JP7626698B2 - Anti-cd40 binding molecules with engineered fc domains and therapeutic uses thereof - Google Patents

Description

RELATED APPLICATIONS This application claims the benefit of International Patent Application No. PCT/CN2018/0108285, filed Sep. 28, 2018, which is incorporated herein by reference in its entirety.

Cluster of differentiation 40 (CD40) is a costimulatory protein of antigen-presenting cells (APCs) that is required for their activation. CD40 is a member of the tumor necrosis factor (TNF) receptor superfamily and is essential for a variety of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. In addition, CD40 is found on the surface of tumor cells, such as B lymphomas, and in approximately 70% of all solid tumors. Its activation has been shown to reverse tolerance to tumor-specific antigens and result in antigen-specific antitumor immunity.

Fc receptors (FcRs) are a family of immune cell surface proteins that can bind to the Fc portion of antibodies. There are several different types of Fc receptors, including Fcγ receptors, Fcα receptors, Fcε receptors, and neonatal Fc receptors (FcRn), with different binding activities for IgG, IgA, IgE, and IgG antibodies, respectively. The Fcγ receptor subfamily includes FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIB (CD32c), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). FcγRI has high binding affinity for IgG1 and IgG3 antibodies, while the other FcγRs have low binding affinity for IgG antibodies.

Different types of Fc receptors play different roles in the immune system. For example, FcγRIII receptors expressed on NK cells and macrophages bind to antibodies attached to infected cells or invading pathogens and trigger antibody-mediated phagocytosis (ADCP) or antibody-dependent cell-mediated cytotoxicity (ADCC) of immune cells, leading to the elimination of the infected cells or invading pathogens. On the other hand, FcγRIIB receptors expressed on B cells and dendritic cells can down regulate the activity of immune cells when they bind to IgG antibodies.

Therapies involving activated immune cells are a promising approach to eliminate diseased cells, such as cancer cells. However, such therapeutic approaches often raise safety concerns. For example, overactivated immune cells can lead to unwanted cytotoxicity and cause tissue damage. Therefore, it is of great interest to develop new immunotherapies that are both effective and safe.

The present disclosure is based, at least in part, on the design of CD40 binding molecules (e.g., CD40 agonists or CD40 antagonists) that include engineered (variant) Fc regions (e.g., engineered Fc regions of IgG1, IgG2, or IgG4 molecules) that exhibit desired hinge flexibility and favorable Fc receptor binding activity and/or selectivity, e.g., enhanced binding affinity and/or selectivity for FcγRIIB or substantially reduced binding affinity for one or more FcγR receptors. Such CD40 binding molecules can be used to modulate (e.g., selectively enhance or selectively inhibit) an immune response in a subject in need of treatment.

Thus, one aspect of the disclosure provides a CD40 binding molecule (e.g., a CD40 agonist or a CD40 antagonist) that includes a CD40 binding moiety and an antibody heavy chain constant region that includes an engineered Fc region that includes at least one mutation at any of positions 220-331, e.g., any of positions 228-329, compared to the wild-type Fc region counterpart. The numbering system follows the EU index.

In some embodiments, the CD40 binding portion is the extracellular domain of CD40L, which may include the amino acid sequence of SEQ ID NO: 140. A CD40 binding molecule that includes such a CD40 binding portion may be a CD40 agonist.

In other embodiments, the CD40 binding portion can be an anti-CD40 antibody fragment. In one embodiment, the anti-CD40 antibody fragment comprises a heavy chain comprising a heavy chain variable region linked to any of the engineered Fc regions described herein, and a light chain comprising a light chain variable region and a light chain constant region.

In some cases, the variant Fc region may have enhanced binding affinity and/or selectivity for FcγRIIB compared to the wild-type counterpart. In other instances, the variant Fc region may have substantially reduced binding affinity for one or more FcγR receptors. In some instances, the variant Fc region has reduced or no binding activity for all FcγR receptors.

In some embodiments, the CD40 binding molecule may comprise a variant Fc region of an IgG1 molecule (e.g., a human IgG1 molecule), which may include mutations including one or more of the following: (a) an amino acid substitution or deletion within positions 233-238 (e.g., 234-238), (b) a substitution at position 265, (c) a substitution at position 267, (d) a substitution at position 297, (e) a substitution at position 328, a substitution at position 329; or a combination thereof. Alternatively, or in addition, the Fc variant of the IgG1 molecule may include one or more mutations at positions 220, 226, 229, 238, 273, 327, 330, and/or 331. In one example, the mutations include a substitution at any of positions 233-236, a deletion at one or more of positions 236-238, or a combination thereof. For example, substitutions at positions 233-235 can include E233P, L234V, L234F, L234A, L235A, and/or L235E. In one example, the mutation includes a deletion at one or more of positions 236-238. In another example, the substitution at position 238 can be P238S and the substitution at position 265 can be D265A, or a combination thereof. In yet another example, the substitution is at position 267 and is S267E. In a further example, the substitution is at position 329 and is P329G. In additional examples, the mutation can include a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 265 can be D265A and the substitution at position 297 can be N297A. Further, one or more substitutions at positions 220, 226, 229, 327, 330, and 331 can be C220S, C226S, C229S, A327G, A330S, and P331S. Exemplary variant Fc regions derived from human IgG1 can be one of G1m1, G1m2, G1m17, G1m27, G1mAA, G1mAAG, G1N297A, G1m240, and G1m40.

In other embodiments, the CD40 binding molecule may comprise an Fc region of an IgG2 molecule (e.g., a human IgG2 molecule), which may include mutations including one or more of the following: (a) a deletion of one or more of positions 237 and 238, (b) a substitution at position 265, (b) a substitution at position 267, (c) a substitution at position 297, (d) a substitution at position 328; or a combination thereof. In some examples, the deletion is at position 237, or at both positions 237 and 238. Alternatively, or in addition, the Fc variant of the IgG2 molecule may include at least one mutation including a substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331. For example, the substitution at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 can be P233E, V234A, V234L, A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S. In one example, the substitution is at position 267 and is S267E. In another example, the substitution is at position 328 and is L328F. In additional examples, the mutation can include a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 265 can be D265A and the substitution at position 297 can be N297A. In some specific examples, the variant Fc region derived from IgG2 can be one of G2m1, G2m17, G2m18, G2m19, G2m20, G2m27, G2m28, G2m29, G2m2040, G2m43, G2G4, G2mAA, and G2m40.

In yet other embodiments, the CD40 binding molecule may comprise an Fc region of an IgG4 molecule (e.g., a human IgG4 molecule), which may include (a) an amino acid residue substitution at position 228, (b) a substitution or deletion at any one of positions 235-238, (c) a substitution at position 265, (d) a substitution at position 267, (e) a substitution at position 297, (e) a substitution at position 328, or a combination thereof. In one example, the substitution at position 228 is S228P. In another example, the deletion is at one or more of positions 236, 237, and 238 (e.g., at both positions 236 and 237). Alternatively, or in addition, the Fc variant of the IgG4 molecule may include a substitution at one or more of positions 233-235, 237, and 273. For example, the substitutions at one or more of positions 233-235, 237, and 273 can be E233P, F234V, F234A, L235S, L235E, L235A, G237A, and V273E. In another example, the substitution at position 267 is S267E. In a further example, the substitution at position 328 is L328F. In additional examples, the mutation can include a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 265 can be D265A and the substitution at position 297 can be N297A. In some specific examples, the variant Fc region is one of G4m1, G4m2, G4m20, G4m28, G4m30, G4m41, G4m42, G4m46, G4mPE, G4mAA, and G4m40.

Any of the variant Fc regions described herein may exhibit enhanced binding activity and/or enhanced selectivity for FcγRIIB compared to a wild-type Fc region. Alternatively, the variant Fc regions described herein may have reduced or no binding activity for any of the FcγR receptors. In some cases, the variant Fc regions may exhibit reduced binding affinity for FcγRIIB. Alternatively, or in addition, the variant Fc regions bind to FcRn.

The anti-CD40 antibody fragment in any of the CD40 binding molecules described herein may be a human or humanized antibody. In some embodiments, the anti-CD40 antibody is an agonistic antibody. In some examples, the anti-CD40 antibody comprises a heavy chain complementarity determining region (HC CDR) the same as that of SEQ ID NO: 128, or a heavy chain complementarity determining region (HC CDR) the same as that of any one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2, and/or a light chain complementarity determining region (LC CDR) the same as that of SEQ ID NO: 129, or a light chain complementarity determining region (LC CDR) the same as that of any one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2. In one example, the antibody fragment may comprise a heavy chain variable region of SEQ ID NO: 128 and/or a light chain variable region of SEQ ID NO: 129. In other examples, the antibody fragment may contain the same heavy chain variable region and/or the same light chain variable region as one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2.

Further provided herein are pharmaceutical compositions comprising any of the CD40 binding molecules described herein and a pharma- ceutically acceptable carrier. Such pharmaceutical compositions can be used to selectively modulate (e.g., selectively activate or selectively inhibit) an immune response in a subject.

In yet another aspect, the present disclosure provides a method for selectively modulating (e.g., selectively activating or selectively inhibiting) an immune response in a subject, the method comprising administering to a subject in need thereof an effective amount of a CD40 binding molecule described herein.

In any of the methods described herein, the subject may be a human patient suffering from or suspected of suffering from cancer, and the CD40 binding molecule may be a CD40 agonist. Exemplary cancers include lung cancer, gastric cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, colorectal cancer, sarcoma, bone cancer, lymphoma, and hematological cancer.

Alternatively, the subject may be a human patient suffering from or suspected of suffering from an immune-related disorder, and the CD40 binding molecule may be a CD40 antagonist. Exemplary immune disorders include autoimmune diseases, immune deficiencies, or allergies. In some embodiments, the target disease for treatment is an autoimmune disease.

In yet another aspect, the present disclosure provides an isolated anti-CD40 antibody that binds to the same epitope of CD40 as a reference antibody selected from the group consisting of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2, or competes with the reference antibody for binding to the epitope.

In some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR1), a heavy chain complementarity determining region 2 (CDR2), and a heavy chain complementarity determining region 3 (CDR3), which collectively are at least 85% identical to the respective heavy chain CDRs of the reference antibody, and/or the antibody comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, which collectively are at least 85% identical to the respective light chain CDRs of the reference antibody.

In another embodiment, the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 collectively contain no more than 10 amino acid mutations compared to the respective heavy chain CDRs of the reference antibody, and/or the light chain CDR1, light chain CDR2, and light chain CDR3 collectively contain no more than 10 amino acid mutations compared to the respective light chain CDRs of the reference antibody.

In one embodiment, the antibody comprises a heavy chain variable region that is at least 85% identical to the heavy chain variable region of a reference antibody, and/or a light chain variable region that is at least 85% identical to the light chain variable region of a reference antibody.

In some embodiments, the heavy chain variable region comprises no more than 10 amino acid residue mutations compared to the heavy chain variable region of a reference antibody, and/or a light chain variable region comprises no more than 10 amino acid mutations compared to the light chain variable region of a reference antibody.

In some examples, the anti-CD40 antibodies disclosed herein may contain the same heavy chain variable region CDRs as the reference antibody and/or the same light chain variable region CDRs as the reference antibody.

Any of the anti-CD40 antibodies described herein can be human or humanized. Pharmaceutical compositions comprising any of the anti-CD40 antibodies described herein and a pharma- ceutical acceptable carrier are also provided herein.

Additionally, the disclosure features an isolated nucleic acid or set of nucleic acids that collectively encode any of the CD40 binding molecules or any of the anti-CD40 antibodies described herein. The nucleic acid or set of nucleic acids can be located in one or two vectors, e.g., expression vectors. Host cells containing such vector(s) are also provided herein.

