Antibodies and methods of use

Abstract

The present invention relates to an antibody against SLAMF6 (SLAM family member 6), also known as NTB-A or CD352, and other therapeutic proteins, nucleic acids encoding these antibodies and therapeutic proteins, a method for producing the antibodies and other therapeutic proteins, and a method for treating diseases such as cancer using the antibodies against SLAMF6 and other therapeutic proteins.

Description

Technology Field

[0001] Technology field

[0002] The present invention relates to an antibody capable of binding to the SLAMF6 protein and its uses.

[0003] Introduction

[0004] An aspect of the present invention includes an antibody directed against SLAMF6 (SLAM family member 6), also known as NTB-A or CD352, and other therapeutic proteins, nucleic acids encoding such antibody and therapeutic proteins, a method for producing the antibody and other therapeutic proteins, and a method for treating a disease such as cancer using the antibody directed against SLAMF6 and other therapeutic proteins. Background Technology

[0005] The target antigen SLAMF6 is a single-pass type I membrane protein and a member of the immunoglobulin superfamily and CD2 subfamily (J Exp Med. 2001 Aug 6;194(3):235-46). Its activity is controlled by the presence or absence of small cytoplasmic adapter proteins, SH2D1A / SAP and / or SH2D1B / EAT-2. This protein induces cytolytic activity only in natural killer (NK) cells expressing high surface densities of natural cytotoxic receptors (J. Exp. Med. 194:235-246(2001)). Positive signaling in NK cells induces the phosphorylation of VAV1. NK cell activation appears to depend on SH2D1B rather than SH2D1A. Together with SLAMF1, SLAMF6 controls the positive selection of thymic natural killer T (NKT) cell lineages and the transition between subsequent expansion and differentiation. SLAMF6 also promotes T cells to differentiate into helper T cell Th17 phenotypes, thereby increasing IL-17 secretion; this co-stimulatory activity requires SH2D1A (J. Immunol. 177:3170-3177(2006)). This further promotes RORC recruitment to the IL-17 promoter (J. Biol. Chem. 287:38168-38177(2012)). Together with SLAMF1 and CD84 / SLAMF5, SLAMF6 may be a negative regulator of the humoral immune response. In the absence of SH2D1A / SAP, SLAMF6 CD4 + It can transmit negative signals to T cells and NKT cells. It also negatively regulates germinal center formation by inhibiting T-cell:B-cell adhesion; this function may be related to the increased association with PTPN6 / SHP-1 via ITSM in the absence of SH2D1A / SAP.

[0006] WO2008 / 027739 discloses an anti-NTB-A antibody and a pharmaceutical composition comprising such antibody. It also discloses a method of using such antibody to treat diseases such as hematological malignancies characterized by binding to NTB-A and the expression of NTB-A.

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

[0008] WO2015 / 104711 discloses compositions and methods for improved T cell modulation in vitro and in vivo, and for the treatment of cancer and other pathological conditions. More specifically, embodiments of the present invention relate to the use of soluble NTB-A polypeptide or agents thereof for the treatment of cancer patients, the prevention and treatment of cytopenia in susceptible patients, and the in vitro preparation of improved cell compositions.

[0009] An aspect of the present invention comprises a specific antibody directed to SLAMF6, a bispecific antibody directed to SLAMF6 and a tumor-associated antigen, a nucleic acid encoding such an antibody of the present invention, a host cell comprising such a nucleic acid encoding such an antibody of the present invention, a method for producing an antibody of the present invention, and a method for treating a human cancer, including but not limited to diseases 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, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy.

[0010] An antibody that binds to SLAMF6 (SEQ No. 11) is described herein. Preferably, said antibody binds to the extracellular domain of SLAMF6 (SEQ No. 12). An aspect of the invention comprises an antibody or an antigen-binding fragment thereof that binds to an epitope on the SLAMF6 protein recognized by the antibody described herein, or cross-competes for binding with the antibody described herein, and preferably possesses at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the binding affinity of the antibody described herein for human SLAMF6. In some embodiments, the antibody is an isolated antibody.

[0011] An aspect of the present invention comprises an antibody or an antigen-binding fragment thereof that binds to SLAMF6, wherein the antibody comprises 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. In some embodiments, the antibody or antigen-binding fragment further comprises a light chain variable region comprising a CDR-L1 sequence comprising either one of SEQ ID NO. 8 and SEQ ID NO. 16; a CDR-L2 sequence comprising either one of SEQ ID NO. 9 or SEQ ID NO. 17; and a CDR-L3 sequence comprising the sequence of SEQ ID NO. 10.

[0012] In some embodiments, the antibody binding to SLAMF6 or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region comprising one of eight combinations of heavy chain and light chain CDRs as presented in Table 1.

[0013] Heavy chain variable region CDR Light chain variable region CDR CDR-H1 CDR-H2 CDR-H3 CDR-L1 CDR-L2 CDR-L3 1 Sequence No. 5 Sequence number 6 Sequence number 7 Sequence number 8 Sequence number 9 Sequence number 10 2 Sequence No. 5 Sequence number 15 Sequence No. 7 Sequence number 8 Sequence number 9 Sequence number 10 3 Sequence No. 5 Sequence number 6 Sequence number 7 Sequence number 16 Sequence number 17 Sequence number 10 4 Sequence No. 5 Sequence number 15 Sequence number 7 Sequence number 16 Sequence number 17 Sequence number 10 5 Sequence No. 5 Sequence number 6 Sequence number 7 Sequence number 16 Sequence number 9 Sequence number 10 6 Sequence No. 5 Sequence number 15 Sequence number 7 Sequence number 16 Sequence number 9 Sequence number 10 7 Sequence No. 5 Sequence number 6 Sequence number 7 Sequence number 8 Sequence number 17 Sequence number 10 8 Sequence No. 5 Sequence number 15 Sequence number 7 Sequence number 8 Sequence number 17 Sequence number 10

[0014] In some preferred embodiments, the 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. In another preferred embodiment, the 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 a light chain variable region comprising 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 of the present invention or its antigen-binding fragment comprises a variable CDR compared to the parent antibody described herein. Accordingly, the present invention provides a variant antibody or its antigen-binding fragment comprising a variant variable region of a parent antibody, wherein the parent antibody or its antigen-binding fragment comprises a CDR-H1 sequence comprising the sequence of SEQ ID NO. 5; a CDR-H2 sequence comprising the sequence of SEQ ID NO. 15; a heavy chain variable region comprising a CDR-H3 sequence comprising the sequence of SEQ ID NO. 7; and a CDR-L1 comprising the sequence of SEQ ID NO. 16; and a CDR-L2 comprising the sequence of SEQ ID NO. 17. A light chain variable region comprising CDR-L3 including the sequence of SEQ ID NO. 10, wherein in one embodiment, the variant antibody or its antigen-binding fragment has 1, 2, 3, 4, 5, or 6 amino acid substitutions, additions, and / or deletions in any one or more of the set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, having 1 to 5 or 1 to 4 or 1 to 3 substitution additions or deletions for a specific use; or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, additions, and / or deletions in the set, wherein the antibody, or its antigen-binding fragment, has a specific binding to SLAMF6. Preferably, the variation is a substitution, and preferably, the substitution is a conservative substitution or a substitution that returns an amino acid in the variable region to a corresponding amino acid from the human germline.In a further embodiment, the variant antibody of the present invention or its antigen-binding fragment comprises a CDR-H1 sequence comprising the sequence of SEQ ID NO. 5; a CDR-H2 sequence comprising the sequence of SEQ ID NO. 15; a heavy chain variable region comprising the CDR-H3 sequence comprising the sequence of SEQ ID NO. 7; and a light chain variable region comprising a CDR-L1 sequence comprising the sequence of SEQ ID NO. 16; a CDR-L2 sequence comprising the sequence of SEQ ID NO. 17; and a CDR-L3 sequence comprising the sequence of SEQ ID NO. 10, wherein one or more of the CDR sequences are modified to be approximately 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the cited corresponding CDR parent sequence.

[0016] In some embodiments, an antibody or an antigen-binding fragment thereof is provided that comprises a heavy chain variable region described 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 SEQ ID NO. 1 or SEQ ID NO. 13, and / or a light chain variable region described in SEQ ID NO. 2 or SEQ ID NO. 14, or a sequence 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, an antibody or an antigen-binding fragment thereof is provided, comprising a heavy chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions, additions, and / or deletions compared to SEQ ID NO. 1 or SEQ ID NO. 13, and / or a light chain variable region comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions compared to SEQ ID NO. 2 or SEQ ID NO. 14. Preferably, the variation comprises substitutions, more preferably conservative substitutions.

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

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

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

[0020] In one embodiment, a full-length antibody is provided comprising a heavy chain sequence including SEQ ID NO. 18 and a light chain sequence including SEQ ID NO. 19.

[0021] In a further aspect of the present invention, an antibody or its antigen-binding fragment that specifically binds to SLAMF6 is provided, said antibody or its antigen-binding fragment comprises three heavy chain CDRs of SEQ ID NO. 1 or SEQ ID NO. 13 and three light chain CDRs of SEQ ID NO. 2 or SEQ ID NO. 14, said CDRs are defined by the Kabat or Chothia numbering system. Preferably, said antibody or its antigen-binding fragment that specifically binds to SLAMF6 comprises three heavy chain CDRs of SEQ ID NO. 13 and 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. Those skilled in the art will readily understand that SEQ ID NOs 18-19 are full-length heavy and light chain sequences containing regions other than the variable region required to generate full-length functional antibodies, namely the constant and Fc regions. Those skilled in the art will understand that the sequences are humanized by replacing amino acids from the variable region of the organism to which the antibody is to be produced with amino acids from the human germline sequence in an effort to minimize the immunogenic effects of the antibody when administered to a human individual. Most amino acid substitutions occur in the framework region, but a number of amino acids from the CDR at non-critical positions may be substituted; such substitutions preferably revert to amino acids that are conserved in nature or amino acids present in the corresponding human germline at specific positions. In the case of the present invention, the amino acids from the substituted CDR have been identified using a structural model to distinguish between paratopic faces and non-paratopic residues in the CDR region. This allows the antibody to be humanized to a higher degree than that achieved by simple CDR grafting.

[0023] In the present invention, SEQ ID NO. 13 comprises two amino acid substitutions in CDR2 (SEQ ID NO. 15) compared to CDR2 (SEQ ID NO. 6) of SEQ ID NO. 1. Specifically, KQ substitution at position 16 of SEQ ID NO. 6; and DG substitution at position 17 of SEQ ID NO. 6.

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

[0025] Those skilled in the art will understand that these humanized sequences do not represent different alternative antibodies when compared to the parent antibody, but merely relate to the same antibody having the same characteristics that has been modified using structural modeling to correspond more closely to the human germline in order to minimize immunogenicity.

[0026] In some embodiments, the antibody or antigen-binding fragment has a binding affinity (K) of 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, or less. D has ).

[0027] In some embodiments, the antibody, or antigen-binding fragment, is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the heavy chain variable region contains a framework sequence. In some embodiments, at least a portion of the framework sequence contains a human consensus framework sequence. In some embodiments, the light chain variable region contains a framework sequence. In some embodiments, at least a portion of the framework sequence contains a human consensus framework sequence.

[0028] Alternative strategies to mitigate antibody effector function have also been used, including the substitution of residues at the antibody lower hinge, such as L234A and L235A (LALA). These residues form part of the Fc-γ receptor binding site on the CH2 domain, and the exchange of these residues between antibody isoforms with greater or lower effector function has confirmed their importance in ADCC. Alanine substitution of these sites is effective in reducing ADCC in both human and rat antibodies, but this substitution is 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, which results in a reduction in effector function and ADCC activity.

[0029] In some embodiments, the antibody, or antigen-binding fragment, is a silenced engineered IgG1 antibody or antigen-binding fragment with reduced or no binding to one or more Fc receptors. In 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 cell cytotoxicity. In some embodiments, the antibody or antigen-binding fragment may 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. Those skilled in the art will understand that the terms induce and / or enhance may mean inducing and / or enhancing cytokine release by immune cells, inducing and / or enhancing proliferation of said immune cells, or inducing and / or enhancing apoptotic activity. It will be readily apparent to those skilled in the art that the terms induce or induce, as used in the context of the present invention, mean causing the activation of immune cells or increasing the activation of immune cells beyond the level of activation shown in the absence of the antibody or antigen-binding fragment. As used herein, the term enhance means increasing the level of activation 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 the 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, one or more additional binding targets are immunomodulatory molecules. In one embodiment, the additional binding target is PD-L1. In one embodiment, the antibody or its antigen-binding fragment is divalent. In another embodiment, the antibody or its antigen-binding fragment is tetravalent. In another embodiment, the antibody or its antigen-binding fragment is trivalent.

