Anti-CD40 binding molecules with engineered Fc domains and therapeutic uses thereof

Abstract

The present disclosure provides CD40 binding molecules comprising engineered human IgG domains, e.g., human IgG1, IgG2, and IgG4 variants with mutations in the hinge domain, which exhibit altered binding activity to one or more Fey receptors. Also described herein are methods for using the CD40 binding molecules to selectively activate or inhibit an immune response in a subject.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of International Patent Application No. PCT / CN2018 / 0108285, filed September 28, 2018, which is incorporated by reference in its entirety. [Background technology]

[0002] 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.

[0003] Fc receptors (FcRs) are a family of immune cell surface proteins that can bind 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.

[0004] 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 infected cells or invading pathogens. Meanwhile, FcγRIIB receptors expressed on B cells and dendritic cells can down regulate the activity of immune cells when they bind to IgG antibodies.

[0005] 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 result in unwanted cytotoxicity and cause tissue damage. Therefore, it is of great interest to develop new immunotherapies that are both effective and safe. Summary of the Invention

[0006] 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 immune responses (e.g., to selectively enhance or selectively inhibit immune responses) in subjects in need of treatment.

[0007] Thus, one embodiment of the present disclosure provides a CD40 binding molecule (e.g., a CD40 agonist or a CD40 antagonist) comprising a CD40 binding moiety and an antibody heavy chain constant region comprising 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 corresponding wild-type Fc region. The numbering system is according to the EU index.

[0008] In some embodiments, the CD40 binding moiety is the extracellular domain of CD40L, which may comprise the amino acid sequence of SEQ ID NO: 140. CD40 binding molecules that comprise such CD40 binding moieties may be CD40 agonists.

[0009] 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.

[0010] 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 examples, the variant Fc region may have substantially reduced binding affinity for one or more FcγR receptors. In some examples, the variant Fc region has low or no binding activity for all FcγR receptors.

[0011] In some embodiments, a 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, an Fc variant of an 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, the substitution at positions 233-235 may 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 may be P238S and the substitution at position 265 may 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 may include a substitution at position 265, a substitution at position 297, or a combination thereof. For example, the substitution at position 265 may be D265A and the substitution at position 297 may be N297A. Additionally, one or more of the 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.

[0012] In other embodiments, the CD40 binding molecule may comprise an Fc region of an IgG2 molecule (e.g., a human IgG2 molecule), which may comprise 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 combinations 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 comprise 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 particular 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.

[0013] 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 an 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 an additional example, 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 particular examples, the variant Fc region is one of G4m1, G4m2, G4m20, G4m28, G4m30, G4m41, G4m42, G4m46, G4mPE, G4mAA, and G4m40.

[0014] Any of the variant Fc regions described herein may exhibit enhanced binding activity and / or enhanced selectivity to FcγRIIB compared to the wild-type Fc region. Alternatively, the variant Fc regions described herein may have reduced or no binding activity to any of the FcγR receptors. In some cases, the variant Fc region may exhibit reduced binding affinity to FcγRIIB. Alternatively, or in addition, the variant Fc region binds to FcRn.

[0015] The anti-CD40 antibody fragment in any of the CD40 binding molecules described herein may be of a human antibody or a 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) identical to that of SEQ ID NO: 128, or a heavy chain complementarity determining region (HC CDR) identical to that of any one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2, and / or a light chain complementarity determining region (LC CDR) identical to that of SEQ ID NO: 129, or a light chain complementarity determining region (LC CDR) identical to 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 can include the same heavy chain variable region and / or the same light chain variable region as one of 19G6D6, 36G7B8, 13F1A7, 9F12D9, and 17C5C2.

[0016] 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.

[0017] In yet another aspect, the 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 as described herein.

[0018] 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 blood cancer.

[0019] 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 of treatment is an autoimmune disease.

[0020] In yet another aspect, the 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In some embodiments, the heavy chain variable region comprises a light chain variable region that contains no more than 10 amino acid residue mutations compared to the heavy chain variable region of a reference antibody, and / or no more than 10 amino acid mutations compared to the light chain variable region of a reference antibody.

[0025] In some examples, an anti-CD40 antibody disclosed herein may contain the same heavy chain variable region CDRs as a reference antibody and / or the same light chain variable region CDRs as a reference antibody.

[0026] Any of the anti-CD40 antibodies described herein can be a human or humanized antibody. Also provided herein are pharmaceutical compositions comprising any of the anti-CD40 antibodies described herein and a pharma- ceutical acceptable carrier.

[0027] Additionally, the present 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.

[0028] The present disclosure also features pharmaceutical compositions comprising one or more of the CD40 binding molecules described herein and / or one or more of the anti-CD40 antibodies disclosed herein for use in treating a targeted disorder as described 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 targeted disorder.

