Antibodies and Methods of Use

Abstract

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

Description

[Technical field]

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

[0002] Introduction Embodiments of the present invention include antibodies and other therapeutic proteins against SLAM family member 6 (SLAMF6), also known as NTB-A or CD352, nucleic acids encoding such antibodies and therapeutic proteins, methods of preparing antibodies and other therapeutic proteins, and methods of treating diseases such as cancer by using antibodies and other therapeutic proteins against SLAMF6. [Background technology]

[0003] The target antigen SLAMF6 is a single-pass type I membrane protein, a member of the immunoglobulin superfamily and the CD2 subfamily (J Exp Med. 2001 Aug 6;194(3):235-46). Its activity is regulated by the presence or absence of small cytoplasmic adaptor proteins, SH2D1A / SAP and / or SH2D1B / EAT-2. This protein induces cytolytic activity only in natural killer cells (NK) that express a high surface density of the natural cytotoxic receptor (J. Exp. Med. 194:235-246(2001)). Positive signaling in NK cells implies phosphorylation of VAV1. NK cell activation appears to depend on SH2D1B and not on SH2D1A. Together with SLAMF1, SLAMF6 controls the transition between positive selection and subsequent proliferation and differentiation of the thymocyte natural killer T (NKT) cell lineage.

[0004] SLAMF6 also promotes T cell differentiation towards the helper T cell Th17 phenotype leading to increased IL-17 secretion, and its costimulatory activity requires SH2D1A (J. Immunol. 177:3170-3177(2006)). Furthermore, it promotes recruitment of RORC to IL-17 promoters (J. Biol. Chem. 287:38168-38177(2012)). Together with SLAMF1 and CD84 / SLAMF5, SLAMF6 may be a negative regulator of humoral immune responses. In the absence of SH2D1A / SAP, SLAMF6 inhibits CD4 + It can transmit negative signals to T and NKT cells and negatively regulates germinal center formation by inhibiting T cell:B cell adhesion, a function that suggests its increased association with PTPN6 / SHP-1, possibly via ITSM, in the absence of SH2D1A / SAP.

[0005] WO2008 / 027739 discloses anti-NTB-A antibodies and pharmaceutical compositions containing said antibodies, and also describes methods of using said antibodies to bind to NTB-A and treat diseases, such as hematological malignancies, characterized by expression of NTB-A.

[0006] WO2014 / 100740 and WO2017 / 004330 disclose antibodies, including antibody-drug conjugates, that specifically bind to NTB-A, and methods of using them to detect or regulate the activity of NTB-A-expressing cells. Also disclosed are methods of treating diseases associated with NTB-A-expressing cells, such as multiple myeloma, non-Hodgkin's lymphoma, and acute myeloid leukemia.

[0007] WO2015 / 104711 describes compositions and methods for improved ex vivo and in vivo T cell regulation and for the treatment of cancer and other pathologies. More specifically, embodiments of the present invention are directed to the use of water-soluble NTB-A polypeptides or agonists thereof for the treatment of cancer patients, the prevention and treatment of cytopenias in susceptible patients, and the ex vivo preparation of improved cell compositions. Summary of the Invention

[0008]

[0023] Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and tumor associated antigens, nucleic acids encoding such antibodies of the invention, host cells encoding the antibodies of the invention, methods for preparing the 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0009] The antibodies described herein provide antibodies that bind to SLAMF6 (SEQ ID NO:11). Preferably, the antibodies bind to the extracellular domain of SLAMF6 (SEQ ID NO:12). Aspects of the invention include antibodies, or antigen-binding fragments thereof, that bind to an epitope on the SLAMF6 protein recognized by the antibodies described herein or cross-compete for binding with the antibodies described herein, and 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 for human SLAMF6 of the antibodies described herein. In some embodiments, the antibody is an isolated antibody.

[0010] Aspects of the invention include an antibody or antigen-binding fragment thereof that binds to SLAMF6, the antibody comprising a heavy chain variable region comprising a CDR-H1 sequence comprising the sequence of SEQ ID NO:5, a CDR-H2 sequence comprising the sequence of SEQ ID NO:6 or SEQ ID NO:15, and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7, and in some embodiments the antibody or antigen-binding fragment further comprises a light chain variable region comprising at least one CDR sequence selected from the group consisting of a CDR-L1 comprising any one of the sequences of SEQ ID NO:8 or SEQ ID NO:16, a CDR-L2 comprising any one of the sequences of SEQ ID NO:9 or SEQ ID NO:17, and a CDR-L3 comprising the sequence of SEQ ID NO:10.

[0011] In some embodiments, an antibody or antigen-binding fragment thereof that binds to SLAMF6 comprises a heavy chain variable region and a light chain variable region that comprise one of the eight combinations of heavy and light chain CDRs as shown in Table 1.

[0012] [Table 1]

[0013] In some preferred embodiments, an antibody or antigen-binding fragment thereof that binds to SLAMF6 comprises a heavy chain variable region comprising 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 comprising CDR-L1 comprising SEQ ID NO:8, CDR-L2 comprising SEQ ID NO:9, and CDR-L3 comprising SEQ ID NO:10.

[0014] In another preferred embodiment, an antibody or antigen-binding fragment thereof that binds to SLAMF6 comprises a heavy chain variable region comprising CDR-H1 comprising SEQ ID NO:5, CDR-H2 comprising SEQ ID NO:15, and CDR-H3 comprising SEQ ID NO:7, and CDR-L1 comprising SEQ ID NO:16, CDR-L2 comprising SEQ ID NO:17, and CDR-L3 comprising SEQ ID NO:10.

[0015] In a further aspect, the antibody or antigen-binding fragment thereof of the present invention comprises variable CDRs compared to the parent antibody described herein.Thus, the present invention provides a mutant antibody or antigen-binding fragment thereof comprising a mutant variable region of a parent antibody, the parent antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising 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, and in one embodiment the mutant antibody or antigen-binding fragment thereof is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acid substitutions, additions, and / or deletions in a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3, collectively, having 1 to 5, or 1 to 4, or 1 to 3 substitutions, additions, or deletions for a particular use, and having 1, 2, 3, 4, 5, or 6 amino acid substitutions, additions, and / or deletions, wherein the antibody or antigen-binding fragment thereof retains specific binding to SLAMF6. Preferably, the variations are substitutions, and preferably the substitutions are conservative substitutions or substitutions to revert an amino acid in the variable region to the corresponding amino acid from human germline. In a further embodiment, the variant antibody or antigen-binding fragment thereof of the invention comprises a light chain variable region comprising 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 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 said CDR sequences are modified to be about 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the corresponding parent CDR sequences listed above.

[0016] In some embodiments, an antibody or antigen-binding fragment thereof is provided that comprises a heavy chain variable region set forth in SEQ ID NO:1 or SEQ ID NO:13, or a sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:13, and / or a light chain variable region set forth in SEQ ID NO:2 or SEQ ID NO:14, or a sequence that is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO:2 or SEQ ID NO:14. In a further embodiment, there is provided an antibody or antigen-binding fragment thereof comprising a heavy chain variable region that comprises 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 that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid substitutions and / or deletions compared to SEQ ID NO:2 or SEQ ID NO:14. Preferably, the variants comprise substitutions, more preferably conservative substitutions.

[0017] It will further be apparent that the amino acid substitutions, additions and / or deletions may be within the framework regions and / or within the CDRs.

[0018] In one embodiment, an antibody or antigen-binding fragment thereof is provided that comprises a heavy chain variable region comprising the sequence of SEQ ID NO:1, and a light chain variable region comprising the sequence of SEQ ID NO:2.

[0019] In one embodiment, an antibody or antigen-binding fragment thereof is provided that comprises a heavy chain variable region comprising the sequence of SEQ ID NO:13, and a light chain variable region comprising the sequence of SEQ ID NO:14.

[0020] 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.

[0021] In yet another embodiment of the invention, there is provided an antibody or antigen-binding fragment thereof that specifically binds to SLAMF6, comprising the three heavy chain CDRs of SEQ ID NO:1 or SEQ ID NO:13 and the three light chain CDRs of SEQ ID NO:2 or SEQ ID NO:14, wherein the CDRs are defined by Kabat or by the Chothia numbering system. Preferably, said antibody or antigen-binding fragment thereof that specifically binds to SLAMF6 comprises the three heavy chain CDRs of SEQ ID NO:13 and the three light chain CDRs of SEQ ID NO:14, as defined by the Kabat or Chothia numbering system.

[0022] SEQ ID NOs: 13-19 are humanized antibody sequences based on the sequences of SEQ ID NOs: 1 and 2. One skilled in the art will readily appreciate that SEQ ID NOs: 18-19 are full-length heavy and light chain sequences including regions other than the variable regions necessary to produce a full-length functional antibody, i.e., the constant region and the Fc region. One skilled in the art will appreciate that the sequences are humanized to minimize the immunogenic effects of the antibodies when they are administered to a human subject by replacing amino acids from the variable regions of the organism in which the antibody is produced with amino acids from human germline sequences. While the majority of amino acid substitutions occur in the framework regions, a number of amino acids from the CDRs occurring at non-critical positions may be replaced, such substitutions preferably being conservative in nature or reverting the amino acid at a particular position to the amino acid present in the corresponding human germline. In this case, the replaced amino acids from the CDRs were identified by using structural models to distinguish between paratopic and non-paratopic residues in the CDR regions. This allows the antibody to be humanized to a higher degree than simple CDR grafting.

[0023] In the present invention, SEQ ID NO:13 contains two amino acid substitutions in CDR2 (SEQ ID NO:15) when 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 a DG substitution at position 17 of SEQ ID NO:6.

[0024] In the present invention, SEQ ID NO:14 contains three amino acid substitutions in CDR1 (SEQ ID NO:16) when compared to CDR1 of SEQ ID NO:2 (SEQ ID NO:8). Specifically, there is an SQ substitution at position 1 of SEQ ID NO:8; an SQ substitution at position 4 of SEQ ID NO:8; and an SD substitution at position 5 of SEQ ID NO:8. SEQ ID NO:14 further contains one amino acid substitution in CDR2 (SEQ ID NO:17) when compared to CDR2 of SEQ ID NO:2 (SEQ ID NO:9). Specifically, there is an ST substitution at position 7 of SEQ ID NO:9.

[0025] It will be understood by those skilled in the art that these humanized sequences do not represent different, alternative antibodies when compared to the parent antibody, but rather relate to the same antibodies with the same properties that have simply been modified using structural modeling to more closely correspond to the human germline in order to minimize immunogenicity.

[0026] In some embodiments, the antibody or antigen-binding fragment has a binding affinity (K D ).

[0027] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody. In some aspects, 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.

[0028] Alternative strategies have also been employed to alleviate antibody effector function, including substitution of residues in the antibody lower hinge, such as L234A and L235A (LALA). These residues form part of the Fc-gamma receptor binding site on the CH2 domain, and exchange of these residues between antibody isotypes had greater or less effector function, identifying their importance in ADCC. Alanine substitutions at these positions are effective in reducing ADCC in both human and mouse antibodies, but these substitutions are less effective in reducing CDC activity (Lo M et al, J Biol Chem. 2017 Mar 3;292(9):3900-3908). In some embodiments, the antibody or antigen-binding fragment is an Fc variant engineered to reduce binding to the Fc gamma receptor, resulting in reduced effector function and ADCC activity.

[0029] In some embodiments, the antibody or antigen-binding fragment is an Fc-silenced engineered IgG1 antibody or an antigen-binding fragment that has reduced or no binding to one or more Fc receptors, hi another embodiment, the antibody is an IgG4 antibody.