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

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

[0034] In another aspect, a vector comprising one or more of the nucleic acids of the present invention is provided.

[0035] In another aspect of the present invention, a host cell is provided that contains one or more nucleic acid(s) encoding the heavy chain and / or light chain, or both, of the antibody of the present invention. In some embodiments, the host cell is grown under conditions in which the nucleic acid(s) are expressed. In other embodiments, a method for recovering the antibody of the present invention is provided.

[0036] An aspect of the present invention comprises a method for producing an antibody or an antigen-binding fragment thereof, the method comprising the step of culturing a host cell under conditions in which the antibody or the antigen-binding fragment thereof is expressed in the host cell, and optionally, the step of isolating said antibody or antigen-binding fragment.

[0037] An aspect of the present invention comprises a pharmaceutical composition comprising an antibody or antigen-binding fragment as described herein and a pharmaceutically acceptable carrier.

[0038] In some embodiments, the pharmaceutical composition or the pharmaceutical further comprises an effective amount of a second therapeutic agent.

[0039] In a further aspect of the present invention, a method for treating a disorder is provided, the method comprising the step of administering an antibody or antigen-binding fragment of the present invention that binds to SLAMF6 (SEQ No. 11) to a patient in need thereof. In one embodiment, the disorder is cancer.

[0040] In addition, a cancer treatment method is provided comprising the step of administering an effective amount of the antibody or antigen-binding fragment of the present invention to an individual in need thereof.

[0041] 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.

[0042] Preferably, the heavy chain variable region includes CDR-H1 including SEQ ID NO. 5, CDR-H2 including SEQ ID NO. 15, and CDR-3 including SEQ ID NO. 7.

[0043] Preferably, the light chain variable region comprises CDR-L1 including SEQ ID NO. 16, CDR-L2 including SEQ ID NO. 17, and CDR-L3 including SEQ ID NO. 10.

[0044] In some embodiments, a method for treating cancer is provided, wherein the antibody or antigen-binding fragment of the present invention is administered to a patient in need thereof, wherein the antibody or antigen-binding fragment of the present invention induces and / or enhances an immune response, e.g., a cytotoxic T-cell response and / or an NK cell response.

[0045] In additional embodiments, the antibody or its antigen-binding fragment comprises a bispecific or multispecific antibody or its antigen-binding fragment that binds to SLAMF6 (SEQ No. 11) and a tumor-specific antigen.

[0046] In some embodiments, 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, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancies.

[0047] According to a further aspect of the present invention, an antibody or antigen-binding fragment of the present invention for use in prevention or therapy is provided.

[0048] Preferably, the antibody or antigen-binding fragment is intended for use in the prevention or treatment of cancer.

[0049] According to a further aspect of the present invention, the use of an antibody or antigen-binding fragment according to the present invention in the manufacture of a drug for treating cancer is provided.

[0050] In some embodiments, the cancer according to the prior aspect 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, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancies. Brief explanation of the drawing

[0051] Figure 1a shows the amino acid sequence of the heavy chain variable region of the parent murine 1B3 antibody (Sequence No. 1). Figure 1b shows the amino acid sequence (Sequence No. 2) of the variable light chain region of the parent murine 1B3 antibody. Figure 2a shows the amino acid sequence (Sequence No. 13) of the heavy chain variable region of the humanized antibody Hu_1B3. Figure 2b shows the amino acid sequence (sequence number 14) of the light chain variable region of the humanized antibody Hu_1B3. Figure 3a shows the sequence alignment of the heavy chain variable region of the 1B3 parent murine antibody sequence and the humanized heavy chain variable region 1B3 sequence. Figure 3b shows the sequence alignment of the light chain variable region of the 1B3 parent murine antibody sequence and the humanized light chain variable region Hu_1B3 sequence. Figure 4 shows the specific dose-related binding of antibody 1B3 to 1B3-expressing Raji cells. Figure 5 shows the enhanced ability of antibody Hu_1B3 to mediate IFNγ production upon T cell activation compared to SLAMF6 antibodies of other clinical stages. Figure 6 shows that antibody 1B3 can induce tumor-infiltrating lymphocytes (TILs) isolated from primary NSCLC tumor samples to generate interferon gamma in in vitro analysis. Figure 7 shows that antibody 1B3 can induce TILs isolated from primary breast cancer samples to generate interferon gamma in an in vitro analysis. This analysis shows that antibody 1B3 has enhanced activity compared to pembrolizumab. Figure 8 shows that antibody 1B3 can induce TILs isolated from primary colorectal cancer samples to generate interferon gamma in an in vitro analysis. This analysis shows that antibody 1B3 has enhanced activity compared to pembrolizumab. Figure 9 shows that activation of SLAMF6 present in isolated T cells using antibody Hu_1B3 induces proliferation of CD8+ T cells. Figure 10 is an MLR analysis and shows that the antibody Hu_1B3 can induce DC-mediated T cell activation, as highlighted by the increase in IFNγ release. Figure 11 shows that the anti-SLAMF6 antibody Hu_1B3 can induce activated T cells to produce granzyme B in a dose-dependent manner. Figure 12 shows that the antibody Hu_1B3 upregulates perforin expression in CD8+ T cells in a dose-dependent manner. Figure 13 shows that the Hu_1B3 antibody was blocked from binding to the receptor on the surface of PBMC in the presence of the human SLAMF6 ECD-mIgG2a Fc fusion protein. Figure 14 shows that the humanized antibody Hu_1B3 is internalized into SLAMF6-expressing cells at a significantly lower rate than the anti-SLAMF6 antibody produced by Seattle Genetics. Figures 15 and 16 show that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against SKBR-3 cells when used with an anti-her2 anti-CD3 bispecific antibody. Figure 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 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 shows that after activating lymphocytes with various antibodies for 96 hours, the antibody HU_1B3 kills significantly more SKBR-3 cells than urelumab (anti-CD137) or isoforms. Figure 20 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against SKBR-3 cells in a dose-dependent manner when used with a fixed concentration of anti-Her2 / anti-CD3 bispecific antibody. Figures 21a and 21b show that the addition of Hu_1B3 in the absence of an activated bispecific antibody also enhances lymphocyte cytotoxicity against SKBR-3 cells. Specific details for implementing the invention

[0052] An aspect of the present invention comprises an antibody directed to SLAMF6, a nucleic acid encoding such an antibody, a host cell comprising such nucleic acid encoding an antibody of the present invention, a method for producing an anti-SLAMF6 antibody, and a method for treating human cancers including but not limited to diseases such as SLAMF6-mediated disorders, e.g., small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy.

[0053] Note that, as used in this application and the appended claims, the singular form includes plural references unless the context clearly indicates otherwise. It is further noted that the claims may be drafted to exclude any optional elements. As such, this statement is intended to serve as a basis for the use of such exclusive terms, such as “only,” “only,” etc., in connection with the citation of claim elements or the use of “negative” limitations.

[0054] As will be apparent to those skilled in the art upon reading this disclosure, each individual aspect described and illustrated herein has distinct components and characteristics that can be easily separated from or combined with the characteristics of any other various aspects without departing from the scope or spirit of the invention. Any cited method may be performed in the order of the cited events or in any other logically possible order.

[0055] definition

[0056] To interpret this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa.

[0057] As used herein, the term "SLAMF6" refers to any natural SLAMF6 protein from any vertebrate source, including mammals such as primates (e.g., humans, primates, and rodents (e.g., mice and rats)), unless otherwise indicated. SLAMF6 proteins may also be referred to as SLAMF6-like proteins. The amino acid sequence of human SLAMF6 is provided herein at SEQ ID NO. 11.

[0058] The term "SLAMF6" includes not only "full-length" untreated SLAMF6 but also any form of SLAMF6 produced by processing in cells. The term also includes naturally occurring variants of SLAMF6, e.g., splice variants, allelic variants, and isoforms. Specifically, the term includes naturally occurring cleaved or secreted forms of the SLAMF6 polypeptide (e.g., extracellular domain sequences). The SLAMF6 polypeptides described herein may be isolated from various sources, e.g., human tissue types or other sources, or may be produced by recombinant or synthetic methods. "Natural sequence SLAMF6 polypeptide" includes polypeptides having the same amino acid sequence as the corresponding naturally derived SLAMF6 polypeptide. Such natural sequence SLAMF6 polypeptides may be isolated from nature or produced by recombinant or synthetic means. The term "SLAMF6 epitope" as used herein refers to an epitope that comprises one or more of the CDR sequences described herein and / or is bound by an antibody exemplified by the binding profile of an anti-SLAMF6 antibody as exemplified in the Examples.

[0059] The term “antibody” is used in its broadest sense and specifically encompasses, for example, a single anti-SLAMF6 monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), anti-SLAMF6 antibody compositions having polyepitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies insofar 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 fragments insofar as they exhibit the desired biological or immunological activity, diabodies, and single-domain antibodies (sdAbs). 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, the antibody contains at least six sets of CDRs defined herein; it will be understood by those skilled in the art that these include, but are not limited to, traditional antibodies (including both monoclonal and polyclonal antibodies), humanized, human and / or chimeric antibodies, antibody fragments, engineered antibodies (e.g., using amino acid modifications as summarized below), multispecific antibodies (including bispecific antibodies), and other analogs known in the art and discussed herein.

[0060] It will be understood that in other embodiments, the term antibody as used herein refers to a structure that does not contain six CDRs; and includes, but is not limited to, Nanobody®, Unibody®, and scFv fragments.

[0061] The terms “anti-SLAMF6 antibody,” “SLAMF6 antibody,” or “antibody binding to SLAMF6” refer to an antibody capable of binding to SLAMF6 with sufficient affinity to be useful as a diagnostic and / or therapeutic agent in targeting SLAMF6. In certain embodiments, the anti-SLAMF6 antibody binds to an epitope of SLAMF6 conserved among SLAMF6 from different species.

[0062] "Isolated antibodies" are antibodies identified, separated, and / or recovered from their environmental components. The environmental contaminants are substances that may interfere with the therapeutic use of the antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0063] With respect to the binding of antibodies to target molecules, the terms “specific binding,” “specifically binding to,” or “specific to a specific polypeptide or an epitope on a specific polypeptide target” refer to a binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule by comparing it to the binding of a control molecule, which is a molecule of similar structure that generally does not have binding activity.

[0064] The term "antagonist" is used in its broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of natural SLAMF6 polypeptide. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural SLAMF6 polypeptide, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an antagonist of SLAMF6 polypeptide may include the steps of contacting the SLAMF6 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities generally associated with the SLAMF6 polypeptide.

[0065] The term "agonist" is used in its broadest sense and includes any molecule that enhances the biological activity of the natural SLAMF6 polypeptide. Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence variants of the SLAMF6 ligand polypeptide, peptides, antisense oligonucleotides, small organic molecules, etc. A method for identifying an agonist of the SLAMF6 polypeptide may include the steps of contacting the SLAMF6 polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities generally associated with the SLAMF6 polypeptide.

[0066] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0067] The terms “prediction” and “prognostic prediction” as used herein are also interchangeable in the sense that the prediction or prognostic method allows the person performing the method to select patients who are considered more likely to respond to treatment with an anticancer agent including an anti-SLAMF6 antibody (usually prior to treatment, but not necessarily).

[0068] SLAMF6 protein

[0069] According to UNIPROT, SLAMF6 is a single-pass type I membrane protein of the immunoglobulin superfamily and CD2 subfamily. This protein consists of an extracellular domain of amino acids 22–226 of SEQ ID NO. 11, a transmembrane region of amino acids 227–247, and a cytoplasmic region of amino acids 248–331.

[0070] In some embodiments, the antibody of the present invention binds to human SLAMF6. As used herein, “human SLAMF6” or “human SLAMF6 protein” refers to the protein of SEQ ID NO. 11 as defined herein.

