/ml were incubated with either CD20 expressing

WIL-2S cells or CD28 CHO cells. Binding was detected using FITC conjugated goat anti-human IgG at 1:100. Panel A shows the binding to WIL-2S cells, and panel B shows the binding to CD28 CHO cells.

Figure 13 shows the results of an alternative binding assay generated by the molecules used for Figure 12. In this case, the fusion proteins were first bound to

WIL-2S CD20 expressing cells, and binding was then detected with CD28mIgG (5 qg/ml)and FITC anti-mouse reagent at 1:100. These results demonstrate the simultaneous binding to both CD20 and CD28 in the same molecule.

Figure 14 shows results obtained using another multispecific fusion construct variant. In this case, modifications were made in the specificity for BD2, so that the

V regions for the G28-1 antibody were used to create a CD37 specific binding domain. Shown are two graphs which illustrate the relative ability of CD20 and/or CD37 antibodies to block the binding of the [2H7-sss-IgG-Hx-G28-l] multispecific fusion protein to Ramos or BJAB cells expressing the CD20 and CD37 targets. Each cell type was preincubated with either the CD20 specific antibody (25 gg/ml) or the

CD37 specific antibody (10 pg/ml) or both reagents (these are mouse anti-human reagents) prior to incubation with the multispecific fusion protein. Binding of the

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WO 2007/146968 PCT/US2007/071052 multispecific fusion protein was then detected with a FITC goat anti-human IgG reagent at 1:100, (preadsorbed to mouse to eliminate cross-reactivity).

Figure 15 shows the results of an ADCC assay performed with BJAB target cells, PBMC effector cells, and with the CD20-hIgG-CD37 specific fusion protein as the test reagent. For a full description of the procedure see the appropriate example. The graph plots the concentration of fusion protein versus the % specific killing at each dosage tested for the single specificity SMIP reagents, and for the [2H7-ssshIgG-STDl-G28-l] LH and HL variants. Each data series plots the dose-response effects for one of these single specificity or multispecific single-chain fusion proteins.

Figure 16 shows a table tabulating the results of a co-culture experiment where PBMC were cultured in the presence of TRU 015, G28-1 SMIP, both molecules together, or the [2H7-sss-IgG-H7-G28-lHL] variant. The fusion proteins were used at 20 pg/ml, and incubated for 24 hours or 72 hours. Samples were then stained with CD3 antibodies conjugated to FITC, and either CD 19 or CD40 specific antibodies conjugated to PE, then subjected to flow cytometry. The percentage of cells in each gate was then tabulated.

Figure 17 shows two columnar graphs of the effects on B cell line apoptosis after 24 hour incubation with the [2H7-sss-hlgG-H7-G28-l HL] molecule or control single CD20 and/or CD37 specificity SMIPs alone or in combination. The percentage of annexin V-propidium iodide positive cells is plotted as a function of the type of test reagent used for the coincubation experiments, Panel A shows the results obtained using Ramos cells, and panel B shows those for Daudi cells. Each single CD20 or CD37 directed SMIP is shown at the concentrations indicated; in addition, where combinations of the two reagents were used, the relative amount of each reagent is shown in parentheses. For the multispecific CD20-CD37 fusion protein, concentrations of 5, 10, and 20 pg/ml were tested.

Figure 18 shows two graphs ofthe [2H7-hIgG-G19-4] molecule variants and their binding to either CD3 expressing cells (Jurkats) or CD20 expressing cells (WIL2S). The molecules include [2H7-sss-hIgG-STDl-G19-4 HL], LH, and [2H7-csc30 hIgG-STDl-G19-4 HL], Protein A purified fusion proteins were titrated from 20 pg/ml down to 0.05 pg/ml, and the binding detected using FITC goat anti-human IgG

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2016231617 23 Sep 2016 at 1:100. MFI (mean fluorescence intensity) is plotted as a function of protein concentration.

Figure 19 shows the results of ADCC assays performed with the [2H7-hIgGSTD1-G19-4 HL] molecule variants with either an SSS hinge or a CSC hinge, BJAB target cells, and either total human PBMC as effector cells or NK cell depleted PBMC as effector cells. Killing was scored as a function of concentration of the multispecific fusion proteins. The killing observed with these molecules was compared to that seen using G19-4, TRU 015, or a combination of these two reagents. Each data series plots a different test reagent, with the percent specific killing plotted as a function of protein concentration.

Figure 20 shows the percentage of Ramos B-cells that stained positive with Annexin V (Ann) and/or propidium iodide (PI) after overnight incubation with each member of a matrix panel of B-cell antibodies (2 pg/ml) in the presence, or absence, of an anti-CD20 antibody (present at 2 pg/ml where added). Goat-anti-mouse secondary antibody was always present at a two-fold concentration ratio relative to other antibodies (either matrix antibody alone, or matrix antibody and anti-CD20 antibody). Vertically striped bars - matrix antibody (2 pg/ml) denoted on X-axis and goat anti-mouse antibody (4 pg/ml). Horizontally striped bars - matrix antibody (2 pg/ml) denoted on X-axis, anti-CD20 antibody (2 pg/ml), and goat anti-mouse antibody (4 pg/ml). The “2^nd step” condition served as a control and involved the addition of goat anti-mouse antibody at 4 pg/ml (vertically striped bar) or 8 pg/ml (horizontally striped bar), without a matrix antibody or anti-CD20 antibody. “CL II” (MHC class II) in the figures refers to a monoclonal antibody cross-reactive to HLA DR, DQ and DP, i.e., to MHC Class II antigens.

Figure 21 shows the percentage of Ramos B-cells that stained positive with

Annexin V (Ann) and/or propidium iodide (PI) after overnight incubation with each member of a matrix panel of B-cell antibodies (2 pg/ml) in the presence, or absence, of an anti-CD79b antibody (present at either 0.5 or 1.0 pg/ml where added). See the description of Figure 20 for identification of “CL II” and “2^nd step” samples.

Vertically striped bars - matrix antibody (2 pg/ml) and goat anti-mouse antibody (4 pg/ml); horizontally striped bars - matrix antibody (2 pg/ml), anti-CD79b antibody

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2016231617 23 Sep 2016 (1.0 pg/ml) and goat anti-mouse antibody (6 pg/ml); stippled bars - matrix antibody (2 pg/ml), anti-CD79b antibody (0.5 μg/ml) and goat anti-mouse antibody (5 μg/ml).

Figure 22 shows the percentage of Ramos B-cells that stained positive with Annexin V (Ann) and/or propidium iodide (PI) after overnight incubation with each member of a matrix panel of B-cell antibodies (2 μg/ml) in the presence, or absence, of an anti-CL II antibody (present at either 0.25 or 0.5 pg/ml where added). See the description of Figure 20 for identification of “CL II” and “2^nd step” samples.

Vertically striped bars - matrix antibody (2 pg/ml) and goat anti-mouse antibody (4 pg/ml); horizontally striped bars - matrix antibody (2 pg/ml), anti-CL II antibody (0.5 pg/ml) and goat anti-mouse antibody (5 pg/ml); stippled bars - matrix antibody (2 pg/ml), anti-CL II antibody (0.25 pg/ml) and goat anti-mouse antibody (4.5 pg/ml).

Figure 23 shows the percentage of DHL-4 B-cells that stained positive with Annexin V (Ann) and/or propidium iodide (PI) after overnight incubation with each member of a matrix panel of B-cell antibodies (2 pg/ml) in the presence, or absence, of an anti-CD22 antibody (present at 2 pg/ml where added). See the description of Figure 20 for identification of “CL II” and “2^nd step” samples. Solid bars - matrix antibody (2 pg/ml) and goat anti-mouse antibody (4 pg/ml); slant-striped bars matrix antibody (2 pg/ml), anti-CD22 antibody (2 pg/ml) and goat anti-mouse antibody (8 pg/ml).

Figure 24 provides a graph demonstrating direct growth inhibition of lymphoma cell lines Su-DHL6 (triangles) and DoHH2 (squares) by free CD20 SMIP (closed symbols) or monospecific CD20xCD20 scorpion (open symbols).

Figure 25 is a graph showing direct growth inhibition of lymphoma cell lines Su-DHL-6 (triangles) and DoHH2 (squares) by free anti-CD37 SMIP (closed symbols) or monospecific anti-CD37 scorpion (open symbols).

Figure 26 presents a graph showing direct growth inhibition of lymphoma cell lines Su-DHL-6 (triangles) and DoHH2 (squares) by a combination of two different monospecific SMIPs (closed symbols) or by a bispecific CD20-CD37 scorpion (open symbols).

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Figure 27 is a graph revealing direct growth inhibition of lymphoma cell lines Su-DHL-6 (triangles) and WSU-NHL (squares) by free CD20 SMIP and CD37 SMIPcombination (closed symbols) or bispecific CD20xCD37 scorpion (open symbols).

Figure 28 provides histograms showing the cell-cycle effects of scorpions.

Samples of DoHH2 lymphoma cells were separately left untreated, treated with SMIP 016 or treated with the monospecific CD37 x CD37 scorpion. Open bars: sub-Gi phase of the cell cyle; black bars: Go/Gi phase; shaded: S phase; and striped: G2/M phase.

Figure 29 presents graphs of data establishing that treatment of lymphoma cells with scorpions resulted in increased signaling capacity compared to free SMIPs, as measured by calcium ion flux.

Figure 30 provides graphs demonstrating scorpion-dependent cellular cytotoxicity

Figure 31 shows graphs of data indicating that scorpions mediate

Complement Dependent Cytotoxicity.

Figure 32 provides data in graphical form showing comparative ELISA binding of a SMIP and a scorpion to low- (B) and high-affinity (A) isoforms of FcyRIII (CD 16).

Figure 33 presents graphs establishing the binding of a SMIP and a scorpion to low (A)- and high (B)-affinity allelotypes of FcyRIII (CD 16) in the presence of target cells.

Figure 34 is a histogram showing the expression level of a CD20 x CD20 scorpion in two experiments (flask 1 and flask 2) under six different culturing conditions. Solid black bars: flask 1; striped bars: flask 2.

Figure 35 provides a histogram showing the production yield of a CD20 x CD 3 7 scorpion.

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Figure 36 presents SDS-PAGE gels (under reducing and non-reducing conditions) of a SMIP and a scorpion.

Figure 37 provides a graph showing that scorpions retain the capacity to bind to target cells. Filled squares: CD20 SMIP; filled triangles: CD37 SMIP; filled circles: humanized CD20 (2Lm20-4) SMIP; open diamond: CD37 x CD37 monospecific scorpion; open squares: CD20 x CD37 bi-specific scorpion; and open triangles: humanized CD20 (2Lm20-4) x humanized CD20 (2Lm20-4) scorpion.

Figure 38 contains graphs showing the results of competitive binding assays establishing that both N- and C-terminal scorpion binding domains participate in target cell binding.

Figure 39 presents data in the form of graphs showing that scorpions have lower off-rates than SMIPs.

Figure 40 shows a graph establishing that scorpions are stable in serum in vivo, characterized by a reproducible,sustained circulating half-life for the scorpion.

Figure 41 provides a dose-response graph for a CD20 x CD37 bispecific scorpion, demonstrating the in vivo efficacy of scorpion administration.

Figure 42 shows target B-cell binding by a monospecific CD20xCD20 scorpion (SO 129) and glycovariants.

Figure 43 provides graphs illustrating CD20xCD20 scorpions (parent and 20 glycovariants) inducing ADCC-mediated killing of BJAB B-cells.

Figure 44 shows a gel revealing the effects on scorpion stability arising from changes in the scorpion linker, including changing the sequence of that linker and extending the linker by adding an H7 sequence to the linker, indicated by a “+” in the H7 line under the gel.

Figure 45 shows the binding to WIL2S cells of a CD20xCD20 scorpion (SO 129) and scorpion linker variants thereof.

Figure 46 shows the direct cell killing of a variety of B-cells by a CD20xCD20 scorpion and by a CD20 SMIP.

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Figure 47 reveals the direct cell killing of additional B-cell lines by a monospecific CD20xCD20 scorpion.

Figure 48 shows the direct cell killing capacities of each of two monospecific scorpions, i.e., CD20xCD20 and CD37xCD37, and a bispecific CD20xCD37 scorpion, the latter exhibiting a different form of kill curve.

Figure 49 graphically depicts the response of Su-DHL-6 B-cells to each of a CD20xCD20 (SO 129), a CD37xCD37, and a CD20xCD37 scorpion.

Figue 50 shows the capacity of a bispecific CD19xCD37 scorpion and Rituxan® to directly kill Su-DHL-6 B-cells.

Figure 51 provides histograms showing the direct killing of DHL-4 B-cells by a variety of CD20-binding scorpions and SMIPs, as well as by Rituxan®, as indicated in the figure. Blue bars: live cells; maroon bars on the right of each pair: Annexin+/PI+.

Figure 52 provides a graphic depiction of the direct cell killing of various

CD20-binding scorpions and SMIPs, as well as by Rituxan®, as indicated in the figure.

Figure 53 provides graphs of the ADCC activity induced by various CD20binding scorpions and SMIPs, as indicated in the figure, as well as by Rituxan®.

Figure 54 provides graphs of the CDC activity induced by vawrious CD2020 binding scorpions and SMIPs, as indicated in the figure, as well as by Rituxan®.

Figure 55 provides histograms showing the Ieveis of Clq binding to CD20binding scorpions bound to Ramos B-cells.

Figure 56 provides scatter plots of FACS analyses showing the loss of mitochondrial membrane potential attributable to CD20-binding scorpions (2Lm2025 4x2Lm20-4 and 01 lx2Lm20-4) and Rituxan®, relative to controls (upper panel);

histograms of the percentage of cells with disrupted mitochondrial membrane potential (disrupted MMP: black bars) are shown in the lower panel.

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Figure 57 provides histograms showing the relative lack of caspase 3 activation by CD20-binding scorpions (2Lm20-4x2Lm20-4 and 01 lx2Lm20-4), Rituximab, CD95, and controls.

Figure 58 provides a composite of four Western blot analyses of Poly (ADP5 ribose) Polymerase and caspases 3, 7, and 9 from B-cells showing little degradation of any of these proteins attributable to CD20-binding scorpions binding to the cells.

Figure 59 is a gel electrophoretogram of B-cell chromosomal DNAs showing the degree of fragmentation attributable to CD20-binding scorpions binding to the cells.

Figure 60 is a gel electrophoretogram of immunoprecipitates obtained with each of an anti-phosphotyrosine antibody and an anti-SYK antibody. The immunoprecipitates were obtained from lysates of B-cells contacted with CD20binding scorpions, as indicated in the figure.

Figure 61 provides combination index plots of CD20-binding scorpions in combination therapies with each of doxorubicin, vincristine and rapamycin.

DETAILED DESCRIPTION

The present invention provides compositions of relatively small peptides having at least two binding regions or domains, which may provide one or more binding specificities, derived from variable binding domains of immunoglobulins, such as antibodies, disposed terminally relative to an effector domain comprising at least part of an immunoglobulin constant region (i.e., a source from which a constant sub-region, as defined herein, may be derived), as well as nucleic acids, vectors and host cells involved in the recombinant production of such peptides and methods of using the peptide compositions in a variety of diagnostic and therapeutic applications, including the treatment of a disorder as well as the amelioration of at least one symptom of such a disorder. The peptide compositions advantageously arrange a second binding domain C-terminal to the effector domain, an arrangement that unexpectedly provides sterically unhindered or less hindered binding by at least two binding domains of the peptide, while retaining an effector function or functions of the centrally disposed effector domain.

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The first and second binding domains of the multivalent peptides according to the invention may be the same (i.e., have identical or substantially identical amino acid sequences and be monospecific) or different (and be multispecific). Although different in terms of primary structure, the first and second binding domains may recognize and bind to the same epitope of a target molecule and would therefore be monospecific. In many instances, however, the binding domains will differ structurally and will bind to different binding sites, resulting in a multivalent, multispecific protein. Those different binding sites may exist on a single target molecule or on different target molecules. In the case of the two binding molecules recognizing different target molecules, those target molecules may exist, e.g., on or in the same structure (e.g., the surface of the same cell), or those target molecules may exist on or in separate structures or locales. For example, a multispecific binding protein according to the invention may have binding domains that specifically bind to target molecule on the surfaces of distinct cell types. Alternatively, one binding domain may specifically bind to a target on a cell surface and the other binding domain may specifically bind to a target not found associated with a cell, such as an extracellular structural (matrix) protein or a free (e.g., soluble or stromal) protein.

The first and second binding domains are derived from one or more regions of the same, or different, immunoglobulin protein structures such as antibody molecules.

The first and/or second binding domain may exhibit a sequence identical to the sequence of a region of an immunoglobulin, or may be a modification of such a sequence to provide, e.g., altered binding properties or altered stability. Such modifications are known in the art and include alterations in amino acid sequence that contribute directly to the altered property such as altered binding, for example by leading to an altered secondary or higher order structure for the peptide. Also contemplated are modified amino acid sequences resulting from the incorporation of non-native amino acids, such as non-native conventional amino acids, unconventional amino acids and imino acids. In some embodiments, the altered sequence results in altered post-translational processing, for example leading to an altered glycosylation pattern.

Any of a wide variety of binding domains derived from an immunoglobulin or immunoglobulin-like polypeptide (e.g., receptor) are contemplated for use in

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WO 2007/146968 PCT/US2007/071052 scorpions. Binding domains derived from antibodies comprise the CDR regions of a Vl and a Vh domain, seen, e.g., in the context of using a binding domain from a humanized antibody. Binding domains comprising complete Vl and Vh domains derived from an antibody may be organized in either orientation. A scorpion according to the invention may have any of the binding domains herein described.

For scorpions having at least one binding domain recognizing a B-cell, exemplary scorpions have at least one binding domain derived from CD3, CD 10, CD 19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85,

CD86, CD89, CD98, CD126, CD127, CDwl30, CD138 or CDwl50. In some embodiments, the scorpion is a multivalent binding protein comprising at least one binding domain having a sequence selected from the group consisting of SEQ ID NOS: 2,4, 6, 103, 105, 107 and 109. In some embodiments, a scorpion comprises a binding domain comprising a sequence selected from the group consisting of any of

SEQ ID NOS: 332-345. In some embodiments, a scorpion comprises a binding domain comprising a sequence derived from immunoglobulin Vl and Vh domains, wherein the sequence is selected from the group consisting of any of SEQ ID NOS: 355-365. The invention further contemplates scorpions comprising a binding domain that has the opposite orientation of Vl and Vh having sequences deducible from any of SEQ IDNOS:355-365.

For embodiments in which either, or both, of the binding domains are derived from more than one region of an immunoglobulin (e.g., an Ig Vl region and an Ig Vh region), the plurality of regions may be joined by a linker peptide. Moreover, a linker may be used to join the first binding domain to a constant sub-region. Joinder of the constant sub-region to a second binding domain (i.e., binding domain 2 disposed towards the C-terminus of a scorpion) is accomplished by a scorpion linker. These scorpion linkers are preferably between about 2-45 amino acids, or 2-38 amino acids, or 5-45 amino acids. For example, the Hl linker is 2 amino acids in length and the STD2 linker is 38 amino acids in length, Beyond general length considerations, a scorpion linker region suitable for use in the scorpions according to the invention includes an antibody hinge region selected from the group consisting of IgG, IgA, IgD and IgE hinges and variants thereof. For example, the scorpion linker may be an antibody hinge region selected from the group consisting of human IgGl, human

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IgG2, human IgG3, and human IgG4, and variants thereof. In some embodiments, the scorpion linker region has a single cysteine residue for formation of an interchain disulfide bond. In other embodiments, the scorpion linker has two cysteine residues for formation of interchain disulfide bonds. In some embodiments, a scorpion linker region is derived from an immunoglobulin hinge region or a C-lectin stalk region and comprises a sequence selected from the group consisting of SEQ ID NOS: 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,231,233,235,237, 239, 241,243,245,247,249,251,253, 255, 257, 259,261, 263,265,267,269,271,273,275, 277, 279, 281, 287, 289, 297,

305,307,309, 310, 311, 313, 314, 315, 316,317, 318, 319, 320, 321, 322, 323, 324,

325, 326,327, 328, 329, 330,331,346, 351, 352, 353, 354, 373, 374, 375, 376 and 377. More generally, any sequence of amino acids identified in the sequence listing as providing a sequence derived from a hinge region is contemplated for use as a scorpion linker in the scorpion molecules according to the invention. In addition, a scorpion linker derived from an Ig hinge is a hinge-like peptide domain having at least one free cysteine capable of participating in an interchain disulfide bond. Preferably, a scorpion linker derived from an Ig hinge peptide retains a cysteine that corresponds to the hinge cysteine disposed towards the N-terminus of that hinge. Preferably, a scorpion linker derived from an IgG 1 hinge has one cysteine or has two cysteines corresponding to hinge cysteines. Additionally, a scorpion linker is a stalk region of a Type II C-lectin molecule. In some embodiments, a scorpion comprises a scorpion linker having a sequence selected from the group consisting of SEQ ID NOS:373-377.

The centrally disposed constant sub-region is derived from a constant region of an immunoglobulin protein. The constant sub-region generally is derived from a

C_H2 portion of a Ch region of an immunoglobulin in the abstract, although it may be derived from a Ch2-Ch3 portion. Optionally, the constant sub-region may be derived from a hinge-Cm or hinge-CH2-CH3 portion of an immunoglobulin, placing a peptide corresponding to an Ig hinge region N-terminal to the constant sub-region and disposed between the constant sub-region and binding domain 1. Also, portions of the constant sub-region may be derived from the Ch regions of different immunoglobulins. Further, the peptide corresponding to an Ig CH3 may be truncated, leaving a C-terminal amino acid sequence selected from the group consisting of SEQ ID NOS:366-371, It is preferred, however, that in embodiments in which a scorpion

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WO 2007/146968 PCT/US2007/071052 hinge is a hinge-like peptide derived from an immunoglobulin hinge, that the scorpion linker and the constant sub-region be derived from the same type of immunoglobulin. The constant sub-region provides at least one activity associated with a Ch region of an immunoglobulin, such as antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), protein A binding, binding to at least one Fc receptor, reproducibly detectable stability relative to a protein according to the invention except for the absence of a constant sub-region, and perhaps placental transfer where generational transfer of a molecule according to the invention would be advantageous, as recognized by one of skill in the art. As with the above-described binding domains, the constant sub-region is derived from at least one immunoglobulin molecule and exhibits an identical or substantially identical amino acid sequence to a region or regions of at least one immunoglobulin. In some embodiments, the constant sub-region is modified from the sequence or sequences of at least one immunoglobulin (by substitution of one or more non-native conventional or unconventional, e.g., synthetic, amino acids or imino acids), resulting in a primary structure that may yield an altered secondary or higher order structure with altered properties associated therewith, or may lead to alterations in post-translational processing, such as gly cosy lation.

For those binding domains and constant sub-regions exhibiting an identical or substantially identical amino acid sequence to one or more immunoglobulin polypeptides, the post-translational modifications of the molecule according to the invention may result in a molecule modified relative to the immunoglobulin(s) serving as a basis for modification. For example, using techniques known in the art, a host cell may be modified, e.g. a CHO cell, in a manner that leads to an altered polypeptide glycosylation pattern relative to that polypeptide in an unmodified (e.g., CHO) host cell.

Provided with such molecules, and the methods of recombinantly producing them in vivo, new avenues of targeted diagnostics and therapeutics have been opened to allow, e.g., for the targeted recruitment of effector cells of the immune system (e.g., cytotoxic T lymphocytes, natural killer cells, and the like) to cells, tissues, agents and foreign objects to be destroyed or sequestered, such as cancer cells and infectious agents. In addition to localizing therapeutic cells to a site of treatment, the peptides

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2016231617 23 Sep 2016 are useful in localizing therapeutic compounds, such as radiolabeled proteins.

Further, the peptides are also useful in scavenging deleterious compositions, for example by associating a deleterious composition, such as a toxin, with a cell capable of destroying or eliminating that toxin (e.g., a macrophage). The molecules of the invention are useful in modulating the activity of binding partner molecules, such as cell surface receptors. This is shown in Figure 17 where apoptotic signaling through CD20 and/or CD37 is markedly enhanced by a molecule of the present invention.

The effect of this signaling is the death of the targeted cell. Diseases and conditions where the elimination of defined cell populations is beneficial would include infectious and parasitic diseases, inflammatory and autoimmune conditions, malignancies, and the like. One skilled in the art would recognize that there is no limitation of the approach to the enhancement of apoptotic signaling. Mitotic signaling and signaling leading to differentiation, activation, or inactivation of defined cell populations can be induced by molecules of the present invention through the appropriate selection of binding partner molecules. Further consideration of the disclosure of the invention will be facilitated by a consideration of the following express definitions of terms used herein.

A “single-chain binding protein” is a single contiguous arrangement of covalently linked amino acids, with the chain capable of specifically binding to one or more binding partners sharing sufficient determinants of a binding site to be detectably bound by the single-chain binding protein. Exemplary binding partners include proteins, carbohydrates, lipids and small molecules.

For ease of exposition, “derivatives” and “variants” of proteins, polypeptides, and peptides according to the invention are described in terms of differences from proteins and/or polypeptides and/or peptides according to the invention, meaning that the derivatives and variants, which are proteins/polypeptides/peptides according to the invention, differ from underivatized or non-variant proteins, polypeptides or peptides of the invention in the manner defined. One of skill in the art would understand that the derivatives and variants themselves are proteins, polypeptides and peptides according to the invention.

An “antibody” is given the broadest definition consistent with its meaning in the art, and includes proteins, polypeptides and peptides capable of binding to at least

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WO 2007/146968 PCT/US2007/071052 one binding partner, such as a proteinaceous or non-proteinaceous antigen. An “antibody” as used herein includes members of the immunoglobulin superfamily of proteins, of any species, of single- or multiple-chain composition, and variants, analogs, derivatives and fragments of such molecules. Specifically, an “antibody” includes any form of antibody known in the art, including but not limited to, monoclonal and polyclonal antibodies, chimeric antibodies, CDR-grafted antibodies, humanized antibodies, single-chain variable fragments, bi-specific antibodies, diabodies, antibody fusions, and the like.

A “binding domain” is a peptide region, such as a fragment of a polypeptide 10 derived from an immunoglobulin (e.g., an antibody), that specifically binds one or more specific binding partners. If a plurality of binding partners exists, those partners share binding determinants sufficient to detectably bind to the binding domain. Preferably, the binding domain is a contiguous sequence of amino acids.

An “epitope” is given its ordinary meaning herein of a single antigenic site,

i.e., an antigenic determinant, on a substance (e.g., a protein) with which an antibody specifically interacts, for example by binding. Other terms that have acquired wellsettled meanings in the immunoglobulin (e.g., antibody) art, such as a “variable light region,” variable heavy region,” “constant light region,” constant heavy region,” “antibody hinge region,” “complementarity determining region,” “framework region,” “antibody isotype,” “Fc region,” “single-chain variable fragment” or “scFv,” “diabody,” “chimera,” “CDR-grafted antibody,” “humanized antibody,” “shaped antibody,” “antibody fusion,” and the like, are each given those well-settled meanings known in the art, unless otherwise expressly noted herein.

Terms understood by those in the art as referring to antibody technology are each given the meaning acquired in the art, unless expressly defined herein.

Examples of such terms are “Vl” and “Vh”, referring to the variable binding region derived from an antibody light and heavy chain, respectively; and Cl and Ch, referring to an “immunoglobulin constant region,” i.e., a constant region derived from an antibody light or heavy chain, respectively, with the latter region understood to be further divisible into C_Hi, Ch2, C_H3 and Ch4 constant region domains, depending on the antibody isotype (IgA, IgD, IgE, IgG, IgM) from which the region was derived. CDR means “complementarity determining region.” A “hinge region” is derived

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2016231617 23 Sep 2016 from the amino acid sequence interposed between, and connecting, the Chi and Ch2 regions of a single chain of an antibody, which is known in the art as providing flexibility, in the form of a “hinge,” to whole antibodies.

A “constant sub-region” is a term defined herein to refer to a peptide, polypeptide, or protein sequence that corresponds to, or is derived from, one or more constant region domains of an antibody. Thus, a constant sub-region may include any or all of the following domains: a Chi domain, a hinge region, a Ch2 domain, a Ch3 domain (IgA, IgD, IgG, IgE, and IgM), and a Ch4 domain (IgE, IgM). A constant sub-region as defined herein, therefore, can refer to a polypeptide region corresponding to an entire constant region of an antibody, or a portion thereof. Typically, a constant sub-region of a polypeptide, or encoding nucleic acid, of the invention has a hinge, Ch2 domain, and Ch3 domain.

An “effector function” is a function associated with or provided by a constant region of an antibody. Exemplary effector functions include antibody-dependent cell15 mediated cytotoxicity (ADCC), complement activation and complement-dependent cytotoxicity (CDC), Fc receptor binding, and increased plasma half-life, as well as placental transfer. An effector function of a composition according to the invention is detectable; preferably, the specific activity of the composition according to the invention for that function is about the same as the specific activity of a wild-type antibody with respect to that effector function, i.e., the constant sub-region of the multivalent binding molecule preferably has not lost any effector function relative to a wild-type antibody]

A “linker” is a peptide, or polynucleotide, that joins or links other peptides or polynucleotides. Typically, a peptide linker is an oligopeptide of from about 2-50 amino acids, with typical polynucleotide linkers encoding such a peptide linker and, thus, being about 6-150 nucleotides in length. Linkers join the first binding domain to a constant sub-region domain. An exemplary peptide linker is (Gly4Ser)3. A scorpion linker is used to join the C-terminal end of a constant sub-region to a second binding domain. The scorpion linker may be derived from an immunoglobulin hinge region or from the stalk region of a type II C-lectin, as described in greater detail below.

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A “target” is given more than one meaning, with the context of usage defining an unambiguous meaning in each instance. In its narrowest sense, a “target” is a binding site, i.e., the binding domain of a binding partner for a peptide composition according to the invention. In a broader sense, “target” or “molecular target” refers to the entire binding partner (e.g., a protein), which necessarily exhibits the binding site. Specific targets, such as “CD20,” “CD37,” and the like, are each given the ordinary meaning the term has acquired in the art. A “target cell” is any prokaryotic or eukaryotic cell, whether healthy or diseased, that is associated with a target molecule according to the invention. Of course, target molecules are also found unassociated with any cell (i.e., a cell-free target) or in association with other compositions such as viruses (including bacteriophage), organic or inorganic target molecule carriers, and foreign objects.

Examples of materials with which a target molecule may be associated include autologous cells (e.g., cancer cells or other diseased cells), infectious agents (e.g., infectious cells and infectious viruses), and the like. A target molecule may be associated with an enucleated cell, a cell membrane, a liposome, a sponge, a gel, a capsule, a tablet, and the like, which may be used to deliver, transport or localize a target molecule, regardless of intended use (e.g., for medical treatment, as a result of benign or unintentional provision, or to further a bioterrorist threat). “Cell-free,” “virus-free,” “carrier-free,” “object-free,” and the like refer to target molecules that are not associated with the specified composition or material.

“Binding affinity” refers to the strength of non-covalent binding of the peptide compositions of the invention and their binding partners. Preferably, binding affinity refers to a quantitative measure of the attraction between members of a binding pair,

An “adjuvant” is a substance that increases or aids the functional effect of a compound with which it is in association, such as in the form of a pharmaceutical composition comprising an active agent and an adjuvant. An “excipient” is an inert substance used as a diluent in formulating a pharmaceutical composition. A “carrier” is a typically inert substance used to provide a vehicle for delivering a pharmaceutical composition.

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2016231617 23 Sep 2016 “Host cell” refers to any cell, prokaryotic or eukaryotic, in which is found a polynucleotide, protein or peptide according to the invention.

“Introducing” a nucleic acid or polynucleotide into a host cell means providing for entry of the nucleic acid or polynucleotide into that cell by any means known in the art, including but not limited to, in vitro salt-mediated precipitations and other forms of transformation of naked nucleic acid/polynucleotide or vector-borne nucleic acid/polynucleotide, virus-mediated infection and optionally transduction, with or without a “helper” molecule, ballistic projectile delivery, conjugation, and the like.

“Incubating” a host cell means maintaining that cell under environmental conditions known in the art to be suitable for a given purpose, such as gene expression. Such conditions, including temperature, ionic strength, oxygen tension, carbon dioxide concentration, nutrient composition, and the like, are well known in the art.

“Isolating” a compound, such as a protein or peptide according to the invention, means separating that compound from at least one distinct compound with which it is found associated in nature, such as in a host cell expressing the compound to be isolated, e.g, by isolating spent culture medium containing the compound from the host cells grown in that medium.

An “organism in need” is any organism at risk of, or suffering from, any disease, disorder or condition that is amenable to treatment or amelioration with a composition according to the invention, including but not limited to any of various forms of cancer, any of a number of autoimmune diseases, radiation poisoning due to radiolabeled proteins, peptides and like compounds, ingested or internally produced toxins, and the like, as will become apparent upon review of the entire disclosure. Preferably, an organism in need is a human patient.

“Ameliorating” a symptom of a disease means detectably reducing the severity of that symptom of disease, as would be known in the art. Exemplary symptoms include pain, heat, swelling and joint stiffness.

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Unless clear from context, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein, with each referring to at least one contiguous chain of amino acids. Analogously, the terms “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably unless it is clear from context that a particular, and non-interchangeable, meaning is intended.

“Pharmaceutically acceptable salt” refers to salts of the compounds of the present invention derived from the combination of such compounds and an organic or inorganic acid (acid addition salts) or an organic or inorganic base (base addition salts).

Using the terms as defined above, a general description of the various aspects of the invention is provided below. Following the general description, working examples are presented to provide supplementary evidence of the operability and usefulness of the invention disclosed herein.

Proteins and polypeptides

In certain embodiments of the invention, there are provided any of the hereindescribed multivalent binding proteins with effector function, including binding domain-immunoglobulin fusion proteins, wherein the multivalent binding protein or peptide with effector function comprises two or more binding domain polypeptide sequences. Each of the binding domain polypeptide sequences is capable of binding or specifically binding to a target(s), such as an antigen(s), which target(s) or antigen(s) may be the same or may be different. The binding domain polypeptide sequence may be derived from an antigen variable region or it may be derived from immunoglobulin-like molecules, e.g., receptors that fold in ways that mimic immunoglobulin molecules. The antibodies from which the binding domains are derived may be antibodies that are polyclonal, including monospecific polyclonal, monoclonal (mAbs), recombinant, chimeric, humanized (such as CDR-grafted), human, single-chain, catalytic, and any other form of antibody known in the art, as well as fragments, variants or derivatives thereof. In some embodiments, each of the binding domains of the protein according to the invention is derived from a complete variable region of an immunoglobulin. In preferred embodiments, the binding domains are each based on a human Ig variable region. In other embodiments, the protein is derived from a fragment of an Ig variable region. In such embodiments, it

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2016231617 23 Sep 2016 is preferred that each binding domain polypeptide sequence correspond to the sequences of each of the complementarity determining regions of a given Ig variable region. Also contemplated within the invention are binding domains that correspond to fewer than all CDRs of a given Ig variable region, provided that such binding domains retain the capacity to specifically bind to at least one target.

The multivalent binding protein with effector function also has a constant subregion sequence derived from an immunoglobulin constant region, preferably an antibody heavy chain constant region, covalently juxtaposed between the two binding domains in the multivalent binding protein with effector function.

The multivalent binding protein with effector function also has a scorpion linker that joins the C-terminal end of the constant sub-region to the N-terminal end of binding domain 2. The scorpion linker is not a helical peptide and may be derived from an antibody hinge region, from a region connecting binding domains of an immunoglobulin, or from the stalk region of type II C-lectins. The scorpion tinker may be derived from a wild-type hinge region of an immunoglobulin, such as an

IgGl, IgG2, IgG3, lgG4, IgA, IgD or an IgE hinge region. In other embodiments, the invention provides multivalent binding proteins with altered hinges. One category of altered hinge regions suitable for inclusion in the multivalent binding proteins is the category of hinges with an altered number of Cysteine residues, particularly those Cys residues known in the art to be involved in interchain disulfide bond formation in immunoglobulin counterpart molecules having wild-type hinges. Thus, proteins may have an IgGl hinge in which one of the three Cys residues capable of participating in interchain disulfide bond formations is missing. To indicate the Cysteine substructure of altered hinges, the Cys subsequence is presented from N- to C-terminus.

Using this identification system, the multivalent binding proteins with altered IgG hinges include hinge structures characterized as cxc, xxc, ccx, xxc, xcx, cxx, and xxx. The Cys residue may be either deleted or substituted by an amino acid that results in a conservative substitution or a non-conservative substitution. In some embodiments, the Cysteine is replaced by a Serine. For proteins with scorpion linkers comprising

IgGl hinges, the number of cysteines corresponding to hinge cysteines is reduced to 1 or 2, preferably with one of those cysteines corresponding to the hinge cysteine disposed closest to the N-terminus of the hinge.

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For proteins with scorpion linkers comprising IgG2 hinges, there may be 0, 1, 2, 3, or 4 Cys residues. Forscorpion linkers comprising altered IgG2 hinges containing 1, 2 or 3 Cys residues, all possible subsets of Cys residues are contemplated. Thus, for such linkers having one Cys, the multivalent binding proteins may have the following Cys motif in the hinge region: cxxx, xcxx, xxcx, or xxxc. For scorpion linkers comprising IgG2 hinge variants having 2 or 3 Cys residues, all possible combinations of retained and substituted (or deleted) Cys residues are contemplated. For multivalent binding proteins with scorpion linkers comprising altered IgG3 or altered IgG4 hinge regions, a reduction in Cys residues from 1 to one less than the complete number of Cys residues in the hinge region is contemplated, regardless of whether the loss is through deletion or substitution by conservative or non-conservative amino acids (e.g,, Serine). In like manner, multivalent binding proteins having a scorpion linker comprising a wild-type IgA,

IgD or IgE hinge are contemplated, as are corresponding altered hinge regions having a reduced number of Cys residues extending from 0 to one less than the total number of Cys residues found in the corresponding wild-type hinge. In some embodiments having an IgGl hinge, the first, or N-terminal, Cys residue of the hinge is retained.

For proteins with either wild-type or altered hinge regions, it is contemplated that the multivalent binding proteins will be single-chain molecules capable of forming homo20 multimers, such as dimers, e.g., by disulfide bond formation. Further, proteins with altered hinges may have alterations at the termini of the hinge region, e.g., loss or substitution of one or more amino acid residues at the N-terminus, C-terminus or both termini of a given region or domain, such as a hinge domain, as disclosed herein.

In another exemplary embodiment, the constant sub-region is derived from a constant region that comprises a native, or an engineered, IgD hinge region. The wild-type human IgD hinge has one cysteine that forms a disulfide bond with the light chain in the native IgD structure. In some embodiments, this IgD hinge cysteine is mutated (e.g., deleted) to generate an altered hinge for use as a connecting region between binding domains of, for example, a bispecific molecule. Other amino acid changes or deletions or alterations in an IgD hinge that do not result in undesired hinge inflexibility are within the scope of the invention. Native or engineered IgD hinge regions from other species are also within the scope of the invention, as are humanized native or engineered IgD hinges from non-human species, and (other non

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IgD) hinge regions from other human, or non-human, antibody isotypes, (such as the llama IgG2 hinge).

The invention further comprehends constant sub-regions attached to scorpion linkers that may be derived from hinges that correspond to a known hinge region, such as an IgG 1 hinge or an IgD hinge, as noted above. The constant sub-region may contain a modified or altered (relative to wild-type) hinge region in which at least one cysteine residue known to participate in inter-chain disulfide bond linkage is replaced by another amino acid in a conservative substitution (e.g., Ser for Cys) or a nonconservative substitution. The constant sub-region does not include a peptide region or domain that corresponds to an immunoglobulin Chi domain.

Alternative hinge and linker sequences that can be used as connecting regions are from portions of cell surface receptors that connect immunoglobulin V-like or immunoglobulin C-like domains. Regions between Ig V-like domains where the cell surface receptor contains multiple Ig V-like domains in tandem, and between Ig C15 like domains where the cell surface receptor contains multiple tandem Ig C-like regions are also contemplated as connecting regions. Hinge and linker sequences are typically from 5 to 60 amino acids long, and may be primarily flexible, but may also provide more rigid characteristics. In addition, linkers frequently provide spacing that facilitates minimization of steric hindrance between the binding domains. Preferably, these hinge and linker peptides are primarily a helical in structure, with minimal β sheet structure. The preferred sequences are stable in plasma and serum and are resistant to proteolytic cleavage. The preferred sequences may contain a naturally occurring or added motif such as the CPPC motif that confers a disulfide bond to stabilize dimer formation. The preferred sequences may contain one or more glycosylation sites. Examples of preferred hinge and linker sequences include, but are not limited to, the interdomain regions between the Ig V-like and Ig C-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, and CD244.

The constant sub-region may be derived from a camelid constant region, such as either a llama or camel IgG2 or IgG3.

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Specifically contemplated is a constant sub-region having the Ch2-Ch3 region from any Ig class, or from any IgG subclass, such as IgGl (e.g., human IgGl). In preferred embodiments, the constant sub-region and the scorpion linker derived from an immunoglobulin hinge are both derived from the same Ig class. In other preferred embodiments, the constant sub-region and the scorpion linker derived from an immunoglobulin hinge are both derived from the same Ig sub-class. The constant sub-region also may be a CH3 domain from any Ig class or subclass, such as IgGl (e.g., human IgGl), provided that it is associated with at least one immunoglobulin effector function.

The constant sub-region does not correspond to a complete immunoglobulin constant region (i.e., CHi-hinge-CH2-CH3) of the IgG class. The constant sub-region may correspond to a complete immunoglobulin constant region of other classes., IgA constant domains, such as an IgAl hinge, an IgA2 hinge, an IgA Ch2 and an IgA Chs domains with a mutated or missing tailpiece are also contemplated as constant sub15 regions. Further, any light chain constant domain may function as a constant subregion, e.g., Cr or any Cl. The constant sub-region may also include JH or JK, with or without a hinge. The constant sub-region may also correspond to engineered antibodies in which, e.g., a loop graft has been constructed by making selected amino acid substitutions using an IgG framework to generate a binding site for a receptor other than a natural FcR (CD16, CD32, CD64, FcERl), as would be understood in the art. An exemplary constant sub-region of this type is an IgG Ch2-Ch3 region modified to have a CD89 binding site.

This aspect of the invention provides a multivalent binding protein or peptide having effector function, comprising, consisting essentially of, or consisting of (a) an

N-terminally disposed binding domain polypeptide sequence derived from an immunoglobulin that is fused or otherwise connected to (b) a constant sub-region polypeptide sequence derived from an immunoglobulin constant region, which preferably includes a hinge region sequence, wherein the hinge region polypeptide may be as described herein, and may comprise, consist essentially of, or consist of, for example, an alternative hinge region polypeptide sequence, in turn fused or otherwise connected to (c) a C-terminally disposed second native or engineered binding domain polypeptide sequence derived from an immunoglobulin.

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The centrally disposed constant sub-region polypeptide sequence derived from an immunoglobulin constant region is capable of at least one immunological activity selected from the group consisting of antibody dependent cell-mediated cytotoxicity, CDC, complement fixation, and Fc receptor binding, and the binding domain polypeptides are each capable of binding or specifically binding to a target, such as an antigen, wherein the targets may be the same or different, and may be found in effectively the same physiological environment (e.g., the surface of the same cell) or in different environments (e.g., different cell surfaces, a cell surface and a cell-free location, such as in solution).

This aspect of the invention also comprehends variant proteins or polypeptides exhibiting an effector function that are at least 80%, and preferably 85%, 90%, 95% or 99% identical to a multivalent protein with effector function of specific sequence as disclosed herein.

Polynucleotides

The invention also provides polynucleotides (isolated or purified or pure polynucleotides) encoding the proteins or peptides according to the invention, vectors (including cloning vectors and expression vectors) comprising such polynucleotides, and cells (e.g., host cells) transformed or transfected with a polynucleotide or vector according to the invention. In encoding the proteins or polypeptides of the invention, the polynucleotides encode a first binding domain, a second binding domain and an Fc domain, all derived from immunoglobulins, preferably human immunoglobulins. Each binding domain may contain a sequence corresponding to a full-length variable region sequence (either heavy chain and/or light chain), or to a partial sequence thereof, provided that each such binding domain retains the capacity to specifically bind. The Fc domain may have a sequence that corresponds to a full-length immunoglobulin Fc domain sequence or to a partial sequence thereof, provided that the Fc domain exhibits at least one effector function as defined herein. In addition, each of the binding domains may be joined to the Fc domain via a linker peptide that typically is at least 8, and preferably at least 13, amino acids in length. A preferred linker sequence is a sequence based on the Gly^Ser motif, such as (Gly4Ser)3.

Variants of the multivalent binding protein with effector function are also comprehended by the invention. Variant polynucleotides are at least 90%, and

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2016231617 23 Sep 2016 preferably 95%, 99%, or 99.9% identical to one of the polynucleotides of defined sequence as described herein, or that hybridizes to one of those polynucleotides of defined sequence under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C. The polynucleotide variants retain the capacity to encode a multivalent binding protein with effector function.

The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015

M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42°C. See Sambrook et al.,

Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, N.Y. 1989).

More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agent) may also be used; however, the rate of hybridization will be affected. In instances wherein hybridization of deoxyoligonucleotides is concerned, additional exemplary stringent hybridization conditions include washing in 6x SSC, 0.05% sodium pyrophosphate at 37°C (for 1420 base oligonucleotides), 48°C (for 17-base oligonucleotides), 55°C (for 20-base oligonucleotides), and 60°C (for 23-base oligonucleotides).

In a related aspect of the invention, there is provided a method of producing a polypeptide or protein or other construct of the invention, for example, including a multivalent binding protein or peptide having effector function, comprising the steps of (a) culturing a host cell as described or provided for herein under conditions that permit expression of the construct; and (b) isolating the expression product, for example, the multivalent binding protein or peptide with effector function from the host cell or host cell culture.

Constructs

The present invention also relates to vectors, and to constructs prepared from known vectors, that each include a polynucleotide or nucleic acid of the invention,

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WO 2007/146968 PCT/US2007/071052 and in particular to recombinant expression constructs, including any of various known constructs, including delivery constructs, useful for gene therapy, that include any nucleic acids encoding multivalent, for example, multispecific, including bispecific, binding proteins and polypeptides with effector function, as provided herein;

to host cells which are genetically engineered with vectors and/or other constructs of the invention and to methods of administering expression or other constructs comprising nucleic acid sequences encoding multivalent, for example, multispecific, including bi-specific, binding proteins with effector function, or fragments or variants thereof, by recombinant techniques.

Various constructs of the invention including multivalent, for example, multispecific binding proteins with effector function, can be expressed in virtually any host cell, including in vivo host cells in the case of use for gene therapy, under the control of appropriate promoters, depending on the nature of the construct (e.g., type of promoter, as described above), and on the nature of the desired host cell (e.g., postmitotic terminally differentiated or actively dividing; e.g., maintenance of an expressible construct as an episome or integrated into the host cell genome).

Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described, for example, in Sambrook, et al., Molecular Cloning:

A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989). Exemplary cloning/expression vectors include, but are not limited to, cloning vectors, shuttle vectors, and expression constructs, that may be based on plasmids, phagemids, phasmids, cosmids, viruses, artificial chromosomes, or any nucleic acid vehicle suitable for amplification, transfer, and/or expression of a polynucleotide contained therein that is known in the art. As noted herein, in preferred embodiments of the invention, recombinant expression is conducted in mammalian cells that have been transfected, transformed or transduced with a nucleic acid according to the invention. See also, for example, Machida, CA., Viral Vectors for Gene Therapy: Methods and Protocols; Wolff, JA, Gene Therapeutics: Methods and Applications of Direct Gene Transfer (Birkhauser 1994); Stein, U and Walther, W (eds., Gene Therapy of

Cancer: Methods and Protocols (Humana Press 2000); Robbins, PD (ed.), Gene Therapy Protocols (Humana Press 1997); Morgan, JR (ed.), Gene Therapy Protocols (Humana Press 2002); Meager, A (ed.), Gene Therapy Technologies,

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Applications and Regulations: From Laboratory to Clinic (John Wiley & Sons Inc. 1999); MacHida, CA and Constant, JG, Viral Vectors for Gene Therapy: Methods and Protocols (Humana Press 2002);New Methods Of Gene Therapy For Genetic Metabolic Diseases NIH Guide, Volume 22, Number 35, October 1, 1993. See also U.S. Pat. Nos. 6,384,210; 6,384,203; 6,384,202; 6,384,018; 6,383,814; 6,383,811; 6,383,795; 6,383,794; 6,383,785; 6,383,753; 6,383,746; 6,383,743; 6,383,738; 6,383,737; 6,383,733; 6,383,522; 6,383,512; 6,383,481; 6,383,478; 6,383,138; 6,380,382; 6,380,371; 6,380,369; 6,380,362; 6,380,170; 6,380,169; 6,379,967; and 6,379,966.

Typically, expression constructs are derived from plasmid vectors. One preferred construct is a modified pNASS vector (Clontech, Palo Alto, CA), which has nucleic acid sequences encoding an ampicillin resistance gene, a polyadenylation signal and a T7 promoter site. Other suitable mammalian expression vectors are well known (see, e.g., Ausubel et al., 1995; Sambrook etal., supra·, see also, e.g., catalogues from Invitrogen, San Diego, CA; Novagen, Madison, WI; Pharmacia, Piscataway, NJ). Presently preferred constructs may be prepared that include a dihydrofolate reductase (DHFR)-encoding sequence under suitable regulatory control, for promoting enhanced production levels of the multivalent binding protein with effector function, which levels result from gene amplification following application of an appropriate selection agent (e.g., methotrexate).

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, as described above. A vector in operable linkage with a polynucleotide according to the invention yields a cloning or expression construct. Exemplary cloning/expression constructs contain at least one expression control element, e.g,, a promoter, operably linked to a polynucleotide ofthe invention. Additional expression control elements, such as enhancers, factor-specific binding sites, terminators, and ribosome binding sites are also contemplated in the vectors and cloning/expression constructs according to the invention. The heterologous structural sequence of the polynucleotide according to the invention is assembled in appropriate phase with translation initiation and termination sequences. Thus, for example, the

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WO 2007/146968 PCT/US2007/071052 multivalent binding protein-encoding nucleic acids as provided herein may be included in any one of a variety of expression vector constructs as a recombinant expression construct for expressing such a protein in a host cell. In certain preferred embodiments the constructs, are included in formulations that are administered in vivo. Such vectors and constructs include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA, such as vaccinia, adenovirus, fowl pox virus, and pseudorabies, or replication deficient retroviruses as described below. However, any other vector may be used for preparation of a recombinant expression construct, and in preferred embodiments such a vector will be replicable and viable in the host.

The appropriate DNA sequence(s) may be inserted into a vector, for example, by a variety of procedures. In general, a DNA sequence is inserted into an appropriate restriction endonuclease cleavage site(s) by procedures known in the art.

Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are contemplated. A number of standard techniques are described, for example, in Ausubel et al. (1993 Current Protocols in Molecular Biology, Greene Publ. Assoc. Inc. & John Wiley &

Sons, Inc., Boston, MA); Sambrook et al. (1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory, Plainview, NY); Maniatis et al. (1982 Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, NY); Glover (Ed.) (1985 DNA Cloning Vol. I and II, IRL Press, Oxford, UK); Hames and Higgins (Eds.), (1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK); and elsewhere.

The DNA sequence in the expression vector is operatively linked to at least one appropriate expression control sequence (e.g., a constitutive promoter or a regulated promoter) to direct mRNA synthesis. Representative examples of such expression control sequences include promoters of eukaryotic cells or their viruses, as described above. Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the

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WO 2007/146968 PCT/US2007/071052 appropriate vector and promoter is well within the level of ordinary skill in the art, and preparation of certain particularly preferred recombinant expression constructs comprising at least one promoter or regulated promoter operably linked to a nucleic acid encoding a protein or polypeptide according to the invention is described herein.

Transcription of the DNA encoding proteins and polypeptides of the invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Gene therapies using the nucleic acids of the invention are also contemplated, comprising strategies to replace defective genes or add new genes to cells and/or tissues, and is being developed for application in the treatment of cancer, the correction of metabolic disorders and in the field of immunotherapy. Gene therapies of the invention include the use of various constructs of the invention, with or without a separate carrier or delivery vehicle or constructs, for treatment of the diseases, disorders, and/or conditions noted herein. Such constructs may also be used as vaccines for treatment or prevention of the diseases, disorders, and/or conditions noted herein. DNA vaccines, for example, make use of polynucleotides encoding immunogenic protein and nucleic acid determinants to stimulate the immune system against pathogens or tumor cells. Such strategies can stimulate either acquired or innate immunity or can involve the modification of immune function through cytokine expression. In vivo gene therapy involves the direct injection of genetic material into a patient or animal, typically to treat, prevent or ameliorate a disease or symptoms associated with a disease. Vaccines and immune modulation are systemic therapies. With tissue-specific in vivo therapies, such as those that aim to treat cancer, localized gene delivery and/or expression/targeting systems are preferred. Diverse gene therapy vectors that target specific tissues are known in the art, and procedures have been developed to physically target specific tissues, for example, using catheterbased technologies, all of which are contemplated herein.

Ex vivo approaches to gene therapy are also contemplated herein and involve the removal, genetic modification, expansion and re-administration of a subject’s, e.g., human patient's, own cells. Examples include bone marrow transplantation for

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2016231617 23 Sep 2016 cancer treatment or the genetic modification of lymphoid progenitor cells. Ex vivo gene therapy is preferably applied to the treatment of cells that are easily accessible and can survive in culture during the gene transfer process (such as blood or skin cells).

Useful gene therapy vectors include adenoviral vectors, lentiviral vectors,

Adeno-associated virus (AAV) vectors, Herpes Simplex Virus (HSV) vectors, and retroviral vectors. Gene therapies may also be carried out using naked DNA, liposome-based delivery, lipid-based delivery (including DNA attached to positively charged lipids), electroporation, and ballistic projection.

In certain embodiments, including but not limited to gene therapy embodiments, the vector may be a viral vector such as, for example, a retroviral vector. Miller et al., 1989 BioTechniques 7:980; Coffin and Varmus, 1996 Retroviruses, Cold Spring Harbor Laboratory Press, NY. For example, retroviruses from which the retroviral plasmid vectors may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

Retroviruses are RNA viruses which can replicate and integrate into the genome of a host cell via a DNA intermediate. This DNA intermediate, or provirus, may be stably integrated into the host cell DNA. According to certain embodiments of the present invention, an expression construct may comprise a retrovirus into which a foreign gene that encodes a foreign protein is incorporated in place of normal retroviral RNA. When retroviral RNA enters a host cell coincident with infection, the foreign gene is also introduced into the cell, and may then be integrated into host cell DNA as if it were part of the retroviral genome. Expression of this foreign gene within the host results in expression of the foreign protein.

Most retroviral vector systems that have been developed for gene therapy are based on murine retroviruses. Such retroviruses exist in two forms, as free viral particles referred to as virions, or as proviruses integrated into host cell DNA. The virion form of the virus contains the structural and enzymatic proteins of the

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2016231617 23 Sep 2016 retrovirus (including the enzyme reverse transcriptase), two RNA copies of the viral genome, and portions of the source cell plasma membrane containing viral envelope glycoprotein. The retroviral genome is organized into four main regions: the Long Terminal Repeat (LTR), which contains czs-acting elements necessary for the initiation and termination of transcription and is situated both 5 ’ and 3 ’ to the coding genes, and the three genes encoding gag, pol, and env. These three genes, gag, pol, and env, encode, respectively, internal viral structures, enzymatic proteins (such as integrase), and the envelope glycoprotein (designated gp70 and pl5e) which confers infectivity and host range specificity of the virus, as well as the “R” peptide of undetermined function.

Separate packaging cell lines and vector-producing cell lines have been developed because of safety concerns regarding the uses of retroviruses, including uses in expression constructs. Briefly, this methodology employs the use of two components, a retroviral vector and a packaging cell line (PCL). The retroviral vector contains long terminal repeats (LTRs), the foreign DNA to be transferred and a packaging sequence (y). This retroviral vector will not reproduce by itself because the genes which encode structural and envelope proteins are not included within the vector genome. The PCL contains genes encoding the gag, pol, and env proteins, but does not contain the packaging signal “y.” Thus, a PCL can only form empty virion particles by itself Within this general method, the retroviral vector is introduced into the PCL, thereby creating a vector-producing cell line (VCL). This VCL manufactures virion particles containing only the foreign genome of the retroviral vector, and therefore has previously been considered to be a safe retrovirus vector for therapeutic use.

A “retroviral vector construct” refers to an assembly which is, within preferred embodiments of the invention, capable of directing the expression of a sequence(s) or gene(s) of interest, such as multivalent binding protein-encoding nucleic acid sequences. Briefly, the retroviral vector construct must include a 5' LTR, a tRNA binding site, a packaging signal, an origin of second strand DNA synthesis and a 3'

LTR. A wide variety of heterologous sequences may be included within the vector construct including, for example, sequences which encode a protein (e.g., cytotoxic protein, disease-associated antigen, immune accessory molecule, or replacement

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WO 2007/146968 PCT/US2007/071052 gene), or which are useful as a molecule itself (e.g., as a ribozyme or antisense sequence).

Retroviral vector constructs of the present invention may be readily constructed from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see, e.g., RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such retroviruses may be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; Rockville, Maryland), or isolated from known sources using commonly available techniques. Any of the above retroviruses may be readily utilized in order to assemble or construct retroviral vector constructs, packaging cells, or producer cells of the invention, given the disclosure provided herein and standard recombinant techniques (e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Kunkle, 1985 Proc.

Natl. Acad. Sci. (USA) 52:488).

Suitable promoters for use in viral vectors generally may include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., 1989 Biotechniques 7:980-990, or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters that may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and BI9 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein, and may be from among either regulated promoters or promoters as described above.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-ΑΜ, PA12, T19-14X, VT19-17-H2, \|/CRE, xj/CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, 1:5-14 (1990). The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO₄ precipitation. In one

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WO 2007/146968 PCT/US2007/071052 alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the multivalent binding proteins with effector function. Such retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the protein or polypeptide. Eukaryotic cells that may be transduced include, but are not limited to, embryonic stem cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, circulating peripheral blood mononuclear and polymorphonuclear cells including myelomonocytic cells, lymphocytes, myoblasts, tissue macrophages, dendritic cells, Kupffer cells, lymphoid and reticuloendothelial cells of the lymph nodes and spleen, keratinocytes, endothelial cells, and bronchial epithelial cells.

Host cells

A further aspect of the invention provides a host cell transformed or transfected with, or otherwise containing, any of the polynucleotides or cloning/expression constructs of the invention. The polynucleotides and cloning/expression constructs are introduced into suitable cells using any method known in the art, including transformation, transfection and transduction. Host cells include the cells of a subject undergoing ex vivo cell therapy including, for example, ex vivo gene therapy. Eukaryotic host cells contemplated as an aspect of the invention when harboring a polynucleotide, vector, or protein according to the invention include, in addition to a subject’s own cells (e.g., a human patient’s own cells), VER.O cells, HeLa cells, Chinese hamster ovary (CHO) cell lines (including modified CHO cells capable of modifying the glycosylation pattern of expressed multivalent binding molecules, see Published US Patent Application No. 2003/0115614 Al), incorporated herein by reference, COS cells (such as COS-7), W138, BHK, HepG2,3T3, RIN, MDCK, A549, PC12, K562, HEK293 cells, HepG2 cells, N cells, 3T3 cells, Spodoptera frugiperda cells (e.g., Sf9 cells), Saccharomyces cerevisiae cells, and any other eukaryotic cell known in the art to be useful in expressing, and optionally isolating, a protein or peptide according to the invention. Also contemplated are prokaryotic cells, including but not limited to, Escherichia coli, Bacillus subtilis,

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Salmonella typhimurium, a Streptomycete, or any prokaryotic cell known in the art to be suitable for expressing, and optionally isolating, a protein or peptide according to the invention. In isolating protein or peptide from prokaryotic cells, in particular, it is contemplated that techniques known in the art for extracting protein from inclusion bodies may be used. The selection of an appropriate host is within the scope of those skilled in the art from the teachings herein.

The engineered host cells can be cultured in a conventional nutrient medium modified as appropriate for activating promoters, selecting transformants, or amplifying particular genes. The culture conditions for particular host cells selected for expression, such as temperature, pH and the like, will be readily apparent to the ordinarily skilled artisan. Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, 1981 Cell 23:175, and other cell lines capable of expressing a compatible vector, for example, the 027, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and, optionally, enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5’ flanking nontranscribed sequences, for example as described herein regarding the preparation of multivalent binding protein expression constructs. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Introduction of the construct into the host cell can be effected by a variety of methods with which those skilled in the art will be familiar, including but not limited to, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis et al., 1986 Basic Methods in Molecular Biology).

In one embodiment, a host cell is transduced by a recombinant viral construct directing the expression of a protein or polypeptide according to the invention. The transduced host cell produces viral particles containing expressed protein or polypeptide derived from portions of a host cell membrane incorporated by the viral particles during viral budding.

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Pharmaceutical compositions

In some embodiments, the compositions of the invention, such as a multivalent binding protein or a composition comprising a polynucleotide encoding such a protein as described herein, are suitable to be administered under conditions and for a time sufficient to permit expression of the encoded protein in a host cell in vivo or in vitro, for gene therapy, and the like. Such compositions may be formulated into pharmaceutical compositions for administration according to well known methodologies. Pharmaceutical compositions generally comprise one or more recombinant expression constructs, and/or expression products of such constructs, in combination with a pharmaceutically acceptable carrier, excipient or diluent. Such carriers will be nontoxic to recipients at the dosages and concentrations employed.

For nucleic acid-based formulations, or for formulations comprising expression products according to the invention, about 0.01 pg/kg to about 100 mg/kg body weight will be administered, for example, by the intradermal, subcutaneous, intramuscular or intravenous route, or by any route known in the art to be suitable under a given set of circumstances. A preferred dosage, for example, is about 1 pg/kg to about 1 mg/kg, with about 5 pg/kg to about 200 pg/kg particularly preferred.

It will be evident to those skilled in the art that the number and frequency of administration will be dependent upon the response of the host. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remingtons Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985). For example, sterile saline and phosphate-buffered saline at physiological pH may be used. Preservatives, stabilizers, dyes and the like may be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and esters ofy-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In addition, antioxidants and suspending agents may be used. Id. The compounds of the present invention may be used in either the free base or salt forms, with both forms being considered as being within the scope of the present invention.

The pharmaceutical compositions that contain one or more nucleic acid constructs of the invention, or the proteins corresponding to the products encoded by such nucleic acid constructs, may be in any form which allows for the composition to be administered to a patient. For example, the composition may be in the form of a

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WO 2007/146968 PCT/US2007/071052 solid, liquid or gas (aerosol). Typical routes of administration include, without limitation, oral, topical, parenteral (e.g,, sublingually or buccally), sublingual, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal, intracavemous, intrathecal, intrameatal, intraurethral injection or infusion techniques. The pharmaceutical composition is formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of one or more compounds of the invention in aerosol form may hold a plurality of dosage units.

For oral administration, an excipient and/or binder may be present. Examples are sucrose, kaolin, glycerin, starch dextrins, sodium alginate, carboxymethylcellulose and ethyl cellulose. Coloring and/or flavoring agents may be present. A coating shell may be employed.

The composition may be in the form of a liquid, e.g., an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to one or more binding domain-immunoglobulin fusion construct or expressed product, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included,

A liquid pharmaceutical composition as used herein, whether in the form of a solution, suspension or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents;

antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of

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WO 2007/146968 PCT/US2007/071052 tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

It may also be desirable to include other components in the preparation, such as delivery vehicles including, but not limited to, aluminum salts, water-in-oil emulsions, biodegradable oil vehicles, oil-in-water emulsions, biodegradable microcapsules, and liposomes. Examples of immunostimulatory substances (adjuvants) for use in such vehicles include N-acetylmuramyl-L-alanine-D10 isoglutamine (MDP), lipopolysaccharides (LPS), glucan, IL-12, GM-CSF, gamma interferon and IL-15.

While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration and whether a sustained release is desired. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic galactide) may also be employed as carriers for the pharmaceutical compositions of this invention. Suitable biodegradable microspheres are disclosed, for example, in U.S, Patent Nos. 4,897,268 and 5,075,109. In this regard, it is preferable that the microsphere be larger than approximately 25 microns.

Pharmaceutical compositions may also contain diluents such as buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates (e.g., glucose, sucrose or dextrins), chelating agents (e.g., EDTA), glutathione and other stabilizers and excipients.

Neutral buffered saline or saline mixed with nonspecific serum albumin are exemplary appropriate diluents. Preferably, product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents.

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The pharmaceutical compositions according to the invention also include stabilized proteins and stable liquid pharmaceutical formulations in accordance with technology known in the art, including the technology disclosed in Published US Patent Application No. 2006/0008415 Al, incorporated herein by reference. Such technologies include derivatization of a protein, wherein the protein comprises a thiol group coupled to N-acetyl-L-cysteine, N-ethyl-maleimide, or cysteine.

As described above, the subject invention includes compositions capable of delivering nucleic acid molecules encoding multivalent binding proteins with effector function. Such compositions include recombinant viral vectors, e.g., retroviruses (see

WO 90/07936, WO 91/02805, WO 93/25234, WO 93/25698, and WO 94/03622), adenovirus (see Berkner, 1988 Biotechniques 6:616-627; Li et al., 1993 Hum. Gene Ther. 4:403-409; Vincent et al., Nat. Genet. 5:130-134; and Kolls et al., 1994 Proc. Natl, Acad. Sci. USA 91:215-219), pox virus (see U.S. Patent No. 4,769,330; U.S. Patent No. 5,017,487; and WO 89/01973)), recombinant expression construct nucleic acid molecules complexed to a polycationic molecule (see WO 93/03709), and nucleic acids associated with liposomes (see Wang et al., 1987 Proc. Natl. Acad. Sci. USA 84:7851). In certain embodiments, the DNA may be linked to killed or inactivated adenovirus (see Curiel et al., 1992 Hum. Gene Ther. 3:147-154; Cotton et al., 1992 Proc. Natl. Acad. Sci. USA 59:6094). Other suitable compositions include

DNA-ligand (see Wu et al., 1989 J. Biol. Chem. 264·. 16985-16987) and lipid-DNA combinations (see Feigner et al., 1989 Proc. Natl. Acad. Sci. USA 84:7413-7417).

In addition to direct in vivo procedures, ex vivo procedures may be used in which cells are removed from a host (e.g., a subject, such as a human patient), modified, and placed into the same or another host animal. It will be evident that one can utilize any of the compositions noted above for introduction of constructs of the invention, either the proteins/polypeptides or the nucleic acids encoding them into tissue cells in an ex vivo context. Protocols for viral, physical and chemical methods of uptake are well known in the art.

Generation of antibodies

Polyclonal antibodies directed toward an antigen polypeptide generally are produced in animals (e.g., rabbits, hamsters, goats, sheep, horses, pigs, rats, gerbils, guinea pigs, mice, or any other suitable mammal, as well as other non-mammal

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2016231617 23 Sep 2016 species) by means of multiple subcutaneous or intraperitoneal injections of antigen polypeptide or a fragment thereof and an adjuvant. Adjuvants include, but are not limited to, complete or incomplete Freund's adjuvant, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, and dinitrophenol. BCG (bacilli CalmetteGuerin) and Corynebacterium parvum are also potentially useful adjuvants. It may be useful to conjugate an antigen polypeptide to a carrier protein that is immunogenic in the species to be immunized; typical carriers include keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Also, aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for anti-antigen polypeptide antibody titer using conventional techniques. Polyclonal antibodies may be utilized in the sera from which they were detected, or may be purified from the sera using, e.g., antigen affinity chromatography.

Monoclonal antibodies directed toward antigen polypeptides are produced using any method which provides for the production of antibody molecules by continuous cell lines in culture. For example, monoclonal antibodies may be made by the hybridoma method as described in Kohler et al., Nature 256:495 [1975]; the human B-cell hybridoma technique (Kosbor et al., Immunol Today 4:72, 1983 ; Cote et al., Proc Natl Acad Sci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985).

When the hybridoma technique is employed, myeloma cell lines may be used. Cell lines suited for use in hybridoma-producing fusion procedures preferably do not produce endogenous antibody, have high fusion efficiency, and exhibit enzyme deficiencies that render them incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NSl/l.Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and

S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210;

and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.

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In an alternative embodiment, human antibodies can be produced from phagedisplay libraries (Hoogenboom et al., J. Mol, Biol. 227: 381 [1991]; Marks et al., J. Mol. Biol. 222: 581, see also U.S. Patent No. 5,885,793), ). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice. One such technique is described in PCT Application No. PCT/US98/17364, filed in the name of Adams et al., which describes the isolation of high affinity and functional agonistic antibodies for MPL- and msk-receptors using such an approach. In this approach, a complete repertoire of human antibody genes can be created by cloning naturally rearranged human V genes from peripheral blood lymphocytes as previously described (Mullinax, et al., Proc. Natl. Acad. Sci.(USA) 87: 8095-8099 [1990]).

Alternatively, an entirely synthetic human heavy chain repertoire can be created from unrearranged V gene segments by assembling each human VH segment with D segments of random nucleotides together with a human J segment (Hoogenboom, et at, J. Mol. Biot 227:381-388 [1992]). Likewise, a light chain repertoire can be constructed by combining each human V segment with a J segment (Griffiths, et al, EMBO J. 13:3245-3260 [1994]). Nucleotides encoding the complete antibody (i.e., both heavy and light chains) are linked as a single-chain Fv fragment and this polynucleotide is ligated to a nucleotide encoding a filamentous phage minor coat protein. When this fusion protein is expressed on the surface of the phage, a polynucleotide encoding a specific antibody can be identified by selection using an immobilized antigen.

Beyond the classic methods of generating polyclonal and monoclonal antibodies, any method for generating any known antibody form is contemplated. In addition to polyclonals and monoclonals, antibody forms include chimerized antibodies, humanized antibodies, CDR-grafted antibodies, and antibody fragments and variants.

Variants and Derivatives of Specific Binding Agents

In one example, insertion variants are provided wherein one or more amino acid residues supplement a specific binding agent amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within

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2016231617 23 Sep 2016 internal regions of the specific binding agent amino acid sequence. Variant products of the invention also include mature specific binding agent products, i.e., specific binding agent products wherein leader or signal sequences are removed, and the resulting protein having additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from a specific protein.

Polypeptides with an additional methionine residue at position -1 (e.g., Met-1multivalent binding peptides with effector function) are contemplated, as are polypeptides of the invention with additional methionine and lysine residues at positions -2 and -1 (Met-2-Lys-1 -multivalent binding proteins with effector function).

Variants of the polypeptides of the invention having additional Met, Met-Lys, or Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.

The invention also embraces specific polypeptides of the invention having additional amino acid residues which arise from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position -1 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated, including those wherein histidine tags are incorporated into the amino acid sequence, generally at the carboxy and/or amino terminus of the sequence.

In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a polypeptide of the invention are removed. Deletions can be effected at one or both termini of the polypeptide, or from removal of one or more residues within the amino acid sequence. Deletion variants necessarily include all fragments of a polypeptide according to the invention.

Antibody fragments refer to polypeptides having a sequence corresponding to at least part of an immunoglobulin variable region sequence. Fragments may be generated, for example, by enzymatic or chemical cleavage of polypeptides corresponding to full-length antibodies. Other binding fragments include those generated by synthetic techniques or by recombinant DNA techniques, such as the

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2016231617 23 Sep 2016 expression of recombinant plasmids containing nucleic acid sequences encoding partial antibody variable regions. Preferred polypeptide fragments display immunological properties unique to, or specific for, a target as described herein.

Fragments of the invention having the desired immunological properties can be prepared by any of the methods well known and routinely practiced in the art.

In still another aspect, the invention provides substitution variants of multivalent binding polypeptides having effector function. Substitution variants include those polypeptides wherein one or more amino acid residues in an amino acid sequence are removed and replaced with alternative residues. In some embodiments, the substitutions are conservative in nature; however, the invention embraces substitutions that ore also non-conservative. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A (see WO 97/09433, page 10, published March 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below.

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Table A

Conservative Substitutions I
SIDE CHAIN	CHARACTERISTIC	AMINO ACID
Aliphatic	Non-polar	GAPILV
	Polar - uncharged	STMNQ
	Polar - charged	DEKR
Aromatic		HF WY
Other		NQDE

Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:NY (1975), pp.71-77] as set out in Table B, immediately below.

Table B

Conservative Substitutions II
SIDE CHAIN	CHARACTERISTIC	AMINO ACID
Non-polar (hydrophobic)	A. Aliphatic:	ALI VP
	B. Aromatic	FW
	C. Sulfur-containing	M
	D. Borderline	G
Uncharged-polar	A. Hydroxyl	STY
	B. Amides	NQ
	C. Sulfhydryl	C
	D. Borderline	G
Positively Charged (Basic)		KRH
Negatively Charged (Acidic)		DE

Conservative Substitutions II

SIDE CHAIN CHARACTERISTIC AMINO ACID

Non-po lar (hydrophobic)

A. Aliphatic: A L I V P

B. Aromatic:

FW

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C. Sulfur-containing:

D. Borderline:

Uncharged-polar

A. Hydroxyl:

B. Amides:

C. Sulfhydryl:

D. Borderline:

Positively Charged (Basic)

Negatively Charged (Acidic)

STY

NQ

C

G

KRH

DE

The invention also provides derivatives of specific binding agent polypeptides. Derivatives include specific binding agent polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life of a specific binding agent polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.

The invention further embraces multivalent binding proteins with effector function that are covalently modified or derivatized to include one or more watersoluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol, as described U.S. Patent Nos: 4,640,835, 4,496,689,

4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other useful polymers known in the art include monomethoxy-polyethylene glycol, dextran, cellulose, and other carbohydrate-based polymers, poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures of

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WO 2007/146968 PCT/US2007/071052 these polymers. Particularly preferred are polyethylene glycol (PEG) -derivatized proteins. Water-soluble polymers may be bonded at specific positions, for example at the amino terminus of the proteins and polypeptides according to the invention, or randomly attached to one or more side chains of the polypeptide. The use of PEG for improving therapeutic capacities is described in US Pat. No. 6, 133, 426 to Gonzales, et al.

Target Sites for Immunoglobulin Mutagenesis

Certain strategies are available to manipulate inherent properties of an antigenspecific immunoglobulin (e.g., an antibody) that are not available to non10 immunoglobulin-based binding molecules. A good example of the strategies favoring, e.g., antibody-based molecules, over these alternatives is the in vivo modulation of the affinity of an antibody for its target through affinity maturation, which takes advantage of the somatic hypermutation of immunoglobulin genes to yield antibodies of increasing affinity as an immune response progresses.

Additionally, recombinant technologies have been developed to alter the structure of immunoglobulins and immunoglobulin regions and domains. Thus, polypeptides derived from antibodies may be produced that exhibit altered affinity for a given antigen, and a number of purification protocols and monitoring screens are known in the art for identifying and purifying or isolating these polypeptides. Using these known techniques, polypeptides comprising antibody-derived binding domains can be obtained that exhibit decreased or increased affinity for an antigen. Strategies for generating the polypeptide variants exhibiting altered affinity include the use of sitespecific or random mutagenesis of the DNA encoding the antibody to change the amino acids present in the protein, followed by a screening step designed to recover antibody variants that exhibit the desired change, e.g., increased or decreased affinity relative to the unmodified parent or referent antibody.

The amino acid residues most commonly targeted in mutagenic strategies to alter affinity are those in the complementarity-determining region (CDR) or hypervariable region of the light and the heavy chain variable regions of an antibody.

These regions contain the residues that physicochemically interact with an antigen, as well as other amino acids that affect the spatial arrangement of these residues. However, amino acids in the framework regions of the variable domains outside the

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CDR regions have also been shown to make substantial contributions to the antigenbinding properties of an antibody, and can be targeted to manipulate such properties. See Hudson, P.J. Curr. Opin. Biotech., 9: 395-402 (1999) and references therein.

Smaller and more effectively screened libraries of antibody variants can be 5 produced by restricting random or site-directed mutagenesis to sites in the CDRs that correspond to areas prone to “hyper-mutation” during the somatic affinity maturation process. See Chowdhury, et al., Nature Biotech., 17: 568-572 (1999) and references therein. The types of DNA elements known to define hyper-mutation sites in this manner include direct and inverted repeats, certain consensus sequences, secondary structures, and palindromes. The consensus DNA sequences include the tetrabase sequence Purine-G-Pyrimidine-A/T (i.e., A or G - G - C or T - A or T) and the serine codon AGY (wherein Y can be C or T).

Thus, another aspect of the invention is a set of mutagenic strategies for modifying the affinity of an antibody for its target. These strategies include mutagenesis of the entire variable region of a heavy and/or light chain, mutagenesis of the CDR regions only, mutagenesis of the consensus hypermutation sites within the CDRs, mutagenesis of framework regions, or any combination of these approaches (“mutagenesis” in this context could be random or site-directed). Definitive delineation of the CDR regions and identification of residues comprising the binding site of an antibody can be accomplished though solving the structure of the antibody in question, and the antibody:ligand complex, through techniques known to those skilled in the art, such as X-ray crystallography, Various methods based on analysis and characterization of such antibody crystal structures are known to those of skill in the art and can be employed to approximate the CDR regions. Examples of such commonly used methods include the Rabat, Chothia, AbM and contact definitions.

The Rabat definition is based on sequence variability and is the most commonly used definition to predict CDR regions. Johnson, et al., Nucleic Acids Research, 28: 214-8 (2000). The Chothia definition is based on the location of the structural loop regions. (Chothia et al,, J. Mol. Biol., 196: 901-17 [1986]; Chothia et al., Nature, 342: 877-83 [1989].) The AbM definition is a compromise between the Rabat and Chothia definitions. AbM is an integral suite of programs for antibody structure modeling produced by the Oxford Molecular Group (Martin , et al., Proc.

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Natl. Acad. Sci (USA) 86:9268-9272 [1989]; Rees, et al., ABMTM, a computer program for modeling variable regions of antibodies, Oxford, UK; Oxford Molecular, Ltd.). The AbM suite models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods An additional definition, known as the contact definition, has been recently introduced. See MacCallum et al., J. Mol. Biol., 5:732-45 (1996). This definition is based on an analysis of the available complex crystal structures.

By convention, the CDR domains in the heavy chain are typically referred to as Hl, H2 and H3, and are numbered sequentially in order moving from the amino terminus to the carboxy terminus. The CDR regions in the light chain are typically referred to as Ll, L2 and L3, and are numbered sequentially in order moving from the amino terminus to the carboxy terminus.

The CDR-H1 is approximately 10 to 12 residues in length and typically starts 4 residues after a Cys according to the Chothia and AbM definitions, or typically 5 residues later according to the Kabat definition. The Η1 is typically followed by a Trp, typically Trp-Val, but also Trp-lle, or Trp-Ala. The length of Hl is approximately 10 to 12 residues according to the AbM definition, while the Chothia definition excludes the last 4 residues.

The CDR-H2 typically starts 15 residues after the end of Hl according to the

Kabat and AbM definitions. The residues preceding H2 are typically Leu-Glu-TrpIle-Gly but there are a number of variations. H2 is typically followed by the amino acid sequence Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala. According to the Kabat definition, the length of H2 is approximately 16 to 19 residues, where the AbM definition predicts the length to be typically 9 to 12 residues.

The CDR-H3 typically starts 33 residues after the end of H2 and is typically preceded by the amino acid sequence Cys-Ala-Arg. H3 is typically followed by the amino acid Gly. The length of H3 ranges from 3 to 25 residues

The CDR-L1 typically starts at approximately residue 24 and will typically follow a Cys. The residue after the CDR-L1 is always Trp and will typically begin one of the following sequences: Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln, or Trp-TyrLeu. The length of CDR-L1 is approximately 10 to 17 residues.

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The CDR-L2 starts approximately 16 residues after the end of LI. It will generally follow residues Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. The length of CDR-L2 is approximately 7 residues.

The CDR-L3 typically starts 33 residues after the end of L2 and typically 5 follows a Cys. L3 is typically followed by the amino acid sequence Phe-Gly-XXXGly. The length of L3 is approximately 7 to 11 residues.

Various methods for modifying antibodies have been described in the art, including, e.g., methods of producing humanized antibodies wherein the sequence of the humanized immunoglobulin heavy chain variable region framework is 65% to

95% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Each humanized immunoglobulin chain will usually comprise, in addition to the CDRs, amino acids from the donor immunoglobulin framework that are, e.g., capable of interacting with the CDRs to effect binding affinity, such as one or more amino acids that are immediately adjacent to a CDR in the donor immunoglobulin or those within about 3 angstroms, as predicted by molecular modeling. The heavy and light chains may each be designed by using any one or all of various position criteria, When combined into an intact antibody, humanized immunoglobulins are substantially non-immunogenic in humans and retain substantially the same affinity as the donor immunoglobulin to the antigen, such as a protein or other compound containing an epitope.

