Antibodies and methods of use

Abstract

The present invention relates to antibodies and other therapeutic proteins directed against SLAM family member 6 (SLAMF6), also known as NTB-A or CD352, nucleic acids encoding such antibodies and therapeutic proteins, methods of making antibodies and other therapeutic proteins, and methods of treating diseases (e.g., cancer) by using antibodies and other therapeutic proteins directed against SLAMF6.

Description

Field of the Invention

[0001] The present invention relates to antibodies capable of binding to SLAMF6 protein and uses thereof.

[0002] introduction

[0003] Aspects of the invention include antibodies and other therapeutic proteins directed against SLAM family member 6 (SLAMF6), also known as NTB-A or CD352, nucleic acids encoding such antibodies and therapeutic proteins, methods of making the antibodies and other therapeutic proteins, and methods of treating diseases (e.g., cancer) by using antibodies and other therapeutic proteins directed against SLAMF6. Background Art

[0004] The target antigen SLAMF6 is a single-pass type I membrane protein and a member of the immunoglobulin superfamily and the CD2 subfamily (J Exp Med. 2001 Aug 6; 194(3):235-46). Its activity is controlled by the presence or absence of the small cytoplasmic adaptor proteins SH2D1A / SAP and / or SH2D1B / EAT-2. The protein triggers cytolytic activity only in natural killer (NK) cells that express a high surface density of natural cytotoxicity receptors (J. Exp. Med.

[0005] 194:235-246 (2001)). Positive signal transduction in NK cells indicates phosphorylation of VAV1. NK cell activation appears to depend on SH2D1B rather than SH2D1A. In conjunction with SLAMF1, SLAMF6 controls the transition between positive selection and subsequent expansion and differentiation of the thymocyte natural killer T (NKT) cell lineage. SLAMF6 also promotes T cell differentiation into the helper T cell Th17 phenotype, leading to increased IL-17 secretion; costimulatory activity requires SH2D1A (J.Immunol.177:3170-3177 (2006)). It further promotes the recruitment of RORC to the IL-17 promoter (J.Biol.Chem.287:38168-38177 (2012)). In conjunction with SLAMF1 and CD84 / SLAMF5, SLAMF6 may be a negative regulator of humoral immune responses. In the absence of SH2D1A / SAP, SLAMF6 can direct its expression to CD4 + It also negatively regulates germinal center formation by inhibiting T cell:B cell adhesion; this function may implicate increased association with PTPN6 / SHP-1 via ITSM in the absence of SH2D1A / SAP.

[0006] WO2008 / 027739 discloses anti-NTB-A antibodies and pharmaceutical compositions comprising such antibodies. Methods of using such antibodies to bind NTB-A and treat diseases, such as hematological malignancies, characterized by NTB-A expression are also described.

[0007] WO2014 / 100740 and WO2017 / 004330 disclose antibodies that specifically bind to NTB-A, including antibody-drug conjugates, and methods for detecting or regulating the activity of cells expressing NTB-A using these antibodies. Methods for treating diseases associated with cells expressing NTB-A, such as multiple myeloma, non-Hodgkin's lymphoma, and acute myeloid leukemia are also disclosed.

[0008] WO2015 / 104711 describes compositions and methods for improved T cell regulation in vitro and in vivo and for treating cancer and other pathologies. More specifically, embodiments of the present invention relate to the use of soluble NTB-A polypeptides or agonists thereof for treating cancer patients, for preventing and treating cytopenias in susceptible patients, and for the ex vivo preparation of improved cell compositions. Summary of the invention

[0009] Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and a tumor-associated antigen, nucleic acids encoding such antibodies of the invention, host cells comprising such nucleic acids encoding antibodies of the invention, methods of making antibodies of the invention, and methods for treating diseases such as human cancers, including but not limited to small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma) skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers and gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

[0010] Antibodies that can bind to SLAMF6 (SEQ ID NO: 11) are described herein. Preferably, the antibody binds to the extracellular domain of SLAMF6 (SEQ ID NO: 12). Aspects of the present invention include antibodies or antigen-binding fragments thereof that bind to an epitope on the SLAMF6 protein recognized by the antibodies described herein, or that cross-compete for binding with the antibodies described herein, and that preferably retain at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the binding affinity of the antibodies described herein for human SLAMF6. In some embodiments, the antibody is an isolated antibody.

[0011] Aspects of the present invention include an antibody or antigen-binding fragment thereof that can bind to SLAMF6, the antibody comprising a heavy chain variable region, the heavy chain variable region comprising: a CDR-H1 sequence comprising a sequence of SEQ ID NO: 5; a CDR-H2 sequence comprising a sequence of SEQ ID NO: 6 or SEQ ID NO: 15; and a CDR-H3 sequence comprising a sequence of SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding fragment further comprises a light chain variable region, the light chain variable region comprising at least one CDR sequence selected from the group consisting of: a CDR-L1 comprising any one of SEQ ID NO: 8 or SEQ ID NO: 16; a CDR-L2 comprising any one of SEQ ID NO: 9 or SEQ ID NO: 17; and a CDR-L3 comprising a sequence of SEQ ID NO: 10.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof that binds to SLAMF6 comprises a heavy chain variable region and a light chain variable region, which comprises one of the eight combinations of heavy chain and light chain CDRs shown in Table 1.

[0013] Table 1

[0014]

[0015] In some preferred embodiments, the antibody or antigen-binding fragment thereof that can bind to SLAMF6 comprises a heavy chain variable region, wherein the heavy chain variable region comprises: CDR-H1 comprising SEQ ID NO: 5; CDR-H2 comprising SEQ ID NO: 6; and CDR-H3 comprising SEQ ID NO: 7; and a light chain variable region, wherein the light chain variable region comprises: CDR-L1 comprising SEQ ID NO: 8; CDR-L2 comprising SEQ ID NO: 9; and CDR-L3 comprising SEQ ID NO: 10.

[0016] In other preferred embodiments, the antibody or its antigen-binding fragment that can bind to SLAMF6 comprises a heavy chain variable region, which comprises: CDR-H1 comprising SEQ ID NO: 5; CDR-H2 comprising SEQ ID NO: 15; and CDR-H3 comprising SEQ ID NO: 7; and a light chain variable region, which comprises: CDR-L1 comprising SEQ ID NO: 16; CDR-L2 comprising SEQ ID NO: 17; and CDR-L3 comprising SEQ ID NO: 10.

[0017] On the other hand, the antibody or antigen-binding fragment thereof of the present invention comprises a variant CDR compared to the parent antibody described herein. Therefore, the present invention provides a variant antibody or antigen-binding fragment thereof, which comprises a variant variable region of a parent antibody, wherein the parent antibody or antigen-binding fragment thereof comprises a heavy chain variable region, the heavy chain variable region comprising: a CDR-H1 sequence comprising a sequence of SEQ ID NO: 5; a CDR-H2 sequence comprising a sequence of SEQ ID NO: 15; and a CDR-H3 sequence comprising a sequence of SEQ ID NO: 7; and a light chain variable region, the light chain variable region comprising: a CDR-L1 comprising a sequence of SEQ ID NO: 16; a CDR-L2 comprising a sequence of SEQ ID NO: 17; and a CDR-L3 comprising a sequence of SEQ ID NO: 18. NO:10 sequence CDR-L3, and wherein, in one embodiment, the variant antibody or its antigen-binding fragment has 1, 2, 3, 4, 5 or 6 amino acid substitutions, additions and / or deletions in any one or more of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 groups; or has a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions, additions and / or deletions in any one or more of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 groups; wherein 1 to 5 or 1 to 4 or 1 to 3 substitutions, additions or deletions have specific uses, and wherein the antibody or its antigen-binding fragment retains binding specificity to SLAMF6. Preferably, the variation is a substitution, preferably, the substitution is a conservative substitution, or a substitution that returns the amino acid in the variable region to the corresponding amino acid of the human germline. In other embodiments, a variant antibody or antigen-binding fragment thereof of the present invention comprises: a CDR-H1 sequence comprising the sequence of SEQ ID NO:5; a CDR-H2 sequence comprising the sequence of SEQ ID NO:15; and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7; and a light chain variable region comprising: a CDR-L1 comprising the sequence of SEQ ID NO:16; a CDR-L2 comprising the sequence of SEQ ID NO:17; and a CDR-L3 comprising the sequence of SEQ ID NO:10, wherein one or more of the CDR sequences are altered so that they are approximately 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding parent CDR sequence described above.

[0018] In some embodiments, an antibody or an antigen-binding fragment thereof is provided, which comprises a heavy chain variable region described in SEQ ID NO:1 or SEQ ID NO:13, or a sequence approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:1 or SEQ ID NO:13, and / or a light chain variable region described in SEQ ID NO:2 or SEQ ID NO:14, or a sequence approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:2 or SEQ ID NO:14. In other embodiments, an antibody or antigen-binding fragment thereof is provided, comprising a heavy chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 1 or SEQ ID NO: 13, and / or a light chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 2 or SEQ ID NO: 14. Preferably, the variant comprises a substitution, more preferably a conservative substitution.

[0019] It will further be apparent that amino acid substitutions, additions and / or deletions may be within the framework regions and / or within the CDRs.

[0020] In one embodiment, an antibody or an antigen-binding fragment thereof is provided, which comprises a heavy chain variable region, wherein the heavy chain variable region contains the sequence of SEQ ID NO:1, and a light chain variable region, wherein the light chain variable region contains the sequence of SEQ ID NO:2.

[0021] In one embodiment, an antibody or an antigen-binding fragment thereof is provided, which comprises a heavy chain variable region, wherein the heavy chain variable region contains the sequence of SEQ ID NO:13, and a light chain variable region, wherein the light chain variable region contains the sequence of SEQ ID NO:14.

[0022] In one embodiment, a full-length antibody is provided, comprising a heavy chain sequence comprising SEQ ID NO:18 and a light chain sequence comprising SEQ ID NO:19.

[0023] In another aspect of the present invention, an antibody or antigen-binding fragment thereof that can specifically bind to SLAMF6 is provided, wherein the antibody or antigen-binding fragment thereof contains 3 heavy chain CDRs of SEQ ID NO: 1 or SEQ ID NO: 13, and 3 light chain CDRs of SEQ ID NO: 2 or SEQ ID NO: 14, wherein the CDRs are defined by the Kabat or Chothia numbering system. Preferably, the antibody or antigen-binding fragment thereof that can specifically bind to SLAMF6 comprises 3 heavy chain CDRs of SEQ ID NO: 13 and 3 light chain CDRs of SEQ ID NO: 14 defined by the Kabat or Chothia numbering system.

[0024] SEQ ID NO: 13-19 are humanized antibody sequences based on the sequences of SEQ ID NO: 1 and 2. It will be readily understood by those skilled in the art that SEQ ID NO: 18-19 are full-length heavy and light chain sequences including non-variable region regions (e.g., constant regions and Fc regions) required to produce full-length functional antibodies. It will be appreciated by those skilled in the art that these sequences can be humanized by replacing the amino acids of the variable regions of the organism producing the antibodies with amino acids of human germline sequences, the replacement being performed in a manner that minimizes the immunogenic effects of these antibodies when administered to human subjects. Most amino acid substitutions occur in the framework region, however, many amino acids present in non-critical positions of the CDRs can also be substituted; such substitutions are preferably conservative in nature or return the amino acids at specific positions to the amino acids present in the corresponding human germline. In the present case, the amino acids from the substituted CDRs have been identified using structural models to distinguish between paratope facing residues and non-paratope residues in the CDR region. This allows antibodies to be humanized to a higher degree than simple CDR transplantation.

[0025] In the present invention, SEQ ID NO: 13 comprises two amino acid substitutions in CDR2 (SEQ ID NO: 15) compared to CDR2 (SEQ ID NO: 6) of SEQ ID NO: 1. Specifically, there is a KQ substitution at position 16 of SEQ ID NO: 6; and there is a DG substitution at position 17 of SEQ ID NO: 6.

[0026] In the present invention, SEQ ID NO: 14 comprises 3 amino acid substitutions in CDR1 (SEQ ID NO: 16) compared to CDR1 (SEQ ID NO: 8) of SEQ ID NO: 2. Specifically, there is a SQ substitution at position 1 of SEQ ID NO: 8; there is a SQ substitution at position 4 of SEQ ID NO: 8; and there is a SD substitution at position 5 of SEQ ID NO: 8. Compared to CDR2 (SEQ ID NO: 9) of SEQ ID NO: 2, SEQ ID NO: 14 further comprises 1 amino acid substitution in CDR2 (SEQ ID NO: 17). Specifically, there is a ST substitution at position 7 of SEQ ID NO: 9.

[0027] One skilled in the art will appreciate that these humanized sequences do not represent different, alternative antibodies when compared to the parent antibody, but rather relate to the same antibody with the same characteristics, which have merely been altered to more closely correspond to the human germline (using structural modeling to minimize immunogenicity).

[0028] In some embodiments, the antibody or antigen-binding fragment has a binding affinity (K of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM or less. D ).

