Bispecific antibodies specific for PD1 and TIM3

Abstract

The present invention relates to bispecific antibodies specific for PD1 and TIM3. The present invention relates to bispecific antibodies comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, in particular to the following bispecific antibodies, wherein the bispecific antibodies bind to TIM3 with a lower binding affinity than the binding to PD1. The present invention further relates to methods of generating these molecules and methods of using them.

Description

[0001] This application is a divisional application of the invention application with a filing date of September 29, 2016, Chinese application number 201680057531.1, and invention name “Bispecific antibody specific for PD1 and TIM3”. Field of the Invention

[0002] The present invention relates to bispecific antibodies comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, particularly bispecific antibodies that bind to TIM3 with a lower affinity than that for PD1. The present invention further relates to methods of producing these molecules and methods of using them. Background of the Invention

[0003] The importance of the immune system in the protection against cancer is based on its ability to detect and destroy abnormal cells. However, some tumor cells can escape the immune system by causing an immunosuppressive state (Zitvogel et al., Nature Reviews Immunology 6 (2006), 715-727). An example of the mechanism of immunosuppression present in hosts carrying tumors is to promote T cell dysfunction or exhaustion. Due to their ability to selectively recognize peptides derived from proteins in all cell compartments; their ability to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells; also known as cytotoxic T lymphocytes (CTL)); and their ability to coordinate a variety of immune responses (by CD4+ helper T cells) (this integration of acquired and innate effector mechanisms), T cells have been the main focus of therapeutic manipulations of endogenous anti-tumor immunity efforts. Exhausted T cells fail to respond to antigen stimulation and proliferate and exert effector functions such as cytotoxicity and cytokine secretion. Further studies have identified that exhausted T cells are characterized by the persistent expression of the inhibitory molecule PD-1 (programmed cell death protein 1) and that blocking the interaction between PD-1 and PD-L1 (PD-1 ligand) can reverse T cell exhaustion and restore antigen-specific T cell responses in mice infected with LCMV (Barber et al., Nature 439 (2006), 682-687). However, targeting the PD-1 / PD-L1 pathway alone does not always lead to the reversal of T cell exhaustion (Gehring et al., Gastroenterology 137 (2009), 682-690), indicating that other molecules may be involved in T cell exhaustion (Sakuishi, J. Experimental Med. 207 (2010), 2187-2194).

[0004] TIM-3 is a molecule initially identified as selectively expressed on Th1 and Tc1 cells that secrete IFN-γ (Monney et al., Nature 415 (2002), 536-541). The interaction between TIM-3 and its ligand, galectin-9, triggers cell death in TIM-3+T cells. Thus, both TIM-3 and PD-1 can function as negative regulators of T cell responses. TIM-3 has been shown to mark the most suppressed or most dysfunctional populations in CD8+T cells in preclinical models of both solid and hematological malignancies (Sakuishi, J. Experimental Med. 207 (2010), 2187-2194; Zhou, Blood 117 (2011), 4501-4510; Majeti R et al., PNAS, 106 (2009), 3396-3401). In these models, all CD8+TIM-3+T cells co-express PD1, and these dual-expressing cells exhibit defects in both cell cycle progression and effector cytokine production [interleukin (IL) -2, TNF, and IFN-γ] than cells expressing PD1 alone. Thus, the TIM-3 pathway may cooperate with the PD-1 pathway to promote the occurrence of severe functional impairment phenotypes in CD8+T cells in cancer. Therefore, it is expected that the combined targeting of the TIM-3 and PD1 pathways is highly effective in controlling tumor growth.

[0005] TIM3 is a human protein belonging to the immunoglobulin superfamily and the protein TIM family. In humans, similar to mice, TIM-3 is expressed on T cells and phagocytic cells such as macrophages and dendritic cells. TIM3 binding to protein ligands (such as galectin-9) can inhibit Th1 responses via apoptosis-induced mechanisms, and thus lead to induction of peripheral tolerance. Reduction of human TIM3 expression by siRNA or inhibition of human TIM3 by blocking antibodies increases interferon α secretion from CD4-positive T cells, supporting the inhibitory effect of TIM3 in human T cells. In phagocytes, TIM3 also functions as a receptor for recognizing apoptotic cells. Analysis of clinical samples from patients with autoimmune diseases showed that TIM3 is not expressed in CD4-positive cells. In particular, compared to clones derived from normal healthy individuals, TIM3 expression levels are lower and IFN-γ secretion levels are higher in T cell clones derived from the cerebrospinal fluid of patients with multiple sclerosis (Koguchi K et al., J Exp Med. 203 (2006) 1413-1418). There are reports on the relationship between TIM-3 and allergic diseases or asthma (WO96 / 27603 and WO2003 / 063792).

[0006] Examples of anti-TIM3 monoclonal antibodies include anti-human TIM3 rat monoclonal antibody (clone 344823, manufactured by R&D Systems) and anti-human TIM-3 mouse monoclonal antibody (clone F38-2E2, manufactured by R&D Systems). WO2013 / 06490 relates to anti-TIM3 antibodies and immunoconjugates thereof that exhibit rapid internalization for use in treating cancer and reducing inflammation. US2012 / 189617 relates to anti-TIM-3 antibodies that exhibit higher effector activity, such as antibody-dependent cellular cytotoxicity (ADCC activity), for use in diseases involving human TIM3-expressing cells.

[0007] Programmed cell death protein 1 (PD-1 or CD279) is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is a cell surface receptor expressed on activated B cells, T cells, and myeloid cells (Okazaki et al. (2002) Curr. Opin. Immunol. 14:391-779-82; Bennett et al. (2003) J Immunol 170:711-8). PD-1 is a monomeric type 1 transmembrane protein composed of an immunoglobulin variable-like extracellular domain and a cytoplasmic domain containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Activated T cells transiently express PD1, but persistent overexpression of PD1 and its ligand, PDL1, promotes immune exhaustion, leading to persistent viral infection, tumor escape, and increased infection and mortality. PD1 expression is induced by antigen recognition via the T cell receptor, and its expression is primarily maintained by continuous T cell receptor signaling. After prolonged antigen exposure, the PD1 locus fails to remethylate, which promotes continued overexpression. Blocking the PD1 pathway can restore exhausted T cell function in cancer and chronic viral infection (Sheridan, Nature Biotechnology 30 (2012), 729-730). Monoclonal antibodies against PD-1 have been described, for example, WO 2003 / 042402, WO 2004 / 004771, WO 2004 / 056875, WO 2004 / 072286, WO 2004 / 087196, WO 2006 / 121168, WO 2006 / 133396, WO 2007 / 005874, WO 2008 / 083174, WO 2008 / 156712, WO 2009 / 024531, WO 2009 / 014708, WO 2009 / 101611, WO 2009 / 114335, WO 2009 / 154335, WO 2010 / 027828,WO 2010 / 027423, WO 2010 / 029434, WO 2010 / 029435, WO 2010 / 036959, WO 2010 / 063011, WO 2010 / 089411, WO 2011 / 066342, WO 2011 / 110604, WO 2011 / 110621, WO 2012 / 145493, WO 2013 / 014668, WO 2014 / 179664, and WO 2015 / 112900.

[0008] It has also been shown that blocking both PD1 and TIM3 can restore antibacterial immune responses, for example in patients with acute alcoholic hepatitis (AAH). Due to chronic endotoxin exposure, lymphocytes from these patients express high levels of immunosuppressive receptors, generate lower levels of interferon gamma, and have elevated IL10 production. These effects can be reversed by blocking PD1 and TIM3, which increases the antimicrobial activity of T cells and neutrophils (Markwick et al, Gastroenterology 148 (2015), 590-602).

[0009] Bispecific antibodies against TIM3 and PD1 for immunotherapy in chronic immune conditions have been described in WO 2011 / 159877. However, there is a need to provide new bispecific antibodies that not only bind to PD1 and TIM3 simultaneously and thus selectively target T cells expressing both PD1 and TIM3, but also avoid blocking other cells such as innate immune cells, e.g., TIM3 on naive dendritic cells (DCs) and monocytes. The bispecific antibodies of the present invention not only effectively block PD1 and Tim3 on T cells that overexpress both PD1 and TIM3, but also have high selectivity for these cells and thus can avoid the side effects caused by the administration of highly active TIM3 antibodies. SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises

[0011] The VH domain comprises

[0012] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0013] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0014] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0015] The VL domain contains

[0016] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,

[0017] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0018] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0019] And the second antigen binding site that specifically binds to TIM3 comprises

[0020] (a) VH domain, which comprises

[0021] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0022] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and

[0023] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0024] The VL domain contains

[0025] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11 or SEQ ID NO: 12,

[0026] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0027] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0028] (b) a VH domain comprising

[0029] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0030] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0031] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0032] The VL domain contains

[0033] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0034] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0035] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or

[0036] (c) a VH domain comprising

[0037] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29,

[0038] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0039] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31; and

[0040] The VL domain contains

[0041] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32,

[0042] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and

[0043] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0044] In one aspect, the bispecific antibody comprising a first antigen binding site that specifically binds PD1 and a second antigen binding site that specifically binds TIM3 is bivalent.

[0045] In another aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with low affinity and binds to PD1 with high affinity. In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with a binding affinity that is at least 50 times lower than that of the binding to PD1, more particularly at least 100 times lower than that of the binding to PD1. In a preferred embodiment, the binding affinity (K D ) is determined using a surface plasmon resonance assay (as described, for example, in Example 12).

[0046] In another aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the first antigen binding site that specifically binds to PD1 comprises

[0047] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 43 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or

[0048] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or

[0049] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or

[0050] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or

[0051] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49,

[0052] And the second antigen binding site that specifically binds to TIM3 comprises

[0053] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, or

[0054] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, or

[0055] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, or

[0056] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or

[0057] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or

[0058] (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or

[0059] (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0060] (h) a VH domain comprising the amino acid sequence of SEQ ID NO: 35 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

[0061] In one specific aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the first antigen binding site that specifically binds to PD1 comprises amino acids comprising SEQ ID NO: 45.

[0062] The VH domain of the TIM3 comprises a VL domain comprising an amino acid sequence of SEQ ID NO: 46, and the second antigen binding site that specifically binds to TIM3 comprises an amino acid sequence of SEQ ID NO: 15.

[0063] The VH domain comprising the amino acid sequence of SEQ ID NO: 16 and the VL domain comprising the amino acid sequence of SEQ ID NO: 16, or the VH domain comprising the amino acid sequence of SEQ ID NO: 25 and the VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0064] In particular, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein

[0065] The first antigen binding site that specifically binds to PD1 comprises

[0066] The VH domain comprises

[0067] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0068] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0069] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0070] The VL domain contains

[0071] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40,

[0072] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0073] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0074] And the second antigen binding site that specifically binds to TIM3 comprises

[0075] The VH domain comprises

[0076] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0077] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0078] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0079] The VL domain contains

[0080] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0081] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0082] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0083] More particularly, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, and the second antigen-binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0084] In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with a binding affinity that is at least 50-fold lower than the binding to PD1, more particularly at least 100-fold lower than the binding to PD1.

[0085] In another aspect, the bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 is a human, humanized or chimeric antibody. In particular, it is a humanized or chimeric antibody.

[0086] In another aspect, the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising the antigen binding site that specifically binds to PD1, and a second Fab fragment comprising the antigen binding site that specifically binds to TIM3.

[0087] In particular, the Fc domain is an IgG domain, more particularly an IgG1 Fc domain or an IgG4 Fc domain.

[0088] In one aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In particular, the Fc domain is of the human IgG1 subclass and comprises the amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0089] In another aspect, the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the Fc domain comprises a modification that promotes the association of the first and second subunits of the Fc domain.

[0090] In one aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding site that specifically binds PD1 and a second antigen-binding site that specifically binds TIM3, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to a knob-into-hole approach. In a specific aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0091] In another aspect, the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein in one of the Fab fragments, the variable domains VL and VH are replaced with each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a specific aspect, the bispecific antibody is as follows, wherein in the first Fab fragment comprising the antigen binding site that specifically binds to PD1, the variable domains VL and VH are replaced with each other.

[0092] In another aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein in one of the Fab fragments, the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index). In a specific aspect, the bispecific antibody is as follows, wherein in the second Fab fragment comprising the antigen-binding site that specifically binds to TIM3, the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R), or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0093] In another aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising

[0094] (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 50, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 52,

[0095] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 51, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 53, or

[0096] (b) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 54, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 56,

[0097] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 55, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 57, or

[0098] (c) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 58, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 60,

[0099] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 59, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 61, or

[0100] (d) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 62, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 64,

[0101] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 63, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 65, or

[0102] (e) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 66, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 68,

[0103] A second heavy chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:67, and a second light chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:69.

[0104] In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising

[0105] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 50, and a first light chain comprising the amino acid sequence of SEQ ID NO: 52,

[0106] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a second light chain comprising the amino acid sequence of SEQ ID NO: 53, or

[0107] (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 54, a first light chain comprising the amino acid sequence of SEQ ID NO: 56,

[0108] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and a second light chain comprising the amino acid sequence of SEQ ID NO: 57, or

[0109] (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 58, a first light chain comprising the amino acid sequence of SEQ ID NO: 60,

[0110] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61, or

[0111] (d) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 62, a first light chain comprising the amino acid sequence of SEQ ID NO: 64,

[0112] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63, and a second light chain comprising the amino acid sequence of SEQ ID NO: 65, or

[0113] (e) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a first light chain comprising the amino acid sequence of SEQ ID NO: 68,

[0114] A second heavy chain comprising the amino acid sequence of SEQ ID NO:67, and a second light chain comprising the amino acid sequence of SEQ ID NO:69.

[0115] According to another aspect of the present invention, polynucleotides encoding bispecific antibodies as previously described herein are provided. The present invention further provides vectors, particularly expression vectors, comprising the polynucleotides of the present invention and prokaryotic or eukaryotic host cells comprising the polynucleotides or vectors of the present invention. In some embodiments, the host cell is a eukaryotic cell, particularly a mammalian cell.

[0116] In another aspect, provided is a method for producing a bispecific antibody as described herein comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising the steps of: a) transforming a host cell with a vector comprising a polynucleotide encoding the bispecific antibody, b) culturing the host cell under conditions suitable for expression of the bispecific antibody, and c) recovering the bispecific antibody from the culture. The present invention also encompasses bispecific antibodies produced by the methods of the present invention.

[0117] The present invention further provides a pharmaceutical composition comprising a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3 as described herein, and at least one pharmaceutically acceptable excipient.

[0118] The present invention also encompasses a bispecific antibody as described herein comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, or a pharmaceutical composition comprising the bispecific antibody for use as a medicament.

[0119] In another aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3 as described herein, or a pharmaceutical composition comprising the bispecific antibody, for use in

[0120] i) regulating immune responses, such as restoring T cell activity,

[0121] ii) stimulate immune response or function,

[0122] iii) treatment of infection,

[0123] iv) treating cancer,

[0124] v) delaying cancer progression,

[0125] vi) Prolonging the survival of patients suffering from cancer.

[0126] In one aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 as described herein, or a pharmaceutical composition comprising the bispecific antibody, for treating a disease in an individual in need thereof. In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, or a pharmaceutical composition comprising the bispecific antibody, for treating cancer. In another specific aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, or a pharmaceutical composition comprising the bispecific antibody, for regulating an immune response. In another aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, or a pharmaceutical composition comprising the bispecific antibody, for treating chronic viral infections.

[0127] Also provided are uses of a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 as described herein for use in the manufacture of a medicament for treating a disease in an individual in need thereof, particularly for the manufacture of a medicament for treating cancer, and a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a composition comprising a pharmaceutically acceptable form of a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 as described herein. In one specific aspect, the disease is cancer. In another specific aspect, the disease is a chronic viral infection. In another aspect, a method for modulating an immune response in an individual is provided, comprising administering to the individual a therapeutically effective amount of a composition comprising a pharmaceutically acceptable form of a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 as described herein. In any of the above aspects, the individual is preferably a mammal, particularly a human.

[0128] The present invention also provides a bispecific antibody as described herein comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, or a pharmaceutical composition comprising the bispecific antibody, for use in preventing or treating cancer, wherein the bispecific antibody is administered in combination with a chemotherapeutic agent, radiation and / or other agents for cancer immunotherapy.

[0129] Furthermore, provided is a method for inhibiting tumor cell growth in an individual, comprising administering to the individual an effective amount of a bispecific antibody as described herein comprising a first antigen-binding site that specifically binds to PD-1 and a second antigen-binding site that specifically binds to TIM3 to inhibit tumor cell growth. The individual is preferably a mammal, particularly a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0130] Figure 1 : Blockade of PD1 with chimeric PD1-0103 strongly enhances IFNγ secretion from allogeneic stimulated primary human T cells.

[0131] Figure 2 : Blockade of PD1 with chimeric PD1-0103 strongly enhances interferon gamma (IFN-γ) secretion by allogeneic stimulated primary human T cells.

[0132] Figure 3 : Blockade of PD1 with chimeric PD1-0103 strongly enhances tumor necrosis factor alpha (TNF) secretion from allogeneic stimulated primary human T cells.

[0133] Figures 4A-4B : Figure 4A) the frequency of CD4 T cells producing granzyme B; Figure 4B ) The amount of IFN-γ in MLR supernatants detected by absorbance (optical density, OD) in the presence of increasing concentrations of different anti-PD-1 antibodies.

[0134] Figure 5 : Figure 5 A) Effect of PD1 / PD-L1 blockade on reactivation of suppressed T cell receptor signaling in the presence of different anti-PD-1 antibodies. Figure 5 B) Effect of PD1 / PD-L1 blockade on reactivation of suppressed T cell receptor signaling in the presence of different anti-PD-1 antibodies.

[0135] Figure 6 Schematic diagram of the FRET assay for simultaneous binding of anti-PD1 / Tim3 bispecific antibodies to recombinant cells

[0136] Figures 7A-7B : Induction of FRET after treatment / binding of different bispecific PD1TIM3 antibodies on cells expressing PD1 and TIM3: HEK293 cells doubly transfected with PD1 SNAP Tim3 CLIP were stained with 100 nM SNAP-Lumi4-Tb (Cisbio) and 100 nM Clip-Red (Cisbio) in Tag-Lite buffer (Cisbio) at 37°C for 1 hour. After washing, the labeled cells were incubated with the indicated bispecific anti-PD1 / Tim3 antibodies [0-10 nM] ( Figure 7B The cells were incubated with the humanized bispecific variants shown in Figure 2 for 1 hour at 4°C, after which time-resolved fluorescence was measured at 665 / 620 nm using a BMG Pherastar reader (depicted are the mean + / - SD of the FRET signal [ratio 665 / 620 nm*10,000], n=3). Figure 7A : 1+1 format (antibodies PD1TIM3_0389 and PD1TIM3_0168) compared to 2+2 construct (PD1TIM3_0358+PD1TIM3_0359), Figure 7B : Humanized bispecific variants PD1TIM3_0476 and PD1TIM3_0477.

[0137] Figures 8A-8B: FRET assay for simultaneous binding of anti-PD1 / TIM3 bispecific antibody 1+1PD1TIM3-0168: SNAP-tagged PD1 and CLIP-tagged TIM3 cells were labeled with 100 nM SNAP-Lumi4-Tb and 100 nM Clip-Red (as previously described). After washing, labeled cells were incubated with bispecific anti-PD1 / TIM3 antibody #0168 [indicated concentrations] at 4°C for 1 hour, after which time-resolved fluorescence was measured at 665 / 620 nm using a BMG Pherastar reader (black line). To highlight the specificity of the FRET signal induced by the bispecific antibody, anti-PD1 monoclonal antibody (#0165; see Figure 8A ) or anti-TIM3 blocking antibody (#0018, see Figure 8B ) competition with either of the two antibodies resulted in almost complete blockade of the FRET signal (grey curve). Treatment with anti-PD1 antibody alone did not result in FRET induction (dotted line, left panel only).

[0138] Figure 9 :

[0139] Figure 9 A: Bispecific 1+1 PD1 TIM3-0389 showed the same binding ratio to positive CD4+ T cells (PD1+, TIM3+) compared to chimeric TIM3_0028 (chi0028) and humanized TIM3-0438 (0438), but less binding to monocytes, NK cells and CD3+ T cells.

[0140] Figure 9 B: Bispecific 1+1 PD1 TIM3-0389 showed significantly increased MFI of binding to positive CD4+ T cells (PD1+, TIM3+) compared to chimeric TIM3_0028 (chi0028) and humanized Tim3-0438 (0438).

[0141] Figure 9 C: Bispecific 1+1 PD1 TIM3-0168 showed no difference in binding to positive CD4+ T cells (PD1+, TIM3+) compared to chimeric TIM3_0018 (Tim3-chi0018) and humanized TIM3-0434 (0434).

[0142] Figure 9 D: Bispecific 1+1 PD1 TIM3-0168 showed only slightly elevated MFI binding to positive CD4+ T cells (PD1+, Tim3+).

[0143] Figure 9 E and Figure 9F: Anti-TIM3 antibody TIM3-0038 shows binding to both monocytes and CD4+ T cells.

[0144] Figure 9 G and Figure 9 H: Bispecific 1+1 PD1 TIM3-0166 (based on chimeric PD1-0103 / Tim3-0038) shows strongly reduced binding to monocytes (compared to the parental anti-TIM3 antibody TIM3_0038, see Figure 9 E and 9F) while retaining strong binding to CD4+ T cells.

[0145] Figure 10 : Figure 10 A to 10D: Bispecific 1+1 PD1 TIM3-0166 (based on chimeric PD1-0103 / TIM3-0038) shows reduced internalization on activated CD4+ T cells and on activated NK cells compared to bispecific 2+2 PD1 TIM3-0321 (also based on chimeric PD1-0103 / TIM3-0038, but with two antigen binding sites for PD and two for TIM3) and compared to the parental TIM3-0038 antibody

[0146] Figure 11A : Analysis over time showed higher membrane localization of both bispecific and PD1 antibodies compared to intracellular clustering of TIM3 antibodies.