In one example, methods for the production of antibodies, and antibody fragments, are described that have binding specificity similar to a parent antibody, but which have increased human characteristics. Humanized antibodies are obtained by chain shuffling using, for example, phage display technology and a polypeptide comprising the heavy or light chain variable region of a non-human antibody specific for an antigen of interest, which is then combined with a repertoire of human complementary (light or heavy) chain variable regions. Hybrid pairings which are specific for the antigen of interest are identified and human chains from the selected pairings are combined with a repertoire of human complementary variable domains (heavy or light). In another embodiment, a component of a CDR from a non-human antibody is combined with a repertoire of component parts of CDRs from human antibodies. From the resulting library of antibody polypeptide dimers, hybrids are

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WO 2007/146968 PCT/US2007/071052 selected and may used in a second humanizing shuffling step; alternatively, this second step is eliminated if the hybrid is already of sufficient human character to be of therapeutic value. Methods of modification to increase human character are known in the art.

Another example is a method for making humanized antibodies by substituting a CDR amino acid sequence for the corresponding human CDR amino acid sequence and/or substituting a FR amino acid sequence for the corresponding human FR amino acid sequences.

Yet another example provides methods for identifying the amino acid residues 10 of an antibody variable domain that may be modified without diminishing the native affinity of the antigen binding domain while reducing its immunogenicity with respect to a heterologous species and methods for preparing these modified antibody variable regions as useful for administration to heterologous species.

Modification of an immunoglobulin such as an antibody by any of the 15 methods known in the art is designed to achieve increased or decreased binding affinity for an antigen and/or to reduce immunogenicity of the antibody in the recipient and/or to modulate effector activity levels. In one approach, humanized antibodies can be modified to eliminate glycosylation sites in order to increase affinity of the antibody for its cognate antigen (Co, et at, Mol. Immunol. 30:1361-1367 [1993]). Techniques such as “reshaping,” hyperchimerization,” and “veneering/resurfacing” have produced humanized antibodies with greater therapeutic potential. Vaswami, et al., Annals of Allergy, Asthma, & Immunol 81:105 (1998); Roguska, et al., Prot. Engineer. 9:895-904 (1996)]. See also US Pat. No. 6,072,035, which describes methods for reshaping antibodies. While these techniques diminish antibody immunogenicity by reducing the number of foreign residues, they do not prevent anti-idiotypic and anti-allotypic responses following repeated administration of the antibodies. Alternatives to these methods for reducing immunogenicity are described in Gilliland etal., J. Immunol. 62(6):3663-71 (1999).

In many instances, humanizing antibodies results in a loss of antigen binding capacity. It is therefore preferable to “back mutate” the humanized antibody to include one or more of the amino acid residues found in the original (most often

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WO 2007/146968 PCT/US2007/071052 rodent) antibody in an attempt to restore binding affinity of the antibody. See, for example, Saldanha et al., Mol. Immunol. 36:709-19 (1999).

Glycosylation of immunoglobulins has been shown to affect effector functions, structural stability, and the rate of secretion from antibody-producing cells (see Leatherbarrow et al., Mol. Immunol. 22:407 (1985), incorporated herein by reference). The carbohydrate groups responsible for these properties are generally attached to the constant regions of antibodies. For example, glycosylation of IgG at Asn 297 in the Ch2 domain facilitates full capacity of the IgG to activate complementdependent cytolysis (Tao et al., J. Immunol. 143:2595 (1989)). Glycosylation of IgM at Asn 402 in the Ch3 domain, for example, facilitates proper assembly and cytolytic activity of the antibody (Muraoka et al., J. Immunol. 142:695 (1989)). Removal of glycosylation sites at positions 162 and 419 in the Chi and Ch3 domains of an IgA antibody led to intracellular degradation and at least 90% inhibition of secretion (Taylor et al., Wall, Mol. Cell. Biol. 8:4197 (1988)). Accordingly, the molecules of the invention include mutationally altered immunoglobulins exhibiting altered glycosylation patterns by mutation of specific residues in, e.g., a constant sub-region to alter effector function. See Co et al., Mol, Immunol. 30:1361-1367 (1993), Jacquemon et al., J. Thromb. Haemost. 4:1047-1055 (2006), Schuster et al,, Cancer Res. 65:7934-7941 (2005), and Warnock et al., Biotechnol Bioeng. 92:831-842 (2005), each incorporated herein by reference.

The invention also includes multivalent binding molecules having at least one binding domain that is at least 80%, preferably 90% or 95% or 99% identical in sequence to a known immunoglobulin variable region sequence and which has at least one residue that differs from such immunoglobulin variable region, wherein the changed residue adds a glycosylation site, changes the location of one or more glycosylation site(s), or preferably removes a glycosylation site relative to the immunoglobulin variable region. In some embodiments, the change removes an Nlinked glycosylation site in a an immunoglobulin variable region framework, or removes an N-linked glycosylation site that occurs in the immunoglobulin heavy chain variable region framework in the region spanning about amino acid residue 65 to about amino acid residue 85, using the numbering convention of Co et al., J. Immunol. 148: 1149, (1992).

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Any method known in the art is contemplated for producing the multivalent binding molecules exhibiting altered glycosylation patterns relative to an immunoglobulin referent sequence. For example, any of a variety of genetic techniques may be employed to alter one or more particular residues. Alternatively, the host cells used for production may be engineered to produce the altered glycosylation pattern. One method known in the art, for example, provides altered glycosylation in the form of bisected, non-fucosylated variants that increase ADCC. The variants result from expression in a host cell containing an oligosaccharidemodifying enzyme. Alternatively, the Potelligent technology of BioWa/Kyowa

Hakko is contemplated to reduce the fucose content of glycosylated molecules according to the invention. In one known method, a CHO host cell for recombinant immunoglobulin production is provided that modifies the glycosylation pattern of the immunoglobulin Fc region, through production of GDP-fucose. This technology is available to modify the glycosylation pattern of a constant sub-region of a multivalent binding molecule according to the invention.

In addition to modifying the binding properties of binding domains, such as the binding domains of immunoglobulins, and in addition to such modifications as humanization, the invention comprehends the modulation of effector function by changing or mutating residues contributing to effector function, such as the effector function of a constant sub-region. These modifications can be effected using any technique known in the art, such as the approach disclosed in Presta et al., Biochem. Soc. Trans. 30:487-490 (2001), incorporated herein by reference. Exemplary approaches would include the use of the protocol disclosed in Presta et al. to modify specific residues known to affect binding in one or more constant sub-regions corresponding to FCyRI, FCyRlI, FCyRIlI, FCaR, and FCcR.

In another approach, the Xencor XmAb technology is available to engineer constant sub-regions corresponding to Fc domains to enhance cell killing effector function. See Lazar et al., Proc. Natl. Acad. Sci. (USA) 103(11):4005-4010 (2006), incorporated herein by reference. Using this approach, for example, one can generate constant sub-regions optimized for FcyR specificity and binding, thereby enhancing cell killing effector function.

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Production of multivalent binding proteins with effector function

A variety of expression vector/host systems may be utilized to contain and express the multivalent binding protein (with effector function) of the invention.

These systems include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, cosmid, or other expression vectors; yeast transformed with yeast expression or shuttle vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant multivalent binding protein productions include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and HEK293 cells. Exemplary protocols for the recombinant expression of the multivalent binding protein are described herein below.

An expression vector can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, a promoter, enhancer, or factor-specific binding site, (2) a structural or sequence that encodes the binding agent which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant multivalent binding protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final multivalent binding protein.

For example, the multivalent binding proteins may be recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. This system also relies on the pre-pro-alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol. The secreted multivalent binding peptide may be purified

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2016231617 23 Sep 2016 from the yeast growth medium by, e.g., the methods used to purify the peptide from bacterial and mammalian cell supernatants.

Alternatively, the cDNA encoding the multivalent binding peptide may be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego,

CA). This vector can be used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in SF9 protein-free medium and to produce recombinant protein. The multivalent binding protein can be purified and concentrated from the medium using a heparin-Sepharose column (Pharmacia, Piscataway, NJ). Insect systems for protein expression, such as the SF9 system, are well known to those of skill in the art. In one such system, Autographa califomica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes in the Spodoptera frugiperda cells or in Trichoplusia larvae. The multivalent binding peptide coding sequence can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the multivalent binding peptide will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can be used to infect S. frugiperda cells or Trichoplusia larvae in which peptide is expressed (Smith et al., J Virol 46: 584, 1983; Engelhard et al., Proc Nat Acad Sci (USA) 91: 3224-7, 1994).

In another example, the DNA sequence encoding the multivalent binding peptide can be amplified by PCR and cloned into an appropriate vector, for example, pGEX-3X (Pharmacia, Piscataway, NJ). The pGEX vector is designed to produce a fusion protein comprising glutathione-S-transferase (GST), encoded by the vector, and a multivalent binding protein encoded by a DNA fragment inserted into the cloning site of the vector. The primers for the PCR can be generated to include for example, an appropriate cleavage site. Where the multivalent binding protein fusion moiety is used solely to facilitate expression or is otherwise not desirable as an attachment to the peptide of interest, the recombinant multivalent binding protein fusion may then be cleaved from the GST portion of the fusion protein. The pGEX30 3X/multivalent binding peptide construct is transformed into E. coli XL-1 Blue cells (Stratagene, La Jolla CA), and individual transformants isolated and grown. Plasmid DNA from individual transformants is purified and may be partially sequenced using

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2016231617 23 Sep 2016 an automated sequencer to confirm the presence of the desired multivalent binding protein-encoding nucleic acid insert in the proper orientation.

The fused multivalent binding protein, which may be produced as an insoluble inclusion body in the bacteria, can be purified as follows. Host cells can be harvested by centrifugation; washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate can be cleared by sonication, and cell debris can be pelleted by centrifugation for 10 minutes at 12,000 X g. The multivalent binding protein fusion-containing pellet can be resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 minutes at 6000g. The pellet can be resuspended in standard phosphate buffered saline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺. The multivalent binding protein fusion can be further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al,).

The gel is soaked in 0.4 M K.C1 to visualize the protein, which is excised and electroeluted in gel-running buffer lacking SDS. If the GST/multivalent binding peptide fusion protein is produced in bacteria as a soluble protein, it can be purified using the GST Purification Module (Pharmacia Biotech).

The multivalent binding protein fusion is preferably subjected to digestion to cleave the GST from the multivalent binding peptide of the invention. The digestion reaction (20-40 pg fusion protein, 20-30 units human thrombin (4000 U/mg (Sigma) in 0.5 ml PBS) can be incubated 16-48 hours at room temperature and loaded on a denaturing SDS-PAGE gel to fractionate the reaction products. The gel can be soaked in 0.4 M KC1 to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of the multivalent binding peptide can be confirmed by amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, CA). Alternatively, the identity can be confirmed by performing HPLC and/or mass spectrometry of the peptides.

Alternatively, a DNA sequence encoding the multivalent binding peptide can be cloned into a plasmid containing a desired promoter and, optionally, a leader sequence (see, e.g., Better et al., Science, 240:1041-43,1988). The sequence of this construct can be confirmed by automated sequencing. The plasmid can then be transformed into a suitable E. coli strain, such as strain MCI061, using standard

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2016231617 23 Sep 2016 procedures employing CaCk incubation and heat shock treatment of the bacteria (Sambrook et al.). The transformed bacteria can be grown in LB medium supplemented with carbenicillin or another suitable form of selection as would be known in the art, and production of the expressed protein can be induced by growth in a suitable medium. If present, the leader sequence can effect secretion of the multivalent binding peptide and be cleaved during secretion. The secreted recombinant protein can be purified from the bacterial culture medium by the methods described herein below.

Mammalian host systems for the expression of the recombinant protein are 10 well known to those of skill in the art and are preferred systems. Host cell strains can be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like, have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure the correct modification and processing of the foreign protein.

It is preferable that the transformed cells be used for long-term, high-yield protein production and, as such, stable expression is desirable. Once such cells are transformed with vectors that preferably contain at least one selectable marker along with the desired expression cassette, the cells are grown for 1-2 days in an enriched medium before being switched to selective medium. The selectable marker is designed to confer resistance to selection and its presence allows growth and recovery of cells that successfully express the foreign protein, Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.

A number of selection systems can be used to recover the cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-, hgprtor aprt- cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate; gpt, which confers

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WO 2007/146968 PCT/US2007/071052 resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside G418 and confers resistance to chlorsulfuron; and hygro, which confers resistance to hygromycin. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a visual indication for identification of transformants include anthocyanins, β-glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

Purification of Proteins

Protein purification techniques are well known to those of skill in the art.

These techniques involve, at one level, the crude fractionation of the polypeptide and non-polypeptide fractions. Having separated the multivalent binding polypeptide from at least one other protein, the polypeptide of interest is purified, but further purification using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity) is frequently desired.

Analytical methods particularly suited to the preparation of a pure multivalent binding peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; and isoelectric focusing. Particularly efficient methods of purifying peptides are fast protein liquid chromatography and HPLC.

Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded multivalent binding protein or peptide. The term purified multivalent binding protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the multivalent binding protein or peptide is purified to any degree relative to its naturally obtainable state. A purified multivalent binding protein or peptide therefore also refers to a multivalent binding protein or peptide, free from the environment in which it may naturally occur.

Generally, purified will refer to a multivalent binding protein composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term substantially purified is used, this designation refers to a multivalent binding protein composition in which the multivalent binding protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%,

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2016231617 23 Sep 2016 about 70%, about 80%, about 90%, about 95%, about 99% or more of the protein, by weight, in the composition.

Various methods for quantifying the degree of purification of the multivalent binding protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific binding activity of an active fraction, or assessing the amount of multivalent binding polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a multivalent binding protein fraction is to calculate the binding activity of the fraction, to compare it to the binding activity of the initial extract, and to thus calculate the degree of purification, herein assessed by a -fold purification number. The actual units used to represent the amount of binding activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed multivalent binding protein or peptide exhibits a detectable binding activity.

Various techniques suitable for use in multivalent binding protein purification are well known to those of skill in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like, or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified multivalent binding protein.

There is no general requirement that the multivalent binding protein always be provided in its most purified state. Indeed, it is contemplated that less substantially multivalent binding proteins will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in greater purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree

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2016231617 23 Sep 2016 of relative purification may have advantages in total recovery of multivalent binding protein product, or in maintaining binding activity of an expressed multivalent binding protein.

It is known that the migration of a polypeptide can vary, sometimes 5 significantly, with different conditions of SDS/PAGE (Capaldi et al., Biochem.

Biophys. Res. Comm., 76:425, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified multivalent binding protein expression products may vary.

Effector cells

Effector cells for inducing, e.g., ADCC, ADCP (antibody-dependent cellular phagocytosis), and the like, against a target cell include human leukocytes, macrophages, monocytes, activated neutrophils, activated natural killer (NK) cells, and eosinophils. Effector cells express FcaR (CD89), FcyRI, FcyRII, FcyRIII, and/or FcsRl and include, for example, monocytes and activated neutrophils. Expression of

FcyRI, e.g., has been found to be up-regulated by interferon gamma (IFN-γ). This enhanced expression increases the cytotoxic activity of monocytes and neutrophils against target cells. Accordingly, effector cells may be activated with (IFN-γ) or other cytokines (e.g., TNF-α or β, colony stimulating factor, IL-2) to increase the presence of FcyRI on the surface of the cells prior to being contacted with a multivalent protein of the invention,

The multivalent proteins of the invention provide an antibody effector function, such as antibody-dependent effector cell-mediated cytotoxicity (ADCC), for use against a target cell. Multivalent proteins with effector function are administered alone, as taught herein, or after being coupled to an effector cell, thereby forming an activated effector cell. An activated effector cell is an effector cell, as defined herein, linked to a multivalent protein with effector function, also as defined herein, such that the effector cell is effectively provided with a targeting function prior to administration.

Activated effector cells are administered in vivo as a suspension of cells in a physiologically acceptable solution. The number of cells administered is on the order of 10⁸-l0⁹, but will vary depending on the therapeutic purpose. In general, the

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101 amount will be sufficient to obtain localization of the effector cell at the target cell, and to provide a desired level of effector cell function in that locale, such as cell killing by ADCC and/or phagocytosis. The term physiologically acceptable solution, as used herein, is intended to include any carrier solution which stabilizes the targeted effector cells for administration in vivo including, for example, saline and aqueous buffer solutions, solvents, antibacterial and antifungal agents, isotonic agents, and the like.

Accordingly, another aspect of the invention provides a method of inducing a specific antibody effector function, such as ADCC, against a cell in a subject, comprising administering to the subject a multivalent protein (or encoding nucleic acid) or activated effector cell in a physiologically acceptable medium. Routes of administration can vary and suitable administration routes will be determined by those of skill in the art based on a consideration of case-specific variables and routine procedures, as is known in the art.

Cell-free effects

Cell-free effects are also provided by the multivalent molecules of the invention, e.g., by providing a CDC functionality. The complement system is a biochemical cascade of the immune system that helps clear foreign matter such as pathogens from an organism, It is derived from many small plasma proteins that work together in inducing cytolysis of a target cell by disrupting the target cell's plasma membrane. The complement system consists of more than 35 soluble and cell-bound proteins, 12 of which are directly involved in the complement pathways. The proteins are active in three biochemical pathways leading to the activation of the complement system: the classical complement pathway, the alternate complement pathway, and the mannose-binding lectin pathway. Antibodies, in particular the IgG 1 class, can also fix complement. A detailed understanding of these pathways has been achieved in the art and will not be repeated here, but it is worth noting that complement-dependent cytotoxicity is not dependent on the interaction of a binding molecule with a cell, e.g., a B cell, of the immune system. Also worth noting is that the complement system is regulated by complement regulating proteins. These proteins are present at higher concentrations in the blood plasma than the complement proteins. The complement regulating proteins are found on the surfaces of self-cells,

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2016231617 23 Sep 2016 providing a mechanism to prevent self-cells from being targeted by complement proteins. It is expected that the complement system plays a role in several diseases with an immune component, such as Barraquer-Simons Syndrome, Alzheimer's disease, asthma, lupus erythematosus, various forms of arthritis, autoimmune heart disease, and multiple sclerosis. Deficiencies in the terminal pathway predispose an individual to both autoimmune disease and infections (particularly meningitis).

Diseases, disorders and conditions

The invention provides a multivalent binding proteins with effector function, and variant and derivative thereof, that bind to one or more binding partners and those binding events are useful in the treatment, prevention, or amelioration of a symptom associated with a disease, disorder or pathological condition, preferably one afflicting humans. In preferred embodiments of these methods, the multivalent (and multispecific) binding protein with effector function associates a cell bearing a target, such as a tumor-specific cell-surface marker, with an effector cell, such as a cell of the immune system exhibiting cytotoxic activity. In other embodiments, the multispecific, multivalent binding protein with effector function specifically binds two different disease-, disorder- or condition-specific cell-surface markers to ensure that the correct target is associated with an effector cell, such as a cytotoxic cell of the immune system. Additionally, the multivalent binding protein with effector function can be used to induce or increase antigen activity, or to inhibit antigen activity. The multivalent binding proteins with effector function are also suitable for combination therapies and palliative regimes.

In one aspect, the present invention provides compositions and methods useful for treating or preventing diseases and conditions characterized by aberrant levels of antigen activity associated with a cell. These diseases include cancers and other hyperproliferative conditions, such as hyperplasia, psoriasis, contact dermatitis, immunological disorders, and infertility, A wide variety of cancers, including solid tumors and leukemias are amenable to the compositions and methods disclosed herein. Types of cancer that may be treated include, but are not limited to:

adenocarcinoma of the breast, prostate, and colon; all forms of bronchogenic carcinoma of the lung; myeloid; melanoma; hepatoma; neuroblastoma; papilloma; apudoma; choristoma; branchioma; malignant carcinoid syndrome; carcinoid heart

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103 disease; and carcinoma (e.g., Walker, basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, Krebs 2, merkel cell, mucinous, non-small cell lung, oat cell, papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, and transitional cell). Additional types of cancers that may be treated include: histiocytic disorders;

leukemia; histiocytosis malignant; Hodgkin’s disease; immunoproliferative small; non-Hodgkin’s lymphoma; plasmacytoma; reticuloendotheliosis; melanoma; chondroblastoma; chondroma; chondrosarcoma; fibroma; fibrosarcoma; giant cell tumors; histiocytoma; lipoma; liposarcoma; mesothelioma; myxoma; myxosarcoma; osteoma; osteosarcoma; chordoma; craniopharyngioma; dysgerminoma; hamartoma;

mesenchymoma; mesonephroma; myosarcoma; ameloblastoma; cementoma;

odontoma; teratoma; thymoma; trophoblastic tumor. Further, the following types of cancers are also contemplated as amenable to treatment: adenoma; cholangioma; cholesteatoma; cyclindroma; cystadenocarcinoma; cystadenoma; granulosa cell tumor; gynandroblastoma; hepatoma; hidradenoma; islet cell tumor; Leydig cell tumor; papilloma; sertoli cell tumor; theca cell tumor; leimyoma; leiomyosarcoma; myoblastoma; myomma; myosarcoma; rhabdomyoma; rhabdomyosarcoma; ependymoma; ganglioneuroma; glioma; medulloblastoma; meningioma; neurilemmoma; neuroblastoma; neuroepithelioma; neurofibroma; neuroma; paraganglioma; paraganglioma nonchromaffin. The types of cancers that may be treated also include, but are not limited to, angiokeratoma; angiolymphoid hyperplasia with eosinophilia; angioma sclerosing; angiomatosis; glomangioma; hemangioendothelioma; hemangioma; hemangiopericytoma; hemangiosarcoma; lymphangioma; lymphangiomyoma; lymphangiosarcoma; pinealoma; carcinosarcoma; chondrosarcoma; cystosarcoma phyllodes; fibrosarcoma;

hemangiosarcoma; leiomyosarcoma; leukosarcoma; liposarcoma;

lymphangiosarcoma; myosarcoma; myxosarcoma; ovarian carcinoma; rhabdomyosarcoma; sarcoma; neoplasms; nerofibromatosis; and cervical dysplasia. The invention further provides compositions and methods useful in the treatment of other conditions in which cells have become immortalized or hyperproliferative due to abnormally high expression of antigen.

Exemplifying the variety of hyperproliferative disorders amenable to the compositions and methods of the invention are B-cell cancers, including B-cell lymphomas (such as various forms of Hodgkin's disease, non-Hodgkins lymphoma

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104 (NHL) or central nervous system lymphomas), leukemias (such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), Hairy cell leukemia and chronic myoblastic leukemia) and myelomas (such as multiple myeloma). Additional B cell cancers include small lymphocytic lymphoma, B-cell pro lymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, solitary plasmacytoma of bone, extraosseous plasmacytoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt’s lymphoma/Ieukemia, B-cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder.

Disorders characterized by autoantibody production are often considered autoimmune diseases. Autoimmune diseases include, but are not limited to: arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, responses associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham’s chorea, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including

Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including hypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia

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105 (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection, graft versus host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathies, seronegative spondyloarthropathies, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM) and

Sheehan’s syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), polyarteritis nodosa (PAN) ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis,

Celiac sprue (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans.

Rheumatoid arthritis (RA) is a chronic disease characterized by inflammation of the joints, leading to swelling, pain, and loss of function. Patients having RA for an extended period usually exhibit progressive joint destruction, deformity, disability and even premature death. Beyond RA, inflammatory diseases, disorders and

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2016231617 23 Sep 2016 conditions in general are amenable to treatment, prevention or amelioration of symptoms (e.g., heat, pain, swelling, redness) associated with the process of inflammation, and the compositions and methods of the invention are beneficial in treating, preventing or ameliorating aberrant or abnormal inflammatory processes, including RA.

Crohn's disease and a related disease, ulcerative colitis, are the two main disease categories that belong to a group of illnesses called inflammatory bowel disease (IBD). Crohn's disease is a chronic disorder that causes inflammation of the digestive or gastrointestinal (GI) tract. Although it can involve any area of the GI tract from the mouth to the anus, it most commonly affects the small intestine and/or colon. In ulcerative colitis, the GI involvement is limited to the colon. Crohn’s disease may be characterized by antibodies against neutrophil antigens, i.e., the perinuclear anti-neutrophil antibody (pANCA), and Saccharomyces cervisiae, i.e. the anti-Saccharomyces cerevisiae antibody (ASCA). Many patients with ulcerative colitis have the pANCA antibody in their blood, but not the ASCA antibody, while many Crohn's patients exhibit ASCA antibodies, and not pANCA antibodies. One method of evaluating Crohn’s disease is using the Crohn’s disease Activity Index (CDAI), based on 18 predictor variables scores collected by physicians. CDAI values of 150 and below are associated with quiescent disease; values above that indicate active disease, and values above 450 are seen with extremely severe disease [Best et al., Development of a Crohn's disease activity index. Gastroenterology 70:439-444 (1976)]. However, since the original study, some researchers use a 'subjective value’ of 200 to 250 as an healthy score.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease caused by recurrent injuries to blood vessels in multiple organs, including the kidney, skin, and joints. In patients with SLE, a faulty interaction between T cells and B-cells results in the production of autoantibodies that attack the cell nucleus. There is general agreement that autoantibodies are responsible for SLE, so new therapies that deplete the B-cell lineage, allowing the immune system to reset as new B-cells are generated from precursors, would offer hope for long lasting benefit in SLE patients.

Multiple sclerosis (MS) is also an autoimmune disease. It is characterized by inflammation of the central nervous system and destruction of myelin, which insulates

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107 nerve cell fibers in the brain, spinal cord, and body. Although the cause of MS is unknown, it is widely believed that autoimmune T cells are primary contributors to the pathogenesis of the disease. However, high levels of antibodies are present in the cerebral spinal fluid of patients with MS, and some theories predict that the B-cell response leading to antibody production is important for mediating the disease.

Autoimmune thyroid disease results from the production of autoantibodies that either stimulate the thyroid to cause hyperthyroidism (Graves' disease) or destroy the thyroid to cause hypothyroidism (Hashimoto’s thyroiditis). Stimulation of the thyroid is caused by autoantibodies that bind and activate the thyroid stimulating hormone (TSH) receptor. Destruction of the thyroid is caused by autoantibodies that react with other thyroid antigens.

Additional diseases, disorders, and conditions amenable to the benefits provided by the compositions and methods of the invention include Sjogren's syndrome is an autoimmune disease characterized by destruction of the body’s moisture-producing glands. Further, immune thrombocytopenic purpura (ITP) is caused by autoantibodies that bind to blood platelets and cause their destruction, and this condition is suitable for application of the materials and methods of the invention. Myasthenia Gravis (MG), a chronic autoimmune neuromuscular disorder characterized by autoantibodies that bind to acetylcholine receptors expressed at neuromuscular junctions leading to weakness of the voluntary muscle groups, is a disease having symptoms that are treatable using the composition and methods of the invention, and it is expected that the invention will be beneficial in treating and/or preventing MG. Still further, Rous Sarcoma Virus infections are expected to be amenable to treatment, or amelioration of at least one symptom, with the compositions and methods of the invention.

Another aspect of the present invention is using the materials and methods of the invention to prevent and/or treat any hyperproliferative condition of the skin including psoriasis and contact dermatitis or other hyperproliferative disease.

Psoriasis, is characterized by autoimmune inflammation in the skin and is also associated with arthritis in 30% of cases, as well as scleroderma, inflammatory bowel disease, including Crohn's disease and ulcerative colitis. It has been demonstrated that patients with psoriasis and contact dermatitis have elevated antigen activity

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108 within these lesions (Ogoshi et al., J. Inv. Dermatol., 110:818-23 [1998]). The multispecific, multivalent binding proteins can deliver a cytotoxic cell of the immune system, for example, directly to cells within the lesions expressing high levels of antigen. The multivalent, e.g., multispecific, binding proteins can be administered subcutaneously in the vicinity of the lesions, or by using any of the various routes of administration described herein and others which are well known to those of skill in the art.

Also contemplated is the treatment of idiopathic inflammatory myopathy (IIM), including dermatomyositis (DM) and polymyositis (PM). Inflammatory myopathies have been categorized using a number of classification schemes. Miller’s classification schema (Miller, Rheum Dis Clin North Am. 20:811-826, 1994) identifies 2 idiopathic inflammatory myopathies (ΠΜ), polymyositis (PM) and dermatomyositis (DM).

Polymyositis and dermatomyositis are chronic, debilitating inflammatory diseases that involve muscle and, in the case of DM, skin. These disorders are rare, with a reported annual incidence of approximately 5 to 10 cases per million adults and 0.6 to 3.2 cases per million children per year in the United States (Targoff, Curr Probl Dermatol. 1991, 3:131 -180). Idiopathic inflammatory myopathy is associated with significant morbidity and mortality, with up to half of affected adults noted to have suffered significant impairment (Gottdiener et al., Am J Cardiol. 1978,41:1141-49). Miller (Rheum Dis Clin North Am. 1994,20:811-826 and Arthritis and Allied Conditions, Ch. 75, Eds. Koopman and Moreland, Lippincott Williams and Wilkins, 2005) sets out five groups of criteria used to diagnose IIM, i.e., Idiopathic Inflammatory Myopathy Criteria (IIMC) assessment, including muscle weakness, muscle biopsy evidence of degeneration, elevation of serum levels of muscleassociated enzymes, electromagnetic triad of myopathy, evidence of rashes in dermatomyositis, and also includes evidence of autoantibodies as a secondary criteria.

IIM associated factors, including muscle-associated enzymes and autoantibodies include, but are not limited to, creatine kinase (CK), lactate dehydrogenase, aldolase, C-reactive protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and antinuclear autoantibody (ANA), myositisspecific antibodies (MSA), and antibody to extractable nuclear antigens.

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Preferred autoimmune diseases amenable to the methods of the invention include Crohn's disease, Guillain-Barre syndrome (GBS; also known as acute inflammatory demyelinating polyneuropathy, acute idiopathic polyradiculoneuritis, acute idiopathic polyneuritis and Landry's ascending paralysis), lupus erythematosus, multiple sclerosis, myasthenia gravis, optic neuritis, psoriasis, rheumatoid arthritis, hyperthyroidism (e.g., Graves' disease), hypothyroidism (e.g., Hashimoto's disease), Ord’s thyroiditis (a thyroiditis similar to Hashimoto's disease), diabetes mellitus (type 1), aplastic anemia, Reiter's syndrome, autoimmune hepatitis, primary biliary cirrhosis, antiphospholipid antibody syndrome (APS), opsoclonus myoclonus syndrome (OMS), temporal arteritis (also known as giant cell arteritis), acute disseminated encephalomyelitis (ADEM), Goodpasture's syndrome, Wegener's granulomatosis, coeliac disease, pemphigus, canine polyarthritis, warm autoimmune hemolytic anemia. In addition, the invention contemplates methods for the treatment, or amelioration of a symptom associated with, the following diseases, endometriosis, interstitial cystitis, neuromyotonia, scleroderma, vitiligo, vulvodynia, Chagas' disease leading to Chagasic cardiopathy (cardiomegaly), sarcoidosis, chronic fatigue syndrome, and dysautonomia.

The complement system is believed to play a role in many diseases with an immune component, such as Alzheimer's disease, asthma, lupus erythematosus, various forms of arthritis, autoimmune heart disease and multiple sclerosis, all of which are contemplated as diseases, disorders or conditions amenable to treatment or symptom amelioration using the methods according to the invention.

Certain constant sub-regions are preferred, depending on the particular effector function or functions to be exhibited by a multivalent single-chain binding molecule. For example, IgG (IgGl, 2, or 3) and IgM are preferred for complement activation, IgG of any subtype is preferred for opsonization and toxin neutralization; IgA is preferred for pathogen binding; and IgE for binding of such parasites as worms.

By way of example, FcRs recognizing the constant region of IgG antibodies have been found on human leukocytes as three distinct types of Fey receptors, which are distinguishable by structural and functional properties, as well as by antigenic

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110 structures detected by CD monoclonal antibodies. They are known as FcyRI, FcyRII, and FcyRIII, and are differentially expressed on (overlapping) subsets of leukocytes.

FcgRI (CD64), a high-affinity receptor expressed on monocytes, macrophages, neutrophils, myeloid precursors and dendritic cells, comprised isoforms la and lb. FcgRI has a high affinity for monomeric human IgGl and IgG3. Its affinity for IgG4 is about 10 times lower, while it does not bind IgG2. FcgRI does not show genetic polymorphism.

FcyRII (CD32), comprised of isoforms 11a, lib 1, llb2, llb3 and 11c, is the most widely distributed human FcyR type, being expressed on most types of blood leukocytes, as well as on Langerhans cells, dendritic cells and platelets. FcyRII is a low-affinity receptor that only binds aggregated IgG. It is the only FcyR class able to bind IgG2. FcyRIIa shows genetics polymorphism, resulting in two distinct allotypes, FcyRlla-H131 and FcyRlla-R131, respectively. This functional polymorphism is attributable to a single amino acid difference: a histidine (H) or an arginine (R) residue at position 131, which is critical for IgG binding. FcyRIIa readily binds human

IgG and IgG3 and appears not to bind IgG4. The FcyRlla-H131 has a much higher affinity for complexed IgG2 than the FcyRlia-R131 allotype.

FcyRIII (CD16) has two isoforms or allelotypes, both of which are able to bind IgGl and IgG3. The FcyRIIa, with an intermediate affinity for IgG, is expressed on macrophages, monocytes, natural killer (NK) cells and subsets of T cells.

FcyRIIIb is a low-affinity receptor for IgG, selectively expressed on neutrophils. It is a highly mobile receptor with efficient collaboration with other membrane receptors. Studies with myeloma IgG dimers have shown that only IgG 1 and IgG3 bind to FcyRIIIb (with low affinity), while no binding of IgG2 and IgG4 has been found. The

FcyRIIIb bears a co-dominant, bi-allelic polymorphism, the allotypes being designated NA1 (Neutrophil Antigen) and NA2.

Yet another aspect of the invention is use of the materials and methods of the invention to combat, by treating, preventing or mitigating the effects of, infection, resulting from any of a wide variety of infectious agents. The multivalent, multispecific binding molecules of the invention are designed to efficiently and effectively recruit the host organism’s immune system to resist infection arising from

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111 a foreign organism, a foreign cell, a foreign virus or a foreign inanimate object. For example, a multispecific binding molecule may have one binding domain that specifically binds to a target on an infectious agent and another binding domain that specifically binds to a target on an Antigen Presenting Cell, such as CD 40, CD80,

CD86, DC-SIGN, DEC-205, CD83, and the like). Alternatively, each binding domain of a multivalent binding molecule may specifically bind to an infectious agent, thereby more effectively neutralizing the agent. In addition, the invention contemplates multispecific, multivalent binding molecules that specifically bind to a target on an infectious agent and to a non-cell-associated binding partner, which may be effective in conjunction with an effector function of the multispecific binding molecule in treating or preventing infection arising from an infectious agent.

Infectious cells contemplated by the invention include any known infectious cell, including but not limited to any of a variety of bacteria (e.g., pathogenic E. coli,

S. typhimurium, P. aeruginosa, B. anthracis, C. botulinum, C. difficile, C. perfringens,

H. pylori, V. cholerae, and the like), mycobacteria, mycoplasma, fungi (including yeast and molds), and parasites (including any known parasitic member of the Protozoa, Trematoda, Cestoda andNematoda). Infectious viruses include, but are not limited to, eukaryotic viruses (e.g., adenovirus, bunyavirus, herpesvirus, papovavirus, paramyxovirus, picomavirus, poxvirus, reovirus, retroviruses, and the like) as well as bacteriophage. Foreign objects include objects entering an organism, preferably a human, regardless of mode of entry and regardless of whether harm is intended. In view of the increasing prevalence of multi-drug-resistant infectious agents (e.g., bacteria), particularly as the causative agents of nosocomial infection, the materials and methods of the invention, providing an approach to treatment that avoids the difficulties imposed by increasing antibiotic resistance.

Diseases, conditions or disorders associated with infectious agents and amenable to treatment (prophylactic or therapeutic) with the materials and methods disclosed herein include, but are not limited to, anthrax, aspergillosis, bacterial meningitis, bacterial pneumoniae (e.g., chlamydia pneumoniae), blastomycosis, botulism, brucellosis, candidiasis, cholera, ciccidioidomycosis, cryptococcosis, diahhreagenic, enterohemorrhagic or enterotoxigenic E. coli, diphtheria, glanders, histoplasmosis, legionellosis, leprosy, listeriosis, nocardiosis, pertussis, salmonellosis,

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112 scarlet fever, sporotrichosis, strep throat, toxic shock syndrome, traveler’s diarrhea, and typhoid fever.

Additional aspects and details of the invention will be apparent from the following examples, which are intended to be illustrative rather than limiting.

Example 1 describes recombinant cloning of immunoglobulin heavy and light chain variable regions. Example 2 describes the construction of Small Modular ImmunoPharmaceuticals. Example 3 describes the construction of a prototype cassette for a multivalent binding protein with effector function. Example 4 describes binding and expression studies with this initial prototype molecule.

Example 5 describes construction of alternative constructs derived from this initial prototype molecule where the sequence of the linker region between the EFD and BD2 was changed in both length and sequence. In addition, it describes alternative forms where the orientation of V regions in binding domain 2 were also altered. Example 6 describes subsequent binding and functional studies on these alternative constructs with variant linker forms, identifying a cleavage in the linker region in several of these derivative forms, and the new sequence variants developed to address this problem. Example 7 describes the construction of an alternative preferred embodiment of the multispecific, multivalent fusion proteins, where both BD1 and BD2 bind to antigens on the same cell type (CD20 and CD37), or another multispecific fusion protein where the antigen binding specificity for BD2 has been changed to human CD3 instead of CD28. Example 8 describes the binding and functional studies performed with the CD20-hIgG-CD37 multispecific constructs. Example 9 describes the binding and functional studies with the CD20-hIgG-CD3 multivalent fusion protein constructs. Example 10 discloses multivalent binding molecules having linkers based on specific regions of the extracellular domains of members of the immunoglobulin superfamily. Example 11 discloses assays for identifying binding domains expected to be effective in multivalent binding molecules in achieving at least one beneficial effect identified as being associated with such molecules (e.g., disease treatment).

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Example 1

Cloning of Immunoglobulin Heavy and Light Chain Variable Regions

Any methods known in the art can be used to elicit antibodies to a given antigenic target. Further, any methods known in the art can be used to clone the immunoglobulin light and/or heavy chain variable regions, as well as the constant sub-region of an antibody or antibodies. The following method provides an exemplary cloning method.

A. Isolation of Total RNA

To clone the immunoglobulin heavy and light chain variable regions, or the constant sub-region, total RNA is isolated from hybridoma cells secreting the appropriate antibody. Cells (2xl0⁷) from the hybridoma cell line are washed with lx PBS and pelleted via centrifugation in a 12 x 75 mm round bottom polypropylene tube (Falcon no. 2059). TRlzol™ Total RNA Isolation Reagent (Gibco BRL, Life Technologies, Cat no. 15596-018) is added (8 ml) to each tube and the cells are lysed via repeated pipetting. The lysate is incubated for 5 minutes at room temperature prior to the addition of 1.6 ml (0.2 x volume) of chloroform and vigorous shaking for 15 seconds. After standing 3 minutes at room temperature, the lysates are centrifuged at 9,000 rpm for 15 minutes in a 4°C pre-chilled Beckman JA-17 rotor in order to separate the aqueous and organic phases. The top aqueous phase (about 4.8 ml) is transferred into a new tube and mixed gently with 4 ml of isopropanol. After a 10 minute incubation at room temperature, the RNA is precipitated by centrifugation at 9,000 rpm in a 4'C JA-17 rotor for 11 minutes. The RNA pellet is washed with 8 ml of ice-cold 75% ethanol and re-pelleting by centrifugation at 7,000 x rpm for 7 minutes in a JA-17 rotor at 4’C. The ethanol wash is decanted and the RNA pellets are air-dried for 10 minutes. The RNA pellets are resuspended in 150 μΐ of diethylpyrocarbonate (DEPC)-treated ddH₂O containing 1 μΐ of RNase Inhibitor (Catalog No. 799017; Boehringer Mannheim/Roche) per 1 ml of DEPC-treated ddH₂O. The pellets are resuspended by gentle pipetting and are incubated for 20 minutes at 55 °C. RNA samples are quantitated by measuring the OD₂60nm of diluted aliquots (1.0 OD_{260 nm} unit = 40 μg/ml RNA).

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B. Rapid Amplification of cDNA Ends

5’ RACE is carried out to amplify the ends of the heavy and light chain variable regions, or the constant sub-region. The 5’ RACE System for Rapid Amplification of cDNA Ends Kit version 2.0 (Life Technologies, cat. no. 18374-058) is used according to the manufacturer’s instructions. Degenerate 5’ RACE oligonucleotide primers are designed to match, e.g., the constant regions of two common classes of mouse immunoglobulin heavy chains (IgGl and IgG2b) using the oligonucleotide design program Oligo version 5.1 (Molecular Biology Insights, Cascade CO). Primers are also designed to match the constant region of the mouse

IgG kappa light chain. This is the only class of immunoglobulin light chain, so no degeneracy is needed in the primer design. The sequences of the primers are as follows:

Name Sequence SEQ ID NO

Heavy Chain GSP1 ’AGGTGCTGGAGGGGACAGTCACTGAGCTGC3 ’ 7

Nested Heavy Chain ’GTCACWGTCACTGRCTCAGGGAARTAGC3 ’ 8 (W = A or T; R = A or G)

Light Chain GSP 1 ’GGGTGCTGCTCATGCTGTAGGTGCTGTCTTTGC3' 0

Nested Light Chain 5 ’CAAGAAGCACACGACTG

AGGCACCTCCAGATG3’ 10

5’ Race Abridged Anchor Primer

5’GGCCACGCGTCGACTAGTACGG

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GNNGGGNNGGGNNG3 ’

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To amplify the mouse immunoglobulin heavy chain component, the reverse transcriptase reaction is carried in a 0.2 ml thin-walled PCR tube containing 2.5 pmoles of heavy chain GSP1 primer (SEQ ID NO: 7), 4 μg of total RNA isolated from a suitable hybridoma clone (e.g., either clone 4A5 or clone 4B5), and 12 μΐ of DEPC treated ddH2O. Likewise, for the mouse light chain component, the reverse transcriptase reaction is carried out in a 0.2 ml thin-walled PCR tube containing 2.5 pmoles of a light chain GSP1 primer (SEQ ID NO: 9), 4 μg of total RNA from a suitable hybridoma clone (e.g., either clone 4A5 or clone 4B5), and 12 μΐ of DEPC treated ddH2O.

The reactions are carried out in a PTC-100 programmable thermal cycler (MJ research Inc,, Waltham, MA). The mixture is incubated at 70°C for 10 minutes to denature the RNA and then chilled on wet ice for 1 minute. The tubes are centrifuged briefly in order to collect moisture from the lids of the tubes. Subsequently, the following components are added to the reaction: 2.5 μΐ of lOx PCR buffer (200 mM Tris-HCl, pH 8.4, 500 mM KC1), 2.5 μΐ of 25 mM MgCl₂, 1 μΐ of 10 mM dNTP mix, and 2.5 μΐ of 0.1 M DTT. After mixing each tube by gentle pipetting, the tubes are placed in a PTC-100 thermocycler at 42’C for 1 minute to pre-warm the mix.

Subsequently, 1 μΐ (200 units) of Superscript™ II Reverse Transcriptase (GibcoBRL; cat no. 18089-011) is added to each tube, gently mixed by pipetting, and incubated for 45 minutes at 42’C. The reactions are cycled to 70°C for 15 minutes to terminate the reaction, and then cycled to 37°C. RNase mix (1 μΐ) is then added to each reaction tube, gently mixed, and incubated at 37’C for 30 minutes.

The first-strand cDNA generated by the reverse transcriptase reaction is purified with the GlassMAX DNA Isolation Spin Cartridge (Gibco-BRL) according to the manufacturer’s instructions. To each first-strand reaction, 120 μΐ of 6 M Nal binding solution is added. The cDNA/Nal solution is then transferred into a GlassMAX spin cartridge and centrifuged for 20 seconds at 13,000 x g. The cartridge inserts are carefully removed and the flow-through is discarded from the tubes. The spin cartridges are then placed back into the empty tubes and 0.4 ml of cold (4’C) lx

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116 wash buffer is added to each spin cartridge. The tubes are centrifuged at 13,000 x g for 20 seconds and the flow-through is discarded. This wash step is repeated three additional times. The GlassMAX cartridges are then washed 4 times with 0.4 ml of cold (4°C) 70% ethanol. After the flow-through from the final 70% ethanol wash is discarded, the cartridges are placed back in the tubes and centrifuged at 13,000 x g for an additional 1 minute in order to completely dry the cartridges. The spin cartridge inserts are then transferred to a fresh sample recovery tube where 50 μΐ of 65°C (preheated) DEPC-treated ddH₂O is quickly added to each spin cartridge. The cartridges are centrifuged at 13,000 x g for 30 seconds to elute the cDNA.

C, Terminal Deoxynucleotidvl Transferase (TdTl Tailing

For each first-strand cDNA sample, the following components are added to a 0.2 ml thin-walled PCR tube: 6.5 μΐ of DEPC-treated ddlUO, 5.0 μΐ of 5x tailing buffer, 2.5 μΐ of 2 mM dCTP, and 10 μΐ of the appropriate GlassMAX-purified cDNA sample. Each 24 μΐ reaction is incubated 2-3 minutes in a thermal cycler at 94°C to denature the DNA, and chilled on wet ice for 1 minute. The contents of the tube are collected by brief centrifugation. Subsequently, 1 μΐ of terminal deoxynucleotidyl transferase (TdT) is added to each tube. The tubes are mixed via gentle pipetting and incubated for 10 minutes at 37°C in a PTC-100 thermal cycler. Following this 10 minute incubation, the TdT is heat inactivated by cycling to 65 °C for 10 minutes. The reactions are cooled on ice and the TdT-tailed first-strand cDNA is stored at -20°C.

D. PCR of dC-tailed First-Strand cDNA

Duplicate PCR amplifications (two independent PCR reactions for each dCtailed first-strand cDNA sample) are performed in a 50 μΐ volume containing 200 μΜ dNTPs, 0.4 μΜ of 5’ RACE Abridged Anchor Primer (SEQ ID NO: 11), and 0.4 μΜ of either Nested Heavy Chain GSP2 (SEQ ID NO: 8) or Nested Light Chain GSP2 (SEQ ID NO: 10), 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 50 mM KC1, 5 μΐ of dC-tailed cDNA, and 5 units of Expand™ Hi-Fi DNA polymerase (Roche/

Boehringer Mannheim GmbH, Germany). The PCR reactions are amplified using a “Touch-do wn/Touch-up” annealing temperature protocol in a PTC-100 programmable thermal cycler (MJ Research Inc.) with the following conditions: initial denaturation of 95°C for 40 seconds, 5 cycles at 94’C for 20 seconds, 61 °C 2°C/cycle for 20 seconds, 72°C for 40 seconds + 1 second/cycle, followed by 5 cycles

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117 at 94’C for 25 seconds, 53°C + l’C/cycle for 20 seconds, 72’C for 46 seconds + 1 second/cycle, followed by 20 cycles at 94’C for 25 seconds, 55’C for 20 seconds,

72°C for 51 seconds + 1 second/cycle, and a final incubation of 72’C for 5 minutes.

E. TOPO TA-Cloning

The resulting PCR products are gel-purified from a 1,0% agarose gel using the

QIAQuick Gel purification system (QIAGEN Inc,, Chatsworth, CA), TA-cloned into pCR2.1 using the TOPO TA Cloning® kit (invitrogen, San Diego, CA, cat. no. K4550-40), and transformed into E. coli TOP10F’ cells (Invitrogen), according to manufacturers’ instructions. Clones with inserts are identified by blue/white screening according to the manufacturer’s instructions, where white clones are considered positive clones. Cultures of 3.5 ml liquid Luria Broth (LB) containing 50 pg/ml ampicillin are inoculated with white colonies and grown at 37°C overnight (about 16 hours) with shaking at 225 rpm.

The QIAGEN Plasmid Miniprep Kit (QIAGEN Inc., cat, no. 12125) is used to purify plasmid DNA from the cultures according to the manufacturer’s instructions. The plasmid DNA is suspended in 34 μΐ of lx TE buffer (pH 8.0) and then positive clones sequenced as previously described by fluorescent dideoxy nucleotide sequencing and automated detection using AB1 Big Dye Terminator 3.1 reagents at 1:4-1:8 dilutions and analyzed using an ABI 3100 DNA sequencer. Sequencing primers used include T7 (5’GTAATACGACTCACTATAGG3’; SEQ ID NO: 12) and Ml3 Reverse (5’CAGGAAACAGCTATGACC3’; SEQ ID NO: 13) primers. Sequencing results will confirm that the clones correspond to mouse IgG sequences.

F, De novo gene synthesis using overlapping oligonucleotide extension PCR

This method involves the use of overlapping oligonucleotide primers and PCR using either a high fidelity DNA polymerase or a mix of polymerases to synthesize an immunoglobulin V-region or other gene. Starting at the middle of the V-region sequence, 40-50 base primers are designed such that the growing chain is extended by 20-30 bases, in either direction, and contiguous primers overlap by a minimum of 20 bases. Each PCR step requires two primers, one priming on the anti-sense strand (forward or sense primer) and one priming on the sense strand (reverse or anti-sense primer) to create a growing double-stranded PCR product. During primer design,

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118 changes can be made in the nucleotide sequence of the final product to create restriction enzyme sites, destroy existing restriction enzyme sites, add flexible linkers, change, delete or insert bases that alter the amino acid sequence, optimize the overall DNA sequence to enhance primer synthesis and conform to codon usage rules for the organism contemplated for use in expressing the synthetic gene.

Primer pairs are combined and diluted such that the first pair are at 5 μΜ an each subsequent pair has a 2-fold greater concentration up to 80 μΜ. One pL from each of these primer mixes is amplified in a 50 pL PCR reaction using Platinum PCR SuperMix-High Fidelity (Invitrogen, San Diego, CA, cat. no. 12532-016). After a 210 minute initial denaturation at 94°C, 30 cycles of PCR are performed using a cycling profile of94’C for 20 seconds, 60’C for 10 seconds; and 68’C for 15 seconds. PCR products are purified using Qiaquick PCR Purification columns (Qiagen Inc., cat. no. 28704) to remove excess primers and enzyme. This PCR product is then reamplified with the next set of similarly diluted primer pairs using PCR conditions exactly as described above, but increasing the extension time of each cycle to 68°C for 30 seconds. The resultant PCR product is again purified from primers and enzymes as described above and TOPO-TA cloned and sequenced exactly as described in section E above.

Example 2

Construction of Small Modular ImmunoPharmaceuticals (SMIPs)

A multispecific, multivalent binding protein with effector function was constructed that contained a binding domain 1 in the form of a single-chain recombinant (murine/human) scFv designated 2H7 (VL-linker-VH). The scFv 2H7 is a small modular immunopharmacaceutical (SMIP) that specifically recognizes CD20. The binding domain was based on a publicly available human CD20 antibody sequence GenBank Accession Numbers, Ml7953 for VH, and Ml7954 for VL. CD20-specific SMIPs are described in co-owned US Patent Publications 2003/133939, 2003/0118592 and 2005/0136049, incorporated herein in their entireties by reference. The peptide linker separating VL and VH was a 15-amino acid linker encoding the sequence: Asp-Gly₃Ser-(Gly4Ser)₂. Binding domain 1 was located at

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119 the N-terminus of the multispecific binding protein, with the C-terminus of that domain linked directly to the N-terminus of a constant sub-region containing a hinge, Ch2 and Ch3 domains (in amino-to-carboxy orientation). The constant sub-region was derived from an IgGl antibody, which was isolated by PCR amplification of human

IgGl from human PBMCs. The hinge region was modified by substituting three Ser residues in place of the three Cys residues present in the wild type version of the human IgGl hinge domain, encoded by the 15 amino acid sequence: EPKSCDKTHTCPPCP (SEQ ID NO: 14; the three Cys residues replaced by Ser residues are indicated in bold). In alternative embodiments, the hinge region was modified at one or more of the cysteines, so that SSS and CSC type hinges were generated. In addition, the final proline was sometimes substituted with a serine as well as the cysteine substitutions.

The C-terminal end of the Cm domain was covalently attached to a series of alternative linker domains juxtaposed between the constant sub-region C-terminus and the amino terminus of binding domain 2. Preferred multivalent binding proteins with effector function will have one of these linkers to space the constant sub-region from binding domain 2, although the linker is not an essential component of the compositions according to the invention, depending on the folding properties of BD2. For some specific multivalent molecules, the linker might be important for separation of domains, while for others it may be less important. The linker was attached to the N-terminal end of scFv 2E12 ((VH-hnker-Vi.), which specifically recognizes CD28. The linker separating the VH and VL domains of the scFv 2E12 part of the multivalent binding molecule was a 20-amino acid linker (Gly4Ser)4, rather than the standard (Gy4Ser)3 linker usually inserted between V domains of an scFv. The longer linker was observed to improve the binding properties of the 2el2 scFv in the VH-VL orientation.

The multispecific, multivalent binding molecule as constructed contained a binding domain 1, which comprises the 2E12 leader peptide sequence from amino acids 1-23 of SEQ ID NO: 171; the 2H7 murine anti-human CD20 light chain variable region, which is reflected at position 24 in SEQ ID NO: 171; an Asp-Gly3-Ser(Gly4Ser)2 linker, beginning at residue 130 in SEQ ID NO: 171, the 2H7 murine antihuman CD20 heavy chain variable region with a leucine to serine (VHL1 IS) amino

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120 acid substitution at residue 11 in the variable domain for VH, and which has a single serine residue at the end of the heavy chain region (i.e., VTVS where a canonical sequence would be VTVSS) (Genbank Acc. No. Ml7953), and interposed between the two binding domains BD1 (2H7) and BD2 (2E12) is a human IgGl constant sub5 region, including a modified hinge region comprising a “CSC” or an “SSS” sequence, and wild-type Ch2 and Ch3 domains. The nucleotide and amino acid sequences of the multivalent binding protein with effector function are set out in SEQ ID NOS: 228 and 229 for the CSC forms, respectively and SEQ ID NOS: 170 and 171, for the SSS forms.

Stably expressing cell lines were created by transfection via electroporation of either uncut or linearized, recombinant expression plasmid into Chinese hamster ovary cells (CHO DG44 cells) followed by selection in methotrexate containing medium. Bulk cultures and master wells producing the highest level of multivalent binding protein were amplified in increasing levels of methotrexate, and adapted cultures were subsequently cloned by limiting dilution. Transfected CHO cells producing the multivalent binding protein were cultured in bioreactors or wave bags using serum-free medium obtained from JRH Biosciences (Excell 302, cat. no. 143241000M, supplemented with 4 mM glutamine (Invitrogen, 25030-081), sodium pyruvate (Invitrogen 11360-070, diluted to IX), non-essential amino acids (Invitrogen, 11140-050, final dilution to IX), penicillin-streptromycin 100 IU/ml (Invitrogen, 15140-122), and recombulin insulin at 1 pg/ml (Invitrogen, 97-503311). Other serum free CHO basal medias may also be used for production, such as CDCHO, and the like.

Fusion protein was purified from spent CHO culture supernatants by Protein A affinity chromatography. The multivalent binding protein was purified using a series of chromatography and filtration steps, including a virus reduction filter. Cell culture supernatants were filtered, then subjected to protein A Sepharose affinity chromatography over a GE Healthcare XK 16/40 column. After binding of protein to the column, the column was washed in dPBS, then 1.0 M NaCl, 20 mM sodium phosphate pH 6.0, and then 25 mM NaCl, 25 mN NaOAc, pH 5.0 to remove nonspecific binding proteins. Bound protein was eluted from the column in 100 mM Glycine (Sigma), pH 3.5, and brought to pH 5.0 with 0.5 M 2-(N-Morpholino) ethanesulfonic acid (MES), pH 6.0. Protein samples were concentrated to 25 mg/ml

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121 in preparation for GPC purification. Size exclusion chromatography was performed on a GE Healthcare AKTA Explorer 100 Air apparatus, using a GE healthcare XK column and Superdex 200 preparative grade (GE healthcare).

The material was then concentrated and formulated with 20 mM sodium 5 phosphate and 240 mM sucrose, with a resulting pH of 6.0. The composition was filtered before filling into sterile vials at various concentrations, depending on the amount of material recovered.

Example 3

Construction of Scorpion Expression Cassette

A nucleic acid containing the synthetic 2H7 scFv (anti-CD20; SEQ ID NO: 1) linked to a constant sub-region as described in Example 2 has been designated TRU015. TRU-015 nucleic acid, as well as synthetic scFv 2E12 (anti-CD28 VL-VH; SEQ ID NO: 3) and synthetic scFv 2E12 (anti-CD28 VH-VL; SEQ ID NO: 5) nucleic acids encoding small modular immunopharmaceuticals, were used as templates for PCR amplification of the various components of the scorpion cassettes The template, or scaffold, for binding domain 1 and the constant sub-region was provided by TRU-015 (the nucleic acid encoding scFv 2H7 (anti-CD20) linked to the constant sub-region) and this template was constructed in the expression vector pD18. The above-noted nucleic acids containing scFv 2E12 in either of two orientations (Vl-Vh and Vh-Vl) provided the coding region for binding domain 2.

TRU 015 SSS hinge CHiCm. far BD2/Linker Insertion

A version of the synthetic 2H7 scFv IgGl containing the SSS hinge was used to create a scorpion cassette by serving as the template for addition of an EcoRI site to replace the existing stop codon and Xbal site. This molecule was amplified by PCR using primer 9 (SEQ ID NO: 23; see Table 1) and primer 87 (SEQ ID NO: 40; see Table 1) as well as a Platinum PCR High Fidelity mix (Invitrogen), The resultant 1.5 Kbp fragment was purified and cloned into the vector pCR2.1-TOPO (Invitrogen), transformed into E. coli strain TOP 10 (Invitrogen), and the DNA sequence verified.

Table 1

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SEQ ID

No. Name Sequence 5'-3' NO.

PCR Primers

GCGATAAAGCTTGCCGCCATGGAA

1	hVK3L-F3H3	GCACCAGCGCAGCTTCTCTTCC ACCAGCGCAGCTTCTCTTCCTCCTG	15
2	hVK3L-F2	CTACTCTGGCTCCCAGATACCACCG GGCTCCCAGATACCACCGGTCAAAT	16
3	hVK3L-F1-2H7VL	TGTTCTCTCCCAGTCTCCAG GCGATAGCTAGCCAGGCTTATCTAC	17
4	2H7VH-NheF	AGCAGTCTGG GCGATAGCTAGCCCCACCTCCTCCA	18
5	G4S-NheR	GATCCACCACCGCCCGAG GCGTACTCGAGGAGACGGTGACCGT	19
6	015VH-XhoR	GGTCCCTGTG GCAGTCTCGAGCGAGCCCAAATCTTG	20
7	G1H-C-XHO	TGACAAAACTC GCAGTCTCGAGCGAGCCCAAATCTTC	21
8	G1H-S-XHO	TGACAAAACTC GCGTGAGAATTCTTACCCGGAGACAGG	22
9	CH3R-EcoR1	GAGAGGCTC GCGACGTCTAGAGTCATTTACCCGGAG	23
10	G1-XBA-R	ACAGG AATTATGGTGGCGGTGGCTCGGGCGGT	24
11	G4SLinkR1-S	GGTGGATCTGGAGGAGGTGGGAGTGGG AATTCCCACTCCCACCTCCTCCAGATCCA	25
12	G4SLinkR1-AS	CCACCGCCCGAGCCACCGCCACCAT GCGTGTCTAGATTAACGTTTGATTTCCAG	26
13	2E12VLXbaR	CTTGGTG GCGATGAATTCTGACATTGTGCTCACCCA	27
14	2E12VLR1F	ATCTCC GCGATGAATTCTCAGGTGCAGCTGAAGGA	28
15	2E12VHR1F	GTCAG GCGAGTCTAGATTAAGAGGAGACGGTGAC	29
16	2E12VHXbaR	TGAGGTTC	30
17	2e12VHdXbaF1	GGGTCTGGAGTGGCTGGGAATGATATG	31
18	2e12VHdXbaR1	ATTCCCAGCCACTCCAGACCCTTTCCTG	32
19	IgBsrGIF	GAGAACCACAGGTGTACACCCTG	33
20	IgBsrGIR Sequencing Primers	GCAGGGTGTACACCTGTGGTTCTCG	34
82	M13R	CAGGAAACAGCTATGAC	35
83	M13F	GTAAAACGACGGCCAGTG	36
84	T7	GTAATACGACTCACTATAGG	37
85	pD18F-17	AACTAGAGAACCCACTG	38
86	pD18F-20	GCTAACTAGAGAACCCACTG	39
87	pD18F-1	ATACGACTCACTATAGGG	40
88	pD18R-s	GCTCTAGCATTTAGGTGAC	41
89	CH3seqF1	CATGAGGCTCTGCACAAC

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SEQ ID

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No.	Name	Sequence 5'-3'	42
90	CH3seqF2	CCTCTACAGCAAGCTCAC	43
91	CH3seqR1	GGTTCTTGGTCAGCTCATC	44
92	CH3seqR2	GTGAGCTTGCTGTAGAGG	45

Table 1. Oligonucleotide primers used to construct CD20-CD28 scorpion cassette. Primers are separated into 2 groups, PCR and Sequencing. PCR primers were used to construct the cassette and sequencing primers were used to confirm the DNA sequence of all intermediates and final constructs.

n2H7 Vk and human/Vp-leader sequence fusion

Oligonucleotide-directed PCR mutagenesis was used to introduce an Agel (ACCGGT) restriction site at the 5’ end of the coding region for TRU 015 VK and an

Nhe I (GCTAGC) restriction site at the 3’ end of the coding region for the (G4S)3 linker using primers 3 and 5 from Table 1. Since primer 3 also encodes the last 6 amino acids of the human VK3 leader (gb:X01668), overlapping PCR was used to sequentially add the N-terminal sequences of the leader including a consensus Kozak box and HinDIII (AAGCTT) restriction site using primers 1, 2 and 5 from Table 1, n2H7 IgGl SSS hwe-CH;Cnj Construction

Primers 4 and 6 (SEQ ID NOS: 18 and 20, respectively; Table 1) were used to re-amplify the TRU-015 Vh with an Nhel site 5' to fuse with the Vk for TRU-015 and an Xho I (5’-CTCGAG-3’) site at the 3' end junction with the IgGl hinge-CHzCm domains. Likewise, the IgGl hinge-Cn2-Cii3 region was amplified using primers 8 and 9 from Table 1, introducing a 5' Xho I site and replacing the existing 3' end with an EcoRI (5’-GAATTC-3’) site for cloning, and destroying the stop codon to allow translation of Binding Domain 2 attached downstream of the CH3 domain.. This version of the scorpion cassette is distinguished from the previously described cassette by the prefix n.

In addition to the multivalent binding protein described above, a protein according to the invention may have a binding domain, either binding domain 1 or 2 or both, that corresponds to a single variable region of an immunoglobulin.

Exemplary embodiments of this aspect of the invention would include binding

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2016231617 23 Sep 2016 domains corresponding to the Vh domain of a camelid antibody, or a single modified or unmodified V region of another species antibody capable of binding to the target antigen, although any single variable domain is contemplated as useful in the proteins of the invention.

2E12 VL-VH and VH-VL constructions

In order to make the 2E12 scFvs compatible with the cassette, an internal Xba I (5’-TCTAGA-3’) site had to be destroyed using overlapping oligonucleotide primers 17 and 18 from Table 1. These two primers in combination with primer pairs 14/16 (VL-VH) or 13/15 (VH-VL) were used to amplify the two oppositely oriented binding domains such that they both carried EcoRI and Xbal sites at their 5' and 3' ends, respectively. Primers 13 and 16 also encode a stop codon (TAA) immediately in front of the Xba I site.

2H7 SSS IgGl 2el2 LH/HL Construction

Effector Domain- Binding Domain 2 Linker addition. (STD linkers - STD I and STD2)

Complementary primers 11 and 12 from Table 1 were combined, heated to 70°C and slow-cooled to room temperature to allow annealing of the two strands. 5' phosphate groups were added using T4 polynucleotide kinase (Roche) in IX Ligation buffer with ImM ATP(Roche) using the manufacturer's protocol. The resulting doublestranded linker was then ligated into the EcoRI site between the coding regions for the

IgGl Ch3 terminus and the beginning of Binding Domain 2 using T4 DNA ligase (Roche). The resultant DNA constructs were screened for the presence of an EcoRI site at the linker-BD2 junction and the nucleotide sequence GAATTA at the Ch3linker junction. The correct STD 1 linker construct was then re-digested with EcoRI and the linker ligation repeated to produce a molecule that had a linker composed of two (STD 2) identical iterations of the Lxl sequence. DNA constructs were again screened as above.

Example 4

Expression studies

Expression studies were performed on the nucleic acids described above that encode multivalent binding proteins with effector function. Nucleic acids encoding

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125 multivalent binding proteins were transiently transfected into COS cells and the transfected cells were maintained under well known conditions permissive for heterologous gene expression in these cells. DNA was transiently transfected into COS cells using PEI or DEAE-Dextran as previously described (PEI= Boussif O. et al., PNAS 92: 7297-7301, (1995), incorporated herein by reference; Pollard H. et al., JBC 273: 7507-7511, (1998), incorporated herein by reference). Multiple independent transfections of each new molecule were performed in order to determine the average expression level for each new form. For transfection by PEI, COS cells were plated onto 60 mm tissue culture plates in DMEM/10%FBS medium and incubated overnight so that they would be approximately 90% confluent on the day of transfection. Medium was changed to serum free DMEM containing no antibiotics and incubated for 4 hours. Transfection medium (4ml/plate) contained serum free DMEM with 50 pg PEI and 10-20 ug DNA plasmid of interest. Transfection medium was mixed by vortexing, incubated at room temperature for 15 minutes, and added to plates after aspirating the existing medium. Cultures were incubated for 3-7 days prior to collection of supernatants. Culture supernatants were assayed for protein expression by SDS-PAGE, Western blotting, binding verified by flow cytometry, and function assayed using a variety of assays including ADCC, CDC, and coculture experiments.

SDS-PAGE Analysis and Western Blotting Analysis

Samples were prepared either from crude culture supernatants (usually 30 μΐ/well) or purified protein aliquots, containing 8 ug protein per well, and 2X Tris-Glycine SDS Buffer (Invitrogen) was added to a IX final concentration. Ten (10) μΐ SeeBlue Marker (Invitrogen, Carlsbad, CA) were run to provide MW size standards. The multivalent binding (fusion) protein variants were subjected to SDS-PAGE analysis on 4-20% Novex Tris-glycine gels (Invitrogen, San Diego, CA). Samples were loaded using Novex Tris-glycine SDS sample buffer (2X) under reducing or nonreducing conditions after heating at 95°C for 3 minutes, followed by electrophoresis at 175V for 60 minutes. Electrophoresis was performed using IX Novex Tris-Glycine

SDS Running Buffer (Invitrogen).

After electrophoresis, proteins were transferred to PVDF membranes using a semi-dry electroblotter apparatus (Ellard, Seattle, WA) for 1 hour at 100 mAmp. Western

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126 transfer buffers included the following three buffers present on saturated Whatman filter paper, and stacked in succession: no. 1 contains 36.34 g/liter Tris, pH 10.4, and 20% methanol; no. 2 contains 3.02 g/liter Tris, pH 10.4, and 20% methanol; and no. 3 contains 3.03 g/liter Tris, pH 9.4, 5.25 g/liter ε-amino caproic acid, and 20% methanol. Membranes were blocked in BLOTTO=5% nonfat milk in PBS overnight with agitation. Membranes were incubated with HRP conjugated goat anti-human IgG (Fc specific, Caltag) at 5 ug/ml in BLOTTO for one hour, then washed 3 times for 15 minutes each in PBS-0.5% Tween 20. Wet membranes were incubated with ECL solution for 1 minute, followed by exposure to X-omat film for 20 seconds.

Figure 2 shows a Western Blot of proteins expressed in COS cell culture supernatant (30 μΐ/well) electrophoresed under non-reducing conditions. Lanes are indicated with markers 1-9 and contain the following samples: Lane 1 (cut off= See Blue Markers, kDa are indicated to the side of the blot. Lane 2= 2H7-sssIgG P238S/P331S-STD1 2el2 VLVH; lane 3= 2H7-sssIgG P238S/P331 S-STDl-2e 12 VHVL, Lane 4=2H715 sssIgG P238S/P33IS-STD2-2el2 VLVH; Lane 5=2H7-sssIgG P238S/P331S-STD22el2 VHVL; Lane 6=2el2 VLVH SMIP; Lane 7=2el2 VHVL SMIP; Lane 8=2H7 SMIP. 2H7 in these constructs is always in the VlVh orientation, sssIgG indicates the identity of the hinge/Iinker located at linker position 1 as shown in Figure 5, P238S/P33 IS indicates the version of human IgGl with mutations from wild type (first aa listed) to mutant (second aa listed) and the amino acid position at which they occur in wild type human IgGl Ch2 and CH3 domains, STD1 indicates the 20-aminoacid (18 + restriction site) linker located in linker position 2 as shown in Figure 5, and STD2 indicates the 38 amino acid (36+restriction site) linker located in linker position 2 as shown in Figure 6.

Binding Studies

Binding studies were performed to assess the bispecific binding properties of the CD20/CD28 multispecific, multivalent binding peptides. Initially, WIL2-S cells were added to 96 well plates and centrifuged to pellet cells. To the seeded plates, CD20/CD28 purified protein was added, using two-fold titrations across the plate from 20 μg/ml down to 0.16 μg/ml. A two-fold dilution series of TRU-015 (source of binding domain 1) purified protein was also added to seeded plate wells, the

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127 concentration of TRU-015 extending from 20 pg/ml down to 0.16 pg/ml. One well containing no protein served as a background control.

Seeded plates containing the proteins were incubated on ice for one hour. Subsequently, the wells were washed once with 200 μΐ 1% FBS in PBS. Goat anti5 human antibody labeled with FITC (Fc Sp) at 1:100 was then added to each well, and the plates were again incubated on ice for one hour. The plates were then washed once with 200 μΐ 1% FBS in PBS and the cells were re-suspended in 200 μΐ 1% FBS and analyzed by FACS.

To assess the binding properties of the anti-CD28 peptide 2E12 VhVl, CD2810 expressing CHO cells were plated by seeding in individual wells of a culture plate.

The CD20/CD28 purified protein was then added to individual wells using a two-fold dilution scheme, extending from 20 pg/ml down to 0.16 pg/ml. The 2E12IgG-VHVL SMIP purified protein was added to individual seeded wells, again using a two-fold dilution scheme, i.e., from 20 pg/ml down to 0.16 pg/ml. One well received no protein to provide a background control. The plates were then incubated on ice for one hour, washed once with 200 μΐ 1% FBS in PBS, and goat anti-human antibody labeled with FITC (Fc Sp, CalTag, Burlingame, CA) at 1.100 was added to each well. The plates were again incubated on ice for one hour and subsequently washed once with 200 μΐ 1% FBS in PBS. Following re-suspension of the cells in 200 μΐ 1% FBS,

FACS analysis was performed. The results showed that multivalent binding proteins with the N-terminal CD20 binding domain 1 bound CD20; those proteins having the C-terminal CD28 binding domain 2 in the N-Vh-Vl-C orientation also bound CD28.

The expressed proteins were shown to bind to CD20 presented on WIL-2S cells (see Figure 3) and to CD28 presented on CHO cells (refer to Figure 3) by flow cytometry (FACS), thereby demonstrating that either BD1 or BD2 could function to bind the specific target antigen. Each data set on the graphs in Figure 3 shows the binding of serial dilutions of the different multivalent binding (fusion) proteins over the titration ranges indicated. The data obtained using these initial constructs indicate that multivalent binding (fusion) proteins with the binding domain 2 version using

2e 12 in the VHVL orientation express better and bind better to CD28 than the form in the VLVH orientation at equivalent concentrations.

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Figure 4 shows a graphical presentation of the results of binding studies performed with purified proteins from each of these transfections/constructs. The figure shows binding profiles of the proteins to CD20 expressing W1L-2S cells, demonstrating that the multivalent molecule binds to CD20 as well as the single specificity SM1P at the same concentration. The top and bottom panels for Figure 5 show the binding profiles of the BD2 specificity (2el2=CD28) to CD28 CHO cells. For binding of binding domain 2 to CD28, the orientation of the V regions affected binding of the 2el2. 2H7-sss-hIgG-STDl-2el2 multivalent binding proteins with the 2el2 in the VH-VL (HL) orientation showed binding at a level equivalent to the single specificity SMIP, while the 2el2 LH molecule showed less efficient binding at the same concentration.

Example 5

Construction of Various Linker Forms of the Multivalent Fusion Proteins.

This example describes the construction of the different linker forms listed in the table shown in Figure 6.

Construction of Ch)-BD2 linkers Hl through H7

To explore the effect of Ch3-BD2 linker length and composition on expression 20 and binding of the scorpion molecules, an experiment was designed to compare the existing molecule 2H7sssIgGl-Lxl-2el2HL to a larger set of similar constructs with different linkers. Using 2H7sssIgGl-Lxl-2el2HL as template, a series of PCR reactions were performed using the primers listed in Oligonucleotide Table 2, which created linkers that varied in length form 0 to 16 amino acids. These linkers were constructed as nucleic acid fragments that spanned the coding region for Ch3 at the BsrGI site to the end of the nucleic acid encoding the linker-BD2 junction at the EcoRI site.

Table 2

SEQ ID

No. Name Sequence 5'-3' NO.

PCR Primers

L1-11R

GCGATAGAATTCCCAGATCCACCACCGCCCGA GCCACCGCCACCATAATTC 46

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	GCGATAGAATTCCCAGAGCCACCGCCACCATA
2	L1-6R	ATTC GCGTATGAATTCCCCGAGCCACCGCCACCCTTA	47
3	L3R	CCCGGAGACAGG GCGTATGAATTCCCAGATCCACCACCGCCCGAG	48
4	L4R	CCACCGCCACCCTTAC GCGTATGAATTCCCGCTGCCTCCTCCCCCAGATC	49
5	L5R	CACCACCGCC	50
6	IgBsrGIF	GAGAACCACAGGTGTACACCCTG GCGATAGAATTCGGACAAGGTGGACACCCCTTAC	51
7	L-CPPCPR	CCGGAGAGAGGGAGAG	52

Table 2. Sequences of primers used to generate CH3-BD2 linker variants.

Figure 6 diagrams the schematic structure of a multivalent binding (fusion) protein and shows the orientation of the V regions for each binding domain, the sequence present at linker position 1 (only the Cys residues are listed), and the sequence and identifier for the linker(s) located at linker position 2 of the molecules.

Example 6

Binding and Functional Studies With Variant Linker Forms of the 2H7-IgG-2el2 Prototype Multivalent Fusion Proteins.

This example shows the results of a series of expression and binding studies on the “prototype” 2H7-sssIgG-Hx-2el 2 VHVL construct with various linkers (HlH7) present in the linker position 2. Each of these proteins was expressed by large15 scale COS transient transfection and purification of the molecules using protein A affinity chromatography, as described in the previous examples. Purified proteins were then subjected to analyses including SDS-PAGE, Western blotting, binding studies analyzed by flow cytometry, and functional assays for biological activity.

Binding Studies Comparine the Different BD2 Orientations

Binding studies were performed as described in the previous examples, except that protein A-purified material was used, and a constant amount of binding (fusion) protein was used for each variant studied, i.e., 0.72 ug/ml. Figure 7 shows a columnar graph comparing the binding properties of each linker variant and 2el2 orientation variant to both CD20 and CD28 target cells. H1-H6 refer to constructs with the Hl25 H6 linkers and 2el2 in the VH-VL orientation. L1-L6 refer to constructs with the HlH6 linkers and 2el2 in the VL-VH orientation. The data demonstrate that the binding

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130 domain 2 specificity for 2el2 binds much more efficiently when present in the HL orientation (samples H1-H6) than when in the LH orientation (samples L1-L6). The effect of linker length is complicated by the discovery, as shown in the next set of figures, that molecules with the longer linkers contain some single-specificity cleaved molecules which are missing the CD28 binding specificity at the carboxy terminus. Other experiments were performed which address the binding of selected linkers, with the results shown in Figures 10, 12, and 13.

SDS-PAGE Analysis of purified H1-H7 Linker Variants

Samples were prepared from purified protein aliquots, containing 8 gg protein per 10 well, and 2X Tris-Glycine SDS Buffer (Invitrogen) was added to a IX final concentration. For reduced samples/gels, 10X reducing buffer was added to IX to samples plus Tris-Glycine SDS buffer. Ten (10) μΐ SeeBlue Marker (Invitrogen, Carlsbad, CA) was run to provide MW size standards. The multivalent binding (fusion) protein variants were subjected to SDS-PAGE analyses on 4-20% Novex

Tris-glycine gels (Invitrogen, San Diego, CA). Samples were loaded using Novex

Tris-glycine SDS sample buffer (2X) under reducing or non-reducing conditions after heating at 95°C for 3 minutes, followed by electrophoresis at 175V for 60 minutes. Electrophoresis was performed using IX Novex Tris-Glycine SDS Running Buffer (Invitrogen). Gels were stained after electrophoresis in Coomassie SDS PAGE R-250 stain for 30 minutes with agitation, and destained for at least one hour. Figure 8 shows the nonreduced and reduced Coomassie stained gels of the [2H7-sss-hIgG P238S/P331S-Hx-2el2 VHVL] multivalent binding (fusion) protein variants, along with TRU-015 and 2el2 HLSMIP as control samples. As the linker length is increased, the amount of protein running at approximately SMIP size (or 52 kDa) increases. The increase in the amount of protein in this band corresponds with a decrease in the amount of protein in the upper band running at about 90 kDa. The gel data indicate that the full-length molecule is being cleaved at or near the linker, to generate a molecule which is missing the BD2 region. A smaller BD2 fragment is not present, indicating (1) that the nucleotide sequence within the linker sequence may be creating a cryptic splice site that removes the smaller fragment from the spliced RNA transcript, or (2) that the protein is proteolyrically cleaved after translation of the fullsize polypeptide, and that the smaller BD2 fragment is unstable, i.e., susceptible to proteolytic processing. Western blotting of some of these molecules indicates that the

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131 proteins all contain the CD20 BD1 sequence, but the smaller band is missing the CD28 BD2 reactivity. No smaller band migrating at ’’bare” scFv size (25-27 kDa) was observed on any gels or blots, indicating that this smaller peptide fragment is not present in the samples.

Western Blot Binding of BD1 and BD2 bv 2H7 specific Fab or CD28mIg

Figure 9 shows the results of Western blotting of the 2H7-sss-hIgG-H6 multivalent binding (fusion) proteins compared to each single-specificity SMIP.

Electrophoresis was performed under non-reducing conditions, and without boiling samples prior to loading. After electrophoresis, proteins were transferred to PVDF membranes using a semi-dry electroblotter apparatus (Ellard, Seattle, WA) for 1 hour at 100 mAmp. Membranes were blocked in BLOTTO (5% nonfat milk in PBS) overnight with agitation. Figure 9A: Membranes were incubated with the AbyD02429.2, a Fab directed to the 2H7 antibody, at 5 qg/ml in BLOTTO for one hour, then washed 3 times for 5 minutes each in PBS-0.5% Tween 20. Membranes were then incubated in 6X His-HRP for one hour at a concentration of 0.5 pg/ml. Blots were washed three times for 15 minutes each in PBST. Wet membranes were incubated with ECL solution for 1 minute, followed by exposure to X-omat film for 20 seconds.

Figure 9B: Membranes were incubated with CD28Ig (Ancell, Bayport, MN) at 10 pg/ml in BLOTTO, then washed three times for 15 minutes each in PBS-0.5% Tween 20. Membranes were then incubated in goat anti-mouse HRP conjugate (CalTag, Burlingame, CA) at 1:3000 in BLOTTO. Membranes were washed three times, for 15 minutes each, then incubated in ECL solution for 1 minute, followed by exposure to X-omat film for 20 seconds. The results from the Western blots indicated that the

CD28 binding domain was present in the multivalent “monomer” fraction migrating at approximately 90 kDa, and in higher order forms. No band was detectable migrating at the position expected for a single SMIP or bare scFv size fragment.

When the CD20 anti-idiotype Fab was used, a SMIP-sized fragment was detected, indicating the presence of a peptide fragment containing (2H7-sss-hIgG), and missing the CD28 scFv BD2 portion of the fusion protein.

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Binding Studies on Selected Linkers

Figure 10 shows the results of binding studies performed on the purified 2H7-ssshIgG-Hx-2el2 fusion proteins. Binding studies were performed to assess the bispecific binding properties ofthe CD20/CD28 multispecific binding peptides.

Initially, WIL2-S cells were plated using conventional techniques. To the seeded plates, CD20/CD28 purified protein was added, using two-fold titrations across the plate from 20 pg/ml down to 0.16 pg/ml. A two-fold dilution series of TRU-015 (source of binding domain 1) purified protein was also added to seeded plate wells, the concentration of TRU-015 extending from 20 pg/ml down to 0.16 pg/ml. One well containing no protein served as a background control.