[0029] The details of one or more embodiments of the invention are set forth in the description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims. [Brief description of the drawings]

[0030] [Figure 1A]FIG. 1A 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. 1A: Binding affinity to FcγRI. [Figure 1B] 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. [Figure 1C] 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. [Figure 1D] FIG. 1D: Binding affinity for FcγRIIIA. [Diagram 2]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. [Figure 3A] 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 are 0.3 μg / ml, 1 μg / ml, 3 μg / ml, 10 μg / ml, and 30 μg / ml from left to right. The two rightmost bars ("Secondary only" and "Blank") serve as controls. FIG 3A: Binding affinity to FcγRIIB. [Figure 3B] FIG. 3B is a graph showing the binding activity of a number of 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. 3B: Binding affinity to FcγRI. [Figure 3C] FIG. 3C: Binding affinity for FcγRIIA. 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 for FcγRIIA. [Figure 3D]FIG. 3D: Binding affinity for FcγRIIIA. 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. 3D: Binding affinity for FcγRIIIA. [Figure 4A] 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. [Figure 4B] FIG. 4B: 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. [Figure 4C]FIG. 4C: 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. [Figure 4D] FIG. 4D: 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. [Figure 4E] FIG. 4E: 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. [Figure 4F]FIG. 4F: 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. [Figure 5A] 5A and 5B show a set of bar graphs depicting the activity of an exemplary anti-CD40 antibody 383 IgG variant, as shown in the activation of human dendritic cells (DCs) from healthy donors by the antibody in solution (FIGS. 5A and 5B) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D). DC activation is shown by the signal bar graph of IL-8 in the culture supernatant. [Figure 5B] 5A and 5B show a set of bar graphs depicting the activity of an exemplary anti-CD40 antibody 383 IgG variant, as shown in the activation of human dendritic cells (DCs) from healthy donors by the antibody in solution (FIGS. 5A and 5B) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D). DC activation is shown by the signal bar graph of IL-8 in the culture supernatant. [Figure 5C] 5A and 5B show a set of bar graphs depicting the activity of an exemplary anti-CD40 antibody 383 IgG variant, as shown in the activation of human dendritic cells (DCs) from healthy donors by the antibody in solution (FIGS. 5A and 5B) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D). DC activation is shown by the signal bar graph of IL-8 in the culture supernatant. [Figure 5D]5A and 5B show a set of bar graphs depicting the activity of an exemplary anti-CD40 antibody 383 IgG variant, as shown in the activation of human dendritic cells (DCs) from healthy donors by the antibody in solution (FIGS. 5A and 5B) or in co-culture with CHO cells expressing human FcγRIIB (FIGS. 5C and 5D). DC activation is shown by the signal bar graph of IL-8 in the culture supernatant. [Figure 6A] 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: A number of anti-CD40 antibody 383 IgG variants as indicated. [Figure 6B] 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. [Figure 7A] 7A-7C are graphs showing tumor growth curves of various anti-CD40 antibody 383 IgG variants in mouse tumor models. Figure 7A: Graph showing tumor volume change at various time points after antibody treatment in homozygous B-hCD40 C57BL6 mice. Mean tumor size ± SEM is shown. [Figure 7B] Figure 7B: Tumor growth curves of various anti-CD40 antibody 383 IgG variants in mouse tumor models. Figure 7B: Serum alanine transaminase (ALT, a liver enzyme released into serum upon liver injury) levels after antibody treatment in homozygous B-hCD40 C57BL6 mice. Mean tumor size ± SEM is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Provided herein are CD40 binding molecules (e.g., CD40 agonists or CD40 antagonists) that comprise 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, which 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).

[0032] Such CD40 binding molecules are expected to show unexpected and superior therapeutic activity.For example, CD40 binding molecules with variant Fc regions that have enhanced binding affinity to FcγRIIB are expected to show enhanced agonist activity, and CD40 binding molecules with variant Fc regions that have enhanced binding specificity to FcγRIIB are expected to show higher tumor selectivity compared to wild-type counterparts.In addition, CD40 binding molecules with low or no binding affinity to FcγR receptors are useful for activating CD40-positive immune cells in tumor microenvironment and / or blocking CD40-positive immune cells in immune disorders.

[0033] Thus, described herein are approaches for designing CD40 binding molecules comprising a CD40 binding portion and an engineered Fc region (Fc variant) that may be of an IgG molecule (e.g., an IgG1, IgG2, and IgG4 molecule, such as human IgG1, human IgG2, and human IgG4 molecule), and their use for modulating immune responses. Such Fc variants may have enhanced binding affinity to 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 to 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 examples, 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.

[0034] I.CD40 binding molecule Described herein are CD40 binding molecules comprising a CD40 binding moiety linked to an engineered Fc region having modified 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.

[0035] (i)CD40 binding part The CD40 binding moiety described herein can be any peptide or polypeptide that binds to CD40, for example, human CD40. CD40 is an immune cell receptor that is well known in the art. For example, NCBI GenBank accession numbers P25942.1 and AAB08705.1 provide information on human and mouse CD40, respectively. Provided below is the amino acid sequence of an exemplary human CD40 polypeptide.

[0036] Human CD40: MVRLPLQCVLWGCLLTAVHPEPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDT WNRETHCHQHKYCDPNGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSD TICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLRALVVIPIIFGILFAILL VLVFIKKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ (SEQ ID NO: 141) CD40 polypeptides from other species are known in the art and can be obtained from publicly available genetic databases, such as GenBank, using either the human or mouse sequence as a query.

[0037] In some embodiments, the CD40 binding moiety can be a polypeptide comprising the extracellular domain of 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.

[0038] 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, such as GenBank, using the human sequence as a query.

[0039] In other embodiments, the CD40 binding moieties described herein can comprise an anti-CD40 antibody. 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., a human or non-human mammal (e.g., a primate, such as a mouse, rat, rabbit, monkey, etc.).

[0040] 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. H ) and optionally a light chain variable region (V L ) are usually involved in binding the antigen (in this case CD40).

[0041] V H and V L The 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"). H and V L is typically composed of three CDRs and four FRs, arranged from amino terminus 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 definition, the Chothia definition, the AbM definition, and / or the contact definition, 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, USDepartment 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. Mol. Biol. 273:927-948, and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs).