[0147] Antibody names in the figure: TIM3 (chi18-A647 = chimeric TIM3_0018 labeled with AlexaA647), a-TIM3 (chi28-A647 = chimeric TIM3_0028 labeled with AlexaA647), Bispec (0168-A647 = 1+1PD1TIM3_0168 labeled with AlexaA647 (based on chimeric PD1-0103 / TIM3-0018)), Bispec (0389-A647 = 1+1PD1TIM3_0389 labeled with Alexa 647 (based on chimeric PD1-0103 / TIM3-0028)) and a-PD1 (0165-A488 = chimeric PD1-0103 labeled with Alexa488).

[0148] Anti-PD1 and the bispecific 1+1 PD1 TIM3_0389 (Bispec 0389) showed only very slow internalization, even after 3 hours, while another bispecific 1+1 PD1 TIM3_0168 (Bispec 0168) internalized more strongly. aTIM3 Ab 0028 showed even stronger internalization, and aTIM3-0018 showed the least internalization.

[0149] Figure 11B Chimeric PD1-0103 (aPD1-0165) showed only poor internalization, while high-affinity chimeric TIM3_0018 (aTim3-chi18) was strongly internalized after TIM3 binding, even after 15 minutes. Internalization of the low-affinity binder, chimeric TIM3_0028 (aTIM3-chi28), was slightly reduced. Bispecific 1+1 AB 0168 (composed of high-affinity binder aPD1-0165 and high-affinity aTIM3-0018) showed a greater reduction in internalization. Bispecific 1+1 AB 0389 (composed of high-affinity binder chimeric PD1-0103 (aPD1-0165) and low-affinity chimeric TIM3_0028 (aTIM3-0028)) showed a very strong reduction in internalization. This is likely due to bivalent binding to PD1 and TIM3, where high-affinity binding to PD1 retains the antibody on the cell surface.

[0150] Figure 12A : PD1-TIM3 bispecific antibody 1+1 PD1TIM3_0168 (based on the efficacy of chimeric PD1-0103 / TIM3-0018 (=AB 0168) compared with chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0018 (=TIM3-chi18) and their combinations.

[0151] Figure 12B : PD1-TIM3 bispecific antibody 1+1 PD1TIM3_0389 (based on the efficacy of chimeric PD1-0103 / TIM3-0028 (=Bispec AB 0389) compared with chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0028 (=TIM3-chi28) and their combinations.

[0152] Figure 12C : The efficacy of the PD1-TIM3 bispecific antibody 1+1 PD1-0103 / Ky8213 (generated similarly to the description of 1+1 CrossMab in Example 1 based on chimeric PD1-0103 / anti-TIM3 Ky8213 from US20120189617 (see antibody 8213, e.g., Example 33)) was compared with chimeric PD1-0103 (= PD1-0165) and anti-TIM3-Ky8213 (from US20120189617 (see antibody 8213, e.g., Example 33)) and their combinations.

[0153] Figure 12D: Efficacy of the PD1-TIM3 bispecific antibody 1+1 PD1TIM3_0389 (based on chimeric PD1-0103 / TIM3-0028 (=Bispec AB 0389 (1+1))) compared to the PD1-TIM3 bispecific antibody 2+2 PD1TIM3_0358 (based on chimeric PD1-0103 / TIM3-0028 (=Bispec AB 0358 (2+2))), and chimeric PD1-0103 (=PD1-0165) and chimeric TIM3_0028 (=TIIM3-chi28) and their combinations.

[0154] Figure 13 Treatment with the PD1-TIM3 bispecific antibody 1+1 PD1TIM3_0476 significantly increased the ability of CD4 T cells to release IFN-γ compared to treatment with either PD1 or TIM3 antibodies alone, and even compared to treatment with a combination of the parental antibody PD1_0376 and the antibody TIM3_0438. CD4 T cells were co-cultured with tumor cell lines expressing MHC II. The PD1-TIM3 bispecific antibody 1+1 PD1TIM3_0476 was tested against the PD1 antibodies aPD1_0376, MDX-1106 (nivolumab), and MK-3475 (pembrolizumab), against the TIM3 antibodies aTIM3_0438 and Kyowa-8213 (as disclosed in WO 2011 / 155697), and against a combination of the anti-PD1 antibody aPD1-0376 and the anti-TIM3 antibody aTIM3_0438.

[0155] Figure 14 : Figure 14 A and 14B: Results of efficacy experiments comparing PD1-TIM3 bispecific antibody 1+1 (0476) with either PD1 or TIM3 antibody alone in immunosuppressed female mice (NOG) challenged with MKN45 cells and supplied with PBMCs from healthy human donors. Figure 14 A and 14B. The graph presents the measured mean tumor size (within the treatment group) over a 30-day period, including the standard error of the mean tumor size. The curve with solid circles corresponds to the growth of tumor size without treatment (vehicle). Figure 14 A shows tumor growth at lower doses (1.5 mg / kg antibody PD1_0376, 1.5 mg / kg nivolumab, 1.5 mg / kg antibody TIM3_0438 or 3 mg / kg bispecific antibody 1+1 PD1TIM3_0476). Figure 14Tumor growth at higher doses (5 mg / kg antibody PD1_0376, 5 mg / kg nivolumab, 5 mg / kg antibody Tim3_0438 or 10 mg / kg bispecific antibody 1+1 PD1TIM3_0476) is shown in B. Detailed Description of the Invention

[0156] definition

[0157] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly used in the art to which the invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0158] As used herein, the term "antigen binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. Examples of antigen binding molecules are antibodies, antibody fragments and scaffold antigen binding proteins.

[0159] The term "antibody" is used herein in the broadest sense to encompass various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific and multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0160] The term "monoclonal antibody" as used herein refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies comprising the group are identical and / or bind to the same epitope, with the exception of possible variant antibodies, e.g., antibodies containing naturally occurring mutations or produced during the production of a monoclonal antibody preparation, which variants are typically present in small amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0161] As used herein, the term "monospecific" antibody refers to an antibody with one or more binding sites, each of which is bound to the same epitope of the same antigen. The term "bispecific" means that an antibody can specifically bind to at least two different antigenic determinants, for example, two binding sites formed by a pair of antibody heavy chain variable domains (VH) and antibody light chain variable domains (VL) that are each free to bind to different epitopes on different antigens or the same antigen. Such bispecific antibodies are 1+1 formats. Other bispecific antibody formats are 2+1 formats (comprising two binding sites for a first antigen or epitope and one binding site for a second antigen or epitope) or 2+2 formats (comprising two binding sites for a first antigen or epitope and two binding sites for a second antigen or epitope). Typically, a bispecific antibody comprises two antigen binding sites, and each antigen binding site is specific for different antigenic determinants.

[0162] The term "valence" as used in this application means the presence of a specific number of binding sites in an antigen binding molecule. Because of this, the terms "bivalent", "tetravalent" and "hexavalent" mean that there are two binding sites, four binding sites and six binding sites in an antigen binding molecule, respectively. The bispecific antibodies according to the present invention are at least "bivalent" and can be "trivalent" or "multivalent" (e.g., "tetravalent" or "hexavalent"). In a specific aspect, the antibody of the present invention has two or more binding sites and is bispecific. That is, the antibody can be bispecific, even in the case of having more than two binding sites (i.e., the antibody is tetravalent or multivalent). In particular, the present invention relates to bispecific bivalent antibodies with one binding site for each antigen to which they specifically bind.

[0163] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein and refer to antibodies with a structure that is substantially similar to the structure of natural antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG class antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two light chains and two heavy chains bound by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also referred to as a heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also referred to as a light chain constant region. Antibody heavy chains can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further divided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Antibody light chains can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0164] "Antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies, triabodies, tetrabodies, cross-Fab fragments; linear antibodies; single-chain antibody molecules (e.g., scFv); multispecific antibodies and single-domain antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see, for example, Plückthun, cited in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments having two antigen binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Hudson et al. also describe triabodies and tetrabodies in Nat Med 9, 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1). In addition, an antibody fragment comprises a single-chain polypeptide having VH domain characteristics or VL domain characteristics, wherein the VH domain characteristics are capable of assembling together with the VL domain into a functional antigen-binding site, wherein the VL domain characteristics are capable of assembling together with the VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0165] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the variable domains of the heavy and light chains, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain. Thus, as used herein, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains have free sulfhydryl groups. Pepsin treatment produces a F(ab')2 fragment having two antigen-binding sites (two Fab fragments) and a portion of the Fc region.

[0166] The term "crossover Fab fragment" or "xFab fragment" or "exchange Fab fragment" refers to a Fab fragment in which the variable regions or constant regions of the heavy and light chains are exchanged. Two different chain compositions of the exchange Fab molecule are possible and are included in the bispecific antibodies of the present invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the exchange Fab molecule comprises a peptide chain consisting of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain consisting of a heavy chain variable region (VH) and a light chain constant region (CL). Such an exchange Fab molecule is also called a CrossFab (VLVH) On the other hand, when the constant regions of the Fab heavy and light chains are exchanged, the exchange Fab molecule contains a peptide chain consisting of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain consisting of the light chain variable region (VL) and the heavy chain constant region (CH1). This exchange Fab molecule is also called CrossFab. (CLCH1) .

[0167] "Single-chain Fab fragments" or "scFab" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide having at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a natural disulfide bond between the CL domain and the CH1 domain. In addition, these single-chain Fab molecules can be further stabilized by inserting cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to create interchain disulfide bonds.

[0168] "Exchange single-chain Fab fragments" or "x-scFabs" are polypeptides consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH and VL together form an antigen-binding site that specifically binds to an antigen, and wherein the linker is a polypeptide having at least 30 amino acids. In addition, these x-scFab molecules can be further stabilized by the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering) to create interchain disulfide bonds.

[0169] A "single-chain variable fragment (scFv)" is an antibody heavy chain variable region (V) joined by a short linker peptide of 10 to about 25 amino acids. H ) and light chain variable region (V L The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can be V H The N-terminus of V L The antibody fragment is a fragment of a polypeptide that is cleaved from the C-terminus of the antibody or vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original antibody. ScFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. In addition, antibody fragments comprise single-chain polypeptides having characteristics of a VH domain, i.e., capable of assembling together with a VL domain into a functional antigen-binding site, or characteristics of a VL domain, i.e., capable of assembling together with a VH domain into a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0170] "Scaffold antigen binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigen binding domains, see, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13:695-701 (2008). In one aspect of the present invention, the scaffold antigen binding protein is selected from CTLA-4 (Evibody), lipocalin (Anticalin), a molecule derived from protein A, such as the Z domain of protein A (Affibody), the A domain (Avimer / Maxibody), serum transferrin (trans-body); a designed ankyrin repeat protein (DARPin), a variable domain of an antibody light chain or heavy chain (single domain antibody, sdAb), a variable domain of an antibody heavy chain (nanoantibody, aVH), a V NAR Fragment, fibronectin (AdNectin), C-type lectin domain (Tetranectin); novel antigen receptor β-lactamase variable domain (V NAR fragments), human γ-crystallin or ubiquitin (Affilin molecule); Kunitz-type domains of human protease inhibitors, microbodies such as proteins from the knottin family, peptide aptamers and fibronectin (adnectin).

[0171] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28 family receptor expressed primarily on CD4+ T cells. Its extracellular domain has an Ig-like variable domain fold. Loops corresponding to antibody CDRs can be substituted with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as evibodies (e.g., US Pat. No. 7,166,697 B1). Evibodies are approximately the same size as the isolated variable regions of antibodies (e.g., domain antibodies). For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001). Lipocalins are a family of extracellular proteins that transport small, hydrophobic molecules such as steroids, bilirubin, retinoids, and lipids. They have a rigid β-sheet secondary structure with multiple loops at the open end of the pyramidal structure that can be engineered to bind different target antigens. Anticalins range in size from 160 to 180 amino acids and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), US7250297B1 and US20070224633. Affimers are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. The domain consists of a three-helix bundle of approximately 58 amino acids. Libraries were generated by randomization of surface residues. For further details, see ProteinEng.Des.Sel.2004,17,455-462 and EP 1641818A1. Avimers are multidomain proteins derived from the A domain scaffold family. The native domain of approximately 35 amino acids adopts a defined disulfide bond structure. Diversity is generated by varying the natural variation exhibited by the A domain family. For further details, see Nature Biotechnology 23(12), 1556-1561(2005) and Expert Opinion on Investigational Drugs 16(6), 909-917(June 2007). Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind different target antigens by inserting peptide sequences in permissive surface loops. Examples of engineered transferrin scaffolds include Trans-bodies. For further details, see J.Biol.Chem 274, 24066-24073(1999). Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and a β-turn.By randomizing the residues in the first α-helix and β-turn of each repeat, they can be engineered to bind to different target antigens. Their binding interface can be increased by increasing the number of modules (affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100 (4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

[0172] Single domain antibodies are antibody fragments composed of a single monomeric variable antibody domain. The first single domain is derived from the variable domain of the antibody heavy chain from camelids (nanoantibodies or V H H fragment). In addition, the term single-domain antibody includes the autonomous human heavy chain variable domain (aVH) or VH derived from sharks. NAR Fragments. Fibronectin is a scaffold that can be engineered to bind to an antigen. Adnectins consist of a backbone composed of the native amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). The three loops at one end of the β-sandwich can be engineered to enable the adnectin to specifically recognize the therapeutic target of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), US20080139791, WO2005056764, and US6818418B1. Peptide aptamers are combinatorial recognition molecules composed of a constant backbone protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted in the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005). Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length that contain 3-4 cysteine ​​bridges—examples of microproteins include KalataBI and conotoxins and cysteine ​​knot peptides (knottins). Microproteins have loops that can be engineered to include up to 25 amino acids without affecting the overall folding of the microprotein. For more details of engineered cysteine ​​knot peptide domains, see WO2008098796.

[0173] An "antigen-binding molecule that binds to the same epitope as a reference molecule" refers to an antigen-binding molecule that blocks 50% or more of the binding of the reference molecule to its antigen in a competition assay, whereas conversely, the reference molecule blocks 50% or more of the binding of the antigen-binding molecule to its antigen in a competition assay.

[0174] As used herein, the term "antigen binding site" refers to a part of an antigen binding molecule that specifically binds to an antigenic determinant. More particularly, the term "antigen binding site" refers to a part of an antibody that includes a region that specifically binds to part or all of an antigen and is complementary. In the case of a large antigen, an antigen binding molecule may only bind to a specific portion of the antigen, which is called an epitope. The antigen binding site can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding site includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding site can bind to its antigen and block or partially block its function. The antigen binding site specifically binding to PD1 or TIM-3 includes antibodies and fragments thereof as further defined herein. In addition, the antigen binding site can include a scaffold antigen binding protein, such as a binding domain based on a designed repeat protein or a designed repeat domain (see, for example, WO 2002 / 020565).

[0175] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope", and refers to a site on the polypeptide macromolecule to which an antigen binding moiety is combined to form an antigen binding moiety-antigen complex (e.g., a continuous segment of amino acids or a conformational configuration consisting of different regions of discontinuous amino acids). Useful antigenic determinants can be found, for example, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other pathological cells, on the surface of immune cells, and are free in serum and / or in the extracellular matrix (ECM). Proteins used as antigens herein can be any native form of protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. In a specific embodiment, the antigen is a human protein. In the case of referring to a specific protein herein, the term includes "full-length" unprocessed protein and any form of protein produced by processing in cells. The term also encompasses naturally occurring variants of proteins, for example, splice variants or allelic variants.

[0176] "Specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific interactions. The ability of antigen binding molecules to bind to specific antigens can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, for example, surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17,323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)). In one embodiment, the degree of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the antigen binding molecule's binding to the antigen, as measured by, for example, SPR. In certain embodiments, the molecule bound to the antigen has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 -7 M or smaller, such as 10 -7 M to 10 -13 M, for example 10 -9 M to 10 -13 The dissociation constant (Kd) of the

[0177] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Therefore, equivalent affinities can comprise different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by conventional methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0178] As used herein, the term "high affinity" for an antibody refers to an antibody that has a 10 -9 M or smaller and even more special is 10 -10 The term "low affinity" for an antibody refers to an antibody with a Kd of 10 -8 or higher Kd.

[0179] An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

[0180] The terms “a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3,” “a bispecific antibody that specifically binds to PD1 and TIM-3,” and “a bispecific antigen-binding molecule specific for PD1 and TIM-3” are used interchangeably herein and refer to a bispecific antibody that is capable of binding to PD1 and TIM-3 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent in targeting PD1 and TIM-3.

[0181] The term "PD1", also known as programmed cell death protein 1, is a 288 amino acid type I membrane protein that was first described in 1992 (Ishida et al., EMBO J., 11 (1992), 3887-3895). PD-1 is a member of the expanded CD28 / CTLA-4 T cell regulator family and has two ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). The structure of the protein includes an extracellular IgV domain, followed by a transmembrane region and an intracellular tail. The intracellular tail contains two phosphorylation sites located in the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switching motif, suggesting that PD-1 negatively regulates TCR signals. This is consistent with the binding of SHP-1 and SHP-2 phosphatases to the cytoplasmic tail of PD-1 after ligand binding. Although PD-1 is not expressed on naive T cells, it is upregulated after T cell receptor (TCR)-mediated activation and is observed on both activated and exhausted T cells (Agata et al., Int. Immunology 8 (1996), 765-772). These exhausted T cells have a dysfunctional phenotype and are unable to respond appropriately. Although PD-1 has a relatively wide expression pattern, its most important role is probably as a co-inhibitory receptor on T cells (Chinai et al., Trends in Pharmacological Sciences 36 (2015), 587-595). Current treatment approaches therefore focus on blocking the interaction of PD-1 with its ligand to enhance T cell responses. The terms "programmed cell death 1", "programmed cell death 1", "protein PD-1", "PD-1", PD1", "PDCD1", "hPD-1" and "hPD-I" can be used interchangeably and include variants, isoforms, species homologs of human PD-1, and analogs having at least one common epitope with PD-1. The amino acid sequence of human PD1 is shown in UniProt (www.uniprot.org) accession number Q15116 (SEQ ID NO: 89).

[0182] The terms "anti-PD1 antibody" and "antibody comprising an antigen binding site that binds to PD1" refer to an antibody that is capable of binding to PD1, particularly a PD1 polypeptide expressed on the surface of a cell, with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting PD1. In one embodiment, the extent of binding of the anti-PD1 antibody to an unrelated, non-PD1 protein is less than about 10% of the binding of the antibody to PD1, as determined, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assay using a biosensor system such as In certain embodiments, the antigen binding protein that binds to human PD1 has a binding affinity for binding to human PD1 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M)K D In a preferred embodiment, the corresponding K D Values ​​were determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human PD1 (PD1-ECD) (for PD1 binding affinity). The term "anti-PD1 antibody" also encompasses bispecific antibodies capable of binding to PD1 and a second antigen.

[0183] The term "TIM3," an abbreviation for "T cell immunoglobulin and mucin domain-containing molecule 3," also known as TIM-3, HAVCR2, KIM-3, TIMD3, and FLJ14428, refers to a T helper cell type 1-specific cell surface protein that regulates macrophage activation and the severity of inflammatory conditions. TIM3 is also associated with cancer, particularly cancer stem cells. The nucleotide and protein sequences of TIM3 are known for many species. For example, the human amino acid sequence can be found under Uniprot accession number Q8TDQ0 (SEQ ID NO: 93). The human protein is characterized by an extracellular domain comprising an Ig-like domain and a mucin domain (further comprising O-linked and N-linked glycosylation sites), comprising approximately amino acids 22-202, a transmembrane domain (amino acids 203-223), and an intracellular (cytoplasmic) domain (amino acids 224-301). For the human TIM3 protein shown as SEQ ID NO: 93, the extracellular domain comprises approximately amino acids 22-202, the transmembrane domain comprises approximately amino acids 203-223, and the cytoplasmic domain comprises approximately amino acids 224- 301. The term "TIM3" includes variants, isoforms, species homologs, and analogs of human TIM3 that share at least one common epitope with TIM3.

[0184] The terms "anti-TIM3 antibody" and "antibody comprising an antigen binding site that binds TIM3" refer to antibodies that are capable of binding to TIM3, particularly a TIM3 polypeptide expressed on the surface of a cell, with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting TIM3. In one embodiment, the extent of binding of the anti-TIM3 antibody to an unrelated, non-TIM3 protein is less than about 10% of the binding of the antibody to TIM3, as determined, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance assay using a biosensor system such as In certain embodiments, the antigen binding protein that binds to human TIM3 has a binding affinity for binding to human TIM3 of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, such as 10 -7 M to 10 -13 M, for example 10 -9 M to 10 -13 M)K D In a preferred embodiment, the corresponding K D The values ​​were determined in a surface plasmon resonance assay using the extracellular domain (ECD) of human TIM3 (TIM3-ECD) (for TIM3 binding affinity). The term "anti-TIM3 antibody" also encompasses bispecific antibodies capable of binding to TIM3 and a second antigen.

[0185] "Blocking" antibodies or "antagonistic" antibodies inhibit or reduce the biological activity of the antigen to which it binds. In some embodiments, blocking antibodies or antagonistic antibodies substantially or completely inhibit the biological activity of the antigen. For example, the bispecific antibodies of the present invention block signaling via PD-1 and TIM-3, thereby restoring the functional response of T cells to antigen stimulation (e.g., proliferation, cytokine production, target cell killing) from a dysfunctional state.

[0186] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain involved in binding an antigen-binding molecule to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0187] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence and / or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs), the latter of which have the highest sequence variability and / or are involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Hypervariable regions (HVRs) are also called complementarity determining regions (CDRs), and these terms are used interchangeably herein to refer to portions of the variable region that form the antigen binding region. This particular region has been described by Kabat et al. in US Patent. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al. in J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared to each other. However, the application of any definition referring to the CDR of an antibody or its variants is expected to be within the scope of the terms defined and used herein. Appropriate amino acid residues encompassing the CDRs defined in each of the references cited above are listed in Table A below for comparison. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of an antibody, one skilled in the art can routinely determine which residues comprise a particular CDR.