[0029] In some embodiments, the antibody or antigen binding fragment is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized or human antibody. In some embodiments, the heavy chain variable region comprises a framework sequence. In some embodiments, at least a portion of the framework sequence comprises a human consensus framework sequence. In some embodiments, the light chain variable region comprises a framework sequence. In some embodiments, at least a portion of the framework sequence comprises a human consensus framework sequence.

[0030] Alternative strategies have also been used to mitigate antibody effector functions, including substitutions of residues in the lower hinge of the antibody (e.g., L234A and L235A (LALA)). These residues form part of the Fc-γ receptor binding site on the CH2 domain, and the exchange of these residues between antibody isotypes with more or less effector functions determines their importance in ADCC. Although alanine substitutions at these sites can effectively reduce ADCC in human and mouse antibodies, these substitutions are less effective in reducing CDC activity (Lo M et al., J Biol Chem. March 3, 2017; 292 (9): 3900-3908). In some embodiments, the antibody or antigen-binding fragment is an Fc variant engineered to reduce binding to the FCγ receptor, and it results in reduced effector function and ADCC activity.

[0031] In some embodiments, the antibody or antigen binding fragment is an Fc-silent engineered IgG1 antibody or antigen binding fragment having reduced or no binding to one or more Fcs. In another embodiment, the antibody is an IgG4 antibody.

[0032] In some embodiments, the antibody or antigen binding fragment is a bispecific antibody or antigen binding fragment that mediates T cell cytotoxicity and / or NK cell cytotoxicity. In some embodiments, the antibody or antigen binding fragment can induce and / or enhance the activation of immune cells. In one embodiment, the immune cell is preferably a T cell. In another embodiment, the immune cell is preferably a NK cell. It will be understood by those skilled in the art that the term induction and / or enhancement may refer to induction and / or enhancement of cytokine release of immune cells and / or induction and / or enhancement of the proliferation and / or induction and / or enhancement of the immune cells. It is obvious to those skilled in the art that the term induction used herein or induction refers to causing the activation of immune cells, or increasing the activation of immune cells to a higher level of activation than that seen when there is no antibody or antigen binding fragment. The term enhancement used in this article refers to increasing the activation level of activated immune cells.

[0033] In some embodiments, the antibody or antigen binding fragment is a bispecific or multispecific antibody or antigen binding fragment that can bind to the SLAMF6 protein (e.g., SEQ ID NO: 11) and bind to one or more other binding targets, preferably the other binding targets are one or more tumor antigens. In another embodiment, one or more other binding targets are immunomodulatory molecules. In one embodiment, the other binding target is PD-L1. In one embodiment, the antibody or its antigen binding fragment is divalent. In another embodiment, the antibody or its antigen binding fragment is tetravalent. In another embodiment, the antibody or its antigen binding fragment is trivalent.

[0034] In some embodiments, the antigen binding fragment is selected from the group consisting of: Fab, Fab', F(ab)2, F(ab')2, Fv, FVTCR, scFv, dAb and single domain antibody.

[0035] In another aspect of the present invention, one or more nucleic acids encoding heavy chains of antibodies of the present invention and / or light chains of antibodies of the present invention are provided. It should be understood that the heavy and light chains of antibodies of the present invention can be on a single nucleic acid molecule, or can be encoded together by two separate nucleic acid molecules.

[0036] In another aspect, a vector comprising one or more nucleic acids of the invention is provided.

[0037] In another aspect of the present invention, a host cell comprising one or more nucleic acids encoding the heavy chain and / or light chain of an antibody of the present invention or both is provided. In some embodiments, the host cell is grown under conditions expressing the nucleic acid. In other embodiments, a method for reclaiming an antibody of the present invention is provided.

[0038] Aspects of the invention include methods of making an antibody or antigen-binding fragment thereof, the methods comprising: culturing a host cell under conditions wherein the antibody or antigen-binding fragment is expressed in the host cell, and, optionally, isolating the antibody or antigen-binding fragment.

[0039] Aspects of the invention include pharmaceutical compositions comprising an antibody or antigen-binding fragment as described herein and a pharmaceutically acceptable carrier.

[0040] In some embodiments, the pharmaceutical composition or medicament further comprises an effective amount of a second therapeutic agent.

[0041] In another aspect of the present invention, a method of treating a disease is provided, the method comprising: administering to a patient in need thereof an antibody or antigen-binding fragment of the present invention that binds to SLAMF6 (SEQ ID NO: 11). In one embodiment, the disease is cancer.

[0042] In another aspect, a method for treating cancer is provided, comprising administering an effective amount of the antibody or antigen-binding fragment of the present invention to a subject in need thereof.

[0043] In one embodiment, the antibody or antigen-binding fragment comprises a heavy chain variable region comprising: CDR-H1 comprising SEQ ID NO:5; CDR-H2 comprising SEQ ID NO:6 or SEQ ID NO:15, and CDR-H3 comprising SEQ ID NO:7, and a light chain variable region comprising: CDR-L1 comprising SEQ ID NO:8 or SEQ ID NO:16, CDR-L2 comprising SEQ ID NO:9 or SEQ ID NO:17, and CDR-L3 comprising SEQ ID NO:10.

[0044] Preferably, the heavy chain variable region comprises: CDR-H1 comprising SEQ ID NO:5, CDR-H2 comprising SEQ ID NO:15 and CDR-3 comprising SEQ ID NO:7.

[0045] Preferably, the light chain variable region comprises: CDR-L1 comprising SEQ ID NO:16, CDR-L2 comprising SEQ ID NO:17 and CDR-L3 comprising SEQ ID NO:10.

[0046] In some embodiments, a method for treating cancer is provided, wherein an antibody or antigen-binding fragment of the present invention is administered to a patient in need thereof, and wherein the antibody or antigen-binding fragment of the present invention can induce and / or enhance an immune response, e.g., a cytotoxic T cell response and / or a NK cell response.

[0047] In another embodiment, the antibody or antigen-binding fragment thereof comprises:

[0048] A bispecific or multispecific antibody or an antigen-binding fragment thereof comprising SLAMF6 (SEQ ID NO: 11) and a tumor-specific antigen.

[0049] In some embodiments, the cancer is selected from small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors, and hematological malignancies.

[0050] According to another aspect of the present invention, there is provided the antibody or antigen-binding fragment of the present invention for use in prevention or treatment.

[0051] Preferably, the antibodies or antigen-binding fragments are used to prevent or treat cancer.

[0052] According to another aspect of the present invention, there is provided use of the antibody or antigen-binding fragment according to the present invention in the preparation of a medicament for treating cancer.

[0053] In some embodiments, the cancer according to the preceding aspects is selected from small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors and hematological malignancies. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1a The amino acid sequence of the parental murine 1B3 antibody heavy chain variable region is shown (SEQ ID NO: 1).

[0056] Figure 1bThe amino acid sequence of the parental murine 1B3 antibody light chain variable region is shown (SEQ ID NO: 2).

[0057] Figure 2a The amino acid sequence of the heavy chain variable region of the humanized antibody Hu_1B3 is shown (SEQ ID NO: 13).

[0058] Figure 2b The amino acid sequence of the light chain variable region of the humanized antibody Hu_1B3 is shown (SEQ ID NO: 14).

[0059] Figure 3a Shown is a sequence alignment of the heavy chain variable region of the 1B3 parent murine antibody sequence and the humanized heavy chain variable region 1B3 sequence.

[0060] Figure 3b Shown is a sequence alignment of the light chain variable region of the 1B3 parental murine antibody sequence and the humanized light chain variable region Hu_1B3 sequence.

[0061] Figure 4 Specific dose-related binding of antibody 1B3 to 1B3-expressing Raji cells was demonstrated.

[0062] Figure 5 The ability of antibody Hu_1B3 to mediate IFNγ production after T cell activation was shown to be enhanced compared to another clinical-stage antibody, SLAMF6.

[0063] Figure 6 Antibody 1B3 was shown to induce interferon gamma production in an ex vivo assay by tumor infiltrating lymphocytes (TILs) isolated from primary NSCLC tumor samples.

[0064] Figure 7 Antibody 1B3 was shown to induce TILs isolated from primary breast cancer samples to produce interferon gamma in an ex vivo assay. The assay demonstrated that antibody 1B3 had enhanced activity compared to pembrolizumab.

[0065] Figure 8 Antibody 1B3 was shown to induce TILs isolated from primary colorectal cancer samples to produce interferon gamma in an ex vivo assay. The assay demonstrated that antibody 1B3 had enhanced activity compared to pembrolizumab.

[0066] Fig. 9 It was shown that activation of SLAMF6 present on isolated T cells using antibody Hu_1B3 induced CD8+ T cell proliferation.

[0067] Fig.10 is an MLR assay and shows that antibody Hu_1B3 can induce DC-mediated T cell activation as highlighted by an increase in IFNγ release.

[0068] Fig.11 It was shown that the anti-SLAMF6 antibody Hu_1B3 could induce activated T cells to produce granzyme B in a dose-dependent manner.

[0069] Fig.12 Antibody Hu_1B3 was shown to upregulate perforin expression on CD8+ T cells in a dose-dependent manner.

[0070] Fig.13 It was shown that in the presence of human SLAMF6ECD-mIgG2aFc fusion protein, Hu_1B3 antibody was blocked from binding to receptors on the surface of PBMCs.

[0071] Fig.14 The humanized antibody Hu_1B3 was shown to be internalized into SLAMF6-expressing cells significantly less than the anti-SLAMF6 antibody produced by Seattle Genetics.

[0072] Fig.15 and 16 It was shown that the addition of Hu_1B3 enhanced lymphocyte cytotoxicity against SKBR-3 cells when used together with the anti-her2 anti-CD3 bispecific antibody.

[0073] Fig.17 It was shown that the addition of Hu_1B3 enhanced lymphocyte cytotoxicity against HCT116 cells when used together with the anti-her2 anti-CD3 bispecific antibody.

[0074] Fig.18 It was shown that the addition of Hu_1B3 enhanced lymphocyte cytotoxicity against MDA-MB-231 cells when used together with the anti-her2 anti-CD3 bispecific antibody.

[0075] Fig.19 It was shown that antibody HU-1B3 caused significantly more SKBR-3 cell death than Urelumab (anti-CD137) or isotype 1 after 96 hours of lymphocyte activation with various antibodies.

[0076] Fig. 20 It was shown that the addition of Hu-1B3 enhanced lymphocyte cytotoxicity against SKBR-3 cells in a dose-dependent manner when used together with a fixed concentration of anti-Her2 / anti-CD3 bispecific antibody.

[0077] Fig.21a and 21b It was shown that the addition of Hu-1B3 in the absence of an enabling bispecific antibody also enhanced lymphocyte cytotoxicity against SKBR-3 cells. DETAILED DESCRIPTION

[0078] Aspects of the invention include antibodies against SLAMF6, nucleic acids encoding such antibodies, host cells comprising such nucleic acids encoding antibodies of the invention, methods of making anti-SLAMF6 antibodies and methods of treating diseases, such as SLAMF6-mediated diseases, such as human cancers, including but not limited to small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma) skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers and gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

[0079] It is noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise. It is also noted that the claims may be drafted to exclude any optional elements. Thus, this statement is intended to serve as antecedent basis for use of the terminology "solely," "only," and the like in connection with the recitation of claim elements or use of a "negative" limitation.

[0080] As will be apparent to those skilled in the art after reading this disclosure, each individual aspect described and illustrated herein has discrete components and features that can be conveniently separated or combined with the features of any other multiple aspects without departing from the scope and spirit of the invention. Any method mentioned can be performed in the order of events mentioned or in any other sequence that is logically feasible.

[0081] definition

[0082] 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 and vice versa.

[0083] Unless otherwise indicated, the term "SLAMF6" as used herein refers to any native SLAMF6 protein from any vertebrate source, including mammals, such as primates (e.g., humans, primates and rodents (e.g., mice and rats)). SLAMF6 protein may also be referred to as SLAMF6-like protein. The amino acid sequence of human SLAMF6 is provided herein in SEQ ID NO: 11.

[0084] The term "SLAMF6" includes "full-length" unprocessed SLAMF6 and any form of SLAMF6 produced by processing in cells. The term also includes naturally occurring variants of SLAMF6, such as splice variants, allelic variants and isoforms. The term specifically includes naturally occurring truncated or secreted forms (e.g., extracellular domain sequences) of SLAMF6 polypeptides. The SLAMF6 polypeptides described herein can be separated from various sources, such as from human tissue types or other sources, or prepared by recombinant or synthetic methods. "Native sequence SLAMF6 polypeptides" include polypeptides having the same amino acid sequence as the corresponding SLAMF6 polypeptides derived from nature. Such native sequence SLAMF6 polypeptides can be separated from nature or can be produced by recombinant or synthetic means. As used herein, the term "SLAMF6 epitope" refers to an epitope bound by an antibody comprising at least one or more CDR sequences described herein, and / or an epitope exemplified by the binding spectrum of the anti-SLAMF6 antibodies as shown in the examples.

