NZ716697B2 - Bi-specific monovalent diabodies that are capable of binding to gpa33 and cd3, and uses thereof - Google Patents

Abstract

The present invention is directed to bi-specific diabodies that comprise two polypeptide chains and which possess at least one binding site specific for an epitope of CD3 and one binding site specific for an epitope of gpA33 (i.e., a "gpA33 x CD3 bi-specific diabody"). The present invention also is directed to bi-specific diabodies that comprise an immunoglobulin Fc Domain ("bi- specific Fc diabodies") and are composed of three polypeptide chains and which possess at least one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3 (i.e., a "gpA33 x CD3 bi-specific Fc diabody"). The bi-specific diabodies and bi-specific Fc diabodies of the present invention are capable of simultaneous binding to gpA33 and CD3. The invention is directed to pharmaceutical compositions that contain such bi-specific diabodies or such bi-specific Fc diabodies. The invention is additionally directed to methods for the use of such diabodies in the treatment of cancer and other diseases and conditions. directed to bi-specific diabodies that comprise an immunoglobulin Fc Domain ("bi- specific Fc diabodies") and are composed of three polypeptide chains and which possess at least one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3 (i.e., a "gpA33 x CD3 bi-specific Fc diabody"). The bi-specific diabodies and bi-specific Fc diabodies of the present invention are capable of simultaneous binding to gpA33 and CD3. The invention is directed to pharmaceutical compositions that contain such bi-specific diabodies or such bi-specific Fc diabodies. The invention is additionally directed to methods for the use of such diabodies in the treatment of cancer and other diseases and conditions.

Description

Title of the Invention: Bi-Specific Monovalent Diabodies That Are e Of Binding to gpA33 And CD3, And Uses Thereof Cross-Reference to Related Applications: This Application claims priority to United States Patent Applications No. 61/869,528 (filed on August 23, 2013; pending) and 61/907,691 (filed on November 22, 2013; g), and to European Patent Application No. 13198859 (filed on December 20, 2013), each of which applications is herein incorporated by reference in its entirety.

Reference to Sequence Listing: This application includes one or more Sequence Listings pursuant to 37 CPR. 1.821 et seq., which are disclosed in both paper and computer-readable media, and which paper and computer-readable disclosures are herein incorporated by reference in their entirety.

Background of the Invention: Field of the Invention: The present invention is dircctcd to bi-spccific monovalent diabodics that comprise two ptide chains and which possess one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3 (i.e., a “gpA33 X CD3 bi-specific monovalent y"). The present invention also is directed to bi- specific monovalent diabodies that comprise an immunoglobulin Fc Domain (“bi- specific monovalent Fc diabodies”) and are composed of three polypeptide chains and which possess one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3 (i.e., a “gpA33 x CD3 bi-specific monovalent Fc y”). The bi-specific monovalent diabodies and bi-specific monovalent Fc diabodics of the present invention are capable of simultaneous binding to gpA33 and CD3. The ion is directed to pharmaceutical compositions that contain such bi- specific monovalent ies or such cific lent Fc diabodies. The invention is additionally directed to methods for the use of such diabodies in the treatment of cancer and other diseases and conditions.

Description of Related Art: I. gpA33 Colorectal cancer is among the most common malignancies of the Western world and is a leading cause of cancer deaths (Silverberg, E. et al. (1989) “Cancer Statistics, 1989,” CA Cancer J Clin. 39(1):3-20). One potentially useful target for colon cancer is the 43kD transmembrane glycoprotein A33 (gpA33) ((Heath, J .K. et al. (1997) “The Human A33 Antigen Is A Transmembrane Glycoprotein And A Novel Member Of The Immunoglobulin Supeifiimily,” Proc. Natl. Acad. Sci. (USA) 94(2):469-474; Ritter, G. et a1. (1997) “Characterization 0f Posttranslational cations Of Human A33 n, A Novel Palmitoylated Surface Glycoprotein Of Human Gastrointestinal Epithelium,’ 9 Biochem. Biophys. Res. Commun. 236(3):682-686). gpA33 was first discovered through raising monoclonal murinc antibodies against the human pancreatic carcinoma derived cell line ASPCI. One antibody (MAb A33) was found to react with a surface cell protein of 43 kDa, which was therefore designated “gpA33” (Wong, N.A. et a]. (2006) “EpCAM and gpA33 Are Markers OfBarrett’s Metaplasia,” J. Clin. Pathol. 260-263). gpA33 is a embrane protein of the onal adhesion molecule family; Abud, H.E. et al. (2000) “The Murine A33 Antigen Is Expressed At Two Distinct Sites During Development, The [CM Of The Blastocyst And The Intestinal Epithelium,” Mech. Dev. 98(1-2):111-114; Barendswaard, E.C. et al. (1998) “Rapid And Specific Targeting 0f Monoclonal dy A33 To A Colon Cancer Xenograft 1n Nude Mice,” Int. J. Oncol. 45-53; Panjideh, H. et a1. (2008) “Biodistribution And Efficacy 0f[1311]A33scFv::CDy, A Recombinant Antibody-Enzyme Protein For Colon Cancer,” Int. J. Oncol. 925-930). Although the functional significance of the A33 antigen is not yet understood, it has been shown to e colonic mucosal repair in an animal model of colitis and is homogeneously expressed in >95% of all colorectal carcinomas. A33 expression is uniform across both disease stage and degree of ogical differentiation, and the n is not detectably secreted 0r shed into the blood stream (Infante, J .R. et a1. (2013) “Safety, Pharmacokinetics And Pharmacocbinamics Of The 33 FlllL1’-Hlllllan Monoclonal Antibody, KRN330, In Patients With Advanced Colorectal Cancer,” Eur. J. Cancer. 49(6):1169-1175; Panjideh, H. et al. (2008) “Biodistribution And Efficacy 0f [1311]A33scFv::CDy, A Recombinant Antibody-Enzyme Protein For Colon Cancer,”lnt. J. Oncol. 32(4):925- 930). Conversely, only a few instances of non-gastrointestinal A33 antigen expression have been identified (Johnstone, C.N. et al. (2000) “Characterization 0f Mouse A33 Antigen, A Definitive Marker For Basolateral Surfaces Of inal Epithelial Cells,” Am. J. l. Gastrointest. Liver Physiol. 279(3):GSOO-GSIO).

In light of the highly restricted expression of the A33 antigen, researchers have explored the possibility of treating A33-associated s with antibodies (Infante, J .R. et al. (2013) “Safety, Pharmacokinetics And Pharmacodynamics Of The Anti-A33 Fully-Human Monoclonal Antibody, KRN330, In Patients With Advanced Colorectal Cancer,” Eur. J. Cancer. 49(6):] 169-1 175; Ackerman, M.E. et al. (2008) “A33 n Displays Persistent Surface Expression,’’ Cancer Immunol.

Immunother. 57(7):1017-1027;Barendswaard, E.C. et al. (2001) “Relative Therapeutic Eliicacy Of (125)1- And (I 3 I)I-Labeled onal Antibody A33 In A Human Colon Cancer Xenograft,” J. Nucl. Med. 42(8):1251-1256; Carrasquillo, J.A. et al. (2011) “('124)I-huA33 Antibody PET 0f ctal Cancer,” J. Nucl. Med. 52(8):]173-1180; Chong, G. et al. (2005) “Phase I Trial 0fl311—HuA33 In Patients With Advanced ctal Carcinoma,” Clin. Cancer Res. ll(13):4818-4826; Dcckcrt, P.M. et al. (2000) acokinetics And Microdistribution OfPolyethylene Glycol-Modified Humanizea’ A33 Antibody Targeting Colon Cancer Xenogra/ts,” Int.

J. Cancer. 87(3):382-390; Johnston, A.P. et al. (2012) “Targeting Cancer Cells: Controlling The Binding And Internalization 0fAntibodv-Functionalized Capsules” ACS Nano. 667-6674; Koppe, MJ. et al. (2005) "Radioimmunotherapy And Colorectal ,” Br. J. Surg. Mar;92(3):264-276; Sakamoto, J. et al. (2006) “A Phase I Radioimmunolocalization Trial OfHumanized Monoclonal Antibody HuA33 In ts With Gastric Carcinoma,” Cancer Sci. 97(11):l248-1254; Scott, A.M. et al. (2005) “A Phase I Trial Q/‘Humanized onal Antibody A33 In Patients With Colorectal Carcinoma: Biodistribution, Pharmacokinetics, And Quantitative Tumor Uptake,” Clin. Cancer Res. :4810-4817; Tschmelitsch, J. et al. (1997) “Enhanced Antitumor Activity 0f Combination Radioimmunotherapy (ml-Labeled Monoclonal Antibody A33) With Chemotherapy (Fluorouracil),”Canccr Res. 57(11):2181-2186). Likewise fragments of such antibodies have also been ted for their potential therapeutic role (Coelho, V. et al. (2007) “Design, Construction, And In Vitro Analysis Of A33scFv::CDy, A Recombinant Fusion Protein For Antibody—Directed Enzyme Prodrug y In Colon Cancer,” Int. J. Oncol. 31(4):951-957). 11. CD3 CD3 is a T cell co-reccptor composed of four distinct chains (Wucherpfennig, KW. et al. (2010) “Structural y Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation 0f Signaling,” Cold Spring Harb. Perspect. Biol. 2(4):a005140; pages 1-14; Chetty, R. et al. (1994) “CD3: Structure, Function And The Role Oflmmunostaining In Clinical Practice,” J. . 173:303-307).

In mammals, the CD3 complex contains a CD37 chain, a CD36 chain, and two CD38 chains. These chains associate with a molecule known as the T cell receptor (TCR) in order to generate an tion signal in T lymphocytes. In the absence of CD3, TCRS do not assemble properly and are degraded (Thomas, S. et al. (2010) “Molecular Immunology Lessons Franz eutic T-Cell Receptor Gene Transfer,” Immunology l29(2):l70—177). CD3 is found bound to the nes of all mature T cells, and in virtually no other cell type (see, Janeway, C.A. et al. (2005) In: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE,” 6th ed.

Garland Science Publishing, NY, pp. 214- 216; Sun, Z. J. et al. (2001) nisms Contributing To T Cell Receptor Signaling And Assembly ed By The Solution Structure Of An Ectodomain Fragment Of The CD3c:y Heterodimer,” Cell :9l3-923; Kuhns, M.S. et al. (2006) “Deconstructing The Form And Function OfThe TCR/CD3 Complex,” Immunity. 2006 Fcb;24(2): 133-139). 111. Bi-Specific Diabodies The ability of an intact, unmodified antibody (e.g., an IgG) to bind an epitope of an antigen s upon the presence of variable domains on the immunoglobulin light and heavy chains (i.e., the VL and VH domains, respectively). The design of a y is based on the single chain Fv construct (scFv) (see, e.g., Holligcr et al. (1993) “’Diabodies Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl.

Acad. Sci. (U.S.A.) 90:6444-6448; US Patent ation No. 2004/0058400 (Hollinger et al.); US 220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like ific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin- Like Growth Factor Receptor For Enhanced Antitumor Activity,” .1. Biol. Chem. 280(20):19665-19672; WO 02/0278] (Mertens et al.); n. T. et al. (2004) “Covalent Disul/ide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications,” Protein Eng. Des Sel. l7(l):21- 27; Wu, A. et al. (2001) merization OfA Chimeric Anti-CD20 Single Chain Fv- Fv Fusion Protein Is Mediated Through Variable Domain Exchange,” Protein Engineering 14(2):]025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion 0/ Human Fc Region,” Abstract 3P-683, J. m. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding system,” Protein Eng. l3(8):583-588; Baeuerle, RA. et al. (2009) “Bispecific T-Cell Engaging Antibodies For Cancer Therapy,” Cancer Res. 69(12):494l-4944).

Interaction of an antibody light chain and an antibody heavy chain and, in particular, interaction of its VL and VH domains forms one of the epitope binding sites of the antibody. In contrast, the scFv construct comprises a VL and VH Domain of an antibody contained in a single ptide chain wherein the domains are ted by a flexible linker of sufficient length to allow self-assembly of the two domains into a functional epitope binding site. Where self-assembly of the VL and VH domains is rendered impossible due to a linker of cient length (less than about 12 amino acid residues), two of the scFv constructs ct with one another other to form a bivalent molecule in which the VL of one chain associates with the VI-I of the other (reviewed in Marvin et al. (2005) “Recombinant Approaches To IgG- Like Bispecific Antibodies, " Acta Pharmacol. Sin. 262649-658). l dies are capable of binding to only one epitope species (i.e., mono-specific), although they can bind multiple copies of that species (i.e., exhibiting bi-valency or multi-valency). The art has noted the capability to produce diabodies that differ from such natural antibodies in being capable of binding two or more different epitope species (i.e., exhibiting bi-specifieity or multispecifieity in addition to bi-valency or multi-valency) (see, e.g., Holliger et al. (1993) “’Diabodies Small Bivalent And Bispecific Antibody Fragments,” Proc. Natl. Acad. Sci. (U.S.A.) 90:6444-6448; US 2004/0058400 (Hollinger et al.); US 2004/0220388 (Mertens et al.); Alt et al. (1999) FEBS Lett. 454(1-2):90-94; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like Bispeci/ic Antibody To Both The Epidermal Growth Factor Receptor And The n-Like Growth Factor Receptor For Enhanced Antitumor Activity,” J. Biol. Chem. 280(20):19665-19672; WO 02/02781 (Mertens et al.); Mcrtcns, N. et al., “New Recombinant Bi- and Trispecific Antibody Derivatives,” In: NOVEL FRONTIERS IN THE PRODUCTION OF COMPOUNDS FOR BIOMEDICAL USE, A.

VanBroekhoven et al. (Eds.), Kluwer Academic Publishers, Dordrecht, The Netherlands (2001), pages 195-208; Wu, A. et al. (2001) “Multimerization OfA Chimeric 020 Single Chain Fv-Fv Fusion n Is Mediated h le Domain Exchange,” Protein Engineering l4(2):1025-1033; Asano et al. (2004) “A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion OfHuman Fc Region,” ct , J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding ,” Protein Eng. 13(8):583-588; Bacuerlc, RA. et al. (2009) cific T-Cell Engaging Antibodies For Cancer Therapy,,3 Cancer Res. 69(12):4941-4944).

The provision of non-monospecific diabodies provides a significant advantage: the capacity to ate and co-localize cells that express different epitopes. Bivalent diabodies thus have anging applications including therapy and immunodiagnosis. Bi-valency allows for great flexibility in the design and engineering of the diabody in various applications, providing enhanced avidity to multimeric antigens, the cross-linking of differing antigens, and ed targeting to specific cell types relying on the presence of both target antigens. Due to their increased valency, low iation rates and rapid clearance from the circulation (for ies of small size, at or below ~50 kDa), diabody molecules known in the art have also shown particular use in the field of tumor g (Fitzgerald et al. (1997) “Improved Tumour Targeting By ltide Stabilized Diabodies Expressed In Pichia pastoris, " Protein Eng. 10:1221). Of particular importance is the co-ligating of differing cells, for example, the cross-linking of cytotoxic T cells to tumor cells (Staerz " et al. (1985) “Hybrid Antibodies Can Target Sites For Attack By T Cells, Nature 8-631, and er et a]. (1996) “Specific Killing 0fLymphoma Cells By xic T-Cells Mediated By A Bispecific y, " Protein Eng. 305).

Diabody epitope binding domains may also be ed to a surface determinant of any immune effector cell such as CD3, CD16, CD32, or CD64, which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells.

In many studies, diabody binding to effector cell determinants, e.g., Fey ors (FcyR), was also found to activate the effector cell (Holligcr et al. (1996) “Specific Killing 0fLymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody, " Protein Eng. 9:299-305; Holliger et a]. (1999) “Carcinoembljtonic Antigen (CEA)- Specific T-cell Activation In Colon Carcinoma Induced By Anti-CD3 X Anti-CEA Bispecific Diabodies And B7 X Anti-CEA Bispecific Fusion Proteins, " Cancer Res. 59:2909-2916; WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; ). Normally, effector cell activation is triggered by the binding of an antigen bound antibody to an effector cell via Fc-FcyR interaction; thus, in this regard, diabody les of the invention may exhibit Ig-like functionality independent of whether they comprise an Fc Domain (e.g., as assayed in any or function assay known in the art or exemplified herein (e.g., ADCC assay». By cross-linking tumor and effector cells, the diabody not only brings the effector cell within the proximity of the tumor cells but leads to effective tumor killing (see " e.g., Cao et a1. (2003) “Bispecific Antibody Conjugates In Therapeutics, Adv. Drug. Deliv. Rev. 55:171-197).

However, the above advantages come at salient cost. The formation of such non-monospecific ies requires the successful assembly of two or more distinct and different polypeptides (i.e., such ion requires that the diabodies be formed through the heterodimerization of different polypeptide chain species). This fact is in contrast to mono-specific diabodies, which are formed through the homodimerization of identical polypeptide chains. Because at least two dissimilar polypeptides (i. 6., two polypeptide species) must be provided in order to form a non-monospeeific diabody, and because homodimerization of such polypeptides leads to inactive molecules (Takemura, S. et al. (2000) “Construction Of A Diabody (Small inant Bispecific Antibody) Using A Refolding System,” Protein Eng. 13(8):583-588), the production of such polypeptides must be accomplished in such a way as to t covalent bonding between polypeptides of the same species (Takemura, S. et al. (2000) “Construction OfA Diabody (Small Recombinant Bispeczfic Antibody) Using A Refolding ,” Protein Eng. 13(8):583-588). The art has therefore taught the valent association of such polypeptides (see, e.g., Olafsen et al. (2004) “Covalent Disulfide-Linked Anti-CEA y Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications, " Prot. Engr. Des. Sel. 17:21-27; Asano et al. (2004) “A Diabody For Cancer lmmunotherapy And Its Functional ement By Fusion Of Human Fc Region,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S. et al. (2000) “Construction Of A Diabody (Small Recombinant Bispeci/ic Antibody) Using A Rejolding system,” Protein Eng. l3(8):583-588; Lu, D. et al. (2005) “A Fully Human Recombinant IgG-Like iflc Antibody To Both The Epidermal Growth Factor Receptor And The Insulin—Like Growth Factor Receptor For Enhanced Antitumor ty,” J. Biol. Chem. ): 19665-19672).

However, the art has recognized that bi-spccific monovalcnt diabodics composed of non-covalently-assoeiated polypeptides are unstable and readily dissociate into non-functional monomers (see, e.g., Lu, D. et al. (2005) “A Fully Human inant IgG-Like Bispeci/ic Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced mor ty,” J. Biol. Chem. 280(20):19665-19672).

In the face of this challenge, the art has succeeded in developing stable, covalently bonded heterodimeric non-monospecific diabodics (see, e.g., WO 2006/113665; WO/2008/157379; WO 2010/080538; WO 2012/018687; WO/2012/162068; Johnson, S. et al. (2010) “Effector Cell Recruitment With Novel Fv-Based finity Re-Targeting Protein Leads To Potent Tumor sis And In Vivo B-Cell Depletion,” J. Molcc. Biol. 399(3):436-449; Veri, MC. et al. (2010) peutic Control OfB Cell Activation Via Recruitment Ochgamma Receptor lIb (CD328) Inhibitory Function With A Novel Bispecific Antibody Scaffold,” Arthritis Rheum. 62(7):]933-1943; Moore, P.A. et al. (2011) “Application Of Dual Affinity Retargeting Molecules To e Optimal Redirected T-Cell Killing 0f B-Cell Lymphoma,” Blood ):4542-4551; US Patent Publications No. 2012/0294796 and 2013/0149236). Such approaches e engineering one or more cysteine es into each of the employed polypeptide species. For example, the addition of a cysteine residue to the C-terminus of such constructs has been shown to allow disulfide bonding between the polypeptide chains, stabilizing the resulting heterodimer t interfering with the binding characteristics of the bivalent molecule.

Diabodies and other immunoglobulins have been described purporting to have specificity for either or both of gpA33 and CD3 (see, e.g., US Patent Publications No. 2012/0014957; 2012/0034160; 2012/0087858; 2012/0189541; 2012/0195900; 2012/0201746; 2012/0237442; 2012/0263722; 258108; and 2012/0276608).

Notwithstanding such success, the production of stable, functional heterodimeric, non-monospccific diabodies can be further improved by the careful consideration and placement of the domains employed in the polypeptide chains. The present invention is thus directed to the ion of specific polypeptides that are particularly designed to form, via covalent bonding, heterodimeric diabodies and heterodimeric Fc diabodies that are capable of simultaneously binding gpA33 and CD3.

Summary of the Invention: The ion is directed to “gpA33 X CD3 bi-spccific monovalcnt diabodies.” In particular embodiments, the diabodies of the present invention further have a domain of an immunoglobulin Fc region (i.e., an “Fe Domain”) (“gpA33 X CD3 bi-specific monovalcnt Fc diabodies”) or an Albumin-Binding Domain (“ABD”) (“gpA33 X CD3 bi-specific monovalent diabodies with ABD”) to extend half-life in vivo. The gpA33 X CD3 bi-spccific monovalent diabodies of the invention and the gpA33 X CD3 bi-speeific monovalent Fc ies of the ion comprise two different polypeptide chains that associate with one another in a heterodimcrie manner to form one binding site specific for an e of gpA33 and one binding site c for an epitope of CD3. The gpA33 X CD3 bi-specific monovalent diabodies and gpA33 X CD3 bi-specific monovalent Fc ies of the invention are thus lent in that they are capable of binding to only one copy of an epitope of gpA33 and to only one copy of an epitope of CD3, but bi-specifie in that a single diabody is able to bind simultaneously to the e of gpA33 and to the epitope of CD3.