[0030] 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 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 cells are preferably T cells. In another embodiment, the immune cells are preferably NK cells. The skilled artisan will understand that the term induce and / or enhance can refer to inducing and / or enhancing cytokine release by immune cells, and / or inducing and / or enhancing proliferation of said immune cells, and / or inducing and / or enhancing cell killing activity. It will be readily apparent to the skilled artisan that the term "induce" or "induction" as used in the present context means causing activation of immune cells or increasing activation of immune cells above the activation level seen in the absence of the antibody or antigen-binding fragment. The term "enhance" as used in the present context refers to increasing the activation level of already activated immune cells.

[0031] In some embodiments, the antibody or antigen-binding fragment is a bispecific or multispecific antibody or antigen-binding fragment that binds to SLAMF6 protein (e.g., SEQ ID NO:11) and binds to one or more additional binding targets, preferably said additional binding targets are one or more tumor antigens. In further embodiments, the one or more additional binding targets are immune modulatory molecules. In one embodiment, the additional binding target is PD-L1. In one embodiment, the antibody or antigen-binding fragment is bivalent. In another embodiment, the antibody or antigen-binding fragment is tetravalent. In another embodiment, the antibody or antigen-binding fragment is trivalent.

[0032] In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a Fab', a F(ab)2, a F(ab')2, a Fv, a FVTCR, a scFv, a dAb, and a single domain antibody.

[0033] In another aspect of the invention, one or more nucleic acids are provided encoding the heavy chain of an antibody of the invention and / or the light chain of an antibody of the invention. It will be understood that the heavy and light chains of an antibody of the invention can be encoded together on a single nucleic acid molecule or by two separate nucleic acid molecules.

[0034] In another aspect, a vector comprising one or more of the nucleic acids of the invention is provided. In another aspect of the invention, a host cell is provided that contains one or more nucleic acids encoding the heavy and / or light chains, or both, of an antibody of the invention. In some embodiments, the host cell is grown under conditions in which the nucleic acids are expressed. In other embodiments, a method for recovering an antibody of the invention is provided.

[0035] Aspects of the invention include methods of making an antibody, or antigen-binding fragment thereof, comprising culturing a host cell under conditions whereby the antibody, or antigen-binding fragment, is expressed in the host cell, and optionally isolating the antibody or antigen-binding fragment.

[0036] Aspects of the invention include pharmaceutical compositions comprising an antibody or antigen-binding fragment as described herein and a pharma- ceutically acceptable carrier. In certain embodiments, the pharmaceutical composition or medicament further comprises an effective amount of a second therapeutic agent.

[0037] In a further aspect of the invention, a method of treating a disorder is provided, the method comprising administering to a patient in need thereof an antibody or antigen-binding fragment of the invention that binds to SLAMF6 (SEQ ID NO: 11). In one embodiment, the disorder is cancer.

[0038] In a further aspect, there is provided a method of treating cancer comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment of the invention.

[0039] 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.

[0040] Preferably, the heavy chain variable region comprises a CDR-H1 comprising SEQ ID NO:5, a CDR-H2 comprising SEQ ID NO:15, and a CDR-3 comprising SEQ ID NO:7.

[0041] 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.

[0042] In some embodiments, methods of treating cancer are provided, in which an antibody or antigen-binding fragment of the invention is administered to a patient in need thereof, wherein said antibody or antigen-binding fragment of the invention induces and / or enhances an immune response, e.g., a cytotoxic T cell response and / or a NK cell response.

[0043] In a further embodiment, the antibody or antigen-binding fragment thereof comprises a bispecific or multispecific antibody or antigen-binding fragment thereof that binds to SLAMF6 (SEQ ID NO:11) and a tumor-specific antigen.

[0044] In some embodiments, the cancer is selected from the group consisting of 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0045] According to a further aspect of the invention there is provided an antibody or antigen-binding fragment of the invention for use in prophylaxis or therapy.

[0046] Preferably, the antibody or antigen-binding fragment is for use in the prevention or treatment of cancer.

[0047] According to a further aspect of the invention there is provided the use of an antibody or antigen-binding fragment according to the invention in the manufacture of a medicament for the treatment of cancer. In some embodiments, the cancer according to the previous aspects is selected from the group consisting of 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies. [Brief description of the drawings]

[0048] [Figure 1] Figure 1a shows the amino acid sequence of the variable region of the heavy chain of the parent murine 1B3 antibody (SEQ ID NO: 1), and Figure 1b shows the amino acid sequence of the variable region of the light chain of the parent murine 1B3 antibody (SEQ ID NO: 2). [Diagram 2] Figure 2a shows the amino acid sequence of the heavy chain variable region of humanized antibody Hu_1B3 (SEQ ID NO: 13), and Figure 2b shows the amino acid sequence of the light chain variable region of humanized antibody Hu_1B3 (SEQ ID NO: 14). [Diagram 3] Figure 3a shows a sequence alignment of the heavy chain variable region of the 1B3 parent mouse antibody sequence and the humanized heavy chain variable region 1B3 sequence. Figure 3b shows a sequence alignment of the light chain variable region of the 1B3 parent mouse antibody sequence and the humanized light chain variable region Hu_1B3 sequence. [Figure 4] FIG. 4 shows the specific dose-dependent binding of antibody 1B3 to 1B3 expressing Raji cells. [Diagram 5] FIG. 5 shows that antibody Hu_1B3 has enhanced ability to mediate IFNγ production upon T cell activation compared to another clinical stage SLAMF6 antibody. [Figure 6] FIG. 6 shows that antibody 1B3 can induce tumor infiltrating lymphocytes (TILs) isolated from primary NSCLC tumor samples to produce interferon gamma in an ex vivo assay. [Figure 7] Figure 7 shows that antibody 1B3 can induce TILs isolated from primary breast cancer samples to produce interferon gamma in an ex vivo assay, which shows that antibody 1B3 has enhanced activity when compared to pembrolizumab. [Figure 8] Figure 8 shows that antibody 1B3 can induce TILs isolated from primary colorectal cancer samples to produce interferon gamma in an ex vivo assay, which shows that antibody 1B3 has enhanced activity when compared to pembrolizumab. [Figure 9] FIG. 9 shows that activation of SLAMF6 present on isolated T cells with antibody Hu_1B3 induces proliferation of CD8+ T cells. [Figure 10] FIG. 10 is an MLR assay showing that antibody Hu_1B3 is able to induce DC-mediated T cell activation, as highlighted by increased IFNy release. [Figure 11] FIG. 11 shows that anti-SLAMF6 antibody Hu_1B3 can induce activated T cells to produce granzyme B in a dose-dependent manner. [Figure 12] FIG. 12 shows that antibody Hu_1B3 dose-dependently upregulates perforin expression on CD8+ T cells. [Figure 13] FIG. 13 shows that the Hu_1B3 antibody is blocked from binding to receptors on the surface of PBMCs in the presence of human SLAMF6 ECD-mIgG2a Fc fusion protein. [Figure 14]FIG. 14 shows that humanized antibody Hu_1B3 incorporation into SLAMF6-expressing cells is significantly less than the anti-SLAMF6 antibody produced by Seattle Genetics. [Figure 15] FIG. 15 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against SKBR-3 cells when used together with anti-her2 anti-CD3Bi specific antibodies. [Figure 16] FIG. 16 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against SKBR-3 cells when used together with anti-her2 anti-CD3Bi specific antibodies. [Figure 17] FIG. 17 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against HCT116 cells when used with an anti-her2 anti-CD3 bispecific antibody. [Figure 18] FIG. 18 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against MDA-MB-231 cells when used with an anti-her2 anti-CD3 bispecific antibody. [Figure 19] FIG. 19 shows that after activation of lymphocytes with various antibodies for 96 hours, antibody HU_1B3 caused significantly more death of SKBR-3 cells than either urelumab (anti-CD137) or the isotypes. [Figure 20] FIG. 20 shows that the addition of Hu_1B3 dose-dependently enhances lymphocyte cytotoxicity against SKBR-3 cells when used with a fixed concentration of anti-Her2 / anti-CD3 bispecific antibody. [Figure 21] Figures 21a and 21b show that addition of Hu_1B3 in the absence of an enabling bispecific antibody also enhances lymphocyte cytotoxicity against SKBR-3 cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Embodiments of the invention include antibodies against SLAMF6, nucleic acids encoding such antibodies, host cells containing such nucleic acids encoding the antibodies of the present application, methods for preparing anti-SLAMF6 antibodies, and methods for treating SLAMF6-mediated disorders, 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0050] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. As such, it should be further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prior basis for the use of exclusive terminology such as "sole," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0051] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has distinct components and features which may be readily separated or combined with the features of any of the other several aspects without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0052] definition For the purposes of interpreting this specification, the following definitions will apply and wherever appropriate, terms used in the singular will also include the plural and vice versa.

[0053] The term "SLAMF6", as used herein, unless otherwise specified, refers to any naturally occurring SLAMF6 protein from any vertebrate source, including mammals such as primates (e.g., humans, primates, and rodents (e.g., mice and rats)). SLAMF6 proteins are also referred to as SLAMF6-like proteins. The amino acid sequence of human SLAMF6 is provided herein in SEQ ID NO:11.

[0054] The term "SLAMF6" encompasses "full-length" unprocessed SLAMF6 as well as any form of SLAMF6 resulting from processing within a cell. The term also encompasses naturally occurring variants of SLAMF6, such as splice variants, allelic variants, and isoforms. The term encompasses naturally occurring truncated or secreted forms of SLAMF6 polypeptides (e.g., extracellular domain sequences). The SLAMF6 polypeptides described herein can be isolated from a variety of sources, such as human tissue types or another source, or can be prepared by recombinant or synthetic methods. A "native sequence SLAMF6 polypeptide" includes a polypeptide having the same amino acid sequence as the corresponding SLAMF6 polypeptide from nature. Such native sequence SLAMF6 polypeptides can be isolated from nature or produced by recombinant or synthetic means. The term "SLAMF6 epitope" as used herein refers to an epitope bound by an antibody comprising at least one or more of the CDR sequences described herein and / or exemplified by the binding profile of an anti-SLAMF6 antibody as shown in the Examples.

[0055] The term "antibody" is used in the broadest sense and specifically includes, for example, a single anti-SLAMF6 monoclonal antibody (including agonist, antagonist, neutralizing, full-length or complete monoclonal antibodies), anti-SLAMF6 antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain anti-SLAMF6 antibodies, and antigen-binding fragments of anti-SLAMF6 antibodies, including Fab, Fab', F(ab')2, and Fv and FV-TCR fragments, diabodies, single domain antibodies (sdAbs), so long as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" herein. Antibodies may be chimeric, human, humanized and / or affinity matured. In some embodiments, antibodies will be understood by those of skill in the art to comprise at least a set of six CDRs as defined herein, including, but not limited to, conventional antibodies (including both monoclonal and polyclonal antibodies), humanized, human and / or chimeric antibodies, antibody fragments, engineered antibodies (e.g., with amino acid modifications as outlined below), multispecific antibodies (including bispecific antibodies), and other analogs known in the art and discussed herein.

[0056] In other embodiments, it will be understood that the term "antibody" as used herein refers to structures that do not contain the six CDRs, including, but not limited to, Nanobodies®, Unibodies®, and scFv fragments.

[0057] The terms "anti-SLAMF6 antibody," "SLAMF6 antibody," or "antibody that binds SLAMF6" refer to an antibody that can bind to SLAMF6 with sufficient affinity such that it is useful as a diagnostic and / or therapeutic agent that targets SLAMF6. In certain embodiments, the anti-SLAMF6 antibody binds to an epitope of SLAMF6 that is conserved among SLAMF6 from different species.