The present disclosure also features pharmaceutical compositions for use in treating a target disorder described herein that include one or more of the CD40 binding molecules and/or one or more of the anti-CD40 antibodies disclosed herein, or the use of such CD40 binding molecules and/or anti-CD40 antibodies for the manufacture of a medicament for use in treating a target disorder.

The details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

1A-1C are graphs depicting the binding activity of various anti-CD40 antibody 383 IgG variants against various types of Fcγ receptors expressed on CHO-K1 cells at various concentrations as indicated. The concentrations of each IgG variant from left to right are 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG 1A: Binding affinity to FcγRI. FIG. 1B is a graph showing the binding activity of various anti-CD40 antibody 383 IgG variants against various types of Fcγ receptors expressed on CHO-K1 cells at various concentrations as indicated. The concentrations of each IgG variant from left to right are 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG. 1B: Binding affinity to FcγRII. FIG. 1C is a graph showing the binding activity of various anti-CD40 antibody 383 IgG variants against various types of Fcγ receptors expressed on CHO-K1 cells at various concentrations as indicated. The concentrations of each IgG variant from left to right are 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG. 1C: Binding affinity to FcγRIIB. FIG. 1D: Binding affinity for FcγRIIIA. 1 is a graph showing stimulation of human CD40 activation in a reporter assay by multiple anti-CD40 antibody 383 IgG variants as indicated by IL8 secretion, with concentrations of each IgG variant corresponding to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, 1 μg/ml, 3 μg/ml, and 10 μg/ml, from left to right. 3A-3D are graphs showing the binding activity of multiple anti-CD40 antibody 383 IgG variants against different types of Fcγ receptors expressed on CHO-K1 cells. The concentrations of each anti-CD40 antibody 383 IgG variant from left to right are 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG 3A: Binding affinity to FcγRIIB. 3A-3C are graphs showing the binding activity of multiple anti-CD40 antibody 383 IgG variants against different types of Fcγ receptors expressed on CHO-K1 cells. The concentrations of each anti-CD40 antibody 383 IgG variant from left to right are 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG 3B: Binding affinity to FcγRI. 3C is a graph showing the binding activity of multiple anti-CD40 antibody 383 IgG variants against various types of Fcγ receptors expressed on CHO-K1 cells. The concentrations of each anti-CD40 antibody 383 IgG variant from left to right are 0.3 μg/ml, 1 μg/ml, 3 μg/ml, 10 μg/ml, and 30 μg/ml. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG 3C: Binding affinity to FcγRIIA. FIG. 3D: Binding affinity for FcγRIIIA. 4A-4D are graphs showing stimulation of human CD40 activation, as indicated by IL8 secretion in a reporter assay, by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond, from left to right, to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4A: IgG variants IgG1m2, IgG2m20, IgG1m40, IgG4m40. 4A-4C are graphs showing stimulation of human CD40 activation, as indicated by IL8 secretion in a reporter assay, by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond, from left to right, to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4B: IgG variants IgG1m240, IgG2m2040, IgG4m41, IgG4m42, IgG2m43, IgG2m44, and IgG4mAA. 4A-4C are graphs showing stimulation of human CD40 activation, as indicated by IL8 secretion in a reporter assay, by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond, from left to right, to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4C: IgG variants IgG1N297A, IgG2mAA, IgG2G4, IgG4mPE, and IgG1mAAG. 4A-4D are graphs showing stimulation of human CD40 activation as indicated by IL8 secretion in a reporter assay by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond from left to right to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4D: IgG variants IgG1m47, IgG1m48, IgG1m49, IgG1m50, and IgG1mAA. 4A-4D are graphs showing stimulation of human CD40 activation, as indicated by IL8 secretion in a reporter assay, by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond, from left to right, to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4E: IgG variants IgG4m46 and IgG4m30. 4A-4D are graphs showing stimulation of human CD40 activation, as indicated by IL8 secretion in a reporter assay, by a number of anti-CD40 antibody 383 IgG variants in solution and/or in co-culture with FcγRIIB expressing cells. Concentrations of each IgG variant correspond, from left to right, to 0.003 μg/ml, 0.01 μg/ml, 0.03 μg/ml, 0.1 μg/ml, 0.3 μg/ml, and 1 μg/ml. The dashed lines indicate the baseline reporter signal for each assay. FIG. 4F: IgG variants IgG1m27, IgG2m19, IgG2m1, IgG4m2, and IgG4m20. 5A and 5B ) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D ). DC activation is indicated by the signal bar graph of IL-8 in the culture supernatant. 5A and 5B ) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D ). DC activation is indicated by the signal bar graph of IL-8 in the culture supernatant. 5A and 5B ) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D ). DC activation is indicated by the signal bar graph of IL-8 in the culture supernatant. 5A and 5B ) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D ). DC activation is indicated by the signal bar graph of IL-8 in the culture supernatant. 6A-C are graphs showing the binding activity of anti-CD40 antibody 383 IgG variants against human CD40 expressed on CHO-K1 cells. The concentrations of each IgG variant from left to right are 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. The three rightmost bars ("Herceptin", "Secondary only", and "Blank") serve as controls. FIG 6A: Multiple anti-CD40 antibody 383 IgG variants as indicated. FIG. 6B: Binding activity of anti-CD40 antibody 383 IgG variants against human CD40 expressed on CHO-K1 cells. Concentrations of each IgG variant from left to right are 0.01 μg/ml, 0.1 μg/ml, 1 μg/ml, and 10 μg/ml. The three rightmost bars ("Herceptin", "Secondary only", and "Blank") serve as controls. FIG. 6B: Multiple anti-CD40 antibody 383 IgG variants as indicated. 7A-7C are graphs showing tumor growth curves of different anti-CD40 antibody 383 IgG variants shown in mouse tumor models. Figure 7A: Graph showing tumor volume change at different time points after antibody treatment in homozygous B-hCD40 C57BL6 mice. Mean tumor size ± SEM is shown. 7A-7C are graphs showing tumor growth curves of various anti-CD40 antibody 383 IgG variants in mouse tumor models.B: Graph showing serum alanine transaminase (ALT, a liver enzyme released in serum upon liver injury) levels after antibody treatment in homozygous B-hCD40 C57BL6 mice. Mean tumor size ± SEM is shown.

Provided herein are CD40 binding molecules (e.g., CD40 agonists or CD40 antagonists) that include a CD40 binding portion (e.g., the extracellular domain of a CD40 ligand or an anti-CD40 antibody fragment) and an engineered (variant) Fc region, and may have altered binding affinity and/or specificity for one or more Fc receptors, e.g., enhanced binding affinity for FcγRIIB, enhanced binding specificity for FcγRIIB, and/or substantially reduced binding affinity for one or more FcγR receptors (e.g., low or no binding activity for all FcγR receptors).

Such CD40 binding molecules are expected to exhibit unexpected and superior therapeutic activity. For example, CD40 binding molecules with variant Fc regions having enhanced binding affinity for FcγRIIB are expected to exhibit enhanced agonist activity, and CD40 binding molecules with variant Fc regions having enhanced binding specificity for FcγRIIB are expected to exhibit higher tumor selectivity compared to their wild-type counterparts. Furthermore, CD40 binding molecules with low or no binding affinity for FcγR receptors are useful for activating CD40-positive immune cells in the tumor microenvironment and/or blocking CD40-positive immune cells in immune disorders.

Thus, described herein are approaches for designing CD40 binding molecules that include a CD40 binding portion and an engineered Fc region (Fc variant), which may be of an IgG molecule (e.g., an IgG1, IgG2, and IgG4 molecule, such as human IgG1, human IgG2, and human IgG4 molecules), and their use to modulate immune responses. Such Fc variants may have enhanced binding affinity for FcγRIIB (CD32B) and/or enhanced binding selectivity compared to other Fc receptors, such as FcγRIII (CD16), compared to their wild-type counterparts. Alternatively, such Fc variants may have substantially reduced binding affinity for one or more FcγR receptors (e.g., all FcγR receptors) compared to their wild-type counterparts. By "substantially reduced" it is meant that the binding affinity of the Fc variant to an FcγR receptor is at least 60% lower (e.g., 70% lower, 80% lower, 90% lower, 95% lower, 98% lower, or 99% lower) than the binding affinity of its wild-type counterpart to the same FcγR receptor. In some instances, the Fc variant may have low or no binding affinity to all FcγR receptors, i.e., the binding affinity is undetectable by conventional assays or the binding affinity is substantially lower and no significant biological activity is elicited.

I. CD40 Binding Molecules Described herein are CD40 binding molecules comprising a CD40 binding moiety linked to an engineered Fc region having altered binding affinity and/or specificity for one or more Fc receptors, as described herein. Such CD40 binding molecules can be CD40 agonists that can induce CD40/CD40L-mediated signaling upon binding to cell surface CD40. Alternatively, the CD40 binding molecules described herein are CD40 antagonists that can inhibit CD40/CD40L-mediated signaling upon binding to cell surface CD40. CD40 binding molecules are useful for modulating (selectively modulating) immune responses when administered to a subject in need of treatment.

(i) CD40 Binding Moiety The CD40 binding moiety described herein can be any peptide or polypeptide that binds to CD40, e.g., human CD40. CD40 is an immune cell receptor that is well known in the art. For example, NCBI GenBank Accession Nos. P25942.1 and AAB08705.1 provide information regarding human and mouse CD40, respectively. Provided below is the amino acid sequence of an exemplary human CD40 polypeptide.

Human CD40:
MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT
WNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSD
TICEPCPVGFFSNVSSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILL
VLVFIKKVAKKPTNKAPPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ ID NO: 141)
CD40 polypeptides from other species are known in the art and can be obtained from publicly available genetic databases, e.g., GenBank, using either the human or mouse sequence as a query.

In some embodiments, the CD40 binding moiety can be a polypeptide that includes the extracellular domain of the CD40 ligand (CD40L or CD154). CD40L is a membrane glycoprotein expressed on the surface of T cells. This molecule has been shown to stimulate B cell proliferation and secretion of immunoglobulins in the presence of cytokines. Additionally, CD40L can induce cytokine production and tumoricidal activity in peripheral blood monocytes. The sequence of CD40L and its extracellular domain are well known in the art. For example, NCBI GenBank Accession No. NP_000065.1 (extracellular domain, amino acids 47-261) provides information regarding human CD40L. Provided below is an exemplary amino acid sequence of the extracellular domain of human CD40L.

Human CD40L (extracellular domain):
HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL (SEQ ID NO: 140)
CD40L polypeptides from other species are known in the art and can be obtained from publicly available genetic databases, eg, GenBank, using the human sequence as a query.

In other embodiments, the CD40 binding moieties described herein can include anti-CD40 antibodies. As used herein, the term "anti-CD40 antibody" refers to any antibody capable of binding to a CD40 polypeptide, which may be of any suitable source, e.g., human or non-human mammalian (e.g., primates such as mice, rats, rabbits, monkeys, etc.).

The anti-CD40 antibodies described herein comprise a heavy chain comprising a heavy chain variable domain linked to any of the Fc variants described herein, and optionally a light chain comprising a light chain variable region and a light chain constant region. The heavy chain variable region ( _VH ) and optionally the light chain variable region ( _VL ) are typically responsible for binding the antigen, in this case CD40.

The _VH and _VL regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions"("CDRs"), interspersed with more conserved regions known as "framework regions"("FRs"). Each _VH and _VL is typically composed of three CDRs and four FRs, arranged from amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat, Chothia, AbM, and/or contact definitions, all of which are well known in the art. See, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al. , (1989) Nature 342:877, Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948, and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp. mrc. ac. uk and bioinf. org. uk/abs.