The gpA33 x CD3 bi-specific monovalent diabodies of the invention are ed of two ptide chains (a “first” and a “second” polypeptide chain), which are covalently bonded to one another, for example by disulfide bonding of cysteine residues located within each polypeptide chain. The gpA33 X CD3 bi- speeific monovalent Fc diabodies of the invention are composed of three polypeptide chains (a “first,” “second” and “third” polypeptide chain), wherein the first and second polypeptide chains are ntly bonded to one another and the first and third polypeptide chains are covalently bonded to one another. The bi-specifie monovalent diabodies and bi-speeific monovalent Fe diabodies of the present invention are capable of simultaneous binding to gpA33 and CD3. The invention is directed to such gpA33 X CD3 bi-specific monovalent ies and bi-specific monovalent gpA33 X CD3 Fe diabodies, and to pharmaceutical compositions that contain such bi- specific monovalent diabodies or such bi-specific monovalent Fe diabodies. The invention is additionally directed to methods for the use of such diabodies in the treatment of cancer and other diseases and conditions.

In , the invention provides a bi-specific monovalent diabody, wherein the bi-specific monovalent diabody is capable of specific binding to an epitope of gpA33 and to an epitope of CD3, wherein the bi-specific monovalent diabody comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains are covalently bonded to one another, and wherein: A. the first polypeptide chain comprises, in the N-terminal to inal direction: i. a Domain 1, comprising a sub-Domain (IA), which comprises a VL Domain of a monoclonal dy capable of binding to CD3 (VLcm) (SEQ ID NO:5); and a sub-Domain (18), which comprises a VH Domain of a monoclonal antibody capable of binding to gpA33 (VngAgg) (SEQ ID NO:27); wherein the mains (1A) and (18) are separated from one another by a peptide linker (SEQ ID NO:1); ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID NO:4) or an E-coil Domain (SEQ ID NO:3), wherein the Domain 2 is ted from the Domain 1 by a peptide linker (SEQ ID NO:2); B. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a sub-Domain (1A), which comprises a VL Domain of a onal dy capable of binding to gpA33 (VLgpAgg) (SEQ ID NO:26) and a sub-Domain (18), which comprises a VB Domain of a monoclonal antibody capable of binding to CD3 (VHcm) (SEQ ID , wherein the mains (1A) and (18) are separated from one another by a peptide linker (SEQ ID NO:1); ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID NO:3) or a K-coil Domain (SEQ ID NO:4), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:2); and wherein the Domain 2 of the first polypeptide chain and the Domain 2 of the second polypeptide chain are not both E-coil Domains or both K-coil Domains; and wherein: (a) the VL Domain of the first polypeptide chain and the VH Domain of the second polypeptide chain form an Antigen Binding Domain capable of specific binding to an epitope of CD3; and (b) the VH Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain form an Antigen Binding Domain capable of specific binding to an epitope of gpA33.

The invention additionally concerns the embodiment of the above-described bi-specific monovalent diabody wherein the first polypeptide chain or the second polypeptide chain comprises, an Albumin-Binding Domain (SEQ ID NO:34), linked C-terminally to Domain 2 or N-terminally to Domain 1A via a Linker 3 (SEQ ID NO:32).

The invention additionally concerns a bi-specific monovalent Fc y, wherein the bi-specific monovalent Fc diabody is e of specific binding to an epitope of gpA33 and to an epitope of CD3, and possesses an IgG Fc Domain, wherein the bi-specific monovalent Fc diabody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first and second polypeptide chains are covalently bonded to one another and the first and third ptide chains are covalently bonded to one another, and wherein: A. the first polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a main (1A), which ses a VL Domain of a monoclonal dy capable of binding to gpA33 (VLgpA33) (SEQ ID NO:26) and a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCD3) (SEQ ID NO:25), wherein the sub-Domains (1A) and (1B) are separated from one another by a peptide linker (SEQ ID NO:1); ii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID NO:3) or a K-coil Domain (SEQ ID NO:4), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:2); and iii. a Domain 3, comprising a sub-Domain (3A), which comprises a cysteine-containing e (Peptide 1) (SEQ ID NO:39) and a sub- Domain (3B), which comprises a polypeptide portion of an IgG Fc Domain having CH2 and CH3 s of an IgG immunoglobulin Fc Domain; wherein the Domains 3 and 2 are separated from one another by a spacer peptide (Linker 5) (GGG); B. the second polypeptide chain comprises, in the inal to C-terminal direction: a Domain 1, sing a sub-Domain (1A), which comprises a VL Domain of a monoclonal dy e of binding to CD3 (VLcm) (SEQ ID NO:5), and a sub-Domain (18), which comprises a VH Domain of a monoclonal antibody capable of binding to gpA33 (VngAgg) (SEQ ID N0:27); wherein the sub-Domains (1A) and (18) are separated from one r by a peptide linker (SEQ ID NO:1); ii. a Domain 2, wherein the Domain 2 is a K-eoil Domain (SEQ ID NO:4) or an E-eoil Domain (SEQ ID NO:3), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:2); and wherein the Domain 2 of the first polypeptide chain and the Domain 2 of the second polypeptide chain are not both E-eoil Domains or both K-coil Domains; and C. the third polypeptide chain comprises, in the N-tcrminal to C-terminal direction, a Domain 3 comprising: (1) a sub-Domain (3A), which comprises a cysteine-containing peptide (Peptide l) (SEQ ID NO:39); and (2) a sub-Domain (3B), which comprises a ptide portion of an IgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fe Domain; and wherein: (a) the polypeptide portions of the IgG Fc domains of the first and third polypeptide chain form the IgG Fc Domain; (b) the VL Domain of the first ptide chain and the VH Domain of the second polypeptide chain form an Antigen g Domain capable of c binding to an epitope of CD3; and (c) the VH Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain form an Antigen Binding Domain capable of specific binding to an epitope of gpA33.

The invention additionally concerns a bi-specifie monovalent Fe diabody, wherein the bi-specifie monovalent Fe diabody is capable of specific g to an epitope of gpA33 and to an epitope of CD3, and possesses an IgG Fe Domain, wherein the bi-specifie monovalent Fe diabody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first and second ptide chains are covalently bonded to one another and the first and third ptide chains are covalently bonded to one another, and wherein: A. the first polypeptide chain comprises, in the N-terminal to C-terminal ion: i. a Domain 3, comprising a sub-Domain (3A), which comprises a cysteine-containing e (Peptide 1) (SEQ ID NO:39) and a sub- Domain (3B), which ses a polypeptide portion of an lgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fe Domain; ii. a Domain 1, comprising a sub—Domain (1A), which ses a VL Domain of a monoclonal antibody capable of binding to gpA33 (VLgpAgg) (SEQ ID NO:26) and a sub-Domain (18), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHcm) (SEQ ID NO:25), wherein the sub-Domains (1A) and (18) are separated from one another by a peptide linker (SEQ ID NO:1); wherein the Domains 1 and 3 are separated from one another by a spacer peptide (Linker 4) (SEQ ID NO:38); iii. a Domain 2, wherein the Domain 2 is an E-coil Domain (SEQ ID NO:3) or a K-coil Domain (SEQ ID NO:4), n the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:2); and B. the second polypeptide chain comprises, in the N-tcrminal to C-tcrminal direction: i. a Domain 1, comprising a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD3 (VLcm) (SEQ ID NO:5); and a sub-Domain (18), which comprises a VB Domain of a monoclonal dy capable of binding to gpA33 (VHgIJAgg) (SEQ ID NO:27); wherein the sub-Domains (1A) and (1B) are separated from one another by a peptide linker (SEQ ID NO:1); ii. a Domain 2, wherein the Domain 2 is a K-coil Domain (SEQ ID NO:4) or an E-eoil Domain (SEQ ID NO:3), wherein the Domain 2 is separated from the Domain 1 by a peptide linker (SEQ ID NO:2); and wherein the Domain 2 of the first polypeptide chain and the Domain 2 of the second polypeptide chain are not both E-coil Domains or both K-coil Domains; and C. the third polypeptide chain comprises, in the N-terminal to C-terminal direction, a Domain 3 comprising: (1) a main (3A), which comprises a cysteine-containing peptide (Peptide 1) (SEQ ID NO:39); and (2) a sub-Domain (3B), which comprises a polypeptide portion of an IgG Fe Domain having CH2 and CH3 domains of an IgG immunoglobulin Fe Domain; and wherein: (a) the ptide portions of the IgG Fe domains of the first and third ptide chain form the IgG Fc Domain; (b) the VL Domain of the first polypeptide chain and the VH Domain of the second polypeptide chain form an Antigen Binding Domain capable of specific binding to an epitope of CD3; and (c) the VB Domain of the first polypeptide chain and the VL Domain of the second polypeptide chain form an Antigen Binding Domain capable of c binding to an e of gpA33.

The invention further concerns the embodiments of any of the above- described bi-specific monovalent Fc diabodies wherein the sub-Domain (3B) of the first polypeptide chain comprises a sequence different from that of the sub-Domain (3B) ofthe third ptide chain.

The ion further concerns the embodiments of such above-described bi— specific monovalent Fe diabodies wherein the main (3B) of the first polypeptide chain has the amino acid sequence of SEQ ID NO:40, and the sub- Domain (3B) of the third polypeptide chain has the amino acid sequence of SEQ ID NO:41.

The invention further concerns the embodiments of such above-described bi- specific monovalent Fc diabodies wherein the sub-Domain (3B) of the first polypeptide chain has the amino acid ce of SEQ ID NO:41, and the sub- Domain (3B) of the third polypeptide chain has the amino acid sequence of SEQ ID NO:40.

The invention further concerns the ments of such described bi— specific monovalent Fe diabodies wherein the Domain 3 of the first polypeptide chain and/or the Domain 3 of the third polypeptide chain comprises a variant CH2-CH3 sequence that exhibits altered binding to an Fey receptor.

The invention r concerns the embodiments of any of the abovedescribed bi-speeific monovalent diabodies or of any of the described bi- specific monovalent Fc ies, wherein the Domain 2 of the first ptide chain comprises an E-coil (SEQ ID NO:3), and the Domain 2 of the second polypeptide chain comprises a K-coil (SEQ ID NO:4).

The invention further concerns the embodiments of any of the abovedescribed bi-specific monovalent diabodies or of any of the above-described bi- speeific monovalent Fe diabodies, wherein the Domain 2 of the first polypeptide chain comprises a K-coil (SEQ ID NO:4), and the Domain 2 of the second ptide chain comprises an E-eoil (SEQ ID NO:3).

The invention further concerns a bi-specific monovalent diabody, wherein the bi-specific monovalent diabody is capable of specific binding to an epitope of CD3 and to an epitope of gpA33, wherein the bi-spccific monovalent diabody comprises: (1) a first polypeptide chain having the amino acid sequence of SEQ ID NO:28, and a second polypeptide chain having the amino acid sequence of SEQ ID NO:30; or (2) a first ptide chain having the amino acid sequence of SEQ ID NO:35, and a second polypeptide chain having the amino acid sequence of SEQ ID NO:30; wherein the first and the second polypeptide chains are covalently bonded to one another by a disulfide bond. —l6- The invention further concerns a cific monovalent Fe diabody, wherein the bi-specific monovalent Fe diabody is capable of specific binding to an c of CD3 and to an e of gpA33, and possesses an IgG Fc Domain, wherein the bi- specific monovalent Fc diabody comprises: (1) a first ptide chain having the amino acid sequence of SEQ ID NO:42, a second polypeptide chain having the amino acid sequence of SEQ ID NO:44, and a third polypeptide chain having the amino acid sequence of SEQ ID NO:46; or (2) a first polypeptide chain having the amino acid sequence of SEQ ID NO:48, a second polypeptide chain having the amino acid sequence of SEQ ID NO:28, and a third polypeptide chain having the amino acid sequence of SEQ ID NO:46; wherein the first and the second polypeptide chains are covalently bonded to one another by a first disulfide bond and the first and third polypeptide chains are covalently bonded to one another by a second disulfide bond.

The invention r concerns a pharmaceutical composition comprising any of the above-described bi-specifie monovalent diabodies or any of the above- deseribed bi-speeific monovalent Fe ies; and a physiologically acceptable carrier.

The invention r concerns the use of the above-described pharmaceutical composition in the treatment of a cancer characterized by the expression of gpA33, and especially such use wherein the cancer is colorectal cancer, colon cancer, gastric cancer or pancreatic cancer.

The invention further concerns a cell that expresses a polypeptide chain of any of the above-described cific monovalent diabodies or any of the above- described bi-specific monovalent Fe ies, as well as a polynueleotide that encodes such expressed polypeptide.

The ion further concerns a cell that expresses an antibody or a polypeptide portion or fragment thereof, wherein the antibody binds to gpA33, and wherein the antibody or polypeptide portion or fragment thereof comprises: (1) CDRl (SEQ ID NO: 14), CDR2 (SEQ ID NO:15) and CDR3 (SEQ ID NO:16) ofa light chain of an anti-human gpA33 antibody; (2) CDRl (SEQ ID NO:18), CDR2 (SEQ ID NO:19) and CDR3 (SEQ ID NO:20) of a heavy chain of an anti—human gpA33 antibody; or (3) both (1) and (2).

Brief Description of the Drawings: Figure 1 rates the structures of the first and second polypeptide chains of a two chain gpA33 X CD3 bi-specific monovalent diabody of the present invention.

Figures 2A and 28 illustrate the structures of two versions of the first, second and third polypeptide chains of a three chain gpA33 x CD3 cific monovalent Fe diabody of the present invention (Version 1, Figure 2A; Version 2, Figure ZB).

Figure 3 demonstrates that the diabodies of the present invention are capable of simultaneously binding to CD3 and to gpA33.

Figure 4 illustrates the ability of the diabodies of the present invention to treat cancer. Colorectal or pancreatic cancer cells were ted in the presence of the gpA33 X CD3 bi-specific monovalent diabody (“DART-l) and either human PBMC (E:T = 25:1) or activated human T cells (E:T = 10:1), and cytotoxicity was measured (Figure 4A (Colon CSCL colorectal cells), Figure 4B 05 colorectal , and Figure 4C (ASPC-l pancreatic cancer cells).

Figures SA-SF show that activation of CD8 T cells occurred in the presence of the CD3 cifie monovalent diabody (“DART-l) only in the presence of cancer cells (Figures 5A-5C: CD8 T cells + c010205 cells (Figure 5A), CD8 T cells + ASPC-l cells (Figure SB), CD8 T cells alone (Figure 5C); Figures 5D-5F: CD4 T cells + c010205 cells (Figure 5D), CD4 T cells + ASPC-l cells (Figure 5E), CD8 T cells alone (Figure SF).

Figures 6A-6D demonstrate that gpA33 X CD3 bi-specific monovalent diabodies (DART-1 and DART-2) mediated equivalent cytotoxieity for SW948 —l8- colorectal adenocarcinoma cells (Figure 6A) and 5 cells (Figure 6B) and ColoZOS-Luc cells (Figure 6C), and that neither diabody mcdiatcd cytotoxicity of thc gpA33-negative cancer cell line, HCTl 16 (Figure 6D).

Figures 7A-7D demonstrate the ability of the gpA33 x CD3 bi-specific monovalent diabody (DART-2), the gpA33 x CD3 bi-specific monovalent diabody having an Albumin-Binding Domain (DART-2 with ABD “w/ABD”) and the gpA33 X CD3 bi-specific monovalent diabody having an globulin IgG Fc Domain 2 with Fe “w/Fc”) to promote the cytotoxicity of cancer cells in the presence of human or cynomolgus monkey PBMCs.

Figure 8 demonstrates the in vivo ability of the gpA33 X CD3 bi-specific monovalent diabody (DART-1) to decrease tumor volume in a murine ColoZOS colon cancer model.

Figures 9A-9D shows tumor imaging data ofNOD scid gamma (NSG) micc ted with Col0205 cells two days afier receiving Vehicle e 9A) or the gpA33 X CD3 bi—specific monovalent diabody (DART—1) (Figure 9B), and 12 days after receiving Vehicle e 9C) or the DART-1 (Figure 9D).

Figure 10 demonstrates the in vivo y of the gpA33 X CD3 bi-specific monovalent diabody (DART-1) to decrease tumor volume in a murine ASPC-l pancreatic cancer model.

Figure 11 shows the ability of the gpA33 X CD3 bi-specific monovalent diabody having an immunoglobulin IgG Fc Domain (DART-2 w/Fc Version I) to mcdiatc a dramatic reduction in tumor volumc in an in vivo colon cancer model.

Figure 12 shows the ability of the gpA33 X CD3 bi-specific monovalent diabody having an immunoglobulin IgG Fc Domain (DART-2 w/Fc Version 1) to e a reduction in tumor volume in an in viva colon cancer model even at extremely low doses.

Figure 13 shows the pharmacokinetics of the gpA33 X CD3 bi-specific monovalent diabody (DART-2), and gpA33 x CD3 bi-specific monovalent diabody having an immunoglobulin IgG Fe Domain (DART-2 w/Fc Version 1) diabodies in cynomolgus monkeys.

Figures 14A-14B show SPR analysis of the binding of DART-2 w/Fc Version 1 to immobilized human and cynomolgus monkey CD3. The black dashed lines represent the global fit to a 1:1 Langmuir model of binding curves obtained at DART-2 w/Fc concentrations of 0, 6.25, 12.5, 25, 50 or 100 nM. The data are representative of three independent experiments.

Figures ISA-153 show SPR analysis of the binding of DART-2 w/Fc Version 1 to captured human and lgus monkey gpA33. The black dashed lines represent the global fit to a 1:1 ir model of binding curves obtained at DART-2 w/Fe Version 1 concentration of 0, 6.25, 12.5, 25, 50 or 100 nM. The data are representative of three independent experiments.

Detailed Description of the ion: The present invention is directed to cific monovalent diabodies that comprise two ptide chains and which possess one binding site c for an epitope of gpA33 and one binding site specific for an epitope of CD3 (i.e., a “gpA33 x CD3 bi-specific monovalent diabody”). The present invention also is directed to bi- spccific monovalent diabodies that comprise an immunoglobulin Fc Domain (“bi- specific monovalent Fe diabodies”) and are ed of three polypeptide chains and which possess one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3 (126., a “gpA33 x CD3 bi-specific monovalent Fe diabody”). The bi-specific monovalent diabodies and cific monovalent Fe diabodies the present invention are capable of simultaneous binding to gpA33 and CD3. The invention is directed to pharmaceutical compositions that contain such bi- c monovalent diabodies or such bi-specific monovalent Fc diabodies. The ion is additionally directed to methods for the use of such diabodies in the treatment of cancer and other diseases and conditions.

The gpA33 X CD3 bi-specific monovalent diabodies of the present invention are composed of two ptide chains that ate with one another to form one binding site specific for an epitope of gpA33 and one binding site specific for an epitope of CD3. The individual polypeptide chains of the diabody are covalently bonded to one another, for example by disulfide bonding of cysteine residues located within each polypeptide chain. Each polypeptide chain contains an Antigen Binding Domain of a Light Chain le Domain, an Antigen g Domain of a Heavy Chain Variable Domain and a heterodimerization Domain. An intervening linker peptide (Linker l) separates the Antigen Binding Domain of the Light Chain Variable Domain from the Antigen Binding Domain of the Heavy Chain Variable Domain.

The Antigen Binding Domain of the Light Chain Variable Domain of the first polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a first functional antigen binding site that is specific for the first antigen (i.e., either gpA33 or CD3). se, the Antigen Binding Domain of the Light Chain Variable Domain of the second polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the first polypeptide chain in order to form a second onal antigen binding site that is specific for the second n (i.e., either gpA33 or CD3, depending upon the identity of the first antigen). Thus, the selection of the Antigen Binding Domain of the Light Chain le Domain and the n Binding Domain of the Heavy Chain Variable Domain of the first and second polypeptide chains are coordinated, such that the two ptide chains collectively comprise Antigen g Domains of Light and Heavy Chain le Domains capable of binding to gpA33 and CD3.

The gpA33 X CD3 bi-specific monovalcnt Fe diabodics of the present invention are composed of a first polypeptide chain, a second polypeptide chain and a third polypeptide chain. The first and second polypeptide chains associate with one another to form one binding site c for an epitope of gpA33 and one binding site specific for an epitope of CD3. The first polypeptide chain and the third ptide chain associate with one another to form an immunoglobulin Fe Domain. The first and second polypeptide chains of the bi-speeific monovalcnt Fc y are covalently bonded to one another, for example by disulfide bonding of cysteine residues located within each polypeptide chain. The first and third polypeptide chains are covalently bonded to one another, for example by disulfide bonding of cysteine residues located within each polypeptide chain. The first and second polypeptide .21. chains each n an Antigen Binding Domain of a Light Chain Variable Domain, an Antigen Binding Domain of a Heavy Chain Variable Domain and a dimerization Domain. An ening linker peptide (Linker 1) separates the Antigen Binding Domain of the Light Chain Variable Domain from the Antigen Binding Domain of the Heavy Chain Variable Domain. The Antigen Binding Domain of the Light Chain Variable Domain of the first polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the second polypeptide chain in order to form a first functional antigen binding site that is specific for the first antigen (i.e., either gpA33 or CD3). Likewise, the Antigen Binding Domain of the Light Chain Variable Domain of the second polypeptide chain interacts with the Antigen Binding Domain of the Heavy Chain Variable Domain of the first polypeptide chain in order to form a second functional n binding site that is c for the second antigen (i.e., cithcr gpA33 or CD3, depending upon the identity of the first antigen). Thus, the selection of the Antigen Binding Domain of the Light Chain Variable Domain and the n Binding Domain of the Heavy Chain le Domain of the first and second polypeptide chains are coordinated, such that the two polypeptide chains collectively comprise Antigen Binding Domains of light and Heavy Chain Variable Domains capable of binding to gpA33 and CD3.

The first and third polypeptide chains each contain a cysteine-containing peptide (Peptide 1) SEQ ID NO:39: and some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete immunoglobulin Fc Domain and a cysteine- containing peptide. The some or all of the CH2 Domain and/or the some or all of the CH3 Domain ate to form the immunoglobulin Fc Domain of the bi-specific monovalent Fc ies of the present invention. The first and third polypeptide chains of the bi-specific monovalent Fc diabodies of the present invention are covalently bonded to one another, for example by disulfide bonding of cysteine residues located within the cysteine-containing e of the polypeptide chains.