[0058] An "isolated antibody" is one that has been identified and separated and / or recovered from components of its environment. Contaminating components of that environment are materials that would interfere with therapeutic uses of the antibody, and can include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0059] With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds" or "specific" to a particular polypeptide or epitope on a particular polypeptide target means binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule (which is generally a molecule of similar structure that does not have binding activity).

[0060] 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 native SLAMF6 polypeptide. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native SLAMF6 polypeptide, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an antagonist of SLAMF6 polypeptide may include contacting SLAMF6 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with SLAMF6 polypeptide.

[0061] The term "agonist" is used in the broadest sense and includes any molecule that enhances the biological activity of a native SLAMF6 polypeptide. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants, such as SLAMF6 ligand polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an agonist of an SLAMF6 polypeptide may include contacting the SLAMF6 polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the SLAMF6 polypeptide.

[0062] "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0063] The terms "predictive" and "prognostic" as used herein are also interchangeable when it means that a method for prediction or prognosis allows the practitioner of the method to select (usually, but not necessarily, prior to treatment) patients deemed more likely to respond to treatment with anti-cancer drugs, including anti-SLAMF6 antibodies.

[0064] SLAMF6 protein According to UNIPROT, SLAMF6 is a single-pass type I membrane protein of the immunoglobulin superfamily and the CD2 subfamily. The protein consists of an extracellular domain between amino acids 22-226 of SEQ ID NO:11, one transmembrane region between amino acids 227-247, and one cytoplasmic region between amino acids 248-331.

[0065] In some embodiments, the antibodies of the invention bind to human SLAMF6. As used herein, "human SLAMF6" or "human SLAMF6 protein" refers to the protein of SEQ ID NO: 11, as defined herein.

[0066] Antibodies according to embodiments of the invention may, in some cases, cross-react with SLAMF6 proteins from species other than human. For example, to facilitate preclinical and toxicology testing, antibodies of the invention may cross-react with murine or primate SLAMF6 proteins. Alternatively, in certain embodiments, antibodies may be specific for human SLAMF6 proteins and may not exhibit species or other types of non-human cross-reactivity.

[0067] antibody Aspects of the present invention include anti-SLAMF6 antibodies, generally therapeutic and / or diagnostic antibodies, as described herein. Antibodies that find use in the methods of the present invention can take any of a number of formats as described herein, including traditional antibodies as well as antibody derivatives, antigen-binding fragments and mimetics, as further described herein. In some embodiments, the antibody has one or more CDRs selected from the set of six CDRs defined herein (including a small number of amino acid changes as described herein). As reviewed above, the term "antibody" as used herein refers to a variety of structures.

[0068] In some embodiments, IgG isotype is used in the present invention. In one embodiment, Fc-silenced IgG1 isotype antibody is used. In another embodiment, IgG4 isotype antibody is used.

[0069] The amino-terminal portion of each chain of an antibody contains a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. In the variable region, three loops come together for each of the heavy and light chain V domains to form the antigen-binding site. Each loop is called a complementarity determining region (hereafter referred to as "CDR") and is where the variation in amino acid sequence is most pronounced. By "variable", we mean that certain segments of the variable region differ greatly in sequence between antibodies. The variability within the variable region is not evenly distributed. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by short regions of extreme variability called "hypervariable regions", each consisting of 9-15 amino acids.

[0070] Each VH and VL is composed of three hypervariable regions ("complementarity determining regions", "CDRs") and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0071] The hypervariable regions generally consist of amino acid residues from 24-34 (CDR-L1; "L" is light chain), 50-56 (CDR-L2) and 89-97 (CDR-L3) in the light chain variable region and from about 31-35B (CDR-H1; "H" is heavy chain, 50-65 (CDR-H2) and 95-102 (CDR-H3) in the heavy chain variable region, Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and / or those residues that form the hypervariable loops (e.g., residues 26-32 (CDR-L1), 50-52 (CDR-L2) and 91-96 (CDR-L3) in the light chain variable region, and 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.

[0072] Throughout this specification, the Kabat numbering system is generally used when referring to residues in the variable domains (approximately residues 1-107 for light chain variable regions and residues 1-113 for heavy chain variable regions) (e.g., Kabat et al., (1991) supra).

[0073] The CDRs contribute to the formation of the antigen-binding site of an antibody, more specifically, the epitope-binding site. An antigen may have multiple epitopes.

[0074] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig) domain" herein is meant a region of an immunoglobulin that has a distinct tertiary structure. Of interest in the present invention are the heavy chain domains that include the constant heavy (CH) domain and the hinge domain. In the context of IgG antibodies, each IgG isotype has three CH regions.

[0075] Another type of Ig domain in a heavy chain is the hinge region. By "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" herein is meant a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.

[0076] Of particular interest in the present invention is the Fc region. As used herein, "Fc" or "Fc region" or "Fc domain" refers to a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain and optionally a portion 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 N-terminal to these domains. For IgA and IgM, Fc can include the J chain. For IgG, the Fc domain includes 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 can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl terminus, where the numbering follows the EU index as in Kabat. In some embodiments, amino acid modifications are made to the Fc region, eg, to alter binding to one or more FcγR or FcRn receptors.

[0077] In some embodiments, the antibody is full-length. By "full-length antibody" herein is meant the structure that constitutes the natural biological formation of an antibody, including variable and constant regions, optionally including one or more modifications as outlined herein.

[0078] Alternatively, antibodies can be in a variety of structures including, but not limited to, antigen-binding fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), chimeric antibodies, humanized antibodies, antibody melts (sometimes referred to as "antibody conjugates"), and antigen-binding fragments of each, structures which rely on the use of a set of CDRs are included within the definition of "antibody."

[0079] In one embodiment, the antibody is an antigen-binding fragment. Specific antigen-binding antibody fragments include (i) Fab fragments consisting of VL, VH, CL and CH1 domains, (ii) Fd fragments consisting of VH and CH1 domains, (iii) Fv fragments consisting of the VL and VH domains of a single antibody, (iv) dAb fragments consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, which are incorporated by reference in their entirety), (v) isolated CDR regions, (vi) F(ab')2 fragments, bivalent fragments comprising two linked Fab fragments, (vii) single chain Fv molecules (scFv), in which the VH and VL domains are linked by a peptide linker that allows the two domains to bind and form an antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. USA, which are incorporated by reference in their entirety). 85:5879-5883), (viii) bispecific single chain Fvs (WO 03 / 11161, which is incorporated by reference in its entirety), and (ix) "diabodies" or "triabodies", multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, which are incorporated by reference in their entireties).

[0080] Chimeric and Humanized Antibodies In some embodiments, the antibodies may be mixtures from different species, e.g., chimeric and / or humanized antibodies, i.e., in the present invention, CDR sets may be used with framework and constant regions other than those specifically described by the sequences herein.

[0081] In general, "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from multiple species; for example, a "chimeric antibody" typically contains a variable region from a mouse (or rat, as the case may be) and a constant region from a human. A "humanized antibody" generally refers to a non-human antibody that has variable domain framework regions exchanged for sequences found in a human antibody. Generally, in a humanized antibody, the entire antibody, except for the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs are encoded in part or in whole by nucleic acids derived from a non-human organism and grafted onto the beta-sheet framework of a human antibody variable region to create an antibody whose specificity is determined by the engrafted CDRs. The creation of such antibodies is described, for example, in WO 92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are fully incorporated 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 bind to two (or more) different antigens, or different epitopes on the same antigen. Diabodies can be produced in a variety of ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, fully incorporated by reference), for example, chemically or from hybrid hybridomas.

[0082] In one embodiment, the antibody is a minibody. Minibodies are minimized antibody-like proteins that contain scFvs linked to a CH3 domain. Hu et al., 1996, Cancer Res. 56:3055-3061, fully incorporated by reference. In some cases, the scFv can be linked to the Fc region and can include part or the entire hinge region. Note that minibodies are included within the definition of "antibody" even though they do not have a complete set of CDRs.

[0083] The antibodies of the present invention are generally isolated or recombinant. In some embodiments, the antibodies of the invention are recombinant, isolated or substantially pure proteins. An "isolated" protein is free from at least some of the substances with which it is normally associated in its natural state, for example constituting at least about 5% by weight, or at least about 50% by weight, of the total protein in a given sample. It is understood that an isolated protein may constitute 5-99.9% by weight of the total protein content, depending on the circumstances. For example, the protein may be made at a much higher concentration by using an inducible promoter or a high expression promoter, such that the protein is made at an increased concentration level. In the case of a recombinant protein, the definition includes the production of the antibody in a wide variety of organisms and / or host cells known in the art in which it is not naturally produced. Typically, an isolated polypeptide is prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to SLAMF6 is substantially free of antibodies that specifically bind to antigens other than SLAMF6.

[0084] Isolated monoclonal antibodies with different specificities can be combined in well-defined compositions. Thus, for example, the antibodies of the present invention can be optionally and individually included or excluded from a formulation, as discussed further below.

[0085] Specific binding to a particular antigen or epitope is, for example, 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 K for antigens or epitopes greater than or equal to M D In one embodiment, the antibody has the formula: D refers to the off-rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a 20-, 50-, 100-, 500-, 1000-, 5000-, 10,000-, or more fold lower K for the antigen or epitope compared to a control molecule. D would have

[0086] Specific binding to a particular antigen or epitope can also be achieved by, for example, A or K a refers to the association rate of a particular antibody-antigen interaction, a K for an antigen or epitope that is at least 20-, 50-, 100-, 500-, 1000-, 5000-, or 10,000-fold greater than that for a control. A or K a This can be demonstrated by an antibody having the formula:

[0087] Standard assays for evaluating the binding ability of an antibody to SLAMF6 can be performed at the protein or cell level and are known in the art, including, for example, ELISA, Western blot, RIA, Octet®, BIAcore® assays and flow cytometry analysis. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibody can also be evaluated by standard assays known in the art, such as by Biacore® or Octet® system analysis.

[0088] SLAMF6 antibody The present invention provides SLAMF6 antibodies that bind to an SLAMF6 polypeptide or a portion thereof. An example of an SLAMF6 amino acid sequence is provided in SEQ ID NO:11. The subject SLAMF6 antibodies can induce or enhance immune cell activation, e.g., 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, "SLAMF6 antibodies."

[0089] In some embodiments, the subject SLAMF6 antibodies can induce and / or enhance cytokine release or proliferation upon contact with T cells, particularly CD4+ or CD8+ T cells expressing SLAMF6 on their surface. Cytokine release or T cell proliferation in this context can be measured in several ways. In one embodiment, the SLAMF6 antibodies of the invention are contacted with activated T cells using a standard assay such as ELISA. In a further embodiment, the subject SLAMF6 antibodies can induce and / or enhance NK cell activation and killing.

[0090] In one embodiment, the antibody is an antibody comprising the following CDRs; in addition, as discussed below, these CDR sequences may also contain a limited number of amino acid variants, as previously described: [Table 2]

[0091] In some embodiments, the antibody comprises the amino acid sequence of at least one or more of the 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 the amino acid sequence of one or more of the CDR sequences provided in SEQ ID NOs: 5, 15, 7, 16, 17, and 10.

[0092] Also disclosed herein are variable heavy and light chains, as well as full-length heavy and light chains (e.g., including constant regions) comprising the CDR sets of the present invention. As will be understood by those skilled in the art, the CDR sets of the present invention can be incorporated into mouse, humanized, or human constant regions (including framework regions). Embodiments of the present invention include heavy and light chain variable regions that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy and light chain variable region sequences (SEQ ID NO: 13) and light chain variable region sequences (SEQ ID NO: 14) disclosed herein.