[0042] In some embodiments, the anti-CD40 antibodies described herein are capable of binding to CD40 and inhibiting 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) provides a measure of the potency of an inhibitor. app or K i,app ) 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.

[0043] Antibody K i, app The apparent Ki value can be determined by measuring the inhibitory effect of various concentrations of 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 the modified Morrison equation (Equation 1) provides an estimate of the apparent Ki value. For competitive inhibitors, the Ki app is K i, app It can be obtained from the y-intercept extracted from linear regression analysis of a plot of versus substrate concentration.

[0044]

number

[0045] In some embodiments, the anti-CD40 antibodies described herein have a Ki 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 for the target antigen or antigen epitope. app In some embodiments, any of the anti-CD40 antibodies may have a Ki value of the antibody to the target antigen or its antigenic epitope. appTo reduce the

[0046] 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 by the assays described herein.

[0047] 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 isolated from an antibody library).

[0048] 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 non-human immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) 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 comprise substantially all of at least one, and typically two, variable domains, where 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 comprise 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. A humanized antibody may also be affinity matured.

[0049] Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is H and V LThe variable regions of the human VV 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, a human VVV having an amino acid sequence that is homologous to the amino acid sequence of the parent non-human antibody is generated. H and V L The chain is the parent V as a search query. H and V L The sequence is used to identify the human V H and V L An acceptor gene is selected.

[0050] The CDR regions in the selected human acceptor gene can be replaced with the CDR regions from the 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.

[0051] 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 longer duration, and / or with greater affinity to a particular target antigen than to alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with higher affinity, avidity, more readily, and / or with longer 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 longer duration than it binds to other antigens or other epitopes within the same antigen. By this definition, it is also understood 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.

[0052] In some embodiments, an anti-CD40 antibody as described herein has suitable binding affinity for a target antigen (e.g., CD40) or an antigenic epitope thereof. As used herein, "binding affinity" refers to the apparent association constant or K A Refers to. A is the dissociation constant (K D The anti-CD40 antibodies described herein have a binding affinity of at least 10 to a target antigen or antigenic epitope. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 Binding affinity (K D The increase in binding affinity is due to the K D The higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a decrease in the K A (or a number K D ) for binding the first antigen. A (or a smaller number K D In such cases, the antibody has specificity for a first antigen (e.g., a first protein in a first conformation or a mimetic thereof) compared to a second antigen (e.g., the same first protein, or a second protein in a second conformation or a 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 In some embodiments, any of the anti-CD40 antibodies may be further affinity matured to increase the binding affinity of the antibody for the target antigen or its antigenic epitope.

[0053] 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 evaluating 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, obtaining a quantitative measure of affinity, as determined using methods such as, for example, ELISA or FACS analysis, is sufficient. A and can therefore be used for comparison, such as to determine whether a higher affinity, e.g., 2-fold higher, is to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., in vitro or in vivo assay, so that K A It is not necessary to make a precise determination of the

[0054] An example anti-CD40 antibody, 383, is provided below. H and V L Amino acid sequence, CDRs are shown in bold). V H : [ka] V L : [ka] Further exemplary anti-CD40 antibodies are provided below (CDRs are shown in bold):

[0055] 19G6D6 V H :

change

change

change

change

change

change

change

change

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change

[0056] 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.

[0057] "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. An epitope may be linear, which is typically 6-15 amino acids in length. Alternatively, an epitope may be conformational. The epitope 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., comprising fewer than three non-overlapping amino acid residues, fewer 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.

[0058] In some embodiments, an anti-CD40 antibody disclosed herein comprises 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, an anti-CD40 antibody comprises 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.

[0059] Also within the scope of the present 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. Functional variants have substantially the same V and VD as any one of the exemplary anti-CD40 antibodies. H and V L For example, it can contain 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 an antibody that binds to the same epitope of CD40 with substantially similar affinity (e.g., K D(having a value of 0.01 to 0.15). Alternatively, or in addition, the amino acid residue mutation is a conservative amino acid residue substitution. As used herein, "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, FMAusubel, 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.

[0060] 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 those 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.

[0061] 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.

[0062] 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, modified as in 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 with 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 utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0063] (ii) Engineered Fc Region The CD40 binding molecules described herein comprise a modified heavy chain constant region, where the Fc domain has been modified to modulate its binding affinity and / or specificity to an Fc receptor, such as FcγR.

[0064] 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 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 examples, an Fc fragment that "selectively binds" or "specifically binds" to a target Fc receptor (e.g., FcγRIIB) may not bind to other Fc receptors (i.e., cannot bind in a normal manner). In other embodiments, the variant Fc fragment does not bind to any FcγR.

[0065] The relative binding affinities of IgG1, IgG2, and IgG4 to different Fc receptors are shown in Table 1 below. [Table 1]

[0066] The Fc variants described herein can have enhanced selectivity for FcγRIIB compared to their wild-type counterparts (wild-type parent Fc region into which mutations are introduced to generate 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 counterparts 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.

[0067] In some embodiments, the Fc variants described herein have suitable binding affinity for FcγRIIB, e.g., enhanced 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 Refers to. A is the dissociation constant (K D The Fc variants described herein have a reciprocal of at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M, or a lower binding affinity (K D The increase in binding affinity can be expressed as K D The higher affinity binding of an Fc fragment to a first Fc receptor compared to a second Fc receptor corresponds to a decrease in the K A (or a number K D ) for binding the first Fc receptor A (or a smaller number KD 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) for FcγRIIB compared to the binding affinity for FcγRIII (either FcγRIIIA or FcγRIIIB). A or smaller K D The difference in binding affinity (e.g., for specificity or other comparisons) is 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 It could double.

