Multispecific antibodies against CD40 and CD137

Abstract

To provide multispecific antibodies binding to human CD40 and human CD137, to provide methods of preparing such multispecific antibodies, and to provide methods of using such multispecific antibodies for therapeutic or other purposes.SOLUTION: Provided is a multispecific antibody comprising: (I) a first antigen-binding region binding to human CD40 which comprises CDR sequences with specific amino acid sequences; and (II) a second antigen-binding region binding to human CD137.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to multispecific antibodies that bind to CD40 and CD137 and to uses of such multispecific antibodies, particularly for the treatment of cancer. [Background technology]

[0002] Background of the Invention CD40 is a member of the tumor necrosis factor (TNF) receptor (TNFR) family and is known as a costimulatory protein found on a variety of cell types. CD40 is constitutively expressed by antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages. It can also be expressed by endothelial cells, platelets, smooth muscle cells, fibroblasts, and epithelial cells. Consistent with its widespread expression on normal cells, CD40 is also expressed on a wide range of tumor cells.

[0003] Together with costimulatory signals (from CD80 and / or CD86), peptide antigens associated with MHC class II molecules are activated by antigen-specific CD4 + When presented to T cells, CD4 + T cells are activated and DC licensing factors CD40 ligand (CD40L) and lymphotoxin-α1β2 (LTα1β2) are upregulated. Activated antigen-specific CD4 + Expression of CD40L and LTα1β2 on T cells induces signaling through CD40 and the LTβ receptor (LTβR), which allows DCs to differentiate into CD8 +These enable the induction of T cell responses. CD40 signaling results in the production of interleukin-12 (IL-12) and upregulation of CD70, CD86, 4-1BB ligand (4-1BBL), OX40 ligand (OX40L), and GITR ligand (GITRL), whereas LTβR signaling leads to the production of type I interferon (IFN). The signaling system that controls the activity of nuclear factor kappa B (NF-kB) is responsive to virtually all TNFR superfamily members. Pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) also contribute to these events. MHC class I-restricted peptides induce CD8 + Upon antigen stimulation of T cells, CD27, 4-1BB, OX40, and glucocorticoid-inducible TNFR-related protein (GITR) are upregulated. + Stimulation of these receptors on T cells by their cognate TNF superfamily ligands in conjunction with IL-12 and type I IFNs results in robust CD8 + T cell activation, proliferation, and effector function, and CD8 +The formation and maintenance of T cell memory occurs. CD40 antibodies exert various actions: killing CD40-expressing tumor cells by inducing antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), or antibody-dependent cell-mediated phagocytosis (ADCP); inducing cell signaling to directly induce apoptosis or growth arrest; or by enabling APCs to promote anti-cancer immune responses independent of CD40 expression on tumor cells. Antibodies that bind to CD40 can trigger CD40 on APCs to prime effector cytotoxic T lymphocytes (CTLs), inducing the release of IL-2 by these cells and indirectly activating NK cells. Antibodies that stimulate CD40 have been disclosed in the prior art, including CP-870,893, a human IgG2 antibody (WO 2003 / 040170 (Patent Document 1)); dacetuzumab, a humanized IgG1 antibody (WO 2000 / 075348 (Patent Document 2)); and Chi Lob 7 / 4, a chimeric IgG1 antibody (US 2009 / 0074711 (Patent Document 3)). In addition, a CD40 antagonistic antibody, lucatumumab, a human IgG1 antibody, has also been disclosed (WO 2002 / 028481 (Patent Document 4)).

[0004] CD137 (4-1BB) is also a member of the TNFR family. + T cells and CD4 +CD137 is a costimulatory molecule on the surface of T cells, regulatory T cells (Tregs), natural killer T cells (NK(T) cells), B cells, and neutrophils. In T cells, CD137 is not constitutively expressed but is induced upon T cell receptor (TCR) activation (e.g., on tumor-infiltrating lymphocytes (TILs)) (Gros et al., J. Clin Invest 2014;124(5):2246-59). Stimulation via its natural ligand 4-1BBL or agonistic antibodies results in signaling using TRAF-2 and TRAF-1 as adaptors. Initial signaling by CD137 involves K-63 polyubiquitination, which ultimately leads to activation of the nuclear factor (NF)-kB pathway and the mitogen-activated protein (MAP) kinase pathway. Signaling enhances T cell costimulation, proliferation, cytokine production, and maturation, leading to CD8 + T cell survival is extended. Agonistic antibodies against CD137 have been shown to promote T cell-mediated anti-tumor control in various preclinical models (Murillo et al., Clin Cancer Res 2008;14(21):6895-906 (Non-Patent Document 2)). Antibodies that stimulate CD137 can induce T cell survival and proliferation, thereby enhancing anti-tumor immune responses. Antibodies that stimulate CD137 have been disclosed in the prior art, including urelumab, a human IgG4 antibody (AU2004279877 (Patent Document 5)), and utomilumab, a human IgG2 antibody (Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733 (Non-Patent Document 3)).

[0005] Westwood JA, et al., Leukemia Research 38 (2014), 948-954 (Non-Patent Document 4) discloses "Combination anti-CD137 and anti-CD40 antibody therapy in murine myc-driven hematological cancers."

[0006] US20090074711 (Patent Document 3) discloses "Human therapies using chimeric agonistic anti-human CD40 antibodies."

[0007] However, despite these and other advances in the art, there remains a need for multispecific antibodies that can bind both CD40 and CD137 and simultaneously bind to CD40-expressing APCs and CD137-expressing T cells, thereby bringing these cell types into close proximity, i.e., activate both cell types and efficiently induce anti-tumor immunity. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2003 / 040170 [Patent Document 2] WO2000 / 075348 [Patent Document 3] US2009 / 0074711 [Patent Document 4] WO2002 / 028481 [Patent Document 5] AU2004279877 [Non-patent literature]

[0009] [Non-Patent Document 1] Gros et al., J. Clin Invest 2014;124(5):2246-59 [Non-patent document 2] Murillo et al., Clin Cancer Res 2008;14(21):6895-906 [Non-patent document 3] Fisher et al. 2012 Cancer Immunol. Immunother. 61: 1721-1733 [Non-patent document 4] Westwood JA, et al., Leukemia Research 38 (2014), 948-954 Summary of the Invention

[0010] The present inventors have identified a multispecific antibody that binds to both CD40 and CD137 and is capable of inducing activation of T cells and APCs.

[0011] Thus, in one aspect, the present invention relates to a multispecific antibody comprising (i) a first antigen-binding region that binds to human CD40 and (ii) a second antigen-binding region that binds to human CD137.

[0012] In some embodiments, the present invention relates to such multispecific antibodies, in which the first antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy and light chain variable regions comprising specific amino acid sequences, optionally with mutations, or comprises the amino acid sequences of an antibody that competes with or has the specificity of an antibody comprising such specific amino acid sequences. In a particular embodiment, the first antigen-binding region comprises heavy and light chain variable sequences comprising CDR1, CDR2, and CDR3 of anti-CD40 antibody 001, or competes with or has the specificity of such an antibody.

[0013] In some embodiments, the invention relates to such multispecific antibodies, wherein the second antigen-binding region comprises heavy and light chain variable sequences whose CDR1, CDR2, and CDR3 comprise or provide specific amino acid sequences, optionally with mutations, or the amino acid sequences of an antibody that competes with or has the specificity of an antibody comprising such specific amino acid sequences. In particular embodiments, the second antigen-binding region comprises heavy and light chain variable sequences comprising CDR1, CDR2, and CDR3 of anti-CD137 antibody 001, 002, 003, 004, 005, 006, 007, 008, 009, 010, 011, or 012, or competes with or has the specificity of any such antibody.

[0014] These and other aspects and embodiments, including nucleic acids encoding the amino acid sequences of such multispecific antibodies; expression vectors comprising such nucleic acids; host cells comprising such nucleic acids or expression vectors; compositions comprising such multispecific antibodies; such compositions for use in treating cancer or other diseases; methods for making such multispecific antibodies; and diagnostic methods and kits based on such multispecific antibodies, are described in further detail below. [The present invention 1001] (I) a first antigen-binding region that binds to human CD40, (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs:1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs:4, YTS, and SEQ ID NO:5, respectively; (b) the heavy chain and light chain variable regions CDR1, CDR2, and CDR3 described in (a), each having a total of 1 to 12 mutations; and (c) (i) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that competes for human CD40 binding with an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b), and / or (ii) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that has specificity for CD40 of an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b). The first antigen-binding region comprises heavy and light chain variable regions CDR1, CDR2 and CDR3 selected from the group consisting of: (II) a second antigen-binding region that binds to human CD137; A multispecific antibody comprising: [The present invention 1002] 1001. The multispecific antibody of the present invention, wherein said first antigen-binding region comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, said second antigen-binding region comprises a second VH sequence and a second VL sequence, and said first and second VH sequence and VL sequence each comprise three CDR sequences, CDR1, CDR2 and CDR3, respectively, and four framework sequences, FR1, FR2, FR3 and FR4, respectively. [The present invention 1003] Any of the multispecific antibodies of the invention as described above, wherein the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS and SEQ ID NO: 5, respectively. [The present invention 1004] The multispecific antibody of any of claims 1002 to 1003, wherein the VH sequence of the first antigen-binding domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NOs: 117 and 6. [The present invention 1005] The multispecific antibody of any of claims 1002 to 1004, wherein the VL sequence of the first antigen-binding domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NOs: 121 and 7. [The present invention 1006] 1006. The multispecific antibody of any of claims 1002 to 1005, wherein the VH sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 117, and the VL sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 121. [The present invention 1007] Any of the multispecific antibodies of the invention, wherein the second antigen-binding region binds to cynomolgus monkey CD137. [The present invention 1008] Any of the multispecific antibodies of the invention, wherein the second antigen-binding region binds to human CD137 (SEQ ID NO:92) to a greater extent than it binds to mutant human CD137 (SEQ ID NO:93). [The present invention 1009] The multispecific antibody of any of claims 1001 to 1007, wherein the second antigen-binding region binds to human CD137 (SEQ ID NO: 92) to a greater extent than to mutant human CD137 (SEQ ID NO: 94). [The present invention 1010] Any of the multispecific antibodies of the invention as described above, wherein the second antigen-binding region binds to human CD137 (SEQ ID NO:92) to a similar extent as it binds to mutant human CD137 (SEQ ID NO:95). [The present invention 1011] Binding to the human CD137 (SEQ ID NO: 92) and the mutant human CD137 (SEQ ID NOs: 93, 94 and 95, respectively) preparing shuffled constructs derived from human CD137 in which protein domains of human CD137 are replaced with corresponding domains of CD137 from different species, using human CD137 and CD137 from different species as reference constructs, transducing cells with plasmids encoding the reference constructs or shuffled constructs, respectively, and measuring the binding of antibodies to each of these CD137 constructs by flow cytometry; Any of the multispecific antibodies of the present invention 1008 to 1010, determined by the above method. [The present invention 1012] the second antigen-binding region comprises: (a) heavy chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 64, 65 and 66, respectively, and light chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 67, 68 and 69, respectively; (b) the heavy and light chain variable regions CDR1, CDR2, and CDR3 described in (a), each having a total of 1 to 12 mutations; and (c) (i) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that competes for human CD137 binding with an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b), and / or (ii) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that has specificity for CD137 of an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b). The multispecific antibody of any one of claims 1001 to 1008 and 1010 to 1011, comprising heavy chain and light chain variable regions CDR1, CDR2 and CDR3 selected from the group consisting of: [The present invention 1013] Any of the multispecific antibodies of the present inventions 1001 to 1008 and 1010 to 1012, wherein the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NOs: 123 and 69. [The present invention 1014] Any of the multispecific antibodies of the present inventions 1001 to 1008 and 1010 to 1013, wherein the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NOs: 127 and 70. [The present invention 1015] Any of the multispecific antibodies of the present inventions 1001 to 1008 and 1010 to 1014, wherein the VH sequence and VL sequence of the second antigen-binding region comprise amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 123 and SEQ ID NO: 127, respectively. [The present invention 1016] the second antigen-binding region comprises: (a) heavy chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 36, 37 and 38, respectively, and light chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 39, 39S and 40, respectively; (b) the heavy and light chain variable regions CDR1, CDR2, and CDR3 described in (a), each having a total of 1 to 12 mutations; and (c) (i) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that competes for human CD137 binding with an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b), and / or (ii) the heavy and light chain variable regions CDR1, CDR2 and CDR3 of an antibody that has specificity for CD137 of an antibody comprising the heavy and light chain variable regions CDR1, CDR2 and CDR3 of (a) or (b). The multispecific antibody of any one of claims 1001 to 1007 and 1009 to 1011, comprising heavy chain and light chain variable regions CDR1, CDR2 and CDR3 selected from the group consisting of: [The present invention 1017] Any of the multispecific antibodies of the present inventions 1001 to 1007, 1009 to 1011, and 1016, wherein the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO:41. [The present invention 1018] Any of the multispecific antibodies of the present inventions 1001 to 1007, 1009 to 1011, and 1016 to 1017, wherein the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 42. [The present invention 1019] Any of the multispecific antibodies of the present inventions 1001 to 1007, 1009 to 1011, and 1016 to 1018, wherein the VH and VL sequences of the second antigen-binding region comprise amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 41 and SEQ ID NO: 42, respectively. [The present invention 1020] The first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and the second antigen-binding region comprises: (a) heavy chain variable regions CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65 and 66, respectively, and light chain variable regions CDR1, CDR2 and CDR3 having the sequences set forth in SEQ ID NOs: 67, 68 and 69, respectively; or (b) heavy chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 36, 37 and 38, respectively, and light chain variable regions CDR1, CDR2 and CDR3 having the sequences shown in SEQ ID NOs: 39, 39S and 40, respectively; The multispecific antibody of any one of 1004 to 1019 of the present invention, comprising: [The present invention 1021] The multispecific antibody of any of claims 1004 to 1020, wherein the FR1, FR2, FR3 and FR4 framework sequences of the VH and VL sequences of the first and / or second antigen-binding domain have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3 and FR4 framework sequences of said VH and VL sequences, respectively. [The present invention 1022] Any of the multispecific antibodies of the invention, wherein the antibody comprises (I) a first binding arm comprising the first antigen-binding region, and (II) a second binding arm comprising the second antigen-binding region. [The present invention 1023] 1022. The multispecific antibody of the invention, wherein said first binding arm comprises a first heavy chain constant sequence and said second binding arm comprises a second heavy chain constant sequence. [The present invention 1024] (I) the first binding arm comprises a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence; (II) the second binding arm comprises a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence; The multispecific antibody of any one of 1022 to 1023 of the present invention. [The present invention 1025] 1024. The multispecific antibody of the invention, wherein said first light chain further comprises a first light chain constant (CL) sequence and said second light chain further comprises a second light chain constant (CL) sequence. [The present invention 1026] Any of the multispecific antibodies of the present invention, wherein the first antigen-binding region is derived from a mouse antibody. [The present invention 1027] Any of the multispecific antibodies of the present invention, wherein the first antigen-binding region is derived from a humanized antibody. [The present invention 1028] The multispecific antibody of any of 1022 to 1027, wherein the first binding arm is derived from a full-length antibody. [The present invention 1029] 1029. The multispecific antibody of any of claims 1022 to 1028, wherein the first binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. [The present invention 1030] Any of the multispecific antibodies of the present invention, wherein the second antigen-binding region is derived from a rabbit antibody. [The present invention 1031] Any of the aforementioned multispecific antibodies of the invention, wherein the second antigen-binding region is derived from a humanized antibody. [The present invention 1032] The multispecific antibody of any of 1022 to 1031, wherein the second binding arm is derived from a full-length antibody. [The present invention 1033] The multispecific antibody of any of claims 1022 to 1032, wherein the second binding arm is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. [The present invention 1034] Any of the aforementioned multispecific antibodies of the invention, wherein each of the first and second antigen-binding regions is derived from a humanized antibody. [This invention 1035] The multispecific antibody of any of 1022 to 1034, wherein each of the first and second binding arms is derived from a full-length antibody. [The present invention 1036] 1036. The multispecific antibody of any of claims 1022 to 1035, wherein each of the first and second binding arms is derived from a full-length IgG1, λ (lambda) or IgG1, κ (kappa) antibody. [This invention 1037] The multispecific antibody of any of claims 1022 to 1036, wherein the first and second heavy chains are of an IgG isotype having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. [The present invention 1038] The multispecific antibody of any of claims 1024 to 1037, wherein each of the first and second heavy chains comprises at least a hinge region, a CH2 region and a CH3 region. [This invention 1039] 1038. A multispecific antibody of the invention, wherein the CH3 regions of the first and second heavy chains comprise asymmetric mutations. [The present invention 1040] The multispecific antibody of any of claims 1024 to 1039, wherein the first heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering, and the second heavy chain has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in the human IgG1 heavy chain according to EU numbering, and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions. [The present invention 1041] (i) in the first heavy chain, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L, and in the second heavy chain, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R; or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering is R, and in the second heavy chain, the amino acid at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering is L; The multispecific antibody of the present invention. [The present invention 1042] comprising a first and a second heavy chain; induces Fc-mediated effector functions to a lesser extent compared to multispecific antibodies comprising the same first and second antigen-binding regions and two heavy chains comprising human IgG1 hinge, CH2 and CH3 regions; Any of the multispecific antibodies of the present invention. [This invention 1043] 1042. The multispecific antibody of the invention, wherein said first and second heavy chains have been modified such that said multispecific antibody induces Fc-mediated effector function to a lesser extent compared to an otherwise identical multispecific antibody comprising unmodified first and second heavy chains. [This invention 1044] 1044. The multispecific antibody of any of claims 1042 to 1043, wherein said Fc-mediated effector function is measured by binding to an Fcγ receptor, binding to C1q, or induction of Fc-mediated cross-linking of FcR. [This invention 1045] The multispecific antibody of the invention 1044, wherein said Fc-mediated effector function is measured by binding to C1q. [The present invention 1046] 1045. A multispecific antibody of the invention, wherein the constant sequences of said first and second heavy and light chains are modified such that C1q binding to said multispecific antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to a wild-type bispecific antibody, and wherein C1q binding is determined by ELISA. [This invention 1047] Any of the multispecific antibodies of the invention, wherein said antibody comprises a first and a second heavy chain, and in at least one of said first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297 and P331 according to EU numbering in a human IgG1 heavy chain are not L, L, D, N and P, respectively. [This invention 1048] 1047. A multispecific antibody of the invention, wherein in said first and second heavy chains, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively. [This invention 1049] 1048. A multispecific antibody of the invention, wherein in said first and second heavy chains, the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain according to EU numbering are F, E and A, respectively. [The present invention 1050] positions in both the first and second heavy chains corresponding to positions L234, L235, and D265 of a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively; and (i) the first heavy chain has an L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering and the second heavy chain has an R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering; or (ii) the first heavy chain has R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; The multispecific antibody of the present invention. [This invention 1051] the positions in both the first and second heavy chains corresponding to positions L234 and L235 of a human IgG1 heavy chain according to EU numbering are F and E, respectively; and (i) the first heavy chain has an L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering and the second heavy chain has an R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering; or (ii) the first heavy chain has R at the position corresponding to K409 of the human IgG1 heavy chain according to EU numbering, and the second heavy chain has L at the position corresponding to F405 of the human IgG1 heavy chain according to EU numbering; The multispecific antibody of the present invention. [This invention 1052] Any of the multispecific antibodies of the invention which are capable of cross-linking a first cell expressing human CD40 and a second cell expressing human CD137. [This invention 1053] 1052. The multispecific antibody of the invention, wherein said cross-linking is determined by an assay using a first cell line expressing human CD40 and a second cell line expressing human CD137, and wherein either the first or second cell line comprises a reporter construct that produces a measurable reporter upon NF-κB activation. [This invention 1054] Any of the multispecific antibodies of the present invention which induce and / or enhance the proliferation of T cells. [This invention 1055] The T cells are CD4 + T cells and / or CD8 + The multispecific antibody of the present invention 1054, which is a T cell. [The present invention 1056] 1056. The multispecific antibody of any of claims 1054 to 1055, wherein said induction or enhancement of T cell proliferation is determined by suboptimal activation of T cells in a PBMC pool. [This invention 1057] 1056. A multispecific antibody of the invention, wherein suboptimal activation is determined by titrating the concentration of anti-CD3 antibody added to a PBMC pool, measuring T cell proliferation, and selecting a concentration of anti-CD3 antibody that results in less T cell proliferation but allows for further enhancement of T cell proliferation. [This invention 1058] Any of the multispecific antibodies of the present inventions 1054 to 1055, wherein T cell proliferation is measured by co-culturing T cells expressing a specific T cell receptor (TCR) with dendritic cells (DCs) presenting the corresponding antigen on the major histocompatibility complex recognized by the TCR. [This invention 1059] the T cells are tumor-infiltrating lymphocytes (TILs); The induction or enhancement of TIL proliferation is determined by incubating a human tumor sample with interleukin 2 (IL-2) and the antibody, and recovering and counting TILs after about 10 to about 14 days of incubation. The multispecific antibody of any one of 1054 to 1055 of the present invention. [The present invention 1060] The multispecific antibody of the present invention 1059, wherein said human tumor is a melanoma or a non-small cell lung cancer (NSCLC) tumor. [The present invention 1061] A bispecific antibody comprising a second antigen-binding region of any one of claims 1001 to 1002, wherein the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 99, 100, and 101, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 102, GVS, and SEQ ID NO: 103, respectively. The multispecific antibody of any of claims 1054 to 1060, which induces or enhances greater proliferation of T cells compared to the multispecific antibody of claim 10. [The present invention 1062] Any of the antibodies of the present invention, which are bispecific antibodies. [This invention 1063] A nucleic acid encoding one or more amino acid sequences of the present invention 1001 to 1062. [This invention 1064] A nucleic acid encoding a multispecific antibody defined in any one of 1001 to 1062 of the present invention. [This invention 1065] An expression vector comprising any one of the nucleic acids of the present inventions 1063 to 1064. [The present invention 1066] A host cell comprising a nucleic acid of any one of 1063 or 1064 of the present invention or an expression vector of 1065 of the present invention. [This invention 1067] The host cell of the invention 1066, which is a recombinant eukaryotic, recombinant prokaryotic, or recombinant microbial host cell. [The present invention 1068] A composition comprising the multispecific antibody of any of the present inventions 1001 to 1062, the nucleic acid of any of the present inventions 1063 and 1064, the expression vector of the present invention 1065, or the host cell of any of the present inventions 1066 and 1067. [The present invention 1069] The composition of the present invention 1068, which is a pharmaceutical composition. [The present invention 1070] The pharmaceutical composition of the present invention 1069 further comprising a pharmaceutically acceptable carrier. [This invention 1071] A multispecific antibody of any of the present inventions 1001 to 1062, a nucleic acid of the present invention 1063 or 1064, an expression vector of the present invention 1065, a host cell of the present invention 1066 or 1067, a composition of the present invention 1068, or a pharmaceutical composition of the present invention 1069 or 1070, for use as a pharmaceutical. [This invention 1072] A multispecific antibody of any of claims 1001 to 1062, a nucleic acid of claim 1063 or 1064, an expression vector of claim 1065, a host cell of claim 1066 or 1067, a composition of claim 1068, or a pharmaceutical composition of claim 1069 or 1070, for use in treating a disease. [This invention 1073] The multispecific antibody, nucleic acid, expression vector, host cell, or composition for use according to the present invention 1072, wherein said disease is an infectious disease. [This invention 1074] The multispecific antibody, nucleic acid, expression vector, host cell, or composition for use according to the present invention 1072, wherein the disease is cancer. [This invention 1075] The multispecific antibody, nucleic acid, expression vector, host cell, or composition for use in accordance with the present invention 1074, wherein the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, colon cancer, kidney cancer, cervical cancer, and prostate cancer, e.g., non-small cell lung cancer (NSCLC) or melanoma. [This invention 1076] A method for treating a disease, comprising the step of administering to a subject in need thereof a multispecific antibody of any of the present inventions 1001 to 1062, a nucleic acid of the present invention 1063 or 1064, an expression vector of the present invention 1065, a host cell of the present invention 1066 or 1067, a composition of the present invention 1068, or a pharmaceutical composition of the present invention 1069 or 1070. [This invention 1077] Use of any of the multispecific antibodies of the present inventions 1001 to 1062, the nucleic acid of the present invention 1063 or 1064, the expression vector of the present invention 1065, the host cell of the present invention 1066 or 1067, the composition of the present invention 1068, or the pharmaceutical composition of the present invention 1069 or 1070 in the manufacture of a medicine. [This invention 1078] 8. The method or use of any of claims 1071 to 1077, for use in combination with one or more further therapeutic agents, such as chemotherapeutic agents. [This invention 1079] (a) providing a first antibody comprising an Fc region comprising a first CH3 region; (b) providing a second antibody comprising a second Fc region comprising a second CH3 region; a step in which the first antibody is a CD40 antibody comprising two first antigen-binding regions of any one of 1001 to 1062 of the present invention, and the second antibody is a CD137 antibody comprising two second antigen-binding regions of any one of 1001 to 1062 of the present invention, or vice versa, and the sequences of the first and second CH3 regions are different, and the heterodimer interaction between the first and second CH3 regions is stronger than the homodimer interaction between each of the first and second CH3 regions; (c) incubating the first antibody with the second antibody under reducing conditions; and (d) obtaining the bispecific CD40×CD137 antibody 1062. A method for producing a bispecific antibody of the present invention, comprising: [The present invention 1080] A method for detecting whether cross-linking between CD40-expressing cells and CD137-expressing cells occurs in a sample obtained from a patient, such as a blood sample, a lymph node sample, or a bone marrow sample, when any of the multispecific antibodies of the present inventions 1001 to 1062 is administered, comprising: (i) contacting the sample with any one of the multispecific antibodies of the present inventions 1001 to 1062 under conditions that allow the formation of a complex between the multispecific antibody and CD40-expressing cells and CD137-expressing cells; and (ii) analyzing whether a complex is formed; The method comprising: [This invention 1081] 1. A kit for detecting cross-linking between CD40-expressing cells and CD137-expressing cells in a sample obtained from a patient, such as a blood sample, a lymph node sample, or a bone marrow sample, comprising: (i) any one of the multispecific antibodies 1001 to 1062 of the present invention; and (ii) Instructions for use of the kit The kit comprises: [This invention 1082] An anti-idiotype antibody that binds to the second or the first and second antigen-binding regions defined in any one of 1001 to 1062 of the present invention. [Brief explanation of the drawings]