[0093] In some embodiments, the present invention provides antibodies that bind to or cross-compete with the same epitope on human SLAMF6 as the SLAMF6 monoclonal antibodies of the present invention described herein (i.e., antibodies that have the ability to cross-compete for binding to SLAMF6 protein with the monoclonal antibodies of the present invention described herein). It will be understood that for an antibody to be considered to cross-compete, it will not necessarily completely block binding of the reference antibody. In some embodiments, 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%.

[0094] antibody modification The present invention further provides variant antibodies, sometimes referred to as "antibody derivatives" or "antibody analogs." That is, there are many modifications that can 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.).

[0095] By "variant" is meant a polypeptide sequence that differs from that of a parent polypeptide by virtue of at least one amino acid modification. In some embodiments, the parent polypeptide is any of the full-length variable heavy or light chains 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: 5-10, 15, 16 or 17. In some embodiments, the amino acid modification can include substitutions, insertions and / or deletions, the former being often preferred. In some embodiments, the substitutions can be conservative substitutions.

[0096] In general, the variant can include any number of modifications as long as the antibody function still exists as described herein.For example, the antibody should specifically bind to human SLAMF6.Similarly, when amino acid variants are generated in the Fc region, for example, the variant antibody should maintain the receptor binding function required for the specific use or display of the antibody.

[0097] A "variant" of the subject antibody can have amino acid mutations in either one or more of the recited CDR sequences, one or more framework regions, or one or more constant regions of the antibody (e.g., Fc region), as described herein.

[0098] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications are generally utilized compared to the parent sequence, and the objective is often to modify function with a minimal number of modifications. In some embodiments, there are 1-5 (1, 2, 3, 4 or 5) modifications (e.g., individual amino acid substitutions, insertions and / or deletions), with 1-2, 1-3 and 1-4 also finding use in many embodiments. For example, in some embodiments, one or more of the CDR sequences of the antibody of the invention may individually contain one or more, e.g., 1, 2, 3, 4 or 5 amino acid modifications, preferably 1-4, 1-3, 1 or 2 modifications. Generally, no more than 4, 5, 6, 7, 8, 9 or 10 changes are made within a set of CDRs.

[0099] It is noted that the number of amino acid modifications may be desirable, for example, to have from 1 to 5 modifications in the Fc region of the wild-type or engineered protein, and from 1 to 5 modifications in the Fv region, for example. The variant polypeptide sequence will preferably have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the parent sequence (e.g., the variable region sequence, the constant region sequence, and / or the CDRs and / or the heavy and light chain sequences of, for example, antibody 1B3).

[0100] As used herein, an "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a particular position in a parent polypeptide sequence with another amino acid, and as used herein, an "amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. As used herein, an "amino acid deletion" or "deletion" refers to the removal of an amino acid at a particular position in a parent polypeptide sequence.

[0101] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to generate a variant. Generally, a parent polypeptide, as used herein, is a 1B3 polypeptide, e.g., a 1B3 V polypeptide. H Chain or V l or the CDR sequences. Thus, as used herein, "parent antibody" refers to an antibody that is modified to generate a variant antibody.

[0102] As used herein, "wild-type" or "WT" or "naive" refers to an amino acid or nucleic acid sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid or nucleic acid sequence that has not been intentionally modified.

[0103] By "variant (mutant) Fc region" herein is meant an Fc sequence that differs from a wild-type Fc sequence by virtue of at least one amino acid modification. Fc variant can refer to the Fc polypeptide itself, a composition comprising an Fc variant polypeptide, or an amino acid sequence.

[0104] In some embodiments, the anti-SLAMF6 antibody of the present invention is composed of a mutated Fc domain. As known in the art, the Fc region of an antibody interacts with a number of Fc receptors and ligands, conferring an array of important functional capabilities, referred to as effector functions. Appropriate modifications can be made at one or more positions, particularly for specific amino acid substitutions that reduce or silence binding to Fc receptors.

[0105] In addition to the modifications outlined above, other modifications can be made, for example, the molecule can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, fully incorporated by reference).

[0106] Moreover, modification with cysteine ​​is particularly useful in antibody-drug conjugate (ADC) applications, which are described further below. In some embodiments, the constant region of an antibody can be engineered to contain one or more cysteines that are specifically "thiol-reactive," allowing for more specific and controlled placement of drug moieties. See, for example, U.S. Patent No. 7,521,541, the entirety of which is incorporated herein by reference.

[0107] In addition, various covalent modifications of antibodies may be made, as outlined below. Covalent modifications of antibodies are included within the scope of the present invention and are generally, but not necessarily, performed post-translationally. For example, some types of covalent modifications of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0108] In addition, as will be appreciated by those of skill in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can all be added to the antibodies (as well as other compositions of the invention).

[0109] bispecific molecule In another aspect, the present invention includes bispecific and multispecific molecules comprising the anti-SLAMF6 antibody of the present invention or a fragment thereof. The antibody or antigen-binding fragment thereof of the present invention can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to two different binding sites or target molecules. In some embodiments, the antibody or antigen-binding fragment thereof of the present invention can be derivatized or linked to at least two functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for a receptor), to generate a multispecific molecule that binds to at least three different binding sites or target molecules. To generate a bispecific or multispecific molecule of the present invention, the antibody of the present invention can be functionally (e.g., by chemical coupling, genetic fusion, non-covalent binding or otherwise) linked to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, to generate a bispecific or multispecific molecule.

[0110] Thus, 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 that bound by the first binding specificity, or the second target epitope may be present on a different target protein than that 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 not presented by the cell presenting the first target epitope. As used herein, the term "binding specificity" refers to a moiety comprising at least one antibody variable domain.

[0111] 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 both SLAMF6-expressing effector cells (e.g., SLAMF6-expressing cytotoxic T cells) and tumor cells expressing the second target epitope.

[0112] In one embodiment, a bispecific antibody of the invention can have a total of two or three antibody variable domains, where a first portion of the bispecific antibody is capable of recruiting the activity of human immune effector cells by specifically binding to an effector antigen located on a human immune effector cell, the effector antigen being SLAMF6, and the first portion consists of at least one antibody variable domain, and a second portion of the bispecific antibody is capable of specifically binding to a target antigen other than the effector antigen, the target antigen being located on a target cell other than a human immune effector cell, and the second portion comprises at least one antibody variable domain.

[0113] In embodiments of the invention in which the binding protein is multispecific, the molecule can further comprise a third binding specificity in addition to the anti-tumor binding specificity and the anti-SLAMF6 binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) moiety, e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby increasing the immune response against the target cell. An "anti-enhancement factor moiety" can be an antibody, a functional antibody fragment, a ligand that binds to a given molecule, e.g., an antigen or receptor, thereby resulting in an enhancement of the effect of a binding determinant against the target cell antigen. An "anti-enhancement factor moiety" can bind to a target cell antigen. Alternatively, the anti-enhancement factor moiety can bind to an entity different from the one to which the first and second binding specificities bind. For example, the anti-enhancement factor moiety can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cells resulting in an increased immune response against the target cell).

[0114] In one embodiment, the bispecific protein of the invention comprises as binding specificity at least one antibody, or antigen-binding fragment thereof (including, for example, Fab, Fab', F(ab')2, Fv, FVTCR, Fd, dAb or single chain Fv). The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv or single chain construct as described in U.S. Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

[0115] In some embodiments, antibodies that can be used in the bispecific molecules of the invention are rat, murine, human, chimeric or humanized monoclonal antibodies.

[0116] Binding of the bispecific molecule to its specific target can be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of protein-antibody complexes of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0117] In one embodiment of the present invention, the bispecific antibody is a tetravalent antibody containing four antigen-binding regions. In a preferred embodiment, the antibody comprises two Fab domains targeting a first antigen, each consisting of a heavy and a light Fab region. These are arranged in the same conformation as the Fabs of natural IgG. The antibody further comprises two chimeric Fab domains targeting a second antigen, each consisting of two chimeric polypeptide domains comprising a chimeric "heavy" chain comprising a variable heavy domain, which is linked via its C-terminus to the N-terminus of the constant region of the alpha or beta chain of the T-cell receptor (TCR), and a chimeric "light" chain comprising a variable light domain, which is linked via its C-terminus to the N-terminus of the constant region of the beta or alpha chain of the TCR, respectively. The chimeric "heavy" and "light" chains are arranged in a chimeric Fab domain, which is linked to the native Fab domain by joining the C-terminus of the constant region of the TCR alpha or beta chain of the chimeric "heavy" chain to the N-terminus of the variable region of the heavy chain of the native Fab domain. Thus, the overall symmetric structure produces a bispecific antibody that targets each of two different antigens in a bivalent manner, and thus a native Fab domain targeting a first antigen and a chimeric Fab domain targeting a second antigen are present in both arms of such a tetravalent antibody.

[0118] In another embodiment, the chimeric Fab domain is positioned proximal to the Fc domain such that the C-terminus of the constant region of the α or β chain of the constant region of the TCR comprising the chimeric "heavy" chain is attached to the N-terminus of the natural hinge, and the natural Fab domain is positioned at the tip of the Fc domain such that the C-terminus of the CH1 domain of the heavy chain comprising the natural Fab domain is attached to the N-terminus of the variable heavy chain comprising the chimeric Fab domain.

[0119] In another embodiment, an asymmetric trivalent format is used, composed of two different antibody arms, such that one arm has a single Fab domain (natural or chimeric) and the second arm of the bispecific antibody has both Fab domains (natural and chimeric) as described above. Heterodimerization of the two different arms is enabled via antibody engineering in the Fc domain, as described in the art (e.g., knobs-into-wholes, electrostatic steering, etc.).

[0120] In yet another embodiment, an asymmetric bivalent format composed of two different antibody arms may be used, one arm carrying a single Fab domain, natural or chimeric, and the other arm carrying a single Fab binding domain, chimeric or natural, respectively. Heterodimerization of the two different arms is possible via antibody engineering in the Fc domain (e.g., knobs-into-holes, electrostatic steering, etc.) that is well described in the art.

[0121] Glycosylation Another type of covalent modification is altered glycosylation. For example, an aglycosylated antibody (i.e., an antibody lacking glycosylation) can be generated. Glycosylation can be altered, for example, to increase the affinity of the antibody for the antigen. Such carbohydrate modification can be achieved, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the removal of one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. Such glycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail by Co et al. in U.S. Pat. Nos. 5,714,350 and 6,350,861, and can be achieved by removing the asparagine at position 297.

[0122] Another type of covalent modification of antibodies involves conjugating the antibody to various non-proteinaceous polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, for example, as described in Nektar Therapeutics' 2005-2006 PEG catalog (available on the Nektar website), U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337, all of which are fully incorporated by reference. Additionally, as is known in the art, amino acid substitutions can be made at various positions within the antibody to facilitate the addition of polymers such as PEG. See, for example, U.S. Patent Application Publication No. 2005 / 0114037, which is entirely incorporated by reference.

[0123] In further embodiments, for example, in the use of the antibodies of the present invention for diagnostic or detection purposes, the antibodies may contain a label. As used herein, "labeled" means that the compound has at least one moiety, element, isotope, or chemical compound attached to it to allow for detection of the compound as described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland.

[0124] Methods for producing the antibodies of the present invention The present invention further provides methods for producing the disclosed anti-SLAMF6 antibodies. These methods include culturing host cells containing isolated nucleic acids encoding the 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, when the antibodies of the present invention are full-length traditional antibodies, for example, host cells contain nucleic acids encoding heavy and light chain variable regions, which can be cultured under conditions such that the antibodies can be produced and isolated.