[0068] 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): [ka]

[0069] 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: VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERRTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 143) Amino acid sequence of mutant mouse IgG1mDANA Fc fragment: VDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 144) Amino acid sequence of mutant mouse IgG2a1mDANA Fc fragment: VDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERRTISKPKGSVRAPQVYVLPPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 145)

[0070] In some embodiments, the Fc variants described herein are human IgG1, G2, or G4 Fc variants that contain 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 examples, one or more mutations can be located in the upper portion of the hinge domain. Alternatively, or in addition, 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 can 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.

[0071] 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 the Fc variants described herein.

[0072] Alternatively, or in addition, the human Fc variants may contain one or more amino acid substitutions at the top, bottom, or both of the hinge domain. For example, the Fc variants may contain 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) 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 contain deletions at one or more of positions 236-238 (e.g., 236, 237, 238, or any combination thereof). Any of the mutations in the hinge domain 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.

[0073] 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 variant 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.

[0074] 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 an IgG2 molecule 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).

[0075] In still 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).

[0076] 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, for example, D265A, S267E, V273E, N297A, L328F, and / or P329G.

[0077] In some cases, any of the Fc variants disclosed herein may further comprise a mutation, e.g., a substitution, at position 309 (e.g., V309L). Such Fc variants may be derived from an IgG2 molecule (e.g., human IgG2). In some embodiments, a 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.

[0078] In some embodiments, an Fc variant described herein may comprise an amino acid sequence 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).

[0079] In one example, the amino acid residue substitutions in the Fc variants described herein are conservative amino acid residue substitutions.

[0080] 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.

[0081] Sequence alignment of human IgG1 variants compared to wild-type human IgG1 (from top to bottom, SEQ ID NOs: 70 to 88) 、 163~168 and 215 ): [ka]

[0082] Sequence alignment of human IgG2 variants compared to wild type human IgG2 (from top to bottom, SEQ ID NOs: 89-105 and 169-173): [ka]

[0083] 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) [ka]

[0084] The amino acid sequences of wild-type human IgG1, IgG2, and IgG4 Fc fragments, and some exemplary hIgG1, hIgG2, and hIgG4 Fc variants (from position 221 onwards based on EU numbering) are shown below.

[0085] Amino acid sequence of wild type human IgG1 Fc fragment: VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1)

[0086] Amino acid sequence of wild type human IgG2 Fc fragment: VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 2)

[0087] Amino acid sequence of wild type human IgG4 Fc fragment: VDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 3)

[0088] Amino acid sequence of human IgG4 S228P Fc variant: VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4)

[0089] Amino acid sequences of exemplary human IgG1 Fc variants: G1m1: VDKKVEPKSCDKTHTCPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5) G1m2: VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6) G1m-2: VDKKVEPKCCVECPPCPAPELLSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 178) G1m240 VDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 179) G1-N297A VDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 180) G1m47 VDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 181) G1m48 VDKKVEPKSsDKTHTsPPsPAPELLGGsSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 182) G1m49 VDKKVEPKSCDKTHTsPPsPAPpvaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 183) G1m50 VDKKVEPKSCDKTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 184)

[0090] 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: VDKTVERKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 26) G2m5: VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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: VDKTVERKCCVECPPCPAPPVASVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEEQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 VDKTVERKCCVECPPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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)

[0091] 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)

[0092] 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, G4mPE. Alternatively, or in addition, Fc variants may have enhanced selectivity for FcγRIIB compared to their wild-type counterparts, such as 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 to FcRn. These Fc variants can be used to construct therapeutic agents described herein that can crosslink immune receptors and FcγRIIB receptors.

[0093] Alternatively, certain Fc variants described herein may have selectivity for FcγRIIB and / or have significantly lower 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.

[0094] The change in binding affinity / specificity of exemplary Fc variants compared to their wild-type counterparts is provided in Tables 2-4 below. "N / A" indicates no data available. If 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. If 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 determined by the same assay under the same experimental conditions, and the variation is significant (e.g., biologically significant) as known to those skilled in the art. If 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 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%). [Table 2] [Table 3] [Table 4]

[0095] The Fc variants described herein can be designed according to the guidance provided herein and produced via conventional recombinant techniques. Their binding affinity and specificity to various Fc receptors can be determined by conventional methods. See also the examples below.

[0096] 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 in the context of 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 may have the same V-terminal sequence as antibody 383. H and V L CDRs. In some examples, the CD40 binding moiety may comprise 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 will 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 agonist activity and residual FcγRIIB binding activity. Too high CD40 agonist activity and / or FcγRIIB binding activity is expected to induce side effects such as cytotoxicity.

[0097] In other specific examples, the CD40 binding molecules disclosed herein include an Fc variant domain derived from IgG2 (e.g., maintaining the IgG2 hinge) and may include 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 include additional mutations at one or more positions involved in binding to Fc receptors, for example, at 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 agonist 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 may have the same V domain as antibody 383. H and V L CDRs. In some examples, the CD40 binding moiety may comprise 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 will 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).

[0098] In addition to the FACS binding assay described herein, the activity of Fc variants on Fcγ receptors can be examined by alternative assays. For example, a CD40 reporter assay with or without co-culture of FcγRIIB-expressing cells can be used to demonstrate the effect 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 reporter assay may indicate a positive result when FACS detectable binding is minimal or not evident.