Seeded plates containing the proteins were incubated on ice for one hour. Subsequently, the wells were washed once with 200 pi 1% FBS in PBS. Goat antihuman antibody labeled with FITC (Fc Sp) at 1:100 was then added to each well, and the plates were again incubated on ice for one hour. The plates were then washed once with 200 pi 1% FBS in PBS and the cells were re-suspended in 200 pi 1% FBS and analyzed by FACS.

To assess the binding properties of the anti-CD28 peptide 2E12 VhVl, CD28expressing CHO cells were plated by seeding in individual wells of a culture plate. The CD20/CD28 purified protein was then added to individual wells using a two-fold dilution scheme, extending from 20 pg/ml down to 0.16 pg/ml. The 2E12IgGvHvL SMIP purified protein was added to individual seeded wells, again using a two-fold dilution scheme, i.e., from 20 pg/ml down to 0.16 pg/ml. One well received no protein to provide a background control. The plates were then incubated on ice for one hour, washed once with 200 pi 1% FBS in PBS, and goat anti-human antibody labeled with FITC (Fc Sp) at 1:100 was added to each well. The plates were again incubated on ice for one hour and subsequently washed once with 200 pi 1% FBS in PBS. Following re-suspension of the cells in 200 pi 1% FBS, FACS analysis was performed. The expressed proteins were shown to bind to CD20 presented on WIL2S cells (see Figure 10A) and to CD28 presented on CHO cells (refer to Figure 10B) by flow cytometry (FACS), thereby demonstrating that either BD1 or BD2 could function to bind the specific target antigen. In addition, the linker used (H1-H6) was not found to significantly affect binding avidity to target antigen.

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SEC Fractionation of Multivalent Binding (Fusion) Proteins. The binding (fusion) protein was purified from cell culture supernatants by protein A Sepharose affinity chromatography over a GE Healthcare XK 16/40 column. After binding of protein to the column, the column was washed in dPBS, then 1.0 M NaCI, 20 mM sodium phosphate pH 6.0, and then 25 mM NaCI, 25 mN NaOAc, pH 5.0, to remove nonspecific binding proteins. Bound protein was eluted from the column in 100 mM Glycine (Sigma), pH 3.5, and brought to pH 5.0 with 0.5 M 2-(N-Morpholino) ethanesulfonic acid (MES), pH 6.0, Protein samples were concentrated to 25 mg/ml using conventional techniques in preparation for GPC purification. Size exclusion chromatography (SEC) was performed on a GE Healthcare AKTA Explorer 100 Air apparatus, using a GE healthcare XK column and Superdex 200, preparative grade (GE healthcare).

Figure 12 shows a table summarizing the results of SEC fractionation of the different binding (fusion) proteins. With increasing linker length, the complexity of the molecules in solution also increases, making it difficult to isolate peak of interest, or POI from higher order forms by HPLC. The H7 linker seems to resolve much of this complexity into a more homogeneous form in solution, so that the soluble forms migrate mostly as a single POI at approximately 172 kDa,

Additional Binding Studies

A second series of experiments was performed (see Figures 12 and 13) with a smaller subset of multivalent binding (fusion) proteins, this time comparing linkers H3, H6, and H7. The data demonstrate that the binding level drops more significantly for CD28 than for CD20 binding, but both drop slightly as linker length increases. Further, the data showed that the H7 linker exhibited the highest level of binding to both antigens. These data were obtained using protein A-purified multivalent binding (fusion) proteins, but were not further purified by SEC, so multiple forms of the molecules may have been present in solution. The results also indicated that the truncated form may have been less stable than the true multivalent polypeptide, since the binding curves do not appear to fully reflect the significant amount of single specificity form present in solution for linker H6.

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Demonstration of Multispecific Binding From a Single Molecule

An alternative binding assay was performed (see Figure 13), where binding to CD20 on the surface of WIL-2S cells was detected with a reagent specific for the CD28 BD2, thereby demonstrating that simultaneous binding may occur to both target antigens, engaging both BD1 and BD2 on the same multispecific binding (fusion) protein (refer to Figure 12) This assay demonstrates the multispecific binding property of the proteins.

Example 7

Construction of Multispecific Binding (Fusion) Proteins With Alternative Specificities in BD2

In addition to the prototype CD20-CD28 multispecific binding molecule, two other forms were made with alternative binding domain 2 regions, including CD37 and CD3 binding domains. The molecules were also made with several of the linker domains described for the [2H7-sss-IgG-Hx/STDx-2el2 HL] multispecific binding (fusion) proteins. The construction of these additional multispecific binding (fusion) molecules are described below.

Anti-CD37 Binding Domain Construction

Table 3

No.	Name	Sequence ACTGCTGCAGCTGGACCGCGCT	SEQ ID NO.
23	G281LH-NheR	AGCTCCGCCGCCACCCGAC GGCGGAGCTAGCGCGGTCCAGC	53
24	G281LH-NheF	TGCAGCAGTCTGGACCTG GCGATCACCGGTGACATCCAGAT	54
25	G281-LH-LPinF	GACTCAGTCTCCAG GCGATACTCGAGGAGACGGTGAC	55
26	G2814_H-HXhoR	TGAGGTTCCTTGAC GCGATCGAATTCAGACATCCAGAT	56
27	G281-LH-LEcoF	GACTCAGTCTCCAG GCGATTCTAGATTAGGAAGAGACG	57
28	G281-LH-HXbaR	GTGACTGAGGTTCCTTGAC GCGATAACCGGTGCGGTCCAGCTG	58
29	G281-HL-HF	CAGCAGTCTGGAC GACCCACCACCGCCCGAGCCACCG CCACCAGAAGAGACGGTGACTGAGG	59 60
30	G281-HL-HR3	TTC ACTCCCGCCTCCTCCTGATCCGCCG
31	G281-HL-HR2	CCACCCGACCCACCACCGCCCGAG GAGTCATCTGGATGTCGCTAGCACTC	61
32	G281-HL-HNheR	CCGCCTCCTCCTGATC	62
33	G281-HL-LNheF	ATCAGGAGGAGGCGGGAGTGCTAGC

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	GACATCCAGATGACTCAGTC GCGATACTCGAGCCTTTGATCTCCAG	63
34	G281-HL-LXhoR	TTCGGTGCCTC GCGATATCTAGACTCAACCTTTGATCT	64
35	G281-HL-LXbaR	CCAGTTCGGTGCCTC GCGATAGAATTCGCGGTCCAGCTGCA	65
36	G281-HL-EcoF	GCAGTCTGGAC	66

Table 3. Oligonucleotide primers used to generate G28-1 anti-CD37 binding domains for both SMIP molecules and scorpions.

The G28-1 scFv (SEQ ID NO: 102) was converted to the G28-1 LH SMIP by PCR using the primers in Table X above. Combining primers 23 and 25 with 10 ng G28-1 scFv, the VK was amplified for 30 cycles of 94C, 20 seconds, 58C, 15 seconds, 68C, 15 seconds using Platinum PCR Supermix Hi-Fidelity PCR mix (Invitrogen, Carlsbad, CA) in an ABI 9700 Thermalcycler. The product of this PCR had the restriction sites PinAI (Agel) at the 5' end of the VK and Nhel at the end of the scFv (G4S)3 linker. The VH was similarly altered by combining primers 24 and 26 with 10 ng G28-1 scFv in a PCR run under the identical conditions as with the VK above. This PCR product had the restriction sites Nhel at the 5' end of the VH and

Xhol at the 3' end. Because significant sequence identity overlap was engineered into primers 23 and 24, the VK and VH were diluted 5-fold, then added at a 1:1 ratio to a PCR using the flanking primers 25 and 26 and a full-length scFv was amplified as above by lengthening the 68C extension time from 15 seconds to 45 seconds. This PCR product represented the entire G28-1 scFv as a PinAI-XhoI fragment and was purified by MinElute column (Qiagen,) purification to remove excess primers, enzymes and salts. The eluate was digested to completion with PinAI (Invitrogen)and Xhol (Roche) in IX H buffer (Roche,) at 37C for 4 hours in a volume of 50 pL. The digested PCR product was then electrophoresed in a 1% agarose gel, the fragment was removed from the gel and re-purified on a MinElute column using buffer QG and incubating the gel-buffer mix at 50C for 10 minutes with intermittent mixing to dissolve the agarose after which the purification on the column was identical for primer removal post-PCR. 3 pL PinAI-Xhol digested G28-1 LH was combined with 1 pL PinAI-Xhol digested pD18-n2H7sssIgGl SMIP in a 10 pL reaction with 5 pL 2X LigaFast Ligation Buffer (Promega, Madison, WI) and 1 pL T4 DNA ligase (Roche), mixed well and incubated at room temperature for 10 minutes. 3 pL of this ligation was then transformed into competent TOP 10 (Invitrogen) using the

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136 manufacturer's protocol. These transformants were plated on LB agar plates with 100 pg/ml carbenicillin(Teknova,) and incubated overnight at 37C. After 18 hours of growth, colonies were picked and inoculated into 1 ml T-Broth (Teknova,) containing 100 pg/ml carbenicillin in a deep well 96-well plate and grown overnight in a 37C shaking incubator. After 18-24 hours of growth, DNA was isolated from each overnight culture using the QIAprep 96 Turbo Kit (Qiagen) on the BioRobot8000 (Qiagen). 10 pL from each clone was then digested with both Hindlll and Xhol restriction enzymes in IX B buffer in a 15 pL reaction volume. The digested DNA was electrophoresed on 1% agarose E-gels (Invitrogen, CA) for restriction site analysis. Clones that contained a Hindlll-Xhol fragment of the correct size were sequence verified. The G28-1 HL SMIP was constructed in a similar manner by placing a PinAI site on the 5' end and a (G4S)4 linker ending in an Nhe I site of the G28-1 VH using primers 29, 30 31 and 32 from Table X above. The VK was altered by PCR using primers 33 and 34 from Table X such that an Nhel site was introduced at the 5' end of the VK and Xhol at the 3' end. These PCRs were then combined as above and amplified with the flanking primers 29 and 34 to yield an intact G28-1 scFv DNA in the VH-VL orientation which was cloned into PinAI-XhoI digested pD18-(n2H7)sssIgGl SMIP exactly as with the G28-1 LH SMIP,

2H7sssIgGl-STDl-G28-l LH/HL Construction

Using the G28-1 LH and G28-1 HL SMIPs as templates, the LH and HL antiCD37 binding domains were altered by PCR such that their flanking restriction sites were compatible with the scorpion cassette. An EcoRI site was introduced at the 5' end of each scFv using either primer 27 (LH) or 36 (HL) and a stop codon/ Xbal site at the 3' end using either primer 28 (LH) or 35 (HL). The resulting DNAs were cloned into EcoRI-Xbal digested pD 18-2H7sssIgG-STD 1.

2H7sssIgG 1 -Hx-G28-1 HL Construction

2H7sssIgGl-Hx-2el2 HL DNAs were digested with BsrGI and EcoRI and the 325 bp fragment consisting of the C-terminal end of the IgGl and linker. These were substituted for the equivalent region in 2H7sssIgGl-STD 1-G19-4 HL by removal of the STD 1 linker using BsrGI-EcoRI and replacing it with the corresponding linkers from the 2H7sssIgGl-Hx-2el2 HL clones.

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Anti-CD 3 Binding domain Construction

Table 4

No.	Name	Sequence	SEQ ID NO.
37	194-LH-LF1	GCGTATGAACCGGTGACATCCAGAT GACACAGACTACATC	67
38	194-LF2	ATCCAGATGACACAGACTACATCCTC CCTGTCTGCCTCTCTGGGAGACAG	68
39	194-LF3	GTCTGCCTCTCTGGGAGACAGAGTCA CCATCAGTTGCAGGGCAAGTCAGGAC	69
40	194-LF4	GTTGCAGGGCAAGTCAGGACATTCGC AATTATTTAAACTGGTATCAGCAG	70
41	194-LF5	ATTTAAACTGGTATCAGCAGAAACCAG ATGGAACTGTTAAACTCCTGATC	71
42	194-LF6	GAACTGTTAAACTCCTGATCTACTACA CATCAAGATTACACTCAGGAGTC	72
43	194-LF7	CAAGATTACACTCAGGAGTCCCATCAA GGTTCAGTGGCAGTGGGTCTGGAAC	73
44	194-LR7	CAGGTTGGCAATGGTGAGAGAATAATC TGTTCCAGACCCACTGCCACTGAAC	74
45	194-LR6	GCAAAAGTAAGTGGCAATATCTTCTGGT TGCAGGTTGGCAATGGTGAGAG	75
46	194-LR5	GAACGTCCACGGAAGCGTATTACCC TGTTGGCAAAAGTAAGTGGCAATATC	76
47	194-LR4	CGTTTGGTTACCAGTTTGGTGCCTCCAC CGAACGTCCACGGAAGCGTATTAC	77
48	194-LR3	ACCACCGCCCGAGCCACCGCCACC CCGTTTGGTTACCAGTTTGGTG	78
49	194-LR2	GCTAGCGCTCCCACCTCCTCCAGATCCA CCACCGCCCGAGCCACCGCCAC	79
50	194-LH-LR1	GTTGCAGCTGGACCTCGCTAGCGCT CCCACCTCCTCCAGATC	80
51	194-LH-HF1	GATCTGGAGGAGGTGGGAGCGCTAGC GAGGTCCAGCTGCAACAGTCTGGACCTG	81
52	194-HF2	AGCTGCAACAGTCTGGACCTGAACT GGTGAAGCCTGGAGCTTCAATGAAG	82
53	194-HF3	AGCCTGGAGCTTCAATGAAGATTTCC TGCAAGGCCTCTGGTTACTCATTC	83
54	194-HF4	GCAAGGCCTCTGGTTACTCATTCACT GGCTACATCGTGAACTGGCTGAAGCAG	84
55	194-HF5	ATCGTGAACTGGCTGAAGCAGAGCC ATGGAAAGAACCTTGAGTGGATTGGAC	85
56	194-HF6	GAACCTTGAGTGGATTGGACTTATTA ATCCATACAAAGGTCTTACTACCTAC	86
57	194-HR6	AATGTGGCCTTGCCCTTGAATTTCTG GTTGTAGGTAGTAAGACCTTTGTATG	87
58	194-HR5	CATGTAGGCTGTGCTGGATGACTTGT CTACAGTTAATGTGGCCTTGCCCTTG	88
59	194-HR4	ACTGCAGAGTCTTCAGATGTCAGACTG AGGAGCTCCATGTAGGCTGTGCTGGATG	89
60	194-HR3	ACCATAGTACCCAGATCTTGCACAG TAATAGACTGCAGAGTCTTCAGATGTC	90
61	194-HR2	GCGCCCCAGACATCGAAGTACCAGTC CGAGTCACCATAGTACCCAGATCTTG	91
62	194-LH-HR1	GCGAATACTCGAGGAGACGGTGACCG TGGTCCCTGCGCCCCAGACATCGAAG	92
63	194-HL-HF1	GCGTATGAACCGGTGAGGTCCAGC

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138
	TGCAACAGTCTGGACCTG ACCGCCACCAGAGGAGACGGTGACCGT	93
64	194-HL-HR1	GGTCCCTGCGCCCCAGACATCGAAGTAC ACCTCCTCCAGATCCACCACCGCCCG	94
65	194-HL-HRO	AGCCACCGCCACCAGAGGAGACGGTG GCGGGGGAGGTGGCAGTGCTAGCGA	95
66	194-HL-LF1	CATCCAGATGACACAGACTACATC GCGAATACTCGAGCGTTTGGTTACCA	96
67	194-HL-LR3Xho	GTTTGGTG GCGATATCTAGATTACCGTTTGGTTAC	97
68	194-HL-LR3Xba	CAGTTTGGTG GCGTATGAGAATTCAGAGGTCCAGCTG	98
69	194-HL-HF1R1	CAACAGTCTGGACCTG GCGTATGAGAATTCTGACATCCAGA	99
70	194-LH-LF1R1	TGACACAGACTACATC GCGTATCTAGATTAGGAGACGGTGACC	100
71	194-LH-HR1Xba	GTGGTCCCTGCGCCCCAGACATCGAAG	101

Table 4. Oligonucleotides used to generate anti-CD3 binding domain from the G19-4 scFv sequence.

The G19-4 binding domain was synthesized by extension of overlapping oligonucleotide primers as described previously. The tight chain PCR was done in two steps, beginning by combining primers 43/44,42/45,41/46 and 40/47 at concentrations of 5uM, 10 μΜ, 20 μΜ and 40 μΜ respectively, in Platinum PCR

Supermix Hi-Fidelity for 30 cycles of 94°C, 20 seconds, 60°C, 10 seconds, 68°C, 15 seconds. 1 pL of the resultant PCR product was reamplified using a primer mix of 39/48 (10 μΜ), 38/49 (20 μΜ) and 37/50 (40 μΜ) for the LH or 66/67 (40 μΜ) for the HL orientation, using the same PCR conditions with the exception of the 68C extension which was increased to 25 seconds. The VK in the LH orientation was bounded by PinAI at the 5' end and Nhel at the 3' end, while the HL orientation had Nhel at the 5' end and Xhol at the 3’ end.

To synthesize the heavy chain, primer mixes with the same concentrations as above were prepared by combining primers 56/57, 55/58, 54/59 and 53/60 for the first PCR step. In the second PCR, primers 52/61 (20 μΜ) and 51/62 (50 μΜ) were amplified with 1 μΐ from the first PCR using the same PCR conditions as with the second PCR of the light chain to make the LH orientation with Nhel at the 5' end and Xhol at the 3' end. Primers 52/61(10 μΜ), 63/64 (20 μΜ), 63 (20 μΜ)/65 (40 μΜ) and 63(20 μΜ)/5 (80 μΜ) were combined in a second PCR with luL from the previous PCR to create the heavy chain in the HL orientation with PinAI at the 5' end and Nhel at the 3' end. As with previous constructs, sufficient overlap was designed

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139 into the primers centered around the Nhel site such that the G19-4 LH was synthesized by combining the heavy and light chain PCRs in the LH orientation and reamplifying with the flanking primers, 37 and 62 and the G19-4 HL was synthesized by combining the HL PCRs and re-amplifying with primers 63 and 67.

Full-length G19-4 LH/HL PCR products were separated by agarose gel electrophoresis, excised from the gel and purified with Qiagen MinElute columns as described earlier. These DNAs were then TOPO-cloned into pCR2.1 (Invitrogen), transformed into TOP 10 and colonies screened First by EcoRI fragment size, then by DNA sequencing. G19-4 LH/HL were then cloned into pD18-IgGl via PinAI-XhoI for expression in mammalian cells.

2H7ss$IeGl-STD 1-G 19-4 LH/HL Construction

Using the G19-4 LH and G19-4 HL SMIPs as templates, the LH and HL antiCD3 binding domains were altered by PCR such that their flanking restriction sites were compatible with the scorpion cassette. An EcoRI site was introduced at the 5’ end of each scFv using either primer 27 (LH) or 36 (HL) and a stop codon/ Xbal site at the 3' end using either primer 28 (LH) or 35 (HL). The resulting DNAs were cloned into EcoRI-Xbal digested pD18-2H7sss!gG-STDl.

2H7sssIgG 1 -Hx-G 19-4 HL Construction

2H7sssIgGl-Hx-2el2 HL DNAs were digested with BsrGI and EcoRI and the

325 bp fragment consisting of the C-terminal end of the IgG 1 and linker. These were substituted for the equivalent region in 2H7sssIgGl-STD 1-G 19-4 HL by removal of the STD1 linker using BsrGI-EcoRI and replacing it with the corresponding linkers from the 2H7sssIgGl-Hx-2el2 HL clones.

Apparent from a consideration of the variety of multivalent binding proteins disclosed herein are features of the molecules that are amenable to combination in forming the molecules of the invention. Those features include binding domain 1, a constant sub-region, including a hinge or hinge-like domain, a linker domain, and a binding domain 2. The intrinsic modularity in the design of these novel binding proteins makes it straightforward for one skilled in the art to manipulate the DNA sequence at the N-terminal and/or C-terminal ends of any desirable module such that it can be inserted at almost any position to create a new molecule exhibiting altered or

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140 enhanced functionality compared to the parental molecule(s) from which it was derived. For example, any binding domain derived from a member of the immunoglobulin superfamily is contemplated as either binding domain 1 or binding domain 2 of the molecules according to the invention. The derived binding domains include domains having amino acid sequences, and even encoding polynucleotide sequences, that have a one-to-one correspondence with the sequence of a member of the immunoglobulin superfamily, as well as variants and derivatives that preferably share 80%, 90%, 95%, 99%, or 99.5% sequence identity with a member of the immunoglobulin superfamily. These binding domains (1 and 2) are preferably linked to other modules of the molecules according to the invention through linkers that may vary in sequence and length as described elsewhere herein, provided that the linkers are sufficient to provide any spacing and flexibility necessary for the molecule to achieve a functional tertiary structure. Another module of the multivalent binding proteins is the hinge region, which may correspond to the hinge region of a member of the immunoglobulin superfamily, but may be a variant thereof, such as the “CSC” or “SSS” hinge regions described herein. Also, the constant sub-region comprises a module of the proteins according to the invention that may correspond to a sub-region of a constant region of an immunoglobulin superfamily member, as is typified by the structure of a hinge-Cn2-CH3 constant sub-region. Variants and derivatives of constant sub-regions are also contemplated, preferably having amino acid sequences that share 80%, 90%, 95%, 99%, or 99.5% sequence identity with a member of the immunoglobulin superfamily.

Exemplary primary structures of the features of such molecules are presented in Table 5, which discloses the polynucleotide and cognate amino acid sequence of illustrative binding domains 1 and 2, as well as the primary structure of a constant sub-region, including a hinge or hinge-like domain, and a linker that may be interposed, e.g,, between the C-terminal end of a constant sub-region and the Nterminal end of a binding domain 2 region of a multivalent binding protein.

Additional exemplars of the molecules according to the invention include the above30 described features wherein, e.g., either or both of binding domains 1 and 2 comprise a domain derived from a Vl or VL-like domain of a member of the immunoglobulin superfamily and a Vh or Vn-hke domain derived from the same or a different member of the immunoglobulin superfamily, with these domains separated by a linker typified

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I4t by any of the linkers disclosed herein. Contemplated are molecules in which the orientation of these domains is Vl-Vh or Vh*Vl for BD1 and/or BD2. A more complete presentation of the primary structures of the various features of the multivalent binding molecules according to the invention is found in the table appended at the end of this disclosure. The invention further comprehends polynucleotides encoding such molecules.

Table 5

^rijadlftS gSwsWSSB w			S#Si8fe
2H7 LH	atggattttcaagtgcagattttcag cttcctgctaatcagtgcttcagtca taatgtccagaggacaaattgttctc tcccagtctccagcaatcctgtctgc atctccaggggagaaggtcacaatga cttgcagggccagctcaagtgtaagt tacatgcactggtaccagcagaagcc aggatcctcccccaaaccctggattt atgccccatccaacctggcttctgga gtccctgctcgcttcagtggcagtgg gtctgggacctcttactctctcacaa tcagcagagtggaggctgaagatgct gccacttattactgccagcagtggag ttttaacccacccacgttcggtgctg ggaccaagctggagctgaaagatggc ggtggctcgggcggtggtggatctgg aggaggtgggagctctcaggcttatc tacagcagtctggggctgagtcggtg aggcctggggcctcagtgaagatgtc ctgcaaggcttctggctacacattta ccagttacaatatgcactgggtaaag cagacacctagacagggcctggaatg gattggagctatttatccaggaaatg gtgatacttcctacaatcagaagttc aagggcaaggccacactgactgtaga caaatcctccagcacagcctacatgc agctcagcagcctgacatctgaagac tctgcggtctatttctgtgcaagagt ggtgtactatagtaactcttactggt acttcgatgtctggggcacagggacc acggtcaccgtctct	mdfqvqifsfllisasvimsrgqivls qspailsaspgekvtmtcrasssvsym hwyqqkpgsspkpwiyapsnlasgvpa rfsgsgsgtsysltisrveaedaatyy cqqwsfnpptfgagtklelkdgggsgg ggsggggssqaylqqsgaesvrpgasv kmsckasgytftsynmhwvkqtprqgl ewigaiypgngdtsynqkfkgkatltv dkssstaymqlssltsedsavyfcarv vyysnsywyfdvwgtgttvtvs	1 (2)

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J ' > ' V	:J'' : 2: :/1 .jp-r,;' ΉΗ ; % S5?		B
2el2 LH	atggattttcaagtgcagattttcag cttcctgctaatcagtgcttcagtca taatgtccagaggagtcgacattgtg ctcacccaatctccagcttctttggc tgtgtctctaggtcagagagccacca tctcctgcagagccagtgaaagtgtt gaatattatgtcacaagtttaatgca gtggtaccaacagaaaccaggacagc cacccaaactcctcatctctgctgct agcaacgtagaatctggggtccctgc caggtttagtggcagtgggtctggga cagactttagcctcaacatccatcct gtggaggaggatgatattgcaatgta tttctgtcagcaaagtaggaaggttc catggacgttcggtggaggcaccaag ctggaaatcaaacggggtggcggtgg atccggcggaggtgggtcgggtggcg gcggatctcaggtgcagctgaaggag tcaggacctggcctggtggcgccctc acagagcctgtccatcacatgcaccg tctcagggttctcattaaccggctat ggtgtaaactgggttcgccagcctcc aggaaagggtctggagtggctgggaa tgatatggggtgatggaagcacagac tataattcagctctcaaatccagact atcgatcaccaaggacaactccaaga gccaagttttcttaaaaatgaacagt ctgcaaactgatgacacagccagata ctactgtgcccgagatggttatagta actttcattactatgttatggactac tggggtcaaggaacctcagtcaccgt ctcctct	MDFQVQIFSFLLISASVIMSRGVDIVL TQSPASLAVSLGQRATISCRASESVEY YVTSLMQWYQQKPGQPPKLLISAASNV ESGVPARFSGSGSGTDFSLNIHPVEED DIAMYFCQQSRKVPWTFGGGTKLEIKR GGGGSGGGGSGGGGSQVQLKESGPGLV APSQSLS1TCTVSGFSLTGYGVNWVRQ PPGKGLEWLGMIWGDGSTDYNSALKSR LSITKDNSKSQVFLKMNSLQTDDTARY YCARDGYSNFHYYVMDYWGQGTSVTVS S	3 (4)
2el2 HL	atggattttcaagtgcagattttcag cttcctgctaatcagtgcttcagtca taatgtccagaggagtccaggtgcag ctgaaggagtcaggacctggcctggt ggcgccctcacagagcctgtccatca catgcaccgtctcagggttctcatta accggctatggtgtaaactgggttcg ccagcctccaggaaagggtctggagt ggctgggaatgatatggggtgatgga agcacagactataattcagctctcaa atccagactatcgatcaccaaggaca actccaagagccaagttttcttaaaa atgaacagtctgcaaactgatgacac agccagatactactgtgcccgagatg gttatagtaactttcattactatgtt atggactactggggtcaaggaacctc agtcaccgtctcctctgggggtggag gctctggtggcggtggatccggcgga ggtgggtcgggtggcggcggatctga cattgtgctcacccaatctccagctt ctttggctgtgtctctaggtcagaga gccaccatctcctgcagagccagtga aagtgttgaatattatgtcacaagtt taatgcagtggtaccaacagaaacca	MDFQVQIFSFLLISASVIMSRGVQVQL KESGPGLVAPSQSLSITCTVSGFSLTG YGVNWVRQPPGKGLEWLGMIWGDGSTD YNSALKSRLSITKDNSKSQVFLKMNSL QTDDTARYYCARDGYSNFHYYVMDYWG QGTSVTVSSGGGGSGGGGSGGGGSGGG GSDIVLTQSPASLAVSLGQRATISCRA SE SVEY YVT S LMQWYQQKPGQ P PKLLI SAASNVESGVPARFSGSGSGTDFSLNI HPVEEDDIAMYFCQQSRKVPWTFGGGT KLEIKR	5 (6)

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- * - · }f 1 *j:	;»#ί.Ί3!-.'' 7Λξκΐ4' «Β5£WBa:issii<eis®iO:««s	. \ V v·. ,. i-. . /γ· · -'•A , v '•’’N -
	ggacagccacccaaactcctcatctc tgctgctagcaacgtagaatctgggg tccctgccaggtttagtggcagtggg tctgggacagactttagcctcaacat ccatcctgtggaggaggatgatattg caatgtatttctgtcagcaaagtagg aaggttccatggacgttcggtggagg caccaagctggaaatcaaacgt
G28-1 LH	accggtgacatccagatgactcagtc tccagcctccctatctgcatctgtgg gagagactgtcaccatcacatgtcga acaagtgaaaatgtttacagttattt ggcttggtatcagcagaaacagggaa aatctcctcagctcctggtctctttt gcaaaaaccttagcagaaggtgtgcc atcaaggttcagtggcagtggatcag gcacacagttttctctgaagatcagc agcctgcagcctgaagattctggaag ttatttctgtcaacatcattccgata atccgtggacgttcggtggaggcacc gaactggagatcaaaggtggcggtgg ctcgggcggtggtgggtcgggtggcg gcggatctgctagcgcagtccagctg cagcagtctggacctgagctggaaaa gcctggcgcttcagtgaagatttcct gcaaggcttctggttactcattcact ggctacaatatgaactgggtgaagca gaataatggaaagagccttgagtgga ttggaaatattgatccttattatggt ggtactacctacaaccggaagttcaa gggcaaggccacattgactgtagaca aatcctccagcacagcctacatgcag ctcaagagtctgacatctgaggactc tgcagtctattactgtgcaagatcgg tcggccctatggactactggggtcaa ggaacctcagtcaccgtctcgag	DIQMTQSPASLSASVGETVTITCRTSE NVY SYLAWYQQKQGKS PQLLVS FAKTL AEGVPSRFSGSGSGTQFSLKISSLQPE DSGSYFCQHHSDNPWTFGGGTELEIKG GGGSGGGGSGGGGSASAVQLQQSGPEL EKPGASVKISCKASGYSFTGYNMNWVK QNNGKSLEWIGNIDPYYGGTTYNRKFK GKATLTVDKSSSTAYMQLKSLTSEDSA VYYCARSVGPMDYWGQGTSVTVS	102 (103)
G28-1 HL	accggtgaggtccagctgcaacagtc tggacctgaactggtgaagcctggag cttcaatgaagatttcctgcaaggcc tctggttactcattcactggctacat cgtgaactggctgaagcagagccatg gaaagaaccttgagtggattggactt attaatccatacaaaggtcttactac ctacaaccagaaattcaagggcaagg ccacattaactgtagacaagtcatcc agcacagcctacatggagctcctcag tctgacatctgaagactctgcagtct attactgtgcaagatctgggtactat ggtgactcggactggtacttcgatgt ctggggcgcagggaccacggtcaccg tctcctctggtggcggtggctcgggc ggtggtggatctggaggaggtgggag cgggggaggtggcagtgctagcgaca tccagatgacacagactacatcctcc ctgtctgcctctctgggagacagagt caccatcagttgcagggcaagtcagg	EVQLQQSGPELVKPGASMKIsckasgy SFTGYIVNWLKQSHGKNLEWIGLINPY KGLTTYNQKFKGKATLTVDKS S S TAYM ELLSLTSEDSAVYYCARSGYYGDSDWY FDVWGAGTTVTVSSGGGGSGGGGSGGG GSGGGGSASDIQMTQTTSSLSASLGDR VTISCRASQDIRNYLNWYQQKPDGTVK LLIYYTSRLHSGVPSRFSGSGSGTDYS LTIANLQPEDIATYFCQQGNTLPWTFG GGTKLVTKRS	104 (105)

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-fyyyy . * J - * » - - ir.	. Α??ΐ: ;Ήλ:-° ' :':/ .................................’ΛαχΙ.?.......	ft Ming-Aftid'Sequence ~ / . - ' V; , '; . · ... .*., .- .	SjHj J3&' L ' UiLdiL* til < sequence
	acattcgcaattatttaaactggtat cagcagaaaccagatggaactgttaa actcctgatctactacacatcaagat tacactcaggagtcccatcaaggttc agtggcagtgggtctggaacagatta ttctctcaccattgccaacctgcaac cagaagatattgccacttacttttgc caacagggtaatacgcttccgtggac gttcggtggaggcaccaaactggtaa ccaaacgctcgag
G19-4 LH	accggtgacatccagatgacacagac tacatcctccctgtctgcctctctgg gagacagagtcaccatcagttgcagg gcaagtcaggacattcgcaattattt aaactggtatcagcagaaaccagatg gaactgttaaactcctgatctactac acatcaagattacactcaggagtccc atcaaggttcagtggcagtgggtctg gaacagattattctctcaccattgcc aacctgcaaccagaagatattgccac ttacttttgccaacagggtaatacgc ttccgtggacgttcggtggaggcacc aaactggtaaccaaacggggtggcgg tggctcgggcggtggtggatctggag gaggtgggagcgctagcgaggtccag ctgcaacagtctggacctgaactggt gaagcctggagcttcaatgaagattt cctgcaaggcctctggttactcattc actggctacatcgtgaactggctgaa gcagagccatggaaagaaccttgagt ggattggacttattaatccatacaaa ggtcttactacctacaaccagaaatt caagggcaaggccacattaactgtag acaagtcatccagcacagcctacatg gagctcctcagtctgacatctgaaga ctctgcagtctattactgtgcaagat ctgggtactatggtgactcggactgg tacttcgatgtctggggcgcagggac cacggtcaccgtctcctcgag	DIQMTQTTSSLSASLGDRVTISCRASQ DIRNYLNWYQQKPDGTVKLLIYYTSRL HSGVPSRFSGSGSGTDYSLTIANLQPE DIATYFCQQGNTLPWTFGGGTKLVTKR GGGGSGGGGSGGGGSASEVQLQQSGPE LVKPGASMKISCKASGYSFTGYIVNWL KQSHGKNLEWIGLINPYKGLTTYNQKF KGKATLTVDKSSSTAYMELLSLTSEDS AVYYCARSGYYGDSDWYFDVWGAGTTV TVSS	106 (107)
G19-4 HL	accggtgaggtccagctgcaacagtc tggacctgaactggtgaagcctggag cttcaatgaagatttcctgcaaggcc tctggttactcattcactggctacat cgtgaactggctgaagcagagccatg gaaagaaccttgagtggattggactt attaatccatacaaaggtcttactac ctacaaccagaaattcaagggcaagg ccacattaactgtagacaagtcatcc agcacagcctacatggagctcctcag tctgacatctgaagactctgcagtct attactgtgcaagatctgggtactat ggtgactcggactggtacttcgatgt ctggggcgcagggaccacggtcaccg tctcctctggtggcggtggctcgggc ggtggtggatctggaggaggtgggag cgctagcgacatccagatgacacaga	EVQLQQSGPELVKPGASMKISCKASGY SFTGYIVNWLKQSHGKNLEWIGLINPY KGLTTYNQKFKGKATLTVDKSSSTAYM ELLSLTSEDSAVYYCARSGYYGDSDWY FDVWGAGTTVTVSSGGGGSGGGGSGGG GSASDIQMTQTTSSLSASLGDRVTISC RASQDIRNYLNWYQQKPDGTVKLLIYY TSRLHSGVPSRFSGSGSGTDYSLTIAN LQPEDIATYFCQQGNTLPWTFGGGTKL VTKRS	108 (109)

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Binding Domain	Nucleotide Sequence	toin« Acid Sequence	8--5: ;3©;: . -W$, 5..:9: dawni/ftA;! . . . , . /.. V > λ/J? %4ϋ Λ “ ‘A Τ- Λ / Λ sequence
	ctacatcctccctgtctgcctctctg . gg ag acagag tcacea t cagt tg ca g ggcaagtcaggacattcgcaattatt taaactggtatcagcagaaaccagat ggaactgttaaactcctgatctacta | cacatcaagattacactcaggagtcc ! catea agg11eagtggc agt ggg t et ( gg aacagatt a ttetctea ccat1gc caacctgcaaccagaagatattgcca cttacttttgccaacaaggtaatacg Cttecgtggaeg ttcggtggaggcac caaaetggtaa ccaaac gct egag

.Hinge Region	Hgglegtide Sequence	Amino Acid Sequence	(aaloc .edd'
sss(s) hlgGl	gagcccaaatcttctgacaaaact cacacatctccaccgagctca	EPKSSDKTHTSPPSS	230	(231)
CSC(s) hlgGl	gagcccaaatcttgtgacaaaact cacacatctccaccgtgctca	EPKSCDKTHTSPPCS	232	(233)
ssc(s) hlgGl	gagcccaaatcttctgacaaaact cacacatctccaccgtgctca	EPKSSDKTHTSPPCS	110	(111)
see(s) “ hlgGl	gagcccaaatcttctgacaaaact cacacatgtccaccgtgctca	EPKSSDKTHTCPPCS	112	(113)
css(s) — hlgGl	gagcccaaatcttgtgacaaaact cacacatctccaccgagctca	EPKSCDKTHTSPPSS	114	(115)
scs(s)hlgGl	gagcccaaatcttgtgacaaaact cacacatgtccaccgagctca	EPKSSDKTHTCPPSS	116	(117)
ccc(s)hlgGl	gagcccaaatcttgtgacaaaact cacacatgtccaccgtgctca	EPKSCDKTHTSPPCS	118-	(119)
ccc(p)hlgGl	gagcccaaatcttgtgacaaaact cacacatgtccaccgtgccca	EPKSCDKTHTSPPCP	120	¢121)
sss(p)hlgGl	gagcccaaatcttctgacaaaact cacacatctccaccgagccca	EPKSSDKTHTSPPSP	122	(123)
esc(p)hlgGl	gagcccaaatcttgtgacaaaact cacacatctccaccgtgccca	EPKSCDKTHTSPPCP	124	¢125)
ssc(p)hlgGl	gagcccaaatcttctgacaaaact cacacatctccaccgtgccca	EPKSSDKTHTSPPCP	126	¢127)
see(p)hlgGl	gagcccaaatcttctgacaaaact cacacatgtccaccgtgccca	EPKSSDKTHTCPPCP	128	¢129)
css(p)hlgGl	gagcccaaatcttgtgacaaaact cacacatctccaccgagccca	EPKSCDKTHTSPPSP	130	(131)
scs(p)hlgGl	gagcccaaatcttgtgacaaaact cacacatgtccaccgagccca	EPKSSDKTHTCPPSP	132	(133)
seppep	agttgtccaccgtgccca	SCPPCP	134	¢135) .
EFD	liucleotide sequence..........	Amino acid Sequence	{awiAo.. acid j ' sequence)
hlgGl {P238S) Ch2Ch3	gcacctgaactcctgggtggatcg tcagtcttcctcttccccccaaaa cccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagac	APELLGGSSVFLFPPKPKDTLMIS RT PEVTCVWD VS HE D PEVKFNWY VDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQ PRE PQVYT	142	¢143)

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	cctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataat gccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtg gtcagcgtcctcaccgtcctgcac caggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaa gccctcccagcccccatcgagaaa acaatctccaaagccaaagggcag ccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggag agcaatgggcagccggagaacaac tacaagaccacgcctcccgtgctg gactccgacggctccttcttcctc tacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcag aagagcctctccc tgtctccgggtaaatga	LPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG K
hlgGl (P331S) CH2CH3	gcacctgaactcctgggtggaccg tcagtcttcctcttccccccaaaa cccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagac cctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataat gccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtg gtcagcgtcctcaccgtcctgcac caggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaa gccctcccagcctccatcgagaaa acaatctccaaagccaaagggcag ccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggag agcaatgggcagccggagaacaac tacaagaccacgcctcccgtgctg gactccgacggctccttcttcctc tacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcag aagagcctctccc tgtctccgggtaaatga	APELLGGPSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTK PREEQYN S TYRV VSVLTVLHQDWLNGKEYKCKVSNK ALPASIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG K	144	(145)
hlgGl (P238S/ P331S) Ch2CH3	gcacctgaactcctgggtggatcg tcagtcttcctcttccccccaaaa cccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtg gtggtggacgtgagccacgaagac cctgaggtcaagttcaactggtac gtggacggcgtggaggtgcataat gccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtg gtcagcgtcctcaccgtcctgcac	APELLGGSSVFLFPPKPKDTLMIS RTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRV VSVLTVLHQDWLNGKEYKCKVSNK ALPASIEKTIS KAKGQ PRE PQVYT LPPSRDELTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG K	146	(147)

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	caggactggctgaatggcaaggag tacaagtgcaaggtctccaacaaa gccctcccagcctccatcgagaaa acaatctccaaagccaaagggcag ccccgagaaccacaggtgtacacc ctgcccccatcccgggatgagctg accaagaaccaggtcagcctgacc tgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggag agcaatgggcagccggagaacaac tacaagaccacgcctcccgtgctg gactccgacggctccttcttcctc tacagcaagctcaccgtggacaag agcaggtggcagcaggggaacgtc ttctcatgctccgtgatgcatgag gctctgcacaaccactacacgcag aagagcctctccctgtctccgggt aaatga
XIttkes·		' - fe&iuc Acid , i- = ΛΙ- | | ζ > 1 . 4 ,	,,
STDl	aattatggtggcggtggctegggc ggtggtggatctggaggaggtggg agtgggaattct	NYGGGGSGGGGSGGGGSGNS	140	(149)
STD2	aattatggtggcggtggctegggc ggtggtggatctggaggaggtggg agtgggaattatggtggcggtggc tcgggcggtggtggatctggagga ggtgggagtgggaattct	nyggggsggggsggggsgnygggg SGGGGSGGGGSGNS	150	(151)
Hl	aattet	NS	152	(153)
H2	ggtggeggtggctcggggaa11 c t	GGGGSGNS	154	(155)
H3	aattatggtggcggtggctctggg aattet	NYGGGGSGNS	156	(157)
H4	ggtggcggtggctcgggcggtggt ggatctgggaattct	GGGGSGGGGSGNS	158	(159)
H5	aattatggtggcggtggctegggc ggtggtggatctgggaattct	NYGGGGSGGGGSGNS	160	(161)
H6	ggtggcggtggctcgggcggtggt ggatctgggggaggaggcagcggg aattet	GGGGSGGGGSGGGGSGNS	162	(163)
H7	gggtgtccaccttgtccgaattct	GCPPCPNS	164	(165)
(G4S)3	ggtggcggtggatccggcggaggt gggtcgggtggcggcggatct	GGGSGGGSGGGS	166	(167)
(G4S) 4	ggtggcggtggctcgggcggtggt ggatctggaggaggtgggagcggg ggaggtggcagt	GGGSGGGSGGGSGGGGS	168	(169)

Table 5. Primary structures (polynucoleotide and cognate amino acid sequences) of exemplary features of multivalent binding molecules.

Example 8

Binding and Functional Studies with Alternative Multispecific Fusion Proteins

Experiments that parallel the experiments described above for the prototypical CD20-IgG-CD28 multispecific binding (fusion) molecule were conducted for each of

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148 the additional multivalent binding molecules described above. In general, the data obtained for these additional molecules parallel the results observed for the prototype molecule. Some of the salient results of these experiments are disclosed below.

Figure 14 shows results of blocking studies performed on one of the new molecules where both BD1 and BD2 bind to target antigens on the same cell or cell type, in this case, CD20 and CD37. This multispecific, multivalent binding (fusion) protein was designed with binding domain 1 binding CD20 (2H7; VLVH orientation), and binding domain 2 binding CD37, G28-1 VL-VH (LH) or VH-VL (HL). The experiment was performed in order to demonstrate the multispecific properties of the protein.

Blocking Studies:Ramos or BJAB B lymphoblastoid cells (2.5x10^s) were preincubated in 96-well V-bottom plates in staining medium (PBS with 2% mouse sera) with murine anti-CD20 (25 pg/ml) antibody, or murine anti-CD37 (10 gg/ml) antibody, both together or staining medium alone for 45 minutes on ice in the dark. Blocking antibodies were pre-incubated with cells for 10 minutes at room temperature prior to addition of the multispecific binding (fusion) protein at the concentration ranges indicated, usually from 0.02 gg/ml to 10 gg/ml, and incubated for a further 45 minutes on ice in the dark. Cells were washed 2 times in staining medium, and incubated for one hour on ice with Caltag (Burlingame, CA) FITC goat anti-human IgG (1:100) in staining medium, to detect binding of the multispecific binding (fusion) proteins to the cells. The cells were then washed 2 times with PBS and fixed with 1% paraformaldehyde (cat. no. 19943, USB, Cleveland, Ohio). The cells were analyzed by flow cytometry using a FACsCalibur instrument and CellQuest software (BD Biosciences, San Jose, CA). Each data series plots the binding of the 2H7-ssshIgG-STDl-G28-l HL fusion protein in the presence of CD20, CD37, or both CD20 and CD37 blocking antibodies. Even though this experiment used one of the cleaved linkers, only the presence of both blocking antibodies completely eliminates binding by the multispecific binding (fusion) protein, demonstrating that the bulk of the molecules possess binding function for both CD20 and CD37. The data were similar for two cell lines tested in panels A and B, Ramos and BJAB, where the CD20 blocking antibody was more effective than the CD37 blocking antibody at reducing the level of binding observed by the multispecific binding (fusion) protein.

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ADCC Assays

Figure 15 shows the results of ADCC assays performed on the CD20-CD37 multispecific binding (fusion) proteins. ADCC assays were performed using BJAB lymphoblastoid B cells as targets and human PBMC as effector cells. BJAB cells were labeled with 500 pCi/ml ⁵¹Cr sodium chromate (250 pCi/pg) for 2 hours at 37°C in IMDM/10%FBS. The labeled cells were washed three times in RPMI.10% FBS and resuspended at 4xl0⁵ cells/ml in RPMI. Heparinized, human whole blood was obtained from anonymous, in-house donors and PBMC isolated by fractionation over Lymphocyte Separation Media (LSM, ICN Biomedical) gradients. Buffy coats were harvested and washed twice in RPMI/10% FBS prior to resuspension in RPMI/10%

FBS at a final concentration of 5x10⁶ cells/ml. Cells were counted by trypan blue exclusion using a hemacytometer prior to use in subsequent assays. Reagent samples were added to RPMI medium with 10% FBS at 4 times the final concentration and three 10 fold serial dilutions for each reagent were prepared. These reagents were then added to 96-well U-bottom plates at 50 μΐ/well for the indicated final concentrations. The ^slCr-labeled BJAB cells were added to the plates at 50 μΐ/well (2xl0⁴ cells/well). The PBMCs were then added to the plates at 100 μΐ/well (5x10⁵ cells/well) for a final ratio of 25:1 effector (PBMC):target (BJAB). Effectors and targets were added to medium alone to measure background killing. The ⁵¹Cr-labeled cells were added to medium alone to measure spontaneous release of ^5ICr and to medium with 5% NP40 (cat. no.28324, Pierce, Rockford, IL) to measure maximal release of ⁵¹Cr. Reactions were set up in triplicate wells of a 96-well plate. Multispecific binding (fusion) proteins were added to wells at a final concentration ranging from 0.01 μg/ml to 10μβ/πι1, as indicated on the graphs. Each data series plots a different multispecific binding (fusion) protein or the corresponding single specificity SMIPs at the titration ranges described. Reactions were allowed to proceed for 6 hours at 37°C in 5% CO₂ prior to harvesting and counting. Twenty-five μΐ of the supernatant from each well were then transferred to a Luma Plate 96 (cat. no. 6006633, Perkin Elmer, Boston, Mass) and dried overnight at room temperature.

CPM released was measured on a Packard TopCounNXT. Percent specific killing was calculated by subtracting (cpm {mean of triplicate samples) of sample - cpm spontaneous release)/(cpm maximal release-cpm spontaneous release) xlOO. Data are plotted as % specific killing versus protein concentration. The data demonstrate that

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150 the multispecific binding (fusion) protein is able to mediate ADCC activity against cells expressing the target antigen(s) as well as the single specificity SMIPs for CD20 and/or CD37, but does not show augmentation in the level of this effector function.

Co-culture Experiments

Figure 16 shows the results of experiments designed to look at other properties of this type of multispecific binding (fusion) protein, where having two binding domains against targets expressed on the same cell or cell type might result in synergistic effects by signaling/binding through the two surface receptors bound. The co-culture experiments were performed using PBMC isolated as described for the

ADCC assays above.. These PBMC were resuspended in culture medium at 2x10⁶ cells/ml in a final volume of 500 μΐ/well, and cultured alone or incubated with single specificity SMIPs for CD20, CD37, CD20+CD37, or the multispecific binding (fusion) protein using the H7 linker, [2H7-sss-IgG-H7-G28-l HL]. Each of the test reagents was added at a final concentration of 20 pg/ml. After 24 hours of culture, no real differences were seen in the % of B cells in culture; however, when the cells were subjected to flow cytometry, cell clumping was visible in the FWD X 90 staining pattern for the cultures containing the multispecific binding (fusion) protein, indicating that the B cells expressing the two target antigens were engaged in homotypic adhesion. After 72 hours in culture, the multispecific binding (fusion) protein resulted in the death of almost all the B cells present. The combination of the two single-specificity SMIPs also drastically decreased the percentage of B cells, but not to the level seen with the multispecific binding molecule. These data suggest that engaging both binding domains for CD20 and CD 3 7 on the same multispecific molecule, results in homotypic adhesion between B cells and may also result in binding of both CD20 and CD37 antigens on the same cell. Without wishing to be bound by theory, the synergistic effect in eliminating target cells may be due (1) to the binding through binding domains 1 and 2 on the same cell types, and/or (2) to interactions of the effector function domain (constant sub-region) of the multivalent binding molecules with monocytes or other cell types in the PBMC culture that result in delayed killing. The kinetics of this killing effect are not rapid, taking more than 24 hours to be achieved, indicating that it is may be a secondary effect, requiring production of cytokines or other molecules prior to the effects being observed.

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Apoptosis Assays

Figure 17 shows the results of experiments designed to explore the induction of apoptosis after treatment of B cell lines with either the [2H7-sss-hIgG-H7-G28-l HL] multispecific, multivalent binding (fusion) proteins or the single specificity

CD20 and/or CD37 SMIPS, alone and in combination with one another. Ramos cells (panel A; ATCC No. CRL-1596), and Daudi cells (panel B; ATCC No. CCL-213) were incubated overnight (24 hours) at 37°C in 5% CO₂ in Iscoves (Gibco) complete medium with 10% FBS at 3 X 10⁵ cells/ml and 5,10, or 20 gg/ml fusion proteins.

For combination experiments with the single specificity SMIPs, the proteins were used at the following concentrations: TRU-015 (CD20 directed SMIP)=10 pg/ml, with 5 pg/ml G28-1 LH (CD37 directed SMIP). Alternatively, TRU-015=20 pg/ml was combined with G28-1 LH at 10 pg/ml. Cells were then stained with Annexin VFITC and propidium iodide using the BD Pharmingen Apoptosis Detection Kit I cat. no. 556547), and processed according to kit instructions. The cells were gently vortexed, incubated in the dark at room temperature for 15 minutes, and diluted in 400 μΐ binding buffer prior to analysis. Samples were analyzed by flow cytometry on a FACsCalibur (Becton Dickinson) instrument using Cell Quest software (Becton Dickinson). The data are presented as columnar graphs plotting the percentage of Annexin V/propidium iodide positive cells versus type of treatment. Clearly, the multispecific binding (fusion) protein is able to induce a significantly higher level of apoptotic death in both cell lines than the single specificity reagents, even when used together. This increased functional activity reflects an interaction of the coordinate binding of BD1 and BD2 (specific for CD20 and CD37) receptors on the target cells.

Example 9

Binding and Functional Properties of2H7-hlgG~G19-4 Multispecific Binding (Fusion) Proteins

This example describes the binding and functional properties of the 2H7hIgG-G19-4 multi specific fusion proteins. The construction of these molecules is described in Example 7. Expression and purification are as described in previous Examples.

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Binding experiments were performed as described for previous molecules, except that the target cells used to measure CD3 binding were Jurkat cells expressing CD3 on their surface. Refer to Figure 18, where the top graph shows binding curves obtained for binding of the CD20-CD3 multispecific molecules to Jurkat cells using purified proteins serially diluted from 20 to 0.05 pg/ml. The HL orientation of the G19-4 specificity seems to bind better to the CD3 antigen than does the LH orientation. The lower panel shows the binding curves obtained for the BD1, the binding domain recognizing CD20. All of the molecules bind well, and at a level nearly equivalent to a single specificity SMIP for CD20.

ADCC Assays

For the data presented in Figure 19, ADCC assays were performed as described in the previous Example. In this case, the fusion proteins were all 2H7hlgG-G 19-4 variants or combinations of the single-specificity SMIPs (2H7, specific for CD20) or antibodies (G19-4, specific for CD3). In addition, for the data presented in the lower panel of Figure 19, NK cells were depleted from PBMC prior to use, by magnetic bead depletion using a MACS (Miltenyi Biotec, Auburn, CA) column separation apparatus and NK cell-specific CD 16 magnetic microbeads (cat no.: 130045-701). The data presented in the two panels demonstrate that all of the CD20hIgG-CD3 multispecific molecules mediate ADCC, regardless of whether NK cells are depleted or total PBMC are used in the assay. For the TR.U 015 or combinations of G19-4 and TRU015, only cultures containing NK cells could mediate ADCC. G19-4 did not work well in either assay against BJAB targets, which do not express CD3, although G19-4 may have bound to CD3 expressing NK T cells and activated these cells in the first assay shown. The killing observed in the lower panel for the multispecific binding (fusion) proteins is probably mediated through activation of cytotoxicity in the T cell population by binding CD3, against the BJAB targets expressing the CD 20 antigen. This killing activity appears to be relatively insensitive to the dosage of the molecules over the concentration ranges used, and is still significantly different from the other molecules tested, even at a concentration of 0.01 ug/ml.

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Example 10

Multivalent Binding Molecules

Other embodiments include linker domains derived from immunoglobulins. More specifically, the source sequences for these linkers are sequences obtained by comparing regions present between the V-like domains or the V- and C-like domains of other members of the immunoglobulin superfamily. Because these sequences are usually expressed as part of the extracellular domain of cell surface receptors, they are expected to be more stable to proteolytic cleavage, and should also not be immunogenic. One type of sequence that is not expected to be as useful in the role of a linker for the multivalent binding (fusion) proteins is the type of sequence expressed on surface-expressed members of the -Ig superfamily, but that occur in the intervening region between the C-like domain and the transmembrane domain. Many of these molecules have been observed in soluble form, and are cleaved in these intervening regions close to the cell membrane, indicating that the sequences are more susceptible to cleavage than the rest of the molecule.

The linkers described above are inserted into either a single specificity SMIP, between the binding domain and the effector function domain, or are inserted into one of the two possible linker positions in a multivalent binding (fusion) protein, as described herein.

A complete listing of the sequences disclosed in this application is appended, and is incorporated herein by reference in its entirety. The color coding indicating the sequence of various regions or domains of the particular polynucleotides and polypeptides are useful in identifying a corresponding region or domain in the sequence of any of the molecules disclosed herein.

Example 11

Screening matrix for scorpion candidates targeting B-cells Introduction

As a means of identifying combinations of paired monoclonal antibody binding domains that would most likely yield useful and potent multivalent binding molecules, or scorpions, against a target population, a series of monoclonal antibodies against B cell antigens was tested in a combination matrix against B cell lines

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154 representing various non Hodgkin’s lymphomas. To ensure that all possible pairwise comparisons of antibodies known or expected to bind to the cell of interest are assayed, a two-dimensional matrix of antibodies may be used to guide the design of studies using a given cell type. Monoclonal antibodies against numerous B cell antigens known by their cluster designations (CDs) are recorded in the left column. Some of these antibodies (designated by the antigen(s) to which they specifically bind), i.e., CD19, CD20, CD21, CD22, CD23, CD30, CD37, CD40, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, and CL II (MHC Class II), were incubated, alone or in combination with other members of this monoclonal antibody set, with antigen-positive target cells. The variable domains of these antibodies are contemplated as binding domains in exemplary embodiments of the multivalent binding molecules. Using the knowledge in the art and routine procedures, those of skill in the art are able to identify suitable antibody sequences (nucleic acid encoding sequences as well as amino acid sequences), for example in publicly available databases, to generate a suitable antibody or fragment thereof (e.g., by hybridizationbased cloning, PCR, peptide synthesis, and the like), and to construct multivalent binding molecules using such compounds. Sources of exemplary antibodies from which binding domains were obtained as described herein are provided in Table 6. Typically, a cloning or synthesis strategy that realizes the CDR regions of an antibody chain will be used, although any antibody, fragment thereof, or derivative thereof that retains the capacity to specifically bind to a target antigen is contemplated.

Stated in more detail, the cloning of heavy and/or light chain variable regions of antibodies from hybridomas is standard in the art. There is no requirement that the sequence of the variable region of interest be known in order to obtain that region using conventional cloning techniques. See, e.g., Gilliland et al., Tissue Antigens

47(1): 1-20 (1996). To prepare single-chain polypeptides comprising a variable region recognizing a murine or human leukocyte antigen, a method was devised for rapid cloning and expression that yielded functional protein within two to three weeks of RNA isolation from hybridoma cells. Variable regions were cloned by poly-G tailing the first-strand cDNA followed by anchor PCT with a forward poly-C anchor primer and a reverse primer specific for the constant region sequence. Both primers contain flanking restriction endonuclease sites for insertion into pUC19. Sets of PCR primers for isolation of murine, hamster and rat Vl and Vh genes were generated. Following

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155 determination of consensus sequences for a specific Vl and Vh pair, the Vl and Vh genes were linked by DNA encoding an intervening peptide linker (typically encoding (Gly4Ser)₃) and the Vt-linker-VH gene cassettes were transferred into the pCDM8 mammalian expression vector, The constructs were transfected into COS cells and sFvs were recovered from conditioned culture medium supernatant. This method has been successfully used to generate functional sFv to human CD2, CD3, CD4, CD8, CD28, CD40, CD45 and to murine CD3 and gp39, from hybridomas producing murine, rat, or hamster antibodies. Initially, the sFvs were expressed as fusion proteins with the hinge-Cm-Cra domains of human IgGl to facilitate rapid characterization and purification using goat anti-human IgG reagents or protein A.

Active sFv could also be expressed with a small peptide, e.g., a tag, or in a tailless form. Expression of CD3 (G19-4) sFv tailless forms demonstrated increased cellular signaling activity and revealed that sFvs have potential for activating receptors.

Alternatively, identification of the primary amino acid sequence of the variable domains of monoclonal antibodies can be achieved directly, e.g., by limited proteolysis of the antibody followed by N-terminal peptide sequencing using, e.g., the Edman degradation method or by fragmentation mass spectroscopy. N-terminal sequencing methods are well known in the art. Following determination of the primary amino acid sequence, the variable domains, a cDNA encoding this sequence is assembled by synthetic nucleic acid synthesis methods (e.g., PCR) followed by scFv generation. The necessary or preferred nucleic acid manipulation methods are standard in the art.

Fragments, derivatives and analogs of antibodies, as described above, are also contemplated as suitable binding domains. Further, any of the constant sub-regions described above are contemplated, including constant sub-regions comprising any of the above-described hinge regions. Additionally, the multivalent single-chain binding molecules described in this example may include any or all of the linkers described herein.

Monoclonal antibodies were initially exposed to cells and then cross-linked using a goat anti-mouse second-step antibody (2^nd step). Optionally, one could crosslink the antibodies prior to contacting cells with the antibodies, e.g., by cross-linking the antibodies in solution. As another alternative, monoclonal antibodies could be

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156 cross-linked in a solid phase by adsorbing onto the plastic bottom of tissue culture wells or “trapped” on this plastic by means of goat anti-mouse antibody adsorbed to the plastic, followed by plate-based assays to evaluate, e.g., growth arrest or cell viability.

Inversion of phosphatidylserine from the cytosolic side of the cell membrane to the exterior cell surface of that plasma membrane is an accepted indicator of proapoptotic events. Progression to apoptosis leads to loss of cell membrane integrity, which can be detected by entry of a cell-impermeant intercalating dye, e.g., propidium iodide (PI). Following cell exposure to monoclonal antibodies alone or in combination, a dual, pro-apoptotic assay was performed and treated cell populations were scored for cell surface-positive annexin V (ANN) and/or PI inclusion.

Annexin V binding /Propidium iodide internalization analysis

Cells and cell culture conditions. Experiments were performed to examine the effect of cross-linking two different monoclonal antibodies against targets expressed on four human B-cell lines. Effects on cell lines were measured by determining levels of ANN and/or PI staining following exposure. The human B cell lines BJAB, Ramos (ATCC//CRL-1596), Daudi (ATCC#CCL-213), and DHL-4 (DSM2#ACC495) were incubated for 24 hours at 37°C in 5% CO2 in Iscoves (Gibco) complete medium with 10% FBS. Cells were maintained at a density between 2-8 x 10⁵ cells/ml and a viability typically >95% prior to study.

Experiments were conducted at a cell density of 2 x 10⁵ cells/ml and 2 pg/ml of each comparative monoclonal antibody from a matrix against B-cell antigens.

Each comparator monoclonal antibody was added at 2 pg/ml alone or individually when combined with each matrix monoclonal antibody, also at 2 gg/ml. Table 6 lists the catalog number and sources of monoclonal antibodies used in these experiments. For cross-linking these monoclonal antibodies in solution, goat anti-mouse IgG (Jackson Labs catalog no. 115-001-008) was added to each well at a concentration ratio of 2:1 (goat anti-mouse: each monoclonal antibody), e.g., a well with only one monoclonal antibody at 2 pg/ml would have goat anti-mouse added to a final concentration of 4 pg/ml, while wells with both comparator monoclonal antibody (2 pg/ml) and a monoclonal antibody from the matrix (2 pg/ml) would have 8 pg/ml of goat anti-mouse antibody added to the well.

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After 24 hours of incubation at 37°C in 5% CO2, cells were stained with Annexin V-FITC and propidium iodide using the BD Pharmingen Annexin V-FITC Apoptosis Detection Kit I (#556547). Briefly, cells were washed twice with cold PBS and resuspended in “binding buffer” at 1 x 10⁶ cells/ml. One hundred microliters of the cells in binding buffer were then stained with 5 μΐ of Annexin V-FITC and 5 μΐ of propidium iodide. The cells were gently mixed and incubated in the dark at room temperature for 15 minutes. Four hundred micro liters of binding buffer were then added to each sample. The samples were then read on a FACsCalibur (Becton Dickinson) and analyzed using Cell Quest software (Becton Dickinson).

Table 6

Name	Catalog number	Commercial supplier
Anti-CD 19	#C2269-74	US Biological (Swampscott, MA)
Anti-CD20	#169-820	Ancell Coro (Bavoort. MN)
Anti-CD21	#170-820	Ancell Coro (Bavoort. MN)
Anti-CD22	#171-820	Ancell Coro (Bayport, MN)
Anti-CD23	#172-820	Ancell Coro (Bavoort, MN)
Anti-CD30	#179-820	Ancell Coro (Bavoort, MN)
Anti-CD37	#186-820	Ancell Coro (Bavoort, MN)
Anti-CD40	#300-820	Ancell Coro (Bavoort. MN)
Anti-CD70	#222-820	Ancell Coro (Bavoort, MN)
Anti-CD72	#C2428-41B1	US Biological (Swampscott. MA)
Anti-CD79a	#235-820	Ancell Coro (Bavoort, MN)
Anti-CD79b	#301-820	Ancell Coro (Bavoort, MN)
Anti-CD80	#110-820	Ancell Coro (Bavoort, MN)
Anti-CD81	#302-820	Ancell Coro (Bavoort, MN)
Anti-CD 8 6	#307-820	Ancell Coro (Bavoort. MN)
Anti-CL II DR, DO, DP	#131-820	Ancell Coro (Bavoort, MN)

Table 6. Antibodies against B cell antigens used in this study and their sources.

Addition of the cross-linking antibody (e.g., goat anti-mouse antibody) to 15 monoclonal antibody A alone resulted in increased cell sensitivity, suggesting that a multivalent binding molecule, or scorpion, constructed with two binding domains recognizing the same antigen would be effective at increasing cell sensitivity.

Without wishing to be bound by theory, this increased sensitivity could be due to antigen clustering and altered signaling. TNF receptor family members, for example, require homo-multimerization for signal transduction and scorpions with equivalent binding domains on each end of the molecule could facilitate this interaction. The

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158 clustering and subsequent signaling by CD40 is an example of this phenomenon in the B cell system.

As shown in Figures 20,21 and 22, the addition of monoclonal antibody A and monoclonal antibody B against different antigens will produce additive or in some combinations greater than additive (i.e., synergistic) pro-apoptotic effects on treated cells. In Figure 20, for example, the combination of anti-CD20 with monoclonal antibodies against other B cell antigens all resulted, to varying extents, in increased cell sensitivity. Some combinations, such as anti-CD20 combined with anti-CD 19 or anti-CD20 combined with anti-CD21, however, produced greater than additive pro-apoptotic effects, indicating that multivalent binding molecules or scorpions composed of these binding domains should be particularly effective at eliminating transformed B cells. Referring to Figure 20, the percentage of cells exhibiting pro-apoptotic activities when exposed to anti-CD 20 antibody alone is about 33% (vertically striped bar corresponding to “20,” i.e., the anti-CD20 antibody);

the percentage of pro-apoptotic cells upon exposure to anti-CD 19 antibody is about 12% (vertically striped bar in Fig. 20 corresponding to “19,” i.e., the anti-CD19 antibody); and the percentage of pro-apoptotic cells upon exposure to both anti-CD20 and anti-CD 19 antibodies is about 73% (horizontally striped bar in Fig. 20 corresponding to “19”). The 73% of pro-apoptotic cells following exposure to both antibodies is significantly greater than the 45% (33% + 12%) sum of the effects attributable to each individual antibody, indicating a synergistic effect attributable to the anti-CD 19 and anti-CD 20 antibody pair. Useful multivalent binding molecules include molecules in which the two binding domains lead to an additive effect on Bcell behavior as well as multivalent binding molecules in which the two binding domains lead to synergistic effects on B-cell behavior. In some embodiments, one binding domain will have no detectable effect on the measured parameter of cell behavior, with each of the paired binding domains contributing to distinct aspects of the activities of the multivalent binding molecule, such as a multispecific, multivalent binding molecule (e.g., binding domain A binds to a target cell and promotes apoptosis while binding domain B binds to a soluble therapeutic such as a cytotoxin). Depending on the design of a multivalent binding molecule, the issue of the type of combined effect (additive, synergistic, or inhibitory) of the two binding domains on a target cell may not be relevant because one of the binding domains is specific for a

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Exemplary binding domain pairings producing additive, synergistic or inhibitory effects, as shown in Figures 20-23, are apparent from Tables 7 and 8.

Table 7 provides quantitative data extracted from each of Figures 20-23 in terms of the percentage of cells staining positive for ANN and/or PI. Table 8 provides calculations using the data of Table 7 that provided a basis for determining whether the interaction of a given pair of antibodies yielded an additive, synergistic, or inhibitory effect, again as assessed by the percentage of cells staining positive for

ANN and/or PI.

Table 7

Name	Anti-CD20	Anti-CD79b	Anti-CL Π	Anti-CD22
Anti-CD 19	13/73*	18/76/66	14/47/46	12/11
Anti-CD20	33/NA	42/94/92	33/71/76	28/33
Anti-CD21	14/75	22/50/76	18/24/40	11/11
Anti-CD22	8/55	12/39/33	12/19/17	10/12
Anti-CD23	8/41	12/63/55	14/22/17	10/12
Anti-CD30	8/38	14/72/61	12/56/61	10/11
Anti-CD37	15/45	19/92/86	20/60/62	19/20
Anti-CD40	10/48	12/44/30	13/21/28	14/13
Anti-CD70	9/40	12/56/39	15/21/15	10/10
Anti-CD72	NA	16/60/64	30/78/63	17/17
Anti-CD79a	21/66	43/42/50	28/55/51	14/14
Anti-CD79b	46/88	70/70/68	45/80/76	26/16

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Name	Anti-CD20	Anti-CD79b	Anti-CL II	Anti-CD22
Anti-CD80	7/41	14/35/30	15/19/17	11/11
Anti-CD81	14/65	25/86/83	25/54/43	19/20
Anti-CD86	7/38	16/58/42	15/24/18	14/11
Anti- CL II	53/77	52/96/98	47/52/43	72/57

In columns 2-4 of Table 7, the numerical values reflect the heights of histogram bars in Figures 20-22, respectively, with the first number in each cell denoting the height of a vertically striped bar, the second number denoting the height of a horizontally striped bar and, where present, the third number reflecting the height of a stippled bar.

In column 5, the first number reflects the height of a solid bars and the second number reflects the height of a slant-striped bar in Figure 23.

Table 8

Name	Anti-CD20	Anti-CD79b	Anti-CL II	Anti-CD22
Anti-CD 19	S: 13+33=46*	A: 18+56=74	S:14+26=40	I: 12+10=22
		A: 18+43=61	S:14+18=32
Anti-CD20	NA	A: 42+56=98	S:33+26=59	A/I:28+10=38
		A:42+43=85	S:33+18=51
Anti-CD21	S: 14+33=47	I: 22+56=78	I: 18+26=44	I: 11+10=21
		S: 22+43=65	A: 18+18=36
Anti-CD22	S: 8+33=41	I: 12+56=68	I: 12+26=38	NA
		I: 12+43=55	I: 12+18=30
Anti-CD23	A: 8+33=41	A:12+56=68	I: 14+26=40	I: 10+10=20
		A:12+43=55	I: 14+18=32

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Name	Anti-CD20	Anti-CD79b	Anti-CL II	Anti-CD22
Anti-CD30	A: 8+33=41	A:14+56=70	S: 12+26=38	I: 10+10=20
		A:14+43=57	S: 12+18=30
Anti-CD37	A: 15+33=48	S:19+56=75	S: 20+26=46	I: 19+10=29
		S:19+43=62	S: 20+18=38
Anti-CD40	A/S: 10+33=43	I: 12+56=68	I; 13+26=39	I: 14+10=24
		I: 12+43=55	A:13+18=31
Anti-CD70	A:9+33=42	I: 12+56=68	I: 15+26=41	I: 10+10=20
		I: 12+43=55	I: 15+18=33
Anti-CD72	NA	1:16+56=72	S; 30+26=56	I: 17+10-27
		A:16+43=59	S: 30+18=48
Anti-CD79a	S: 21+33=54	I: 43+56=99	A:28+26=54	I: 14+10=24
		I: 43+43=86	A:28+18=46
Anti-CD79b	S: 46+33=79	NA	S:45+26=71	I: 26+10=36
			S:45+18=63
Anti-CD80	A:7+33=40	I: 14+56=70	I: 15+26=41	I: 11+10=21
		I; 14+43=57	I: 15+18=33
Anti-CD 81	S:14+33=47	A:25+56=81	A: 25+26=51	I: 19+10=29
		S:25+43=68	A:25+18=43
Anti-CD86	A: 7+33=40	I: 16+56=72	I: 15+26=41	I; 14+11=25
		I: 16+43=59	I: 15+18=33

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Name	Anti-CD20	Anti-CD79b	Anti-CL II	Anti-CD22
Anti- CL II	I: 53+33=86	A: 52+56=108 A: 52+43=95	NA	I; 72+10=82

“A” means an “additive” effect was observed “S” means a “synergistic” effect was observed “1” means an “inhibitory” effect was observed ^Equation schematic: A+B=C, where “A” is the percent ANN and/or PI positive cells due to matrix antibody alone, “B” is the percent ANN and/or PI positive cells due to the common antibody (anti-CD20 for Fig. 20, anti-CD79b for Fig. 21, anti-CLII for Fig. 22, and anti-CD22 for Fig. 23), and “C” is the expected additive effect. (See Table 7, above, for the quantitative data corresponding to Figures 20-23.) Where two equations are present in a cell, the upper equation reflects results use of the higher indicated concentration of common antibody; the lower equation reflects use of the lower indicated concentration of common antibody.

In some embodiments, the two binding domains interact in an inhibitory, additive or synergistic manner in sensitizing (or de-sensitizing) a target cell such as a B cell. Figure 23 shows the protective, or inhibitory, effects resulting from combining anti-CD22 antibody with strongly pro-apoptotic monoclonal antibodies such as the anti-CD79b antibody or anti-MHC class II (i.e., anti-CL II) antibody. For example, Figure 23 and Table 7 show that anti-CD22 antibody alone induces no more than about 10% of cells to exhibit pro-apoptotic behavior (solid bar corresponding to “22” in Fig. 23) and anti-CD79b induces about 26% pro-apoptotic cells (solid bar corresponding to “CD79b” in Fig. 23). In combination, however, anti-CD22 and antiCD79b induce only about 16% pro-apoptotic cells (slant-striped bar corresponding to “79b” in Fig. 23). Thus, the combined antibodies induce 16% pro-apoptotic cells, which is less than the 38% sum of the individual effects attributable to anti-CD22 (12%) and anti-CD79b (26%). Using this approach, an inspection of Figure 23 and/or Tables 7-8 reveals that anti-CD22 antibody, and by extension a multispecific, multivalent binding molecule comprising an anti-CD22 binding domain, when used in separate combination with each of the following antibodies (or corresponding binding domains): anti-CD19, anti-CD20, anti-CD21, anti-CD23, anti-CD30, anti-CD37, antiCD40, anti-CD70, anti-CD72, anti-CD79a, anti-CD79b, anti-CD80, anti-CD81, antiCD86 and anti-MHC class II antibodies/binding domains, will result in an inhibited overall effect.

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Without wishing to be bound by theory, the data can be interpreted as indicating that anti-CD22 antibody, or a multispecific, multivalent binding molecule comprising an anti-CD22 binding domain, will protect against, or mitigate an effect of, any of the antibodies listed immediately above. More generally, a multispecific, multivalent binding molecule comprising an anti-CD22 binding domain will inhibit the effect arising from interaction with any of CD19, CD20, CD21, CD23, CD30, CD37, CD40, CD70, CD72, CD 79a, CD79b, CD80, CD81, CD86, and MHC class II molecules. It can be seen in Figure 23 and Table 8 that anti-CD22 antibody, and by extension a binding domain comprising an anti-CD22 binding domain, will function as an inhibitor or mitigator of the activity of any antibody/binding domain recognizing a B-cell surface marker such as a CD antigen. Multivalent binding molecules, including multispecific, multivalent binding molecules, are expected to be useful in refining treatment regimens for a variety of diseases wherein the activity of a binding domain needs to be attenuated or controlled.

In addition to the inhibitory, additive or synergistic combined effect of two binding domains interacting with a target cell, typically through the binding of cellsurface ligands, the experimental results disclosed herein establish that a given pair of binding domains may provide a different type of combined effect depending on the relative concentrations of the two binding domains, thereby increasing the versatility of the invention. For example, Table 8 discloses that anti-CD21 and anti-CD79b interact in an inhibitory manner at the higher tested concentration of anti-CD79b, but these two antibodies interact in a synergistic manner at the lower tested concentration of anti-CD79b. Although some embodiments will use a single type of multivalent binding molecule, i.e., a monospecific, multivalent binding molecule, comprising,

e.g., a single CD21 binding domain and a single CD79b binding domain, the invention comprehends mixtures of multivalent binding molecules that will allow adjustments of relative binding domain concentrations to achieve a desired effect, such as an inhibitory, additive or synergistic effect. Moreover, the methods of the invention encompass use of a single multivalent binding molecule in combination with another binding molecule, such as a conventional antibody molecule, to adjust or optimize the relative concentrations of binding domains. Those of skill in the art will be able to determine useful relative concentrations of binding domains using standard

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164 techniques (e.g., by designing experimental matrices of two dilution series, one for each binding domain).

Without wishing to be bound by theory, it is recognized that the binding of one ligand may induce or modulate the surface appearance of a second ligand on the same cell type, or it may alter the surface context of the second ligand so as to alter its sensitivity to binding by a specific binding molecule such as an antibody or a multivalent binding molecule.

Although exemplified herein using B cell lines and antigens, these methods to determine optimally effective multivalent binding molecules (i.e., scorpions) are applicable to other disease settings and target cell populations, including other normal cells, their aberrant cell counterparts including chronically stimulated hematopoietic cells, carcinoma cells and infected cells.

Other signaling phenotypes such as Ca²⁺mobilization; tyrosine phosphoregulation; caspase activation; NF-κΒ activation; cytokine, growth factor or chemokine elaboration; or gene expression (e.g., in reporter systems) are also amenable to use in methods of screening for the direct effects of monoclonal antibody combinations.

As an alternative to using a secondary antibody to cross-link the primary antibodies and mimic the multivalent binding molecule or scorpion structure, other molecules that bind the Fc portion of antibodies, including soluble Fc receptors, protein A, complement components including Clq, mannose binding lectin, beads or matrices containing reactive or cross-linking agents, bifunctional chemical crosslinking agents, and adsorption to plastic, could be used to cross-link multiple monoclonal antibodies against the same or different antigens.

Example 12

Multivalent Binding Protein with Effector Function, or Scorpion, Structures

The general schematic structure of a scorpion polypeptide is H2N-binding domain 1-scorpion linker-constant sub-region-binding domain 2. scorpions may also have a hinge-like region, typically a peptide region derived from an antibody hinge, disposed N-terminal to binding domain 1. In some scorpion embodiments, binding

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2016231617 23 Sep 2016 domain 1 and binding domain 2 are each derived from an immunoglobulin binding domain, e.g., derived from a Vl and a Vh- The Vl and a Vh are typically joined by a linker. Experiments have been conducted to demonstrate that scorpion polypeptides may have binding domains that differ from an immunoglobulin binding domain, including an Ig binding domain from which the scorpion binding domain was derived, by amino acid sequence differences that result in a sequence divergence of typically less than 5%, and preferably less than 1%, relative to the source Ig binding domain.

Frequently, the sequence differences result in single amino acid changes, such as substitutions. A preferred location for such amino acid changes is in one or more regions of a scorpion binding domain that correspond, or exhibit at least 80% and preferably 85% or 90%, sequence identity to an Ig complementarity determining region (CDR) of an I g binding domain from which the scorpion binding domain was derived. Further guidance is provided by comparing models of peptides binding the same target, such as CD20. With respect to CD20, epitope mapping has revealed that the 2H7 antibody, which binds CD20, recognizes the Ala-Asn-Pro-Ser (ANPS) motif of CD20 and it is expected that CD20-binding scorpions will also recognize this motif. Amino acid sequence changes that result in the ANPS motif being deeply embedded in a pocket formed of scorpion binding domain regions corresponding to Ig CDRs are expected to be functional binders of CD20. Modeling studies have also revealed that scorpion regions corresponding to CDR3 (Vl), CDR1 -3 (Vh) contact CD20 and changes that maintain or facilitate these contacts are expected to yield scorpions that bind CD20.

In addition to facilitating interaction of a scorpion with its target, changes to the sequences of scorpion binding domains (relative to cognate Ig binding domain sequences) that promote interaction between scorpion binding domain regions that correspond to Ig Vl and Vh domains are contemplated. For example, in a CD20binding scorpion region corresponding to Vl, the sequence SYIV may be changed by substituting an amino acid for Val (V33), such as His, resulting in the sequence SYIH. This change is expected to improve interaction between scorpion regions corresponding to Vl and Vh domains. Further, it is expected that the addition of a residue at the N-terminus of a scorpion region corresponding to Vh-CDR3 will alter the orientation of that scorpion region, likely affecting its binding characteristics,

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166 because the N-terminal Ser of Vh-CDR3 makes contact with CD20. Routine assays will reveal those orientations that produce desirable changes in binding characteristics. It is also contemplated that mutations in scorpion regions corresponding to Vh-CDR2 and/or Vh-CDR3 will create potential new contacts with a target, such as CD20. For example, based on modeling studies, it is expected that substitutions of either Y105 and W106 (found in the sequence NSYW) in a region corresponding to Vh-CDR3 will alter the binding characteristics of a scorpion in a manner amenable to routine assay for identifying scorpions with modified binding characteristics. By way of additional example, it is expected that an alteration in the sequence of a scorpion binding domain corresponding to an Ig VL-CDR3, such as the Trp (W) in the sequence CQQW, will affect binding. Typically, alterations in a scorpion region corresponding to an Ig CDR will be screened for those scorpions exhibiting an increase in affinity for the target.

Based on the model structure of the humanized CD20 scFv binding domain

20-4, on the published information relating to the CD20 extracellular loop structure (Du, et ah, J Biol. Chem, 282(20):15073-80 (2007)), and on the CD20 binding epitope recognized by the mouse 2H7 antibody (which was the source of CDRs for the humanized 20-4 scFv binding domain), mutations were engineered in the CDR regions of the 2Lm20-4x2Lm20-4 scorpion with the aim of improving the affinity of its binding to CD20. First, the mutations were design to influence the 20-4 CDR conformation and to promote more efficient binding to the CD20 extracellular loop. Second, the introduced changes were designed to provide new intermolecular interactions between the 2Lm20-4x2Lm20-4 scorpion and its target. These mutations include: VL CDR1 V33H i.e., a substitution of His for Val at position 33 of CDR1 in the VL region), VL CDR3 W90Y, VH CDR2 D57E, VH CDR3 insertion of V after residue S99, VH CDR3 Y101K, VH CDR3 N103G, VH CDR3 N104G, and VH CDR3 Y105D. Due to expected synergistic effects of combining some of theses mutations, 11 mutants were designed, combining different mutations as shown in Table 9 (residues introduced by mutation are bolded and underscored).