[0125] The variable heavy and light chains of the antibodies of the invention are disclosed herein (both protein and nucleic acid sequences); as understood in the art, they can be readily augmented to produce full-length heavy and light chains. H When DNA fragments encoding V and VL segments are provided, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. H A DNA fragment containing a nucleic acid encoding is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are ligated such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0126] V H The isolated DNA encoding the region is H The DNA encoding the heavy chain constant region was then separated into another DNA molecule (C H 1. C H 2 and C H3) to obtain a full-length heavy chain gene. 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, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In a preferred embodiment, the heavy chain constant region is an IgG1 or IgG4 constant region. In the case of a Fab fragment heavy chain gene, the V H The DNA encoding the heavy chain C H It can be operably linked to another DNA molecule encoding only one constant region.

[0127] The isolated DNA encoding the VL region is L The DNA encoding the light chain constant region is then coupled to another DNA molecule encoding the light chain constant region, L The light chain constant region can be converted to a full-length light chain gene (and a Fab light chain gene) by operably linking it to the rat light chain constant region. The sequences of rat light 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, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing 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.

[0128] To generate a polynucleotide sequence encoding an scFv antibody fragment,H - and VL coding DNA fragments are VL and V H The sequences include VL and VL linked by a flexible linker. H The fragment may be operably linked to another fragment encoding a flexible linker, e.g., another fragment encoding the amino acid sequence (Gly4-Ser)3, so that it may be expressed as a contiguous single-chain protein having the domain (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).

[0129] Aspects of the invention include nucleic acids encoding the antibodies of the invention. Such polynucleotides encode both the variable and constant regions of each of the heavy and light chains, although other combinations are contemplated by the invention in accordance with the compositions described herein. Aspects of the invention include oligonucleotide fragments derived from the disclosed polynucleotides, and nucleic acid sequences complementary to these polynucleotides.

[0130] Polynucleotides according to embodiments of the present invention may be in the form of or include RNA, DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA. In some embodiments, the DNA molecule may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or the non-coding (antisense) strand. The coding sequence encoding the polypeptide may be identical to the coding sequence provided herein, or may be a different coding sequence that, as a result of redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA provided herein.

[0131] In some embodiments, the nucleic acid encoding the antibody of the present invention is incorporated into an expression vector that may be extrachromosomal or designed to integrate 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, transcriptional and translational control sequences, promoters, ribosome binding sites, enhancers, origins of replication, etc.), or other components (such as selection genes), all of which are operably linked as is well known in the art. In some cases, two nucleic acids are used, each placed in a different expression vector (e.g., heavy chain in a first expression vector, light chain in a second expression vector), or they may be placed in the same expression vector. Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of host cell, the expression level of the desired protein, and the like.

[0132] In general, the nucleic acid and / or expression can be introduced into a suitable host cell to generate a recombinant host cell using any method (e.g., transformation, transfection, electroporation, infection) suitable for the selected host cell, such that the nucleic acid molecule is operably linked to one or more expression control elements (e.g., in a vector, in a construct generated by intracellular processing, integrated into the host cell genome). The resulting recombinant host cell can be maintained under suitable conditions 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.

[0133] Mammalian cell lines available as hosts for expression 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 other cell lines. 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.

[0134] For general methods for antibody molecular biology, expression, purification, and screening, see, e.g., U.S. Pat. Nos. 4,816,567, 4,816,397, 6,331,415, and 7,923,221, as well as Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0135] Pharmaceutical Compositions Aspects of the invention include compositions, e.g., pharmaceutical compositions, containing one or more (or a combination) of antibodies, or antigen-binding fragments thereof, of the invention, formulated with a pharma- ceutically acceptable carrier. Such compositions can contain one or a combination (e.g., two or more different) of antibodies or bispecific molecules of the invention. For example, pharmaceutical compositions of the invention can contain a combination of antibodies that bind to different epitopes on a target antigen or have complementary activities.

[0136] The pharmaceutical composition of the present invention can also be administered in combination therapy, i.e., in combination with other drugs.For example, the combination therapy can include the antibody of the present invention in combination with at least one other antitumor agent, or anti-inflammatory or immunosuppressant agent.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 antibody of the present invention.

[0137] As used herein, "pharmaceutical acceptable carrier" includes 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, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0138] The pharmaceutical compound of the present invention may include one or more pharma- ceutically acceptable salts. "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects (e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). The pharmaceutical composition of the present invention may also include a pharma- ceutically acceptable antioxidant. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Proper fluidity is maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0139] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It is also desirable to include isotonic agents in the composition, such as sugars, sodium chloride, and the like. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0140] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of the above-mentioned media and agents for pharmaceutically active substances is known in the art.Except where any conventional media or agent is incompatible with active compound, its use in the pharmaceutical composition of the present invention is contemplated.Additional active compounds can also be incorporated into the composition.

[0141] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, "dosage unit form" refers to physically discrete units suitable as a single dose for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The description of the dosage unit form of the present invention is oriented by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technology of compounding such active compound for the treatment of individual susceptibility.

[0142] For administration of antibodies, dosages can range from 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 usually 0.01 to 5 mg / kg of host body weight. For example, dosages can 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 to 5 mg / kg or within the range of 1 to 10 mg / kg. Examples of treatment regimens include daily, 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 invention include 1 mg / kg body weight, 3 mg / kg, 5 mg / kg, or 10 mg / kg body weight via intravenous administration, with the antibody being given using one of the following dosing regimens: (i) weekly for six doses, then monthly; (ii) weekly; (iii) 3 mg / kg body weight once, followed by 1 mg / kg body weight weekly.

[0143] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the ranges indicated. In some embodiments, the antibody is administered multiple times. The interval between single doses can be, for example, weekly, monthly, every three months, or yearly. The intervals can be irregular, as indicated by measuring the blood concentration of the antibody against 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, about 25-300 μg / ml.

[0144] In some embodiments, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary according to the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency can vary according to 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 may continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses are sometimes required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement of the symptoms of the disease. The patient can then be administered a prophylactic regimen.

[0145] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be modified to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.The dosage level selected depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, or its ester, salt, or amide, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical field.

[0146] A "therapeutically effective dose" of the antibody 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 periods, or prevention of functional impairment or disability due to disease affliction. For example, 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%, even more preferably at least about 80%, at least about 90%, or at least about 95% compared to an untreated subject. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a subject. One of skill in the art can determine such amounts based on factors such as the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0147] The compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. Preferred routes of administration for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The term "parenteral administration" as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0148] Alternatively, the antibodies of the invention can be administered via a parenteral route, such as a topical, epidermal or mucosal route, for example, intranasally, orally, intravaginally, rectally, sublingually or topically.

[0149] Active compound can be prepared with carriers that protect compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Many methods for preparing the above formulations are patented or generally known to those skilled in the art (see Sustained and Controlled Release Drug Delivery Systems (1978) JR Robinson, ed., Marcel Dekker, Inc., NY).

[0150] In certain embodiments, the monoclonal antibody of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compound of the present invention crosses the BBB (if desired), it can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that are selectively transported to specific cells or organs, thus enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate 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); K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; See also IJ Fidler (1994) Immunomethods 4:273.

[0151] Uses and Methods The antibodies, antibody compositions and methods of the invention have numerous in vitro and in vivo diagnostic and therapeutic utilities, including the diagnosis and treatment of immune-mediated disorders.

[0152] In some embodiments, these molecules can be administered to cells in culture, in vitro or ex vivo, or to human subjects, e.g., in vivo, to treat, prevent and / or diagnose various disorders. As used herein, the term "subject" 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 the antibody of the present invention is administered together with another agent, the two can be administered sequentially or simultaneously.

[0153] Given the specific binding of the antibodies of the present invention to SLAMF6, the antibodies of the present invention can be used to specifically detect SLAMF6 expression on the surface of immune cells and can further be used to purify SLAMF6 via immunoaffinity purification.

[0154] Furthermore, given the expression of SLAMF6 on immune cells, the antibodies, antibody compositions and methods of the invention can be used to treat subjects having a tumorigenic disease, e.g., a disease 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0155] 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0156] In further embodiments, the antibodies of the invention are used in the manufacture of a medicament for the treatment of cancer such as, for example, 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0157] In one embodiment, the antibodies of the invention (e.g., monoclonal antibodies, antibody fragments, Nanobodies™, multispecific and bispecific molecules and compositions, etc.) can be used to detect the amount (level) of SLAMF6, or the amount of immune cells containing SLAMF6 on their membrane surface, which can then be correlated with specific disease symptoms for diagnosis.

[0158] In another embodiment, antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention can be initially tested for binding activity relevant to therapeutic or diagnostic use in vitro. For example, compositions of the invention can be tested using the flow cytometry assays described in the Examples below.

[0159] In some embodiments, the antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention have additional utility in the treatment and diagnosis of disease. 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 immune cell activation; mediating phagocytosis or ADCC of cells in the presence of human effector cells expressing SLAMF6, or blocking binding of SLAMF6 ligand to SLAMF6.

[0160] In certain embodiments, antibodies (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) are used in vivo to treat, prevent or diagnose various diseases. Examples of relevant diseases include human cancer tissues 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, uterine 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, stomach cancer, glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies, among others.

[0161] Suitable routes of administration of the antibody compositions (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) of the invention in vivo and in vitro are well known to those of skill in the art and can be selected by those of skill in the art. 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, as well as the concentration and / or formulation of the antibody composition.

[0162] As mentioned above, the antibody of the present invention can be co-administered with one or more additional therapeutic agents, such as immunostimulants, cytotoxic agents, radiotoxic agents or immunosuppressants. The antibody can be linked to the agent (as an immunoconjugate) or 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, such as anti-cancer therapies, such as radiation therapy. Such therapeutic agents include, among others, anti-tumor drugs such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea, which are themselves only effective at levels that are toxic or subtoxic to the patient. Other agents suitable for co-administration with the antibody of the present invention include other agents used in the treatment of cancer, such as Avastin (registered trademark), 5FU and gemcitabine. The co-administration of the anti-SLAMF6 antibody or antigen-binding fragment thereof with a chemotherapeutic agent provides two anti-cancer agents that act through different mechanisms to produce a cytotoxic effect on human tumor cells. Such co-administration can solve problems caused by the development of resistance to the drugs or changes in the antigenicity of tumor cells.

[0163] 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 with 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 range from 10 8 ~10 9 The order of magnitude may be sufficient, but varies depending on the purpose of treatment.

[0164] Treatment with target-specific effector cells can be performed in conjunction with other techniques. For example, antitumor therapy using the compositions of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) and / or effector cells equipped with these compositions can be combined with chemotherapy. In addition, combination immunotherapy can be used to target two different cytotoxic effector populations for tumor cell rejection.

[0165] The bispecific and multispecific molecules of the invention can also be used to modulate FcγR or the amount of FcγR on effector cells, such as by capping and removing receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose.

[0166] Embodiments of the invention include kits comprising the antibody compositions of the invention (e.g., monoclonal antibodies, bispecific or multispecific molecules) and instructions for their use, e.g., in the treatment of cancer. The kits can further contain one or more additional reagents, such as immunosuppressive reagents, cytotoxic or radiotoxic agents, or one or more additional antibodies of the invention (e.g., antibodies with complementary activity that bind to an epitope in the SLAMF6 antigen different from the first antibody).

[0167] Thus, a patient treated with an antibody composition of the invention may additionally be administered (before, simultaneously with, or after administration of the antibody of the invention) another therapeutic agent, such as a cytotoxic or radiotoxic agent, which enhances or augments the therapeutic effect of the antibody.