[0015] [Figure 1] Sequence alignment of human CD137, African elephant CD137, and wild boar CD137. Amino acids in African elephant CD137 or wild boar CD137 that differ from those in the human sequence are highlighted in black. [Figure 2] CD137 shuffle constructs containing African elephant (shuffle 5) CD137 domains or wild boar (shuffles 1-4, 6) CD137 domains. [Figure 3] Expression of CD137 shuffle constructs in HEK293-T17 cells. HEK293-T17 cells were transfected with the CD137 shuffle constructs. Cell surface expression of the constructs was measured by flow cytometry using a polyclonal anti-CD137 antibody that recognizes human CD137, wild boar CD137, and African elephant CD137. [Figure 4-1] Binding of CD137 antibody clones to CD137 shuffle constructs expressed in HEK293-T17 cells. HEK293-T17 cells were transfected with CD137 shuffle constructs, human CD137 (hCD137 wild type), and African elephant boar CD137, as indicated. Binding of various CD137 antibody clones to these constructs expressed in HEK293-T17 cells was measured by flow cytometry. Staining with a polyclonal anti-CD137 antibody is shown as a control. [Figure 4-2] See illustration in Figure 4-1. [Figure 5] A matrix-like mixing grid used for automated bispecific antibody discovery. Parent antibodies can be plated as shown. They can then be combined using a simple matrix-like mixing grid to obtain bispecific antibodies. Directed Fab arm exchange can then be performed to obtain bispecific antibodies. [Figure 6]Expression of CD40 and CD137 on the cell surface of stably transduced HEK293-NFK-gfp-luc and K562 cells. NF-κB / 293 / GFP-Luc cells (A) and K562 cells (B) were stably transduced with CD40 or CD137. Surface expression of CD40 (left panel) and CD137 (right panel) was measured by flow cytometry (white curve: control without antibody; gray curve: antibody staining). [Figure 7A] Analysis of bispecific antibodies simultaneously targeting CD40 and CD137 (CD40 × CD137). Bispecific antibodies targeting CD40 and CD137 (CD40-FEAL × CD137-FEAR) were tested in duplicate by reporter assay (A–L: CD40-001 × CD137-001 to CD40-001 × CD137-012). CD137 activation was measured based on the luciferase activity (relative luminescence units, RLU) of CD137-transduced NF-κB / 293 / GFP-Luc (HEK293_NFK_CD137_gfp_luc) upon incubation with K562 cells transduced with the indicated bispecific antibody and CD40 for transactivation (K562_CD40) or wild-type K562 cells (K562_wildtype) as a control. CD40 activation was measured based on the luciferase activity (RLU) of CD40-transfected NF-κB / 293 / GFP-Luc (HEK293_NFK_CD40_gfp_luc) upon incubation with K562 cells transduced with the indicated bispecific antibody and CD137 for transactivation (K562_CD137) or wild-type K562 cells (K562_wildtype) as a control. Two monospecific monovalent antibodies with one irrelevant arm (b12-FEAL×CD137-FEAR, b12-FEAL×CD40-FEAR) were used as controls for the bispecific CD40×CD137 antibodies. [Figure 7B] See illustration in Figure 7A. [Figure 7C] See illustration in Figure 7A. [Figure 7D] See illustration in Figure 7A. [Figure 7E] See illustration in Figure 7A. [Figure 7F] See illustration in Figure 7A. [Figure 7G] See illustration in Figure 7A. [Figure 7H] See illustration in Figure 7A. [Figure 7I] See illustration in Figure 7A. [Figure 7J] See illustration in Figure 7A. [Figure 7K] See illustration in Figure 7A. [Figure 7L] See illustration in Figure 7A. [Figure 8A] Induction of CD8+ T cell proliferation by CD40×CD137 bispecific antibodies in a non-antigen-specific T cell assay. CFSE-labeled PBMCs were incubated with CD40×CD137 bispecific antibodies or a monospecific monovalent control antibody for 4 days. CD8+ cell proliferation was measured by flow cytometry. Data shown are CFSE plots (A) showing CD8+ T cell proliferation induced by 0.02 μg / mL of each indicated bispecific antibody and control antibody, as well as dividing cell rates and proliferation indices calculated using FlowJo software for CD40-001-FEAL×CD137-005-FEAR (B), CD40-001-FEAL×CD137-009-FEAR (C), CD40-001-FEAL×CD137-003-FEAR (D), and CD40-001-FEAL×CD137-011-FEAR (E). [Figure 8B] See illustration in Figure 8A. [Figure 8C] See illustration in Figure 8A. [Figure 8D] See illustration in Figure 8A. [Figure 8E] See illustration in Figure 8A. [Figure 9A]Enhancement of CD8+ T cell proliferation by CD40×CD137 bispecific antibodies in an antigen-specific T cell assay. CFSE-labeled T cells transfected with claudin-6-specific TCRs were incubated with immature DCs electroporated with claudin-6 IVT-RNA for 5 days in the presence or absence of CD40×CD137 bispecific antibodies or control antibodies. CD8+ T cell proliferation was measured by flow cytometry. Data shown are CFSE plots (A) showing CD8+ T cell proliferation induced by 0.02 μg / mL of each indicated bispecific antibody and control. The dividing cell rate and proliferation index were calculated using FlowJo software for each indicated bispecific antibody (B) and CD40-001-FEAL×CD137-005-FEAR and control antibodies (C), and CD40-001-FEAL×CD137-009-FEAR and control antibodies (D) at the indicated concentrations. Proliferation index curves for each indicated bispecific antibody at serial dilutions ranging from 6.4 × 10-5 to 5 μg / mL are also shown (E). Curves were analyzed by nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). EC50 values ​​for induction of T cell proliferation for CD40-001-FEAL x CD137-005-FEAR and CD40-001-FEAL x CD137-009-FEAR were 0.005 μg / mL and 0.030 μg / mL, respectively. [Figure 9B] See illustration in Figure 9A. [Figure 9C] See illustration in Figure 9A. [Figure 9D] See illustration in Figure 9A. [Figure 9E] See illustration in Figure 9A. [Figure 10]Induction of CD8+ T cell proliferation by humanized CD40xCD137 bispecific antibody in a non-antigen-specific T cell assay. CFSE-labeled PBMCs were incubated with humanized CD40xCD137 bispecific antibody, parental bispecific antibody, or IgG1 control antibody for 4 days. CD8+ T cell proliferation was measured by flow cytometry. Data shown are dividing cell rate and proliferation index calculated by FlowJo software. (nd = not determined) [Figure 11] Enhancement of CD8+ T cell proliferation by humanized CD40×CD137 bispecific antibody in an antigen-specific T cell assay. CFSE-labeled T cells transfected with a claudin-6-specific TCR were incubated with immature DCs electroporated with claudin-6 IVT-RNA for 4 days in the presence or absence of a humanized CD40×CD137 bispecific antibody (BisG1-CD40-001-H6LC1-FEAL×CD137-009-HC7LC2-FEAR), parental bispecific antibody, or an IgG1 control antibody. CD8+ T cell proliferation was measured by flow cytometry. Data shown are the dividing cell rate and proliferation index for the indicated antibody, calculated using FlowJo software. (nd = not determined) [Figure 12] Ex vivo expansion of TILs from human melanoma tissue resections with CD40×CD137 bispecific antibody. Tumor fragments derived from resected tissue were cultured with 100 U / mL IL-2 and the indicated concentrations of CD40×CD137 bispecific antibody (BisG1-CD40-001-FEAL×CD137-009-FEAR). After 14 days of culture, cells were harvested and analyzed by flow cytometry. Relative viable TIL counts per sample (normalized to 1,000 counting beads measured) are shown. Each data point represents one well and represents the expansion of TILs from two tumor fragments analyzed in one FACS tube. The line indicates the average of five samples measured. [Figure 13]Ex vivo expansion of TILs from human non-small cell lung cancer (NSCLC) tissue resections by CD40×CD137 bispecific antibody. Tumor fragments derived from resected NSCLC tissue were cultured with 10 U / mL IL-2 and the indicated concentrations of CD40×CD137 bispecific antibody (BisG1-CD40-001-FEAL×CD137-009-FEAR). After 10 days of culture, cells were harvested and analyzed by flow cytometry. Relative viable TIL counts per sample (normalized to 1,000 counting beads measured) are shown. Each data point represents one well and represents the expansion of TILs from two tumor fragments analyzed in one FACS tube. The line indicates the average of five samples measured.

[0016] (Table 1) Sequence TIFF2025186475000001.tif117170TIFF2025186475000002.tif233170TIFF2025186475000003.tif230170TIFF2025186475000004.tif2371 70TIFF2025186475000005.tif235170TIFF2025186475000006.tif235170TIFF2025186475000007.tif237170TIFF2025186475000008.tif244 170TIFF2025186475000009.tif243170TIFF2025186475000010.tif206170TIFF2025186475000011.tif236170TIFF2025186475000012.tif236170TIFF2025186475000013.tif236170TIFF2025186475000014.tif238170TIFF2025186475000015.tif242170*Amino acid positions 118 to 447 according to EU numbering DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention definition The term "CD40" as used herein refers to CD40, also known as tumor necrosis factor receptor superfamily member 5 (TNFRSF5), which is a receptor for the ligand TNFSF5 / CD40L. CD40 is known to transduce signals via TRAF6 and MAP3K8 that activate ERK in macrophages and B cells, resulting in the induction of immunoglobulin secretion by B cells. Other synonyms used for CD40 include, but are not limited to, B cell surface antigen CD40, Bp50, CD40L receptor, and CDw40. In one embodiment, CD40 is human CD40, whose UniProt accession number is P25942. The sequence of human CD40 is also set forth in SEQ ID NO: 115. Amino acids 1-20 of SEQ ID NO: 115 correspond to the signal peptide of human CD40; amino acids 21-193 of SEQ ID NO: 115 correspond to the extracellular domain of human CD40; the remainder of the protein, i.e., amino acids 194-215 and 216-277 of SEQ ID NO: 115, are the transmembrane and cytoplasmic domains, respectively.

[0018] The term "CD137" as used herein refers to CD137(4-1BB), also known as tumor necrosis factor receptor superfamily member 9 (TNFRSF9), which is a receptor for the ligand TNFSF9 / 4-1BBL. CD137(4-1BB) is thought to be involved in T cell activation. Other synonyms for CD137 include, but are not limited to, 4-1BB ligand receptor, CDw137, T cell antigen 4-1BB homolog, and T cell antigen ILA. In one embodiment, CD137(4-1BB) is human CD137(4-1BB), whose UniProt accession number is Q07011. The sequence of human CD137 is also shown in SEQ ID NO: 92. Amino acids 1-23 of SEQ ID NO: 92 correspond to the signal peptide of human CD137; amino acids 24-186 of SEQ ID NO: 92 correspond to the extracellular domain of human CD137; the remainder of the protein, i.e., amino acids 187-213 and 214-255 of SEQ ID NO: 92, are the transmembrane and cytoplasmic domains, respectively.

[0019] The term "chimeric antibody" as used herein refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term used to refer to various types of antibody modifications, and methods for antibody engineering are well known to those skilled in the art. Specifically, chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies may be genetically or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibody production may be performed by methods other than those described herein. Chimeric monoclonal antibodies for human therapeutic applications have been developed to reduce the potential antibody immunogenicity of non-human antibodies, such as rodent antibodies. These typically include a non-human (e.g., mouse or rabbit) variable region specific for an antigen of interest, and human constant heavy and light antibody chain domains. The term "variable region" or "variable domain" as used in the context of chimeric antibodies refers to the region comprising the CDRs and framework regions of both the heavy and light immunoglobulin chains, as described below.

[0020] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to have a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody CDRs that together form the antigen-binding site into homologous human acceptor framework regions (FRs) (see WO92 / 22653 and EP0629240). To fully restore the binding affinity and specificity of the parent antibody, it may be necessary to replace the human framework regions with framework residues from the parent antibody (i.e., non-human antibody) (backmutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, framework regions of primarily human origin, optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a constant region that is entirely human. Optionally, additional amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with favorable characteristics such as affinity and biochemical properties, and / or additional amino acid mutations may be introduced into the constant region.

[0021] As used herein, a protein "derived from" another protein, e.g., a parent protein, means that one or more amino acid sequences of the protein are identical to or similar to one or more amino acid sequences in the other protein or parent protein. For example, in an antibody, binding arm, antigen-binding region, or constant region derived from another or parent antibody, binding arm, antigen-binding region, or constant region, one or more amino acid sequences are identical to or similar to the amino acid sequence of the other or parent antibody, binding arm, antigen-binding region, or constant region. Examples of such one or more amino acid sequences include, but are not limited to, the amino acid sequences of the VH CDR and VL CDR, and / or one or more or all of the framework regions, VH region, VL region, CL region, hinge region, or CH region. For example, a humanized antibody can be described herein as "derived from" a non-human parent antibody, meaning that at least the VL CDR and VH CDR sequences are identical to or similar to the VH CDR and VL CDR sequences of the non-human parent antibody. A chimeric antibody may be described herein as "derived from" a non-human parent antibody, which typically means that the VH and VL sequences may be identical to or similar to those of the non-human parent antibody. Another example is a binding arm or antigen-binding region, which may be described herein as "derived from" a particular parent antibody, which typically means that the binding arm or antigen-binding region comprises VH and / or VL CDRs, or VH and / or VL sequences, that are identical to or similar to those of the binding arm or antigen-binding region of the parent antibody. However, as described elsewhere herein, amino acid modifications, such as mutations, may be made elsewhere, such as in the CDRs, constant region, or antibody, binding arm, or antigen-binding region, to introduce desired characteristics.When used in the context of one or more sequences derived from a first or parent protein, a "similar" amino acid sequence preferably has at least about 50%, e.g., at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 97%, 98%, or 99% sequence identity.

[0022] Non-human antibodies can be generated in several different species, including mouse, rabbit, chicken, guinea pig, llama, and goat.

[0023] Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). Other techniques for producing monoclonal antibodies can be used, such as viral or oncogenic transformation of B lymphocytes, or phage display techniques using libraries of antibody genes, and such methods are well known to those skilled in the art.

[0024] Hybridoma production in such non-human species is a very well-established procedure. Immunization protocols and techniques for isolating splenocytes from immunized animals / non-human species for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0025] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0026] Human monoclonal antibodies can be generated using transgenic or transchromosomic mice carrying parts of the human immune system instead of the mouse system.

[0027] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain (abbreviated as "HC") typically comprises a heavy chain variable region (herein referred to as V H or VH) and a heavy chain constant region (referred to herein as C H The heavy chain constant region is typically composed of three domains: C, ... H 1. C H 2, and C H 3. Heavy chains typically may further include a hinge region. Each light chain (abbreviated as "LC") typically includes a light chain variable region (herein V L or VL) and a light chain constant region (referred to herein as C LThe light chain constant region is typically composed of one domain: C L It consists of: V H Area and V L The regions can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in sequence and / or in the shape of structured loops), also called complementarity determining regions (CDRs), interrupted by regions of high degree of conservation called framework regions (FRs). H and V L Typically, a CDR is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196 , 901-917 (1987). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign (program version 4.9.1; 2013-12-19) (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Research 2010;38, D301-307; see also the internet http address www.imgt.org / ).