Table 9

V_LCDR1

V_l CDR3

V_HCDR2

V_H CDR3

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RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYYSNYWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SVYYGGYWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSDWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYYSGGDWYFDL
RASSSVSYIV	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYKSNSYWYFDL
RASSSVSYIV	QQWSFNPPT	AIYPGNGETSYNQKFKG	SYYSNSYWYFDL
RASSSVSYIV	QQYSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSYWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGDTSYNQKFKG	SYKSNSDWYFDL
RASSSVSYIH	QQWSFNPPT	AIYPGNGETSYNQKFKG	SYYSNSDWYFDL
RASSSVSYIH	QQYSFNPPT	AIYPGNGDTSYNQKFKG	SYYSNSDWYFDL
RASSSVSYIH	QQYSFNPPT	AIYPGNGETSYNQKFKG	SYKSGGDWYFDL

Mutations were introduced into binding domains of the CD20xCD20 scorpion (2Lm20-4x2Lm20-4) by PCR mutagenesis using primers encoding the altered sequence region. After sequence confirmation, DNA fragments encoding the 2Lm205 4 scFv fragments with corresponding mutations were cloned into a conventional expression vector containing a coding region for the constant sub-region of a scorpion, resulting in a polynucleotide containing the complete DNA sequence of new versions of the 2Lm20-4x2Lm20-4 scorpion. The variants of the 2Lm20-4x2Lm20-4 scorpion with CDR mutations were produced by expression in a transient COS cell system and purified through Protein A and size-exclusion (SEC) chromatography.

The binding properties of 2Lm20-4x2Lm20-4 scorpion variants were evaluated by FACS analysis using primary B-cells and the WIL2-S B-lymphoma cell line.

Other mutants have also been generated using a similar approach to optimize CD20 binding domains. The CD20 SMIP designated TRU015 served as a substrate for generating mutants and, unless noted to the contrary, all domains were human domains. The following mutants were found to contain useful and functional CD20

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168 binding domains. The 018008 molecule contained a substitution of Q (single-letter amino acid code) for S at position 27 of CDR1 in VL, a substitution of S for T at position 28 in CDR1 of VH and a substitution of L for V at position 102 in CDR3 of VH. The following partial scorpion linker sequences, corresponding to the CCCP sequence in an IgG 1 hinge, were separately combined with the mutated VL and VH: CSCS, SCCS and SCCP, consistent with the modular design of scorpions. The 018009 molecule contained a substitution of Q for S at position 27 of CDR1 of VL, a substitution of S for T at position 28 of CDR1 of VH and substitutions of S for V at position 96, L for V at position 102 and deletion of the V at position 95, all in CDR3 of VH. The same scorpion linkers sub-sequences described above as being found in the scorpion linkers used in 018008 were used in 018009. The 018010 molecule contained substitutions of a Q for S at position 27, an I for M at position 33 and a V for H at position 34, all in CDR1 of VL, along with an S for T substitution at position 28 of CDR1 of VH and an L for V substitution at position 102 in CDR3 of VH.

Scorpion linkers defined by the CSCS and SCCS sub-sequences were used with

018010. 018011 contained the same mutations in CDR1 of VL and in CDR1 of VH as described for 018010, along with deletion of V at position 95, substitution of S for V at position 96 and substitution of L for V at postion 102, all in CDR3 of VH. Scorpion linkers defined by the CSCS, SCCS and SCCP sub-sequences were used in

018011 molecules. The 018014 VL was an unmutated mouse VL, with a human VH containing the S for T change at 28 in CDR1 and the L for V change at 102 in CDR3. 018015 also contained an unmutated mouse VL along with a human VH containing an S for T change at 28 of CDR1 and, in CDR3, a deletion of V at 95, substitution of S for V at 96, and substitution of L for V at 102, The 2Lm5 molecule had a Q for S at

27in CDR1 of VL, an F for Y at 27 and an S for T at 30, both in CDR1 of VH, as well as deletion of the V at 95, S for V at 96 and L for V at 102, all in CDR3 of VH. Scorpion linkers defined by the CSCS, SCCS and SCCP were separately used in each of 018014 and 018015. 2Lm5-l was the same as 2Lm5 except 2Lm5-l had no mutations in CDR1 of VH, and only a scorpion linker defined by the CSSS sub30 sequence was used. 2Lm6-l had the mutations of 2Lm5 and a substitution of T for S at 92 and S for F at 93 in CDR3 of VL, and only the scorpion linker defined by the CSSS sub-sequence was used. The only mutations in 2Lml6 were the mutations in CDR3 of VH listed above for 2Lm5-l. Scorpion linkers defined by the sub-sequences CSCS, SCCS, and SCCP were separately used in 2Lml6. 2Lml6-l substituted Q for

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S at 27 in CDR1 of VL and substituted T for S at 92, and S for F at 93, both in CDR3 of VL, and, in CDR3 of VH, deleted V at 95, substituted S for V at 96 and substituted L for V at 102; only the scorpion linker defined by the CSSS sub-sequence was used. 2Lml9-3 substituted Q for S at 27,1 for M at 33, and V for H at 34, all in CDR1 of

VL, along with the mutations in CDR3 of VH listed for 2Lml6-l. Scorpion linkers defined by the sub-sequences CSCS, SCCS, and SCCP were separately used in 2Lml9-3. The 2Lm20-4 molecule contained an I for M at 33 and a V for H at 34, both in CDR1 of VL, along with the mutations in CDR3 of VH listed for 2Lml6-l. For 2Lm5-l, 2Lm6-l, 2Lml6,2Lml6-l, 2Lml9-3, and 2Lm20-4, there also was an S for L substitution at position 11 in the framework region of VH. Scorpion linkers defined by the CSCS, SCCS and SCCP sub-sequences were separately used in 2Lm20-4. Finally, the substitution of S for P at position 331 was present in the following mutants: 018008 with the scorpion linker defined by CSCS, 018009 with each of scorpion linkers defined by CSCS and SCCP, 018010 with the scorpion linker defined by CSCS, 018011 with the scorpion linker defined by SCCP, 018014 with the scorpion linker defined by CSCS, 018015 with the scorpion linker defined by CSCS, 2Lml6 with scorpion linkers defined by any of CSCS, SCCS, and SCCP, 2Lml9-3 with a scorpion linker defined by CSCS or SCCP, and 2Lm20-4 with a scorpion linker defined by CSCS or SCCP.

In addition, changes in the length of a linker joining two regions of a binding domain, such as regions of a scorpion binding domain that correspond to an Ig Vl and Vh, are contemplated. For example, removal of a C-terminal Asp in interdomain linkers where it is found is expected to affect the binding characteristics of a scorpion, as is a substitution of Gly for Asp.

Also contemplated are scorpions that have a scorpion linker (interposed Cterminal to the constant sub-region and N-terminal to binding domain 2) that is lengthened relative to a hinge region of an Ig, with amino acid residues being added C-terminal to any cysteine in the scorpion that corresponds to an Ig hinge cysteine, with the scorpion cysteine being capable of forming an interchain disulfide bond.

Scorpions containing these features have been constructed and are characterized below.

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Efforts were undertaken to improve the expression, stability and therapeutic potency of scorpions through the optimization of the scorpion linker covalently joining the constant sub-region and the C-terminally disposed binding domain 2. The prototypical scorpion used for optimization studies contained an anti-CD20 scFV (binding domain 1) fused N-terminal to the constant sub-region derived from IgGl Ch2 and Ch3, with a second anti-CD20 scFv fused C-terminal to that constant subregion. This scorpion, like immunoglobulin molecules, is expected to associate through the constant region (or sub-region) to form a homodimeric complex with peptide chains linked by disulfide bonds. To obtain high level of expression of a stable, tetravalent molecule with high affinity for its CD20 target, the scorpion linker between the constant sub-region and the second binding domain must accommodate the following considerations. First, steric hindrance between the homologous binding domains carried by the two scFv fragments (one scFv fragment on each of two scorpion monomers) should be minimized to facilitate maintenance of the native conformations of each binding domain. Second, the configurations and orientations of binding domains should allow productive association of domains and high-affinity binding of each binding domain to its target. Third, the scorpion linker itself should be relatively protease-resistant and non-immunogenic.

In the exemplary CD20xCD20 scorpion construct SOI 29, the C-terminus of

Ch3 and the second anti-CD20 scFV domain were linked by the 2H7 scorpion linker, a peptide derived from, and corresponding to, a fragment of a natural human hinge sequence of IgGl. The 2H7 scorpion linker served as a base for design efforts using computer-assisted modeling that were aimed at improving the expression of scorpions and improving the binding characteristics of the expressed molecules,

To analyze the 2H7 scorpion linker, the 3-dimensional structure of a dimeric form of the human IgGl hinge was modeled using Insight II software. The crystal structure of anti-CD20 scFV in the Vh-Vl orientation was chosen as a reference structure for the 20-4 binding domains (RCSB Protein Data Bank entry code: 1A14). In intact IgGl, the hinge connects the C-terminus of theCm domain to the N-terminus of the Ch2 domain, with the configuration of each domain being such that hinge cysteine residues can pair to form a homodimer. In the exemplary scorpion molecule, the hinge-derived 2H7 linker connected the C-terminal end of the scorpion domain

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171 derived from the IgG 1 Ch3 domain to the N-terminal end of that portion of scorpion binding domain 2 derived from an IgGl Vh2 domain. Using a 3-D modeled structure of the Vh-Vl scFV, expectations of the optimal distance between the C-terminal ends of the 2H7 linkers was influenced by three considerations. First, hinge stability must be maintained, and stability is aided by dimerization, e.g., homodimerization, which means that the hinge cysteines must be able to pair in the presence of the two folded binding domains. Second, two binding domains, e.g., scFVs, must accommodate the 2H7 linker C-termini without steric interference in order to allow for proper protein folding. Third, the CDRs of each binding domain should be able to face the same direction, as in a native antibody, because each binding domain of the prototypical scorpion can bind adjacent receptors (CD20) on the same cell surface. Given these considerations, the distance between the two N-terminal ends of scFvs is expected to be approximately 28A. The distance between the C-terminal ends of the theoretically designed 2H7 linkers in dimeric scorpion forms is expected to be about 16A. To accommodate the distances expected to be needed for optimizing the performance of a scorpion, the C-terminus of the 2H7 linker was extended by at least 3 amino acids. Such an extension is expected to allow for the formation of disulfide bonds between 2H7 linker cysteine residues, to allow for proper folding of the C-terminal binding domain 2, and to facilitate a correct orientation of the CDRs. In addition, in intact

IgGl, due to the presence of the Chi and Vli domains between the hinge and binding domains, the distance between the binding domains carried by the two chains is further increased and is expected to further favor the cross-linking of adjacent receptors on the same cell surface. In view of the considerations described above, a set of linkers with different lengths was designed (Table 10). To minimize immunogenicity, natural residues present at the N-terminal end of the Ch2 domain (Ala-Pro-Glu-Leu or APEL) were used to lengthen the 2H7 scorpion linker by sequence addition to the C-terminus of the scorpion linker. The longer constructs contained one or multiple (Gly4Ser) linker units known to be protease-resistant and flexible.

The CD20xCD20 scorpion constructs containing extended scorpion linkers between the Ch3 domain of the constant sub-region and the C-terminal scFv binding domain were constructed using PCR mutagenesis and subcloned into a conventional mammalian expression vector. The effect of linker length on CD20xCD20 scorpion

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2016231617 23 Sep 2016 expression could be analyzed be comparing the yield of secreted protein in transient expression experiments using COS or HEK293 cells, or by analysis of protein synthesis and accumulation in the cells by Western blot analyses or pulse-chase studies with [35]S-labeled methionine/cysteine.

Table 10

Construct Number	Scorpion linker core (2H7) sequence	Extension sequence	Extended scorpion linker sequence
1	GCPPCPNS	APEL	GCPPCPNSAPEL
2	GCPPCPNS	APELGGGGS	GCPPCPNS APELGGGGS
3	GCPPCPNS	APELGGGGSGGGGS	GCPPCPNS APELGGGGSGGGGS
4	GCPPCPNS	APELGGGGSGGGGSGGGGS	GCPPCPNS APELGGGGSGGGGSGGGGS

Glycosylated scorpions are also contemplated and, in this context, it is contemplated that host cells expressing a scorpion may be cultured in the presence of a carbohydrate modifier, which is defined herein as a small organic compound, preferably of molecular weight less than 1000 daltons, that inhibits the activity of an enzyme involved in the addition, removal, or modification of sugars that are part of a carbohydrate attached to a polypeptide, such as occurs during N-linked carbohydrate maturation of a protein. Glycosylation is a complex process that takes place in the endoplasmic reticulum (“core glycosylation”) and in the Golgi bodies (“terminal glycosylation”). A variety of glycosidase and/or mannosidase inhibitors provide one or more of desired effects of increasing ADCC activity, increasing Fc receptor binding, and altering glycosylation pattern. Exemplary inhibitors include, but are not

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173 limited to, castanospermine and kifunensine. The effects of expressing scorpions in the presence of at least one such inhibitor are disclosed in the following example.

Example 13

Scorpion protein expression levels and characterization

Scorpion protein expression levels were determined and the expressed proteins were characterized to demonstrate that the protein design led to products having practical benefits. A monospecific CD20xCD20 scorpion and a bispecific CD20xCD37 scorpion were expressed in CHO DG44 cells in culture using conventional techniques.

Basal level, stable expression of the CD20xCD20 scorpion SO 129 (21m204x21m20-4) in CHO DG44 cells cultured in the presence of various feed supplements was observed as shown in Fig. 34. All culture media contained 50 nM methotrexate, a concentration that maintained copy number of the scorpion-encoding polynucleotide. The polynucleotide contained a coding region for the scorpion protein that was not codon-optimized for expression in CHO DG44 cells. The polynucleotide was introduced into cells using the pDl 8 vector Apparent from Fig. 34, expression levels of about 7-46 μg/ml were obtained.

Expression levels following amplification of the polynucleotide encoding a bispecific CD20xCD37 scorpion were also determined. The pD18 vector was used to clone the CD20xCD37 scorpion coding region and the plasmid was introduced into CHO DG44 cells. Amplification of the encoding polynucleotide was achieved using the t/A/r-methotrexate technique known in the art, where increasing concentrations of MTX are used to select for increased copy number of the Dihydrofolate Reductase gene (dhfr), which leads to co-amplification of the tightly linked polynucleotide of interest. Fig. 35 shows that stable expression levels of about 22-118 pg/ml of the bispecific CD20xCD37 scorpion were typically observed. Variability in yield was seen under different conditions, including methotrexate concentration used for amplification, but these variables are amenable to optimization by those of skill in the art. A variety of other scorpion molecules described herein were also subjected to expression analyses in CHO and/or COS cells, with the results provided in Table 11, below. These results demonstrate that significant yields of scorpion proteins can be

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174 obtained using conventional techniques and routine optimization of the amplification technique.

Expressed proteins were also characterized by SDS-PAGE analysis to assess the degrees of homogeneity and integrity of the expressed proteins and to confirm molecular weight of monomeric peptides. The denaturing polyacrylamide gels (420% Tris Glycine) were run under reducing and non-reducing conditions. The results presented in Fig. 36 reveal single protein bands for each of a 2Lm20-4 SCC SMIP and SI 000 (CD20(21m20-4)xCD20(21m20-4) monospecific scorpion- SO 126) of the expected monomeric molecular weights under reducing conditions. These data establish that SMIPs and scorpions are amenable to purification in an intact form. Under non-reducing conditions, a trace amount of a peptide consistent with the expected size of a monomeric SMIP was seen, with the vast majority of the protein appearing in a single well-defined band consistent with a dimeric structure. Under these non-reducing conditions, the monospecific scorpion protein showed a single well-defined band of a molecular weight consistent with a dimeric structure. The dimeric structures for both the SMIP and the scorpion are consistent with their monomeric structures, each of which contains a hinge-like scorpion linker containing at least one Cysteine capable of participating in disulfide bond formation.

The effect of scorpion linkers on the expression and integrity of scorpions was also assessed, and results are shown in Table 12, This table lists scorpion linker variants of the monospecific CD20xCD20 (2Lm20-4x2Lm20-4) SOI 29 scorpion and the CD20xCD28 S0033 scorpion (2H7sccpIgGl-H7-2el2), their integrity as single chain molecules, and their transient expression levels in COS cells relative to the parent scorpion SO 129 or S0033, as appropriate, with an H7 linker (set as 100%).

Table 13 provides data resulting from an evaluation of scorpion linker variants incorporated into the CD20xCD20 scorpion, along with analogous data for the CD20xCD28 scorpion. Table 13 provides data resulting from an evaluation of S0129 variants containing scorpion linkers that are not hinge-like linkers containing at least one Cysteine capable of disulfide bond formation; rather, the scorpion linkers in these molecules are derived from Type II C-lectin stalks. Apparent from the data presented in Table 13 is that hinge-like scorpion linkers may be associated with scorpions expressed at higher or lower levels than an unmodified parent scorpion linker in

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2016231617 23 Sep 2016 transient expression assays. Further, some of the linker variants exhibit greater resistance to proteolytic cleavage than the unmodified parent linker, a concern for all or almost all expressed proteins. The data of Table 13 show that non-hinge-like linkers such as linkers derived from the stalk region of Type II C-Iectins are found in scorpions that exhibit binding characteristics that vary slightly from scorpions containing hinge-like scorpion linkers. Additionally, the scorpion containing a nonhinge-like scorpion linker exhibits effector function (ADCC) that either equals or exceeds the ADCC associated with scorpions having hinge-like scorpion linkers.

Table 11

MwjlgSBSm jiSKSl ITBCT 1-π J aWKAH

Wait» | Stsjwnc» ; Θη*®γ vsrhtrtSs-t» seq' i based . t-M? jWSLSLBPC

Nw. ;aS&igisps . W...iO

MS) ·α«>5 W'^iWiT

TT; j com-sh?

iijs ! “gf ‘WHBSt&SuB’oK iRiiuiWse«pf¥wr' Tw®'

MS'S iiXSLSS.

_hSS gaS&SEsPs”

Ί·15<ί iGKSS.Sf.^ri5 ' 'sw? 1 &k$l%5k;......

-*8 tolW“

EYi.WSOP&ltSNS'

SSRWBS» —Hie

SV*'.-.·.· .V.<\WA.W. .,Vt\SA\S'«\’iS'i'iSV.»V»«'t'»·»··'

JOWHSasWiAiliS -T.....tfjjg

SVSERPFPPHS............ ‘['wii:

......................

SxfiflSSfliiir· 1« COS <ΡΛ·«jutes; at O iOStis^s flask;aflaSva S> frSISiMfl? <X·) ‘ΏΜΜφβ ibswseat ’Κ^-ίΑί&ϊΛ'ί-ι sfliii;· «μ». issnd rsM.'risjtif fc*nd{«*. *·-'!· Sff&iiflatipd

Table 12

Linker Name	S0129 (2Lm20-4 x 2Lm20-4) linker variants - aa seq	Changes in CH3?1	Linker seq. based on	20x20 Expression	20x20 Cleavage? 3
H7	GCPPCPNS	N	H7	100	-
H8	GSPPSPNS	N	H7	107	+
H9	GSPPSPNS	Y	H7	142	-
H10	EPKSTDKTHTCPPCPNS	N	lgG1 hinqe	98	-
H11	EPKSTDKTHTSPPSPNS	N	lgG1 hinge	126	+
H16	LSVKADFLTPSIGNS	N	CD80	174	+
H17	LSVKADFLTPSISCPPCPNS	N	CD80 + K7	113	+
H18	LSVLANFSQPEIGNS	N	CD86	165	++
H19	LSVLANFSQPEISCPPCPNS	N	CD86 + H7	161	4

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H20	LKIQERVSKPKISNS	N	CD2	115	+++
H21	LKIQERVSKPKISCPPCPNS	N	CD2 + H7	90	+++
H22	LNVSERPFPPHIQNS	N	CD22	149	++
H23	LDVSERPFPPHIQSCPPCPNS	N	CD22 + H7	121	++
H24	REQLAEVTLSLKANS	N	CD80	145	++
H25	REQLAEVTLSLKACPPCPNS	N	CD80 + H7	98	+
H26	RIHQMNSELSVLANS	N	CD86	170	++
H27	RIHQMNSELSVLACPPCPNS	N	CD86 + H7	154	++
H28	DTKGKNVLEKIFSNS	N	CD2	153	+
H30	LPPETQESQEVTLNS	N	CD22	78	+
H32	RIHLNVSERPFPPNS	N	CD22	184	++
H33	RIHLNVSERPFPPCPPCPNS	N	CD22 + H7	74
H36	GCPPCPGGGGSNS	N	H7	110
H40	GCPPCPANS	Y	H7	110	+
H41	GCPPCPANS	Y	H7	102	-
H42	GCPPCPNS	Y	H7	99	-
H44	GGGASCPPCPGNS	Y	H7	108	+
H45	GGGASCPPCAGNS	Y	H7	107	-
H46	GGGASCPPCANS	Y	H7	98	-
H47	LSVKADFLTPSIGNS	Y	CD80	141	-
H48	ADFLTPSIGNS	N	CD80	137	-
H50	LSVLANFSQPEIGNS	Y	CD86	21	-
H51	LSVLANFSQPEIGNS	Y	CD86	110	-
H52	SQPEIVPISNS	Y	CD86	95	-
H53	SQPEIVPISCPPCPNS	Y	CD86 + H7	95	-
H54	SVLANFSQPEISCPPCPNS	Y	CD86 + H7	72	+/-
H55	RIHQMNSELSVLANS	Y	CD86	118	+
H56	QMNSELSVLANS	Y	CD86	130	-
H57	VSERPFPPNS	Y	CD22	118	-
H58	KPFFTCGSADTCPNS	Y	CD72	103	-
H59	KPFFTCGSADTCPNS	Y	CD72	94	-
H60	QYNCPGQYTFSMNS	Y	CD69	>100a	-
H61	EPAFTPGPNIELQKDSDCNS	Y	CD94	>100	-
H62	QRHNNSSLNTRTQKARHCNS	Y	NKG2A	>100	-
H63	NSLFNQEVQIPLTESYCNS	Y	NKG2D	>100	-
'Additional changes to the end of CH3 such as 1-9 aa deletion and/or codon optimization
“Transient expression in COS (6W plates), relative to S0129-H7 parent (%)
JCleavage product(s) observed by SDS-PAGE/silver stain:

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	-=none, +=faint band, ++=major band(s), +++>50% cleaved
°H60-H63 variants compared by estimation of recovery of protein purified from COS spent media.

Table 13

Protein s	Descriptio n	Productio n Yield (ug protein purified/m 1 sup)	% POI (M.wt in Kd by MALS )	Improve ment over S0129wt POI	Binding to Ramos	ADCC assay	Sequence of scorpion linker
S0129w t	H7 linker	1.6	67 (167)	-	-	-	GCPPC
SO 129CD69	CD69 stalk	2.9	66 (167)	1.8	Weaker than S0129wt	♦Slightly better than S0l29wt POI	QYNCPGQYTF SM
S0129- CD72	CD72 truncated stalk	2.0	69 (165)	1.2	Similar to S0129wt	♦Slightly better than S0129wt POI	PFFTCGSADTC
SO129CD94	CD94 stalk	2.9	67 (171)	1.8	Similar to S0129wt	* Slightly better than S0129wt POI	EPAFTPGPNIE LQKDSDC
SO 129NKG2 A	NKG2A stalk	2.5	93 (170)	2.2	Slightly better than S0129wt	Similar to S0129wt POI	QRHNNSSLNT RTQKARHC
SO129NKG2 D	NKG2D stalk	1.9	70 (166)	1.2	Similar to S0129wt	*Slightly better than S0129wt POI	NSLFNQEVQIP LTESYC

As noted in the preceding example, production by expression of scorpions in cultures containing a carbohydrate modifier is contemplated. In exemplary embodiments, castanospermine (MW 189.21) is added to the culture medium to a final concentration of about 200 μΜ (corresponding to about 37.8 pg/mL), or concentration ranges greater than about 10,20, 30,40, 50, 60, 70, 80,90, 100, 110, 120,130, 140, or 150 μΜ, and up to about 300, 275, 250, 225,200, 175, 150, 125, 100, 75, 60, or 50 pg/mL. For example, ranges of 10-50, or 50-200, or 50-300, or 100-300, or 150-250 μΜ are contemplated. In other exemplary embodiments, DMJ, for example DMJ-HC1 (MW 199.6) is added to the culture medium to a final concentration of about 200 μΜ (corresponding to about 32.6 pg DMJ/mL), or

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178 concentration ranges greater than about 10,20,30,40,50,60, 70, 80,90, 100, 110, 120,130, 140, or 150 μΜ, and up to about 300,275, 250, 225,200, 175, 150, 125,

100,75, 60, or 50 pg/mL. For example, ranges of 10-50, or 50-200, or 50-300, or 100-300, or 150-250 μΜ are contemplated. In other exemplary embodiments, kifunensine (MW 232.2) is added to the culture medium to a final concentration of about 10 μΜ (corresponding to about 2.3 μg/mL), or concentration ranges greater than about 0.5,1,2, 3,4, 5, 6,7, 8, 9 or 10 μΜ, and up to about 50,45,40,35, 30,25, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 μΜ. For example, ranges of 1-10, or 1-25, or 150, or 5-10, or 5-25, or 5-15 μΜ are contemplated.

In one experiment, a monospecific CD20xCD20 scorpion (SO 129) was expressed in cells cultured in 200 μΜ castanospermine (SO 129 CS200) or 10 μΜ (excess) kifunensine (S0129 KF 10) and the binding, or staining, of WIL2S cells by the expressed scorpion was measured, as shown in Fig. 42. In comparative binding studies, moreover, a glycosylated SO 129 scorpion bound CD 16 (FCyRIII) approximately three times better than the unglycosylated SO 129 scorpion.

In another study, the ADCC-mediated killing of BJAB B-cells by humanized CD20xCD20 scorpion (S0129) was explored. The results shown in Fig. 43 establish that the scorpion, when expressed in cells being cultured in the presence of either castanospermine or kifunensine, led to significantly more potent ADCC-mediatd

BJAB B-cell death for a given concentration of scorpion exposure.

Example 14

Scorpion binding

a. Domain spacing

Bispecific scorpions are capable of binding at least two targets simultaneously, 25 utilizing the pairs of binding domains at the N- and C-terminus of the molecule. In so doing, for cell-surface targets, the composition can cross-link or cause the physical co-approximation of the targets. It will be appreciated by those skilled in the art that many receptor systems are activated upon such cross-linking, resulting in signal induction causing changes in cellular phenotype. The design of the compositions disclosed herein was intended, in part, to maximize such signaling and to control the resultant phenotype.

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Approximate dimensions of domains of the scorpion compositions, as well as expectations of interdomain flexibility in terms of ranges of interdomain angles, are known and were considered in designing the scorpion architecture. For scorpions using scFv binding domains for binding domains 1 and 2 (BD1 and BD2), an IgGl N5 terminal hinge (Hl), and the H7 PIMS linker described herein, the binding domain at the N-terminus and the binding domain at the C-terminus may be maximally about 150-180A apart and minimally about 20-30A apart. Binding domains at the Nterminus may be maximally about 90-100A apart and minimally about 10-20A apart (Deisenhofer, et al., 1976, Hoppe-Seyler’s Z. Physiol. Chem. Bd. 357, S. 435-445;

Gregory, et al., 1987, Mol. Immunol. 24(8):821-9.; Poljak, et al., 1973, Proc. Natl. Acad. Sci., 1973, 70: 3305-3310; Bongini, et al., 2004, Proc. Natl. Acad. Sci. 101: 6466-6471; Kienberger, et al., 2004, EMBO Reports, 5: 579 - 583, each incorporated herein by reference). The choice of these dimensions was done in part to allow for receptor-receptor distances of less than about 50A in receptor complexes bound by the scorpion as distances less than this may be optimal for maximal signaling of certain receptor oligomers (Paar, et al., 2002, J. Immunol., 169: 856-864, incorporated herein by reference) while allowing for the incorporation of Fc structures required for effector function.

The binding domains at the N- and C-terminus of scorpions were designed to be flexible structures to facilitate target binding and to allow for a range of geometries of the bound targets. It will also be appreciated by those skilled in the art that flexibility between the N- or C-terminal binding domains (BD1 and BD2, respectively) and between the binding domains and the Fc domain of the molecule, as well as the maximal and minimal distances between receptors bound by BD1 and/or

BD2, can be modified, for example by choice of N-terminal hinge domain (Hl) and, by structural analogy, the more C-terminally located scorpion linker domain (H2).

For example hinge domains from IgGl, IgG2, lgG3, IgG4, IgE, IgA2, synthetic hinges and the hinge-like Ch2 domain of IgM show different degrees of flexibility, as well as different lengths. Those skilled in the art will understand that the optimal choice of Hl and scorpion linker (H2) will depend upon the receptor system(s) the scorpion is designed to interact with as well as the desired signaling phenotype induced by scorpion binding.

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In some embodiments, scorpions have a scorpion linker (H2) that is a hingelike linker corresponding to an Ig hinge, such as an IgGl hinge. These embodiments include scorpions having an amino acid sequence of the scorpion hinge that is an Nterminally extended sequence relative to, e.g., the H7 sequence or the wild-type IgGl hinge sequence. Exemplary scorpion linkers of this type would have the sequence of the H7 hinge N-termanally extended by H2N-APEL(x)_y-CO2H, where x is a unit of the Gly4Ser linker and y is a number between 0 and 3. Exemplifying the influence of the scorpion linker on scorpion stability is a study done using two scorpions, a bispecific CD20xCD28 scorpion and a monospecific CD20xCD20 scorpion. For each of these two scorpion designs, a variety of scorpion linkers were inserted. In particular, scorpion linkers Hl6 and Hl7, which primarily differ in that Hl7 has the sequence of Hl 6 with the sequence of H7 appended at the C-terminus, and scorpion linkers Hl8 and 19, in which analogously the sequence of H7 is appended at the Cterminus of Hl 8 in generating Hl 9. For each of the two scorpion backbones (20x28 and 20x20), each of the four above-described scorpion linkers were inserted at the appropriate location. Transient expression of these constructs was obtained in COS cells and the scorpion proteins found in the culture supernatants were purified on protein A/G-coated wells (Pierce SEIZE IP kit). Purified proteins were fractionated on SDS-PAGE gels and visualized by silver stain. Inspection of Fig. 44 reveals that the additional H7 sequence in the scorpion linker adds to the stability of each type of scorpion linker and each type of scorpion protein. In other words, appending H7 to the C-terminus of either H16 or H18 added to the stability of the scorpion molecule, and this observation held regardless of whether the scorpion was CD20xCD28 or CD20xCD20. In terms of target binding, the scorpion proteins having the

CD20xCD20 architecture exhibited similar binding properties to the parent monospecific humanized CD20xCD20 scorpion SO 129, as shown in Fig. 45.

Beyond the preceding embodiments, however, it may be desirable to prevent bound receptors from approaching within about 50A of each other to intentionally create submaximal signals (Paar, et al., J. Immunol., 169: 856-864). In such a case, choices of Hl and Scorpion linker (H2) that are shorter and less flexible than those described above would be expected to be appropriate.

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The same spacing considerations apply to scorpion linkers that are not hingelike. These scorpion linkers are exemplified by the class of peptides having the amino acid sequence of a stalk region of a C-lectin. Exemplary scorpion hinges comprising a C-lectin stalk region are scorpion hinges derived from the CD72 stalk region, the

CD94 stalk region, and the NKG2A stalk region. Scorpions containing such scorpion hinges were constructed and characterized in terms of expression, susceptibility to cleavage, and amenability to purification. The data are presented in Table 14.

Table 14

Linker Name	G,S Codon optimization1	End of CH3	SOI 29 Scorpion Linker variants amino acid seq	Linker seq. based on	Expression (%S0129)!	Cleavage1	Bonch-top purification %POI
H7	N	K	GCPPCPNS	H7	100	-	70
H60	Y(17)	K	GCPPCPNS	H7	114	-	ND
H61	Y(15)	K	GCPPCPNS	H7	90	-	66
H62	N	G	QRH NN SSLNTRTQKARHCPNS	NKG2A Stalk	129	-	89
H63	Y(17)	G	QRH NN SSLNTRTQKARHCPNS	NKG2A Stalk	100	-	85
H64	Y(15)	G	QRH NN SSLNTRTQKARHCPNS	NKG2A stalk	81	-	83
H65	N	G	EPAFTPGPNIELQKDSDCRNS	CD94 stalk	133	-	66
H66	Y{17)	G	EPAFTPGPNIELQKDSDCPNS	CD94 stalk	200	-	64
H67	Y(15)	G	EPAFTPGPNIELQKDSDCPNS	CD94 stalk	129		65
H68	N	G	RTRYLQVSQQLQQTN RVLEVTNSSLRQQLR LKITQLGQSAEDLQGSRRELAQSQEALQVEQ RAHQAAEGQLQACQADRQKTKETLQSEEQ QRRALEQKLSNMENRLKPFFTCGSADTC	CD72 full stalk	110	-	75

‘Codon optimization of Gly^Ser linker, with (17) or without (15) restriction site ’Estimate of expression in COS based on recovery of protein in benchtop purification ’Cleavage produces) observed by SDS-PAGE/Coomassie Blue stain of purified protein

b._ Binding of N- and C-terminal binding domains

Both N- and C-terminal domains participate in target cell binding

The target cell binding abilities of a CD20 SMIP (TRU015), a CD37 SMIP (SMIP016), a combination of CD20 and CD37 SMIPS (TRU015+SMIP016), and the

CD20xCD37 bispecific scorpion (015x016), were assessed by measuring the capacity of each of these molecules to block the binding of an antibody specifically competing for binding to the relevant target, either CD37 or CD20. The competing antibodies were FITC-labeled monoclonal anti-CD37 antibody or PE-labeled monoclonal antiCD20 antibody, as appropriate. Ramos B-cells provided the targets.

Ramos B-cells at 1.2xl0⁷/ml in PBS with 5% mouse sera (#100-113, Gemini

Bio-Products, West Sacramento, CA) (staining media) were added to 96-well Vbottom plates (25 μΐ/well). The various SMIPs and scorpions were diluted to 75

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182 qg/ml in staining media and 4-fold dilutions were performed to theconcentrations indicated in Fig. 38. The diluted compounds were added to the plated cells in addition to media alone for control wells. The cells were incubated for 10 minutes with the compounds and then FITC anti-CD37 antibody (#186-040, Ancell, Bayport,

MN) at 5 pg/ml and PE anti-CD20 antibody (#555623, BD Pharmingen, San Jose,

CA) at 3 pg/ml (neet) were added together to the wells in 25 μΐ staining media. The cells were incubated on ice in the dark for 45 minutes and then washed 2.5 times with PBS. Cells were fixed with 1% paraformaldehyde (#19943 1 LT, USB Corp, Cleveland, OH) and then run on a FACs Calibur (BD Biosciences, San Jose, CA).

The data were analyzed with Cell Quest software (BD Biosciences, San Jose, CA).

The results shown in Fig. 38 establish that all SMIPs, SMIP combinations and scorpions containing a CD20 binding site successfully competed with PE-labeled antiCD 20 antibody for binding to Ramos B-cells (upper panel); all SMIPs, SMIP combinations and scorpions containing a CD37 binding site successfully competed with FITC-Iabeled anti-CD 37 antibody for binding to Ramos B-cells (lower panel). The bispecific CD20xCD37 scorpion, therefore, was shown to have operable N- and C-terminal binding sites for targets on B-cells.

c. Cell-surface persistence

An investigation of the cell-surface persistence of bound SMIPs and scorpions (monospecific and bispecific) on the surface of B-cells revealed that scorpions exhibited greater cell-surface persistence than SMIPs. Ramos B-cells at 6xl0⁶ /ml (3xlO⁵/well) in staining media (2.5% goat sera, 2.5% mouse sera in PBS) were added to 96-well V-bottom plates. Test reagents were prepared at two-fold the final concentration in staining media by making a 5-fold serial dilution of a 500 nM initial stock and then were added 1:1 to the Ramos B-cells, In addition, media controls were also plated. The cells were incubated in the dark, on ice, for 45 minutes. The plates were then washed 3.5 times with cold PBS. The secondary reagent, FITC goat antihuman IgG (#H 10501, Caltag/Invitrogen, Carlsbad,CA) was then added at a 1:100 dilution in staining media. The cells were incubated for 30 minutes in the dark, on ice. Cells were then washed 2.5 times by centrifugation with cold PBS, fixed with a 1% paraformaldehyde solution (#19943 1 LT ,USB Corp, Cleveland, OH) and then run on a FACs Calibur (BD Biosciences, San Jose, CA), The data were analyzed with

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CellQuest software (BD Biosciences, San Jose, CA). Results of the data analysis are presented in Fig. 37, which shows the binding of several SMIPs, a monospecific CD20xCD20 scorpion and a bispecific CD20xCD37 scorpion to their targets on Ramos B cells.

Two tubes of Ramos B-cells (7xl0⁵/ml) were incubated for 30 minutes on ice with each of the two compounds being investigated, i.e., a humanized CD20 (2Lm204) SMIP and a humanized CD20xCD20 (2Lm20-4x2Lm20-4) scorpion, each at 25 μg/ml in Iscoves media with 10% FBS. At the end of the incubation period, both tubes were washed 3 times by centrifugation. One tube of cells was then plated into

96-well flat-bottom plates at 2xl0⁵ cells/well in 150 μΐ of Iscoves media with one plate then going into the 37°C incubator and the other plate incubated on ice. The second tube of each set was resuspended in cold PBS with 2% mouse serum and 1% sodium azide (staining media) and plated into a 96-well V-bottom plate at 2xl0⁵ cells/well for immediate staining with the secondary antibody, i.e., FITC goat anti15 human IgG (#1410501, Caltag/Invitrogen, Carlsbad, CA). The secondary antibody was added at a 1:100 final dilution in staining media and the cells were stained on ice, in the dark, for 30 minutes. Cells were then washed 2.5 times with cold PBS, and fixed with 1% paraformaldehyde (#19943 1 LT, USB Corp, Cleveland, OH).

At the time points designated in Fig, 39, samples were harvested from the 9620 well flat-bottom plates, incubated at either 37°C or on ice, and placed into 96-well Vbottom plates (2x10^s cells/well). The cells were washed once with cold staining media, resuspended, and the secondary antibody was added at a final dilution of 1:100 in staining media. These cells were incubated on ice, in the dark, for 30 minutes. The cells were then washed 2.5 times by centrifugation in cold PBS, and subsequently fixed with 1% paraformaldehyde. The samples were run on a FACS Calibur (BD Biosciences, San Jose, CA) and the data was analyzed with CellQuest software (BD Biosciences, San Jose, CA). Results presented in Fig. 39 demonstrate that the binding of a SMIP and a scorpion to the surface of B-cells persists for at least six hours, with the monospecific hu CD20xCD20 (2Lm20-4x2Lm20-4) scorpion persisting to a greater extent than the hu CD20 (2Lm20-4) SMIP.

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Example 15

Direct cell killing by monospecific and bispecfic scorpions

Experiments were conducted to assess the capacity of monospecific and bispecific scorpion molecules to directly kill lymphoma cells, i.e., to kill these cells without involvement of ADCC or CDC. In particular, the Su-DHL-6 and DoHH2 lymphoma cell lines were separately subjected to a monospecific scorpion, i.e., a CD20xCD20 scorpion or a CD37xCD37 scorpion, or to a bispecific CD20xCD37 scorpion.

Cultures of Su-DHL-6, DoHH2, Rec-1, and WSU-NHL lymphoma cells were established using conventional techniques and some of these cultures were then individually exposed to a monospecific CD20 SMIP, a monospecific scorpion (CD20xCD20 or CD37xCD37), or a bispecific scorpion (CD20xCD37 or CD19xCD37). The exposure of cells to SMIPs or scorpions was conducted under conditions that did not result in cross-linking. The cells remained in contact with the molecules for 96 hours, after which growth was measured by detection of ATP, as would be known in the art. The cell killing attributable to the CD20 SMIP and the CD20xCD20 monospecific scorpion are apparent in Fig. 24 and Table 15. The cell killing capacity of the CD37xCD37 monospecific scorpion is apparent from Fig. 25 and Table 15, the ability of the CD20xCD37 bispecific scorpion to kill lymphoma cells is apparent from Fig. 26 and Table 15, and the capacity of the CD19xCD37 bispecific scorpion to kill lymphoma cells is evident from Fig. 27 and Table 15. Data were pooled from three independent experiments and points represent the mean ± SEM. ICso values in Table 15 were determined from the curves in Figs. 24,25, and 26, as noted in the legend to Table 15, and are defined as the concentration resulting in 50% inhibition compared to untreated cultures. The data in the figures and table demonstrate that scorpions are greater than 10-fold more potent in killing these cell lines than the free SMIP using the same binding domains.

Table 15

	Cell Line
IC50 (nM)	SU-DHL-6	DoHH2	WSU-NHL

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	Cell Line
CD20 SMIP*	>100	60	NA
CD20xCD20 scorpion*	0.3	4.0	NA
CD37 SMIP**	>100	>100	NA
CD37xCD37 scorpion**	10	1,2	NA
CD20 SMIP and CD37 SMIP***	6	2	NA
CD20xCD37 scorpion***	0.05	0.05	NA
CD 19 SMIP and CD37 SMIP****	0.16	NA	0.40
CD19xCD37 scorpion****	0.005	NA	0.04

* Data derived from Fig. 24.

** Data derived from Fig. 25.

*** Data derived from Fig. 26.

**** Data derived from Fig, 27.

Additional experiments with the humanized CD20xCD20 scorpion SO 129 were conducted in Su-DHL-4, Su-DHL-6, DoHH2, Rec-1, and WSU-NHL cells. The results are presented in Fig. 46 and Fig. 47. The data provided in these figures extends the findings discussed above in showing that scorpions have the capacity to directly kill a variety of cell lines.

The above findings were extended to other monospecific and bispecific scorpions, with each scorpion demonstrating capacity to directly kill B cells. DoHH2 B-cells were exposed in vitro to the monospecific CD20xCD20 scorpion, a

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186 monospecific CD37xCD37 scorpion, or a bispecific CD20xCD37 scorpion. The results presented in Fig. 48 demonstrate that bispecific scorpions have kill curves that are different in form from monospecific scorpions.

Culturing Su-DHL-6 cells in the presence of 70 nM CD20xCD20 scorpion 5 (SO 129), CD20xCD37 scorpion, or CD37xCD37 scorpion also led to direct B-cell killing in an in vitro environment (Fig. 49). Consistently, Su-DHL-6 cells exposed to either a bispecific CD19xCD37 scorpion or to Rituxan® led to direct cell killing, with the bispecific scorpion exhibiting lethality at lower doses, as revealed in Fig. 50.

Another demonstration of direct cell killing was provided by exposing DHL-4 cells to four independent monospecific scorpions recognizing CD20. Two versions of CD20xCD20 scorpion were designed to incorporate two 20-4 binding domains (204x20-4 and SO 129) and the second two incorporate a hybrid of the 011 and 20-4 binding domains. All four of the independently constructed and purified versions of the two CD20xCD20 scorpion designs, (20-4x20-4 and SO 129) and hybrid (011x20-4 and 011 x20-4AAsp), efficiently killed the DHL-4 cells in a direct manner. For this study, DHL-4 cells were treated in vitro with 1 pg/ml of the indicated proteins for 24 hours. Cells were then stained with Annexin V and Propidium Iodide, early and late markers of cell death, respectively, and cell populations were quantified by FACS,

The results presented in Fig. 51 establish the direct killing capacity of each of the

CD20xCD20 constructs as evidenced by increased staining shown in black bars. In addition, the results demonstrate that the hybrid 011x20-4 proteins exhibited a slight increase in direct cell killing relative to 20-4x20-4-based scorpions, despite the fact that each of these scorpions monospecifically recognized CD20. In a separate set of experiments, the dose-response of the four independent scorpion constructs was determined by FACS analysis of Annexin V- and Propidium Iodide-stained cell populations. The results, shown in Fig. 52, demonstrate dose-responsive increases in cell death resulting from treatment of the DHL-4 cells with each of the independent scorpion constructs.

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IS?

Example 16

Accessory functions mediated by scorpions (ADCC & CDC) a, Scorpion-dependent cellular cytotoxicity

Experiments were conducted to determine whether scorpions would mediate 5 the killing of BJAB B lymphoma cells. BJAB B lymphoma cells were observed to be killed with CD20 and/or CD37 scorpions.

Initially, lxl0⁷/ml BJAB B-cells were labeled with 500 qCi/ml ⁵¹Cr sodium chromate (#CJS1, Amersham Biosciences, Piscataway, NJ) for 2 hours at 37°C in Iscoves media with 10% FBS. The ⁵¹Cr-loaded BJAB B cells were then washed 3 times in RPMI media with 10% FBS and resuspended at 4xlO^s/ml in RPMI.

Peripheral blood mononuclear cells (PBMC) from in-house donors were isolated from heparinized whole blood via centrifugation over Lymphocyte Separation Medium (#50494, MP Biomedicals, Aurora, Oh), washed 2 times with RPMI media and resuspended at 5xl0⁶/ml in RPMI with 10% FBS. Reagent samples were added to

RPMI media with 10% FBS at 4 times the final concentration and three 10-fold serial dilutions for each reagent were prepared. These reagents were then added to 96-well U- bottom plates at 50 μΐ/well to the indicated final concentrations. The ⁵¹Cr-labeled BJAB were then added to the plates at 50 μΐ/well (2xl0⁴/well). The PBMC were then added to the plates at 100 μΐ/well (5xl0⁵/well) for a final ratio of 25:1 effectors (PBMC):target (BJAB). Effectors and targets were added to media alone to measure background killing. The ⁵¹Cr-labeled BJAB were added to media alone to measure spontaneous release of ⁵¹Cr and to media with 5% NP40 (#28324, Pierce, Rockford, Ill) to measure maximal release of ^5ICr. The plates were incubated for 6 hours at 37°C in 5%CC>2. Fifty μΐ (25 μΐ would also be suitable) of the supernatant from each well were then transferred to a LumaPlate-96 (#6006633, Perkin Elmer, Boston, Mass) and dried overnight at room temperature.

After drying, radioactive emissions were quantitated as cpm on a Packard TopCount-NXT. Sample values were the mean of triplicate samples. Percent specific killing was calculated using the following equation: % Kill = ((sample - spontaneous release)/(maximal release - spontaneous release)) x 100. The plots in Fig. 30 show that BJAB B cells were killed by monospecific scorpions CD20xCD20 and CD37xCD37. The combination of CD20 SMIP and CD37 SMIP also killed BJAB B

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188 cells. These results demonstrate that scorpions exhibit scorpion-dependent cellular cytotoxicity and it is expected that this functionality is provided by the constant subregion of the scorpion, providing ADCC activity.

b. Scorpion role in complement-dependent cytotoxicity

Experiments also demonstrated that scorpions have Complement-Dependent

Cytotoxicity (CDC) activity. The experiment involved exposure of Ramos B-cells to CD19 and/or CD37 SMIPs and scorpions, as described below and as shown in Fig.

31.

The experiment was initiated by adding from 5 to 2,5 x 10⁵ Ramos B-cells to 10 wells of 96-well V-bottomed plates in 50 μΐ of Iscoves media (no FBS). The test compounds in Iscoves, (or Iscoves alone) were added to the wells in 50 μΐ at twice the indicated final concentration. The cells and reagents were incubated for 45 minutes at 37°C. The cells were washed 2.5 times in Iscoves with no FBS and resuspended in Iscoves with human serum (# Al 13, Quidel, San Diego, CA) in 96-well plates at the indicated concentrations. The cells were then incubated for 90 minutes at 37°C. The cells were washed by centrifugation and resuspended in 125 μΐ cold PBS. Cells were then transferred to FACs cluster tubes (#4410, CoStar, Coming, NY) and 125 μΐ PBS with propidium iodide (# P-16063, Molecular Probes, Eugene, OR) at 5 μg/ml was added. The cells were incubated with the propidium iodide for 15 minutes at room temperature in the dark and then placed on ice, quantitated, and analyzed on a

FACsCalibur with CellQuest software (Becton Dickinson). The results presented in Fig. 31 establish that the CD19 SMIP, but not the CD37 SMIP, exhibits CDC activity, with a combination of the two SMIPs exhibiting approximately the same level of CDC activity as CD 19 SMIP alone. The CD19xCD37 scorpion, however, exhibited significantly greater CDC activity than either SMIP alone or in combination, establishing that the scorpion architecture provides a greater level of Complementdependent Cytotoxicity than other molecular designs.

c. ADCC/CDC activity of CD20xCD2Q monospecific scorpions

Three distinct CD20xCD20 monospecific scorpions were examined for ADCC and CDC functionality, along with appropriate controls. ADCC was assayed using conventional techniques, and the results are presented in Fig. 53. Apparent from the

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Figure is the appreciable, but not identical, ADCC activity associated with each of the tested CD20xCD20 monospecific scorpions.

To assess CDC, Ramos B-cell samples (4x10^s) were incubated with each of the CD20xCD20 scorpions (0, 0.5,5, 50 and 500 nM) and serum (10%) for 3.5 hour at 37°C. Cell death was assessed by 7-AAD staining and FACS analysis. The results are presented in Fig. 54, which reveals that the scorpions exhibit some CDC activity. In a similar experiment, Ramos B-cell samples (4x10⁵) were incubated with CD20xCD20 scorpion protein (5, 50, 100 nM) and serum (10%) for 2 hour at 37°C. Cells were washed 2X and incubated with anti-human Clq FITC antibody. Bound

Clq was assessed by FACS analysis and the results are presented in Fig. 55. These results are consistent with the results presented in Fig. 54 that each of the CD20xCD20 monospecific scorpions was associated with some CDC activity, although less activity than was associated with a CD20 SMIP.

d. Interactions of scorpions with FgyRIII

ELISA studies showed that scorpions bound to FcyRIII (CD 16) low (a low affinity isoform or allelotype) at increased levels in the absence of target cells.

ELISA plates were initially coated with either low- or high-affinity CD16mIgG using conventional techniques. The ability of this immobilized fusion protein to capture either a CD20 SMIP or a CD20xCD20 monospecific scorpion was assessed. Bound

SMIPs and scorpions were detected with goat anti-human IgG (HRP) secondary antibody and mean fluorescence intensity (MFI) was determined. PBS alone (negative control) is shown as a single point. The results are presented in Fig. 32A (capture by CD 16 high affinity isoform fusion) and 32B (capture by CD 16 low affinity iso form fusion). Apparent from a consideration of Figs. 32A and 32B is that both CD20 SMIP and CD20xCD20 monospecific scorpion showed increased binding to both the high- and low-affinity CD 16 isoform fusions, with the CD20xCD20 scorpion showing a dramatic increase in binding to the low affinity isoform fusion with increasing protein concentration.

The binding of scorpions to the FcyRIII isoforms in the presence of target cells was also assessed. The data show the increased binding of scorpions to both FcyRIII (CD 16) low- and high-affinity isoforms or allelotypes in the presence of target cells with increasing protein concentration.

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In conducting the experiment, CD20-positive target cells were exposed to CD20 SMIPs or CD20xCD20 monospecific scorpions under conditions that allowed the binding of the SMIP or scorpion to the CD20-positive target cell. Subsequently, the SMIP- or scorpion-bearing target cell was exposed to either CD 16 high- or low5 affinity isoform tagged with mouse IgFc. A labeled goat anti-mouse Fc was then added as a secondary antibody to label the immobilized CD 16 tagged with the mouse IgFc. Cells were then detected using flow cytometry on a FACs Calibur (BD Biosciences, San Jose, CA) and analyzed with Cell Quest software (BD Biosciences, San Jose, CA). As shown in Fig. 33, increased concentrations of each of the CD20

SMIP and the CD20xCD20 monospecific scorpion led to increased binding to the CD 16 isoforms in the presence of target cells, with the increase in binding of the CD20xCD20 scorpion being more significant than the increased binding seen with the CD20 SMIP.

Example 17

Cell-cycle effects of scorpions on target lymphoma cells

The cell-cycle effects of scorpions were assessed by exposing lymphoma cells to SMIPs, monospecific scorpions and bispecific scorpions. More particularly, DoHH2 lymphoma cells (0,5 x 10⁶) were treated for 24 hours with 0.4 nM rituximab, CD20xCD37 scorpion, TRU-015 (CD20 SMIP) + SMIP-016 combination (0.2 nM each), 100 nM SMIP-016 or 100 nM CD37xCD37 scorpion. These concentrations respresent about 10-fold more than the IC50 value ofthe scorpion in a 96-hour growth inhibition assay (see Figs. 24-27). Cultures were labeled for 20 minutes at 37°C with 10 μΜ BrdU (bromodeoxyuridine). Following fixation, cells were stained with antiBrdU-FITC antibody and counterstained with propidium iodide. Values in Fig. 28 are the mean +/- SD of 4 replicate cultures from 2-3 independent experiments. All sample data were analyzed at the same time and pooled for presentation using both the BrdU and PI incorporation dot plots. Plots demonstrate that a major effect of scorpion treatment is a depletion of cells in S-phase, as well as an increase in the Gq/Gi compartment.

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Example 18

Physiological effects of scorpions

a. Mitochondrial potential

CD20xCD20 scorpions induced loss of mitochondrial membrane potential in 5 DHL4 B-cells, as revealed in a JC-1 assay. JC-1 is a cationic carbocyanine dye that exhibits potential-dependent accumulation in the mitochondria (Mitoprobe® JC-1 Assay Kit for Flow Cytometry from Molecular Probes). JC-1 is more specific to the mitochondrial membrane than the plasma membrane and is used to determine changes in mitochondrial membrane potential. Accumulation in mitochondria is indicated by a fluorescence shift from green (529nm) to red (590nm).

In conducting the experiment, DHL-4 B-cells (5xl0⁵ cells/ml) were initially cultured in 24-well plates and treated for 24 hours with 1 pg/ml CD20xCD20 scorpion, Rituximab, IgG control antibody, or 5 μΜ staurosporine at 37°C, 5%CO2, in a standard tissue-culture incubator. JC-1 dye (10 μΐ/ml, 2 μΜ final concentration) was added and cells were incubated for another 30 minutes at 37°C. Cells were harvested by centrifugation (5minutes at 1200 rpm), washed with 1ml PBS, and resuspended in 500 μΐ PBS. Cells were analyzed by flow cytometry (FACSCalibur, BD) with 488 nM excitation and 530 nM and 585 nM emission filters. For the representative scatter plots shown in Fig. 56, red fluorescence was measured on the

Y-axis and green fluorescence was measured on the X-axis. Depolarization of the mitochondrial membrane was measured as a decrease in red fluorescence, as seen in the positive control CCCP (carbonyl cyanide 3-chlorophenylhydrazone), a known mitochondrial membrane potential disrupter. To confirm that JC-1 was responsive to changes in membrane potential, DHL-4 B-cells were treated with two concentrations of CCCP (50μΜ and 250μΜ) for 5 minutes at 37°C, 5%CO₂. An additional positive control was cells treated with the broad-spectrum kinase inhibitor staurosporine to induce apoptosis. The results shown in Fig. 56 are dot-plot graphs of 10,000 counts, with red fluorescence plotted on the Y-axis and green fluorescence plotted on the Xaxis. A summary histogram of the percentage of cells with disrupted mitochondrial membrane potential (disrupted MMP: black bars) is shown in Fig 56. These results demonstrate that treatment with either the 20-4x20-4 scorpion or the 011x20-4

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192 scorpion generated a decrease in the mitochondrial membrane potential associated with cell death.

b. Calcium flux

Scorpion molecules were analyzed for influences on cell signaling pathways, using Ca^ mobilization, a common feature of cell signaling, as a measure therefor.

SU-DHL-6 lymphoma cells were labeled with Calcium 4 dye and treated with the test molecules identified below. Cells were read for 20 seconds to determine background fluorescence, and then SMIPs/scorpions were added (first dashed line in Fig. 28) and fluorescence was measured out to 600 seconds. At 600 seconds, an 8-fold excess of cross-linked goat-anti-human F(ab)’2 was added and fluorescence was measured for a further 300 seconds. Panel (A) of Fig. 28 shows the results obtained with a combination of CD20 SMIP and CD37 SMIP (red line); or obtained with a CD20xCD37 bispecific scorpion (black line), compared with unstimulated cells (blue line). In panel B of Fig. 28, the results of treating cells withCD20 SMIP alone (red line) resulted in Ca⁺⁺ mobilization, but this was not as robust as the signal generated by the monospecific CD20xCD20 scorpion (black line). The Ca⁺⁺ mobilization plots of Fig. 28 represent the fluorescence from triplicate wells treated with equimolar amounts of scorpion and SMIP/SMIP combinations.

c. Caspases 3.7 and 9

The ability of CD20-binding scorpions to directly kill B-cells as evidenced by increased Annexin V and Propidium Iodide staining and the loss of mitochondrial membrane potential led to an further investigation of additional apoptosis-related effects of CD20-binding scorpions in B-cells. The approach taken was to perform Apol assays on DHL-4 B-cells exposed to CD20xCD20 scorpions or appropriate controls. The Apol assay is based on a synthetic peptide substrate for caspase 3 and 7. The assay components are available from Promega (Apo-ONE® Homogeneuous Caspase-3/7 Assay). Caspase-mediated cleavage of the labeled peptide Z-DEVDRhodamine 110 releases the fluorescent rhodamine 110 label, which is measured using 485 nm excitation and 530nm detection.

In the experiment, 100 μΐ DHL-4 B-cells (lxl0⁶ cells/ml) were plated in black

96-well flat-bottom tissue culture plates and treated for 24 or 48 hours with 1 μg/ml

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CD20xCD20 scorpion, Rituximab, an IgG control antibody, or 5 μΜ staurosporine at 37°C, 5%CO2 in a standard tissue-culture incubator. (Staurosporine is a smallmolecule, broad-spectrum protein kinase inhibitor that is known in the art as a potent inducer of classical apoptosis in a wide variety of cell types.) After 24 or 48 hours,

100 μΐ of the 100-fold diluted substrate was added to each well, gently mixed for one minute on a plate shaker (300 rpm) and incubated at room temperature for two hours. Fluorescence was measured using 485 nM excitation and 527 nM emission filter (Fluoroskan Ascent FL, Thermo Labsystems). Graphs showing average fluorescent intensity of triplicate treatments plus/minus standard deviation after 24 hours and 48 hours (24 hours only for staurosporine) are presented in Fig. 57. These results establish that CD20-binding scorpions do not directly kill B-cells by an apoptotic pathway involving activation of caspase 3/7.

The results obtained in the Apo-1 assay were confirmed by Western blot analyses designed to detect pro-caspase cleavage resulting in activated caspase or to detect cleavage of PARP (Poly (ADP-Ribose) Polymerase), a protein known to be cleaved by activated caspase 3. DHL-4 B-cells were exposed to a CD 20 binding scorpion or a control for 4,24, or 48 hours and cell lysates were fractionated on SDSPAGE and blotted for Western analyses using conventional techniques. Fig. 58 presents the results in the form of a collection of Western blots. The bottom three

Westerns utilized anti-caspase antibodies to detect shifts in molecular weight of the caspase enzyme, reflecting proteolytic activation. For caspases 3, 7, and 9, there was no evidence of caspase activation by any of the CD20-binding molecules. Staurosporine served as a positive control for the assay, and induced pro-caspase cleavage to active caspase for each of caspases 3,7 and 9. The fourth Western blot shown in Fig. 58 reveals that PARP, a known substrate of activated caspase 3, was not cleaved, consistent with a failure of CD20-binding scorpions to activate caspase 3.

The results of all of these experiments are consistent in showing that caspase 3 activation is not a significant feature of the direct cell killing of DHL-4 B-cells induced by CD20 binding scorpions.

In addition, a time series study was conducted to determine the effect of CD20 binding proteins, including a CD20xCD20 scorpion, on Caspase 3. DoHH2 or SuDHL-6 B-cells were incubated with lOnM CD20 binding protein (SO 129 scorpion,

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2Lm20-4 SMIP, or Rituxan®) +/- soluble CD16 Ig (40nM), soluble CD 16 Ig alone, or media. The cells were cultured in complete RPMI with 10% FBS at 3xl0⁵/well/300 μΐ and harvested at 4 hours, 24 hours or 72 hours. The 72-hour time-point samples were plated in 500 μΐ of the test agent. Cells were washed with PBS and then stained for intracellular active caspase-3 using the BD Pharmingen Caspase 3, Active Form, mAB Apoptosis Kit:FITC (cat no.55048, BD Pharmingen, San Jose, CA). Briefly, after 2 additional washes in cold PBS, the cells were suspended in cold cytofix/cytoperm solution and incubated on ice for 20 minutes. Cells were then washed by centrifugation, aspirated, and washed two times with Perm/Wash buffer at room temperature. The samples were then stained with 20 μΐ FITC-anti-caspase 3 in 100 μΐ of Perm-Wash buffer at room temperature in the dark for thirty minutes. The samples were then washed two times with Perm-Wash buffer, and resuspended in 500 μΐ of Perm-Wash buffer. Washed cells were then transferred to FACs tubes and run on a FACs Calibur (BD Biosciences, San Jose, CA) and analyzed with Cell Quest software (BD Biosciences, San Jose, CA). The results are shown in Table 16.

Table 16

Molecule (10 nM)	Percentage Caspase-3 positive cells	Percentage in live gate
	4 hours	24 hours	48 hours	4 hours	24 hours	48 hours
RTXN	7	25	7	75	53	56
and CD 16 hi (4X)	27	47	21	79	60	43
CD20 SMIP (2Lm20-4)	5	5	10	89	85	81
and CD 16 hi	28	54	21	61	60	41
Humanized CD20xCD20 scorpion	7	13	14	69	68	61

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(S00129)
and CD 16 hi	30	31	15	67	75	72
Media	7	5	9	89	82	80
and CD 16 hi	6	5	9	91	83	80

The results of all of these experiments are consistent in showing that there is limited activation of caspase 3 in the absence of CD 16, which does not implicate caspase 3 activation as a significant feature of the direct cell killing induced by CD20 binding scorpions.

d. DNA fragmentation

Induction of classical apoptotic signaling pathways ultimately results in condensation and fragmented degradation of chromosomal DNA. To determine whether CD20-binding scorpions directly killed B-cells through a classical apoptotic mechanism, the state of B-cell chromosomal DNA was examined following exposure of the cells to CD20-binding scorpions, or controls. Initially, DHL-4 B-cells were treated in vitro for 4, 24 or 48 hours with a CD20-binding molecule, i.e., the monospecific CD20xCD20 (2Lm20-4x2Lm20-4) scorpion, the CD20xCD20 (01 lx2Lm20-4) scorpion, or Rituximab, or with a control. Subsequently, cells were lysed and chromosomal DNA was purified using conventional techniques. The chromosomal DNA was then size-fractionated by gel electrophoresis. The gel electrophoretogram shown in Fig. 59 reveals a lack of DNA fragmentation that demonstrated that the cell death generated by CD20-binding scorpions was not mediated by a classical apoptotic pathway. Staurosporine-treated cells were used as positive control in these assays.

e. SYK phosphorylation

SYK is a phospho-regulated protein with several phosphorylation sites that functions as a transcriptional repressor. SYK is localized to the cell nucleus, but is capable of rapid relocation to the membrane upon activation. For activation, SYK must retain its nuclear localization sequence. Activated SYK has a role in suppressing breast cancer tumors and SYK is activated by pro-apoptotic signals such

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196 as ionizing radiation, BCR ligation and MHC class II cross-linking. Further, SYK has been shown to affect the PLC-γ and Ca^H' pathways. Given these observations, the capacity of CD20-binding scorpions to affect SYK was investigated.

DHL-6 B-cells were exposed to a bispecific CD20xCD37 scorpion for 0, 5, 7 5 or 15 hours and the cells were lysed. Lysates were immunoprecipitated with either an anti-phosphotyrosine antibody or with an anti-SYK antibody. Immunoprecipitates were fractionated by gel electrophoresis and the results are shown in Fig. 60.

Apparent from an inspection of Fig. 60 is the failure of the bispecific CD20xCD37 scorpion to induce phosphorylation of SYK, thereby activating it. Consistent with the above-described studies on caspase activation and chromosomal DNA fragmentation, it does not appear that CD20-binding scorpions directly kill B-cells using a classic apoptotic pathway, such as the caspase-dependent pathway. While not wishing to be bound by theory, it is expected that the CD20-binding scorpions directly kill B-cells through a caspase-, and SYK-, independent pathway that does not prominently feature chromosomal DNA fragmentation, at least not on the same time frame as fragmentation occurs during caspase-dependent apoptosis.

Example 19

Scorpion applications

a. In vivo activity of scorpions

The activity of scorpions was also assessed using a mouse model.

Measurements of scorpion activity in vivo involved administration of 10-300 pg scorpion and subsequent time-series determinations of serum concentrations of that scorpion. Results of these studies, presented as serum concentration curves for each of two bispecific scorpions (i.e., S0033, a CD20xCD27 scorpion and a CD20xCD37 scorpion) from three-week pharmacokinetic studies in mice are presented in Fig. 40. The data in Fig. 40 show that it took at least 500 hours after administration before the serum levels of each of the two bispecific scorpions fell back to baseline levels. Thus, scorpions show serum stability and reproducible, sustained circulating half-lives invivo.

The in vivo efficacy of scorpions was also assessed. An aggressive Ramos xenograft model was used in parallel experiments with SMIPs versus historical

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197 immunoglobulin controls. The survival curves provided in Fig. 41 reveal that administration of 10 μg bispecific scorpion had negligible influence on survival, but administration of 100-300 μg had significant positive effect on the survival of mice bearing Ramos xenografts,

b. Combination therapies it is contemplated that scorpions will find application in the prevention, treatment or amelioration of a symptom of, a wide variety of conditions affecting man, other mammals and other organisms. For example, CD20-binding scorpions are expected to be useful in treating or preventing a variety of diseases associated with excessive or aberrant B-cells. In fact, any disease amenable to a treatment involving the depletion of B-cells would be amenable to treatment with a CD20-binding scorpion. In addition, scorpions, e.g,, CD20-binding scorpions, may be used in combination therapies with other therapeutics. To illustrate the feasibility of a wide variety of combination therapies, the monospecific CD20xCD20 scorpion (SO 129) was administered to Su-DHL-6 B-cells in combination with doxorubicin, vincristine or rapamycin. Doxorubicin is a topoisomerase II poison that interferes with DNA biochemistry and belongs to a class of drugs contemplated for anti-cancer treatment. Rapamycin (Sirolimus) is a macrolide antibiotic that inhibits the initiation of protein synthesis and suppresses the immune system, finding application in organ transplantation and as an anti-proliferative used with coronary stents to inhibit or prevent restenosis. Vincristine is a vinca alkaloid that inhibits tubule formation and has been used to treat cancer.

The experimental results shown in Fig. 61 are presented as Combination Index values for each combination over a range of effect levels. The interactions of the monospecific CD20xCD20 scorpion SO 129 are different for each drug class, while with Rituxan® (RTXN) the plots forms are similar. The effect seen with doxorubicin at high concentrations may reflect a shift towards monovalent binding. The data establish that CD20-binding scorpions may be used in combination with a variety of other therapeutics and such combinations would be apparent to one of skill in the art in view of the present disclosure.

Variations on the structural themes for multivalent binding molecules with effector function, or scorpions, will be apparent to those of skill in the art upon review

198

2016231617 23 Sep 2016

Of the present disclosure, and such variant structures are within the scope of the invention.

The entire disclosure in the complete specification of our Australian Patent No. 2007257692 and Australian Patent Application No. 2014200661 are by these crossreferences incorporated into the present application.

8214656_1 (GHMatters) P79767.AJ.2

199

2016231617 03 Aug 2018

Claims (15)

000 <210> <211> <212> <213> 301 PRT Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H42(AA) 35 <400> 301 Gly Cys Pro Pro Cys Pro Asn Ser 5089144_1 (GHMatters) P79767.AU.1 5/02/14 488 2016231617 23 Sep 2016 <210> 302 <400> 302 000 <210> 303 <400> 303 000 <210> 304 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> Linker H44 (PN) <400> 304 ggaggagcta gttgtccacc ttgtcccggg 36 <210> 305 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide 5089144_1 (GHMatters) P79767.AU.1 5/02/14 489 2016231617 23 Sep 2016 <220> <223> Linker H44 (AA) <400> 305 Gly Gly Ala Ser Cys Pro Pro Cys Pro Gly Asn Ser 15 10 <210> 306 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> Linker H45 (PN) <400> 306 ggaggagcca gttgtccacc ttgtgccggg aattct 36 <210> 307 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide 5089144_1 (GHMatters) P79767.AU.1 5/02/14 490 2016231617 23 Sep 2016 <220> <223> Linker H45 (AA) <400> 307 Gly Gly Ala Ser Cys Pro Pro Cys Ala Gly Asn Ser 15 10 <210> <211> <212> <213> 308 DNA Artificial sequence 15 <220> <223> Synthetic polynucleotide <220> <223> Linker H46 <400> 308 ggaggagcca gttgtccacc ttgtgcgaat tct 33 25 <210> <211> <212> <213> 309 11 PRT Artificial sequence 30 <220> <223> Synthetic peptide <220> <223> Linker H46 35 <400> 309 Gly Gly Ala Ser Cys Pro Pro Cys Ala Asn Ser 5089144_1 (GHMatters) P79767.AU.1 5/02/14 491 2016231617 23 Sep 2016 000 <210> 296 <211> 27 <212> DNA <213> Artificial sequence 5089144_1 (GHMatters) P79767.AU.1 5/02/14 486 2016231617 23 Sep 2016 <220> <223> Synthetic polynucleotide 5 <220> <223> Linker H40 (PN) <400> 296 10 ggatgtccac cttgtcccgc gaattct 27 <210> 297 15 <211> 9 <212> PRT <213> Artificial sequence <220> 20 <223> Synthetic peptide <220> <223> Linker H40 (AA) <400> 297 Gly Cys Pro Pro Cys Pro Ala Asn Ser 30 1 5 <210> 298 <400> 298 35 000 <210> 299 5089144_1 (GHMatters) P79767.AU.1 5/02/14 487 2016231617 23 Sep 2016 <211> 9 <212> PRT <213> Artificial sequence 000 <210> 295 <400> 295 000 <210> 294 <400> 294 000 <210> 293 <400> 293 000 <210> 292 <400> 292 000 <210> 291 <400> 291 000 <210> 240 <211> 51 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> Linker Hll (PN) <400> 240 20 gagcccacat ctaccgacaa aactcacaca tctccaccca gcccgaattc t 51 <210> <211> <212> <213> 241 PRT Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> Linker Hll (AA) Glu Pro Thr Ser Thr Asp Lys Thr His Thr Ser Pro Pro Ser 35 <400> 241 5089144_1 (GHMatters) P79767.AU.1 5/02/14 462 2016231617 23 Sep 2016 Pro Asn 000 <210> 239 The claims defining the invention are as follows:

1 5 10 <210> 372 <211> 5 <212> PRT <213> Artificial sequence 15 <220> <223> Synthetic peptide <220> 20 <223> Scorpion linker

<400> 372

Gly Cys Pro Pro Cys

25 1 5 <210> 373 <211> 12 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

534

2016231617 23 Sep 2016 <223> Scorpion linker <400> 373

Gin Tyr Asn Cys Pro Gly Gin Tyr Thr Phe Ser Met 15 10 <210>

<211>

<212>

<213>

374

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Scorpion linker <400> 374

Pro Phe Phe Thr Cys Gly Ser Ala Asp Thr Cys 15 10 <210>

<211>

<212>

<213>

375

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Scorpion linker

5089144_1 (GHMatters) P79767.AU.1 5/02/14

535

2016231617 23 Sep 2016 <400> 375

Glu Pro Ala Phe Thr Pro Gly Pro Asn lie Glu Leu Gin Lys 5 Asp Ser

15 10 15

Asp Cys

<210> 376 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Scorpion linker

25 <400> 376

Gin Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gin Lys Ala Arg

15 10 15

Hi s Cys <210> 377 <211> 17

5089144_1 (GHMatters) P79767.AU.1 5/02/14

536

2016231617 23 Sep 2016

<212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Scorpion linker <400> 377

Asn Ser Leu Phe Asn Gin Glu Val Gin lie Pro Leu Thr Glu Ser Tyr

15 1 5 10

Cys <210>

<211>

<212>

378

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VL CDR1 (TRU-015) <400> 378

Arg Ala Ser Ser Ser Val Ser Tyr Met 1 5

His

5089144_1 (GHMatters) P79767.AU.1 5/02/14

537

2016231617 23 Sep 2016

<210> 379 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> anti-CD20 VH CDR3 (TRU-015)

1 5

<210> 347 <211> 4 <212> PRT <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

511

2016231617 23 Sep 2016 <223> Synthetic peptide

<220> <223> Extension sequence <400> 347

Ala Pro Glu Leu

1 5

1 5 <210>

<211>

<212>

317

PRT <213> Artificial sequence

1 5 <210> 310 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H47 <400> 310 Leu Ser Val Lys Ala Asp Phi Ser 1 5 <210> 311 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

30 <223> Linker H48 <400> 311

Lys Ala Asp Phe Leu Thr Pro Ser lie Gly Asn Ser 35 1 5 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

492

2016231617 23 Sep 2016 <210> 312

<400> 312 000 <210> 313 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Linker H50 <400> 313

Leu Ser Val Leu Ala Asn Phe Ser Gin Pro Glu lie Gly Asn 2 0 Ser

15 10 <210>

<211>

<212>

<213>

314

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H51 <400> 314

5089144_1 (GHMatters) P79767.AU.1 5/02/14

493

2016231617 23 Sep 2016

Leu Ser Val Leu Ala Asn Phe Ser Gin Ser

Pro Glu lie Gly Asn <210>

<211>

<212>

<213>

315

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H52 <400> 315

Ser Gin Pro Glu He Val Pro He Ser Asn Ser <210>

<211>

<212>

<213>

316

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H53

35 <400> 316

Ser Gin Pro Glu He Val Pro

He Ser Cys

Pro Pro Cys Pro

5089144_1 (GHMatters) P79767.AU.1 5/02/14

494

2016231617 23 Sep 2016

Asn Ser

1 5 <210>

<211>

<212>

<213>

262

DNA

Artificial sequence

1 5 <210>

<211>

<212>

<213>

258

DNA

Artificial sequence

1 5

Ser <210> 244 <211> 51 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H13 (PN) <400> 244 gagcccacat ctaccgacaa aactcacaca tctccaccca gcccgaattc t 51 <210>

<211>

<212>

<213>

245

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H13 (AA)

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Asn <400> 245

5089144_1 (GHMatters) P79767.AU.1 5/02/14

464

2016231617 23 Sep 2016

Ser <210> 246 <211> 36 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

15 <223> Linker H15 (PN) <400> 246 ggcggtggtg gctcctgtcc accttgtccg aattct 36 <210> 247 <211> 12 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H15 (AA) <400> 247

Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Asn Ser 15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

465

2016231617 23 Sep 2016

<210> 248 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucle< <220> <223> Linker H16 (PN) <400> 248

15 ctgtctgtga aagctgactt cctcactcca tccatcggga attct 45 <210>

<211>

<212>

249

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H16 (AA) <400> 249

Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser lie Gly Asn Ser

35 1 5 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

466

2016231617 23 Sep 2016 <210>

<211>

<212>

<213>

250

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H17 (PN) <400> 250 ctgtctgtga aagctgactt cctcactcca tccatctcct gtccaccttg cccgaattct 60

<210> 251 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Linker H17 (AA)

30 <400> 251

Leu Ser Val Lys Ala Asp Phe Leu Thr Pro Ser lie Ser Cys Pro Pro

15 10

Cys Pro Asn Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

467

2016231617 23 Sep 2016 <210> 252 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H18 (PN) <400> 252 ctgtctgtgc tcgctaactt cagtcagcca gagatcggga attct 45 <210> 253 <211> 15 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide

30 <220>

<223> Linker H18 (AA) <400> 253

35 Leu Ser Val Leu Ala Asn Phe Ser Gin Pro Glu lie Gly Asn Ser

15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

468

2016231617 23 Sep 2016 <210>

<211>

<212>

<213>

254

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H19 (PN) <400> 254 ctgtctgtgc tcgctaactt cagtcagcca gagatctcct gtccaccttg cccgaattct 60

20 <210> 255 <211> <212> <213> 20 PRT Artificial sequence 25 <220> <223> Synthetic peptide 30 <220> <223> Linker H19 (AA)

<400> 255

Leu Ser Val Leu Ala Asn Phe Ser Gin Pro Glu lie Ser Cys 35 Pro Pro

15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

469

2016231617 23 Sep 2016

Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

256

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H20 (PN) <400> 256 ctgaaaatcc aggagagggt cagtaagcca aagatctcga attct 45 <210>

<211>

<212>

<213>

257

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H20 (AA)

35 <400> 257

Leu Lys lie Gin Glu Arg Val Ser Lys Pro Lys He Ser Asn

5089144_1 (GHMatters) P79767.AU.1 5/02/14

470

2016231617 23 Sep 2016

Ser

1 5

1 5

Asp Thr Thr Gly Ala Val Gin Leu Gin Gin Ser Gly Pro Glu Ser Glu

20 25 30

Lys Pro Gly Ala Ser Val Lys lie Ser Cys Lys Ala Ser Gly Tyr Ser

35 40 45

Phe Thr Gly Tyr Asn Met Asn Trp Val Lys Gin Asn Asn Gly Lys Ser

50 55 60

Leu Glu Trp He Gly Asn He Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr

65 70 75

5089144_1 (GHMatters) P79767.AU.1 5/02/14

330

2016231617 23 Sep 2016

Asn Ser Arg Ser Lys Phe Lys 85 Gly Gly Thr Ala Tyr Met Gin Ser Ala 100 Val Tyr Tyr Cys Ala Arg Gly Gin 115 Gly Thr Ser Val Thr Val Gly Gly 130 Gly Ser Gly Gly Gly Gly Asp lie 145 150 160 Gin Met Thr Gin Ser Pro Glu Thr 165 175 Val Thr He Thr Cys Arg Leu Ala 180 Trp Tyr Gin Gin Lys Gin

Lys Ala Thr Leu Thr Val Asp Lys

90 95

Leu Lys Ser Leu Thr Ser Glu Asp

105 110

Ser Val Gly Pro Met Asp Tyr Trp

120 125

Ser Ser Gly Gly Gly Gly Ser Gly

135 140

Ser Gly Gly Gly Gly Ser Ala Ser

155

Ala Ser Leu Ser Ala Ser Val Gly

170

Thr Ser Glu Asn Val Tyr Ser Tyr

185 190

Gly Lys Ser Pro Gin Leu Leu Val

5089144_1 (GHMatters) P79767.AU.1 5/02/14

331

195

205

2016231617 23 Sep 2016

Ser Phe

200

1 RR

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

35 195 200 205

5089144_1 (GHMatters) P79767.AU.1 5/02/14

261

2016231617 23 Sep 2016

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

15 255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

5089144_1 (GHMatters) P79767.AU.1 5/02/14

262

2016231617 23 Sep 2016

1 5 <210> 168 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> Linker(G4S)4

5089144_1 (GHMatters) P79767.AU.1 5/02/14

111

2016231617 23 Sep 2016 <400> 168 ggtggcggtg gctcgggcgg tggtggatct ggaggaggtg ggagcggggg aggtggcagt 60

<210> <211> <212> 10 <213> 169 17 PRT Artificial sequence <220> <223> Synthetic peptide 15 <220> <223> Linker(G4S)4 <400> 169

20 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly

15 10

25 Ser <210> 170 <211> 2337 <212> DNA 30 <213> Artificial sequence <220> <223> Synthetic polynucleotide 35 <220> <223> 2H7sssIgGl-STDl-2el2HL (DNA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

112

2016231617 23 Sep 2016

<400> 170 aagcttgccg cagtgcttca ccatggattt 60 tcaagtgcag attttcagct tcctgctaat 5 gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct 10 ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc 15 tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag 20 cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 25 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 30 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac 35 gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga

5089144_1 (GHMatters) P79767.AU.1 5/02/14

113

2016231617 23 Sep 2016

gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt 5 ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga acatccccac cctcttcccc cgagctcagc 900 acctgaactc ctggggggac cgtcagtctt 10 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg 15 gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg 20 gtcctcaccg caaggtctcc tcctgcacca 1140 ggactggctg aatggcaagg agtacaagtg aacaaagccc gcagccccga tcccagcccc 1200 catcgagaaa acaatctcca aagccaaagg 25 gaaccacagg ccaggtcagc tgtacaccct 1260 gcccccatcc cgggatgagc tgaccaagaa 30 ctgacctgcc ggagagcaat tggtcaaagg 1320 cttctatccc agcgacatcg ccgtggagtg gggcagccgg cggctccttc agaacaacta 1380 caagaccacg cctcccgtgc tggactccga 35 ttcctctaca cgtcttctca gcaagctcac 1440 cgtggacaag agcaggtggc agcaggggaa tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 ctccctgtct ccgggtaaga aggtgggagt gggaattctc ctcacagagc ctgtccatca aaactgggtt cgccagcctc tggaagcaca gactataatt caagagccaa gttttcttaa ctgtgctcga gatggttata aacctcagtc accgtctcct tgggtcgggt ggcggcggat gtctctaggt cagagagcca cacaagttta atgcagtggt tgctgctagc aacgtagaat agactttagc

1500 attatggtgg

1560 aggtgcagct

1620 catgcaccgt

1680 caggaaaggg

1740 cagctctcaa

1800 aaatgaacag

1860 gtaactttca

1920 ctgggggtgg

1980 ctgacattgt

2040 ccatctcctg

2100 accaacagaa

2160 ctggggtccc

2220 cggtggctcg gaaggagtca ctcagggttc tctggagtgg atccagacta tctgcaaact ttactatgtt aggctctggt gctcacccaa cagagccagt accaggacag tgccaggttt ggcggtggtg ggacctggcc tcattaaccg ctgggaatga tcgatcacca gatgacacag atggactact ggcggtggat tctccagctt gaaagtgttg ccacccaaac agtggcagtg gatctggagg tggtggcgcc gctatggtgt tatggggtga aggacaactc ccagatacta ggggtcaagg ccggcggagg ctttggctgt aatattatgt tcctcatctc ggtctgggac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

115

2016231617 23 Sep 2016 ctcaacatcc atcctgtgga ggaggatgat attgcaatgt atttctgtca gcaaagtagg 2280

-1-l· o

o f*fc o

CD

CM

3AlllS0d Id JO/P^UB NNV lU90J9d

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

28/67

2016231617 23 Sep 2016

FIG. 23

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

29/67

FIG. 24

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

30/67

FIG. 25

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

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31/67

FIG. 26

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

32/67

FIG. 27

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

33/67

FIG. 23

A.