[0168] In other embodiments, the subject may be additionally treated with an agent that modulates, e.g., enhances or inhibits, expression or activity of Fcγ or Fcγ receptors, e.g., by treating the subject with a cytokine. Preferred cytokines for administration during multispecific molecule therapy include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).

[0169] The compositions (e.g., antibodies, multispecific and bispecific molecules) of the present invention can also be used to target cells expressing FcγR or SLAMF6, for example, to label such cells. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the present invention provides a method for localizing ex vivo or in vitro cells expressing Fc receptors, such as FcγR or SLAMF6. The detectable label can be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

[0170] In certain embodiments, the invention provides a method of detecting the presence of SLAMF6 antigen in a sample or a method of measuring the amount of SLAMF6 antigen comprising contacting a sample and a control sample with a monoclonal antibody or antigen-binding fragment thereof that specifically binds to SLAMF6 under conditions that allow for the formation of a complex between the antibody or portion thereof and SLAMF6. The formation of the complex is then detected, where a difference in complex formation between the sample compared to the control sample indicates the presence of SLAMF6 antigen in the sample.

[0171] In other embodiments, the present invention provides methods for treating immune-mediated inflammatory diseases 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, uterine 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, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal gland cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies.

[0172] All references cited herein are incorporated by reference in their entirety, including, but not limited to, all articles, publications, patents, patent applications, presentations, textbooks, reports, manuscripts, pamphlets, books, internet postings, journal articles, periodicals, product fact sheets, etc. The discussion of references herein is intended merely to summarize the assertions made by their authors and is not an admission that any reference constitutes prior art, nor that applicants reserve the right to challenge the accuracy and pertinence of the cited references.

[0173] Although the foregoing invention has been described in some detail by way of illustration and example, for clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

[0174] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The following examples, sequences and figures are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the procedures described without departing from the spirit of the invention.

[0175] Working Example: Example 1: Antibody generation and screening. Hybridoma generation Recombinant ECD protein was used to immunize mice for the generation of mouse Fab against SLAMF6 ECD (SEQ ID NO: 12) at Alere, San Diego.-SLAMF6-hum. ECD splenocytes from immunized mice were used to generate a library of fabs using industry standard techniques.

[0176] Secondary Screening Fab supernatants were tested for binding to SLAMF6 protein expressed on the surface of Raji cells and activated PBMCs. Supernatants were diluted 1:10 in FACS buffer.

[0177] Example 2: Structural characterization of monoclonal antibodies against SLAMF6 The cDNA sequences encoding the heavy and light chain variable regions of the monoclonal antibody 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 screen can be seen in Figure 1.

[0178] The nucleotide and amino acid sequences of the heavy chain variable region of 1B3 are shown in SEQ ID NOs:3 and 1, respectively.

[0179] The nucleotide and amino acid sequences of the light chain variable region of 1B3 are shown in SEQ ID NOs: 4 and 2, respectively.

[0180] Further analysis of the 1B3 VH sequence using the Kabat system of CDR region determination led to the delineation of the heavy chain CDR1, CDR2 and CDR3 regions as shown in SEQ ID NOs: 5, 6 and 7, respectively. Figure la shows the 1B3 VH sequence with CDR1, CDR2 and CDR3 boxed.

[0181] Further analysis of the 1B3 VL sequence using the Kabat system of CDR region determination led to the delineation of the light chain CDR1, CDR2 and CDR3 regions, as shown in SEQ ID NOs: 8, 9 and 10, respectively. Figure lb shows the 1B3 VL sequence with CDR1, CDR2 and CDR3 boxed.

[0182] Example 3: Specificity of Fab Supernatant Monoclonal Antibodies to SLAMF6 Measured by Flow Cytometric Analysis 5×10 6Raji cells were 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. Test antibodies were diluted to 30 nM / l in FACS buffer and increasing amounts were added to each well as shown in Figure 4, which was incubated on ice for 30 minutes. The cells were then washed once with FACS buffer (DPBS, 2% FBS), pelleted, washed and resuspended in FACS buffer. Secondary goat anti-mouse antibody was diluted to 1 μg.ml and 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 200 ul FACS buffer. Samples were read using a Guava Easycyte Plus HT flow cytometer and results were analyzed using the Guava Cytosoft software suite.

[0183] As can be seen in FIG. 4, antibody 1B3 exhibits dose-specific dependent binding to SLAMF6-expressing Raji cells.

[0184] Example 4: Ability of anti-SLAMF6 antibodies to activate T cells and stimulate IFNγ production 96-well non-tissue culture plates were coated with 250ng / ml OKT3 and different anti-SLAMF6 antibody / isotype concentrations overnight at 4°C. Plates were washed twice with PBS and subsequently blocked for 30 minutes with R10 medium (RPMI with 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin). After blocking, 100,000 T cells were resuspended in 100μl R10 medium and added onto the plates (T cells isolated from PBMCs using Miltenyi kit code: 130-096-535 according to the manufacturer's instructions). Plates were incubated at 37°C for 72 hours and supernatants were collected and diluted for use in IFNγ ELISA assays.

[0185] IFNγ was measured using the IFN-gamma DuoSet ELISA kit (R&D Systems product no. DY285B) according to the manufacturer's instructions. result

[0186] The humanized antibody Hu_1B3 showed increased activity in OKT3 preactivated T cells, reflected by increased IFNγ production at low antibody concentrations, compared to the Seatle Genetic antibody against SLAMF6 described in WO2017 / 004330 (Figure 5). This induction of IFNγ production indicates that antibodies directed against SLAMF6 have therapeutic efficacy in patients with suppressed immune systems.

[0187] Example 5: Humanization of antibody 1B3. We have humanized the murine 1B3 monoclonal antibody using CDR grafting technology. To guide the humanization process and help decide whether to preserve the parent murine residues or replace them with their human germline counterparts, we constructed a homology molecule model of the Fv of the 1B3 murine monoclonal antibody.

[0188] The definition of the CDRs is based on the Kabat nomenclature. The selection of the human framework acceptor regions onto which the 1B3 rat CDR regions were grafted was achieved by searching the IMGT mouse and human V gene databases with the 1B3 mouse variable region sequences as input using IgBLAST, a tool developed at NCBI to facilitate the analysis of immunoglobulin V region sequences. The strategy applied was to use human germline sequences, which are naturally occurring human sequences that do not contain the specific somatic mutations found in individual human antibody sequences.

[0189] Heavy Chain Design The amino acid sequence of the VH isolated from the murine 1B3 hybridoma (CDR regions are shown in bold according to the Kabat numbering scheme) is shown below. [ka]

[0190] Selection of human framework receptor VH regions Selection of the human framework acceptor VH region onto which the 1B3 mouse CDR regions were grafted was achieved by searching the IMGT human VH gene database using IgBLAST with the mouse VH region amino acid sequence as input. The closest matching entry was identified based on sequence alignment of the parent antibody against the human germline. Identification of the optimal human germline as acceptor was based on ordered criteria of sequence identity between the frameworks as defined by Kabat, and / or identity and / or compatibility of interchain interface residues and supporting loops with the canonical conformation of the parent CDRs. Human germline IGHV1-2*02 was selected as the most suitable heavy chain.

[0191] Designing IGHV1-2*02 human germline using the framework acceptor region Humanized version The mouse CDRs (bold) as defined by the Kabat nomenclature were grafted into IGHV1-2*02, resulting in the following detailed sequence: Many residues are framework mouse residues (outside the CDR residues), i.e., conserved from the parental mouse 1B3 VH sequence, and have been conserved because they may be structurally important to maintain full activity of the antibody. [ka]

[0192] 86.7% identity (85 identical residues out of a total of 98 residues in the V genes) with the humanized version (OBT577-12-VHB) using the IGHV1-2*02 human germline.

[0193] Light Chain Design The amino acid sequence of mouse 1B3 VL (highlighting the CDR regions as defined by the Kabat nomenclature) is shown below. [ka]

[0194] Selection of human framework receptor VL domains Selection of the human framework acceptor VL region onto which the 1B3 VL mouse CDR regions were grafted was achieved 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 sequence alignment of the parent antibody against the human germline. Identification of the optimal human germline as acceptor was based on ordered criteria of sequence identity between the frameworks as defined by Kabat, and identity and / or compatibility (compatibility) of the interchain interface residues and supporting loops with the canonical conformation of the parent CDR. From this analysis, the human germline, IGKV1-33*01, was considered the best choice for the human framework acceptor region. Therefore, this human germline was used to design the humanized version.

[0195] Designed using IGKV1-33*01 human germline as the framework acceptor region Humanized version The murine CDRs, as defined by Kabat numbering, were grafted into IGKV1-33*01, giving the following detailed sequence: A number of structurally important residues have been retained to maintain full activity of the antibody. This resulted in a humanized version with 86.3% identity (82 out of 95 amino acid residues). [ka] 86.3% identity (82 / 95) between the humanized version and the IGKV1-33*01 human germline

[0196] Example 6 ELISPOT with tumor-infiltrating lymphocytes Primary tumor-derived tumor infiltrating lymphocytes (TILs) from NSCLC (Figure 6), breast cancer (Figure 7) or CRC (Figure 8) tumors were stimulated with murine 1B3 or pembrolizumab at 10 μg / ml and OKT3 at 1 μg / ml diluted in complete IMDM culture medium for 96 hours. After stimulation, TILs were harvested, counted and plated at 100000 cells / well on IFNγ ELISPOT plates (Mabtech). Plates were incubated at 37°C for 24 hours and then developed according to the manufacturer's instructions. Spot counts were read using an ImmunoSpot® Series 5 ELISPOT analyzer and data were analyzed using GraphPad Prism software.

[0197] FIG. 6 shows that antibody 1B3 activates NSCLC-derived TILs, as reflected by significantly higher IFNγ production than the isotype antibody.

[0198] FIG. 7 shows that antibody 1B3 activates significantly higher numbers of breast cancer-derived TILs to produce IFNγ than pembrolizumab.

[0199] FIG. 8 shows that antibody 1B3 activates significantly higher numbers of colorectal cancer-derived TILs to produce IFNγ than pembrolizumab.

[0200] Example 7 Binding affinity of humanized 1B3 antibody Biacore T-200 binding affinity experiments were performed at 25°C. Flow cells 2, 3 and 4 of a CM5 chip were coated with a maximum amount of 500 RU of goat anti-human IgG. Test antibodies were captured on flow cells 2, 3 and 4. Flow cell 1 was kept blank and used for reference subtraction. Antigen was flowed over the chip. Binding of antigen to antibody was monitored in real time. The observed k on and k off Therefore, KD was determined.

[0201] [Table 3]

[0202] Example 8 In vitro proliferation assay using anti-SLAMF6 antibody 1B3 method. Non-tissue culture treated 96-well plates (BD Falcon, USA) were coated with anti-human CD3 (eBioscience, USA) antibody alone at 250ng / ml or humanized anti-SLAMF6 antibody (Hu_1B3) or isotype control antibody at concentrations of 0, 0.2, 0.4, 0.6, 0.9, 1.2ug / ml and incubated overnight at 4°C. The next day, plates were washed and blocked with AIM V media. T cells isolated from PBMCs generated from healthy donors were stained with cell proliferation dye eFluor™ 670 (eBioscience, USA), washed and plated onto antibody-coated plates (100,000 cells / well in 100μl) in AIM V (Thermofisher Scientific, USA) media with FCS, penicillin-streptomycin and cultured for 72 hours at 37°C in a tissue culture incubator. On day 3, cells were harvested and stained with FITC labeled anti-human CD8, Brilliant Violet 711™ labeled anti-human CD4, PE labeled anti-human CD69 (Biolegend, USA), and the fixable viability dye eFluor™ 506 (eBioscience, USA). Samples were analyzed on an Attune NxT flow cytometer (Thermofisher Scientific, USA) and data were analyzed using FlowJo software (TreeStar, USA).