[0188] Table A: CDR Definition 1

[0189] CDR Kabat Chothia <![CDATA[AbM 2 ]]> <![CDATA[V H CDR1]]> 31-35 26-32 26-35 <![CDATA[V H CDR2]]> 50-65 52-58 50-58 <![CDATA[V H CDR3]]> 95-102 95-102 95-102 <![CDATA[V L CDR1]]> 24-34 26-32 24-34 <![CDATA[V L CDR2]]> 50-56 50-52 50-56 <![CDATA[V L CDR3]]> 89-97 91-96 89-97

[0190] 1 The numbering of all CDR definitions in Table A follows the numbering convention proposed by Kabat et al. (see below).

[0191] 2 "AbM" with a lowercase "b" as used in Table A refers to CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

[0192] Kabat et al. also defined a numbering system that can be applied to the variable region sequence of any antibody. One of ordinary skill in the art can clearly assign this "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system proposed by Kabat et al. in US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise indicated, reference to specific amino acid residue position numbers in antibody variable regions is based on the Kabat numbering system.

[0193] In addition to the CDR1 in VH, CDRs generally include amino acid residues that form hypervariable loops. CDRs also include "specificity determination residues" or "SDRs" that are residues that contact antigens. SDRs are included in CDR regions referred to as abbreviated CDRs (or a-CDRs). Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front.Biosci.13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues (e.g., FR residues) in the variable domain are numbered herein according to Kabat et al. above.

[0194] "Framework" or "FR" refers to the variable domain residues excluding the hypervariable region (HVR) residues. The variable domain FR is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences typically appear in the following order in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0195] For the purposes of this paper, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0196] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0197] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, several of which are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0198] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of the variable domains in at least one (typically two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has been humanized. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been additionally modified or altered based on the constant region of the original antibody to produce properties according to the present invention, particularly those antibodies with respect to C1q binding and / or Fc receptor (FcR) binding.

[0199] A "human" antibody is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source utilizing human antibody libraries or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0200] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a group of substantially homogeneous antibodies, i.e., the individual antibodies constituting the group are identical and / or bind to the same epitope, except for example containing naturally occurring mutations or possible variant antibodies occurring during the generation of monoclonal antibody preparations, such variants generally exist in very small amounts. Unlike polyclonal antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a group of substantially homogeneous antibodies, and should not be interpreted as requiring antibodies to be generated by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be generated by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and such methods and other exemplary methods for generating monoclonal antibodies are described herein.

[0201] As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In particular, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. The amino acid sequence of the heavy chain is always presented with a C-terminal lysine, but variants without a C-terminal lysine are included in the present invention.

[0202] IgG Fc region comprises IgG CH2 and IgG CH3 domains. The "CH2 domain" of human IgG Fc region generally extends from the amino acid residue at about position 231 to the amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein can be a native sequence CH2 domain or a variant CH2 domain. The "CH3 domain" is included in the residue segment from the C-terminus to the CH2 domain in the Fc region (i.e., from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG). The CH3 region herein can be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain with a "protrusion" ("section") introduced in one chain and a corresponding "cavity" ("hole") introduced in the other chain; see U.S. Patent No. 5,821,333, which is expressly incorporated herein by reference). As described herein, such variant CH3 domains can be used to promote heterodimerization of two different antibody heavy chains. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0203] " Knot into hole " technology is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method includes introducing a protrusion ("knot") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide so that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing the small amino acid side chains at the interface of the first polypeptide with larger side chains (such as tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (such as alanine or threonine), a compensatory cavity with the same or similar size as the protrusion is generated in the interface of the second polypeptide. Protrusions and cavities can be produced by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A, and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit of the Fc domain comprising the knob modification further comprises the amino acid substitution S354C, and the subunit of the Fc domain comprising the hole modification further comprises the amino acid substitution Y349C. The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0204] "A region equivalent to an immunoglobulin Fc region" is intended to include naturally occurring allelic variants of the immunoglobulin Fc region, as well as variants that have produced substitutions, additions, or deletions but do not substantially reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular toxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the immunoglobulin Fc region without substantial loss of biological function. These variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, for example, Bowie, JU et al., Science 247: 1306-10 (1990)).

[0205] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptor), and B cell activation.

[0206] An "activating Fc receptor" is an Fc receptor that, upon engagement by the Fc region of an antibody, elicits a signaling event that stimulates cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). A specific activating Fc receptor is human FcγRIIIa (see UniProt accession number P08637, version 141).

[0207] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S) n , (SG4) n or G4 (SG4) n Peptide linkers, wherein "n" is generally a number between 1 and 10, usually 2 to 4, especially 2.

[0208] By "fused" or "linked" is meant that the components (eg, the antigen binding site and the Fc domain) are linked directly by peptide bonds or via one or more peptide linkers.

[0209] The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0210] "Percentage (%) of amino acid sequence identity relative to a reference polypeptide (protein) sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after alignment and introduction of gaps (if necessary, to achieve maximum sequence identity, and without considering any conservative substitutions as part of sequence identity). Alignment for determining percentages of amino acid sequence identity can be achieved with various methods well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN.SAWI or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences. However, for the purposes of this article, amino acid sequence identity % values ​​were generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, and the source code has been filed with the U.S. Copyright Office (US Copyright Office, Washington DC, 20559) along with user files, and is registered in the U.S. Copyright Office with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. in South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and are not changed. When ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A relative to (with, or to) a given amino acid sequence B (or alternatively, the % amino acid sequence identity that a given amino acid sequence A has or contains relative to (with, or to) a given amino acid sequence B) is calculated as follows:

[0211] Multiply 100 by the fraction X / Y

[0212] wherein X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in a program alignment of A and B, and wherein Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein were obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0213] In certain aspects, amino acid sequence variants of the bispecific antibodies of the present invention provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the bispecific antibody. Amino acid sequence variants of bispecific antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the molecule, or by peptide synthesis. These modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct has the desired characteristics, such as antigen binding. The sites of interest for substitution-type mutagenesis include HVRs and frameworks (FRs). Conservative substitutions are provided under the heading "Preferred Substitutions" in Table B and are further described below with reference to amino acid side chain categories (1) to (6). Amino acid substitutions can be introduced into the molecule of interest and screened for products with the desired activity, such as retained / enhanced antigen binding, reduced immunogenicity, or enhanced ADCC or CDC.

[0214] Table B

[0215]

[0216]

[0217] Amino acids can be grouped according to common side chain properties:

[0218] (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

[0219] (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

[0220] (3) Acidic: Asp, Glu;

[0221] (4) Basic: His, Lys, Arg;

[0222] (5) Residues that affect chain orientation: Gly, Pro;

[0223] (6) Aromatic: Trp, Tyr, Phe.

[0224] Non-conservative substitutions entail exchanging a member of one of these classes for another class.

[0225] The term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are present in one or more hypervariable region residues of a parent antigen binding molecule (e.g., humanized or human antibody). Typically, the resulting variants selected for further study will have changes (e.g., improvements) and / or will have certain biological properties of the parent antigen binding molecule substantially retained in terms of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antigen binding molecule. Exemplary substitution variants are affinity matured antibodies, which can be readily produced using, for example, affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated, and the variant antigen binding molecules are displayed on phage and screened for specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions can be carried out in one or more HVRs, as long as these changes do not substantially reduce the ability of the antigen binding molecule to bind to antigens. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in HVR. A useful method for identifying antibody residues or regions that may be targeted for mutagenesis is referred to as "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that demonstrate functional sensitivity for the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antigen binding molecule complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they possess desired characteristics.

[0226] Amino acid sequence insertions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, as well as intersequence insertions of single or multiple amino acid residues. Examples of terminal insertions include bispecific antibodies with an N-terminal methionyl residue. Other insertion variants of the molecule include fusions to the N-terminus or C-terminus of a polypeptide to increase the serum half-life of the bispecific antibody.

[0227] In some aspects, the bispecific antibodies provided herein are changed to increase or reduce the degree to which the antibody is glycosylated. By changing the amino acid sequence to produce or remove one or more glycosylation sites, the glycosylation variants of the molecule can be easily obtained, for example, the carbohydrate attached to the Fc domains can be changed. The natural antibodies produced by mammalian cells generally include the biantennary oligosaccharides of the side chains, which are generally connected to the Asn297 of the CH2 domains in the Fc region by an N bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose attached to the GlcNAc in the " stem " of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modification can be carried out in the bispecific antibodies of the present invention to produce variants with some improved properties. In one aspect, variants of bispecific antibodies with a carbohydrate structure lacking the fucose attached (directly or indirectly) to the Fc region are provided. Such fucosylation variants may have enhanced ADCC function, see, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.) or US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Other variants of the bispecific antibodies of the present invention include those with bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. These variants may have reduced fucosylation and / or enhanced ADCC function, see, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have enhanced CDC function and are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0228] In some aspects, it may be desirable to produce a cysteine ​​engineered variant of the bispecific antibody of the present invention, such as "thioMAb", in which one or more residues of the molecule are replaced by cysteine ​​residues. In a specific embodiment, the substituted residue appears in an accessible position of the molecule. By replacing those residues with cysteine, reactive thiol groups are thus positioned at accessible sites of the antibody, and can be used to conjugate the antibody with other modules (such as drug modules or linker-drug modules) to produce immunoconjugates. In certain embodiments, any one or more of the following residues can be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antigen binding molecules can be produced according to the description in, for example, U.S. Patent No. 7,521,541.

[0229] In some aspects, the bispecific antibodies provided herein can be further modified to contain other non-protein modules known in the art and readily available. Modules suitable for antibody derivatization include but are not limited to water-soluble polymers. Non-limiting examples of water-soluble polymers include but are not limited to polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polyoxypropylene / ethylene oxide copolymers, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. Polymer can have any molecular weight and can be side chain or non-side chain. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be identical or different molecules. Generally, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the bispecific antibody derivative is to be used therapeutically under defined conditions, etc.

[0230] In another aspect, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam, NW et al., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). The radiation can be of any wavelength, including but not limited to wavelengths that do not damage normal cells, but that heat the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

[0231] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0232] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide can contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds found in peptide nucleic acids (PNA)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0233] With respect to "isolated" nucleic acid molecules or polynucleotides, it is meant a nucleic acid molecule, DNA or RNA, that has been removed from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered to be isolated for the purposes of the present invention. Other examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially purified) polynucleotides in solution form. Isolated polynucleotides include polynucleotide molecules contained in cells that normally contain polynucleotide molecules, but the polynucleotide molecules exist extrachromosomally or at a chromosomal position different from their natural chromosomal position. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention also include such molecules produced synthetically. In addition, the polynucleotides or nucleic acids can be or can include regulatory elements, such as promoters, ribosome binding sites or transcription terminators.

[0234] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to 5 point mutations per every 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or, alternatively, multiple nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These alterations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed independently between residues in the reference sequence or in a group or multiple consecutive groups within the reference sequence. In practice, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention can be routinely determined using known computer programs such as ALIGN-2, as discussed above for polypeptides.

[0235] The term "expression cassette" refers to a recombinant or synthetically produced polynucleotide having a series of specific nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, in addition to other sequences, a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette of the present invention comprises a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0236] The term "vector" or "expression vector" is synonymous with "expression construct" and refers to a DNA molecule used to introduce a specific gene operably associated therewith into a target cell and to direct the expression of the specific gene. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of the host cell into which it has been introduced. The expression vectors of the present invention include expression cassettes. The expression vectors allow the transcription of large amounts of stable mRNA. Once the expression vector enters the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cell's transcription and / or translation machinery. In one embodiment, the expression vector of the present invention includes an expression cassette comprising a polynucleotide sequence encoding a bispecific antigen binding molecule of the present invention or a fragment thereof.

[0237] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acids have been introduced, including the progeny of these cells. Host cells include "transformants" and "transformed cells," which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. Progeny may not be completely identical to the parent cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected in the original transformed cells are included herein. A host cell is any type of cell system that can be used to produce the bispecific antigen binding molecules of the present invention. In particular, the host cell is a prokaryotic or eukaryotic host cell. Host cells include cultured cells, such as cultured mammalian cells, such as CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name a few, as well as cells included in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0238] An "effective amount" of an agent is that amount necessary to cause a physiological change in the cells or tissues to which it is administered.

[0239] A "therapeutically effective amount" of a pharmaceutical agent (e.g., a pharmaceutical composition) refers to an amount effective to achieve the desired therapeutic or preventive result, at dosages and for periods of time. For example, a therapeutically effective amount of an agent eliminates, reduces, delays, minimizes, or prevents the adverse effects of a disease.

[0240] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0241] The term "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the preparation is administered.

[0242] "Pharmaceutically acceptable excipient" refers to an ingredient in a pharmaceutical composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, or preservatives.

[0243] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0244] As used herein, "treatment" (and grammatical variations thereof) refers to a clinical intervention that attempts to alter the natural course of a disease in a treated individual, and can be used prophylactically or during clinical pathology. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis. In some embodiments, the molecules of the invention are used to delay the development of a disease or slow the progression of a disease.

[0245] As used herein, the term "cancer" refers to a proliferative disease, such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung cancer (NSCL), bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the stomach, colon cancer, breast cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, hepatocellular carcinoma, biliary tract cancer, central nervous system (CNS) tumors, chordoma, brain stem glioma, glioblastoma multiforme, astrocytoma, neurilemoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

[0246] Bispecific antibodies of the present invention

[0247] The present invention provides novel bispecific antibodies comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3, having particularly advantageous properties such as manufacturability, stability, binding affinity, biological activity, specific targeting of certain T cells, targeting efficiency, and reduced toxicity. In particular, these are bispecific antibodies that bind to PD1 with high affinity and to TIM3 with low affinity.

[0248] A. Exemplary Bispecific Antibodies Binding PD1 and TIM-3

[0249] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein

[0250] The first antigen binding site that specifically binds to PD1 comprises

[0251] The VH domain comprises

[0252] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0253] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0254] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0255] The VL domain contains

[0256] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40;

[0257] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0258] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0259] And the second antigen binding site that specifically binds to TIM3 comprises

[0260] (a) VH domain, which comprises

[0261] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0262] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and

[0263] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0264] The VL domain contains

[0265] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11 or SEQ ID NO: 12,

[0266] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0267] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0268] (b) a VH domain comprising

[0269] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0270] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0271] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0272] The VL domain contains

[0273] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0274] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0275] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or

[0276] (c) a VH domain comprising

[0277] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29,

[0278] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0279] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31; and

[0280] The VL domain contains

[0281] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32,

[0282] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and

[0283] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0284] In one specific aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein

[0285] The first antigen binding site that specifically binds to PD1 comprises

[0286] The VH domain comprises

[0287] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0288] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0289] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0290] The VL domain contains

[0291] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40;

[0292] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0293] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0294] And the second antigen binding site that specifically binds to TIM3 comprises

[0295] The VH domain comprises

[0296] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0297] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and

[0298] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0299] The VL domain contains

[0300] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 12,

[0301] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0302] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6.

[0303] In another specific aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein

[0304] The first antigen binding site that specifically binds to PD1 comprises

[0305] The VH domain comprises

[0306] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0307] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0308] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0309] The VL domain contains

[0310] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40;

[0311] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0312] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0313] And the second antigen binding site that specifically binds to TIM3 comprises

[0314] The VH domain comprises

[0315] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0316] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0317] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0318] The VL domain contains

[0319] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0320] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0321] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0322] In yet another aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein

[0323] The first antigen binding site that specifically binds to PD1 comprises

[0324] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 43 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or

[0325] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or

[0326] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or

[0327] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or

[0328] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49,

[0329] And the second antigen binding site that specifically binds to TIM3 comprises

[0330] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, or

[0331] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, or

[0332] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, or

[0333] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or

[0334] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or

[0335] (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or

[0336] (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0337] (h) a VH domain comprising the amino acid sequence of SEQ ID NO: 35 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

[0338] In another aspect, the bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3 is a human, humanized or chimeric antibody. In particular, it is a humanized antibody.

[0339] In one aspect, provided is a humanized, bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein

[0340] The first antigen binding site that specifically binds to PD1 comprises

[0341] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or

[0342] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or

[0343] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or

[0344] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49,

[0345] And the second antigen binding site that specifically binds to TIM3 comprises

[0346] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, or

[0347] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or

[0348] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or

[0349] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.

[0350] In a specific aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, and the second antigen-binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16 or a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0351] In particular, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein

[0352] The first antigen binding site that specifically binds to PD1 comprises

[0353] The VH domain comprises

[0354] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0355] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0356] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0357] The VL domain contains

[0358] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40;

[0359] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0360] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0361] And the second antigen binding site that specifically binds to TIM3 comprises

[0362] The VH domain comprises

[0363] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0364] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0365] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0366] The VL domain contains

[0367] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0368] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0369] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22.

[0370] More particularly, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and the second antigen-binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0371] More specifically, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, and the second antigen-binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0372] In yet another aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 and SEQ ID NO: 49, and the second antigen-binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.

[0373] In one aspect, the bispecific antibody comprising a first antigen binding site that specifically binds PD1 and a second antigen binding site that specifically binds TIM3 is bivalent. This means that the bispecific antibody comprises one antigen binding site that specifically binds PD1 and one antigen binding site that specifically binds TIM3 (1+1 format).

[0374] In another aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with low affinity and binds to PD1 with high affinity. In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with a binding affinity that is at least 50 times lower than that of the binding to PD1, more particularly at least 100 times lower than that of the binding to PD1. In a preferred embodiment, the binding affinity (K D ) is determined using a surface plasmon resonance assay (e.g., as described in Example 12).

[0375] In one aspect, provided herein is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein

[0376] The first antigen binding site that specifically binds to PD1 with high affinity comprises

[0377] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 43 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or

[0378] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or

[0379] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or

[0380] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or

[0381] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49,

[0382] And the second antigen binding site that specifically binds to TIM3 with low affinity comprises

[0383] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or

[0384] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or

[0385] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28.

[0386] In a specific aspect, provided is a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the first antigen-binding site that specifically binds to PD1 with high affinity comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, and the second antigen-binding site that specifically binds to TIM3 with low affinity comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0387] In another aspect, the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising the antigen binding site that specifically binds to PD1, and a second Fab fragment comprising the antigen binding site that specifically binds to TIM3.

[0388] Particularly, the Fc domain is an IgG domain, more particularly an IgG1 Fc domain or an IgG4 Fc domain.

[0389] In another aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising

[0390] (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 50, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 52,

[0391] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 51, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 53, or

[0392] (b) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 54, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 56,

[0393] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 55, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 57, or

[0394] (c) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 58, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 60,

[0395] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 59, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 61, or

[0396] (d) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 62, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 64,

[0397] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 63, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 65, or

[0398] (e) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 66, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 68,

[0399] A second heavy chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:67, and a second light chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:69.

[0400] In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising

[0401] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 50, and a first light chain comprising the amino acid sequence of SEQ ID NO: 52,

[0402] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a second light chain comprising the amino acid sequence of SEQ ID NO: 53, or

[0403] (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 54, a first light chain comprising the amino acid sequence of SEQ ID NO: 56,

[0404] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and a second light chain comprising the amino acid sequence of SEQ ID NO: 57, or

[0405] (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 58, a first light chain comprising the amino acid sequence of SEQ ID NO: 60,

[0406] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61, or

[0407] (d) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 62, a first light chain comprising the amino acid sequence of SEQ ID NO: 64,

[0408] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63, and a second light chain comprising the amino acid sequence of SEQ ID NO: 65, or

[0409] (e) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a first light chain comprising the amino acid sequence of SEQ ID NO: 68,

[0410] A second heavy chain comprising the amino acid sequence of SEQ ID NO:67, and a second light chain comprising the amino acid sequence of SEQ ID NO:69.

[0411] More specifically, the present invention provides a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 62, a first light chain comprising the amino acid sequence of SEQ ID NO: 64, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63, and a second light chain comprising the amino acid sequence of SEQ ID NO: 65.

[0412] In another specific aspect, provided is a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a first light chain comprising the amino acid sequence of SEQ ID NO: 68, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a second light chain comprising the amino acid sequence of SEQ ID NO: 69.

[0413] In another aspect, the bispecific antibody comprising a first antigen binding site that specifically binds PD1 and a second antigen binding site that specifically binds TIM3 is tetravalent. In one aspect, the bispecific antibody comprises two antigen binding sites that specifically bind PD1 and two antigen binding sites that specifically bind TIM3 (2+2 format).

[0414] In one aspect, the bispecific antibody of the invention comprises

[0415] (a) two light chains and two heavy chains of an antibody comprising two Fab fragments comprising an antigen binding site that specifically binds to TIM3, and

[0416] (b) two additional Fab fragments comprising an antigen binding site that specifically binds to PD1, wherein each of the additional Fab fragments is linked to the C-terminus of the heavy chain of (a) via a peptide linker.

[0417] In a specific aspect, the peptide linker is (G4S)4. In another aspect, the two additional Fab fragments comprising an antigen binding site that specifically binds to PD1 are crossover Fab fragments in which the variable domains VL and VH are replaced with each other and the VL-CH chains are each connected to the C-terminus of the heavy chain of (a) via a peptide linker.

[0418] In a specific aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, comprising

[0419] (a) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 70, a first light chain comprising the amino acid sequence of SEQ ID NO: 71, and a second light chain comprising the amino acid sequence of SEQ ID NO: 72, or

[0420] (b) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 73, a first light chain comprising the amino acid sequence of SEQ ID NO: 74, and a second light chain comprising the amino acid sequence of SEQ ID NO: 75, or

[0421] (c) two heavy chains each comprising the amino acid sequence of SEQ ID NO: 76, a first light chain comprising the amino acid sequence of SEQ ID NO: 77, and a second light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0422] Fc domain modifications that reduce Fc receptor binding and / or effector function

[0423] In certain aspects, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can be comprised of a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0424] The next section describes preferred aspects of the bispecific antigen binding molecules of the present invention comprising Fc domain modifications that reduce Fc receptor binding and / or effector function. In one aspect, the present invention relates to a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly Fcγ receptors. In particular, the Fc domain is of the human IgG1 subclass having amino acid mutations L234A, L235A, and P329G (numbering according to the Kabat EU index).