In some embodiments, the anti-CD40 antibodies described herein can bind to CD40 and inhibit the activity of CD40 by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more, including any increment therein). The apparent inhibition constant (Ki ^app or K _i,app ), which provides a measure of the potency of an inhibitor, is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-CD40 antibodies described herein can be determined by routine methods known in the art.

The K _i, ^app value of an antibody can be determined by measuring the inhibitory effect of various concentrations of the antibody on the extent of the reaction (e.g., enzyme activity), and fitting the change in the pseudo-first-order rate constant (v) as a function of inhibitor concentration to a modified Morrison equation (Equation 1) provides an estimate of the apparent K i value. For competitive inhibitors, the K i ^app can be obtained from the y-intercept extracted from a linear regression analysis of a plot of K _i, ^app versus substrate concentration.

where A corresponds to v _o /E, the initial velocity of the enzyme reaction in the absence of inhibitor (v _o ) divided by the total enzyme concentration (E).

In some embodiments, the anti-CD40 antibodies described herein may have a Ki ^app value for a target antigen or antigenic epitope of 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 pM or less. In some embodiments, any of the anti-CD40 antibodies may be further affinity matured to lower the Ki ^app of the antibody for the target antigen or antigenic epitope thereof.

In some cases, the anti-CD40 antibody can inhibit signaling induced by CD40/CD40L interaction by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more, including any increment therein). Such inhibitory activity can be determined by conventional methods or assays described herein.

The antibodies described herein may be murine, rat, human, or of any other origin (including chimeric or humanized antibodies). Such antibodies are not naturally occurring, i.e., are not produced in an animal without human action (e.g., by immunizing such an animal with the desired antigen or a fragment thereof, or by being isolated from an antibody library).

In some embodiments, the anti-CD40 antibody is a humanized antibody, which may have one or more of the elements or features described below or elsewhere herein. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from a non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's CDRs are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In some cases, a humanized antibody may contain substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody will also optimally contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that are altered relative to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the _VH and _VL variable regions of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, human _VH and _VL chains having amino acid sequences that are homologous to the amino acid sequences of the parent non-human antibody are identified from any antibody gene database using the parent _VH and _VL sequences as search queries. Human _VH and _VL acceptor genes are then selected.

The CDR regions in the selected human acceptor gene can be replaced with CDR regions from a parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important for interactions with the CDR regions can be used to replace the corresponding residues in the human acceptor gene.

In some embodiments, the anti-CD40 antibodies described herein specifically bind to a corresponding target antigen or epitope thereof, e.g., a CD40 antigen or epitope. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts more frequently, more rapidly, with greater duration, and/or with greater affinity with a particular target antigen than with alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (CD40) or an antigenic epitope therein is an antibody that binds to this target antigen with higher affinity, avidity, more readily, and/or with greater duration than it binds to other antigens or other epitopes within the same antigen. It is understood by this definition that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some examples, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes within the same antigen (i.e., only baseline binding activity can be detected by conventional methods). In some embodiments, the anti-CD40 antibodies described herein specifically bind to CD40. Alternatively, or in addition, the anti-CD40 antibodies described herein specifically bind to human CD40 or a fragment thereof compared to their mouse counterpart, or vice versa (e.g., have at least 10-fold higher binding affinity for one antigen than the other, as determined in the same assay under the same assay conditions). In other examples, the anti-CD40 antibodies described herein may cross-react with human and non-human CD40 (e.g., mouse), e.g., the difference in binding affinity for human and non-human CD40 is less than 5-fold, e.g., less than 2-fold, or substantially similar.

In some embodiments, an anti-CD40 antibody as described herein has suitable binding affinity to a target antigen (e.g., CD40) or antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K _A. K _A is the reciprocal of the dissociation constant (K _D ). An anti-CD40 antibody described herein has a binding affinity (K D ) to a target antigen or antigenic epitope of at least 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , 10 ⁻¹⁰ M, or lower. An increase in binding affinity corresponds to a decrease in K _D . Higher affinity binding of an antibody to a first antigen compared to a second _{antigen can be indicated by a higher K A (} or lower numerical value K _D ) for binding the first antigen than the K _A (or numerical value K _D ) for binding the second antigen. In such cases, the antibody has specificity for a first antigen (e.g., a first protein in a first conformation or mimetic thereof) compared to a second antigen (e.g., the same first protein, or a second protein in a second conformation or mimetic thereof). The difference in binding affinity (e.g., for specificity or other comparison) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 ^5- fold. In some embodiments, any of the anti-CD40 antibodies can be further affinity matured to increase the binding affinity of the antibody for the target antigen or antigenic epitope thereof.

Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following formula:
[Bound] = [Free] / (Kd + [Free])
However, it is not necessarily necessary to make an exact determination of _K, as simply obtaining a quantitative measure of affinity, as determined using methods such as, for example, ELISA or FACS analysis, may be proportional to K and can therefore be used for comparison, such as determining whether a higher affinity, e.g., two-fold higher, is to obtain a qualitative measure of _affinity , or to obtain an inference of affinity by activity in, for example, a functional assay, e.g., an in vitro or in vivo assay.

Provided below is an example anti-CD40 antibody 383 ( _VH and _VL amino acid sequences, CDRs are shown in bold).
_VH :
_VL :
Further exemplary anti-CD40 antibodies are provided below (CDRs are shown in bold):

19G6D6
_VH :
_VL :
36G7B8
_VH :
_VL :
13F1A7
_VH :
_VL :
9F12D9
_VH :
_VL :
17C5C2
_VH :
_VL :

In some embodiments, the anti-CD40 antibodies described herein bind to the same epitope as any of the exemplary antibodies described herein or compete with the exemplary antibodies for binding to the CD40 antigen.

"Epitope" refers to a site on a target antigen that is recognized and bound by an antibody. The site may be composed entirely of amino acid components, entirely of chemical modifications of amino acids of a protein (e.g., glycosyl moieties), or a combination thereof. Overlapping epitopes include at least one common amino acid residue. Epitopes may be linear, which are typically 6-15 amino acids in length. Alternatively, epitopes may be conformational. The epitopes to which an antibody binds can be determined by routine techniques, such as epitope mapping methods (see, e.g., the discussion below). An antibody that binds to the same epitope as an exemplary antibody described herein may bind to the exact same epitope or to a substantially overlapping epitope (e.g., including less than three non-overlapping amino acid residues, less than two non-overlapping amino acid residues, or only one non-overlapping amino acid residue) as the exemplary antibody. Whether two antibodies compete with each other for binding to a cognate antigen can be determined by competition assays well known in the art.

In some embodiments, the anti-CD40 antibodies disclosed herein comprise the same heavy and light chain CDRs as those of (a) SEQ ID NO:128 and SEQ ID NO:129, (b) SEQ ID NO:130 and SEQ ID NO:131 (19G6D6), (c) SEQ ID NO:132 and SEQ ID NO:133 (36G7B8), (d) SEQ ID NO:134 and SEQ ID NO:135 (13F1A7), (e) SEQ ID NO:136 and SEQ ID NO:137 (9F12D9), or (f) SEQ ID NO:138 and SEQ ID NO:139 (17C5C2). In some examples, the anti-CD40 antibodies comprise a heavy chain variable domain of SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, or SEQ ID NO:138, and/or a light chain variable domain of SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139.

Also within the scope of this disclosure are functional variants of any one of the exemplary anti-CD40 antibodies disclosed herein. Such functional variants are substantially similar to any one of the exemplary anti-CD40 antibodies, both structurally and functionally. A functional variant comprises substantially the same _VH and _VL CDRs as any one of the exemplary anti-CD40 antibodies. For example, it may comprise only up to 10 (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acid residue mutations in the entire CDR region (collectively) of the antibody, and binds to the same epitope of CD40 with substantially similar affinity (e.g., has a _KD value in the same order of magnitude). Alternatively, or in addition, the amino acid residue mutations are conservative amino acid residue substitutions. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to one of ordinary skill in the art, such as those found in references that summarize such methods, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include those made among amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the anti-CD40 antibodies described herein may comprise heavy chain CDRs that, in combination (collectively), have at least 80% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percentage therein) sequence identity to the heavy chain CDRs of SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, or SEQ ID NO:138. Alternatively, or in addition, the anti-CD40 antibodies may comprise light chain CDRs that, collectively, have at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percentage therein) sequence identity to those of SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:139.

In some embodiments, the anti-CD40 antibodies described herein may comprise a heavy chain variable domain that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percentage therein) identical to SEQ ID NO: 128, 130, 132, 134, 136, or 138 and/or a light chain variable domain that is at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% and any incremental percentage therein) identical to SEQ ID NO: 129, 131, 133, 135, 137, or 139.

The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, as modified by Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10,1990. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

(ii) Engineered Fc Regions The CD40 binding molecules described herein comprise a modified heavy chain constant region, in which the Fc domain has been modified to modulate its binding affinity and/or specificity to an Fc receptor, such as FcγR.

In some embodiments, the Fc variants in the CD40 binding molecules described herein have enhanced FcγRIIB selectivity compared to their wild-type counterparts. Selectivity for FcγRIIB, selective binding to FcγRIIB, or Fc fragments with specific binding to FcγRIIB are terms well understood in the art. A molecule is said to exhibit "selective binding" or "specific binding" if it reacts more frequently, more rapidly, with a longer duration, and/or with greater affinity to a particular target antigen (e.g., FcγRIIB receptor) than it reacts with another target (e.g., FcγRIII receptor). An Fc fragment "specifically binds" to an Fc receptor if it binds with higher affinity, avidity, more readily, and/or with a longer duration than it binds to other Fc receptors. For example, an Fc fragment that specifically (or preferentially) binds to FcγRIIB is an Fc fragment that binds to this Fc receptor with higher affinity, avidity, more readily, and/or with a longer duration than it binds to other Fc receptors. By this definition, it is also understood that, for example, an Fc fragment that selectively or specifically binds to a first Fc receptor may or may not specifically or preferentially bind to a second Fc receptor. Thus, "selective binding," "specific binding," or "preferential binding" does not necessarily require (although it can include) exclusive binding. In some instances, an Fc fragment that "selectively binds" or "specifically binds" to a target Fc receptor (e.g., FcγRIIB) may not bind to (i.e., cannot bind in a normal manner to) other Fc receptors. In other embodiments, the variant Fc fragment does not bind to any FcγR.

The relative binding affinities of IgG1, IgG2, and IgG4 to different Fc receptors are shown in Table 1 below.

The Fc variants described herein can have enhanced selectivity for FcγRIIB compared to their wild-type counterparts (the wild-type parent Fc region into which mutations are introduced to produce the Fc variant). The relative binding activity of such Fc variants to FcγRIIB versus other Fc receptors (e.g., FcγRIII) is higher than the relative binding activity of the wild-type counterpart to FcγRIIB versus other Fc receptors (e.g., FcγRIII). The Fc variants can have enhanced binding activity to FcγRIIB and/or decreased binding activity to another Fc receptor, e.g., FcγRIII. In some embodiments, the Fc variants described herein can have decreased binding activity to both FcγRIIB and another Fc receptor (e.g., FcγRIII), however, the level of decreased binding activity to other Fc receptors (e.g., FcγRIII) is greater than the level of decreased binding activity to FcγRIIB.