[0203] FIG. 9 shows that stimulating SLAMF6 present on isolated T cells with anti-SLAMF6 antibody in the presence of CD3 significantly increases T cell proliferation compared to isotype or CD3 alone.

[0204] Example 9 One-way mixed lymphocyte reaction (MLR) with allogeneic PBMC using anti-SLAMF6 antibody Hu_1B3. method: T cells isolated from one donor (donor 1) were resuspended in RPMI 1640 medium containing 10% supplemented bovine serum and 2 mM L-glutamine (culture medium).

[0205] Cryopreserved PBMCs from a second donor (Donor 2) were treated with 50ug / ml mitomycin C at a density of 2E6 cells / ml in culture medium. Cells were treated for 90 minutes at 37°C before removing the mitomycin C by washing with culture medium.

[0206] 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 various concentrations of humanized antibody Hu_1B3 and control solutions and cultured for 6 days in a total volume of 100ul of medium / well. Isotype was used as a negative control as well as anti-CD137 mAb and was included in the comparison.

[0207] Culture supernatants were harvested on day 6 and analyzed for IFN-γ concentrations by ELISA (R&D Systems: DY285B) according to the manufacturer's instructions. Figure 10 shows that the anti-SLAMF6 antibody Hu_1B3 shows a dose-dependent increase in cytokine release, indicating that T cells are activated by the presence of the antibody, and that SLAMF6 activation results in higher cytokine release from isolated T cells than activation via CD137.

[0208] Example 10 SLAMF6-Mediated Granzyme B and Perforin Release from T Cells PBMC isolation As a first step in T cell isolation, PBMCs are isolated from the buffy coat (Leucopak from Stanford Blood Center). Blood is diluted in PBS in a 1:4 ratio (10 ml blood + 30 ml PBS) and 30 ml of diluted blood is carefully placed on 15 ml of Ficoll-Hypaque (GE-Healthcare cat.No.17-1440-03). The tubes are centrifuged in a sorvall centrifuge at 400g (1400 rpm) for 30 min at RT with the brake off. Monocytes are separated by density gradient. Approximately 10 ml of the cellular white fraction from all tubes are pooled into a single 50 ml tube, washed and counted using a Cellometer Auto 2000. The PBMCs generated from the buffy coat are used for T cell isolation.

[0209] T cell isolation Pan T cell isolation from PBMCs is a negative selection process in which all remaining immune cell subsets, except for 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 buffy coats are washed and resuspended in FACS sorting buffer (0.1% BSA in PBS) at a concentration of 2.5e7 cells / mL, Pan T cell biotin-antibody cocktail is added to the cells, the antibody-cell suspension is mixed using a 1 ml pipette, and incubated on ice for 5 minutes. Pan T cell microbead isolation cocktail is added to the mixture and incubated on ice for an additional 10 minutes. MACS separation method is used to isolate naive T cells. The PBMC biotin-labeled microbead mixture is run over an LS column (Miltneyi Biotec, Cat#130-042-401) in a high-field MACS separator and the flow-through depleted of non-T cells is collected, washed once with FACS sorting buffer and resuspended in AIMV complete medium with 5% FBS.

[0210] Granzyme B ELISA For the Granzyme B functional assay, non-tissue culture treated 96-well plates are coated with human Hu_1B3 or isotype control antibody in combination with 250ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific Cat#16-0031-85). Each test antibody is coated in triplicate at 2:3 dilutions for a 10-point titration starting at 4.2μg / ml in 100μl PBS. Plates are sealed and incubated overnight at 4°C. On the day of Granzyme B functional assay setup, plates are washed and blocked with AIMV complete medium with 5% FCS for 20 minutes to reduce non-specific binding.

[0211] Granzyme B immunoassay is set up using Human Granzyme B DuoSet ELISA kit from R&D Systems (Cat# DY2906-05). Nunc-immunoassay plates are coated with Granzyme B capture antibody diluted 1:60 in PBS and plates are incubated overnight at 4°C. The next day plates are washed with ELISA wash buffer (PBS+0.05% tween20) and blocked in PBS with 1% BSA. Standards are added to each well after blocking with 100 μl of sample diluted 1:60 in dilution buffer and incubated overnight at 4°C. Plates were developed the following day and OD values ​​were captured on a VersaMax tunable microplate reader. Granzyme B release is quantified and plotted using Graphpad Prism 8 software.

[0212] conclusion Granzyme-mediated apoptosis is one of the major mechanisms used by cytotoxic lymphocytes to eliminate transformed cells. The data indicate that antibody Hu_1B3 can induce cytotoxic functions important for tumor suppression. Antibody Hu_1B3 dose-dependently increased granzyme B from activated T cells (Figure 11) and increased EC 50 is 0.51 μg / ml.

[0213] Perforin intracellular assay Perforin Intracellular Quantification Assay Setup: Coat non-tissue culture treated 96-well plates with human Hu_1B3 or isotype control antibody in combination with 250ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific Cat#16-0031-85). Coat each test antibody at 2:3 dilutions for a 10-point titration in 100μl of PBS starting at 4.2μg / ml. Seal plates and incubate overnight at 4°C. On the day of Granzyme B functional assay setup, wash plates and block with AIMV complete medium with 5% FCS for 20 minutes to reduce non-specific binding. For this assay, add 200,000 pan T cells per well in 100μl of AIMV culture medium with 5% FBS and culture for 3 days at 37°C in a tissue culture incubator. On the day of the assay, a protein transport inhibitor cocktail (Thermofisher Scientific, Cat# 00-4980-93) is added to all wells and incubated for 4 hours to prevent perforin transport to the extracellular space. Cells are harvested and stained for T cell surface markers CD3, CD4, and CD8, followed by intracellular perforin staining using the FIX&PERM™ Cell Permeabilization Kit (Thermofisher Scientific, Cat# GAS-004). Cells were analyzed on an Attune NxT FACS analyzer (Thermofisher Scientific, MD) and data were analyzed with FlowJo software (BD Biosciences, San Jose).

[0214] conclusion Perforin-mediated necrosis is another major mechanism of killing induced by cytotoxic T lymphocytes, and Hu_1B3 inhibited it with an EC 50 , which dose-dependently enhances perforin upregulation in CD8+ T cells (Figure 12).

[0215] Example 11 Competitive Binding Assay to Evaluate the Binding Epitope of Antibody 1B3 Cryopreserved human PBMCs were thawed and washed once with suspension in FACS buffer (DPBS with 2% FCS) followed by centrifugation at 1200 rpm for 5 min to pellet the cells and discard the supernatant (same method used for subsequent washes). Cells were dispensed into 96-well assay plates at 100,000 cells per well in FACS buffer and then washed again. Next, cells were blocked for 1 h with SLAMF6 ECD-mIgG2a Fc fusion protein 100 nM or 300 nM for SLAMF6 receptor blocking in 100 μl FACS buffer on ice for 1 h before washing. Humanized Hu_1B3 antibody or human IgG1 isotype control was titrated in 1 / 3 serial dilutions at a top concentration of 10 nM and then added to the blocked PBMCs and cells were incubated on ice for 1 h followed by two washes. Secondary antibody, goat anti-human-IgG-RPE (Southern Biotech, Ref: 2040-05), at a concentration of 1 μg / mL in FACS buffer was then applied to the treated cells for 30 minutes on ice. One of the untreated wells containing cells was also stained with the secondary antibody, and the other was left unstained to act as a control for secondary antibody binding. After this final incubation, the cells were washed again twice and the final cell pellet was resuspended in FACS buffer. The mean fluorescence intensity of the secondary antibody was measured for each sample using an Attune NxT Flow Cytometer (ThermoFischer Scientific) in a 96-well plate format according to industry standard protocols, and the raw data was analyzed using FlowJo analysis software.

[0216] As seen in Figure 13, the Hu_1B3 antibody was blocked from binding to the receptor on the surface of PBMC in the presence of human SLAMF6 ECD-mIgG2a Fc fusion protein, indicating that Hu_1B3 competitively binds to human SLAMF6 ECD. Therefore, it is believed that Hu_1B3 binds to the homodimerization epitope of SLAMF6 and enhances cytotoxic T cell function through the SAP-mediated activation pathway by functioning as an agonist antibody.

[0217] Example 12 Internalization of Hu_1B3 antibody RAJI, human Burkitt lymphoma cells (Cat No CCL-86, American Type Culture Collection [ATCC], Manassas VA) were grown in RPMI-1640 medium (Cellgro, Cat No 10-041-CM, Mediatech, Manassas VA) supplemented with 10% fetal bovine serum (HyClone* Cosmic Calf Serum, Cat No SH30087-03, Thermo Scientific, Waltham, MA) and 1% sodium pyruvate (Cellgro, Cat. # 25-000-Cl) using industry standard aseptic techniques. RAJI cells were grown at 5 × 10 per well in 24-well glass-bottom culture plates. 5 Cells were plated at a density of 100x and allowed to grow in growth medium for 48 hours at 37°C. Wells were prepared after 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours and 24 hours for the following samples: secondary antibody only control, human IgG isotype control, antibody Hu_1B3, clinical anti-SLAMF6 antibody from Seattle genetics (positive control). Secondary only control wells and no antibody control wells were used as fluorescence controls.

[0218] All antibody incubations and washing steps were subsequently performed on ice with ice-cold reagents. Culture medium was aspirated from wells and washed twice with IF buffer (Dulbecco's Phosphate Buffered Saline (DPBS, Thermo Scientific, Waltham VA, Cat. # SH30028-03)) + 2% FBS). Primary antibodies (purified OBT humanized Hu_1B3; isotype or positive control antibodies (Seattle genetics) were diluted to 2ug / ml in IF buffer and 200μl was applied to appropriate wells for 15 minutes. The same volume of IF buffer alone was added to wells designated for secondary antibody control. Secondary antibodies (goat anti-human IgG-Alexa Fluor 488, Invitrogen Cat. #A11013) were diluted to a concentration of 2ug / mL in IF buffer and added to the primary incubation for an additional 15 minutes.

[0219] After primary and secondary antibody labeling, human IgG isotype control, secondary antibody only control, and test and positive controls after 0 min were processed. Cells were washed twice with IF buffer and placed on ice in a second 24-well plate containing 4% paraformaldehyde (4% diluted in half with DPBS, Cat No 19943, Affymetrix, Santa Clara, CA) to stop internalization and fix cells.

[0220] The remaining cells were washed twice with IF buffer and 1 mL of warmed growth medium was added to each well before placing in a 37° C. incubator. Cells at 0, 0.5, 1, 2, 4, and 24 h were fixed in paraformaldehyde on ice as described for the control samples.

[0221] All cells remained in fixative for at least 15 min on ice. Cells were washed with IF buffer and coverslips were added to each well with 2–3 drops of Prolong Gold Anti-Fade reagent + DAPI (Cat No P-36931, Invitrogen, Grand Island, NY) as a nuclear counter stain. Cell images were acquired using a Leica microscope (Leica DMI600B), a Leica monochrome camera (Leica DFC350FX), a filter set for DAPI and Alexa Fluor488 and an oil immersion lens 63x. Images were saved in TIFF format and analyzed using ImageJ.