[0425] The Fc domain confers favorable pharmacokinetic properties to the bispecific antibodies of the present invention, including a long serum half-life, which contributes to good accumulation in target tissues and a favorable tissue-blood distribution ratio. However, at the same time, it may result in unwanted targeting of the bispecific antibodies of the present invention to cells expressing Fc receptors rather than preferred antigen-bearing cells. Thus, in specific embodiments, the Fc domain of the bispecific antibodies of the present invention exhibits reduced binding affinity for Fc receptors and / or reduced effector function compared to a native IgG Fc domain, particularly an IgG1 Fc domain or an IgG4 Fc domain. More particularly, the Fc domain is an IgG1 Fc domain.

[0426] In one such aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising the Fc domain) exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the binding affinity for Fc receptors compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain), and / or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5% of the effector function compared to a native IgG1 Fc domain (or a bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain). In one aspect, the Fc domain (or the bispecific antigen binding molecule of the invention comprising the Fc domain) does not substantially bind to an Fc receptor and / or induce effector function. In a specific aspect, the Fc receptor is an Fcγ receptor. In one aspect, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically human FcγRIIIa. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In one aspect, the effector function is one or more of CDC, ADCC, ADCP, and cytokine secretion. In a specific aspect, the effector function is ADCC. In one aspect, the Fc domain exhibits a binding affinity to the neonatal Fc receptor (FcRn) that is substantially similar to that of the native IgG1 Fc domain. Substantially similar binding to FcRn is achieved when the Fc domain (or the bispecific antigen binding molecule of the invention comprising said Fc domain) exhibits more than about 70%, particularly more than about 80%, more particularly more than about 90% of the binding affinity of a native IgG1 Fc domain (or the bispecific antigen binding molecule of the invention comprising a native IgG1 Fc domain) to FcRn.

[0427] In a specific aspect, the Fc domain is engineered to have reduced binding affinity for Fc receptors and / or reduced effector function compared to a non-engineered Fc domain. In a specific aspect, the Fc domain of the bispecific antigen binding molecule of the present invention comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to Fc receptors and / or effector function. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one aspect, the amino acid mutations reduce the binding affinity of the Fc domain to Fc receptors. In another aspect, the amino acid mutations reduce the binding affinity of the Fc domain to Fc receptors by at least 2 times, at least 5 times, or at least 10 times. In one aspect, the bispecific antigen binding molecule of the present invention comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to Fc receptors compared to a bispecific antibody of the present invention comprising a non-engineered Fc domain. In a specific aspect, the Fc receptor is an Fcγ receptor. In other aspects, the Fc receptor is a human Fc receptor. In one aspect, the Fc receptor is an inhibitory Fc receptor. In a specific aspect, the Fc receptor is an inhibitory human Fcγ receptor, more specifically human FcγRIIB. In some aspects, the Fc receptor is an activating Fc receptor. In a specific aspect, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa, most particularly human FcγRIIIa. Preferably, the binding to each of these receptors is reduced. In some aspects, the binding affinity to complement components, particularly the binding affinity to C1q, is also reduced. In one aspect, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or the bispecific antigen binding molecule of the present invention comprising the Fc domain) exhibits a non-engineered form of the Fc domain (or the bispecific antigen binding molecule of the present invention comprising the non-engineered form of the Fc domain) for FcRn binding affinity of more than about 70%, substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is retained. The Fc domain or the bispecific antigen binding molecule of the present invention comprising the Fc domain can exhibit such affinity of more than about 80% and even more than about 90%. In certain embodiments, the Fc domain of the bispecific antigen binding molecule of the present invention is engineered to have reduced effector function compared to the non-engineered Fc domain.The reduced effector function may include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling that induces apoptosis, reduced dendritic cell maturation, or reduced T cell priming.

[0428] Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant in which residues 265 and 297 are substituted with alanine ( U.S. Patent No. 7,332,581 ). Certain antibody variants with improved or reduced binding to FcRs have been described (see, e.g., U.S. Patent No. 6,737,056 ; WO 2004 / 056312 , and Shields, R.L. et al., J. Biol. Chem. 276 (2001) 6591-6604 ).

[0429] In one aspect of the invention, the Fc domain comprises amino acid substitutions at positions E233, L234, L235, N297, P331, and P329. In some aspects, the Fc domain comprises amino acid substitutions L234A and L235A ("LALA"). In one such embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In one aspect, the Fc domain comprises an amino acid substitution at position P329. In a more specific aspect, the amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises an amino acid substitution at position P329 and another amino acid substitution selected from the group consisting of E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A, and P329G ("P329G LALA"). The amino acid substitution combination "P329G LALA" almost completely eliminates Fcγ receptor binding of human IgG1 Fc domains, as described in PCT patent application No. WO 2012 / 130831 A1. The document also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function). Such antibodies are IgG1 with mutations L234A and L235A or with mutations L234A, L235A and P329G (numbering according to the EU index of Kabat et al, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0430] In one aspect, the bispecific antibodies of the invention comprise (all positions according to the EU index as in Kabat) (i) a homodimeric Fc region of human IgG1 subclass, optionally with the mutations P329G, L234A and L235A, or (ii) a homodimeric Fc region of human IgG4 subclass, optionally with the mutations P329G, S228P and L235E, or (iii) a homodimeric Fc region of human IgG1 subclass, optionally with the mutations P329G, L234A and L235E. A, L235A, I253A, H310A, and H435A, or optionally with the mutations P329G, L234A, L235A, H310A, H433A, and Y436A, or (iv) a heterodimeric Fc region wherein one Fc region polypeptide comprises the mutation T366W and the other Fc region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc region polypeptide comprises the mutations T366W and Y349C and the other (v) a heterodimeric Fc region of human IgG1 subclass, wherein both Fc region polypeptides comprise mutations P329G, L234A and L235A and one Fc region polypeptide comprises mutations T366W and S354C; (v) a heterodimeric Fc region of human IgG1 subclass, wherein both Fc region polypeptides comprise mutations P329G, L234A and L235A and one Fc region polypeptide comprises mutation T366W and S354C; 366W, and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or one of the Fc-region polypeptides comprises the mutations T366W and Y349C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or one of the Fc-region polypeptides comprises the mutations T366W and S354C, and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0431] In one aspect, the Fc domains are IgG4 Fc domains. In a more specific embodiment, the Fc domains are IgG4 Fc domains comprising amino acid substitutions at position S228 (Kabat numbering), specifically amino acid substitutions S228P. In a more specific embodiment, the Fc domains are IgG4 Fc domains comprising amino acid substitutions L235E and S228P and P329G. Such amino acid substitutions reduce the Fab arm exchange of IgG4 antibodies in vivo (see Stubenrauch et al., Drug Metabolism and Disposition 38, 84-91 (2010)). Thus, in one aspect, provided is a bispecific antibody comprising (all positions according to the EU index as in Kabat) a heterodimeric Fc-region of human IgG4 subclass, wherein both Fc-region polypeptides comprise the mutations P329G, S228P and L235E and one Fc-region polypeptide comprises the mutation T366W and the other Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or wherein one Fc-region polypeptide comprises the mutations T366W and Y349C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V, and S354C, or wherein one Fc-region polypeptide comprises the mutations T366W and S354C and the other Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C.

[0432] Antibodies with extended half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005 / 0014934. The neonatal Fc receptor (FcRn) is responsible for the transfer of maternal IgG to the fetus (Guyer, R. Let al., J. Immunol. 117 (1976) 587-593 and Kim, J. K. et al., J. Immunol. 24 (1994) 2429-2434). These antibodies comprise an Fc region with one or more substitutions that improve Fc region binding to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ). See also Duncan, AR and Winter, G., Nature 322 (1988) 738-740; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0433] The binding to Fc receptors can be easily determined, for example, by ELISA or by using surface plasmon resonance (SPR) of a standard instrument such as a BIAcore instrument (GE Healthcare), and Fc receptors can be obtained, for example, by recombinant expression. Suitable such binding assays are described herein. Alternatively, the binding affinity of Fc domains or cell-activating bispecific antigen binding molecules comprising Fc domains for Fc receptors can be assessed using a cell line known to express specific Fc receptors, such as human NK cells expressing Fc γ IIIa receptors. The effector functions of the bispecific antibodies of the present invention comprising Fc domains can be measured by methods known in the art. Suitable assays for measuring ADCC are described herein. Other examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al., Proc. Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assays can be used (see, e.g., ACTI TM non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); CytoTox Non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for this assay include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model disclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

[0434] Fc domain modifications that promote heterodimerization

[0435] The bispecific antigen binding molecule of the present invention comprises the different antigen binding sites that merge with one or another of the two subunits in the Fc domains, so the two subunits in the Fc domains can be included in two different polypeptide chains. The recombinant coexpression of these polypeptides and subsequent dimerization result in several possible combinations of two polypeptides. In order to improve the productive rate and the purity of the bispecific antibody of the present invention in recombinant production, it is therefore advantageous to introduce a modification that promotes the combination of the desired polypeptide in the Fc domains of the bispecific antigen binding molecule of the present invention.

[0436] Therefore, in a specific aspect, the present invention relates to a bispecific antibody comprising a first antigen-binding site that specifically binds PD1 and a second antigen-binding site that specifically binds TIM3, wherein the Fc domain comprises modifications that promote association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one aspect, the modifications are in the CH3 domain of the Fc domain.

[0437] In a specific aspect, the modification is a so-called "knob-into-hole" modification, comprising a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. Thus, the present invention relates to a bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein according to the knob-into-hole approach, the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole. In a specific aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0438] The "knot into cavity" technique is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al. Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method comprises introducing a protrusion ("knot") at the interface of a first polypeptide and introducing a corresponding cavity ("cavity") into the interface of a second polypeptide such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. Protrusions are constructed by replacing small amino acid side chains at the interface of a first polypeptide with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is generated at the interface of the second polypeptide.

[0439] Therefore, in one aspect, in the CH3 domains of the first subunit of the Fc domains of the bispecific antigen binding molecule of the present invention, amino acid residues are replaced with amino acid residues with larger side chain volume, thereby producing a projection in the cavity that can be positioned in the CH3 domains of the second subunit in the CH3 domains of the first subunit; and in the CH3 domains of the second subunit of the Fc domains, amino acid residues are replaced with amino acid residues with smaller side chain volume, thereby producing a cavity in which the projection in the CH3 domains of the first subunit can be positioned. Projections and cavities can be produced by changing the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis, or by peptide synthesis. In a specific aspect, in the CH3 domains of the first subunit of the Fc domains, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domains of the second subunit of the Fc domains, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one aspect, in the second subunit of the Fc domain, the threonine residue at position 366 is additionally replaced by a serine residue (T366S), and the leucine residue at position 368 is additionally replaced by an alanine residue (L368A).

[0440] In yet another aspect, in the first subunit of the Fc domains, the serine residue at position 354 is additionally replaced with a cysteine ​​residue (S354C), and in the second subunit of the Fc domains, the tyrosine residue at position 349 is additionally replaced with a cysteine ​​residue (Y349C). Introducing these two cysteine ​​residues results in the formation of a disulfide bridge between the two subunits of the Fc domains, which further stabilizes the dimer (Carter (2001), J Immunol Methods 248, 7-15). In a specific aspect, the first subunit of the Fc domains comprises amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domains comprises amino acid substitutions Y349C, T366S and Y407V (numbering is according to Kabat EU index).

[0441] However, alternatively or additionally, other knob-into-hole technologies may be used as described in EP 1 870 459. In one embodiment, the multispecific antibody comprises the mutations R409D and K370E in the CH3 domain of the "knob-chain" and the mutations D399K and E357K in the CH3 domain of the "hole-chain" (numbering according to the Kabat EU index).

[0442] In one aspect, the bispecific antibody comprises a T366W mutation in the CH3 domain of the “knuckle chain” and mutations T366S, L368A and Y407V in the CH3 domain of the “hole chain” and additionally mutations R409D and K370E in the CH3 domain of the “knuckle chain” and mutations D399K and E357K in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

[0443] In one aspect, the bispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains, or the multispecific antibody comprises the mutations Y349C and T366W in one of the two CH3 domains and the mutations S354C, T366S, L368A and Y407V in the other of the two CH3 domains and additionally the mutations R409D and K370E in the CH3 domain of the “knob chain” and the mutations D399K and E357K in the CH3 domain of the “hole chain” (numbering according to the Kabat EU index).

[0444] In an alternative aspect, modifications that promote association of the first and second subunits of the Fc domain include modifications that mediate electrostatic manipulation effects, for example, as described in PCT Publication No. WO 2009 / 089004. Generally, this approach involves replacing one or more amino acid residues at the interface of the two Fc domain subunits with charged amino acid residues such that homodimer formation is electrostatically unfavorable but heterodimerization is electrostatically favorable.

[0445] In addition to the "knob-into-hole technology", other technologies for modifying the CH3 domain of the heavy chain of a multispecific antibody to enhance heterodimerization are known in the art. These technologies are encompassed herein as alternatives to the "knob-into-hole technology" in combination with bispecific antibodies, particularly those described in WO 96 / 27011, WO 98 / 050431, EP 1870459, WO 2007 / 110205, WO 2007 / 147901, WO 2009 / 089004, WO 2010 / 129304, WO 2011 / 90754, WO 2011 / 143545, WO 2012 / 058768, WO 2013 / 157954 and WO 2013 / 096291.

[0446] In one aspect, in bispecific antibodies, heterodimerization of the first and second heavy chains of a multispecific antibody is supported using the approach described in EP 1870459. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3 / CH3 domain interface between the first and second heavy chains.

[0447] Thus, in this aspect, in the tertiary structure of the multispecific antibody, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain form an interface between the CH3 domains of the respective antibodies, wherein the amino acid sequence of the CH3 domain of the first heavy chain and the amino acid sequence of the CH3 domain of the second heavy chain both comprise a set of amino acids located within the interface in the tertiary structure of the antibody, wherein the first amino acid from the set of amino acids in the interface in the CH3 domain of one heavy chain is replaced with a positively charged amino acid and the second amino acid from the set of amino acids in the interface in the CH3 domain of the other heavy chain is replaced with a negatively charged amino acid. Bispecific antibodies according to this aspect are also referred to herein as "CH3 (+ / -) engineered bispecific antibodies" (wherein the abbreviation + / - represents the oppositely charged amino acids introduced in the respective CH3 domains).

[0448] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is selected from K, R and H, and the negatively charged amino acid is selected from E or D.

[0449] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is selected from K and R, and the negatively charged amino acid is selected from E or D.

[0450] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the positively charged amino acid is K and the negatively charged amino acid is E.

[0451] In one aspect, in the CH3(+ / -) engineered bispecific antibody, the amino acid R at position 409 is replaced with D and the amino acid K at position 370 is replaced with E in the CH3 domain of one heavy chain, and the amino acid D at position 399 is replaced with K and the amino acid E at position 357 is replaced with K in the CH3 domain of the other heavy chain (numbering is according to the Kabat EU index).

[0452] In one aspect, heterodimerization of the first and second heavy chains of a multispecific antibody is supported using the method described in WO 2013 / 157953. In one embodiment, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with K, and the amino acid L at position 351 in the CH3 domain of the other heavy chain is replaced with D (numbering according to the Kabat EU index). In another embodiment, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with K and the amino acid L at position 351 is replaced with K, and the amino acid L at position 351 in the CH3 domain of the other heavy chain is replaced with D (numbering according to the Kabat EU index).

[0453] In another aspect, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with K and the amino acid L at position 351 is replaced with K, and the amino acid L at position 351 in the CH3 domain of the other heavy chain is replaced with D (numbering according to the Kabat EU index). In addition, the CH3 domain of the other heavy chain comprises at least one of the following substitutions: amino acid Y at position 349 is replaced with E, amino acid Y at position 349 is replaced with D, and amino acid L at position 368 is replaced with E (numbering according to the Kabat EU index). In one embodiment, amino acid L at position 368 is replaced with E (numbering according to the Kabat EU index).

[0454] In one aspect, the method described in WO 2012 / 058768 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one aspect, the amino acid L at position 351 in the CH3 domain of one heavy chain is replaced with Y and the amino acid Y at position 407 is replaced with A, and the amino acid T at position 366 in the CH3 domain of the other heavy chain is replaced with A and the amino acid K at position 409 is replaced with F (numbering is according to the Kabat EU index). In another embodiment, in addition to the above-mentioned replacements, at least one of amino acid positions 411 (originally T), 399 (originally D), 400 (originally S), 405 (originally F), 390 (originally N) and 392 (originally K) is also replaced in the CH3 domain of the other heavy chain (numbering is according to the Kabat EU index). Preferred replacements are:

[0455] - replacing the amino acid T at position 411 with an amino acid selected from the group consisting of N, R, Q, K, D, E and W (numbering according to the KabatEU index),

[0456] - replacing the amino acid D at position 399 with an amino acid selected from the group consisting of R, W, Y, and K (numbering according to the Kabat EU index),

[0457] - replacing the amino acid S at position 400 with an amino acid selected from the group consisting of E, D, R and K (numbering according to the Kabat EU index),

[0458] - substitution of the amino acid F at position 405 with an amino acid selected from the group consisting of I, M, T, S, V and W (numbering according to the KabatEU index;

[0459] - substitution of the amino acid N at position 390 with an amino acid selected from the group consisting of R, K and D (numbering according to the Kabat EU index; and

[0460] - Substitution of the amino acid K at position 392 with an amino acid selected from the group consisting of V, M, R, L, F and E (numbering according to the KabatEU index).

[0461] In another aspect, a bispecific antibody is engineered according to WO 2012 / 058768, i.e., in the CH3 domain of one heavy chain, the amino acid L at position 351 is replaced with Y and the amino acid Y at position 407 is replaced with A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced with V and the amino acid K at position 409 is replaced with F (numbering according to the Kabat EU index). In another embodiment of the multispecific antibody, in the CH3 domain of one heavy chain, the amino acid Y at position 407 is replaced with A, and in the CH3 domain of the other heavy chain, the amino acid T at position 366 is replaced with A and the amino acid K at position 409 is replaced with F (numbering according to the Kabat EU index). In the last of the above embodiments, in the CH3 domain of the other heavy chain, the amino acid K at position 392 is replaced with E, and the amino acid T at position 411 is replaced with E, and the amino acid D at position 399 is replaced with R and the amino acid S at position 400 is replaced with R (numbering according to the Kabat EU index).

[0462] In one aspect, the method described in WO 2011 / 143545 is used to support heterodimerization of the first heavy chain and the second heavy chain of the multispecific antibody. In one aspect, amino acid modifications in the CH3 domains of the two heavy chains are introduced at positions 368 and / or 409 (numbering according to the Kabat EU index).

[0463] In one aspect, heterodimerization of the first and second heavy chains of a bispecific antibody is supported using the method described in WO 2011 / 090762. WO 2011 / 090762 relates to amino acid modifications according to the "knob-into-hole" (KiH) technique. In one embodiment, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with W, and the amino acid Y at position 407 in the CH3 domain of the other heavy chain is replaced with A (numbering is according to the Kabat EU index). In another embodiment, the amino acid T at position 366 in the CH3 domain of one heavy chain is replaced with Y, and the amino acid Y at position 407 in the CH3 domain of the other heavy chain is replaced with T (numbering is according to the Kabat EU index).

[0464] In one aspect, the method described in WO 2009 / 089004 is used to support heterodimerization of the first heavy chain and the second heavy chain of the bispecific antibody. In one embodiment, the amino acid K or N at position 392 in the CH3 domain of one heavy chain is replaced with a negatively charged amino acid (in one embodiment, with E or D, in a preferred embodiment, with D), and the amino acid D at position 399 in the CH3 domain of the other heavy chain, the amino acid E or D at position 356, or the amino acid E at position 357 is replaced with a positively charged amino acid (in one embodiment, with K or R, in a preferred embodiment, with K, in a preferred embodiment, the amino acid at position 399 or 356 is replaced with K) (numbering is according to the Kabat EU index). In a further embodiment, in addition to the above replacements, the amino acid K or R at position 409 in the CH3 domain of one heavy chain is replaced with a negatively charged amino acid (in one embodiment, with E or D, in a preferred embodiment, with D) (numbering is according to the Kabat EU index). In an even further aspect, in addition to or instead of the above substitutions, amino acid K at position 439 and / or amino acid K at position 370 in the CH3 domain of one heavy chain are each independently replaced with a negatively charged amino acid (in one embodiment, with E or D, in a preferred embodiment, with D) (numbering according to the Kabat EU index).

[0465] In one aspect, heterodimerization of the first and second heavy chains of a multispecific antibody is supported using the approach described in WO 2007 / 147901. In one embodiment, in the CH3 domain of one heavy chain, amino acid K at position 253 is replaced with E, amino acid D at position 282 is replaced with K, and amino acid K at position 322 is replaced with D, and in the CH3 domain of the other heavy chain, amino acid D at position 239 is replaced with K, amino acid E at position 240 is replaced with K, and amino acid K at position 292 is replaced with D (numbering according to the Kabat EU index).

[0466] The C-terminus of the heavy chain of the bispecific antibodies reported herein can be a complete C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C-terminal amino acid residues are removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG.

[0467] In one aspect of all aspects reported herein, the bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to the Kabat EU index). In one embodiment of all aspects reported herein, the bispecific antibody comprising a heavy chain comprising a C-terminal CH3 domain as specified herein comprises a C-terminal glycine residue (G446, numbering according to the Kabat EU index).

[0468] Modifications in the Fab domain

[0469] In one aspect, the present invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to TIM3, wherein in one of the Fab fragments, either the variable domains VH and VL or the constant domains CH1 and CL are exchanged. The bispecific antibody is prepared according to Crossmab technology.

[0470] Multispecific antibodies with domain replacement / swap in one binding arm (CrossMab VH-VL or CrossMab CH-CL) are described in detail in WO 2009 / 080252 and Schaefer, W. et al, PNAS, 108 (2011) 11187-1191. They clearly reduce side products caused by mispairing of a light chain directed against a first antigen with an incorrect heavy chain directed against a second antigen (compared to approaches without such domain swaps).

[0471] In a specific aspect, the present invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to TIM3, wherein in one of the Fab fragments, the variable domains VL and VH are replaced with each other so that the VH domain is part of the light chain and the VL domain is part of the heavy chain. In a specific aspect, the bispecific antibody is one in which the variable domains VL and VH are replaced with each other in the first Fab fragment comprising the antigen binding site that specifically binds to PD1.