In some embodiments, the Fc variants described herein have suitable binding affinity for FcγRIIB, e.g., enhanced, as compared to the wild-type parent Fc from which the Fc variant is derived. As used herein, "binding affinity" refers to the apparent association constant or K _{A. K A} is the reciprocal of the dissociation constant (K _D ). The Fc variants described herein may have a binding affinity (K _D ) for FcγRIIB of at least 10 ⁻⁵ , 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , 10 ⁻¹⁰ M, or lower. An increase in binding affinity corresponds to a decrease in K _D . Higher affinity binding of an Fc fragment to a first Fc receptor compared to a second Fc receptor may be indicated by a higher K _A (or lower numerical value K _D ) for binding the first Fc receptor than the K _A (or numerical value K _D ) for binding the second Fc receptor. In such cases, the Fc variant has specificity for a first Fc receptor compared to a second Fc receptor. In some embodiments, the Fc variants described herein have a higher binding affinity (higher K or smaller K) to FcγRIIB compared to the binding affinity to FcγRIII (either FcγRIIIA or FcγRIIIB). The difference in binding affinity (e.g., for specificity or other comparisons ₎ can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5 _, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 ^5- fold.

In some embodiments, the Fc variants described herein may be designed, for example, by mutating one or more amino acid residues of a wild-type human IgG1, IgG2, or IgG4 Fc fragment against the amino acid residues of the corresponding mouse IgG, e.g., mouse IgG1. A sequence comparison of human and mouse IgG (hIgG and mIgG, respectively) is shown below (from top to bottom, SEQ ID NOs: 60-64, each representing a combination of the corresponding Fc region fragments 211-245, 260-278, and 320-332):

The amino acid sequences of wild-type mouse IgG1 and IgG2 Fc fragments, and exemplary Fc variants with reduced FcγR binding are shown below.
Amino acid sequence of wild type mouse IgG1 Fc fragment:
VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 142)
Amino acid sequence of wild type mouse IgG2a Fc fragment:
VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLSSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 143)
Amino acid sequence of mutant mouse IgG1 mDANA Fc fragment:
VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 144)
Amino acid sequence of mutant mouse IgG2a1 mDANA Fc fragment:
VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPKIKDVLMISLSSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 145)

In some embodiments, the Fc variants described herein are human IgG1, G2, or G4 Fc variants that include one or more mutations (e.g., amino acid substitutions, deletions, or additions) in the hinge domain of the Fc fragment. Human IgG contains a CPPC or CPSC core motif in the hinge domain (positions 226-229 according to the EU index). Positions 216-225 are considered the upper portion of the hinge domain, and positions 230-238 are considered the lower portion of the hinge domain. The numbering system used herein is in accordance with the EU index unless explicitly indicated. In some instances, the one or more mutations can be located in the upper portion of the hinge domain. Alternatively, or in addition, the one or more mutations can be located in the lower portion of the hinge domain. In some embodiments, the Fc region of any of the anti-CD40 molecules disclosed herein may contain at least one mutation at any of positions 220-331, preferably at any of positions 228-329. Such Fc variants may have altered binding affinity and/or specificity for one or more of the Fc receptors (e.g., FcγIIB, FcγRI, FcγRIIA, or FcγRIIIA) compared to their native counterparts.

In some embodiments, mutations to human IgG Fc can be made according to the corresponding amino acid residues in the hinge domain of mouse IgG1. For example, mouse IgG1 does not contain a GGP motif at positions 236-238. Thus, one or more residues of this GGP motif can be deleted from a human IgG1, IgG2, or IgG4 Fc fragment to produce an Fc variant described herein.

Alternatively, or in addition, the human Fc variants may comprise one or more amino acid substitutions at the top, bottom, or both of the hinge domain. For example, the Fc variants may comprise one or more amino acid substitutions at one or more of positions 233, 234, 235, and/or 236. Such amino acid substitutions may be combined with one or more deletions of the GGP motif (236-238) as described herein. These mutations may be introduced into a human IgG2 or IgG4 Fc fragment to produce the Fc variants described herein. In some examples, the Fc variants described herein comprise a deletion at one or more of positions 236-238 (e.g., 236, 237, 238, or any combination thereof).
Any of the hinge domain mutations described herein may be combined with mutations (e.g., amino acid substitutions) at one or more positions involved in interactions with Fc receptors. Such positions include, but are not limited to, positions 265, 267, 273, 297, and 327-331, or combinations thereof. Exemplary amino acid substitutions at these positions include D265A, S267E, V273E, N297A, L328F, P329G, A330S, and/or P331S.

In some embodiments, the Fc variants disclosed herein may be derived from an IgG1 molecule (e.g., human IgG1) and include one or more mutations at one or more of positions 220, 226, 229, 234-238, 265, 267, 297, and 327-331. For example, the Fc variants may include a substitution or deletion within positions 234-238, a substitution at position 265 (e.g., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 297 (e.g., N297A), a substitution at position 328 (e.g., L328F), a substitution at position 329 (e.g., P329G), or a combination thereof. Alternatively, or in addition, it may include substitutions at one or more of positions 220 (e.g., C220S), 226 (e.g., C226S), 229 (e.g., C229S), 327 (e.g., A327G), 330 (e.g., A330S), and 331 (e.g., P331S). In some cases, an Fc variant derived from an IgG1 molecule may include a deletion at one or more of positions 236-238.

In some embodiments, the Fc variants disclosed herein may be derived from an IgG2 molecule (e.g., human IgG2) and include one or more mutations at one or more of positions 233-235, 237-238, 265-268, 273, 297, 328, 330, and 331. In some examples, such Fc variants may include a deletion within positions 237-238 (e.g., a deletion at position 237 or a deletion at both positions 237 and 238), a substitution at position 265 (e.g., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 297 (e.g., N297A), a substitution at position 328 (e.g., L328F), or a combination thereof. Alternatively, or in addition, Fc variants derived from IgG2 molecules may include substitutions at one or more of positions 233-235, 237, 238, 268, 273, 330, and 331 (e.g., P233E, V234A, V234L, A235L, A235S, G237A, P238S, H268A, H268Q, V273E, A330S, and P331S).

In yet other embodiments, the Fc variants disclosed herein may be derived from an IgG4 molecule (e.g., human IgG4) and include one or more mutations at one or more of positions 228, 233-238, 265, 267, 273, 297, and 328. In some examples, such Fc variants may include a substitution at position 228 (e.g., S228P), a substitution or deletion at any of positions 235-238 (e.g., one of positions 236-238, e.g., a deletion at positions 236 and 237), a substitution at position 265 (e.g., D265A), a substitution at position 267 (e.g., S267E), a substitution at position 273 (e.g., V273E), a substitution at position 297 (e.g., N297A), a substitution at position 328 (e.g., L328F), or a combination thereof. Alternatively, or in addition, the Fc variant may include substitutions at one or more of positions 233-235 and 237 (e.g., E233P, F234V, F234A, L235S, L235E, L235A, and G237A).

Also within the scope of this disclosure are Fc variants derived from IgG2 or IgG4 molecules that contain one or more mutations at positions 265, 267, 273, 297, 328, and/or 329. Such mutations may include amino acid substitutions at one or more of these positions, e.g., D265A, S267E, V273E, N297A, L328F, and/or P329G.

In some cases, any of the Fc variants disclosed herein may further include a mutation, e.g., a substitution, e.g., V309L, at position 309. Such Fc variants may be derived from an IgG2 molecule (e.g., human IgG2). In some embodiments, the mutation at position 309 may be combined with a mutation at one or more of positions 234, 268, 330, and 331. An example of such an Fc variant includes G2m43.

In some embodiments, the Fc variants described herein may comprise an amino acid sequence that is at least 85% (e.g., 90%, 95%, 98%, 99%, or more) identical to its wild-type counterpart (e.g., a wild-type human IgG1, IgG2, or IgG4 Fc fragment described herein).

In one example, the amino acid residue substitutions in the Fc variants described herein are conservative amino acid residue substitutions.

Provided below are sequence alignments showing exemplary positions where mutations can be introduced into hIgG1, hIgG2, and IgG4 to produce various Fc variants for the present disclosure.

Sequence alignment of human IgG1 variants compared to wild-type human IgG1 (from top to bottom, SEQ ID NOs: 70-88 , 163-168 and 215 ):

Sequence alignment of human IgG2 variants compared to wild-type human IgG2 (from top to bottom, SEQ ID NOs: 89-105 and 169-173):

Sequence alignment of human IgG4 variants compared to wild-type human IgG4 (from top to bottom, SEQ ID NOs: 65, 106-127, and 174-177)

The amino acid sequences of wild-type human IgG1, IgG2, and IgG4 Fc fragments, as well as some exemplary hIgG1, hIgG2, and hIgG4 Fc variants (starting at position 221 according to EU numbering) are shown below:

Amino acid sequence of wild type human IgG1 Fc fragment:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

Amino acid sequence of wild type human IgG2 Fc fragment:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

Amino acid sequence of wild type human IgG4 Fc fragment:
VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3)

Amino acid sequence of human IgG4 S228P Fc variant:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4)

Amino acid sequences of exemplary human IgG1 Fc variants:
G1m1:
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)
G1m2:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6)
G1m-2:
VDKKVEPKCCVECPPCAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)
G1m-4:
VDKKVEPKYGPPCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8)
G1m5:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 9)
G1m7:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 10)
G1m8:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)
G1m9:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12)
G1m15:
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13)
G1m17:
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 14)
G1m18:
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15)
G1m19:
VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 16)
G1m25:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 17)
G1m27:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)
G1m28:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 19)
G1m29:
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 20)
G1mAA:
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 21)
G1mAG:
VDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22)
G1m40:
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 160)
G1m45
VDKKVEPKSCDKTHTCPPCPAPPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 178)
G1m240
VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179)
G1 N297A
VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 180)
G1m47
VDKKVEPKSCDKTHTCPPCPAPPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 181)
G1m48
VDKKVEPKSsDKTHTsPPsPAPELLGGsSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 182)
G1m49
VDKKVEPKSCDKTHTsPPsPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 183)
G1m50
VDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 184)

Amino acid sequences of exemplary human IgG2 Fc variants:
G2m1:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 23)
G2m-1:
VDKTVERKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 24)
G2m2:
VDKTVERKCCVECPPCPAPPFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 25)
G2m-4:
VDKTVERKYGPPCPPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26)
G2m5:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 27)
G2m7:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28)
G2m8:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 29)
G2m9:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30)
G2m10:
VDKTVERKCCVECPPCPAPEVSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 31)
G2m15:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 32)
G2m17:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33)
G2m18:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 34)
G2m19:
VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 35)
G2m20:
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 36)
G2m27:
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 37)
G2m28:
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38)
G2m40:
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 161)
G2m43
VDKTVERKCCVECPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 185)
G2m44
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSqEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPSsIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 186)
G2mAA
VDKTVERKCCVECPPCPAPPaaaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 213)
G2m2040
VDKTVERKCCVECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 187)
G2G4
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 188)

Amino acid sequences of exemplary human IgG4 Fc variants:
G4m1:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 39)
G4m-1:
VDKRVESKSCDKTHTPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 40)
G4m2:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 41)
G4m-2:
VDKRVESKCCVEPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 42)
G4m3:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 43)
G4m4:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 44)
G4m5:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 45)
G4m7:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 46)
G4m8:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47)
G4m9:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 48)
G4m10:
VDKRVESKYGPPCPPCPAPEVSSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 49)
G4m17:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 50)
G4m18:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:51)
G4m19:
VDKRVESKYGPPCPPCPAPEFLSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:52)
G4m20:
VDKRVESKYGPPCPPCPAPEFLGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:53)
G4m25:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 54)
G4m27:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 55)
G4m28:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 56)
G4m29:
VDKRVESKYGPPCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGFPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 57)
G4m30:
VDKRVESKYGPPCPPCPAPEFLPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 58)
G4mPE:
VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 59)
G4m40:
VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 162)
G4m41
VDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 189)
G4m42
VDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 190)
G4m46
VDKRVESKYGPPCPPCPAPEALGapsVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 191)
G4mAA
VDKRVESKYGPPCPPCPAPEAaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 192)

The Fc variants described herein may exhibit enhanced binding activity to FcγRIIB compared to their wild-type counterparts. Examples include G2m2, G2m5, G2m7, G2m8, G2m9, G2m19, G2m44, G1m7, G1m9, G1m27, G1m45, G1m47, G1m49, G1m50, G4m7, G4m46, and G4mPE. Alternatively, or in addition, the Fc variants may have enhanced selectivity for FcγRIIB compared to their wild-type counterparts, e.g., G2m1, G2m20, G2m43, G2m44, G2G4, G2mAA, G1m2, G1m15, G1m17, G1m18, G1m19, G1m27, G1m28, G1m29, G4m1, G4m2, G4m7, G4m8, G4m9, G4m20, G4m25, G4m27, G4m28, G4m30, G4m46, and G4mPE. Such Fc variants may retain binding activity for FcRn. These Fc variants can be used to construct therapeutics described herein that can crosslink immune receptors and FcγRIIB receptors.