[0071] The antibody according to an embodiment of the present invention may cross-react with SLAMF6 proteins of non-human species in certain cases. For example, to facilitate preclinical and toxicity testing, the antibody of the present invention may cross-react with murine or primate SLAMF6 proteins. Alternatively, in certain embodiments, the antibody may be specific to human SLAMF6 proteins and may not exhibit species- or other types of non-human cross-reactivity.

[0072] antibodies

[0073] Aspects of the present invention include anti-SLAMF6 antibodies as described herein, generally therapeutic and / or diagnostic antibodies. The antibodies used in the methods of the present invention may take any number of formats as described herein, including traditional antibodies as well as antibody derivatives, antigen-binding fragments, and mimics as further described herein. In some embodiments, the antibody has one or more CDRs selected from a set of six CDRs as defined herein (including a few amino acid changes as described herein). As discussed above, various structures referred to as "antibodies" as used herein.

[0074] In some embodiments, IgG isoforms are used in the present invention. In one embodiment, an Fc-silenced IgG1 isoform antibody is used. In another embodiment, an IgG4 isoform antibody is used.

[0075] The amino-terminal portion of each chain of the antibody contains a variable region of about 100 to 110 amino acids or more, which is primarily responsible for antigen recognition. In the variable region, three loops converge for each of the V domains of the heavy and light chains to form the antigen binding site. Each loop is referred to as the complementarity-determining region (hereinafter referred to as "CDR"), where variation in the amino acid sequence is most significant. "Variable" refers to the fact that specific segments of the variable region differ widely in sequence between antibodies. Variability within the variable region is not uniformly distributed. Instead, the V region consists of relatively invariable stretches called framework regions (FRs) of 15 to 30 amino acids, separated by shorter regions of extreme variability called "hypervariable regions," which are each 9 to 15 amino acids or longer.

[0076] Each VH and VL consists of three supervariable regions ("complementary determination region", "CDR") and four FRs, arranged in the following order from amino-terminal to carboxy-terminal: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0077] The hypervariable region generally includes amino acid residues around amino acid residues 24–34 (CDR-L1; "L" indicates light chain), 50–56 (CDR-L2), and 89–97 (CDR-L3) in the light chain variable region, and amino acid residues around 31–35B (CDR-H1; "H" indicates 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 such residues form a hypervariable loop (e.g., residues 26–32 (CDR-L1), 50–52 (CDR-L2), and 91–96 (CDR-L3) in the light chain variable region). and forms 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 present invention are described below.

[0078] Throughout this specification, the Kabat numbering system is generally used to refer to residues in the variable domain (approximately, residues 1-107 in the light chain variable domain and residues 1-113 in the heavy chain variable domain) (e.g., Kabat et al., (1991)).

[0079] CDRs contribute to the antigen-binding of antibodies, or more specifically, the formation of epitope binding sites. A single antigen may have one or more epitopes.

[0080] In the IgG subclass of immunoglobulins, there are several immunoglobulin domains in the heavy chain. As described herein, "immunoglobulin (Ig) domain" refers to a region of immunoglobulin having a distinct tertiary structure. Of interest in the present invention are heavy chain domains including constant heavy chain (CH) domains and hinge domains. In the context of IgG antibodies, IgG isoforms each have three CH regions.

[0081] Another type of Ig domain of the heavy chain is the hinge region. As used herein, "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" refers to a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody.

[0082] Of particular interest in the present invention is the Fc region. As used herein, "Fc," "Fc region," or "Fc domain" refers to a polypeptide comprising a constant region of an antibody excluding a first constant region immunoglobulin domain, and in some cases, a portion of a 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-terminus for these domains. In the case of IgA and IgM, Fc may include a J chain. In the case of IgG, the Fc domain comprises the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is typically defined to include residues C226 or P230 at its carboxyl-terminus, where numbering follows EU indices such as those in kavat. In some embodiments, amino acid modifications are made to the Fc region to alter binding to, for example, one or more FcγR receptors or FcRn receptors.

[0083] In some embodiments, the antibody is full-length. As defined herein, "full-length antibody" means a structure constituting the natural biological form of an antibody that includes variable and constant regions and optionally includes one or more modifications as defined herein.

[0084] Alternatively, antibodies may be various structures, each including but not limited to antigen-binding fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and each antigen-binding fragment. Structures that depend on the use of a CDR set are included within the definition of “antibody.”

[0085] In one embodiment, the antibody is an antigen-binding fragment. The specific antigen-binding antibody fragment is (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains, (ii) an Fd fragment consisting of VH and CH1 domains, and (iii) an Fv fragment consisting of VL and VH domains of a monoclonal antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, incorporated whole), (v) an isolated CDR region, (vi) a divalent F(ab')2 fragment containing two linked Fab fragments, (vii) a single-stranded Fv molecule (scFv) in which a VH domain and a VL domain are connected by a peptide linker that associates the two domains to form an antigen-binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated whole), (viii) a bispecific single-stranded Fv (WO 03 / 11161, incorporated herein by reference), and (ix) a multivalent or multispecific constructed by gene fusion It includes, but is not limited to, fragments of “diabadi” or “triabody” (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all of which are incorporated by reference).

[0086] Chimeric antibodies and humanized antibodies

[0087] In some embodiments, the antibody may be a mixture from different species, e.g., a chimeric antibody and / or a humanizing antibody. That is, in the present invention, the CDR set may be used as a framework and invariant region other than those specifically described herein by sequence.

[0088] Generally, both "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, "chimeric antibodies" traditionally include variable region(s) from mice (or in some cases rats) and constant region(s) from humans. "Humanized antibodies" generally refer to non-human antibodies in which the variable-domain framework region is exchanged for a sequence found in a human antibody. Generally, in humanized antibodies, the entire antibody, excluding the CDR, is encoded by a polynucleotide of human origin or is identical to such antibodies except within its CDR. A CDR encoded in part or in whole by a nucleic acid originating from a non-human organism is grafted into the beta-sheet framework of the human antibody variable region to produce an antibody, and its specificity is determined by the grafted CDR. The production of such antibodies is described, for example, in WO 92 / 11018, Jones, 1986, Nature 321:522-525, and Verhoeyen et al., 1988, Science 239:1534-1536, all incorporated by reference in their entirety. In one embodiment, the antibody of the present invention may be a multispecific antibody, particularly a bispecific antibody, and is also sometimes referred to as a “diabadi.” These are antibodies that bind to two (or more) different antigens, or to different epitopes on the same antigen. Diabadi may be prepared in various ways known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, incorporated by reference in its entirety), for example, chemically or from a hybridoma.

[0089] In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein containing a scFv connected to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061, full text incorporated by reference). In some cases, the scFv may be connected to an Fc region and may contain some or all of a hinge region. It should be noted that the minibody is included within the definition of an "antibody" despite not having a complete set of CDRs.

[0090] The antibodies of the present invention are generally isolated or recombined.

[0091] In some embodiments, the antibody of the present invention is a recombinant protein, an isolated protein, or a substantially pure protein. The “isolated” protein is not accompanied by at least some of the material that normally associates in nature, 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 the isolated protein may constitute 5 to 99.9% by weight of the total protein content depending on the circumstances. For example, the protein may be produced at significantly higher concentrations through the use of an inducible promoter or a high-expression promoter, thereby allowing the protein to be produced at increased concentration levels. In the case of a recombinant protein, the definition involves the production of antibodies in various organisms and / or host cells known in the art that do not occur naturally. Typically, the isolated polypeptide will be produced by at least one purification step. “Isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, isolated antibodies that specifically bind to SLAMF6 practically do not have antibodies that specifically bind to antigens other than SLAMF6.

[0092] Isolated monoclonal antibodies having different specificities can be combined in widely defined compositions. Thus, for example, antibodies of the present invention may be included in or excluded from formulations optionally and individually as further discussed below.

[0093] Specific binding to a specific antigen or epitope is, for example, at least about 10 to the antigen or epitope -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, alternatively at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or K in excess of it D It can be represented by an antibody having, and the above K D K refers to the dissociation rate of a specific antibody-antigen interaction. Typically, antibodies that bind specifically to an antigen exhibit a K value 20, 50, 100, 500, 1000, 5,000, 10,000-fold or higher lower for the antigen or epitope compared to a control molecule. D will have.

[0094] In addition, specific binding to a specific antigen or epitope is, for example, K that is at least 20, 50, 100, 500, 1000, 5,000, 10,000-fold or greater than that for the epitope compared to the control for the antigen or epitope A or K a It can be represented by an antibody having, and the above K A or K a refers to the assembly rate of a specific antibody-antigen interaction.

[0095] Standard assays for evaluating the binding ability of antibodies to SLAMF6 can be performed at the protein or cellular level and are known in the art, including, for example, ELISA, Western blot, RIA, Octet®, BIAcore® assays, and flow cytometry. Suitable assays are described in detail in the Examples. The binding kinetics of the antibody (e.g., binding affinity) can also be evaluated by standard assays known in the art, such as Biacore® or Octet® system assays.

[0096] SLAMF6 antibody

[0097] The present invention provides an SLAMF6 antibody that binds to an SLAMF6 polypeptide or a portion thereof. An example of an SLAMF6 amino acid sequence is provided in SEQ ID NO. 11. The target SLAMF6 antibody may enhance the immune response in a tumor by inducing or enhancing immune cell activation, e.g., T cell activation and / or NK cell activation. These antibodies are referred to herein as "anti-SLAMF6" antibodies or, for ease of description, "SLAMF6 antibodies."

[0098] In some embodiments, the target SLAMF6 antibody may induce and / or enhance cytokine release or proliferation upon contact with T cells, particularly CD4+ or CD8+ T cells expressing SLAMF6 on their surface. In this context, cytokine release or T cell proliferation may be measured in various ways. In one embodiment, the SLAMF6 antibody of the present invention is contacted with activated T cells using a standard assay such as ELISA. In further embodiments, the target SLAMF6 antibody may induce and / or enhance NK cell activation and apoptosis.

[0099] In one embodiment, the antibody is an antibody comprising the following CDR; furthermore, as discussed below, these CDR sequences may also contain a limited number of amino acid variants as previously described:

[0100] CDR Sequence number 1B3_VH_CDR1 Sequence No. 5 1B3_VH_CDR2 Sequence number 15 1B3_VH_CDR3 Sequence number 7 1B3_VL_CDR1 Sequence number 16 1B3_VL_CDR2 Sequence number 17 1B3_VL_CDR3 Sequence number 10

[0101] In some embodiments, the antibody comprises at least one amino acid sequence 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 about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identical to one or more amino acid sequences of the CDR sequences provided in SEQ ID NOs. 5, 15, 7, 16, 17 and 10. The present invention also discloses variable heavy chains and light chains comprising the CDR set of the present invention, as well as full-length heavy chains and light chains (e.g., also including invariant regions). As understood by those skilled in the art, the CDR set of the present invention may be contained within a murine, humanized, or human invariant region (including a framework region). An aspect of the present invention comprises a heavy chain variable region and a light chain variable region that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the heavy chain variable region sequence (Sequence No. 13) and light chain variable region sequence (Sequence No. 14) disclosed herein.

[0102] In some embodiments, the present invention provides an antibody that cross-competes with the SLAMF6 monoclonal antibody of the present invention described herein, or binds to or cross-competes with the same epitope on human SLAMF6 (i.e., an antibody having the ability to cross-compete with the monoclonal antibody of the present invention described herein for binding to the SLAMF6 protein). It will be understood that when an antibody is considered to cross-compete, it does not necessarily block the binding of the reference antibody completely. In some embodiments, the binding of the reference antibody is reduced by about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 97, 98, or 99% or more.

[0103] Antibody modification

[0104] The present invention further provides variant antibodies sometimes referred to as “antibody derivatives” or “antibody analogs.” That is, there are a number of variations that may be made of the antibodies of the present invention, including but not limited to amino acid modifications in the CDR (affinity maturation), amino acid modifications in the Fc region, glycosylated variants, and other types of covalent modifications (e.g., in the case of attachment of a drug conjugate).

[0105] "Variant" means a polypeptide sequence that differs from that of the parent polypeptide by at least one amino acid modification. In some embodiments, the parent polypeptide is a full-length variable heavy or light chain listed in SEQ ID NOs 1 or 2, 13 or 14, or is one or more of the CDR sequences disclosed in any one of SEQ ID NOs 5 to 10, 15, 16 or 17. In some embodiments, the amino acid modification may include substitutions, insertions and / or deletions, the former being preferred in many cases. In some embodiments, the substitution may be a conservative substitution.