[0472] In another aspect and to further improve correct pairing, a bispecific antibody comprising a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to TIM3 can contain different charged amino acid substitutions (so-called "charged residues"). These modifications are introduced in the cross-linked or non-cross-linked CH1 and CL domains.

[0473] In a specific aspect, the present invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to TIM3, wherein in one of the Fab fragments, the amino acid at position 124 in the constant domain CL is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index). In a specific aspect, the bispecific antibody is one wherein in the second Fab fragment comprising an antigen binding site that specifically binds to TIM3, the amino acid at position 124 in the constant domain CL is independently substituted with lysine (K), arginine (R), or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0474] In a specific aspect, the present invention relates to a bispecific antibody comprising a first Fab fragment that specifically binds to PD1 and a second Fab fragment that specifically binds to TIM3, wherein the amino acid at position 123 (EU numbering) in one of the CL domains is replaced with arginine (R) and the amino acid at position 124 (EU numbering) is replaced with lysine (K), and wherein the amino acids at position 147 (EU numbering) and position 213 (EU numbering) in one of the CH1 domains are replaced with glutamic acid (E). In a specific aspect, the bispecific antibody is one in which the amino acid at position 123 (EU numbering) in the second Fab fragment comprising an antigen-binding site that specifically binds to TIM3 is replaced with arginine (R) and the amino acid at position 124 (EU numbering) is replaced with lysine (K), and wherein the amino acids at position 147 (EU numbering) and position 213 (EU numbering) in one of the CH1 domains are replaced with glutamic acid (E).

[0475] In yet another aspect, the bispecific antibody is a bivalent antibody comprising

[0476] a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

[0477] b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other.

[0478] The antibody under a) does not contain the modification reported under b) and the heavy chain and the light chain under a) is an isolated chain.

[0479] In the antibody under b) within the light chain the variable light domain VL is replaced with the variable heavy domain VH of said antibody, and in the heavy chain the variable heavy domain VH is replaced with the variable light domain VL of said antibody.

[0480] In one aspect, (i) the amino acid at position 124 (numbering according to Kabat) in the constant domain CL of the first light chain under a) is substituted with a positively charged amino acid, and wherein the amino acid at position 147 or the amino acid at position 213 (numbering according to the Kabat EU index) in the constant domain CH1 of the first heavy chain under a) is substituted with a negatively charged amino acid, or (ii) the amino acid at position 124 (numbering according to Kabat) in the constant domain CL of the second light chain under b) is substituted with a positively charged amino acid, and wherein the amino acid at position 147 or the amino acid at position 213 (numbering according to the Kabat EU index) in the constant domain CH1 of the second heavy chain under b) is substituted with a negatively charged amino acid.

[0481] In another aspect, (i) the amino acid at position 124 in the constant domain CL of the first light chain under a) is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a preferred embodiment, with lysine (K) or arginine (R)), and wherein the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the first heavy chain under a) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index), or (ii) the amino acid at position 124 in the constant domain CL of the second light chain under b) is independently substituted with lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) (in a preferred embodiment, with lysine (K) or arginine (R)), and wherein the amino acid at position 147 or the amino acid at position 213 in the constant domain CH1 of the second heavy chain under b) is independently substituted with glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0482] In one aspect, the amino acids at positions 124 and 123 in the constant domain CL of the second heavy chain are substituted with K (numbering according to the Kabat EU index).

[0483] In one aspect, the amino acid at position 123 is substituted with R and the amino acid at position 124 is substituted with K in the constant domain CL of the second heavy chain (numbering according to the Kabat EU index).

[0484] In one aspect, the amino acids at positions 147 and 213 in the constant domain CH1 of the second light chain are substituted with E (numbering according to the EU index as in Kabat).

[0485] In one aspect, the amino acids at positions 124 and 123 in the constant domain CL of the first light chain are substituted with K, and the amino acids at positions 147 and 213 in the constant domain CH1 of the first heavy chain are substituted with E (numbering according to the Kabat EU index).

[0486] In one aspect, the amino acid at position 123 in the constant domain CL of the first light chain is substituted with R and the amino acid at position 124 is substituted with K, and in the constant domain CH1 of the first heavy chain the amino acids at positions 147 and 213 are both substituted with E (numbering according to the Kabat EU index).

[0487] In one aspect, the amino acids at positions 124 and 123 in the constant domain CL of the second heavy chain are replaced by K, and wherein in the constant domain CH1 of the second light chain the amino acids at positions 147 and 213 are replaced by E, and in the variable domain VL of the first light chain the amino acid at position 38 is replaced by K, the amino acid at position 39 in the variable domain VH of the first heavy chain is replaced by E, the amino acid at position 38 in the variable domain VL of the second heavy chain is replaced by K, and the amino acid at position 39 in the variable domain VH of the second light chain is replaced by E (numbering is according to the Kabat EU index).

[0488] In one aspect, the bispecific antibody is a bivalent antibody comprising

[0489] a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

[0490] b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the variable domains VL and VH of the second light chain and the second heavy chain are replaced with each other, and wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

[0491] The antibody under a) does not contain the modifications reported under b) and the heavy and light chains under a) are separated chains. In the antibody under b), in the light chain, the variable light chain domain VL is replaced with the variable heavy chain domain VH of the antibody, and the constant light chain domain CL is replaced with the constant heavy chain domain CH1 of the antibody; and in the heavy chain, the variable heavy chain domain VH is replaced with the variable light chain domain VL of the antibody, and the constant heavy chain domain CH1 is replaced with the constant light chain domain CL of the antibody.

[0492] In one aspect, the bispecific antibody is a bivalent antibody comprising

[0493] a) a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and

[0494] b) a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

[0495] The antibody under a) does not contain the reported modifications under b) and the heavy and light chains under a) are separate chains. In the antibody under b), the constant light chain domain CL in the light chain is replaced with the constant heavy chain domain CH1 of the antibody; and the constant heavy chain domain CH1 in the heavy chain is replaced with the constant light chain domain CL of the antibody.

[0496] In one aspect, the multispecific antibody is a multispecific antibody comprising

[0497] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0498] b) one, two, three or four single-chain Fab fragments that specifically bind to one to four further antigens (i.e. the second and / or third and / or fourth and / or fifth antigen, preferably specifically bind to one further antigen, i.e. the second antigen),

[0499] The single-chain Fab fragment under b) is fused to the full-length antibody under a) at the C- or N-terminus of the heavy or light chain of the full-length antibody via a peptide linker.

[0500] In one aspect, one or two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the heavy or light chain via a peptide linker.

[0501] In one aspect, one or two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the heavy chain via a peptide linker.

[0502] In one aspect, one or two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of the light chain via a peptide linker.

[0503] In one aspect, two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of each heavy or light chain via a peptide linker.

[0504] In one aspect, two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of each heavy chain via a peptide linker.

[0505] In one aspect, two identical single-chain Fab fragments that bind to a second antigen are fused to the full-length antibody at the C-terminus of each light chain via a peptide linker.

[0506] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0507] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains,

[0508] b) a first polypeptide consisting of:

[0509] ba) an antibody heavy chain variable domain (VH), or

[0510] bb) antibody heavy chain variable domain (VH) and antibody constant domain 1 (CH1),

[0511] wherein the first polypeptide is fused at the N-terminus of its VH domain to the C-terminus of one of the two heavy chains of the full-length antibody via a peptide linker,

[0512] c) a second polypeptide consisting of:

[0513] ca) antibody light chain variable domain (VL), or

[0514] cb) an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL),

[0515] The second polypeptide is fused to the C-terminus of the other heavy chain of the full-length antibody at the N-terminus of the VL domain via a peptide linker, and the antibody heavy chain variable domain (VH) of the first polypeptide and the antibody light chain variable domain (VL) of the second polypeptide together form an antigen binding site that specifically binds to the second antigen.

[0516] In one aspect, the antibody heavy chain variable domain (VH) of the polypeptide under b) and the antibody light chain variable domain (VL) of the polypeptide under c) are linked and stabilized via an interchain disulfide bridge by introducing a disulfide bond between the following positions:

[0517] (i) heavy chain variable domain position 44 versus light chain variable domain position 100, or

[0518] (ii) heavy chain variable domain position 105 versus light chain variable domain position 43, or

[0519] (iii) heavy chain variable domain position 101 versus light chain variable domain position 100 (numbering is according to the Kabat EU index throughout).

[0520] Techniques for introducing non-native disulfide bridges for stabilization are described, for example, in WO 94 / 029350; Rajagopal, V., et al., Prot. Eng. (1997) 1453-1459; Kobayashi, H., et al., Nucl. Med. Biol. 25 (1998) 387-393; and Schmidt, M., et al., Oncogene 18 (1999) 1711-1721. In one embodiment, the disulfide bond between the variable domains of the polypeptides under optional b) and c) is between position 44 of the heavy chain variable domain and position 100 of the light chain variable domain. In one embodiment, the disulfide bond between the variable domains of the polypeptides under optional b) and c) is between position 105 of the heavy chain variable domain and position 43 of the light chain variable domain (numbering is always according to Kabat). In one embodiment, trivalent, bispecific antibodies without disulfide stabilization between the variable domains VH and VL of said optional single chain Fab fragment are preferred.

[0521] In one aspect, the bispecific antibody is a trispecific or tetraspecific antibody comprising

[0522] a) a first light chain and a first heavy chain of a full-length antibody that specifically binds to a first antigen, and

[0523] b) a second (modified) light chain and a second (modified) heavy chain of a full-length antibody that specifically binds to a second antigen, wherein the variable domains VL and VH are replaced with each other, and / or wherein the constant domains CL and CH1 are replaced with each other, and

[0524] c) wherein one to four antigen-binding peptides that specifically bind to one or two additional antigens (ie, the third and / or fourth antigens) are fused to the C- or N-terminus of the light or heavy chain of a) and / or b) via a peptide linker.

[0525] The antibody under a) does not contain the modification reported under b) and the heavy chain under a) and the light chain are separated chains.

[0526] In one aspect, the trispecific or tetraspecific antibody comprises under c) one or two antigen-binding peptides that specifically bind to one or two additional antigens.

[0527] In one aspect, the antigen binding peptide is selected from the group of a scFv fragment and a scFab fragment.

[0528] In one aspect, the antigen binding peptide is a scFv fragment.

[0529] In one aspect, the antigen binding peptide is a scFab fragment.

[0530] In one aspect, the antigen binding peptide is fused to the C-terminus of the heavy chain of a) and / or b).

[0531] In one aspect, the trispecific or tetraspecific antibody comprises under c) one or two antigen-binding peptides that specifically bind to a further antigen.

[0532] In one aspect, the trispecific or tetraspecific antibody comprises two identical antigen-binding peptides under c) that specifically bind to a third antigen. In a preferred embodiment, these two identical antigen-binding peptides are fused to the C-termini of the heavy chains of a) and b) via the same peptide linker. In a preferred embodiment, either of the two identical antigen-binding peptides is a scFv fragment or a scFab fragment.

[0533] In one aspect, the trispecific or tetraspecific antibody comprises two antigen-binding peptides under c) that specifically bind to a third and a fourth antigen. In one embodiment, the two antigen-binding peptides are fused to the C-termini of the heavy chains of a) and b) via the same peptide linker. In a preferred embodiment, either of the two antigen-binding peptides is a scFv fragment or a scFab fragment.

[0534] In one aspect, the bispecific antibody is a bispecific, tetravalent antibody comprising

[0535] a) two light chains and two heavy chains of an antibody that specifically binds to a first antigen (and comprises two Fab fragments),

[0536] b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are each fused to either the C-terminus or the N-terminus of the heavy chain of a) via a peptide linker, and

[0537] The following modifications were made in this Fab fragment:

[0538] (i) in the two Fab fragments of a), or in the two Fab fragments of b), the variable domains VL and VH are replaced with each other, and / or the constant domains CL and CH1 are replaced with each other, or

[0539] (ii) in the two Fab fragments of a) the variable domains VL and VH are replaced with each other, and the constant domains CL and CH1 are replaced with each other, and in the two Fab fragments of b) the variable domains VL and VH are replaced with each other, or the constant domains CL and CH1 are replaced with each other, or

[0540] (iii) in the two Fab fragments of a) the variable domains VL and VH are replaced with each other, or the constant domains CL and CH1 are replaced with each other, and in the two Fab fragments of b) the variable domains VL and VH are replaced with each other, and the constant domains CL and CH1 are replaced with each other, or

[0541] (iv) the variable domains VL and VH are replaced with each other in the two Fab fragments of a), and the constant domains CL and CH1 are replaced with each other in the two Fab fragments of b), or

[0542] (v) The constant domains CL and CH1 are replaced with each other in the two Fab fragments of a), and the variable domains VL and VH are replaced with each other in the two Fab fragments of b).

[0543] In one aspect, said additional Fab fragments are each fused via a peptide linker to either the C-terminus of the heavy chain of a) or the N-terminus of the heavy chain of a).

[0544] In one aspect, said additional Fab fragments are each fused to either C-terminus of the heavy chain of a) via a peptide linker.

[0545] In one aspect, said additional Fab fragments are each fused to the N-terminus of the heavy chain of a) via a peptide linker.

[0546] In one aspect, the following modifications are performed in the Fab fragments: in the two Fab fragments in a) or in the two Fab fragments in b), the variable domains VL and VH are replaced with each other, and / or the constant domains CL and CH1 are replaced with each other.

[0547] In one aspect, the bispecific antibody is a tetravalent antibody comprising:

[0548] a) a (modified) heavy chain of a first antibody that specifically binds to a first antigen and comprises a first VH-CH1 domain pair, wherein the N-terminus of the second VH-CH1 domain pair of the first antibody is fused to the C-terminus of the heavy chain via a peptide linker,

[0549] b) two light chains of the first antibody described in a),

[0550] c) a (modified) heavy chain of a second antibody that specifically binds to a second antigen and comprises a first VH-CL domain pair, wherein the N-terminus of the second VH-CL domain pair of the second antibody is fused to the C-terminus of the heavy chain via a peptide linker, and

[0551] d) Two (modified) light chains of the second antibody of c) , each comprising a CL-CH1 domain pair.

[0552] In one aspect, the bispecific antibody comprises:

[0553] a) the heavy and light chains of a first full-length antibody that specifically binds to a first antigen, and

[0554] b) a heavy chain and a light chain of a second full length antibody that specifically binds to a second antigen, wherein the N-terminus of the heavy chain is linked to the C-terminus of the light chain via a peptide linker.

[0555] The antibody under a) does not contain the modification reported under b) and the heavy chain and the light chain are separated chains.

[0556] In one aspect, the bispecific antibody comprises

[0557] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0558] b) an Fv fragment that specifically binds to a second antigen and comprises a VH2 domain and a VL2 domain, wherein the two domains are linked to each other via a disulfide bridge,

[0559] Wherein only the VH2 domain or the VL2 domain is fused to the heavy chain or light chain of the full-length antibody that specifically binds to the first antigen via a peptide linker.

[0560] In this bispecific antibody the heavy and light chains under a) are separate chains.

[0561] In one aspect, the other of the VH2 domain or the VL2 domain is not fused via a peptide linker to the heavy or light chain of the full length antibody that specifically binds to the first antigen.

[0562] In all aspects reported herein, the first light chain comprises a VL domain and a CL domain and the first heavy chain comprises a VH domain, a CH1 domain, a hinge region, a CH2 domain and a CH3 domain.

[0563] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0564] a) two Fab fragments that specifically bind to a first antigen,

[0565] b) a CrossFab fragment that specifically binds to a second antigen, wherein the CH1 and CL domains are exchanged with each other,

[0566] c) an Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain,

[0567] wherein the C-termini of the CH1 domains of the two Fab fragments are linked to the N-termini of the heavy chain Fc region polypeptide, and wherein the C-terminus of the CL domain of the CrossFab fragment is linked to the N-terminus of the VH domain of one of the Fab fragments.

[0568] In one aspect, the bispecific antibody is a trivalent antibody comprising

[0569] a) two Fab fragments that specifically bind to a first antigen,

[0570] b) a CrossFab fragment that specifically binds to a second antigen, wherein the CH1 and CL domains are exchanged with each other,

[0571] c) an Fc region comprising a first Fc region heavy chain and a second Fc region heavy chain,

[0572] wherein the C-terminus of the CH1 domain of the first Fab fragment is linked to the N-terminus of one of the heavy chain Fc region polypeptides and the C-terminus of the CL domain of the CrossFab fragment is linked to the N-terminus of the other heavy chain Fc region polypeptide, and wherein the C-terminus of the CH1 domain of the second Fab fragment is linked to the N-terminus of the VH domain of the first Fab fragment or the N-terminus of the VH domain of the CrossFab fragment.

[0573] In one aspect, the bispecific antibody comprises

[0574] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0575] b) a Fab fragment that specifically binds to a second antigen and comprises a VH2 domain and a VL2 domain, comprising a heavy chain fragment and a light chain fragment, wherein in the light chain fragment the variable light chain domain VL2 is replaced by the variable heavy chain domain VH2 of said antibody, and in the heavy chain fragment the variable heavy chain domain VH2 is replaced by the variable light chain domain VL2 of said antibody,

[0576] wherein the heavy chain Fab fragment is inserted between the CH1 domain of one of the heavy chains of the full-length antibody and the respective Fc region of the full-length antibody, and the N-terminus of the light chain Fab fragment is connected to the C-terminus of the light chain of the full-length antibody that is paired with the heavy chain of the full-length antibody into which the heavy chain Fab fragment is inserted.

[0577] In one aspect, the bispecific antibody comprises

[0578] a) a full-length antibody that specifically binds to a first antigen and consists of two antibody heavy chains and two antibody light chains, and

[0579] b) a Fab fragment that specifically binds to a second antigen and comprises a VH2 domain and a VL2 domain, comprising a heavy chain fragment and a light chain fragment, wherein within the light chain fragment, the variable light chain domain VL2 is replaced with the variable heavy chain domain VH2 of said antibody, and within the heavy chain fragment, the variable heavy chain domain VH2 is replaced with the variable light chain domain VL2 of said antibody, and wherein the C-terminus of the heavy chain fragment of the Fab fragment is linked to the N-terminus of one of the heavy chains of the full-length antibody and the C-terminus of the light chain fragment of the Fab fragment is linked to the N-terminus of the light chain of the full-length antibody that is paired with the heavy chain of the heavy chain fragment to which the Fab fragment is conjugated in the full-length antibody.

[0580] polynucleotides

[0581] The present invention further provides isolated polynucleotides encoding the bispecific antibodies or fragments thereof as described herein.

[0582] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide of the present invention is RNA, for example in the form of messenger RNA (mRNA). The RNA of the present invention can be single-stranded or double-stranded.

[0583] B. Recombination Methods

[0584] Recombinant methods and compositions can be used to generate the bispecific antibodies provided herein, for example, as described in U.S. Patent No. 4,816,567. In one aspect, isolated nucleic acids encoding the bispecific antibodies described herein are provided. Such nucleic acids can encode the amino acid sequence of VL comprising antigen binding sites that specifically bind to PD1 and TIM-3 and / or the amino acid sequence of VH comprising antigen binding sites that specifically bind to PD1 and TIM-3 (e.g., in the light and / or heavy chains of the antibody). In another aspect, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another aspect, a host cell comprising such nucleic acids is provided. In one such aspect, the host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising antibody VL and an amino acid sequence comprising antibody VH, or (2) a first vector and a second vector, wherein the first vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VL and the second vector comprises a nucleic acid encoding an amino acid sequence comprising antibody VH. In one aspect, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one aspect, a method for generating a bispecific antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody under conditions suitable for antibody expression, as provided above, and optionally recovering the antibody from the host cell (or host cell culture fluid).

[0585] For the recombinant production of bispecific antibodies comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as described herein, nucleic acids encoding bispecific antibodies are isolated (e.g., as described above) and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding antibody heavy and light chains).

[0586] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237; US 5,789,199; and US 5,840,523. See also Charlton, KA, In: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, which describes expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the bacterial cell slurry in a soluble fraction and can be further purified.

[0587] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22: 1409-1414 (2004); and Li, H. et al., Nat. Biotech. 24: 210-215 (2006).

[0588] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0589] Plant cell cultures can also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429 (which describe plant cell cultures for producing antibodies in transgenic plants). TM technology).

[0590] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines include monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Graham, FL et al., J. Gen Virol. 36:59-74 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, for example, in Mather, JP, Biol. Reprod. 23:243-252 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather, JP et al., Annals of NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR - CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77: 4216-4220 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0591] C. Assay

[0592] The bispecific antibodies provided herein comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3 can be identified, screened, or characterized by their physical / chemical properties and / or biological activities by various assays known in the art.

[0593] 1. Affinity Assay

[0594] According to the surface plasmon resonance (SPR) method proposed in the examples, standard instruments such as The affinity of the bispecific antigen-binding molecules, antibodies and antibody fragments provided herein to the corresponding antigen can be determined using a ELISA instrument (GE Healthcare), and a receptor or target protein that can be obtained by, for example, recombinant expression. Specific illustrative and exemplary embodiments for measuring binding affinity are described in Examples 1b, 5 or 12. According to one aspect, using K was measured by surface plasmon resonance using a T100 instrument (GE Healthcare) at 25 °C. D .

[0595] 2. Binding assays and other assays

[0596] In one aspect, the antibodies of the invention are tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, etc. The binding of the bispecific antibodies provided herein to the corresponding recombinant antigen or cells expressing the antigen can be assessed by ELISA as described in Example 12.

[0597] In another aspect, the present invention provides a cell-based TR-FRET assay for determining the simultaneous binding of a bispecific antibody format to two different receptors present on a cell. The selected Tag-lite technology is a combination of classic TR-FRET (time-resolved fluorescence resonance energy transfer) and SNAP-tag technology (e.g., New England Biolabs, CISBIO), which allows labeling of antigens present on the cell surface with fluorescent donor or acceptor dyes. The assay is described in Example 13.

[0598] In yet another aspect, fresh PBMCs are used in binding assays to show binding to different PBMCs such as monocytes, NK cells and T cells.