[0028] Unless otherwise stated or contradicted by the context, references to amino acid positions in the constant region herein are based on EU numbering (Edelman et al., Proc Natl Acad Sci U.S.A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0029] The term "antibody" (Ab), as used herein, refers to a molecule comprising at least one antibody variable domain, e.g., an immunoglobulin heavy chain variable region, or an immunoglobulin heavy and light chain variable region, or a fragment thereof, or a derivative of either, which molecule has the ability to specifically bind to an antigen, e.g., under typical physiological conditions, and has a half-life of a significant period, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days, or more, or any other suitable period defined functionally (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the antibody binding to the antigen and / or a period sufficient for the antibody to recruit effector activity). Specifically, an antibody may be an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof. The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with antigens. The constant regions of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component of the classical pathway of complement activation. As indicated above, the term antibody herein includes, unless otherwise specified or clearly contradicted by the context, antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to antigens. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) Fab' or Fab fragments, i.e., V L Domain, V H Domain, C L domain, and C H (ii) a monovalent fragment consisting of one domain, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) an F(ab')2 fragment, i.e., a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V HDomain and C H (iv) an Fd fragment consisting essentially of one domain; (iv) a V of one arm of an antibody L Domains and V H (v) an Fv fragment consisting essentially of a V domain; H These include dAb fragments (Ward et al., Nature 341, 544-546 (1989)), which essentially consist of domains and are also called domain antibodies (Holt et al., Trends Biotechnol. 2003 Nov;21(11):484-90); (vi) camelids or nanobodies (Revets et al., Expert Opin Biol Ther. 2005 Jan;5(1):111-24); and (vii) isolated complementarity-determining regions (CDRs). Additionally, the two domains of the Fv fragment, V, are also known as Fv fragments. L and V H are encoded by separate genes, but V L Area and V HThese regions can be linked, using recombinant methods, by synthetic linkers that allow them to be produced as a single protein chain in which they pair to form monovalent molecules (known as single-chain antibodies or single-chain Fvs (scFvs); see, e.g., Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term antibody unless otherwise noted or clearly indicated by context. Generally, such fragments are included within the meaning of antibody, but collectively and each independently, they are unique features of the present invention and exhibit different biological properties and utilities. These and other useful antibody fragments in the present invention, as well as bispecific forms of such fragments, are discussed further herein. The term antibody, unless otherwise specified, should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to antigens, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. The antibodies generated can have any isotype and / or subclass.

[0030] Conventional antibodies, eg antibodies raised in any species, are usually monospecific, bivalent antibodies, meaning that they contain two antigen-binding regions that bind to the same epitope.

[0031] As used herein, the term "multispecific antibody" refers to an antibody having different antigen-binding regions defined by different antibody sequences. Thus, a multispecific antibody may have two, three, four, five, or more different antigen-binding regions. Examples of multispecific antibodies include antibodies having two different antigen-binding regions, i.e., bispecific antibodies.

[0032] Examples of multispecific antibodies comprising three or more different antigen-binding regions include, but are not limited to, (i) bispecific antibodies in which an additional single-chain variable fragment (scFv) is linked to the Fc portion (Weidle et al., Cancer Genomics Proteomics. 2013 Jan-Feb;10(1):1-18), (ii) fusion proteins consisting of three or more scFvs (triabodies, tetrabodies; Chames et al., FEMS Microbiol Lett. 2000 Aug 1;189(1):1-8), and (iii) fusion proteins linked to scFvs (Kermer et al. Mol Cancer Ther. 2014 Jan;13(1):112-21).

[0033] The term "bispecific antibody" as used herein refers to an antibody that has two different antigen-binding regions defined by different antibody sequences.

[0034] As used herein, unless contradicted by context, the term "Fab arm" or "arm" refers to one heavy-light chain pair and is used synonymously herein with "half molecule."

[0035] The term "binding arm comprising an antigen-binding region" refers to an antibody molecule or fragment comprising an antigen-binding region. Thus, the binding arm can comprise, for example, the six CDR sequences of the VH and VL, the VH and VL sequences, a Fab or Fab' fragment, or a Fab arm.

[0036] As used herein, unless contradicted by context, the term "Fc region" refers to the region of an antibody comprising at least a hinge region, a CH2 domain, and a CH3 domain.

[0037] As used herein, the term "isotype" refers to a particular type of immunoglobulin encoded by HC (e.g., IgG, IgD, IgA, IgE, and IgM) genes or LC (kappa, κ or lambda, λ) genes. Each isotype can have several subclasses, e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0038] The term "monovalent antibody" means in the present invention that an antibody molecule is capable of binding to a single molecule of antigen and is therefore incapable of antigen cross-linking.

[0039] A "CD40 antibody" or "anti-CD40 antibody" is an antibody, as described above, that specifically binds to the antigen CD40.

[0040] A "CD137 antibody" or "anti-CD137 antibody" is an antibody, as described above, that specifically binds to the antigen CD137.

[0041] A "CD40xCD137 antibody" or "anti-CD40xCD137 antibody" is a bispecific antibody that contains two different antigen-binding regions, one of which specifically binds to the antigen CD40 and one of which specifically binds to the antigen CD137.

[0042] The terms "specifically bind," "specifically binding," "specific binding," or other similar expressions refer to the ability of an antibody to bind preferentially to a particular antigen over other antigens or to a particular portion (epitope) of an antigen over other portions of the same antigen.

[0043] As used herein, the term "binding" in the context of antibody binding to a given antigen or epitope typically refers to a binding of about 10 to 150 s, as measured by surface plasmon resonance (SPR) technology on a BIAcore3000 instrument, for example, using the antibody as the ligand and the antigen as the analyte, or vice versa. -7 M or less, e.g., about 10 -8M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even smaller K D for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D K which is at least 10 times lower, such as at least 100 times lower, for example at least 1,000 times lower, such as at least 10,000 times lower, for example at least 100,000 times lower, D The higher affinity amount binds to a given antigen with an affinity equivalent to the antibody's K D is determined by the antibody's K D is very small (i.e., the antibody is highly specific), the amount by which the affinity for the antigen is higher than the affinity for a nonspecific antigen can be at least 10,000 times.

[0044] As used herein, the term "k d " (sec -1 ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is known as k off Also called value.

[0045] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0046] Two antibodies have the "same specificity" if they bind to the same antigen and to the same epitope. Whether a tested antibody recognizes the same epitope as a certain antigen-binding antibody, i.e., whether the antibodies bind to the same epitope, can be tested by various methods well known to those skilled in the art.

[0047] The competition between antibodies can be detected by cross-blocking assay.For example, competitive ELISA assay can be used as cross-blocking assay.For example, target antigen can be coated on the well of a microtiter plate, and antigen-binding antibody and candidate competitive test antibody can be added.The amount of antigen-binding antibody that binds to the antigen in the well is indirectly correlated with the binding ability of the candidate competitive test antibody that competes with it for binding to the same epitope.Specifically, the greater the affinity of the candidate competitive test antibody for the same epitope, the smaller the amount of antigen-binding antibody that binds to the well coated with the antigen.The amount of antigen-binding antibody that binds to the well can be measured by labeling the antibody with a detectable or measurable label.

[0048] An antibody that competes with another antibody, e.g., an antibody comprising a heavy chain variable region and a light chain variable region described herein, for binding to an antigen, or that has the specificity for the antigen of another antibody, e.g., an antibody comprising a heavy chain variable region and a light chain variable region described herein, may be a variant of the heavy chain variable region and / or light chain variable region described herein, i.e., an antibody that contains modifications and / or a degree of identity in the CDRs described herein.

[0049] As used herein, an "isolated multispecific antibody" is intended to refer to a multispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to CD40 and CD137 is substantially free of monospecific antibodies that specifically bind to CD40 or CD137).

[0050] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groups of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, for example, amino acid residues that are effectively blocked or masked by a peptide that specifically binds to an antigen (in other words, the amino acid residues are within the sphere of influence of the peptide that specifically binds to the antigen).

[0051] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition, which display a single binding specificity and affinity for a particular epitope.

[0052] As used herein, the term "heterodimeric interaction between a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and a second CH3 region in a first CH3 / second CH3 heterodimeric antibody.

[0053] As used herein, the term "homodimeric interaction between a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric antibody, and the interaction between a second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric antibody.

[0054] As used herein, the term "homodimeric antibody" refers to an antibody comprising two first Fab arms or half molecules, wherein the amino acid sequences of the Fab arms or half molecules are the same.

[0055] As used herein, the term "heterodimeric antibody" refers to an antibody comprising a first Fab arm and a second Fab arm or half molecule, wherein the amino acid sequences of the first and second Fab arms or half molecules are different. Specifically, the CH3 regions, or antigen-binding regions, or the CH3 regions and antigen-binding regions of the first and second Fab arms / half molecules are different.

[0056] The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, such as a cysteine ​​residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

[0057] The present invention also relates to the V of the bispecific antibodies of the examples. L area, V H Also provided are multispecific antibodies, such as bispecific antibodies, that comprise functional variants of a V region, or one or more CDRs. L , V H , or functional variants of the CDRs, still enable each antigen-binding region of the bispecific antibody to retain at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody, and in some cases, such bispecific antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent bispecific antibody.

[0058] Typically, such functional variants retain a significant degree of sequence identity to the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., percent identity = number of identical positions / total number of positions × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0059] In the present invention, the following notation is used to describe mutations unless otherwise specified: the name of the mutated amino acid, followed by the mutated position number, followed by the content of the mutation. Thus, if the mutation is a substitution, the name of the amino acid substituting the previous amino acid is included; if an amino acid is deleted, it is indicated by *; if the mutation is an addition, the added amino acid is included after the original amino acid. The name of the amino acid may be a one-letter code or a three-letter code. Thus, for example, substitution of lysine at position 409 with arginine is represented as K409R, substitution of lysine at position 409 with any amino acid is represented as K409X, deletion of lysine at position 409 is represented as K409*, and addition of P after lysine at position K409 is represented as K409KP.

[0060] Exemplary variants include those that differ from the VH and / or VL and / or CDRs of a parent sequence primarily by conservative substitutions; for example, 12, e.g., 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions.

[0061] In the present invention, conservative substitutions may be defined as substitutions within the classes of amino acids set out below.

[0062] Amino acid residue classes for conservative substitutions: Acidic residues: Asp (D) and Glu (E) Basic residues: Lys (K), Arg (R), and His (H) Hydrophilic uncharged residues: Ser (S), Thr (T), Asn (N), and Gln (Q) Aliphatic uncharged residues: Gly (G), Ala (A), Val (V), Leu (L), and Ile (I) Non-polar, uncharged residues: Cys (C), Met (M), and Pro (P) Aromatic residues: Phe (F), Tyr (Y), and Trp (W)

[0063] The first and / or second antigen-binding regions of the present invention may also be variants of the first and / or second antigen-binding regions, respectively, disclosed herein.

[0064] Those skilled in the art are familiar with methods for introducing modifications and that some amino acids in CDR sequences can be modified, for example, by amino acid substitution, to, for example, increase the affinity of the antibody for the target antigen, reduce the potential immunogenicity of non-human antibodies used in humans, and / or increase the yield of antibodies expressed by host cells. Such modifications can be introduced without affecting the epitope of the target antigen to which the antibody binds.

[0065] The term "recombinant host cell" (or simply "host cell" or "cell"), as used herein, is intended to refer to a cell into which a nucleic acid such as an expression vector, e.g., a nucleic acid such as an expression vector encoding a multispecific antibody of the invention, has been introduced. Recombinant host cells include, for example, transfectomas, e.g., CHO cells, CHO-S cells, HEK cells, HEK293 cells, HEK-293F cells, Expi293F cells, PER.C6 cells, or NS0 cells, and lymphocytic cells.

[0066] The term "treatment" refers to the administration of an effective amount of a therapeutically active multispecific antibody of the invention to relieve, ameliorate, inhibit or eliminate (cure) the symptoms or disease state.

[0067] The term "effective amount" or "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of a multispecific antibody may vary depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the multispecific antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the multispecific antibody or fragment thereof are outweighed by the therapeutically beneficial effects.

[0068] The term "anti-idiotypic antibody" refers to an antibody that recognizes unique determinants normally associated with the antigen-binding site of an antibody.

[0069] In the present invention, the term "induce Fc-mediated effector functions to a low extent" as used in reference to antibodies, including multispecific antibodies, means that the extent to which the antibody induces Fc-mediated effector functions (such functions are particularly selected from the list of IgG Fc receptor (Fc gamma R, FcγR) binding, C1q binding, ADCC, or CDC) is lower than that of a human IgG1 antibody comprising (i) the same CDR sequences, particularly comprising the same first and second antigen-binding regions as the antibody, and (ii) two heavy chains comprising the hinge region, CH2 region, and CH3 region of human IgG1.

[0070] Fc-mediated effector function can be measured based on binding to FcγR, binding to C1q, or induction of Fc-mediated cross-linking by FcγR.

[0071] Further aspects and embodiments of the invention The present invention relates to molecules that comprise two different antigen-binding regions, one of which has specificity for human CD40 and one of which has specificity for human CD137.

[0072] In certain embodiments, the molecule may be a multispecific antibody.

[0073] Accordingly, the present invention relates to a multispecific antibody comprising (i) a first antigen-binding region that binds to human CD40 and (ii) a second antigen-binding region that binds to human CD137.

[0074] As demonstrated by the inventors of the present invention, bispecific antibodies according to the present invention can induce intracellular signaling upon binding to CD40 expressed on one cell and CD137 expressed on another cell. Thus, multispecific antibodies according to the present invention can transactivate two different cells. In humans, CD40 is expressed on several cells, including antigen-presenting cells (APCs) such as dendritic cells, whereas CD137 is expressed on T cells and other cells. Therefore, multispecific antibodies, such as bispecific antibodies according to the present invention, that bind to CD40 and CD137 can simultaneously bind to APCs and T cells that express these receptors. Without being bound by theory, multispecific antibodies, such as bispecific antibodies according to the present invention, can thus (i) mediate cell-cell interaction between APCs and T cells through receptor binding and (ii) simultaneously activate both CD40 and CD137, which is initially induced by receptor clustering upon cross-linking and cell-cell interaction and is not necessarily dependent on the agonistic activity of the monospecific, bivalent parent antibody. Thus, these transactivating multispecific antibodies, like bispecific antibodies, can exert costimulatory activity in the context of APC:T cell interactions and induce T cell responses against tumor cells. Thus, this mechanism of action can enable APCs to present various tumor-specific antigens to T cells, mirroring the activation of natural T cells through antigen presentation by activated APCs. Without being limited by theory, costimulatory activity can result in one or more of the following: (i) activation of only specific T cells (i.e., those in contact with APCs), as opposed to any T cells; (ii) reactivation of exhausted T cells through potent costimulation via activated APCs and CD137 agonism; and (iii) antigen stimulation of T cells by inducing antigen presentation by activated APCs and simultaneously agonizing CD137.

[0075] Thus, multispecific, eg bispecific, antibodies of the invention may be used to treat diseases that would benefit from T cell activation, such as cancer.

[0076] In one embodiment the multispecific antibody according to the invention comprises: (I) a first antigen-binding region that binds to human CD40, comprising CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 3 or a sequence with up to four amino acid variations in SEQ ID NO: 3, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 5 or a sequence with up to four amino acid variations in SEQ ID NO: 5; and (b) (i) a heavy chain variable region CDR3 and a light chain variable region CDR3 of an antibody that competes for human CD40 binding with an antibody comprising the heavy chain variable region CDR3 and the light chain variable region CDR3 described in (a), and / or (ii) has the specificity for CD40 of an antibody comprising the heavy chain variable region CDR3 and the light chain variable region CDR3 described in (a), and (II) A second antigen-binding domain that binds to human CD137 Includes:

[0077] In a further embodiment, the first antigen-binding region may further comprise a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 1 or the sequence shown in SEQ ID NO: 1 with up to two amino acid modifications, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 2 or the sequence shown in SEQ ID NO: 2 with up to two amino acid modifications; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 4 or the sequence shown in SEQ ID NO: 4 with up to two amino acid modifications, and / or a light chain variable region CDR2 having the sequence YTS or the sequence shown in YTS with up to two amino acid modifications.

[0078] Thus, in one aspect, the present invention provides a method for treating a pulmonary arthritis, comprising: (I) a first antigen-binding region that binds to human CD40, comprising CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a), each having a total of 1 to 12 mutations; and (c) (i) CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of an antibody that competes for human CD40 binding with an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and / or (ii) has the specificity for CD40 of an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and (II) A second antigen-binding domain that binds to human CD137 The present invention relates to a multispecific antibody comprising:

[0079] In a further embodiment, the first antigen-binding region comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, and the second antigen-binding region comprises a second heavy chain variable (VH) sequence and a second light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively.

[0080] In a further embodiment, the multispecific antibody comprises (I) a first binding arm comprising a first antigen-binding region and (II) a second binding arm comprising a second antigen-binding region.

[0081] In one embodiment, the first binding arm comprises a first antigen-binding region and a first heavy chain constant sequence, and the second binding arm comprises a second antigen-binding region and a second heavy chain constant sequence.

[0082] In a further embodiment, (i) the first binding arm comprises a first antigen-binding region, wherein the first binding arm comprises a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence, and (ii) the second binding arm comprises a second antigen-binding region, wherein the second binding arm comprises a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence.

[0083] In a further embodiment, the first light chain further comprises a first light chain constant (CL) sequence and the second light chain further comprises a second light chain constant (CL) sequence.

[0084] In one embodiment, the first binding arm comprises a first Fab arm comprising a first antigen-binding region, and the second binding arm comprises a second Fab arm comprising a second antigen-binding region.

[0085] In one embodiment, the first and second antigen-binding regions of the multispecific antibody according to the invention are derived from a humanized antibody. In one embodiment, the first and second binding arms may be derived from a humanized antibody.

[0086] In one embodiment, the first binding arm and the second binding arm of the multispecific antibody according to the invention are derived from a full-length antibody.

[0087] In one embodiment, the first binding arm and the second binding arm of the multispecific antibody according to the invention are derived from a full-length IgG1, λ (lambda) antibody or an IgG1, κ (kappa) antibody.

[0088] In one embodiment, the first binding arm and the second binding arm are derived from a monoclonal antibody.

[0089] In one embodiment, the first and second heavy chains of the multispecific antibody according to the invention are of the IgG isotype. The subclasses of the first and second heavy chains may be independently selected from the group consisting of, for example, IgG1, IgG2, IgG3, and IgG4. In one embodiment, the first and second heavy chains are of the same IgG subclass, for example, IgG1.

[0090] In one embodiment, the multispecific antibody according to the invention is an isolated antibody.

[0091] In further embodiments, each of the first and second heavy chains comprises at least one hinge region, CH2 region, and CH3 region, hi further embodiments, the CH3 regions of the first and second heavy chains comprise asymmetric mutations.

[0092] In one embodiment, the multispecific antibody according to the invention is a bispecific antibody.

[0093] In one embodiment, a multispecific antibody according to the invention is capable of cross-linking one cell, e.g., a first cell, that expresses human CD40 with another cell, e.g., a second cell, that expresses human CD137.

[0094] In one embodiment, cross-linking is confirmed by an assay using a first cell line expressing human CD40 and a second cell line expressing human CD137, wherein either the first cell line or the second cell line contains a reporter construct that results in the production of a measurable reporter upon NF-κB activation.

[0095] In one embodiment, the first cell may be an antigen-presenting cell and the second cell may be a CD4 + T cells or CD8 + It may be a T cell, such as a T cell.

[0096] A variety of methods may be used to confirm cross-linking of a first cell expressing CD40 and a second cell expressing CD137, and the present invention is not limited to any particular method.

[0097] In one embodiment, cross-linking can be confirmed by a reporter assay, e.g., as described in Example 4. Briefly, the assay comprises the steps of co-culturing a reporter cell line expressing a first target antigen and transduced with a reporter gene (e.g., luciferase) driven by an NF-κB response element, with a second cell line expressing a second target antigen; adding a multispecific antibody according to the present invention at a concentration of 100 ng / mL to 10,000 ng / mL to the cell co-culture; and measuring reporter gene expression, e.g., luciferase production, wherein the first target antigen is human CD40 and the second target antigen is human CD137, or vice versa.

[0098] In this assay, a multispecific antibody capable of inducing cross-linking of CD40 and CD137 expressed on different cells results in measurable activation of the first target antigen based on reporter gene expression upon activation of the NF-κB pathway.

[0099] In one embodiment, a multispecific antibody according to the present invention may be able to induce reporter gene expression that occurs upon NF-κB activation only upon addition of a second cell line expressing a second target antigen that does not have an NF-κB reporter gene.

[0100] In one embodiment, a multispecific antibody according to the present invention may be able to induce higher levels of reporter gene expression upon NF-κB activation when a second cell line expressing a second target antigen that does not have an NF-κB reporter gene is added compared to the addition of a second cell line that does not express the second target antigen.