[0085] The term "antibody" is used in the broadest sense, specifically covering, for example, a single anti-SLAMF6 monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), an anti-SLAMF6 antibody composition with multi-epitope specificity, a polyclonal antibody formed by at least two complete antibodies, a single-chain anti-SLAMF6 antibody and an antigen-binding fragment of an anti-SLAMF6 antibody (including Fab, Fab', F(ab')2 and Fv and FV-TCR fragments, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological or immunological activity), multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity). The term "immunoglobulin (Ig)" is used interchangeably with the term "antibody" herein. Antibodies can be chimeric, human, humanized and / or affinity matured. It will be understood by those of ordinary skill in the art that, in some embodiments, a minimal form of an antibody comprises a set of 6 CDRs as defined herein; they include, but are not limited to, traditional antibodies (including monoclonal and polyclonal antibodies), humanized, human and / or chimeric antibodies, antibody fragments, engineered antibodies (e.g., with amino acid modifications as described below), multispecific antibodies (including bispecific antibodies), and other analogs known in the art and discussed herein.

[0086] It should be understood that in other embodiments, the term antibody used herein refers to a structure that does not contain 6 CDRs; including but not limited to and scFv fragments.

[0087] The term "anti-SLAMF6 antibody", "SLAMF6 antibody" or "antibody that binds to SLAMF6" refers to an antibody that is capable of binding to SLAMF6 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting SLAMF6. In certain embodiments, the anti-SLAMF6 antibody binds to a SLAMF6 epitope that is conserved in SLAMF6 from different species.

[0088] An "isolated antibody" is an antibody that has been identified, separated and / or recovered from components of its environment. Contaminants of its environment are substances that may interfere with the therapeutic use of the antibody and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes.

[0089] In the context of antibody binding to a target molecule, the terms "specific binding", "specifically binds to" or "is specific for" refer to binding that is detectably distinct from non-specific interactions, relative to a specific polypeptide or epitope on a specific polypeptide target. For example, specific binding can be measured by determining the binding of a molecule and comparing it to the binding of a control molecule, which is typically a structurally similar molecule that has no binding activity.

[0090] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of a natural SLAMF6 polypeptide. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural SLAMF6 polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an antagonist of a SLAMF6 polypeptide may include contacting a SLAMF6 polypeptide with a candidate antagonist molecule, and measuring a detectable change in one or more biological activities typically associated with a SLAMF6 polypeptide.

[0091] The term "agonist" is used in the broadest sense and includes any molecule that enhances the biological activity of a natural SLAMF6 polypeptide. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of SLAMF6 ligand polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying a SLAMF6 polypeptide agonist may include: contacting a SLAMF6 polypeptide with a candidate agonist molecule, and measuring a detectable change in one or more biological activities typically associated with a SLAMF6 polypeptide.

[0092] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0093] The terms "predictive" and "prognostic" as used herein are also interchangeable, meaning that a predictive or prognostic method allows one practicing the method to select patients who are considered (usually prior to treatment, but not necessarily) to be more likely to respond to treatment with an anti-cancer drug (including anti-SLAMF6 antibodies).

[0094] SLAMF6 protein

[0095] According to UNIPROT, SLAMF6 is a single-pass type I membrane protein of the immunoglobulin superfamily and CD2 subfamily. The protein consists of an extracellular domain between amino acids 22-226 of SEQ ID NO: 11, a transmembrane region between amino acids 227-247 and a cytoplasmic region between amino acids 248-331.

[0096] In some embodiments, the antibodies of the invention can bind to human SLAMF6. As used herein, "human

[0097] "SLAMF6" or "human SLAMF6 protein" refers to the protein having SEQ ID NO: 11 as defined herein.

[0098] In some cases, antibodies according to embodiments of the present invention may cross-react with SLAMF6 proteins from non-human species. For example, to facilitate preclinical and toxicology testing, antibodies of the present invention may cross-react with mouse or primate SLAMF6 proteins. Alternatively, in certain embodiments, antibodies may be specific to human SLAMF6 proteins and may not show species or other types of non-human cross-reactivity.

[0099] Antibody

[0100] Aspects of the present invention include anti-SLAMF6 antibodies, generally therapeutic and / or diagnostic antibodies, as described herein. Antibodies that can be used in the methods of the present invention can take any of the various forms as described herein, including traditional antibodies and antibody derivatives, antigen binding fragments and mimetics, as further described herein. In some embodiments, the antibody has one or more CDRs selected from a group of 6 CDRs as defined herein (including a small amount of amino acid changes as described herein). As mentioned above, the term "antibody" used herein refers to a variety of structures.

[0101] In some embodiments, the present invention uses an IgG isotype. In one embodiment, an Fc-silent IgG1 isotype antibody is used. In another embodiment, an IgG4 isotype antibody is used.

[0102] The amino terminal portion of each chain of the antibody contains a variable region of about 100-110 or more amino acids, which is mainly responsible for antigen recognition. In the variable region, each V domain of the heavy chain and the light chain gathers three loops to form an antigen binding site. Each loop is called a complementary determining region (hereinafter referred to as "CDR"), in which the variation of the amino acid sequence is most significant. "Variable" refers to the fact that some fragments of the variable region have very different sequences between antibodies. The variability within the variable region is not evenly distributed. On the contrary, the V region consists of the following: a less variable segment of about 15-30 amino acids called a framework region (FR), and a highly variable shorter region of 9-15 amino acids or longer in length, called a "hypervariable region" that separates these FRs.

[0103] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0104] The hypervariable region generally includes amino acid residues 24-34 (CDR-L1; "L" for light chain), 50-56 (CDR-L2), and 89-97 (CDR-L3) from the light chain variable region, and about 31-35B (CDR-H1; "H" for heavy chain), 50-65 (CDR-H2), and 95-102 (CDR-H3) from the heavy chain variable region; Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed., Public Health Service, National Institutes of Health. Health), Bethesda, MD (1991) and / or those residues that form a hypervariable loop (e.g., residues 26-32 (CDR-L1), 50-52 (CDR-L2), and 91-96 (CDR-L3) in the light chain variable region and residues 26-32 (CDR-H1), 53-55 (CDR-H2), and 96-101 (CDR-H3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Specific CDRs of the invention are described below.

[0105] In this specification, the Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1-107 for the light chain variable domain and residues 1-113 for the heavy chain variable domain) (eg, Kabat et al., supra (1991)).

[0106] The CDRs contribute to the formation of the antigen binding site, or more specifically, the epitope binding site of an antibody.A single antigen may have more than one epitope.

[0107] In the IgG subclass of immunoglobulin, there are several immunoglobulin domains in the heavy chain. In this article, "immunoglobulin (Ig) domain" refers to the immunoglobulin region with different tertiary structures. The present invention is interested in the heavy chain domain, including constant weight (CH) domain and hinge domain. In the case of IgG antibodies, IgG isotypes each have three CH regions.

[0108] Another type of Ig domain of the heavy chain is the hinge region. "Hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein refers to the flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

[0109] Of particular interest to the present invention is the Fc region. As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising an antibody constant region, excluding the first constant region immunoglobulin domain, and in some cases, part of the hinge. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include a J chain. For IgG, the Fc domain includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues C226 or P230 at its carboxyl terminus, where numbering is according to the EU index in Kabat. In some embodiments, amino acid modifications are made to the Fc region, for example, to alter binding to one or more FcγR receptors or to the FcRn receptor.

[0110] In some embodiments, the antibody is full length. "Full length antibody" herein refers to the structure that constitutes the natural biological form of an antibody, including variable and constant regions, optionally including one or more modifications as outlined herein.

[0111] Alternatively, the antibody can be a variety of structures, including but not limited to antigen-binding fragments, monoclonal antibodies, bispecific antibodies, miniantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as "antibody conjugates"), and antigen-binding fragments of each. Structures that rely on the use of a set of CDRs are included in the definition of "antibody".

[0112] In one embodiment, the antibody is an antigen binding fragment. Specific antigen binding antibody fragments include, but are not limited to, (i) a Fab fragment consisting of a VL, VH, CL and CH1 domain, (ii) a Fd fragment consisting of a VH and CH1 domain, (iii) a Fv fragment consisting of a VL and VH domain of a single antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341:544-546, incorporated herein by reference in its entirety), which consists of a single variable region, (v) an isolated CDR region, (vi) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments, (vii) a single-chain Fv molecule (scFv), in which the VH domain and the VL domain are connected by a peptide linker that allows the two domains to unite to form an antigen binding site (Bird et al., 1988, Science 341:544-546, incorporated herein by reference in its entirety). 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, which is incorporated herein by reference in its entirety)), (viii) bispecific single chain Fv (WO 03 / 11161, which is incorporated herein by reference) and (ix) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol.

[0113] 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc.Natl.Acad.Sci.USA

[0114] 90:6444-6448, each of which is incorporated herein by reference in its entirety).

[0115] Chimeric and humanized antibodies

[0116] In some embodiments, the antibody may be a mixture of antibodies from different species, such as chimeric antibodies and / or humanized antibodies. That is, in the present invention, the CDR set may be used with framework regions and constant regions other than those specifically described in the sequences herein.

[0117] In general, "chimeric antibodies" and "humanized antibodies" both refer to antibodies that incorporate regions from multiple species. For example, "chimeric antibodies" traditionally include variable regions from mice (or rats, in some cases) and constant regions from humans. "Humanized antibodies" generally refer to non-human antibodies that have replaced the variable domain framework region with sequences present in human antibodies. Typically, in humanized antibodies, the entire antibody except the CDR is encoded by a human polynucleotide, or, except for the region within its CDR, it is identical to such antibodies. CDR, partially or entirely encoded by a nucleic acid derived from a non-human organism, is transplanted into the β-folded framework of the variable region of a human antibody to produce an antibody, and its specificity is determined by the grafted CDR. The production of such antibodies is described in, for example, WO 92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated herein by reference. In one embodiment, the antibodies of the present invention may be multispecific antibodies, particularly bispecific antibodies, sometimes also referred to as "diabodies". These are antibodies that can bind to two (or more) different antigens or different epitopes on the same antigen. Diabodies can be made in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449, all incorporated herein by reference), for example, chemically prepared or prepared from hybrid hybridomas.

[0118] In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein comprising a scFv connected to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated herein by reference in its entirety. In some cases, the scFv may be attached to the Fc region and may include some or the entire hinge region. It should be noted that although a minibody does not have a complete set of CDRs, it is still included in the definition of "antibody".

[0119] The antibodies of the invention are generally isolated or recombinant.

[0120] In some embodiments, the antibody of the present invention is a recombinant protein, an isolated protein or a substantially pure protein. "Isolated" protein is not accompanied by at least some of the materials usually associated with it in its natural state, such as those constituting at least about 5% or at least about 50% of the total protein weight in a given sample. It should be understood that, depending on different circumstances, isolated protein can account for 5 to 99.9% by weight of the total protein content. For example, protein can be prepared with significantly higher concentrations using an inducible promoter or a high expression promoter, thereby preparing a protein with increased concentration levels. In the case of a recombinant protein, this definition includes producing antibodies in a variety of organisms and / or host cells known in the art, wherein the antibody is not naturally produced in the organism and / or host cell. Typically, an isolated polypeptide will be prepared by at least one purification step. "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities. For example, an isolated antibody that specifically binds to SLAMF6 is substantially free of antibodies that specifically bind to other antigens other than SLAMF6.

[0121] Isolated monoclonal antibodies with different specificities can be combined into well-defined compositions.Thus, for example, an antibody of the invention can be optionally and individually included or excluded from a formulation, as discussed further below.

[0122] Specific binding to a particular antigen or epitope can be demonstrated, for example, by the following characteristics: D is at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or higher, where K D Refers to the off-rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a K for that antigen or epitope. D 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more lower than a control molecule.

[0123] Likewise, specific binding for a particular antigen or epitope can be demonstrated, for example, by the following: A or K aAt least 20-fold, 50-fold, 100-fold, 500-fold, 1000-fold, 5,000-fold, 10,000-fold or more greater than a control for the epitope, wherein K A or K a Refers to the binding rate of a specific antibody-antigen interaction.

[0124] Standard assays for assessing the ability of antibodies to bind SLAMF6 can be performed at the protein or cellular level and are known in the art, including, for example, ELISA, Western blot, RIA, The binding kinetics (e.g., binding affinity) of the antibodies can also be determined by standard assays known in the art, such as by or System analysis and evaluation.

[0125] SLAMF6 Antibody

[0126] The present invention provides SLAMF6 antibodies that bind to a SLAMF6 polypeptide or a portion thereof. An example of a SLAMF6 amino acid sequence is provided in SEQ ID NO: 11. The subject SLAMF6 antibodies can induce or enhance immune cell activation, such as T cell activation and / or NK cell activation, to enhance the immune response in tumors. These antibodies are referred to herein as "anti-SLAMF6" antibodies, or, for ease of description, as "SLAMF6 antibodies".

[0127] In some embodiments, the subject SLAMF6 antibody can induce and / or enhance cytokine release or proliferation when contacting T cells, particularly CD4+ or CD8+ T cells expressing SLAMF6 on their surface. In this case, cytokine release or T cell proliferation can be measured in a variety of ways. In one embodiment, a standard test such as ELISA is used to contact the SLAMF6 antibody of the present invention with activated T cells. In another embodiment, the subject SLAMF6 antibody can induce and / or enhance NK cell activation and killing.