EZI Sub G1 S

Gq/Gi @IG2/M

E=3SubG1 S

IGq/G! ^G2/M

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

34/67

FIG. 29

Time (seconds)

Time (seconds)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

35/67

FIG. 30

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

36/67

FIG. 31

-•-CD37 SMIP

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

37/67

MFI

FIG. 32A

C016 (HIGH) BINDING ELISA

Protein (ug/mL)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

38/67

FIG. 32B

CD16 (LOW) BINDING ELISA

Protein (ug/mL)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

39/67

FIG. 33

CD16 Lo binding to WIL2S bound SMiP/Scorpion

SUBSTITUTE SHEET (RULE 26)

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PCT/US2007/071052

2016231617 23 Sep 2016

40/67

FIG. 34

A 9 C Ο E F G Feed Condition

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

Φ .X to £

CO io

CM o >» co

41/67

IO <*>

¢:

Q. u O 0 (0 to >< « © u_ CM co c

1/67

FIG. 1

BD2

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/TJS2007/071052

2016231617 23 Sep 2016

1. A single-chain protein comprising from amino to carboxy terminus:

(a) a first binding domain derived from an immunoglobulin-like molecule or the variable regions of an immunoglobulin;

(b) a Fc region comprising a domain derived from an immunoglobulin Ch2 domain, wherein said Fc region does not comprise a domain derived from an immunoglobulin Chi domain;

(c) a linker peptide of at least 5 amino acids; and (d) a second binding domain derived from an immunoglobulin-like molecule or the variable regions of an immunoglobulin, wherein the first or second binding domain binds 41BB/TNFRSF9; and wherein the first binding domain and the second binding domain recognize different molecular targets.

2 0 Asp Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

329

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H66

35 <400> 329

Glu Pro Ala Phe Thr Pro Gly Pro Asn He

Glu Leu Gin Lys

5089144_1 (GHMatters) P79767.AU.1 5/02/14

501

2016231617 23 Sep 2016

Asp Ser

2 0 Asp Thr Thr Gly Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val 20 25 30

25 Lys Pro Gly Ala Ser Met Lys lie Ser Cys Lys Ala Ser Gly Tyr Ser 35 40 45

30 Phe Thr Gly Tyr He Val Asn Trp Leu Lys Gin Ser His Gly Lys Asn 50 55 60 35 Leu Glu Trp He Gly Leu He Asn Pro Tyr Lys Gly Leu Thr

Thr Tyr

65 70 75

5089144_1 (GHMatters) P79767.AU.1 5/02/14

346

2016231617 23 Sep 2016

2 0 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

340 345 350

25 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

355 360 365

30 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

370 375 380

35 Ala Leu Pro Ala Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin

385 390 395

5089144_1 (GHMatters) P79767.AU.1 5/02/14

317

2016231617 23 Sep 2016

400

Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp 5 Glu Leu

405 410

415

2 0 aga 2283 <210>

<211>

<212>

<213>

197

754

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7sssIgGl-H7-G194 HL (w/2el2 leader) (AA)

35 <220>

<221> misc_feature <222> (1)..(22)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

282

2016231617 23 Sep 2016 <223>

<220>

<221>

.2 '-£3

Ό

O % V

VV W %Λ>

% % % % ^z *

Cell Line/MTX concentration oooooooo v c\ o co ω n c\ % % ^ζς>

2/67

FIG. 2

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

2. A protein according to claim 1, wherein the first and/or second binding domain is a single-chain variable antibody fragment (scFv).

*=3» (H Aea l^e lvu/6n

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

42/67

FIG. 36

Noft-Reduced Reduced

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

43/67

FIG. 37

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

44/67

FIG. 38

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

45/67

FIG. 39

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

46/67

FIG. 40

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052 kO o

<N

Ph <L>

47/67

VO <N

I» kO <N kO o

<N

FIG. 41 % Survival

20x37 Scorpion Dose Response in a Ramos Xenograft Model (TRU.PC.0102)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

48/67

FIG. 42

Target Cell Binding of SO129 Parent and Glycovariants

Geo MFI

CONCENTRATION (nM)

WIL2S staining with 2lm2G-4, and SO129 +/- carbohydrate modification Kifunensine@10 uM (excess), Castanospermine@200 uM

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

49 / 67 <*>

d ί

s o

o o

o

Protein [nM]

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

50/67

FtG.44

X XIIIXXX iA -A -A -i. —A |_3b —lr t£> Go ^: M O> <0 ilOO O)

-kPa r· ' ipuji n t

98 &__** W

64 ' i'J •I.

20x28 20x20

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

51/67 io

T- σ>

O CM

73 στ a: m s to ϊ Ο O) T- v T to

Η ω I X T X X

Η Η H O

EC

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

52/67

FIG. 46

Activity of 20x20 Scorpion (closed) vs SMIP (open)

SU-DHL-6

DoHH2

Rec-1

SU-DHL-6

DoHH2

Rec-1

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/0710S2

2016231617 23 Sep 2016

53/67

Activity of 20x20 scorpion vs NHL cell lines

FIG. 47 «-SU-DHL-4 * SU-DHL-6 ▼ DoHH2 ♦ WSU-NHL • Rec-1

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

54/67

FIG. 48

Activity vs DoHH2 ▼ 20x20 0 20x37 □ 37x37

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

55/67

FIG. 49

SU-DHL-6 Response to Molecules

Time (sec)

70 nM S0129 — 70 nM 20x37

- 70 nM 37x37 — anti-IgM

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

56/67

FIG. 50 □ RTXN a 19x37

Concentration (nM)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

57/67

FIG. 51

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

58/67

FIG. 52 ug/ml

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

59167

FIG. 53

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

60/67

FIG. 54

0 I-1-1-1-10.01 0.1 1 10 100

1000

AB cone. (nM)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968 PCT/US2007/071052

2016231617 23 Sep 2016

61 167

FIG. 55

AB Cone. (nM)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

62/67

FIG. 56

Rituximab 20-4x20-4 011x20-4

100 so « 70 S

O 60 nJZji 50 £«

O

0 *

KJ Intact MMP — Disrupted MMP

O §

ΓΊ o

s £

ja co o

in

O.

O

O

Q o

in

CN

CL

O o

o

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052 o

Ph

63/67

CD (Z>

CD

CN

CD

CN o

(N

FIG. 57

IgG Rituximab 20-4x20-4 011x20-4 Staurosporine Control 5uM

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

PARP

64/67

FIG. 58

Caspase 3

Caspase 9

Caspase 7

C R 4 11 st C R 4 11 st C R 11 st

Lanes:

C - control IgG R = Rituximab 4 « 20-4x20-4 11 =011x20-4 st - steurosporine

3/67

FIG. 3

BINDING OF PROTEIN EXPRESSED IN COS SUPERNATANTS TO CELLS EXPRESSING TARGET ANTIGENS

A. BINDING OF COS SUPERNATANTS TO WIL-2S CELLS

(=□ 2h7-sss-hlgG-STD1*2e12 LH 2h7-sss-blgG-STD1-2e12 HL 2h7lgG SMIP mock

B. BINDING OF COS SUPERNATANTS TO CD28 OHO CELLS 2e12 SMIPs vs. 2H7-2e12 SCORPIONS

CO 2h7-sss-hlgG-STD1-2e12 LH Ξ 2h7-$ss-hlgG-STD1-2e12 HL g=§S2e12LH SMIP ^2e12HLSMIP Hi mock

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

3. A protein according to claim 2, wherein the scFv comprises SEQ ID NO: 2, 4, 6, 103, 105, 107 or 109.

4 hours 24 hours 48 hours

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

65/67 σ>

V) d

LL eutjodsojneis

7Ό7Χ40

-4— 2H7-sss-hlgG-STD1-G19-4LH 2H7-csc-hlgG-STD1-G19-4 HL —A—G19-4 —h— G19-4 + TRU01S —TRU015 —·— media

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

25/67

2016231617 23 Sep 2016

FIG. 20

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

26/67

PCT/US2007/071052

2016231617 23 Sep 2016 mini!

I11H1IIIIII smsaasaffliig < >

CM 10

IIIIIIIIHIIIIIIIIIIIIItIHIIIIIIIfllltllll [lllllllllllltlllflllllllllllllllllllllltlt _:*ϊ*ΐί<ίΐ*ί*ίίΐ*ΐ*ΐΐΐΐ<*ίϊΐ*<νΐ*τ+>' iiiiiiiiiitimiiiiriitfiiiii iiiiiiiiidiiiuiiuiu

ΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ,ΙΙΙΙΠΙΙ Itlllllll lllltllllllllllllll Ι1ΙΙΙΙΙΙ11ΙΙΠΙΙΙ co

ΙΙΙΠΙΪΠΗΝΠΙΚΗΠΠΙΠΠΠΠΙΙΗΙΗΙΙΙΗΙΙΙΠΠΠΗΠΙΠΓ “«ΜββΜ at

CM d

¢:

— Illllllllll tlllllllH llllll1111111Itl t> 111 LI Itlll 11111

C\l r*

IllllllfllUllllllllUIIIIIIIIIIIIIIIIIIIIIIIIIIIIJ

11111111111)1111 tUIIUIIIIIIU HllllllllllllHHIIIIIIIIIIIIIHIIIIinillllllllllt liillUKHIIIimilllUIIHIIIllllllllllllllllllllllllllllllllll o

co

Itllllllllllllllllllllllllllllllllllllllllllllllllllllll·

CM iiitiiiititifiiiiHiriiiiiiiiiiiiiiiiiiiiuuii

Xi'Mf'iiifi’SfifWiic+rti'tiWiiiiiiiitfeirifitii.tiiiivMtirtiiiiUtiHi.iWtit

IHlIlimilllllllllllllllllllllllllllltlllllllllllllllllllllllllllllllllllllllllllll _ -iti-.i-i-W.v.ii-iW.iii^i'eSixi-i-i'iiitiWitiYi'iAis.illllllllllllllllllllllllllllllllllllimilllllllllllllllllllllllllti ©

co eAjiisod id JO/pus NNV % o

CM <n

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

27/67 ms

O

III I1UI1II

T5 CX CM CO

I .* t/jf «V * t #**'*.'* Λ» t** fc > fc**

11111111111111111 I H111111111111U1111111 HUH

r nt

CD

CO

ΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ.Ι , 1 C 1 / , / ' ί * ί ' ; ' j ' / / / Ϊ f i ⁵ Ζ ¹ ί ' ί ' ί 1 f ' / ¹ ? ' # ^fc t f f * 7λ-7λ ίϊΰϊ ΓιΊΐΊϊ'ιϊ ιΪιινηι’ιηιΪπι ΪΪιηπιΪι η »ι ι ιίιί ϊιιϊή'ιιιΐιιιιιιιιιιιιιιιι ε

CM

CM o

u.

CD

CO }<» ziz}rt>Jrt*}z}>i ζϊζΐζΐζ» Iziztzjzi, 5>ί#»}ζ1

4— 2H7-sss-hlgG-STD1-G19-4LH -·— 2H7-csc-hlgG-STD1-G19-4 HL A—G19-4 —G19-4 + TRU015 *— TRU015 —·— media

B. ADCC Activity in NK Cell Depiated PBMC Effector Cultures Using BJAB Targets (30:1, E:T) and 2H7-G194 Fusion Proteins

CONCENTRATION (ug/mL)

4/67

FIG. 4

BINDING OF PURIFIED PROTEIN FROM COS CELLS TO WIL2-S: TRU015 vs. 2H7-SSSIgG-STD1-2e12 MULTISPECIFIC FUSION PROTEINS GOATANTI-KUMAN FITC 1:100

EVENTS

- TRU0155ug/mL to⁴

FL1-H

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

4. A protein according to any one of claims 1 to 3, wherein the first and/or second binding domain comprises chimeric, humanized, or human immunoglobulin variable regions.

5 <400> 369

Gin Lys Ser 1 <210>

<211>

<212>

<213>

370

PRT

Artificial sequence <220>

<223> Synthetic peptide

20 <220>

<223> Partial CH3 sequence <400> 370

25 Gin Lys Ser Leu Ser Leu 1 5

<210> 371 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

533

2016231617 23 Sep 2016 <220>

<223> Partial CH3 sequence <400> 371

Gin Lys Ser Leu Ser

5 1 5 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Glu Thr Ser Tyr

5 <221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3

5 20 25 30

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Asn Ser Tyr Trp Tyr

5 <400> 358

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Gly Gly Asp Trp Tyr

Phe Asp Leu 50

<210> 359 <211> 51 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

35 <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3

5089144_1 (GHMatters) P79767.AU.1 5/02/14

521

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL 5 CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 359

Arg Ala Ser Ser Ser Val Ser Tyr lie Val Xaa Gin Gin Trp Ser Phe

15 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Lys Ser Asn Ser Tyr Trp Tyr

Phe Asp Leu 50

5089144_1 (GHMatters) P79767.AU.1 5/02/14

522

2016231617 23 Sep 2016 <210> 360 <211> 51 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3

15 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDRl and VL CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH 25 CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 360

35 Arg Ala Ser Ser Ser Val Ser Tyr lie Val Xaa Gin Gin Trp Ser Phe

15 10 15

5089144_1 (GHMatters) P79767.AU.1 5/02/14

523

2016231617 23 Sep 2016

Asn Pro Pro Thr Xaa Ala lie Tyr Pro Gly Asn Gly Glu Thr Ser Tyr

5 35 40 45

Phe Asp Leu 50 <210>

<211>

<212>

<213>

358

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL CDR3

30 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature

5089144_1 (GHMatters) P79767.AU.1 5/02/14

520

2016231617 23 Sep 2016 <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3

5 <220>

<223> anti-CD20 VH CDR3 <400> 345

Ser Tyr Lys Ser Gly Gly Asp <210>

<211>

<212>

<213>

346

PRT

Artificial sequence <220>

<223> Synthetic peptide

Trp Tyr Phe Asp Leu <220>

<223> Scorpion linker core (2H7) sequence <400> 346

Gly Cys Pro Pro Cys Pro Asn Ser

5 Gin Gin Tyr Ser Phe Asn Pro Pro Thr 1 5 <210> 336

5 Asp Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

330

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H67 <400> 330

Glu Pro Ala Phe Thr Pro Gly Pro Asn lie Glu Leu Gin Lys Asp Ser

25 1 5 10 15

Asp Cys Pro Asn Ser

20 <210> 331 <211> 118 <212> PRT <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

502

2016231617 23 Sep 2016 <223> Synthetic peptide <220>

<223> Linker H68 <400> 331

Arg Thr Arg Tyr Leu Gin Val Ser Gin Gin Leu Gin Gin Thr Asn Arg

5 <220>

<223> Linker H63 <400> 326

Asn Ser Leu Phe Asn Gin Glu Val Gin lie Pro Leu Thr Glu Ser Tyr

15 10 15

15 Cys Asn Ser <210> 327

20 <211> 21 <212> PRT <213> Artificial sequence <220>

25 <223> Synthetic peptide <400> 327

Gin Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gin Lys 30 Ala Arg

15 10

His Cys Pro Asn Ser 35 20

5089144_1 (GHMatters) P79767.AU.1 5/02/14

500

2016231617 23 Sep 2016

<210> 328 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5 <220>

<223> Synthetic peptide <220>

<221> misc_feature <223> Linker H41 <400> 299

15 Gly Cys Pro Pro Cys Pro Ala Asn Ser 1 5 <210> 300 <400> 300

5 <220>

<223> Synthetic peptide <220>

5 <220>

<223> Linker H30 (AA) <400> 277

5 <400> 275

Asp Thr Lys Gly Lys Asn Val Leu Glu Lys lie Phe Asp Ser Cys Pro

15 10

Pro Cys Pro Asn Ser 20 <210> 276 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H30 (PN) <400> 276 ctgccacctg agacacagga gagtcaagaa gtcaccctga attct 45

<210> 277 <211> 15 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

480

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide

5 Asp Thr Lys Gly Lys Asn Val Leu Glu Lys lie Phe Ser Asn Ser

15 10 <210>

<211>

<212>

<213>

274

DNA

Artificial sequence

15 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H29 (PN) <400> 274 gataccaaag ggaagaacgt cctcgagaag atcttcgact cctgtccacc ttgcccgaat 60 tct

<210> 275 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

479

2016231617 23 Sep 2016 <220>

<223> Linker H29 (AA)

5 Ser <210> 242 <211> 51 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H12 (PN) <400> 242 gagcccacat ctaccgacaa aactcacaca tctccaccca gcccgaattc t 51

<210> 243 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H12 (AA) <400> 243

Glu Pro Thr Ser Thr Asp Lys Thr His Thr Cys Pro Pro Cys

5089144_1 (GHMatters) P79767.AU.1 5/02/14

463

2016231617 23 Sep 2016

Pro Asn

5 <400> 239

5 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H9 (PN) <400> 236 tctccacctt ctccgaattc t 21 <210>

<211>

<212>

<213>

237

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H9 (AA) <400> 237

Ser Pro Pro Ser Pro Asn Ser 1 5

35 <210> 238 <400> 238

5089144_1 (GHMatters) P79767.AU.1 5/02/14

461

2016231617 23 Sep 2016

5 655

Gly Gly Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala

5 10 Gly Val Gly Pro 545 560 Leu Ser 530 Phe Pro Val Ser Ala Leu Pro Thr Ser Gly 550 Gin 535 Tyr Ser Gly Leu Val Ser Asn He Trp 555 Thr 540 Val Cys Arg Thr Gin Gly Lys Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr 15 565 570 575 Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys 20 Asp Asn 580 585 590 Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr 25 Asp Asp 595 600 605 Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr 30 610 615 620 Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser 35 625 630 635 640

5089144_1 (GHMatters) P79767.AU.1 5/02/14

456

2016231617 23 Sep 2016

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

645 650

5 <220>

<221> misc_feature <222> (661)..(772) <223> VL2

5 <223>

<220>

<221>

<222>

5 Ser Leu Gin Thr 530 Ser Leu Ser He Thr 535 Cys Thr Val Ser Gly 540 Phe Ser Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu 10 545 550 555 560 Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr Asn 15 Ser Ala 565 570 575 20 Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val 580 585 590 25 Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr 595 600 605 30 Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr 610 615 620 35 Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly

Gly Ser

625 630 635

5089144_1 (GHMatters) P79767.AU.1 5/02/14

444

2016231617 23 Sep 2016

640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 5 Ser Asp

645 650

655

5 Thr Asp 305 320 Pro Pro Glu Val Thr Cys 310 Val Glu Val Lys Phe Asn Trp Tyr Asn Ala 10 335 325 Lys Thr Lys Pro Arg Glu Glu 15 Val Val 340 Ser Val Leu Thr Val Leu His 20 Glu Tyr 355 Lys Cys Lys Val Ser Asn Lys 25 Lys Thr 370 375 He Ser Lys Ala Lys Gly Gin 30 Thr Leu 385 390 400 35 Pro Pro Ser Arg Asp Glu Leu Thr Cys

Val Val Asp Val Ser His Glu

315

Val Asp Gly Val Glu Val His

330

Gin Tyr Asn Ser Thr Tyr Arg

345 350

Gin Asp Trp Leu Asn Gly Lys

360 365

Ala Leu Pro Ala Pro lie Glu

380

Pro Arg Glu Pro Gin Val Tyr

395

Thr Lys Asn Gin Val Ser Leu

410

405

5089144_1 (GHMatters) P79767.AU.1 5/02/14

442

2016231617 23 Sep 2016

415

Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp 5 Glu Ser

420 425 430

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 10 Leu Asp

435 440 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 15 Lys Ser

450 455 460

Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His 2 0 Glu Ala

465 470 475

480

25 Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

485 490

495

30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

500 505 510

35 Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro

515 520 525

5089144_1 (GHMatters) P79767.AU.1 5/02/14

443

2016231617 23 Sep 2016

5 Phe Ser Ser Arg Gly Ser Gly 85 Ser Gly Thr Ser Tyr 90 Ser Leu Thr lie Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Phe 10 100 105 110 Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Asp Gly 15 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ser Gin Ala 20 130 135 140 Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val 25 145 150 155 160 Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 30 Asn Met 165 170 175 35 His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp He

Gly Ala

180 185 190

5089144_1 (GHMatters) P79767.AU.1 5/02/14

440

2016231617 23 Sep 2016 lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly

195 200 205

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

225 230 235

240

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr

245 250

255

Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp 25 Lys Thr

260 265 270

His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly 30 Pro Ser

275 280 285

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He 35 Ser Arg

290 295 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

441

2016231617 23 Sep 2016

5 tttcattact atgttatgga ctactggggt caaggaacct cagtcaccgt ctcctctggg 1920 ggtggaggct ctggtggcgg tggatccggc ggaggtgggt cgggtggcgg cggatctgac 1980 attgtgctca cccaatctcc agcttctttg gctgtgtctc taggtcagag agccaccatc 2040 tcctgcagag ccagtgaaag tgttgaatat tatgtcacaa gtttaatgca 15 gtggtaccaa 2100 cagaaaccag gacagccacc caaactcctc atctctgctg ctagcaacgt agaatctggg 2160

20 gtccctgcca ggtttagtgg cagtgggtct gggacagact ttagcctcaa catccatcct 2220 gtggaggagg atgatattgc aatgtatttc tgtcagcaaa gtaggaaggt tccatggacg 2280 ttcggtggag gcaccaagct ggaaatcaaa cgttaatcta ga 2322

30 <210> 227 <211> 767 <212> PRT <213> Artificial sequence

35 <220>

<223> Synthetic polypeptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

437

2016231617 23 Sep 2016 <220>

<223> n2H7sss!gGl-H6-2el2HL (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221> misc_feature <222> (21) . . (126) <223> VL <220>

<221> misc_feature <222> (127)..(142) <223> Linker <220>

<221> misc_feature <222> (143)..(264) <223> VH <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge <220>

<221> misc_feature <222> (497)..(514) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (515)..(635) <223> VH2

5089144_1 (GHMatters) P79767.AU.1 5/02/14

438

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (636)..(655) <223> Linker2 <220>

<221> misc_feature <222> (656)..(767) <223> VL2 <400> 227

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp 15 Leu Pro

15 10

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He 20 Leu Ser

20 25 30

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 25 Ser Ser

35 40 45

Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 30 Pro Lys

50 55 60

Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 35 Ala Arg

65 70 75

5089144_1 (GHMatters) P79767.AU.1 5/02/14

439

2016231617 23 Sep 2016

5 Thr Asp 305 320 Pro Pro Glu Val Thr Cys 310 Val Val Val Asp Val 315 Ser His Glu 10 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 15 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 20 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 25 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 370 375 380 30 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 385 390 395 400 35 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu

5089144_1 (GHMatters) P79767.AU.1 5/02/14

430

2016231617 23 Sep 2016

Thr Cys

415

Leu Val Lys Gly Glu Ser

420

Asn Gly Gin Pro Leu Asp

435

Ser Asp Gly Ser Lys Ser

450

Arg Trp Gin Gin

Glu Ala

465

480

Leu His Asn His Gly Lys

30 495

Asn Tyr Gly Gly Ser Gin

35 500

410

Asp lie Ala Val Glu Trp

425 430

Lys Thr Thr Pro Pro Val

445

Ser Lys Leu Thr Val Asp

460

Ser Cys Ser Val Met His

475

Ser Leu Ser Leu Ser Pro

490

Gly Gly Gly Ser Gly Asn

505 510

405

Phe Tyr Pro Ser

Glu Asn Asn Tyr

440

Phe Phe Leu Tyr

455

Gly Asn Val Phe

470

Tyr Thr Gin Lys

485

Gly Gly Ser Gly

5089144_1 (GHMatters) P79767.AU.1 5/02/14

431

2016231617 23 Sep 2016

Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser

515 520 525

Leu Ser lie Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly

530 535 540

Val Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly

545 550 555

560

Met He Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser 565 570 20 575

Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val Phe Leu 25 Lys 580 585 590

Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg 30 595 600 605

Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly Gin 35 610 615 620

5089144_1 (GHMatters) P79767.AU.1 5/02/14

432

2016231617 23 Sep 2016

Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

625 630 635

640

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Leu

645 650

655

Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr

660 665 670

He Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu

675 680 685

Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He

690 695 700

Ser Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly

705 710 715

720

Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn He His Pro Val Glu Glu

725 730

735

5089144_1 (GHMatters) P79767.AU.1 5/02/14

433

2016231617 23 Sep 2016

Asp Asp lie Ala Met Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp

740 745 750

Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Arg 755 760

<210> 226 <211> 2322 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> n2H7sss!gGl-H6- -2el2HL (DNA) <400> 226 aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtcaaattgt tctctcccag tctccagcaa tcctgtctgc atctccaggg 120 gagaaggtca caatgacttg cagggccagc tcaagtgtaa gttacatgca ctggtaccag 180 cagaagccag gatcctcccc caaaccctgg atttatgccc catccaacct ggcttctgga 240 gtccctgctc gcttcagtgg cagtgggtct gggacctctt actctctcac aatcagcaga 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

434

2016231617 23 Sep 2016

gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa 5 ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct 10 ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag 15 cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac 20 tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt 25 gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca 30 ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc aaggacaccc ggacgtgagc tcatgatctc 960 ccggacccct gaggtcacat gcgtggtggt 35 cacgaagacc gcataatgcc ctgaggtcaa 1020 gttcaactgg tacgtggacg gcgtggaggt aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

435 gaatggcaag gagtacaagt gcaaggtctc

2016231617 23 Sep 2016 cgtcctcacc gtcctgcacc caacaaagcc ctcccagccc agaaccacag gtgtacaccc cctgacctgc ctggtcaaag tgggcagccg gagaacaact cttcctctac agcaagctca atgctccgtg atgcatgagg tccgggtaag ggtggcggtg ttctcaggtg cagctgaagg catcacatgc accgtctcag gcctccagga aagggtctgg taattcagct ctcaaatcca cttaaaaatg

1080 aggactggct

1140 ccatcgagaa

1200 tgcccccatc

1260 gcttctatcc

1320 acaagaccac

1380 ccgtggacaa

1440 ctctgcacaa

1500 gctcgggcgg

1560 agtcaggacc

1620 ggttctcatt

1680 agtggctggg

1740 gactatcgat

1800 aacaatctcc ccgggatgag cagcgacatc gcctcccgtg gagcaggtgg ccactacacg tggtggatct tggcctggtg aaccggctat aatgatatgg caccaaggac aaagccaaag ctgaccaaga gccgtggagt ctggactccg cagcagggga cagaagagcc gggggaggag gcgccctcac ggtgtaaact ggtgatggaa aactccaaga ggcagccccg accaggtcag gggagagcaa acggctcctt acgtcttctc tctccctgtc gcagcgggaa agagcctgtc gggttcgcca gcacagacta gccaagtttt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

436

2016231617 23 Sep 2016 aacagtctgc aaactgatga cacagccaga tactactgtg ctcgagatgg ttatagtaac 1860

5 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1140 ctcccagccc ccatcgagaa aacaatctcc aaagccaaag ggcagccccg agaaccacag 1200 io gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa 15 tgggcagccg 1320 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

20 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaag 1500 aattatggtg gcggtggctc gggcggtggt ggatctggga attctcaggt gcagctgaag 1560 gagtcaggac ctggcctggt ggcgccctca cagagcctgt ccatcacatg 30 caccgtctca 1620 gggttctcat taaccggcta tggtgtaaac tgggttcgcc agcctccagg aaagggtctg 1680

35 gagtggctgg gaatgatatg gggtgatgga agcacagact ataattcagc tctcaaatcc 1740

5089144_1 (GHMatters) P79767.AU.1 5/02/14

424

2016231617 23 Sep 2016 agactatcga tcaccaagga caactccaag agccaagttt tcttaaaaat gaacagtctg 1800 caaactgatg acacagccag atactactgt gctcgagatg gttatagtaa 5 ctttcattac 1860 tatgttatgg actactgggg tcaaggaacc tcagtcaccg tctcctctgg gggtggaggc 1920 io tctggtggcg gtggatccgg cggaggtggg tcgggtggcg gcggatctga cattgtgctc 1980 acccaatctc cagcttcttt ggctgtgtct ctaggtcaga gagccaccat ctcctgcaga 2040 gccagtgaaa gtgttgaata ttatgtcaca agtttaatgc agtggtacca acagaaacca 2100 ggacagccac ccaaactcct catctctgct gctagcaacg tagaatctgg 20 ggtccctgcc 2160 aggtttagtg gcagtgggtc tgggacagac tttagcctca acatccatcc tgtggaggag 2220

25 gatgatattg caatgtattt ctgtcagcaa agtaggaagg ttccatggac gttcggtgga 2280 ggcaccaagc tggaaatcaa acgttaatct aga 2313

<210> 225 <211> 764 <212> PRT <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

425

2016231617 23 Sep 2016 <223> Synthetic polypeptide <220>

<223> n2H7sssIgGl-H5-2el2HL (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221> misc_feature <222> (21) . . (126) <223> VL <220>

<221> misc_feature <222> (127)..(142) <223> Linker <220>

<221> misc_feature <222> (143)..(264) <223> VH <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge <220>

<221> misc_feature <222> (497)..(511) <223> EFD-BD2 Linker <220>

<221> misc_feature

5089144_1 (GHMatters) P79767.AU.1 5/02/14

426

2016231617 23 Sep 2016

<222> (512) . . (632) <223> VH2 <220> <221> misc feature <222> (633)..(652) <223> Linker2 <220> <221> misc feature <222> (653) . . (764) <223> VL2 <400> 225

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser 20 25 30

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser 35 40 45

Val Pro Ser Lys 50 Tyr Met His Trp Tyr 55 Gin Gin Lys Pro Gly 60 Ser Ser Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

427

2016231617 23 Sep 2016

65 70 80 5 Phe Ser Gly Ser Gly Ser Ser Arg 85 10 Val Glu Ala Glu Asp Ala Ser Phe 100 15 Asn Pro Pro Thr Phe Gly Asp Gly 115 20 Gly Gly Ser Gly Gly Gly Gin Ala 130 25 Tyr Leu Gin Gin Ser Gly Ser Val 145 150 160 30 Lys Met Ser Cys Lys Ala Asn Met 165 175 35 His Trp Val Lys Gin Thr

Gly Thr Ser Tyr Ser Leu Thr lie

Ala Thr Tyr Tyr Cys Gin Gin Trp

105 110

Ala Gly Thr Lys Leu Glu Leu Lys

120 125

Gly Ser Gly Gly Gly Gly Ala Ser

135 140

Ala Glu Leu Val Arg Pro Gly Ala

155

Ser Gly Tyr Thr Phe Thr Ser Tyr

170

Pro Arg Gin Gly Leu Glu Trp He

5089144_1 (GHMatters) P79767.AU.1 5/02/14

428

180

185

190

2016231617 23 Sep 2016

Gly Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly

195 200 205

5 415

Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser

420 425 430

Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

435 440 445

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

450 455 460

Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465 470 475

480

Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro 30 Gly Lys

485 490

495

35 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser Gin Val Gin

500 505 510

5089144_1 (GHMatters) P79767.AU.1 5/02/14

419

2016231617 23 Sep 2016

Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser

515 520 525 lie Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn

530 535 540

Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met He

545 550 555

560

Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu

565 570

575

Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys 25 Met Asn

580 585 590

Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg 30 Asp Gly

595 600 605

Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly Gin 35 Gly Thr

610 615 620

5089144_1 (GHMatters) P79767.AU.1 5/02/14

420

2016231617 23 Sep 2016

Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

625 630 635

640

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Leu Thr Gin

645 650

655

Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr 15 He Ser

660 665 670

Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu 20 Met Gin

675 680 685

Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He 25 Ser Ala

690 695 700

Ala Ser Asn Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly 30 Ser Gly

705 710 715

720

35 Ser Gly Thr Asp Phe Ser Leu Asn He His Pro Val Glu Glu Asp Asp

725 730

5089144_1 (GHMatters) P79767.AU.1 5/02/14

421

2016231617 23 Sep 2016

735 lie Ala Met Tyr Phe Cys Gin Gin Ser Arg Lys 5 Thr Phe

740 745

Gly Gly Gly Thr Lys Leu Glu He Lys Arg 10 755 760 <210> 224 <211> 2313 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> n2H7sssIgGl-H5-2el2HL (DNA) <400> 224

25 aagcttgccg ccatggaagc accagcgcag cttctcttcc ctggctccca 60 gataccaccg gtcaaattgt tctctcccag tctccagcaa atctccaggg 120 gagaaggtca caatgacttg cagggccagc tcaagtgtaa ctggtaccag 180 cagaagccag gatcctcccc caaaccctgg atttatgccc 35 ggcttctgga 240 gtccctgctc gcttcagtgg cagtgggtct gggacctctt

Val Pro Trp

750 tcctgctact tcctgtctgc gttacatgca catccaacct actctctcac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

422

2016231617 23 Sep 2016

aatcagcaga 300 gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc aaggacaccc ggacgtgagc tcatgatctc 960 ccggacccct gaggtcacat gcgtggtggt cacgaagacc gcataatgcc ctgaggtcaa 1020 gttcaactgg tacgtggacg gcgtggaggt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

423

2016231617 23 Sep 2016 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1080

5 <220>

<223> n2H7sss!gGl-H4-2el2HL (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221> misc_feature <222> (21) . . (126) <223> VL <220>

<221> misc_feature <222> (127)..(142) <223> Linker <220>

<221> misc_feature <222> (143)..(264) <223> VH <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge <220>

<221> misc_feature <222> (497) . . (509) <223> EFD-BD2 Linker

5089144_1 (GHMatters) P79767.AU.1 5/02/14

414

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (510)..(630) <223> VH2 <220>

<221> misc_feature <222> (631)..(650) <223> Linker2 <220>

<221> misc_feature <222> (651)..(762) <223> VL2 <400> 223

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

20 1 5 10

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser

25 20 25 30

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

30 35 40 45

Val Ser Tyr Met His Pro Lys

Trp Tyr Gin Gin Lys

Pro Gly Ser Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

415

2016231617 23 Sep 2016

Pro Trp lie Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

65 70 75

5 Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys 515 520 525

5 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys

405 410

415

5 Pro Ala 65 80 Trp Arg He Tyr Ala Pro 70 Ser Phe Ser Gly Ser Gly Ser Gly 10 Ser Arg 85 Val Glu Ala Glu Asp Ala Ala 15 Ser Phe 100 Asn Pro Pro Thr Phe Gly Ala 20 Asp Gly 115 Gly Gly Ser Gly Gly Gly Gly 25 Gin Ala 130 135 Tyr Leu Gin Gin Ser Gly Ala 30 Ser Val 145 150 160 35 Lys Met Ser Cys Lys Ala Ser

Asn Met

165

Asn Leu Ala Ser Gly Val Pro

Thr Ser Tyr Ser Leu Thr lie

Thr Tyr Tyr Cys Gin Gin Trp

105 110

Gly Thr Lys Leu Glu Leu Lys

120 125

Ser Gly Gly Gly Gly Ala Ser

140

Glu Leu Val Arg Pro Gly Ala

155

Gly Tyr Thr Phe Thr Ser Tyr

170

5089144_1 (GHMatters) P79767.AU.1 5/02/14

404

2016231617 23 Sep 2016

175

His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie 5 Gly Ala

180 185 190

He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe 10 Lys Gly

195 200 205

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 15 Met Gin

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 2 0 Ala Arg

225 230 235

240

25 Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr

245 250

255

Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr

260 265 270

His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

405

275

285

2016231617 23 Sep 2016

280

Val Ser Phe Arg 290 Leu Phe Pro Pro Lys 295 Pro Lys Asp Thr Leu 300 Met He Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 370 375 380 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

Thr Leu

5089144_1 (GHMatters) P79767.AU.1 5/02/14

406

2016231617 23 Sep 2016

385

400

390

395

5 735

Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys

5 <220>

<221> misc_feature <222> (505)..(625) <223> VH2

5 Cys Asp Ala Tyr Arg 595 Asp Trp Gly Gin Gly Gly Ser 10 610 Gly Gly Gly Gly Ser Asp 15 625 640 lie Val Leu Thr 20 Gly Gin 655 25 Arg Ala Thr He Tyr Val 660 30 Thr Ser Leu Met Pro Lys 675 Leu Leu He Ser 35 Ala Arg 690

Gly Tyr Ser Asn Phe

600

Thr Ser Val Thr Val

615

Ser Gly Gly Gly Gly

630

Gin Ser Pro Ala Ser

645

Ser Cys Arg Ala Ser

665

Gin Trp Tyr Gin Gin

680

Ala Ala Ser Asn Val

695

His Tyr Tyr Val Met

605

Ser Ser Gly Gly Gly

620

Ser Gly Gly Gly Gly

635

Leu Ala Val Ser Leu

650

Glu Ser Val Glu Tyr

670

Lys Pro Gly Gin Pro

685

Glu Ser Gly Val Pro

700

5089144_1 (GHMatters) P79767.AU.1 5/02/14

385

2016231617 23 Sep 2016

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn lie His Pro 705 710 715 5 720

Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys Gin Gin Ser Arg Lys 10 725 730 735 Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

15 Arg

740 745 750

<210> 218 20 <211> 2292 <212> DNA <213> Artificial sequence <220> 25 <223> Synthetic polynucleotide <220> <223> n2H7sss!gGl-H2-2el2HL (DNA) 30 <400> 218 aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtcaaattgt tctctcccag tctccagcaa tcctgtctgc 35 atctccaggg 120 gagaaggtca caatgacttg cagggccagc tcaagtgtaa gttacatgca

5089144_1 (GHMatters) P79767.AU.1 5/02/14

386

2016231617 23 Sep 2016

ctggtaccag 180 cagaagccag ggcttctgga gatcctcccc 240 caaaccctgg atttatgccc catccaacct gtccctgctc aatcagcaga gcttcagtgg 300 cagtgggtct gggacctctt actctctcac gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

387

2016231617 23 Sep 2016

aaggacaccc ggacgtgagc tcatgatctc 960 ccggacccct gaggtcacat gcgtggtggt 5 cacgaagacc gcataatgcc ctgaggtcaa 1020 gttcaactgg tacgtggacg gcgtggaggt 10 aagacaaagc cgtcctcacc cgcgggagga 1080 gcagtacaac agcacgtacc gtgtggtcag gtcctgcacc caacaaagcc aggactggct 1140 gaatggcaag gagtacaagt gcaaggtctc 15 ctcccagccc agaaccacag ccatcgagaa 1200 aacaatctcc aaagccaaag ggcagccccg gtgtacaccc cctgacctgc tgcccccatc 1260 ccgggatgag ctgaccaaga accaggtcag 20 ctggtcaaag tgggcagccg gcttctatcc 1320 cagcgacatc gccgtggagt gggagagcaa 25 gagaacaact cttcctctac acaagaccac 1380 gcctcccgtg ctggactccg acggctcctt agcaagctca atgctccgtg ccgtggacaa 1440 gagcaggtgg cagcagggga acgtcttctc 30 atgcatgagg tccgggtaag ctctgcacaa 1500 ccactacacg cagaagagcc tctccctgtc ggtggcggtg tggcctggtg gctcggggaa 1560 ttctcaggtg cagctgaagg agtcaggacc 35 gcgccctcac aaccggctat agagcctgtc 1620 catcacatgc accgtctcag ggttctcatt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

388

2016231617 23 Sep 2016 ggtgtaaact gggttcgcca gcctccagga aagggtctgg agtggctggg aatgatatgg 1680 ggtgatggaa gcacagacta taattcagct ctcaaatcca gactatcgat 5 caccaaggac 1740 aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga cacagccaga 1800 io tactactgtg ctcgagatgg ttatagtaac tttcattact atgttatgga ctactggggt 1860 caaggaacct cagtcaccgt ctcctctggg ggtggaggct ctggtggcgg tggatccggc 1920 ggaggtgggt cgggtggcgg cggatctgac attgtgctca cccaatctcc agcttctttg 1980 gctgtgtctc taggtcagag agccaccatc tcctgcagag ccagtgaaag 20 tgttgaatat 2040 tatgtcacaa gtttaatgca gtggtaccaa cagaaaccag gacagccacc caaactcctc 2100

25 atctctgctg ctagcaacgt agaatctggg gtccctgcca ggtttagtgg cagtgggtct 2160 gggacagact ttagcctcaa catccatcct gtggaggagg atgatattgc aatgtatttc 2220 tgtcagcaaa gtaggaaggt tccatggacg ttcggtggag gcaccaagct ggaaatcaaa 2280 cgttaatcta ga 35 2292

5089144_1 (GHMatters) P79767.AU.1 5/02/14

389

2016231617 23 Sep 2016 <210>

<211>

<212>

<213>

219

757

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> n2H7sss!gGl-H2-2el2HL (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221> misc_feature <222> (21) . . (126) <223> VL <220>

<221> misc_feature <222> (127)..(142) <223> Linker <220>

<221> misc_feature <222> (143)..(264) <223> VH <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

390

2016231617 23 Sep 2016 <221> misc_feature <222> (497)..(504) <223> EFD-BD2 Linker

5 Leu Gly 495 His Lys Asn His Tyr 485 Thr Gin Lys Ser Leu 490 Ser Leu Ser Pro Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro 10 500 505 510 Ser Gin Ser Leu Ser He Thr Cys Thr Val Ser Gly Phe Ser Leu Thr 15 515 520 525 Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu 20 530 535 540 Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala 25 545 550 555 560 Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser 30 Gin Val 565 570 575 35 Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg

Tyr Tyr

580 585 590

5089144_1 (GHMatters) P79767.AU.1 5/02/14

384

2016231617 23 Sep 2016

5 10 Lys Lys lie Thr 385 400 Cys Thr 370 Ser Leu Lys Val Ser Lys Asn Gly 390 Lys 375 Gin Ala Pro Leu Arg Pro Ala Pro 380 Gin He Val Glu Tyr Lys Ala Glu Pro 395 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 15 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp 20 Glu Ser 420 425 430 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 25 435 440 445 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 30 450 455 460 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His

Glu Ala

35 465 470 475

480

5089144_1 (GHMatters) P79767.AU.1 5/02/14

383

2016231617 23 Sep 2016

5 cagtggta 2048 <210> 217 <211> 751 <212> PRT 10 <213> Artificial sequence <220> <223> Synthetic polypeptide 15 <220> <223> n2H7sssIgGl-Hl-2el2HL <220> <221> misc feature 20 <222> (1)··(20) <223> Leader <220> <221> misc feature 25 <222> (21) . . (126) <223> VL <220> <221> misc feature 30 <222> (127) . . (142) <223> Linker <220> <221> misc feature 35 <222> (143) . . (264) <223> VH

5089144_1 (GHMatters) P79767.AU.1 5/02/14

378

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge <220>

<221> misc_feature <222> (497) . . (498) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (499) . . (619) <223> VH2 <220>

<221> misc_feature <222> (620)..(639) <223> Linker2 <220>

<221> misc_feature <222> (640)..(751) <223> VL2 <400> 217

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

30 1 5 10

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser

35 20 25 30

5089144_1 (GHMatters) P79767.AU.1 5/02/14

379

2016231617 23 Sep 2016

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

35 40 45

Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys

50 55 60

Pro Trp lie Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

65 70 75

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg

85 90

Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Phe

100 105 110

Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Asp Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ser Gin Ala

130 135 140

Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

5089144_1 (GHMatters) P79767.AU.1 5/02/14

380

Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

Asn Met

165 170

175

2016231617 23 Sep 2016

Ser Val

145

160

150

155

His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie Gly Ala

180 185 190

He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly

195

200

205

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

225 230 235

240

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr

245 250

255

5089144_1 (GHMatters) P79767.AU.1 5/02/14

381

2016231617 23 Sep 2016

Gly Lys Thr Thr Thr Val 260 Thr Val Ser Ser Glu 265 Pro Lys Ser Ser Asp 270 His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys

Glu Tyr

355 360 365

5089144_1 (GHMatters) P79767.AU.1 5/02/14

382

2016231617 23 Sep 2016

5 Gly Gin Pro Pro Lys Leu Leu lie Ser Ala Ala Ser Asn Val Glu Ser

565 570

575

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

580 585 590

Leu Asn He His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys

595 600 605

Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

610 615 620

Glu He Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

625 630 635

640

Gly Gly Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala

645 650

35 655

5089144_1 (GHMatters) P79767.AU.1 5/02/14

373

2016231617 23 Sep 2016

Pro Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser Gly Phe Ser Leu

660 665 670

Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu

675 680 685

Glu Trp Leu Gly Met He Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser

690 695 700

Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser Gin

715

705

720

710

Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr

725 730

25 735

Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp

740 745 750

Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

755

760 <210> 216

5089144_1 (GHMatters) P79767.AU.1 5/02/14

374

2016231617 23 Sep 2016

<211> 2048 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> n2H7sss!gGl-Hl- -2el2HL (DNA) <400> 216 aagcttgccg ctggctccca ccatggaagc 60 accagcgcag cttctcttcc tcctgctact gataccaccg atctccaggg gtcaaattgt 120 tctctcccag tctccagcaa tcctgtctgc gagaaggtca ctggtaccag caatgacttg 180 cagggccagc tcaagtgtaa gttacatgca cagaagccag ggcttctgga gatcctcccc 240 caaaccctgg atttatgccc catccaacct gtccctgctc aatcagcaga gcttcagtgg 300 cagtgggtct gggacctctt actctctcac gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

375

2016231617 23 Sep 2016

cactgggtaa agcagacacc tagacagggc ctggaatgga ttggagctat ttatccagga 600 5 aatggtgata cttcctacaa tcagaagttc aagggcaagg ccacactgac tgtagacaaa 660 tcctccagca cagcctacat gcagctcagc agcctgacat ctgaagactc tgcggtctat 720 10 ttctgtgcaa gagtggtgta ctatagtaac tcttactggt acttcgatgt ctggggcaca 780 gggaccacgg tcaccgtctc gagcgagccc aaatcttctg acaaaactca 15 cacatcccca 840 ccgagcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 900 20 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 960 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1020 25 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1080 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc 30 caacaaagcc 1140 ctcccagccc ccatcgagaa aacaatctcc aaagccaaag ggcagccccg agaaccacag 1200 35 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260

5089144_1 (GHMatters) P79767.AU.1 5/02/14

376

2016231617 23 Sep 2016 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1320 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt 5 cttcctctac 1380 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440 io atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaag 1500 aattctcagg tgcagctgaa ggagtcagga cctggcctgg tggcgccctc acagagcctg 1560 tccatcacat gcaccgtctc agggttctca ttaaccggct atggtgtaaa ctgggttcgc 1620 cagcctccag gaaagggtct ggagtggctg ggaatgatat ggggtgatgg 20 aagcacagac 1680 tataattcag ctctcaaatc cagactatcg atcaccaagg acaactccaa gagccaagtt 1740

25 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca gatactactg tgctcgagat 1800 ggttatagta actttcatta ctatgttatg gactactggg gtcaaggaac ctcagtcacc 1860 gtctcctctg ggggtggagg ctctggtggc ggtggatccg gcggaggtgg gtcgggtggc 1920 ggcggatctg acattgtgct cacccaatct ccagcttctt tggctgtgtc 35 tctaggtcag 1980 agagccacca tctcctgcag agccagtgaa agtgttgaat attatgtcac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

377

2016231617 23 Sep 2016 aagtttaatg 2040

5 Asn Asp Pro Gly Pro 115 Thr Phe Gly Gly Gly Ser Gly Gly Gly 10 Gin Ala 130 Tyr Leu Gin Gin Ser Gly 15 Ser Val 145 150 160 20 Lys Met Ser Cys Lys Ala Asn Met 165 175 25 His Trp Val Lys Gin Thr Gly Ala 180 30 He Tyr Pro Gly Asn Gly Lys Gly 195 35 Lys Ala Thr Leu Thr Val Met Gin

105 110

Ala Gly Thr Lys Leu Glu Leu Lys

120 125

Gly Ser Gly Gly Gly Gly Ala Ser

135 140

Ala Glu Leu Val Arg Pro Gly Ala

155

Ser Gly Tyr Thr Phe Thr Ser Tyr

170

Pro Arg Gin Gly Leu Glu Trp lie

185 190

Asp Thr Ser Tyr Asn Gin Lys Phe

200 205

Asp Lys Ser Ser Ser Thr Ala Tyr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

369

210 215 220

2016231617 23 Sep 2016

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 5 Ala Arg 225 230 235 240

5 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

15 10 15

5 <220>

<221> misc_feature <222> (127)..(142) <223> Linker

5 gtaaactggg ttcgccagcc tccaggaaag ggtctggagt ggctgggaat gatatggggt 2100 gatggaagca cagactataa ttcagctctc aaatccagac tatcgatcac caaggacaac 2160 tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac agccagatac 2220 tactgtgctc gagatggtta tagtaacttt cattactatg ttatggacta 15 ctggggtcaa 2280 ggaacctcag tcaccgtctc ctcttaatct aga 2313 <210> 215 <211> 764 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

30 <223> n2H7sss!gGl-STD2-2el2LH (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

366

2016231617 23 Sep 2016 <221> misc_feature <222> (21) . . (126) <223> VL

5 10 Ser Lys Arg Glu 465 480 Asp Gly Ser Phe Gly Phe Asn 470 Leu 455 Val Tyr Phe Ser Ser Lys Cys Leu Ser 475 Thr 460 Val Val Met Asp His Ser Gin 450 Trp Ala Gin Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 15 485 490 495 Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 Gly Gly 500 505 510 Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly 25 Leu Val 515 520 525 Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser Gly 30 Phe Ser 530 535 540 Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly

35 Lys Gly

545 550 555

560

5089144_1 (GHMatters) P79767.AU.1 5/02/14

360

2016231617 23 Sep 2016

Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr Asn 5 565 570 575

Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser 10 Lys Ser 580 585 590

Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr 15 Ala Arg 595 600 605

Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr 2 0 Val Met 610 615 620

Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Gly 25 Gly 625 Gly 630 635 640

30 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 655 645 650 35 Ser Asp He Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val

Ser Leu

5089144_1 (GHMatters) P79767.AU.1 5/02/14

361

660

665

670

2016231617 23 Sep 2016

Gly Gin Arg Ala Thr lie Ser Cys Arg Ala Ser Glu Ser Val 5 Glu Tyr

675 680 685

Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly 10 Gin Pro

690 695 700

Pro Lys Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser Gly Val Pro

705 710 715

720

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu 20 Asn He

725 730

735

25 His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys Gin Gin Ser

740 745 750

30 Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

755 760 765

35 Arg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

362

2016231617 23 Sep 2016

<210> 214 <211> 2313 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> n2H7sssIgGl-STD2-2el2LH (DNA) <400> 214 aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60 gataccaccg gtcaaattgt tctctcccag tctccagcaa tcctgtctgc atctccaggg 120 gagaaggtca caatgacttg cagggccagc tcaagtgtaa gttacatgca ctggtaccag 180 cagaagccag gatcctcccc caaaccctgg atttatgccc catccaacct ggcttctgga 240 gtccctgctc gcttcagtgg cagtgggtct gggacctctt actctctcac aatcagcaga 300 gtggaggctg aagatgctgc cacttattac tgccagcagt ggagttttaa cccacccacg 360 ttcggtgctg ggaccaagct ggagctgaaa gatggcggtg gctcgggcgg tggtggatct 420 ggaggaggtg gagctagcca ggcttatcta cagcagtctg gggctgagct ggtgaggcct 480

5089144_1 (GHMatters) P79767.AU.1 5/02/14

363

2016231617 23 Sep 2016

ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag 5 cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac 10 tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc 15 ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca 20 ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc aaggacaccc ggacgtgagc tcatgatctc 960 ccggacccct gaggtcacat gcgtggtggt 25 cacgaagacc gcataatgcc ctgaggtcaa 1020 gttcaactgg tacgtggacg gcgtggaggt 30 aagacaaagc cgtcctcacc cgcgggagga 1080 gcagtacaac agcacgtacc gtgtggtcag gtcctgcacc caacaaagcc aggactggct 1140 gaatggcaag gagtacaagt gcaaggtctc 35 ctcccagccc agaaccacag ccatcgagaa 1200 aacaatctcc aaagccaaag ggcagccccg gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

364 cagcgacatc gccgtggagt gggagagcaa

2016231617 23 Sep 2016 cctgacctgc ctggtcaaag tgggcagccg gagaacaact cttcctctac agcaagctca atgctccgtg atgcatgagg tccgggtaag aattatggtg tgggaattct gacattgtgc gagagccacc atctcctgca gcagtggtac caacagaaac cgtagaatct ggggtccctg caacatccat cctgtggagg ggttccatgg acgttcggtg cggcggaggt gggtcgggtg cctggtggcg

1260 gcttctatcc

1320 acaagaccac

1380 ccgtggacaa

1440 ctctgcacaa

1500 gcggtggctc

1560 tcacccaatc

1620 gagccagtga

1680 caggacagcc

1740 ccaggtttag

1800 aggatgatat

1860 gaggcaccaa

1920 gcggcggatc

1980 gcctcccgtg gagcaggtgg ccactacacg gggcggtggt tccagcttct aagtgttgaa acccaaactc tggcagtggg tgcaatgtat gctggaaatc tcaggtgcag ctggactccg cagcagggga cagaagagcc ggatctggag ttggctgtgt tattatgtca ctcatctctg tctgggacag ttctgtcagc aaacggggtg ctgaaggagt acggctcctt acgtcttctc tctccctgtc gaggtgggag ctctaggtca caagtttaat ctgctagcaa actttagcct aaagtaggaa gcggtggatc caggacctgg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

365

2016231617 23 Sep 2016 ccctcacaga gcctgtccat cacatgcacc gtctcagggt tctcattaac cggctatggt 2040

5 Lys Val Thr Val Lys Pro 340 Arg Glu Glu Gin Tyr 345 Asn Ser Thr Tyr Arg 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 10 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 15 370 375 380 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 20 385 390 395 400 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu 25 Thr Cys 405 410 415 30 Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser 420 425 430 35 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

Leu Asp

435 440 445

5089144_1 (GHMatters) P79767.AU.1 5/02/14

359

2016231617 23 Sep 2016

5 Leu Ala 225 240 Ser Arg Ser Leu Thr Ser 230 Glu Asp Ser Ala Val 235 Tyr Phe Cys Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr 245 250 10 255 Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr 15 260 265 270 His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 20 275 280 285 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 25 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 30 305 310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

35 Asn Ala

325 330

335

5089144_1 (GHMatters) P79767.AU.1 5/02/14

358

2016231617 23 Sep 2016

5 <220> <221> <222> <223> misc feature (128) . . (142) Linker 10 <220> <221> <222> <223> misc feature (143)..(264) VH 15 <220> <221> <222> <223> misc feature (265) . . (279) Hinge 20 <220> <221> <222> <223> misc feature (497) . . (516) EFD-BD2 Linker 25 <220> <221> <222> <223> misc feature (517) . . (637) VH2 30 <220> <221> <222> <223> misc feature (638)..(657) Linker2 35 <220> <221> <222> <223> misc feature (658)..(769) VL2 <400> 213

5089144_1 (GHMatters) P79767.AU.1 5/02/14

355

2016231617 23 Sep 2016

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

15 10 15

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser

20 25 30

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

35 40 45

Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys

50 55 60

Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

65 70 75

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg

30 85 90 95

Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Phe

35 100 105 110

5089144_1 (GHMatters) P79767.AU.1 5/02/14

356

2016231617 23 Sep 2016

Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Asp Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ser Gin Ala 130 135 140 Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val 145 150 155 160 Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met 165 170 175 His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp He Gly Ala 180 185 190 lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly 195 200 205 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin 210 215 220

5089144_1 (GHMatters) P79767.AU.1 5/02/14

357

2016231617 23 Sep 2016

5 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaag 1500 aattatggtg gcggtggctc gggcggtggt ggatctggag gaggtgggag tgggaattct 1560 caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag 15 cctgtccatc 1620 acatgcaccg tctcagggtt ctcattaacc ggctatggtg taaactgggt tcgccagcct 1680

20 ccaggaaagg gtctggagtg gctgggaatg atatggggtg atggaagcac agactataat 1740 tcagctctca aatccagact atcgatcacc aaggacaact ccaagagcca agttttctta 1800 aaaatgaaca gtctgcaaac tgatgacaca gccagatact actgtgctcg agatggttat 1860 agtaactttc attactatgt tatggactac tggggtcaag gaacctcagt 30 caccgtctcc 1920 tctgggggtg gaggctctgg tggcggtgga tccggcggag gtgggtcggg tggcggcgga 1980

35 tctgacattg tgctcaccca atctccagct tctttggctg tgtctctagg tcagagagcc 2040

5089144_1 (GHMatters) P79767.AU.1 5/02/14

353

2016231617 23 Sep 2016 accatctcct gcagagccag tgaaagtgtt gaatattatg tcacaagttt aatgcagtgg 2100 taccaacaga aaccaggaca gccacccaaa ctcctcatct ctgctgctag 5 caacgtagaa 2160 tctggggtcc ctgccaggtt tagtggcagt gggtctggga cagactttag cctcaacatc 2220 io catcctgtgg aggaggatga tattgcaatg tatttctgtc agcaaagtag gaaggttcca 2280 tggacgttcg gtggaggcac caagctggaa atcaaacgtt aatctaga 2328

<210> 213 <211> 769 <212> PRT 20 <213> Artificial sequence <220> <223> Synthetic polypeptide 25 <220> <223> n2H7sss!gGl-STDl-2el2HL

30 <220> <221> misc feature <222> (1)··(20) <223> Leader 35 <220> <221> misc feature <222> (21) . . (127) <223> VL

5089144_1 (GHMatters) P79767.AU.1 5/02/14

354

2016231617 23 Sep 2016

5 305

320

Ser His Glu Asp 10 Gly Val

335

Glu Val His Asn 15 Asn Ser

340

Thr Tyr Arg Val 2 0 Trp Leu

355

Asn Gly Lys Glu 25 Pro Ala

370

Arg Thr Pro Glu Val Thr

310

Pro Glu Val Lys Phe Asn

325 330

Ala Lys Thr Lys Pro Arg

345

Val Ser Val Leu Thr Val

360

Tyr Lys Cys Lys Val Ser

375

Cys Val Val Val

315

Trp Tyr Val Asp

Glu Glu Gin Tyr

350

Leu His Gin Asp

365

Asn Lys Ala Leu

380

Pro He Glu Lys Glu Pro

Thr He Ser Lys Ala Lys Gly Gin Pro Arg

385 390 395

400

Gin Val Tyr Asn Gin

Thr Leu Pro

Pro Ser Arg Asp Glu

Leu Thr Lys

405

410

5089144_1 (GHMatters) P79767.AU.1 5/02/14

349

2016231617 23 Sep 2016

415

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 5 lie Ala 420 425 430

Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys 10 Thr Thr 435 440 445

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 15 Lys Leu 450 455 460

Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser 2 0 Cys Ser 465 470 475 480 25 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser

Leu Ser

485 490

495

Leu Ser Pro Gly Lys 500

<210> 212 <211> 2328 <212> DNA

5089144_1 (GHMatters) P79767.AU.1 5/02/14

350

2016231617 23 Sep 2016

<213> Artificial sequence 5 <220> <223> Synthetic polynucleotide <220> <223> n2H7sssIgGl-STDl-2el2HL (DNA) <400> 212 10 aagcttgccg ctggctccca ccatggaagc 60 accagcgcag cttctcttcc tcctgctact gataccaccg atctccaggg gtcaaattgt 120 tctctcccag tctccagcaa tcctgtctgc 15 gagaaggtca ctggtaccag caatgacttg 180 cagggccagc tcaagtgtaa gttacatgca 20 cagaagccag ggcttctgga gatcctcccc 240 caaaccctgg atttatgccc catccaacct gtccctgctc aatcagcaga gcttcagtgg 300 cagtgggtct gggacctctt actctctcac 25 gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg 30 ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct 35 ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag cactgggtaa agcagacacc tagacagggc ctggaatgga ttggagctat

5089144_1 (GHMatters) P79767.AU.1 5/02/14

351 tcagaagttc aagggcaagg ccacactgac

2016231617 23 Sep 2016 ttatccagga aatggtgata tgtagacaaa tcctccagca tgcggtctat ttctgtgcaa ctggggcaca gggaccacgg cacatcccca ccgagcccag cccaaaaccc aaggacaccc ggacgtgagc cacgaagacc gcataatgcc aagacaaagc cgtcctcacc gtcctgcacc caacaaagcc ctcccagccc agaaccacag gtgtacaccc cctgacctgc ctggtcaaag tgggcagccg

600 cttcctacaa

660 cagcctacat

720 gagtggtgta

780 tcaccgtctc

840 cacctgaact

900 tcatgatctc

960 ctgaggtcaa

1020 cgcgggagga

1080 aggactggct

1140 ccatcgagaa

1200 tgcccccatc

1260 gcttctatcc

1320 gcagctcagc ctatagtaac gagcgagccc cctgggggga ccggacccct gttcaactgg gcagtacaac gaatggcaag aacaatctcc ccgggatgag cagcgacatc agcctgacat tcttactggt aaatcttctg ccgtcagtct gaggtcacat tacgtggacg agcacgtacc gagtacaagt aaagccaaag ctgaccaaga gccgtggagt ctgaagactc acttcgatgt acaaaactca tcctcttccc gcgtggtggt gcgtggaggt gtgtggtcag gcaaggtctc ggcagccccg accaggtcag gggagagcaa

5089144_1 (GHMatters) P79767.AU.1 5/02/14

352

2016231617 23 Sep 2016 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1380

5 Asn lie Trp Tyr Tyr 195 Gin Gin Lys Pro Asp 200 Gly Thr Val Lys Leu 205 Leu Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 10 210 215 220 Gly Ser Gly Thr Asp Tyr Ser Leu Thr He Ala Asn Leu Gin Pro Glu 15 225 230 235 240 Asp He Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro 20 Trp Thr 245 250 255 25 Phe Gly Gly Gly Thr Lys Leu Val Thr Lys Arg Ser Ser Glu Pro Lys 260 265 270 30 Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 275 280 285 35 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

Asp Thr

290 295 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

348

2016231617 23 Sep 2016

Leu Met lie Ser Asp Val

5 Asn Gin Lys Phe Lys 85 Gly Lys Ala Thr Leu 90 Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala 10 100 105 110 Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr 15 115 120 125 Phe Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly 20 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Asp 25 145 150 155 160 lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu 30 Gly Asp 165 170 175 35 Arg Val Thr He Ser Cys Arg Ala Ser Gin Asp He Arg Asn

Tyr Leu

180 185 190

5089144_1 (GHMatters) P79767.AU.1 5/02/14

347

2016231617 23 Sep 2016

5 tcatgctccg tgatgcatga ggctctgcac aaccactaca cctctccctg 1500 tctccgggta agtgactcta ga 1522 <210>

<211>

<212>

209

500

PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide

20 <220>

<223> G19-4 LH SMIP (AA) <220>

<221>

<222>

<223>

misc_feature (1)··(20) Leader <220>

<221>

<222>

<223>

misc_feature (21)..(128) VL <220>

<221> misc_feature <222> (129)..(145) <223> Linker ggcagcaggg cgcagaagag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

337

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (146) . . (267) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <400> 209

Met Glu Ala Pro Ala Gin Leu Leu Pro 15 1 5

Asp Thr Thr Gly Asp He Gin Leu Ser 20 20

Ala Ser Leu Gly Asp Arg Val Gin Asp 25 35

He Arg Asn Tyr Leu Asn Trp Thr Val 30 50 55

Lys Leu Leu He Tyr Tyr Thr Pro Ser 35 65 70 80

Leu Phe Leu Leu Leu Leu Trp

Met Thr Gin Thr Thr Ser Ser

25 30

Thr lie Ser Cys Arg Ala Ser

40 45

Tyr Gin Gin Lys Pro Asp Gly

Ser Arg Leu His Ser Gly Val

5089144_1 (GHMatters) P79767.AU.1 5/02/14

338

2016231617 23 Sep 2016

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ala 85 90 5 Asn Leu Gin Pro Glu Asp He Ala Thr Tyr Phe Cys Gin Gin Gly Asn 100 105 110 10 Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Val Thr Lys Arg 115 120 125 15 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 130 135 140 20 Ser Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly 145 150 155 25 160 Ala Ser Met Lys He Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly 30 165 170 175 Tyr He Val Asn Trp Leu Lys Gin Ser His Gly Lys Asn Leu

35 Glu Trp

180

185

190

5089144_1 (GHMatters) P79767.AU.1 5/02/14

339

2016231617 23 Sep 2016 lie Gly Leu He Asn Pro Tyr Lys Gly Leu Thr Thr Tyr Asn Gin Lys

195

200

205

Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala

210

215

220

Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr

225

240

230

235

Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val

245

250

255

Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ser Glu Pro 25 Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 30 Leu Leu

275 280 285

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 Thr Leu

290 295 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

340

2016231617 23 Sep 2016

Met lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

305 310 315

320

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

325 330

335

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn 15 Ser Thr

340 345 350

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp 2 0 Leu Asn

355 360 365

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 25 Ala Pro

370 375 380

He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu 30 Pro Gin

385 390 395

400

35 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val

405 410

5089144_1 (GHMatters) P79767.AU.1 5/02/14

341

2016231617 23 Sep 2016

415

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie 5 Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr 10 Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 15 Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys 20 Ser Val

465 470 475

480

25 Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu

485 490

495

Ser Pro Gly Lys

500 <210> 210 <211> 1525 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

342

2016231617 23 Sep 2016

<220> <223> Synthetic polynucleotide 5 <220> <223> G19- <400> 210 -4 HL SMIP (DNA) 10 aagcttgccg ctggctccca ccatggaagc 60 accagcgcag cttctcttcc tcctgctact gataccaccg gcctggagct gtgaggtcca 120 gctgcaacag tctggacctg aactggtgaa 15 tcaatgaaga cgtgaactgg tttcctgcaa 180 ggcctctggt tactcattca ctggctacat 20 ctgaagcaga atacaaaggt gccatggaaa 240 gaaccttgag tggattggac ttattaatcc cttactacct caagtcatcc acaaccagaa 300 attcaagggc aaggccacat tgactgtaga 25 agcacagcct ctattactgt acatggagct 360 cctcagtctg acatctgaag actctgcagt gcaagatctg cgcagggacc ggtactatgg 420 tgactcagac tggtacttcg atgtctgggg 30 acagtcaccg aggaggtggg tctcctctgg 480 cggcggtggc tcgggcggtg gtggatctgg 35 agcgctagcg tctgggagac acatccagat 540 gacacagact acatcctccc tgtctgcctc agagtcacca ctggtatcag tcagttgcag 600 ggcaagtcag gacattcgca attatttgaa

5089144_1 (GHMatters) P79767.AU.1 5/02/14

343

2016231617 23 Sep 2016

cagaaaccag acactcagga atggaactgt 660 taaactcctg atctactaca catcaagact 5 gtcccatcaa cattgccaac ggttcagtgg 720 cagtgggtct ggaacagatt attctctcac 10 ctgcaaccag tccctggacg aagatattgc 780 cacttacttt tgccaacagg gtaatacact ttcggtggag ttctgacaaa ggaccaaact 840 ggtgaccaaa cgctcgagcg agcccaaatc 15 actcacacat agtcttcctc gcccaccgtg 900 cccagcacct gaactcctgg gtggaccgtc ttccccccaa cacatgcgtg aacccaagga 960 caccctcatg atctcccgga cccctgaggt 20 gtggtggacg ggacggcgtg tgagccacga 1020 agaccctgag gtcaagttca actggtacgt 25 gaggtgcata gtaccgtgtg atgccaagac 1080 aaagccgcgg gaggagcagt acaacagcac gtcagcgtcc caagtgcaag tcaccgtcct 1140 gcaccaggac tggctgaatg gcaaggagta 30 gtctccaaca caaagggcag aagccctccc 1200 agcccccatc gagaaaacca tctccaaagc ccccgagaac caagaaccag cacaggtgta 1260 caccctgccc ccatcccggg atgagctgac 35 gtcagcctga ggagtgggag cctgcctggt 1320 caaaggcttc tatccaagcg acatcgccgt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

344

2016231617 23 Sep 2016 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1380 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ⁵ ggggaacgtc 1440 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1500 io ctgtctccgg gtaagtgact ctaga 1525 <210>

<211>

<212>

<213>

211

501

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> G19-4 HL SMIP (AA)

25 <220>

<221> misc_feature <222> (1) . . (20) <223> Leader

30 <220>

<221> misc_feature <222> (21) . . (142) <223> VH

35 <220>

<221> misc_feature <222> (143)..(159)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

345

2016231617 23 Sep 2016

<223> Linker <220> <221> misc feature 5 <222> (160)..(267) <223> VL <220> <221> misc feature 10 <222> (270) . . (284) <223> Hinge <400> 211 15 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10

5 Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

210 215 220

5 <220>

<221> misc_feature <222> (268) . . (283) <223> Hinge

5 <220> <223> G28- <400> 206 -1 HL SMIP (DNA) aagcttgccg ctggctccca ccatggaagc 60 accagcgcag cttctcttcc tcctgctact 10 gataccaccg gcctggcgct gtgcggtcca 120 gctgcagcag tctggacctg agtcggaaaa tcagtgaaga tatgaactgg tttcctgcaa 180 ggcttctggt tactcattca ctggctacaa 15 gtgaagcaga ttattatggt ataatggaaa 240 gagccttgag tggattggaa atattgatcc 20 ggtactacct caaatcctcc acaaccggaa 300 gttcaagggc aaggccacat tgactgtaga ggcacagcct ctattactgt acatgcagct 360 caagagtctg acatctgagg actctgcagt 25 gcaagatcgg cgtctcttct tcggccctat 420 ggactactgg ggtcaaggaa cctcagtcac ggtggcggtg aggcgggagt gctcgggcgg 480 tggtgggtcg ggtggcggcg gatcaggagg 30 gctagcgaca gggagagact tccagatgac 540 tcagtctcca gcctccctat ctgcatctgt 35 gtcaccatca gtatcagcag catgtcgaac 600 aagtgaaaat gtttacagtt atttggcttg aaacagggaa agaaggtgtg aatctcctca 660 gctcctggtc tcttttgcaa aaaccttagc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

327

2016231617 23 Sep 2016

ccatcaaggt cagcagcctg tcagtggcag 720 tggatcaggc acacagtttt ctctgaagat 5 cagcctgaag gtggacgttc attctggaag 780 ttatttctgt caacatcatt ccgataatcc 10 ggtggaggca tgacaaaact ccgaactgga 840 gatcaaaggc tcgagcgagc ccaaatcttc cacacatgcc cttcctcttc caccgtgccc 900 agcacctgaa ctcctgggtg gaccgtcagt 15 cccccaaaac atgcgtggtg ccaaggacac 960 cctcatgatc tcccggaccc ctgaggtcac gtggacgtga cggcgtggag gccacgaaga 1020 ccctgaggtc aagttcaact ggtacgtgga 20 gtgcataatg ccgtgtggtc ccaagacaaa 1080 gccgcgggag gagcagtaca acagcacgta 25 agcgtcctca gtgcaaggtc ccgtcctgca 1140 ccaggactgg ctgaatggca aggagtacaa tccaacaaag agggcagccc ccctcccagc 1200 ccccatcgag aaaaccatct ccaaagccaa 30 cgagaaccac gaaccaggtc aggtgtacac 1260 cctgccccca tcccgggatg agctgaccaa agcctgacct gtgggagagc gcctggtcaa 1320 aggcttctat ccaagcgaca tcgccgtgga 35 aatgggcagc cgacggctcc cggagaacaa 1380 ctacaagacc acgcctcccg tgctggactc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

328

2016231617 23 Sep 2016 ttcttcctct acagcaagct caccgtggac aagagcaggt gaacgtcttc 1440 tcatgctccg tgatgcatga ggctctgcac aaccactaca 5 cctctccctg 1500 tctccgggta agtgactcta ga 1522 <210> 207 <211> 500 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

20 <223> G28-1 HL SMIP (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221>

<222>

<223>

misc_feature (21) . . (136) VH ggcagcaggg cgcagaagag <220>

<221>

<222>

<223>

misc_feature (137)..(158) Linker <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

329

2016231617 23 Sep 2016 <221> misc_feature <222> (159) . . (266) <223> VL

5 Gly Asn He Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe

195 200 205

5 Arg Phe Ser Gly lie Ser

5 <221> misc_feature <222> (145)..(260) <223> VH <220>

5 Lys Pro 415 Ala Pro Lys Gly Gin 405 Pro Arg Glu Pro Gin 410 Val Tyr Thr Leu Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 10 Leu Val 420 425 430 Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser 15 Asn Gly 435 440 445 Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 20 Ser Asp 450 455 460 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 25 Arg Trp 465 470 475 480 Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 30 Leu His 485 490 495 35 Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

500 505 510

5089144_1 (GHMatters) P79767.AU.1 5/02/14

310

2016231617 23 Sep 2016

<210> 202 <211> 1518 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> 2el2-sss-IgGl LH SMIP (DNA) <400> 202 atggattttc cataatgtcc aagtgcagat 60 tttcagcttc ctgctaatca gtgcttcagt agaggagtcg tctaggtcag acattgtgct 120 cacccaatct ccagcttctt tggctgtgtc agagccacca aagtttaatg tctcctgcag 180 agccagtgaa agtgttgaat attatgtcac cagtggtacc tgctagcaac aacagaaacc 240 aggacagcca cccaaactcc tcatctctgc gtagaatctg ctttagcctc gggtccctgc 300 caggtttagt ggcagtgggt ctgggacaga aacatccatc aagtaggaag ctgtggagga 360 ggatgatatt gcaatgtatt tctgtcagca gttccatgga cggtggatcc cgttcggtgg 420 aggcaccaag ctggaaatca aacggggtgg ggcggaggtg aggacctggc ggtcgggtgg 480 cggcggatct caggtgcagc tgaaggagtc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

311

2016231617 23 Sep 2016

ctggtggcgc ctcattaacc cctcacagag 540 cctgtccatc acatgcaccg tctcagggtt 5 ggctatggtg gctgggaatg taaactgggt 600 tcgccagcct ccaggaaagg gtctagagtg atatggggtg atcgatcacc atggaagcac 660 agactataat tcagctctca aatccagact 10 aaggacaact tgatgacaca ccaagagcca 720 agttttctta aaaatgaaca gtctgcaaac 15 gccagatact tatggactac actgtgctcg 780 agatggttat agtaactttc attactatgt tggggtcaag ttctgacaaa gaacctcagt 840 caccgtctcc tctgatcagg agcccaaatc 20 actcacacat agtcttcctc ccccaccgtc 900 cccagcacct gaactcctgg ggggaccgtc ttccccccaa cacatgcgtg aacccaagga 960 caccctcatg atctcccgga cccctgaggt 25 gtggtggacg ggacggcgtg tgagccacga 1020 agaccctgag gtcaagttca actggtacgt 30 gaggtgcata gtaccgtgtg atgccaagac 1080 aaagccgcgg gaggagcagt acaacagcac gtcagcgtcc caagtgcaag tcaccgtcct 1140 gcaccaggac tggctgaatg gcaaggagta 35 gtctccaaca caaagggcag aagccctccc 1200 agcccccatc gagaaaacaa tctccaaagc ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

312

2016231617 23 Sep 2016 acatcgccgt caagaaccag 1260 gtcagcctga cctgcctggt caaaggcttc tatcccagcg ggagtgggag 1320 agcaatgggc agccggagaa caactacaag accacgcctc ctccgacggc 1380 tccttcttcc tctacagcaa gctcaccgtg gacaagagca i° ggggaacgtc 1440 ttctcatgct ccgtgatgca tgaggctctg cacaaccact gagcctctcc 1500 ccgtgctgga ggtggcagca acacgcagaa

15 ctgtctccgg gtaaatga 1518

<210> 203 20 <211> 505 <212> PRT <213> Artificial sequence <220> 25 <223> Synthetic polypeptide <220> <223> 2el2-sss-IgGl LH SMIP 30 <220> <221> misc feature <222> (1) · · (23) <223> Leader 35 <220> <221> misc feature <222> (24)..(135)

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<223> VL <220> <221> misc feature 5 <222> (136)..(150) <223> Linker <220> <221> misc feature 10 <222> (151)..(271) <223> VH <220> <221> misc feature 15 <222> (274) . . (288) <223> Hinge <400> 203 20 Met Asp Phe Gin Val Gin He Ala Ser 1 5

25 Val Pro He Ala Met Ser 20 Arg Gly Val 30 Ser Leu Ala Val Ser Leu Gly Arg Ala 35 35 Ser Glu Ser Val Glu Tyr Tyr Tyr Gin 50 55

Phe Ser Phe Leu Leu lie Ser

5 180 185 190

Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro

5 Gly Lys Gly Leu Glu Trp Leu 70 Gly Met He Trp 75 Gly Asp Gly Ser 65 80 Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn 10 85 90 Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp 15 100 105 110 Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr 20 115 120 125 Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser 25 130 135 140 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 30 145 150 155 160 Gly Gly Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser

35 Leu Ala

165 170

175

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2016231617 23 Sep 2016

Val Ser Leu Gly Gin Arg Ala Thr lie Ser Cys Arg Ala Ser Glu Ser

5 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1380 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1440 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1500 cagaagagcc tctccctgtc tccgggtaaa tga

15 1533 <210> 201 <211> 510 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide 25 <220> <223> 2el2-sss-IgGl HL SMIP <220> 30 <221> misc feature <222> (1)··(22) <223> Leader <220> 35 <221> misc feature <222> (24) . . (144) <223> VH

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<220> <221> <222> misc feature (145)..(164) 5 <223> Linker <220> <221> misc feature <222> (165) . . (276) 10 <223> VL <220> <221> misc feature <222> (279) . . (293) 15 <223> Hinge

<400> 201

Met Asp Phe Gin Val Gin lie

20 Ala Ser 1 5

25 Val Gly He Pro Met Ser 20 Arg Gly Val Gly Leu Val Ala Pro Ser Gin 30 Val Ser 35 Gly Phe Ser Leu Thr Gly Tyr 35 Pro Pro 50 55

Phe Ser Phe Leu Leu He Ser

5 660 665 670

Ser Cys Arg Ala Ser Gin Asp He Arg Asn Tyr Leu Asn Trp Tyr Gin

5 Glu Asp Glu Val 335 Pro Glu Val 325 Lys Phe Asn Trp Tyr 330 Val Asp Gly Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 340 345 350 10 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 355 360 365 15 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 370 375 380 20 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 25 400

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 30 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala

35 Val Glu

420 425 430

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2016231617 23 Sep 2016

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435

440

445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450

455

460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465

480

470

475

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485

490

495

Pro Gly Lys Gly Cys Pro Pro Cys Pro Asn Ser Glu Val Gin 25 Leu Gin

500 505 510

Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys lie Ser

515

520

525

Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr He Val Asn Trp Leu

530

535

540

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2016231617 23 Sep 2016

Lys Asn 545 560 Gin Pro Ser His Gly Lys Asn 550 Leu Glu Trp lie 555 Gly Leu He Tyr Lys Gly Leu Thr Thr Tyr Asn Gin Lys Phe Lys Gly Lys Ala Thr 565 570 575 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser 580 585 590 Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 595 600 605 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 610 615 620 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 625 630 635 640 Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Asp He Gin

35 Met Thr

645 650

655

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Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr lie

5 Lys Asn 545 560 Gin Pro Ser His Gly Lys 550 Asn Leu Glu Trp He 555 Gly Leu He Tyr Lys Gly Leu Thr Thr Tyr Asn Gin Lys Phe Lys Gly Lys 10 Ala Thr 565 570 575 15 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser 580 585 590 20 Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 595 600 605 25 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr Thr 610 615 620 30 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 625 630 635 640 35 Gly Gly Gly Ser Ala Ser Asp lie Gin Met Thr Gin Thr Thr