Alternatively, certain Fc variants described herein may have selectivity for FcγRIIB and/or appreciably low or no binding activity for any FcγR. Examples include G1m2, G1m25, G1m40, G1mAAG, G1m240, G2m1, G2m20, G2m40, G2m2040, G4m5, G4m18, G4m19, G4m20, G4m30, G4m40, G4m41, G4m42. Such Fc variants may retain binding activity for FcRn. Therapeutic agents (e.g., antibodies) containing such Fc variants may be able to crosslink immune receptors and FcγRIIB receptors due to the avidity effect.

The changes in binding affinity/specificity of exemplary Fc variants compared to their wild-type counterparts are provided in Tables 2-4 below. "N/A" indicates no data available. When the binding activity of an Fc variant is found to be "unchanged" compared to its wild-type counterpart, it means that there is no significant variation in binding activity between the Fc variant and the wild-type counterpart as shown by the same assay under the same experimental conditions. When the binding activity of an Fc variant is "increased" or "decreased" compared to its wild-type counterpart, it means that the binding activity of the Fc variant is higher or lower than the binding activity of the wild-type counterpart as determined by the same assay under the same experimental conditions, and the variation is significant (e.g., biologically significant) as known to those of skill in the art. When the binding activity of an Fc variant is "slightly increased" or "slightly decreased" compared to its wild-type counterpart, it means that the binding activity of the Fc variant is higher or lower than the binding activity of the wild-type counterpart as determined by the same assay under the same experimental conditions, and the variation is statistically significant, but at a limited level (e.g., up to 10%).

The Fc variants described herein can be designed following the guidance provided herein and produced via routine recombinant techniques. Their binding affinity and specificity for various Fc receptors can be determined by routine methods. See also the examples below.

In certain examples, the CD40 binding molecules (anti-CD40 molecules) disclosed herein may retain the hinge region of the IgG1 parent and include one or more mutations (e.g., one or more deletions at positions 236-238 relative to mouse IgG1, as discussed above) that result in reduced binding affinity to Fc receptors such as FcγR2B, but maintain residual binding activity to the Fc receptor. Exemplary Fc variants with such properties include, but are not limited to, G1m2, G4m20, G4m30, and G4m46. The CD40 binding portion of such anti-CD molecules may be derived from an agonist anti-CD40 antibody. Such a CD40 binding portion may be derived from the anti-CD40 antibody 383 disclosed herein. In some examples, the CD40 binding portion includes the same _VH and _VL CDRs as antibody 383. In some examples, the CD40 binding portion may include the same VH and/or VL chains as antibody 383 (SEQ ID NOs: 128 and 129, respectively). Alternatively, the CD40 binding moiety may be a functional variant of antibody 383, as disclosed herein. Such CD40 binding molecules would have several advantageous features, as shown in the examples below. Examples include enhanced anti-tumor efficacy with improved therapeutic window and reduced in vivo side effects (e.g., less liver toxicity), which may be due to baseline CD40 agonistic activity and residual FcγRIIB binding activity. Too high CD40 agonistic activity and/or FcγRIIB binding activity would be expected to induce side effects such as cytotoxicity.

In other specific examples, the CD40 binding molecules disclosed herein comprise an Fc variant domain derived from IgG2 (e.g., maintaining the IgG2 hinge) and may comprise one or more mutations (e.g., deletions or substitutions) at one or more of positions 236-238 relative to the murine counterpart to reduce binding affinity to Fc receptors, particularly binding affinity to FcγRIIB. In some cases, such Fc variants have a deletion at one or more of positions 236-238. Alternatively, or in addition, Fc variants may have substitutions at positions 234, 237, 238, 268, or combinations thereof. Such Fc variants may comprise an additional mutation at one or more positions involved in binding to Fc receptors, e.g., position 265 (e.g., D265A). Fc variants may maintain residual binding activity to Fc receptors, such as FcγRIIB (e.g., G2m20 and G2m43). Alternatively, the Fc variant may not bind to an Fc receptor such as FcγRIIB (e.g., G2m40). The CD40 binding portion of such an anti-CD molecule may be derived from an agonistic anti-CD40 antibody. Such a CD40 binding portion may be derived from the anti-CD40 antibody 383 disclosed herein. In some cases, the CD40 binding portion comprises the same _VH and _VL CDRs as antibody 383. In some examples, the CD40 binding portion may comprise the same VH and/or VL chains as antibody 383 (SEQ ID NOs: 128 and 129, respectively). Alternatively, the CD40 binding portion may be a functional variant of antibody 383, as disclosed herein. Such a CD40 binding molecule would have several advantageous features, as shown in the examples below. Examples include enhanced anti-tumor efficacy with improved therapeutic window and reduced in vivo side effects (e.g., less liver toxicity), which may result from a combination of baseline CD40 agonist activity and residual FcγRIIB binding activity (e.g., 383-IghuG2m20) or a combination of relatively high CD40 agonist activity and no binding to FcγRIIB (e.g., 383-IghuG2m40).

In addition to the FACS binding assays described herein, the activity of Fc variants on Fcγ receptors can be examined with alternative assays. For example, a CD40 reporter assay, with or without co-culture of FcγRIIB-expressing cells, can be used to demonstrate the effects of receptor cross-linking and activation. Due to avidity effects resulting from simultaneous binding of antibodies to two targets (e.g., CD40 and FcγRIIB), this assay is more sensitive to detect FcγRIIB binding. A positive result may be indicated by a reporter assay when detectable binding by FACS is minimal or not evident.

II. Preparation of CD40 Binding Molecules Containing Fc Variants The CD40 binding molecules described herein can be prepared by conventional methodologies, e.g., recombinant techniques. Some examples are provided below.

For CD40 binding molecules that include the extracellular domain of CD40L, the coding sequence for the CD40L extracellular domain can be fused in frame with the coding sequence for a suitable Fc variant. The coding sequence for the entire CD40 binding molecule can be cloned into a suitable expression vector and introduced into a suitable host cell for protein expression.

Antibodies that bind to CD40 can be prepared by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, antibodies specific for CD40 or its extracellular domain can be produced by conventional hybridoma technology. A full-length target receptor or a fragment thereof can be optionally coupled to a carrier protein such as KLH and used to immunize a host animal to generate antibodies that bind to the antigen. The route and schedule of immunization of the host animal generally follows established conventional techniques for stimulating and producing antibodies, as further described herein. General techniques for producing mouse, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including a human, or antibody-producing cells therefrom may be engineered to serve as the basis for the production of mammalian hybridoma cell lines, including humans. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally with an amount of an immunogen, including those described herein.

Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497, or modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. Generally, this technique involves fusing myeloma cells with lymphoid cells using a fusogen such as polyethylene glycol or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to eliminate unhybridized parent cells. Any of the media described herein, with or without serum, can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative to the cell fusion technique, EBV-immortalized B cells can be used to produce the anti-immune cell receptor monoclonal antibodies described herein. Hybridomas are expanded and subcloned as necessary, and supernatants are assayed for anti-immunogen activity by conventional immunological tests (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of the parent hybridoma that produce monoclonal antibodies capable of modulating the activity of a target immune cell receptor. Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from the culture medium or body fluids, if desired, by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by treating the preparation over an adsorbent made of the immunogen attached to a solid phase, and eluting or releasing the desired antibody from the immunogen. Immunization of a host animal with a target antigen or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (linking through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR (where R and R1 are different alkyl groups), can yield a population of antibodies (e.g., monoclonal antibodies).

If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) can be sequenced and the polynucleotide sequence then cloned into a vector to further construct the anti-CD40 antibody described herein. The sequence encoding the antibody of interest can be maintained in the vector within a host cell, which can then be propagated and frozen for future use.

Alternatively, the polynucleotide sequence may be used for genetic engineering to "humanize" the antibody or to improve the affinity (affinity maturation) or other properties of the antibody. For example, the constant region may be engineered to be more similar to human constant regions to avoid an immune response if the antibody is to be used in human clinical trials and treatments. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for the target antigen and greater potency in inhibiting or activating the activity of an immune cell receptor. It will be apparent to one of skill in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity for a target receptor.

In other embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or stronger immune response can also be used to generate humanized or human antibodies. Examples of such technology include the Xenomouse® from Amgen, Inc. (Fremont, Calif.) and the HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, N.J.). In another alternative, antibodies can be made recombinantly by phage display or yeast technology. See, for example, U.S. Pat. Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150, and Winter et al. , (1994) Annu. Rev. Immunol. 12:433-455.

Alternatively, antibody library techniques such as phage display techniques (McCafferty et al., (1990) Nature 348:552-553), yeast display techniques, or mammalian cell display techniques can be used to isolate antibodies, such as human antibodies, specific for target immune receptors.

Methods for constructing humanized antibodies are also well known in the art. See, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the _VH and _VL variable regions of a parent non-human antibody are subjected to three-dimensional molecular modeling analysis according to methods known in the art. Framework amino acid residues predicted to be important for the formation of the correct CDR structure are then identified using the same molecular modeling analysis. In parallel, human VH and VL chains having amino acid sequences that are homologous to the amino acid sequences of the parent non-human antibody are identified from any antibody gene database using the parent VH and VL sequences as search queries. Human VH and VL acceptor genes are then selected.

The CDR regions in the selected human acceptor gene can be replaced with CDR regions from a parent non-human antibody or a functional variant thereof. If necessary, residues in the framework regions of the parent chain that are predicted to be important for interactions with the CDR regions (see discussion above) can be used to replace the corresponding residues in the human acceptor gene.

Once an antibody capable of binding to a target immune cell receptor is obtained, the coding sequence for its heavy chain can be fused in frame with the coding sequence for a suitable Fc variant that may selectively bind FcgRIIB or that does not bind FcγR, e.g., any of the Fc variants described herein via conventional recombinant techniques. In some cases, the antibody is first investigated for its agonistic effect in activating the immune cell receptor to which it binds. Such agonistic antibodies can be selected to generate the CD40 binding molecules described herein to enhance the agonistic effect.

In other examples, an antibody is first investigated for its antagonistic effect in inhibiting the immune cell receptor to which it binds. Such antagonist antibodies can be selected to generate CD40 binding molecules described herein to down-regulate immune responses. Alternatively, Fc variants with low or no binding activity to Fc receptors can be selected to generate CD40 antagonists.