[0128] In one embodiment, the antibody is an antibody comprising the following CDRs; in addition, as described below, these CDR sequences may also contain a limited number of amino acid variants as described above:

[0129] CDR SEQ ID NO: 1B3_VH_CDR1 SEQ ID NO:5 1B3_VH_CDR2 SEQ ID NO:15 1B3_VH_CDR3 SEQ ID NO:7 1B3_VL_CDR1 SEQ ID NO:16 1B3_VL_CDR2 SEQ ID NO:17 1B3_VL_CDR3 SEQ ID NO:10

[0130] In some embodiments, the antibody comprises an amino acid sequence of at least one or more CDR sequences provided in SEQ ID NOs: 5, 15, 7, 16, 17, and 10. In some embodiments, the antibody comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence of one or more CDR sequences provided in SEQ ID NOs: 5, 15, 7, 16, 17, and 10.

[0131] Also disclosed herein are variable heavy chains and light chains comprising the CDR sets of the invention, as well as full-length heavy chains and light chains (e.g., also comprising constant regions). As will be appreciated by those skilled in the art, the CDR sets of the invention may be incorporated into murine, humanized or human constant regions (including framework regions). Aspects of the invention include heavy chain variable regions and light chain variable regions that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the heavy chain variable region sequences (SEQ ID NO: 13) and light chain variable region sequences (SEQ ID NO: 14) disclosed herein.

[0132] In some embodiments, the present invention provides antibodies that bind to the same epitope on human SLAMF6 as the SLAMF6 monoclonal antibodies of the present invention described herein or cross-compete with the SLAMF6 monoclonal antibodies of the present invention described herein (i.e., antibodies that have the ability to cross-compete with the monoclonal antibodies of the present invention described herein for binding to the SLAMF6 protein). It should be understood that for an antibody that is considered to be cross-competitive, it does not necessarily completely block the binding of the reference antibody. In some embodiments, the binding of the reference antibody is reduced by at least about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 98 or 99%.

[0133] Antibody modification

[0134] The present invention further provides variant antibodies, sometimes referred to as "antibody derivatives" or "antibody analogs." That is, many modifications may be made to the antibodies of the present invention, including but not limited to amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, and other types of covalent modifications (e.g., for attachment of drug conjugates, etc.).

[0135] "Variant" refers to a polypeptide sequence that differs from the sequence of a parent polypeptide due to at least one amino acid modification. In some embodiments, the parent polypeptide is a full-length variable heavy or light chain listed in SEQ ID NO: 1 or 2, 13 or 14, or one or more of the CDR sequences disclosed in any of SEQ ID NO: 5 to 10, 15, 16 or 17. In some embodiments, the amino acid modification may include substitution, insertion and / or deletion, the former being preferred in many cases. In some embodiments, the substitution may be a conservative substitution.

[0136] In general, variants may include any number of modifications, as long as the antibody still functions, as described herein. For example, the antibody should still specifically bind to human SLAMF6. Similarly, for example, if amino acid variants are generated within the Fc region, the variant antibody should retain the receptor binding function required for the specific application or indication of the antibody.

[0137] "Variants" of a subject antibody can be made having amino acid variations as described herein in one or more of the listed CDR sequences, one or more framework regions, or one or more constant regions (eg, in the Fc region of an antibody).

[0138] In some embodiments, 1,2,3,4,5,6,7,8,9 or 10 amino acid modifications are generally used compared to the parent sequence, because the general goal is to change the function with the least number of modifications. In some embodiments, there are 1 to 5 (1,2,3,4 or 5) modifications (e.g., single amino acid substitutions, insertions and / or deletions), wherein 1-2,1-3 and 1-4 modifications are also found to be useful in many embodiments. For example, in some embodiments, one or more CDR sequences of antibodies of the present invention can comprise one or more, e.g., 1,2,3,4 or 5 amino acid modifications alone, preferably 1-4,1-3,1 or 2 modifications. Generally, no more than 4,5,6,7,8,9 or 10 changes are used in one group of CDRs.

[0139] It should be noted that the number of amino acid modifications may be within a functional domain: for example, it may be desirable to have 1-5 modifications in the Fc region of a wild-type or engineered protein, and, for example, 1-5 modifications in the Fv region. The variant polypeptide sequence will preferably be at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96, 97%, 98% or 99% identical to the parent sequence (e.g., variable region sequence, constant region sequence and / or heavy and light chain sequence and / or CDR of antibody 1B3).

[0140] As used herein, "amino acid substitution" or "substitution" refers to replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid. As used herein, "amino acid insertion" or "insertion" refers to adding an amino acid at a specific position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to removing an amino acid at a specific position in a parent polypeptide sequence.

[0141] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide" or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. Generally, the parent polypeptide used herein may refer to a 1B3 polypeptide, such as 1B3V H or V L Therefore, "parent antibody" as used herein refers to an antibody that has been modified to produce a variant antibody.

[0142] "Wild type" or "WT" or "native" herein refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0143] "Variant Fc region" refers herein to an Fc sequence that differs from that of a wild-type Fc sequence by virtue of at least one amino acid modification. Fc variant may refer to the Fc polypeptide itself, a composition or an amino acid sequence comprising an Fc variant polypeptide.

[0144] In some embodiments, the anti-SLAMF6 antibodies of the present invention are composed of variant Fc domains. As known in the art, the Fc region of an antibody interacts with many Fc receptors and ligands, providing a series of important functional capabilities, known as effector functions. Suitable modifications can be made at one or more positions, particularly to reduce or silence specific amino acid substitutions that bind to Fc receptors.

[0145] In addition to the modifications outlined above, other modifications may be made. For example, the molecule may be stabilized by the introduction of disulfide bonds linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245, incorporated herein by reference in its entirety).

[0146] In addition, modification of cysteine ​​is particularly useful in antibody-drug conjugate (ADC) applications, as further described below. In some embodiments, the constant region of an antibody can be engineered to contain one or more cysteines that are particularly "sulfhydryl-reactive", thereby allowing for more specific and controlled positioning of the drug moiety. See, e.g., U.S. Patent No. 7,521,541, which is incorporated herein by reference in its entirety.

[0147] In addition, antibodies may be subjected to a variety of covalent modifications, as described below.

[0148] Covalent modifications of antibodies are included within the scope of the present invention and are usually, but not always, performed post-translationally. For example, by reacting specific amino acid residues of the antibody with an organic derivatizing agent that reacts with selected side chains or N- or C-terminal residues, several types of covalent modifications of the antibody are introduced into the molecule.

[0149] In addition, as will be appreciated by those skilled in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can be added to the antibodies (as well as other compositions of the invention).

[0150] Bispecific molecules

[0151] On the other hand, the present invention includes bispecific and multispecific molecules comprising anti-SLAMF6 antibodies of the present invention or fragments thereof. The antibody of the present invention or its antigen-binding portion thereof can be derivatized or connected to another functional molecule, such as another peptide or protein (such as another antibody or ligand of a receptor) to produce a bispecific molecule that binds two different binding sites or target molecules. In some embodiments, the antibody of the present invention or its antigen-binding portion thereof can be derivatized or connected to at least two functional molecules, such as other peptides or proteins (such as other antibodies or ligands of receptors) to produce multispecific molecules bound to at least three different binding sites or target molecules. In order to produce bispecific or multispecific molecules of the present invention, the antibody of the present invention can be functionally connected (for example, by chemical coupling, genetic fusion, non-covalent association or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, thereby producing bispecific or multispecific molecules.

[0152] Therefore, the present invention includes bispecific molecules comprising at least one first binding domain for a first target epitope (i.e., SLAMF6) and a second binding domain for a second target epitope. The second target epitope may be present on the same target protein as the target epitope bound by the first binding specificity; or the second target epitope may be present on a different target protein than the target epitope bound by the first binding specificity. The second target epitope may be present on the same cell as the first target epitope (i.e., SLAMF6); or the second target epitope may be present on a target that is not displayed by a cell that displays the first target epitope. As used herein, the term "binding specificity" refers to a portion comprising at least one antibody variable domain.

[0153] In another embodiment of the invention, the second target epitope is present on a tumor cell.Thus, aspects of the invention include bispecific molecules capable of binding to effector cells expressing SLAMF6 (eg, cytotoxic T cells expressing SLAMF6) and tumor cells expressing a second target epitope.

[0154] In one embodiment, the bispecific antibody of the present invention may have a total of two or three antibody variable domains, wherein the first part of the bispecific antibody is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on human immune effector cells, wherein the effector antigen is SLAMF6, and the first part is composed of at least one antibody variable domain, and the second part of the bispecific antibody is capable of specifically binding to a target antigen other than the effector antigen, wherein the target antigen is located on a target cell other than the human immune effector cell, and the second part comprises at least one antibody variable domain.

[0155] In one embodiment of the present invention where the binding protein is multispecific, in addition to the anti-tumor binding specificity and the anti-SLAMF6 binding specificity, the molecule may also include a third binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) portion, for example, a molecule that binds to a surface protein involved in cytotoxic activity to increase the immune response to a target cell. An "anti-enhancement factor portion" may be an antibody, a functional antibody fragment, or a ligand that binds to a given molecule (e.g., an antigen or a receptor), thereby resulting in an enhanced effect of the binding determinant for a target cell antigen. An "anti-enhancement factor portion" may be combined with a target cell antigen. Alternatively, the anti-enhancement factor portion may be combined with an entity different from the entity that is combined with the first and second binding specificities. For example, the anti-enhancement factor portion may be combined with a cytotoxic T cell (e.g., through CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an enhanced immune response to a target cell).

[0156] In one embodiment, the bispecific protein of the present invention comprises at least one antibody or its antigen binding fragment as binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, FVTCR, Fd, dAb or single-chain Fv. The antibody can also be a light chain or heavy chain dimer, or any minimal fragment thereof, such as the Fv or single-chain construct described in U.S. Pat. No. 4,946,778, the contents of which are expressly incorporated herein by reference.

[0157] In some embodiments, antibodies useful in the bispecific molecules of the invention are rat, murine, human, chimeric or humanized monoclonal antibodies.

[0158] Binding of a bispecific molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, biological assays (e.g., growth inhibition), or Western blot assays. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by using a labeled reagent (e.g., antibody) specific for the complex of interest.

[0159] In one embodiment of the present invention, bispecific antibodies are tetravalent antibodies comprising four antigen-binding regions. In a preferred embodiment, the antibody comprises two Fab domains targeting the first antigen, and each is composed of heavy chain and light chain Fab regions. They are arranged in the same conformation as the Fab of natural IgG. The antibody also comprises two chimeric Fab domains targeting the second antigen, which are composed of two chimeric polypeptide domains, each of which comprises a chimeric "heavy" chain and a chimeric "light" chain, respectively, wherein the chimeric "heavy" chain comprises a variable heavy chain domain, wherein the variable heavy chain domain is connected to the N-terminal of the constant region of the α or β chain of the T cell receptor (TCR) by its C-terminus, and the chimeric "light" chain comprises a variable light chain domain, wherein the variable light chain domain is connected to the N-terminal of the constant region of the β or α chain of the TCR by its C-terminus. Chimeric "heavy" chain and "light" chain are arranged into chimeric Fab domains, which are connected to the natural Fab domains by connecting the C-terminus of the constant region of the α or β chain of the TCR of the chimeric "heavy" chain to the N-terminus of the variable region of the heavy chain of the natural Fab domain. Thus, the overall symmetrical structure results in a bispecific antibody that targets each of two different antigens in a bivalent manner, such that a native Fab domain targeting the first antigen and a chimeric Fab domain targeting the second antigen are present on both arms of such a tetravalent antibody.

[0160] In another embodiment, the chimeric Fab domain is located proximal to the Fc domain such that the C-terminus of the constant region of the alpha or beta chain of the TCR constant region comprising the chimeric "heavy" chain is linked to the N-terminus of the native hinge, and the native Fab domain is located distal to the Fc domain such that the C-terminus of the heavy chain CH1 domain comprising the native Fab domain is linked to the N-terminus of the variable heavy chain comprising the chimeric Fab domain.

[0161] In another embodiment, an asymmetric trivalent format is used, which comprises two different antibody arms, whereby one arm has a single Fab domain (natural or chimeric), and the second arm of the bispecific antibody has two Fab domains (natural and chimeric), as described above. The heterodimerization of the two different arms is achieved by antibody engineering in the Fc domain, as described in the art (e.g., knobs-into-wholes, electrostatic steering, etc.).

[0162] In yet another embodiment, an asymmetric bivalent format can be used, which comprises two different antibody arms, whereby one arm has a single Fab domain (native or chimeric), and the other arm also has a single Fab binding domain (chimeric or native). Heterodimerization of the two different arms is achieved by antibody engineering in the Fc domain, as fully described in the art (e.g., buttons, electrostatic steering, etc.).