[0101] In one embodiment the multispecific antibody is a bispecific antibody, which in one embodiment comprises: (i) is capable of inducing reporter gene expression when added to a co-culture of a reporter cell line expressing CD137 and a second cell line expressing CD40, or (ii) when added to a co-culture of a reporter cell line expressing CD137 and a second cell line expressing CD40, it is capable of inducing greater reporter gene expression than a reference bispecific antibody comprising the same second antigen-binding region that binds to human CD137, wherein the first antigen-binding region of the reference bispecific antibody binds to an unrelated target antigen, e.g., the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 99, 100, and 101, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 102, GVS, and 103, respectively;

[0102] In one embodiment the multispecific antibody is a bispecific antibody, which in one embodiment comprises: (i) is capable of inducing reporter gene expression when added to a co-culture of a reporter cell line expressing CD40 and a second cell line expressing CD137, or (ii) when added to a co-culture of a reporter cell line expressing CD40 and a second cell line expressing CD137, it is capable of inducing greater reporter gene expression than a reference bispecific antibody comprising the same first antigen-binding region that binds to human CD40, wherein the second antigen-binding region of the reference bispecific antibody comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 99, 100, and 101, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 102, GVS, and 103, respectively.

[0103] In one embodiment, the multispecific antibody according to the invention induces and / or enhances T cell proliferation, e.g., the T cells are CD4 + T cells and / or CD8 + T cells.

[0104] A variety of methods for determining or measuring T cell proliferation may be used, and the present invention is not limited to any particular method.

[0105] In one embodiment, induction or enhancement of T cell proliferation is measured by a non-antigen-specific T cell proliferation assay, e.g., as described in Example 5. Thus, induction and / or enhancement of T cell proliferation can be confirmed based on suboptimal activation of T cells in a PBMC pool (peripheral blood mononuclear lymphocytes). Suboptimal activation can be confirmed by gradually varying the concentration of anti-CD3 antibody added to the PBMC pool, measuring T cell proliferation, and selecting an anti-CD3 antibody concentration that results in a low level of T cell proliferation but allows for further enhancement of T cell proliferation. This concentration depends on the PBMC donor and is determined for each donor before the assay is performed.

[0106] In one embodiment, the induction or enhancement of T cell proliferation is confirmed by activating T cells in PBMCs with said suboptimal concentration of anti-CD3 antibody, contacting the PBMCs with the multispecific antibody, and measuring T cell proliferation. In a further embodiment, the PBMCs may be labeled with CFSE, contacting the PBMCs with the multispecific antibody may be performed by incubation for 4 days, and T cell proliferation may be measured by flow cytometry.

[0107] Inducing a certain response or effect, e.g., "inducing T cell proliferation," may mean that there was no such response or effect, e.g., T cell proliferation, before the induction, but it may also mean that there was a certain level of response or effect, e.g., T cell proliferation, before the induction, and that the response or effect, e.g., T cell proliferation, is enhanced after the induction. Thus, "inducing" also includes "enhancing."

[0108] T cell proliferation can also be measured using a test antigen of interest, for example, by an antigen-specific T cell proliferation assay described in Example 6. Thus, the induction and / or enhancement of T cell proliferation can be measured by co-culturing T cells expressing a TCR specific to a peptide of the test antigen presented in the major histocompatibility complex (MHC) with DCs presenting the corresponding peptide recognized by the TCR in the MHC. For example, the T cells can be CD8+ T cells, and the MHC can be MHC class I, or the T cells can be CD4+ T cells, and the MHC can be MHC class II. T cells expressing a specific TCR can be produced by transduction with mRNA encoding the TCR. DCs presenting the corresponding peptide can be produced by transduction with mRNA encoding the antigen. Co-culturing TCR-positive T cells with antigen-presenting cells induces T cell proliferation; the degree of proliferation can depend on the antigen density presented by DCs and / or the strength of the costimulatory signal. In one embodiment, T cell proliferation can be measured by such an antigen-specific T cell assay using CFSE-labeled T cells, adding the antibody to be tested, and measuring T cell proliferation by flow cytometry after 4 days.

[0109] In one embodiment, the induction or enhancement of T cell proliferation is measured using tumor-infiltrating lymphocytes (TILs) in an ex vivo expansion assay, e.g., as described in Example 11. The effect of a multispecific antibody of the invention on the induction or enhancement of TIL proliferation can be assessed by incubating a human tumor sample with interleukin-2 (IL-2) and the antibody, and recovering and counting viable TILs about 10 to about 14 days after incubation. In this case, the induction or enhancement of TIL proliferation can be measured by comparison with an appropriate control, e.g., a human tumor sample incubated without any multispecific antibody or incubated with a reference (control) multispecific antibody. For example, a sample of human tumor tissue can be isolated, e.g., by biopsy or from a surgical specimen, washed in serum-free medium, and tumor pieces about 1 to 2 mm in diameter can be placed in a culture dish or well, e.g., one or two tumor pieces in 1 mL of appropriate medium, and incubated at 37°C. An appropriate medium can be, for example, serum-free medium (e.g., X-VIVO 15) supplemented with 10% human serum albumin, 1% penicillin / streptomycin, 1% fungizone, and IL-2 at a concentration ranging from 10 to 100 U / mL, e.g., 10 U / mL or 100 U / mL. Multispecific antibodies can then be added to the TIL medium at appropriate concentrations. Cell cultures can be optionally split during this period, if necessary. After a total culture period of 10 to 14 days, TILs can be harvested and enumerated, for example, by flow cytometry using anti-CD3, anti-CD4, anti-CD8, anti-CD56, and anti-7-AAD antibodies to detect viable CD4+ and CD8+ T cells and NK cells.

[0110] In one embodiment, the multispecific antibody is a bispecific antibody, which bispecific antibody comprises a second antigen-binding region according to any aspect or embodiment described herein, but wherein the first antigen-binding region induces and / or enhances greater proliferation of T cells compared to a reference bispecific antibody comprising heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 99, 100, and 101, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 102, GVS, and 103, respectively.

[0111] In one embodiment, the multispecific antibody is a bispecific antibody, which bispecific antibody comprises a first antigen-binding region according to any aspect or embodiment described herein, but wherein the second antigen-binding region induces and / or enhances greater proliferation of T cells compared to a reference bispecific antibody comprising heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 99, 100, and 101, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 102, GVS, and 103, respectively.

[0112] Binding to CD40 As described above, the multispecific antibody according to the present invention comprises a first antigen-binding region that binds to human CD40.

[0113] In one embodiment, the multispecific antibody according to the invention comprises a first antigen-binding region that binds to human CD40, said first antigen-binding region comprising: (a) a heavy chain variable region CDR3 having the sequence shown in SEQ ID NO: 3 or a sequence with up to four amino acid alterations in SEQ ID NO: 3, and a light chain variable region CDR3 having the sequence shown in SEQ ID NO: 5 or a sequence with up to four amino acid alterations in SEQ ID NO: 5; (b) (i) a heavy chain variable region CDR3 and a light chain variable region CDR3 of an antibody that competes for human CD40 binding with an antibody comprising the heavy chain variable region CDR3 and the light chain variable region CDR3 described in (a), and / or (ii) has the specificity for CD40 of an antibody comprising the heavy chain variable region CDR3 and the light chain variable region CDR3 described in (a). The heavy chain variable region CDR3 and the light chain variable region CDR3 are selected from the group consisting of:

[0114] In a further embodiment, the first antigen-binding region may further comprise a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 1 or the sequence shown in SEQ ID NO: 1 with up to two amino acid modifications, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 2 or the sequence shown in SEQ ID NO: 2 with up to two amino acid modifications; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 4 or the sequence shown in SEQ ID NO: 4 with up to two amino acid modifications, and / or a light chain variable region CDR2 having the sequence YTS or the sequence shown in YTS with up to two amino acid modifications.

[0115] In one embodiment, the multispecific antibody according to the invention comprises a first antigen-binding region that binds to human CD40, said first antigen-binding region comprising: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a), each having a total of 1 to 12 mutations; and (c) (i) CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of an antibody that competes for human CD40 binding with an antibody comprising the heavy and light chain variable regions of (a) or (b), and / or (ii) CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of an antibody that has the specificity for CD40 of an antibody comprising the heavy and light chain variable regions of (a) or (b). The heavy chain variable region and the light chain variable region comprise CDR1, CDR2, and CDR3 selected from the group consisting of:

[0116] In one embodiment, the first antigen-binding region comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively.

[0117] In one embodiment, the first antigen-binding region comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively.

[0118] In one embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively. Thus, the first antigen-binding region may comprise CDR1, CDR2, and CDR3 of heavy and light chain variable regions having the sequences of the CD40 antibody shown in Table 1.

[0119] Examples of antibodies comprising such a first antigen-binding region are the chimeric antibodies Chi Lob 7 / 4 and CD40-001 disclosed herein.

[0120] In another embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively, with a total of 1 to 12 mutations, e.g., 1 to 11 mutations, 1 to 10 mutations, 1 to 8 mutations, 1 to 7 mutations, 1 to 6 mutations, 1 to 5 mutations, 1 to 4 mutations, 1 to 3 mutations, or 1 to 2 mutations.

[0121] In one embodiment, the mutations may be amino acid substitutions, such as conservative amino acid substitutions.

[0122] In one embodiment, the mutations may be distributed across the CDR1, CDR2, and CDR3 of VH and the CDR1, CDR2, and CDR3 of VL, such that each of the CDR3s of VH and VL contains a maximum of three mutations, and each of the CDR2 and CDR1 of VH and VL contains a maximum of two amino acid mutations.

[0123] In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively, with a total of 1 to 12 mutations, wherein the CDR3 of VH and VL each comprises up to 3 amino acid mutations, and the CDR1 and CDR2 of VH and VL each comprise up to 2 amino acid mutations.

[0124] In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively, with a total of 1 to 10, for example 1 to 8, mutations, wherein the VH and VL CDR1, CDR2, and CDR3 each contain up to 2 amino acid mutations.

[0125] In a further embodiment, the first antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively, with a total of 1 to 6 mutations, wherein the VH and VL CDR1, CDR2, and CDR3 each contain up to 1 amino acid mutation.

[0126] Those skilled in the art are familiar with methods for introducing mutations and the fact that some amino acids in a CDR sequence can be mutated, for example, by amino acid substitution, to, for example, increase the affinity of the antibody for the target antigen, reduce the potential immunogenicity of a non-human antibody used in humans, and / or increase the yield of the antibody expressed by a host cell. Such mutations can be introduced without affecting the target epitope to which the antibody binds.

[0127] In another embodiment, the first antigen-binding region (i) competes for human CD40 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b), and / or (ii) comprises CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that has the specificity for CD40 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b).

[0128] In a further embodiment, the first antigen-binding region comprises a heavy chain variable region and a light chain variable region of an antibody that (i) competes for human CD40 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b), and / or (ii) has the specificity for CD40 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b).

[0129] In this context, the term " compete " refers to the competition between two antibodies for binding to target antigen.If two antibodies do not interfere with each other in binding to target antigen, these antibodies are non-competitive, which indicates that these antibodies do not bind to the same part of target antigen, i.e., epitope.The method for testing the antibody competition for binding to target antigen is well known to those skilled in the art.An example of this method is the so-called cross-competition assay, which can be carried out for example as ELISA or by flow cytometry.

[0130] For example, an ELISA-based assay can be performed by coating ELISA plate wells with each antibody, adding and incubating a competing antibody and the His-tagged extracellular domain of the target antigen, and determining whether the added antibody inhibits the binding of the His-tagged protein to the coated antibody by adding a biotin-labeled anti-His antibody followed by streptavidin-polyHRP, and then developing the reaction with ABTS and measuring the absorbance at 405 nm. For example, a flow cytometry assay can be performed by incubating cells expressing the target antigen with an excess of unlabeled antibody, incubating the cells with a suboptimal concentration of biotin-labeled antibody, followed by incubation with fluorescently labeled streptavidin, and analyzing by flow cytometry.

[0131] In one embodiment, the VH sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NO: 117 and SEQ ID NO: 6, for example, SEQ ID NO: 117.

[0132] In one embodiment, the VL sequence of the first antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to at least one of SEQ ID NO: 121 and SEQ ID NO: 7, for example, SEQ ID NO: 121.

[0133] In one embodiment, the VH and VL sequences of the first antigen-binding region comprise sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0134] In one embodiment, the VH and VL sequences of the first antigen-binding region comprise sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 117 and SEQ ID NO: 121, respectively.

[0135] In one embodiment, the VH and VL sequences differ only in the non-CDR sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0136] In one embodiment, the VH and VL sequences differ only in the non-CDR sequences shown in SEQ ID NO: 117 and SEQ ID NO: 121, respectively.

[0137] In one embodiment, the VH and VL sequences differ only in the framework sequences.

[0138] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of said VH and VL sequences.

[0139] In one embodiment, each of the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to each of the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 7.

[0140] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 7, and the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of the first antigen-binding region have a total of 1 to 12 mutations compared to the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions having the sequences shown in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively. In a further embodiment, the mutations may be as described above.

[0141] In yet another embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 7, and the first antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy chain and light chain variable regions having the sequences shown in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively.

[0142] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 117 and the VL sequence set forth in SEQ ID NO: 121, and the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of the first antigen-binding region have a total of 1 to 12 mutations compared to the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively. In a further embodiment, the mutations may be as described above.

[0143] In yet another embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 117 and the VL sequence set forth in SEQ ID NO: 121, and the first antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy chain and light chain variable regions having the sequences set forth in SEQ ID NOs: 1, 2, 3, 4, YTS, and 5, respectively.

[0144] In one embodiment, the VH sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:117.

[0145] In one embodiment, the VL sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:121.

[0146] In a further embodiment, the VH and VL sequences of the first antigen-binding region comprise the amino acid sequences of SEQ ID NO: 117 and SEQ ID NO: 121, respectively.

[0147] In one embodiment, the VH sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:6.

[0148] In one embodiment, the VL sequence of the first antigen-binding region comprises the amino acid sequence of SEQ ID NO:7.

[0149] In a further embodiment, the VH and VL sequences of the first antigen-binding region comprise the amino acid sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively.

[0150] In one embodiment, a multispecific antibody according to the invention may comprise a first binding arm comprising a first antigen-binding region of any aspect or embodiment herein.

[0151] In one embodiment, the multispecific antibody according to the invention comprises a first binding arm comprising a first antigen-binding region and a first heavy chain constant sequence.

[0152] In one embodiment, a multispecific antibody according to the invention comprises a first binding arm comprising a first antigen-binding region, said first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence.

[0153] In one embodiment, the first light chain further comprises a first light chain constant (CL) sequence.

[0154] In a further embodiment, the first heavy chain comprises at least one of a hinge region, a CH2 region, and a CH3 region.

[0155] In certain embodiments, the multispecific antibody according to the invention comprises a first Fab arm comprising a first antigen-binding region.

[0156] In one embodiment, the first antigen-binding region may be derived from a mouse antibody.

[0157] In one embodiment, the first antigen-binding region may be derived from a chimeric antibody such as Chi Lob7 / 4.

[0158] In one embodiment, the first antigen-binding region may be derived from a humanized antibody.

[0159] In one embodiment, the first binding arm may be derived from a full-length antibody.

[0160] In one embodiment, the first binding arm may be derived from a full-length IgG1, λ (lambda) antibody or an IgG1, κ (kappa) antibody.

[0161] In one embodiment, the first binding arm may be derived from a monoclonal antibody.

[0162] In one embodiment, the first heavy chain may be of the IgG isotype, optionally selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0163] In one embodiment, the first binding arm may be derived from an antibody comprising an HC comprising SEQ ID NO: 118 and an LC comprising SEQ ID NO: 122, optionally comprising one or more mutations in the constant region of the HC, e.g., 1 to 10, e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5 mutations.

[0164] In one embodiment, the first binding arm comprises an HC comprising SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120 and an LC comprising SEQ ID NO: 122.

[0165] Binding to CD137 The multispecific antibody according to the invention comprises a second antigen-binding region that binds to human CD137.

[0166] In a further embodiment, the second antigen-binding region also binds to cynomolgus monkey CD137.

[0167] In one embodiment, the second antigen-binding region comprises a heavy chain variable region CDR3 and a light chain variable region CDR3 selected from the group consisting of: (a) a heavy chain variable region CDR3 having the sequence shown in SEQ ID NO: 10 or a sequence with up to four amino acid alterations in SEQ ID NO: 10, and a light chain variable region CDR3 having the sequence shown in SEQ ID NO: 12 or a sequence with up to four amino acid alterations in SEQ ID NO: 12 (CD137 clone 001); (b) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 17 or a sequence with up to four amino acid alterations in SEQ ID NO: 17, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 19 or a sequence with up to four amino acid alterations in SEQ ID NO: 19 (CD137 clone 002); (c) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 24 or a sequence with up to four amino acid alterations in SEQ ID NO: 24, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 26 or a sequence with up to four amino acid alterations in SEQ ID NO: 26 (CD137 clone 003); (d) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 31 or a sequence with up to four amino acid alterations in SEQ ID NO: 31, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 33 or a sequence with up to four amino acid alterations in SEQ ID NO: 33 (CD137 clone 004); (e) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 38 or a sequence with up to four amino acid alterations in SEQ ID NO: 38, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 40 or a sequence with up to four amino acid alterations in SEQ ID NO: 40 (CD137 clone 005); (f) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 45 or a sequence with up to four amino acid alterations in SEQ ID NO: 45, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 47 or a sequence with up to four amino acid alterations in SEQ ID NO: 47 (CD137 clone 006); (g) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 52 or a sequence with up to four amino acid alterations in SEQ ID NO: 52, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 54 or a sequence with up to four amino acid alterations in SEQ ID NO: 54 (CD137 clone 007); (h) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 59 or a sequence with up to four amino acid alterations in SEQ ID NO: 59, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 61 or a sequence with up to four amino acid alterations in SEQ ID NO: 61 (CD137 clone 008); (i) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 66 or a sequence with up to four amino acid alterations in SEQ ID NO: 66, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 68 or a sequence with up to four amino acid alterations in SEQ ID NO: 68 (CD137 clone 009); (j) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 73 or a sequence with up to four amino acid alterations in SEQ ID NO: 73, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 75 or a sequence with up to four amino acid alterations in SEQ ID NO: 75 (CD137 clone 010); (k) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 80 or a sequence with up to four amino acid alterations in SEQ ID NO: 80, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 82 or a sequence with up to four amino acid alterations in SEQ ID NO: 82 (CD137 clone 011); (l) a heavy chain variable region CDR3 having the sequence set forth in SEQ ID NO: 87 or a sequence with up to four amino acid alterations in SEQ ID NO: 87, and a light chain variable region CDR3 having the sequence set forth in SEQ ID NO: 89 or a sequence with up to four amino acid alterations in SEQ ID NO: 89 (CD137 clone 012); and (m)(i) The heavy chain variable region CDR3 and light chain variable region CDR3 of an antibody that competes for human CD137 binding with an antibody comprising the heavy chain variable region CDR3 and light chain variable region CDR3 of any one of (a) to (l), and / or (ii) has the specificity for CD137 of an antibody comprising the heavy chain variable region CDR3 and light chain variable region CDR3 of any one of (a) to (l).