Ser Ser

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2016231617 23 Sep 2016

655

645

650

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr He Ser Cys Arg 5 Ala Ser 660 665 670 Gin Asp He Arg Asn Tyr Leu Asn Trp Tyr Gin Gin Lys Pro 10 Asp Gly 675 680 685 Thr Val Lys Leu Leu He Tyr Tyr Thr Ser Arg Leu His Ser 15 Gly Val 690 695 700 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser 20 Leu Thr 705 710 715 720 25 lie Ala Asn Leu Gin Pro Glu Asp He Ala Thr Tyr Phe Cys

Gin Gin

725

730

735

Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Val Thr

740 745 750

Lys Arg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<210> 198 <211> 2298 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H7-G281 HL (DNA) <400> 198 aagcttgccg cagtgcttca ccatggattt 60 tcaagtgcag attttcagct tcctgctaat gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

291

2016231617 23 Sep 2016

aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 5 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 10 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 15 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga 20 acatccccac cctcttcccc cgtgcccagc 900 acctgaactc ctggggggat cgtcagtctt 25 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg 30 cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg gtcctcaccg caaggtctcc tcctgcacca 1140 ggactggctg aatggcaagg agtacaagtg 35 aacaaagccc gcagccccga tcccagcccc 1200 catcgagaaa accatctcca aagccaaagg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

292

2016231617 23 Sep 2016 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260 ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ccgtggagtg 5 ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380 io ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500 ccgggtaagg ggtgtccacc ttgtccgaat tctgaggtcc agctgcaaca gtctggacct 1560 gaactggtga agcctggagc ttcaatgaag atttcctgca aggcctctgg 20 ttactcattc 1620 actggctaca tcgtgaactg gctgaagcag agccatggaa agaaccttga gtggattgga 1680

25 cttattaatc catacaaagg tcttactacc tacaaccaga aattcaaggg caaggccaca 1740 ttaactgtag acaagtcatc cagcacagcc tacatggagc tcctcagtct gacatctgaa 1800 gactctgcag tctattactg tgcaagatct gggtactatg gtgactcgga ctggtacttc 1860 gatgtctggg gcgcagggac cacggtcacc gtctcctctg gtggcggtgg 35 ctcgggcggt 1920 ggtggatctg gaggaggtgg gagcggggga ggtggcagtg ctagcgacat

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2016231617 23 Sep 2016 ccagatgaca 1980 cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 2040 agtcaggaca ttcgcaatta tttaaactgg tatcagcaga aaccagatgg aactgttaaa 2100 ctcctgatct actacacatc aagattacac tcaggagtcc catcaaggtt io cagtggcagt 2160 gggtctggaa cagattattc tctcaccatt gccaacctgc aaccagaaga tattgccact 2220

15 tacttttgcc aacagggtaa tacgcttccg tggacgttcg gtggaggcac caaactggta 2280 accaaacggt aatctaga 2298 <210> 199 <211> 759 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide

30 <220>

<223> 2H7sssIgGl-H7-G281 HL (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader

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2016231617 23 Sep 2016 <220>

<221> misc feature <222> (23)..(128) 5 <223> VL1

<220> <221> misc feature <222> (129) . . (144) 10 <223> Linker

<220> <221> misc feature <222> (145)..(265) 15 <223> VH1

<220> <221> misc feature <222> (268)..(282) 20 <223> Hinge

<220> <221> misc feature <222> (500)..(507) 25 <223> EFD-BD2 Linker

<220> <221> misc feature <222> (508)..(629) 30 <223> VH2

<220> <221> misc feature <222> (630)..(651) 35 <223> Linker2

<220> <221> misc feature <222> (630)..(651) 35 <223> Linker2 <220>

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2016231617 23 Sep 2016

<221> mi sc feature <222> (652) . . (759) <223> VL2 5 <400> 199

5 Glu Glu 335 Asp Val Pro Glu Val 325 Lys Phe Asn Trp Tyr Val Asp Gly Val 330 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 10 Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 15 Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 20 Pro lie 370 375 380 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 25 Gin Val 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin 30 Val Ser 405 410 415 35 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala

Val Glu

420 425 430

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Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435

440

445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450

455

460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465

480

470

475

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 20 Leu Ser

485 490

495

25 Pro Gly Lys Gly Cys Pro Pro Cys Pro Asn Ser Glu Val Gin Leu Gin

500 505 510

30 Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys lie Ser

515 520 525

35 Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr He Val Asn Trp Leu

530 535 540

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5 Ser Gin Arg Trp Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 110 100 105 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 10 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 15 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala 145 150 155 20 160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 25 165 170 175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu 30 Trp lie 180 185 190 Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin

35 Lys Phe

195 200 205

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Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys 20 Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Cys Pro Ala Pro Glu Leu 25 Leu Gly

275 280 285

Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 30 Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 Ser His

305 310 315

320

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

5 <222>

<223>

<220>

<221>

5 ctgtctgcct ctctgggaga cagagtcacc atcagttgca gggcaagtca ggacattcgc 2040 aattatttaa actggtatca gcagaaacca gatggaactg ttaaactcct gatctactac 2100 acatcaagat tacactcagg agtcccatca aggttcagtg gcagtgggtc tggaacagat 2160 tattctctca ccattgccaa cctgcaacca gaagatattg ccacttactt 15 ttgccaacag 2220 ggtaatacgc ttccgtggac gttcggtgga ggcaccaaac tggtaaccaa acggtaatct 2280

5 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

5 <220>

<221> misc_feature <222> (23) . . (128) <223> VL

5 gaaccacagg tgtacaccct gcccccatcc cgggatgagc ccaggtcagc 1260 ctgacctgcc tggtcaaagg cttctatcca agcgacatcg ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc cggctccttc 1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc 15 cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc ctccctgtct 1500

20 ccgggtaagg ggtgtccacc ttgtccgaat tctcaggtgc gtcagggcct 1560 ggcagcgtgg cgccctcaca gagcctgtcc atcacatgca gttctcatta 1620 accggctatg gtgtaaactg ggttcgccag cctccaggaa gtggctggga 1680 atgatatggg gtgatggaag cacagactat aattcagctc 30 actatcgatc 1740 accaaggaca actccaagag ccaagttttc ttaaaaatga aactgatgac 1800 aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct agctgaagga ccgtctcagg agggtctgga tcaaatccag acagtctgca

35 acagccagat actactgtgc tcgagatggt tatagtaact tgttatggac 1860 ttcattacta

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2016231617 23 Sep 2016 tactggggtc tggtggcggt ggatccggcg ccaatctcca gcttctttgg cagtgaaagt gttgaatatt acagccaccc aagctcctca gtttagtggc agtgggtctg tgatattgca atgtatttct caccaagctg gaaatcaaac

2301 aaggaacctc

1920 gaggtgggtc

1980 ctgtgtctct

2040 atgtcacaag

2100 tctctgctgc

2160 ggacagactt

2220 gtcagcaaag

2280 gttaatctag agtcaccgtc gggtggcggc aggtcagaga tttaatgcag tagcaacgta tagcctcaac taggaaggtt <210>

<211>

<212>

<213>

195

760

PRT

Artificial sequence <220>

<223> Synthetic polypeptide tcctctgggg ggatctgaca gccaccatct tggtaccaac gaatctgggg atccatcctg ccatggacgt <220>

<223> 2H7sscIgGl-H7-2el2HL (w/2el2 linker gtggaggctc ttgtgctcac cctgcagagc agaaaccagg tccctgccag tggaggagga tcggtggagg (AA) <220>

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2016231617 23 Sep 2016 <221> misc_feature <222> (1)..(22) <223> Leader

5 Lys Arg 770 <210> 194 <211> 2301 <212> DNA 10 <213> Artificial sequence <220> <223> Synthetic polynucleotide 15 <220> <223> 2H7sscIgGl-H7-2el2HL (DNA) <400> 194 aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat 20 cagtgcttca 60 gtcataatgt ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120 25 ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180 taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240 30 tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300 agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag 35 ttttaaccca 360 cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc

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gggcggtggt 420 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga acatccccac cctcttcccc cgtgcccagc 900 acctgaactc ctggggggat cgtcagtctt ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg gtcctcaccg caaggtctcc tcctgcacca 1140 ggactggctg aatggcaagg agtacaagtg

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2016231617 23 Sep 2016 aacaaagccc tcccagcccc catcgagaaa accatctcca gcagccccga 1200

5 Ser Gly 545 560 Leu Lys Thr Gly Tyr Gly 550 Val Asn Trp Val Arg 555 Gin Pro Pro Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr 10 575 565 570 Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn 15 Ser Lys 580 585 590 Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp 20 Thr Ala 595 600 605 Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr 25 Tyr Val 610 615 620 Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser 30 Gly Gly 625 630 635 640 35 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

Gly Gly

645 650

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2016231617 23 Sep 2016

655

Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala 5 Val Ser

660 665 670

Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser 10 Val Glu

675 680 685

Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro 15 Gly Gin

690 695 700

Pro Pro Lys Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser Gly Val

705 710 715

720

Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 25 Leu Asn

725 730

735

30 He His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys Gin Gin

740 745 750

35 Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He

755 760 765

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5 Glu Glu 335 Asp Val Pro Glu Val 325 Lys Phe Asn Trp Tyr Val Asp Gly Val 330 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 10 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 15 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 20 370 375 380 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 25 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin 30 Val Ser 405 410 415 35 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala

Val Glu

420 425 430

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2016231617 23 Sep 2016

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 25 Gly Gly

500 505 510

Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro 30 Gly Leu

515 520 525

Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser 35 Gly Phe

530 535 540

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2016231617 23 Sep 2016

5 Ser Gin Arg Trp Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 110 100 105 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 10 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 15 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala 145 150 155 20 160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 25 165 170 175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu 30 Trp lie

180

5 <222>

<223>

<220>

<221>

5 lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gin

660 665 670

5 <220>

<221> misc_feature <222> (23) . . (128) <223> VL

5 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu lie 755 760

Lys Arg 765

5 Ser Gly Leu Tyr 530 Ser He Thr Cys Thr 535 Val Ser Gly Phe Ser 540 Leu Thr Gly Val Asn Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu 10 545 550 555 560 Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala 15 Leu Lys 565 570 575 20 Ser Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val Phe Leu 580 585 590 25 Lys Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala 595 600 605 30 Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly 610 615 620 35 Gin Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser

Gly Gly

625 630 635

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640

Gly Gly Ser Gly 5 lie Val

655

5 He Ser 305 320 Ser Arg His Thr Pro Glu 310 Val Thr Cys Val Val Val 315 Asp Val Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 10 335 325 330 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 15 Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 20 Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 25 Pro lie 370 375 380 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 30 Gin Val 385 390 395 400 35 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin

Val Ser

405 410

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415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala 5 Val Glu

420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 10 Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 15 Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 20 Val Met

465 470 475

480

25 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser

500 505 510

35 Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin

515 520 525

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5 85 90

Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp

5 <220>

<221> misc_feature <222> (1)..(22) <223> Leader

5 625

640

Gly Gly Gly Gly Ser 10 Thr Gin

645

655

15 Ser Pro Ala Ser Leu lie Ser

660

Cys Arg Ala Ser Glu 20 Met Gin

675

Trp Tyr Gin Gin Lys 25 Ser Ala

690

Ala Ser Asn Val Glu 30 Ser Gly

705

720

Ser Gly Gly Gly Gly

630

Gly Gly Gly Gly Ser

650

Ala Val Ser Leu Gly

665

Ser Val Glu Tyr Tyr

680

Pro Gly Gin Pro Pro

695

Ser Gly Val Pro Ala

710

Ser Leu Asn He His

730

Ser Gly Gly Gly

635

Asp He Val Leu

Gin Arg Ala Thr

670

Val Thr Ser Leu

685

Lys Leu Leu He

700

Arg Phe Ser Gly

715

Pro Val Glu Glu

35 Ser Gly Thr Asp Phe Asp Asp

725

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735 lie Ala Met Tyr Phe Cys Gin Gin Thr Phe

740

Ser Arg Lys

745

Gly Gly Gly Thr Lys Leu Glu He Lys Arg

5 405 410

415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu

5 10 Gly Leu lie Ser 305 320 Pro Met 290 Ser His Ser Arg Val Thr Phe Pro Leu Glu 310 Phe 295 Val Pro Thr Pro Cys Lys Val Pro Val 315 Lys Asp 300 Thr Val Val Asp Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 15 325 330 335 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 20 Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 25 Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 30 Pro He 370 375 380 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 35 Gin Val 385 390 395 400

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Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

5 Pro Val 65 80 Lys Pro Pro Trp He Tyr 70 Ala Ala Arg Phe Ser Gly Ser Gly Thr lie 10 85 Ser Arg Val Glu Ala Glu Asp Gin Trp 15 100 Ser Phe Asn Pro Pro Thr Phe Leu Lys 20 115 Asp Gly Gly Gly Ser Gly Gly Ser Ser 25 130 135 Gin Ala Tyr Leu Gin Gin Ser Gly Ala 30 145 150 160 Ser Val Lys Met Ser Cys Lys Ser Tyr

35 165

175

Pro Ser Asn Leu Ala Ser Gly

Ser Gly Thr Ser Tyr Ser Leu

Ala Ala Thr Tyr Tyr Cys Gin

105 110

Gly Ala Gly Thr Lys Leu Glu

120 125

Gly Gly Ser Gly Gly Gly Gly

140

Gly Ala Glu Ser Val Arg Pro

155

Ala Ser Gly Tyr Thr Phe Thr

170

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Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys 30 Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu 35 Leu Gly

275 280 285

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5 610 615 620

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

5 Pro Leu Gly Lys Lys Gly 500 Gly Gly Glu Ser Gly Pro Gly Leu 10 He Thr 515 Cys Thr Val Ser Gly Phe 15 Trp Val 530 Arg Gin Pro Pro Gly Lys 20 Trp Gly 545 550 560 25 Asp Gly Ser Thr Asp Tyr Ser He 565 575 30 Thr Lys Asp Asn Ser Lys Ser Leu 580 35 Gin Thr Asp Asp Thr Ala

Tyr Ser

595

Gly Ser Gly Asn Ser Gin Val Gin

505 510

Val Ala Pro Ser Gin Ser Leu Ser

520 525

Ser Leu Thr Gly Tyr Gly Val Asn

535 540

Gly Leu Glu Trp Leu Gly Met lie

555

Asn Ser Ala Leu Lys Ser Arg Leu

570

Ser Gin Val Phe Leu Lys Met Asn

585 590

Arg Tyr Tyr Cys Ala Arg Asp Gly

600 605

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Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val

5 400

Tyr Thr Leu Pro Pro Val Ser

5 175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp 30 Val Trp

245 250

255

35 Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

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Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 25 Thr Tyr

340 345 350

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 30 Asn Gly

355 360 365

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 35 Pro He

370 375 380

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Glu Lys Thr lie Ser

Gin Val

385

5 10 Ser Ser Pro Val 65 80 Ser Val Ser Trp Tyr He Met His Trp Pro Tyr Ser Gin Asn Gin Leu 75 Lys 60 Ala Pro Ser Gly Gly Ser 50 Lys Pro Pro Tyr 70 55 Ala Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie 15 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 20 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 25 115 120 125 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 30 130 135 140 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro

Gly Ala

35 145 150 155

160

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

5 Arg Tyr Tyr Val Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His 605 Tyr 595 600 Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Gly Gly 10 610 615 620 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 15 625 630 635 640 Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala 20 Val Ser 645 650 655 25 Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser Val Glu 660 665 670 30 Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin 675 680 685 35 Pro Pro Lys Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser Gly Val 690 695 700

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Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Leu Asn 5 705 710 715 720

He His Pro Val Glu Glu Asp Asp He Ala Met 10 Gin Gin 725 730 735 Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly

15 Glu lie

740 745

Asp Phe Ser

Tyr Phe Cys

Thr Lys Leu

750

Lys Arg

<210> 184 <211> 2301 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H2-2el2HL (DNA) <400> 184

aagcttgccg ccatggattt tcaagtgcag attttcagct cagtgcttca 60 tcctgctaat

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gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct 5 ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc 10 tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc 15 agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 20 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 25 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 30 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 35 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga ccacggtcac cgtctctgat caggagccca aatcttctga

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2016231617 23 Sep 2016 caaaactcac

840 acatccccac cgagcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 900 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg io cgtggaggtg 1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080

15 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140 aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg 25 ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa 30 cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

35 ccgggtaagg gtggcggtgg ctcggggaat tctcaggtgc agctgaagga gtcaggacct 1560

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2016231617 23 Sep 2016 ggcctggtgg gttctcatta accggctatg gtggctggga atgatatggg actatcgatc accaaggaca aactgatgac acagccagat tgttatggac tactggggtc tggtggcggt ggatccggcg ccaatctcca gcttctttgg cagtgaaagt gttgaatatt acagccaccc aaactcctca gtttagtggc agtgggtctg tgatattgca atgtatttct caccaagctg gaaatcaaac cgccctcaca

1620 gtgtaaactg

1680 gtgatggaag

1740 actccaagag

1800 actactgtgc

1860 aaggaacctc

1920 gaggtgggtc

1980 ctgtgtctct

2040 atgtcacaag

2100 tctctgctgc

2160 ggacagactt

2220 gtcagcaaag

2280 gttaatctag gagcctgtcc ggttcgccag cacagactat ccaagttttc tcgagatggt agtcaccgtc gggtggcggc aggtcagaga tttaatgcag tagcaacgta tagcctcaac taggaaggtt atcacatgca cctccaggaa aattcagctc ttaaaaatga tatagtaact tcctctgggg ggatctgaca gccaccatct tggtaccaac gaatctgggg atccatcctg ccatggacgt ccgtctcagg agggtctgga tcaaatccag acagtctgca ttcattacta gtggaggctc ttgtgctcac cctgcagagc agaaaccagg tccctgccag tggaggagga tcggtggagg

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2301 <210>

<211>

<212>

<213>

185

760

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7sssIgGl-H2-2el2HL (w/2el2 leader) (AA)

15 <220>

<221> misc_feature <222> (1)..(22) <223> Leader

20 <220>

<221> misc_feature <222> (23)..(127) <223> VL

25 <220>

<221> misc_feature <222> (128)..(144) <223> Linker

30 <220>

<221> misc_feature <222> (145)..(265) <223> VH

35 <220>

<221> misc_feature <222> (268)..(282)

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2016231617 23 Sep 2016 <223> Hinge <220>

<221> misc_feature <222> (500)..(507) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (508)..(628) <223> VH2 <220>

<221>

<222>

<223>

misc_feature (629)..(648) Linker2 <220>

<221>

<222>

<223>

misc_feature (649)..(760) VL2 <400> 185

25 Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

15 10

30 Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He

35 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

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5 His Leu 495 Glu Ser Ala Leu His 485 Asn His Tyr Thr Gin 490 Lys Ser Leu Ser Pro Gly Lys Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu 10 500 505 510 Val Ala Pro Ser Gin Ser Leu Ser He Thr Cys Thr Val Ser Gly Phe 15 515 520 525 Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly Lys 20 530 535 540 Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr 25 545 550 555 560 Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn 30 Ser Lys 565 570 575 35 Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp

Thr Ala

580 585 590

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5 acatccccac cgagcccagc acctgaactc ctggggggac cgtcagtctt cctcttcccc 900 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg 15 tgtggtcagc 1080 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140

20 aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga 30 cggctccttc 1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440

35 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

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2016231617 23 Sep 2016 ccgggtaaga attctcaggt gcagctgaag gagtcaggac ctggcctggt ggcgccctca 1560 cagagcctgt ccatcacatg caccgtctca gggttctcat taaccggcta 5 tggtgtaaac 1620 tgggttcgcc agcctccagg aaagggtctg gagtggctgg gaatgatatg gggtgatgga 1680 io agcacagact ataattcagc tctcaaatcc agactatcga tcaccaagga caactccaag 1740 agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccag atactactgt 1800 gctcgagatg gttatagtaa ctttcattac tatgttatgg actactgggg tcaaggaacc 1860 tcagtcaccg tctcctctgg gggtggaggc tctggtggcg gtggatccgg 20 cggaggtggg 1920 tcgggtggcg gcggatctga cattgtgctc acccaatctc cagcttcttt ggctgtgtct 1980

25 ctaggtcaga gagccaccat ctcctgcaga gccagtgaaa gtgttgaata ttatgtcaca 2040 agtttaatgc agtggtacca acagaaacca ggacagccac ccaaactcct catctctgct 2100 gctagcaacg tagaatctgg ggtccctgcc aggtttagtg gcagtgggtc tgggacagac 2160 tttagcctca acatccatcc tgtggaggag gatgatattg caatgtattt 35 ctgtcagcaa 2220 agtaggaagg ttccatggac gttcggtgga ggcaccaagc tggaaatcaa

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2016231617 23 Sep 2016 acgttaatct 2280 aga

2283 <210>

<211>

<212>

<213>

183

754

PRT

Artificial sequence <220>

<223> Synthetic polypeptide

15 <220>

<223> 2H7sssIgGl-Hl-2el2HL (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH

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2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (268) . . (281) <223> Hinge <220>

<221> misc_feature <222> (500)..(501) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (502)..(622) <223> VH2 <220>

<221> misc_feature <222> (623)..(642) <223> Linker2 <220>

<221> misc_feature <222> (643)..(754) <223> VL2 <400> 183

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

30 1 5 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He

35 20 25 30

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Leu Ala Ser Ser Ala 35 Ser Pro Gly Glu Lys 40 Val Thr Met Thr Cys 45 Arg Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro

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Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

Ser Tyr

170

2016231617 23 Sep 2016

Gly Ala

145

160

175

165

150

155

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

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Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 30 Thr Tyr

340 345 350

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 35 Asn Gly

355 360 365

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Lys Pro 5 Glu He 370 Tyr Lys Cys Lys Val 375 Ser Asn Lys Ala Leu 380 Pro Ala Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 10 400

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 15 415 405 410

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala 2 0 Val Glu 420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 25 Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

30 Thr Val

450 455 460

Asp Lys Ser Arg Val Met

Trp Gin Gin

Gly Asn Val

Phe Ser Cys Ser

465 470 475

480

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5 Gly Gly Gly Gly Gly Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly 515 520 525

5 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

405 410

415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu

420 425 430

15 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

20 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

25 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Gly 500 505 510

5 Pro Val 65 80 Lys Pro Pro Trp He Tyr 70 Ala Pro Ser Asn Leu 75 Ala Ser Gly 10 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie 85 90 15 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 20 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 25 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 30 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala 145 150 155 160 35 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

Ser Tyr

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165

170

175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu 5 Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin 10 Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 15 Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 2 o Phe Cys

225 230 235

240

25 Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

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2016231617 23 Sep 2016

275

Gly Ser Ser Val Phe 5 Leu Met

290 lie Ser Arg Thr Pro 10 Ser His

305

320

15 Glu Asp Pro Glu Val Glu Val

325

335

His Asn Ala Lys Thr Thr Tyr

340

Arg Val Val Ser Val Asn Gly

355

Lys Glu Tyr Lys Cys Ser He

370

280

Leu Phe Pro Pro Lys

295

Glu Val Thr Cys Val

310

Lys Phe Asn Trp Tyr

330

Lys Pro Arg Glu Glu

345

Leu Thr Val Leu His

360

Lys Val Ser Asn Lys

375

Lys Ala Lys Gly Gin

285

Pro Lys Asp Thr

300

Val Val Asp Val

315

Val Asp Gly Val

Gin Tyr Asn Ser

350

Gin Asp Trp Leu

365

Ala Leu Pro Ala

380

Pro Arg Glu Pro

Glu Lys Thr He Ser Gin Val

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

385

400

390

395

5 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

Pro lie

370 375 380

2016231617 23 Sep 2016

Asn Gly

360

5 Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys

Ser Ser

260 265 270

2016231617 23 Sep 2016

Val Trp

255

245

250

5 atccatcctg tggaggagga tgatattgca atgtatttct gtcagcaaag taggaaggtt 2340 ccatggacgt tcggtggagg caccaagctg gaaatcaaac gttaatctag a

5 735

Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp

5 10 Gly Asp Ala Phe 625 640 Thr lie 610 Met Gly Asp Phe Ser Leu Asn 615 He His Pro Val Glu 620 Pro Glu Trp Asp Thr Tyr Phe Lys Val 635 Cys Gin 630 Gin Ser Arg Gly Gly Thr Lys Leu Glu He Lys Arg Gly Gly Gly Gly Ser Gly Gly 15 645 650 655 Gly Gly Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Lys Glu 20 Ser Gly 660 665 670 Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser He Thr Cys 25 Thr Val 675 680 685 Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg 30 Gin Pro 690 695 700 Pro Gly Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly Asp

35 Gly Ser

705 710 715

720

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2016231617 23 Sep 2016

Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser lie Thr Lys Asp

725 730

5 Pro Gly Lys Asn 500 Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 505 510 Gly Gly Gly Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly 515 520 525 10 Ser Gly Gly Gly Gly Ser Gly Asn Ser Asp lie Val Leu Thr Gin Ser 530 535 540 15 Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys 545 550 555 20 560

Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp 25 575 565 570

Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser 30 Ala Ala 580 585 590 Ser Asn Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

35 Gly Ser

595 600 605

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5 Tyr Ala Gin Ala Gin Lys 580 Pro Gly Ser Asn Val Glu Ser Gly Gly Ser 10 595 Gly Thr Asp Phe Ser Leu Asp He 15 610 Ala Met Tyr Phe Cys Gin Phe Gly 20 625 630 640 Gly Gly Thr Lys Leu Glu 25 Gly Gly 645 655 30 Gly Gly Ser Gly Gly Gly Ser Gly 660 35 Pro Gly Leu Val Ala Pro Thr Val

675

Gin Pro Pro Lys Leu Leu lie Ser

585 590

Val Pro Ala Arg Phe Ser Gly Ser

600 605

Asn He His Pro Val Glu Glu Asp

615 620

Gin Ser Arg Lys Val Pro Trp Thr

635

He Lys Arg Gly Gly Gly Gly Ser

650

Gly Ser Gin Val Gin Leu Lys Glu

665 670

Ser Gin Ser Leu Ser He Thr Cys

680 685

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Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro

690 695 700

Pro Gly Lys Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser

705 710 715

720

Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr 15 Lys Asp

725 730

735

20 Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp

740 745 750

25 Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His

755 760 765

30 Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser

770 775 780

35 Ser 785

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <210>

<211>

<212>

<213>

178

785

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

5 Asp Lys Ser Arg Trp Gin 470 Gin Gly Asn Val Phe 475 Ser Cys Ser Val 465 480 Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 10 495 485 490 Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly 15 Ser Gly 500 505 510 Gly Gly Gly Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly 20 Gly Gly 515 520 525 Ser Gly Gly Gly Gly Ser Gly Asn Ser Asp lie Val Leu Thr 25 Gin Ser 530 535 540 Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys 30 545 550 555 560 Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met

35 Gin Trp

565 570

575

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2016231617 23 Sep 2016

5 Arg Asn Val Gly Val 355 Ser Val Leu Thr Val 360 Leu His Gin Asp Trp 365 Leu Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 370 375 380 10 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 15 400

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 20 415 405 410

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala 25 Val Glu 420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 30 Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

35 Thr Val

450 455 460

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2016231617 23 Sep 2016

5 255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300

He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 35 Thr Tyr

340 345 350

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2016231617 23 Sep 2016

5 ccgggtaaga attatggtgg cggtggctcg ggcggtggtg gatctggagg aggtgggagt 1560 gggaattatg gtggcggtgg ctcgggcggt ggtggatctg gaggaggtgg gagtgggaat 1620 tctgacattg tgctcaccca atctccagct tctttggctg tgtctctagg tcagagagcc 1680 accatctcct gcagagccag tgaaagtgtt gaatattatg tcacaagttt 15 aatgcagtgg 1740 taccaacaga aaccaggaca gccacccaaa ctcctcatct ctgctgctag caacgtagaa 1800

20 tctggggtcc ctgccaggtt tagtggcagt gggtctggga cagactttag cctcaacatc 1860 catcctgtgg aggaggatga tattgcaatg tatttctgtc agcaaagtag gaaggttcca 1920 tggacgttcg gtggaggcac caagctggaa atcaaacggg gtggcggtgg atccggcgga 1980 ggtgggtcgg gtggcggcgg atctcaggtg cagctgaagg agtcaggacc 30 tggcctggtg 2040 gcgccctcac agagcctgtc catcacatgc accgtctcag ggttctcatt aaccggctat 2100

35 ggtgtaaact gggttcgcca gcctccagga aagggtctgg agtggctggg aatgatatgg 2160

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 ggtgatggaa gcacagacta taattcagct ctcaaatcca gactatcgat caccaaggac 2220 aactccaaga gccaagtttt cttaaaaatg aacagtctgc aaactgatga 5 cacagccaga 2280 tactactgtg ctcgagatgg ttatagtaac tttcattact atgttatgga ctactggggt 2340 io caaggaacct cagtcaccgt ctcctcttaa tctaga

2376 <210> 177 15 <211> 785 <212> PRT <213> Artificial sequi <220> 20 <223> Synthetic polypi <220> <223> 2H7sssIgGl-STD2 25 <220> <221> misc feature <222> (1)··(22) <223> Leader 30 <220> <221> misc feature <222> (23) . . (129) <223> VL 35 <220> <221> misc feature <222> (130)..(144)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

157

2016231617 23 Sep 2016

<223> Linker <220> <221> misc feature 5 <222> (145)..(265) <223> VH <220> <221> misc feature 10 <222> (268)..(282) <223> Hinge <220> <221> misc feature 15 <222> (500)..(537) <223> EFD-BD2 Linker <220> <221> misc feature 20 <222> (538)..(649) <223> VL2 <220> <221> misc feature 25 <222> (650)..(664) <223> Linker2 <220> <221> misc feature 30 <222> (665)..(785) <223> VH2 <400> 177

35 Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu lie Ser Ala Ser

15 10

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Val Ala Leu Ala lie Met Ser Arg Gly Gin 20 He Val Leu Thr Ser Met Gin Thr Ser Cys 45 Pro 30 Arg He Ser Ser Ala 35 Lys 40 25 Val Ser Pro Gly Glu Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu

Leu Lys

115 120 125

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2016231617 23 Sep 2016

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

15 175

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

5 580

Asp Phe Ser Leu Asn Ala Met

5 Arg Asn Val Gly Val 355 Ser Val Leu Thr Val 360 Leu His Gin Asp Trp 365 Leu Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser lie 370 375 380 10 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 15 400

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 20 415 405 410

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala 25 Val Glu 420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 30 Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

35 Thr Val

450 455 460

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

5 <220> <221> <222> <223> misc feature (145)..(265) VH 10 <220> <221> <222> <223> misc feature (268)..(282) Hinge 15 <220> <221> <222> <223> misc feature (500)..(519) EFD-BD2 Linker 20 <220> <221> <222> <223> misc feature (520)..(631) VL2 25 <220> <221> <222> <223> misc feature (632)..(646) Linker2 30 <220> <221> <222> <223> misc feature (647)..(767) VH2 <400> 175

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu lie Ser 35 Ala Ser

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2016231617 23 Sep 2016

Val Ala lie He Met Ser 20 Arg Gly Gin He Val 25 Leu Ser Gin Ser Pro 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu

Leu Lys

115 120 125

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2016231617 23 Sep 2016

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

15 175

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260

265

270

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

275 280 285

Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 35 Thr Tyr

340 345 350

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2016231617 23 Sep 2016

5 740 745 750

Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

5 Gly Ala He Tyr Lys Phe

195

5 Ala Arg Phe Ser Thr lie

5 Gly Val Leu Ser 530 Val Ala Pro Ser Gin 535 Ser Leu Ser lie Thr 540 Cys Thr Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro 10 545 550 555 560 Gly Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly Asp Gly 15 Ser Thr 565 570 575 20 Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn 580 585 590 25 Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp 595 600 605 30 Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr 610 615 620 35 Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val

Ser Ser

625 630 635

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2016231617 23 Sep 2016

640

Gly Gly Gly Gly 5 Ser Gly

655

5 He Ser 305 320 Ser Arg His Thr Pro Glu 310 Val Thr Cys Val Val Val 315 Asp Val Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 10 335 325 330 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 15 Thr Tyr 340 345 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 20 Asn Gly 355 360 365 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 25 Ser lie 370 375 380 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 30 Gin Val 385 390 395 400 35 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin

Val Ser

405 410

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2016231617 23 Sep 2016

415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala 5 Val Glu

420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 10 Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 25 Leu Ser

485 490

495

30 Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

500 505 510

35 Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro

515 520 525

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2016231617 23 Sep 2016

5 85 90

Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp

5 Lys Pro Glu He 370 Tyr Lys Cys Lys Val 375 Ser Asn Lys Ala Leu 380 Pro Ala Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 10 385 390 395 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin 15 Val Ser 405 410 415 20 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu 420 425 430 25 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 30 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460 35 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser

Val Met

465 470 475

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2016231617 23 Sep 2016

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 5 Leu Ser 485 490 495 10 Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 500 505 510 Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser 15 Gly Pro 515 520 525 Gly Leu Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr 20 Val Ser 530 535 540 Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin 25 Pro Pro 545 550 555 560 30 Gly Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly Asp Gly

Ser Thr

565

570

Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser lie Thr Lys Asp Asn

575

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2016231617 23 Sep 2016

580 585 590

Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr 5 Asp Asp 595 600 605

Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe 10 His Tyr 610 615 620

Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val 15 Ser Ser 625 630 635 640

20 Gly Gly Ser Gly 655 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 645 650 25 Gly Gly Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala 660 665 670 30 Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser 675 680 685 35 Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin

Lys Pro

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2016231617 23 Sep 2016

690

695

700

Gly Gin Pro Pro Lys Leu Leu lie Ser Ala Ala Ser Asn Val Glu Ser

705

720

710

715

5 260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

5 Leu Ala Ser Ser Ala 35 Ser Pro Gly Glu Lys Val 40 Thr Met Thr Cys 45 Arg Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 10 50 55 60 Pro Lys Pro Trp lie Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 15 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 20 Thr He 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 25 Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu 30 Leu Lys 115 120 125 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

35 Ser Ser

130 135 140

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2016231617 23 Sep 2016

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu 15 Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin 2 0 Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr 25 Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr 3 o Phe Cys

225 230 235

240

35 Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

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2016231617 23 Sep 2016

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

5 aaggttccat ggacgttcgg tggaggcacc aagctggaaa tcaaacgtta atctaga 2337 <210>

<211>

<212>

<213>

171

772

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7sssIgGl-STDl-2el2HL (AA)

20 <220>

<221> misc_feature <222> (1) . . (22) <223> Leader

25 <220>

<221> misc_feature <222> (23) . . (128) <223> VL

30 <220>

<221> misc_feature <222> (129)..(144) <223> Linker

35 <220>

<221> misc_feature <222> (145)..(265)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <223> VH <220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

misc_feature (268) . . (281) Hinge misc_feature (500)..(519) EFD-BD2 linker misc_feature (520)..(640) VH2 misc_feature (641)..(660) Linker2 misc_feature (661)..(772) VL2 <400> 171

30 Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

15 10

35 Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He

20 25 30

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2016231617 23 Sep 2016

5 Gly Gly Gly Gly Ser Gly Asn Ser 1 5 <210> 156 10 <211> 30 <212> DNA <213> Artificial sequence <220> 15 <223> Synthetic primer <220> <223> Linker H3 (PN) 20 <400> 156 aattatggtg gcggtggctc tgggaattct 30 25 <210> 157 <211> 10 <212> PRT <213> Artificial sequence 30 <220> <223> Synthetic peptide <220> <223> Linker H3 (AA) 35 <400> 157

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Asn Tyr Gly Gly Gly Gly Ser Gly Asn Ser 15 10 <210> 158 <211> 39 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> Linker H4 (PN) <400> 158 ggtggcggtg gctcgggcgg tggtggatct gggaattct 39 <210>

<211>

<212>

<213>

159

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H4 (AA) <400> 159

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser 35 1 5 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<210> 160 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> Linker H5 (PN) <400> 160

aattatggtg gcggtggctc gggcggtggt ggatctggga attct 45 <210>

<211>

<212>

<213>

161

PRT

Artificial sequence <220>

<223> Synthetic peptide

25 <220>

<223> Linker H5 (AA) <400> 161

30 Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Asn Ser

15 10

35 <210> 162 <211> 54 <212> DNA

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> Linker H6 (PN) <400> 162

5 <400> 153

Asn Ser 1 <210> 154 <211> 24 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

20 <223> Linker H2 (PN) <400> 154 ggtggcggtg gctcggggaa ttct 24

<210> 155 <211> 8 <212> PRT 30 <213> Artificial sequence <220> <223> Synthetic primer 35 <220> <223> Linker H2 (AA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

105

2016231617 23 Sep 2016 <400> 155

5 Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

5 Ala Pro 1 Pro Lys Glu Leu Leu 5 Gly Gly Ser Ser Val 10 Phe Leu Phe Pro Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr 10 Cys Val 20 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 15 Trp Tyr 35 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 20 Glu Glu 50 55 60 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 25 Leu His 65 70 75 80 30 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 35 Ala Leu Pro Ala Ser lie Glu Lys Thr He Ser Lys Ala Lys

Gly Gin

100 105 110

5089144_1 (GHMatters) P79767.AU.1 5/02/14

100

2016231617 23 Sep 2016

Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

115

120

125

Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130

135

140

Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn

145

160

150

155

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 2 0 Phe Leu

165 170

175

25 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val

180 185 190

30 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys 35 210 215

5089144_1 (GHMatters) P79767.AU.1 5/02/14

101

2016231617 23 Sep 2016

<210> 148 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> STD1 (DNA) <400> 148

aattatggtg gcggtggctc gggcggtggt ggatctggag gaggtgggag tgggaattct 60

<210> 149 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> STD1 (AA) <400> 149

Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

15 10 15

Ser Gly Asn Ser 20

5089144_1 (GHMatters) P79767.AU.1 5/02/14

102

2016231617 23 Sep 2016

<210> 150 <211> 114 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer

<220>

<223> STD2 (DNA)

15 <400> 150 aattatggtg gcggtggctc gggcggtggt ggatctggag gaggtgggag tgggaattat 60 ggtggcggtg ttct gctcgggcgg tggtggatct ggaggaggtg 114 ggagtgggaa <210>

<211>

<212>

151

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> STD2 (AA)

35 <400> 151

Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly

Ser Gly Gly

5089144_1 (GHMatters) P79767.AU.1 5/02/14

103

2016231617 23 Sep 2016

Gly Gly

15 10

5 Gin Asp Trp Leu Asn Lys

5 <400> 138 ctcgagt 7

5 Ser Cys Pro Pro Cys Pro 1 5 <210> 136 <211> 7 <212> DNA 10 <213> <220> Artificial sequence <223> Synthetic primer 15 <400> 136 tgatcag 7

20 <210> 137 <211> 2 <212> PRT <213> Artificial sequence 25 <220> <223> Synthetic peptide <400> 137

30 Asp Gin 1 <210>

<211>

<212>

<213>

138

DNA

Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer

5 <223> ssc(p)-h!gGl (DNA) <400> 133

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser Pro

15 10 <210> 134 <211> 18 <212> DNA <213> Artificial sequence <220>

<221> misc_feature <223> ssc(p) (DNA) <220>

<223> Synthetic primer <400> 134 agttgtccac cgtgccca 18

<210> 135 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 135

5 <221> misc_feature <223> css(p)-hlgGl (AA) <400> 131

5 <220>

<221> misc_feature <223> see(p)-hlgGl (AA) <400> 129

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro

15 10 <210> 130 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> css(p)-hlgGl (DNA) <400> 130 gagcccaaat cttgtgacaa aactcacaca tctccaccga gccca 45

<210> 131 <211> 15 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide <220>

5 <220>

<221> misc_feature <223> csc(p)-hIgGl (AA) <400> 125

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Pro

15 10 <210> 126 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> ssc(p)-hIgGl (DNA) <400> 126 gagcccaaat cttctgacaa aactcacaca tctccaccgt gccca 45

35 <210> 127 <211> 15 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<221> misc_feature <223> ssc(p)-hlgGI (AA)

5 gagcccaaat cttgtgacaa aactcacaca tgtccaccgt gccca 45

<210> 121 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<400> 121

20 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

15 10 <210>

<211>

<212>

<213>

122

DNA

Artificial sequence

30 <220>

<223> Synthetic primer <400> 122 gagcccaaat cttctgacaa aactcacaca tctccaccga gccca 35 45

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 123 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 123

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro

15 10 <210> 124 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> csc(p)-hIgGl (DNA) <400> 124 gagcccaaat cttgtgacaa aactcacaca tctccaccgt gccca 45

<210> 125 <211> 15 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide

5 145

160

Ser Cys Arg Ala 10 Tyr Gin

175

15 Gin Lys Pro Asp Ser Arg

180

20 Leu His Ser Gly Gly Thr

195

25 Asp Tyr Ser Leu Ala Thr

210

30 Tyr Phe Cys Gin Gly Gly 225 240

Thr Lys Leu Val

Ser Leu Ser Ala Ser

150

Ser Gin Asp lie Arg

165

Gly Thr Val Lys Leu

185

Val Pro Ser Arg Phe

200

Thr lie Ala Asn Leu

215

Gin Gly Asn Thr Leu

230

Thr Lys Arg Ser 245

Leu Gly Asp Arg Val

155

Asn Tyr Leu Asn Trp

170

Leu lie Tyr Tyr Thr

190

Ser Gly Ser Gly Ser

205

Gin Pro Glu Asp lie

220

Pro Trp Thr Phe Gly

235

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 110 <211> 45 5 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer 10 <220> <221> misc feature <223> ssc(s)-hIgGl (DNA) 15 <400> 110 gagcccaaat cttctgacaa aactc; 45 <210> 111 20 <211> 15 <212> PRT <213> Artificial sequence <220> 25 <223> Synthetic peptide <220> <221> misc feature 30 <223> ssc(s)-hIgGl (AA) <400> 111 Glu Pro Lys Ser Ser Asp Lys 35 Ser 1 5

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210> 112 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> see(s)-hlgGl (DNA) <400> 112 gagcccaaat cttctgacaa aactcacaca tgtccaccgt getea 45 <210>

<211>

<212>

113

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<221> misc_feature <223> see(s)-hlgGl (AA) <400> 113

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35 Ser

15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210> 114 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> css(s)-hIgGl (DNA) <400> 114 gagcccaaat cttgtgacaa aactcacaca tctccaccga gctca 45 <210>

<211>

<212>

115

PRT <213> Artificial sequence

25 <220>

<223> Synthetic peptide <220>

<221> misc_feature <223> css(s)-h!gGl (AA) <400> 115

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser 35 Ser

15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210> 116 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<221> misc_feature <223> scs(s)-hIgGl (DNA) <400> 116 gagcccaaat cttgtgacaa aactcacaca tgtccaccga gctca 45 <210>

<211>

<212>

117

PRT <213> Artificial sequence

25 <220>

<223> Synthetic peptide <220>

<221> misc_feature <223> scs(s)-h!gGl (AA) <400> 117

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Ser 35 Ser

15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 118 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <400> 118

gagcccaaat cttgtgacaa aactcacaca tgtccaccgt gctca 45

<210> 119 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

25 <400> 119

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Ser

15 10

<210> 120 <211> 45 <212> DNA <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <223> Synthetic primer <400> 120

5 lie Val Trp He

5 <211> 248 <212> PRT <213> Artificial sequence <220>

5 <220>

<221> misc_feature <222> (109)..(125) <223> Linker

5 35 40 45

Ser Phe Ala Lys Ser Gly

Thr Leu Ala Glu

Gly Val

Pro Ser Arg Phe

Ser Gly Ser Gly Thr Gin Phe Ser Leu Lys lie Ser Ser Leu Gin Pro

65 70 75

Glu Asp Ser Gly Ser Tyr Phe Cys Gin His His Ser Asp Asn 20 Pro Trp

85 90

Thr Phe Gly Gly Gly Thr Glu Leu Glu He Lys Gly Gly Gly Gly Ser

25 100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Ala Val Gin Leu

30 115 120 125

Gin Gin Lys He

Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala

Ser Val

130

135

140

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Ser Asn 145 160 Cys Trp Lys Ala Ser Gly Tyr 150 Ser Phe Thr Gly Tyr Asn 155 Met Val Lys Gin Asn Asn Gly Lys Ser Leu Glu Trp He Gly Asn lie Asp 165 170 175 Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe Lys Gly Lys Ala 180 185 190 Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin Leu Lys 195 200 205 Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Val 210 215 220 Gly Pro Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val

Ser

225 230 235 <210>

<211>

<212>

<213>

104

767

DNA

Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<220> <223> Synthetic polynucleotide 5 <220> <223> G28- <400> 104 -1 VHVL (DNA) accggtgagg agcttcaatg tccagctgca 60 acagtctgga cctgaactgg tgaagcctgg 10 aagatttcct ctggctgaag gcaaggcctc 120 tggttactca ttcactggct acatcgtgaa 15 cagagccatg aggtcttact gaaagaacct 180 tgagtggatt ggacttatta atccatacaa acctacaacc atccagcaca agaaattcaa 240 gggcaaggcc acattaactg tagacaagtc 20 gcctacatgg ctgtgcaaga agctcctcag 300 tctgacatct gaagactctg cagtctatta tctgggtact gaccacggtc atggtgactc 360 ggactggtac ttcgatgtct ggggcgcagg 25 accgtctcct tgggagcggg ctggtggcgg 420 tggctcgggc ggtggtggat ctggaggagg 30 ggaggtggca gtctgcctct gtgctagcga 480 catccagatg acacagacta catcctccct ctgggagaca ttatttaaac gagtcaccat 540 cagttgcagg gcaagtcagg acattcgcaa 35 tggtatcagc atcaagatta agaaaccaga 600 tggaactgtt aaactcctga tctactacac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 cactcaggag tcccatcaag gttcagtggc agtgggtctg ttctctcacc 660 attgccaacc tgcaaccaga agatattgcc acttactttt 5 taatacgctt 720 ccgtggacgt tcggtggagg caccaaactg gtaaccaaac 767 <210> 105 <211> 253 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

20 <223> G28-1 VHVL (AA) <220>

<221> misc_feature <222> (1)..(121) <223> VH <220>

<221> misc_feature <222> (122)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(253) <223> VL <400> 105 gaacagatta gccaacaggg gctcgag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Glu Val Gin Leu Gly Ala 1

Ser Met Lys lie Gly Tyr lie Val Asn Trp Trp lie

Gly Leu lie Asn Lys Phe

Lys Gly Lys Ala Ala Tyr 65

25 80

Gin Gin Ser Gly Pro

Ser Cys Lys Ala Ser

Leu Lys Gin Ser His

Pro Tyr Lys Gly Leu

Thr Leu Thr Val Asp

Glu Leu Val Lys Pro

5 <213> Artificial sequence <220>

<223> Synthetic polypeptide

5 <220>

<223> Synthetic primer <220>

<223> 194-HF2 <400> 82 agctgcaaca gtctggacct gaactggtga agcctggagc ttcaatgaag 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HF3 <400> 83 agcctggagc ttcaatgaag atttcctgca aggcctctgg ttactcattc 50

<210> 84 <211> 53 <212> DNA <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <223> Synthetic primer <220>

<223> 194-HF4 <400> 84 gcaaggcctc tggttactca ttcactggct acatcgtgaa ctggctgaag cag 53 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HF5 <400> 85 atcgtgaact ggctgaagca gagccatgga aagaaccttg agtggattgg ac 52

<210> 86 <211> 52 <212> DNA 30 <213> Artificial sequence <220> <223> Synthetic primer 35 <220> <223> 194-HF6

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 86 gaaccttgag tggattggac ttattaatcc atacaaaggt cttactacct ac 52 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HR6 <400> 87 aatgtggcct tgcccttgaa tttctggttg taggtagtaa gacctttgta tg 52 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HR5 <400> 88 catgtaggct gtgctggatg acttgtctac agttaatgtg gccttgccct tg 35 52

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 89 <211> 55 <212> DNA <213> Artificial sequence 5 <220> <223> Synthetic primer <220> 10 <223> 194-HR4 <400> 89 actgcagagt cttcagatgt cagac ggatg 55 15 <210> 90 <211> 52 <212> DNA 20 <213> Artificial sequence <220> <223> Synthetic primer 25 <220> <223> 194-HR3 <400> 90 accatagtac ccagatcttg cacag 30 52 <210> 91 <211> 52 35 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer <220>

<223> 194-HR2 <400> 91 gcgccccaga catcgaagta ccagtccgag tcaccatagt acccagatct tg 52 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LH-HR1 <400> 92 gcgaatactc gaggagacgg tgaccgtggt ccctgcgccc cagacatcga ag 52

<210> 93 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> 194-HL-HF1

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 93 gcgtatgaac cggtgaggtc cagctgcaac agtctggacc tg 42 <210> 94 <211> 55 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HL-HR1 <400> 94 accgccacca gaggagacgg tgaccgtggt ccctgcgccc cagacatcga 20 agtac 55 <210>

<211>

<212>

DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HL-HRO <400> 95

35 acctcctcca gatccaccac cgcccgagcc accgccacca gaggagacgg tg 52

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 96 <211> 49 <212> DNA 5 <213> Artificial sequence <220> <223> Synthetic primer 10 <220> <223> 194-HL-LF1 <400> 96 gcgggggagg tggcagtgct agcga 15 49 <210> 97 <211> 34 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer 25 <220> <223> 194-HL-LR3Xho <400> 97 30 gcgaatactc gagcgtttgg ttacc 34 <210> 98 35 <211> 37 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer <220>

<223> 194-HL-LR3Xba <400> 98 gcgatatcta gattaccgtt tggttaccag tttggtg 37 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-HL-HF1R1 <400> 99 gcgtatgaga attcagaggt ccagctgcaa cagtctggac ctg 43

<210> 100 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <223> 194-LH-LF1R1 <400> 100 gcgtatgaga attctgacat ccagatgaca cagactacat c 5 41 <210>

<211>

<212>

<213>

101

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LH-HRlXba <400> 101

20 gcgtatctag attaggagac ggtgaccgtg gtccctgcgc cccagacatc gaag 54 <210>

<211>

<212>

<213>

102

725

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> G28-1 VLVH (DNA)

35 <400> 102 accggtgaca tccagatgac tcagtctcca gcctccctat ctgcatctgt gggagagact 60

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

gtcaccatca catgtcgaac aagtgaaaat gtttacagtt atttggcttg gtatcagcag 120 5 aaacagggaa aatctcctca gctcctggtc tcttttgcaa aaaccttagc agaaggtgtg 180 ccatcaaggt tcagtggcag tggatcaggc acacagtttt ctctgaagat cagcagcctg 240 10 cagcctgaag attctggaag ttatttctgt caacatcatt ccgataatcc gtggacgttc 300 ggtggaggca ccgaactgga gatcaaaggt ggcggtggct cgggcggtgg 15 tgggtcgggt 360 ggcggcggat ctgctagcgc agtccagctg cagcagtctg gacctgagct ggaaaagcct 420 20 ggcgcttcag tgaagatttc ctgcaaggct tctggttact cattcactgg ctacaatatg 480 aactgggtga agcagaataa tggaaagagc cttgagtgga ttggaaatat tgatccttat 540 25 tatggtggta ctacctacaa ccggaagttc aagggcaagg ccacattgac tgtagacaaa 600 tcctccagca cagcctacat gcagctcaag agtctgacat ctgaggactc 30 tgcagtctat 660 tactgtgcaa gatcggtcgg ccctatggac tactggggtc aaggaacctc agtcaccgtc 720 35 tcgag 725

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210>

<211>

<212>

103

239

PRT

5 <400> 77 cgtttggtta ccagtttggt gcctccaccg aacgtccacg gaagcgtatt ac 52 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LR3 <400> 78 accaccgccc gagccaccgc caccccgttt ggttaccagt ttggtg 46

<210> 79 <211> 50 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> 194-LR2 <400> 79

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 gctagcgctc ccacctcctc cagatccacc accgcccgag ccaccgccac 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence

5 <223> Synthetic primer <220>

<223> 194-LR6

5 gttgcagggc aagtcaggac attcgcaatt atttaaactg gtatcagcag 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LF5

20 <400> 71 atttaaactg gtatcagcag aaaccagatg gaactgttaa actcctgatc 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence

30 <220>

<223> Synthetic primer <220>

<223> 194-LF6 <400> 72 gaactgttaa actcctgatc tactacacat caagattaca ctcaggagtc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LF7 <400> 73 caagattaca ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga ac 52 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LR7 <400> 74 caggttggca atggtgagag aataatctgt tccagaccca ctgccactga ac 52 <210> 75 <211> 50

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <212> DNA <213> Artificial sequence <220>

5 <220>

<223> 194-LF2 <400> 68 atccagatga cacagactac atcctccctg tctgcctctc tgggagacag 10 50

15 <210> <211> <212> <213> 69 52 DNA Artificial sequence <220> <223> Synthetic primer 20 <220> <223> 194-LF3 25 <400> 69

gtctgcctct ctgggagaca gagtcaccat cagttgcagg gcaagtcagg ac 52

30 <210> 70 <211> 50 <212> DNA <213> Artificial sequence 35 <220> <223> Synthetic primer

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> 194-LF4 <400> 70

5 <400> 47 gcgatagaat tcccagagcc accgccacca taattc 36

<210> 48 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> L3R <400> 48 gcgtatgaat tccccgagcc accgc 45 <210> 49 <211> 49 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> L4R <400> 49

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 gcgtatgaat tcccagatcc accaccgccc gagccaccgc cacccttac 49

<210> 50 <211> 44 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> L5R <400> 50 gcgtatgaat tcccgctgcc tcctc 44 <210> 51 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> IgBsrGIF <400> 51 gagaaccaca ggtgtacacc ctg 23 <210> 52

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <211> 50 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> L-CPPCPR <400> 52 gcgatagaat tcggacaagg tggacacccc ttacccggag acagggagag 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> G281LH-NheR <220>

<223> Synthetic primer <400> 53 actgctgcag ctggaccgcg ctagctccgc cgccacccga c 41

<210> 54 <211> 40 <212> DNA <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <223> Synthetic primer <220>

<223> G281LH-NheF <400> 54 ggcggagcta gcgcggtcca gctgcagcag tctggacctg 40 <210> 55 <211> 37 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> G281-LH-LPinF <400> 55 gcgatcaccg gtgacatcca gatgactcag tctccag 37

<210> 56 <211> 37 <212> DNA 30 <213> Artificial sequence <220> <223> Synthetic primer 35 <220> <223> G281-LH-HXhoR

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 56 gcgatactcg aggagacggt gactgaggtt ccttgac 37 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> G281-LH-LEcoF <400> 57 gcgatcgaat tcagacatcc agatgactca gtctccag 38 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer

30 <220>

<223> G281-LH-HXbaR <400> 58 gcgattctag attaggaaga gacggtgact gaggttcctt gac 35 43

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 59 <211> 37 <212> DNA <213> Artificial sequence 5 <220> <223> Synthetic primer 10 <220> <223> G281-HL-HF <400> 59 gcgataaccg gtgcggtcca gctgc 15 37 <210> 60 <211> 52 <212> DNA 20 <213> Artificial sequence <220> <223> Synthetic primer 25 <220> <223> G281-HL-HR3 <400> 60 gacccaccac cgcccgagcc accgc 30 52 <210> 61 <211> 49 35 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer <220>

<223> G281-HL-HR2 <400> 61 actcccgcct cctcctgatc cgccgccacc cgacccacca ccgcccgag 49 <210> 62 <211> 42 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> G281-HL-HNheR <400> 62 gagtcatctg gatgtcgcta gcactcccgc ctcctcctga tc 42

<210> 63 <211> 45 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> G281-HL-LNheF

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 63 atcaggagga ggcgggagtg ctagcgacat ccagatgact cagtc 45 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer

15 <220>

<223> G281-HL-LXhoR <400> 64 gcgatactcg agcctttgat ctccagttcg gtgcctc 20 37 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> G281-HL-LXhoR <400> 65

35 gcgatatcta gactcaacct ttgatctcca gttcggtgcc tc 42

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 66 <211> 37 <212> DNA 5 <213> Artificial sequence <220> <223> Synthetic primer 10 <220> <223> G281-HL-EcoF <400> 66 gcgatagaat tcgcggtcca gctgc 15 37 <210> 67 <211> 40 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer 25 <220> <223> 194-LH-LF1 <400> 67 30 gcgtatgaac cggtgacatc cagat 40 <210> 68 35 <211> 50 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer

5 atacgactca ctataggg 18

<210> 41 10 <211> 19 <212> DNA <213> Artificial sequence

<220> 15 <223> Synthetic primer

<220> <223> pD18R-s 20 <400> 41

gctctagcat ttaggtgac 19

25 <210> 42 <211> 18 <212> DNA <213> Artificial sequence

30 <220> <223> Synthetic primer

<220> <223> CH3seqFl 35 <400> 42

catgaggctc tgcacaac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <210>

<211>

<212>

DNA <213> Artificial sequence <220>

5 1 5 10 <210> 15 <211> 46 <212> DNA <213> Homo sapiens <220>

<223> hVK3L-F3H3 <400> 15 gcgataaagc ttgccgccat ggaagcacca gcgcagcttc tcttcc 46

<210> 16 <211> 50 <212> DNA <213> Homo sapiens <220> <223> hVK3LF2

30 <400> 16 accagcgcag cttctcttcc tcctgctact ctggctccca gataccaccg 50

35 <210> 17 <211> 45 <212> DNA

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <213> Homo sapiens <220>

<223> hVK3LF12HVL <400> 17 ggctcccaga taccaccggt caaattgttc tctcccagtc tccag 45 <210> 18 <211> 35 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

20 <223> 2H7VHNheF <400> 18 gcgatagcta gccaggctta tctacagcag tctgg 35

<210> 19 <211> 43 <212> DNA 30 <213> Artificial sequence <220> <223> Synthetic primer 35 <220> <223> G4SNheR

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 19 gcgatagcta gccccacctc ctccagatcc accaccgccc gag 43 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 015VHXhoR <400> 20 gcgtactcga ggagacggtg accgtggtcc ctgtg 35 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer

30 <220>

<223> GIHC-Xho <400> 21 gcagtctcga gcgagcccaa atcttgtgac aaaactc 35 37

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 22 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> GIH-S-Xho <400> 22 gcagtctcga gcgagcccaa atctl 37 <210> 23 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> CH3REcoRl <400> 23 gcgtgagaat tcttacccgg agaci 36 <210> 24 <211> 32 <212> DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer <220>

<223> G1 XBZ-R <400> 24 gcgacgtcta gagtcattta cccggagaca gg 32 <210> 25 <211> 54 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> G4SLinkRl-S <400> 25 aattatggtg gcggtggctc gggcggtggt ggatctggag gaggtgggag tggg 54

<210> 26 <211> 54 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> G4SLinkRl-AS

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 26 aattcccact cccacctcct ccagatccac caccgcccga ccat 54 gccaccgcca <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer

15 <220>

<223> 2E12VLXbaR <400> 27 gcgtgtctag attaacgttt gatttccagc ttggtg 20 36 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 2E12VLR1F <400> 28

35 gcgatgaatt ctgacattgt gctcacccaa tctcc 35

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 29 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer <220> <223> 2E12VHR1F <400> 29

gcgatgaatt ctcaggtgca gctgaaggag tcag 34 <210> 30 <211> 37 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> 2E12VHXbaR <400> 30

30 gcgagtctag attaagagga gacggtgact gaggttc 37 <210>

<211>

<212>

DNA <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic primer 5 <220> <223> 2el2VHdXbaFl <400> 31 gggtctggag tggctgggaa tgatatg 10 27

15 <210> <211> <212> <213> 32 28 DNA Artificial sequence <220> <223> Synthetic primer 20 <220> <223> 2el2VHdXbaRl <400> 32 25 attcccagcc actccagacc ctttcctg 28

<210> 33 30 <211> 23 <212> DNA <213> Artificial sequence <220> 35 <223> Synthetic primer <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<223> IgBsrGIF <400> 33

gagaaccaca ggtgtacacc ctg 5 23

<210> 34 <211> 25 10 <212> DNA <213> Artificial sequence

<220> <223> Synthetic primer 15 <220> <223> IgBsrGIR <400> 34

20 gcagggtgta cacctgtggt tctcg 25

<210> 35 25 <211> 17 <212> DNA <213> Artificial sequence

<220> 30 <223> Synthetic primer

<220> <223> M13R 35 <400> 35

caggaaacag ctatgac 17

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 36 <211> 18 5 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer 10 <220> <223> M13F <400> 36 15 gtaaaacgac ggccagtg 18 <210> 37 20 <211> 20 <212> DNA <213> Artificial sequence <220> 25 <223> Synthetic primer <220> <223> T7 30 <400> 37 gtaatacgac tcactatagg 20 35 <210> 38 <211> 17 <212> DNA

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<213> Artificial sequence <220> <223> Synthetic primer 5 <220> <223> pD18F-17 <400> 38 10 aactagagaa cccactg 17

<210> 39 15 <211> 20 <212> DNA <213> Artificial sequence <220> 20 <223> Synthetic primer <220> <223> pD18F-20 25 <400> 39 gctaactaga gaacccactg 20

30 <210> 40 <211> 18 <212> DNA <213> Artificial sequence 35 <220> <223> Synthetic primer

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> pD18F-l <400> 40

5 gtcacwgtca ctgrctcagg gaartagc 28 <210> 9 <211> 33 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> Light Chain GSP1 <400> 9 gggtgctgct catgctgtag gtgctgtctt tgc 33 <210> 10 <211> 32 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> Nested Light Chain <400> 10 caagaagcac acgactgagg cacctccaga tg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<210> 11 5 <211> 36 <212> DNA <213> <220> Artificial sequence 10 <223> <220> Synthetic primer <223> Race Abridged Anchor 15 <220> <221> misc feature <222> (24)..(25) <223> n is a, c, g, or t 20 <220> <221> misc feature <222> (29) . . (30) <223> n is a, c, g, or t 25 <220> <221> misc feature <222> (34) . . (35) <223> n is a, c, g, or t 30 <400> 11 ggccacgcgt cgactagtac gggnnc 36 35 <210> 12 <211> 20 <212> DNA

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> T7 primer <400> 12

5 Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

260 265 270

5 145

160

Gly Gly Gly Ser 10 Leu Ala

175

15 Val Ser Leu Gly Glu Ser

180

20 Val Glu Tyr Tyr Lys Pro

195

25 Gly Gin Pro Pro Glu Ser

210

30 Gly Val Pro Ala Phe Ser 225 240

Leu Asn lie His Phe Cys

Ser Gly Gly Gly Gly

150

Asp lie Val Leu Thr

165

Gin Arg Ala Thr lie

185

Val Thr Ser Leu Met

200

Lys Leu Leu lie Ser

215

Arg Phe Ser Gly Ser

230

Pro Val Glu Glu Asp

Ser Gly Gly Gly Gly

155

Gin Ser Pro Ala Ser

170

Ser Cys Arg Ala Ser

190

Gin Trp Tyr Gin Gin

205

Ala Ala Ser Asn Val

220

Gly Ser Gly Thr Asp

235

Asp lie Ala Met Tyr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

245 250

255

5 Gly Leu Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser 35 40 45

5 1 5 10 15

Val lie Met Ser Arg Gly Val Asp lie Val Leu Thr Gin Ser Pro Ala

5 <223> anti-CD20 (2H7) LH (AA) <220>

<221>

<222>

<223>

misc_feature (1)··(22) Leader <220>

<221>

<222>

<223>

misc_feature (23)..(128) VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH <400> 2

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu lie Ser 30 Ala Ser

15 10

Val lie Met Ser Arg Gly Gin lie Val Leu Ser Gin Ser Pro Ala lie

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly 5 Ser Ser 50 55 60 Pro Lys Pro Trp lie Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 10 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 15 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 20 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 25 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro 30 Gly Ala 145 150 155 160 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

35 Ser Tyr

165 170

175

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Asn Met His Trp Trp lie

180

Gly Ala lie Tyr Lys Phe

195

5 <120> Single-Chain Multivalent Binding Proteins with

Effector

Function

<130> 30906/41393C 10 <160> 379 <170> Patentln version 3.3 15 <210> 1 <211> 795 <212> DNA <213> Artificial sequence 20 <220> <223> Synthetic polynucleotide <220> <223> anti-CD20 (2H7) LH (DNA) 25 <400> 1 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt

cataatgtcc 60

30 agaggacaaa ttgttctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120 gtcacaatga cttgcagggc cagctcaagt gtaagttaca tgcactggta ccagcagaag 180 o o ccaggatcct cccccaaacc ctggatttat gccccatcca acctggcttc tggagtccct 240

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

gctcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag cagagtggag 300 5 gctgaagatg ctgccactta ttactgccag cagtggagtt ttaacccacc cacgttcggt 360 gctgggacca agctggagct gaaagatggc ggtggctcgg gcggtggtgg atctggagga 420 10 ggtgggagct ctcaggctta tctacagcag tctggggctg agtcggtgag gcctggggcc 480 tcagtgaaga tgtcctgcaa ggcttctggc tacacattta ccagttacaa 15 tatgcactgg 540 gtaaagcaga cacctagaca gggcctggaa tggattggag ctatttatcc aggaaatggt 600 20 gatacttcct acaatcagaa gttcaagggc aaggccacac tgactgtaga caaatcctcc 660 agcacagcct acatgcagct cagcagcctg acatctgaag actctgcggt ctatttctgt 720 25 gcaagagtgg tgtactatag taactcttac tggtacttcg atgtctgggg cacagggacc 780 acggtcaccg tctct 30 795

<210> 2 <211> 265 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Synthetic polypeptide <220>

5/67

FIG. 5

256

A. BINDING OF PURIFIED PROTEIN FROM COS CELLS TO CD28CHO: 2e12 HL SMIP vs. 2H7-SSSIgG-STD1-2e12 HL MULTISPECIFIC FUSION PROTEIN

EVENTS Ml

5. A protein according to any one of claims 1 to 4, wherein the first and/or second binding domain comprises a light chain immunoglobulin variable region (VL1) and a heavy chain immunoglobulin variable region (VH1), wherein said variable regions are positioned in a VH1-VL1 oraVLl-VHl orientation.

6/67

FIG. 6 A

TABLE IDENTIFYING FUNCTIONAL ELEMENTS OF MULTISPECIFIC FUSION PROTEINS

BD1 Binding Domain 1 Linker 1 EFD Effector Function Domain Linker 2 ID .inker .ength aa) .(nicer ^Jlus Ft. Site CH3 COOH Linker 2 Sequence (- restriction site) Fusion Junction with BD2 (L2RS- QVQ 2e12) BD2 Binding Domain 2 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CR2-CH3 STD 20 PGK MYGGGGSGGGGSGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CR2-CH3 STD2 38 PGK NYGGGGSGGGGSGGGGSG NYGGGGSGGGGSGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CH2-CH3 H1 L1=2e12 Vl-Vh 2<RS) PGK NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CH2-CH3 H2 L2=2e12 Vl-Vh 8 PGK GGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CH2-CH3 H3 L3=2e12 Vl-Vh 10 PGK MYGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CA2-CH3 H4 L4=2e12 Vl-Vh 13 PGK GGGGSGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CR2-CH3 H5 L5=2e12 Vl-Vh 15 PGK MYGGGGSGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CH2-CH3 H6 L6=2e12 Vl-Vh 18 PGK GGGGSGGGGSGGGGSG NSQVQ 2e12 Vh-Vl 2H7 Vl-Vh Modified hlgGI hinge ccc->sss hlgGI- CH2-CH3 H7 8 PGK GCPPCP NS QVQ 2e12 Vh-Vl

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

6. A protein according to any one of claims 1 to 5, wherein the immunoglobulin-like molecule is a receptor.

8481915_1 (GHMatters) P79767.AU.2

200

2016231617 03 Aug 2018

7-03X140

7Ό3Χ7-03 qeoitxn)!H |oj;uoo

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968 PCT/US2007/071052

2016231617 23 Sep 2016

66/67

FIG. SO

DHL6 Cells Treated with 20x37 Scorpion _ί AntiP-TyrlP . Anti Syk IP _r

Ladder T=0 T=5 T=7 T=15 T=0 T=5 T=7 T=15

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052 τ—Η

Ο (Μ

Ρη <υ (Ζ <Ν

Ο γ—Η

ΓΩ <Ν r—Η

Ο <Ν

67/67

Effect Level

SUBSTITUTE SHEET (RULE 26)

2016231617 23 Sep 2016

SEQUENCE LISTING <110> Thompson et al.

7-03X7-07 qeiuixruia jOJJUOO auijodsojnejs 7-0 3^X140 7-07X7-03 qsujixnjiy |OJ|UOO auuodsojneis

7/67

FIG. 6B (1 of 5)

Constructs

tiune BD1 Hinge EH> iLrnlrer SD2 2H?-5SS-hlgG-S7Dl-2e12LH 2H7 (VL-VH) hlgGl-SSS hlgGor MgG JP238SJP331S1 |STD1 2e12 (VL-VH, 2W-5ss-blgG-S7D1-2e12 HL 2H7 (VL-VH) hlgGl-SSS hlgGor hlgG (P23BSP331S1 ISTD1 2e12 (VH-VL, 2H7-«s-hlgG-STD2-2e12LH 2H7 (VL-VH, hlgGl-SSS hlgG or hlgG (P23BS/P331S1 ISTO2 2e12 (VL-VH) 2H7-sss-h!gG-STD2-2el2 HL 2H7 (VL-VH) hlgGl-SSS h|gG or hlgG ; IP238SP331S1 ST02 2e12 (VH-VL) 2H7-csc-hlgGSTD1-2Bl2LH ZH7 (VL-VH, hlgGl -esc hlgGor hlgG (P2^8SP331S1 STD1 2e 12 (VL-VH) 2H7-csoWgG-STD2-2e12lH 2H7 (VL-VH, hlgGl -esc hlgG or hlgG IP23BS/P331S1 STD2 2el2 (VL-VH) ZH7-csc-hlgG-STDl-2el2 HL 2H7 (VL-VH, hlgGl -esc hlgG or hlgG IP238&P331S1 STD1 2el2 (VL-VH) 2H?-cse-MgG.STD2-2e12HL ptf (VL-VH, hlgGl-csc hlgG or hlgG /P238S/PJI31S1 ISTD2 2e12(VHVl) 2H7-sss-hlgG-H1-2el2 LH 2H7 (VL-VH) hlgGl -SSS hlgG or hlgG IP238S1P331SI H1 2e12 (VL-VH) 2H7-c$&hlgG-H1-2e12LH |2H7 (VL-VH, hlgGl-esc MgGorhigG fP23BS/P331Sl HI 2e12{\t-W) 2H7-«s4ilgGH1-2e12 HL 2H7(H-VH, hlgGl-SSS hlgG or hlgG iP238S/P33lSl HI 2e12 (VH-VL) 2H7-cr*higG-H1-2e1? HL (VL-VH) hlgGl-csc MgGorhigG 1P238S/P331S1 H1 2e1Z (VH-VL) 2K7-«s-hlgGH2-2e12LH J2H7 (VL-VH, hlgGl-SSS hlgG or hlgG fR23SSIE33ia , HZ . 2e12(W-VL) 2H7-osc-blgG-H2-2e12 LH 2H7 (VL-VH, hlgGl-csc hlgG or hlgG IP238S/P331SI JH2 2e12 (VL-VH, 2H7-s»-hlgG-H2-2e12HL I2H7 (VL-VH, hlgGl-SSS . hlgG or MgG IP238S/P331S1 H2 2e12(VH-Vl) ZH?-csc-h|gG-H2-2e12LH 2H7(VL-W) IlgGl-esc hlgG or hlgG ’P738S/P331SI HZ 2o12 (VH-VL, I !H7-sss-hlgG-H3-2e12 LH 2H7 (VL-VH, ilgGl -SSS IlgGor MgG P23BSP331S1 H3 2e12 (VL-VH, ... -......... . H7-csc-hlgGH3-2e12LH 2H7 (VL-VH) 1 IgGl -esc ilgGorhlgG P238S/P331S1 H3 2e12 (VL-VH, 2 H7-s$s-hlgG-H3-2el2 HL 2H7 (VL-VH, h IgGl-SSS . IgG or hlgG P238SP331S1 H3 2el2 (VH-VL, 2 H7-csc-hlgG-H3-2e12 IH 2H7 (VL-VH, h lgG1-esc t IgG or hlgG ^:>?38S/P331SI H3 Ze12 (VH-VL) • 2 H7-sss-WgGH4-2e12LH 2H7 (VL-VH) h IgGl-SSS h IgG or hlgG ^23eSP337SI H4 2e12 (VL-VH, 2I H7-csc-MgG-H4-2e12LH 2H7 (VL-VH) hl gGI-csc If IgG or hlgG >238S/P331S) H4 >e12 (VL-VH, 2l· l7-sss-hlgG-H4-2e12 HL ZH7 (VL-VH, hl gGI-SSS h IF gG or hlgG 238S/P331S1 H4 !e12 (VH-VL) 21 !7-csc-hlgG-H4-2e12LH j 2H7 (VL-VH, hlj ,61 -esc hl (£ gG or hlgG 23fi&F33ia. H4 >e12 (VH-VL,

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

7. A protein according to any one of claims 1 to 6, wherein the binding domain that does not bind to 4-1BB/TNFRSF9 binds to a target selected from the group consisting of a tumor antigen, a B-cell target, a TNF receptor superfamily member, a Hedgehog family member, a receptor tyrosine kinase, a proteoglycan-related molecule, a TGF-beta superfamily member, a Wnt-related molecule, a receptor ligand, a T-cell target, a Dendritic cell target, an NK cell target, a monocyte/macrophage cell target and an angiogenesis target.

8/67

FIG. 6B(2of5)

N»mt BD1 Hmjft EFD Linker BDZ 2H7-sss-hlgG-H5-2e12 LH 2H7JVL-VH) WgGl-SSS hlgGorhlgG IP23BSIP331SI H5 2e12 (VL-VH) 2H7-cse-hlgG-H5-2e12 LH 2H7(Vl-VH) hlgG1-csc hlgGorhlgG . (P238SIP331SI H5 2e12 (VL-VH) 2H7-sss-hlgG-H5-2e12 HL .. 2H7 (VL-VH, hlgG1-$SS hlgGorhlgG IP238SP331S1 H5 2e12 (VH-VL) 2H7-csc-hlgG-H5-26l2 LH 2H7(VL-VH) hlgGI-csc hlgGorhlgG . (P238SIP331S1 HS 2e12 (VH-VL) 2H7-sss-tilgG-H5-2e12 LH 2H7 (VL-VH) higGl-SSS hlgGorhlgG (P238SP331S1 H6 2e12 (VL-VH) 2H7-csc-hlgG-H6-2e12LH 2H7 (VL-VH, hlgGl-csc hlgGorhlgG ./P23SSIP331S) HS 2el2{VL-VH) 2H7-sss4ilgG-H6-2e12 HL 2H7 (VL-VH) hlgG1-SSS hlgGorhlgG 1P238SP331SI HS 2o12 (VH-VL) 2H7-csc-hlgG-H6-2e12 LH 2H7(VL-VH) hlgG1 -esc HlgGorhlgG IP23HSIP331S1 H6 2e12(VH-Vt) 2H7-sss-hlgG-H7-2e12 LH 2H7 (VL-VH) hlgG1-$SS hlgGorhlgG (P238SE331SL H7 2e12 (VL-VH) 2H7-csc-hlgG-H7*2e12LH 2H7(VL-VH) htgG1-csc hlgGorhlgG fP23BSF33lSI- H7 2e12(VL-VH) 2H7-sss-hig6-H7-2e12 HL 2H7 (VL-VH, hlgGI-SSS hlgGorhlgG (P23SSP331SL. H7 2el2 (VH-VL) 2H?-csc-hlgG-H7«2el2LH 2H7 (VL-VH) h|gGl -esc hlgGorhlgG rraaspaaisi H7 2e12 (VH-VL) 2H7-sss-hlgG-$TD1-G28-1 LH 2H7 (VL-VH! hlgGI-SSS hlgGorhlgG fP23flS/P331Sl ST01 G28-1 (VL-VH, 2H7-sss-htgG-STD1-G2&-1 HL 2H7 (VL-VH) hlgG1-SSS hlgGorhlgG P238S1P331S1 STD1 628-1 (VL-VH) 2H7-sss-MgG-STD2-G28-1 LH 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG IE238SE3215t STD2 G28-1 (VL-VH) 2K7-sss-hlgG-STD2-G28-1 HL 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG fp93BSP331Sl STD2 G2B-1 (VH-VL) ?H7-ac-higG-STO1-G2&-1 LH ?H7(VL-VH, ilgGI-csc hlgGorhlgG !P23flSE331SI . ST01 G28-1 (VL-VH) 1 !H7-cse-hlgG-STD2-G28-1 LH H7 (VL-VH) tlgGl -esc ilgG or hlgG P23BSP331SL STO2 G28-1 (VH-VL) 1 H7-csc-hlgG-S7Dl-G28-1 HL 2 H7 (VL-VH) 1 IgGl-esc ilgG or hlgG E2MS/P331S1.. STD1 G28-1 (VL-VH, 2 H7-csc-hlgG-STO2-G28-1 K 2 H7 (VL-VH) h IgGI-csc ... i ilgG or h|gG passmsi STD2 G28-1 (VH-VL, 2 H7-sss-hlgG-H1-G28-1 LH 2I 17 (VL-VH) h lgG1-SSS h I IgG or hlgG paflaraas).. H1 G28-1 (VL-VH) 2 H7-csc-hlgG-H1-G28-1 LH 21 i7 (VL-VH) h gG1 -esc h (I IgG or hlgG =>238S/P331S1 1 G28-1 (VH-VL) 2 17-sss-hlgG-H1-G28-1 HL 21 17 (VL-VH, h gGUSSS h ii IgG or hlgG 23BSIE221S1 11 G28-1 (VL-VH) 21 17-c$c-hlgG-H1-G28-1 HL 2H 7 (VL-VH) hl gG1 -esc h .If gG or hlgG >238S1F331SI . 11 328-1 (VH-VL) 21 l7-sss4ilgG-H2-G28-1 LH 2H 7 (VL-VH) hl jGI-SSS h IE gGorhlgG 1 !23flSf331S!...... 12 < 328-1 (VL-VH)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

8. A protein according to claim 7, wherein the tumor antigen is selected from the group consisting of SQUAMOUS CELL CARCINOMA ANTIGEN 1, SQUAMOUS CELL CARCINOMA ANTIGEN 2, Ovarian carcinoma antigen CA125, MUCIN 1, CTCL tumor antigen sel-1, CTCL tumor antigen sel4-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-l, CTCL tumor antigen se37-2, CTCL tumor antigen se57-l, CTCL tumor antigen se89-l, Prostate-specific membrane antigen, 5T4 oncofetal trophoblast glycoprotein, Orf73 Kaposi's sarcoma-associated herpesvirus, MAGE-CI, MAGE-B1 ANTIGEN, MAGE-B2 ANTIGEN, MAGE-2 ANTIGEN, MAGE-4a antigen, MAGE-4b antigen, Colon cancer antigen NY-CO-45, Lung cancer antigen NY-LU- 12 variant A, Cancer associated surface antigen, Adenocarcinoma antigen ART1, Paraneoplastic associated braintestis-cancer antigen, Neuro-oncological ventral antigen 2, Hepatocellular carcinoma antigen gene 520, TUMOR-ASSOCIATED ANTIGEN CO-029, Tumor-associated antigen MAGE-X2, Synovial sarcoma X breakpoint 2, Squamous cell carcinoma antigen recognized by T cell, Serologically defined colon cancer antigen 1, Serologically defined breast cancer antigen NYBR-15, Serologically defined breast cancer antigen NY-BR- 16, Chromogranin A; parathyroid secretory protein 1, DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6.