[0099] 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.

[0100] In the case of a CD40 binding molecule that comprises 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.

[0101] 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 to CD40 or its extracellular domain can be produced by conventional hybridoma technology. The 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 described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells therefrom can be engineered to serve as the basis for the production of mammalian hybridoma cell lines, including humans. Typically, a host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an amount of an immunogen, including those described herein.

[0102] 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, DW, 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. In general, this technique involves fusing myeloma cells and lymphoid cells using fusogens 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 secreting monoclonal antibodies.As another alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce anti-immune cell receptor monoclonal antibodies described herein.Hybridomas are optionally propagated and subcloned, and supernatants are assayed for anti-immunogen activity by conventional immunological testing methods (e.g., radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

[0103] Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of parent hybridomas that produce monoclonal antibodies that can modulate the activity of target immune cell receptors. Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture medium or body fluids, if necessary, 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 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, for example, maleimidobenzoyl sulfosuccinimide ester (linking via cysteine ​​residues), N-hydroxysuccinimide (via 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).

[0104] 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 antibodies described herein. The sequence encoding the antibody of interest can be maintained in a vector within a host cell, which can then be propagated and frozen for future use.

[0105] Alternatively, the polynucleotide sequence can 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 can be engineered to be more similar to the human constant region to avoid immune response when the antibody is 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 immune cell receptors. It will be clear to those skilled in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity for the target receptor.

[0106] 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 more desirable (e.g., fully human antibodies) or stronger immune responses can also be used to generate humanized or human antibodies. Examples of such technology include Xenomouse® from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies can be recombinantly produced by phage display or yeast technology. See, for example, U.S. Patent 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.

[0107] Alternatively, antibody library technologies such as phage display technology (McCafferty et al., (1990) Nature 348:552-553), yeast display technology, or mammalian cell display technology can be used to isolate antibodies, such as human antibodies, specific for a target immune receptor.

[0108] Methods for constructing humanized antibodies are also well known in the art. See, e.g., Queen et al., Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989). In one example, the V of a parent non-human antibody is H and V L The variable regions of the VH and VL chains are subjected to three-dimensional molecular modeling analysis according to methods known in the art.Then, the framework amino acid residues predicted to be important for the formation of the correct CDR structure are identified using the same molecular modeling analysis.In parallel, the 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.Then, human VH and VL acceptor genes are selected.

[0109] The CDR regions in the selected human acceptor gene can be replaced with the CDR regions from the 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 above) can be used to replace the corresponding residues in the human acceptor gene.

[0110] Once an antibody capable of binding to a target immune cell receptor is obtained, the coding sequence of its heavy chain can be fused in frame with the coding sequence of a suitable Fc variant that may selectively bind to FcgRIIB or not bind to 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 of 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.

[0111] In other examples, the antibody is first examined for its antagonistic effect of inhibiting the immune cell receptor to which it binds. Such antagonistic antibodies can be selected to generate the CD40 binding molecules described herein to downregulate immune responses. Alternatively, Fc variants with low or no binding activity to Fc receptors can be selected to generate CD40 antagonists.

[0112] The resulting antibody molecule or CD40 binding molecule described herein can be produced via conventional recombinant techniques, as exemplified below. The nucleic acids encoding the heavy and light chains of the antibody or CD40 binding molecule polypeptide described herein can be cloned into one 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 prompter. 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.

[0113] In some instances, 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 to allow the formation of the antibody.

[0114] In general, the nucleic acid sequence encoding one or all chains of the antibody can be operably linked to a suitable promoter and cloned into a suitable expression vector using methods known in the art. For example, the nucleotide sequence and the 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 depends on the type of host cell used to produce the antibody.

[0115] 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.

[0116] Regulatable promoters can also be used. Such regulatable promoters include those that use the lac repressor from E. coli [Brown, M. et al., Cell, 49:603-612 (1987)] and 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)] as a transcriptional modulator to regulate transcription from mammalian cell promoters with lac operators. Other systems include FK506 dimers, VP16 or p65, using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad.

[0117] Regulatable promoters containing repressors with operons can be used. In one embodiment, the lac repressor from E. coli can function as a transcriptional modulator to regulate transcription from mammalian cell promoters with lac operators [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)]. A minimal promoter containing tetO from the major immediate early promoter of human cytomegalovirus (hCMV) is used in combination with the tetracycline repressor (tetR) with the transcriptional activator (VP16) to create a tetR-tet operator system to control gene expression in mammalian cells, creating the tetR-mammalian cell transcriptional activator fusion protein tTa (tetR-VP16). In one embodiment, a tetracycline-inducible switch is used. When the tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter, only the tetracycline repressor (tetR), but not the tetR-mammalian cell transcription factor fusion derivative, can function as a strong 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 regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992), Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0118] In addition, the vectors 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.

[0119] Examples of polyadenylation signals useful in practicing 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.

[0120] 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 cells can be cultured under suitable conditions for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered by the cultured cells (e.g., from the cells or culture supernatant) via conventional methods, such as affinity purification. If desired, the polypeptide chains of the antibody can be incubated under suitable conditions for a suitable period of time to allow for production of the antibody.

[0121] In some embodiments, the method for preparing the antibodies described herein includes 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 that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions to allow the formation of the antibody.

[0122] 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, such as 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 the expression of the polypeptide chains of the antibody. 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.

[0123] 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.

[0124] The biological activity of the antibodies described herein can be verified using assays known in the art or described herein.

[0125] 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.