The resulting antibody molecules or CD40 binding molecules described herein can be produced via conventional recombinant techniques, as exemplified below. The nucleic acids encoding the heavy and light chains of the polypeptides of the antibodies or CD40 binding molecules described herein can be cloned into an expression vector, with each nucleotide sequence operably linked to a suitable promoter. In one example, each of the nucleotide sequences encoding the heavy and light chains is operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences for the heavy and light chains.

In some examples, the nucleotide sequences encoding the two chains of an antibody are cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each of them can be isolated from the host cell that expresses it, and the isolated heavy and light chains can be mixed and incubated under suitable conditions that allow the formation of the antibody.

In general, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule that can pair with each other and join together with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector/promoter will depend on the type of host cell to be used to produce the antibody.

A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, Simian Virus 40 (SV40) early promoter, E. coli lac UV5 promoter, and herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli as a transcriptional modulator to regulate transcription from mammalian cell promoters having the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)], those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992), Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998), Shockelt, P., et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astrodiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

A regulatable promoter containing a repressor can be used with the operon. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from a mammalian cell promoter with a lac operator [M. Brown et al., Cell, 49:603-612 (1987)], Gossen and Bujard (1992), [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] to create a tetR-tet operator system to control gene expression in mammalian cells. A minimal promoter containing tetO derived from the major immediate early promoter of human cytomegalovirus (hCMV) is used to create the tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16). In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is appropriately positioned downstream of the TATA element of the CMV IE promoter, only the tetracycline repressor (tetR), but not the tetR-mammalian cell transcription factor fusion derivative, can function as a potent transmodulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its tunable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

In addition, the vector can include, for example, some or all of the following: a selectable marker gene, such as a neomycin gene, for selecting stable or transient transfectants in mammalian cells, an enhancer/promoter sequence from the immediate early gene of human CMV for high levels of transcription, transcription termination and RNA processing signals from SV40 for mRNA stability, the SV40 polyoma origin of replication and ColE1 for proper episomal replication, an internal ribosome binding site (IRES), a versatile multiple cloning site, and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art.

Examples of polyadenylation signals useful for carrying out the methods described herein include, but are not limited to, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cell can be cultured under conditions suitable for expression of the antibody or any polypeptide chains thereof. Such antibodies or polypeptide chains thereof can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, e.g., affinity purification. If desired, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time to allow production of the antibody.

In some embodiments, the methods for preparing the antibodies described herein include a recombinant expression vector encoding both the heavy and light chains of the antibodies described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under suitable conditions to allow expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If desired, the two chains recovered from the host cells can be incubated under suitable conditions to allow formation of the antibody.

In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-immune cell receptor antibody and the other encoding the light chain of the same antibody. Both of the two recombinant expression vectors can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, e.g., calcium phosphate-mediated transfection. Alternatively, each of the expression vectors can be introduced into a suitable host cell. Positive transformants can be selected and cultured under suitable conditions that allow expression of the antibody polypeptide chains. If the two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium and then incubated under suitable conditions that allow the formation of the antibody. If the two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a Protein A or Protein G-bound matrix.

The biological activity of the antibodies described herein can be verified using assays known in the art or described herein.

III. Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising any of the CD40 binding molecules or anti-CD40 antibodies described herein, and the use of such to modulate immune responses elicited by CD40/CD40L signaling. The CD40 binding molecules described herein or the anti-CD40 antibodies also described herein can be used to treat diseases such as cancer or immune-related disorders.

The CD40 binding molecules or anti-CD40 antibodies described herein can be mixed with a pharma- ceutically acceptable carrier (excipient) to form a pharmaceutical composition for use in treating a target disease. By "acceptable," it is meant that the carrier must be compatible with (and preferably be able to stabilize) the active ingredient of the composition and not deleterious to the subject being treated. Pharmaceutically acceptable excipients (carriers) include buffers, which are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions used in the methods of the invention may be in the form of lyophilized formulations or aqueous solutions and may contain pharma- ceutically acceptable carriers, excipients, or stabilizers. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K.E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, and the like. The surfactants may include proteins such as amine, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and/or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

In some examples, the pharmaceutical compositions described herein include liposomes containing the antibody (or encoding nucleic acid), which can be prepared by methods known in the art, such as those described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

The CD40 binding molecules, anti-CD40 antibodies, or encoding nucleic acid(s) can also be entrapped in microcapsules, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, prepared, for example, by coacervation techniques or by interfacial polymerization. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical compositions described herein can be formulated in sustained release form. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, these matrices being in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or for administration by inhalation or insufflation.

To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical carriers, such as conventional tablet ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums, as well as other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition, such that the composition may be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill may contain an inner dosage and an outer dosage component, the latter in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

Suitable surfactants include non-ionic agents such as polyoxyethylene sorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80 or 85), among others. Compositions containing a surfactant conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added as required.

Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™, and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition or it can be dissolved in oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and form an emulsion upon mixing with phospholipids (e.g., egg phospholipids, soybean phospholipids, or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, can be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g., 5-20%. The fat emulsion contains fat droplets of 0.1 to 1.0 μm, especially 0.1 to 0.5 μm, and has a pH in the range of 5.5 to 8.0.

The emulsion composition may be prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect.

Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by use of a gas. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices which deliver the formulation in an appropriate manner.

IV. Therapeutic Applications Any of the CD40 binding molecules or anti-CD40 antibodies disclosed herein can be used to modulate (e.g., enhance or inhibit) the immune response against invading pathogens and/or diseased cells, such as cancer cells.

To practice the methods disclosed herein, an effective amount of the pharmaceutical compositions described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and milled powders.

The subject treated by the methods described herein can be a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

In some cases, the subject is a human patient suffering from or at risk of a cell-mediated disease or disorder, such as cancer, including, but not limited to, lung cancer, stomach cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, or colorectal cancer. Further exemplary cancers include breast cancer, biliary tract cancer, bladder cancer, brain cancer, including glioblastoma and medulloblastoma, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, stomach cancer, hematological tumors, including acute lymphocytic leukemia and myeloid leukemia, e.g., B-cell CLL, T-cell acute lymphoblastic leukemia/lymphoma, hairy cell leukemia, chronic myelogenous leukemia, multiple myeloma, AIDS-related leukemia and adult T-cell leukemia/lymphoma, intraepithelial neoplasia, including Bowen's disease and Paget's disease, liver cancer, lung cancer, lymphomas, including Hodgkin's disease and lymphocytic lymphoma, neuroblastoma, squamous epithelial tumors, including lymphomas, including lymphocytic lymphomas, neuroblastoma, squamous epithelial tumors, and leukemias. Cancers that may be treated include, but are not limited to, oral cancer, including cancer, ovarian cancer, including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma, skin cancer, including melanoma, Merkel cell carcinoma, Kaposi's sarcoma, basal cell carcinoma, and squamous cell carcinoma, testicular tumors, including seminoma, non-seminoma (teratoma, choriocarcinoma), stromal tumors, embryonic tumors such as germ cell tumors, thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma, and renal cancer, including adenocarcinoma and Wilms' tumor. Subjects suffering from cancer can be identified by routine medical examinations, e.g., laboratory tests, organ function tests, CT scans, or ultrasound. In some embodiments, the subjects treated by the methods described herein may be human cancer patients who have undergone or are undergoing anti-cancer therapy, e.g., chemotherapy, radiation therapy, immunotherapy, or surgery.

In another example, the subject is a human patient suffering from or at risk of an immune-related disorder. Immune-related disorder refers to a dysfunction of the immune system, including autoimmune diseases, immune deficiencies, and/or allergies. In one embodiment, the immune-related disorder is an autoimmune disease. Examples of immune-related disorders include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), myasthenia gravis (MG), Graves' disease, idiopathic thrombocytopenic purpura (ITP), Guillain-Barré syndrome, autoimmune myocarditis, membranous glomerulonephritis, diabetes mellitus, type I or type II diabetes, multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, gastritis, celiac disease, leukoplakia, hepatitis, primary biliary cirrhosis, inflammatory bowel disease, spondyloarthropathy, experimental autoimmune encephalomyelitis. , immune neutropenia, juvenile onset diabetes, and immune responses associated with cytokines, delayed hypersensitivity mediated by T lymphocytes typically seen in tuberculosis, sarcoidosis, and polymyositis, polyarteritis nodosa, cutaneous vasculitis, pemphigus, pemphigoid, Goodpasture's syndrome, Kawasaki's disease, systemic sclerosis, antiphospholipid syndrome, Sjogren's syndrome, graft-versus-host (GVH) disease, and immune thrombocytopenia. Subjects suffering from immune-related disorders can be identified, for example, by routine medical examinations, such as laboratory tests. In some embodiments, subjects treated by the methods described herein can be human subjects with immune-related disorders who have received or are receiving immune-related disorder treatments, such as immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory drugs, and/or analgesics.

As used herein, an "effective amount" refers to the amount of each active agent required to confer a therapeutic effect on a subject, either alone or in combination with one or more other active agents. In some embodiments, the therapeutic effect is to modulate (e.g., activate) a target immune receptor, thereby inducing or enhancing an immune response mediated by the receptor. Determining whether an amount of a composition described herein achieves a therapeutic effect will be apparent to one of skill in the art. As will be recognized by those of skill in the art, an effective amount will vary depending on individual patient parameters, including the particular condition being treated, the severity of the condition, age, physical condition, size, sex, and weight, duration of treatment, the nature of concomitant therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of the medical practitioner. These factors are well known to those of skill in the art and can be addressed with only routine experimentation. In general, it is preferred to use the maximum dose of each component or combination thereof, i.e., the highest safe dose according to sound medical judgment.

Empirical considerations such as half-life generally contribute to the determination of dosage. For example, molecules compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. Dosing frequency can be determined and adjusted over the course of therapy and is generally, but not necessarily, based on the treatment and/or suppression and/or amelioration and/or delay of the targeted disease/disorder. Alternatively, sustained release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one example, the dosage of a CD40 binding molecule or anti-CD40 antibody described herein can be empirically determined in an individual who has received one or more doses of the antibody. The individual is given incremental doses of the agonist. Indicators of disease/disorder can be followed to assess the effectiveness of the agonist.

In general, for administration of any of the therapeutic agents, such as CD40 binding molecules or anti-CD40 antibodies described herein, the initial candidate dose may be about 2 mg/kg. For purposes of this disclosure, a typical daily dose may range anywhere from about 0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors described above. For repeated administration over several days or more, depending on the condition, treatment is sustained until desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms. An exemplary dosing regimen includes administering an initial dose of about 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg of the antibody, or a maintenance dose of about 1 mg/kg every other week thereafter. However, other dosing regimens may be useful depending on the pattern of pharmacokinetic decay the physician wishes to achieve. For example, dosing from 1 to 4 times per week is contemplated. In some embodiments, dosages ranging from about 3 μg/mg to about 2 mg/kg (e.g., about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg, about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) can be used. In some embodiments, the dosing frequency is once every week, every 2 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, or every 10 weeks, or once every month, every 2 months, or every 3 months, or more. The progress of this therapy is easily monitored by conventional techniques and assays. The dosing regimen (including the therapeutic method used) can vary over time.

In some embodiments, a dose ranging from about 0.3 to 5.00 mg/kg can be administered to a normal weight adult patient. In some examples, the dosage of a therapeutic agent such as an antibody described herein can be 10 mg/kg. The particular dosing regimen, i.e., dose, timing, and repetition, will vary depending on the particular individual and that individual's medical history, as well as the characteristics of the individual agent (such as the half-life of the agent and other considerations known in the art).