[0599] In another aspect, competition assays can be used to identify antibodies that compete with specific antibodies or antigen binding sites for binding to targets, respectively. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) bound by a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3 according to the present invention. Detailed exemplary methods for locating epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0600] In an exemplary competition assay, immobilized PD1 or TIM3 is incubated in a solution containing a first labeled antibody (which binds to PD1 or TIM3) and a second unlabeled antibody (which is to be tested for its ability to compete with the first antibody for binding to PD1 or TIM3). The second antibody may be present in the hybridoma supernatant. As a control, immobilized PD1 or TIM3 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to PD1 or TIM3, excess unbound antibody is removed and the amount of label associated with immobilized PD1 or TIM3 is measured. If the amount of label associated with immobilized PD1 or TIM3 in the test sample is substantially reduced relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to PD1 or TIM3. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0601] 3. Activity Assay

[0602] In one aspect, an assay is provided for identifying bispecific antibodies having biological activity comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM3. The biological activity can include, for example, enhancing the activation and / or proliferation of different immune cells, particularly T cells, secretion of immunoregulatory cytokines such as IFNγ or TNF-α, blocking the PD1 pathway, blocking the TIM3 pathway, and the ability to kill tumor cells. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0603] In certain aspects, the antibodies of the present invention are tested for such biological activities. In one aspect, provided is an immune cell assay that measures the activation of lymphocytes from one individual (donor X) against lymphocytes from another individual (donor Y). Mixed lymphocyte reaction (MLR) can demonstrate the effect of blocking the PD1 pathway on lymphocyte effector cells. The T cells in this assay are tested for activation and their IFN-γ secretion in the presence or absence of the bispecific antibodies of the present invention. This assay is described in more detail in Example 16.

[0604] D. Immunoconjugates

[0605] The invention also provides immunoconjugates comprising a bispecific antibody of the invention conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes.

[0606] In one aspect, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425). 235B1); auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58: 2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13: 477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16: 358-362 (2006); Torgov et al., Bioconj. Chem. 16: 717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97: 829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0607] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis toxin, scutellaria baicalensis toxin, scutellaria scabra ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[0608] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to generate radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 ,Bi 212 , P 32 , Pb 212 When the radioconjugate is used for detection, it may contain a radioactive atom for scintigraphic studies, such as Tc 99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0609] Conjugates of the antibody and cytotoxic agent can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0610] The immunoconjugates or ADCs herein specifically encompass, but are not limited to, conjugates prepared with the following cross-linkers, including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology Inc., Rockford, IL, USA).

[0611] E. Methods and compositions for diagnosis and detection

[0612] In certain aspects, any of the bispecific antibodies provided herein comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3 may be useful for detecting the presence of both PD1 and TIM3 in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as AML stem cell cancer cells.

[0613] In one aspect, a bispecific antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of both PD1 and TIM3 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with a bispecific antibody described herein under conditions that allow the bispecific antibody to bind to both PD1 and TIM3, and detecting whether a complex is formed between the bispecific antibody and the two antigens. Such methods can be in vitro or in vivo methods. In one embodiment, a bispecific antibody is used to select subjects suitable for treatment with a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3, for example, where PD1 and TIM3 are biomarkers for selecting patients.

[0614] In certain aspects, labeled bispecific antibodies are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase (which are coupled to enzymes such as HRP that oxidize dye precursors using hydrogen peroxide), lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, or stable free radicals.

[0615] F. Pharmaceutical Compositions, Formulations, and Routes of Administration

[0616] In yet another aspect, the present invention provides a pharmaceutical composition comprising any of the bispecific antibodies provided herein that comprises a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3, for use, for example, in any of the following treatment methods. In one embodiment, the pharmaceutical composition comprises any of the bispecific antibodies provided herein and at least one pharmaceutically acceptable excipient. In another embodiment, the pharmaceutical composition comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0617] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of one or more bispecific antibodies dissolved or dispersed in a pharmaceutically acceptable excipient. The phrase "pharmaceutical or pharmaceutically acceptable" refers to molecular entities and compositions that are generally non-toxic to recipients at the dosages and concentrations employed, i.e., do not produce adverse, allergic, or other adverse reactions when properly administered to animals, such as humans. In accordance with the present disclosure, the preparation of pharmaceutical compositions comprising at least one bispecific antibody and optionally additional active ingredients will be known to those skilled in the art, as exemplified by Remington's Pharmaceutical Sciences, 18th edition, Mack Printing Company, 1990, which is incorporated herein by reference. Specifically, the composition is a lyophilized formulation or an aqueous solution. As used herein, as known to those of ordinary skill in the art, "pharmaceutically acceptable excipients" include any and all of solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, stabilizers, and combinations thereof.

[0618] Parenteral compositions include those designed for administration by injection, such as subcutaneous, intradermal, intralesional, intravenous, intraarterial, intramuscular, intrathecal or intraperitoneal injection. For injection, the bispecific antibodies of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks solution, Ringer's solution or physiological saline buffer. The solution can contain a preparaton, such as a suspending agent, a stabilizer and / or a dispersant. Alternatively, the bispecific antibody may be in powder form to be composed with a suitable carrier (e.g., sterile pyrogen-free water) before use. As needed, a sterile injectable solution is prepared by incorporating the fusion protein of the present invention in an appropriate solvent into a variety of other ingredients listed below in the desired amount. Sterility can be easily achieved, for example, by filtering through a sterile filtration membrane. Typically, dispersions are prepared by incorporating various sterilized active ingredients into a sterile carrier containing a basic dispersion medium and / or other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered liquid medium. The liquid medium should be appropriately buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose before injection. The composition must be stable under the conditions of manufacture and storage and remain resistant to the contaminating effects of antimicrobials such as bacteria and fungi. It should be understood that endotoxin contamination should be kept to a minimum at a safe level, for example, below 0.5 ng / mg protein. Suitable pharmaceutically acceptable excipients include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, Such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of highly concentrated solutions. In addition, suspensions of these active compounds can be prepared as appropriate oily injection suspensions.Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes.

[0619] Active ingredient can be embedded in microcapsules prepared, for example, by coacervation technology or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse emulsions. Such technology is disclosed in Remington's Pharmaceutical Sciences (18th Ed. Mack Printing Company, 1990). Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide, the matrix being in the form of shaped articles, such as films, or microcapsules. In a particular embodiment, the absorption of the injectable composition can be prolonged by using an agent that delays absorption in the composition, such as aluminum monostearate, gelatin or a combination thereof.

[0620] Exemplary pharmaceutically acceptable excipients herein also include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International, Inc. Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0621] Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation including a histidine-acetate buffer.

[0622] In addition to the aforementioned compositions, bispecific antibodies can also be formulated as depot formulations. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, bispecific antibodies can be formulated with suitable polymeric materials or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as slightly soluble derivatives, such as slightly soluble salts.

[0623] Pharmaceutical compositions comprising the bispecific antibodies of the present invention can be prepared by conventional mixing, dissolving, emulsifying, encapsulating, embedding, or lyophilizing processes. Pharmaceutical compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing of the protein into a pharmaceutically usable formulation. The appropriate formulation depends on the chosen route of administration.

[0624] Bispecific antibodies can be formulated into compositions in free acid or base, neutral, or salt form. Pharmaceutically acceptable salts are salts that substantially retain the biological activity of the free acid or base. These salts include acid addition salts, such as those formed with free amino groups of the protein composition or with inorganic acids such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide; or organic bases such as isopropylamine, trimethylamine, histidine, or procaine. Pharmaceutical salts have higher solubility in aqueous and other protic solvents than the corresponding free base forms.

[0625] When required for the particular indication being treated, the compositions herein may also contain more than one active ingredient, preferably, the active ingredients have complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0626] Formulations for in vivo administration are generally sterile. Sterility can be readily accomplished, for example, by filtration through sterile filtration membranes.

[0627] G. Methods of Treatment and Compositions

[0628] Any bispecific antibody provided herein that comprises a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3 can be used in therapeutic methods.

[0629] For use in therapeutic methods, bispecific antibodies comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3 as defined herein can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the dosing regimen, and other factors known to practitioners.

[0630] In one aspect, a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as defined herein is provided for use as a medicament. In further aspects, a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as defined herein is provided for use in treating a disease, in particular for treating cancer. In certain embodiments, a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 is provided for use in a method of treatment. In one embodiment, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as described herein for use in treating a disease in an individual in need thereof. In certain embodiments, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 for use in a method of treating an individual having a disease, the method comprising administering a therapeutically effective amount of the bispecific antibody to the individual. In certain embodiments, the disease to be treated is cancer. In another aspect, the disease to be treated is a chronic viral infection, such as HIV, HBV, HCV, HSV1, CMV, LCMV or EBV. The subject, patient, or "individual" in need of treatment is generally a mammal, more particularly a human.

[0631] In another aspect, the present invention provides a bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as defined herein for use in the manufacture or preparation of a medicament for treating a disease in an individual in need thereof. In one embodiment, the medicament is for use in a method of treating a disease, the method comprising administering a therapeutically effective amount of the medicament to an individual having the disease.

[0632] In certain aspects, the disease to be treated is a proliferative disorder, particularly cancer. Examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferative disorders that can be treated using the bispecific antibodies comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 according to the present invention include, but are not limited to, neoplasms located in the abdomen, bones, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary glands, testicles, ovaries, thymus, thyroid gland), eyes, head and neck, nervous system (central and peripheral), lymphatic system, pelvic cavity, skin, soft tissue, spleen, chest, and urogenital system. Precancerous conditions or lesions and cancer metastasis are also included. In some aspects, the cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, head and neck cancer. In still other aspects, the cancer is selected from the group consisting of carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In another aspect, the cancer to be treated is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0633] In yet another aspect, the disease to be treated is a chronic viral infection. The term "chronic viral infection" refers to a condition in which a subject is afflicted or infected with a chronic virus. Examples of chronic viral infections include human immunodeficiency virus (HIV), hepatitis B virus infection (HBV), hepatitis C virus infection (HCV), herpes simplex virus 1 (HSV1), cytomegalovirus (CMV), lymphocytic choriomeningitis virus (LCMV), or Epstein-Barr virus (EBV).

[0634] The skilled artisan will readily recognize that in many cases, a bispecific antibody may not provide a cure, but may only provide a partial benefit. In some embodiments, a physiological change that has some benefit is also considered therapeutically beneficial. Thus, in some embodiments, the amount of a bispecific antibody that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

[0635] In yet another aspect, the present invention provides a method for treating a disease in an individual, comprising administering to the individual a therapeutically effective amount of a bispecific antibody of the present invention comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3. In one embodiment, a composition comprising the bispecific antibody of the present invention in a pharmaceutically acceptable form is administered to the individual. In certain embodiments, the disease to be treated is a proliferative disorder. In a specific embodiment, the disease is cancer. In certain embodiments, the method further comprises administering to the individual a therapeutically effective amount of at least one additional therapeutic agent, for example, an anticancer agent if the disease to be treated is cancer. In another aspect, the disease is a chronic viral infection. The "individual" according to any of the above embodiments may be a mammal, preferably a human.

[0636] For the prevention or treatment of disease, the appropriate dosage of the bispecific antibody of the present invention comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the route of administration, the patient's weight, the type of fusion protein, the severity and course of the disease, whether the bispecific antibody is administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's clinical history, and response to the fusion protein, and the decision of the attending physician. In any case, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulse infusions.

[0637] A bispecific antibody comprising a first antigen binding site that specifically binds to PD1 and a second antigen binding site that specifically binds to TIM-3 as defined herein is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, a bispecific antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) may be an initial candidate dose for administration to the patient, for example, whether by one or more separate administrations or by continuous infusion. Depending on the factors mentioned above, a typical daily dose may be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment will generally continue until the desired symptoms of the disease are suppressed. An exemplary dosage of the bispecific antibody will be in the range of about 0.005 mg / kg to about 10 mg / kg. In other examples, the dosage can also include per administration from about 1 μg / kg body weight, about 5 μg / kg body weight, about 10 μg / kg body weight, about 50 μg / kg body weight, about 100 μg / kg body weight, about 200 μg / kg body weight, about 350 μg / kg body weight, about 500 μg / kg body weight, about 1 mg / kg body weight, about 5 mg / kg body weight, about 10 mg / kg body weight, about 50 mg / kg body weight, about 100 mg / kg body weight, about 200 mg / kg body weight, about 350 mg / kg body weight, about 500 mg / kg body weight, to about 1000 mg / kg body weight or more, and any range derivable therein. In the example of a derivable range of the numbers listed herein, it can be administered based on the above numbers with a range of about 5 mg / kg body weight to about 100 mg / kg body weight, about 5 μg / kg body weight to about 500 mg / kg body weight, etc. Therefore, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg or 10 mg / kg (or any combination thereof) can be administered to the patient. Such dosage can be administered intermittently, for example, weekly or once every three weeks (for example, so that the patient receives about 2 to about 20, or for example, about 6 dosages of fusion protein). An initial higher loading dose can be administered, followed by one or more lower dosages. However, other dosage regimens may be useful. It is easy to monitor the progress of this therapy by conventional techniques and assays.

[0638] A bispecific antibody comprising a first antigen-binding site that specifically binds PD1 and a second antigen-binding site that specifically binds TIM-3, as defined herein, is typically used in an amount effective to achieve its intended purpose. For use in treating or preventing a disease condition, the bispecific antibody of the invention or a pharmaceutical composition thereof is administered or applied in a therapeutically effective amount. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0639] For systemic administration, the therapeutically effective dose can be initially estimated based on in vitro assays (e.g., cell culture assays). A dose can then be formulated in animal models to achieve the IC50, including that determined in cell culture. 50 Such information can be used to more accurately determine useful doses in humans.

[0640] Initial dosages can also be estimated based on in vivo data (eg, animal models) using techniques well known in the art. Based on animal data, one of ordinary skill in the art can readily optimize dosing for humans.

[0641] The dosage and interval can be adjusted individually to provide a plasma level of the bispecific antibody sufficient to maintain the therapeutic effect. The patient dosage for administration by injection is generally about 0.1 to 50 mg / kg / day, generally about 0.5 to 1 mg / kg / day. The therapeutically effective plasma level can be achieved by administering multiple doses per day. The level in the plasma can be measured, for example, by HPLC.

[0642] In cases of local administration or selective uptake, the effective local concentration of the bispecific antibody may not be related to plasma concentration. One skilled in the art will be able to optimize the therapeutically effective local dose without undue experimentation.

[0643] The therapeutically effective dose of the bispecific antibodies described herein will generally provide therapeutic benefit without causing substantial toxicity. The toxicity and therapeutic efficacy of the fusion protein can be determined by standard pharmaceutical procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD 50 (a dose lethal to 50% of the population) and ED 50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 Bispecific antibodies that exhibit a large therapeutic index are preferred. In one embodiment, the bispecific antibodies according to the present invention exhibit a high therapeutic index. Data obtained from cell culture assays and animal studies can be used in formulating a dosage range suitable for use in humans. The dose is preferably within a circulating concentration range that includes the ED with little or no toxicity. 50 The dosage may vary within this range depending on a variety of factors, such as the dosage form employed, the route of administration used, the condition of the subject, etc. The exact formulation, route of administration, and dosage can be selected by the individual physician taking into account the patient's condition (see, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1, which is incorporated herein by reference in its entirety).

[0644] The attending physician who treats a patient with the bispecific antibody of the present invention knows how and when to terminate, interrupt or adjust the administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is insufficient (excluding toxicity), the attending physician will also know to adjust the treatment to a higher level. In the management of the condition of interest, the size of the administered dose will vary with the severity of the condition to be treated, the route of administration, etc. The severity of the condition can be evaluated, for example, in part by standard prognostic evaluation methods. In addition, the dosage and possible dosage frequency will also vary according to the age, weight and response of the individual patient.

[0645] Other medications and treatments

[0646] As previously described herein, bispecific antibodies comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3 can be administered in combination with one or more other agents in therapy. For example, the fusion protein of the present invention can be co-administered with at least one additional therapeutic agent. The term "therapeutic agent" encompasses any agent that can be used to treat a symptom or disease in an individual in need of such treatment. Such additional therapeutic agent can comprise any active ingredient suitable for the specific indication being treated, preferably, the active ingredients having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is another anticancer agent.

[0647] Such other agents are suitably combined in an amount effective for the intended purpose. The effective amount of such other agents depends on the amount of the fusion protein used, the type of disease or treatment, and other factors discussed above. Usually with the same dosage and route of administration as described herein, or about 1% to 99% of the dosage described herein, or with any dosage determined to be appropriate empirically / clinically and using the antigen binding molecules of the present invention by any route.

[0648] Such combination therapies described above encompass both combined administration (wherein two or more therapeutic agents are included in the same or separate compositions) and separate administration, in which case administration of the bispecific antibody can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant.

[0649] H. Products

[0650] In another aspect of the present invention, an article of manufacture is provided that comprises materials useful for treating, preventing, and / or diagnosing the conditions described above. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition that is effective for treating, preventing, and / or diagnosing the condition, either alone or in combination with another composition, and may have a sterile access port (e.g., the container may be a vial or intravenous solution bag with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is a bispecific antibody as previously defined herein that comprises a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM-3.

[0651] The label or package insert indicates that the composition is used to treat a selected condition. Furthermore, the article of manufacture may include (a) a first container containing a composition comprising a bispecific antibody of the invention; and (b) a second container containing a composition comprising another cytotoxic or otherwise therapeutic agent. The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a specific condition.

[0652] Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0653] Table C (sequence):

[0654]

[0655]

[0656]

[0657]

[0658]

[0659]

[0660]

[0661]

[0662]

[0663]

[0664]

[0665] List the specific embodiments of the present invention below:

[0666] 1. A bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein

[0667] The first antigen binding site that specifically binds to PD1 comprises

[0668] The VH domain comprises

[0669] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 37,

[0670] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and

[0671] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and

[0672] The VL domain contains

[0673] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 40;

[0674] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 41, and

[0675] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42;

[0676] And the second antigen binding site that specifically binds to TIM3 comprises

[0677] (a) VH domain, which comprises

[0678] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 1,

[0679] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and

[0680] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 3; and

[0681] The VL domain contains

[0682] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11 or SEQ ID NO: 12,

[0683] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0684] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 6; or

[0685] (b) a VH domain comprising

[0686] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 17,

[0687] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 18, and

[0688] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 19; and

[0689] The VL domain contains

[0690] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 20,

[0691] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 21, and

[0692] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 22; or

[0693] (c) a VH domain comprising

[0694] (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 29,

[0695] (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, and

[0696] (iii) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 31; and

[0697] The VL domain contains

[0698] (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 32,

[0699] (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 33, and

[0700] (iii) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 34.

[0701] 2. A bispecific antibody comprising a first antigen-binding site that specifically binds to PD1 and a second antigen-binding site that specifically binds to TIM3, wherein the bispecific antibody binds to TIM3 with a binding affinity that is at least 50-fold lower than the binding to PD1, more particularly at least 100-fold lower than the binding to PD1.

[0702] 3. A bispecific antibody as defined herein, wherein

[0703] The first antigen binding site that specifically binds to PD1 comprises

[0704] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 43 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or

[0705] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or

[0706] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or

[0707] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or

[0708] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49,

[0709] And the second antigen binding site that specifically binds to TIM3 comprises

[0710] (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, or

[0711] (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, or

[0712] (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, or

[0713] (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or

[0714] (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 23 and a VL domain comprising the amino acid sequence of SEQ ID NO: 24, or

[0715] (f) a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26, or

[0716] (g) a VH domain comprising the amino acid sequence of SEQ ID NO: 27 and a VL domain comprising the amino acid sequence of SEQ ID NO: 28, or

[0717] (h) a VH domain comprising the amino acid sequence of SEQ ID NO: 35 and a VL domain comprising the amino acid sequence of SEQ ID NO: 36.

[0718] 4. A bispecific antibody as defined herein, wherein

[0719] The first antigen binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46,

[0720] And the second antigen binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0721] 5. A bispecific antibody as defined herein, wherein

[0722] The first antigen binding site that specifically binds to PD1 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46,

[0723] And the second antigen binding site that specifically binds to TIM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 25 and a VL domain comprising the amino acid sequence of SEQ ID NO: 26.

[0724] 6. The bispecific antibody as defined herein before, wherein the bispecific antibody is a human, humanized or chimeric antibody.

[0725] 7. The bispecific antibody as defined herein, wherein the bispecific antibody comprises an Fc domain, a first Fab fragment comprising the antigen binding site that specifically binds to PD1, and a second Fab fragment comprising the antigen binding site that specifically binds to TIM3.

[0726] 8. The bispecific antibody as defined herein before, wherein the Fc domain is an IgG, in particular an IgG1 Fc domain or an IgG4 Fc domain.

[0727] 9. The bispecific antibody as defined herein before, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fc receptors, in particular Fcγ receptors.

[0728] 10. The bispecific antibody as defined herein before, wherein the Fc domain is of human IgG1 subclass, with the amino acid mutations L234A, L235A and P329G (numbering according to Kabat EU index).

[0729] 11. The bispecific antibody as defined herein before, wherein the Fc domain comprises a modification promoting association of the first and the second subunit of the Fc domain.

[0730] 12. The bispecific antibody as defined herein before, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to the knob-into-hole approach.

[0731] 13. The bispecific antibody as defined herein before, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering) and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V (numbering according to the Kabat EU index).

[0732] 14. The bispecific antibody as defined herein before, wherein in one of the Fab fragments the variable domains VL and VH replace each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

[0733] 15. The bispecific antibody as defined herein before, wherein in the first Fab fragment comprising the antigen binding site that specifically binds to PD1, the variable domains VL and VH are replaced with each other.

[0734] 16. The bispecific antibody as defined herein, wherein in one of the Fab fragments the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0735] 17. The bispecific antibody as defined herein, wherein in the second Fab fragment comprising the antigen binding site that specifically binds to TIM3, the amino acid at position 124 in the constant domain CL is independently replaced by lysine (K), arginine (R) or histidine (H) (numbering according to the Kabat EU index), and the amino acids at positions 147 and 213 in the constant domain CH1 are independently replaced by glutamic acid (E) or aspartic acid (D) (numbering according to the Kabat EU index).