[0126] 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 the active ingredient of the composition (and preferably be able to stabilize the active ingredient) and not be 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. KE. Hoover.

[0127] The pharmaceutical compositions used in the methods of the present invention may contain pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzylammonium 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).

[0128] In some examples, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids) that 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. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition that includes phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[0129] The CD40 binding molecules, anti-CD40 antibodies, or encoding nucleic acid(s) may also be entrapped in microcapsules, such as 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).

[0130] 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 antibodies, and these matrices are in the form of shaped articles, such as 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 consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0131] Pharmaceutical compositions used for in vivo administration must be sterile.This can be easily achieved, for example, by filtration through a sterile filtration membrane.Therapeutic antibody compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0132] 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.

[0133] 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.

[0134] Suitable surfactants include, inter alia, 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). 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- ceutical acceptable vehicles, may be added as required.

[0135] 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%, for example 5-20%, of oil. Fat emulsions contain fat droplets of 0.1-1.0 μm, especially 0.1-0.5 μm, and have a pH in the range of 5.5-8.0.

[0136] The emulsion composition may be prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0137] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, as well as powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic effect.

[0138] Compositions, preferably in sterile pharma- ceutically acceptable solvents, can be nebulized by the use of gases. Nebulized solutions can be breathed directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0139] IV. Therapeutic Applications Any of the CD40 binding molecules or anti-CD40 antibodies disclosed herein can be used to modulate (eg, enhance or inhibit) the immune response against invading pathogens and / or diseased cells, such as cancer cells.

[0140] To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, 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.

[0141] The subject treated by the methods described herein may be a mammal, more preferably a human, including, but not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

[0142] 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, including lymphocytic lymphomas, ... Cancers that may be used 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, such as 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, such as chemotherapy, radiation therapy, immunotherapy, or surgery.

[0143] In other examples, 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 disease, immunodeficiency, and / or allergy. 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-Barre 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-related disorders include, but are not limited to, 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, 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, the subject treated by the methods described herein can be a human subject with an immune-related disorder who has received or is receiving immune-related disorder treatment, for example, immunosuppressive mediation, hormone replacement therapy, blood transfusions, anti-inflammatory drugs, and / or analgesics.

[0144] As used herein, "effective amount" refers to the amount of each active agent required to provide a therapeutic effect to a subject, 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 concurrent 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.

[0145] 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. The frequency of administration 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 improvement and / or delay of the target disease / disorder. Alternatively, sustained release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0146] In one example, the dosage of the CD40 binding molecules or anti-CD40 antibodies described herein can be empirically determined in individuals who have received one or more doses of the antibody. Individuals are given increasing doses of the agonist. Disease / disorder indicators can be followed to assess the effectiveness of the agonist.

[0147] 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 mentioned 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, a dosage range of 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 dosage 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 dosage regimen (including the treatment used) can vary over time.

[0148] 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 well known in the art.

[0149] 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.

[0150] As used herein, the term "treating" 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, improving, ameliorating, or affecting the disorder, a symptom of the disease, or a predisposition to a disease or disorder.

[0151] 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. This delay can 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 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 produce statistically significant results.

[0152] "Onset" or "progression" of a disease refers to the initial symptoms and / or subsequent progression of the disease. Onset of a disease can be detected and evaluated 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.

[0153] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to activate the activity of the target receptor in vivo by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0154] Depending on the type of disease or site of disease to be treated, the pharmaceutical composition can be administered to the subject using conventional methods known to those skilled in the medical field. 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. In addition, 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.

[0155] Injectable compositions may contain various 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 injection, water-soluble antibodies may be administered by drip 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.

[0156] 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.

[0157] 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.

[0158] The effectiveness of treatment of a target disease / disorder can be assessed by methods well known in the art.

[0159] 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.

[0160] When co-administered with an additional therapeutic agent, the preferred therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0161] 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 antiproliferative / mitotic inhibitors including rifabutin, taxol, 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, nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), antiproliferative / mitotic inhibitors such as trazenes-dacarbazinine (DTIC); antiproliferative / mitotic inhibitors 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 (antimigratory agent); secretion inhibitors (breveldin); 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 inhibitors, 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;They are mitochondrial dysfunction inducers and caspase activators; chromatin disruptors;

[0162] For examples of additional useful agents, see also Physician's Desk Reference, 59th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al., Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0163] V.Kit 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.

[0164] 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 to treat, delay onset, or alleviate the target disease, such as those described herein. The kit can further include instructions for selecting 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.

[0165] The instructions for use of the therapeutic agent generally include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. The 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.

[0166] 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 can be provided.

[0167] The kit of the present invention is 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 specific devices, such as inhalers, nasal administration devices (e.g., nebulizers) or infusion devices, such as mini-pumps, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced 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 with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a therapeutic agent as described herein.

[0168] The kit may optionally provide additional components such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0169] 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. Elis, ed., 1998) Academic Press, Animal Cell Culture (R. I. Freshney, ed., 1987), Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Oberts, 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 CCBlackwell, eds.), Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), Current Protocols in Molecular Biology (FMAusubel, et al., eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994), Current Protocols in Immunology (JEColigan et al. al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA 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 JDCapra, eds., Harwood Academic Publishers, 1995).