The formation of heterodimers of the first and second polypeptide chains of the bi-spccific monovalent diabody or bi-specific monovalent Fc diabody can be driven by the heterodimerization domains. Such s include GVEPKSC (SEQ ID NO:54) (or VE PKSC; SEQ ID NO:55) on one polypeptide chain and GFNRGEC (SEQ ID NO:56) (or ; SEQ ID NO:57) on the other polypeptide chain (U82007/0004909). Alternatively, such domains can be engineered to n coils of opposing charges. The heterodimerization Domain of one of the polypeptide chains comprises a sequence of at least six, at least seven or at least eight positively charged amino acids, and the dimerization Domain of the other polypeptide chain comprises a sequence of at least six, at least seven or at least eight negatively charged amino acids. For example, the first or the second heterodimerization Domain may comprise a sequence comprising eight vely charged amino acids and the other of the heterodimerization domains may comprise a sequence comprising eight negatively charged amino acids. The positively charged amino acid may be lysine, arginine, histidine, etc. and/or the vely charged amino acid may be glutamic acid, aspartic acid, etc. The positively charged amino acid is preferably lysine and/or the negatively d amino acid is preferably glutamic acid.

The bi-specific monovalent diabodics and bi-specific monovalent Fc diabodics of the present invention are engineered so that such first and second polypeptide chains covalently bond to one another via cysteine residues along their . Such cysteine residues may be introduced into the intervening linker that separates the VL and VB domains of the polypeptides. Alternatively, and more ably, a second peptide (Linker 2) is introduced into each polypeptide chain, for example, at the amino-terminus of the polypeptide chains or at a position that places Linker 2 n the heterodimerization Domain and the n Binding Domain of the Light Chain Variable Domain or Heavy Chain Variable Domain.

As indicated above, gpA33 is expressed by colorectal cells. Antibodies capable of immunospccifically binding to gpA33 are capablc of binding to such cells.

CD3 is expressed on T cells. Thus, antibodies capable of immunospecifieally binding to both gpA33 and CD3 are capable of targeting T cells to colorectal and other cancer cells that express gpA33 (e.g., colon carcinoma cells, pancreatic cancer cells, etc.) and of thus providing an improved therapy for such cancers.

]. Preferred gpA33 x CD3 Bi-Specific Monovalent Diabodies of the Present Invention A. gpA33 x CD3 Bi-Specifie Monovalent Diabodies One ment of the present invention s to gpA33 X CD3 bi-speeific lent ics that are composed of a first polypeptide chain and a second polypeptide chain, whose sequences permit the ptide chains to covalently bind to each other to form a covalently-associated complex that is capable of simultaneously binding to both gpA33 and CD3.

The first polypeptide chain of preferred gpA33 x CD3 bi-specific lent diabodies comprise, in the N-terminal to C-tenninal direction, an N— terminus, the VL Domain of a monoclonal dy capable of binding to either CD3 or gpA33 (i.e., either VLcm or VLgpAgg), a first intervening spacer peptide (Linker 1), a VH Domain of a monoclonal antibody capable of binding to either gpA33 (if such first polypeptide chain contains VLcm) or CD3 (if such first polypeptide chain contains VLgpkzg), a cysteine-containing second intervening spacer peptide (Linker 2), a heterodimer—promoting Domain and a C-terminus (Figure 1).

The second ptide chain of preferred gpA33 X CD3 bi-specific monovalent diabodies comprises, in the N-terminal to C-terminal direction, an N- teiminus, a VL Domain of a monoclonal dy capable of binding to either gpA33 or CD3 (i.e., either VLgpA33 or VLcm, depending upon the VL Domain selected for the first polypeptide chain of the diabody), an intervening linker peptide (Linker 1), a VI-I Domain of a monoclonal antibody capable of g to either CD3 (if such second polypeptide chain contains VLgpA33) or CD3 (if such second polypeptide chain contains VLCDg), a cysteine-containing spacer peptide (Linker 2), a heterodimer- promoting Domain, and a C-terminus (Figure 1).

The VL Domain of the first polypeptide chain of preferred gpA33 X CD3 bi- speeifie lent diabodics interacts with the VH Domain of the second polypeptide chain of preferred gpA33 X CD3 bi-specific monovalent diabodies in order to form a first functional antigen binding site that is specific for a first antigen (i.e., either CD3 or . Likewise, the VL Domain of the second polypeptide chain cts with the VH Domain of the first polypeptide chain in order to form a second functional antigen binding site that is specific for a second antigen (i.e., either gpA33 or CD3, depending upon the identity of the first antigen). Thus, the selection of the VL and VH s of the first and second polypeptide chains are nated, such that the two polypeptide chains of preferred gpA33 x CD3 bi-specific monovalent diabodies tively comprise VL and VH domains capable of binding to gpA33 and CD3 (i.e., they comprise VLcm/VHcm and VLgpAgg/VngAgg).

Most preferably, the length of the intervening linker peptide (Linker l, which separates such VL and VB domains) is selected to substantially or completely t the VL and VH domains of the polypeptide chain from binding to one another. Thus the VL and VH domains of the first polypeptide chain are substantially or completely incapable of binding to one r. Likewise, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to one another.

A preferred intervening spacer e (Linker 1) has the ce (SEQ ID N021): GGGSGGGG.

The cysteine-containing second intervening spacer peptide (Linker 2) will contain 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide r 2) has the sequence is SEQ ID NO:2: GGCGGG.

The heterodimer-promoting domains of the first and second polypeptides differ from one another and are designed to associate with one another so as to promote ation of the first and second polypeptide chains. Thus, in a preferred embodiment, one of these polypeptide chains will be engineered to contain a heterodimer—promoting “E-eoil” Domain (SEQ ID NO:3): EVAALEKEVAALEKEVAALEKEVAALEK whose residues will form a negative charge at pH 7, while the other of the two polypeptide chains will be engineered to contain a heterodimer-promoting “K-coil” Domain (SEQ ID NO:4): EVAALEEEVAALEEEVAALEEEVAALEE whose residues will form a positive charge at pH 7. The presence of such charged domains promotes association between the first and second polypeptides, and thus fosters heterodimerization. It is rial which coil is provided to which chain, as long as the coils ed on the first and second polypeptide chains differ so as to foster heterodimerization between such chains. 1. The gpA33 x CD3 Bi-Specific Monovalent Diabody, “DART-1” The first and second polypeptide chains of a preferred gpA33 X CD3 bi- specific monovalent diabody, ated herein as “DART-1” comprise polypeptide domains having the following sequences: The VL Domain of an antibody that binds CD3 (VLcm) (SEQ ID NO:5): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK RAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG The Antigen Binding Domain of VLCD3 comprises CDRl having the sequence: (SEQ ID NO:6) RSSTGAVTTSNYAN; CDR2 having the sequence (SEQ ID N0:7): GTNKRAP; and CDR3 having the ce (SEQ ID N0:8): ALWYSNLWV.

The VH Domain of an dy that binds CD3 (VHCD3) (SEQ ID N029): EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKY NNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVS WFAYWGQGTLVTVSS The Antigen Binding Domain of VHcm comprises: CDRl having the sequence (SEQ ID N0:l0): TYAMN; CDRZ having the sequence (SEQ ID N0:ll) RIRSKYNNYATYYADSVKD; and CDR3 having the ce (SEQ ID NO:12): HGNFGNSYVSWFAY.

The VL Domain of a murine antibody that binds gpA33 (VLgpAgg) (SEQ ID N0:l3): QIVLTQSPAIMSASPGERVTMTCSARSSISFMYWYQQKPGSSPRLLIYDTSNLAS GVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPLTFGSGTKLELK The Antigen Binding Domain of g comprises CDRl having the sequence (SEQ ID NO:14): SARSSISFMY; CDR2 having the sequence (SEQ ID NO:15): DTSNLAS; and CDR3 having the sequence (SEQ ID NO:16): QQWSSYPLT.

The VH Domain of a murine antibody that binds gpA33 (VngA33) (SEQ ID QVQLQQSGPELVKPGASVKISCKASGYTFSGSWMNWVKQRPGQGLEWIGRIYPGD GETNYNGKFKDKATLTADKSSTTAYMELSSLTSVDSAVYFCARIYGNNVYFDVWG AGTTVTVSS The Antigen Binding Domain of VngA33 comprises CDRl having the sequence (SEQ ID NO:18): GSWMN; CDR2 having the sequence (SEQ ID NO:19): RIYPGDGETNYNGKFKD; and CDR3 having the ce (SEQ ID NO:20): IYGNNVYFDV.

The first intervening spacer peptide (Linker ]) has the sequence (SEQ ID NO:1): GGGSGGGG. The cysteine-containing spacer peptide (Linker 2) has the sequence is SEQ ID NO:2: GGCGGG.

The heterodimer-promoting Domain of the first polypeptide chain is the “E- coil” Domain (SEQ ID NO:3). The heterodimer-promoting Domain of the second polypeptide chain is the “K-coil” Domain (SEQ ID NO:4).

Thus, the first polypeptide chain of DART-1 has the sequence (SEQ ID NO:21): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK RAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG GGGSGGGGQVQLQQSGPELVKPGASVKISCKASGYTFSGSWMNWVKQRPGQGLEW IGRIYPGDGETNYNGKFKDKATLTADKSSTTAYMELSSLTSVDSAVYFCARIYGN NVYFDVWGAGTTVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK As will be appreciated, residues 1-110 of SEQ ID NO:21 are the VL Domain of an antibody that binds CD3 (VLcm) (SEQ ID NO:5); residues 111-118 of SEQ ID NO:21 are the first intervening spacer e (Linker l) (SEQ ID NO:1); residues 7 of SEQ ID NO:21 are the VH Domain of a murine dy that binds gpA33 (VngA33) (SEQ ID NO:17), residues 238-243 of SEQ ID NO:21 are the ne-containing spacer peptide (Linker 2’) (SEQ ID NO:2) and residues 244- 271 of SEQ ID NO:21 are the heterodimer-promoting “E-coil” Domain (SEQ ID N0:3).

A preferred polynueleotide that s the first polypeptide chain of DART-l has the sequence (SEQ ID NO:22): caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtga ccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattg ggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaa agggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccg ctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctct gtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctggga ggtggtggatccggcggaggtggacaggtccagctgcagcagtctggacctgagc tggtgaagcctggggcctcagtgaagatttcctgcaaagcttcaggctacacatt cagtggctcttggatgaactgggtgaagcagaggcctggacagggtcttgagtgg attggacggatctaccctggagatggagaaactaactacaatgggaagtttaagg acaaggccacactgactgcagacaaatcatccaccacagcctacatggagctcag gacctctgtggactctgcggtctatttctgtgcaagaatctatggtaat tacttcgatgtctggggcgcagggaccacggtcaccgtgtcttccggag gatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaa ggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaa The second polypeptide chain of DART-1 has the sequence (SEQ ID NO:23): QIVLTQS S PGERVTMTCSARS S I SFMYWYQQKPGSSPRLLIYDTSNLAS GVPVRFSGSGSGTSYSLT I SRMEAEDAATYYCQQWSSY PLTFGSGTKLELKRGGG SGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR IRSKYNNYATYYADSVKDRFT I SRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFG NSYVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE As will be appreciated, residues 1-107 of SEQ ID NO:23 are the VL Domain of a murine antibody that binds gpA33 (VLgPAgg) (SEQ ID NO:13); es 108-115 of SEQ ID NO:23 are the first intervening spacer peptide (Linker l) (SEQ ID NO:1); residues 116-240 of SEQ ID NO:23 are the VB Domain of an antibody that binds CD3 (VHCD3) (SEQ ID NO:9), residues 241-246 of SEQ ID NO:23 are the cysteine-containing spacer peptide (Linker 2) (SEQ ID NO:2) and residues 247- 274 of SEQ ID NO:23 are the heterodimer-promoting “K-coil” Domain (SEQ ID NO:4).

A preferred polynucleotide that s the second polypeptide chain of DART-1 has the ce (SEQ ID NO:24): caaattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaggg tcaccatgacctgcagtgccaggtcaagtataagtttcatgtactggtaccagca gaagccaggatcctcccccagactcctgatttatgacacatccaacctggcttct ggagtccctgttcgcttcagtggcagtgggtctgggacctcttattctctcacaa tcagccgaatggaggctgaagatgctgccacttattactgccagcagtggagtag ttacccactcacgttcggttctgggaccaagctggagctgaaacggggtggagga tccggcgqagchgagaggtgcagctggtggagtctqggggaqgcttggtccagc ctggagggtccctgagactctcctgtgcagcctctggattcaccttcaacacata gaattgggtccgccaggctccagggaaggggctggagtgggttgcaagg atcaggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggata gattcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacag cctgaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggc aattcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgt cttccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgc tttgaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag 2. The gpA33 x CD3 Bi-Specific Monovalent Diabody, “DARTJ” The first and second polypeptide chains of a second preferred gpA33 x CD3 bi-specific monovalent diabody, designated herein as “DART-2,” comprise polypeptide domains having the following sequences: The VL Domain of an antibody that binds CD3 (VLCD3') (SEQ ID N0:5): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK RAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG The Antigen Binding Domain of VLcDg comprises CDR] having the sequence: (SEQ ID NO:6) RSSTGAVTTSNYAN; CDR2 having the sequence (SEQ ID NO:7): GTNKRAP; and CDR3 having the sequence (SEQ ID NO:8'): ALWYSNLWV The VH Domain of an antibody that binds CD3 (VI-1cm) (SEQ ID : EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRIRSKY NNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVS WFAYWGQGTLVTVSS The Antigen Binding Domain of VHcm comprises CDRl having the ce (SEQ ID NO:10): TYAMN; CDR2 having the sequence (SEQ ID NO:11): RIRSKYNNYATYYADSVKD; and CDR3 having the sequence:(SEQ ID NO:12) HGNFGNSYVSWFAY.

The above-discussed murine antibody that binds to human gpA33 was humanized to provide the VL and VH domains of preferred diabody DART-2. These humanized s are as follows: The VL Domain of a humanized antibody that binds gpA33 (VLgpAgg) (SEQ ID N0:26): DIQLTQSPSFLSASVGDRVTITCSARSSISFMYWYQQKPGKAPKLLIYDTSNLAS SGSGSGTEFTLTISSLEAEDAATYYCQQWSSYPLTFGQGTKLEIK The Antigen Binding Domain of VLgpAzg comprises CDRl having the sequence (SEQ ID NO:14): SARSSISFMY; CDR2 having the sequence (SEQ ID NO:15): DTSNLAS; and CDR3 having the sequence (SEQ ID NO:16): QQWSSYPLT.

The VH Domain of a zcd antibody that binds gpA33 ) (SEQ ID NO:27): QVQLVQSGAEVKKPGASVKVSCKASGYTFTGSWMNWVRQAPGQGLEWIGRIYPGD GKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARIYGNNVYFDVWG QGTTVTVSS The Antigen Binding Domain of VngA33 Comprises CDRl having the sequence (SEQ ID NO:18): GSWMN; CDR2 having the sequence (SEQ ID NO:19): RIYPGDGETNYNGKFKD; and CDR3 having the sequence (SEQ ID NO:20): IYGNNVYFDV.

Thc first intervening spaccr pcptidc (Linkcr 1) has thc scqucncc (SEQ ID N0:l): GGGSGGGG. The cysteine-containing spacer peptide (Linker 2) has the sequence is SEQ ID NO:2: GGCGGG.

The heterodimer-promoting Domain of the first polypeptide chain is the “E- coil” Domain (SEQ ID NO:3). The heterodimer-promoting Domain of the second polypeptide chain is the “K-coil” Domain (SEQ ID NO:4).

Thus, the first polypeptide chain of DART-2 has the sequence (SEQ ID NO:28): QAVVTQE PSLTVS PGGTVTLTCRS STGAVTTSNYANWVQQKPGQAPRGLI GGTNK RAPWT PARFSGSLLGGKAALT I TGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG GGQVQLVQSGAEVKKPGASVKVSCKASGYTFTGSWMNWVRQAPGQGLEW I GRIYPGDGETNYNGKFKDRVT I TADKSTSTAYMELSSLRSEDTAVYYCARIYGN NVYFDVWGQGTTVTVS SGGCGGGEVAALEKEVAALEKEVAALEKEVAALEK As will be appreciated, residues 1-110 of SEQ ID NO:28 are the VL Domain of an antibody that binds CD3 (VLcm) (SEQ ID NO:5); rcsiducs 111-118 of SEQ ID NO:28 are the first intervening spacer peptide (Linker l) (SEQ ID NO:1); residues 119-237 of SEQ ID NO:28 are the VB Domain of an antibody that binds gpA33 (VngA33) (SEQ ID NO:27), residues 238-243 of SEQ ID NO:28 are the cysteine-containing spacer peptide r 2) (SEQ ID NO:2) and residues 244-271 of SEQ ID NO:28 are the heterodimer-promoting “E-coil” Domain (SEQ ID NO:3).

A preferred polynucleotide that encodes the first polypeptide chain of DART-2 has the cc (SEQ ID NO:29): caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtga ccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattg ggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaa agggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccg ctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctct gtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctggga ggtggtggatccggcggaggtggacaggtccagctggtccagagcggggccgaag tcaaaaaacccggagcaagcgtgaaggtctcctgcaaagcatcaggctatacatt tacaggcagctggatgaactgggtgaggcaggctccaggacagggactggagtgg atcgggcgcatctaccctggagacggcgaaactaactataatggaaagttcaaag tgaccatcacagccgataagtctactagtaccgcctacatggagctgag ctccctgcggtctgaagataccgccgtctactattgcgctagaatttacggaaac aatgtctattttgacgtgtgggggcagggaacaactgtgactgtctcctccggag gcggtggagaagtggccgcactggagaaagaggttgctgctttggagaa ggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaa The second ptide chain of DART-2 has the sequence (SEQ ID NO:30): DI QLTQS PS FL SASVGDRVTI TCSARS S I SFMYWYQQKPGKAPKLLI YDTSNLAS GVPSRFSGSGSGTEFTLT I SSLEAEDAATYYCQQWSSY PLTFGQGTKLE I KGGGS GGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRI RSKYNNYATYYADSVKDRFT I SRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGN S YVSWFAYWGQGTLVTVS SGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE As will be appreciated, residues 1-106 of SEQ ID NO:30 are the VL Domain of an antibody that binds gpA33 Q (SEQ ID NO:26'); residues 107- 114 of SEQ ID NO:30 are the first intervening spacer peptide (Linker I) (SEQ ID NO:1); residues 115-239 of SEQ ID NO:30 are the VB Domain of an antibody that binds CD3 (VHcm) (SEQ ID NO:25), residues 240-245 of SEQ ID NO:30 are the cysteine-containing spacer peptide (Linker 2) (SEQ ID NO:2) and residues 246-273 of SEQ ID NO:30 are the heterodimer-promoting “K-coil” Domain (SEQ ID NO:4).

A preferred polynucleotide that encodes the second ptide chain of DART-2 has the scqucncc (SEQ ID NO:31): gacattcagctgactcagtccccctcttttctgtccgcatccgtcggagatcgag tgactattacttgctctgctaggtcctcaatcagcttcatgtactggtatcagca gaagcccggcaaagcacctaagctgctgatctacgacacaagcaacctggcctcc ggggtgccatctcggttctctggcagtgggtcaggaactgagtttaccctgacaa ttagctccctggaggctgaagatgccgctacctactattgccagcagtggagcag ctatcctctgaccttcggacaggggactaaactggaaatcaagggtggaggatcc qgcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctg gagggtccctgagactctcctgtgcagcctctggattcaccttcagcacatacgc tatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatc aggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggatagat tcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcct gaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaat gtgtcttggtttgcttattggggacaggggacactggtgactgtgtctt ccggaggatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgcttt gaaagagaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag 3. The gpA33 x CD3 Bi-Speeific Monovalent Diabody Having An Albumin-Binding Domain (ABD) (“DART-2 w/ABD”) In another embodiment of the invention, the gpA33 x CD3 bi-specific monovalent y will comprise an Albumin-Binding Domain (“ABD”) (gpA33 x CD3 bi-specific monovalent diabody with ABD”).

As disclosed in WO 2012/018687, in order to e the in vivo pharmacokinetic properties of diabody molecules, the molecules may be modified to contain a polypeptide portion of a serum-binding protein at one or more of the termini of the diabody le. Most preferably, such ptide portion of a serum- binding protein will be led at the C-terminus of the diabody molecule. A particularly preferred polypeptide portion of a serum-binding protein for this purpose is the albumin binding domain (ABD) from streptococcal protein G. The albumin binding domain 3 (ABD3) of protein G of Streptococcus strain 0148 is particularly prcfcrrcd.

The albumin binding domain 3 (ABD3) of n G of Streptococcus strain G148 ts of 46 amino acid residues forming a stable thrcc-hclix bundle and has broad albumin g specificity (Johansson, M.U. et a]. (2002) “Structure, Specificity And Mode 0f Interaction For Bacterial Albumin—Binding Modules,” J.

Biol. Chem. 277(10):8ll4-8120). Albumin is the most abundant protein in plasma and has a half-life of 19 days in humans. Albumin ses several small molecule binding sites that permit it to non-covalently bind to other proteins and thereby extend their serum ives.