9/67

FIG. 6S (3 of 5)

Name BD1 Hinge EFD Linker B02 2H7-csc-hlgG-H2-G28-1 LH 2H7 (VL-VH, hlgGI -esc hlgGorhlgG (P238S/P331S1 H2 G28-1 (VH-VL) 2H7-sss-hlgG-H2-G28-1 HL 2H7 (VL-VH) hlgGI-SSS hlgGahlgG . (P238S/P331S1 H2 628-1 (VL-VH) 2H7-csc-blgG-H2-G28-1 LH 2H7 (VL-VH) hlgGI -esc hlgGorhlgG IP238SIP331S1 H2 G28-1 (VH-VL) 2H7-sss-hlgG-H3-G28-l LH 2H7 (VL-VH) hlgGI -SSS hlgGorhlgG IP238SP331S1 H3 628-1 (VL-VH) 2H7-csc-hlgG-H3-G28-1 LH 2H7 (VL-VH) hlgGI-esc hlgGorhlgG iP238SP331Sl H3 G28-1 (VH-VL) 2H7-sss4ilgG-H3-G28-1 HL 2H7 (VL-VH, hlgGI-SSS hlgGorhlgG fP238SF331Sl H3 628-1 (VL-VH, 2H7-csc-hlgG-H3-G28-l LH 2H7 (VL-VH) h|gG1-esc hlgGorhlgG (P238S/P331S1 H3 G28-1 (VH-VL) 2K7-s5S+lgG-H4-G28-1 LH 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG 1P238&P331S1 H4 628-1 (VL-VH) 2H7-csc-hlgG-H4-G28-UH 2H7 (VL-VH) hlgGI-esc hlgGorhlgG /P238SP331S1 H4 G28-1 (VH-VL) 2H7-$ss-hlgS-H4-G26-1 HL 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG £E238SE3315LJ H4 G28-1 (VL-VH) 2H7-OC-hlgG+H-G28-1 LH 2H7 (VL-VH) hlgGI-esc hlgGorhlgG IP238SP331SI. H4 628-1 (VH-VL) 2H7-sss-hlgG-H5-G28-1 LH 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG fP238SlP3^1Sl H5 ..... G28-1 (VH-VL, 2H7-sSS-hlgG-H5-G26-1 LH 2H7 (VL-VH) hlgGI-esc hlgGorhlgG IP238SP331S1 H5 G28-1 (VH-VL) 2H7-sss-hlgG-H5-G28-1 LH 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG IF238SP331S1— HS G2B-1(VH-VL) 2H7-csc4>lgG-H5-G2B-1 LH 2H7 (VL-VH, hlgGI-esc hlgGorhlgG rP238SP331SI H5 G2B-1 (VH-VL) 2H7-sss-li!gG-H6-G28-1 LH 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG fP23BSJP331Sl H6 G28-1 (VL-VH) 2H7-csc-b|gG-H6-G28-1 LH 2H7 (VL-VH) hlgGI-esc hlgGorhlgG ie2MS/raaisi H6 G2B-1 (VL-VH) 2H7-SSS-hlgG-H5-G28-1 HL 2H7 (VL-VH) hlgGI-SSS hlgGorhlgG (P238S/P331S1— H6 <326-1(VH-VL) 2H7-csc-h1gG-H6-G28-1 LH JH7 (VL-VH, hlgGI-esc hlgGorhlgG (P238S1P331S1 H6 G28-1 (VH-VL) 2H7-sss4ilgG-H7-G2B-1 LH !H7 (VL-VH) hlgGI ’SSS hlgGorhlgG IP238SZP331S1 H7 G26-1 (VL-VH) 2H7-csc-hlgG-H7-G2B-1 LH J H7 (VL-VH, ilgGl-csc ... hlgGorhlgG fP236SP331St H7 G28-1 (VL-VH) 2H7-sss-hlgG-H7-G28-1 HL 2 H7 (VL-VH, i lgG1-SSS hlgGorhlgG P238S/P331S1 H7 628-1 (VH-VL) 2H7-csc-hlgG-H7-G28-l LH 2 H7 (VL-VH, igGI-csc IgG or MgG IP238SZP331S1 H7 628-1(VH-VL) 2H7-sss-hlgG-STO1-G19-4 LH .2 H7 (VL-VH, Ii IgGl -SSS A IlgGorhlgG P238S/P331S1 STD1 G19-4 (VL-VH) 2H7-sss-hlgG-SlD1-GlW HL 2 47 (VL-VH) h IgGl -SSS 1 ilgGorhlgG P23BSE331S!... ST01 G19-4 (VH-VL,

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

9. A protein according to any one of claims 1 to 8, wherein the Fc region further comprises an immunoglobulin hinge region.

10 20 25 30

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Asn Ser Asp Trp Tyr

15 35 40 45

Phe Asp Leu 50 <210>

<211>

<212>

364

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL

5089144_1 (GHMatters) P79767.AU.1 5/02/14

528

2016231617 23 Sep 2016

CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3

15 <400> 364

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Tyr Ser Phe

20 1 5 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

25 20 25 30

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Asn Ser Asp Trp Tyr

30 35 40 45

Phe Asp Leu 50 <210> 365 <211> 51

5089144_1 (GHMatters) P79767.AU.1 5/02/14

529

2016231617 23 Sep 2016 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL 15 CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 365

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Tyr Ser Phe

15 10 15

Asn Pro Pro Ser Tyr

Thr Xaa Ala

He Tyr Pro Gly

Asn Gly Glu Thr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

530

2016231617 23 Sep 2016

Asn Gin Lys Phe Lys Gly Xaa Ser Trp Tyr

Tyr Lys Ser Gly Gly Asp

Phe Asp Leu 10 50

<210> 366 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> partial CH3 sequence

25 <400> 366

Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 15 10

<210> 367 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

531

2016231617 23 Sep 2016 <220>

<223> Partial CH3 sequence <400> 367

Gin Lys Ser Leu Ser Leu Ser Pro Gly 1 5 <210>

<211>

<212>

368

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Partial CH3 sequence <400> 368

Gin <210>

<211>

<212>

369

PRT <213> Artificial sequence

35 <220>

<223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

532

2016231617 23 Sep 2016 <220>

<223> Partial CH3 sequence

10 <400> 361

Arg Ala Ser Ser Ser Val Ser Tyr lie Val Xaa Gin Gin Tyr Ser Phe

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Asn Ser Tyr Trp Tyr

Phe Asp Leu 50

<210> 362 <211> 51 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

525

2016231617 23 Sep 2016 <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL 10 CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 362

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

15 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

20 25 30

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Lys Ser Asn Ser Asp Trp Tyr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

526

2016231617 23 Sep 2016

Phe Asp Leu 5 50 <210>

<211>

<212>

363

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3

20 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH 30 CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3

5089144_1 (GHMatters) P79767.AU.1 5/02/14

527

2016231617 23 Sep 2016 <400> 363

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

10 35 40 45

Phe Asp Leu 50 <210>

<211>

<212>

<213>

361

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL CDR3

35 <220>

<221> misc_feature <222> (21) . . (21)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

524

2016231617 23 Sep 2016 <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

10 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL CDR3 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH 2 0 CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 357

30 Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

15 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

20 25 30

5089144_1 (GHMatters) P79767.AU.1 5/02/14

519

2016231617 23 Sep 2016

Asn Gin Lys Phe Lys Gly Xaa Ser Tyr Tyr Ser Asn Ser Asp Trp Tyr

10 <400> 353

Gly Cys Pro Pro Cys Pro Asn Ser Ala Pro Glu Leu Gly Gly Gly Gly

15 10 15

Ser Gly Gly Gly Gly Ser 20 <210>

<211>

<212>

<213>

354

PRT

Artificial sequence <220>

<223> Synthetic peptide

30 <220>

<223> Extended scorpion linker <400> 354

35 Gly Cys Pro Pro Cys Pro Asn Ser Ala Pro Glu Leu Gly Gly Gly Gly

15 10 15

5089144_1 (GHMatters) P79767.AU.1 5/02/14

515

2016231617 23 Sep 2016

Ser Gly Gly Gly Gly Ser Gly <210> 355 <211> 51 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide

Gly Gly Gly Ser <220>

<223> VL CDR1-X-VL CDR3-X- VH CDR2-X-VH CDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDRl and VL CDR3

25 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH 35 CDR3 <400> 355

5089144_1 (GHMatters) P79767.AU.1 5/02/14

516

2016231617 23 Sep 2016

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

15 10

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

Asn Gin Lys Phe Lys Gly Xaa Ser Val Tyr Tyr Ser Asn Tyr Trp Tyr

Phe Asp Leu 50 <210> 356 <211> 51 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide

30 <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3 <220>

<221> misc_feature <222> (11)..(11) <223> Xaa = a range of amino acids between VL CDR1 and VL CDR3

5089144_1 (GHMatters) P79767.AU.1 5/02/14

517

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (21) . . (21) <223> Xaa = a range of amino acids between VL CDR3 and VH CDR2 <220>

<221> misc_feature <222> (39) . . (39) <223> Xaa = a range of amino acids between VH CDR2 and VH CDR3 <400> 356

Arg Ala Ser Ser Ser Val Ser Tyr lie His Xaa Gin Gin Trp Ser Phe

15 10 15

Asn Pro Pro Thr Xaa Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr

Asn Gin Lys Phe Lys Gly Xaa Ser Val Tyr Tyr Gly Gly Tyr Trp Tyr

Phe Asp Leu 50

35 <210> 357 <211> 51 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

518

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> VLCDR1-X-VLCDR3-X- VHCDR2-X-VHCDR3

10 <400> 351

Gly Cys Pro Pro Cys Pro Asn Ser Ala Pro Glu Leu <210>

<211>

<212>

<213>

352

PRT

Artificial sequence <220>

<223> Extended scorpion linker <400> 352

Gly Cys Pro Pro Cys Pro Asn Ser Ala Gly Gly

Pro Glu Leu Gly Gly

Ser

35 <210> 353 <211> 22 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

514

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Extended scorpion linker

10 1

<210> 348 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Extension sequence

25 <400> 348

Ala Pro Glu Leu Gly Gly Gly Gly Ser 1 5

<210> 349 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

512

2016231617 23 Sep 2016 <220>

<223> Extension sequence <400> 349

Ala Pro Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 15 10 <210>

<211>

<212>

<213>

350

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Extension sequence <400> 350

Ala Pro Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

15 10

Gly Gly Ser

35 <210> 351 <211> 12 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

513

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Extended scorpion linker

10 <211> 17 <212> PRT <213> Artificial sequence <220>

15 <223> Synthetic peptide <220>

<223> anti-CD-20 VH CDR2 <400> 336

Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys

Gly <210> 337 <211> 17 <212> PRT <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

506

2016231617 23 Sep 2016 <223> Synthetic peptide <220>

<223> anti-CD-20 VH CDR2 <400> 337

Ala lie Tyr Pro Gly Asn Gly Glu Phe Lys

Thr Ser Tyr Asn Gin Lys

Gly <210>

<211>

<212>

338

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VH CDR3

30 <400> 338

Ser Val Tyr Tyr Ser Asn Tyr Trp Tyr Phe Asp Leu 15 10 <210> 339 <211> 12

5089144_1 (GHMatters) P79767.AU.1 5/02/14

507

2016231617 23 Sep 2016 <212> PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VH CDR3 <400> 339

Ser Val Tyr Tyr Gly Gly Tyr Trp Tyr Phe Asp Leu 15 10 <210>

<211>

<212>

<213>

340

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VH CDR3 <400> 340

Ser Tyr Tyr Ser Asn Ser Asp Trp Tyr Phe Asp Leu 15 10

35 <210> 341 <211> 12 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

508

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VH CDR3 <400> 341

Ser Tyr Tyr Ser Gly Gly Asp Trp Tyr Phe Asp Leu <210>

<211>

<212>

<213>

342

PRT

Artificial sequence <220>

<223> Synthetic peptide

25 <220>

<223> anti-CD20 VH CDR3 <400> 342

Ser Tyr Lys Ser Asn Ser Tyr Trp Tyr Phe Asp Leu

<210> 343 <211> 12 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

509

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide <220>

<223> anti-CD20 VH CDR3 <400> 343

Ser Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Leu 15 10 <210>

<211>

<212>

344

PRT <213> Artificial sequence

20 <220>

<223> Synthetic peptide <220>

<223> anti-CD-20 VH CDR3 <400> 344

Ser Tyr Lys Ser Asn Ser Asp Trp Tyr Phe Asp Leu

<210> 345 <211> 12 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

510

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide

10 1 5 10 15

Val Leu Glu Val Thr Asn Ser Ser Leu Arg Gin Gin Leu Arg Leu Lys

15 20 25 30 lie Thr Gin Leu Gly Gin Ser Ala Glu Asp Leu Gin Gly Ser Arg Arg

20 35 40 45

Glu Leu Ala Gin Ala Gin Glu Ala Leu Gin Val Glu Gin Arg Ala His

50 55 60

Gin Ala Ala Glu Gly Gin Leu Gin Ala Cys Gin Ala Asp Arg Gin Lys

65 70 75

Thr Lys Glu Leu Glu

Thr Leu Gin Ser Glu Glu Gin Gin Arg Arg Ala

5089144_1 (GHMatters) P79767.AU.1 5/02/14

503

2016231617 23 Sep 2016

Gin Lys Leu Ser Asn Met Glu Asn Arg Leu Lys Pro Phe Phe Thr Cys

100 105 110

Gly Ser Ala Asp Thr Cys 115 <210>

<211>

<212>

<213>

332

PRT

Artificial sequence <220>

<223> Synthetic peptide

20 <220>

<223> anti-CD-20 VL CDRl <400> 332

Arg Ala Ser Ser Ser Val Ser Tyr lie His

<210> 333 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

504

2016231617 23 Sep 2016 <220>

<223> anti-CD-20 VL CDR1 <400> 333

Arg Ala Ser Ser Ser Val Ser Tyr lie Val <210>

<211>

<212>

<213>

334

PRT

Artificial sequence

15 <220>

<223> Synthetic peptide <220>

<223> anti-CD-20 VL CDR3 <400> 334

Gin Gin Trp Ser Phe Asn Pro Pro Thr 1 5

<210> 335 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

505

2016231617 23 Sep 2016 <223> anti-CD-20 VL CDR3 <400> 335

10 <220>

<223> Linker H65 <400> 328

15 Glu Pro Ala Phe Asp Ser 1

Thr Pro Gly Pro Asn lie Glu Leu Gin Lys

10 <210> 325 <211> 21 <212> PRT <213> Artificial sequence 15 <220> <223> Synthetic peptide

<220> 20 <223> Linker H62 <400> 325

Gin Arg His Asn Asn Ser Ser Leu Asn Thr Arg Thr Gin Lys 25 Ala Arg

15 10 15

His Cys Pro Asn Ser 30 20

<210> 326 <211> 19 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

499

2016231617 23 Sep 2016 <220>

<223> Synthetic peptide

10 <220>

<223> Synthetic peptide <220>

<223> Linker H54 <400> 317

Ser Val Leu Ala Asn Phe Ser Gin Pro Glu lie Ser Cys Pro Pro Cys

15 10 15

Pro Asn Ser

<210> 318 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

495

2016231617 23 Sep 2016 <223> Linker H55 <400> 318

Arg lie His Gin Met Asn Ser Glu Leu Ser

Ser Val Leu Ala Asn <210>

<211>

<212>

<213>

319

PRT

Artificial sequence

15 <220>

<223> Synthetic peptide <220>

<223> Linker H56 <400> 319

Gin Met Asn Ser Glu Leu Ser Val Leu Ala Asn Ser 15 10 <210>

<211>

<212>

<213>

320

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H57

5089144_1 (GHMatters) P79767.AU.1 5/02/14

496

2016231617 23 Sep 2016 <400> 320

Val Ser Glu Arg Pro Phe Pro Pro Asn Ser <210>

<211>

<212>

<213>

321

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H58 <400> 321

Lys Pro Phe Phe Thr Cys Gly Ser Ala Asp Thr Cys Pro Asn Ser

15 10

<210> 322 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H59 <400> 322

5089144_1 (GHMatters) P79767.AU.1 5/02/14

497

2016231617 23 Sep 2016

Lys Pro Phe Phe Thr Cys Gly Ser Ala Asp Thr Cys Pro Asn Ser

15 10 <210>

<211>

<212>

<213>

323

PRT

Artificial sequence <220>

<223> Synthetic peptide

15 <220>

<223> Linker H60 <400> 323

Gin Tyr Asn Cys Pro Gly Gin Tyr Thr Phe Ser Met Asn Ser <210>

<211>

<212>

324

PRT <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H61 <400> 324

5089144_1 (GHMatters) P79767.AU.1 5/02/14

498

2016231617 23 Sep 2016

Glu Pro Ala Phe Thr Pro Gly Pro Asn lie Glu Leu Gin Lys Asp Ser

15 10 15

Asp Cys Pro Asn Ser 20

10 <223> Linker H33 (AA) <400> 283

Arg lie His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro Cys 15 Pro Pro

15 10

Cys Pro Asn Ser 20 20

<210> 284 25 <400> 000 284 <210> 285 30 <400> 000 285 <210> 286 35 <400> 000 286

5089144_1 (GHMatters) P79767.AU.1 5/02/14

484

2016231617 23 Sep 2016

<210> 287 <400> 287 000 <210> 288 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H36 (PN) <400> 288 gggtgtccac cttgtccagg cggtc 39 <210> 289 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

<223> Linker H36 (AA)

35 <400> 289

Gly Cys Pro Pro Cys Pro Gly Gly Gly Gly Ser Asn Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

485

2016231617 23 Sep 2016 <210> 290 <400> 290

10 <400> 279

Leu Pro Pro Glu Thr Gin Glu Ser Gin Glu Val Thr Leu Ser Cys Pro

15 10

Pro Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

280

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H32 (PN) <400> 280 cggattcacc tgaacgtgtc cgagaggccc tttcctccga attct 45

5089144_1 (GHMatters) P79767.AU.1 5/02/14

482

2016231617 23 Sep 2016

<210> 281 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220> <223> Linker H32 (AA) <400> 281

15 Arg lie His Leu Asn Val Ser Glu Arg Pro Phe Pro Pro Asn Ser

15 10 <210>

<211>

<212>

<213>

282

DNA

Artificial sequence

25 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H33 (PN) <400> 282 cggattcacc tgaacgtgtc cgagaggccc tttcctccct gtccaccctg cccgaattct 60 <210> 283

5089144_1 (GHMatters) P79767.AU.1 5/02/14

483

2016231617 23 Sep 2016 <211> 20 <212> PRT <213> Artificial sequence

10 Leu Pro Pro Glu Thr Gin Glu Ser Gin Glu Val Thr Leu Asn Ser

15 10 <210>

<211>

<212>

<213>

278

DNA

Artificial sequence

20 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H31 (PN) <400> 278 ctgccacctg agacacagga gagtcaagaa gtcaccctgt cctgtccacc ttgcccgaat 60 tct

35 <210> 279 <211> 21 <212> PRT

5089144_1 (GHMatters) P79767.AU.1 5/02/14

481

2016231617 23 Sep 2016 <213> Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H31 (AA)

10 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H23 (PN) <400> 262 ctggatgtga gtgagaggcc ttttcctcca cacatccagt cctgtccacc ttgcccgaat 60 tct

25 <210> <211> <212> <213> 263 21 PRT Artificial sequence 30 <220> <223> Synthetic peptide <220> <223> Linker H23 (AA) 35 <400> 263

5089144_1 (GHMatters) P79767.AU.1 5/02/14

473

2016231617 23 Sep 2016

Leu Asp Val Ser Glu Arg Pro Phe Pro Pro His lie Gin Ser Cys Pro

15 10

Pro Cys Pro Asn Ser 20 <210> 264 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H24 (DNA) <400> 264 cgggaacagc tggcagaggt cactttgagc ttgaaagcga attct 45

<210> 265 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H24 (AA) <400> 265

5089144_1 (GHMatters) P79767.AU.1 5/02/14

474

2016231617 23 Sep 2016

Arg Glu Gin Leu Ala Glu Val Thr Leu Ser Leu Lys Ala Asn Ser

15 10 <210> 266 <211> 60 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H25 (PN) <400> 266

20 cgggaacagc tggcagaggt cactttgagc gtgaaagctt gtccaccctg cccgaattct 60

<210> 267 25 <211> 20 <212> PRT <213> Artificial sequence <220> 30 <223> Synthetic peptide

<220> <223> Linker H25 (AA) 35 <400> 267

5089144_1 (GHMatters) P79767.AU.1 5/02/14

475

2016231617 23 Sep 2016

Arg Glu Gin Leu Ala Glu Val Thr Leu Ser Leu Lys Ala Cys Pro Pro

15 10

Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

268

DNA

Artificial sequence

15 <220>

<223> Synthetic polynucleotide <220>

<223> Linker H26 (PN) <400> 268 cggattcacc agatgaactc cgagttgagc gtgctcgcga attct 45 <210>

<211>

<212>

<213>

269

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H26 (AA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

476

2016231617 23 Sep 2016 <400> 269

Arg lie His Gin Met Asn Ser Glu Leu Ser Val Leu Ala Asn 5 Ser

15 10 <210>

<211>

<212>

<213>

270

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H27 (PN) <400> 270 cggattcacc agatgaactc cgagttgagc gtgctcgctt gtccaccctg cccgaattct 60

<210> 271 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H27 (AA) <400> 271

5089144_1 (GHMatters) P79767.AU.1 5/02/14

477

2016231617 23 Sep 2016

Arg lie His Gin Met Asn Ser Glu Leu Ser Val Leu Ala Cys Pro Pro

15 10

Cys Pro Asn Ser 20 <210>

<211>

<212>

<213>

272

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H28 (PN) <400> 272 gataccaaag ggaagaacgt cctcgagaag atcttctcga attct 45

<210> 273 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide

<220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

478

2016231617 23 Sep 2016 <223> Linker H28 (AA) <400> 273

10 <220>

<223> Synthetic polypeptide <220>

<223> Linker H21 (PN) <400> 258 ctgaaaatcc aggagagggt cagtaagcca aagatctcct gtccaccttg cccgaattct 60

<210> 259 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <220> <223> Linker H21 (AA) <400> 259

35 Leu Lys lie Gin Glu Arg Val Ser Lys Pro Lys He Ser Cys Pro Pro

15 10 15

5089144_1 (GHMatters) P79767.AU.1 5/02/14

471

2016231617 23 Sep 2016

Cys Pro Asn Ser 20 <210> 260 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polypeptide

15 <220>

<223> Linker H22 (PN) <400> 260 ctggatgtga gtgagaggcc ttttcctcca cacatccaga attct 20 45 <210>

<211>

<212>

<213>

261

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H22 (AA)

35 <400> 261

Leu Asp Val Ser Glu Arg Pro Phe Pro Pro His lie Gin Asn

5089144_1 (GHMatters) P79767.AU.1 5/02/14

472

2016231617 23 Sep 2016

Ser

10 <211> 45 <212> DNA <213> Artificial sequence <220>

15 <223> Synthetic polynucleotide <220>

<223> ccc(s) (DNA)

20 <400> 232 gagcccaaat cttgtgacaa aactcacaca tctccaccgt gctca 45

25 <210> <211> <212> <213> 233 15 PRT Artificial sequence 30 <220> <223> Synthetic polypeptide <220> <223> ccc(s) (AA) 35 <400> 233

5089144_1 (GHMatters) P79767.AU.1 5/02/14

459

2016231617 23 Sep 2016

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Cys Ser

15 10 <210>

<211>

<212>

<213>

234

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> Linker H8 (PN) <400> 234 gggtctccac cttctccgaa ttct 24 <210>

<211>

<212>

<213>

235

PRT

Artificial sequence <220>

<223> Synthetic peptide <400> 235

Gly Ser Pro Pro Ser Pro Asn Ser 1 5 <210> 236

5089144_1 (GHMatters) P79767.AU.1 5/02/14

460

2016231617 23 Sep 2016 <211> 21 <212> DNA <213> Artificial sequence

10 <210> 230 <211> 45 <212> DNA 15 <213> Artificial sequence <220> <223> Synthetic polynucleotide 20 <220> <223> scs(s) -hlgGl (DNA) <400> 230 gagcccaaat cttctgacaa aactcacaca 25 45 <210> 231 <211> 15 30 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide 35 <220> <223> scs(s) -hlgGl (AA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

458

2016231617 23 Sep 2016 <400> 231

Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser 5 Ser

15 10 <210> 232

10 660 665 670

Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser

15 675 680 685

Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro

690 695 700

Gly Gin Pro Pro Lys Glu Ser

Leu Leu

He

Ser Ala Ala

Ser Asn Val

705 710 715

720

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 30 Phe Ser

725 730

735

35 Leu Asn He His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys

740 745 750

5089144_1 (GHMatters) P79767.AU.1 5/02/14

457

2016231617 23 Sep 2016

Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

755 760 765

Glu lie Lys Arg 770

10 <400> 229

15 Met Ala 1 Asp Ser Phe Gin Val 5 Gin He Phe Ser Phe 10 Leu Leu He Ser Val lie Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 20 25 30 20 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 25 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 30 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 35 80

5089144_1 (GHMatters) P79767.AU.1 5/02/14

451

2016231617 23 Sep 2016

Ala Arg Phe Thr lie

Ser Arg Val Gin Trp

Ser Phe Asn Leu Lys

115

Asp Gly Gly Ser Ser

130

Ser Gly Ser Gly Ser

Glu Ala Glu Asp Ala

100

Pro Pro Thr Phe Gly

120

Gly Ser Gly Gly Gly

135

Gly Thr Ser Tyr Ser Leu

Ala Thr Tyr Tyr Cys Gin

105 110

Ala Gly Thr Lys Leu Glu

125

Gly Ser Gly Gly Gly Gly

140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

30 175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp He

35 180 185 190

5089144_1 (GHMatters) P79767.AU.1 5/02/14

452

2016231617 23 Sep 2016

Gly Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

20 255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Cys

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Cys Ser Ala Pro Glu Leu Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

453

2016231617 23 Sep 2016 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 10 335

15 His Thr Asn Tyr Ala Lys 340 Thr Lys Pro Arg Glu 345 Glu Gin Tyr Asn Ser 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 355 360 365 20 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He 370 375 380 25 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 30 400 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin

Val Ser

35 405 410

415

5089144_1 (GHMatters) P79767.AU.1 5/02/14

454

2016231617 23 Sep 2016

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu

420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly 30 Ser Gly

500 505 510

Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser 35 Gly Pro

515 520 525

5089144_1 (GHMatters) P79767.AU.1 5/02/14

455

2016231617 23 Sep 2016

10 <223>

<220>

<221>

<222>

15 <223>

<220>

<221>

<222>

20 <223>

misc_feature (1)··(22) Leader misc_feature (23)..(128) VL misc_feature (129) . . (144) Linker misc_feature (145)..(265)

VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(519) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (520)..(640) <223> VH2 <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

450

2016231617 23 Sep 2016 <221> misc_feature <222> (641)..(660) <223> Linker2

10 lie Val Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gin

660 665 670

15 Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val

675 680 685

20 Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys

690 695 700

25 Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala Arg

705 710 715

720

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn He His Pro

725 730

735

Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys Gin Gin Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

445

740

745

750

2016231617 23 Sep 2016

Arg Lys

Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 5 Arg

755 760 765 <210> 228 10 <211> 2337 <212> DNA <213> Artificial sequence <220>

15 <223> Synthetic polynucleotide <220>

<223> 2H7sssIgGl-H7-G281 HL (DNA)

20 <400> 228 aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60 gtcataatgt ccagaggaca aattgttctc tcccagtctc cagcaatcct 25 gtctgcatct 120 ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180

30 taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240 tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300 agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360

5089144_1 (GHMatters) P79767.AU.1 5/02/14

446

2016231617 23 Sep 2016

cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 5 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc 10 aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 15 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 20 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt 25 ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttgtga acatccccac cctcttcccc cgtgctcagc 900 acctgaactc ctggggggac cgtcagtctt 30 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg 35 cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

447

2016231617 23 Sep 2016 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140 aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg 5 gcagccccga 1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260 io ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct 20 ctccctgtct 1500 ccgggtaaga attatggtgg cggtggctcg ggcggtggtg gatctggagg aggtgggagt 1560

25 gggaattctc aggtgcagct gaaggagtca ggacctggcc tggtggcgcc ctcacagagc 1620 ctgtccatca catgcaccgt ctcagggttc tcattaaccg gctatggtgt aaactgggtt 1680 cgccagcctc caggaaaggg tctggagtgg ctgggaatga tatggggtga tggaagcaca 1740 gactataatt cagctctcaa atccagacta tcgatcacca aggacaactc 35 caagagccaa 1800 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccagatacta

5089144_1 (GHMatters) P79767.AU.1 5/02/14

448 ttactatgtt atggactact ggggtcaagg

2016231617 23 Sep 2016 ctgtgctcga gatggttata aacctcagtc accgtctcct tgggtcgggt ggcggcggat gtctctaggt cagagagcca cacaagttta atgcagtggt tgctgctagc aacgtagaat agactttagc ctcaacatcc gcaaagtagg aaggttccat atctaga

1860 gtaactttca

1920 ctgggggtgg

1980 ctgacattgt

2040 ccatctcctg

2100 accaacagaa

2160 ctggggtccc

2220 atcctgtgga

2280 ggacgttcgg

2337 aggctctggt gctcacccaa cagagccagt accaggacag tgccaggttt ggaggatgat tggaggcacc ggcggtggat tctccagctt gaaagtgttg ccacccaaac agtggcagtg attgcaatgt aagctggaaa ccggcggagg ctttggctgt aatattatgt tcctcatctc ggtctgggac atttctgtca tcaaacgtta <210> 229 <211> 772 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7cscIgGl-STDl-2el2HL (w/2E12 leader) (AA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

449

2016231617 23 Sep 2016 <220>

<221>

<222>

10 Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin

210 215 220

15 Leu Ser Ser Leu Ala Arg 225 240

Val Val Tyr Tyr Gly Thr

255

Gly Thr Thr Val Lys Thr

260

His Thr Ser Pro Pro Ser

275

Val Phe Leu Phe

Thr Ser Glu Asp Ser Ala

230

Ser Asn Ser Tyr Trp Tyr

245 250

Thr Val Ser Ser Glu Pro

265

Pro Ser Pro Ala Pro Glu

280

Pro Pro Lys Pro Lys Asp

Val Tyr Phe Cys

235

Phe Asp Val Trp

Lys Ser Ser Asp

270

Leu Leu Gly Gly

285

Thr Leu Met He

5089144_1 (GHMatters) P79767.AU.1 5/02/14

429

Ser Arg

290 295 300

2016231617 23 Sep 2016

10 Phe Ser Ser Arg Gly Ser Gly 85 Ser Gly Thr Ser Tyr 90 Ser Leu Thr He Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Phe 100 105 110 15 Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Asp Gly 115 120 125 20 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Ser Gin Ala 130 135 140 25 Tyr Leu Gin Gin Ser Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val 145 150 155 30 160 Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

Asn Met

35 165 170

175

5089144_1 (GHMatters) P79767.AU.1 5/02/14

416

2016231617 23 Sep 2016

His Trp Gly Ala lie Tyr Lys Gly

Lys Ala Met Gin

210

Leu Ser Ala Arg 225

20 240

Val Lys Gin

180

Pro Gly Asn

195

Thr Leu Thr

Ser Leu Thr

Thr Pro Arg

Gly Asp Thr

200

Val Asp Lys

215

Ser Glu Asp

230

Gin Gly Leu

185

Ser Tyr Asn

Ser Ser Ser

Ser Ala Val

235

Glu Trp He

190

Gin Lys Phe

205

Thr Ala Tyr

220

Tyr Phe Cys

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr 25 245 250 255

Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp 30 Lys Thr

260 265 270

His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

35 275 280 285

5089144_1 (GHMatters) P79767.AU.1 5/02/14

417

2016231617 23 Sep 2016

Val Ser Phe Arg 290 Leu Phe Pro Pro Lys 295 Pro Lys Asp Thr Leu 300 Met He Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 370 375 380 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

Thr Leu

385 390 395

400

5089144_1 (GHMatters) P79767.AU.1 5/02/14

418

2016231617 23 Sep 2016

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys

405 410

10 Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg 530 535 540

15 Gin Gly 545 560 Pro Asp Pro Gly Lys Gly Leu Glu 550 Trp Leu Gly 555 Met He Trp 20 Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr 565 570 575 25 Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin 580 585 590 30 Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn 595 600 605 35 Phe His Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

408

615

620

Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

Gly Gly

625 630 635

640

2016231617 23 Sep 2016

Val Thr 610

Gly Ser Gly Gly Gly Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala

645 650

655

Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala

660 665 670

Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin

675 680 685

Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser Asn

690 695 700

Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

705 710 715

720

Asp Phe Ser Leu Asn He His Pro Val Glu Glu Asp Asp He

5089144_1 (GHMatters) P79767.AU.1 5/02/14

409

2016231617 23 Sep 2016

Ala Met

725 730

735

Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Gly Gly

740 745

Thr Lys Leu Glu lie Lys Arg 755

15 <210> 222 <211> 2307 <212> DNA <213> Artificial sequence

20 <220>

<223> Synthetic polynucleotide <220>

<223> n2H7sssIgGl-H4-2el2HL (DNA) <400> 222 aagcttgccg ccatggaagc accagcgcag cttctcttcc ctggctccca 60

30 gataccaccg gtcaaattgt tctctcccag tctccagcaa atctccaggg 120 gagaaggtca caatgacttg cagggccagc tcaagtgtaa ctggtaccag 180 cagaagccag gatcctcccc caaaccctgg atttatgccc ggcttctgga 240

Thr Phe Gly

750 tcctgctact tcctgtctgc gttacatgca catccaacct

5089144_1 (GHMatters) P79767.AU.1 5/02/14

410

2016231617 23 Sep 2016

gtccctgctc aatcagcaga gcttcagtgg 300 cagtgggtct gggacctctt actctctcac 5 gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa 10 ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct 15 ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat 20 aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac 25 tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt 30 gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc 35 aaggacaccc ggacgtgagc tcatgatctc 960 ccggacccct gaggtcacat gcgtggtggt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

411

2016231617 23 Sep 2016 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1020 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag 5 cgtcctcacc 1080 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1140 io ctcccagccc ccatcgagaa aacaatctcc aaagccaaag ggcagccccg agaaccacag 1200 gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgacctgc 1260 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1320 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt 20 cttcctctac 1380 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1440

25 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaag 1500 ggtggcggtg gctcgggcgg tggtggatct gggaattctc aggtgcagct gaaggagtca 1560 ggacctggcc tggtggcgcc ctcacagagc ctgtccatca catgcaccgt ctcagggttc 1620 tcattaaccg gctatggtgt aaactgggtt cgccagcctc caggaaaggg 35 tctggagtgg 1680 ctgggaatga tatggggtga tggaagcaca gactataatt cagctctcaa

5089144_1 (GHMatters) P79767.AU.1 5/02/14

412

2016231617 23 Sep 2016 atccagacta 1740 tcgatcacca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 1800 gatgacacag ccagatacta ctgtgctcga gatggttata gtaactttca ttactatgtt 1860 atggactact ggggtcaagg aacctcagtc accgtctcct ctgggggtgg io aggctctggt 1920 ggcggtggat ccggcggagg tgggtcgggt ggcggcggat ctgacattgt gctcacccaa 1980

15 tctccagctt ctttggctgt gtctctaggt cagagagcca ccatctcctg cagagccagt 2040 gaaagtgttg aatattatgt cacaagttta atgcagtggt accaacagaa accaggacag 2100 ccacccaaac tcctcatctc tgctgctagc aacgtagaat ctggggtccc tgccaggttt 2160 agtggcagtg ggtctgggac agactttagc ctcaacatcc atcctgtgga 25 ggaggatgat 2220 attgcaatgt atttctgtca gcaaagtagg aaggttccat ggacgttcgg tggaggcacc 2280

30 aagctggaaa tcaaacgtta atctaga 2307 <210> 223

35 <211> 762 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

413

2016231617 23 Sep 2016 <220>

<223> Synthetic polypeptide

10 Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser

420 425 430

15 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

435 440 445

20 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

450 455 460

25 Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

465

480

470

475

Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

485

490

Asn Tyr Gly Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu

495

5089144_1 (GHMatters) P79767.AU.1 5/02/14

407

2016231617 23 Sep 2016

Lys Glu

500 505 510

10 <220>

<221> misc_feature <222> (1) . . (20) <223> Leader

15 <220>

<221> misc_feature <222> (21) . . (126) <223> VL

20 <220>

<221> misc_feature <222> (127)..(142) <223> Linker

25 <220>

<221> misc_feature <222> (143)..(264) <223> VH

30 <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge

35 <220>

<221> misc_feature <222> (497) . . (506)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

402

2016231617 23 Sep 2016

<223> EFD-BD2 Linker <220> <221> misc feature 5 <222> (507)..(627) <223> VH2 <220> <221> misc feature 10 <222> (628) . . (647) <223> Linker2 <220> <221> misc feature 15 <222> (648)..(759) <223> VL2 <400> 221 20 Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10

25 Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser 20 25 30

30 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser 35 40 45 35 Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser

Pro Lys

50 55 60

5089144_1 (GHMatters) P79767.AU.1 5/02/14

403

2016231617 23 Sep 2016

10 740 745 750

Leu Glu He Lys Arg 755

<210> 220 <211> 2298 <212> DNA 20 <213> Artificial sequence <220> <223> Synthetic polynucleotide 25 <220> <223> n2H7sss!gGl-H3-2el2HL (DNA) <400> 220 aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact 30 ctggctccca 60 gataccaccg gtcaaattgt tctctcccag tctccagcaa tcctgtctgc atctccaggg 120 35 gagaaggtca caatgacttg cagggccagc tcaagtgtaa gttacatgca ctggtaccag 180

5089144_1 (GHMatters) P79767.AU.1 5/02/14

398

2016231617 23 Sep 2016

cagaagccag ggcttctgga gatcctcccc 240 caaaccctgg atttatgccc catccaacct 5 gtccctgctc aatcagcaga gcttcagtgg 300 cagtgggtct gggacctctt actctctcac gtggaggctg cccacccacg aagatgctgc 360 cacttattac tgccagcagt ggagttttaa 10 ttcggtgctg tggtggatct ggaccaagct 420 ggagctgaaa gatggcggtg gctcgggcgg 15 ggaggaggtg ggtgaggcct gagctagcca 480 ggcttatcta cagcagtctg gggctgagct ggggcctcag ttacaatatg tgaagatgtc 540 ctgcaaggct tctggctaca catttaccag 20 cactgggtaa ttatccagga agcagacacc 600 tagacagggc ctggaatgga ttggagctat aatggtgata tgtagacaaa cttcctacaa 660 tcagaagttc aagggcaagg ccacactgac 25 tcctccagca tgcggtctat cagcctacat 720 gcagctcagc agcctgacat ctgaagactc 30 ttctgtgcaa ctggggcaca gagtggtgta 780 ctatagtaac tcttactggt acttcgatgt gggaccacgg cacatcccca tcaccgtctc 840 gagcgagccc aaatcttctg acaaaactca 35 ccgagcccag cccaaaaccc cacctgaact 900 cctgggggga ccgtcagtct tcctcttccc aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

399 gttcaactgg tacgtggacg gcgtggaggt

2016231617 23 Sep 2016 ggacgtgagc cacgaagacc gcataatgcc aagacaaagc cgtcctcacc gtcctgcacc caacaaagcc ctcccagccc agaaccacag gtgtacaccc cctgacctgc ctggtcaaag tgggcagccg gagaacaact cttcctctac agcaagctca atgctccgtg atgcatgagg tccgggtaag aattatggtg aggacctggc ctggtggcgc ctcattaacc ggctatggtg gctgggaatg

960 ctgaggtcaa

1020 cgcgggagga

1080 aggactggct

1140 ccatcgagaa

1200 tgcccccatc

1260 gcttctatcc

1320 acaagaccac

1380 ccgtggacaa

1440 ctctgcacaa

1500 gcggtggctc

1560 cctcacagag

1620 taaactgggt

1680 gcagtacaac gaatggcaag aacaatctcc ccgggatgag cagcgacatc gcctcccgtg gagcaggtgg ccactacacg tgggaattct cctgtccatc tcgccagcct agcacgtacc gagtacaagt aaagccaaag ctgaccaaga gccgtggagt ctggactccg cagcagggga cagaagagcc caggtgcagc acatgcaccg ccaggaaagg gtgtggtcag gcaaggtctc ggcagccccg accaggtcag gggagagcaa acggctcctt acgtcttctc tctccctgtc tgaaggagtc tctcagggtt gtctggagtg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

400

2016231617 23 Sep 2016

atatggggtg atggaagcac agactataat tcagctctca aatccagact atcgatcacc 1740 aaggacaact ccaagagcca agttttctta aaaatgaaca gtctgcaaac tgatgacaca 1800 gccagatact actgtgctcg agatggttat agtaactttc attactatgt tatggactac 1860 tggggtcaag gaacctcagt caccgtctcc tctgggggtg gaggctctgg tggcggtgga 1920 tccggcggag gtgggtcggg tggcggcgga tctgacattg tgctcaccca atctccagct 1980 tctttggctg tgtctctagg tcagagagcc accatctcct gcagagccag tgaaagtgtt 2040 gaatattatg tcacaagttt aatgcagtgg taccaacaga aaccaggaca gccacccaaa 2100 ctcctcatct ctgctgctag caacgtagaa tctggggtcc ctgccaggtt tagtggcagt 2160 gggtctggga cagactttag cctcaacatc catcctgtgg aggaggatga tattgcaatg 2220 tatttctgtc agcaaagtag gaaggttcca tggacgttcg gtggaggcac caagctggaa 2280 atcaaacgtt aatctaga 2298

<210> 221 <211> 759

5089144_1 (GHMatters) P79767.AU.1 5/02/14

401

2016231617 23 Sep 2016 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> n2H7sss!gGl-H3-2el2HL (AA)

10 <220>

<221> misc_feature <222> (626)..(645) <223> Linker2

15 <220>

<221> misc_feature <222> (646)..(757) <223> VL2

20 <400> 219

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

15 10

Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

391

2016231617 23 Sep 2016

Pro Lys 50

Pro Ala 65 80 Trp Arg He Tyr Ala Pro 70 Phe Ser Gly Ser Gly Ser Ser Arg 85 Val Glu Ala Glu Asp Ala Ser Phe 100 Asn Pro Pro Thr Phe Gly Asp Gly 115 Gly Gly Ser Gly Gly Gly Gin Ala 130 Tyr Leu Gin Gin Ser Gly Ser Val 145 150 160 Lys Met Ser Cys Lys Ala

Asn Leu Ala Ser Gly Val Pro

Thr Ser Tyr Ser Leu Thr lie

90 95

Thr Tyr Tyr Cys Gin Gin Trp

105 110

Gly Thr Lys Leu Glu Leu Lys

120 125

Ser Gly Gly Gly Gly Ala Ser

140

Glu Leu Val Arg Pro Gly Ala

155

Gly Tyr Thr Phe Thr Ser Tyr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

392

His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

Gly Ala

180 185 190

2016231617 23 Sep 2016

Asn Met

175

165

170

He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe Lys Gly

195 200 205

Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gin

210 215 220

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg

225 230 235

240

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp Gly Thr

245 250

30 255

Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr

35 260 265 270

5089144_1 (GHMatters) P79767.AU.1 5/02/14

393

2016231617 23 Sep 2016

His Pro Thr Ser Ser 275 Pro Pro Ser Pro Ala 280 Pro Glu Leu Leu Gly 285 Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg 290 295 300 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 305 310 315 320 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 325 330 335 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 370 375 380 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

394

2016231617 23 Sep 2016

Thr Leu

385

400

Pro Pro Ser Arg Thr Cys

415

Leu Val Lys Gly Glu Ser

420

Asn Gly Gin Pro Leu Asp

435

Ser Asp Gly Ser Lys Ser

450

Arg Trp Gin Gin

Glu Ala

465

30 480

Leu His Asn His Gly Lys

495

390

Asp Glu Leu Thr

405

Phe Tyr Pro Ser

Glu Asn Asn Tyr

440

Phe Phe Leu Tyr

455

Gly Asn Val Phe

470

Tyr Thr Gin Lys

485

395

Lys Asn Gin Val Ser Leu

410

Asp lie Ala Val Glu Trp

425 430

Lys Thr Thr Pro Pro Val

445

Ser Lys Leu Thr Val Asp

460

Ser Cys Ser Val Met His

475

Ser Leu Ser Leu Ser Pro

490

5089144_1 (GHMatters) P79767.AU.1 5/02/14

395

2016231617 23 Sep 2016

Gly Gly Ser Gly

Pro Gly Thr Val

Ser Gly Gin Pro

530

Pro Gly Gly Ser 545

20 560

Thr Asp Lys Asp

25 575

Asn Ser Thr Asp

Asp Thr Phe His

Gly Gly Ser

500

Leu Val Ala

515

Phe Ser Leu

Lys Gly Leu

Tyr Asn Ser

565

Lys Ser Gin

580

Ala Arg Tyr

595

Gly Asn Ser

Pro Ser Gin

520

Thr Gly Tyr

535

Glu Trp Leu

550

Ala Leu Lys

Val Phe Leu

Tyr Cys Ala

600

Gin Val Gin

505

Ser Leu Ser

Gly Val Asn

Gly Met lie

555

Ser Arg Leu

570

Lys Met Asn

585

Arg Asp Gly

Leu Lys Glu

510

He Thr Cys

525

Trp Val Arg

540

Trp Gly Asp

Ser He Thr

Ser Leu Gin

590

Tyr Ser Asn

605

5089144_1 (GHMatters) P79767.AU.1 5/02/14

396

2016231617 23 Sep 2016

Tyr Tyr Val Met Asp Tyr Val Ser 610 5 Ser Gly Gly Gly Gly Ser Gly Ser 625 630 640 10 Gly Gly Gly Gly Ser Asp Ser Leu 645 15 655

Ala Val Ser Leu Gly Gin Ser Glu 20 660

Ser Val Glu Tyr Tyr Val Gin Lys 25 675

Pro Gly Gin Pro Pro Lys Val Glu 30 690

Ser Gly Val Pro Ala Arg Asp Phe 35 705 710 720

Trp Gly Gin Gly Thr Ser Val Thr

615 620

Gly Gly Gly Gly Ser Gly Gly Gly

635 lie Val Leu Thr Gin Ser Pro Ala

650

Arg Ala Thr He Ser Cys Arg Ala

665 670

Thr Ser Leu Met Gin Trp Tyr Gin

680 685

Leu Leu He Ser Ala Ala Ser Asn

695 700

Phe Ser Gly Ser Gly Ser Gly Thr

715

5089144_1 (GHMatters) P79767.AU.1 5/02/14

397

2016231617 23 Sep 2016

Ser Leu Asn lie His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe

725 730

10 Val Gly 255 Val Thr Tyr Tyr Ser 245 Asn Ser Tyr Trp Tyr 250 Phe Asp Val Trp 15 Gly Thr Thr Val Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr 260 265 270 20 His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 275 280 285 25 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg 290 295 300

30 Thr Asp 305 320 Pro Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 310 315 35 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

Asn Ala

5089144_1 (GHMatters) P79767.AU.1 5/02/14

370

2016231617 23 Sep 2016

335 325 330 Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 340 345 350 Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 355 360 365 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro He Glu Lys Thr 370 375 380 lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu 385 390 395 400 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys 405 410 415 Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser 420 425 430 Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

5089144_1 (GHMatters) P79767.AU.1 5/02/14

371

2016231617 23 Sep 2016

Leu Asp 435 Ser Asp Gly Ser Phe Phe Lys Ser 450 Arg Trp Gin Gin Gly Asn Glu Ala 465 470 480 Leu His Asn His Tyr Thr Gly Lys 485 495 Asn Tyr Gly Gly Gly Gly Gly Gly 500 Ser Gly Asn Ser Asp He Leu Ala 515 Val Ser Leu Gly Gin Arg Glu Ser 530 Val Glu Tyr Tyr Val Thr Lys Pro

440 445

Leu Tyr Ser Lys Leu Thr Val Asp

455 460

Val Phe Ser Cys Ser Val Met His

475

Gin Lys Ser Leu Ser Leu Ser Pro

490

Ser Gly Gly Gly Gly Ser Gly Gly

505 510

Val Leu Thr Gin Ser Pro Ala Ser

520 525

Ala Thr lie Ser Cys Arg Ala Ser

535 540

Ser Leu Met Gin Trp Tyr Gin Gin

5089144_1 (GHMatters) P79767.AU.1 5/02/14

372

2016231617 23 Sep 2016

545

560

550

555

10 Asp Thr Thr Gly Gin lie Val Leu Ser Gin Ser Pro Ala He Leu Ser

20 25 30

15 Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

35 40 45

20 Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys

50 55 60

25 Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

65 70 75

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg

85 90 95

Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Phe

5089144_1 (GHMatters) P79767.AU.1 5/02/14

368

100

2016231617 23 Sep 2016

10 <220>

<221> misc_feature <222> (143)..(264) <223> VH

15 <220>

<221> misc_feature <222> (265) . . (279) <223> Hinge

20 <220>

<221> misc_feature <222> (497) . . (516) <223> EFD-BD2 Linker

25 <220>

<221> misc_feature <222> (517)..(628) <223> VL2

30 <220>

<221> misc_feature <222> (629)..(643) <223> Linker

35 <220>

<221> misc_feature <222> (644)..(764)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

367

2016231617 23 Sep 2016 <223> VH2 <400> 215

10 Ser Gly Thr Gin Phe Ser Leu Lys lie Ser Ser Leu Gin Pro Glu Asp

225 230 235

240

Ser Gly Ser Tyr Phe Cys Gin His His Ser Asp Asn Pro Trp Thr Phe

245 250

255

Gly Gly Gly Thr Glu Leu Glu He Lys Gly Ser Ser Glu Pro Lys Ser

260 265 270

Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

275 280 285

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

290 295 300

Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

5089144_1 (GHMatters) P79767.AU.1 5/02/14

332

2016231617 23 Sep 2016

Val Ser

305

320

His Glu Asp Pro Val Glu

335

Val His Asn Ala Ser Thr

340

Tyr Arg Val Val Leu Asn

355

Gly Lys Glu Tyr Ala Pro

370 lie Glu Lys Thr

Pro Gin

385

30 400

Val Tyr Thr Leu Gin Val

35 415

310

Glu Val Lys Phe

325

Lys Thr Lys Pro

Ser Val Leu Thr

360

Lys Cys Lys Val

375

He Ser Lys Ala

390

Pro Pro Ser Arg

405

315

Asn Trp Tyr Val Asp Gly

330

Arg Glu Glu Gin Tyr Asn

345 350

Val Leu His Gin Asp Trp

365

Ser Asn Lys Ala Leu Pro

380

Lys Gly Gin Pro Arg Glu

395

Asp Glu Leu Thr Lys Asn

410

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val

420 425 430

Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

450 455 460

Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val

465 470 475

480

Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu

485 490

25 495

Ser Pro Gly Lys

500 <210> 208 <211> 1522 <212> DNA <213> Artificial sequence

<220>

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<223> Synthetic polynucleotide 5 <220> <223> G19- <400> 208 -4 LH SMIP (DNA) aagcttgccg ctggctccca ccatggaagc 60 accagcgcag cttctcttcc tcctgctact 10 gataccaccg ctctctggga gtgacatcca 120 gatgacacag actacatcct ccctgtctgc gacagagtca aaactggtat ccatcagttg 180 cagggcaagt caggacattc gcaattattt 15 cagcagaaac attacactca cagatggaac 240 tgttaaactc ctgatctact acacatcaag 20 ggagtcccat caccattgcc caaggttcag 300 tggcagtggg tctggaacag attattctct aacctgcaac gcttccgtgg cagaagatat 360 tgccacttac ttttgccaac agggtaatac 25 acgttcggtg gggcggtggt gaggcaccaa 420 actggtaacc aaacggggtg gcggtggctc ggatctggag acctgaactg gaggtgggag 480 cgctagcgag gtccagctgc aacagtctgg 30 gtgaagcctg attcactggc gagcttcaat 540 gaagatttcc tgcaaggcct ctggttactc 35 tacatcgtga tggacttatt actggctgaa 600 gcagagccat ggaaagaacc ttgagtggat aatccataca aaggtcttac tacctacaac cagaaattca agggcaaggc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

cacattaact 660 gtagacaagt tgaagactct catccagcac 720 agcctacatg gagctcctca gtctgacatc gcagtctatt cttcgatgtc actgtgcaag 780 atctgggtac tatggtgact cggactggta tggggcgcag tgacaaaact ggaccacggt 840 caccgtctcc tcgagcgagc ccaaatcttc cacacatgcc cttcctcttc caccgtgccc 900 agcacctgaa ctcctgggtg gaccgtcagt cccccaaaac atgcgtggtg ccaaggacac 960 cctcatgatc tcccggaccc ctgaggtcac gtggacgtga cggcgtggag gccacgaaga 1020 ccctgaggtc aagttcaact ggtacgtgga gtgcataatg ccgtgtggtc ccaagacaaa 1080 gccgcgggag gagcagtaca acagcacgta agcgtcctca gtgcaaggtc ccgtcctgca 1140 ccaggactgg ctgaatggca aggagtacaa tccaacaaag agggcagccc ccctcccagc 1200 ccccatcgag aaaaccatct ccaaagccaa cgagaaccac gaaccaggtc aggtgtacac 1260 cctgccccca tcccgggatg agctgaccaa agcctgacct gtgggagagc gcctggtcaa 1320 aggcttctat ccaagcgaca tcgccgtgga aatgggcagc cgacggctcc cggagaacaa 1380 ctacaagacc acgcctcccg tgctggactc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 ttcttcctct acagcaagct caccgtggac aagagcaggt gaacgtcttc 1440

10 <400> 207

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

10 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

15 Met Gin Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

225 230 235

240

Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gin Gly Thr Ser Val

245 250

255

Thr Val Ser Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro

260 265 270

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

275 280 285

Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro

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Glu Val

290 295 300 5 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 305 310 315 320 10 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 325 330 335 15 Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 340 345 350 20 Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 355 360 365 25 Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala 370 375 380 30 Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro

Ser Arg

385 390 395

35 400

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Asp Lys 415 Glu Gly Leu Thr Lys 405 Asn Gin Val Ser Leu 410 Thr Cys Leu Val Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 420 425 430

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 435 440 445

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 450 455 460

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 465 470 475 480 Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys

<210> 206 485 <211> 1522 <212> DNA <213> Artificial sequence

<220>

<223> Synthetic polynucleotide

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2016231617 23 Sep 2016

10 Ser Leu Gin Pro His Ser

100

15 Asp Asn Pro Trp Lys Gly

115

20 Gly Gly Gly Ser Ala Ser

130

25 Ala Val Gin Leu Gly Ala 145 160

Ser Val Lys He Gly Tyr

175

Asn Met Asn Trp

Ser Gly Ser Gly Thr

Glu Asp Ser Gly Ser

105

Thr Phe Gly Gly Gly

120

Gly Gly Gly Gly Ser

135

Gin Gin Ser Gly Pro

150

Ser Cys Lys Ala Ser

165

Val Lys Gin Asn Asn

Gin Phe Ser Leu Lys

90 95

Tyr Phe Cys Gin His

110

Thr Glu Leu Glu He

125

Gly Gly Gly Gly Ser

140

Glu Leu Glu Lys Pro

155

Gly Tyr Ser Phe Thr

170

Gly Lys Ser Leu Glu

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180

185

190

2016231617 23 Sep 2016

Trp lie

10 <221> misc_feature <222> (261) . . (275) <223> Hinge <400> 205

Met Glu Ala Pro Ala Gin Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

15 10

Asp Thr Thr Gly Asp lie Gin Met Thr Gin Ser Pro Ala Ser Leu Ser

20 25 30

Ala Ser Val Gly Glu Thr Val Thr He Thr Cys Arg Thr Ser Glu Asn

35 40 45

Val Tyr Ser Tyr Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro

50 55 60

Gin Leu Leu Val Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser

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10 Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

420 425 430

15 Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn

435 440 445

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 2 0 Phe Leu

450 455 460

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val

465

480

470

475

30 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin

485 490

495

Lys Ser Leu Ser Leu Ser Pro Gly Lys

500

505

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2016231617 23 Sep 2016

<210> 204 <211> 1498 5 <212> DNA <213> Artificial sequence 10 <220> <223> Synthetic polynucleotide <220> <223> G28-1 LH SMIP (DNA) <400> 204 15 aagcttgccg ccatggaagc accagcgcag cttctcttcc tcctgctact ctggctccca 60 20 gataccaccg gtgacatcca gatgactcag tctccagcct ccctatctgc atctgtggga 120 gagactgtca ccatcacatg tcgaacaagt gaaaatgttt acagttattt ggcttggtat 180 25 cagcagaaac agggaaaatc tcctcagctc ctggtctctt ttgcaaaaac cttagcagaa 240 ggtgtgccat caaggttcag tggcagtgga tcaggcacac agttttctct gaagatcagc 300 30 agcctgcagc ctgaagattc tggaagttat ttctgtcaac atcattccga taatccgtgg 360 35 acgttcggtg gaggcaccga actggagatc aaaggtggcg gtggctcggg cggtggtggg 420 tcgggtggcg gcggatctgc tagcgcagtc cagctgcagc agtctggacc tgagctggaa 480

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2016231617 23 Sep 2016

aagcctggcg cttcagtgaa gatttcctgc aaggcttctg gttactcatt cactggctac 540 aatatgaact gggtgaagca gaataatgga aagagccttg agtggattgg aaatattgat 600 ccttattatg gtggtactac ctacaaccgg aagttcaagg gcaaggccac attgactgta 660 gacaaatcct ccagcacagc ctacatgcag ctcaagagtc tgacatctga ggactctgca 720 gtctattact gtgcaagatc ggtcggccct atggactact ggggtcaagg aacctcagtc 780 accgtctcga gcgagcccaa atcttctgac aaaactcaca catgcccacc gtgcccagca 840 cctgaactcc tgggtggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 900 atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 960 gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 1020 cgggaggagc agtacaacag cacgtaccgt gtggtcagcg tcctcaccgt cctgcaccag 1080 gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 1140 atcgagaaaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 1200 cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct

5089144_1 (GHMatters) P79767.AU.1 5/02/14

320

2016231617 23 Sep 2016 ggtcaaaggc 1260 ttctatccaa gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 1320 aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 1380 gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat io gcatgaggct 1440 ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaagtg actctaga 1498 <210> 205 <211> 492 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

25 <223> G28-1 LH SMIP (AA) <220>

<221> misc_feature <222> (1) . . (20) <223> Leader <220>

<221> misc_feature <222> (21) . . (127) <223> VL <220>

<221> misc feature

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <222> (128)..(144) <223> Linker <220>

10 15

Asp He Val Leu Thr Gin Ser

25 30

Gin Arg Ala Thr He Ser Cys

40 45

Val Thr Ser Leu Met Gin Trp

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2016231617 23 Sep 2016

Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Ser Ala Ala Ser Asn

65 70 75

Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser 10 Gly Thr

85 90

Asp Phe Ser Leu Asn He His Pro Val Glu Glu Asp Asp He 15 Ala Met

100 105 110

Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly 2 0 Gly Gly

115 120 125

Thr Lys Leu Glu He Lys Arg Gly Gly Gly Gly Ser Gly Gly 25 Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Lys Glu Ser Gly 30 Pro Gly

145 150 155

160

35 Leu Val Ala Pro Ser Gin Ser Leu Ser He Thr Cys Thr Val Ser Gly

165 170

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2016231617 23 Sep 2016

175

Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro 5 Pro Gly

180 185 190

Lys Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp

195 200 205

Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser

210 215 220

Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr

225 230 235

240

Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His 25 Tyr Tyr

245 250

255

30 Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Asp

260 265 270

35 Gin Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro

275 280 285

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2016231617 23 Sep 2016

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

290 295 300

Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val

305 310 315

320

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 15 Trp Tyr

325 330

335

10 195 200 205

Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser

210

215

220

Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

225

240

230

235

Leu Asn He His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys

245

250

255

Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

260

265

270

Glu

His

He Lys Arg Asp Gin Glu Pro Lys Thr

Ser Ser Asp Lys

Thr

275

280

285

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe

290 295 300

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro

305 310 315

320

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

325 330

335

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 20 Lys Thr

340 345 350

Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val 25 Ser Val

355 360 365

Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr 3 0 Lys Cys

370 375 380

Lys Val Ser Asn Lys Ala Leu Pro Ala Ser He Glu Lys Thr 35 He Ser

385 390 395

400

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

10 15

Gin Val Gin Leu Lys Glu Ser

25 30

Ser Leu Ser He Thr Cys Thr

40 45

Gly Val Asn Trp Val Arg Gin

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2016231617 23 Sep 2016

10 <223> 2el2-sss-IgGl HL SMIP (DNA) <400> 200 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60 agaggagtcc aggtgcagct gaaggagtca ggacctggcc tggtggcgcc ctcacagagc 120 ctgtccatca catgcaccgt ctcagggttc tcattaaccg gctatggtgt 20 aaactgggtt 180 cgccagcctc caggaaaggg tctagagtgg ctgggaatga tatggggtga tggaagcaca 240

25 gactataatt cagctctcaa atccagacta tcgatcacca aggacaactc caagagccaa 300 gttttcttaa aaatgaacag tctgcaaact gatgacacag ccagatacta ctgtgctcga 360 gatggttata gtaactttca ttactatgtt atggactact ggggtcaagg aacctcagtc 420 accgtctcct ctgggggtgg aggctctggt ggcggtggat ccggcggagg 35 tgggtcgggt 480 ggcggcggat ctgacattgt gctcacccaa tctccagctt ctttggctgt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

gtctctaggt 540 cagagagcca cacaagttta ccatctcctg 600 cagagccagt gaaagtgttg aatattatgt atgcagtggt tgctgctagc accaacagaa 660 accaggacag ccacccaaac tcctcatctc aacgtagaat agactttagc ctggggtccc 720 tgccaggttt agtggcagtg ggtctgggac ctcaacatcc gcaaagtagg atcctgtgga 780 ggaggatgat attgcaatgt atttctgtca aaggttccat tcaggagccc ggacgttcgg 840 tggaggcacc aagctggaaa tcaaacgtga aaatcttctg cctgggggga acaaaactca 900 cacatcccca ccgtccccag cacctgaact tcgtcagtct ccggacccct tcctcttccc 960 cccaaaaccc aaggacaccc tcatgatctc gaggtcacat gttcaactgg gcgtggtggt 1020 ggacgtgagc cacgaagacc ctgaggtcaa tacgtggacg gcagtacaac gcgtggaggt 1080 gcataatgcc aagacaaagc cgcgggagga agcacgtacc gaatggcaag gtgtggtcag 1140 cgtcctcacc gtcctgcacc aggactggct gagtacaagt aacaatctcc gcaaggtctc 1200 caacaaagcc ctcccagcct ccatcgagaa aaagccaaag ccgggatgag ggcagccccg 1260 agaaccacag gtgtacaccc tgcccccatc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1320

10 675 680 685

Gin Lys Pro Asp Gly Thr Val Lys Leu Leu He Tyr Tyr Thr Ser Arg

15 690 695 700

Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

705 710 715

720

Asp Tyr Ser Leu Thr He Ala Asn Leu Gin Pro Glu Asp He 25 Ala Thr

725 730

735

30 Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Trp Thr Phe Gly Gly Gly

740 745 750

Thr Lys Leu Val

Thr Lys Arg

755

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2016231617 23 Sep 2016 <210> 200 <211> 1533 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

10 Met Ala 1 Asp Ser Phe Gin Val 5 Gin He Phe Ser Phe 10 Leu Leu He Ser Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 20 25 30 15 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 20 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 25 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 30 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

Thr lie

35 85 90 95

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Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp

100 105 110

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

25 175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

30 180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

35 195 200 205

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2016231617 23 Sep 2016

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

15 255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

275 280 285

Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

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2016231617 23 Sep 2016

10 Met Ala 1 Asp Ser Phe Gin Val 5 Gin He Phe Ser Phe 10 Leu Leu He Ser Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 15 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 20 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 25 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 30 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

35 Thr lie

85 90 95

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2016231617 23 Sep 2016

10 <222> <223>

<220>

<221>

15 <222>

<223>

Leader misc_feature (23)..(128) VL misc_feature (129) . . (144)

Linker misc_feature (145)..(265)

VH1 <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(507) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (508)..(629) <223> VH2 <220>

<221> misc_feature <222> (630)..(646) <223> Linker2

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<220> <221> mi sc feature <222> (647) .. (754) <223> VL2 <400> 197

10 Asp Gly Ser Thr Ser lie

575

Thr Lys Asp Asn Ser Leu

580

Gin Thr Asp Asp Tyr Ser

595

Asn Phe His Tyr Ser Val

610

Thr Val Ser Ser

Gly Gly

625

640

Gly Gly Ser Gly

535

Gly Lys Gly Leu Glu Trp

550

Asp Tyr Asn Ser Ala Leu

565 570

Ser Lys Ser Gin Val Phe

585

Thr Ala Arg Tyr Tyr Cys

600

Tyr Val Met Asp Tyr Trp

615

Gly Gly Gly Gly Ser Gly

630

Gly Gly Gly Ser Asp He

540

Leu Gly Met He

555

Lys Ser Arg Leu

Leu Lys Met Asn

590

Ala Arg Asp Gly

605

Gly Gin Gly Thr

620

Gly Gly Gly Ser

635

Val Leu Thr Gin

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr lie Ser

Cys Arg

660 665 670

2016231617 23 Sep 2016

Ser Pro

655

645

650

Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr

675 680 685

Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser

690 695 700

Asn Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly

705 710 715

720

Thr Asp Phe Ser Leu Asn He His Pro Val Glu Glu Asp Asp He Ala

725 730

30 735

Met Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly

35 740 745 750

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2016231617 23 Sep 2016

Gly Thr Lys Leu Glu lie Lys Arg 755 760

<210> 196 <211> 2283 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H7-G194 HL (DNA) <400> 196 aagcttgccg cagtgcttca ccatggattt 60 tcaagtgcag attttcagct tcctgctaat gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc

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279

2016231617 23 Sep 2016

ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc 5 aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 10 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac 15 gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt 20 ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga acatccccac cctcttcccc cgtgcccagc 900 acctgaactc ctggggggat cgtcagtctt 25 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg 30 gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg 35 gtcctcaccg caaggtctcc tcctgcacca 1140 ggactggctg aatggcaagg agtacaagtg aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

280 gcccccatcc cgggatgagc tgaccaagaa

2016231617 23 Sep 2016 gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca tgctccgtga ctccctgtct ccgggtaagg gtctggacct gaactggtga ttactcattc actggctaca gtggattgga cttattaatc caaggccaca ttaactgtag gacatctgaa gactctgcag ctggtacttc gatgtctggg ctcgggcggt

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 tgcatgaggc

1500 ggtgtccacc

1560 agcctggagc

1620 tcgtgaactg

1680 catacaaagg

1740 acaagtcatc

1800 tctattactg

1860 gcgcagggac

1920 cttctatcca caagaccacg cgtggacaag tctgcacaac ttgtccgaat ttcaatgaag gctgaagcag tcttactacc cagcacagcc tgcaagatct agcgacatcg cctcccgtgc agcaggtggc cactacacgc tctgaggtcc atttcctgca agccatggaa tacaaccaga tacatggagc gggtactatg ccgtggagtg tggactccga agcaggggaa agaagagcct agctgcaaca aggcctctgg agaaccttga aattcaaggg tcctcagtct gtgactcgga cacggtcacc gtctcctctg gtggcggtgg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

281

2016231617 23 Sep 2016 ggtggatctg gaggaggtgg gagcgctagc gacatccaga tgacacagac tacatcctcc 1980

10 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

15 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Gly Cys Pro Pro Cys Pro Asn Ser Gin Val Gin Leu Lys

500 505 510

Glu Ser Gly Pro Gly Ser Val Ala Pro Ser Gin Ser Leu Ser lie Thr

515 520 525

Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn

5089144_1 (GHMatters) P79767.AU.1 5/02/14

276

2016231617 23 Sep 2016

Trp Val 530

Arg Gin Pro Pro 5 Trp Gly

545

560

10 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 340 345 350

15 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 355 360 365

20 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 370 375 380

25 Glu Gin 385 400 Lys Val Thr He Ser Lys 390 Ala Lys Gly Gin Pro 395 Arg Glu Pro 30 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 405 410 415 35 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala

5089144_1 (GHMatters) P79767.AU.1 5/02/14

275

420

425

430

2016231617 23 Sep 2016

Val Glu

10 Met Asp Phe Gin Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser 1 5 10

15 Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 20 25 30

20 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45

25 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60

30 Pro Val 65 80 Lys Pro Pro Trp He Tyr 70 Ala Pro Ser Asn Leu 75 Ala Ser Gly 35 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

Thr lie

5089144_1 (GHMatters) P79767.AU.1 5/02/14

272

2016231617 23 Sep 2016

Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 5 Gin Trp

100 105 110

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu 10 Leu Lys

115 120 125

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 15 Ser Ser

130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro 2 0 Gly Ala

145 150 155

160

25 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

5089144_1 (GHMatters) P79767.AU.1 5/02/14

273

2016231617 23 Sep 2016

195

Lys Gly Lys Ala Thr 5 Ala Tyr

210

Met Gin Leu Ser Ser l o Phe Cys

225

240

15 Ala Arg Val Val Tyr Val Trp

245

255

Gly Thr Gly Thr Thr Ser Ser

260

Asp Lys Thr His Thr Leu Gly

275

30 Gly Ser Ser Val Phe Leu Met

290

35 lie Ser Arg Thr Pro Ser His 305

200

Leu Thr Val Asp Lys

215

Leu Thr Ser Glu Asp

230

Tyr Ser Asn Ser Tyr

250

Val Thr Val Ser Asp

265

Ser Pro Pro Cys Pro

280

Leu Phe Pro Pro Lys

295

Glu Val Thr Cys Val

310

205

Ser Ser Ser Thr

220

Ser Ala Val Tyr

235

Trp Tyr Phe Asp

Gin Glu Pro Lys

270

Ala Pro Glu Leu

285

Pro Lys Asp Thr

300

Val Val Asp Val

315

5089144_1 (GHMatters) P79767.AU.1 5/02/14

274

2016231617 23 Sep 2016

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 5 Glu Val 325 330 335

10 <220>

<221> misc_feature <222> (129)..(144) <223> Linker

15 <220>

<221> misc_feature <222> (145)..(265) <223> VH

20 <220>

<221> misc_feature <222> (268)..(282) <223> Hinge

25 <220>

<221> misc_feature <222> (500)..(507) <223> EFD-BD2 Linker

30 <220>

<221> misc_feature <222> (508)..(628) <223> VH2

35 <220>

<221> misc_feature <222> (629)..(648)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

271

2016231617 23 Sep 2016 <223> Linker2

<220> <221> misc feature 5 <222> (649) ..(760) <223> VL2 <400> 195

10 Met Ala 1 Asp Ser Phe Gin Val 5 Gin He Phe Ser Phe 10 Leu Leu He Ser Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 15 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 20 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 25 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 30 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

35 Thr lie

85 90 95

5089144_1 (GHMatters) P79767.AU.1 5/02/14

260

2016231617 23 Sep 2016

10 <222> <223>

<220>

<221>

15 <222>

<223>

Leader misc_feature (23)..(128) VL misc_feature (129) . . (144)

Linker misc_feature (145)..(265)

VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(517) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (518)..(638) <223> VH2 <220>

<221> misc_feature <222> (639)..(658) <223> Linker2

5089144_1 (GHMatters) P79767.AU.1 5/02/14

259

2016231617 23 Sep 2016

<220> <221> mi sc feature <222> (659) ..(770) <223> VL2 <400> 193

10 Arg Ala Thr He Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Tyr Val

675 680 685

15 Thr Ser Leu Met Gin Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys

690 695 700

20 Leu Leu He Ser Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ala Arg

705 710 715

720

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn He His Pro

725

730

735

Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys Gin Gin Ser Arg Lys

740 745 750

Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys

5089144_1 (GHMatters) P79767.AU.1 5/02/14

254

2016231617 23 Sep 2016

Arg

755 760 765 5 <210> 192 <211> 2331 <212> DNA <213> Artificial sequence 10 <220> <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H6-2el2HL (DNA) 15 <400> 192 aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat cagtgcttca 60 20 gtcataatgt ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120 ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180 25 taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc caacctggct 240 tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc 30 tctcacaatc 300 agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag ttttaaccca 360 35 cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420

5089144_1 (GHMatters) P79767.AU.1 5/02/14

255

2016231617 23 Sep 2016

ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc 5 aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 10 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac 15 gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt 20 ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga acatccccac cctcttcccc cgagcccagc 900 acctgaactc ctggggggac cgtcagtctt 25 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg 30 gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg 35 gtcctcaccg caaggtctcc tcctgcacca 1140 ggactggctg aatggcaagg agtacaagtg aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

256 gcccccatcc cgggatgagc tgaccaagaa

2016231617 23 Sep 2016 gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca tgctccgtga ctccctgtct ccgggtaagg cagcgggaat tctcaggtgc gagcctgtcc atcacatgca ggttcgccag cctccaggaa cacagactat aattcagctc ccaagttttc ttaaaaatga tcgagatggt tatagtaact agtcaccgtc

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 tgcatgaggc

1500 gtggcggtgg

1560 agctgaagga

1620 ccgtctcagg

1680 agggtctgga

1740 tcaaatccag

1800 acagtctgca

1860 ttcattacta

1920 cttctatccc caagaccacg cgtggacaag tctgcacaac ctcgggcggt gtcaggacct gttctcatta gtggctggga actatcgatc aactgatgac tgttatggac agcgacatcg cctcccgtgc agcaggtggc cactacacgc ggtggatctg ggcctggtgg accggctatg atgatatggg accaaggaca acagccagat tactggggtc ccgtggagtg tggactccga agcaggggaa agaagagcct ggggaggagg cgccctcaca gtgtaaactg gtgatggaag actccaagag actactgtgc aaggaacctc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

257 tggtggcggt ggatccggcg gaggtgggtc

2016231617 23 Sep 2016 tcctctgggg gggtggcggc ggatctgaca aggtcagaga gccaccatct tttaatgcag tggtaccaac tagcaacgta gaatctgggg tagcctcaac atccatcctg taggaaggtt ccatggacgt

2331 gtggaggctc

1980 ttgtgctcac

2040 cctgcagagc

2100 agaaaccagg

2160 tccctgccag

2220 tggaggagga

2280 tcggtggagg ccaatctcca cagtgaaagt acagccaccc gtttagtggc tgatattgca caccaagctg <210>

<211>

<212>

<213>

193

770

PRT

Artificial sequence <220>

<223> Synthetic polypeptide gcttctttgg gttgaatatt aaactcctca agtgggtctg atgtatttct gaaatcaaac <220>

<223> 2H7sssIgGl-H6-2el2HL (w/2el2 leader)

35 <220>

<221> misc_feature <222> (1)..(22) ctgtgtctct atgtcacaag tctctgctgc ggacagactt gtcagcaaag gttaatctag a (AA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

258

2016231617 23 Sep 2016 <223>

<220>

<221>

10 His Thr Asn Tyr Ala Lys 340 Thr Lys Pro Arg Glu 345 Glu Gin Tyr Asn Ser 350 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 15 Asn Gly 355 360 365

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 2 0 Pro lie 370 375 380

Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 25 Gin Val 385 390 395 400

30 Tyr Val 415 Thr Ser Leu Pro Pro 405 Ser Arg Asp Glu Leu 410 Thr Lys Asn Gin 35 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala

Val Glu

5089144_1 (GHMatters) P79767.AU.1 5/02/14

251

420

425

430

2016231617 23 Sep 2016

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr 5 Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 10 Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 15 Val Met

465 470 475

480

20 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

500 505 510

Asn Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro

515 520 525

Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser Gly Phe Ser Leu Thr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

252

530 535 540

2016231617 23 Sep 2016

Gly Tyr Leu Glu 545 560 Gly Val Asn Trp 550 Val Arg Gin Pro Pro 555 Gly Lys Gly Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala 565 570 575 Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val 580 585 590 Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr 595 600 605 Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr 610 615 620 Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser 625 630 635 640 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

Ser Asp

5089144_1 (GHMatters) P79767.AU.1 5/02/14

253

2016231617 23 Sep 2016

655

645

650

10 Met Asp Phe Gin Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser 1 5 10

15 Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He 20 25 30

20 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45

25 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60

30 Pro Val 65 80 Lys Pro Pro Trp He Tyr 70 Ala Pro Ser Asn Leu 75 Ala Ser Gly 35 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

Thr lie

5089144_1 (GHMatters) P79767.AU.1 5/02/14

248

2016231617 23 Sep 2016

Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp

100 105 110

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 25 Ser Tyr

165 170

175

30 Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

35 Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

5089144_1 (GHMatters) P79767.AU.1 5/02/14

249

2016231617 23 Sep 2016

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp 15 Val Trp

245 250

255

20 Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

260 265 270

25 Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly

275 280 285

30 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300

35 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

5089144_1 (GHMatters) P79767.AU.1 5/02/14

250

2016231617 23 Sep 2016

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 5 Glu Val 325 330 335

10 <220>

<221> misc_feature <222> (129)..(144) <223> Linker

15 <220>

<221> misc_feature <222> (145)..(265) <223> VH

20 <220>

<221> misc_feature <222> (268)..(282) <223> Hinge

25 <220>

<221> misc_feature <222> (500)..(514) <223> EFD-BD2 Linker

30 <220>

<221> misc_feature <222> (515)..(635) <223> VH2

35 <220>

<221> misc_feature <222> (636)..(655)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

247

2016231617 23 Sep 2016 <223> Linker2

<220> <221> misc feature 5 <222> (656) ..(767) <223> VL2 <400> 191

10 <210> 190 <211> 2322 <212> DNA <213> Artificial sequence 15 <220> <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H5-2el2HL (DNA) <400> 190 20 aagcttgccg ccatggattt tcaagtgcag attttcagct cagtgcttca 60 25 gtcataatgt ccagaggaca aattgttctc tcccagtctc gtctgcatct 120 ccaggggaga aggtcacaat gacttgcagg gccagctcaa catgcactgg 180 30 taccagcaga agccaggatc ctcccccaaa ccctggattt caacctggct 240 tctggagtcc ctgctcgctt cagtggcagt gggtctggga tctcacaatc 300 35 agcagagtgg aggctgaaga tgctgccact tattactgcc ttttaaccca 360

tcctgctaat cagcaatcct gtgtaagtta atgccccatc cctcttactc agcagtggag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

243

2016231617 23 Sep 2016

cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 5 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 10 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 15 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 20 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga 25 acatccccac cctcttcccc cgagcccagc 900 acctgaactc ctggggggac cgtcagtctt 30 ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg 35 cataatgcca tgtggtcagc agacaaagcc 1080 gcgggaggag cagtacaaca gcacgtaccg gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

244

2016231617 23 Sep 2016

caaggtctcc 1140 aacaaagccc gcagccccga tcccagcccc 1200 catcgagaaa acaatctcca aagccaaagg gaaccacagg ccaggtcagc tgtacaccct 1260 gcccccatcc cgggatgagc tgaccaagaa ctgacctgcc ggagagcaat tggtcaaagg 1320 cttctatccc agcgacatcg ccgtggagtg gggcagccgg cggctccttc agaacaacta 1380 caagaccacg cctcccgtgc tggactccga ttcctctaca cgtcttctca gcaagctcac 1440 cgtggacaag agcaggtggc agcaggggaa tgctccgtga ctccctgtct tgcatgaggc 1500 tctgcacaac cactacacgc agaagagcct ccgggtaaga ttctcaggtg attatggtgg 1560 cggtggctcg ggcggtggtg gatctgggaa cagctgaagg catcacatgc agtcaggacc 1620 tggcctggtg gcgccctcac agagcctgtc accgtctcag gcctccagga ggttctcatt 1680 aaccggctat ggtgtaaact gggttcgcca aagggtctgg taattcagct agtggctggg 1740 aatgatatgg ggtgatggaa gcacagacta ctcaaatcca cttaaaaatg gactatcgat 1800 caccaaggac aactccaaga gccaagtttt aacagtctgc ttatagtaac aaactgatga 1860 cacagccaga tactactgtg ctcgagatgg

5089144_1 (GHMatters) P79767.AU.1 5/02/14

245

2016231617 23 Sep 2016 tttcattact ctcctctggg ggtggaggct cggatctgac attgtgctca agccaccatc tcctgcagag gtggtaccaa cagaaaccag agaatctggg gtccctgcca catccatcct gtggaggagg tccatggacg ttcggtggag

2322 atgttatgga

1920 ctggtggcgg

1980 cccaatctcc

2040 ccagtgaaag

2100 gacagccacc

2160 ggtttagtgg

2220 atgatattgc

2280 gcaccaagct ctactggggt tggatccggc agcttctttg tgttgaatat caaactcctc cagtgggtct aatgtatttc ggaaatcaaa <210>

<211>

<212>

<213>

191

767

PRT

Artificial sequence <220>

<223> Synthetic polypeptide caaggaacct ggaggtgggt gctgtgtctc tatgtcacaa atctctgctg gggacagact tgtcagcaaa cgttaatcta <220>

<223> 2H7sssIgGl-H5-2el2HL (w/2el2 leader) cagtcaccgt cgggtggcgg taggtcagag gtttaatgca ctagcaacgt ttagcctcaa gtaggaaggt ga (AA) <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

246

2016231617 23 Sep 2016 <221> misc_feature <222> (1)..(22) <223> Leader

10 Leu Thr Gin Ser Arg Ala

660

15 Thr He Ser Cys Thr Ser

675

20 Leu Met Gin Trp Leu Leu

690

25 He Ser Ala Ala Phe Ser 705 720

Gly Ser Gly Ser Val Glu

735

Glu Asp Asp He

Gly Gly Gly Ser Gly Gly

645 650

Pro Ala Ser Leu Ala Val

665

Arg Ala Ser Glu Ser Val

680

Tyr Gin Gin Lys Pro Gly

695

Ser Asn Val Glu Ser Gly

710

Gly Thr Asp Phe Ser Leu

725 730

Ala Met Tyr Phe Cys Gin

Gly Gly Ser Asp

Ser Leu Gly Gin

670

Glu Tyr Tyr Val

685

Gin Pro Pro Lys

700

Val Pro Ala Arg

715

Asn He His Pro

Gin Ser Arg Lys

5089144_1 (GHMatters) P79767.AU.1 5/02/14

242

750

2016231617 23 Sep 2016

Val Pro

740 745

10 100 105 110

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

15 115 120 125

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

20 130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 30 Ser Tyr

165 170

175

Asn Met His Trp He

Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu

180

185

190

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Gly Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp 20 Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys 25 Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu 30 Leu Gly