[0170] Without further elaboration, it is believed that a person skilled in the art can utilize the present invention to its fullest extent based on the above description. Accordingly, the specific embodiments provided herein should be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0171] 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 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 147) 383-huIgG1m27 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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 WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 151) 383-huIgG4m2 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 152) 383-huIgG4m20 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 153) 383-huIgG4m30 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPPCAPEPFLPSVFLPPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(sequence number 154) 383-msIgG LC DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRC(SEQ ID NO: 155) 383-msIgG1 DANA HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFVDDVEVHTAQTQPREEQFASTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 156) 383-msIgG1 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 157) 383-msIgG2a DANA HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERRTISKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 158) 383-ms IgG2a HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKP CPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERITSKPKGSVRAPQVYVLPPPEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 159)

[0172] Additional Fc variants were made, the amino acid sequences of the heavy chain (HC) are shown below (the light chain (LC) sequences are shown above). 383-huIgG1 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 207) 383-huIgG2 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 208) 383-huIgG4SP HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 209) 383-huIgG1mAA QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 210) 383-huIgG1m40 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVaVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 193) 383-huIgG1m45HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 194) 383-huIgG1m240 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 195) 383-huIgG1 N297A QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYaSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 196) 383-huIgG1m47 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPpvaGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKgLPssIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 197) 383-huIgG1m48 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSsDKT HTsPPsPAPELLGGsSVFLFPKPKDTLMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSREEMTKNQVSLTCLVKGGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGS(request number 198) 383-huIgG1m49 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTsPPsPAPpvaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 199) 383-huIgG1m50 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPEfeGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAsIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 214 ) 383-huIgG1mAAG QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYW GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 211) 383-huIgG2m40 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 200) 383-huIgG2m43 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPaaasSVFLFPPKPKDTLMISRTPEVTCVVVDVSaEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPPSG(request number 201) 383-huIgG2m44 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSqEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVlHQDWLNGKEYKCKVSNKGLPssIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 202) 383-huIgG2mAA HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPaaaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 203) 383-huIgG2m2040HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFASTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 204) 383-huIgG2G4 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKTVERKCCV ECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 205) 383-huIgG4m40 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (sequence number 206) 383-huIgG4m41 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 66) 383-huIgG4m42 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGPPCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 67) 383-huIgG4m46 HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPEaLGaPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKaYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 68) 383-huIgG4mPE HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 212) 383-huIgG4mAA HC QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVESKYGP PCPPCPAPEaaGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 69)

[0173] Example 2. Binding activity of CD40 antibodies with Fc variants to cellular FcγR Measurement To determine the binding activity of CD40 activity in the IgG mutants 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.

[0174] Using the anti-CD40 antibody 383 disclosed herein as an exemplary anti-CD40 moiety, a number of anti-CD40 IgG Fc variants have been designed and constructed according to the disclosure herein, including 383-msIgG1, 383-msIgG1 DANA, 383-msIgG2a, 383-msIgG2a 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.

[0175] For FACS analysis of the 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.

[0176] As shown in Figures 1A-1D and 3A-3D, many anti-CD40 human IgG1, IgG2, and IgG4 variants exhibited binding activity to cell surface expressed FcγRs, whereas others showed no clear findings to the FcγRs tested. A summary of the changes in binding properties is provided above in Tables 2-4.

[0177] Example 3. Fc region engineered CD40 antibodies bind different CD40 Showed agonist activity To measure the agonist activity of the IgG variants, 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 in assay buffer (MEM with 1% FBS) at 1 × 10 4 The cells were diluted to 1000 cells / mL. Cells were added at 100uL / well to give 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 2x final concentration with 100uL / well test sample. The assay plate was incubated for 18-20 hours in a 37°C, 5% CO2 incubator. After 18-20 hours of incubation, 8ul 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. No. 62IL8PEB). Specifically, an assay volume of 16ul was used. Results were read using Time Resolved Fluorescence with a Tecan F200pro and relative light unit data was recorded.

[0178] 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 the secretion of IL8. The magnitude of CD40 activation was influenced by the Fc variant contained therein due to the flexibility of the hinge in each antibody isotype structure.

[0179] 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 their 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.

[0180] Example 4. Characterization of anti-CD40 antibody IgG variants in dendritic cell (DC) functional assays Exemplary anti-CD40 antibody Ig variants, including various Fc mutants, were tested in vitro for CD40 binding activity 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.

[0181] Frozen human PBMCs (Allcells, Cat. No. PB005F) from a healthy donor were thawed and transferred to a 50ml tube containing 25mL of RPMI1640 medium. B cells were isolated using the EasySep™ Human CD14 Positive Selection Kit II (Stemcell, Cat. No. 17858). The PBMC suspension was centrifuged at 250g for 15 minutes, the supernatant was discarded and the cells were resuspended in 1.5mL of EasySep™ buffer. Selection cocktail was added to the samples in 14mL (17x100mm) polystyrene round bottom tubes. 100ul / ml, 150ul for each sample. Cells were transferred from the 50ml tube to a 14mL (17x100mm) polystyrene round bottom tube, mixed and incubated for 15 minutes at room temperature (RT). Magnetic particles were mixed before transferring to the sample. Magnetic particles were transferred to the sample. 50ul / ml, 75ul for each sample. Mix and incubate at room temperature for 10 minutes. Add 8.5 mL of EasySep™ Buffer to make the sample up to 10 mL. Gently pipette up and down 2-3 times to mix. Place the tube in the magnet and incubate for 5 minutes. Remove the magnet and invert the magnet and tube in one continuous motion to pour off the supernatant. Collect this supernatant for B cell enrichment. Repeat the above washing steps two more times. Resuspend the cells in 5 mL of RPMI 1640. Count the cell density.