Thus, the first ptide chain or second ptide chain of a gpA33 x CD3 bi-specific monovalent diabody having an Albumin-Binding Domain contains a third linker (Linker 3), which separates the E-eoil (or K-coil) of such polypeptide chain from the Albumin-Binding Domain. A preferred sequence for such Linker 3 is GGGS (SEQ ID NO:32) or GGGNS (SEQ ID NO:33). A preferred Albumin- Binding Domain (ABD) has the amino acid sequence (SEQ ID NO:34): LAQAKEAAIRELDKYGVS DYYKNL I DNAKSAEGVKAL I DE ILAALP In order to illustrate this aspect of the invention, the first polypeptide chain of the above-described DART-2 was modified to contain an Albumin-Binding Domain, resulting in a gpA33 X CD3 bi-speeific monovalent diabody having an ABD, designated herein as “DART-2 w/ABD.” The first polypeptide chain of such DART-2 w/ABD has the amino acid sequence (SEQ ID NO:35): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK RAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG GGGSGGGGQVQLVQSGAEVKKPGASVKVSCKASGYTFTGSWMNWVRQAPGQGLEW IGRIYPGDGETNYNGKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARIYGN NVYFDVWGQGTTVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGGGS LAQAKEAAIRELDKYGVSDYYKNLIDNAKSAEGVKALIDEILAALP As will be recognized, residues 1-271 of SEQ ID NO:35 are identical to es 1-271 of , and thus provide, in the N-terminal to C-terminal direction, the VL Domain of an antibody that binds CD3 (VLcm) (SEQ ID NO:5); the first intervening spacer peptide (Linker l) (SEQ ID NO:1); the VH Domain of an antibody that binds gpA33 (VngA33) (SEQ ID NO:27), the ne-containing spacer peptide (Linker 2) (SEQ ID NO:2), the heterodimcr-promoting “E-coil” Domain (SEQ ID NO:3) and a C-terminus. Residues 272-275 are Linker 3 (SEQ ID NO:32), and es 276-321 are an Albumin-Binding Domain (SEQ ID NO:34).

A preferred polynucleotide that encodes the first polypeptide chain of DART-2 w/ABD has the ce (SEQ ID NO:36): caggctgtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtga ccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattg gcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaa agggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccg ctctgactattaccggggcacaggccgaggacgaagccgattactattgtgctct tagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctggga gggggtggatccggcggaggtggacaggtccagctggtccagagcggggccgaag tcaaaaaacccggagcaagcgtgaaggtctcctgcaaagcatcaggctatacatt tacaggcagctggatgaactgggtgaggcaggctccaggacagggactggagtgg atcgggcgcatctaccctggagacggcgaaactaactataatggaaagttcaaag accgagtgaccatcacagccgataagtctactagtaccgcctacatggagctgag ctccctgcggtctgaagataccgccgtctactattgcgctagaatttacggaaac aatgtctattttgacgtgtgggggcagggaacaactgtgactgtctcctccggag gatgtggcggtggagaagtggccgcactggagaaagaggttgctgctttggagaa ggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaaggcggcgggtct caggcaaaagaggcagccatccgcgaactggataaatatggcgtgagcg attattataagaacctgattgacaacgcaaaatccgcggaaggcgtgaaagcact gattgatgaaattctggccgccctgcct The second polypeptide chain of DART-2 w/ABD is the same as the above- discussed second polypeptide chain of DART-2 (SEQ ID NO:30).

B. The gpA33 x CD3 Bi-Specific Monovalent Diabodies Having An IgG Fc Domain (“DART-2 w/Fc”) In a further embodiment, the invention provides gpA33 X CD3 bi-specific monovalent diabodies having an IgG Fc Domain. Such diabodies are accordingly referred to herein as “gpA33 X CD3 bi-specific monovalent Fc diabodies.” The PC Domain of the Fc diabodies of the present invention may be either a complete Fc region (e.g., a complete lgG Fc region) or only a fragment of a complete Fc region.

Although the Fc Domain of the bi-speciflc monovalent Fc ies of the present invention may possess the ability to bind to one or more Fc receptors (e.g., FcyR(s)), morc prcfcrably such Fc Domain will cause rcduccd binding to FcyRIA (CD64), FcleiA (CD32A), FC’yRilB (CD328), FcyRillA (CD16a) or FcleilB (CDl6b) (relative to the binding exhibited by a wild-type Fc region) or will substantially eliminate the ability of such Fc Domain to bind to such receptor(s). The PC Domain of the bi-specific monovalent Fc diabodies of the present invention may include some or all of the CH2 Domain and/or some or all of the CH3 Domain of a complete Fc , or may comprise a variant CH2 and/or a variant CH3 sequence (that may include, for e, one or more ions and/or one or more deletions with respect to the CH2 or CH3 s of a complete Fc region). The Fc Domain of the bi- spccific monovalent Fc diabodics of the present invention may comprise non-Fe polypeptide portions, or may comprise portions of non-naturally complete Fc s, or may comprise turally occurring orientations of CH2 and/or CH3 domains (such as, for example, two CH2 domains or two CH3 domains, or in the N-terminal to C-terminal ion, a CH3 Domain linked to a CH2 Domain, etc.).

In a first ment, denoted as on 1” and shown in Figure 2A, the first polypeptide chain of an exemplary gpA33 X CD3 bi-specific monovalent Fc diabody will comprise, in the N-terminal to C-terminal direction, an N-terminus, the VL Domain of a monoclonal antibody capable of binding to either gpA33 or CD3 (i.e., either VLgpAgg or VLCD3), an ening spacer peptide (Linker l), a VH Domain of a monoclonal antibody capable of binding to either gpA33 (if such first polypeptide chain contains VLcm) or CD3 (if such first polypeptide chain ns VLgpmg), a cysteine—containing second intervening spacer peptide (Linker 2), a heterodimer-promoting , a spacer peptide (Linker 5), a cysteine-containing e (Peptide 1), an IgG Fc Domain rably, all or a portion of the CH2 and CH3 domains of an antibody Fc region), and a C-terminus.

In a second embodiment, denoted as “Version 2” and shown in Figure 28, the first ptide chain of an exemplary gpA33 X CD3 bi-specific monovalent Fc diabody will comprise, in the N-terminal to C-terminal direction, an N-terminus, a cysteine-containing peptide de 1), an IgG Fc Domain rably, all or a portion of the CH2 and CH3 domains of an antibody Fc region), an intervening spacer peptide (Linker 4); the VL Domain of a monoclonal antibody capable of binding to either gpA33 or CD3 (i.e., either VLgpA33 or Vchg), an intervening spacer peptide (Linker 1), a VB Domain of a monoclonal antibody capable of binding to either gpA33 (if such first polypeptide chain contains VLcm) or CD3 (if such first polypeptide chain contains VLgPA33), a cysteine-containing second intervening spacer peptide r 2), a heterodimer-promoting Domain, and a C-terminus.

] Preferably, in either embodiment, the Fc Domain of the first polypeptide chain will cause reduced binding to FcyRIA (CD64), FcyRIIA ), FcyRIIB (CD328), FcyRIIIA (CDl6a) or FcyRIlIB (CD16b) (relative to the binding exhibited by a wild-type Fc region) or will substantially eliminate the ability of such Fc Domain to bind to such receptor(s). Fe variants and mutant forms capable of mediating such altered binding are well known in the art and include amino acid substitutions at positions 234 and 235, a substitution at position 265 or a substitution at position 297 (see, for example, US Patent No. 5,624,821, herein incorporated by reference). In a preferred embodiment the CH2 and CH3 Domain includes a tution at position 234 with alanine and 235 with alanine.

The second polypeptide chain of such exemplary gpA33 x CD3 bi-specific lent Fc diabodies (Version 1 and Version 2) will comprise, in the N-terminal to C-terminal direction, an N-terminus, a VL Domain of a monoclonal antibody capable of binding to either gpA33 or CD3 (1'.e._, cithcr VLgng or VLcm, depending upon the VL Domain selected for the first polypeptide chain of the diabody), an intervening linker e (Linker l), a VH Domain of a monoclonal antibody e of binding to either CD3 (if such second polypeptide chain contains VLgpAgg) or CD3 (if such second polypeptide chain contains VLcm), a cysteine-containing spacer peptide (Linker 2), a heterodimer-promoting Domain (preferably a K-coil Domain), and a C-terminus.

The exemplary gpA33 X CD3 bi-specific monovalent Fc ies (Version 1 and Version 2) will additionally se a third ptide chain that will comprise, in the N-terminal to C-terminal direction, an N-terminus, a cysteine- containing peptide (Peptide 1), an IgG Fc Domain rably, all or a portion of the CH2 and CH3 domains of an antibody Fc region) having the same isotypc as that of the Fc Domain of the first polypeptide chain and a C-terminus. Preferably, the Fc Domain of the third polypeptide chain will cause reduced binding to FcyRIA (CD64), A (CD32A), FcleIB (CD328), FcyRIIIA (CDl6a) or FcyRIIIB (CD16b) (relative to the binding ted by a wild-type Fc region) or will substantially eliminate the ability of such Fe Domain to bind to such receptor(s), as discussed above, with respect to the first polypeptide chain of the exemplary gpA33 X CD3 bi- specific lent Fe ies.

The optionally present intervening spacer peptide (Linker 4) will preferably comprise the amino acid sequence (SEQ ID NO:37): APSSS, and more preferably have the amino acid sequence (SEQ ID NO:38): APSSSPME.

The cysteine-containing peptide (Peptide l) of the first and third polypeptide chains may be comprised of the same amino acid sequence or of ent amino acid sequences, and will n 1, 2, 3 or more cysteine residues. A particularly red Peptide 1 has the amino acid sequence (SEQ ID NO:39): DKTHTCPPCP.

The intervening spacer peptide (Linker 1) preferably has the sequence of SEQ ID NO:1_, described above. The cysteine-containing second intervening spacer pcptidc (Linker 2) preferably has the sequence of SEQ ID NO:2, described above.

The heterodimer-promoting Domain of the first and second polypeptide chains of the gpA33 x CD3 bi-specific monovalent Fc diabodies will preferably by the above-described E-coil Domain (SEQ ID NO:3) and K-coil Domain (SEQ ID NO:4), and will be selected so that one of such polypeptide chains possesses an E-coil Domain, s the other possesses a K-coil Domain, as discussed above.

A preferred spacer e (Linker 5) has the sequence GGG.

The CH2 and/or CH3 domains of the first and third polypeptides need not be identical, and advantageously are modified to foster complexing between the two polypeptides. For example, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a ‘knob’, e.g., tryptophan) can be introduced into the CH2 or CH3 Domain such that steric interference will prevent interaction with a rly mutated Domain and will obligate the mutated Domain to pair with a Domain into which a complementary, or accommodating mutation has been engineered,i.e., ‘the hole’ (e.g., a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the bi-specific monovalent Fc diabody molecule, and further, engineered into any portion of the polypeptides chains of said pair. Methods of protein engineeri ng to favor heterodimerization over homodimerization are well known in the art, in ular with respect to the engineering of immunoglobulin-like les, and are encompassed herein (see e.g., Ridgway et al. (1996) “‘Knobs-Into-Holes’ Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization,” Protein Engr. 9:617-621, Atwell et al. (1997) “Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library,” J. Mol. Biol. 270: 26-35, and Xie et al. (2005) “A New Format Of Bispecific dy: Highly Efficient dimerization, Expression And Tumor Cell Lysis,” J. Immunol. Methods -101; each of which is hereby incorporated herein by reference in its entirety). ably the ‘knob’ is engineered into the CH2-CH3 domains of the first polypeptide chain and the ‘hole’ is ered into the CH2-CH3 domains of the third polypeptide chain. Thus, the ‘knob’ will help in preventing the first ptide chain from homodimerizing via its CH2 and/or CH3 domains. As the third polypeptide chain preferably contains the ‘hole’ substitution it will dimerizc with the first polypeptide chain as well as homodimcrizc with itself.

A preferred knob is created by ing an Fe Domain of a native lgG Fc region to contain the modification T366W. A preferred hole is created by modifying an Fe Domain of a native lgG Fc region to contain the ation T3668, L368A and Y407V. To aid in purifying the third polypeptide chain homodimer from the final bi- specific monovalent Fe diabody comprising the first, second and third polypeptide , the protein A binding site of the CH2 and CH3 domains of the third polypeptide chain is preferably mutated by amino acid tution at on 435 (H435R). To aid in purifying the third polypeptide chain homodimer from the final bi-specific monovalent Fc diabody sing the first, second and third polypeptide , the protein A binding site of the CH2 and CH3 domains of the third polypeptide chain is preferably mutated by amino acid substitution. Thus the third polypeptide chain homodimer will not bind to protein A, whereas the bi-specific monovalent Fc diabody will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.

A preferred sequence for the CH2 and CH3 domains of an antibody Fe Domain present in the first polypeptide chain is (SEQ ID NO:40): APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK A preferred sequence for the CH2 and CH3 domains of an antibody Fe Domain present in the third polypeptide chain is (SEQ ID NO:41): APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK 1. DART-2 w/Fc Version 1 The first, second and third polypeptide chains of a preferred gpA33 X CD3 bi-specific monovalent Fc y, designated herein as “DART-2 w/Fc Version I,” comprise polypeptide domains having the following sequences: The first polypeptide chain of such DART-2 w/Fc Version 1 has the amino acid sequence (SEQ ID NO:42): SPSFLSASVGDRVTITCSARSSISFMYWYQQKPGKAPKLLIYDTSNLAS GVPSRFSGSGSGTEFTLTISSLEAEDAATYYCQQWSSYPLTFGQGTKLEIKGGGS GGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVGRI RSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGN SYVSWFAYWGQGTLVTVSSGGCGGGEVAALEKEVAALEKEVAALEKEVAALEKGG GDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK As will be appreciated, residues 1-106 of SEQ ID NO:42 are the VL Domain of an antibody that binds gpA33 (VLgpA33) (SEQ ID NO:26); residues 107- 114 of SEQ ID NO:42 are the first intervening spacer peptide (Linker 1) (SEQ ID NO:1); residues 115-239 of SEQ ID NO:42 are the VH Domain of an antibody that binds CD3 (VHCD3) (SEQ ID NO:25); residues 5 of SEQ ID NO:42 are the ne-containing spacer peptide r 2) (SEQ ID NO:2); residues 3 of SEQ ID NO:42 are the heterodimer-promoting “E-coil” Domain (SEQ ID NO:3); residues 274-276 are the spacer peptide GGG (Linker 5); residues 277-286 are Peptide 1 (SEQ ID NO:39), residues 277-503 are the sequence for the CH2 and CH3 domains of an antibody Fc Domain (SEQ ID NO:40).

A preferred polynucleotide that encodes the first polypeptide chain of DART-2 w/Fc Version 1 has the sequence (SEQ ID NO:43): gacattcagctgactcagtccccctcttttctgtccgcatccgtcggagatcgag tgactattacttgctctgctaggtcctcaatcagcttcatgtactggtatcagca cggcaaagcacctaagctgctgatctacgacacaagcaacctggcctcc ggggtgccatctcggttctctggcagtgggtcaggaactgagtttaccctgacaa ttagctccctggaggctgaagatgccgctacctactattgccagcagtggagcag ctatcctctgaccttcggacaggggactaaactggaaatcaagggtggaggatcc ggcggcggaggcgaggtgcagctggtggagtctgggggaggcttggtccagcctg ccctgagactctcctgtgcagcctctggattcaccttcagcacatacgc tatgaattgggtccgccaggctccagggaaggggctggagtgggttggaaggatc aggtccaagtacaacaattatgcaacctactatgccgactctgtgaaggatagat tcaccatctcaagagatgattcaaagaactcactgtatctgcaaatgaacagcct gaaaaccgaggacacggccgtgtattactgtgtgagacacggtaacttcggcaat tcttacgtgtcttggtttgcttattggggacaggggacactggtgactgtgtctt ccggaggatgtggcggtggagaagtggccgcactggagaaagaggttgctgcttt ggagaaggaggtcgctgcacttgaaaaggaggtcgcagccctggagaaaggcggc ggggacaaaactcacacatgcccaccgtgcccagcacctgaagccgcggggggac cgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggac ccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaag ttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcggg aggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacca ggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctccca gcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacagg tgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgtg gtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaat gggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggct ccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaa cgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaag agcctctccctgtctccgggtaaa The second polypcptidc chain of such DART-2 w/Fc n ] has thc amino acid sequence (SEQ ID : QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNK RAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLG GGGSGGGGQVQLVQSGAEVKKPGASVKVSCKASGYTFTGSWMNWVRQAPGQGLEW IGRIYPGDGETNYNGKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARIYGN NVYFDVWGQGTTVTVSSGGCGGGKVAALKEKVAALKEKVAALKEKVAALKE As will be appreciated, residues 1-110 of SEQ ID NO:44 are the VL Domain of an antibody that binds CD3 (VLcm) (SEQ ID NO:5); residues 111-118 of SEQ ID NO:44 are the first intervening spacer peptide (Linker l) (SEQ ID NO:1); residues 7 of SEQ ID NO:44 are the VH Domain of an antibody that binds gpA33 (VngA33) (SEQ ID NO:27), residues 238-243 of SEQ ID NO:44 are the cysteine-containing spacer peptide (Linker 2) (SEQ ID NO:2) and residues 244-271 of SEQ ID NO:44 are the heterodimer-promoting “K-coil” Domain (SEQ ID NO:4).

A preferred polynucleotide that encodes the second polypeptide chain of DART-2 w/Fc Version 1 has the ce (SEQ ID NO:45): gtggtgactcaggagccttcactgaccgtgtccccaggcggaactgtga ccctgacatgcagatccagcacaggcgcagtgaccacatctaactacgccaattg ggtgcagcagaagccaggacaggcaccaaggggcctgatcgggggtacaaacaaa agggctccctggacccctgcacggttttctggaagtctgctgggcggaaaggccg ctattaccggggcacaggccgaggacgaagccgattactattgtgctct gtggtatagcaatctgtgggtgttcgggggtggcacaaaactgactgtgctggga gggggtggatccggcggaggtggacaggtccagctggtccagagcggggccgaag tcaaaaaacccggagcaagcgtgaaggtctcctgcaaagcatcaggctatacatt tacaggcagctggatgaactgggtgaggcaggctccaggacagggactggagtgg atcgggcgcatctaccctggagacggcgaaactaactataatggaaagttcaaag accgagtgaccatcacagccgataagtctactagtaccgcctacatggagctgag ctccctgcggtctgaagataccgccgtctactattgcgctagaatttacggaaac aatgtctattttgacgtgtgggggcagggaacaactgtgactgtctcctccggag gatgtggcggtggaaaagtggccgcactgaaggagaaagttgctgctttgaaaga gaaggtcgccgcacttaaggaaaaggtcgcagccctgaaagag ] The third polypeptide chain of such DART-2 w/Fc Version 1 has the amino acid sequence (SEQ ID NO:46): DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKS LSLSPGK As will be iated, residues 1-10 of SEQ ID NO:46 are Peptide l (SEQ ID NO:39) and residues 11-227 are the CH2 and CH3 domains of an antibody Fc Domain (SEQ ID NO:41).

A preferred polynucleotide that encodes the third polypeptide chain of DART-2 w/Fc Version 1 has the sequence (SEQ ID : gacaaaactcacacatgcccaccgtgcccagcacCtgaagccgcggggggaccgt cagtcttCCtCttccccccaaaacccaaggacaccctcatgatctcccggacccc tgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggagg agcagtacaacagcacgtaccgtgtggtcagcgtCthaccgtcctgcaccagga ctggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcc gagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgt acaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgagttg cgcagtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatggg cagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctcct tcttcctcgtcagcaagctcaccgtggacaagagcaggtggcagcaggggaacgt Cttctcatgctccgtgatgcatgaggctctgcacaaccgctacacgcagaagagc ctctccctgtctccgggtaaa 2. DART—2 w/Fc n 2 The first, second and third polypeptide chains of a second preferred gpA33 X CD3 bi-specific monovalent Fc diabody, designated herein as “DART-2 w/Fc Version 2,” comprisc polypeptidc domains having thc following ccs. Among other differences, DART-2 w/Fc n 1 differs from DART-2 w/Fc Version 22 in the positioning of the CH2 and CH3 sequences of the first polypeptide chain; these sequences are positioned C-terminal to the VL and VH sequences of DART-2 w/Fc Version 1, whereas they are positioned N-terminal to the VL and VH ces of DART-2 w/Fc Version 2.

The first polypeptide chain of such DART-2 w/Fc n 2 has the amino acid sequence (SEQ ID N0:48): DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNG KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKAPSSSPMEDIQLTQSPSFLSASVGDRVTITCSARSSISFMYWYQQKPG KAPKLLIYDTSNLASGVPSRFSGSGSGTEFTLTISSLEAEDAATYYCQQWSSYPL TFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMNW VRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTE DTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSGGCGGGKVAALKEKVAALKEK VAALKEKVAALKE As will be appreciated, residues 1-10 of SEQ ID N0:48 are Peptide l (SEQ ID NO:39)_; residues 11-227 of SEQ ID N0:48 are the sequence for the CH2 and CH3 domains of an antibody Fe Domain (SEQ ID N0:40); residues 228-235 of SEQ ID N0:48 are ening spacer peptide (Linker 4) (SEQ ID NO:38); residues 236- 341 of SEQ ID N0:48 are the VL Domain of an antibody that binds gpA33 (VLgpA33) (SEQ ID ; residues 342-349 of SEQ ID N0:48 are the first intervening spacer peptide (Linker l) (SEQ ID NO:1); residues 4 of SEQ ID N0:48 are the VH Domain of an antibody that binds CD3 (Vng) (SEQ ID NO:25); residues 475—480 of SEQ ID N0:48 are the cysteine-containing spacer peptide (Linker 2) (SEQ ID NO:2); and residues 481-508 of SEQ ID N0:48 are the heterodimcr- promoting “K-coil” Domain (SEQ ID N014).

The second polypeptide chain of such DART-2 w/Fe Version 2 has the amino acid sequence of the first polypeptide chain of DART-2 (i.e., SEQ ID NO:28) (described above).

The third polypeptide chain of such DART-2 w/Fc Version 2 has the amino acid sequence of SEQ ID N0:46 (described above).