[0163] Glycosylation

[0164] Another type of covalent modification is the change of glycosylation. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be prepared. Glycosylation can be changed, for example, to increase the affinity of the antibody to the antigen. Such carbohydrate modifications can be achieved by, for example, changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions that lead to the elimination of one or more variable region framework glycosylation sites can be performed, thereby eliminating the glycosylation of the site. This aglycosylation can increase the affinity of the antibody to the antigen. This method is described in more detail in U.S. Patents 5,714,350 and 6,350,861 to Co et al., and can be achieved by removing the asparagine at position 297.

[0165] Another type of covalent modification of antibodies includes linking the antibody to various non-protein polymers, including but not limited to various polyols, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in a manner described, for example, in the 2005-2006 PEG Catalog of Nektar Therapeutics (available from the Nektar website) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, which are incorporated herein by reference in their entirety. In addition, as is known in the art, amino acid substitutions can be made at various positions in the antibody to facilitate the addition of polymers such as PEG. See, for example, U.S. Publication No. 2005 / 0114037A1, which is incorporated herein by reference in its entirety.

[0166] In other embodiments, for example, when the antibodies of the invention are used for diagnostic or detection purposes, the antibodies may comprise a label. "Labeled" herein refers to a compound having at least one attached moiety, element, isotope or chemical compound to enable detection of the compound, as described in Molecular Probes Handbook, 6th edition, by Richard P. Haugland, which is expressly incorporated herein by reference in its entirety.

[0167] Methods for producing antibodies of the present invention

[0168] The present invention also provides methods for producing the disclosed anti-SLAMF6 antibodies. These methods include culturing host cells containing isolated nucleic acids encoding antibodies of the present invention. As will be appreciated by those skilled in the art, this can be done in a variety of ways, depending on the nature of the antibody. In some embodiments, in the case where the antibody of the present invention is a full-length conventional antibody, for example, a host cell containing nucleic acids encoding a heavy chain variable region and a light chain variable region can be cultured under conditions that enable the production and separation of the antibody.

[0169] The variable heavy and light chains of the antibodies of the present invention are disclosed herein (protein and nucleic acid sequences); as is understood in the art, these can be readily expanded to produce full-length heavy and light chains. That is, having provided a V encoding a protein as outlined herein, H and V L In the case of DNA fragments encoding V segments, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the V L or V H A DNA fragment of a protein is operably linked to another DNA fragment encoding another protein (e.g., an antibody constant region or a flexible linker). The term "operably linked" used in this context is intended to mean that the two DNA fragments are connected so that the amino acid sequences encoded by the two DNA fragments remain in the reading frame.

[0170] Code V H The isolated DNA encoding the V H The DNA is operably linked to the DNA encoding the heavy chain constant region (C H 1, C H 2 and C H 3) and converted into full-length heavy chain genes. The sequences of rat heavy chain constant region genes are known in the art (see, for example, Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In a preferred embodiment, the heavy chain constant region is IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, the encoding V H The DNA may be operably linked to a heavy chain C H 1 constant region of another DNA molecule.

[0171] Code V L The isolated DNA encoding the V L The DNA is operably linked to the light chain constant region encoding C L Another DNA molecule of the light chain is converted into a full-length light chain gene (and a Fab light chain gene). The sequence of the rat light chain constant region gene is known in the art (see, for example, Kabat, EA et al. (1991) "Hot Immunology Protein Sequences" (Sequences of Proteins of Immunological Interest), Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region is a kappa or lambda constant region.

[0172] In order to construct a polynucleotide sequence encoding a scFv antibody fragment, H and V L The DNA fragment encoding the flexible linker is operably linked to another fragment encoding the amino acid sequence (Gly4-Ser)3, such that V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The regions are connected by flexible linkers (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0173] Aspects of the invention include nucleic acids encoding antibodies of the invention. Such polynucleotides encode the variable and constant regions of each heavy and light chain, but other combinations are contemplated by the invention in light of the compositions described herein. Aspects of the invention include oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides.

[0174] The polynucleotide according to the embodiments of the present invention can be or can include RNA, DNA, cDNA, genomic DNA, nucleic acid analogs and synthetic DNA. In some embodiments, the DNA molecule can be double-stranded or single-stranded, if it is single-stranded, it can be a coding (sense) strand or a non-coding (antisense) strand. The coding sequence of the coded polypeptide can be identical to the coding sequence provided herein or can be different coding sequences, and due to the redundancy or degeneracy of the genetic code, the sequence encodes the polypeptide identical to the DNA provided herein.

[0175] In some embodiments, the nucleic acid encoding the antibody of the present invention is incorporated into an expression vector, which may be extrachromosomal or designed to be integrated into the genome of the host cell into which it is introduced. The expression vector may contain any number of suitable regulatory sequences (including but not limited to transcription and translation control sequences, promoters, ribosome binding sites, enhancers, replication origins, etc.) or other components (selection genes, etc.), all of which are operably connected, as is well known in the art. In some cases, two nucleic acids are used and each is placed in a different expression vector (e.g., a heavy chain in a first expression vector, a light chain in a second expression vector), or they may be placed in the same expression vector. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the selection of host cells, the expression level of the desired protein, and the like.

[0176] In general, nucleic acid and / or expression can be introduced into a suitable host cell to produce a recombinant host cell using any method suitable for the selected host cell (e.g., transformation, transfection, electroporation, infection), so that one or more nucleic acid molecules are operably linked to one or more expression control elements (e.g., in a vector, in a construct produced by processing in a cell, integrated into the host cell genome). The resulting recombinant host cell can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with suitable salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing the encoded polypeptide. In some embodiments, the heavy chain and the light chain are produced in the same host cell. In some embodiments, the heavy chain is produced in one host cell and the light chain is produced in another host cell.

[0177] Mammalian cell lines that can be used as expression hosts are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) (Manassas, VA), including but not limited to Chinese hamster ovary (CHO) cells, HEK293 cells, FS293, Expi293, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and many others. Non-mammalian cells, including but not limited to bacteria, yeast, insects, and plants, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals such as cows or chickens.

[0178] General methods for antibody molecular biology, expression, purification and screening are well known, for example, see U.S. Pat. Nos. 4,816,567, 4,816,397, 6,331,415 and 7,923,221, and Antibody Engineering, Kontermann and Dubel, eds., Springer, Heidelberg, 2001 and 2010 Hayhurst and Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard and Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0179] Pharmaceutical composition

[0180] Aspects of the invention include compositions, such as pharmaceutical compositions, containing one or more (or combinations) of antibodies or antigen-binding portions thereof of the invention formulated with a pharmaceutically acceptable carrier. Such compositions may include a combination of one or more (e.g., two or more different) antibodies or bispecific molecules of the invention. For example, a pharmaceutical composition of the invention may include a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0181] The pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e., in combination with other agents. For example, a combination therapy may include an antibody of the present invention in combination with at least one other anti-tumor agent or anti-inflammatory agent or immunosuppressant. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the antibodies of the present invention.

[0182] As used herein, "pharmaceutically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody or antibody fragment, can be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0183] The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not produce any undesirable toxicological effects (see, for example, Berge, SM et al. (1977) J. Pharm. Sci. 66: 1-19). The pharmaceutical compositions of the present invention may also include a pharmaceutically acceptable antioxidant. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, such as olive oil and injectable organic esters, such as ethyl oleate. For example, by using a coating material (e.g., lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained.

[0184] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersants. Prevention of the presence of microorganisms may be ensured by sterilization steps (see above) and the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, the absorption of injectable pharmaceutical forms may be prolonged by the addition of substances that delay absorption, such as aluminum monostearate and gelatin.

[0185] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, these media or agents should be considered for use in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the compositions.

[0186] The dosage regimen is adjusted to provide the optimal desired effect (e.g., therapeutic effect). For example, it can be a single bolus, divided administration over time, or the dosage can be reduced or increased proportionally according to the urgency of the treatment situation. Particularly advantageous are parenteral compositions formulated in unit dosage form for ease of administration and dosage uniformity. Unit dosage form herein refers to physically discrete units used as a single dose for a subject to be treated, each unit containing a predetermined amount of active compound and a desired drug carrier, which is calculated to produce the desired therapeutic effect. The specifications of the dosage unit form of the present invention depend on or are directly dependent on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations in the field of compounding such active compounds for therapeutic sensitivity in individuals.

[0187] For administration of antibodies, dosage ranges may be about 0.0001 to 100 mg / kg, about 0.001 to 50 mg / kg, about 0.001 to 10 mg / kg, about 0.01 to 10 mg / kg, and more typically 0.01 to 5 mg / kg of host body weight. For example, dosages may be 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.75 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 4 mg / kg body weight, 5 mg / kg body weight, 7.5 mg / kg body weight or 10 mg / kg body weight, or within the range of 0.1-5 mg / kg or 1-10 mg / kg. Exemplary treatment regimens require administration every day, every other day, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months. Preferred dosing regimens for the anti-SLAMF6 antibodies of the present invention include administering the antibody by intravenous administration of 1 mg / kg body weight, 3 mg / kg, 5 mg / kg or 10 mg / kg body weight, using one of the following dosing regimens: (i) six doses per week, followed by monthly administration; (ii) weekly administration; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight per week.

[0188] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the indicated range. In some embodiments, the antibodies are administered in multiple situations. The intervals between single doses can be, for example, weekly, monthly, every three months or annually. The intervals can also be irregular, as indicated by measuring the blood levels of antibodies to the target antigen in the patient. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / ml, and in some methods reaches about 25-300 μg / ml.

[0189] In some embodiments, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency depend on the half-life of the antibody in the patient. In general, human antibodies have the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low doses are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, it is sometimes necessary to use relatively high doses at relatively short intervals until the progression of the disease slows or terminates, preferably until the patient shows partial or complete improvement in symptoms of the disease. Thereafter, a preventive regimen can be administered to the patient.

[0190] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient that is effective to obtain the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient. The selected dosage level depends on a variety of pharmacokinetic factors, including the activity of the particular composition used herein or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and other factors well known in the medical field.

[0191] The "therapeutically effective dose" of the antibodies of the present invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom periods, or the prevention of damage or disability caused by disease affliction. For example, relative to an untreated subject, a "therapeutically effective dose" preferably inhibits cell growth or tumor growth by at least about 10%, at least about 20%, at least about 30%, more preferably at least about 40%, at least about 50%, even more preferably at least about 60%, at least about 70%, and still more preferably at least about 80%, at least about 90% or at least about 95%. The ability of a compound to inhibit tumor growth can be assessed in an animal model system that predicts human tumor efficacy. Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell growth, which inhibition can be measured in vitro by tests known to technicians. A therapeutically effective amount of a therapeutic compound can reduce tumor size, or otherwise improve the symptoms of the subject. One of ordinary skill in the art will be able to determine such an amount based on factors such as the size of the subject, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0192] The compositions of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. It will be appreciated by those skilled in the art that the route and / or mode of administration vary depending on the desired results. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" as used herein means a form of administration other than enteral and topical administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion administration.

[0193] Alternatively, the antibodies of the invention may be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.

[0194] The active compound can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used. Many methods for preparing such preparations have been patented or are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems (1978) JR Robinson, Marcel Dekker, Inc., New York).

[0195] In certain embodiments, the monoclonal antibodies of the present invention can be formulated to ensure proper in vivo distribution. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. In order to ensure that the therapeutic compounds of the present invention pass through the BBB (if necessary), they can be formulated in, for example, liposomes. For methods of making liposomes, see, for example, U.S. Patents 4,522,811; 5,374,548; and 5,399,331. Liposomes may include one or more portions that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (see, for example, VVRanade, 1989, J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0196] Uses and methods

[0197] The antibodies, antibody compositions and methods of the invention have a variety of in vitro and in vivo diagnostic and therapeutic utilities, including the diagnosis and treatment of immune-mediated diseases.

[0198] In some embodiments, these molecules can be administered to cultured cells in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and / or diagnose a variety of diseases. The term "subject" as used herein is intended to include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals, e.g., non-human primates and non-mammals. Preferred subjects include human patients. When an antibody of the invention is administered together with another agent, the two may be administered in either order or simultaneously.

[0199] In view of the specific binding of the antibody of the present invention to SLAMF6, the antibody of the present invention can be used to specifically detect the expression of SLAMF6 on the surface of immune cells. In addition, it can also be used to purify SLAMF6 by immunoaffinity purification.

[0200] In addition, given the expression of SLAMF6 on immune cells, the antibodies, antibody compositions and methods of the invention can be used to treat subjects suffering from tumorigenic disorders, such as disorders characterized by the presence of tumor cells, such as small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, renal cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

[0201] In one embodiment, the antibodies of the invention are used to treat cancer, e.g., small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors and hematological malignancies.

[0202] In other embodiments, the antibodies of the invention are used to prepare a medicament for treating cancer, such as small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors and hematological malignancies.

[0203] In one embodiment, an antibody (eg, a monoclonal antibody, an antibody fragment, a Nanobody TM , multispecific and bispecific molecules and compositions, etc.) can be used to detect the levels of SLAMF6, or the levels of immune cells containing SLAMF6 on their membrane surfaces, which levels can then be correlated with certain disease symptoms for diagnosis.

[0204] In another embodiment, the antibodies of the invention (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) can be initially tested for binding activity associated with in vitro therapeutic or diagnostic uses. For example, the compositions of the invention can be tested using the flow cytometry assay described in the following examples.