[0106] Generally, variants may include any number of modifications as long as the antibody's function, as described herein, still exists. For example, the antibody must still bind specifically to human SLAMF6. Similarly, if amino acid variants are generated within the Fc region, for example, the variant antibody must retain the receptor binding function necessary for the specific application or instruction of the antibody.

[0107] A “variant” of the antibody of the present invention may be formed to have an amino acid variation in one or more of the listed CDR sequences of the antibody as described herein, in one or more of the framework regions, or in one or more of the invariant regions (e.g., the Fc region).

[0108] In some embodiments, since the goal is often to change function with the minimum number of modifications, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications relative to the parent sequence are generally utilized. In some embodiments, there are 1 to 5 modifications (1, 2, 3, 4, or 5) (e.g., individual amino acid substitutions, insertions, and / or deletions), and 1-2, 1-3, and 1-4 are also used in many embodiments. For example, in some embodiments, one or more of the CDR sequences of the antibody of the present invention may individually include one or more, e.g., 1, 2, 3, 4, or 5 amino acid modifications, preferably 1-4, 1-3, 1, or 2 modifications. Generally, 4, 5, 6, 7, 8, 9, or 10 or fewer modifications are made within the CDR set.

[0109] It should be noted that the number of amino acid modifications may be within the functional domain: for example, it may be desirable to have 1 to 5 modifications in the Fc region of the wild-type or engineered protein, as well as 1 to 5 modifications in the Fv region, for example. The variant polypeptide sequence will preferably have about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of identity with respect to the parent sequence (e.g., variable region sequence, constant region sequence, and / or, for example, heavy chain and light chain sequences of antibody 1B3 and / or CDR).

[0110] As used herein, "amino acid substitution" or "substitution" means the replacement of an amino acid with another amino acid at a specific position in the parent polypeptide sequence. As used herein, "amino acid insertion" or "insertion" means the addition of an amino acid at a specific position in the parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" means the removal of an amino acid at a specific position in the parent polypeptide sequence.

[0111] As used herein, "maternal polypeptide," "maternal protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. Generally, as used herein, the maternal polypeptide is the 1B3 polypeptide, e.g., 1B3 V H Alternatively, it may refer to the VL chain or CDR sequence. Accordingly, the term "parent antibody" as used herein means an antibody modified to produce a variant antibody.

[0112] In this document, "wild type," "WT," or "natural" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. WT proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., have amino acid sequences or nucleotide sequences that have not been intentionally modified.

[0113] In this document, the term "variant Fc region" means an Fc sequence that differs from that of the wild-type Fc sequence by at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, a composition containing an Fc variant polypeptide, or an amino acid sequence.

[0114] In some embodiments, the anti-SLAMF6 antibody of the present invention comprises a variant Fc domain. As is known in the art, the Fc region of the antibody interacts with a number of Fc receptors and ligands to provide an array of important functional capabilities referred to as effector functions. Suitable modifications may be made to specific amino acid substitutions at one or more locations that reduce or silence binding, particularly to Fc receptors.

[0115] In addition to the modifications summarized above, other modifications may be made. For example, the molecule may be stabilized by the incorporation of a disulfide crosslink connecting the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, incorporated by reference throughout).

[0116] Additionally, modifications in cysteine ​​are particularly useful for antibody-drug conjugate (ADC) applications described further below. In some embodiments, the constant region of the antibody may be engineered to contain one or more cystes that are particularly "thiol-reactive" to allow for more specific controlled placement of the drug moiety. For example, refer to U.S. Patent No. 7,521,541, the full text of which is incorporated herein by reference. Furthermore, there are various covalent modifications of the antibody that may be made as outlined below.

[0117] Covalent modification of antibodies is included within the scope of the present invention and is generally, but not always, performed after translation. For example, various types of covalent modification of antibodies are introduced into the molecule by reacting specific amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chain or N- or C-terminal residues.

[0118] In addition, as understood by those skilled in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can all be added to antibodies (as well as other compositions of the present invention).

[0119] bispecific molecule

[0120] In another aspect, the present invention comprises bispecific and multispecific molecules comprising the anti-SLAMF6 antibody of the present invention or a fragment thereof. The antibody of the present invention, or its antigen-binding portion, may be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or ligand against a receptor), to produce a bispecific molecule that binds to two different binding sites or target molecules. To produce the bispecific or multispecific molecule of the present invention, the antibody of the present invention may be functionally linked to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimic (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to produce the bispecific or multispecific molecule.

[0121] Accordingly, the present invention comprises a bispecific molecule comprising at least one first binding specificity for a first target epitope (i.e., SLAMF6) and a second binding specificity for a second target epitope. The second target epitope may be present on the same target protein that is bound by the first binding specificity; or the second target epitope may be present on a different target protein that is bound by the first binding specificity. The second target epitope may be present on the same cell as the first target epitope (i.e., SLAMF6); or the second target epitope may be present on a target that is not shown by the cell showing the first target epitope. As used herein, the term “binding specificity” refers to a moiety comprising at least one antibody variable domain.

[0122] In another embodiment of the present invention, the second target epitope is present on a tumor cell. Accordingly, an aspect of the present invention comprises a bispecific molecule capable of binding to both an SLAMF6-expressing effector cell (e.g., an SLAMF6-expressing cytotoxic T cell) and a tumor cell expressing the second target epitope.

[0123] In one embodiment, the bispecific antibody of the present invention may have a total of two or three antibody variable domains, wherein the first portion of the bispecific antibody may mobilize the activity of human immune effector cells by specifically binding to an effector antigen located on human immune effector cells, wherein the effector antigen is SLAMF6, and the first portion is composed of one antibody variable domain, and the second portion of the bispecific antibody may specifically bind to a target antigen other than the effector antigen, wherein the target antigen is located on a target cell other than the human immune effector cells, and the second portion comprises one or two antibody variable domains.

[0124] In an embodiment of the present invention in which the binding protein is multispecific, the molecule may further comprise a third binding specificity in addition to antitumor, binding specificity, and anti-SLAMF6 binding specificity. In one embodiment, the third binding specificity is a molecule that binds to an anti-enhancing factor (EF) portion, for example, a surface protein associated with cytotoxic activity, and thereby increases the immune response against target cells. The "anti-enhancing factor portion" may be an antibody, a functional antibody fragment, or a ligand that binds to a given molecule, for example, an antigen or receptor, thereby causing an enhancement of the binding determinant's effect on target cell antigens. The "anti-enhancing factor portion" may bind to target cell antigens. Alternatively, the anti-enhancing factor portion may bind to an entity different from the entity to which the first and second binding specificities bind. For example, the anti-enhancing factor portion can bind to cytotoxic T-cells (e.g., through CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells that result in an increased immune response to target cells).

[0125] In one embodiment, the bispecific protein of the present invention comprises at least one antibody with binding specificity, or an antigen-binding fragment thereof comprising, for example, Fab, Fab', F(ab')2, Fv, Fd, dAb, or a single-chain Fv. The antibody may also be a light-chain or heavy-chain dimer as described in U.S. Patent No. 4,946,778, the contents of which are expressly incorporated by reference, or any minimum fragment thereof such as Fv or a single-chain construct.

[0126] In some embodiments, the antibodies that can be used for the bispecific molecules of the present invention are murine, human, chimeric, or humanized monoclonal antibodies.

[0127] The binding of a bispecific molecule to its specific target can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific to the complex of interest.

[0128] 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 targets a first antigen and comprises two Fab domains, each consisting of a heavy chain and a light chain Fab region. These are arranged in the same form as in the Fab of natural IgG. The antibody further comprises two chimeric Fab domains targeting a second antigen, which are composed of two chimeric polypeptide domains, each comprising a chimeric "heavy" chain containing a variable heavy chain domain (its C-terminus is connected 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 containing a variable light chain domain (its C-terminus is connected to the N-terminus of the constant region of the beta or alpha chain of the TCR). The chimeric "heavy" chain and "light" chain are arranged within the chimeric Fab domain, which is connected to the natural Fab domain by attaching the C-terminus of the constant region of the alpha- or beta-chain of the TCR of the chimeric "heavy" chain to the N-terminus of the variable region of the heavy chain of the natural Fab domain. Thus, the overall symmetric structure generates a bispecific antibody that targets each of two different antigens in a bivalent manner, so that the natural Fab domain targeting the first antigen and the chimeric Fab domain targeting the second antigen are present in both arms of this quadrivalent antibody.

[0129] In another embodiment, the chimeric Fab domain is located close to the Fc domain so that the C-terminus of the alpha- or beta-chain of the invariant region of the TCR containing the chimeric "heavy" chain is attached to the N-terminus of the natural hinge, and the natural Fab domain is located far from the Fc domain so that the C-terminus of the CH1 domain of the heavy chain containing the natural Fab domain is attached to the N-terminus of the variable heavy chain containing the chimeric Fab domain.

[0130] In another specific embodiment, an asymmetric trivalent format is used, which consists of two different antibody arms, one arm having a single Fab domain (natural or chimeric) and the second arm of the bispecific antibody having both Fab domains (natural and chimeric) as described above. Heterodimerization of the two different arms is possible through antibody manipulation in the Fc domain as described in the art (e.g., knobs-into-wholes, electrostatic steering, etc.).

[0131] In another embodiment, an asymmetric divalent format may be used, which consists of two different antibody arms, one arm having a single Fab domain (natural or chimeric) and the other arm also having a single Fab binding domain (chimeric or natural). Heterodimerization of the two different arms is possible through antibody manipulation in the Fc domain, which is well described in the art (e.g., knobs-into-wholes, electrostatic steering, etc.).

[0132] Glycosylation

[0133] Another type of covalent modification is a change in glycosylation. For example, aglycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced.

[0134] Glycosylation can be modified, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be achieved by resulting in the removal of one or more variable region framework glycosylation sites, thereby removing glycosylation from those sites. Such aglycosylation can increase the affinity of the antibody for an antigen. This approach is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al., and can be achieved by removing asparagine at position 297.

[0135] Other types of covalent modifications of antibodies include linking antibodies to various non-protein polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in a manner set forth, for example, in the 2005-2006 PEG Catalog from Nektar Therapeutics (available on the Nektar website) and U.S. Patents No. 4,640,835; No. 4,496,689; No. 4,301,144; No. 4,670,417; No. 4,791,192, or No. 4,179,337 (all incorporated by reference in their entirety). Additionally, as is known in the art, amino acid substitutions may be made at various positions within the antibody to facilitate the addition of polymers such as PEG. For example, refer to U.S. Publication No. 2005 / 0114037A1, in which the entirety is incorporated by reference.

[0136] In further embodiments, for example, when using the antibody of the present invention for diagnostic or detection purposes, the antibody may include a label. As used herein, "labeled" means having at least one moiety, element, isotope, or chemical compound attached to enable the detection of the compound, as described in Richard P. Haugland’s *The Molecular Probes Handbook, 6th Edition*, which is expressly incorporated herein by reference.

[0137] Method for producing antibodies of the present invention

[0138] The present invention further provides a method for producing the disclosed anti-SLAMF6 antibody. These methods comprise culturing host cells containing isolated nucleic acid(s) encoding the antibody of the present invention. As understood by those skilled in the art, this may be carried out in various ways depending on the properties of the antibody. In some embodiments, where the antibody of the present invention is a full-length conventional antibody, for example, the host cells may contain nucleic acid encoding a heavy chain variable region and the light chain variable region may be cultured and isolated under conditions that allow the antibody to be produced.

[0139] The variable heavy and light chains of the antibodies of the present invention are disclosed herein (both protein and nucleic acid sequences); as is understood in the art, they can be easily enhanced to produce full-length heavy and light chains. That is, V as summarized herein H If DNA fragments encoding the and VL segments are provided, these DNA fragments can be further manipulated, for example, by standard recombinant DNA technology to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene, or a scFv gene. In these manipulations, V L - or V H- A coding DNA fragment is operably linked to another DNA fragment coding for 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 linked such that the amino acid sequences coded by the two DNA fragments remain within the frame.