[0168] In a further embodiment, the second antigen-binding region further comprises CDR1 and CDR2 of a heavy chain region and / or a light chain region selected from the group consisting of: (a) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 8 or a sequence with up to two amino acid alterations in SEQ ID NO: 8, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 9 or a sequence with up to two amino acid alterations in SEQ ID NO: 9; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 11 or a sequence with up to two amino acid alterations in SEQ ID NO: 11, and / or a light chain variable region CDR2 having the sequence KAS or a sequence with up to two amino acid alterations in KAS (CD137 clone 001); (b) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 15 or a sequence with up to two amino acid alterations in SEQ ID NO: 15, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 16 or a sequence with up to two amino acid alterations in SEQ ID NO: 16; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 18 or a sequence with up to two amino acid alterations in SEQ ID NO: 18, and / or a light chain variable region CDR2 having the sequence KAS or a sequence with up to two amino acid alterations in KAS (CD137 clone 002); (c) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 22 or a sequence with up to two amino acid alterations in SEQ ID NO: 22, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 23 or a sequence with up to two amino acid alterations in SEQ ID NO: 23; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 25 or a sequence with up to two amino acid alterations in SEQ ID NO: 25, and / or a light chain variable region CDR2 having the sequence RTS or a sequence with up to two amino acid alterations in RTS (CD137 clone 003); (d) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 29 or a sequence with up to two amino acid alterations in SEQ ID NO: 29, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 30 or a sequence with up to two amino acid alterations in SEQ ID NO: 30; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 32 or a sequence with up to two amino acid alterations in SEQ ID NO: 32, and / or a light chain variable region CDR2 having the sequence GAS or a sequence with up to two amino acid alterations in GAS (CD137 clone 004); (e) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 36 or a sequence with up to two amino acid alterations in SEQ ID NO: 36, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 37 or a sequence with up to two amino acid alterations in SEQ ID NO: 37; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 39 or a sequence with up to two amino acid alterations in SEQ ID NO: 39, and / or a light chain variable region CDR2 having the sequence SAS or a sequence with up to two amino acid alterations in SAS (CD137 clone 005); (f) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 43 or a sequence with up to two amino acid alterations in SEQ ID NO: 43, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 44 or a sequence with up to two amino acid alterations in SEQ ID NO: 44; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 46 or a sequence with up to two amino acid alterations in SEQ ID NO: 46, and / or a light chain variable region CDR2 having the sequence AAS or a sequence with up to two amino acid alterations in AAS (CD137 clone 006); (g) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 50 or a sequence with up to two amino acid alterations in SEQ ID NO: 50, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 51 or a sequence with up to two amino acid alterations in SEQ ID NO: 51; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 53 or a sequence with up to two amino acid alterations in SEQ ID NO: 53, and / or a light chain variable region CDR2 having the sequence KAS or a sequence with up to two amino acid alterations in KAS (CD137 clone 007); (h) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 57 or a sequence with up to two amino acid alterations in SEQ ID NO: 57, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 58 or a sequence with up to two amino acid alterations in SEQ ID NO: 58; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 60 or a sequence with up to two amino acid alterations in SEQ ID NO: 60, and / or a light chain variable region CDR2 having the sequence RAS or a sequence with up to two amino acid alterations in RAS (CD137 clone 008); (i) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 64 or a sequence with up to two amino acid alterations in SEQ ID NO: 64, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 65 or a sequence with up to two amino acid alterations in SEQ ID NO: 65; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 67 or a sequence with up to two amino acid alterations in SEQ ID NO: 67, and / or a light chain variable region CDR2 having the sequence GAS or a sequence with up to two amino acid alterations in GAS (CD137 clone 009); (j) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 71 or a sequence with up to two amino acid alterations in SEQ ID NO: 71, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 72 or a sequence with up to two amino acid alterations in SEQ ID NO: 72; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 74 or a sequence with up to two amino acid alterations in SEQ ID NO: 74, and / or a light chain variable region CDR2 having the sequence KAS or a sequence with up to two amino acid alterations in KAS (CD137 clone 010); (k) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 78 or a sequence with up to two amino acid alterations in SEQ ID NO: 78, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 79 or a sequence with up to two amino acid alterations in SEQ ID NO: 79; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 81 or a sequence with up to two amino acid alterations in SEQ ID NO: 81, and / or a light chain variable region CDR2 having the sequence DTS or a sequence with up to two amino acid alterations in DTS (CD137 clone 011); (l) a heavy chain variable region CDR1 having the sequence shown in SEQ ID NO: 85 or a sequence with up to two amino acid alterations in SEQ ID NO: 85, and / or a heavy chain variable region CDR2 having the sequence shown in SEQ ID NO: 86 or a sequence with up to two amino acid alterations in SEQ ID NO: 86; and / or a light chain variable region CDR1 having the sequence shown in SEQ ID NO: 88 or a sequence with up to two amino acid alterations in SEQ ID NO: 88, and / or a light chain variable region CDR2 having the sequence SAS or a sequence with up to two amino acid alterations in SAS (CD137 clone 012).

[0169] In one embodiment, the second antigen-binding region comprises CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 11, KAS, and SEQ ID NO: 12, respectively (CD137 clone 001); (b) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 15, 16, and 17, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 18, KAS, and SEQ ID NO: 19, respectively (CD137 clone 002); (c) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 22, 23, and 24, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 25, RTS, and SEQ ID NO: 26, respectively (CD137 clone 003); (d) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 29, 30, and 31, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 32, GAS, and SEQ ID NO: 33, respectively (CD137 clone 004); (e) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); (f) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 43, 44, and 45, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 46, AAS, and SEQ ID NO: 47, respectively (CD137 clone 006); (g) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 50, 51, and 52, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 53, KAS, and SEQ ID NO: 54, respectively (CD137 clone 007); (h) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 57, 58, and 59, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 60, 61, and 62, respectively (CD137 clone 008); (i) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009); (j) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 71, 72, and 73, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 74, KAS, and SEQ ID NO: 75, respectively (CD137 clone 010); (k) a heavy chain variable region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 78, 79, and 80, respectively, and a light chain variable region CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 81, DTS, and 82, respectively (CD137 clone 011); (l) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 85, 86, and 87, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 88, SAS, and 89, respectively (CD137 clone 012); (m) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region according to any one of (a) to (l), which have a total of 1 to 12 mutations; and (n)(i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in any of (a) to (m), and / or (ii) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that has the specificity for CD137 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in any of (a) to (m).

[0170] Thus, the second antigen-binding region may comprise the CDR1, CDR2, and CDR3 sequences of the heavy and light chain variable regions of a CD137 antibody shown in Table 1, i.e., CD137 clone 001, CD137 clone 002, CD137 clone 003, CD137 clone 004, CD137 clone 005, CD137 clone 006, CD137 clone 007, CD137 clone 008, CD137 clone 009, CD137 clone 010, CD137 clone 011, or CD137 clone 012. In particular, the second antigen-binding region may comprise the CDR1, CDR2, and CDR3 sequences of the heavy and light chain variable regions from the same CD137 antibody clone, optionally wherein the framework regions are predominantly human-derived framework regions optionally containing one or more amino acid backmutations to non-human amino acid sequences.

[0171] In a further embodiment, the second antigen-binding region comprises the heavy and light chain variable regions of an antibody that (i) competes for human CD137 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy and light chain variable regions described in any of (a) to (m), and / or (ii) has the specificity for CD137 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy and light chain variable regions described in any of (a) to (m).

[0172] In one embodiment, the second antigen-binding region binds to human CD137 (SEQ ID NO: 92) more strongly than it binds to mutant human CD137 (SEQ ID NO: 93). The mutant human CD137 of SEQ ID NO: 93 is also referred to herein as shuffle 6.

[0173] In another embodiment, the second antigen-binding region binds to human CD137 (SEQ ID NO: 92) more strongly than it binds to mutant human CD137 (SEQ ID NO: 94). The mutant human CD137 of SEQ ID NO: 94 is also referred to herein as shuffle 5.

[0174] In a further embodiment, the second antigen-binding region binds to human CD137 (SEQ ID NO: 92) as strongly as it binds to mutant human CD137 (SEQ ID NO: 95). The mutant human CD137 of SEQ ID NO: 95 is also referred to herein as shuffle 4.

[0175] In the present invention, "stronger" means that the affinity of the second antigen-binding region is stronger for human CD137 (SEQ ID NO: 92) than for mutant human CD137 (SEQ ID NO: 93 and SEQ ID NO: 94, shuffle 6 and shuffle 5, respectively). In the absence of binding to mutant CD137, the affinity for human CD137 is considered to be much stronger than for mutant CD137. However, in the presence of binding to mutant CD137, the affinity for human CD137 may be two-fold, for example, three-fold, four-fold, five-fold, or six-fold greater than the affinity for each mutant CD137.

[0176] In the present invention, "comparably strong" means that the affinity of the second antigen-binding region is similar for human CD137 (SEQ ID NO: 92) and for mutant human CD137 (SEQ ID NO: 95, shuffle 4). Specifically, "similar" in this context may mean that the difference between the affinity for human CD137 and the affinity for mutant CD137 is at most 2.5-fold, e.g., 2.2-fold, or 2.0-fold, or 1.8-fold, or 1.75-fold, or 1.5-fold.

[0177] The mutant human CD137 of SEQ ID NO: 93 corresponds to the amino acid sequence of human CD137 in which amino acids 24 to 47 (shuffle 6) have been replaced with the corresponding amino acids from boar CD137.

[0178] Thus, in one embodiment, the second antigen-binding region binds to an epitope of human CD137 that includes or requires one or more of the amino acids L, Q, D, P, C, S, N, C, P, A, G, T, F, C, D, N, N, R, N, Q, I, C, S, and P at positions 24-47 of SEQ ID NO: 92 (corresponding to SEQ ID NO: 129).

[0179] The mutant human CD137 of SEQ ID NO: 94 corresponds to the amino acid sequence of human CD137 in which amino acids 48 to 88 (shuffle 5) have been replaced with the corresponding amino acids from African elephant CD137.

[0180] Thus, in one embodiment, the second antigen-binding region binds to an epitope of human CD137 that includes or requires one or more of the amino acids C, P, P, N, S, F, S, S, A, G, G, Q, R, T, C, D, I, C, R, Q, C, K, G, V, F, R, T, R, K, E, C, S, S, T, S, N, A, E, C, D, and C at positions 48-88 of SEQ ID NO: 92 (corresponding to SEQ ID NO: 130).

[0181] SEQ ID NO: 95, a mutant human CD137, corresponds to the amino acid sequence of human CD137 in which amino acids 59 to 114 (shuffle 4) have been replaced with the corresponding amino acids from African elephant CD137.

[0182] Thus, in one embodiment, the second antigen-binding region does not bind to an epitope of human CD137 that includes or requires one or more of the amino acids T, P, G, F, H, C, L, G, A, G, C, S, M, C, E, Q, D, C, K, Q, G, Q, E, L, T, and K at positions 89 to 114 of SEQ ID NO: 92 (corresponding to SEQ ID NO: 131).

[0183] In one embodiment, binding to mutant CD137 and human CD137 may be performed as a shuffle assay as described in Example 2. That is, shuffle constructs derived from human CD137, in which protein domains of human CD137 are replaced with corresponding domains of CD137 from different species, may be generated, and human CD137 and CD137 from different species may be used as reference constructs to transduce cells with plasmids encoding the reference constructs or shuffle constructs, respectively, and binding of antibodies to each of these CD137 constructs may be measured by flow cytometry, e.g., FACS, to human CD137 (SEQ ID NO: 92) and mutant human CD137 (SEQ ID NOs: 93, 94, and 95).

[0184] If binding to a certain shuffled construct is lost, it indicates that the corresponding region is likely involved in the antibody epitope. Thus, the protein domains of human CD137 that contribute to the epitope of an anti-human CD137 antibody can be determined by shuffle assays. The CD137 proteins from different species used to create shuffled constructs should be selected so that monoclonal anti-human CD137 antibodies do not bind to the entire CD137 protein from these different species (reference constructs).

[0185] Measurement of binding to human CD137 and its variants can specifically be carried out using a monoclonal antibody comprising two second antigen-binding regions according to the present invention.

[0186] In one embodiment, the second antigen-binding region comprises CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 8, 9, and 10, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 11, KAS, and SEQ ID NO: 12, respectively (CD137 clone 001); (b) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 15, 16, and 17, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 18, KAS, and SEQ ID NO: 19, respectively (CD137 clone 002); (c) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); (d) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 43, 44, and 45, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 46, AAS, and SEQ ID NO: 47, respectively (CD137 clone 006); (e) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009); (f) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 71, 72, and 73, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 74, KAS, and SEQ ID NO: 75, respectively (CD137 clone 010); (g) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 85, 86, and 87, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 88, SAS, and SEQ ID NO: 89, respectively (CD137 clone 012); (h) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region according to any one of (a) to (g), which have a total of 1 to 12 mutations; and (i)(i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in any of (a) to (h), and / or (ii) has the specificity for CD137 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in any of (a) to (h).

[0187] Thus, in one embodiment, the second antigen-binding region comprises CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009); (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a), each having a total of 1 to 12 mutations; (c) (i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b), and / or (ii) has the specificity for CD137 of an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b).

[0188] In another embodiment, the second antigen-binding region comprises CDR1, CDR2, and CDR3 of a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a), each having a total of 1 to 12 mutations; and (c) (i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b), and / or (ii) has the specificity for CD137 of an antibody comprising the CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a) or (b).

[0189] In certain embodiments, the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively. Examples of such antibodies include, but are not limited to, the antibody referred to herein as CD137 clone 009.

[0190] In another embodiment, the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively. Examples of such antibodies include, but are not limited to, the antibody referred to herein as CD137 clone 005.

[0191] In another embodiment, the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively, with a total of 1 to 12 mutations, e.g., 1 to 10 mutations, or 1 to 8 mutations, or 1 to 6 mutations, or 1 to 4 mutations, or up to 2 mutations (CD137 clone 009).

[0192] In another embodiment, the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively, with a total of 1 to 12 mutations, for example, 1 to 10 mutations, or 1 to 8 mutations, or 1 to 6 mutations, or 1 to 4 mutations, or 1 to 2 mutations (CD137 clone 005).

[0193] In one embodiment, the mutations may be amino acid substitutions, such as conservative amino acid substitutions.

[0194] In one embodiment, the mutations may be distributed across the CDR1, CDR2, and CDR3 of VH and the CDR1, CDR2, and CDR3 of VL, such that each of the CDR3s of VH and VL contains a maximum of three mutations, and each of the CDR2 and CDR1 of VH and VL contains a maximum of two amino acid modifications.

[0195] Thus, in a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively, with a total of 1 to 12 mutations (CD137 clone 009), wherein the VH and VL CDR3 each comprises up to three amino acid alterations, and the VH and VL CDR1 and CDR2 each comprise up to two amino acid alterations.

[0196] In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively, with a total of 1 to 10, e.g., 1 to 8, mutations (CD137 clone 009), wherein the VH and VL CDR1, CDR2, and CDR3 each contain up to 2 amino acid modifications.

[0197] In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively, with a total of 1 to 6 mutations (CD137 clone 009), wherein the VH and VL CDR1, CDR2, and CDR3 each contain up to 1 amino acid alteration.

[0198] In another embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, 38, 39, SAS, and 40, respectively, with a total of 1 to 12 mutations (CD137 clone 005), wherein CDR3 of VH and VL each comprises up to 3 amino acid alterations, and CDR1 and CDR2 of VH and VL each comprise up to 2 amino acid alterations.

[0199] In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, 38, 39, SAS, and 40, respectively, with a total of 1 to 10, e.g., 1 to 8, mutations (CD137 clone 005), wherein VH and VL CDR1, CDR2, and CDR3 each comprise up to 2 amino acid alterations.

[0200] In a further embodiment, the second antigen-binding region comprises heavy and light chain CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, 38, 39, SAS, and 40, respectively, with a total of 1 to 6 mutations (CD137 clone 005), wherein the VH and VL CDR1, CDR2, and CDR3 each contain up to 1 amino acid alteration.

[0201] In further embodiments, there may be a total of 1 to 12 mutations, for example 1 to 10 mutations, or 1 to 8 mutations, or 1 to 6 mutations, or 1 to 4 mutations, or 1 to 2 mutations; and each CDR sequence contains a maximum of 2 amino acid substitutions.

[0202] Those skilled in the art are familiar with methods for introducing mutations, and that some amino acids in CDR sequences can be mutated, for example, by amino acid substitution, to, for example, increase the affinity of the antibody for the target antigen or reduce the immunogenicity of non-human antibodies used in human therapy. Such mutations can be introduced without affecting the epitope of the target antigen to which the antibody binds.

[0203] In one embodiment, the second antigen-binding region comprises a second heavy chain variable (VH) sequence and a second light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively.

[0204] In one embodiment, the second antigen-binding region comprises a second heavy chain variable (VH) sequence and a second light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively.

[0205] In one embodiment, the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VH sequence shown in SEQ ID NO: 123 (humanized CD137 clone 009) (b) VH sequence shown in SEQ ID NO: 13 (CD137 clone 001) (c) VH sequence shown in SEQ ID NO: 20 (CD137 clone 002) (d) VH sequence shown in SEQ ID NO: 27 (CD137 clone 003) (e) VH sequence shown in SEQ ID NO: 34 (CD137 clone 004) (f) VH sequence shown in SEQ ID NO: 41 (CD137 clone 005) (g) VH sequence shown in SEQ ID NO: 48 (CD137 clone 006) (h) VH sequence shown in SEQ ID NO: 55 (CD137 clone 007) (i) VH sequence shown in SEQ ID NO: 62 (CD137 clone 008) (j) VH sequence shown in SEQ ID NO: 69 (CD137 clone 009) (k) VH sequence shown in SEQ ID NO: 76 (CD137 clone 010) (l) VH sequence shown in SEQ ID NO: 83 (CD137 clone 011) (m) VH sequence shown in SEQ ID NO: 90 (CD137 clone 012).

[0206] In one embodiment, the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VL sequence shown in SEQ ID NO: 127 (humanized CD137 clone 009) (b) VL sequence shown in SEQ ID NO: 14 (CD137 clone 001) (c) VL sequence shown in SEQ ID NO: 21 (CD137 clone 002) (d) VL sequence shown in SEQ ID NO: 28 (CD137 clone 003) (e) VL sequence shown in SEQ ID NO: 35 (CD137 clone 004) (f) VL sequence shown in SEQ ID NO: 42 (CD137 clone 005) (g) VL sequence shown in SEQ ID NO: 49 (CD137 clone 006) (h) VL sequence shown in SEQ ID NO: 56 (CD137 clone 007) (i) VL sequence shown in SEQ ID NO: 63 (CD137 clone 008) (j) VL sequence shown in SEQ ID NO: 70 (CD137 clone 009) (k) VL sequence shown in SEQ ID NO: 77 (CD137 clone 010) (l) VL sequence shown in SEQ ID NO: 84 (CD137 clone 011) (m) VL sequence shown in SEQ ID NO: 91 (CD137 clone 012).

[0207] In one embodiment, the VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VH sequence shown in SEQ ID NO: 123 and VL sequence shown in SEQ ID NO: 127 (humanized CD137 clone 009) (b) VH sequence shown in SEQ ID NO: 13 and VL sequence shown in SEQ ID NO: 14 (CD137 clone 001) (c) VH sequence shown in SEQ ID NO: 20 and VL sequence shown in SEQ ID NO: 21 (CD137 clone 002) (d) VH sequence shown in SEQ ID NO: 27 and VL sequence shown in SEQ ID NO: 28 (CD137 clone 003) (e) VH sequence shown in SEQ ID NO: 34 and VL sequence shown in SEQ ID NO: 35 (CD137 clone 004) (f) VH sequence shown in SEQ ID NO: 41 and VL sequence shown in SEQ ID NO: 42 (CD137 clone 005) (g) VH sequence shown in SEQ ID NO: 48 and VL sequence shown in SEQ ID NO: 49 (CD137 clone 006) (h) VH sequence shown in SEQ ID NO: 55 and VL sequence shown in SEQ ID NO: 56 (CD137 clone 007) (i) the VH sequence shown in SEQ ID NO: 62 and the VL sequence shown in SEQ ID NO: 63 (CD137 clone 008) (j) VH sequence shown in SEQ ID NO: 69 and VL sequence shown in SEQ ID NO: 70 (CD137 clone 009) (k) the VH sequence shown in SEQ ID NO: 76 and the VL sequence shown in SEQ ID NO: 77 (CD137 clone 010) (l) VH sequence shown in SEQ ID NO: 83 and VL sequence shown in SEQ ID NO: 84 (CD137 clone 011) (m) VH sequence shown in SEQ ID NO: 90 and VL sequence shown in SEQ ID NO: 91 (CD137 clone 012).

[0208] In one embodiment, the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VH sequence shown in SEQ ID NO: 123 (humanized CD137 clone 009) (b) VH sequence shown in SEQ ID NO: 41 (CD137 clone 005) (c) VH sequence shown in SEQ ID NO: 69 (CD137 clone 009).

[0209] In one embodiment, the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VL sequence shown in SEQ ID NO: 127 (humanized CD137 clone 009) (b) VL sequence shown in SEQ ID NO: 42 (CD137 clone 005) (c) VL sequence shown in SEQ ID NO: 70 (CD137 clone 009).

[0210] In one embodiment, the VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to an amino acid sequence selected from the group consisting of: (a) VH sequence shown in SEQ ID NO: 123 and VL sequence shown in SEQ ID NO: 127 (humanized CD137 clone 009) (b) VH sequence shown in SEQ ID NO: 41 and VL sequence shown in SEQ ID NO: 42 (CD137 clone 005) (c) VH sequence shown in SEQ ID NO: 69 and VL sequence shown in SEQ ID NO: 70 (CD137 clone 009).

[0211] In one embodiment, the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 41 (CD137 clone 005).

[0212] In one embodiment, the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 69 (CD137 clone 009).

[0213] In one embodiment, the VH sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 123 (humanized CD137 clone 009).

[0214] In one embodiment, the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 42 (CD137 clone 005).

[0215] In one embodiment, the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 70 (CD137 clone 009).

[0216] In one embodiment, the VL sequence of the second antigen-binding region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 127 (humanized CD137 clone 009).

[0217] In one embodiment, the VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 41 and SEQ ID NO: 42 (CD137 clone 005), respectively.

[0218] In one embodiment, the VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identity to SEQ ID NO: 69 and SEQ ID NO: 70 (CD137 clone 009), respectively.