For purposes of this disclosure, the appropriate dosage of the CD40 binding molecules or anti-CD40 antibodies described herein will depend on the particular antibody, multiple antibody and/or non-antibody peptides (or compositions thereof) used, the type and severity of the disease/disorder, whether the CD40 binding molecules or anti-CD40 antibodies are administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to agonists, and the discretion of the attending physician. Typically, the clinician will administer the CD40 binding molecules or anti-CD40 antibodies until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in thrombosis. Methods for determining whether a dosage has produced a desired result will be apparent to one of skill in the art. Administration of one or more CD40 binding molecules or anti-CD40 antibodies may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to the skilled physician. Administration of the CD40 binding molecule or anti-CD40 antibody can be essentially continuous over a preselected period of time, or can be in a series of spaced doses, e.g., either before, during, or after the onset of the target disease or disorder.

As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease/disorder, or a predisposition to a disease/disorder, for the purpose of curing, healing, mitigating, alleviating, altering, relieving, enhancing, ameliorating, or affecting the disorder, symptom of the disease, or predisposition to the disease or disorder.

Alleviating the target disease/disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease. Alleviating the disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of the target disease or disorder means suspending, impeding, slowing, delaying, stabilizing, and/or postponing the progression of the disease. The delay may be for a variety of lengths of time, depending on the history of the disease and/or the individual being treated. A method of "delaying" or alleviating the onset of a disease, or delaying the onset of a disease, is a method that reduces the likelihood of developing one or more symptoms of the disease within a given time frame and/or reduces the severity of the symptoms within a particular time frame, when compared to not using the method. Such comparisons are typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

"Onset" or "progression" of a disease refers to the initial symptoms and/or subsequent progression of a disease. Onset of a disease can be detected and assessed using standard clinical techniques well known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of a symptom. "Onset" includes onset, recurrence, and onset. As used herein, "onset" or "onset" of a target disease or disorder includes initial onset and/or recurrence.

In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to activate the target receptor by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo.

Depending on the type of disease or site of disease being treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the medical arts. The composition can also be administered via other conventional routes, for example, orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. Additionally, the subject can be administered via an injectable depot administration route, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

Injectable compositions may include a variety of carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injections, water-soluble antibodies may be administered by infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

The particular dosing regimen used in the methods described herein, i.e., dosage, timing, and repetition, will depend on the particular subject and the subject's medical history.

In some embodiments, a combination of two or more antibodies, or an antibody and another suitable therapeutic agent, can be administered to a subject in need of treatment. The CD40 binding molecules or anti-CD40 antibodies can also be used in combination with other agents that serve to enhance and/or complement the effectiveness of the agent.

The efficacy of treatment of the target disease/disorder can be evaluated by methods known in the art.

The therapeutic agents described herein can be utilized in combination with other types of treatments for a target disease, such as cancer. Additional anti-cancer therapies include chemotherapy, surgery, radiation therapy, gene therapy, and the like. When a second therapeutic agent is used, such agent can be administered simultaneously or sequentially (in any order) with the CD40 binding molecules or anti-CD40 antibodies described herein.

When co-administered with additional therapeutic agents, the preferred therapeutically effective dose of each agent may be reduced due to additive or synergistic effects.

The treatments of the present disclosure can be combined with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including, but not limited to, GVAX, DC-based vaccines, etc.), or checkpoint inhibitors (including, but not limited to, agents that block CTLA4, PD1, LAG3, TIM3, etc.). Alternatively, the treatments of the present disclosure can be combined with chemotherapeutic agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine), purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilone, and navelbine. disruptor), epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylneuramine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxane Antiproliferative/mitotic inhibitors including ribavirin, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; an enzyme (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents, night flu drugs, Antiproliferative/mitotic inhibitory alkylating agents such as the aryl mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazine (DTIC); antiproliferative/mitotic inhibitory antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin); , carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (tissue plasminogen activator, streptokinase, urokinase, etc.), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents agents); secretory inhibitors (breverdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibition agents, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, prenisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; chromatin disruptors.

For examples of additional useful agents, see also Physician's Desk Reference, 59. sup. th edition, (2005), Thomson P D R, Montvale N. J., Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20. sup. th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md., Braunwald et al., Eds. See Harrison's Principles of Internal Medicine, 15. sup. th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway N.J.

V. Kits The present disclosure also provides kits for use in enhancing a desired immune response using any of the CD40 binding molecules or anti-CD40 antibodies described herein.

In some embodiments, the kit can include instructions for use according to any of the methods described herein. The included instructions can include instructions for administering a therapeutic agent, such as those described herein, to treat, delay onset, or ameliorate the target disease. The kit can further include instructions for screening an individual suitable for treatment based on identifying whether the individual has the target disease. In yet other embodiments, the instructions include instructions for administering a therapeutic agent, such as an antibody, to an individual at risk for the target disease.

Instructions for use of the therapeutic agent generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions provided in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions transmitted on a magnetic or optical storage disk) are also acceptable.

The label or package insert indicates that the composition is used for treating, delaying the onset of, and/or ameliorating the targeted disease or disorder, such as cancer. Instructions for performing any of the methods described herein may be provided.

The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packages for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., a nebulizer), or an infusion device, such as a minipump, are also contemplated. The kits may have a sterile access port (e.g., the container may be an intravenous solution bag, or a vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag, or a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a therapeutic agent as described herein.

The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture that includes the contents of the kit described above.

General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are fully described, for example, in such publications as: Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press, Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons, Methods in Enzymology (Academic Press, Inc.), Handbook of Experimental Immunology (D.M. Weir and C. C. Blackwell, eds.), Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987), Current Protocols in Molecular Biology (FM. Ausubel, et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994), Current Protocols in Immunology (J.E. Coligan et al. al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (C.A. Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and J. D. Capra, eds. , Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the specific embodiments provided herein are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

Example 1. Anti-CD40 Antibodies with Engineered Fc Regions cDNA sequences encoding anti-CD40 antibody variable domains with various heavy chain CH1 and Fc regions or human kappa light chain constant regions were synthesized and cloned. CHO transient expression was performed using plasmids containing the corresponding heavy and light chain sequences. The antibodies were purified by Protein A affinity chromatography. The amino acid sequences of the heavy chain (HC) and light chain (LC) are shown below.
383-huIgG LC
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 146)
383-huIgG1m2 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 147)
383-huIgG1m27 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 148)
383-huIgG2m1 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 149)
383-huIgG2m19 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC VECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGFPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 150)
383-huIgG2m20 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 151)
383-huIgG4m2 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 152)
383-huIgG4m20 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 153)
383-huIgG4m30 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 154)
383-ms IgG LC
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC (SEQ ID NO: 155)
383-msIgG1 DANA HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD YWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 156)
383-msIgG1 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD YWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 157)
383-msIgG2a DANA HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 158)
383-msIgG2a HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSS GVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 159)

Additional Fc variants were made, the amino acid sequences of the heavy chain (HC) are shown below (the light chain (LC) sequence is shown above).
383-huIgG1 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 207)
383-huIgG2 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 208)
383-huIgG4SP HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 209)
383-huIgG1mAA
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 210)
383-huIgG1m40 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVaVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 193)
383-huIgG1m45 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194)
383-huIg G1m240 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 195)
383-huIgG1 N297A
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 196)
383-huIgG1m47 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 197)
383-huIgG1m48 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSsDKT HTsPPsPAPELLGGsSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 198)
383-huIgG1m49 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTsPPsPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 199)
383-huIgG1m50 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 214 )
383-huIgG1mAAG
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGT NYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 211)
383-huIgG2m40 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 200)
383-huIgG2m43 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 201)
383-huIgG2m44 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSqEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSPREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 202)
383-huIgG2mAA HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPaaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 203)
383-huIgG2m2040 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 204)
383-huIgG2G4 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 205)
383-huIgG4m40 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 206)
383-huIgG4m41 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 66)
383-huIgG4m42 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 67)
383-huIgG4m46 HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPEALGaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 68)
383-huIgG4mPE HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 212)
383-huIgG4mAA HC
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 69)

Example 2. Measuring the binding activity of CD40 antibodies with Fc variants to cellular FcγRs To determine the binding activity of CD40 antibodies with IgG variants to cellular Fc receptors, CHO cells were engineered to express human FcγRs (FcγRI, FcγRIIA, FcγRIIB, and FcγRIII) using a lentiviral delivery system known in the art.

Using the anti-CD40 antibody 383 disclosed herein as an exemplary anti-CD40 moiety, 383-msIgG1, 383-msIgG1 DANA, 383-msIgG2a, 383-msIgG2a A number of anti-CD40 IgG Fc variants were designed and constructed in accordance with the disclosure herein, including DANA, 383-hIgG2, control huIgG1, 383-huIgG1m27, 383-huIgG2m19, 383-huIgG1m2, 383-huIgG2m1, 383-huIgG2m20, 383-huIgG4m2, 383-huIgG4m20, 383-huIgG4m30, 383-huIgG1mAA, and 383-huIgG4SP (amino acid sequences shown above). These IgG variants contain mutations in the upper hinge domain, lower hinge domain, or CH2 domain.

For FACS analysis of binding of IgG variants to various FcγRs, CHO cells overexpressing FcγRs were harvested using trypsin-EDTA and suspended in cold staining buffer (3% BSA in PBS). Test IgG variants diluted in staining buffer were added to the cells. The mixture was incubated for 2 h at 4°C, then washed twice with cold staining buffer and resuspended in PE-labeled anti-human IgG, followed by incubation for 2 h at 4°C. The mixture was washed twice with staining buffer and resuspended in 2% PFA in PBS for FACS.

As shown in Figures 1A-1D and 3A-3D, many of the anti-CD40 human IgG1, IgG2, and IgG4 variants exhibited binding activity to cell surface expressed FcγRs, while others showed no clear findings to the FcγRs tested. A summary of the changes in binding characteristics is provided above in Tables 2-4.

Example 3. Fc region engineered CD40 antibodies bind different CD40
To measure the agonistic activity of IgG variants that exhibited agonistic activity, a CD40 reporter assay was developed, which includes reporter cells overexpressing human CD40 T cells. The GS-H2-huCD40 reporter cells were resuspended in assay buffer and cell density and viability were measured with trypan blue. The cell suspension was diluted to ^1x104 cells/mL in assay buffer (MEM with 1% FBS). Cells were added at 100uL/well to a final cell count of 1000 cells/well in the assay plate (Nunc, Cat. No. 167425). Serial working dilutions of test samples were prepared in assay buffer at 2x final concentration. Samples were added to the assay plate at 100uL/well of test sample at 2x final concentration. The assay plate was incubated in a 37°C, 5% _CO2 incubator for 18-20 hours. After 18-20 hours of incubation, 8 ul of supernatant was collected from each well of the assay plate and added to the HTRF detection assay plate (Nunc). Human Interleukin 8 (reporter of CD40 activation) detection assay was performed using a Human IL-8 Assay Kit (Cisbio, Cat# 62IL8PEB). Specifically, an assay volume of 16 ul was used. Results were read using Time Resolved Fluorescence with a Tecan F200pro and relative light unit data was recorded.

As shown in Figure 2 and Figures 4A-4E, all of the anti-CD40 antibody IgG variants tested stimulated human CD40 activation, as evidenced by IL8 secretion. The magnitude of CD40 activation was influenced by the Fc variant contained therein due to the flexibility of the hinge of each antibody isotype structure.

CD40 reporter assays were also performed in co-culture with CHO cells expressing FcγRIIB, and the results are shown in Figures 4A-4D. Binding to CD40 and FcγR2B by antibody molecules tested simultaneously in a microenvironment affects individual binding due to the avidity effect, which refers to the cumulative strength of multiple affinities of individual non-covalent interactions. Antibodies showed increased activity that correlated with binding activity to FcγRIIB. It was noted that antibodies with Fc variants that had weak or no apparent binding to FcγRIIB showed enhanced agonist activity in the co-culture reporter assay, suggesting a higher sensitivity of the reporter assay that may be due to the avidity effect. Thus, the Fc structure of an antibody may affect the agonist activity of antibodies with the same variable domain.