[0140] V H Isolated DNA encoding the region is V as a molecule H -Coding DNA heavy chain invariant region (C H 1, C H 2 and C H It can be converted into a full-length heavy chain gene by operably linking it to another DNA molecule encoding 3). Sequences of rat heavy chain constant regions genes are known in the art (e.g., see 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 containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In one preferred embodiment, the heavy chain constant region is an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, V H -Coding DNA is unique in its heavy chain C H 1. It can be operablely linked to other DNA molecules encoding invariant regions.

[0141] The isolated DNA encoding the VL region is V L - Coding DNA, the light chain invariant region C LIt can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking it to another DNA molecule encoding it. Sequences of rat light chain constant regions are known in the art (e.g., see 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 containing these regions can be obtained by standard PCR amplification. In one preferred embodiment, the light chain constant region is a kappa or lambda constant region.

[0142] To generate a polynucleotide sequence encoding the scFv antibody fragment, V H - and VL-coding DNA fragments are operablely linked to other fragments encoding a flexible linker, for example, an amino acid sequence (Gly4-Ser)3, V H and enables the VL sequences to be expressed as a continuous single-strand protein, and VL and V H The regions are connected by flexible linkers (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0143] An aspect of the present invention comprises nucleic acids encoding the antibodies of the present invention. These polynucleotides encode both variable and constant regions of each heavy chain and light chain, but other combinations are also considered by the present invention according to the compositions described herein. An aspect of the present invention comprises the disclosed polynucleotides and oligonucleotide fragments derived from nucleic acid sequences complementary to these polynucleotides.

[0144] Polynucleotides according to embodiments of the present invention may be in the form of RNA, DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA, or may comprise these. In some embodiments, the DNA molecule may be double-stranded or single-stranded, and if single-stranded, may be a coding (sense) strand or a non-coding (antisense) strand. The coding sequence encoding the polypeptide may be identical to or different from the coding sequence provided herein, and the sequence codes for the same polypeptide as the DNA provided herein as a result of redundancy or degeneracy of the genetic code.

[0145] In some embodiments, the nucleic acid(s) encoding the antibody of the present invention are incorporated into an expression vector, which may be designed to be extrachromosomal or incorporated within the genome of the host cell to be introduced. The expression vector may contain any number of appropriate regulatory sequences (including, but not limited to, transcription and translation control sequences, promoters, ribosome binding sites, enhancers, replication origins, etc.) or other components (such as selected genes), all of which are operably linked as known in the art. In some cases, two nucleic acids are used and each may be placed in different expression vectors (e.g., a heavy chain in a first expression vector and a light chain in a second expression vector), or alternatively, they may be placed in the same expression vector. It will be understood by those skilled in the art that the design of the expression vector(s), including the selection of regulatory sequences, may depend on such factors as the selection of the host cell and the desired expression level of the protein.

[0146] Generally, nucleic acids and / or expressions are introduced into suitable host cells and recombinant host cells can be generated using any method suitable for the selected host cells (e.g., transformation, transfection, electroporation, infection) so that nucleic acid molecule(s) are operably linked to one or more expression regulatory elements (e.g., in a vector, in a construct generated by a cellular process within the host cell genome). The generated recombinant host cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a suitable culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby generating polypeptide(s) encoded by this. In some embodiments, the heavy chain and light chain are generated in the same host cell. In some embodiments, the heavy chain is generated in one host cell and the light chain is generated in another host cell.

[0147] 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, Virginia), including but not limited to Chinese hamster ovary (CHO) cells, HEK 293 cells, FS293, Expi293, NSO cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g., Hep G2), and numerous other cell lines. Non-mammalian cells, including but not limited to bacteria, yeast, insects, and plants, may also be used to express recombinant antibodies. In some embodiments, antibodies may be produced in genetically modified animals such as cattle or chickens.

[0148] General methods for antibody molecular biology, expression, purification, and screening are widely known, for example, see U.S. Patents No. 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.

[0149] Pharmaceutical composition

[0150] An aspect of the present invention comprises a composition, e.g., a pharmaceutical composition, comprising one or more antibodies of the present invention (or a combination thereof) or antigen-binding portion(s) thereof, formulated together with a pharmaceutically acceptable carrier. Such a composition may comprise one or a combination of the antibodies (e.g., two or more different) or bispecific molecules of the present invention. For example, a pharmaceutical composition of the present invention may comprise a combination of antibodies having different epitopes on a target antigen or having complementary activity.

[0151] The pharmaceutical composition of the present invention may also be administered as a combination therapy, that is, in combination with other agents. For example, a combination therapy may include the antibody of the present invention combined with at least one other antitumor agent, or an anti-inflammatory agent or an immunosuppressant. Examples of therapeutic agents that may be used in combination therapy are described in more detail below in the section on the uses of the antibody of the present invention.

[0152] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coating agents, antimicrobial and antifungal agents, isotonic agents, absorption retardants, etc. Preferably, the carriers are 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., the antibody or antibody fragment, may be coated with a material that protects the compound from the action of acids and other natural conditions that may inactivate the compound.

[0153] The pharmaceutical compound of the present invention may comprise one or more pharmaceutically acceptable salts. "Pharmaceutically acceptable salt" refers to a salt that retains the desirable biological activity of the parent compound and does not impart any undesirable toxic effects (see, e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). The pharmaceutical composition of the present invention may also comprise a pharmaceutically acceptable antioxidant. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable fluidity may be maintained, for example, by the use of a coating material such as lecithin, the maintenance of the required particle size in the case of a dispersion, and the use of a surfactant.

[0154] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization procedure and the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Additionally, extended absorption in the injectable pharmaceutical form may be achieved by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0155] 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 such media and formulations for pharmaceutically active substances is known in the art. Unless any conventional media or formulation is incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is considered. A supplementary active compound may also be incorporated into the composition.

[0156] Dose therapy is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, multiple divided doses may be administered over time, or the dose may be reduced or increased in proportion to the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosing unit forms for ease of administration and uniformity of dosage. Dosing unit forms as used herein refer to physical discrete units suitable as a single dose for the individual to be treated; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in conjunction with the required pharmaceutical carrier. The specifications for the dosing unit forms of the present invention depend directly on (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the inherent limitations of the art in preparing such active compounds for the treatment of an individual's sensitivity.

[0157] In the case of antibody administration, the dosage may be in the range of 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 generally 0.01 to 5 mg / kg of host body weight. For example, the dosage may be within the range of 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 0.1-5 mg / kg or 1-10 mg / kg. Exemplary therapeutic regimens involve administration daily, every other day, twice weekly, once weekly, once every two weeks, once every three weeks, once every four weeks, once monthly, once every three months, or once every three to six months. A preferred regimen for the anti-SLAMF6 antibody of the present invention comprises 1 mg / kg body weight, 3 mg / kg, 5 mg / kg, or 10 mg / kg body weight via intravenous administration, wherein the antibody is provided using one of the following administration schedules: (i) administration six times weekly, followed by monthly; (ii) weekly; or (iii) 3 mg / kg body weight followed by 1 mg / kg body weight once weekly.

[0158] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, and in this case, the dosage of each antibody administered falls within the indicated range. In some embodiments, the antibodies are administered multiple times. The interval between single doses may be, for example, weekly, monthly, every three months, or annually. Additionally, the interval may be irregular as indicated by measuring the blood levels of antibodies against the target antigen in the patient. In some embodiments, the dosage is adjusted to achieve plasma antibody concentrations of about 1-1000 μg / ml and, in some methods, about 25-300 μg / ml.

[0159] In some embodiments, antibodies may be administered in sustained-release formulations, in which case less frequent administration is required. The dosage and frequency depend on the half-life of the antibody in the patient. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. The dosage and frequency may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for life. In therapeutic applications, relatively high doses are sometimes required at relatively short intervals until disease progression is reduced or terminated, preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, the patient may receive prophylactic therapy.

[0160] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary to obtain an amount of the active ingredient, composition, and mode of administration effective in achieving a desirable therapeutic response for a specific patient without toxicity to the patient. The selected dosage level will depend on the activity of the specific composition of the present invention used, or its ester, salt, or amide, the route of administration, the time of administration, the elimination rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or substances used in combination with the specific composition used, various pharmacokinetic factors including the age, sex, weight, condition, general health, and previous medical history of the patient to be treated, and similar factors widely known in the medical field.

[0161] The “therapeutic 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 symptom-free periods, or the prevention of damage or disability caused by disease distress. For example, the “therapeutic effective dose” inhibits cell growth or tumor growth by preferably at least about 10%, at least about 20%, at least about 30%, more preferably at least about 40%, at least about 50%, even more preferably at least about 60%, at least about 70%, and even more preferably at least about 80%, at least about 90%, or at least about 95% compared to an untreated individual. The ability of the compound to inhibit tumor growth can be evaluated in animal model systems that predict efficacy in human tumors. Alternatively, these characteristics 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 skilled in the art. A therapeutically effective dose of the therapeutic compound may reduce tumor size or otherwise improve symptoms in the individual. A person skilled in the art may determine these amounts based on these factors, such as the size of the individual, the severity of the individual's symptoms, and the selected specific composition or route of administration.

[0162] The compositions of the present invention may be administered via one or more routes of administration using one or more of the various methods known in the art. As understood by those skilled in the art, the route and / or method of administration will vary depending on the desired result. Preferred routes of administration for the antibodies of the present invention include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes by injection or infusion. The term "parenteral administration" as used herein generally refers to a method of administration other than intestinal and local administration by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intravertebral, epidural, and intrasternal injections and infusions.

[0163] Alternatively, the antibody of the present invention may be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, for example, intranasally, or orally, vaginally, rectally, sublingually, or topically.

[0164] The active compound may be prepared with a carrier that protects the compound against rapid release, such as in controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Many methods for preparing such formulations have been patented or are generally known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems (1978) JR Robinson, ed., Marcel Dekker, Inc., NY).

[0165] In certain embodiments, the monoclonal antibodies of the present invention may be formulated to ensure adequate distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if desired), they may be formulated, for example, into liposomes. For methods of manufacturing liposomes, refer, for example, to U.S. Patents No. 4,522,811; No. 5,374,548; and No. 5,399,331. Liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, thereby enhancing targeted drug delivery (e.g., see VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (e.g., see U.S. Patent No. 5,416,016); Mannoside (Umezawa et al. (1988) Biochem. Biophys. Res. Commun. 153:1038); antibody (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); also containing K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; See IJ Fidler (1994) Immunomethods 4:273.

[0166] Uses and methods

[0167] The antibody, antibody composition, and method of the present invention have numerous in vitro and in vivo diagnostic and therapeutic uses, including the diagnosis and treatment of immune-mediated disorders.

[0168] In some embodiments, these molecules may be administered to cultured cells, in vitro or in vitro, or to human individuals, for example, in vivo, to treat, prevent, and / or diagnose various disorders. As used herein, the term “individual” is intended to include human and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates, and non-mammalians. Preferred individuals include human patients. When the antibodies of the present invention are administered together with other agents, the two substances may be administered sequentially or simultaneously.

[0169] Given the specific binding of the antibody of the present invention to SLAMF6, the antibody of the present invention can be used to specifically detect SLAMF6 expression on the surface of immune cells, and furthermore, can be used to purify SLAMF6 through immunoaffinity purification.

[0170] In addition, when the expression of SLAMF6 on immune cells is provided, the antibody, antibody composition, and method of the present invention may be used to treat an individual with a tumorigenic disorder, for example, a disorder characterized by the presence of tumor cells, 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, cervical cancer, prostate cancer, renal cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy.

[0171] In one embodiment, the antibody of the present invention is used for the treatment of cancer, e.g., small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy.

[0172] In a further embodiment, the antibody of the present invention is used in the manufacture of a drug for the treatment of cancer, e.g., small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancies.

[0173] In one embodiment, the antibody of the present invention (e.g., monoclonal antibody, antibody fragment, Nanobody™, multispecific and bispecific molecules and compositions, etc.) may be used to detect the level of SLAMF6, or the level of immune cells containing SLAMF6 on a membrane surface, and then this level may be linked to specific disease symptoms for diagnosis.

[0174] In other embodiments, antibodies of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) may initially be tested in vitro for binding activity related to therapeutic or diagnostic use. For example, compositions of the present invention may be tested using the flow cytometry method described in the following examples.