[0182] CD14 isolated from human PBMCs + Cells, 1 x 10 6The medium was adjusted to 1000 cells / ml and supplemented with GM-CSF and IL-4 and added to the 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 was changed by transferring the culture supernatant to a centrifuge tube and centrifuged at 250xg for 5 minutes. 10 ml of fresh medium containing GN-CSF and IL-4 was used to suspend the cell pellet in the centrifuge tube and then added to the flask during centrifugation so that the cell pellet thus formed was resuspended in 10 ml of fresh medium. The resuspended cells were transferred back to the same well or flask and incubated for another 2 days. On the fifth day, the above procedure was repeated for medium change. The cells were 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.

[0183] To perform the DC activation assay, take the medium containing the cells from the T75 flask into a 50 ml tube and centrifuge at 500xg for 5 min at room temperature. Discard the supernatant and resuspend the cells in 3 ml of RPMI1640 10% FBS medium. Count the cell density and adjust the cell concentration to 1e6 / ml. Add 0.05 ml of cell suspension to 2.5 x 10 4 Cells / well were added to a 96-well plate. IL-8 and IL12P70 were detected in cell culture supernatants after 24 hours (day 3) using HuIL-12-P70 Detection Kit (Cisbio, Cat. No.: 62HIL12PEH) and HuIL-8 Detection Kit (Cisbio, Cat. No.: 62HIL08PEH). DC activation assays were also performed in co-culture with CHO cells expressing FcγR2B.

[0184] As shown in Figures 5A-5D, the CD40 antibody IgG variants tested stimulated human CD40 activation to varying degrees, as evidenced by the secretion of IL8 from DC cultures following antibody incubation. The magnitude of DC activation was influenced by the Fc variant contained therein, due to the flexibility of the hinge in each antibody isotype structure and its interaction with Fc receptors expressed by DC cultures.

[0185] 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. Simultaneous binding of antibody molecules to CD40 and FcγRIIB in the microenvironment affects their individual binding due to avidity effects that lead to changes in the CD40 agonistic effect of the antibody. The antibodies showed increased activity that correlated with their binding activity to FcγRIIB, consistent with the observations in the reporter assay above. Thus, the Fc structure of an antibody affects the overall agonistic activity of antibodies with the same variable domains.

[0186] Example 5. IgG mutant 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 min. Cells were diluted in cold PBS-BSA (2%) at 5x10 6 The antibodies were resuspended at 0.01, 0.1, 1, and 10ug / ml and dispensed into 100ul / tube. Chimeric anti-CD40 antibodies were diluted 3-fold in PBS-BSA (final concentrations of 0.01, 0.1, 1, and 10ug / ml) and 50ul of each concentration was added to the CHO-CD40 cells. The cell solutions were mixed and incubated for 2 hours at 4°C in the dark. The cells were then washed twice with PBS-BSA. Secondary antibody conjugate (goat F(ab')2 anti-human IgG-Fc(PE), pre-adsorbed (Abcam#ab98596)) at a concentration of 100ul / vial was added, 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 Figure 6A-6B, these antibodies showed high binding activity to human CD40 expressed on the surface of CHO cells.

[0187] Example 6. IgG mutant CD40 antibodies Shows 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 the 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. Tumor size was approximately 150±50 mm 3 Mice were divided into groups when the tumor size was 4-6 weeks after treatment (n=6). CD40 antibodies were administered by intraperitoneal injection, and tumor sizes were measured during weeks 4-6 of antibody treatment. Tumor sizes were measured twice weekly by caliber and expressed as 0.5×length×width. 2 The tumor volume was calculated using the formula: Antitumor efficacy was evaluated between the tumor sizes in the control and antibody-treated groups.

[0188] 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.

[0189] The potential for antibody-induced hepatotoxicity was examined in a mouse model by measuring serum ALT levels after antibody treatment. Human G2 induced a very large increase in ALT, exceeding the upper limit of the assay (>1000 U / L). Validated Fc variants, including G1m2, G2m20, and G2m40, induced low or minimal increases in ALT.

[0190] As an example, 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.

[0191] 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.

[0192] 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.

[0193] From the above description, those 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 usages and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the claims.

[0194] Equivalent While 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 equivalents thereof, 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.

[0195] All definitions and those used herein should be understood to control for any dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0196] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, in some cases including the entire document.

[0197] The indefinite articles "a" and "an," as used herein in the specification and claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0198] 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 specifically identified element 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.

[0199] When used in the present specification and claims, "or" should be understood to have the same meaning as "and / or" 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.

[0200] As used herein in the specification and claims, 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, may optionally be present, whether or not related to the specifically identified elements. 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.

[0201] 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

1. A CD40 binding molecule comprising a heavy chain having the amino acid sequence of SEQ ID NO: 147 and a light chain having the amino acid sequence of SEQ ID NO:

146.

2. 2. An isolated nucleic acid or set of nucleic acids that collectively encodes the CD40 binding molecule of claim 1.

3. A vector or set of vectors collectively comprising the nucleic acid or set of nucleic acids of claim 2.

4. 4. The vector or set of vectors according to claim 3 which is one or more expression vectors.

5. A host cell comprising a vector or set of vectors according to claim 3 or claim 4.

6. A pharmaceutical composition for selectively modulating an immune response in a subject, comprising an effective amount of a CD40 binding molecule of claim 1 and a pharma- ceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the subject has or is suspected of having cancer.

8. 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.

9. A pharmaceutical composition according to 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 described in claim 10, wherein the checkpoint inhibitor is a PD1 inhibitor.

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