[0736] 18. A bispecific antibody as defined herein before, comprising

[0737] (a) a first heavy chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 50, and a first light chain comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 52,

[0738] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 51, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 53, or

[0739] (b) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 54, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 56,

[0740] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 55, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 57, or

[0741] (c) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 58, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 60,

[0742] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 59, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 61, or

[0743] (d) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 62, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 64,

[0744] a second heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 63, and a second light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 65, or

[0745] (e) a first heavy chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 66, a first light chain comprising an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO: 68,

[0746] A second heavy chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:67, and a second light chain comprises an amino acid sequence that has at least 95% sequence identity to the sequence of SEQ ID NO:69.

[0747] 19. A bispecific antibody as defined herein before, comprising

[0748] (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 50, and a first light chain comprising the amino acid sequence of SEQ ID NO: 52,

[0749] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 51, and a second light chain comprising the amino acid sequence of SEQ ID NO: 53, or

[0750] (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 54, a first light chain comprising the amino acid sequence of SEQ ID NO: 56,

[0751] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and a second light chain comprising the amino acid sequence of SEQ ID NO: 57, or

[0752] (c) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 58, a first light chain comprising the amino acid sequence of SEQ ID NO: 60,

[0753] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 59, and a second light chain comprising the amino acid sequence of SEQ ID NO: 61, or

[0754] (d) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 62, a first light chain comprising the amino acid sequence of SEQ ID NO: 64,

[0755] a second heavy chain comprising the amino acid sequence of SEQ ID NO: 63, and a second light chain comprising the amino acid sequence of SEQ ID NO: 65, or

[0756] (e) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, a first light chain comprising the amino acid sequence of SEQ ID NO: 68,

[0757] A second heavy chain comprising the amino acid sequence of SEQ ID NO:67, and a second light chain comprising the amino acid sequence of SEQ ID NO:69.

[0758] 20. A polynucleotide encoding a bispecific antibody as herein before defined.

[0759] 21. A vector, in particular an expression vector, comprising a polynucleotide as defined herein before.

[0760] 22. A prokaryotic or eukaryotic host cell comprising a polynucleotide as herein before defined or a vector as herein before defined.

[0761] 23. A method for producing a bispecific antibody as defined herein before, comprising the steps of:

[0762] a) transforming a host cell with a vector comprising a polynucleotide encoding the bispecific antibody, b) culturing the host cell under conditions suitable for expression of the bispecific antibody, and c) recovering the bispecific antibody from the culture.

[0763] 24. A pharmaceutical composition comprising a bispecific antibody as defined herein before and at least one pharmaceutically acceptable excipient.

[0764] 25. A bispecific antibody as defined herein before or a pharmaceutical composition as defined herein before for use as a medicament.

[0765] 26. A bispecific antibody as defined herein before or a pharmaceutical composition as defined herein before, for use

[0766] i) regulating immune responses, such as restoring T cell activity,

[0767] ii) stimulate immune response or function,

[0768] iii) treatment of infection,

[0769] iv) treating cancer,

[0770] v) delaying cancer progression,

[0771] vi) Prolonging the survival of patients suffering from cancer.

[0772] 27. A bispecific antibody as defined herein before or a pharmaceutical composition as defined herein before for use in preventing or treating cancer.

[0773] 28. A bispecific antibody as defined herein before or a pharmaceutical composition as defined herein before for use in the treatment of a chronic viral infection.

[0774] 29. A bispecific antibody as defined herein before or a pharmaceutical composition as defined herein before for use in preventing or treating cancer, wherein the bispecific antibody is administered in combination with a chemotherapeutic agent, radiation and / or other agents for cancer immunotherapy.

[0775] 30. A method of inhibiting tumor cell growth in an individual, comprising administering to the individual an effective amount of a bispecific antibody as hereinbefore defined to inhibit tumor cell growth. Example

[0776] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced, given the general description provided above.

[0777] Materials and general methods

[0778] General information on the nucleotide sequences of human immunoglobulin light and heavy chains can be found in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The amino acids of the antibody chains are numbered and referred to according to the numbering system of Kabat (Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) as defined above.

[0779] Recombinant DNA technology

[0780] DNA was manipulated using standard methods, such as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

[0781] gene synthesis

[0782] The desired gene segments were prepared from oligonucleotides generated by chemical synthesis. 600-1800 bp long gene segments flanked by single restriction endonuclease cleavage sites were assembled by annealing and ligating oligonucleotides, including PCR amplification, and subsequently cloned into the pGA4 cloning vector based on pPCRScript (Stratagene) via designated restriction sites, such as KpnI / SacI or AscI / PacI. The DNA sequences of the subcloned gene segments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to the instructions provided by Geneart (Regensburg, Germany).

[0783] DNA sequencing

[0784] DNA sequences were determined by double-strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0785] DNA and protein sequence analysis and sequence data management

[0786] Sequence creation, mapping, analysis, annotation, and interpretation were performed using the GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0.

[0787] expression vector

[0788] For expression of the described antibodies, variants of the expression plasmids based on cDNA organization with or without CMV-Intron A promoter or based on genomic organization with CMV promoter were applied to transient expression (eg HEK293) cells.

[0789] In addition to the antibody expression cassette, the vector also contains:

[0790] - an origin of replication, which allows this plasmid to replicate in E. coli, and

[0791] -β-lactamase gene, which confers ampicillin resistance in Escherichia coli.

[0792] The transcription unit of the antibody gene is composed of the following elements:

[0793] - one or more unique restriction sites at the 5' end,

[0794] - the immediate early enhancer and promoter from human cytomegalovirus,

[0795] - followed by, in the case of cDNA tissue, the intron A sequence,

[0796] - 5' untranslated region of a human antibody gene,

[0797] - immunoglobulin heavy chain signal sequence,

[0798] - Humans having immunoglobulin exon-intron organization as cDNA or as genomic organization

[0799] Antibody chains (wild type or with domain swaps),

[0800] - a 3' untranslated region having a polyadenylation signal sequence, and

[0801] - One or more unique restriction sites at the 3' end.

[0802] The fusion gene that comprises antibody chain as described below is produced by PCR and / or gene synthesis, and is assembled by connecting (for example, using the unique restriction site in the corresponding vector) corresponding nucleic acid segment by known recombination method and technology.Verify the nucleotide sequence of subclone by DNA sequencing.For transient transfection, by preparing a large amount of plasmid from the plasmid preparation (Nucleobond AX, Macherey-Nagel) of the Escherichia coli culture through transformation.

[0803] Cell culture technology

[0804] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, JS, Dasso, M., Harford, JB, Lippincott-Schwartz, J. and Yamada, KM (eds.), John Wiley & Sons, Inc. were used.

[0805] Multispecific antibodies were expressed by transient co-transfection of the respective expression plasmids in adherently grown HEK293-EBNA or in suspension grown HEK29-F cells as described below.

[0806] Transient transfection in the HEK293 system

[0807] All antibodies and bispecific antibodies were generated by transient transfection of 293F cells using the Freestyle system (ThermoFisher). Here, 293F cells were cultured in F17 medium, transfected with 293Free (Novagene) and fed with VPA 4mM after 4 hours and 0.6% glucose after 7 and 16 hours. Expi293F was used. TM Expression system kit (ThermoFisher). Here, in Expi293 TM Expi293F was cultured in expression medium TM ExpiFectamine TM The 293 transfection kit was used for transfection according to the manufacturer's instructions. Vh-VL Improved stability and purity of bispecific antibodies and reduced aggregation tendency did not require adjustment of plasmid ratios. Therefore, co-transfection of LC, HC, crossed LC, and crossed HC plasmids was performed using relative plasmid ratios of 1:1:1:1 (for 1+1 CrossMab) or 1:1:1 (for 2+2 CrossMab). Cell supernatants were harvested after 7 days and purified by standard methods.

[0808] Protein determination

[0809] The protein concentration of purified antibodies and derivatives was determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated from the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411-1423.

[0810] Determination of antibody concentration in supernatant

[0811] The concentration of antibodies and derivatives in the cell culture supernatant was assessed by immunoprecipitation with protein A agarose beads (Roche). 60 μL protein A agarose beads were washed three times in TBS-NP40 (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet-P40). Subsequently, 1-15 mL cell culture supernatant was applied to the protein A agarose beads pre-equilibrated in TBS-NP40. After incubation at room temperature for 1 hour, the beads were washed once with 0.5 mL TBS-NP40 on an Ultrafree-MC-filter column (Amicon), washed twice with 0.5 mL 2x phosphate buffered saline (2xPBS, Roche), and simply washed four times with 0.5 mL 100 mM sodium citrate pH 5,0. By adding 35 μl LDS sample buffer (Invitrogen) was used to elute the bound antibody. The sample was mixed with reducing agents or kept unreduced and heated at 70°C for 10 min. Therefore, 5-30 μl was applied to 4-12% Bis-Tris SDS-PAGE (Invitrogen) (MOPS buffer was used for non-reducing SDS-PAGE, with MES buffer with antioxidant running buffer supplement (Invitrogen) was used for reducing SDS-PAGE) and stained with Coomassie blue.

[0812] The concentration of antibodies and derivatives in cell culture supernatants was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind to protein A were applied to an Applied Biosystems Poros A / 20 column in 200 mM KH2PO4, 100 mM sodium citrate, pH 7.4 on an Agilent HPLC 1100 system and eluted from the matrix with 200 mM NaCl, 100 mM citric acid, pH 2.5. The eluted protein was quantified by UV absorbance and peak area integration. A purified standard IgG1 antibody served as a standard.

[0813] Alternatively, the concentration of antibodies and derivatives in cell culture supernatants was measured by sandwich IgG-ELISA. Briefly, StreptaWell high binding streptavidin A-96 well microtiter plates (Roche) were plated with 100 μL / well of 0.1 μg / mL biotinylated anti-human IgG capture molecule F(ab')2<h-Fcγ> BI (Dianova) was coated at room temperature for 1 hour or overnight at 4°C, followed by three washes with 200 μL / well PBS, 0.05% Tween (PBST, Sigma). 100 μL / well of each dilution series of cell culture supernatant containing antibody in PBS (Sigma) was added to the wells and incubated on a microtiter plate shaker at room temperature for 1-2 hours. The wells were washed three times with 200 μL / well PBST and incubated with 100 μl of 0.1 μg / mL F(ab')2 on a microtiter plate shaker.<hFcγ> Bound antibody was detected using POD (Dianova) at room temperature for 1-2 hours. Unbound antibody was washed away three times with 200 μL / well PBST, and bound antibody was detected by adding 100 μL / well ABTS. Absorbance was measured on a Tecan spectrofluorometer at a wavelength of 405 nm (reference wavelength 492 nm).

[0814] Protein purification

[0815] With reference to standard scheme, protein is purified from the cell culture supernatant of filtration.In brief, antibody is applied to protein A sepharose column (GE Healthcare) and washed with PBS.At pH 2.8, realize the elution of antibody, then neutralize sample immediately.By size exclusion chromatography (Superdex 200, GE Healthcare) in PBS or in 20mM histidine, 150mM NaCl (pH 6.0) from monomeric antibody separation aggregated protein.Merge monomeric antibody fraction, use for example MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator to concentrate (if necessary), at-20 ℃ or-80 ℃ freezing and store.Provide partial sample for example for carrying out protein analysis and analytical characterization by SDS-PAGE, size exclusion chromatography (SEC) or mass spectrometry subsequently.

[0816] SDS-PAGE

[0817] Use according to the manufacturer's instructions Pre-Cast gel system (Invitrogen). Specifically, 10% or 4-12% Bis-TRIS Pre-Cast Gel (pH 6.4) and MES (reducing gel, with Antioxidant running buffer additive) or MOPS (non-reducing gel) running buffer.

[0818] Analytical size exclusion chromatography

[0819] The antibody of embodiment 1 is purified by size exclusion chromatography (SEC).Size exclusion chromatography (SEC) is carried out by HPLC chromatography, is used to measure the aggregation and oligomeric state of antibody.In brief, the antibody of protein A purification is applied to 300mM NaCl, 50mM KH on Agilent HPLC 1100 system PO / K HPO On the Tosoh TSKgel G3000SW post in (pH 7.5), or on the Superdex 200 post (GE Healthcare) in the 2x PBS on Dionex HPLC system.By the integration of UV absorbance and peak area, the protein of wash-out is quantitatively measured.With BioRad gel filtration standard 151-1901 as standard.

[0820] Mass spectrometry

[0821] This section describes the characterization of multispecific antibodies with VH / VL exchange (VH / VL CrossMabs), with an emphasis on their correct assembly. The expected primary structure was analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact CrossMabs and deglycosylated / plasmin-digested or alternatively deglycosylated / limited LysC-digested CrossMabs.

[0822] VH / VL CrossMabs were deglycosylated with N-glycosidase F in phosphate or Tris buffer at a protein concentration of 1 mg / ml for 17 hours at 37°C. 100 μg of deglycosylated VH / VLCrossMabs were digested with plasmin or restricted LysC (Roche) for 120 hours at room temperature and 40 minutes at 37°C, respectively. Before mass spectrometry analysis, the sample was desalted on a Sephadex G25 column (GE Healthcare) by HPLC. Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0823] Determine the binding and binding affinity of multispecific antibodies to their respective antigens using surface plasmon resonance (SPR) (BIACORE)

[0824] The binding of the generated antibodies to the respective antigens was investigated using a BIACORE instrument (GE Healthcare Biosciences AB, Uppsala, Sweden) by surface plasmon resonance. Briefly, for affinity measurements, goat anti-human IgG, JIR 109-005-098 antibody was immobilized on a CM5 chip via amine coupling to present antibodies against the respective antigens. Binding was measured at 25°C (or at 37°C) in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween 20, ph 7.4). Antigen (R&D Systems or internal purification) was added to the solution at various concentrations. Binding was measured by injection of antigen for 80 seconds to 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3-10 minutes, and K was estimated using a 1:1 Langmuir binding model. D Negative control data (e.g., buffer curve) were subtracted from the sample curves to correct for system-inherent baseline drift and reduce noise signals. Sensorgrams were analyzed and affinity data were calculated using the respective Biacore evaluation software.

[0825] TIM3 Antibody

[0826] Example 1a: Generation of anti-TIM3 antibodies

[0827] Immunization of mice

[0828] NMRI mice were genetically immunized with a plasmid expression vector encoding full-length human Tim-3 by intradermal application of 100 μg of carrier DNA (plasmid 15304_hTIM3-fl) followed by electroporation (two square pulses of 1000 V / cm, 0.1 ms duration, 0.125 s interval; followed by four square pulses of 287.5 V / cm, 10 ms duration, 0.125 s interval). Mice received six consecutive immunizations on days 0, 14, 28, 42, 56, 70, and 84. Blood was collected and serum was prepared on days 36, 78, and 92 for titer determination by ELISA (see below). Animals with the highest titers were selected for boosting on day 96 with an intravenous injection of 50 μg of recombinant human Tim-3 human Fc chimera, and monoclonal antibodies were isolated by hybridoma technology by fusing splenocytes with myeloma cell lines 3 days after boosting.

[0829] Serum titration determination (ELISA)

[0830] Human recombinant Tim-3 human Fc chimera was immobilized in 96-well NUNC Maxisorp plates at 0.3 μg / ml (100 μl / well) in PBS. The plates were then blocked with 200 μl / well of PBS containing 2% Crotein C. Serial dilutions of antiserum were applied in duplicate at 100 μl / well in PBS containing 0.5% Crotein C. Detection was performed with an HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch / Dianova 115-036-071; 1 / 16,000). For all steps, the plates were incubated at 37°C for 1 hour. Between steps, the plates were washed three times with PBS containing 0.05% Tween 20. Signal was generated by adding soluble BM Blue POD substrate (Roche) at 100 μl / well and stopped by adding 1 M HCl at 100 μl / well. The absorbance at 450 nm was read against 690 nm as a reference. The titer was defined as the dilution of the antiserum that produced a half-maximal signal.

[0831] Example 1b: Characterization of anti-TIM3 antibodies

[0832] ELISA targeting TIM3

[0833] Nunc-MaxiSorp Streptavidin plates (MicroCoat #11974998 / MC1099) were coated with 25 μl / well of Tim3-ECD-His-Biotin (biotinylated with BirA ligase) and incubated at room temperature for 1 hour with shaking at 400 rpm. After washing (3 x 90 μl / well PBST buffer), 25 μl of aTim3 sample or diluted (1:2 step) reference antibody aTim3 F38-2E2 (Biolegend) were added and incubated at room temperature for 1 hour. After washing (3 x 90 μl / well PBST buffer), 25 μl / well of a 1:9000 dilution of sheep anti-mouse POD (GE NA9310V) were added and incubated at room temperature for 1 hour with shaking at 400 rpm. After washing (4 x 90 μl / well PBST buffer), 25 μl / well TMB substrate (Calbiochem, #CL07) was added and incubated until OD 1.5–2.5. The reaction was then stopped by adding 25 μl / well 1N HCl solution. Measurements were taken at 370 / 492 nm.

[0834] ELISA results as EC 50 The values ​​[ng / ml] are listed in Summary Table 1 below.

[0835] Cell ELISA targeting TIM3

[0836] Adherent CHO-K1 cell lines stably transfected with plasmid 15312_hTIM3-fl_pUC_Neo encoding full-length human Tim3 and selected with G418 (neomycin resistance marker on the plasmid) were seeded into 384-well flat-bottom plates at a concentration of 1.2×10E6 cells / ml and grown overnight.

[0837] The next day, 25 μl / well of Tim3 sample or aTim3 reference antibody F38-2E2 without azide (Biolegend, 354004) was added and incubated at 4° C. for 2 hours (to avoid internalization). After washing (3x90 μl / well PBST (BIOTEK washer: program 29, 1x90), the cells were fixed by ejecting the remaining buffer and adding 50 μl / well 0.05% glutaraldehyde: 25% glutaraldehyde (Sigma catalog number: G5882) diluted 1:500 in 1x PBS buffer and incubating at room temperature for 1 hour. After washing (3x90 μl / well PBST (BIOTEK washer: program 21, 3x90 GreinLysin), 25 μl / well secondary antibody for detection (sheep anti-mouse-POD; horseradish POD-linked F(ab')2 fragment; GE NA9310) was added, followed by incubation at room temperature for 2 hours while shaking at 400 rpm. After washing (3x90 μl / well PBST (BIOTEK washer: program 21, 3x90 GreinLysin), 25 μl / well TMB substrate solution (Roche 11835033001) and incubated until OD 1.5–2.5. The reaction was then stopped by adding 25 μl / well of 1N HCl solution. Measurements were taken at 370 / 492 nm.

[0838] The results of cell ELISA were used as “EC 50 The CHO-Tim3" values ​​[ng / ml] are listed in the summary Table 1 below.

[0839] Table 1: Binding affinity of exemplary antibodies (ELISA and BIACORE)

[0840]

[0841] BIAcore characterization of TIM3 antibodies

[0842] The surface plasmon resonance (SPR)-based assay has been used to determine the kinetic parameters of binding between several mouse Tim3 binders and commercial human Tim3 binding references. Therefore, anti-mouse IgG was immobilized to the surface of a (BIAcore) CM5 sensor chip by amine coupling. The sample was then captured and monomeric hu / cy Tim3-ECD and Fc-tagged human Tim3-ECD dimers were bound to them. The sensor chip surface was regenerated after each analysis cycle. Finally, the equilibrium constant K was obtained by fitting the data to a 1:1 Langmuir interaction model. D .

[0843] Approximately 12,000 response units (RU) of 30 μg / ml anti-mouse IgG (GE Healthcare #BR-1008-38) were coupled to spots 1, 2, 4, and 5 of flow cells 1-4 of a CM5 sensor chip in a BIAcore B4000 using an amine coupling kit supplied by GE Healthcare at pH 5.0 (spots 1, 5 are active and spots 2, 4 are reference spots).

[0844] The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4). The flow chamber temperature was set to 25° C. and the sample compartment temperature was set to 12° C. The system was primed with running buffer.

[0845] Samples were injected at a concentration of 200 μg / ml for 30 seconds and bound to spots 1 and 5 on each flow cell, allowing for the measurement of eight samples in parallel. A full range of concentrations (monomeric cynomolgus monkey, monomeric human, and dimeric human Tim3-ECD fused to huFc) were then injected over each sample for 240 seconds, followed by a dissociation time of 30 / 1800 seconds. Each analytical cycle (sample capture, spots 1 and 5 – Tim3 ECD injection) was then regenerated with a 30-second injection of glycine-HCl, pH 1.7. The flow rate for the entire run was set at 30 μl / min.

[0846] Finally, the dual-referenced data were fitted to a 1:1 Langmuir interaction model using BIAcore B4000 evaluation software. The resulting affinities for monomeric human and cynomolgus monkey Tim3, and huFc-fused dimeric human Tim3 are shown in Table 2a. The affinity for the huTim3 dimer is likely influenced by avidity interactions and, therefore, appears stronger than the affinity for monomeric huTim3.

[0847] Table 2a: Binding affinity determined by BIAcore - K obtained by kinetic SPR measurements D-nf indicates no possible fit, most likely due to no or weak binding

[0848]

[0849] Determination of affinity for Tim3 via SPR (chimeric TIM3-0016 variant (0018) and humanized version)

[0850] Protein A was immobilized to the surface of a Biacore CM5 sensor chip via amine coupling. Samples were then captured and hu-Tim3-ECD bound to them. The sensor chip surface was regenerated after each analysis cycle. Equilibrium and kinetic rate constants were then obtained by fitting the data to a 1:1 Langmuir interaction model.

[0851] Approximately 2000 response units (RU) of 20 μg / ml protein A were coupled to spots 1, 2, 4 and 5 of all flow cells of a CM5 sensor chip in a Biacore B4000 instrument using an amine coupling kit supplied by GE Healthcare.

[0852] The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4). The flow chamber temperature was set to 25° C. and the sample compartment temperature was set to 12° C. The system was primed with running buffer.

[0853] Different samples were injected at a concentration of 10 nM for 30 seconds and sequentially bound to points 1 and 5 of all flow cells. Then a full set of monomeric human Tim3-ECD dilutions (600 nM, 200 nM, 66.7 nM, 2 x 22.2 nM, 7.4 nM, 2.5 nM and 2 x 0 nM) were sequentially injected for 300 seconds on each sample. Each antigen injection was followed by a dissociation time of 12 seconds / 1000 seconds and two 30-second regeneration steps using glycine-HCl pH 1.5 solution, the last of which contained a stabilization period after a 20-second injection.