[0175] In some embodiments, the antibodies of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) have additional utility in the treatment and diagnosis of diseases. For example, monoclonal antibodies, multispecific or bispecific molecules may be used to induce one or more of the following biological activities in vivo or in vitro: to induce and / or enhance the activation of immune cells; to mediate phagocytosis or ADCC of cells in the presence of human effector cells expressing SLAMF6; or to block the binding of an SLAMF6 ligand to SLAMF6.

[0176] 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 representing, in particular, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor, and hematological malignancies.

[0177] Suitable routes for administering the antibody compositions of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules and compositions) in vivo and in vitro are widely known in the art and can be selected by those skilled in the art. For example, antibody compositions may be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecules used will depend on the age and body weight of the individual and the concentration and / or formulation of the antibody composition.

[0178] As described above, the antibody of the present invention may be co-administered with one or more additional therapeutic agents, e.g., immunostimulators, cytotoxic agents, radiotoxic agents, or immunosuppressants. The antibody may be linked to a formulation (as an immune complex) or administered separately from the formulation. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the formulation, or may be co-administered with other known therapies, e.g., anticancer therapy, e.g., radiation therapy. Such therapeutic agents include, in particular, anti-neoplastic agents effective only at levels that are toxic or subtoxic to the patient themselves, such as doxorubicin (Adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea. Other formulations suitable for co-administration with the antibody of the present invention include other formulations used in cancer treatment, such as Avastin®, 5FU, and gemcitabine. Co-administration of the anti-SLAMF6 antibody of the present invention or its antigen-binding fragment with a chemotherapy agent provides two anticancer agents that operate through different mechanisms to produce cytotoxic effects on human tumor cells. Such co-administration can address problems caused by the development of resistance to the drug or changes in the antigenicity of tumor cells.

[0179] Target-specific effector cells, for example, effector cells linked to the compositions of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules), may also be used as therapeutic agents. The effector cells for targeting may be human leukocytes, such as macrophages, neutrophils, or monocytes. Other cells include eosinophils, natural killer cells, and other IgG- or IgA-receptor-bearing cells. Preferably, effector cells may be obtained from the target to be treated. Target-specific effector cells may be administered as a suspension of cells in a physiologically acceptable solution. The number of cells administered is 10 8 -10 9It could be around that amount, but it will vary depending on the purpose of treatment.

[0180] Treatment using target-specific effector cells may be performed in combination with other techniques. For example, anti-tumor therapy using the compositions of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) and / or effector cells armed with these compositions may be used in combination with chemotherapy.

[0181] Additionally, combination immunotherapy can be used to direct two distinct groups of cytotoxic effectors toward tumor cell rejection.

[0182] The bispecific and multispecific molecules of the present invention can also be used to regulate FcγR or FcγR levels on effector cells, for example, by capping and removing receptors on the cell surface. Mixtures of anti-Fc receptors can also be used for this purpose.

[0183] An aspect of the present invention comprises a kit comprising an antibody composition of the present invention (e.g., a monoclonal antibody, a bispecific or multispecific molecule) and instructions for its use, for example, in the treatment of cancer. The kit may further contain one or more additional reagents, such as an immunosuppressing reagent, a cytotoxic agent or a radiotoxic agent, or one or more additional antibodies of the present invention (e.g., an antibody having complementary activity that binds to an epitope on an SLAMF6 antigen distinct from the first antibody).

[0184] Accordingly, a patient treated with the antibody composition of the present invention may additionally receive other therapeutic agents that enhance or increase the therapeutic effect of the antibody, such as cytotoxic agents or radiotoxic agents (before, simultaneously with, or after the administration of the antibody of the present invention).

[0185] In other embodiments, the subject may be further treated with an agent that modulates, for example, enhances or inhibits the expression or activity of Fcγ or Fcγ receptors by treating the subject with a cytokine. Cytokines preferred for administration during treatment with a multispecific molecule include granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumor necrosis factor (TNF).

[0186] The compositions of the present invention (e.g., antibodies, multispecific and bispecific molecules) may also be used to target cells expressing FcγR or SLAMF6 to label such cells, for example. For such use, a binder may be linked to a detectable molecule. Thus, the present invention provides a method for localizing cells expressing Fc receptors, such as FcγR or SLAMF6, in vitro or in vitro. The detectable label may be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

[0187] In a specific embodiment, the present invention provides a method for detecting the presence of SLAMF6 antigen in a sample, or a method for measuring the amount of SLAMF6 antigen, comprising the step of contacting a sample and a control sample with a monoclonal antibody or its antigen-binding portion that specifically binds to SLAMF6 under conditions allowing the formation of a complex between an antibody or a portion thereof and SLAMF6. Subsequently, the formation of a complex is detected, wherein a difference in complex formation between the samples compared to a control sample indicates the presence of SLAMF6 antigen in the sample.

[0188] In another embodiment, the present invention provides a method for treating immune-mediated disorders in an individual, e.g., human cancer, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy.

[0189] All references cited herein, including but not limited to all papers, publications, patents, patent applications, presentations, texts, reports, manuscripts, brochures, books, internet posts, journal articles, periodicals, product fact sheets, etc., are incorporated herein by reference in their entirety. The discussion of references herein is merely for the purpose of summarizing the claims of the authors of the literature, and it is not acknowledged that any reference constitutes prior art and that the applicant reserves the right to claim the accuracy and relevance of any cited references.

[0190] Although the foregoing invention has been described in some detail as examples and illustrations for clarity of understanding, it will be readily apparent to those skilled in the art that specific changes and modifications may be made in light of the teachings of the present invention without departing from the spirit or scope of the dependent claims.

[0191] The present invention is further illustrated by the following examples, which should not be construed as further limitations. The following examples, sequences, and drawings are provided to aid in understanding the invention, and the true scope is set forth in the appended claims. It is understood that variations may be made to the procedures presented without departing from the spirit of the invention.

[0192] Examples

[0193] Example 1: Antibody generation and screening.

[0194] Hybridoma generation

[0195] To generate mouse Fabs against human SLAMF6 ECD (SEQ No. 12), recombinant ECD protein was used to immunize mice (Alere, San Diego. -SLAMF6-hum.ECD). A library of Fabs was generated using industry-standard techniques with splenocytes from immunized mice.

[0196] 2nd screening

[0197] Binding of SLAMF6 protein expressed on the surface of Raji cells and activated PBMCs to the Fab supernatant was tested. The supernatant was diluted to one-tenth with FACS buffer.

[0198] Example 2: Structural characteristics of a monoclonal antibody against SLAMF6

[0199] The cDNA sequences encoding the heavy and light chain variable regions of the monoclonal antibody were obtained using standard PCR technology and sequenced using standard DNA sequencing technology. The heavy and light chain variable regions of 1B3 selected in the screening are shown in Figure 1.

[0200] The nucleotide and amino acid sequences of the heavy chain variable region of 1B3 are shown in SEQ ID NOs 3 and 1, respectively.

[0201] The nucleotide and amino acid sequences of the light chain variable region of 1B3 are shown in SEQ ID NOs 4 and 2, respectively.

[0202] Further analysis of the 1B3 VH sequence using the Kavat system for determining CDR regions confirmed the heavy chain CDR1, CDR2, and CDR3 regions as shown in sequence numbers 5, 6, and 7, respectively. Figure 1a shows the 1B3 VH sequence, with CDR1, CDR2, and CDR3 indicated by boxes.

[0203] Further analysis of the 1B3 VL sequence using the Kavat system for determining CDR regions confirmed the light chain CDR1, CDR2, and CDR3 regions as shown in sequence numbers 8, 9, and 10, respectively. Figure 1b shows the 1B3 VL sequence, with CDR1, CDR2, and CDR3 indicated by boxes.

[0204] Example 3: Specificity of Fab supernatant monoclonal antibody against SLAMF6 determined by flow cytometry

[0205] 5x10 6 Raji cells were placed into each well of a 96-well plate and washed 1x 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), 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 added in increasing amounts as shown in Figure 4 to each well incubated on ice for 30 minutes. Then, the cells were washed once with FACS buffer (DPBS, 2% FBS), pelleted, washed, and resuspended in FACS buffer. The secondary goat-anti-mouse antibody was diluted to 1 µg / ml, 100 µl was added to each well, and the plate was incubated on ice for 30 minutes, followed by one wash with FACS buffer (DPBS, 2% FBS). Cells were pelleted and resuspended in 200 µl of FACS buffer. Samples were read using a Guava Easycyte Plus HT flow cytometer, and the results were analyzed using the Guava Cytosoft software suite.

[0206] As can be seen in Figure 4, antibody 1B3 exhibits specific dose-dependent binding to SLAMF6-expressing Raji cells.

[0207] Example 4: Ability of anti-SLAMF6 antibody to activate T cells and stimulate IFNγ production

[0208] 96-well non-tissue culture plates were coated with 250 ng / ml OKT3 and various concentrations of anti-SLAMF6 antibody / isoform overnight at 4°C. The plates were washed twice with PBS and blocked for 30 minutes with R10 medium (RPMI containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin). After blocking, 1 million T cells were resuspended in 100 μl of R10 medium and added to the plates (T cells isolated from PBMC using Miltenyi kit code: 130-096-535 followed the manufacturer's instructions). The plates were incubated at 37°C for 72 hours, the supernatant was collected, and diluted for use in IFNγ ELISA analysis.

[0209] IFNγ was measured using the IFN-gamma DuoSet ELISA kit (R&D systems Cat.#DY285B) following the manufacturer's instructions.

[0210] result

[0211] The humanized antibody Hu_1B3 showed increased IFNγ production at lower antibody concentrations compared to the Seattle Genetics antibody against SLAMF6 described in WO2017 / 004330 (Fig. 5), demonstrating enhanced activity in OKT3 pre-activated T cells. This induction of IFNγ production indicates that the antibody against SLAMF6 will have a therapeutic effect in patients with a suppressed immune system.

[0212] Example 5: Humanization of Antibody 1B3

[0213] Humanization of the murine 1B3 monoclonal antibody was performed using CDR transplantation technology. To induce the humanization process and aid in the decision to preserve the maternal murine residues or replace them with their human germline counterparts, a homologous molecular model of the Fv of the 1B3 murine monoclonal antibody was constructed.

[0214] The definition of CDR is based on the Kabat nomenclature. The selection of human framework receptor regions grafted with 1B3 rat CDR regions was achieved by searching the IMGT murine and human V gene databases using IgBLAST, developed by NCBI to facilitate the analysis of immunoglobulin V region sequences, with the 1B3 murine variable region sequence as the input. The strategy applied was to use human germline sequences, which are natural human sequences that do not contain idiopathic somatic mutations found in individual human antibody sequences.

[0215] Heavy chain design

[0216] The amino acid sequence of VH isolated from mouse 1B3 hybridoma (CDR regions according to Kabat numbering are shown in bold) is shown below:

[0217]

[0218] Selection of the human framework receptor VH region

[0219] The selection of the human framework receptor VH region grafted with the 1B3 murine CDR region was achieved by searching the IMGT human VH gene database using IgBLAST with the murine VH region amino acid sequence as the input. The closest match was identified based on sequence alignment of the maternal antibody against the human germline. The identification of the optimal human germline as a receptor was based on the following sequence criteria: sequence identity across the framework as defined by Kavart, and identity and / or compatibility of the morphological form of the maternal CDR with interchain interface residues and support loops. The human germline IGHV1-2*02 was selected as the most suitable heavy chain.

[0220] Design using the IGHV1-2*02 human germline as the framework receptor region

[0221] Humanized version

[0222] The following detailed sequences were obtained by grafting murine CDRs (in bold) as defined by Kabat nomenclature onto IGHV1-2*02. Many residues are framework murine residues (external CDR residues), i.e., conserved from the parent murine 1B3 VH sequence; these were conserved because they may be structurally important for maintaining the overall activity of the antibody.

[0223]

[0224] 86.7% identity between the humanized version (OBT577-12-VHB) and the IGHV1-2*02 human germline (85 of the total 98 residues of the V gene are identical).

[0225] Light chain design

[0226] The amino acid sequence of mouse 1B3 VL (CDR region defined by Kabat nomenclature is highlighted) is shown below.