Pharmaceutical Compositions The compositions of the invention include bulk drug compositions useful in the manufacture of pharmaceutical compositions (e.g., impure or non-sterile compositions) and pharmaceutical compositions (i.e., compositions that are suitable for administration to a subject or patient) which can be used in the preparation of unit dosage forms. Such compositions comprise a prophylactically or therapeutically ive amount of the gpA33 x CD3 bi-specific lent diabodies or gpA33 X CD3 bi-specific monovalent Fe diabodies disclosed herein and an additional therapeutic agent) and a pharmaceutically acceptable carrier. Preferably, compositions of the invention comprise a prophylactically or therapeutically ive amount of one or more molecules of the invention and a pharmaceutically acceptable carrier.

The invention also encompasses pharmaceutical compositions comprising such gpA33 x CD3 bi-specifie monovalent ies or gpA33 x CD3 bi-specifie monovalent Fc diabodies and a second therapeutic antibody (e.g., a cancer-antigen c monoclonal antibody) that is specific for a particular antigen associated with a cancer, and a pharmaceutically acceptable carrier.

In a specific embodiment, the term “pharmaceutically able” means approved by a regulatory agency of the Federal or a state government or listed in the US. Pharmacopeia or other generally recognized pharmacopeia for use in s, and more particularly in humans. The term “carrier” refers to a diluent, nt (e.g., Freund’s nt (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such ceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, ble or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred r when the pharmaceutical composition is administered intravenously.

Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical ents include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also n minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, sions, emulsion, tablets, pills, capsules, powders, sustained release formulations and the like.

Generally, the ients of compositions of the invention are supplied either separately or mixed er in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compositions of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include, but are not limited to those formed with anions such as those d from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric ides. isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

] The ion also provides a pharmaceutical pack or kit comprising one or more containers filled with such disclosed gpA33 x CD3 bi-specific monovalent diabodies or gpA33 X CD3 bi-specific monovalent Fe diabodies (alone or with additional eutic agent(s)) and such pharmaceutically acceptable carrier.

Additionally, one or more other prophylactic or therapeutic agents useful for the treatment of a disease can also be included in the pharmaceutical pack or kit. The invention also provides a pharmaceutical pack or kit sing one or more ners filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a mental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The t invention provides kits that can be used in the above methods.

In one embodiment, a kit comprises one or more molecules of the ion. In another embodiment, a kit further ses one or more other lactic or therapeutic agents useful for the treatment of a , in one or more containers. In another embodiment, a kit further comprises one or more antibodies that bind one or more antigens associated with a cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

Uses of the Compositions of the Invention The gpA33 x CD3 bi-specific lent diabodies or gpA33 x CD3 bi- spccific monovalent Fc diabodies of the present invention have the ability to treat any disease or condition associated with or characterized by the expression of gpA33.

Thus, without limitation, pharmaceutical compositions comprising such molecules may be employed in the diagnosis or treatment of colon cancers, colorectal cancers, and pancreatic cancers.

Methods of Administration ] The compositions of the present invention may be provided for the treatment, prophylaxis, and amelioration of one or more symptoms associated with a disease, disorder or infection by administering to a subject an effective amount of a pharmaceutical composition of the invention. In a preferred aspect, such compositions are substantially purified (i.e., substantially free from substances that limit its effect or produce undesired ffects). In a specific embodiment, the t is an animal, preferably a mammal such as imate (e.g., bovine, equine, feline, canine, rodent, etc.) or a primate (e.g., monkey such as, a cynomolgus monkey, human, etc.). In a preferred embodiment, the subject is a human.

Various delivery s are known and can be used to administer the compositions of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the antibody or filsion protein, receptor-mediated endocytosis (See, e.g., Wu et al. (1987) “Receptor-Mediated In Vitro Gene Transformation By A e DNA Carrier Susie/n, ” J. Biol. Chem. —46- 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc.

Methods of administering the gpA33 X CD3 cific monovalent diabodies or gpA33 x CD3 bi-spccific monovalent Fc diabodies of the present invention include, but are not limited to, parenteral administration (e.g._, intraderrnal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and l (e.g., intranasal and oral routes). In a specific embodiment, the molecules of the invention are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, for example, by infusion or bolus ion, by absorption through epithelial or mucocutaneous linings , oral mucosa, rectal and intestinal , etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration can also be employed, e.g., by use of an r or nebulizer, and formulation with an aerosolizing agent. See, e.g., US. Patent Nos. 6,019,968; 5,985, 320; 5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. W0 92/ 19244; W0 97/32572; W0 97/44013; W0 98/31346; and W0 99/66903, each of which is incorporated herein by reference in its entirety.

The invention also provides that the gpA33 x CD3 cific monovalent diabodies or gpA33 X CD3 bi-specific monovalent Fc ies of the invention are packaged in a hermetically sealed container such as an e or sachette indicating the quantity of such molecules. In one embodiment, the gpA33 x CD3 bi-spccific monovalent diabodies or gpA33 X CD3 bi-specific lent Fc diabodies of the invention are supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject. Preferably, the gpA33 X CD3 diabodies or gpA33 X CD3 Fc diabodies of the invention are supplied as a dry sterile lyophilized powder in a ically sealed container at a unit dosage of at least 5 pg, more preferably at least 10 pg, at least 15 pg, at least 25 pg, at least 50 pg, at least 100 pg, or at least 200 pg.

The lyophilized gpA33 X CD3 bi-specific lent diabodies or gpA33 X CD3 bi-specific monovalcnt Fe diabodics of the invention should be stored at between 2 and 8°C in their original container and the molecules should be stered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour afier being reconstituted. In an alternative embodiment, gpA33 X CD3 eific monovalcnt diabodies or gpA33 X CD3 bi-specific monovalent Fe diabodics of the invention are supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the molecule, fusion protein, or conjugated molecule.

Preferably, the liquid form of such bi-specific lcnt diabodics or bi-speeific monovalent Fe diabodies is supplied in a hermetically sealed container in which the molecules are present at a concentration of least 1 ug/ml, more ably at least 2.5 rig/ml, at least 5 rig/ml, at least 10 ug/ml, at least 50 ug/ml, or at least 100 [Lg/ml.

The amount of gpA33 X CD3 bi-speeif1c monovalcnt diabodics or gpA33 X CD3 bi-speciflc monovalcnt Fc diabodies of the invention which will be effective in the treatment, prevention or amelioration of one or more symptoms associated with a disorder can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the condition, and should be d according to the judgment of the practitioner and each patient’s stances. Effective doses may be extrapolated from dose-response curves d from in vitro or animal model test systems.

For gpA33 X CD3 bi-specific monovalent diabodies or gpA33 X CD3 bi- speeific monovalcnt Fc diabodics encompassed by the invention, the dosage administered to a patient is typically at least about 0.01 ug/kg, at least about 0.05 ug/kg, at least about 0.1 [lg/kg, at least about 0.2 rig/kg, at least about 0.5 [Lg/kg, at least about 1 [Lg/kg, at least about 2 pig/kg, at least about 3 rig/kg, at least about 5 ug/kg, at least about 10 ug/kg, at least about 20 ug/kg, at least about 30 ug/kg, at least about 50 [Lg/kg, at least about 0.1 mg/kg, at least about 0.15 mg/kg, or more of the subject’s body weight.

The dosage and frequency of administration of the bi-specific lent diabodies or cific lcnt Fe diabodies of the invention may be reduced or altered by enhancing uptake and tissue penetration of the bi-specific monovalent Fc diabodics by modifications such as, for example, lipidation.

] In one embodiment, the dosage of the gpA33 X CD3 bi-specific monovalent diabodics or gpA33 X CD3 Fc bi-spccific monovalent diabodics of the invention administered to a t may be calculated for use as a single agent therapy. In another embodiment the bi-specific monovalent ics or bi-specific lent Fc diabodics of the invention are used in combination with other therapeutic compositions and the dosage administered to a patient are lower than when such bi- specific monovalent diabodies or bi-specific monovalent Fc diabodics are used as a single agent therapy.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention y to the area in need of treatment; this may be achieved by, for example, and not by way of tion, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, ing membranes, such as sialastic membranes, or fibers.

Preferably, when administering a molecule of the invention, care must be taken to use materials to which the molecule does not absorb.

] In another embodiment, the compositions can be delivered in a vesicle, in ular a liposome (See Langer (1990) “New Methods OfDrug Delivery, " Science 249:1527-1533); Treat et al., in Liposomes in the Therapy of ious e and Cancer, Lopez-Berestein and Fidler (eds), Liss, New York, pp. 353- 365 (1989); Lopcz-Bercstcin, ibid., pp. 3 17-327; see generally ibid.).

In yet another embodiment, the compositions can be delivered in a controlled release or sustained release system. Any technique known to one of skill in the art can be used to produce sustained release formulations comprising one or more molecules of the invention. See, e.g., US. Patent No. 4,526,938; PCT publication WO 91/05548; PCT publication WO 98; Ning et al. (1996) “lntratumoral Radioimmunotheraphy Of A Human Colon Cancer aft Using A Sustained—Release Gel," Radiotherapy & Oncology 392179-189, Song et al. (1995) “Antibody Mediated Lung Targeting OfLong-Circulating Emulsions, " PDA Journal of Pharmaceutical Science & logy 502372-397; Cleek et al. (1997) gradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, " Pro. Int‘l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997) “Microencapsulation 0f Recombinant Humanized IMonoclonal Antibody For Local Delivery, " Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of which is incorporated herein by reference in its ty. In one embodiment, a pump may be used in a lled release system (See Langer, supra; Sefton, (1987) “Implantable Pumps, CRC Crit. Rev. Biomed. Eng. 142201-240; Buchwald et al. (1980) “Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis, " Surgery 882507-516; and Saudek et al. (1989) “A Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery, " N. Engl. J.

Med. 321:574—579). In another embodiment, polymeric materials can be used to achieve controlled release of antibodies (see e.g., MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds), CRC Pres, Boca Raton, Florida (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds), Wiley, New York ; Levy et al. (1985) “Inhibition 0f Calcification 0f Bioprosthetic Heart Valves By Local Controlled- e Diphosphonate," Science 228:190-192; During et al. (1989) “Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo terization, ” Ann. Neurol. 253351-356; Howard et al. (1989) “Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits, " J. Neurosurg. 7(1 ):105-1 12); US. Patent No. 377; US. Patent No. 5,916,597; US. Patent No. 5,912,015; US. Patent No. ,989,463; US. Patent No. 5,128,326; PCT Publication No. W0 99/ 15154; and PCT Publication No. WO 99/20253). es of polymers used in sustained e formulations include, but are not limited to, poly(2—hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-Vinyl acetate). poly(methacrylic acid), polyglycolides (PLG), hydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene ), etides (PLA), actide-co-glycolides) (PLGA), and polyorthoesters. In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target (e. g., the lungs), thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, supra, vol. 2, pp. 115-138 (1984)). In r embodiment, polymeric compositions useful as controlled release implants are used according to Dunn et al. (See US. 5,945,155).

This particular method is based upon the therapeutic effect of the in situ controlled release of the ive material from the polymer system. The implantation can generally occur anywhere within the body of the patient in need of therapeutic treatment. In another embodiment, a non-polymeric sustained delivery system is used, y a non-polymeric implant in the body of the subject is used as a drug delivery system. Upon implantation in the body, the c solvent of the implant will dissipate, disperse, or leach fi'om the composition into surrounding tissue fluid, and the non-polymeric material will lly coagulate or precipitate to form a solid, microporous matrix (See US. 5,888,533).

Controlled release systems are discussed in the review by Langer (1990, "New Methods Of Drug Delivery, " Science 249:1527-1533). Any technique known to one of skill in the art can be used to produce sustained e ations comprising one or more therapeutic agents of the invention. See, e.g., US. Patent No. 938; ational ation Nos. WO 91/05548 and WO 96/20698; Ning et al. (1996) “Intratunzoral Radioinmumotheraphy Of A Human Colon Cancer Xenograft Using A Sustained—Release Gel," Radiotherapy & Oncology 39:179-189, Song et al. (1995) “Antibody Mediated Lung Targeting 0f Long-Circulating Emulsions, " PDA Journal of Pharmaceutical Science & Technology 50:372-397; Clcck et al. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, " Pro. lnt’l. Symp. Control. Rel. Bioact. Mater. 24:853-854; and Lam et al. (1997) “ll/[icroencapsulation OfRecombinant Humanized Monoclonal Antibody For Local Delivery," Free. Int’l. Symp. Control Rel. Bioact.

Mater. 242759—760, each of which is incorporated herein by reference in its ty.

In a specific embodiment where the composition of the invention is a nucleic acid encoding a bi-speeifie monovalent diabody or bi-specific monovalent Fe diabody of the invention, the nucleic acid can be administered in vivo to e expression of its encoded bi-speeific monovalent diabody or bi-specific lent Fe diabody, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (See US. Patent No. 4,980,286), or by direct injection, or by use of mieroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell- surfaee receptors or transfeeting , or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (See e.g., Joliot et a]. (1991) “Antennapedia Homeobox Peptide Regulates Neural Morphogenesis, " Proc.

Natl. Acad. Sci. (U.S.A.) 88:]864-1868), etc. Alternatively, a nucleic acid can be uced intracellularly and orated within host cell DNA for expression by homologous recombination.

Treatment of a t with a therapeutically or prophylactically effective amount of the gpA33 x CD3 bi-specific monovalent diabodies or gpA33 x CD3 bi- specifie lent Fe diabodies of the invention can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with molecules of the invention one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. In other embodiments, the pharmaceutical compositions of the invention are stered once a day, twice a day, or three times a day. In other embodiments, the pharmaceutical compositions are administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year or once per year. It will also be appreciated that the effective dosage of the molecules used for treatment may increase or decrease over the course of a particular treatment.

] Having now lly described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the t invention unless specified.

Characteristics of Anti-Human gpA33 Monoclonal Antibody A murine monoclonal dy capable of specific binding to human gpA33 was chimerieized and humanized. The VL and VP] chains of the original murine antibody have the ces of SEQ ID NOs:l3 and 17, respectively. The VL and VH chains of the humanized antibody have the sequences of SEQ ID NOS:26 and 27, respectively.

The Antigen Binding Domain of VLgpAgg comprises CDRl having the sequence (SEQ ID NO:14): SARSSISFMY; CDR2 having the sequence (SEQ ID : DTSNLAS; and CDR3 having the sequence (SEQ ID NO:16): QQWSSYPLT.

The Antigen Binding Domain of VngAgg comprises CDRl having the ce (SEQ ID NO:18): GSWMN; CDR2 having the sequence (SEQ ID NO:19): RIYPGDGETNYNGKFKD; and CDR3 having the sequence (SEQ ID NO:20): IYGNNVYFDV.

Table 1 shows the effect of such alterations on the kinetics of g.

Table l -'_-E-—— Murine mAb l 3.3 x 10 7.5 x 10' Chimeric mAb 1 5.8 x 10 1.4 x 10'» Humanized mAb 1 5.6 x 10 1.9 x 10‘- The data indicates that the modifications resulting in the humanization of the antibody VL and VH domains did not substantially affect gpA33 g kinetics.

Example 2 uction Of gpA33 x CD3 cific Monovalent Diabodies and Fe Diabodies And Control Diabodies Table 2 contains a list of sequences of the polypeptide chains of the preferred gpA33 X CD3 diabodies and gpA33 x CD3 Fc diabodies that were expressed and purified. The diabodies were found to be e of simultaneously g to gpA33 and CD3, as judged by the detection of such simultaneous binding by the exemplary gpA33 x CD3 bi-specifie monovalent diabodies, DART-l and DART-2, and by the exemplary gpA33 x CD3 bi-speeific monovalent Fe y (DART-2 w/Fc). Additionally, a control bi-specific monovalent diabody (“Control DART”) was produced that was bi-specific monovalent for CD3 and FITC, and was found to be capable of simultaneously binding to CD3 and FITC.

Table 2 Substituent Polypeptides (in Diabody the N-Terminal to C-Terminal Direction) gpA33 X CD3 bi-specific lent SEQ ID NO:21 y (DART-l) SEQ ID NO:23 gpA33 x CD3 cific monovalent SEQ ID NO:28 diabody (DART-2) SEQ ID NO:30 gpA33 x CD3 bi-specific monovalent diabody having an Albumin-Binding SEQ ID NO:35 Domain (DART-2 w/ABD) SEQ n) NO:30 Comprises an Albumin-Binding Domain (ABD) for extension of half-life in vivo gpA33 x CD3 bi-specific monovalent diabody having an lgG Fc Domain SEQ ID N0:42 version 1 (DART-2 w/Fc Version 1) SEQ ID NO:44 Comprises an Fc Domain for extension SEQ ID NO:46 of half-life in viva gpA33 x CD3 bi-spccific monovalent diabody having an IgG Fc Domain SEQ ID NO:48 version 2 2 w/Fc Version 2) SEQ ID NO:28 Comprises an Fc Domain for extension SEQ ID NO:46 of half-life in vivo The gpA33 X CD3 bi-specific monovalent diabodies are heterodimers ed of two ptide chains (one chain of each recited sequence) and the gpA33 X CD3 bi-specific monovalent Fc ies arc hctcrotrimcrs composed of three polypeptide chains (one chain of each recited amino acid sequence). s for forming bi-specific monovalent diabodies are provided in , WO 2008/157379, , , and W0 2012/ 162067.

The control CD3 x FITC bi-specific monovalent diabody was found to be capable of simultaneously binding to CD3 and to FITC. The above-described gpA33 X CD3 bi-specific monovalent diabodies and gpA33 x CD3 bi-specific monovalent Fc diabodies were found to be capable of simultaneously binding to gpA33 and to CD3.

In order to demonstrate such simultaneous g, the gpA33 X CD3 bi-specific monovalent diabody DART-l was incubated in the presence of a soluble CD3 fragment that had been immobilized to a solid support. The detection of binding was assessed by the ty of immobilized antibodies to additionally bind gpA33. The results confirm the capacity of the above-described gpA33 X CD3 cific lent diabodies and gpA33 X CD3 bi-spccific monovalent Fc diabodies to e simultaneous binding to gpA33 and CD3 (Figure 3).

Example 3 gpA33 x CD3 Bi-Specific Monovalent Diabodies Are Cytotoxic to Cancer Cells ] The ability of the gpA33 X CD3 bi-specific monovalent diabodies of the present invention to treat cancer was illustrated by incubating colorectal or pancreatic cancer cells in the presence of the gpA33 X CD3 bi-specific lent DART-1 and either human PBMC (E:T = 25:1) or activated human T cells (E:T = 10:1). gpA33 x CD3 bi-specific monovalent diabody DART-1 exhibited potent redirected killing ability with concentrations required to achieve 50% maximal activity (EC50) in the sub-ng/mL to around 1 ng/mL range. In contrast, cytotoxicity was not observed when gpA33-negative cancer cell lines (e.g., HCT116) were ed. The results of the investigation are shown in Figure 4A (colorectal cancer stem-like cells (Colon CSCL cells), Figure 4B (C010205 colorectal , and Figure 4C (ASPC-l pancreatic cancer cells). Results are summarized in Table 3.

Target Cell Line EC50 of gpA33 x EffectorzTarget Max % Killing CD3 Bi-Specific (EzT) Observed Monovalent Diabodv (n mL) Example 4 T cell Activation in the ce of gpA33 x CD3 Bi-Specific Monovalent Diabodies In order to further demonstrate the ability of the diabodies of the t invention to treat cancer, resting human T cells were incubated with the gpA33 X CD3 bi-specific monovalent DART-l in the presence or absence of cancer cells (c010205 or ASPC-l). To characterize T cell activation during gpA33 X CD3 bi-specific monovalent diabody 1)-mcdiated redirected killing process, T cells from redirected killing assays were stained for the T cell activation marker CD25 and analyzed by FACS. CD25 was uprcgulatcd in CD8 (Figures 5A-SB) and CD4 (Figures SD-SE) T cells in a ependent manner indicating that the gpA33 x CD3 bi-specific monovalent diabodies induced T cell activation in the s of redirected killing. Conversely, in the absence of target cells there was no activation of CD8 (Figure 5C) or CD4 (Figure 5F) T cells indicating the gp-A33 x CD3 diabodies do not activate T cells in the absence of target cells. Likewise, CD8 or CD4 T cells were not activated when incubated with target cells and a control cific monovalent diabody (Control DART) (Figures 5A-SB, and Figures 5D-5F, respectively) indicating the ement of cross-linking the T cell and target cell with the gpA33 x CD3 bi-spccific monovalent diabodics.

Example 5 Equivalency of gpA33 x CD3 Bi-Specifie Monovalent y (DART-1) Having Murine uman gpA33 Variable Domain Sequences and gpA33 x CD3 Bi- Specific lent Diabody (DART-2) Having Humanized Anti-Human gpA33 Variable Domain Sequences As discussed above, the gpA33 X CD3 bi-specific monovalent diabody DART-1 contains VLgpAgg and VngAgg domains of a murine monoclonal antibody, whereas the gpA33 X CD3 cific monovalent diabody DART-2 contains humanized VLgpAgg and humanized VngA33 domains of the same murine antibody. In order to demonstrate the ability of the humanized “43%” and Vl-lwm domains to promote T cell ing to gpA33-expressing cancer cells, cancer cells that s gpA33 were incubated in the presence of resting T cells (LDH assay; E:T = 10:1) in the presence of either DART-1, DART-1 or a control bi-spccific monovalent diabody (Control DART). The s of this analysis (shown in Figures 6A-6D) demonstrate that DART-1 and DART-2 mediated equivalent cytotoxicity for SW948 colorectal adenocarcinoma cells (Figure 6A) and c010205 cells (Figure 6B). DART-l and DART-2 both mediated cytotoxicity of a luciferase expressing C010205 cell line which was stably transfected with firefly luciferase gene (luc2) (ColoZOS-Luc), as measured by decreased luminescence (Figure 6C). r DART-1 nor DART-2 mediated cytotoxicity of the gpA33-negative cancer cell line, HCTl 16 (Figure 6D).