[0205] In some embodiments, the antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention have other utilities in the treatment and diagnosis of diseases. For example, monoclonal antibodies, multispecific or bispecific molecules can be used to induce one or more of the following biological activities in vivo or in vitro: inducing and / or enhancing the activation of immune cells; mediating phagocytosis or ADCC of cells in the presence of human effector cells expressing SLAMF6, or preventing SLAMF6 ligands from binding to SLAMF6.

[0206] In one embodiment, antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat, prevent or diagnose a variety of diseases. Examples of relevant diseases include, for example, human cancer tissues representing small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors and hematological malignancies.

[0207] Suitable routes for in vivo and in vitro administration of the antibody compositions of the invention (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) are well known in the art and can be selected by one of ordinary skill. For example, the antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used will depend on the age and weight of the subject and the concentration and / or formulation of the antibody composition.

[0208] As previously described, the antibodies of the present invention can be co-administered with one or more other therapeutic agents such as immunostimulants, cytotoxic agents, radiotoxic agents or immunosuppressants. The antibody can be linked to the agent (in the form of an immune complex) or can be administered separately from the agent. In the latter case (separate administration), the antibody can be administered before, after or simultaneously with the agent, or can be co-administered with other known therapies (e.g., anti-cancer therapies, such as radiotherapy). Such therapeutic agents include anti-tumor agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil and cyclophosphamide hydroxyurea, which themselves are only effective at levels that are toxic or subtoxic to the patient. Other agents suitable for co-administration with the antibodies of the present invention include agents used to treat cancer, such as 5FU and gemcitabine. The co-administration of the anti-SLAMF6 antibody or antigen-binding fragment thereof of the present invention with a chemotherapeutic agent provides two anticancer agents that act through different mechanisms to produce cytotoxic effects on human tumor cells. This co-administration can solve problems caused by drug resistance or changes in tumor cell antigenicity.

[0209] Target-specific effector cells, such as effector cells linked to the compositions of the invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) can also be used as therapeutic agents. Effector cells for targeting can be human leukocytes, such as macrophages, neutrophils or monocytes. Other cells include eosinophils, natural killer cells and other cells carrying IgG or IgA receptors. If necessary, effector cells can be obtained from the subject to be treated. Target-specific effector cells can be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered can be 10 8 -10 9 of magnitude, but will vary depending on the purpose of the treatment.

[0210] Therapies with target-specific effector cells can be combined with other techniques. For example, anti-tumor therapy using the compositions of the invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) and / or effector cells equipped with these compositions can be used in combination with chemotherapy. In addition, combined immunotherapy can be used to direct two different cytotoxic effector populations toward tumor cell rejection.

[0211] The bispecific and multispecific molecules of the invention can also be used to modulate FcγR or FcγR levels on effector cells, for example by capping and eliminating receptors on the cell surface. Anti-Fc receptor cocktails can also be used for this purpose.

[0212] Aspects of the invention include kits comprising antibody compositions of the invention (e.g., monoclonal antibodies, bispecific or multispecific molecules) and instructions for their use, e.g., for treating cancer. The kits may also include one or more other agents, e.g., immunosuppressants, cytotoxic agents or radiotoxic agents, or one or more other antibodies of the invention (e.g., antibodies having complementary activity that binds to an epitope of a SLAMF6 antigen, different from the first antibody).

[0213] Thus, a patient treated with an antibody composition of the invention may additionally be administered (before, simultaneously with, or after administration of an antibody of the invention) another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or amplifies the therapeutic effect of the antibody.

[0214] In other embodiments, the subject can be further treated with an agent that modulates, e.g., enhances or inhibits, the expression or activity of Fcγ or an Fcγ receptor, e.g., by treating the subject with a cytokine. Preferred cytokines for administration during treatment with the multispecific molecule include granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).

[0215] The compositions (e.g., antibodies, multispecific and bispecific molecules) of the invention can also be used to target cells expressing FcγR or SLAMF6, for example, to label such cells. For such uses, the binding agent can be linked to a detectable molecule. Thus, the invention provides methods for localizing cells expressing Fc receptors such as FcγR or SLAMF6 ex vivo or in vitro. The detectable marker can be, for example, a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor.

[0216] In one embodiment, the present invention provides a method for detecting the presence of SLAMF6 antigen in a sample or measuring the amount of SLAMF6 antigen, comprising contacting a sample and a control sample with a monoclonal antibody or an antigen-binding portion thereof (which specifically binds to SLAMF6) under conditions that allow formation of a complex between the antibody or portion thereof and SLAMF6. The formation of the complex is then detected, wherein the difference in complex formation between the sample and the control sample indicates the presence of SLAMF6 antigen in the sample.

[0217] In other embodiments, the present invention provides methods of treating an immune-mediated disorder in a subject, such as human cancer, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine cancer, adrenal cancer, sarcoma, gastrointestinal stromal tumors, neuroendocrine tumors and hematological malignancies.

[0218] All references cited in this specification, including but not limited to all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, manuals, books, Internet postings, journal articles, periodicals, product fact sheets, etc., are hereby incorporated by reference in their entirety. The discussion of the references herein is intended only to summarize the assertions made by their authors, and is not an admission that any reference constitutes prior art, and applicants reserve the right to challenge the accuracy and pertinence of the cited references.

[0219] Although the present invention has been described in detail by way of illustration and example for purposes of illustration, it will be apparent to one skilled in the art, based on the teachings of this invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims.

[0220] The present invention is further illustrated by the following examples, but should not be construed as further limiting. The following examples, sequence listings and accompanying drawings are provided to aid in understanding the present invention, the true scope of the present invention being set forth in the appended claims. It should be understood that the described approach may be modified without departing from the spirit of the present invention.

[0221] Example:

[0222] Example 1: Antibody generation and screening.

[0223] Hybridoma generation

[0224] Recombinant ECD protein was used to immunize mice to generate mouse Fab against hu SLAMF6ECD (SEQ ID NO: 12) at Alere, San Diego. -SLAMF6-hum.ECD splenocytes from immunized mice were used to generate fab libraries using industry standard techniques.

[0225] Secondary screening

[0226] The Fab supernatant was tested for binding to SLAMF6 protein expressed on the surface of Raji cells and activated PBMCs. The supernatant was diluted 1:10 with FACS buffer.

[0227] Example 2: Structural characterization of SLAMF6 monoclonal antibody.

[0228] The cDNA sequences encoding the heavy and light chain variable regions of the monoclonal antibodies were obtained using standard PCR techniques and sequenced using standard DNA sequencing techniques. The heavy and light chain variable regions of 1B3 selected from the screening can be seen in FIG1 .

[0229] The nucleotide and amino acid sequences of the heavy chain variable region of 1B3 are shown in SEQ ID NOs: 3 and 1, respectively.

[0230] The nucleotide and amino acid sequences of the light chain variable region of 1B3 are shown in SEQ ID NOs: 4 and 2, respectively.

[0231] Further analysis of the 1B3 VH sequence using the Kabat system for CDR region determination resulted in the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs: 5, 6 and 7, respectively. Figure 1a The 1B3 VH sequence is shown with CDR1, CDR2 and CDR3 boxed.

[0232] Further analysis of the 1B3 VL sequence using the Kabat system for CDR region determination resulted in the delineation of the light chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs: 8, 9 and 10, respectively. Figure 1b The 1B3 VL sequence is shown with CDR1, CDR2 and CDR3 boxed.

[0233] Example 3: Specificity of Fab supernatant monoclonal antibodies against SLAMF6 determined by flow cytometric analysis.

[0234] 5x10 6 Each Raji cell was placed in each well of a 96-well plate and washed once with FACS buffer (DPBS, 2% FBS). The cells were pelleted by spinning at 1200 rpm for 5 minutes. The pellet was washed once with FACS buffer (DPBS, 2% FBS) and pelleted again by spinning at 1200 rpm for 5 minutes and resuspended in FACS buffer. The test antibody was diluted to 30 nM / l in FACS buffer and added as Figure 4The indicated increments were added to each well incubated on ice for 30 minutes. The cells were then washed once with FACS buffer (DPBS, 2% FBS), pelleted in FACS buffer, washed and resuspended. Goat anti-mouse secondary antibody was diluted to 1 μg.ml, 100 μl was added to each well, the plate was incubated on ice for 30 minutes, and then washed once with FACS buffer (DPBS, 2% FBS). The cells were pelleted and resuspended in 200ul FACS buffer. The samples were read using a Guava Easycyte Plus HT flow cytometer and the results were analyzed using the Guava Cytosoft software suite.

[0235] from Figure 4 As can be seen, antibody 1B3 showed specific dose-dependent binding to SLAMF6-expressing Raji cells.

[0236] Example 4: Ability of anti-SLAMF6 antibodies to activate T cells and stimulate IFNγ production.

[0237] 96-well non-tissue culture plates were coated overnight at 4°C with 250ng / ml OKT3 and different concentrations of anti-SLAMF6 antibodies / isotypes. The plates were washed twice with PBS and then blocked for 30 minutes with R10 medium (RPMI containing 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin). After blocking, 100,000 T cells were resuspended in 100μl R10 medium and added to the plates (T cells were isolated from PBMC using the Miltenyi kit, code: 130-096-535, according to the manufacturer's instructions). The plates were incubated at 37°C for 72 hours, and the supernatants were collected and diluted for IFNγ ELISA assays.

[0238] IFNγ was measured using the IFN-γ DuoSet ELISA kit (R&D systems cat. no. DY285B) according to the manufacturer's instructions.

[0239] result

[0240] Compared to the Seattle Genetics antibody against SLAMF6 described in WO2017 / 004330, the humanized antibody Hu_1B3 showed enhanced activity in OKT3 pre-activated T cells, reflected by increased IFNγ production at lower antibody concentrations ( Figure 5 ). This induction of IFNγ production suggests that antibodies targeting SLAMF6 would have therapeutic effects in patients with suppressed immune systems.

[0241] Example 5: Humanization of Antibody 1B3.

[0242] Humanization of the murine 1B3 monoclonal antibody using CDR grafting technology To guide the humanization process and assist in the decision to retain parental murine residues or replace them with their human germline counterparts, a homologous molecular model of the 1B3 murine monoclonal antibody Fv was constructed.

[0243] The definition of CDRs is based on the Kabat nomenclature. The selection of human framework acceptor regions for grafting 1B3 rat CDR regions was accomplished by searching the IMGT mouse and human V gene databases using IgBLAST, which was developed at NCBI to facilitate the analysis of immunoglobulin V region sequences, with the 1B3 mouse variable region sequence as input. The strategy applied was to use human germline sequences, which are natural human sequences that do not contain specific somatic mutations present in individual human antibody sequences.

[0244] Heavy chain design

[0245] The amino acid sequence of VH isolated from mouse 1B3 hybridoma cells (according to the Kabat numbering scheme, CDR regions are shown in bold) is shown below.

[0246]

[0247] Selection of human framework acceptor VH regions

[0248] The selection of the human framework receptor VH region for transplanting 1B3 mouse CDR region was completed by searching the IMGT human VH gene database using IgBLAST with the mouse VH region amino acid sequence as input. Based on the sequence alignment of the parental antibody and the human germline, the closest matching entry was identified. The identification of the best human germline as receptor was based on the following ordered criteria: the sequence identity of the cross-frame defined by Kabat, and the identity and / or compatibility of the canonical conformation of the interchain interface residues and the supporting loop with the parental CDR. Human germline IGHV1-2*02 was selected as the most suitable heavy chain.

[0249] Design using the IGHV1-2*02 human germline as the framework acceptor region

[0250] Humanized form

[0251] The mouse CDRs (in bold) defined by Kabat nomenclature were transplanted into IGHV1-2*02 to obtain the following detailed sequence. Many residues are framework mouse residues (outside CDR residues), i.e., conserved from the parental mouse 1B3 VH sequence; they are conserved because they are structurally likely to be important for maintaining the full activity of the antibody.

[0252]

[0253] A humanized version (OBT577-12-VHB) with 86.7% identity (85 of a total of 98 residues in the V gene) was used using the IGHV1-2*02 human germline.

[0254] Light chain design

[0255] The amino acid sequence of mouse 1B3 VL (with the CDR regions highlighted as defined by Kabat nomenclature) is shown below.

[0256]

[0257] Selection of human framework acceptor VL regions

[0258] The selection of human framework acceptor VL regions for transplantation of 1B3 VL mouse CDR regions was completed by searching the IMGT human VL gene database using IgBLAST with the mouse VL region amino acid sequence as input. The closest matching entry was identified based on the sequence alignment of the parental antibody with the human germline. The identification of the best human germline as an acceptor was based on the following ordered criteria: sequence identity across the framework defined by Kabat, and identity and / or compatibility of the canonical conformations of the interchain interface residues and supporting loops with the parental CDR. According to this analysis, human germline IGKV1-33*01 appears to be the best choice as a human framework acceptor region. Therefore, this human germline was used for the design of a humanized form.