[0227]

[0228] Selection of Human Framework Receptor VL Regions

[0229] The selection of the human framework receptor VL region to which the 1B3 VL murine CDR region is grafted was achieved by searching the IMGT human VL gene database using IgBLAST with the murine VL region amino acid sequence as the input. Based on sequence alignment of the parent antibody against the human germline, the closest match was identified. The identification of the optimal human germline as a receptor was based on the following sequencing criteria: sequence identity across the framework as defined by Kavart, and identity and / or compatibility of the morphological form of the parent CDR with interchain interface residues and support loops. From this analysis, the human germline IGKV1-33*01 was found to be the best choice as the human framework receptor region. Therefore, this human germline was used in the design of the humanized version.

[0230] Design using IGKV1-33*01 human germline as the framework receptor region

[0231] Humanized version

[0232] A murine CDR as defined by Kabat numbering was grafted onto IGKV1-33*01 to obtain the following detailed sequence. A number of residues structurally important for maintaining the full activity of the antibody were retained. This provided a humanized version with 86.3% identity (82 out of 95 amino acid residues).

[0233]

[0234] 86.3% identity between the humanized version and the IGKV1-33*01 human germline (82 / 95).

[0235] Example 6: ELISPOT using tumor-infiltrating lymphocytes.

[0236] Primary tumor-derived tumor-infiltrating lymphocytes (TILs) from NSCLC (Fig. 6), breast cancer (Fig. 7), or CRC (Fig. 8) tumors were stimulated for 96 hours using OKT3 diluted to 1 μg / ml in 10 μg / ml of murine 1B3 or pembrolizumab and complete IMDM culture medium. After stimulation, TILs were harvested, counted, and plated at 100,000 cells / well on IFNγ ELISPOT plates (Mabtech). The 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.

[0237] Figure 6 shows that antibody 1B3 activates NSCLC-derived TILs, reflecting significantly higher IFNγ production than isotype antibodies.

[0238] Figure 7 shows that antibody 1B3 activates a significantly larger number of breast cancer-derived TILs than pembrolizumab to produce IFNγ.

[0239] Figure 8 shows that antibody 1B3 activates a significantly larger number of colorectal cancer-derived TILs than pembrolizumab to produce IFNγ.

[0240] Example 7. Binding affinity of humanized 1B3 antibody.

[0241] Binding affinity experiments were performed on a Biacore T-200 at 25°C. Flow cells 2, 3, and 4 of the CM5 chip were coated with up to 500 RU of goat anti-human IgG. Test antibodies were captured in flow cells 2, 3, and 4. Flow cell 1 was kept blank and used for reference subtraction. The antigen flowed over the chip. The binding of the antigen to the antibody was monitored in real time. The observed k on and k off KD was determined from.

[0242] Sample K D (M) K A (1 / M) k on (1 / Ms) k off (1 / s) Full X 2 1B3 1.72x10 -9 5.8x10 8 5.41 x 10 5 9.32x 10 -4 0.227

[0243] Example 8. In vitro proliferation assay using anti-SLAMF6 antibody 1B3

[0244] method

[0245] Non-tissue culture-treated 96-well plates (BD Falcon, USA) were coated with 250 ng / ml of anti-human CD3 (eBioscience, USA) antibody alone or in combination with humanized anti-SLAMF6 antibody (Hu_1B3), or with an isotype control antibody at a concentration of 0. 0.2, 0.4, 0.6, 0.9, and 1.2 µg / ml were added and incubated overnight at 4°C. The next day, the plates were washed and blocked with AIM V medium. T cells isolated from PBMCs generated from healthy donors were stained with the cell proliferation dye eFluor™ 670 (eBioscience, USA), washed, and seeded into antibody-coated plates (100,000 cells per well in 100 μl) containing FCS and penicillin-streptomycin in AIM V (Thermofisher Scientific, USA) and cultured in a tissue culture incubator at 37°C for 72 hours. On day 3, cells were collected and stained with FITC-labeled anti-human CD8, Brilliant Violet 711™-labeled anti-human CD4, PE-labeled anti-human CD69 (Biolegend, USA), and the fixation 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).

[0246] Figure 9 shows that stimulation of SLAMF6 present on isolated T cells using an anti-SLAMF6 antibody in the presence of CD3 significantly increases T-cell proliferation compared to isoform or CD3 alone.

[0247] Example 9. Unidirectional mixed lymphocyte reaction (MLR) using allogeneic PBMCs with anti-SLAMF6 antibody Hu_1B3.

[0248] method:

[0249] 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).

[0250] Cryopreserved PBMCs from the second donor (Donor 2) were treated with 50 µg / ml mitomycin C in culture medium at a density of 2E6 cells / ml. After treating the cells at 37°C for 90 minutes, the mitomycin-C was removed by washing with culture medium.

[0251] Then, 100,000 cells from Donor 1 were 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 at a total volume of 100 µl of culture medium / well. The isoform was used as a negative control, as well as the anti-CD137 mAb included for comparison.

[0252] The culture supernatant was harvested on the 6th day, and the concentration of IFN-γ was analyzed by ELISA (R&D Systems: DY285B) according to the manufacturer's instructions.

[0253] Figure 10 shows that the anti-SLAMF6 antibody Hu_1B3 exhibits a dose-related increase in cytokine release, which indicates that T-cells are activated by the presence of the antibody. It also shows that activation of SLAMF6 results in higher cytokine release from isolated T cells than activation via CD137.

[0254] Example 10. SLAMF6 mediated the release of granzyme B and perforin from T cells.

[0255] PBMC simple interest

[0256] As the first step in T cell isolation, PBMCs were isolated from a buffy coat (Leucopak at Stanford Blood Center). Blood was diluted in PBS at a 1:4 ratio (10 ml blood + 30 ml PBS), and 30 ml of the diluted blood was carefully placed on a 15 ml Ficoll-Hypaque (GE-Healthcare cat. No. 17-1440-03). With the brake off, the tube was centrifuged at 400 g (1400 rpm) in a Sorval centrifuge at room temperature for 30 minutes. Monocytes were separated by density gradient. Approximately 10 ml of the cell white fraction from all tubes was collected into a single 50 ml tube, washed, and counted using a Cellometer auto 2000. The PBMCs generated from the buffy coat were used for T cell isolation.

[0257] T cell isolation

[0258] Pan T cell isolation from PBMCs is a negative selection process in which all immune cell subsets, excluding 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 were washed and resuspended in FACS sorting buffer (0.1% BSA in PBS) at a concentration of 2.5 e7 cells / mL. A Pan T cell biotin-antibody cocktail was added to the cells, the antibody-cell suspension was mixed using a 1 mL pipette, and incubated on ice for 5 minutes. A Pan T cell microbead isolation cocktail was added to the mixture and incubated on ice for another 10 minutes. MACS isolation technology was used to isolate untreated T cells. A mixture of biotin-labeled PBMC micro beads was run on an LS column of a high-magnetic field MACS separator (Miltneyi Biotec, Cat# 130-042-401), the non-T cell depleted pass-through was collected, washed once with FACS sorting buffer, and resuspended in AIMV complete medium containing 5% FBS.

[0259] Granzyme B ELISA

[0260] For Granzyme B function analysis, human Hu_1B3 or isotype control antibodies were coated in combination with 250 ng / ml of anti-human CD3 (OKT3 clone) (Thermofisher Scientific Cat#16-0031) on non-tissue culture-treated 96-well plates. Each test antibody was coated at a 2:3 dilution for a triple 10-point titration in 100 μl PBS, starting at 4.2 μg / ml. The plates were sealed and incubated overnight at 4°C. On the day of Granzyme B function analysis setup, the plates were washed and blocked for 20 minutes with AIMV complete medium containing 5% FCS to reduce non-specific binding.

[0261] Granzyme B immunoassay was established using the Human Granzyme B DuoSet ELISA kit from R&D Systems. Nunc-immunoassay plates were coated with a granzyme B capture antibody diluted 1:60 in PBS, and the plates were incubated overnight at 4°C. The next day, the plates were washed with ELISA wash buffer (PBS + 0.05% Tween 20) and blocked with 1% BSA in PBS. After blocking the plates, 100 μl of the sample and standard, diluted 1:60 in dilution buffer, were added to each well, and the plates were incubated overnight at 4°C. The next day, the plates were developed, and OD values ​​were captured using a VersaMax adjustable microplate reader. Granzyme B release was quantified and plotted using Graphpad Prism 8 software.

[0262] conclusion

[0263] Granzyme-mediated apoptosis is one of the major mechanisms used by cytotoxic lymphocytes to eliminate transformed cells. Data show that antibody Hu_1B3 can induce cytotoxic functions important for tumor suppression. Antibody Hu_1B3, at an EC of 0.51 μg / ml 50 This induces a dose-dependent increase in granzyme B from activated T cells (Fig. 11).

[0264] Perforin intracellular analysis

[0265] For the setup of the intracellular quantification assay of Perforin, non-tissue culture-treated 96-well plates were coated with human Hu_1B3 or isomorphic control antibodies combined with 250 ng / ml of anti-human CD3 (OKT3 clone) (Thermofisher Scientific Cat#16-0031-85). Each test antibody was coated with a 2:3 dilution for a 10-point titration in 100 μl PBS, starting at 4.2 μg / ml. The plates were sealed and incubated overnight at 4°C. On the day of the Granzyme B functional assay setup, the plates were washed and blocked for 20 minutes with AIMV complete medium containing 5% FCS to reduce non-specific binding. For this assay, 200,000 PAN T cells were added per well to 100 μl of AIMV culture medium containing 5% FBS and cultured in a tissue culture incubator at 37°C for 3 days. On the day of analysis, a protein transport inhibitor cocktail (Thermofisher Scientific, Cat# 00-4980-93) was added to all wells and incubated for 4 hours to prevent perforin transport into the extracellular space. Cells were collected and stained for T cell surface markers CD3, CD4, and CD8, followed by intracellular perforin staining using the FIX & PERM™ Cell Permeability Kit. Cells were analyzed on an Attune NxT FACS analyzer (Thermofisher Scientific, MD), and data were analyzed using FlowJo software (BD Biosciences, San Jose).

[0266] conclusion

[0267] Perforin-mediated necrosis is another major mechanism of apoptosis induced by cytotoxic T lymphocytes, and Hu_1B3, at an EC of 0.44 μg / ml 50 As a result, it enhanced the upregulation of perforin in CD8+ T cells in a dose-dependent manner (Fig. 12).

[0268] Example 11. Competitive binding analysis to evaluate the binding epitope of antibody 1B3

[0269] Cryopreserved human PBMCs were thawed, suspended in FACS buffer (DPBS containing 2% FCS), washed once, and then centrifuged at 1200 rpm for 5 minutes to pellet the cells and discard the supernatant (the same method was used for subsequent washes). Before washing the cells one more time, they were distributed into 96-well assay plates at a rate of 100,000 cells per well in FACS buffer. Then, the cells were blocked with SLAMF6 ECD-mIgG2a Fc fusion protein at 100 nM or 300 nM for 1 hour in 100 µl of FACS buffer on ice for SLAMF6 receptor blockade, and washed after 1 hour. Humanized Hu_1B3 antibody or human IgG1 isotype control was titrated with serial dilutions from 1 to 3 at a peak concentration of 10 nM, added to blocked PBMCs, incubated on ice for 1 hour, and washed twice. The secondary antibody, goat anti-human IgG-RPE (Southern Biotech, Ref: 2040-05), was applied to the treated cells at a concentration of 1 μg / mL in FACS buffer for 30 minutes on ice. One untreated well containing cells was also stained with the secondary antibody, while the other was left unstained to serve as a control for secondary antibody binding. After this final incubation, the cells were washed twice again, and the final cell pellet was resuspended in FACS buffer. The average fluorescence intensity of the secondary antibody was determined 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 were analyzed using FlowJo analysis software.

[0270] As shown in Figure 13, the Hu_1B3 antibody was blocked from binding to the receptor on the surface of PBMC in the presence of the human SLAMF6 ECD-mIgG2a Fc fusion protein, which indicates that Hu_1B3 competitively binds to human SLAMF6 ECD. Therefore, it can be seen that Hu_1B3 binds to the homodimerization epitope of SLAMF6, acts as an antagonist antibody, and increases cytotoxic T cell function through the SAP-mediated activation pathway.

[0271] Example 12. Internalization of Hu_1B3 antibody

[0272] Using industry-standard aseptic technique, 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).