As shown in Table 4, DART-l and DART-2 exhibited similar equivalent bioactivity against multiple tumor cell lines.

Table 4 Effector/Target LDH Assay Donor T Cell Cross-Reactivity of gpA33 x CD3 Bi-Speeific Monovalent Diabodies, gpA33 x CD3 Bi-Specific Monovalent Diabodies Having an Albumin-Binding Domain and gpA33 x CD3 Bi-Specifie Monovalent ies Having an IgG Fe Domain with PBMCs of Cynomolgus Monkey ] As shown above, the humanized VLgMgg and humanized VngA33 domains of the gpA33 X CD3 bi-specific monovalent diabody DART-2 mediate the cytotoxicity of gpA33-expressing cancer cells in the presence of human T cells. The VLcm and VHcm domains of the gpA33 X CD3 bi-specific monovalent diabodies of the present invention were unexpectedly found to also be capable of binding to the CD3 of cynomolgus monkey T cells and redirect those cells to kill gpA33-expressing cells.

] As shown in s 7A-7D, the gpA33 X CD3 bi-specific monovalent DART-2 diabody, the gpA33 X CD3 bi-specific monovalent diabody having an Albumin-Binding Domain (DART-2 w/ABD) and the gpA33 X CD3 bi-specific monovalent DART-2 diabody having an IgG Fe Domain (DART-2 w/Fc) were all found to be capable of promoting the cytotoxicity of cancer cells in the presence of human or cynomolgus monkey PBMCs. Figures 7A-7B show the ability of‘ the three diabodies to mediate cytotoxicity of S-Luc cells that were incubated with human PBMC, as measured by LDH assay (Figure 7A) or luciferase (Figure 78).

Figures 7C-7D show the corrcsponding ability of thc thrcc diabodics to mediate cytotoxicity of ColoZOS-Luc cells that were incubated with cynomolgus monkey PBMC, as measured by LDH assay (Figure 7A) or luciferase (Figure 78).

As shown in Table 5, the gpA33 x CD3 bi-specific monovalent diabody DART-2 and the gpA33 x CD3 cific monovalent diabody having an Albumin- Binding Domain (DART-2 w/ABD) displayed comparable CTL activity. The bi- specific monovalent diabodies exhibited consistent activity with both human and cynomolgus monkey (cyno) PBMC effector cells.

Table 5 EC50 — CTL Activity (ng/mL) C010205 Target Cells LDH Assay Luciferase Assay Human DART Cyno Human Cyno PBMC PBMC PBMC PBMC gpA33 X CD3 bi-specific 4.09 3.81 2.73 1.55 monovalent diabody (DART-2) gpA33 X CD3 i-spccific diabody having an Albumin-Binding 5.52 4.63 3.07 1.63 Domain (DART-2 w/ABD) Example 7 in vivo Reactivity of gpA33 x CD3 diabody in Murine Colon Tumor Model ] In order to trate the in vivo ability of the gpA33 X CD3 ies of the present invention to provide a treatment for cancer, c010205 cells were co- implanted with activated T cells in immunodeficient NSG (NOD scid gamma) mice no, A. et al. (2008) “Human Acute ia Cells Injected In NOD/Ltsz- SCI'd/IL-2Rgamma Null Mice Generate A Faster And More Eflicien! Disease Compared To Other NOD/Scid-Related s,” Int. J. Cancer :2222-2227; Sanchcz, P.V. et al. (2009) “A Robust Xenotransplantation Modcl ForAcute Myeloid Leukemia,” Leukemia 23(11):2109-2ll7; Racki, WJ. et al. (2010) “NOD-Scid IL2rgammalNu/l) Mouse Model Of Human Skin Transplantation And Allografi Rejection,” Transplantation 89(5):527-536; Choi, B. et al. (2011) “Human B Cell Development And Antibody Production In zed NOD/SCID/IL-L’RyflVu/l) (NSG) Mice Conditioned By Busulfan,” J. Clin. l. 253-264; Sartelet, H. et al. (2012) “Description OfA New Xenograji Model OfMetastatic Neuroblastoma Using lD/IIng Null (NSG) Mice,” In Vivo 26(1):]9-29; Spranger, S. et al. (2012) cid lL—2Rg(null) Mice: A Preclinical Model System To Evaluate Human Dendritic Cell-Based Vaccine Strategies in vivo,” J. Transl. Med. 10:30; von Bonin, M. et al. (2013) “in vivo Expansion QfCo-Transplanted T Cells Impacts 0n Tumor lie-Initiating Activity OfHuman Acute il/Lveloitl Leukemia 1n NSG Mice,” PLoS One. 8(4):e60680).

The gpA33 X CD3 bi-specific monovalent diabody DART-l was administered IV to the mice for once daily for 4 days (QDx4) starting at implantation.

C010205 tumor volume was found to increase in mice receiving the Vchiclc control (Figure 8). r, animals receiving DART-1 were found to exhibit lower or no tumor volume (Figure 8).

Imaging of NSG mice implanted with C010205 cells showed that at day 2 of treatment mice receiving Vehicle (Figure 9A) or the gpA33 X CD3 bi-specific monovalent diabody DART-1 (Figure 98) had significant . However, at day 12 of treatment mice receiving the gpA33 X CD3 bi-specific monovalent diabody DART-1 had dramatically lower tumor volumes (Figure 9D). At day 12 of treatment, mice receiving Vehicle showed increased tumor volume (Figure 9C).

As further evidence of the in vivo ability of thc gpA33 x CD3 diabodics of the present ion to provide a treatment for cancer, the above-described tumor model was conducted using ASPC-l pancreatic tumor cells and activated human T cells (E:T = 1:1). The gpA33 X CD3 bi-specific monovalent y DART-l, a control bi-specific monovalent diabody (Control DART), or Vehicle were administered IV for once daily for 9 days (QDx9) starting at tation. ASPC-l tumor volume was found to se in mice receiving the Vehicle control (Figure ). However, animals receiving DART-l were found to exhibit lower tumor volume, in a dose-dependent manner (Figure 10).

Example 8 Efficacy Determination of gpA33 x CD3 Bi-Specific Monovalent Diabody Having An lgG Fc Domain Version 1 (DART-2 w/Fc n 1) In order to determine the efficacy of the gpA33 x CD3 bi-specific monovalent diabody having an lgG Fe Domain version 1 (DART-2 w/Fc Version 1), mice were infused (using osmotic pumps) for 7 days with the above-described DART- 2 w/Fc Version 1 at various dosage levels. 48 h after pump implantation (i.e., in the presence of a steady-state circulating level of DART-2 w/Fc Version 1), a mixture of C010205 tumor cells and T cells were implanted subcutaneously into the mice, and the extent of tumor growth was monitored. Table 6 summarizes the design of the study; each group contained 8 female mice.

Table 6 Dose Route / Cell Schedule lmplant(s) Vehicle IV/QDxS COLO205 (5 E6) gpA33xCD3 bi-specific 3.1 IP/CIF 5 monovalent diabody having an (5 E6) lgG Fe Domain 2 w/Fc ls (5E6) Version 1) DART-2 w/Fc Version1 . lP/ClF COL0205 (5 E6) hT-cclls (5E6) DART-2 w/Fc Version 1 . lP/ClF COL0205 (5E6) hT-cells (5E6) DART-2 w/Fc Version 1 IP/CIF COLO205 (5 E6) hT-eells (5E6) DART-2 w/Fc Version 1 .o m IV/QDxS COLO205 (5E6) hT-cells (5E6) ] The s of this study are shown in Figure 11, and indicate that the administration of the above-described gpA33 X CD3 cific monovalent diabodies having an lgG Fe Domain (DART-2 w/Fc Version 1) mediated a dramatic reduction in tumor volume at all tested dosages.

In light of the dramatic reduction in tumor volume obtained in the above study, a r study was conducted to assess efficacy at much lower doses. Table 7 summarizes the design of this further study; each group contained 8 female mice.

Table 7 Dose Route / Cell Schedule lmplant(s) Vehicle IV/QDxS COLO205 (5 E6) gpA33xCD3 bi-speeifie 0.2 lP/ClF COLO205 monovalent y having an (5 E6) IgG Fe Domain (DART-2 w/Fc hT-cells (5E6) Version 1) DART-2 w/Fc Version 1 . IP/CIF COLO205 (5E6) hT-eclls (5E6) DART-2 w/Fc Version 1 IP/CIF COLOZOS (5 E6) hT-cells (5E6) DART-2 w/Fe Version 1 IP/CIF 5 (5E6) hT-cells (5E6) DART-2 w/Fc n 1 O U) IV/QDxS COLO205 (5 E6) hT-cells (5E6) The results of this further study are shown in Figure 12. In Figure 12, each symbol denotes an animal that received the indicated dosage of the above-described gpA33 X CD3 bi—specific monovalent diabody having an IgG Fe Domain (DART—2 wi’Fc Version 1) or e. The data show efficacy at all tested dosages.

Example 9 Pharmacokinetic Profile of gpA33 x CD3 cific Monovalent Diabody (DART-2) and gpA33 x CD3 Bi-Specific Monovalent Diabody Having an IgG Fc Domain (DART-2 w/Fc) in lgus Monkey The ability of the VLcm and VH0); domains of the diabodies of the present invention to bind to the CD3 of cynomolgus monkey permits the use of such animals to measure the in viva pharrnacokinetics of the diabodies of the present invention. —61- To measure such cokinetics, the above-described gpA33 x CD3 bi- specific monovalcnt diabody (DART-2) or gpA33 x CD3 bi-speeific monovalcnt diabody having an IgG Fe Domain (DART-2 w/Fc Version 1) were injected into lgus monkeys (10 ug/kg/day) and the concentration of such molecules remaining in the circulation was monitored. Figure 13 shows the result of this study, and indicates that DART-2 and DART-2 w/Fc n 1 t first-order elimination kinetics.

Example 10 SPR Analysis of gpA33 x CD3 Bi-Specific lent Fc Diabody (DART-l w/Fc Version 1) Binding to Human and Cynomolgus Monkey CD3 and gpA33 gpA33 x CD3 bi-specific Fe diabody (DART-2 w/Fc Version 1) binding to soluble versions of human and cynomolgus monkey CD3 receptor was analyzed by SPR on a BIAcore 3000 biosensor (GE, Healthcare). Receptors were immobilized on the CMS sensor chip according to the procedure recommended by the cturer.

Briefly, the carboxyl groups on the sensor chip surface were activated with an injection of a solution containing 0.2M N-ethyl-N-(3dietylamino-propyl) carbodiimide and 0.05M N-hydroxy-suceinimide. Soluble CD3 receptor (1 rig/ml) was then injected over the activated CMS surface in lOmM sodium-acetate, pH 5.0, at flow rate 5 [LL/min, followed by 1 M ethanolamine for vation.

The soluble versions of lgus and human CD3 employed in such analysis were expressed in mammalian cells as a CD38 / CD36 heterodimer, stabilized by oppositely charged heterodimer-promoting E-coil and K-coil sequences at their C- termini. The soluble cynomolgus CD38 contained the first 118 amino acid residues of lgus monkey CD38, with the V35 allele (FN18+) ed by the above- deseribed E-coil Domain (SEQ ID NO:3) at the carboxy terminus. The amino acid sequence of the V35 allele (FN18+) cynomolgus CD38 is (SEQ ID NO:49): MQSGTRWRVL GLCLLSIGVW GQDGNEEMGS ITQTPYQVSI SGTTVILTCS QHLGSEAQWQ HNGKNKEDSG DRLFLPEFSE MEQSGYYVCY PRGSNPEDAS HHLYLKARVC ENCMEMDVMA VATIVIVDIC LLVY YWSKNRKAKA KPVTRGAGAG GRQRGQNKER PPPVPNPDYE PIRKGQQDLY SGLNQRRI The soluble cynomolgus CD35 ned the first 101 amino acid es of lgus monkey CD35 followcd by the above-described K-coil Domain (SEQ ID N0:4) at the carboxy terminus. The amino acid sequence of the cynomolgus CD35 is (SEQ ID : MEHSTFLSGL VLATLLSQVS PFKIPVEELE DRVFVKCNTS VTWVEGTVGT LLTNNTRLDL GKRILDPRGI YRCNGTDIYK DKESAVQVHY VELD PATLAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQ ALLRNDQVYQ PLRDRDDAQY SRLGGNWARN K The two proteins were co-expressed in mammalian CHO-S cells and purified using an anti-E/K-coil mAb coupled to SEPHAROSE®.

The e human CD39. contained residues 1-127 of human CD39. with C119S and C122S, followed by the above-described E-coil Domain (SEQ ID NO:3) at the carboxy terminus. The amino acid sequence of human CD38 is (SEQ ID NO:51): MQSGTHWRVL VGVW GQDGNEEMGG ITQTPYKVSI SGTTVILTCP QYPGSEILWQ HNDKNIGGDE DDKNIGSDED HLSLKEFSEL EQSGYYVCYP RGSKPEDANF YLYLRARVEE NEMEMDVMSV ATIVIVDICI TGGLLLLVYY WSKNRKAKAK PVTRGAGAGG RQRGQNKERP PPVPNPDYEP IRKGQRDLYS GLNQRRI The e human CD35 contained residues 1-101 of human CD35 followed by the above-described K-coil Domain (SEQ ID NO:4) at the carboxy terminus. The two proteins were co-expressed in mammalian CHO-S cells and purified using an anti-E/K-coil affinity column. The amino acid sequence of human CD36 is (SEQ ID NO:52): FKIPIEELE DRVFVNCNTS ITWVEGTVGT LLSDITRLDL. GKRILDPRGI YRCNGTDIYK DKESTVQVHY RMCQSCVELD PATVAGIIVT DVIATLLLAL GVFCFAGHET GRLSGAADTQ ALLRNDQVYQ PLRDRDDAQY SHLGGNWARN The e human gpA33 contained residues 1-235 of human gpA33 with (SEQ ID NO:53) HHHHHH (“6His”) repeats at the y terminal end. The soluble cynomolgus gpA33 contained residues 1-314 of lgus monkey gpA33 Met 1 to Gln 314 with 6 His repeats at the carboxy terminal end. The proteins were expressed in ian CHO-S cells and purified using Ni SEPHAROSE®.

Binding experiments were performed in HBS—EP buffer, which ns 10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA and 0.005% P20 surfactant.

Binding of DART-2 w/Fc Version 1 was analyzed (in duplicate) at concentrations of 0, 6.25, 12.5, 25, 50 and 100 nM, injected for 120 sec at a flow rate of 30 uL/min.

Regeneration of the immobilized receptor surfaces was performed by pulse injection of 10mM e, pH 1.5. Reference curves were obtained by injection of each dilution of DART-2 w/Fc over the treated e with no immobilized protein.

Binding curves at zero concentration were subtracted as a blank. KD values were determined by a global fit of binding curves to the Langmuir 1:1 binding model (BIAevaluationTM sofiware v4.1).

The SPR analysis of gpA33 x CD3 cific Fc y (DART-2 w/Fc Version 1) binding to human and cynomolgus monkey CD3 and gpA33 demonstrated a substantial similarity for the molecules from the two different species (Figures 14A— 14B; Figures ISA-15B). Table 8 provides the equilibrium dissociation constants (KDs) calculated by global fit to a 1:1 Langmuir model y and kinetic nts for DART-2 w/Fc ctions. The KD values of DART-2 w/Fc Version 1 for human and cynomolgus monkey CD3 are nearly identical at 23 and 26 nM, respectively, despite some difference in the l binding responses n the two antigens. Random orientation of antigens with different amino acid sequences directly immobilized on the surface can result in different densities of available binding sites on the surface. The KD values for the interaction of DART-2 w/Fc Version 1 with human and monkey gpA33 are 2.2nM and 12nM, respectively (Table 8). The difference in affinity is the result ofa relatively small decrease in association rate constant and increase in dissociation rate constant for the interaction of DART-2 w/Fe Version 1 with cynomolgus monkey gpA33 (Table 8). The data are averages of three independent experiments in duplicates (SD = rd deviation; h, human; cyno. cynomolgus monkey). —64- Table 8 Equilibrium Dissociation Constants (KD) For The Binding Of DART-2 W/Fc Version 1 To Anti ens From Different S I ecies hCD38/5 l.5(i0.1) x 10 3.5(:l:0.06)x10" 23 32.0 l.3(fl:0.02)x10 3.4(i0.02)x10" 26 $0.6 h- .A33 His 4.2(i0.3) x10 9.0(i0.5)x10' 2.30.2 C o_ A33-His 2.3 i0.2 xlO' 2.8 :0] x10" 12:1.0 All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual ation or patent application was specifically and individually ted to be incorporated by reference in its ty. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or tions of the invention following, in general, the principles of the invention and ing such departures from the present disclosure as come within known or ary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

What Is Claimed Is: Claim 1. A bi-specific diabody, wherein said cific diabody is capable of specific binding to an epitope of gpA33 and to an epitope of CD3, wherein the bi-specific y comprises a first polypeptide chain and a second polypeptide chain, n said first and second polypeptide chains are covalently bonded to one another, and wherein: A. the first polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD3 (VLCD3) and comprising the amino acid sequence of SEQ ID NO:5; and a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to gpA33 (VHgpA33) and comprising the amino acid sequence of SEQ ID NO:27; wherein said mains (1A) and (1B) are separated from one another by a peptide linker comprising the amino acid sequence of SEQ ID NO:1; ii. a Domain 2, wherein said Domain 2 is a K-coil Domain sing the amino acid sequence of SEQ ID NO:4 or an E-coil Domain sing the amino acid sequence of SEQ ID NO:3, wherein said Domain 2 is separated from said Domain 1 by a peptide linker comprising the amino acid sequence of SEQ ID NO:2; B. the second polypeptide chain comprises, in the inal to inal direction: i. a Domain 1, comprising a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to gpA33 (VLgpA33) and comprising the amino acid sequence of SEQ ID NO:26 and a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCD3) and comprising the amino acid sequence of SEQ ID NO:25, wherein said sub-Domains (1A) and (1B) are separated from one another by a peptide linker sing the amino acid sequence of SEQ ID NO:1; ii. a Domain 2, wherein said Domain 2 is an E-coil Domain comprising the amino acid sequence of SEQ ID NO:3 or a K-coil Domain comprising the amino acid sequence of SEQ ID NO:4, wherein said Domain 2 is separated from said Domain 1 by a peptide linker comprising the amino acid ce of SEQ ID NO:2; and wherein said Domain 2 of said first polypeptide chain and said Domain 2 of said second polypeptide chain are not both E-coil Domains or both K-coil Domains; and wherein: (a) said VL Domain of said first polypeptide chain and said VH Domain of said second polypeptide chain form a monovalent Antigen Binding Domain capable of specific binding to said epitope of CD3; and (b) said VH Domain of said first polypeptide chain and said VL Domain of said second polypeptide chain form a monovalent Antigen Binding Domain capable of specific binding to said epitope of gpA33.

Claim 2. The bi-specific y of claim 1, wherein said first ptide chain comprises an Albumin-Binding Domain comprising the amino acid sequence of SEQ ID NO:34, said Albumin-Binding Domain being positioned C-terminally to said Domain 2, and separated from said Domain 2 by a linker comprising the amino acid sequence of SEQ ID NO:32.

Claim 3. A cific Fc diabody, wherein said bi-specific Fc y is capable of specific binding to an epitope of gpA33 and to an epitope of CD3, and possesses an IgG Fc Domain, wherein the cific Fc diabody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein said first and second polypeptide chains are covalently bonded to one another and said first and third polypeptide chains are ntly bonded to one another, and wherein: A. the first ptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a main (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to gpA33 (VLgpA33) comprising the amino acid sequence of SEQ ID NO:26 and a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to CD3 (VHCD3) comprising the amino acid sequence of SEQ ID NO:25, wherein said sub-Domains (1A) and (1B) are separated from one r by a e linker comprising the amino acid sequence of SEQ ID NO:1; ii. a Domain 2, wherein said Domain 2 is an E-coil Domain comprising the amino acid sequence of SEQ ID NO:3 or a K-coil Domain comprising the amino acid sequence of SEQ ID NO:4, wherein said Domain 2 is separated from said Domain 1 by a peptide linker comprising the amino acid sequence of SEQ ID NO:2; and iii. a Domain 3, comprising a sub-Domain (3A), which comprises a cysteine-containing peptide comprising the amino acid sequence of SEQ ID NO:39 and a sub-Domain (3B), which comprises a polypeptide portion of an IgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fc Domain; wherein said Domains 3 and 2 are separated from one r by a spacer peptide having sequence GGG; B. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a sub-Domain (1A), which ses a VL Domain of a monoclonal antibody e of binding to CD3 (VLCD3) comprising the amino acid sequence of SEQ ID NO:5, and a sub-Domain (1B), which comprises a VH Domain of a monoclonal antibody capable of binding to gpA33 (VHgpA33) comprising the amino acid sequence of SEQ ID NO:27; wherein said mains (1A) and (1B) are separated from one another by a peptide linker comprising the amino acid sequence of SEQ ID NO:1; ii. a Domain 2, wherein said Domain 2 is a K-coil Domain comprising the amino acid sequence of SEQ ID NO:4 or an E-coil Domain comprising the amino acid sequence of SEQ ID NO:3, wherein said Domain 2 is separated from said Domain 1 by a peptide linker comprising the amino acid ce of SEQ ID NO:2; and wherein said Domain 2 of said first polypeptide chain and said Domain 2 of said second polypeptide chain are not both E-coil Domains or both K-coil s; and C. the third polypeptide chain comprises, in the N-terminal to C-terminal direction, a Domain 3 comprising: i. a sub-Domain (3A), which comprises a cysteine-containing peptide comprising the amino acid sequence of SEQ ID NO:39; and ii. a sub-Domain (3B), which comprises a polypeptide portion of an IgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fc Domain; and n: (a) said polypeptide portions of the IgG Fc domains of said first and third polypeptide chain form said IgG Fc Domain; (b) said VL Domain of said first polypeptide chain and said VH Domain of said second polypeptide chain form a monovalent Antigen Binding Domain capable of specific binding to said epitope of gpA33; and (c) said VH Domain of said first polypeptide chain and said VL Domain of said second polypeptide chain form a lent Antigen Binding Domain capable of ic binding to said epitope of CD3.