[0259] Design of the framework acceptor region using the IGKV1-33*01 human germline

[0260] Humanized form

[0261] The mouse CDRs defined by Kabat nomenclature were grafted into IGKV1-33*01 to obtain the following detailed sequence. Several residues of structural importance for maintaining full activity of the antibody were retained. This resulted in a humanized form with 86.3% identity (82 of 95 amino acid residues identical).

[0262]

[0263] Humanized form with 86.3% identity (82 / 95) using the IGKV1-33*01 human germline

[0264] Example 6: ELISPOT with tumor infiltrating lymphocytes.

[0265] From NSCLC( Figure 6 ), breast cancer( Figure 7 ) or CRC( Figure 8) tumors were stimulated with 10 μg / ml of murine 1B3 or pembrolizumab and OKT3 diluted to 1 μg / ml in complete IMDM medium for 96 h. TILs were harvested after stimulation, counted and plated at 100,000 cells / well on IFNγ ELISPOT plates (Mabtech). Plates were incubated at 37°C for 24 h and then developed according to the manufacturer's instructions. The data were read on a Series 5 ELISPOT analyzer and analyzed using GraphPad Prism software.

[0266] Figure 6 Antibody 1B3 was shown to activate non-small cell lung cancer-derived TILs, as reflected by significantly higher IFNγ production than the isotype antibody.

[0267] Figure 7 showed that antibody 1B3 activated significantly more breast cancer-derived TILs to produce IFNγ compared to pembrolizumab.

[0268] Figure 8 showed that antibody 1B3 activated significantly more colorectal cancer-derived TILs to produce IFNγ compared with pembrolizumab.

[0269] Example 7: Binding affinity of humanized 1B3 antibody.

[0270] Binding affinity experiments were performed on a Biacore T-200 at 25°C. Flow cells 2, 3 and 4 of the CM5 chip were coated with a maximum of 500 RU of goat anti-human IgG. Test antibodies were captured on flow cells 2, 3 and 4. Flow cell 1 remained blank and was used for reference subtraction. Antigen flowed through the chip. Binding of antigen to antibody was monitored in real time. Based on the observed k on and k off , determine KD.

[0271] Table 2

[0272] sample <![CDATA[K D (M)]]> <![CDATA[K A (1 / M)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[k off (1 / s)]]> <![CDATA[Full X 2 > 1B3 <![CDATA[1.72x10 -9 ]]> <![CDATA[5.8x10 8 ]]> <![CDATA[5.41x 10 5 ]]> <![CDATA[9.32x 10 -4 ]]> 0.227

[0273] Example 8: In vitro proliferation assay using anti-SLAMF6 antibody 1B3.

[0274] method

[0275] Non-tissue culture treated 96-well plates (BD Falcon, USA) were coated with 250 ng / ml anti-human CD3 (eBioscience, USA) antibody alone or in combination with humanized anti-SLAMF6 antibody (Hu_1B3) or isotype control antibody (concentrations of 0.0.2, 0.4, 0.6, 0.9 and 1.2 ug / ml) and incubated overnight at 4°C. The plates were washed the next day and blocked with AIM V medium. T cells isolated from PBMCs generated from healthy donors were stained with the cell proliferation dye eFluor TM 670 (eBioscience, USA), washed and plated on antibody-coated plates (100,000 cells in 100 μl per well) in AIM V (Thermofisher Scientific, USA) medium containing FCS, penicillin-streptomycin, and cultured in a tissue culture incubator at 37°C for 72 hours. On day 3, cells were collected and stained with FITC-labeled anti-human CD8, Brilliant Violet 711 TM tagged anti-human CD4, PE-labeled anti-human CD69 (Biolegend, USA) and the fixable viability dye eFluor TM The samples were analyzed on an attune NxT flow cytometer (Thermofisher Scientific, USA), and the data were analyzed using FlowJo software (TreeStar, USA).

[0276] Fig. 9 It was shown that stimulation of SLAMF6 present on isolated T cells using anti-SLAMF6 antibodies in the presence of CD3 resulted in a significant increase in T cell proliferation compared to the isotype or CD3 alone.

[0277] Example 9: One-way mixed lymphocyte reaction (MLR) using anti-SLAMF6 antibody Hu_1B3 and allogeneic PBMCs.

[0278] method:

[0279] Isolated T cells from one donor (Donor 1) were resuspended in RPMI1640 medium containing 10% supplemented bovine serum and 2 mM L-glutamine (medium).

[0280] Cryopreserved PBMCs from a second donor (Donor 2) were treated with 50 ug / ml mitomycin C at a density of 2E6 cells / ml in culture medium. Cells were treated for 90 minutes at 37°C and then mitomycin C was removed by washing with culture medium.

[0281] 100,000 cells from donor 1 were then combined with 100,000 mitomycin C-treated cells from donor 2 in a 96-well U-bottom plate. The combined cells were treated with different concentrations of humanized antibody Hu-1B3 and control solution and cultured for 6 days in a total volume of 100ul culture medium / well. Isotype was used as a negative control and anti-CD137 mAb was included for comparison.

[0282] Culture supernatants were collected on day 6 and the concentration of IFN-γ was determined by ELISA (R&D Systems: DY285B) according to the manufacturer's instructions.

[0283] Fig.10 The anti-SLAMF6 antibody Hu_1B3 was shown to show a dose-related increase in cytokine release, indicating that T cells were activated by the presence of the antibody. It was also shown that activation of SLAMF6 resulted in higher cytokine release by isolated T cells compared to activation via CD137.

[0284] Example 10: SLAMF6-mediated release of granzyme B and perforin from T cells.

[0285] PBMC isolation

[0286] As the first step of T cell separation, PBMC is separated from the buffy coat (Leucopak of Stanford Blood Center). Blood is diluted in PBS with a ratio of 1:4 (10ml blood+30ml PBS), and then 30ml diluted blood is carefully placed on 15ml Ficoll-Hypaque (GE-Healthcare catalog number 17-1440-03). The tube is centrifuged at room temperature for 30 minutes with 400g (1400rpm) in a sorvall centrifuge, during which the brake is closed. Mononuclear cells are separated by density gradient. The white fractionation part of about 10ml cells from all tubes is pooled in a single 50ml tube, washed and counted using Cellometer auto2000. PBMC produced from the buffy coat is used for T cell separation.

[0287] T cell isolation

[0288] The isolation of pan T cells from PBMC is a negative selection process in which all remaining immune cell subsets except CD4 and CD8 T cells are labeled with biotin-conjugated antibodies and captured by streptavidin-coated microbeads in a high magnetic field. PBMCs isolated from the buffy coat were washed and resuspended in FACS sorting buffer (0.1% BSA in PBS) at a concentration of 2.5e7 cells / mL, and a pan T cell biotin-antibody cocktail was added to the cells, and the antibody-cell suspension was mixed using a 1 ml pipette and incubated on ice for 5 minutes. Pan T Cell MicroBead separation cocktail was added to the mixture and then incubated on ice for another 10 minutes. Untouched T cells were separated using MACS separation technology. The biotinylated PBMC microbead mixture was run over an LS column (Miltneyi Biotec, catalog number 130-042-401) in a high field MACS separator and the flow-through depleted non-T cells were collected, washed once with FACS sorting buffer and then resuspended in AIMV complete medium (containing 5% FBS).

[0289] Granzyme B ELISA

[0290] For the Granzyme B functional assay, non-tissue culture treated 96-well plates were coated with human Hu_1B3 or isotype control antibody in combination with 250ng / ml of anti-human CD3 (OKT3 clone) (Thermofisher Scientific catalog number 16-0031-85). Each test antibody was coated at 4.2μg / ml starting at 2:3 dilutions, 10 point titrations were performed in 100μl PBS, with three replicates. The plates were sealed and incubated overnight at 4°C. On the day of the Granzyme B functional assay setup, the plates were washed and blocked with AIMV complete medium (containing 5% FCS) for 20 minutes to reduce nonspecific binding.

[0291] Granzyme B immunoassay was set up using the Human GranzymeB DuoSet ELISA kit (Catalog No. DY2906-05) from R&D systems. Nunc-immunoassay plates were coated with granzyme B capture antibody diluted 1:60 in PBS, and the plates were incubated overnight at 4°C. The next day, the plates were washed with ELISA wash buffer (PBS + 0.05% Tween 20) and blocked with 1% BSA in PBS. After blocking the plates, 100 μl of samples were diluted 1:60 in dilution buffer, and standards were added to the respective wells and incubated overnight at 4°C. The plates were developed the next day, and OD values ​​were acquired on a VersaMax adjustable microplate reader. Granzyme B release was quantified and plotted using Graphpad Prism 8 software.

[0292] in conclusion

[0293] Granzyme-mediated apoptosis is one of the main mechanisms by which cytotoxic lymphocytes eliminate transformed cells. The data showed that antibody Hu_1B3 was able to induce cytotoxic functions that are critical for tumor suppression. Antibody Hu_1B3 was expressed at an EC of 0.51 μg / ml. 50 Induces a dose-dependent increase in granzyme B from activated T cells ( Fig.11 ).

[0294] Perforin intracellular assay

[0295] Perforin intracellular quantitative test setup, non-tissue culture treated 96-well plates were coated with human Hu_1B3 or isotype control antibody combined with 250ng / ml anti-human CD3 (OKT3 clone) (Thermofisher Scientific catalog number 16-0031-85). Each test antibody started at 4.2μg / ml, coated at a 2:3 dilution, and 10-point titration was performed in 100μl PBS with three replicates. The plate was sealed and incubated overnight at 4°C. On the day of the granzyme B functional test setup, the plate was washed and blocked with AIMV complete medium (containing 5% FCS) for 20 minutes to reduce nonspecific binding. For this test, 200,000 pan T cells were added to each well in 100μl AIMV medium (containing 5% FBS) and cultured at 37°C in a tissue culture incubator for 3 days. On the day of the experiment, protein transport inhibitor cocktail (Thermofisher Scientific, catalog number 00-4980-93) was added to all wells and incubated for 4 hours to prevent perforin from transporting into the extracellular space. Cells were collected and stained for T cell surface markers CD3, CD4 and CD8, and then FIX&PERM TM Intracellular perforin staining was performed using a cell permeabilization kit (Thermofisher Scientific, catalog number GAS-004). Cells were analyzed on an Attune NxT FACS analyzer (Thermofisher Scientific, Maryland), and data were analyzed using FlowJo software (BD Biosciences, San Jose, MD).

[0296] in conclusion

[0297] Perforin-mediated necrosis is another major killing mechanism induced by cytotoxic T lymphocytes, and Hu-1B3 enhanced the upregulation of perforin in CD8+ T cells in a dose-dependent manner ( Fig.12 ), E.C. 50 It is 0.44μg / ml.

[0298] Example 11: Competitive Binding Assay to Evaluate Binding Epitopes of Antibody 1B3

[0299] The human PBMCs of cryopreservation are thawed and washed once by being suspended in FACS buffer (DPBS containing 2% FCS), then centrifuged at 1200rpm for 5 minutes to precipitate cells and discard supernatant (subsequent washing is carried out by the same method). Before washing again, cells are distributed in 96-well test plates with 100,000 cells in each well FACS buffer. Then the cells are blocked for 1 hour with 100nM or 300nM SLAMF6 ECD-mIgG2aFc fusion proteins, for SLAMF6 receptor blocking in 100 μl FACS buffer on ice, for 1 hour, then washed. Then the humanized Hu_1B3 antibody or human IgG1 isotype control with a maximum concentration of 10nM and 1 / 3 serial dilution titration is added to the blocked PBMC, and then the cells are incubated on ice for 1 hour, then washed twice. Then the secondary antibody, goat anti-human IgG-RPE (Southern Biotech, reference number 2040-05) at a concentration of 1 μg / mL in FACS buffer was applied to the treated cells for 30 minutes on ice. A previously untreated well containing cells was also stained with the secondary antibody, and the other was unstained, as a control for the binding of the secondary antibody. After the final incubation, the cells were washed twice again, and the final cell pellet was resuspended in FACS buffer. According to the industry standard protocol, the mean fluorescence intensity of the secondary antibody of each sample was determined in a 96-well plate format using Attune NxT flow cytometer (Thermo Fischer Scientific), and the raw data were analyzed using FlowJo analysis software.

[0300] like Fig.13 As shown in the figure, in the presence of human SLAMF6 ECD-mIgG2a Fc fusion protein, Hu_1B3 antibody was blocked and could not bind to the receptor on the surface of PBMC, indicating that Hu_1B3 competitively binds to human SLAMF6 ECD. Therefore, it can be considered that Hu_1B3 binds to the homodimerization epitope of SLAMF6 and enhances cytotoxic T cell function through the SAP-mediated activation pathway, acting as an agonist antibody.

[0301] Example 12: Internalization of Hu-1B3 Antibody

[0302] RAJI, human Burkitt's lymphoma cells (Catalog # CCL-86, American Type Culture Collection [ATCC], Manassas, VA) were grown in RPMI-1640 medium (Cellgro, Catalog # 10-041-CM, Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (HyClone* Cosmic Calf Serum, Catalog # SH30087-03, Thermo Scientific, Waltham, MA) and 1% sodium pyruvate (Cellgro, Catalog # 25-000-Cl) using industry standard sterile techniques.