[0854] Finally, the double-referenced data were fitted to a 1:1 Langmuir interaction model using Biacore B4000 evaluation software. D The values ​​are shown in Table 2b.

[0855] Determination of affinity for Tim3 via SPR (chimeric TIM3-0028 and humanized versions)

[0856] Anti-human Fc IgG was immobilized to the surface of a (Biacore) CM5 sensor chip via amine coupling. Samples were then captured and huTim3-ECD bound to them. The sensor chip surface was regenerated after each analysis cycle. Equilibrium and kinetic rate constants were finally obtained by fitting the data to a 1:1 Langmuir interaction model.

[0857] Approximately 2500 response units (RU) of 10 μg / ml anti-human Fc IgG (GE Healthcare #BR-1008-39) were coupled to spots 1, 2, 4 and 5 of all flow cells of a CM5 sensor chip in a Biacore B4000 instrument using an amine coupling kit supplied by GE Healthcare.

[0858] The sample and running buffer was HBS-EP+ (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v / v surfactant P20, pH 7.4). The flow chamber temperature was set to 25° C. and the sample compartment temperature was set to 12° C. The system was primed with running buffer.

[0859] Different samples were injected at a concentration of 10 nM for 30 seconds and sequentially bound to points 1 and 5 of all flow cells. A full set of monomeric human Tim3-ECD dilutions (600 nM, 200 nM, 66.7 nM, 2 x 22.2 nM, 7.4 nM, 2.5 nM, and 2 x 0 nM) was then sequentially injected for 300 seconds over each sample. Each antigen injection was followed by a dissociation time of 12 seconds / 700 seconds and two 30-second regeneration steps using a 3 M MgCl solution, the last of which contained an "additional post-injection wash" using running buffer.

[0860] Finally, the double-referenced data were fitted to a 1:1 Langmuir interaction model using Biacore B4000 evaluation software. D The values ​​are shown in Table 2b.

[0861] Table 2b: Binding affinity determined by BIAcore - K obtained by kinetic SPR measurements D value

[0862] sample <![CDATA[huTim3 K D (25℃)[M]]]> Chimeric TIM3-0016 variant (0018) 2.78E-09 TIM3-0433 5.74E-09 TIM3-0434 5.76E-09 Chimeric TIM3-0028 2.35E-07 TIM3-0438 3.05E-07 TIM3-0443 2.87E-07

[0863] Example 2: Generation of anti-TIM3 antibody derivatives

[0864] Chimeric antibody derivatives

[0865] Chimeric Tim3 antibodies were generated by amplifying the variable heavy and light chain regions of anti-TIM3 mouse antibodies Tim3-0016, Tim3-0016 variant (0018), Tim3-0021, Tim3-0022, Tim3-0026, Tim3-0028, Tim3-0030, and Tim3-0033, Tim3-0038 via PCR and cloning them into a heavy chain expression vector as fusion proteins with human IgG1 backbone / human CH1-hinge-CH2-CH3 (with LALA and PG mutations (leucine 234 to alanine, leucine 235 to alanine, proline 329 to glycine) that eliminate effector function) and into a light chain expression vector as fusion proteins with human C-κ. The LC and HC plasmids were then co-transfected into HEK293 cells and purified from the supernatant after 7 days by standard methods for antibody purification.

[0866] Removal of glycosylation site NYT: modification of one HVR-L1 position in Tim3-0016, Tim3_0016 variant (referred to as 0018 or Tim3_0018) by replacing N with Q or S

[0867] Mutations within the variable light chain region of Tim3_0016 and Tim3_0016 variant (0018) were generated by in vitro mutagenesis using the Agilent "Quick Change Lightning Site-Directed Mutagenesis Kit" according to the manufacturer's instructions. Using this method, the asparagine (N) of the glycosylation site motif NYT in the light chain HVR-L1 (SEQ ID NO:4) was replaced with glutamine (Q) (generating SEQ ID NO:11 = Tim3_0016_HVR-L1 variant 1_NQ), or alternatively, the asparagine (N) was replaced with serine (S) (generating SEQ ID NO:12 = Tim3_0016_HVR-L1 variant 2_NS). In both cases, the glycosylation site motif NYT was successfully modified. The LC and HC plasmids encoding the variants were then co-transfected into HEK293 cells and purified from the supernatant after 7 days using standard methods for antibody purification.

[0868] The generated mutants were tested by ELISA on human Tim3, ELISA on cynomolgus monkey Tim3 and cell ELISA on adherent CHO-K1 cells expressing full-length human Tim3.

[0869] Table 3:

[0870]

[0871]

[0872] It was found that all mutants generated showed even more functional binding to human TIM3 (human), cynomolgus monkey TIM3 (cyno) or human TIMR on CHO cells than the parent antibody Tim3_0016 or Tim3_0016 antibody variant Tim3_0018, respectively.

[0873] Humanized antibody derivatives

[0874] Humanization of the VH and VL domains of murine anti-Tim3-0016 variant (0018) and anti-Tim3_0028

[0875] Humanized anti-Tim3 antibody variants Tim3-0433 and Tim3-0434 were generated based on the amino acid sequences of the VH and VL domains of a) the anti-Tim3 antibody Tim3_0016 variant (0018) (an amino acid sequence having 6 HVRs, in which the HVR-L1 variant 2_NS (the glycosylation site was removed by mutation from N to S) was used in the light chain), while humanized anti-Tim3 antibody variants Tim3-0438 and Tim3-0443 were generated based on the amino acid sequences of the VH and VL domains of b) the anti-Tim3 antibody Tim3_0028.

[0876] The humanized amino acid sequences of the heavy and light chain variable regions were retrogradely translated into DNA and the resulting cDNAs were synthesized (GenArt). They were then cloned into a heavy chain expression vector as a fusion protein with the human IgG1 backbone / human CH1-hinge-CH2-CH3 with LALA and PG mutations that abolish effector function (leucine 234 to alanine, leucine 235 to alanine, proline 329 to glycine) or into a light chain expression vector as a fusion protein with human C-κ. The LC and HC plasmids were then co-transfected into HEK293 cells and purified from the supernatant after 7 days using standard methods for antibody purification. The resulting humanized Tim3 antibodies were named as follows:

[0877] Table 4: VH and VL sequences of humanized antibodies

[0878] Humanized antibody of Tim3_0016 variant (0018) VH / SEQ ID NO: VL / SEQ ID NO: Tim3-0433 SEQ ID NO:13 SEQ ID NO:14 Tim3-0434 SEQ ID NO:15 SEQ ID NO:16 Humanized antibody against Tim3_0028 VH / SEQ ID NO: VL / SEQ ID NO: Tim3-0438 SEQ ID NO:25 SEQ ID NO:26 Tim3-0443 SEQ ID NO:27 SEQ ID NO:28

[0879] Table 5: HVR sequences of humanized antibodies

[0880]

[0881]

[0882] Example 3: Effect of human anti-TIM-3 antibodies on cytokine production in mixed lymphocyte reaction (MLR)

[0883] Mixed lymphocyte reactions were used to demonstrate the effects of blocking the TIM-3 pathway on lymphocyte effector cells. T cells in the assay were tested for activation and IFN-γ secretion in the presence or absence of anti-TIM-3 mAbs.

[0884] Human lymphocytes were isolated from the peripheral blood of healthy donors by density gradient centrifugation using Leukosep (Greiner Bio One, 227 288). Briefly, heparinized blood was diluted with 3 volumes of PBS and a 25 ml sample of the diluted blood was layered in a 50 ml Leukosep tube. After centrifugation at 800 x g for 15 minutes (without interruption) at room temperature, the fraction containing lymphocytes was harvested, washed in PBS and used directly in functional assays or resuspended in freezing medium (10% DMSO, 90% FCS) at 1.0E+07 cells / ml and stored in liquid nitrogen. A 1:1 target cell / responder cell ratio was used in the MLR assay (i.e., each MLR culture contained -2.0E+05 PBMCs from each donor in a total volume of 200 μl). Anti-TIM3 monoclonal antibodies Tim3_0016, Tim3_0016 variant (Tim3_0018), Tim3_0021, Tim3_0022, Tim3_0026, Tim3_0028, Tim3_0030, Tim3_0033, Tim3_0038, and F38-2E2 (BioLegend) were added to each culture at various antibody concentrations. Negative controls included either no antibody or an isotype control antibody, while recombinant hu IL-2 (20 EU / ml) was used as a positive control. Cells were cultured at 37°C for 6 days. After day 6, 100 μl of culture medium was removed from each culture for cytokine measurement. Cytokine expression was assessed using the OptEIA IFN-γ levels were measured using an ELISA kit (BD Biosciences).

[0885] The results are shown in Table 6 (IFN-g secretion / release). Anti-TIM-3 monoclonal antibodies promoted T cell activation and IFN-γ secretion in a concentration-dependent manner. Anti-TIM3 antibodies Tim3_0021, Tim3_0022, Tim3_0028, and Tim3_0038 reduced the release of inflammatory cytokines (IFN-γ) more than the F38-2E2 antibody. When compared to the F38-2E2 antibody, Tim3_0016, a variant of Tim3_0016 (Tim3_0018), Tim3_0033, and Tim3_0038 showed similar release. In contrast, cultures containing an isotype control antibody did not show an increase in IFN-γ secretion.

[0886] Table 6a: Percentage of IFNγ release induced by anti-Tim3 antibodies compared to rec hu IL-2 (20 EU / ml) as positive control (=100%) and no antibody as negative control

[0887]

[0888] In further experiments, the EC values ​​of the following chimeric and humanized antibodies (generated as described above) in combination with 0.1 μg / ml anti-PD1 mAb were measured. 50 Values: Chimeric chi_Tim3_018 antibody and its humanized versions Tim3-433 and Tim3-434, chimeric chi_Tim3_028 antibody and its humanized versions Tim3-438 and Tim3-443 were measured using different lymphocyte donor mixtures (D2 and D3, or D1 and D5, respectively). The results are shown in Table 6b.

[0889] Table 6b: ECs induced by anti-Tim3 antibodies (IFN-g secretion / release) 50

[0890] Antibody <![CDATA[EC of donor D2+D3 50 [nM]]]> <![CDATA[EC of donor D1+D5 50 [nM]]]> chi_Tim3_018 3.1 4.2 Tim3-433 3.0 2.4 Tim3-434 1.7 2.6 chi_Tim3_028 2.9 6.4 Tim3-438 1.9 2.7 Tim3-443 3.0 4.7

[0891] PD1 antibodies

[0892] Example 4: Generation of anti-PD-1 antibodies

[0893] Immunization of mice

[0894] NMRI mice were genetically immunized with a plasmid expression vector encoding full-length human PD-1 by intradermal application of 100 ug of vector DNA (plasmid 15300_hPD1-fl) followed by electroporation (two 1000 V / cm rectangular pulses for 0.1 ms with an interval of 0.125 s; followed by four 287.5 V / cm rectangular pulses for 10 ms with an interval of 0.125 s). Mice received six consecutive immunizations on days 0, 14, 28, 42, 56, 70, and 84. Blood was collected and serum was prepared on days 36, 78, and 92 for titer determination by ELISA (see below). Animals with the highest titers were selected for boosting on day 96 by intravenous injection of 50 ug of recombinant human PD1 human Fc chimera, and monoclonal antibodies were isolated by hybridoma technology by fusing splenocytes with myeloma cell lines 3 days after the boost.

[0895] Determination of serum titer (ELISA)

[0896] Human recombinant PD1-human Fc chimera was immobilized in 96-well NUNC Maxisorp plates at 0.3 μg / ml (100 μl / well) in PBS. Plates were then blocked with 200 μl / well of PBS containing 2% Crotein C; duplicate application of serial dilutions of antiserum in PBS containing 0.5% Crotein C (100 μl / well); and detection with an HRP-conjugated goat anti-mouse antibody (Jackson Immunoresearch / Dianova 115-036-071; 1 / 16,000). For all steps, the plates were incubated at 37°C for 1 hour. Between steps, the plates were washed three times with PBS containing 0.05% Tween 20. Signal generation was achieved by adding 100 μl / well of BM Blue POD substrate solution (Roche) and termination was achieved by adding 100 μl / well of 1 M HCl. Absorbance was read at 450 nm, referenced to 690 nm. The titer was defined as the dilution of antiserum that produced a half-maximal signal.

[0897] Example 5: Characterization of anti-PD1 antibodies / binding of anti-PD1 antibodies to human PD1

[0898] ELISA for human PD1

[0899] Nunc Maxisorp streptavidin-coated plates (MicroCoat #11974998001) were coated with 25 μl / well of biotinylated PD1-ECD-AviHis and incubated overnight at 4°C. After washing (3 x 90 μl / well PBST buffer), 25 μl of anti-PD1 sample or reference antibody (human anti-PD1; Roche / mouse anti-PD1; Biolegend; catalog: 329912) was added and incubated at room temperature for 1 hour. After washing (3 x 90 μl / well PBST buffer), 25 μl / well of goat anti-human H+L-POD (JIR, JIR109-036-088) / sheep anti-mouse-POD (GE Healthcare; NA9310) at a dilution of 1:2000 / 1:1000 was added and incubated on a shaker at room temperature for 1 hour. After washing (3 x 90 μl / well PBST buffer), 25 μl / well TMB substrate (Roche cat. no. 11835033001) was added and incubated until OD 2 - 3. Measurements were taken at 370 / 492 nm.

[0900] The ELISA results are listed as EC50 values ​​[ng / ml] in the summary Tables 7 and 8 below.

[0901] Cell ELISA targeting PD1

[0902] Adherent CHO-K1 cell lines stably transfected with plasmid 15311_hPD1-fl_pUC_Neo encoding full-length human PD1 and selected with G418 (neomycin resistance marker on the plasmid) were seeded into 384-well flat-bottom plates at a concentration of 0.01×10E6 cells / ml and grown overnight.

[0903] The next day, 25 μl / well of PD1 sample or human anti-PD1 (Roche) / mouse anti-PD1 (Biolegend; catalog: 329912) reference antibody was added and incubated at 4°C for 2 hours (to prevent internalization). After careful washing (1 x 90 μl / well PBST), cells were fixed by adding 30 μl / well of 0.05% glutaraldehyde (Sigma, catalog number: G5882, 25%) diluted in 1x PBS buffer and incubating at room temperature for 10 minutes. After washing (3 x 90 μl / well PBST), 25 μl / well of secondary antibody for detection was added: goat anti-human H+L-POD (JIR, JIR109-036-088) / sheep anti-mouse-POD (GE NA9310), followed by incubation at room temperature for 1 hour on a shaker. After washing (3 x 90 μl / well PBST), 25 μl / well TMB substrate solution (Roche 11835033001) was added and incubated until OD 1.0-2.0. The plate was measured at 370 / 492 nm.

[0904] The cell ELISA results are listed below in the summary Table 8 as "EC50 CHO-PD1" values ​​[ng / ml].

[0905] ELISA for cynomolgus monkey PD1

[0906] Nunc Maxisorp Streptavidin-coated plates (MicroCoat #11974998001) were coated with 25 μl / well of biotinylated cynoPD1-ECD-biotin and incubated overnight at 4°C. After washing (3 x 90 μl / well PBST buffer), 25 μl of anti-PD1 sample or reference antibody (human anti-PD1; Roche) was added and incubated on a shaker for 1 hour at room temperature. After washing (3 x 90 μl / well PBST buffer), 25 μl / well of goat anti-human H+L-POD (JIR, JIR109-036-088) was added at a 1:1000 dilution and incubated on a shaker for 1 hour at room temperature. After washing (3 x 90 μl / well PBS...

Claims

1. A bispecific antibody comprising: an Fc domain, a first Fab fragment comprising a first antigen binding site that specifically binds to PD1, and a second Fab fragment comprising a second antigen binding site that specifically binds to TIM3, wherein The first antigen binding site that specifically binds to PD1 comprises The VH domain comprises (i) HVR-H1 as shown in the amino acid sequence of SEQ ID NO:37, (ii) HVR-H2 as shown in the amino acid sequence of SEQ ID NO:38, and (iii) HVR-H3 as shown in the amino acid sequence of SEQ ID NO:39; and The VL domain contains (i) HVR-L1 as shown in the amino acid sequence of SEQ ID NO:40, (ii) HVR-L2 as shown in the amino acid sequence of SEQ ID NO:41, and (iii) HVR-L3 as shown in the amino acid sequence of SEQ ID NO:42; And the second antigen binding site that specifically binds to TIM3 comprises The VH domain comprises (i) HVR-H1 as shown in the amino acid sequence of SEQ ID NO: 1, (ii) HVR-H2 as shown in the amino acid sequence of SEQ ID NO:2, and (iii) HVR-H3 as shown in the amino acid sequence of SEQ ID NO:3; and The VL domain contains (i) HVR-L1 as shown in the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:11 or SEQ ID NO:12, (ii) HVR-L2 as shown in the amino acid sequence of SEQ ID NO:5, and (iii) HVR-L3 as shown in the amino acid sequence of SEQ ID NO:

6.

2. The bispecific antibody according to claim 1, wherein The first antigen binding site that specifically binds to PD1 comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 43 and a VL domain comprising the amino acid sequence of SEQ ID NO: 44, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 46, or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 47, or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 48, or (e) a VH domain comprising the amino acid sequence of SEQ ID NO: 45 and a VL domain comprising the amino acid sequence of SEQ ID NO: 49, And the second antigen binding site that specifically binds to TIM3 comprises (a) a VH domain comprising the amino acid sequence of SEQ ID NO: 7 and a VL domain comprising the amino acid sequence of SEQ ID NO: 8, or (b) a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10, or (c) a VH domain comprising the amino acid sequence of SEQ ID NO: 13 and a VL domain comprising the amino acid sequence of SEQ ID NO: 14, or (d) a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO:

16.

3. The bispecific antibody according to claim 1 or 2, wherein The first antigen binding site that specifically binds to PD1 comprises a VH domain comprising an amino acid sequence of SEQ ID NO: 45 and a VL domain comprising an amino acid sequence of SEQ ID NO: 46, And the second antigen binding site that specifically binds to TM3 comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and a VL domain comprising the amino acid sequence of SEQ ID NO:

16.

4. The bispecific antibody according to claim 1, wherein the bispecific antibody is a humanized antibody.

5. The bispecific antibody according to claim 1, wherein the Fc domain is an IgG1 Fc domain or an IgG4 Fc domain.

6. The bispecific antibody according to claim 1, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to an Fc receptor.

7. The bispecific antibody according to claim 1, wherein the Fc domain comprises one or more amino acid substitutions that reduce binding to Fcγ receptors.

8. The bispecific antibody according to claim 1, wherein the Fc domain is of human IgG1 subclass with amino acid mutations L234A, L235A and P329G, numbering according to the Kabat EU index.

9. The bispecific antibody according to claim 1, wherein the Fc domain comprises a modification that promotes association of the first and second subunits of the Fc domain.

10. The bispecific antibody according to claim 1, wherein the first subunit of the Fc domain comprises a knob and the second subunit of the Fc domain comprises a hole according to the knob-into-hole approach.

11. The bispecific antibody according to claim 1, wherein the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W, numbering according to the Kabat EU index, and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S and Y407V, numbering according to the Kabat EU index.

12. The bispecific antibody according to claim 1, wherein in one of the Fab fragments the variable domains VL and VH are replaced with each other, such that the VH domain is part of the light chain and the VL domain is part of the heavy chain.

13. The bispecific antibody according to claim 12, wherein in the first Fab fragment comprising the antigen binding site that specifically binds to PD1, the variable domains VL and VH are replaced with each other.

14. The bispecific antibody according to claim 1, wherein in one of the Fab fragments the amino acid at position 124 in the constant domain CL is independently substituted by lysine (K), arginine (R) or histidine (H), numbering according to the Kabat EU index, and the amino acids at positions 147 and 213 in the constant domain CH1 are independently substituted by glutamic acid (E) or aspartic acid (D), numbering according to the Kabat EU index.

15. The bispecific antibody according to claim 14, wherein in the second Fab fragment comprising the antigen binding site that specifically binds TIM3, the amino acid at position 124 in the constant domain CL is independently replaced by lysine (K), arginine (R) or histidine (H), numbering according to the Kabat EU index, and the amino acids at positions 147 and 213 in the constant domain CH1 are independently replaced by glutamic acid (E) or aspartic acid (D), numbering according to the Kabat EU index.

16. The bispecific antibody according to claim 1, comprising (a) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 54, and a first light chain comprising the amino acid sequence of SEQ ID NO: 56, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 55, and a second light chain comprising the amino acid sequence of SEQ ID NO: 57, or (b) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 66, and a first light chain comprising the amino acid sequence of SEQ ID NO: 68, A second heavy chain comprising the amino acid sequence of SEQ ID NO: 67, and a second light chain comprising the amino acid sequence of SEQ ID NO:

69.

17. A polynucleotide encoding a bispecific antibody according to any one of claims 1 to 16.

18. A vector comprising the polynucleotide according to claim 17.

19. The vector according to claim 18, which is an expression vector.

20. A prokaryotic or eukaryotic host cell comprising a polynucleotide according to claim 17 or a vector according to claim 18 or 19.

21. A method for producing a bispecific antibody according to any one of claims 1 to 16, comprising the steps of: a) transforming a host cell with a vector comprising a polynucleotide encoding the bispecific antibody, b) culturing the host cell under conditions suitable for expression of the bispecific antibody, and c) recovering the bispecific antibody from the culture.

22. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 16 and at least one pharmaceutically acceptable excipient.

23. Use of the bispecific antibody according to any one of claims 1 to 16 or the pharmaceutical composition according to claim 22 for the preparation of a medicament for restoring T cell activity.

24. Use of a bispecific antibody according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 22 for the preparation of a medicament for stimulating an immune response.

25. Use of a bispecific antibody according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 22 for the preparation of a medicament for inhibiting tumor growth.

26. Use of a bispecific antibody according to any one of claims 1 to 16 or a pharmaceutical composition according to claim 22 for the preparation of a medicament for inhibiting tumor growth, wherein the bispecific antibody is administered in combination with a chemotherapeutic agent, radiation and / or other agents for cancer immunotherapy.

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