Claim 4. A bi-specific Fc diabody, wherein said bi-specific Fc diabody is e of specific binding to an epitope of gpA33 and to an epitope of CD3, and possesses an IgG Fc Domain, n the bi-specific Fc diabody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein said first and second polypeptide chains are covalently bonded to one another and said first and third polypeptide chains are covalently bonded to one another, and wherein: A. the first polypeptide chain comprises, in the N-terminal to inal direction: i. a Domain 3, comprising a sub-Domain (3A), which comprises a cysteine-containing peptide comprising the amino acid sequence of SEQ ID NO:39 and a sub-Domain (3B), which comprises a polypeptide portion of an IgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fc Domain; ii. a Domain 1, sing a sub-Domain (1A), which comprises a VL Domain of a onal antibody e of binding to gpA33 (VLgpA33) comprising the amino acid sequence of SEQ ID NO:26 and a main (1B), which comprises a VH Domain of a monoclonal antibody capable of g to CD3 (VHCD3) comprising the amino acid sequence of SEQ ID NO:25, wherein said sub-Domains (1A) and (1B) are separated from one another by a peptide linker comprising the amino acid sequence of SEQ ID NO:1; wherein said Domains 1 and 3 are separated from one another by a spacer peptide comprising the amino acid sequence of SEQ ID NO:38; iii. a Domain 2, wherein said Domain 2 is an E-coil Domain comprising the amino acid sequence of SEQ ID NO:3 or a K-coil Domain comprising the amino acid sequence of SEQ ID NO:4, wherein said Domain 2 is separated from said Domain 1 by a peptide linker sing the amino acid sequence of SEQ ID NO:2; and B. the second polypeptide chain comprises, in the N-terminal to C-terminal direction: i. a Domain 1, comprising a sub-Domain (1A), which comprises a VL Domain of a monoclonal antibody capable of binding to CD3 (VLCD3) comprising the amino acid sequence of SEQ ID NO:5; and a sub-Domain (1B), which ses a VH Domain of a monoclonal antibody e of binding to gpA33 (VHgpA33) comprising the amino acid sequence of SEQ ID NO:27; wherein said sub-Domains (1A) and (1B) are ted from one another by a peptide linker comprising the amino acid sequence of SEQ ID NO:1; ii. a Domain 2, wherein said Domain 2 is a K-coil Domain comprising the amino acid sequence of SEQ ID NO:4 or an E-coil Domain comprising the amino acid sequence of SEQ ID NO:3, wherein said Domain 2 is separated from said Domain 1 by a peptide linker comprising the amino acid sequence of SEQ ID NO:2; and wherein said Domain 2 of said first polypeptide chain and said Domain 2 of said second polypeptide chain are not both E-coil Domains or both K-coil Domains; and C. the third polypeptide chain comprises, in the N-terminal to C-terminal ion, a Domain 3 comprising: (1) a main (3A), which comprises a cysteine-containing peptide comprising the amino acid sequence of SEQ ID NO:39; and (2) a sub-Domain (3B), which comprises a polypeptide portion of an IgG Fc Domain having CH2 and CH3 domains of an IgG immunoglobulin Fc Domain; and wherein: (a) said polypeptide portions of the IgG Fc domains of said first and third polypeptide chain form said IgG Fc Domain; (b) said VL Domain of said first polypeptide chain and said VH Domain of said second polypeptide chain form a monovalent Antigen Binding Domain capable of specific binding to said e of gpA33; and (c) said VH Domain of said first polypeptide chain and said VL Domain of said second polypeptide chain form a monovalent Antigen Binding Domain capable of specific binding to said epitope of CD3.

Claim 5. The cific Fc diabody of any one of claims 3-4, wherein said sub-Domain (3B) of said first polypeptide chain comprises a sequence different from that of said sub-Domain (3B) of said third ptide chain, wherein functionality of said IgG Fc Domain is retained.

Claim 6. The bi-specific Fc diabody of claim 5, n said sub-Domain (3B) of said first polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, and said sub-Domain (3B) of said third polypeptide chain comprises the amino acid sequence of SEQ ID NO:41.

Claim 7. The bi-specific Fc diabody of claim 5, wherein said sub-Domain (3B) of said first polypeptide chain comprises the amino acid sequence of SEQ ID NO:41, and said sub-Domain (3B) of said third polypeptide chain comprises the amino acid sequence of SEQ ID NO:40.

Claim 8. The bi-specific Fc diabody of any one of claims 3-7, wherein said Domain 3 of said first polypeptide chain and/or said Domain 3 of said third polypeptide chain comprises a variant CH2-CH3 sequence that exhibits altered g to an Fcγ receptor.

Claim 9. The bi-specific diabody of any one of claims 1-2 or the bi-specific Fc diabody of any one of claims 3-8, wherein said Domain 2 of said first polypeptide chain comprises an E-coil sing the amino acid sequence of SEQ ID NO:3, and said Domain 2 of said second polypeptide chain comprises a K-coil comprising the amino acid sequence of SEQ ID NO:4.

Claim 10. The cific diabody of any one of claims 1-2 or the bi-specific Fc diabody of any one of claims 3-8, wherein said Domain 2 of said first polypeptide chain comprises a K-coil comprising the amino acid sequence of SEQ ID NO:4, and said Domain 2 of said second polypeptide chain comprises an E-coil comprising the amino acid sequence of SEQ ID NO:3.

Claim 11. A bi-specific y, wherein said bi-specific y is capable of specific binding to an epitope of CD3 and to an epitope of gpA33, wherein said bi-specific diabody comprises: (1) a first ptide chain comprising the amino acid sequence of SEQ ID NO:28, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:30; or (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:35, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:30; wherein said first and said second polypeptide chains are covalently bonded to one another by a disulfide bond.

Claim 12. A bi-specific Fc diabody, wherein said bi-specific Fc diabody is e of specific binding to an epitope of CD3 and to an epitope of gpA33, and possesses an IgG Fc Domain, wherein said bi-specific Fc diabody ses: (1) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:42, a second ptide chain comprising the amino acid ce of SEQ ID NO:44, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:46; or (2) a first polypeptide chain comprising the amino acid sequence of SEQ ID NO:48, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO:28, and a third polypeptide chain comprising the amino acid sequence of SEQ ID NO:46; wherein said first and said second polypeptide chains are ntly bonded to one another by a first disulfide bond and said first and third polypeptide chains are covalently bonded to one another by a second disulfide bond.

Claim 13. A pharmaceutical composition comprising the bi-specific diabody of any one of claims 1-2 or 9-11 or the bi-specific Fc diabody of any one of claims 3-10 or 12; and a physiologically acceptable carrier.

Claim 14. Use of the bispecific diabody of any one of claims 1-2 or 9-11, or of the bispecific Fc diabody of any one of claims 3-10 or 12, or of the pharmaceutical composition of claim 13, in the manufacture of a ment for treating a cancer characterized by the expression of gpA33.

Claim 15. The use of claim 14, wherein said cancer is colorectal cancer, colon cancer, gastric cancer or pancreatic cancer.

Claim 16. An in vitro cell that expresses said first polypeptide chain and said second ptide chain of the bi-specific diabody of any one of claims 1-2 or 9-11, or of the bi-specific Fc diabody of any one of claims 3-10 or 12.

Claim 17. A cleotide that encodes said first polypeptide chain and said second polypeptide chain of the bi-specific diabody of any one of claims 1-2 or 9-11, or of the bi-specific Fc diabody of any one of claims 3-10 or 12. 1/20 - VL K-coil {or E-coil} Polypeptide Chain 1 COOH WNL 'I-C Linker 2 W Linker 2 «W4/! ptide Chain 2 COOH NW—mC E—coil (or K—coil) fl Assembled Diabody COOH Figure 1 2/20 NHL, Polypeptide Chain 1 VL COOH CH3 K»coi| CH2 (or E' coil) Peptide 1 c~c~WV\NVL-c VH Linker 2 Linker S Linker 2 COOH WWWaCW A ptide Chain 2 VH E-coil ;) (or K—coil) Poiypeptide Chain 3 COOH Peptide 1 ”Stir“. c.- c.

CH2 NH? Assembled Fc Diabody (Version 1) “NW\\\ Figure 2A 3/20 Peptide 1 CH2 NH) - c- c «nullllllllmllnum Polypeptide Chain 1 (or E-c.oi|) comm-C Linker ptide Chain 2 E-coil (or K-coil) Peptide i NH, C- C m5:;:-., Poiypeptide Chain 3 --------------Lam” (OOH é Peptide 1 Assembled Fc Diabody (Version 2) Figure 28 4/20 shCD3 e/gpA33 detection °°°°° -I:- gpA33 x CD3 DART-1 400000 -'|'- Control DART 300000 200000 100000 0.001 0.01 0.1 1 10 Conc (pg/ml) Figure 3 /20 Colon CSLC Human PBMC - E:T= 25:1 40 gpA33 x cos DART-1 (%) + Control DART Cytotoxicity 302010 1o—2 10-1 1o0 101 1o2 103 Concentration (nglml) Figure 4A Co|0205 Colorectal Cancer Activated T Cells - :1 -u- gpA33 x CD3 DART-1 40 u (%) + Control DART Cytotoxicity 30201o 1o—2 100 1o2 10‘ Concentration (nglml) Figure 4B ASPC-l - Pancreatic Cancer Activated T Cells - E:T=10:1 {I- gpA33 x CD3 DART-1 (%) + Control DART Cytotoxicity wONO.- O '2 10 ° 10 2 10‘ Concentration (nglml) Figure 4C 6/20 Co|0205 + C08 T Cells -D- gpA33 x CD3 DART-1 150 + l DART C025 '2 10° 102 10“ con(nglml) Figure. 5A ASPC-1 + CD8 T Cells -D- gpA33xCD3 DART-1 150 1— Control DART In 100 '2 10° 102 104 con(nglml) Figure. 53 CD8 T-cells Alone '5' gpA33 x CD3 DART-1 150 -fi- Control DART C025 '2 10° 102 10‘ con(nglml) Figure 5C 7/20 Co|0205 + CD4 T Cells 300 -D- gpA33xCD3 DART-1 250 + l DART '2 10° 102 104 con(nglml) Figure. 5D ASPC-1 + CD4 T Cells 300 -D- gpA33 x CD3 DART-1 250 -fi- Control DART E 200 R 150 '2 10° 102 104 con(nglml) Figure_ 5E CD4 T cells alone 300 -D- gpA33x CD3 DART-1 Control DART 250 + E 200 til 150 '2 10° 102 10‘ con(ng/m|) Figure 5F 8/20 SW948 + Resting T cells (LDH) E:T=10:1 + gpA33 x CD3 DART-2 -§O -D- gpA33 x CD3 DART-1 (‘70) + Control DART Cytotoxicity 0:) ONO—L O '3 10'2 10'1 10° 101 102 103 con(ng/ml) Figure 6A Co|0205 + Resting T cells (LDH) E:T=10:1 + gpA33 x cm DART-2 .5O -D- gpA33 x CD3 DART-1 ("/o) + l DART Cytotoxicity w ONO_I C '3 10'2 10'1 10° 101 102 103 con(ng/ml) Figure GB 9/20 ColoZOS-Luc + Resting T-cells (LUM) E:T=10:1 23000 19000 Luminescence _L_L —\Ol 00 CO CO + Control DART -:I- gpA33 x cos DART-1 -o- gpA33 x cos DART-2 7000 3000 -1000 '3 10'2 10'1 10° 101 102 103 tration (nglml) Figure 6C HCT116 (A33-ve) + Resting T cells (LDH) E:T=10:1 -O- gpA33 x CD3 DART-2 40 -D- gpA33 x CD3 DART-1 (%) 4'- Control DART Cytotoxicity 302010 O a—a—a—a—B—n—Q—I‘é ‘3 10'2 10‘1 10° 101 102 103 104 concentration(nglml) Figure 6D /20 Co|0205-Luc + Human PBMC (LDH) E:T=30:1 24h + l DART '0- gpA33 x CD3 DART-2 Cytotoxicity -D- NO gpA33 x CD3 DART-2 wIABD -°- gpA33 x CD3 DART-2 w/Fc “3 10“ 10'3 10'2 10"| 10° 101 102 Figure 7A Co|0205-Luc + Human PBMC (LUM) E:T=30:1 24h 30000 25000 + Control DART 8 20000 E '0' gpA33 x CD3 DART-2 wch 015000 a: -l:l- gpA33 x cm DART-2 w/ABD .E 1 0000 E gpA33 x CD3 DART-2 3 5000 .5000 105 104 10'3 10'2 10'1 10° 101 102 Figure 7B 11/20 ColoZOS-Luc + Cyno PBMC (LDH) :1 24h 0')O + Control DART gpA33 x CD3 DART-2 wch (%) -D- gpA33 x CD3 DART-2 w/ABD Cytotoxicity MOO-#0!OCO gpA33 x CD3 DART-2 ‘5 10" 10'3 10'2 10'1 10° 101 102 Figure 7C ColoZOS-Luc + Cyno PBMC (LUM) E:T=30:1 24h 15000 4- Control DART 12000 '0' gpA33 x CD3 DART-2 w/Fc Luminescence -D- gpA33 x CD3 DART-2 wIABD 9000 gpA33 x CD3 DART-2 6000 3000 ‘5 104 10'3 10'2 10'1 10° 101 102 Figure 7D 12/20 3000 g 2000 g 1000 0 10 20 30 Study Day + Vehicle -V- CD3 DART-1 (0.02 mg/kg) -A- gpA33xCD3 DART-1 (0.1 mglkg) E} gpA33xCD3 DART-1 (0.5 mg/kg) Figure 8 13/20 Figure 9A: Day 2 g Data (Vehicle): Figure QB: Day 2 Imaging Data (gpA33 x CD3 DART-1 (0.5 mg/kg)): 14/20 Figure 9C: Day 12 Imaging Data le): Figure 9D: Day 12 Imaging Data (gpA33 x CD3 DART-1 (0.5 mg/kg)): /20 (mm3) 600 Volume 400 Tumor 300 iuyp; .- 0 1o 20 so 40 50 Study Day + Vehicle -I- l DART (0.5 mglkg) D- gpA33xCDB DART-1 (0.5 mglkg) fi- gpA33xCD3 DART-1 (0.1 mglkg) V- gpA33xCD3 DART-1 (0.02 mglkg) Figure 10 16/20 4000 3500 (mms) 3000 2530 Volume 2000 Tumor 1500 1000 so 20 30 40 so sway Day ¢++4IH> Vehicte gpA33xCDs DART 2 wch version 1 (3.1 mglkglday) gpA33xCD3 DART 2 wch version 1 (1.5 mglkglday) gpA33xCD3 DART 2 311ch n 1 (0.75 mgikglday) gpA33xCD3 DART 2 WIT-“c version 1 (0.375 mglkglday) gpA33xC03 DART 2 wife version 1 (0.5 mgikglday) Figure 11 17/20 FEE. 95282 88:... ) 8&3 8x35 8%? 8&9: age me v0.2 80.8 m 80.8 we gpA33 x 003 DART-2 wIFc version 1 Figure 12 18/20 (nglmL) tration 96 120 144 Time (hr) [:3 gpA33 x cm DART-2 (Monkey 1) O gpA33 x CD3 DART-2 (Monkey 2) I gpA33 x C03 DART-2 wch Version 1 (Monkey 3) Figure 13 19/20 DART-2 w/Fc Version 1 Binding of Human CD3 Figure 14A «333 $3 33 3:33 3133 3333 33333 3 DART-2 wIFc Version 1 g of Cynomolgus CD3 Figures 14B /20 NE} . m i :fékMM £33}- g; ~§§ i} 58%} 3:3} 3:33 353$} i‘iwm & DART-2 w/Fc Version 1 Binding of Human gpA33 Figures 15A “gs-s DART-2 w/Fc Version 1 Binding of lgus gpA33 Figures 153 18189543_1 CE LISTING <110> MacroGenics, Inc.