[0222] Figure 14 shows that the humanized antibody Hu_1B3 is internalized by SLAMF6 expressing cells significantly less than the clinical SeattleGenetics antibody, indicating that binding to the antibody Hu_1B3 induces a more sustained response in T cells because the receptor remains on the cell surface for a longer period of time, and therefore may be a more effective agonist.

[0223] Example 13 Cytotoxicity Assay SKBr3 HCT116 and MDA-MB-231 were purchased from ATCC. Cell lines were maintained according to the manufacturer's (ATCC) instructions using standard aseptic techniques. 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 with 5% CO2. SKBr3 expresses high copy number Her2 (approximately 5 × 10 6 HCT116 is a colon cancer cell line that expresses low copy numbers of Her2. MDA-MB-231 is a breast cancer cell line that expresses low copy numbers of Her2.

[0224] Target and effector (PBMC) cells were prepared under the following conditions: On the day before the assay, 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 plated at 10,000 cells / well in 96-well tissue culture treated plates. Cells were grown overnight in a 37°C incubator with 5% CO2.

[0225] The day before the assay, 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 following 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.

[0226] Bispecific antibody Cris7-Her2 was used in the cytotoxicity assay. The antibody detects both Her2 and CD3ε. The Cris7-Her2 bispecific antibody was diluted to 100ng / ml and then serially diluted 3-fold to 0.0012ng / ml. Additionally, Cris7-Her2 + agonist antibody was tested. A combination of Cris7-Her2 bispecific antibody and agonist antibody was included to observe enhanced cytotoxicity by agonist antibody. Urelumab, an antibody against 4-1BB, was used as a positive control. Isotype was used as a negative control. Hu_1B3 was the test article. Urelumab, isotype, and Hu_1B3 were used at a final concentration of 2.5ug / ml. All samples were performed in triplicate. Controls included target cells alone or target cells + effector cells. Further controls included target and effector cells plus Hu_1B3 or target and effector cells plus isotype control.

[0227] Cytotoxicity assays were incubated for 48-96 hours depending on the cell line. After incubation, target cell viability was measured. Viability was based on quantification of ATP, which signals the presence of metabolically active cells. The assay was luminescence-based, and Cell Titer-Glo® (Promega, Madison, WI) was the reagent used to measure live cells. Assay conditions were as per the manufacturer's instructions, and all steps were performed at room temperature. Briefly, the 96-well plate and Cell Titer-Glo® reagent were equilibrated to room temperature for 30 minutes. After 30 minutes, the cell culture media was removed and the target cells were washed with 200ul of PBS, and the washing step was repeated one more time. Then, 100ul of Cell Titer-Glo® 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 allow the luminescence signal to stabilize. After 10 min, samples were transferred to an opaque-walled 96-well plate and luminescence was recorded.

[0228] The viability of the target cells was proportional to the luminescence signal generated. All samples were performed in triplicate and the average was calculated for each assay condition. The percent cytotoxicity of the samples was normalized to the control, which was the viability of the target cells in the presence of effector cells (target + effector). To calculate the viability, the luminescence signal of the sample was divided by the luminescence signal of the control. Cytotoxicity was calculated based on the percentage of non-viable cells remaining. As can be seen in Figure 15, in SKBr3 cells, the bispecific antibody alone, or the bispecific antibody + isotype control, has effective cytotoxicity at higher concentrations of 0.8-100ng / ml. The addition of urelumab shifts the cytotoxicity curve slightly to the left, but not by 5%. However, the addition of 2.5ug / ml of Hu_1B3 enhanced the cytotoxicity of SKBR-3 cells by 10-40%.

[0229] FIG. 16 shows that similar results are seen in a second PBMC donor.

[0230] FIG. 17 shows enhanced cytotoxicity by Hu_1B3 against HCT116 cells. HCT116 cells, a colon cancer cell line, express low levels of Her2 on the cell surface. Cytotoxicity assays were performed using PBMCs and HCT116 cells with the T cell-engaging bispecific antibody, Cris7-Her2. As described above, Hu_1B3 was added to the assay to determine whether it enhances the function of T cells or NK cells present in PBMCs, where T cells and NK cells both express the co-activating receptor, SLAMF6. The data show dose-dependent cytotoxicity by the T cell-engaging bispecific antibody, Cris7-Her2, at the various concentrations tested. More importantly, the data show that increased cytotoxicity was observed when Hu_1B3 was added. The EC50 for Cris7-Her2 specific alone or bispecific antibody plus isotype control is approximately 0.25 ng / ml, whereas the EC50 for bispecific antibody plus Hu_1B3 is approximately 0.07 ng / ml, representing an approximately 3.6-fold increase in cytotoxicity.

[0231] FIG. 18 shows enhanced cytotoxicity by Hu_1B3 against MDA-MB-231 cells. MDA-MB-231 cells, a breast cancer cell line, express low levels of Her2 on the cell surface. Cytotoxicity assays were performed using MDA-MB-231 cells, PBMCs, and the T cell engaging bispecific antibody, Cris7-Her2, as described above. Again, Hu_1B3 enhances the cytotoxicity of the Cris7-Her2 bispecific at various concentrations tested. Specifically, a 40% increase in cytotoxicity was observed at 1 ng / mL of Cris7 compared to the isotype control.

[0232] Figure 19 shows that after 96 hours of incubation, the combination of the bispecific with Hu_1B3 showed high levels of killing of SKBR-3 cells even at the lowest levels of bispecific antibody concentration, whereas at these levels, either the bispecific alone or the combination of the bispecific with Urelumab, or the isotype control, caused very low levels or no cell death, suggesting that Hu_1B3 caused potent activation of lymphocytes.

[0233] Figure 20 shows that in the above cytotoxicity assay, the bispecific Cris7-Her2 antibody remained constant (0.0457ng / ml) when the test antibody concentration was titrated at 1 / 3 dilutions ranging from 7.5ug / ml to 0.0034ug / ml. Dose-dependent killing of SKBR-3 cells was observed compared to urelumab or isotype control, further demonstrating the ability of Hu_1B3 to activate cytotoxic lymphocytes.

[0234] Example 14 Cytotoxicity Assay in the Absence of Cris7 Bispecific Another cytotoxicity assay was set up as above, but without the bispecific antibody. For single agent SKBR-3 cytotoxicity testing, only Hu_1B3, isotype control, and urelumab were used. The concentration range used in the assay was 33ug / ml to 0.13ug / ml at 1 / 3 dilution with one donor. For the second donor, the test antibody was further diluted to 0.0152ug / ml (Figure 21b).

[0235] Figures 21a and 21b show that in the absence of the CD3-Her2 bispecific antibody Hu_1B3, lymphocytes can be activated to induce cell death in SKBR-3 cells, in contrast to urelumab or isotype control, which show little cell killing.

[0236] Sequence Listing JPEG0007604401000008.jpg245170JPEG0007604401000009.jpg245170JPEG0007604401000010.jpg245170

Claims

1. An antibody or antigen-binding fragment thereof that binds to SLAMF6, a CDR-H1 sequence comprising SEQ ID NO:5; a CDR-H2 sequence comprising SEQ ID NO:6 or SEQ ID NO:15; a CDR-H3 sequence comprising SEQ ID NO:7; and a heavy chain variable region comprising a CDR-L1 sequence comprising SEQ ID NO:8 or SEQ ID NO:16; a CDR-L2 sequence comprising SEQ ID NO:9 or SEQ ID NO:17; a CDR-L3 sequence comprising SEQ ID NO: 10; a light chain variable region comprising 4. An antibody or antigen-binding fragment comprising:

2. The heavy chain variable region CDR-H1 comprising the sequence of SEQ ID NO:5; CDR-H2 comprising the sequence of SEQ ID NO: 15; CDR-H3 comprising the sequence of SEQ ID NO: 7; The antibody or antigen-binding fragment of claim 1 , comprising:

3. The light chain variable region comprises: CDR-L1 comprising the sequence of SEQ ID NO: 16; CDR-L2 comprising the sequence of SEQ ID NO: 17; CDR-L3 comprising the sequence of SEQ ID NO: 10; 3. The antibody or antigen-binding fragment of claim 1 or claim 2, comprising:

4. An antibody or antigen-binding fragment thereof that binds to SLAMF6, CDR-H1 comprising SEQ ID NO:5; CDR-H2 comprising SEQ ID NO: 15; and CDR-H3 comprising SEQ ID NO:7; and a heavy chain variable region comprising CDR-L1 comprising SEQ ID NO: 16; and CDR-L2 comprising SEQ ID NO: 17; and CDR-L3 comprising SEQ ID NO: 10; and A light chain variable region comprising 4. An antibody or antigen-binding fragment comprising:

5. An antibody, or an antigen-binding fragment thereof, that binds to SLAMF6, i) the three heavy chain CDRs of SEQ ID NO:1 and the three light chain CDRs of SEQ ID NO:2, or ii) the three heavy chain CDRs of SEQ ID NO: 13 and the three light chain CDRs of SEQ ID NO: 14 Including, The CDRs are defined by the Kabat or Chothia numbering system.

6. a heavy chain variable region comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13; a light chain variable region comprising a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 14; The antibody or antigen-binding fragment thereof that binds to SLAMF6 according to claim 4, comprising:

7. The antibody or antigen-binding fragment thereof of claim 6, comprising a heavy chain variable region comprising SEQ ID NO: 13 and a light chain variable region comprising SEQ ID NO:

14.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment is a monoclonal antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment is a chimeric, humanized, or bispecific antibody or antigen-binding fragment.

10. The antibody or antigen-binding fragment thereof of any of claims 1 to 9, wherein the antibody or antigen-binding fragment is an Fc-silenced engineered IgG1 antibody or an antigen-binding fragment having reduced or no binding to one or more Fc receptors.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody or antigen-binding fragment is capable of inducing and / or enhancing T cell cytotoxicity.

12. The antigen-binding fragment may be Fab, Fab', F(ab) ２ , F(ab′) ２ 12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, which is selected from the group consisting of Fv, FV-TCR fragment, and scFv.

13. i) the heavy chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 12, and / or ii) A light chain variable region of the antibody or antigen-binding fragment according to any one of claims 1 to 12. A polynucleotide encoding the

14. An expression vector comprising at least one polynucleotide according to claim 13.

15. i. an expression vector comprising the polynucleotide of claim 13, or ii. a first expression vector comprising a polynucleotide encoding a heavy chain variable region of the antibody or antigen-binding fragment thereof according to claim 13; and a second expression vector comprising a polynucleotide encoding a light chain variable region of the antibody or antigen-binding fragment thereof according to claim 13; A host cell comprising:

16. 20. A method for producing an antibody or antigen-binding fragment thereof, comprising culturing a host cell according to claim 15 under conditions in which the antibody or antigen-binding fragment is expressed in the host cell.

17. The method of claim 16, further comprising isolating the antibody or antigen-binding fragment.

18. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12 and a pharma- ceutically acceptable carrier.

19. 20. Use of an antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to any one of claims 1 to 12 or 18 in the manufacture of a pharmaceutical composition for use in the treatment of cancer.

20. An antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to any one of claims 1 to 12 or 18, for use in the treatment of cancer.

21. 21. The use according to claim 19 or 20, wherein the cancer is selected from the group consisting of non-small cell lung cancer including small cell lung cancer, squamous cell carcinoma and adenocarcinoma, skin cancer including melanoma, breast cancer including TNBC, colorectal cancer, gastric cancer, ovarian cancer, uterine 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, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

22. The use according to any one of claims 19 to 21, wherein the pharmaceutical composition or medicament further comprises an effective amount of a second therapeutic agent.

23. An antibody or antigen-binding fragment thereof, or a pharmaceutical composition according to any one of claims 1 to 12 or 18, for use in therapy or as a medicament.

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