[0303] Raji cells were collected at 5×10 5 The density of cells / well was plated in 24-well cell glass bottom culture plates and allowed to proliferate for 48 hours at 37°C in growth medium. Wells were prepared for the following samples: secondary antibody control only, human IgG isotype control, antibody Hu_1B3, clinical anti-SLAMF6 antibody (from Seattle genetics) (positive control) at 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours and 24 hours. Secondary antibody control wells and no antibody control wells were used as fluorescence controls.

[0304] All antibody incubations and wash steps were then performed on ice with ice-cold reagents. The medium was aspirated from the wells and washed twice with IF buffer (Dulbecco's phosphate-buffered saline (DPBS, Thermo Scientific, Waltham, Virginia, catalog number #SH30028-03) + 2% FBS). The primary antibody (purified OBT humanized Hu_1B3; isotype or positive control antibody (Seattle Genetics)) was diluted to 2ug / mL in IF buffer, and 200μl was added to the appropriate wells for 15 minutes. The same volume of IF buffer was added separately to the wells designated for the secondary antibody control. The secondary antibody (goat anti-human IgG-Alexa Fluor 488, Invitrogen catalog number A11013) was diluted to a concentration of 2ug / mL in IF buffer and then added to the primary incubation for 15 minutes.

[0305] After primary and secondary antibody labeling, human IgG isotype controls, secondary antibody only controls, and test and positive controls (samples at 0 minutes) were processed. Cells were washed twice with IF buffer and then placed in a second 24-well plate on ice containing 4% paraformaldehyde (4% half dilution in DPBS, catalog number 19943, Affymetrix, Santa Clara, CA) to stop internalization and fix the cells.

[0306] The remaining cells were washed twice with IF buffer, 1 mL of warm growth medium was added to each well, and then placed in an incubator at 37° C. At 0 h, 0.5 h, 1 h, 2 h, 4 h, and 24 h, the cells were fixed in paraformaldehyde on ice as described for the control samples.

[0307] All cells were kept fixed on ice for at least 15 minutes. The cells were washed with IF buffer and coverslips were added to each well with 2-3 drops of Prolong Gold Anti-Fade reagent plus DAPI (Catalog No. P-36931, Invitrogen, Grand Island, New York) as a nuclear negative stain. Cell images were obtained using a Leica microscope (Leica DMI600B), a Leica monochrome camera (Leica DFC350FX), DAPI and Alexa Fluor 488 filter sets, and a 63x oil immersion lens. Images were saved in TIFF format and analyzed using ImageJ.

[0308] Fig.14 The humanized antibody Hu_1B3 was shown to be internalized significantly less into SLAMF6-expressing cells than the clinical Seattle Genetics antibody. This suggests that after binding to antibody Hu_1B3, the receptor remains on the cell surface longer and will therefore induce a more durable response in T cells and therefore will be a more effective agonist.

[0309] Example 13: Cytotoxicity test

[0310] SKBr3 HCT116 and MDA-MB-231 were purchased from ATCC. Cell lines were maintained using standard sterile techniques as directed by the manufacturer (ATCC). Cell culture medium consisted of RPMI-1640 containing 2 mM L-glutamine and 25 mM HEPES (Corning), 1% penicillin / streptomycin (Sigma-Aldrich), and 10% heat-inactivated FCS (HyClone). Cell lines were maintained in exponential phase and grown in a 37°C incubator containing 5% CO2. SKBr3 is a breast cancer cell line that expresses a high copy number of Her2 (approximately 5x10 6 copies / cell). HCT116 is a colorectal cancer cell line expressing low copy number of Her2. MDA-MB-231 is a breast cancer cell line expressing low copy number of Her2.

[0311] Target and effector (PBMC) cells were prepared under the following conditions. One day before the experiment, target cells were washed with PBS, incubated with 0.25% trypsin, and resuspended in cell culture medium. Viability and cell concentration were measured by dye exclusion. Target cells were seeded in 96-well tissue culture treated plates at 10,000 cells / well. Cells were grown overnight in a 37°C incubator with 5% CO2.

[0312] The day before the experiment, frozen PBMCs were thawed in a 37°C water bath, washed with cell culture medium, and cultured overnight in a 37°C incubator with 5% CO2. The next day, viability and cell concentration were measured by dye exclusion. PBMCs were added to target cells at an E:T ratio of 10:1. 10,000 target cells were mixed with 100,000 effector cells.

[0313] The bispecific antibody Cris7-Her2 is used for cytotoxicity assays. The antibody can detect Her2 and CD3ε. The Cris7-Her2 bispecific antibody was diluted to 100ng / ml and then serially diluted 3 times to 0.0012ng / ml. In addition, Cris7-Her2+ agonist antibodies were tested. A combination of Cris7-Her2 bispecific antibodies plus agonist antibodies was included to observe the enhanced cytotoxicity of agonist antibodies. Urelumab is an antibody against 4-1BB and is used as a positive control. Isotypes are used as negative controls. Hu_1B3 is a test product. Urelumab, isotypes and Hu_1B3 are used at a final concentration of 2.5ug / ml. All samples were run in triplicate. Controls included target cells alone or target cells plus effector cells. Other controls included target cells and effector cells plus Hu_1B3 or target cells and effector cells plus isotype controls.

[0314] Cytotoxicity assays are performed by incubating the cells for 48-96 hours, depending on the cell line. After incubation, the viability of the target cells is measured. Viability is based on the quantification of ATP, which signals the presence of metabolically active cells. The assay is based on luminescence and (Promega, Madison, WI) was used as a reagent for measuring viable cells. For assay conditions, the manufacturer's instructions were followed, and all steps were performed at room temperature. Briefly, a 96-well plate and The reagents were allowed to equilibrate to room temperature for 30 minutes. After 30 minutes, the cell culture medium was removed and the target cells were washed with 200ul PBS; the washing step was repeated once more. Next, 100ul of Cell The reagent was added to all wells. The plate was placed on an orbital shaker and mixed at 250 rpm for 2 minutes to induce cell lysis. The plate was removed from the orbital shaker and incubated in the dark for 10 minutes to stabilize the luminescent signal. After 10 minutes, the samples were transferred to an opaque-walled 96-well plate and the luminescence was recorded.

[0315] The viability of the target cells is proportional to the luminescent signal generated. All samples were run in triplicate and the average for each test condition was calculated. The percentage of cytotoxicity of the sample was normalized to the control; the control was the survival rate of the target cells in the presence of effector cells (target + effector). To calculate viability, the luminescent signal of the sample was divided by the luminescent signal of the control. Cytotoxicity was calculated based on the percentage of non-viable cells remaining. Fig.15 As shown, for SKBr3 cells, the bispecific antibody alone or the bispecific antibody plus isotype control had effective cytotoxicity at higher concentrations of 0.8-100 ng / ml. The addition of Urecitabine slightly shifted the cytotoxicity curve to the left, but not more than 5%. However, the addition of 2.5ug / ml Hu_1B3 increased the cytotoxicity of SKBR-3 cells by 10%-40%.

[0316] Fig.16 showed that similar results were seen in a second PBMC donor.

[0317] Fig.17 Hu_1B3 is shown to enhance the cytotoxicity of HCT116 cells. HCT116 cells are a colon cancer cell line that expresses low levels of Her2 on the cell surface. HCT116 cells were used, together with PBMC, and T cell-engaged bispecific antibody Cris7-Her2 for cytotoxicity testing. As described above, Hu_1B3 was added to the test to determine whether it enhances the function of T cells or NK cells present in PBMC; both T cells and NK cells express the co-activating receptor SLAMF6. The data show that T cells engage the dose-dependent cytotoxicity of bispecific antibody Cris7-Her2 at different test concentrations. More importantly, the data show that when Hu_1B3 is added, an increase in cytotoxicity is observed. The EC50 of Cris7-Her2 bispecific alone or bispecific antibody + isotype control is about 0.25ng / mL, while the EC50 of bispecific antibody + Hu_1B3 is about 0.07ng / ml. This means that cytotoxicity has increased by about 3.6 times.

[0318] Fig.18Hu_1B3 showed enhanced cytotoxicity to MDA-MB-231 cells. MDA-MB-231 cells are a breast cancer cell line that express low levels of Her2 on the cell surface. Cytotoxicity assays were performed using MDA-MB-231 cells, together with PBMCs, and T cells engaging the bispecific antibody Cris7-Her2, as described above. Similarly, Hu_1B3 enhanced the cytotoxicity of the Cris7-Her2 bispecific at various test concentrations. Specifically, a 40% increase in cytotoxicity was observed at 1 ng / mL of Cris7 compared to the isotype control.

[0319] Fig.19 The results showed that after 96 hours of incubation, the combination of bispecific and Hu_1B3 showed high levels of SKBR-3 cell killing even at the lowest levels of bispecific antibody concentration. However, at these levels, the bispecific alone or the combination of bispecific with urerulumab or isotype control resulted in very low levels or no cell killing, indicating that Hu_1B3 provides strong activation of lymphocytes.

[0320] Fig. 20 The above cytotoxicity assay is shown under experimental conditions where the bispecific Cris 7-Her2 antibody is maintained at a constant level (0.0457 ng / ml) and the concentration of the test antibody is titrated by 1 / 3 dilution in a concentration range of 7.5 ug / ml to 0.0034 ug / ml. Dose-dependent killing of SKBR-3 cells was observed compared to Urecitabine or isotype control, further indicating the ability of Hu-1B3 to activate cytotoxic lymphocytes.

[0321] Example 14: Cytotoxicity test when Cris7 dual specificity is deleted

[0322] Another cytotoxicity assay was set up as described above, but without the bispecific antibody. Single agent cytotoxicity with SKBR-3 was tested using only Hu-1B3, isotype control, and Urecitabine. The concentration range used in the assay was 33ug / ml-0.13ug / ml, with one donor performing a 1 / 3 dilution. For the second donor, the test antibody was further diluted to 0.0152ug / ml ( Figure 21b ).

[0323] Fig.21a and 21b It was shown that Hu-1B3 was able to activate lymphocytes to induce cell killing of SKBR-3 cells in the absence of CD3-Her2 bispecific antibody. This was in contrast to Urecitabine or isotype control, where almost no cell killing was seen.

[0324] Sequence Listing

[0325]

[0326]

[0327]

Claims

1. A multispecific or bispecific antibody or antigen-binding fragment thereof capable of binding to SLAMF6 and one or more other targets, the antibody or antigen-binding fragment comprising: A heavy chain variable region, the heavy chain variable region comprising: CDR-H1 sequence, which is SEQ ID NO:5; The CDR-H2 sequence is SEQ ID NO: 6; and The CDR-H3 sequence is SEQ ID NO:7; and A light chain variable region, the light chain variable region comprising: CDR-L1 sequence, which is SEQ ID NO:8; The CDR-L2 sequence is SEQ ID NO:9; and The CDR-L3 sequence is SEQ ID NO:

10.

2. A multispecific or bispecific antibody or antigen-binding fragment thereof capable of binding to SLAMF6 and one or more other targets, the antibody or antigen-binding fragment comprising: Heavy chain variable region, comprising: CDR-H1, which is SEQ ID NO:5; CDR-H2, which is SEQ ID NO: 15; and CDR-H3, which is SEQ ID NO:7; and Light chain variable region, including: CDR-L1, which is SEQ ID NO: 16; CDR-L2, which is SEQ ID NO: 17; and CDR-L3, which is SEQ ID NO:

10.

3. A multispecific or bispecific antibody or antigen-binding fragment thereof capable of binding to SLAMF6 and one or more other targets, the antibody or antigen-binding fragment comprising: i) the three heavy chain CDRs of SEQ ID NO: 1 and the three light chain CDRs of SEQ ID NO: 2, or ii) 3 heavy chain CDRs of SEQ ID NO: 13 and 3 light chain CDRs of SEQ ID NO: 14; The CDRs are defined by either the Kabat or Chothia numbering systems.

4. A multispecific or bispecific antibody or antigen-binding fragment thereof capable of binding to SLAMF6 and one or more other targets, comprising: a heavy chain variable region, which is SEQ ID NO: 13, and The light chain variable region is SEQ ID NO:

14.

5. The multispecific or bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the one or more other binding targets include PD-L1.

6. The multispecific or bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment is a monoclonal antibody.

7. The multispecific or bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment is a chimeric, humanized or human antibody or antigen-binding fragment.

8. The multispecific or bispecific antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein the antibody or antigen-binding fragment is an Fc-silent engineered IgG1 antibody or antigen-binding fragment having reduced or no binding to one or more Fc receptors.

9. The multispecific or bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is capable of inducing and / or enhancing T cell cytotoxicity.

10. The multispecific or bispecific antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

11. A pharmaceutical composition comprising the multispecific or bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, and a pharmaceutically acceptable carrier.

12. Use of the multispecific or bispecific antibody or antigen-binding fragment thereof or pharmaceutical composition according to any one of claims 1 to 11 in the preparation of a medicament for treating cancer, wherein the cancer is selected from the group consisting of non-small cell lung cancer, breast cancer, and colorectal cancer.

13. The use according to claim 12, wherein the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma, and the breast cancer is TNBC.

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