[0219] In one embodiment, the VH and VL sequences of the second antigen-binding region each comprise an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to SEQ ID NO: 123 and SEQ ID NO: 127 (humanized CD137 clone 009), respectively.

[0220] In one embodiment, the VH and VL sequences differ only in the framework sequences.

[0221] In one embodiment, the respective FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first and / or second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3, and FR4 framework sequences of said VH and VL sequences.

[0222] In one embodiment, the VH and VL sequences differ only in the non-CDR sequences shown in SEQ ID NO: 41 and SEQ ID NO: 42 (CD137 clone 005), respectively.

[0223] In one embodiment, the VH and VL sequences differ only in the non-CDR sequences shown in SEQ ID NO: 69 and SEQ ID NO: 70 (CD137 clone 009), respectively.

[0224] In one embodiment, the VH and VL sequences differ only in the non-CDR sequences shown in SEQ ID NO: 123 and SEQ ID NO: 127 (humanized CD137 clone 009), respectively.

[0225] In one embodiment, the VH and VL sequences differ only in the framework sequences.

[0226] In one embodiment, the respective FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the first and / or second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the respective FR1, FR2, FR3, and FR4 framework sequences of said VH and VL sequences.

[0227] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 41 and the VL sequence shown in SEQ ID NO: 42.

[0228] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 41 and the VL sequence shown in SEQ ID NO: 42, and the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of the second antigen-binding region have a total of 1 to 12 mutations compared to the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions having the sequences shown in SEQ ID NOs: 36, 37, 38, 39, SAS, and 40, respectively. In a further embodiment, the mutations may be as described above.

[0229] In yet another embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 41 and the VL sequence set forth in SEQ ID NO: 42, and the second antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy chain and light chain variable regions having the sequences set forth in SEQ ID NOs: 36, 37, 38, 39, SAS, and 40, respectively.

[0230] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 69 and the VL sequence shown in SEQ ID NO: 70.

[0231] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 69 and the VL sequence set forth in SEQ ID NO: 70, and the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of the second antigen-binding region have a total of 1 to 12 mutations compared to the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively. In a further embodiment, the mutations may be as described above.

[0232] In yet another embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 69 and the VL sequence set forth in SEQ ID NO: 70, and the second antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy chain and light chain variable regions having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively.

[0233] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence shown in SEQ ID NO: 123 and the VL sequence shown in SEQ ID NO: 127.

[0234] In one embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 123 and the VL sequence set forth in SEQ ID NO: 127, and the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of the second antigen-binding region have a total of 1 to 12 mutations compared to the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively. In a further embodiment, the mutations may be as described above.

[0235] In yet another embodiment, the FR1, FR2, FR3, and FR4 framework sequences of the VH and VL sequences of the second antigen-binding region have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the FR1, FR2, FR3, and FR4 framework sequences of the VH sequence set forth in SEQ ID NO: 123 and the VL sequence set forth in SEQ ID NO: 127, and the second antigen-binding region comprises CDR1, CDR2, and CDR3 of heavy chain and light chain variable regions having the sequences set forth in SEQ ID NOs: 64, 65, 66, 67, GAS, and 68, respectively.

[0236] In one embodiment, the VH sequence of the second antigen-binding region comprises SEQ ID NO: 123 (humanized CD137 clone 009).

[0237] In one embodiment, the VL sequence of the second antigen-binding region comprises SEQ ID NO: 127 (humanized CD137 clone 009).

[0238] In one embodiment, the VH and VL sequences of the second antigen-binding region comprise SEQ ID NO: 123 and SEQ ID NO: 127, respectively.

[0239] In one embodiment, the VH sequence of the second antigen-binding region is (a) SEQ ID NO: 41 (CD137 clone 005) (b) SEQ ID NO: 69 (CD137 clone 009) The VH sequence is selected from the group consisting of:

[0240] In one embodiment, the VL sequence of the second antigen-binding region is (a) SEQ ID NO: 42 (CD137 clone 005) (b) SEQ ID NO: 70 (CD137 clone 009) The VL sequence is selected from the group consisting of:

[0241] In one embodiment, the VH and VL sequences of the second antigen-binding region are (a) the VH sequence shown in SEQ ID NO: 41 and the VL sequence shown in SEQ ID NO: 42 (CD137 clone 005); (b) VH sequence shown in SEQ ID NO: 69 and VL sequence shown in SEQ ID NO: 70 (CD137 clone 009) is selected from the group consisting of:

[0242] In one embodiment, the multispecific antibody according to the invention comprises a second binding arm comprising a second antigen-binding region.

[0243] In one embodiment, the multispecific antibody according to the invention comprises a second binding arm comprising a second antigen-binding region and a second heavy chain constant sequence.

[0244] In one embodiment, a multispecific antibody according to the invention comprises a second binding arm comprising a second antigen-binding region, said second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, and a second light chain comprising a second light chain variable (VL) sequence.

[0245] In one embodiment, the second light chain further comprises a second light chain constant sequence.

[0246] In a further embodiment, the second heavy chain comprises at least one of a hinge region, a CH2 region, and a CH3 region.

[0247] In certain embodiments, the multispecific antibody according to the invention comprises a second Fab arm comprising a second antigen-binding region.

[0248] In one embodiment, the second antigen-binding region is derived from a rabbit antibody, such as any of the anti-CD137 clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 disclosed herein, particularly clones 5 and 9.

[0249] In one embodiment, the second antigen-binding region is derived from a chimeric antibody, such as an antibody comprising a variable region derived from any of the anti-CD137 clones 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 disclosed herein, particularly any of clones 5 and 9.

[0250] In one embodiment, the second antigen-binding region is derived from a humanized antibody.

[0251] In one embodiment, the second binding arm is derived from a full-length antibody.

[0252] In one embodiment, the second binding arm is derived from a full-length IgG1, λ (lambda) antibody or an IgG1, κ (kappa) antibody.

[0253] In one embodiment, the second binding arm is derived from a monoclonal antibody.

[0254] In one embodiment, the second heavy chain is of the IgG isotype, optionally having a subclass from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0255] In one embodiment, the first binding arm may be derived from an antibody comprising an HC comprising SEQ ID NO: 124 and an LC comprising SEQ ID NO: 128, optionally comprising one or more mutations in the constant region of the HC, e.g., 1 to 10, e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5 mutations.

[0256] In one embodiment, the first binding arm comprises an HC comprising SEQ ID NO: 124, 125, or 126 and an LC comprising SEQ ID NO: 128.

[0257] Binding to CD40 and CD137 In some embodiments, the present invention relates to a multispecific antibody comprising: (I) a first antigen-binding region that binds to human CD40, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in a), which have a total of 1 to 12 mutations; and (c) (i) CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of an antibody that competes for human CD40 binding with an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and / or (ii) has the specificity for CD40 of an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and (II) a second antigen-binding region that binds to human CD137, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (x) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009); (y) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in x), which have a total of 1 to 12 mutations; and (z) (i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (x) or (y), and / or (ii) has the specificity for CD137 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (x) or (y).

[0258] In another aspect, the present invention relates to a multispecific antibody comprising: (I) a first antigen-binding region that binds to human CD40, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (a) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; (b) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (a), each having a total of 1 to 12 mutations; and (c) (i) CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of an antibody that competes for human CD40 binding with an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and / or (ii) has the specificity for CD40 of an antibody comprising the CDR1, CDR2, and CDR3 of the heavy and light chain variable regions of (a) or (b), and (II) a second antigen-binding region that binds to human CD137, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of: (x) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); (y) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (x), which have a total of 1 to 12 mutations; and (z) (i) CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region of an antibody that competes for human CD137 binding with an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (x) or (y), and / or (ii) has the specificity for CD137 of an antibody comprising CDR1, CDR2, and CDR3 of the heavy chain variable region and light chain variable region described in (x) or (y).

[0259] Thus, in one embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively, and the second antigen-binding region comprises (a) a heavy chain variable region CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and a light chain variable region CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD136 clone 009); or (b) heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences shown in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); Includes:

[0260] In another embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009).

[0261] In another embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005).

[0262] In a further embodiment, the first antigen-binding region of the multispecific antibody according to the invention comprises a first heavy chain variable (VH) sequence and a first light chain variable (VL) sequence, and the second antigen-binding region of the multispecific antibody according to the invention comprises a second heavy chain variable (VH) sequence and a second light chain variable (VL) sequence, each variable sequence comprising three CDR sequences, CDR1, CDR2 and CDR3 respectively, and four framework sequences, FR1, FR2, FR3 and FR4 respectively.

[0263] In a further embodiment, the VH and VL sequences of the first antigen-binding region comprise sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequences of the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 7, respectively; and the VH and VL sequences of the second antigen-binding region comprise sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequences of the VH sequence shown in SEQ ID NO: 41 and the VL sequence shown in SEQ ID NO: 42 (CD137 clone 005), respectively.

[0264] In another further embodiment, the VH and VL sequences of the first antigen-binding region comprise sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequences of the VH sequence shown in SEQ ID NO: 6 and the VL sequence shown in SEQ ID NO: 7, respectively; and the VH and VL sequences of the second antigen-binding region comprise sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to the amino acid sequences of the VH sequence shown in SEQ ID NO: 69 and the VL sequence shown in SEQ ID NO: 70 (CD137 clone 009), respectively.

[0265] In another further embodiment, the VH and VL sequences of the first antigen-binding region comprise sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence shown in SEQ ID NO: 117 and the VL sequence shown in SEQ ID NO: 121, respectively; and the VH and VL sequences of the second antigen-binding region comprise sequences that have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence shown in SEQ ID NO: 123 and the VL sequence shown in SEQ ID NO: 127 (humanized CD137 clone 009), respectively.

[0266] In a particular embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first heavy chain variable sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the first light chain sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and (II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second heavy chain variable sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and the second light chain sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NO: 68, respectively (CD137 clone 009); wherein the first heavy chain and the second heavy chain are of the human IgG1 isotype, the first light chain and the second light chain are of IgG1,κ, and the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering for both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and (a) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is R; or (b) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is L.

[0267] In a further particular embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first heavy chain variable sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the first light chain sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and (II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second heavy chain variable sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively, and the second light chain sequence comprises CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 39, SAS, and SEQ ID NO: 40, respectively (CD137 clone 005); wherein the first heavy chain and the second heavy chain are of the human IgG1 isotype, the first light chain and the second light chain are of IgG1,κ, and the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering for both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and (a) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is R; or (b) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is L.

[0268] In a particular embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first VH sequence and VL sequence have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence set forth in SEQ ID NO: 117 and the VL sequence set forth in SEQ ID NO: 121, respectively; and (II) A second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second VH sequence and VL sequence comprise sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence set forth in SEQ ID NO: 123 and the VL sequence set forth in SEQ ID NO: 127 (humanized CD137 clone 009), respectively.

[0269] In a further specific embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first VH sequence comprises SEQ ID NO: 117 and the first VL sequence comprises SEQ ID NO: 121; and (II) A second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second VH sequence comprises SEQ ID NO: 123 and the second VL sequence comprises SEQ ID NO: 127 (humanized CD137 clone 009).

[0270] In a particular embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first VH sequence and VL sequence comprise sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence set forth in SEQ ID NO: 117 and the VL sequence set forth in SEQ ID NO: 121, respectively; and (II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second VH sequence and VL sequence comprise sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identity to the amino acid sequences of the VH sequence set forth in SEQ ID NO: 123 and the VL sequence set forth in SEQ ID NO: 127 (humanized CD137 clone 009), respectively; wherein the first heavy chain and the second heavy chain are of the human IgG1 isotype, the first light chain and the second light chain are of IgG1,κ, and the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering for both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and (a) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is R; or (b) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is L.

[0271] In a further specific embodiment, the present invention relates to a bispecific antibody comprising: (I) a first binding arm comprising a first heavy chain comprising a first heavy chain variable (VH) sequence and a first heavy chain constant (CH) sequence, and a first light chain comprising a first light chain variable (VL) sequence and a first light chain constant (CL) sequence, wherein the first VH sequence comprises SEQ ID NO: 117 and the first VL sequence comprises SEQ ID NO: 121; and (II) a second binding arm comprising a second heavy chain comprising a second heavy chain variable (VH) sequence and a second heavy chain constant (CH) sequence, wherein the second light chain further comprises a second light chain constant (CL) sequence and a second light chain variable (VL) sequence, wherein the second VH sequence comprises SEQ ID NO: 123 and the second VL sequence comprises SEQ ID NO: 127 (humanized CD137 clone 009); wherein the first heavy chain and the second heavy chain are of the human IgG1 isotype, the first light chain and the second light chain are of IgG1,κ, and the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering for both the first constant heavy chain and the second constant heavy chain are F, E, and A, respectively, and (a) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is R; or (b) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the first constant heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the second constant heavy chain is L.

[0272] In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm that binds to human CD40 and a second binding arm that binds to human CD137, wherein: (i) the first binding arm comprises a heavy chain (HC) amino acid sequence comprising or consisting of SEQ ID NO: 118 and a light chain (LC) amino acid sequence comprising or consisting of SEQ ID NO: 122; and (ii) the second binding arm comprises an HC amino acid sequence that includes or consists of SEQ ID NO: 124 and an LC amino acid sequence that includes or consists of SEQ ID NO: 128; Optionally, SEQ ID NO: 118, SEQ ID NO: 124, or both, include one or more mutations in the constant region of the HC, e.g., 1 to 10, e.g., 1 to 5, e.g., 1, 2, 3, 4, or 5 mutations.

[0273] In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm that binds to human CD40 and a second binding arm that binds to human CD137, wherein: (i) the first binding arm comprises an HC amino acid sequence that includes or consists of SEQ ID NO: 119 and an LC amino acid sequence that includes or consists of SEQ ID NO: 122; and (ii) the second binding arm comprises an HC amino acid sequence comprising or consisting of SEQ ID NO:125 and an LC amino acid sequence comprising or consisting of SEQ ID NO:128;

[0274] In another aspect, the present invention relates to a bispecific antibody comprising a first binding arm that binds to human CD40 and a second binding arm that binds to human CD137, wherein: (i) the first binding arm comprises an HC amino acid sequence that includes or consists of SEQ ID NO: 120 and an LC amino acid sequence that includes or consists of SEQ ID NO: 122; and (ii) the second binding arm comprises an HC amino acid sequence comprising or consisting of SEQ ID NO:126 and an LC amino acid sequence comprising or consisting of SEQ ID NO:128;

[0275] Bispecific morphology In a particular embodiment, the multispecific antibody according to the invention is a bispecific antibody.

[0276] The present invention provides a bispecific CD40xCD137 antibody that can crosslink cells expressing CD40 and cells expressing CD137, such as antigen-presenting cells and T cells, respectively. A specific antigen-binding region can be selected from the range of antibodies or antigen-binding regions provided by the present invention depending on the functional properties desired for a particular application. Many different forms and uses of bispecific antibodies are known in the art and are reviewed in Kontermann; Drug Discovery Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

[0277] Bispecific antibodies according to the invention are not limited to any particular bispecific form or method of making them.

[0278] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody with two binding arms comprising different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, e.g., via two scFvs linked in tandem by an added peptide linker; and (iii) a dual variable domain antibody (DVD-Ig), in which each light and heavy chain comprises two variable domains tandemly linked by a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iv) chemically linked bispecific (Fab') fragments; (v) Tandabs, which are fusions of two single-chain diabodies, resulting in tetravalent bispecific antibodies with two binding sites for each target antigen; (vi) Flexibodies, which are combinations of scFvs and diabodies, resulting in multivalent molecules; (vii) so-called "dock-and-lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when added to Fab, can generate trivalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called Scorpion molecules, which, for example, contain two scFvs fused to both ends of human Fab arms; and (ix) diabodies.

[0279] In one embodiment, the bispecific antibody of the invention is a diabody, crossbody, or bispecific antibody obtained by directed Fab arm exchange (as described in WO2011131746 (Genmab)).

[0280] Examples of different classes of bispecific antibodies include: (i) IgG-like molecules with complementary CH3 domains that force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, in which the two sides of the molecule each comprise an Fab fragment or a portion of an Fab fragment of at least two different antibodies; (iii) IgG-fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or a portion of an Fab fragment; (iv) Fc-fusion molecules, in which a single-chain Fv molecule or stabilized diabody is fused to the constant domain of a heavy chain, an Fc region, or a portion thereof; (v) different These include, but are not limited to, Fab fusion molecules in which Fab fragments are fused together and fused to a heavy chain constant domain, Fc region, or portion thereof; and (vi) ScFv-based and diabody-based antibodies and heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, Fc region, or portion thereof.

[0281] Examples of IgG-like molecules with complementary CH3 domain molecules include triomab / quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called knob-into-hole molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body molecules and PIG body molecules (Pharmabcine, WO2010134666, WO2014081202), strand-exchange engineered domain body (SEEDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), azimetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254), and DuoBody® molecules (Genmab A / S, WO2011131746).

[0282] Examples of recombinant IgG-like dual targeting molecules include dual-targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al. 2009. Science 323, 1610-1614.), cross-linked Mab (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17,501-506.)

[0283] Examples of IgG fusion molecules include dual variable domain (DVD)-Ig molecules (Abbott, US 7,612,181), dual-domain double-head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), as well as BsAb molecules (Zymogenetics, WO2010111625), HERCULES molecules (Biogen Idec, US 007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246), and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0284] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), dual affinity retargeting technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538), and dual (ScFv)2-Fab molecules (National Research Center of Antibody Medicine (China)).

[0285] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al. J. Immunol. 1998 Feb 15;160(4):1677-86), dual-action or bis-Fab molecules (Genentech, Bostrom, et al. 2009. Science 323, 1610-1614), dock-and-lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J. Immunol. 2000 Dec 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).

[0286] Examples of ScFv-based antibodies, diabody-based antibodies, and domain antibodies include bispecific T cell-triggering (BiTE) molecules (Micromet, WO2005061547), tandem diabody molecules (TandAb) (Affimed Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), dual affinity retargeting technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), single-chain diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack, WO2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual-targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010) and dual-targeting heavy chain-only domain antibodies.

[0287] In one embodiment, each of the first and second heavy chains comprises at least one hinge region, a CH2 region, and a CH3 region.

[0288] In further embodiments, the CH3 regions of the first heavy chain and the second heavy chain comprise asymmetric mutations, eg, asymmetric mutations (also referred to herein as modifications), that result in stable heterodimeric antibodies.

[0289] In one embodiment, the bispecific antibody of the present invention comprises a first heavy chain comprising a first CH3 region and a second heavy chain comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the respective homodimeric interactions between the first CH3 region and the second CH3 region. Further details regarding these interactions and how they can be achieved are provided, for example, in WO 2011 / 131746 and WO 2013 / 060867 (Genmab), which are incorporated herein by reference.

[0290] As further described herein, stable bispecific CD40xCD137 antibodies can be obtained in high yields using a specialized method based on one homodimeric parent CD40 antibody and one homodimeric parent CD137 antibody that contain only a few, fairly conservative, asymmetric mutations in their CH3 regions. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at positions that are not identical.

[0291] Thus, in one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the sequences of the CH3 regions of the first and second heavy chains comprise an asymmetric mutation, e.g., a mutation in one of the CH3 regions at a position corresponding to position 405 of the human IgG1 heavy chain according to EU numbering, and a mutation in the other CH3 region at a position corresponding to position 409 of the human IgG1 heavy chain according to EU numbering.

[0292] In one aspect, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain and a second heavy chain, wherein in the first heavy chain at least one amino acid substitution is made at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and in the second heavy chain at least one amino acid substitution is made at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0293] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409 in a human IgG1 heavy chain according to EU numbering, and the second heavy chain comprises an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, 407, and 409 in a human IgG1 heavy chain according to EU numbering, and wherein the first heavy chain and the second heavy chain are not substituted at the same positions.

[0294] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 366, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

[0295] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 368, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407, and 409.

[0296] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 370, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407, and 409.

[0297] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 399, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407, and 409.

[0298] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 405, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409.

[0299] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 407, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 409.

[0300] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid substitution at position 409, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407.

[0301] Thus, in one embodiment of a bispecific antibody defined in any of the embodiments disclosed herein, the sequences of the first and second CH3 regions comprise asymmetric mutations, i.e. mutations at different positions in the two CH3 regions, for example a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region.

[0302] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407. In one such embodiment, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu. In another embodiment thereof, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys.

[0303] and the second heavy chain comprises an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, and Lys at position 409. In another embodiment thereof, the first heavy chain comprises Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain comprises an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and Lys at position 409.

[0304] and the second heavy chain comprises a Leu at position 405 and a Lys at position 409. In another embodiment thereof, the first heavy chain comprises a Phe at position 405 and an Arg at position 409, and the second heavy chain comprises an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and a Lys at position 409. In another embodiment thereof, the first heavy chain comprises a Phe at position 405 and an Arg at position 409, and the second heavy chain comprises a Leu at position 405 and a Lys at position 409.