Moore, Paul Li, Jonathan Chen, Francine n, Leslie Shah, Kalpana Bonvini, Ezio <120> Bi-Specific Monovalent Diabodies That Are Capable Of Binding to gpA33 And CD3, And Uses Thereof <130> 1301.0112PCT <150> US 61/869,528 <151> 201323 <150> US 61/907,691 <151> 201322 <150> EP 13198859 <151> 201320 <160> 57 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 ptide <400> 1 Gly Gly Gly Ser Gly Gly Gly Gly 1 5 <210> 2 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Linker 2 Polypeptide <400> 2 Page 1 18189543_1 Gly Gly Cys Gly Gly Gly 1 5 <210> 3 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> E-Coil Domain <400> 3 Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val 1 5 10 15 Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 25 <210> 4 <211> 28 <212> PRT <213> cial Sequence <220> <223> K-Coil Domain <400> 4 Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val 1 5 10 15 Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 25 <210> 5 <211> 110 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(110) <223> Light Chain Variable Domain of Murine Anti-CD3 Antibody <400> 5 Page 2 18189543_1 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 110 <210> 6 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(14) <223> CDR1 of Light Chain Variable Domain of Murine Anti-CD3 dy <400> 6 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 7 <211> 7 <212> PRT <213> Mus musculus Page 3 18189543_1 <220> <221> MISC_FEATURE <222> (1)..(7) <223> CDR2 of Light Chain Variable Domain of Murine Anti-CD3 Antibody <400> 7 Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 8 <211> 9 <212> PRT <213> Mus us <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR3 of Light Chain Variable Domain of Murine Anti-CD3 Antibody <400> 8 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 9 <211> 125 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(125) <223> Heavy Chain Variable Domain of Murine Anti-CD3 Antibody <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Page 4 18189543_1 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 10 <211> 5 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(5) <223> CDR1 of Heavy Chain Variable Domain of Murine Anti-CD3 Antibody <400> 10 Thr Tyr Ala Met Asn 1 5 <210> 11 <211> 19 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(19) <223> CDR2 of Heavy Chain Variable Domain of Murine D3 Antibody <400> 11 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Page 5 18189543_1 1 5 10 15 Val Lys Asp <210> 12 <211> 14 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(14) <223> CDR3 of Heavy Chain le Domain of Murine Anti-CD3 Antibody <400> 12 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 13 <211> 106 <212> PRT <213> Mus musculus <220> <221> misc_feature <223> Light Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 13 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Page 6 18189543_1 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 14 <211> 10 <212> PRT <213> Mus musculus <220> <221> misc_feature <222> (1)..(10) <223> CDR1 of Light Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 14 Ser Ala Arg Ser Ser Ile Ser Phe Met Tyr 1 5 10 <210> 15 <211> 7 <212> PRT <213> Mus us <220> <221> misc_feature <222> (1)..(7) <223> CDR2 of Light Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 15 Asp Thr Ser Asn Leu Ala Ser 1 5 <210> 16 <211> 9 <212> PRT <213> Mus musculus Page 7 18189543_1 <220> <221> MISC_FEATURE <222> (1)..(9) <223> CDR3 of Light Chain Variable Domain of Murine Anti-gpA33 dy <400> 16 Gln Gln Trp Ser Ser Tyr Pro Leu Thr 1 5 <210> 17 <211> 119 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(119) <223> Heavy Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Gly Ser 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe Asp Val Trp Gly Ala Gly 100 105 110 Page 8 18189543_1 Thr Thr Val Thr Val Ser Ser <210> 18 <211> 5 <212> PRT <213> Mus musculus <220> <221> misc_feature <223> CDR1 of Heavy Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 18 Gly Ser Trp Met Asn 1 5 <210> 19 <211> 17 <212> PRT <213> Mus musculus <220> <221> misc_feature <223> CDR2 of Heavy Chain Variable Domain of Murine pA33 Antibody <400> 19 Arg Ile Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15 <210> 20 <211> 10 <212> PRT <213> Mus musculus <220> <221> misc_feature <223> CDR3 of Heavy Chain Variable Domain of Murine Anti-gpA33 Antibody <400> 20 Page 9 43_1 Ile Tyr Gly Asn Asn Val Tyr Phe Asp Val 1 5 10 <210> 21 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-1 <400> 21 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Gln Gln Ser Gly Pro Glu 115 120 125 Leu Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Ser Gly Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly 145 150 155 160 Page 10 18189543_1 Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr 165 170 175 Asn Tyr Asn Gly Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys 180 185 190 Ser Ser Thr Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Val Asp 195 200 205 Ser Ala Val Tyr Phe Cys Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe 210 215 220 Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Gly Gly Cys 225 230 235 240 Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 245 250 255 Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 270 <210> 22 <211> 813 <212> DNA <213> cial Sequence <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-1 <400> 22 caggctgtgg tgactcagga gccttcactg accgtgtccc gaac tgtgaccctg 60 acatgcagat ccagcacagg gacc acatctaact acgccaattg ggtgcagcag 120 aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 gagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca caaaactgac tgtgctggga ggtggtggat ccggcggagg tggacaggtc 360 cagctgcagc agtctggacc tgagctggtg aagcctgggg cctcagtgaa gatttcctgc 420 Page 11 18189543_1 aaagcttcag gctacacatt cagtggctct tggatgaact gggtgaagca gaggcctgga 480 cagggtcttg agtggattgg ctac cctggagatg gagaaactaa ctacaatggg 540 aagg acaaggccac actgactgca gacaaatcat ccaccacagc ctacatggag 600 ctcagcagcc tgacctctgt tgcg gtctatttct gtgcaagaat ctatggtaat 660 tact tcgatgtctg gggcgcaggg accacggtca ccgtgtcttc cggaggatgt 720 ggcggtggag aagtggccgc actggagaaa gaggttgctg ctttggagaa ggaggtcgct 780 gcacttgaaa aggaggtcgc agccctggag aaa 813 <210> 23 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-1 <400> 23 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Met Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu Ile Tyr 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Leu Lys Arg Gly Gly Gly Ser Gly 100 105 110 Page 12 18189543_1 Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 115 120 125 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 130 135 140 Asn Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 145 150 155 160 Glu Trp Val Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr 165 170 175 Tyr Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser 180 185 190 Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr 195 200 205 Ala Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val 210 215 220 Ser Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225 230 235 240 Gly Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala 245 250 255 Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu 260 265 270 Lys Glu <210> 24 <211> 822 <212> DNA <213> cial Sequence <220> Page 13 43_1 <223> Nucleic Acid Molecule Encoding Second Polypeptide Chain of DART-1 <400> 24 gttc tcacccagtc tccagcaatc atgtctgcat ctccagggga cacc 60 atgacctgca gtgccaggtc aagtataagt ttcatgtact ggtaccagca gaagccagga 120 tcctccccca gactcctgat ttatgacaca tccaacctgg cttctggagt ccctgttcgc 180 ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagccgaat ggaggctgaa 240 gatgctgcca cttattactg ccagcagtgg agtagttacc cactcacgtt cggttctggg 300 accaagctgg agctgaaacg gggtggagga tccggcggag gcggagaggt gcagctggtg 360 gagtctgggg gaggcttggt ccagcctgga gggtccctga gactctcctg tgcagcctct 420 ggattcacct cata cgctatgaat tgggtccgcc aggctccagg gaaggggctg 480 gagtgggttg caaggatcag gtccaagtac aacaattatg caacctacta tgccgactct 540 gtgaaggata ccat ctcaagagat gattcaaaga actcactgta tctgcaaatg 600 aacagcctga aaaccgagga cacggccgtg tattactgtg tgagacacgg taacttcggc 660 aattcttacg tgtcttggtt tgcttattgg ggacagggga cactggtgac ttcc 720 ggaggatgtg gcggtggaaa agtggccgca gaga aagttgctgc tttgaaagag 780 gccg cacttaagga aaaggtcgca gccctgaaag ag 822 <210> 25 <211> 125 <212> PRT <213> Mus musculus <220> <221> MISC_FEATURE <222> (1)..(125) <223> Heavy Chain Variable Domain of Anti-CD3 Antibody <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 25 30 Page 14 18189543_1 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 26 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Light Chain Variable Domain of Humanized pA33 Antibody <400> 26 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Ala Glu Page 15 18189543_1 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 27 <211> 119 <212> PRT <213> Artificial ce <220> <223> Heavy Chain Variable Domain of Humanized Anti-gpA33 Antibody <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Ser 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Page 16 43_1 <210> 28 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-2 <400> 28 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Gly Ser Trp Met Asn Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr Page 17 18189543_1 165 170 175 Asn Tyr Asn Gly Lys Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe 210 215 220 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Cys 225 230 235 240 Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 245 250 255 Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys 260 265 270 <210> 29 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-2 <400> 29 caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg 60 acatgcagat cagg gacc acatctaact attg ggtgcagcag 120 aagccaggac aggcaccaag gatc gggggtacaa acaaaagggc tccctggacc 180 cctgcacggt gaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca caaaactgac tgtgctggga ggtggtggat ccggcggagg tggacaggtc 360 cagctggtcc agagcggggc cgaagtcaaa aaacccggag caagcgtgaa ggtctcctgc 420 aaagcatcag gctatacatt tacaggcagc tggatgaact gggtgaggca ggctccagga 480 Page 18 18189543_1 cagggactgg agtggatcgg gcgcatctac cctggagacg gcgaaactaa ctataatgga 540 aagttcaaag accgagtgac catcacagcc gataagtcta ctagtaccgc ctacatggag 600 tccc tgcggtctga cgcc tatt gcgctagaat ttacggaaac 660 aatgtctatt ttgacgtgtg ggggcaggga acaactgtga ctgtctcctc cggaggatgt 720 ggcggtggag aagtggccgc actggagaaa gaggttgctg ctttggagaa ggaggtcgct 780 gcacttgaaa aggaggtcgc agccctggag aaa 813 <210> 30 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-2 <400> 30 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 40 45 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Page 19 18189543_1 115 120 125 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 130 135 140 Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 145 150 155 160 Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr 165 170 175 Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 180 185 190 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala 195 200 205 Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser 210 215 220 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 225 230 235 240 Gly Cys Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala 245 250 255 Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys 260 265 270 <210> 31 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> c Acid Molecule Encoding Second Polypeptide Chain of DART-2 <400> 31 Page 20 18189543_1 gacattcagc tgactcagtc cccctctttt ctgtccgcat ccgtcggaga tcgagtgact 60 attacttgct ctgctaggtc ctcaatcagc ttcatgtact ggtatcagca cggc 120 aaagcaccta tgat ctacgacaca agcaacctgg cctccggggt gccatctcgg 180 ttctctggca gtgggtcagg aactgagttt accctgacaa ttagctccct ggaggctgaa 240 gatgccgcta cctactattg ccagcagtgg agcagctatc ctctgacctt gggg 300 actaaactgg aaatcaaggg tggaggatcc ggcggcggag gcgaggtgca gctggtggag 360 tctgggggag gcttggtcca gcctggaggg tccctgagac tctcctgtgc tgga 420 ttcaccttca gcacatacgc tatgaattgg gtccgccagg ctccagggaa ggggctggag 480 tgggttggaa ggtc caagtacaac aattatgcaa cctactatgc cgactctgtg 540 aaggatagat tcaccatctc aagagatgat tcaaagaact cactgtatct gcaaatgaac 600 agcctgaaaa ccgaggacac ggccgtgtat tactgtgtga gacacggtaa cttcggcaat 660 tcttacgtgt cttggtttgc ttattgggga caggggacac tggtgactgt gtcttccgga 720 ggatgtggcg gtggaaaagt ggccgcactg aaggagaaag ttgctgcttt gaaagagaag 780 gtcgccgcac aaaa ggtcgcagcc gag 819 <210> 32 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 Polypeptide <400> 32 Gly Gly Gly Ser <210> 33 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 Polypeptide <400> 33 Page 21 18189543_1 Gly Gly Gly Asn Ser 1 5 <210> 34 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Albumin g Domain <400> 34 Leu Ala Gln Ala Lys Glu Ala Ala Ile Arg Glu Leu Asp Lys Tyr Gly 1 5 10 15 Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys Ser Ala Glu 25 30 Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu Pro 40 45 <210> 35 <211> 321 <212> PRT <213> Artificial Sequence <220> <223> First Polypeptide Chain of DART-2 w/ABD <400> 35 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Page 22 43_1 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Gly Ser Trp Met Asn Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr 165 170 175 Asn Tyr Asn Gly Lys Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe 210 215 220 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Cys 225 230 235 240 Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 245 250 255 Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Gly 260 265 270 Page 23 18189543_1 Gly Gly Ser Leu Ala Gln Ala Lys Glu Ala Ala Ile Arg Glu Leu Asp 275 280 285 Lys Tyr Gly Val Ser Asp Tyr Tyr Lys Asn Leu Ile Asp Asn Ala Lys 290 295 300 Ser Ala Glu Gly Val Lys Ala Leu Ile Asp Glu Ile Leu Ala Ala Leu 305 310 315 320 <210> 36 <211> 963 <212> DNA <213> cial Sequence <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-2 w/ABD <400> 36 caggctgtgg tgactcagga gccttcactg accgtgtccc gaac tgtgaccctg 60 acatgcagat ccagcacagg cgcagtgacc aact acgccaattg ggtgcagcag 120 aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca tgac tgtgctggga gggggtggat ccggcggagg tggacaggtc 360 cagctggtcc agagcggggc cgaagtcaaa aaacccggag caagcgtgaa ggtctcctgc 420 aaagcatcag gctatacatt tacaggcagc tggatgaact gggtgaggca ggctccagga 480 cagggactgg agtggatcgg gcgcatctac cctggagacg gcgaaactaa tgga 540 aagttcaaag accgagtgac catcacagcc gataagtcta ccgc ctacatggag 600 ctgagctccc ctga agataccgcc gtctactatt gcgctagaat ttacggaaac 660 aatgtctatt ttgacgtgtg ggggcaggga acaactgtga ctgtctcctc cggaggatgt 720 ggcggtggag aagtggccgc actggagaaa gaggttgctg agaa ggaggtcgct 780 Page 24 18189543_1 gcacttgaaa aggaggtcgc agccctggag aaaggcggcg ggtctctggc ccaggcaaaa 840 gaggcagcca tccgcgaact ggataaatat ggcgtgagcg attattataa gaacctgatt 900 gacaacgcaa aatccgcgga aggcgtgaaa gcactgattg atgaaattct ggccgccctg 960 cct 963 <210> 37 <211> 5 <212> PRT <213> Artificial ce <220> <223> Linker 4 Polypeptide <400> 37 Ala Pro Ser Ser Ser 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker 4 Polypeptide <400> 38 Ala Pro Ser Ser Ser Pro Met Glu 1 5 <210> 39 <211> 10 <212> PRT <213> cial Sequence <220> <223> Peptide 1 <400> 39 Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 Page 25 43_1 <210> 40 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> CH2 and CH3 Domains of Modified Fc Domain of First DART Polypeptide Chain <400> 40 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 145 150 155 160 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Page 26 43_1 165 170 175 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 41 <211> 217 <212> PRT <213> Artificial Sequence <220> <223> CH2 and CH3 Domains of Modified Fc Domain of Third DART Polypeptide Chain <400> 41 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 1 5 10 15 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 25 30 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 40 45 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 65 70 75 80 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90 95 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 100 105 110 Page 27 18189543_1 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 115 120 125 Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro 130 135 140 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 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 Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 180 185 190 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Tyr Thr Gln 195 200 205 Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 <210> 42 <211> 503 <212> PRT <213> Artificial Sequence <220> <223> First ptide Chain of DART-2 w/Fc Version 1 Construct <400> 42 Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 40 45 Page 28 43_1 Asp Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly 100 105 110 Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 115 120 125 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser 130 135 140 Thr Tyr Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 145 150 155 160 Trp Val Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr 165 170 175 Ala Asp Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys 180 185 190 Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala 195 200 205 Val Tyr Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser 210 215 220 Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly 225 230 235 240 Gly Cys Gly Gly Gly Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala 245 250 255 Page 29 43_1 Leu Glu Lys Glu Val Ala Ala Leu Glu Lys Glu Val Ala Ala Leu Glu 260 265 270 Lys Gly Gly Gly Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 275 280 285 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 290 295 300 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 305 310 315 320 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 325 330 335 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 340 345 350 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 355 360 365 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 370 375 380 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 385 390 395 400 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 405 410 415 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 420 425 430 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 435 440 445 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 450 455 460 Page 30 18189543_1 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 465 470 475 480 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 485 490 495 Leu Ser Leu Ser Pro Gly Lys <210> 43 <211> 1509 <212> DNA <213> Artificial Sequence <220> <223> Nucleic Acid Molecule Encoding First Polypeptide Chain of DART-2 w/Fc n 1 Construct <400> 43 gacattcagc tgactcagtc cccctctttt ctgtccgcat ccgtcggaga tcgagtgact 60 attacttgct ctgctaggtc ctcaatcagc tact ggtatcagca gaagcccggc 120 aaagcaccta agctgctgat ctacgacaca agcaacctgg cctccggggt gccatctcgg 180 ttctctggca gtgggtcagg aactgagttt accctgacaa ttagctccct ggaggctgaa 240 gatgccgcta attg ccagcagtgg agcagctatc ctctgacctt cggacagggg 300 actaaactgg aaatcaaggg tggaggatcc ggcggcggag gcgaggtgca gctggtggag 360 tctgggggag tcca gcctggaggg tccctgagac tctcctgtgc agcctctgga 420 ttcaccttca gcacatacgc tatgaattgg gtccgccagg ctccagggaa ggggctggag 480 tgggttggaa ggatcaggtc caagtacaac aattatgcaa atgc cgactctgtg 540 aaggatagat tcaccatctc aagagatgat tcaaagaact cactgtatct gcaaatgaac 600 aaaa ccgaggacac ggccgtgtat tactgtgtga gtaa cttcggcaat 660 tcttacgtgt cttggtttgc ttattgggga caggggacac tggtgactgt gtcttccgga 720 ggatgtggcg gtggagaagt ggccgcactg gagaaagagg ttgctgcttt ggagaaggag 780 gtcgctgcac agga ggtcgcagcc ctggagaaag gcggcgggga caaaactcac 840 acatgcccac cagc acctgaagcc gcggggggac cgtcagtctt cctcttcccc 900 Page 31 18189543_1 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 960 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 1020 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 1080 accg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1140 aacaaagccc tcccagcccc catcgagaaa accatctcca aagg gcagccccga 1200 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1260 tgcc tggtcaaagg tccc agcgacatcg ccgtggagtg ggagagcaat 1320 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cttc 1380 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1440 tgctccgtga tgcatgaggc tctgcacaac cactacacgc gcct ctccctgtct 1500 ccgggtaaa 1509 <210> 44 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Second Polypeptide Chain of DART-2 w/Fc Version 1 Construct <400> 44 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Page 32 43_1 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gly Gly 100 105 110 Gly Ser Gly Gly Gly Gly Gln Val Gln Leu Val Gln Ser Gly Ala Glu 115 120 125 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 130 135 140 Tyr Thr Phe Thr Gly Ser Trp Met Asn Trp Val Arg Gln Ala Pro Gly 145 150 155 160 Gln Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Glu Thr 165 170 175 Asn Tyr Asn Gly Lys Phe Lys Asp Arg Val Thr Ile Thr Ala Asp Lys 180 185 190 Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 195 200 205 Thr Ala Val Tyr Tyr Cys Ala Arg Ile Tyr Gly Asn Asn Val Tyr Phe 210 215 220 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Cys 225 230 235 240 Gly Gly Gly Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys 245 250 255 Glu Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 260 265 270 <210> 45 <211> 813 <212> DNA Page 33 18189543_1 <213> Artificial Sequence <220> <223> Nucleic Acid le Encoding Second Polypeptide Chain of DART-2 w/Fc Version 1 Construct <400> 45 caggctgtgg tgactcagga gccttcactg accgtgtccc caggcggaac tgtgaccctg 60 agat ccagcacagg cgcagtgacc aact acgccaattg ggtgcagcag 120 aagccaggac aggcaccaag gggcctgatc gggggtacaa acaaaagggc tccctggacc 180 cctgcacggt tttctggaag tctgctgggc ggaaaggccg ctctgactat taccggggca 240 caggccgagg acgaagccga ttactattgt gctctgtggt atagcaatct gtgggtgttc 300 gggggtggca caaaactgac tgtgctggga gggggtggat gagg tggacaggtc 360 cagctggtcc agagcggggc cgaagtcaaa aaacccggag caagcgtgaa ggtctcctgc 420 aaagcatcag gctatacatt tacaggcagc tggatgaact gggtgaggca ggctccagga 480 cagggactgg agtggatcgg gcgcatctac cctggagacg gcgaaactaa ctataatgga 540 aagttcaaag accgagtgac catcacagcc gataagtcta ctagtaccgc ctacatggag 600 ctgagctccc tgcggtctga agataccgcc gtctactatt gcgctagaat ttacggaaac 660 aatgtctatt ttgacgtgtg ggggcaggga acaactgtga ctgtctcctc cggaggatgt 720 ggcggtggaa aagtggccgc actgaaggag aaagttgctg ctttgaaaga gaaggtcgcc 780 aagg aaaaggtcgc agccctgaaa gag 813 <210> 46 <211> 227 <212> PRT <213> Artificial ce <220> <223> Third ptide Chain of DART-2 w/Fc Version 1 Construct <400> 46 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 25 30 Page 34 43_1 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn Arg Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Page 35 18189543_1 <210> 47 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid Molecule Encoding Third Polypeptide Chain of DART-2 w/Fc Version 1 Construct <400> 47 gacaaaactc acacatgccc accgtgccca gcacctgaag ccgcgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg atgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct aagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca cacc ccat cccgggagga gatgaccaag 420 aaccaggtca gcctgagttg cgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca agcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcgt cagcaagctc gaca agagcaggtg gcagcagggg 600 aacgtcttct ccgt gatgcatgag gctctgcaca accgctacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 48 <211> 508 <212> PRT <213> cial Sequence <220> <223> First Polypeptide Chain of DART-2 w/Fc Version 2 Construct <400> 48 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Page 36 43_1 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys Ala Pro Ser Ser Ser Pro Met Glu Asp Ile Gln Leu Thr Page 37 43_1 225 230 235 240 Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile 245 250 255 Thr Cys Ser Ala Arg Ser Ser Ile Ser Phe Met Tyr Trp Tyr Gln Gln 260 265 270 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Thr Ser Asn Leu 275 280 285 Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu 290 295 300 Phe Thr Leu Thr Ile Ser Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr 305 310 315 320 Tyr Cys Gln Gln Trp Ser Ser Tyr Pro Leu Thr Phe Gly Gln Gly Thr 325 330 335 Lys Leu Glu Ile Lys Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln 340 345 350 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 355 360 365 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr Ala Met Asn 370 375 380 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg Ile 385 390 395 400 Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val Lys 405 410 415 Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu 420 425 430 Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Val Page 38 18189543_1 435 440 445 Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr Trp 450 455 460 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Cys Gly Gly Gly 465 470 475 480 Lys Val Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu Lys Val 485 490 495 Ala Ala Leu Lys Glu Lys Val Ala Ala Leu Lys Glu 500 505 <210> 49 <211> 198 <212> PRT <213> Macaca fascicularis <220> <221> EATURE <222> (1)..(198) <223> V35 allele (FN18+) of CD3 epsilon <400> 49 Met Gln Ser Gly Thr Arg Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Ile Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr 25 30 Gln Thr Pro Tyr Gln Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 40 45 Cys Ser Gln His Leu Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys 50 55 60 Asn Lys Glu Asp Ser Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu 65 70 75 80 Page 39 18189543_1 Met Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro 85 90 95 Glu Asp Ala Ser His His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn 100 105 110 Cys Met Glu Met Asp Val Met Ala Val Ala Thr Ile Val Ile Val Asp 115 120 125 Ile Cys Ile Thr Leu Gly Leu Leu Leu Leu Val Tyr Tyr Trp Ser Lys 130 135 140 Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly Ala Gly Ala Gly 145 150 155 160 Gly Arg Gln Arg Gly Gln Asn Lys Glu Arg Pro Pro Pro Val Pro Asn 165 170 175 Pro Asp Tyr Glu Pro Ile Arg Lys Gly Gln Gln Asp Leu Tyr Ser Gly 180 185 190 Leu Asn Gln Arg Arg Ile <210> 50 <211> 171 <212> PRT <213> Macaca fascicularis <220> <221> EATURE <222> (1)..(171) <223> CD3 delta <400> 50 Met Glu His Ser Thr Phe Leu Ser Gly Leu Val Leu Ala Thr Leu Leu 1 5 10 15 Ser Gln Val Ser Pro Phe Lys Ile Pro Val Glu Glu Leu Glu Asp Arg 25 30 Page 40 18189543_1 Val Phe Val Lys Cys Asn Thr Ser Val Thr Trp Val Glu Gly Thr Val 40 45 Gly Thr Leu Leu Thr Asn Asn Thr Arg Leu Asp Leu Gly Lys Arg Ile 50 55 60 Leu Asp Pro Arg Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys 65 70 75 80 Asp Lys Glu Ser Ala Val Gln Val His Tyr Arg Met Cys Gln Asn Cys 85 90 95 Val Glu Leu Asp Pro Ala Thr Leu Ala Gly Ile Ile Val Thr Asp Val 100 105 110 Ile Ala Thr Leu Leu Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His 115 120 125 Glu Thr Gly Arg Leu Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg 130 135 140 Asn Asp Gln Val Tyr Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr 145 150 155 160 Ser Arg Leu Gly Gly Asn Trp Ala Arg Asn Lys 165 170 <210> 51 <211> 207 <212> PRT <213> Homo s <220> <221> MISC_FEATURE <222> (1)..(207) <223> CD3 epsilon <400> 51 Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser 1 5 10 15 Page 41 43_1 Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr 25 30 Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr 40 45 Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys 50 55 60 Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp 65 70 75 80 His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr 85 90 95 Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu 100 105 110 Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met 115 120 125 Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu 130 135 140 Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys 145 150 155 160 Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn 165 170 175 Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg 180 185 190 Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile 195 200 205 <210> 52 <211> 150 Page 42 18189543_1 <212> PRT <213> Homo s <220> <221> MISC_FEATURE <222> (1)..(150) <223> CD3 delta <400> 52 Phe Lys Ile Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn Cys 1 5 10 15 Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu Ser 25 30 Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg Gly 40 45 Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser Thr 50 55 60 Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp Pro 65 70 75 80 Ala Thr Val Ala Gly Ile Ile Val Thr Asp Val Ile Ala Thr Leu Leu 85 90 95 Leu Ala Leu Gly Val Phe Cys Phe Ala Gly His Glu Thr Gly Arg Leu 100 105 110 Ser Gly Ala Ala Asp Thr Gln Ala Leu Leu Arg Asn Asp Gln Val Tyr 115 120 125 Gln Pro Leu Arg Asp Arg Asp Asp Ala Gln Tyr Ser His Leu Gly Gly 130 135 140 Asn Trp Ala Arg Asn Lys 145 150 <210> 53 Page 43 18189543_1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> 6His Peptide <400> 53 His His His His His His 1 5 <210> 54 <211> 7 <212> PRT <213> cial Sequence <220> <223> Heterodimerization Domain <400> 54 Gly Val Glu Pro Lys Ser Cys 1 5 <210> 55 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Heterodimerization Domain <400> 55 Val Glu Pro Lys Ser Cys 1 5 <210> 56 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Heterodimerization Domain <400> 56 Gly Phe Asn Arg Gly Glu Cys Page 44 18189543_1 1 5 <210> 57 <211> 6 <212> PRT <213> cial Sequence <220> <223> Heterodimerization Domain <400> 57 Phe Asn Arg Gly Glu Cys 1 5 Page 45