Example 4. Characterization of anti-CD40 antibody IgG variants in dendritic cell (DC) functional assays Exemplary anti-CD40 antibody Ig variants, including various Fc variants, were tested in vitro for CD40 binding and agonist activity as described in the Examples above, and in vivo for anti-tumor efficacy and toxicity as in Example 6. Their ability to activate human dendritic cells was performed as follows.

Frozen human PBMCs (Allcells, Cat. No. PB005F) from healthy donors were thawed and transferred to a 50 ml tube containing 25 mL of RPMI 1640 medium. B cells were isolated using EasySep™ Human CD14 Positive Selection Kit II (Stemcell, Cat. No. 17858). The PBMC suspension was centrifuged at 250 g for 15 minutes, the supernatant was discarded and the cells were resuspended in 1.5 mL of EasySep™ buffer. Selection cocktail was added to the samples in 14 mL (17x100 mm) polystyrene round bottom tubes. 100 ul/ml, 150 ul for each sample. The cells were transferred from the 50 ml tube to a 14 mL (17x100 mm) polystyrene round bottom tube, mixed and incubated at room temperature (RT) for 15 minutes. Mix magnetic particles before transferring to sample. Transfer magnetic particles to sample. 50 ul/ml, 75 ul for each sample. Mix and incubate at room temperature for 10 minutes. Add 8.5 ml EasySep™ buffer to top up sample to 10 mL. Gently pipette up and down 2-3 times to mix. Place tube in magnet and incubate for 5 minutes. Remove magnet and invert magnet and tube in one continuous motion to pour off supernatant. Collect this supernatant for B cell enrichment. Repeat above washing procedure 2 more times. Resuspend cells in 5 mL RPMI 1640. Count cell density.

CD14 ⁺ cells isolated from human PBMCs were adjusted to ^1x106 cells/ml, supplemented with GM-CSF and IL-4, and added to cell culture medium. The cells are incubated for 3 days in a humidified incubator at 37°C and 5% CO2. On the third day, the medium is replaced by transferring the culture supernatant to a centrifuge tube and centrifuged at 250xg for 5 minutes. 10 ml of fresh medium containing GM-CSF and IL-4 is used to suspend the cell pellet in the centrifuge tube, which is then added to the flask during centrifugation so that the cell pellet thus formed is resuspended in 10 ml of fresh medium. The resuspended cells are transferred back to the same well or flask and incubated for another 2 days. On the fifth day, the above procedure is repeated for medium replacement. The cells are further incubated for another 2 days. On the seventh day, immature dendritic cells can be observed and are ready to be used for the desired application.

To perform DC activation assay, the medium containing cells from T75 flasks was taken into a 50 ml tube and centrifuged at 500xg for 5 min at room temperature. The supernatant was discarded and the cells were resuspended in 3 ml of RPMI1640 10% FBS medium. The cell density was counted and the cell concentration was adjusted to 1e6/ml. 0.05 ml of the cell suspension was added to a 96-well plate at 2.5x104 cells/well. IL-8 and IL12P70 were detected in the cell culture supernatant after 24 hours (day ³ ) using HuIL-12-P70 detection kit (Cisbio, Catalog No: 62HIL12PEH) and HuIL-8 detection kit (Cisbio, Catalog No: 62HIL08PEH). DC activation assay was also performed in co-culture with CHO cells expressing FcγR2B.

As shown in Figures 5A-5D, the CD40 antibody IgG variants tested stimulated human CD40 activation to various degrees, as evidenced by the secretion of IL8 from DC cultures after antibody incubation. The magnitude of DC activation was influenced by the Fc variant contained therein, due to the flexibility of the hinge of each antibody isotype structure and its interaction with Fc receptors expressed by DC cultures.

To confirm the effect of Fc-FcγRIIB cross-linking on CD40 activation, DC activation was also performed in co-culture with FcγR2B-expressing CHO cells. When antibody molecules simultaneously bind to CD40 and FcγRIIB in the microenvironment, the individual binding is influenced by avidity effects that lead to changes in the CD40 agonist effect of the antibody. The antibodies showed increased activity that correlated with the binding activity to FcγRIIB, consistent with the observations in the reporter assay above. Thus, the Fc structure of an antibody influences the overall agonist activity of antibodies with the same variable domain.

Example 5. IgG variant CD40 antibodies show similar binding to cellular CD40 FACS was used to assess the binding properties of exemplary anti-CD40 antibody IgG variants. CHO cells overexpressing human CD40 or cynomolgus CD40 were harvested using trypsin-EDTA partial digestion followed by centrifugation at 1000 rpm for 5 minutes. Cells were resuspended in cold PBS-BSA (2%) at ^5x106 /ml and dispensed into 100 ul/tube. Chimeric anti-CD40 antibodies were diluted 3-fold in PBS-BSA (final concentrations of 0.01, 0.1, 1, and 10 ug/ml) and 50 ul of each concentration was added to the CHO-CD40 cells. The cell solutions were mixed and incubated in the dark at 4°C for 2 hours. Cells were then washed twice with PBS-BSA. Secondary antibody conjugate (Goat F(ab')2 anti-human IgG-Fc(PE), pre-adsorbed (Abcam #ab98596)) was added at a concentration of 100 ul/vial, the cells were mixed and incubated for 1 hour at 4°C in the dark. The cells were then washed twice with PBS-BSA, followed by fixation with 2% PFA in PBS and FACS analysis on a FACScaliber. As shown in Figures 6A-6B, these antibodies showed high binding activity to human CD40 expressed on the surface of CHO cells.

Example 6. IgG variant CD40 antibodies
Exhibit Differential Tumor Activity in In Vivo Animal Models Exemplary anti-CD40 antibodies with various Fc variants were tested in vivo in mouse syngeneic tumor models to test how Fc variants may affect efficacy and toxicity of these antibodies. Syngeneic mouse tumor models were developed using human CD40 extracellular domain knock-in C57BL6 mice. Murine colon carcinoma MC38 cells were subcutaneously implanted into homozygous human CD40 knock-in C57BL6 mice on day 0. Mice were grouped when tumor size was approximately 150±50 mm ³ (n=6). CD40 antibodies were administered by intraperitoneal injection and tumor size was measured during 4-6 weeks of antibody treatment. Tumor size was measured twice a week by caliber and calculated as tumor volume using the formula 0.5×length× ^width2 . Antitumor efficacy was evaluated between tumor size in control and antibody treatment groups.

As shown in Figure 7A, mouse IgG2aDANA (Fc null) provided the reference efficacy caused by Fab'2-induced CD40 activation. Human G4m2, G1m2, G2m20 and G2m40 showed superior or comparable efficacy to mIgG2aDANA.

Potential antibody-induced hepatotoxicity was examined in a mouse model by measuring serum ALT levels after antibody treatment. Human G2 showed very large ALT elevations, exceeding the upper limit of the assay (>1000 U/L). Efficient Fc variants, including G1m2, G2m20, and G2m40, showed low or minimal ALT elevations.

As examples, 383-G2m20 and -G2m40 showed high agonist activity in the absence of cross-linking and in vivo efficacy with FcγR interactions (G2m40 is FcγR null in both binding and DC activation assays). Furthermore, the residual FcgRIIB binding activity of G2m20 enhanced efficacy. Fc variants G2m1 and G2m43 are expected to have similar efficacy but improved toxicity window given the similar in vitro activity profiles as G2m20 and G2m40.

As another example, 383-G1m2 showed reduced agonist activity in the absence of cross-linking. Residual in vivo FcgRIIB binding activity enhanced the in vivo agonist activity of this IgG variant, but with minimal hepatotoxicity. Fc variants G1mAA, G1 N279A, G4m20, G4m30, and G4m46 are expected to have similar efficacy and toxicity windows given their similar in vitro activity profiles to G1m2.

Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced with an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is merely an example of a generic series of equivalent or similar features.

From the above description, one skilled in the art can easily ascertain the essential features of the present disclosure, and can make various changes and modifications to adapt the disclosure to various uses and conditions without departing from its spirit and scope. Accordingly, other embodiments are within the scope of the claims.

Equivalents Although several embodiments of the invention have been described and illustrated herein, those skilled in the art will readily envision various other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein, and each such variation and/or modification is deemed to be within the scope of the embodiments of the invention described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and/or configurations will depend on the particular application or applications in which the teachings of the invention are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Thus, the foregoing embodiments are presented by way of example only, and it will be understood that, within the scope of the appended claims and their equivalents, the embodiments of the invention may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. Furthermore, any combination of two or more of such features, systems, articles, materials, kits, and/or methods is within the inventive scope of the present disclosure, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent.

All definitions defined and used herein should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which they are cited, and in some cases may include the entire document.

The indefinite articles "a" and "an," as used herein in the specification and claims, should be understood to mean "at least one," unless expressly indicated to the contrary.

The phrase "and/or" as used herein in the specification and claims should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be interpreted in the same manner, i.e., "one or more" of the elements so conjoined. Other elements other than the elements specifically identified by the "and/or" clause may optionally be present, whether related to the elements specifically identified or not. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising", in one embodiment refers only to A (optionally including elements other than B), in another embodiment refers only to B (optionally including elements other than A), in yet another embodiment refers to both A and B (optionally including other elements), etc.

As used herein and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" should be interpreted as inclusive, i.e., including at least one, but not more than one, of a number or list of elements, and optionally additional list items. Only terms clearly indicated to the contrary, such as "only one" or "exactly one," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein should be interpreted as indicating exclusive alternatives (i.e., "either or but not both") when preceded by a term of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, should have its ordinary meaning as used in the field of patent law.

As used in the specification and claims herein, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but need not include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether or not related to the specifically identified elements, may optionally be present. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and/or B") may refer in one embodiment to at least one A (optionally including more than one A) and no B (and optionally including elements other than B), in another embodiment to at least one B (optionally including more than one B) and no A (and optionally including elements other than A), in yet another embodiment to at least one A (optionally including more than one A) and at least one B (optionally including more than one B) (and optionally including other elements), etc.

It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims (11)

A CD40 binding molecule comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:147 and a light chain comprising the amino acid sequence of SEQ ID NO:146 . 2. An isolated nucleic acid or set of nucleic acids that collectively encodes the CD40 binding molecule of claim 1 . A vector or set of vectors collectively comprising the nucleic acid or set of nucleic acids of claim 2 . 4. The vector or set of vectors according to claim 3 which is one or more expression vectors. A host cell comprising a vector or set of vectors according to claim 3 or claim 4 . A pharmaceutical composition for selectively modulating an immune response in a subject, comprising an effective amount of a CD40 binding molecule described in claim 1 and a pharma- ceutically acceptable carrier. The pharmaceutical composition of claim 6 , wherein the subject has or is suspected of having cancer. 8. The pharmaceutical composition of claim 7 , wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, liver cancer, breast cancer, skin cancer, pancreatic cancer, brain cancer, prostate cancer, bladder cancer, colorectal cancer, sarcoma, bone cancer, lymphoma and blood cancer. The pharmaceutical composition of any one of claims 6 to 8, for use in combination with an immunomodulatory agent, a chemotherapeutic agent, or a combination thereof. 10. The pharmaceutical composition of claim 9, wherein the immunomodulatory agent is a checkpoint inhibitor. 11. The pharmaceutical composition of claim 10, wherein the checkpoint inhibitor is a PD1 inhibitor.