[0305] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain comprises Lys at position 409, a Thr at position 370, and a Leu at position 405. In a further embodiment, the first heavy chain comprises Arg at position 409, and the second heavy chain comprises Lys at position 409, a Thr at position 370, and a Leu at position 405.

[0306] In yet another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises a Lys at position 370, a Phe at position 405, and an Arg at position 409, and the second heavy chain comprises a Lys at position 409, a Thr at position 370, and a Leu at position 405.

[0307] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain comprises Lys at position 409, and a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

[0308] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises an Arg at position 409 and the second heavy chain comprises a Lys at position 409, and a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0309] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409, and the second heavy chain comprises a Lys at position 409, and a) an Ile at position 350 and a Leu at position 405 or b) a Thr at position 370 and a Leu at position 405.

[0310] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409, and the second heavy chain comprises an He at position 350, a Thr at position 370, a Leu at position 405, and a Lys at position 409.

[0311] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409 and the second heavy chain has an amino acid other than Phe at position 405, such as an amino acid other than Phe, Arg, or Gly at position 405; or the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409 and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0312] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain having an amino acid at position 409 other than Lys, Leu, or Met and a second heavy chain having an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr.

[0313] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain having a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and a second heavy chain having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

[0314] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain having a Tyr at position 407 and an Arg at position 409 and a second heavy chain having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and a Lys at position 409.

[0315] In another embodiment, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys. In another embodiment, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407.

[0316] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has Gly, Leu, Met, Asn, or Trp at position 407.

[0317] and the second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, and Lys at position 409.

[0318] and the second heavy chain has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and Lys at position 409.

[0319] and the second heavy chain has a Tyr, Leu, Met, Asn, or Trp at position 407 and a Lys at position 409.

[0320] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has a Tyr at position 407 and an Arg at position 409, and the second heavy chain has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, and Lys at position 409.

[0321] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has a Tyr at position 407 and an Arg at position 409, and the second heavy chain has an Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and a Lys at position 409.

[0322] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has a Tyr at position 407 and an Arg at position 409, and the second heavy chain has a Gly, Leu, Met, Asn, or Trp at position 407 and a Lys at position 409.

[0323] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first heavy chain has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second heavy chain has (i) at position 368, an amino acid other than Phe, Leu, and Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys; or (ii) Trp at position 370, or (iii) at position 399, an amino acid other than Asp, Cys, Pro, Glu, or Gln, e.g., Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys; or (iv) at position 366, an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g., Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys; It has the following characteristics.

[0324] In one embodiment, the first heavy chain has an Arg, Ala, His, or Gly at position 409 and the second heavy chain has (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368; or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg, or Tyr at position 399; or (iv) Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366 It has the following characteristics.

[0325] In one embodiment, the first heavy chain has Arg at position 409 and the second heavy chain has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or (ii) Trp at position 370, or (iii) Phe, His, Lys, Arg, or Tyr at position 399, or (iv) Ala, Asp, Glu, His, Asn, Val, Gln at position 366 It has the following characteristics.

[0326] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain and a second heavy chain, wherein (i) the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the first heavy chain and the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the second heavy chain, or (ii) the amino acid at the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R in the first heavy chain and the amino acid at the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L in the second heavy chain.

[0327] In a further embodiment, the first heavy chain and the second heavy chain are of the human IgG1 isotype.

[0328] In another further embodiment, the first heavy chain and the second heavy chain are of the human IgG2 isotype.

[0329] In another further embodiment, the first heavy chain and the second heavy chain are of the human IgG3 isotype.

[0330] In another embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first heavy chain and a second heavy chain of the human IgG4 isotype, wherein (i) the amino acid at the position corresponding to S228 in the human IgG4 heavy chain according to EU numbering is P in the first heavy chain and the amino acids at the positions corresponding to S228, F405, and R409 in the human IgG4 heavy chain according to EU numbering are P, L, and K, respectively, in the second heavy chain, or (ii) the amino acids at the positions corresponding to S228, F405, and R409 in the human IgG4 heavy chain according to EU numbering are P, L, and K, respectively, in the first heavy chain and the amino acid at the position corresponding to S228 in the human IgG4 heavy chain according to EU numbering is P in the second heavy chain.

[0331] When an amino acid at a particular position in a first heavy chain and / or an amino acid at a particular position in a second heavy chain is referred to herein, such reference should be understood to include embodiments in which the amino acid at a particular position in the first heavy chain is present at the corresponding position in the second heavy chain but not in the first heavy chain, and / or the amino acid at a particular position in the second heavy chain is present at the corresponding position in the first heavy chain but not in the second heavy chain.

[0332] In addition to the amino acid substitutions specified above, the first and second heavy chains may also include further amino acid substitutions, deletions, or insertions relative to the wild-type heavy chain sequence.

[0333] In a further embodiment, the first and second Fab arms (or heavy chain constant domains) comprising the first and second heavy chains comprise, in addition to the specified mutations, CH3 sequences independently selected from the following: (IgG1m(a)) (SEQ ID NO: 106), (IgG1m(f)) (SEQ ID NO: 107), and (IgG1m(ax) (SEQ ID NO: 108).

[0334] In one embodiment, neither the first nor the second heavy chain comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

[0335] In a further embodiment, both the first and second heavy chains comprise a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

[0336] In separate and individual embodiments, one or both of the Fab arms comprises a heavy chain constant region sequence independently selected from SEQ ID NOs: 109, 110, 111, 112, 113, and 116 (see Table 1).

[0337] Methods for preparing bispecific antibodies Conventional methods, such as hybrid hybridoma technology and chemical ligation (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare the bispecific antibodies of the invention. Co-expression of two antibodies consisting of different heavy and light chains in a host cell produces a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated, for example, by affinity chromatography or similar methods.

[0338] Strategies to promote the formation of functional bispecific products when different antibody constructs are coexpressed can also be used, for example, by the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to species-restricted preferential heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimer formation is the "knob-into-hole" strategy, in which a protuberance is introduced into the surface of a first heavy chain polypeptide and a corresponding depression is introduced into a second heavy chain polypeptide, so that the protuberance fits into the depression at the interface between the two heavy chains, promoting heterodimer formation and preventing homodimer formation. The "protuberance" is created by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain. By replacing large amino acid side chains with smaller ones, a compensatory "cavity" of the same or similar size as the protuberance is created at the interface of the second polypeptide (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO 2009 / 089004 (Amgen) describe other strategies for promoting heterodimer formation when different antibody domains are co-expressed in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are substituted with charged amino acids, making homodimer formation electrostatically unfavorable and heterodimerization electrostatically favorable. WO2007110205 (Merck) describes yet another strategy for promoting heterodimerization by exploiting the differences between the CH3 domains of IgA and IgG.

[0339] Preferred methods for preparing the bispecific CD40xCD137 antibodies of the invention include those described in WO 2011 / 131746 and WO 2013 / 060867 (Genmab), which comprise the following steps: (a) providing a first antibody comprising an Fc region comprising a first CH3 region; (b) providing a second antibody comprising a second Fc region comprising a second CH3 region; wherein the first antibody is a CD40 antibody comprising two first antigen-binding regions as described herein and the second antibody is a CD137 antibody comprising two second antigen-binding regions as described herein, or vice versa; and the sequences of the first CH3 region and the second CH3 region are different such that a heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than each homodimeric interaction between the first CH3 region and the second CH3 region; (c) incubating the first antibody with the second antibody under reducing conditions; and (d) Obtaining a bispecific CD40xCD137 antibody.

[0340] In one embodiment, a first antibody is incubated with a second antibody under reducing conditions sufficient to cause disulfide bond isomerization at the cysteines in the hinge region, wherein the heterodimeric interaction of the first and second antibodies in the resulting heterodimeric antibody is such that no Fab arm exchange occurs at 0.5 mM GSH after 24 hours at 37°C.

[0341] Without being limited by theory, in step c), heavy chain disulfide bonds in the hinge regions of the parent antibodies (the first and second antibodies of steps a) and b) are reduced, and the resulting cysteines can then form inter-heavy chain disulfide bonds with cysteine ​​residues of another parent antibody molecule (originally having different specificity). In one embodiment of this method, the reducing conditions of step c) comprise the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises restoring the conditions to non-reducing or less reducing conditions, for example by removing the reducing agent by desalting. In one particular embodiment, the bispecific antibody is produced as follows: equal amounts of two complementary parent antibodies are incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a buffer solution (e.g., PBS or Tris-EDTA) at 31°C for 5 hours; the reduction reaction is stopped by removing the reducing agent 2-MEA using a spin column (e.g., Microcon centrifugal filter, 30k, Millipore) (Labrijn et al. Nature Protocols, Vol. 9 No. 10, p. 2450-2463; 2014). In another particular embodiment, the method is that of Example 3.

[0342] For this method, any of the CD40 antibodies and CD137 antibodies disclosed herein can be used. In certain embodiments, a first antibody and a second antibody that bind to human CD40 and human CD137, respectively, can be selected to obtain a bispecific antibody as described herein.

[0343] In one embodiment of this method, the first antibody and / or the second antibody is a full-length antibody.

[0344] The Fc regions of the first and second antibodies may be of any isotype, including, but not limited to, an IgG isotype having a subclass selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are of the IgG1 isotype. In another embodiment, one of the Fc regions of these antibodies is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an IgG1 Fc region and an IgG4 Fc region.

[0345] In a further embodiment, one of the parent antibodies is engineered not to bind to Protein A, thus allowing the heterodimeric antibody to be separated from the homodimeric parent antibody by passing the product over a Protein A column.

[0346] As mentioned above, the sequences of the first and second CH3 regions of the homodimeric parent antibody are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interactions between the first and second CH3 regions, respectively. Further details regarding these interactions and how they can be achieved are provided in WO 2011 / 131746 and WO 2013 / 060867 (Genmab), which are incorporated herein by reference in their entireties.

[0347] Specifically, stable bispecific CD40xCD137 antibodies can be obtained in high yields using the above-described method of the present invention, which is based on two homodimeric antibodies that bind to CD40 and CD137, respectively, and that contain only a few, fairly conservative, asymmetric mutations in their CH3 regions, meaning that the sequences of the first and second CH3 regions contain amino acid substitutions at positions that are not identical.

[0348] The bispecific antibodies of the present invention can also be obtained by co-expressing constructs encoding the first and second polypeptides in a single cell. Thus, in a further aspect, the present invention relates to a method for producing a bispecific antibody, comprising the steps of: (a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc region comprising a first CH3 region and a first antigen-binding region that binds to human CD40 according to any aspect or embodiment herein; (b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region comprising a second CH3 region and a second antigen-binding region that binds to human CD137 according to any aspect or embodiment herein; wherein the sequences of the first CH3 region and the second CH3 region are different and are such that a heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than each homodimeric interaction between the first CH3 region and the second CH3 region, and wherein the first CH3 region has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and the second CH3 region has a substitution of at least one amino acid at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain according to EU numbering, and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions; Optionally, the first nucleic acid construct and the second nucleic acid construct encode the light chain sequences of the first antibody and the second antibody. (c) co-expressing the first nucleic acid construct and the second nucleic acid construct in a host cell; and (d) Obtaining the heterodimeric protein from the cell culture.

[0349] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells producing the bispecific antibodies of the present invention.

[0350] In one embodiment of the invention, a bispecific antibody is obtained by any of the methods according to the invention.

[0351] Suitable expression vectors containing promoters, enhancers, etc., and suitable host cells for producing antibodies are well known in the art. Examples of host cells include yeast cells, bacterial cells, and mammalian cells, such as CHO cells or HEK cells.

[0352] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first CH3 region and a second CH3 region comprising the sequence of SEQ ID NO: 107 (IgG1m(f)) apart from the specified mutations.

[0353] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein neither the first Fc region nor the second Fc region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.

[0354] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein both the first Fc region and the second Fc region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0355] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein said first Fc region and said second Fc region are human antibody Fc regions.

[0356] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein said first Fc region and said second Fc region, apart from the specified mutations, comprise a sequence independently selected from the group consisting of SEQ ID NOs: 109, 110, 111, 112, 113, and 116.

[0357] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein the first antigen-binding region and the second antigen-binding region are derived from a heavy chain antibody.

[0358] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, and the first antigen-binding region and the second antigen-binding region comprise a first light chain and a second light chain.

[0359] In further embodiments, the co-expression method according to the invention includes any of the other features described above for the in vitro method.

[0360] Inactive form Effector functions mediated by the Fc region of an antibody enable the destruction of foreign entities, e.g., killing pathogens, and the elimination and degradation of antigens. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are initiated by the binding of the Fc region to cells bearing Fc receptors (FcR), whereas complement-dependent cytotoxicity (CDC) and complement-dependent cell-mediated cytotoxicity (CDCC) are initiated by the binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

[0361] Fc-mediated effector functions, such as ADCC and complement activation, have been suggested to contribute to the therapeutic efficacy of monoclonal antibodies used to treat cancer (Weiner et al. Cell 2012, 148:1081-1084).

[0362] Multispecific antibodies, such as bispecific antibodies according to the present invention, can be used to target T cells, e.g., CD4 + T cells and / or CD8 + The antibody binds to CD137 expressed on T cells. For example, simultaneous binding of the antibody to CD40 expressed on antigen-presenting cells (APCs) can stimulate both CD40-expressing APCs and CD137-expressing T cells, thereby increasing, for example, T cell proliferation.

[0363] Generally, binding of an antibody to a target antigen expressed by a cell can result in interaction with an effector molecule, such as an Fc receptor or complement protein, thereby inducing an Fc-mediated effector function, such as ADCC or complement activation, resulting in the killing of the cell expressing the target antigen.

[0364] The use of multispecific antibodies, such as bispecific antibodies according to the present invention, is based on their ability to confer costimulation on APCs and T cells.

[0365] In certain embodiments, it is preferred that the multispecific antibody does not bind to FcRs, such as FcγRs, and therefore does not induce FcR-mediated cross-linking.

[0366] In a further embodiment, it is preferred that the multispecific antibody does not evoke effector function, so as to avoid killing of CD40- and / or CD137-expressing cells.

[0367] In one aspect of the invention, a multispecific CD40xCD137 antibody according to the invention comprises (i) a first binding arm comprising a first heavy chain and a first antigen-binding region and (ii) a second binding arm comprising a second heavy chain and a second antigen-binding region according to any aspect or embodiment described herein.

[0368] In one embodiment, a multispecific antibody according to the present invention comprises a first heavy chain and a second heavy chain, and the antibody induces and / or enhances Fc-mediated effector function to a lesser extent than a multispecific antibody comprising two heavy chains that comprise the same first and second antigen-binding regions as the antibody and that comprise the hinge, CH2, and CH3 regions of human IgG1.

[0369] In one embodiment, a multispecific antibody according to the invention comprises a first antigen-binding region and a second antigen-binding region, and a first heavy chain and a second heavy chain, each heavy chain comprising the hinge, CH2, and CH3 regions of human IgG1, wherein at least one of the first heavy chain and the second heavy chain comprises a modification that makes it less likely to induce and / or enhance Fc-mediated effector function compared to a reference multispecific antibody comprising the same first and second antigen-binding region as the antibody and two heavy chains comprising the hinge, CH2, and CH3 regions of human IgG1 but without the modification.

[0370] In one embodiment, the first and second heavy chains are modified such that the multispecific antibody induces and / or enhances Fc-mediated effector function to a lesser extent than an otherwise identical multispecific antibody comprising unmodified first and second heavy chains.

[0371] In one embodiment, Fc-mediated effector function can be measured based on binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated FcR cross-linking.

[0372] In one embodiment, Fc-mediated effector function is measured based on binding to C1q.

[0373] In one embodiment the first and second heavy and light chain constant sequences have been altered such that C1q binding to the multispecific antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to the wild-type multispecific antibody, when C1q binding is measured by ELISA.

[0374] Human IgG1 is known for its ability to induce Fc-mediated effector functions, whereas other human isotypes such as IgG4 are less able to induce Fc-mediated effector functions.

[0375] The first heavy chain and the second heavy chain may each be of any isotype, including but not limited to, an IgG1 isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and may optionally contain one or more mutations or modifications. In one embodiment, the first heavy chain and the second heavy chain each are of or derived from the IgG4 isotype and optionally contain one or more mutations or modifications. In one embodiment, the first heavy chain and the second heavy chain each are of or derived from the IgG1 isotype and optionally contain one or more mutations or modifications. In another embodiment, one of the heavy chains is of the IgG1 isotype and the other is of the IgG4 type or is derived from each such isotype and optionally contains one or more mutations or modifications.

[0376] In one embodiment, one or both of the first and second heavy chains are such that an antibody comprising two first heavy chains or two second heavy chains is likely to lack effector function. For example, the first and second heavy chains may be of the IgG4 isotype or a non-IgG4 type, such as IgG1, IgG2, or IgG3, that has been mutated to reduce or even eliminate the ability to mediate effector functions such as ADCC compared to the unmutated heavy chains. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177(2):1129-1138 (2006) and Hezareh M, J Virol.; 75(24):12161-12168 (2001). The multispecific antibody according to the present invention may comprise modifications in the first and second heavy chains compared to the wild-type human IgG1 sequence. Multispecific antibodies containing such modifications in the Fc region of the antibody can be inactive or non-activated multispecific antibodies. As used herein, the terms "inactive," "inactive," or "non-activated" refer to an Fc region that is at least unable to bind to any Fcγ (gamma) receptor, unable to bind to C1q, or unable to induce Fc-mediated FcR cross-linking. The inactivity of the Fc region, or the first and / or second heavy chains, of the multispecific antibodies of the present invention can be tested using a bivalent monospecific antibody comprising the Fc region, or two first heavy chains or two second heavy chains. This can also be tested using a multispecific antibody comprising a first heavy chain and a second heavy chain.

[0377] In order to develop therapeutic antibodies, some mutants can be constructed to make the Fc region of antibody inactive in the interaction with Fcγ receptor and C1q.The present invention is not limited to any specific mutation that is related to reducing Fc-mediated effector function.Examples of such mutants are described herein.

[0378] Therefore, amino acids in the Fc region that play a major role in interactions with C1q and Fcγ receptors may be modified. Examples of amino acid positions that may be modified include positions L234, L235, and P331.

[0379] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at at least one position corresponding to L234, L235, and P331 in the human IgG1 heavy chain according to EU numbering may be A, A, and S, respectively (Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst. (D64):700-4). The amino acid substitutions L234F and L235E can also result in an Fc region with reduced interaction with Fcγ receptors and C1q (Canfield et al., 1991, J. Exp. Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in the human IgG1 heavy chain according to EU numbering can be F and E, respectively. The amino acid substitution D265A can reduce binding to all Fcγ receptors and prevent ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at position corresponding to D265 in the human IgG1 heavy chain according to EU numbering can be A. Binding to C1q can be inhibited by mutating positions D270, K322, P329, and P331. Mutation of these positions to either D270A, K322A, P329A, or P331A can render the antibody lacking CDC activity (Idusogie EE, et al., 2000, J Immunol. 164: 4178-84). Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at at least one position corresponding to D270, K322, P329, and P331 in a human IgG1 heavy chain according to EU numbering can be A, A, A, and A, respectively.

[0380] An alternative approach to minimizing the interaction of the Fc region with Fcγ receptors and C1q is to remove glycosylation sites from the antibody. For example, by mutating position N297 to Q, A, or E, the glycosylation site essential for IgG-Fcγ receptor interaction is removed. Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in the human IgG1 heavy chain according to EU numbering can be G, Q, A, or E (Leabman et al., 2013, MAbs; 5(6):896-903). Another alternative approach to minimize the interaction of the Fc region with the Fcγ receptor can be achieved by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (Shields et al., 2001, J. Biol. Chem. (276):6591-604).

[0381] Alternatively, human IgG2 and IgG4 subclasses, although reported to interact with Fcγ receptors, are considered to have inherently weak interactions with C1q and Fcγ receptors (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725). Mutations that suppress these residual interactions can be generated in both isotypes to reduce unwanted side effects associated with FcR binding. In the case of IgG2, these include V234A and G237A, and in the case of IgG4, they include L235E. Thus, in at least one of the first and second heavy chains, e.g., in both the first and second heavy chains, the amino acids at positions corresponding to V234 and G237 in the human IgG2 heavy chain according to EU numbering may be A and A, respectively. In one embodiment, the amino acid at the position corresponding to L235 in the human IgG4 heavy chain according to EU numbering may be E.

[0382] Other approaches to further minimize interactions with Fcγ receptors and C1q in IgG2 antibodies include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19):753-62.

[0383] The hinge region of an antibody may also be important for interaction with Fcγ receptors and complement (Brekke et al., 2006, J Immunol 177:1129-1138; Dall'Acqua WF, et al., 2006, J Immunol 177:1129-1138). Therefore, mutations in or deletions of the hinge region can affect the effector function of an antibody.