275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 Leu Met

290 295 300

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10 <220>

<221> misc_feature <222> (23) . . (128) <223> VL

15 <220>

<221> misc_feature <222> (129)..(144) <223> Linker

20 <220>

<221> misc_feature <222> (145)..(265) <223> VH

25 <220>

<221> misc_feature <222> (268)..(282) <223> Hinge

30 <220>

<221> misc_feature <222> (500)..(512) <223> EFD-BD2 Linker

35 <220>

<221> misc_feature <222> (513)..(633)

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<223> VH2 <220> <221> misc feature <222> (634) . . (653) <223> Linker2 <220> <221> misc feature <222> (654)..(765) <223> VL2 <400> 189

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser 15 Ala Ser 1 5 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro 2 0 Ala He 20 25 30

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 25 Ala Ser 35 40 45

Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly 30 Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly

35 Val Pro

65 70 75

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Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie

10 755 760 <210> 188 <211> 2316 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide 20 <220> <223> 2H7sssIgGl-H4-2el2HL (DNA) <400> 188 25 aagcttgccg ccatggattt tcaagtgcag cagtgcttca 60 gtcataatgt ccagaggaca aattgttctc gtctgcatct 120 30 ccaggggaga aggtcacaat gacttgcagg catgcactgg 180 taccagcaga agccaggatc ctcccccaaa 35 caacctggct 240 tctggagtcc ctgctcgctt cagtggcagt

attttcagct tcccagtctc gccagctcaa ccctggattt gggtctggga

Val Pro Trp

750 tcctgctaat cagcaatcct gtgtaagtta atgccccatc cctcttactc

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tctcacaatc 300 c agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag u cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 10 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 15 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 20 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 25 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga 30 acatccccac cctcttcccc cgagcccagc 900 acctgaactc ctggggggac cgtcagtctt ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg 35 gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg

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2016231617 23 Sep 2016 cataatgcca tgtggtcagc gtcctcaccg caaggtctcc aacaaagccc gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca tgctccgtga ctccctgtct ccgggtaagg ggtgcagctg aaggagtcag atgcaccgtc tcagggttct aggaaagggt ctggagtggc agctctcaaa tccagactat agacaaagcc

1080 tcctgcacca

1140 tcccagcccc

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 tgcatgaggc

1500 gtggcggtgg

1560 gacctggcct

1620 cattaaccgg

1680 tgggaatgat

1740 cgatcaccaa gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatccc caagaccacg cgtggacaag tctgcacaac ctcgggcggt ggtggcgccc ctatggtgta atggggtgat ggacaactcc cagtacaaca aatggcaagg acaatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc ggtggatctg tcacagagcc aactgggttc ggaagcacag aagagccaag gcacgtaccg agtacaagtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct ggaattctca tgtccatcac gccagcctcc actataattc ttttcttaaa

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2016231617 23 Sep 2016 aatgaacagt 1800 ctgcaaactg atgacacagc cagatactac tgtgctcgag atggttatag taactttcat 1860 tactatgtta tggactactg gggtcaagga acctcagtca ccgtctcctc tgggggtgga 1920 ggctctggtg gcggtggatc cggcggaggt gggtcgggtg gcggcggatc io tgacattgtg 1980 ctcacccaat ctccagcttc tttggctgtg tctctaggtc agagagccac catctcctgc 2040

15 agagccagtg aaagtgttga atattatgtc acaagtttaa tgcagtggta ccaacagaaa 2100 ccaggacagc cacccaaact cctcatctct gctgctagca acgtagaatc tggggtccct 2160 gccaggttta gtggcagtgg gtctgggaca gactttagcc tcaacatcca tcctgtggag 2220 gaggatgata ttgcaatgta tttctgtcag caaagtagga aggttccatg 25 gacgttcggt 2280 ggaggcacca agctggaaat caaacgttaa tctaga 2316 <210> 189 <211> 765 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide

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2016231617 23 Sep 2016 <220>

<223> 2H7sssIgGl-H4-2el2HL (AA)

10 420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

15 435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Val Met

Trp Gin Gin

Gly Asn Val

Phe Ser Cys Ser

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 30 Leu Ser

485 490

495

Pro Gly Lys Asn Val Gin

500

505

510

Tyr Gly Gly Gly Gly Ser Gly Asn

Ser Gin

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Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser

515 520 525 lie Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn

530 535 540

Trp Val Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met He

545 550 555

560

Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser 2 0 Arg Leu

565 570

575

25 Ser He Thr Lys Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn

580 585 590

30 Ser Leu Gin Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly

595 600 605

35 Tyr Ser Asn Phe His Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr

610 615 620

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Ser Val Thr Val Ser Gly Ser

10 <220>

<221> misc_feature <222> (1)..(22) <223> Leader

15 <220> <221> misc feature <222> (23)..(128) <223> VL <220> 20 <221> misc feature <222> (129) . . (144) <223> Linker

<220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(509) <223> EFD-BD2 Linker

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

misc_feature (510)..(630) VH2 misc_feature (631)..(650) Linker2 misc_feature (651) . . (762) VL2 <400> 187

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser 20 Ala Ser

15 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro 25 Ala He

20 25 30

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 30 Ala Ser

Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly 35 Ser Ser

50 55 60

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10 Met Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Gly Gly

740 745

Glu Asp Asp

Trp Thr Phe

750

15 Gly Thr Lys Leu Glu lie Lys Arg

755 760 <210> 186 20 <211> 2307 <212> DNA <213> Artificial sequence <220> 25 <223> Synthetic polynucleotide <220> <223> 2H7sssIgGl-H3-2el2HL (DNA) 30 <400> 186 aagcttgccg ccatggattt tcaagtgcag attttcagct cagtgcttca 60 gtcataatgt ccagaggaca aattgttctc tcccagtctc 35 gtctgcatct 120 ccaggggaga aggtcacaat gacttgcagg gccagctcaa catgcactgg 180

tcctgctaat cagcaatcct gtgtaagtta

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taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc 5 tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc 10 agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 15 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 20 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg 25 ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga 30 gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga 35 acatccccac cctcttcccc cgagcccagc 900 acctgaactc ctggggggac cgtcagtctt

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2016231617 23 Sep 2016 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg 5 cgtggaggtg 1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080 io gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140 aacaaagccc tcccagcccc catcgagaaa acaatctcca aagccaaagg gcagccccga 1200 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1260 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg 20 ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1380

25 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500 ccgggtaaga attatggtgg cggtggctct gggaattctc aggtgcagct gaaggagtca 1560 ggacctggcc tggtggcgcc ctcacagagc ctgtccatca catgcaccgt 35 ctcagggttc 1620 tcattaaccg gctatggtgt aaactgggtt cgccagcctc caggaaaggg

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tctggagtgg 1680 ctgggaatga tatggggtga tggaagcaca gactataatt cagctctcaa atccagacta 1740 tcgatcacca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 1800 gatgacacag ccagatacta ctgtgctcga gatggttata gtaactttca ttactatgtt 1860 atggactact ggggtcaagg aacctcagtc accgtctcct ctgggggtgg aggctctggt 1920 ggcggtggat ccggcggagg tgggtcgggt ggcggcggat ctgacattgt gctcacccaa 1980 tctccagctt ctttggctgt gtctctaggt cagagagcca ccatctcctg cagagccagt 2040 gaaagtgttg aatattatgt cacaagttta atgcagtggt accaacagaa accaggacag 2100 ccacccaaac tcctcatctc tgctgctagc aacgtagaat ctggggtccc tgccaggttt 2160 agtggcagtg ggtctgggac agactttagc ctcaacatcc atcctgtgga ggaggatgat 2220 attgcaatgt atttctgtca gcaaagtagg aaggttccat ggacgttcgg tggaggcacc 2280 aagctggaaa tcaaacgtta atctaga 2307

<210> 187 <211> 762

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2016231617 23 Sep 201 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7sssIgGl-H3-2el2HL (w/2el2 leader) (AA)

10 625 630 635

640

Gly Gly Ser Gly Gly Gly Gly Ser Asp lie Val Leu Thr Gin 15 Ser Pro

645 650

655

20 Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg

660 665 670

25 Ala Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr

675 680 685

30 Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser

690 695 700

Asn Val Glu Ser Gly

Ser Gly Val

Pro Ala Arg Phe

Ser Gly Ser Gly

705

710

715

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720

Thr Asp Phe Ser Leu Asn lie His Pro Val Glu 5 lie Ala

725 730

735

10 405

415

Leu Thr Cys Leu Val 15 Val Glu

420

Trp Glu Ser Asn Gly 20 Pro Pro

435

Val Leu Asp Ser Asp 25 Thr Val

450

Asp Lys Ser Arg Trp 30 Val Met

465

480

Lys Ala Lys Gly Gin

390

Ser Arg Asp Glu Leu

410

Lys Gly Phe Tyr Pro

425

Gin Pro Glu Asn Asn

440

Gly Ser Phe Phe Leu

455

Gin Gin Gly Asn Val

470

Asn His Tyr Thr Gin

490

Pro Arg Glu Pro

395

Thr Lys Asn Gin

Ser Asp He Ala

430

Tyr Lys Thr Thr

445

Tyr Ser Lys Leu

460

Phe Ser Cys Ser

475

Lys Ser Leu Ser

35 His Glu Ala Leu His Leu Ser

485

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2016231617 23 Sep 2016

495

10 Ser Gly Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser 530 535 540

15 Gly Cys 545 560 Pro Thr Gly Leu Val Ala 550 Pro Ser Gin Ser Leu 555 Ser He Thr 20 Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin 565 570 575 25 Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly 580 585 590 30 Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys 595 600 605 35 Asp Asn Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu

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Gin Thr

610 615 620 Asp Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe 625 630 635 640 His Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val 645 650 655 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 660 665 670 Ser Gly Gly Gly Gly Ser Asp lie Val Leu Thr Gin Ser Pro Ala Ser 675 680 685 Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala Ser 690 695 700 Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin Gin 705 710 715 720 Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser

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Asn Val

735

725

730

Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Thr Asp

740

745

Phe Ser Leu Asn lie His Pro Val Glu Glu Asp Met Tyr

755 760

Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Gly Thr

770 775

Lys Leu Glu He Lys Arg

785

790 <210>

<211>

<212>

182

2283

DNA <213> Artificial sequence

30 <220>

<223> Synthetic polynucleotide <220>

<223> 2H7sssIgGl-Hl-2el2HL (DNA) <400> 182 aagcttgccg ccatggattt tcaagtgcag attttcagct

Gly Ser Gly

750

Asp He Ala

765

Phe Gly Gly

780 tcctgctaat

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cagtgcttca 60 gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt

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2016231617 23 Sep 2016 ggcacaggga ccacggtcac cgtctctgat caggagccca aatcttctga caaaactcac 840

10 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val

385 390 395

400

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

405 410

415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu

420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser

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Val Met

465

480

470

475

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser 485 490 495 Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 500 505 510 Gly Gly Gly Ser Gly Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly 515 520 525 Ser Gly Gly Gly Gly Ser Gly Asn Ser Gin Val Gin Leu Lys Glu Ser 530 535 540 Gly Pro Gly Leu Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr 545 550 555 560 Val Ser Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val

Arg Gin

565

570

35 575

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Pro Asp Pro Gly Gly Lys 580 Gly Leu 5 Ser Thr Asp Tyr Asn Ser Thr Lys 595 10 Asp Asn Ser Lys Ser Gin Gin Thr 610 15 Asp Asp Thr Ala Arg Tyr Asn Phe 625 630 640 20 His Tyr Tyr Val Met Asp Thr Val 645 25 655

Ser Ser Gly Gly Gly Gly Gly Gly 30 660

Ser Gly Gly Gly Gly Ser Ala Ser 35 675

Glu Trp Leu Gly Met lie Trp Gly

585 590

Ala Leu Lys Ser Arg Leu Ser lie

600 605

Val Phe Leu Lys Met Asn Ser Leu

615 620

Tyr Cys Ala Arg Asp Gly Tyr Ser

635

Tyr Trp Gly Gin Gly Thr Ser Val

650

Ser Gly Gly Gly Gly Ser Gly Gly

665 670

Asp lie Val Leu Thr Gin Ser Pro

680 685

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Leu Ala Val Ser Ala Ser

690

Glu Ser Val Glu

Gin Gin

705

720

Lys Pro Gly Gin Asn Val

15 735

Glu Ser Gly Val Thr Asp

20 740

Leu Gly Gin Arg Ala

695

Tyr Tyr Val Thr Ser

710

Pro Pro Lys Leu Leu

725

Pro Ala Arg Phe Ser

745

Thr lie Ser Cys Arg

700

Leu Met Gin Trp Tyr

715

He Ser Ala Ala Ser

730

Gly Ser Gly Ser Gly

750

Phe Ser Leu Asn He His Pro Val Glu Glu Asp Asp He Ala Met Tyr

25 755 760 765

Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr

770 775 780

Lys Leu Glu He Lys Arg

785

790 <210> 181 <211> 790

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2016231617 23 Sep 2016 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> 2H7sssIgGl(P238S/P331S)-STD2-2el2HL (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1) . . (22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <222> (500)..(537) <223> EFD-BD2 Linker <220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

<220>

<221>

<222>

<223>

misc_feature (538)..(658) VH2 misc_feature (659)..(678) Linker2 misc_feature (679)..(790) VL2 <400> 181

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

15 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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50 55 60

2016231617 23 Sep 2016

10 Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

275 280 285

15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300

20 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr

340 345 350

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu

5089144_1 (GHMatters) P79767.AU.1 5/02/14

182

355

365

10 Ser Val Lys Met Ser Tyr

175

Asn Met His Trp Trp lie

180

Gly Ala He Tyr Lys Phe

195

Lys Gly Lys Ala Ala Tyr

210

Met Gin Leu Ser

Phe Cys

225

240

Ala Arg Val Val

Ser Cys Lys Ala

165

Val Lys Gin Thr

Pro Gly Asn Gly

200

Thr Leu Thr Val

215

Ser Leu Thr Ser

230

Tyr Tyr Ser Asn

Gin Ala Gly Ala

145 150 155

160

Ser Gly Tyr Thr Phe Thr

170

Pro Arg Gin Gly Leu Glu

185 190

Asp Thr Ser Tyr Asn Gin

205

Asp Lys Ser Ser Ser Thr

220

Glu Asp Ser Ala Val Tyr

235

Ser Tyr Trp Tyr Phe Asp

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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10 2391 <210> 180 <211> 790 <212> PRT 15 <213> Artificial sequi <220> <223> Synthetic polypi 20 <220> <223> 2H7sssIgGl-STD2 <220> <221> misc feature 25 <222> (1)··(22) <223> Leader <220> <221> misc feature 30 <222> (23)..(128) <223> VL <220> <221> misc feature 35 <222> (129) . . (144) <223> Linker

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2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(537) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (538)..(658) <223> VH2 <220>

<221> misc_feature <222> (659)..(678) <223> Linker2 <220>

<221> misc_feature <222> (679)..(790) <223> VL2 <400> 180

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

15 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro

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Ala lie

20 25 30

2016231617 23 Sep 2016

Leu Ala Ser Ala Ser Pro Gly Glu Lys 40 Val Thr Met Thr Cys 45 Arg Ser 35 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

Ser Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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130

135

140

Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro

2016231617 23 Sep 2016

10 740 745 750

Asp Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His

15 755 760 765

Tyr Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser

770 775 780

Ser

785

<210> 179 <211> 2391 <212> DNA 30 <213> Artificial sequence <220> <223> Synthetic polynucleotide 35 <220> <223> 2H7sssIgGl-STD2-2el2HL (DNA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<400> 179 aagcttgccg cagtgcttca ccatggattt 60 tcaagtgcag attttcagct tcctgctaat 5 gtcataatgt gtctgcatct ccagaggaca 120 aattgttctc tcccagtctc cagcaatcct 10 ccaggggaga catgcactgg aggtcacaat 180 gacttgcagg gccagctcaa gtgtaagtta taccagcaga caacctggct agccaggatc 240 ctcccccaaa ccctggattt atgccccatc 15 tctggagtcc tctcacaatc ctgctcgctt 300 cagtggcagt gggtctggga cctcttactc agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag 20 cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc 25 ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt 30 aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac 35 gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga

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2016231617 23 Sep 2016 gtctatttct cgatgtctgg ggcacaggga caaaactcac acatccccac cctcttcccc ccaaaaccca cgtggtggtg gacgtgagcc cgtggaggtg cataatgcca tgtggtcagc gtcctcaccg caaggtctcc aacaaagccc gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca tgctccgtga gtgcaagagt

780 ccacggtcac

840 cgagcccagc

900 aggacaccct

960 acgaagaccc

1020 agacaaagcc

1080 tcctgcacca

1140 tcccagcccc

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 tgcatgaggc ggtgtactat cgtctctgat acctgaactc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatccc caagaccacg cgtggacaag tctgcacaac agtaactctt caggagccca ctggggggac cggacccctg ttcaactggt cagtacaaca aatggcaagg acaatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc actggtactt aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg agtacaagtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct

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2016231617 23 Sep 2016 ctccctgtct 1500 ccgggtaaga attatggtgg cggtggctcg ggcggtggtg gatctggagg aggtgggagt 1560 gggaattatg gtggcggtgg ctcgggcggt ggtggatctg gaggaggtgg gagtgggaat 1620 tctcaggtgc agctgaagga gtcaggacct ggcctggtgg cgccctcaca 10 gagcctgtcc 1680 atcacatgca ccgtctcagg gttctcatta accggctatg gtgtaaactg ggttcgccag 1740

15 cctccaggaa agggtctgga gtggctggga atgatatggg gtgatggaag cacagactat 1800 aattcagctc tcaaatccag actatcgatc accaaggaca actccaagag ccaagttttc 1860 ttaaaaatga acagtctgca aactgatgac acagccagat actactgtgc tcgagatggt 1920 tatagtaact ttcattacta tgttatggac tactggggtc aaggaacctc 25 agtcaccgtc 1980 tcctctgggg gtggaggctc tggtggcggt ggatccggcg gaggtgggtc gggtggcggc 2040

30 ggatctgaca ttgtgctcac ccaatctcca gcttctttgg ctgtgtctct aggtcagaga 2100 gccaccatct cctgcagagc cagtgaaagt gttgaatatt atgtcacaag tttaatgcag 2160 tggtaccaac agaaaccagg acagccaccc aaactcctca tctctgctgc tagcaacgta 2220

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2016231617 23 Sep 2016 gaatctgggg tccctgccag gtttagtggc agtgggtctg ggacagactt tagcctcaac 2280

10 <223> 2H7sssIgGl(P238S/P331S)-STD2-2el2LH (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268) . . (281) <223> Hinge

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (500)..(537) <223> EFD-BD2 Linker <220>

<221> misc_feature <222> (538)..(649) <223> VL2 <220>

<221> misc_feature <222> (650)..(664) <223> Linker2 <220>

<221> misc_feature <222> (665)..(785) <223> VH2 <400> 178

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser Ala Ser

25 1 5 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala He

30 20 25 30

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser

35 35 40 45

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2016231617 23 Sep 2016

Ser Ser Ser Ser 50 Val Ser Tyr Met His 55 Trp Tyr Gin Gin Lys 60 Pro Gly Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro 65 70 75 80 Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie 85 90 Ser Arg Val Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp 100 105 110 Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 115 120 125 Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 130 135 140 Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro

Gly Ala

145 150 155

35 160

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2016231617 23 Sep 2016

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

165 170

175

Asn Met His Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu Trp lie

180 185 190

Gly Ala He Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

30 255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys Ser Ser

35 260 265 270

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2016231617 23 Sep 2016

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu Leu Gly

275 280 285

Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

325 330

20 335

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr

340 345 350

Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly

355 360 365

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser He

370 375 380

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2016231617 23 Sep 2016

Glu Gin 385 400 Lys Val Thr He Ser Lys 390 Ala Lys Gly Gin Pro 395 Arg Glu Pro Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser 405 410 415 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu 420 425 430 Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 435 440 445 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 450 455 460 Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met 465 470 475 480 His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser

Leu Ser

35 485 490

495

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2016231617 23 Sep 2016

10 595

Tyr Phe Cys Gin Gin Gly Gly

15 610

Thr Lys Leu Glu lie Gly Gly

20 625

640

Ser Gly Gly Gly Gly 25 Pro Gly

645

655

30 Leu Val Ala Pro Ser Ser Gly

660

Pro Ala Arg Phe Ser

585 lie His Pro Val Glu

600

Ser Arg Lys Val Pro

615

Lys Arg Gly Gly Gly

630

Ser Gin Val Gin Leu

650

Gin Ser Leu Ser lie

665

Gly Ser Gly Ser

590

Glu Asp Asp lie

605

Trp Thr Phe Gly

620

Gly Ser Gly Gly

635

Lys Glu Ser Gly

Thr Cys Thr Val

670

Phe Ser Leu Pro Gly

Thr Gly Tyr

Gly Val Asn

Trp Val Arg Gin

Pro

675

680

685

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2016231617 23 Sep 2016

Lys Gly Leu Glu Trp Leu Gly Met lie Trp Gly Asp Gly Ser Thr Asp 5 690 695 700

Tyr Asn 10 705 720 Asn Ser Ser Ala Leu Lys 710 Ser Arg Leu Ser He 715 Thr Lys Asp Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr 15 725 730 735

Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His 2 0 Tyr Tyr 740 745 750 Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser

25 Ser

755 760 765 <210>

<211>

<212>

176

2376

DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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2016231617 23 Sep 2016

<223> 2H7sssIgGl-STD2-2el2LH (DNA) <400> 176 aagcttgccg ccatggattt tcaagtgcag attttcagct tcctgctaat 5 cagtgcttca 60 gtcataatgt ccagaggaca aattgttctc tcccagtctc cagcaatcct gtctgcatct 120 10 ccaggggaga aggtcacaat gacttgcagg gccagctcaa gtgtaagtta catgcactgg 180 taccagcaga agccaggatc ctcccccaaa ccctggattt atgccccatc 1 s caacctggct 240 tctggagtcc ctgctcgctt cagtggcagt gggtctggga cctcttactc tctcacaatc 300 agcagagtgg aggctgaaga tgctgccact tattactgcc agcagtggag 20 ttttaaccca 360 cccacgttcg gtgctgggac caagctggag ctgaaagatg gcggtggctc gggcggtggt 420 25 ggatctggag gaggtgggag ctctcaggct tatctacagc agtctggggc tgagtcggtg 480 aggcctgggg cctcagtgaa gatgtcctgc aaggcttctg gctacacatt taccagttac 540 30 aatatgcact gggtaaagca gacacctaga cagggcctgg aatggattgg agctatttat 600 ccaggaaatg gtgatacttc ctacaatcag aagttcaagg gcaaggccac 35 actgactgta 660 gacaaatcct ccagcacagc ctacatgcag ctcagcagcc tgacatctga

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2016231617 23 Sep 2016 agactctgcg gtctatttct cgatgtctgg ggcacaggga caaaactcac acatccccac cctcttcccc ccaaaaccca cgtggtggtg gacgtgagcc cgtggaggtg cataatgcca tgtggtcagc gtcctcaccg caaggtctcc aacaaagccc gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca

720 gtgcaagagt

780 ccacggtcac

840 cgagcccagc

900 aggacaccct

960 acgaagaccc

1020 agacaaagcc

1080 tcctgcacca

1140 tcccagcccc

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 ggtgtactat cgtctctgat acctgaactc catgatctcc tgaggtcaag gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatccc caagaccacg cgtggacaag agtaactctt caggagccca ctggggggac cggacccctg ttcaactggt cagtacaaca aatggcaagg acaatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc actggtactt aatcttctga cgtcagtctt aggtcacatg acgtggacgg gcacgtaccg agtacaagtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa

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2016231617 23 Sep 2016 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1500

10 His Leu 495 Glu Ser Ala Leu His 485 Asn His Tyr Thr Gin 490 Lys Ser Leu Ser Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 15 500 505 510 Gly Gly Gly Ser Gly Asn Ser Asp He Val Leu Thr Gin Ser Pro Ala 20 515 520 525 Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala 25 530 535 540 Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin 30 545 550 555 560 Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Ser Ala Ala

35 Ser Asn

565 570

575

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2016231617 23 Sep 2016

Val Glu Ser Gly Val Gly Thr

10 755 760 765 <210>

<211>

<212>

<213>

175

767

PRT

Artificial sequence <220>

<223> Synthetic polypeptide

20 <220>

<223> 2H7sssIgGl(P238S/P331S)-STDl-2el2LH (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker

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2016231617 23 Sep 2016

10 Ser Pro 655 Gly Gly Gly Gly Gly Ser 645 Gin Val Gin Leu 650 Lys Glu Ser Gly Leu Val Ala Pro Ser Gin Ser Leu Ser He Thr Cys Thr Val Ser Gly 15 660 665 670 Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly 20 675 680 685 Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly Asp Gly Ser Thr Asp 25 690 695 700 Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser He Thr Lys Asp Asn Ser 30 705 710 715 720 Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp

35 Asp Thr

725 730

735

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Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr

10 Lys Gly Lys Ala Ala Tyr

210

15 Met Gin Leu Ser Phe Cys 225 240

Ala Arg Val Val Val Trp

255

Gly Thr Gly Thr Ser Ser

260

Asp Lys Thr His Leu Gly

275

Gly Pro Ser Val

185

Pro Gly Asn Gly Asp Thr

200

Thr Leu Thr Val Asp Lys

215

Ser Leu Thr Ser Glu Asp

230

Tyr Tyr Ser Asn Ser Tyr

245 250

Thr Val Thr Val Ser Asp

265

Thr Ser Pro Pro Ser Ser

280

Phe Leu Phe Pro Pro Lys

190

Ser Tyr Asn Gin

205

Ser Ser Ser Thr

220

Ser Ala Val Tyr

235

Trp Tyr Phe Asp

Gin Glu Pro Lys

270

Ala Pro Glu Leu

285

Pro Lys Asp Thr

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2016231617 23 Sep 2016

Leu Met

290 295 300 5 He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 305 310 315 320 10 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 325 330 335 15 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr 340 345 350 20 Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly 355 360 365 25 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro lie 370 375 380 30 Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val 385 390 395 35 400

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Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser

405 410

415

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu

420 425 430

Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

435 440 445

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

450 455 460

Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met

465 470 475

480

His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser

485 490

495

Pro Gly Lys Asn Tyr Gly Gly Gly Gly Ser Gly Gly Gly Gly 35 Ser Gly

500 505 510

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Gly Gly Gly 515 Ser Gly Asn Ser Asp 520 He Val Leu Thr Gin 525 Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr He Ser Cys Arg Ala 530 535 540 Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin 545 550 555 560 Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu He Ser Ala Ala Ser Asn 565 570 575 Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr 580 585 590 Asp Phe Ser Leu Asn lie His Pro Val Glu Glu Asp Asp He Ala Met 595 600 605 Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly

Gly Gly

35 610

615

620

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Thr Lys Leu Glu lie Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly

625 630 635

640

10 Ser Arg Val Glu Gin Trp

100

15 Ser Phe Asn Pro Leu Lys

115

20 Asp Gly Gly Gly Ser Ser

130

25 Gin Ala Tyr Leu Gly Ala 145 160

Ser Val Lys Met Ser Tyr

175

Asn Met His Trp

Gly Ser Gly Ser Gly

Ala Glu Asp Ala Ala

105

Pro Thr Phe Gly Ala

120

Ser Gly Gly Gly Gly

135

Gin Gin Ser Gly Ala

150

Ser Cys Lys Ala Ser

165

Val Lys Gin Thr Pro

Thr Ser Tyr Ser Leu

90 95

Thr Tyr Tyr Cys Gin

110

Gly Thr Lys Leu Glu

125

Ser Gly Gly Gly Gly

140

Glu Ser Val Arg Pro

155

Gly Tyr Thr Phe Thr

170

Arg Gin Gly Leu Glu

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2016231617 23 Sep 2016

Trp lie

180

10 Gly Gly Gly Ser Leu Ala

660

15 Val Ser Leu Gly Glu Ser

675

20 Val Glu Tyr Tyr Lys Pro

690

Gly Gin Pro Pro 25 Glu Ser

705

720

30 Gly Val Pro Ala Phe Ser

735

Leu Asn lie His Phe Cys

Ser Gly Gly Gly Gly Ser

645 650

Asp lie Val Leu Thr Gin

665

Gin Arg Ala Thr lie Ser

680

Val Thr Ser Leu Met Gin

695

Lys Leu Leu lie Ser Ala

710

Arg Phe Ser Gly Ser Gly

725 730

Pro Val Glu Glu Asp Asp

Gly Gly Gly Gly

Ser Pro Ala Ser

670

Cys Arg Ala Ser

685

Trp Tyr Gin Gin

700

Ala Ser Asn Val

715

Ser Gly Thr Asp lie Ala Met Tyr

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2016231617 23 Sep 2016

740

745

Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly 5 Lys Leu

755 760

Glu lie Lys Arg 10 770 <210>

<211>

<212>

<213>

173

2322

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> 2H7sssIgGl-STDl-2el2LH (DNA) <400> 173

25 aagcttgccg ccatggattt tcaagtgcag attttcagct cagtgcttca 60 gtcataatgt gtctgcatct ccaggggaga catgcactgg ccagaggaca

120 aggtcacaat

180 aattgttctc gacttgcagg taccagcaga caacctggct agccaggatc ctcccccaaa

240 tctggagtcc ctgctcgctt cagtggcagt tcccagtctc gccagctcaa ccctggattt gggtctggga

750

Gly Gly Thr

765 tcctgctaat cagcaatcct gtgtaagtta atgccccatc cctcttactc

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tctcacaatc 300 agcagagtgg ttttaaccca aggctgaaga 360 tgctgccact tattactgcc agcagtggag cccacgttcg gggcggtggt gtgctgggac 420 caagctggag ctgaaagatg gcggtggctc ggatctggag tgagtcggtg gaggtgggag 480 ctctcaggct tatctacagc agtctggggc aggcctgggg taccagttac cctcagtgaa 540 gatgtcctgc aaggcttctg gctacacatt aatatgcact agctatttat gggtaaagca 600 gacacctaga cagggcctgg aatggattgg ccaggaaatg actgactgta gtgatacttc 660 ctacaatcag aagttcaagg gcaaggccac gacaaatcct agactctgcg ccagcacagc 720 ctacatgcag ctcagcagcc tgacatctga gtctatttct cgatgtctgg gtgcaagagt 780 ggtgtactat agtaactctt actggtactt ggcacaggga caaaactcac ccacggtcac 840 cgtctctgat caggagccca aatcttctga acatccccac cctcttcccc cgagcccagc 900 acctgaactc ctggggggac cgtcagtctt ccaaaaccca cgtggtggtg aggacaccct 960 catgatctcc cggacccctg aggtcacatg gacgtgagcc cgtggaggtg acgaagaccc 1020 tgaggtcaag ttcaactggt acgtggacgg

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134

2016231617 23 Sep 2016 cataatgcca tgtggtcagc gtcctcaccg caaggtctcc aacaaagccc gcagccccga gaaccacagg ccaggtcagc ctgacctgcc ggagagcaat gggcagccgg cggctccttc ttcctctaca cgtcttctca tgctccgtga ctccctgtct ccgggtaaga aggtgggagt gggaattctg tctaggtcag agagccacca aagtttaatg cagtggtacc tgctagcaac agacaaagcc

1080 tcctgcacca

1140 tcccagcccc

1200 tgtacaccct

1260 tggtcaaagg

1320 agaacaacta

1380 gcaagctcac

1440 tgcatgaggc

1500 attatggtgg

1560 acattgtgct

1620 tctcctgcag

1680 aacagaaacc

1740 gcgggaggag ggactggctg catcgagaaa gcccccatcc cttctatccc caagaccacg cgtggacaag tctgcacaac cggtggctcg cacccaatct agccagtgaa aggacagcca cagtacaaca aatggcaagg acaatctcca cgggatgagc agcgacatcg cctcccgtgc agcaggtggc cactacacgc ggcggtggtg ccagcttctt agtgttgaat cccaaactcc gcacgtaccg agtacaagtg aagccaaagg tgaccaagaa ccgtggagtg tggactccga agcaggggaa agaagagcct gatctggagg tggctgtgtc attatgtcac tcatctctgc

5089144_1 (GHMatters) P79767.AU.1 5/02/14

135

2016231617 23 Sep 2016 gtagaatctg gggtccctgc caggtttagt ggcagtgggt ctttagcctc 1800 aacatccatc ctgtggagga ggatgatatt gcaatgtatt 5 aagtaggaag 1860 gttccatgga cgttcggtgg aggcaccaag ctggaaatca cggtggatcc 1920 io ggcggaggtg ggtcgggtgg cggcggatct caggtgcagc aggacctggc 1980 ctggtggcgc cctcacagag cctgtccatc acatgcaccg ctcattaacc 2040 ggctatggtg taaactgggt tcgccagcct ccaggaaagg gctgggaatg 2100 atatggggtg atggaagcac agactataat tcagctctca 20 atcgatcacc 2160 aaggacaact ccaagagcca agttttctta aaaatgaaca tgatgacaca 2220

25 gccagatact actgtgctcg agatggttat agtaactttc tatggactac 2280 ctgggacaga tctgtcagca aacggggtgg tgaaggagtc tctcagggtt gtctggagtg aatccagact gtctgcaaac attactatgt tggggtcaag gaacctcagt caccgtctcc tcttaatcta 2322 ga

<210> 174 <211> 767 <212> PRT <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

136

2016231617 23 Sep 2016 <223> Synthetic polypeptide <220>

<223> 2H7sssIgGl-STDl-2el2LH (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(519) <223> EFD-BD2 linker <220>

<221> misc_feature

5089144_1 (GHMatters) P79767.AU.1 5/02/14

137

2016231617 23 Sep 2016

<222> (520)..(631) <223> VL2 <220> <221> misc feature <222> (632)..(646) <223> Linker2 <220> <221> misc feature <222> (647)..(767) <223> VH2 <400> 174

Met Ala 1 Asp Ser Phe Gin Val 5 Gin He Phe Ser Phe 10 Leu Leu He Ser Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro Ala lie 20 25 30 Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser 35 40 45 Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly Ser Ser 50 55 60 Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly Val Pro

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2016231617 23 Sep 2016

10 100 105 110

Ser Phe Asn Pro Pro Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

15 115 120 125

Asp Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser

20 130 135 140

Gin Ala Tyr Leu Gin Gin Ser Gly Ala Glu Ser Val Arg Pro Gly Ala

145 150 155

160

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 30 Ser Tyr

165 170

175

Asn Met His Trp lie

Trp Val Lys Gin Thr Pro Arg Gin Gly Leu Glu

180

185

190

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2016231617 23 Sep 2016

Gly Ala lie Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gin Lys Phe

195 200 205

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

210 215 220

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

225 230 235

240

Ala Arg Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val Trp

245 250

255

Gly Thr Gly Thr Thr Val Thr Val Ser Asp Gin Glu Pro Lys 25 Ser Ser

260 265 270

Asp Lys Thr His Thr Ser Pro Pro Ser Ser Ala Pro Glu Leu 30 Leu Gly

275 280 285

Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 Leu Met

290 295 300

5089144_1 (GHMatters) P79767.AU.1 5/02/14

128

2016231617 23 Sep 2016

10 Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser

725

730

735

Leu Asn He His Pro Val Glu Glu Asp Asp He Ala Met Tyr Phe Cys

740

745

750

Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

755

760

765

25 Glu He Lys Arg 770 <210> 172

30 <211> 772 <212> PRT <213> Artificial sequence <220>

35 <223> Synthetic polypeptide <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

124

2016231617 23 Sep 2016 <223> 2H7sssIgGl(P238S/P331S)-STDl-2el2HL (w/2el2 leader) (AA) <220>

<221> misc_feature <222> (1)..(22) <223> Leader <220>

<221> misc_feature <222> (23) . . (128) <223> VL <220>

<221> misc_feature <222> (129)..(144) <223> Linker <220>

<221> misc_feature <222> (145)..(265) <223> VH <220>

<221> misc_feature <222> (268)..(282) <223> Hinge <220>

<221> misc_feature <222> (500)..(519) <223> EFD-BD2 linker <220>

<221> misc_feature <222> (520)..(640) <223> VH2

5089144_1 (GHMatters) P79767.AU.1 5/02/14

125

2016231617 23 Sep 2016 <220>

<221> misc_feature <222> (641)..(660) <223> Linker2 <220>

<221> misc_feature <222> (661)..(772) <223> VL2 <400> 172

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu He Ser 15 Ala Ser 1 5 10

Val He Met Ser Arg Gly Gin He Val Leu Ser Gin Ser Pro 2 0 Ala He 20 25 30

Leu Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg 25 Ala Ser 35 40 45

Ser Ser Val Ser Tyr Met His Trp Tyr Gin Gin Lys Pro Gly 30 Ser Ser 50 55 60

Pro Lys Pro Trp He Tyr Ala Pro Ser Asn Leu Ala Ser Gly 35 Val 65 Pro 70 75 80

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2016231617 23 Sep 2016

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie

10 275 280 285

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

290 295 300 lie Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

305 310 315

320

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 25 Glu Val

325 330

335

30 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr

340 345 350

Arg Val Val Asn Gly

Ser Val Leu Thr Val Leu His Gin Asp Trp Leu

355

360

365

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120

2016231617 23 Sep 2016

10 ggtggcggtg gctcgggcgg tggtggatct gggggaggag gcagcgggaa ttct 54 <210>

<211>

<212>

<213>

163

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker H6 (AA)

25 <400> 163

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

15 10

Asn Ser <210> 164 <211> 24

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109

2016231617 23 Sep 2016

<212> <213> DNA Artificial sequence <220> 5 <223> Synthetic primer <220> <223> Linker H7 (PN) 10 <400> 164 gggtgtccac cttgtccgaa ttct 24

15 <210> <211> <212> <213> 165 8 PRT Artificial sequence 20 <220> <223> Synthetic peptide <220> <223> Linker H7 (AA) 25 <400> 165 Gly Cys Pro Pro Cys Pro Asn 1 5 30 <210> 166 <211> 45 <212> DNA 35 <213> Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

110

2016231617 23 Sep 2016 <223> Synthetic primer <220>

<223> Linker(G4S)3 <400> 166 ggtggcggtg gatccggcgg aggtgggtcg ggtggcggcg gatct 45 <210>

<211>

<212>

<213>

167

PRT

Artificial sequence <220>

<223> Synthetic peptide <220>

<223> Linker(G4S)3 <400> 167

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

10 Gly Gly Ser Gly Asn Ser 35

<210> 152 15 <211> <212> <213> 6 DNA Artificial sequence 20 <220> <223> Synthetic primer <220> <223> Linker Hl (PN) 25 <400> aattct 6 152

30 <210> 153 <211> 2 <212> PRT <213> Artificial sequence 35 <220> <223> Synthetic peptide

5089144_1 (GHMatters) P79767.AU.1 5/02/14

104

2016231617 23 Sep 2016 <220>

<223> Linker Hl (AA)

10 Ala Leu Pro Ala Gly Gin

100

15 Pro Arg Glu Pro Glu Leu

115

20 Thr Lys Asn Gin Tyr Pro

130

25 Ser Asp lie Ala Asn Asn 145 160

Tyr Lys Thr Thr Phe Leu

175

Tyr Ser Lys Leu Asn Val

Asn Gly Lys Glu Tyr

Pro lie Glu Lys Thr

105

Gin Val Tyr Thr Leu

120

Val Ser Leu Thr Cys

135

Val Glu Trp Glu Ser

150

Pro Pro Val Leu Asp

165

Thr Val Asp Lys Ser

Lys Cys Lys Val Ser

90 95 lie Ser Lys Ala Lys

110

Pro Pro Ser Arg Asp

125

Leu Val Lys Gly Phe

140

Asn Gly Gin Pro Glu

155

Ser Asp Gly Ser Phe

170

Arg Trp Gin Gin Gly

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2016231617 23 Sep 2016

180

185

Phe Ser Cys Ser Val Met His Glu Ala Leu His 5 Thr Gin

195 200

Lys Ser Leu Ser Leu Ser Pro Gly Lys 10 210 215

190

Asn His Tyr

205

<210> 144 <211> 654 <212> DNA <213> Homo sapiens <220> <223> hlgGl (P331S) <400> 144 gcacctgaac tcctgggtgg caaggacacc 60 ctcatgatct cccggacccc ccacgaagac 120 cctgaggtca agttcaactg caagacaaag 180 ccgcgggagg agcagtacaa cgtcctgcac 240 caggactggc tgaatggcaa cctcccagcc 300 tccatcgaga aaacaatctc

ccccaaaacc tggacgtgag tgcataatgc gcgtcctcac ccaacaaagc gagaaccaca

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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ggtgtacacc 360 ctgcccccat cccgggatga c. cctggtcaaa 420 u ggcttctatc ccagcgacat ggagaacaac 480 tacaagacca cgcctcccgt 10 cagcaagctc 540 accgtggaca agagcaggtg gatgcatgag 600 15 gctctgcaca accactacac atga 654 <210> 145 20 <211> 217 <212> PRT

<213> Homo sapiens <220>

25 <223> hlgGl (P331S) <400> 145

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 30 Pro Lys

15 10

Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr 35 Cys Val

20 25 30

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Val Trp Val Asp Val Ser His Glu Asp 40 Pro Glu Val Lys Phe 45 Asn Tyr 35 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 Ala Leu Pro Ala Ser lie Glu Lys Thr He Ser Lys Ala Lys Gly Gin 100 105 110 Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu 115 120 125 Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu

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Asn Asn

145 150 155

160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170

175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin 195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 146 <211> 654 <212> DNA <213> Homo sapiens <220> <223> hlgGl (P238S/P331S) <400> 146 gcacctgaac tcctgggtgg atcgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60

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ctcatgatct ccacgaagac cccggacccc 120 tgaggtcaca cctgaggtca agttcaactg gtacgtggac 5 caagacaaag 180 ccgcgggagg cgtcctgcac agcagtacaa 240 cagcacgtac 10 caggactggc cctcccagcc tgaatggcaa 300 ggagtacaag 15 tccatcgaga ggtgtacacc aaacaatctc 360 caaagccaaa ctgcccccat cctggtcaaa cccgggatga 420 gctgaccaag ggcttctatc ccagcgacat cgccgtggag 20 ggagaacaac 480 tacaagacca cagcaagctc cgcctcccgt 540 gctggactcc accgtggaca agagcaggtg gcagcagggg 25 gatgcatgag 600 gctctgcaca atga accactacac 654 gcagaagagc

tgcgtggtgg ggcgtggagg cgtgtggtca tgcaaggtct gggcagcccc aaccaggtca tgggagagca gacggctcct aacgtcttct ctctccctgt tggacgtgag tgcataatgc gcgtcctcac ccaacaaagc gagaaccaca gcctgacctg atgggcagcc tcttcctcta catgctccgt ctccgggtaa <210> 147 <211> 217 <212> PRT <213> Homo sapiens <220>

<223> hlgGl (P238S/P331S)

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2016231617 23 Sep 2016 <400> 147

10 Pro Cys Lys Val Asp Thr 20 Leu Met He Ser Arg 25 Thr Pro Glu Val Thr 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45 15 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 20 Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 25 80

Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 30 85 90 Ala Leu Pro Ala Pro lie Glu Lys Thr He Ser Lys Ala Lys

Gly Gin

35 100 105 110

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Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

115

120

125

Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

130

135

140

Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn

145

160

150

155

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165

170

20 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val

180

185

190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gin

195

200

205

Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 142

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<211> 654 <212> DNA <213> Homo sapiens 5 <220> <223> hlgGl (P238S) <400> 142 10 gcacctgaac caaggacacc tcctgggtgg 60 atcgtcagtc ttcctcttcc ccccaaaacc ctcatgatct ccacgaagac cccggacccc 120 tgaggtcaca tgcgtggtgg tggacgtgag 15 cctgaggtca caagacaaag agttcaactg 180 gtacgtggac ggcgtggagg tgcataatgc 20 ccgcgggagg cgtcctgcac agcagtacaa 240 cagcacgtac cgtgtggtca gcgtcctcac caggactggc cctcccagcc tgaatggcaa 300 ggagtacaag tgcaaggtct ccaacaaagc 25 cccatcgaga ggtgtacacc aaacaatctc 360 caaagccaaa gggcagcccc gagaaccaca ctgcccccat cctggtcaaa cccgggatga 420 gctgaccaag aaccaggtca gcctgacctg 30 ggcttctatc ggagaacaac ccagcgacat 480 cgccgtggag tgggagagca atgggcagcc 35 tacaagacca cagcaagctc cgcctcccgt 540 gctggactcc gacggctcct tcttcctcta accgtggaca gatgcatgag agagcaggtg 600 gcagcagggg aacgtcttct catgctccgt

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa io

atga 654 <210> 143 <211> 217 <212> PRT <213> Homo sapiens <220> <223> hlgGl (P238S) <400> 143 Ala Pre i Glu Leu Leu Gly Gly Ser Ser Val Pro Lys 1 5 10

Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

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10 <210> <211> <212> <213> 139 2 PRT Artificial sequence 15 <220> <223> Synthetic peptide 20 <400> 139 Ser Ser 1

<210> 140 25 <211> 654 <212> DNA <213> Homo sapiens <220> 30 <223> hlgGl wild type <400> 140

gcacctgaac tcctgggtgg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 60 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 120

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cctgaggtca caagacaaag agttcaactg 180 5 ccgcgggagg agcagtacaa cgtcctgcac 240 caggactggc tgaatggcaa cctcccagcc 300 10 cccatcgaga aaacaatctc ggtgtacacc 360 ctgcccccat cccgggatga 15 cctggtcaaa 420 ggcttctatc ccagcgacat ggagaacaac 480 20 tacaagacca cgcctcccgt cagcaagctc 540 accgtggaca agagcaggtg gatgcatgag 600 25 gctctgcaca accactacac atga 654 30 <210> 141 <211> 217 <212> PRT

<213> Homo sapiens

35 <220>

<223> hlgGl wild type

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 141

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 5 1 5 10

10 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Ser Pro Pro Ser Pro

15 10 <210> 132 <211> 45 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<221> misc_feature <223> ssc(p)-hlgGl (DNA) <400> 132 gagcccaaat cttgtgacaa aactcacaca tgtccaccga gccca 45 <210>

<211>

<212>

<213>

133

PRT

Artificial sequence <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <223> Synthetic peptide <220>

<221>

misc feature

10 <400> 127

15 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Pro 10 1 5 <210> 128 <211> 45 <212> DNA 20 <213> Artificial sequence <220> <223> Synthetic primer 25 <220> <221> misc feature <223> see(p)-hlgGI (DNA) <400> 128 30 gagcccaaat cttctgacaa aactcacaca tgtccaccgt gccca 45

<210> 129 <211> 15 <212> PRT <213> Artificial sequence

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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<223> Synthetic peptide

10 Gly Leu Lys Phe

Lys Gly 15 Ala Tyr

20 Met Glu Tyr Cys

25 Ala Arg Val Trp

30 Gly Ala Ser Gly

35 Gly Gly Met Thr

130

Asn Trp Leu Lys

He Asn Pro Tyr

Lys Ala Thr Leu

Leu Leu Ser Leu

Ser Gly Tyr Tyr

100

Gly Thr Thr Val

115

Gly Ser Gly Gly

Gin Ser His Gly

Lys Gly Leu Thr

Thr Val Asp Lys

Thr Ser Glu Asp

Gly Asp Ser Asp

105

Thr Val Ser Ser

120

Gly Gly Ser Ala

135

Lys Asn Leu Glu

Thr Tyr Asn Gin

Ser Ser Ser Thr

Ser Ala Val Tyr

Trp Tyr Phe Asp

110

Gly Gly Gly Gly

125

Ser Asp He Gin

140

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Gin Thr Thr Ser Thr lie

10 <223> Synthetic polypeptide <220>

<223> G19-4 VHVL (AA)

15 <220>

<221> misc_feature <222> (1)..(122) <223> VH

20 <220>

<221> misc_feature <222> (123)..(139) <223> Linker

25 <220>

<221> misc_feature <222> (140)..(248) <223> VL

30 <400> 109

Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

15 10

Ser Met Lys lie Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Gly Tyr

10 <220>

<221> misc_feature <222> (126) . . (247) <223> VH

15 <400> 107

Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

15 10

Asp Arg Val Thr lie Ser Cys Arg Ala Ser Gin Asp lie Arg Asn Tyr

25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu lie

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ala Asn Leu

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Gin Pro

65 70 75

Glu Pro Asp Trp lie Ala Thr 85 Tyr Phe Cys Gin Gin Gly Asn 90 Thr Leu Thr Phe Gly Gly Gly Thr Lys Leu Val Thr Lys Arg Gly Gly Gly Gly 100 105 110 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Glu Val Gin 115 120 125 Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys 130 135 140 He Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr He Val Asn 145 150 155 160

Trp Leu Lys Gin Ser His Gly Lys Asn Leu Glu Trp He Gly Leu He 165 170 35 175

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Asn Pro Tyr Lys Gly Leu Thr Thr Tyr Asn Gin Lys Phe Lys Gly Lys

180

185

190

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu

195

200

205

Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser

210

215

220

Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly

225 230 235

240

Thr Thr Val Thr Val Ser Ser 245

<210> 108 <211> 752 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide <220> <223> G19-4 VHVL (DNA) <400> 108

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accggtgagg agcttcaatg tccagctgca 60 acagtctgga cctgaactgg tgaagcctgg 5 aagatttcct ctggctgaag gcaaggcctc 120 tggttactca ttcactggct acatcgtgaa cagagccatg aggtcttact gaaagaacct 180 tgagtggatt ggacttatta atccatacaa 10 acctacaacc atccagcaca agaaattcaa 240 gggcaaggcc acattaactg tagacaagtc 15 gcctacatgg ctgtgcaaga agctcctcag 300 tctgacatct gaagactctg cagtctatta tctgggtact gaccacggtc atggtgactc 360 ggactggtac ttcgatgtct ggggcgcagg 20 accgtctcct tgggagcgct ctggtggcgg 420 tggctcgggc ggtggtggat ctggaggagg agcgacatcc agacagagtc agatgacaca 480 gactacatcc tccctgtctg cctctctggg 25 accatcagtt tcagcagaaa gcagggcaag 540 tcaggacatt cgcaattatt taaactggta 30 ccagatggaa aggagtccca ctgttaaact 600 cctgatctac tacacatcaa gattacactc tcaaggttca caacctgcaa gtggcagtgg 660 gtctggaaca gattattctc tcaccattgc 35 ccagaagata gacgttcggt ttgccactta 720 cttttgccaa cagggtaata cgcttccgtg ggaggcacca aactggtaac caaacgctcg ag

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

752 <210> 109

10 15

Gly Tyr Ser Phe Thr

Gly Lys Asn Leu Glu

Thr Thr Tyr Asn Gin

Lys Ser Ser Ser Thr

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

30 85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

35 100 105 110

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser

130 135 140

Asp lie Gin Met Thr Gin Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

145 150 155

160

Asp Arg Val Thr lie Ser Cys Arg Ala Ser Gin Asp lie Arg Asn Tyr

165 170

20 175

Leu Asn Trp Tyr Gin Gin Lys Pro Asp Gly Thr Val Lys Leu Leu lie

180 185 190

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr lie Ala Asn Leu Gin Pro

210 215 220

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Glu Asp lie Ala Thr Tyr Phe Cys Gin Gin Gly Asn Thr Leu Pro Trp

225 230 235

240

Thr Phe Gly Gly Gly Thr Lys Leu Val Thr Lys Arg Ser 245 250 <210> 106 <211> 749 <212> DNA <213> Artificial sequence <220>

<223> Synthetic polynucleotide <220>

20 <223> G19-4 VLVH (DNA) <400> 106 accggtgaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 60 gtcaccatca gttgcagggc aagtcaggac attcgcaatt atttaaactg gtatcagcag 120 aaaccagatg gaactgttaa actcctgatc tactacacat caagattaca 30 ctcaggagtc 180 ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tgccaacctg 240

35 caaccagaag atattgccac ttacttttgc caacagggta atacgcttcc gtggacgttc 300

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ggtggaggca tggtggatct ccaaactggt 360 aaccaaacgg ggtggcggtg gctcgggcgg 5 ggaggaggtg actggtgaag ggagcgctag 420 cgaggtccag ctgcaacagt ctggacctga cctggagctt tggctacatc caatgaagat 480 ttcctgcaag gcctctggtt actcattcac 10 gtgaactggc tattaatcca tgaagcagag 540 ccatggaaag aaccttgagt ggattggact 15 tacaaaggtc aactgtagac ttactaccta 600 caaccagaaa ttcaagggca aggccacatt aagtcatcca ctctgcagtc gcacagccta 660 catggagctc ctcagtctga catctgaaga 20 tattactgtg tgtctggggc caagatctgg 720 gtactatggt gactcggact ggtacttcga gcagggacca 749 cggtcaccgt ctcctcgag

<210>

<211>

<212>

<213>

107

247

PRT

Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> G-19-4 VLVH (AA) <220>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <221> misc_feature <222> (1)..(108) <223> VL

10 <220>

<223> G-28-1 VLVH (AA) <220>

<221>

<222>

<223>

misc_feature (1)··(107)

VL <220>

<221>

<222>

<223>

misc_feature (108) . . (124) Linker <220>

<221>

<222>

<223>

misc_feature (125) . . (239) VH <400> 103

30 Asp lie Gin Met Thr Gin Ser Pro Ala Ser Leu Ser Ala Ser Val Gly

15 10

35 Glu Thr Val Thr lie Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr

20 25 30

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Leu Ala Trp Tyr Gin Gin Lys Gin Gly Lys Ser Pro Gin Leu Leu Val

10 <220>

<223> Synthetic primer <220>

<223> 194-LH-LR1 <400> 80 gttgcagctg gacctcgcta gcgctcccac ctcctccaga tc 42 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LH-HF1 <400> 81 gatctggagg aggtgggagc gctagcgagg tccagctgca acagtctgga cctg 54 <210> 82

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <211> 50 <212> DNA <213> Artificial sequence

10 <400> 75 gcaaaagtaa gtggcaatat cttctggttg caggttggca atggtgagag 50 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> 194-LR5 <400> 76 gaacgtccac ggaagcgtat taccctgttg gcaaaagtaa gtggcaatat c 51

<210> 77 <211> 52 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> 194-LR4

10 <223> Synthetic primer <220>

<223> CH3seqF2

15 <400> 43 cctctacagc aagctcac 18 <210>

<211>

<212>

DNA <213> Artificial sequence

25 <220>

<223> Synthetic primer <220>

<223> CH3seqRl <400> 44 ggttcttggt cagctcatc 19 <210>

<211>

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> CH3seqR2 <400> 45 gtgagcttgc tgtagagg 18 <210>

<211>

<212>

<213>

DNA

Artificial sequence <220>

<223> Synthetic primer <220>

<223> L1-11R <400> 46 gcgatagaat tcccagatcc accaccgccc gagccaccgc caccataatt c 51

<210> 47 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic primer

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> L1-6R

10 gtaatacgac tcactatagg 20 <210> 13 <211> 18 <212> DNA <213> Artificial sequence <220>

<223> Synthetic primer <220>

<223> M13 reverse primer <400> 13 caggaaacag ctatgacc 18

30 <210> 14 <211> 15 <212> PRT <213> Homo sapiens

35 <220>

<223> human IgGl hinge domain

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 14

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

10 Glu lie Lys Arg 275

<210> 7 15 <211> 30 <212> DNA <213> Artificial sequence <220> 20 <223> Synthetic primer <220> <223> Heavy Chain GSP1 25 <400> 7 aggtgctgga ggggacagtc actgagctgc 30

30 <210> 8 <211> 28 <212> DNA <213> Artificial sequence 35 <220> <223> Synthetic primer

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <220>

<223> Nested Heavy Chain <400> 8

10 Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro 50 55 60

15 Gly Ser 65 80 Lys Thr Gly Leu Glu Trp 70 Leu Gly Met He Trp 75 Gly Asp Gly 20 Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser lie Thr Lys Asp Asn 85 90 25 Ser Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp 100 105 110 30 Thr Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr 115 120 125 35 Tyr Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val

Ser Ser

130 135 140

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Gly Gly Gly Gly Ser Gly

10 <220>

<221> misc_feature <222> (1) . . (23) <223> Leader

15 <220>

<221> misc_feature <222> (24) . . (144) <223> VH

20 <220>

<221> misc_feature <222> (145)..(164) <223> Linker

25 <220>

<221> misc_feature <222> (165) . . (276) <223> VL

30 <400> 6

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu lie Ser Ala Ser

15 10

Val He Met Ser Arg Gly Val Gin Val Gin Leu Lys Glu Ser

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Gly Pro

20 25 30

10 255

Val Met Asp Tyr Trp Gly Gin Gly Thr Ser Val Thr Val Ser Ser

15 260 265 270 <210>

<211>

<212>

<213>

828

DNA

Artificial sequence <220>

<223> Synthetic polynucleotide <220>

<223> anti-CD28 (2el2) HL (DNA) <400> 5

30 atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60 agaggagtcc aggtgcagct gaaggagtca ggacctggcc tggtggcgcc ctcacagagc 120 ctgtccatca catgcaccgt ctcagggttc tcattaaccg gctatggtgt aaactgggtt 180

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

cgccagcctc tggaagcaca caggaaaggg 240 tctggagtgg ctgggaatga tatggggtga 5 gactataatt caagagccaa cagctctcaa 300 atccagacta tcgatcacca aggacaactc 10 gttttcttaa ctgtgcccga aaatgaacag 360 tctgcaaact gatgacacag ccagatacta gatggttata aacctcagtc gtaactttca 420 ttactatgtt atggactact ggggtcaagg 15 accgtctcct tgggtcgggt ctgggggtgg 480 aggctctggt ggcggtggat ccggcggagg ggcggcggat gtctctaggt ctgacattgt 540 gctcacccaa tctccagctt ctttggctgt 20 cagagagcca cacaagttta ccatctcctg 600 cagagccagt gaaagtgttg aatattatgt 25 atgcagtggt tgctgctagc accaacagaa 660 accaggacag ccacccaaac tcctcatctc aacgtagaat agactttagc ctggggtccc 720 tgccaggttt agtggcagtg ggtctgggac 30 ctcaacatcc gcaaagtagg atcctgtgga 780 ggaggatgat attgcaatgt atttctgtca aaggttccat 828 ggacgttcgg tggaggcacc aagctggaaa tcaaacgt

<210> 6 <211> 276

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <212> PRT <213> Artificial sequence <220>

<223> Synthetic polypeptide <220>

<223> anti-CD28 (2el2) HL (AA)

10 20 25 30

Ser Leu Ala Val Ser Leu Gly Gin Arg Ala Thr lie Ser Cys Arg Ala

15 35 40 45

Ser Glu Ser Val Glu Tyr Tyr Val Thr Ser Leu Met Gin Trp Tyr Gin

50 55 60

Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu lie Ser Ala Ala Ser Asn

65 70 75

Val Glu Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr

30 85 90 95

Asp Phe Ser Leu Asn lie His Pro Val Glu Glu Asp Asp lie Ala Met

35 100 105 110

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

Tyr Phe Cys Gin Gin Ser Arg Lys Val Pro Trp Thr Phe Gly Gly Gly

115 120 125

Thr Lys Leu Glu lie Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly

130 135 140

Ser Gly Gly Gly Gly Ser Gin Val Gin Leu Lys Glu Ser Gly Pro Gly

145 150 155

160

Leu Val Ala Pro Ser Gin Ser Leu Ser lie Thr Cys Thr Val Ser Gly 165 170 20 175

Phe Ser Leu Thr Gly Tyr Gly Val Asn Trp Val Arg Gin Pro Pro Gly 25 180 185 190

Lys Gly Leu Glu Trp Leu Gly Met He Trp Gly Asp Gly Ser Thr Asp 30 195 200 205 Tyr Asn Ser Ala Leu Lys Ser Arg Leu Ser lie Thr Lys Asp

Asn Ser

35 210 215 220

5089144_1 (GHMatters) P79767.AU.1 5/02/14

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Lys Ser Gin Val Phe Leu Lys Met Asn Ser Leu Gin Thr Asp Asp Thr

225 230 235

240

Ala Arg Tyr Tyr Cys Ala Arg Asp Gly Tyr Ser Asn Phe His Tyr Tyr

245 250

10 Lys Gly Lys Ala Ala Tyr

210

Met Gin Leu Ser 15 Phe Cys

225

240

Ala Arg Val Val 20 Val Trp

255

Val Lys Gin Thr

Pro Gly Asn Gly

200

Thr Leu Thr Val

215

Ser Leu Thr Ser

230

Tyr Tyr Ser Asn

245

Pro Arg Gin Gly Leu Glu

185 190

Asp Thr Ser Tyr Asn Gin

205

Asp Lys Ser Ser Ser Thr

220

Glu Asp Ser Ala Val Tyr

235

Ser Tyr Trp Tyr Phe Asp

250

Gly Thr Gly Thr Thr Val Thr Val Ser

25 260 30 <210> 3 <211> 813 <212> DNA <213> Artificial sequence <220> <223> Synthetic polynucleotide 35 <220> <223> anti-CD28 (2el2) LH (DNA)

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016

<400> 3 atggattttc cataatgtcc aagtgcagat 60 tttcagcttc ctgctaatca gtgcttcagt 5 agaggagtcg tctaggtcag acattgtgct 120 cacccaatct ccagcttctt tggctgtgtc 10 agagccacca aagtttaatg tctcctgcag 180 agccagtgaa agtgttgaat attatgtcac cagtggtacc tgctagcaac aacagaaacc 240 aggacagcca cccaaactcc tcatctctgc 15 gtagaatctg ctttagcctc gggtccctgc 300 caggtttagt ggcagtgggt ctgggacaga aacatccatc aagtaggaag ctgtggagga 360 ggatgatatt gcaatgtatt tctgtcagca 20 gttccatgga cggtggatcc cgttcggtgg 420 aggcaccaag ctggaaatca aacggggtgg 25 ggcggaggtg aggacctggc ggtcgggtgg 480 cggcggatct caggtgcagc tgaaggagtc ctggtggcgc ctcattaacc cctcacagag 540 cctgtccatc acatgcaccg tctcagggtt 30 ggctatggtg gctgggaatg taaactgggt 600 tcgccagcct ccaggaaagg gtctggagtg atatggggtg atcgatcacc atggaagcac 660 agactataat tcagctctca aatccagact 35 aaggacaact tgatgacaca ccaagagcca 720 agttttctta aaaatgaaca gtctgcaaac

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 gccagatact actgtgcccg agatggttat agtaactttc attactatgt tatggactac 780 tggggtcaag gaacctcagt caccgtctcc tct 5 813 <210>

<211>

<212>

<213>

271

PRT

Artificial sequence <220>

<223> Synthetic polypeptide

15 <220>

<223> anti-CD28 (2el2) LH (AA) <220>

<221> misc_feature <222> (1) . . (23) <223> Leader <220>

<221> misc_feature <222> (24)..(135) <223> VL <220>

<221> misc_feature <222> (136)..(150) <223> Linker <220>

<221> misc_feature <222> (151)..(271) <223> VH

5089144_1 (GHMatters) P79767.AU.1 5/02/14

2016231617 23 Sep 2016 <400> 4

Met Asp Phe Gin Val Gin lie Phe Ser Phe Leu Leu lie Ser Ala Ser

10 20 30 40 50 60 % Annexin V / Pl Positive

E co £

H

B. Daudl Cells: 2H7-ssS’hlgG’H7-G26-1 HL

TRU-015 (10) + G2B-1 LH (5) ’lllllllllllllllllllllllllllllllllllllllllh ////////// ® TRU-015 (20) + G2B-1 LH (10, w

ΓΤ

Q

G28-1 LH

TRU 015

Media /////////////

0 10 20 30 40 50 60 70 80 % Annexin VI PI Positive □ 5 010 20

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

23/67

FIG. 18

A. Binding of 2H7-hlgG-G19-4 Fusion Proteins to JurRat Cells

2H7-sss-hlgG1-STD1-G194-HL 2H7-csc-hlgG1-STD1-G194HL

2H7-sss-hlgG1-STD1-G194LH g a h FTIC

B. Binding of 2H7-hlgG-G19-4 to WH-2S Cells

TRU015 2H7-sss-hlgG-STD1-G194HL

2H7-csc-tilgG-ST0l-G194LH —2H7-sss-h!gG-STD1-G194LH mock

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

24/67

2016231617 23 Sep 2016

FIG. 19

A. ADCC Activity of 2H7-G19-4 Multispecific Fusion Proteins Against BJAB Targets Using Human PBMC Effectors at 25:1

10/67

FIG. SB (4 of 5)

fame BD1 Hinge ffO Linker SD2 2H7-sss-hlgG-STD2-G194 LH 2H7 (VL-VH) hlgGl-SSS higGorhlgG /P238Sff331Sl STD2 G19-4 (VL-VH) 2H7-sss-hlgG-ST02-G194HL 2H7 (VL-VH, hlgGl -SSS hlgGcrhlgG (Ε23Β5ίΕ33151 ST02 G194 (VH-VL) 2H7-csc-hlgG-STD1-G194LH 2H7 (VL-VH, hlgGl-esc higGorhlgG IP238SIP331S1 STDl 6194 (VL-VH, 2H7-csc-hlgG-STD2-G1W LH 2H7 (VL-VH) hlgGl-esc higGorhlgG 1E238SE331SL. ST02 619-4 (VH-VL) 2H7-csc-hlgG-STD1-G19-4 HL 2H7(VL-VH) hlgGl-esc higGorhlgG . EZ38SIE331SL STDl G194(VL-W) 2H7-csc3itgG-STD2-G1W Hl 2H7(VL-VH) hlgGl-esc htgG orhlgG . 1P236SP331SL STO2 GI94 (VH-VL) 2H7-sss-hlgG-H1-G194 LH 2H7(VL-VH) hlgGl-SSS hlgGcrhlgG fP2Msipaaisi Hl G194 (VL-VH) 2H7-csc-hlgG-H1-G1S-4 LH 2H7(VL’VH) hlgGl-esc hlgGcrhlgG (P23BS/P331SL HI 6194 (VH-VL) 2H7-sss-hlgG-H1-Gl94HL 2H7 (VL-VH) hlgGl-SSS hlgGcrhlgG ms/raaia. HI 6194 (VL-VH, 2H7-csc-hlgG-H1-G194HL 2H7 (VL-VH) hlgGl-esc hlgGcrhlgG IP238STO31S1 HI G194 (VH-VL, 2H7-sss-hlgG-H2-G194 LH 2H7 (VL-VH) hlgGl-SSS hlgGcrhlgG ipamasi- H2 G194 (VL-VH) 2H7-csc-hlgG-H2-G194 LH 2H7 (VL-VH) hlgGl-esc higGorhlgG 1E23W31SL·. H2 G194 (VL-VH) 2H7-sss-hlgG-H2-G194 HL 2H7 (VL-VH, hlgGl-SSS hlgGcrhlgG (P23S5E331S1- H2 G194 (VL-VH) 2H7-csc-WgG-H2-Gl94 LH 2H7(VL-VH) hlgGl-esc higGorhlgG ircaasfp33isi. H2 G194 (VL-VH) 2H7-i$5-h)gG-H3-G19-4 LH 2H7IVL-VH) hlgGl -SSS hlgGcrhlgG IP238SP331SL K3 G194 (VL-VH) 2H7-csc-hlgG-H3-Gl94 LH 2H7 (VL-VH, hlgGl-esc IgGorhlgG fP23flSIE331S) H3 G194 (VH-VL) 2H7-sss-hlgG-H3-G19-4 Hl 2H7 (VL-VH) hlgGl-SSS higGorhlgG P2MSF331S1 H3 gum (vl-vh) 2H7-CSC-bJgG-H3-G194 LH 2H7 (VL-VH) hlgGl-esc higGorhlgG IP238SIP331SL- H3 6194 (VH-VL, 2H7-sss-hlgG-H4-Gl94lH !H7 (VL-VH) hilgGI -SSS hlgGcrhlgG [Ε2382Ε33151 H4 G194 (VL-VH) 2H7-csc-h|gG-H4-Gl94LH !H7 (VL-VH) ilgG1 -esc higGorhlgG PMRS/P331SI H4 G194 (VH-VL, 2H7-5SS-blgG-H4-G19-4 Hl ! H7(VL-VH) ilgGI -SSS higGorhlgG P238SIP331SL. H4 G194 (VL-VH, 2H7-csc-higG-H4-G194 LH 2 H7 (VL-VH) 1 lgG1-esc ilgG or htgG P23flSE331Sl- H4 3194 (VH-VL) 2H?-5SS-hlgG-H5-G194 LH 2 H7 (VL-VH) h IgGl -SSS ilgG orhlgG P238S/P331S) H5 3104 (VL-VH) 2H7-csc-tilgG-H5-G194 LH 2i H7 (VL-VH) h IgGI-csc 1 IgGorhlgG E238SE331SL. H5 < 3104 (VH-VL, 2H7-iS54ilgG-H5-G1WHL 2I 17 (VL-VH) h IgGl -SSS 11 IgGorhlgG £23SS£331SU 45 £ >194 (VL-VH)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

10° 10¹ GOAT ANTI-HUMAN FITC 1:100 — 2e12 HL SMIP 5ug/mL — 2h7-2e12 HL SCORPION 5ug/mL

256-1

B. BINDING OF PURIFIED PROTEIN FROM COS CELLS TO CD28CHO: 2H7-SSSIgG-STD1-2e12 LH vs. 2H7-SSS1gG-STD1-2e12 HL MULTISPECIFIC FUSION PROTEIN GOAT ANTI-HUMAN FITC 1:100

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

10. A protein according to claim 9, wherein the hinge region is a hinge region selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgE, IgA2, synthetic hinge and the hinge-like Ch2 domain of IgM.

11 ιιιιιιιιιιιιιιιιιιιιιιιΐΜΐιιι iiHiiuii iHliuii to σι »# J <O / ? / J ? J *SrS· >/>,><>*

UUiHMiiiinllMllIlIlllllMIIIIIIIIHIIIIIIUtllHilHIllΙΙΙΙΙΙΗΗΗ

Swj.wws»1

BHBB

Η 11IIIIMIIUI JltllH I lllllllllUMlI Jlllll IIIIIUIHH

Hill IIItllllllltllll111111111 tlllllllll till·· mill

CM o

CO co

CM

ΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙΙ|1|Ι|ΙΙΙ1Ι||ΙΙΙΙΙΙ|ΙΙΙΙΙΙΙΙΙΙΙΙ

CM

I I I F «? wyww iiuinniinniiuiiiiiiniiii|iiiiiiimn

11/67

FIG. GB (5 of 5)

Name BD1 Hinge EFD (Joker 802 2H7-csc4ilgG-H5-G1M LH 2H7 (VL-VH) WgG1-csc higGorhlgG H5 G19+(VH-VL) 2H7-sss4ilgG-HB-<51&-4 LH 2H7 (VL-VH) hlgGUSSS higGorhlgG IKMSPMISI- H6 G1W (VL-VH) 2H7<sc-hlgG4lW31WLH 2H7 (VL-VH) NgG1 -esc higGorhlgG H6 G1M (VL-VH) 2H7-s5S-hlgG-HS-G1MHL 2H7 (VL-VH) htgGt-SSS higGorhlgG H6 G1M (VH-VL) 2H7-o»WgG-HMilMlH 2H7 (VL-VH) hlgGI-csc MgGofhlgG mseaaia. H6 G194 (VH-VL) 2H7-ssHtlgG+l7-G1W LH 2H7 (VL-VH) hlgGl-SSS higGorhlgG 1 H7 G1M (VL-VH) 2H7-<50-iilgG-H7-Gl WLH 2H7 (VL-VH) bjgGl -esc higGorhlgG pBsmst H7 G1W (VL-VH) 2H7-sss-hlg6-H7-G1WHl 2H7(Vl-W) hlgGl-SSS higGorhlgG gzasraasL. H7 G1EM (VH-VL) 2H7-cs£-hl§G-H7-G194 LH 2H7 (VL-VH) hlgGI-csc higGorhlgG (EaflaSUSL H7 G1M(WfVL)

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

11. A protein according to claim 10, wherein the hinge region is an IgGl hinge region.

8481915_1 (GHMatters) P79767.AU.2

201

2016231617 03 Aug 2018

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FIG. 7

ALTERING THE LINKER AFTER THE EFD ANDTHE ORIENTATION OF V REGIONS IN BD2 AFFECTS BINDING EFFICIENCY OF MULTISPECIFIC FUSION PROTEINS

BINDING OF 2H7-SSShlgG-L/Hx-2e12 MULTISPECIFIC FUSION PROTEINS WITH VARIOUS LINKERS TO WIL2-S AND CD28 CHO **ALL FUSION PROTEINS USED AT 0.72 ug/mL, PROTEIN A PURIFIED, GENERATED FROM TRANSIENT COS SUPERNATANTS.

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

12. A protein according to claim 11, wherein the hinge region is a human IgGl hinge region with a mutation at one or two cysteine residues.

13/67

FIG. 8

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NON-REOUCED

REDUCED

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

13. A protein according to any one of claims 1 to 12, wherein the Fc region further comprises a domain derived from an immunoglobulin Ch3 domain.

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FIG. 9

Western Blots of Multispecific Fusion Proteins With H6 Linker A. Absence of SMIP or smaller CD28 detectable forms B. Presence of a SMIP sized form using CD20 anti-id Fab

A. Detection of 2e12 BD2 B. Detection of 2H7 CD20 by CD28-murinelgG BD1 by Fab: AbyD02429.2

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

15/67

FIG. 10

A. Binding of Multispecific Fusion Proteins With Variant Linkers to WiL-2S Cells

CONCENTRATION (ug/mL)

B. Binding of Muttispecific Fusion Protein With Variant Linkers to CD28 CHO Cells

—TRU015 ·—H1 —H2 —x— H3 — — H4 H5 F- H6

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

16/67

FIG. 11

Summary of SEC Fractionation of 2H7-ssslgG-2e12 HL Multispecific Fusion Proteins with Variant Linkers

Linker ID Retention Time for POI %POI % Other Forms SDS-PAGE Analysis of Cteevageal EFD/BD2 junction H1 7.620 70.9 29.0 (Yes) H2 7.589 67.5 32.5 (Yes) H3 7.605 68.7 31.4 Yes H4 7.622 (shoulder) 68.0 32.0 Yes H5 7.933/7.680 (doublet with shoulder) 32.42/33.94 33.63 YES H6 7.901/7.69 (doublet with shoulder) 34.5/29.3 36.1 YES H7 7.788 64.2 15.8 (HMW) NO

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

17/67

FIG. 12

Binding of [2H7-sss-hlgG-H_x-2e12 HL] Fusion Proteins with Different Linkers to Cells Expressing Target Antigen for BD1 or BD2

B. CD28 CHO Cells ♦- H3 — H6 H7 —h— gah FITC

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

18/67

FIG. 13

Simultaneous Binding of BD1 and BD2:

Binding of [2H7-sss-blgG-H_x-2e12 HL] Fusion Proteins with H3, H6, and H7 Linkers to WIL-2S Cells can be detected with CD28mlgG + FITC anti-mouse

H3 -e~H6 —A—H7 TRU015 + CO28mlg + g a m FITC —*— 2e12 HL + CD28mlg + g a m FITC —·— gam FITC

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

19/67

FIG. 14

A. Blocking of Binding of 2H7-sss-hlgG-H7-G28-1 HL Protein to Ramos Cells by CD20 and/or CD37 Targeted Antibodies

2H7-sss-hlgG-H7-G28-1 HL alone —— Plus anti-CD20 + anti-CD37 Plus anti-CD20 —*— Plus anti-CD37

B. Blocking of Binding of 2H7-sss-hlgG-H7-G28-1 HL to BJAB Cells by CD20 and/or CD37 Targeted Antibodies

2H7-sss-hlgG-H7-G28-1 HL alone —Pius anii-CD20 + anti-CD37 Plus anti-CD20 —*— Plus anti-CD37

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052 so o

Cl

Oh

20/ 67 (Z)

Cl

O

SO

FIG. 15

CC

Cl

SO o

Cl

ADCC Activity of 2H7-sss-hlgG1-STD1-G28-1 Variants against BJAB Targets

G28-HL

2H7-SSS-hlgG-STD1-G28-1LH

TRU015

2H7-SSS-tilgG-STD1-G28-1HL

G28-LH

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

211 67

FIG. 16

The Percent CD3, CD19, and CD40 Positive Lymphocytes Present in Culture after Incubation with TRU-015, G28-1 SMIP, TRU-015+G28-1 SMIP, or2H7-sss-hlgG1-H7-G28-1 HL

24 Hours CD3* CD19* CM9* Media 77% 8% 7% TRU015 76% 8.5% 7.9% G28-1 SMIP 78% 8. 9% 7.8% TRU015+G28-1 SMIP 76.3% 8.1% 7.5% 2H7-ssshlgG1-G28-1HL (H7) 78.4% 8.9% 7.9% 72 Hours .. .. CD3* CD19* CD40* Media 78.5% 7.9% 7.7% TRU015 85.2% 2.3% 2.7% G28-1 SMIP 86.7% 2.2% 2.3% TRU015+G28-1 SMIP 87.1% 1.1% 1.1% 2H7-ssshlgG1*G28-1 HL (H7) 96.7% .08% .77%

SUBSTITUTE SHEET (RULE 26)

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

22 /67

FIG. 17

The Percent Annexin and/or PI Positive B Cells in Culture after 24 Hour Incubation with Single or Multispecific Fusion Proteins. A. Ramos Cells:

2H7-sss-hlgGH7-G20-1 HL

TRU-015 (10) + G28-1 LH (5)

Φ TRU-015(20,+ G28-1 LH(10) co Q)

G28-1 LH

TRU 015

Media

14. A protein according to any one of claims 1 to 13, wherein the Fc region comprises a human immunoglobulin hinge region, Ch2 domain, and Ch3 domain.

15. A protein according to any one of claims 1 to 14, wherein the linker peptide is between 5 and 45 amino acids in length.

16. A protein according to any one of claims 1 to 15, wherein the linker peptide comprises one or more GGGGS motifs.

17. A protein according to any one of claims 1 to 8, wherein the linker peptide comprises an immunoglobulin core hinge region.

18. A protein according to any one of claims 1 to 8, wherein the linker peptide is derived from a stalk region of a type II C-lectin.

19. A protein according to claim 18, wherein the type II C-lectin is selected from the group consisting of CD69, CD72, CD94, NKG2A, andNKG2D.

20. A protein according to any one of claims 1 to 8, wherein the linker peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 149, 151, 153, 155, 157, 159, 161, 163,

165, 167, 169, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,

263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 287, 289, 297, 305, 307, 309, 310, 311, 313,

314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 346,

373,374,375,376, and 377.

21. A protein according to any one of claims 1 to 8, wherein the linker peptide is derived from an interdomain region between the Ig V-like and Ig C-like region of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, or CD244.

8481915_1 (GHMatters) P79767.AU.2

202

2016231617 03 Aug 2018

22. A protein according to any one of claims 1 to 21, wherein the protein is capable of forming dimers.

23. A protein according to any one of claims 1 to 5, wherein the binding domain that does not bind to 4-1BB/TNFRSF9 binds to a target on a cell of an infectious organism.

24. A protein according to claim 23, wherein the infectious organism is a bacterium, mycobacterium, fungus, or parasite.

25. A protein according to any one of claims 1 to 5, wherein the binding domain that does not bind to 4-1BB/TNFRSF9 binds to a virus.

26. Use of a protein according to any one of claims 23 to 25 in the manufacture of a medicament for treating, preventing, or mitigating an infection resulting from the infectious organism or virus.

27. A method for treating, preventing, or mitigating an infection resulting from an infectious organism or virus comprising administering to a subject in need thereof a protein according to any one of claims 1 to 25.

28. A method for treating a cell proliferation disorder, comprising administering to a subject in need thereof a protein according to any one of claims 1 to 22.

29. A method according to claim 28, wherein the cell proliferation disorder is a cancer or a tumor.

30. A method for treating an autoimmune disease, comprising administering to a subject in need thereof a protein according to any one of claims 1 to 22.

31. A method according to claim 30, wherein the autoimmune disease is rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, asthma, systemic lupus erythematosus (SLE), diabetes, multiple sclerosis, solid organ transplant rejection, or graft versus host disease (GVHD).

32. A nucleic acid encoding a protein according to any one of claims 1 to 25.

8481915_1 (GHMatters) P79767.AU.2

203

2016231617 03 Aug 2018

33. A vector comprising a nucleic acid according to claim 32.

34. A host cell comprising a nucleic acid according to claim 32 or vector according to claim 33, wherein said host cell is not present in a human.

35. A composition comprising a protein according to any one of claims 1 to 25 and a pharmaceutically acceptable carrier.

36. Use of a protein according to any one of claims 1 to 25 in the manufacture of a medicament.

8481915_1 (GHMatters) P79767.AU.2

WO 2007/146968

PCT/US2007/071052

2016231617 23 Sep 2016

15 <400> 379

Val Val Tyr Tyr Ser Asn Ser Tyr Trp Tyr Phe Asp Val 15 10

5089144_1 (GHMatters) P79767.AU.1 5/02/14