[0384] In one embodiment, the multispecific antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of the first and second immunoglobulin heavy chains, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0385] In one embodiment, in both the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively.

[0386] In another embodiment, in at least one of the first and second heavy chains, one or more amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are not L, L, and D, respectively, and the amino acids at positions corresponding to N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0387] In one embodiment, one or both heavy chains contain a mutation that removes an acceptor site for Asn-linked glycosylation or is otherwise engineered to alter glycosylation characteristics. For example, the N297Q mutation can be used in the IgG1 Fc region to remove an Asn-linked glycosylation site. Thus, in a specific embodiment, one or both heavy chains contain an IgG1 wild-type sequence that includes the N297Q mutation.

[0388] As used herein, the term "amino acid corresponding to a position" refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins can be found by alignment with human IgG1. Unless otherwise specified or contradicted by context, amino acids in constant region sequences are numbered herein according to the EU numbering guidelines (as described in Kabat, EA et al., 1991, Sequences of proteins of immunological interest. 5th Edition - US Department of Health and Human Services, NIH publication No. 91-3242, pp. 662, 680, 689). Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is one that aligns with the other amino acid or segment when using a standard sequence alignment program such as ALIGN, ClustalW, or the like, typically with default settings, and has at least 50%, at least 80%, at least 90%, or at least 95% identity to the human IgG1 heavy chain. Methods for aligning sequences or segments in a sequence and thereby determining the corresponding positions in the sequence for the amino acid positions according to the present invention are considered to be well known.

[0389] In the present invention, amino acid positions may be defined as described above.

[0390] The term "amino acid is not" or similar phraseology, when referring to an amino acid in the heavy chain, should be understood to mean that the amino acid is any other amino acid other than the specific amino acid referred to. For example, the amino acid at the position corresponding to L234 in the human IgG1 heavy chain is not L means that the amino acid can be either another natural amino acid other than L or an unnatural amino acid.

[0391] In one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is not D.

[0392] In one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0393] In one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is a hydrophobic amino acid or a polar amino acid.

[0394] The term "hydrophobic" as used herein with respect to amino acid residues refers to amino acid residues selected from the group consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0395] Thus, in one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0396] The term "polar" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of C, D, E, H, K, N, Q, R, S, and T. Thus, in one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0397] In another embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is an aliphatic uncharged amino acid, an aromatic amino acid, or an acidic amino acid.

[0398] The term "aliphatic uncharged" as used herein with respect to amino acid residues refers to any amino acid residue selected from the group consisting of A, G, I, L, and V.

[0399] Thus, in one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, G, I, L, and V.

[0400] The term "aromatic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of F, T, and W.

[0401] Thus, in one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of F, T, and W.

[0402] The term "acidic" as used herein with respect to an amino acid residue refers to any amino acid residue selected from the group consisting of D and E.

[0403] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of D and E.

[0404] Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of D and E.

[0405] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0406] In certain embodiments, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0407] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is not D.

[0408] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is not D.

[0409] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0410] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to D265 in a human IgG1 heavy chain according to EU numbering is not D, and the amino acids at the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0411] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is a hydrophobic amino acid or a polar amino acid.

[0412] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is a hydrophobic amino acid or a polar amino acid.

[0413] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0414] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of C, E, H, K, N, Q, R, S, and T. In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y.

[0415] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0416] In another embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is an aliphatic uncharged amino acid, an aromatic amino acid, or an acidic amino acid.

[0417] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, G, I, L, and V.

[0418] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of F, T, and W.

[0419] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of D and E.

[0420] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0421] In another embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is an aliphatic uncharged amino acid, an aromatic amino acid, or an acidic amino acid.

[0422] Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, G, I, L, and V.

[0423] Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of F, T, and W.

[0424] Thus, in one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of D and E.

[0425] In certain embodiments, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W.

[0426] In a further embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain according to EU numbering is not N.

[0427] In one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain according to EU numbering is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain according to EU numbering is P.

[0428] In one embodiment, in the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position N297 in a human IgG1 heavy chain according to EU numbering is not N.

[0429] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to N297 in a human IgG1 heavy chain according to EU numbering is not N, and the amino acid at the position corresponding to position P331 in a human IgG1 heavy chain according to EU numbering is P.

[0430] In a further embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are not L and L, respectively.

[0431] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0432] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V.

[0433] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in human IgG1 heavy chains according to EU numbering are hydrophobic or polar amino acids.

[0434] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0435] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively.

[0436] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0437] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are not L and L, respectively.

[0438] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are not L and L, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0439] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.

[0440] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0441] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively.

[0442] In certain embodiments, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively.

[0443] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in human IgG1 heavy chains according to EU numbering are aliphatic uncharged amino acids, aromatic amino acids, or acidic amino acids.

[0444] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, G, I, and V, respectively.

[0445] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of F, T, and W, respectively.

[0446] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of D and E, respectively.

[0447] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0448] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E or A and A, respectively.

[0449] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E or A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0450] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at the positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E or A and A, respectively.

[0451] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E or A and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0452] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively.

[0453] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively.

[0454] In one embodiment, in at least one of the first and second heavy chains, at least the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are A and A, respectively.

[0455] In one embodiment, in both the first and second heavy chains, at least the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are A and A, respectively.

[0456] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are not L, L, and D, respectively.

[0457] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0458] In one embodiment, in at least one of the first and second heavy chains, the amino acids corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, Y, V, and W, and the amino acid at the position corresponding to position D265 is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, Y, V, and W.

[0459] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.

[0460] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of: A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are each selected from the group consisting of: A, C, F, G, H, I, M, R, T, V, W, and Y.

[0461] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0462] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0463] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are hydrophobic or polar amino acids.

[0464] In one embodiment, in both the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group of amino acids consisting of A, C, F, G, H, I, L, M, R, T, V, W, and Y, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, F, G, H, I, M, R, T, V, W, and Y, respectively.

[0465] In one embodiment, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group of amino acids consisting of C, D, E, H, K, N, Q, R, S, and T, respectively, and the amino acid at the position corresponding to position D265 in a human heavy chain according to EU numbering is selected from the group consisting of C, E, H, K, N, Q, R, S, and T.

[0466] In certain embodiments, in both the first and second heavy chains, the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, respectively, and the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

[0467] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are aliphatic uncharged amino acids, aromatic amino acids, or acidic amino acids.

[0468] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, G, I, and V, respectively.

[0469] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of F, T, and W, respectively.

[0470] Thus, in one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of D and E, respectively.

[0471] In certain embodiments, in at least one of the first and second heavy chains, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0472] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are not L, L, and D, respectively.

[0473] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are not L, L, and D, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0474] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are aliphatic uncharged amino acids, aromatic amino acids, or acidic amino acids.

[0475] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, G, I, L, and V, and the amino acids at the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of A, G, I, and V, respectively.

[0476] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are selected from the group consisting of D and E, respectively.

[0477] In a particular embodiment, in both the first heavy chain and the second heavy chain, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain according to EU numbering is selected from the group consisting of A, E, F, G, I, L, T, V, and W, and the amino acids at the positions corresponding to L234 and L235 are selected from the group consisting of A, D, E, F, G, I, T, V, and W, respectively.

[0478] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A or A, A, and A, respectively.

[0479] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0480] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A or A, A, and A, respectively.

[0481] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A or A, A, and A, respectively, and the amino acids at positions corresponding to positions N297 and P331 in a human IgG1 heavy chain according to EU numbering are N and P, respectively.

[0482] In certain embodiments, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively.

[0483] In a particularly preferred embodiment, in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively.

[0484] In one embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are A, A, and A, respectively.

[0485] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are A, A, and A, respectively.

[0486] In another embodiment, in at least one of the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are F, E, A, Q, and S, respectively.

[0487] In one embodiment, in both the first heavy chain and the second heavy chain, the amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are F, E, A, Q, and S, respectively.

[0488] In a particular embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 67, GAS, and SEQ ID NOs: 68 (CD137 clone 009), and in at least one of the first and second heavy chains, e.g., in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0489] In another embodiment, the first antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 4, YTS, and SEQ ID NO: 5, respectively; and the second antigen-binding region comprises heavy chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 36, 37, and 38, respectively, and light chain variable regions CDR1, CDR2, and CDR3 having the sequences set forth in SEQ ID NOs: 39, SAS, and 40, respectively (CD137 clone 005), wherein in at least one of the first and second heavy chains, e.g., in both the first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0490] The non-activating Fc region prevents the antibody from interacting with Fc receptors present on blood cells such as monocytes or with C1q to activate the classical complement pathway. Reduced Fc activity was investigated in antibody mutants containing various combinations of amino acid substitutions in the Fc region. Three amino acid substitutions, including the mutations L234F, L235E, and D265A, were introduced into the parent antibody of the present invention. Substitutions at these three amino acid positions were introduced into the K409R IgG1 backbone and / or the F405L IgG1 backbone. The resulting non-activating antibody mutants are designated with the suffix "FEAR" or "FEAL," respectively. The parent antibodies were used to generate bispecific antibodies of the present invention, as described in the Examples.

[0491] In one aspect, the light chain and / or heavy chain of the multispecific antibody according to the present invention may be modified to increase the expression level and / or production yield. In one embodiment, the light chain of the antibody according to the present invention may be modified. Such modifications are known in the art and may be performed, for example, according to the method described in Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004). Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807-14.

[0492] In a further embodiment of the invention, one or both of the antibodies forming part of the multispecific antibody of the invention are engineered to decrease or increase binding to the neonatal Fc receptor (FcRn) in order to manipulate the serum half-life of the multispecific antibody. Techniques for extending or shortening serum half-life are well known in the art. See, for example, Dall'Acqua et al. 2006, J. Biol. Chem., 281:23514-24; Hinton et al. 2006, J. Immunol., 176:346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157-9.

[0493] In one aspect, the multispecific antibody defined in any of the embodiments disclosed herein comprises a first constant heavy chain (HC) and a first constant light chain (LC), wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain of SEQ ID NO: 109 for both the first heavy chain and the second heavy chain are F, E, and A, respectively.

[0494] In one embodiment, the multispecific antibody defined in any of the embodiments disclosed herein comprises a first and a second constant heavy chain (HC) and a first and a second constant light chain (LC), wherein the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain of SEQ ID NO: 109, for both the first and second heavy chains, are F and E, respectively.

[0495] In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein, for both the first and second heavy chains, the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering are F and E, respectively, and (i) for the first heavy chain, the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L and for the second heavy chain, the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R; or (ii) for the first heavy chain, the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering is R and for the second heavy chain, the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering is L.

[0496] In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein, for both the first and second heavy chains, the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the first heavy chain is L, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the second heavy chain is R. Thus, in a further embodiment, the first heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO: 113, and the second heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO: 112.

[0497] In one embodiment, the multispecific antibody comprises a first and a second heavy chain, wherein, for both the first and second heavy chains, the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering for the first heavy chain is R, and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering for the second heavy chain is L. Thus, in a further embodiment, the first heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO: 112, and the second heavy chain comprises the constant heavy chain sequence shown in SEQ ID NO: 113.

[0498] nucleic acid The present invention also relates to nucleic acids encoding one or more of the amino acid sequences according to any aspect or embodiment disclosed herein.

[0499] The present invention also relates to a nucleic acid encoding a multispecific antibody as defined in any aspect or embodiment disclosed herein.

[0500] The present invention also relates to an expression vector comprising a nucleic acid of the invention.

[0501] The present invention also relates to a host cell comprising a nucleic acid or an expression vector according to the invention.

[0502] In one embodiment, the host cell is a recombinant eukaryotic host cell, a recombinant prokaryotic host cell, or a recombinant microbial host cell.

[0503] In a further embodiment, the expression vector further comprises a nucleotide sequence encoding the constant region of the light chain, the heavy chain, or both the light and heavy chain of an antibody, eg, a human antibody.

[0504] The expression vector of the present invention may be any suitable vector (a nucleic acid sequence comprising an appropriate set of expression control elements), including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors. Examples of such vectors include SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmid derivatives, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid is in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compact nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector such as pBR322, pUC19 / 18, or pUC118 / 119, the minimal size nucleic acid vector "midge" (e.g., as described in Schakowski et al., Mol Ther 3, 793 800 (2001)), or a precipitated nucleic acid vector construct, such as a CaP04 precipitated construct (e.g., as described in WO200046147; Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986); Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US 5,589,466 and US 5,973,972).

[0505] In one embodiment, the vector is suitable for expressing a CD40 antibody and / or a CD137 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), and pET vectors (Novagen, Madison WI).

[0506] The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH (reviewed in F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0507] The expression vector may also or alternatively be a vector suitable for expression in mammalian cells, e.g., a vector containing glutamine synthetase as a selectable marker, such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0508] The nucleic acid and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of directing a polypeptide, such as a nascent polypeptide chain, to the periplasmic space or into the cell medium. Such sequences are known in the art and include secretory leader or signal peptides.

[0509] The expression vector may contain or be accompanied by any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer, as well as the RSV promoter, SV40 promoter, SL33 promoter, MMTV promoter, and HIV LTR promoter), an effective poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter as opposed to a constitutive promoter such as CMV IE.

[0510] In one embodiment, an expression vector encoding a CD40 antibody and / or a CD137 antibody may be placed into and / or delivered to a host cell or animal via a viral vector.

[0511] In yet another aspect, the present invention relates to a host cell comprising a first nucleic acid construct and a second nucleic acid construct as specified herein above.

[0512] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the multispecific antibodies of the invention.

[0513] The first CD40-specific antibody may be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, that produces the antibody defined herein. The second CD137-specific antibody may similarly be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, that produces the antibody. Such antibodies may be used to prepare multispecific antibodies according to the present invention. The multispecific antibodies according to the present invention may also be expressed in a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma.

[0514] Exemplary host cells include yeast cells, bacterial cells, plant cells, and mammalian cells, such as CHO cells, CHO-S cells, HEK cells, HEK293 cells, HEK-293F cells, Expi293F cells, PER.C6 cells, or NS0 cells, or lymphocytic cells. For example, in one embodiment, the host cell may contain a first nucleic acid construct and a second nucleic acid construct stably integrated into the cellular genome. In another embodiment, the invention provides a cell containing a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, comprising the above-specified first and second nucleic acid constructs.

[0515] In yet another aspect, the present invention relates to a transgenic non-human animal or a transgenic plant that comprises nucleic acids encoding one or two sets of human heavy chains and human light chains and produces a multispecific antibody of the invention.

[0516] The first CD40-specific antibody and / or the second CD137-specific antibody may also be produced by a hybridoma, a transgenic non-human animal, or a transgenic plant comprising nucleic acids encoding one or two sets of human heavy and human light chains, which animal or plant produces the multispecific antibody or antibodies for use in the multispecific antibody of the invention.

[0517] In one aspect, the invention relates to nucleic acids encoding one or more of the amino acid sequences set forth in Table 1.

[0518] In one aspect, the present invention relates to an expression vector comprising: (i) a nucleic acid sequence encoding the heavy chain sequence of the first binding arm of any one of the embodiments disclosed herein; (ii) a nucleic acid sequence encoding the light chain sequence of the first binding arm of any one of the embodiments disclosed herein; (iii) a nucleic acid sequence encoding the heavy chain sequence of the second binding arm of any one of the embodiments disclosed herein; (iv) a nucleic acid sequence encoding the light chain sequence of the second binding arm of any one of the embodiments disclosed herein; (v) a nucleic acid as described in (i) and a nucleic acid as described in (ii); (vi) a nucleic acid as described in (iii) and a nucleic acid as described in (iv) (vii) A nucleic acid as set forth in (i), (ii), (iii), and (iv).

[0519] In certain embodiments, the nucleic acid may encode a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 of a CD40 antibody listed in Table 1, and may encode a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116.

[0520] In another embodiment, the nucleic acid may encode a heavy chain variable region comprising VH CDR1, VH CDR2, and VH CDR3 of one of the CD137 antibodies listed in Table 1, i.e., any one of clones 001-012, and may encode a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116.

[0521] In separate specific embodiments, the nucleic acid, nucleic acid construct, combination of a first and a second nucleic acid construct, expression vector, or combination of a first and a second expression vector according to the invention may encode: (a) a HC comprising (i) a VH comprising a VH CDR1, VH CDR2, and VH CDR3 of a CD40 antibody of Table 1, and framework regions of predominantly human origin, optionally comprising one or more amino acid backmutations to a non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (b) a HC comprising: (i) a VH comprising a VH CDR1, VH CDR2, and VH CDR3 of one of the CD137 antibodies listed in Table 1, i.e., any one of clones 001-012, and framework regions of predominantly human origin, optionally comprising one or more amino acid backmutations to a non-human amino acid sequence; and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (c) an LC comprising: (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of a CD40 antibody of Table 1, and framework regions of predominantly human origin, optionally comprising one or more amino acid backmutations to a non-human amino acid sequence; and (ii) a light chain constant region having the sequence of SEQ ID NO: 114; (d) an LC comprising: (i) a VL comprising a VL CDR1, VL CDR2, and VL CDR3 of one of the CD137 antibodies listed in Table 1, i.e., any of clones 001-012, and framework regions of predominantly human origin, optionally containing one or more amino acid backmutations to a non-human amino acid sequence; and (ii) a light chain constant region having the sequence of SEQ ID NO: 114; (e) both (a) and (b); (f) both (a) and (c); (g) both (b) and (d); (h) both (c) and (d); or (i) All of (a), (b), (c), and (d).

[0522] In other separate specific embodiments, the nucleic acid, nucleic acid construct, combination of a first and a second nucleic acid construct, expression vector, or combination of a first and a second expression vector according to the invention may encode: (a) a HC comprising (i) a VH comprising a VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NOs: 1, 2, and 3, and framework regions of predominantly human origin, optionally comprising one or more amino acid backmutations to a non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (b) a HC comprising (i) a VH comprising a VH CDR1, VH CDR2, and VH CDR3 of SEQ ID NOs: 64, 65, and 66, and framework regions of predominantly human origin, optionally comprising one or more amino acid backmutations to a non-human amino acid sequence, and (ii) a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (c) an LC comprising (i) a VL comprising a VL CDR1, a VL CDR2, and a VL CDR3 of SEQ ID NO: 4, YTS, and SEQ ID NO: 5, and framework regions of predominantly human origin, optionally containing one or more amino acid backmutations to a non-human amino acid sequence, and (ii) a light chain constant region having the sequence of SEQ ID NO: 114; (d) (i) a VL comprising SEQ ID NO: 67, GAS, and a VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 68, and framework regions of predominantly human origin, optionally containing one or more amino acid backmutations to a non-human amino acid sequence, and (ii) a LC comprising a light chain constant region having the sequence of SEQ ID NO: 114; (e) both (a) and (b); (f) both (a) and (c); (g) both (b) and (d); (h) both (c) and (d); or (i) All of (a), (b), (c), and (d).

[0523] In other separate specific embodiments, the nucleic acid, nucleic acid construct, combination of a first and a second nucleic acid construct, expression vector, or combination of a first and a second expression vector according to the present invention may encode: (a) a VH comprising SEQ ID NO: 117 and a HC comprising a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (b) a VH comprising SEQ ID NO: 123 and a HC comprising a human IgG1 heavy chain having a sequence selected from the group consisting of SEQ ID NOs: 110, 111, 112, 113, and 116; (c) a VL comprising SEQ ID NO: 121 and a light chain constant region having the sequence of SEQ ID NO: 114; (d) a VL comprising SEQ ID NO: 127 and a light chain constant region having the sequence of SEQ ID NO: 114; (e) both (a) and (b); (f) both (a) and (c); (g) both (b) and (d); (h) both (c) and (d); or (i) All of (a), (b), (c), and (d).

[0524] In other separate specific embodiments, the nucleic acid, nucleic acid construct, combination of a first and a second nucleic acid construct, expression vector, or combination of a first and a second expression vector according to the invention may encode: (a) HC containing SEQ ID NO: 118 (CD40-001-HC6, IgG1); (b) HC containing SEQ ID NO: 119 (CD40-001-HC6-FEAL); (c) HC containing SEQ ID NO: 120 (CD40-001-HC6-FEAR); (d) HC containing SEQ ID NO: 124 (CD137-009-HC7); (e) HC containing SEQ ID NO: 125 (CD137-009-HC7-FEAR); (f) HC containing SEQ ID NO: 126 (CD137-009-HC7-FEAL); (g) LC containing SEQ ID NO: 122 (CD40-001-LC1); (h) LC containing SEQ ID NO: 128 (CD137-009-LC2); (i) both (a) and (g); (j) both (b) and (g); (k) both (c) and (g); (l) both (d) and (h); (m) both (e) and (h); (n) both (f) and (h); (o) both (b) and (e); (p) both (c) and (f); (q) both (g) and (h); (r) all of (b), (e), (g), and (h); (s) All of (c), (f), (g), and (h). ...

Claims

[Claim 1] The invention described in the specification and drawings of this application.

Images