[0273] RAJI cells in 24-well glass-bottom cell culture plates, 5 x 10 cells per well 5 Cells were plated at a certain density and allowed to grow in growth medium at 37°C for 48 hours. Wells were prepared for the following samples: at 0, 0.5, 1, 2, 4, and 24 hours, a secondary antibody-only control, a human IgG isotype control, the antibody Hu_1B3, and Seattle Genetics' clinical anti-SLAMF6 antibody (positive control). The secondary antibody-only control wells and antibody-free control wells were used as fluorescence controls.

[0274] All antibody incubation and washing steps were subsequently performed on ice using ice-cold reagents. Culture medium was aspirated from the wells and washed twice with IF buffer (Dulbecco's phosphate-buffered saline) (DPBS, Thermo Scientific, Waltham VA, Cat. # SH30028-03) + 2% FBS. The primary antibody (purified OBT humanized Hu_1B3; isoform or positive control antibody (Seattle Genetics)) was diluted to 2 µg / mL in IF buffer, and 200 μl was applied to the appropriate wells for 15 minutes. Only the same volume of IF buffer was added to the wells designated as the secondary antibody control. The secondary antibody (goat anti-human IgG - Alexa Fluor 488, Invitrogen Cat. #A11013) was diluted to a concentration of 2 µg / mL in IF buffer and added to the primary incubation for 15 minutes.

[0275] After primary and secondary antibody labeling, human IgG isotype controls, secondary antibody-alone controls, and test and positive controls were treated from 0-minute samples. Cells were washed twice with IF buffer, placed in a second 24-well plate containing 4% paraformaldehyde (4% dilution in DPBS, Cat No. 19943, Affymetrix, Santa Clara, CA) on ice to stop internalization, and fixed the cells.

[0276] The remaining cells were washed twice with IF buffer, and 1 mL of warmed growth medium was added to each well before being placed in a 37°C incubator. At 0h, 0.5h, 1h, 2h, 4h, and 24h, the cells were fixed in paraformaldehyde on ice as described for the control sample.

[0277] All cells were kept in the fixative on ice for at least 15 minutes. Cells were washed with IF buffer, and coverslips were added to each well with 2–3 drops of Prolong Gold Anti-Fade reagent and DAPI (Cat No. P-36931, Invitrogen, Grand Island, NY) as a nuclear counterstain. Cell images were acquired using a Leica Microscope (Leica DMI600B), a Leica monochrome camera (Leica DFC350FX), a filter set for DAPI, and an Alexa Fluor 488 and 63x oil immersion lens. Images were saved in TIFF format and analyzed using ImageJ.

[0278] Figure 14 shows that the humanized antibody Hu_1B3 is internalized into SLAMF6-expressing cells at a significantly lower rate than the clinical Seattle Genetics antibody. This indicates that when the antibody Hu_1B3 binds, the receptor remains on the cell surface for a longer period, thereby inducing a more sustained response in T cells and consequently becoming a more effective agonist.

[0279] Example 13. Cytotoxicity Analysis

[0280] cell culture

[0281] SKBr3 HCT116 and MDA-MB-231 were purchased from ATCC. Cell lines were maintained using standard aseptic techniques according to the manufacturer's (ATCC) instructions. The cell culture medium consisted of RPMI-1640 containing 2 mM L-glutamine, 25 mM HEPES (Corning), 1% penicillin / streptomycin (Sigma-Aldrich), and 10% heat-inactivated FCS (HyClone). Cell lines were maintained in the exponential phase and grown in a 37°C incubator containing 5% CO2. SKBr3 is a breast cancer cell line expressing a high copy number of Her2 (~5 x 10⁶). 6(Copy / Cell). HCT116 is a colorectal cancer cell line expressing a low copy number of Her2. MDA-MB-231 is a breast cancer cell line expressing a low copy number of Her2.

[0282] Cytotoxicity Analysis

[0283] Target and effector (PBMC) cells were prepared under the following conditions. The day before analysis, target cells were washed with PBS, incubated with 0.25% trypsin, and resuspended in cell culture medium. Viability and cell concentration were measured by the dye exclusion method. Target cells were plated at 10,000 cells / well in 96-well tissue culture treatment plates. Cells were grown overnight in a 37°C incubator containing 5% CO2.

[0284] On the day before analysis, frozen PBMCs were thawed in a 37°C water bath, washed with cell culture medium, and incubated overnight in a 37°C incubator containing 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.

[0285] The bispecific antibody Cris7-Her2 was used for the cytotoxicity assay. The antibody detects both Her2 and CD3ε. The Cris7-Her2 bispecific antibody was diluted to 100 ng / ml and then three-fold to 0.0012 ng / ml. Additionally, the Cris7-Her2 + agonist antibody combination was tested. By including the combination of the Cris7-Her2 bispecific antibody and the agonist antibody, enhanced cytotoxicity induced by the agonist antibody was observed. Urelumab, an antibody against 4-1BB, was used as a positive control. The isoform was used as a negative control. Hu_1B3 was the subject of the test. Urelumab, the isoform, and Hu_1B3 were used at a final concentration of 2.5 µg / ml. All samples were run in triplicate. The controls included target cells alone or target cells plus effector cells. Additional controls included target cells and effector cells + Hu_1B3, or target cells and effector cells + isomorphic controls.

[0286] Cytotoxicity assays were performed by culturing for 48–96 hours depending on the cell line. After culturing, the viability of target cells was measured. Viability is based on the quantification of ATP, which signals the presence of metabolically active cells. The assay is luminescence-based, and Cell Titer-Glo® (Promega, Madison, WI) was the reagent used to measure living cells. The manufacturer's instructions were followed for assay conditions, and all steps were performed at room temperature. Briefly, the 96-well plate and Cell Titer-Glo® reagent were equilibrated at room temperature for 30 minutes. After 30 minutes, the cell culture medium was removed, and the target cells were washed with 200 µl of PBS; the washing step was repeated once more. Next, 100 µl of Cell Titer-Glo® reagent was added to all wells. The plate was placed in an orbital shaker and mixed at 250 rpm for 2 minutes to induce cell lysis. The plate was removed from the orbital shaker and incubated in the dark for 10 minutes to stabilize the luminescence signal. After 10 minutes, the sample was transferred to an opaque wall 96-well plate and luminescence was recorded.

[0287] The viability of target cells was proportional to the generated luminescence signal. All samples were triplicated, and the average was calculated for each analysis condition. The % cytotoxicity of the samples was normalized relative to the control group; the control group represented the viability of target cells in the presence of effector cells (Target + Effector). To calculate viability, the luminescence signal of the samples was divided by the luminescence signal of the control group. Cytotoxicity was calculated based on the percentage of remaining non-living cells. As shown in Figure 15, for SKBr3 cells, the bispecific antibody alone or the bispecific antibody + isomorphic control exhibited effective cytotoxicity at higher concentrations ranging from 0.8 to 100 ng / ml. The addition of urelumab shifted the cytotoxicity curve slightly to the left, but by less than 5%. However, the addition of 2.5 µg / ml of Hu_1B3 increased the cytotoxicity of SKBR-3 cells by 10–40%.

[0288] Figure 16 shows that similar results were obtained in the second PBMC donor as well.

[0289] Figure 17 shows the enhanced cytotoxicity of Hu_1B3 against HCT116 cells. HCT116 cells, a colon cancer cell line, express low levels of Her2 on their cell surface. Cytotoxicity assays were performed using HCT116 cells and the T-cell binding bispecific antibody Cris7-Her2 in combination with PBMCs. As previously mentioned, Hu_1B3 was added to the assay to determine whether it enhances the function of T cells or NK cells present in PBMCs; both T cells and NK cells express the co-activating receptor SLAMF6. The resulting data show dose-dependent cytotoxicity by the T-cell binding bispecific antibody Cris7-Her2 at various tested concentrations. More importantly, the data indicate that an increase in cytotoxicity was observed upon the addition of Hu_1B3. EC10 for Cris7-Her2 bispecific alone or bispecific antibody + isomorphic control 50 While is approximately 0.25 ng / mL, EC for bispecific antibody + Hu_1B3 50 It is approximately 0.07 ng / ml. This indicates that cytotoxicity increases by about 3.6 times.

[0290] Figure 18 shows the enhanced cytotoxicity of 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 analysis was performed using MDA-MB-231 cells, PBMCs, and the T cell-binding bispecific antibody Cris7-Her2 as described above. Again, Hu_1B3 enhances the cytotoxicity of the Cris7-Her2 bispecific antibody at various concentrations tested. Specifically, compared to the isomorphic control, a 40% increase in cytotoxicity was observed at 1 ng / mL of Cris7.

[0291] Figure 19 shows that after incubation for 96 hours, the combination of the bispecific antibody and Hu_1B3 exhibited high levels of SKBR-3 cell death even at the lowest levels of the bispecific antibody concentration. In contrast, at these levels, the bispecific antibody alone, or the bispecific antibody combined with urelumab or an isotype control, resulted in very low levels of cell death or no cell death, suggesting that Hu_1B3 provides potent activation of lymphocytes.

[0292] Figure 20 shows the cytotoxicity assay under test conditions in which the bispecific Cris 7-Her2 antibody is maintained at a constant level (0.0457 ng / ml) and the concentration of the test antibody is titrated at a 1 / 3 dilution in the range of 7.5 µg / ml to 0.0034 µg / ml. Compared to urelumab or isotype controls, dose-dependent death of SKBR-3 cells was observed, which further demonstrates the ability of Hu_1B3 to activate cytotoxic lymphocytes.

[0293] Example 14. Analysis of cytotoxicity in the absence of Cris7 bispecific antibodies

[0294] Another cytotoxicity assay was set up as previously described, but without the use of bispecific antibodies. It was used to test the cytotoxicity of single agents, Hu_1B3, isotype control, urelumab alone, and SKBR-3. The concentration range used for the assay was 33 µg / ml to 0.13 µg / ml, using one donor and a 1 / 3 dilution. The test antibody was further diluted to 0.0152 µg / ml for a second donor (Fig. 21b).

[0295] Figures 21a and 21b show that in the absence of CD3-Her2 bispecific antibodies, Hu_1B3 can activate lymphocytes to induce apoptosis in SKBR-3 cells. This is in contrast to urelumab or isotype controls, where almost no apoptosis occurs.

[0296] Sequence list order number detail order 1 1B3_VH_aa QVQLKQSGAE LVRPGTSVKV SCKASGYAFT NYLIEWVKQR PGQGLEWIGV INPGSGGTNY NEKFKDKATL TADKSSNTAY MQLSSLTSDD SAVYFCARRG WDYFDYWGQG TTLTVSS 2 1B3_VL_aa QIVLTQSPAL MSTSPGEKVT MTCSASSSVS YIYWFQQKPG SSPKPWIYRT SNLASGVPAR FSGSGSGTSY SLTISSMEAE DAATYYCQQW DNNPYTFGGG TKLEIK 3 1B3_VH_nt CAGGTGCAACTCAAGCAAAGCGGTGCAGAACTGGTGGACCTGGCACATCAGTCAAGGTGTCATGCAAAGCTAGTGGATACGCCTTCACTAACTACCTGATTGAGTGGGTGAAGCAAAGACCTGGTCAGGGTCTGGAATGGATTGGAGTGATCAACCCAGGTAGCGGAGGAACTA ACTACAACGAGAAGTTCAAGGATAAGGCAACTCTGACTGCCGACAAGAGCTCTAACACAGCCTATATGCAACTGTCCAGTCTCACTAGCGATGATTCCGCAGTGTACTTCTGTGCTCGCAGAGGCTGGGACTACTTTGACTACTGGGGTCAAGGAACTACTCTGACAGTGTCCAGC 4 1B3_VL_nt CAGATCGTTCTCACCCAGAGTCCTGCACTGATGTCAACAAGCCCTGGCGAGAAAGTTACAATGACTGCAGTGCATCCTCTTCCGTTTCTTACATCTATTGGTTCCAGCCAGGGAGCTCACCAAAGCCTTGGATCTACAGAACATCCAATCTCTC GCAAGCGGTGTTCCAGCTAGGTTCAGTGGGTCCGGATCAGGCACATCCTACTCTCTGACAATCCCTCCATGGAAGCAGAAGACGCTGCAACCTACTATTGCCAACAGGTGGGACAACAATCCCTACACCTTTGGAGGTGGTACCAAGCTGGAGATCAAG 5 1B3_VH_CDR1_aa NYLIE 6 1B3_VH_CDR2_aa VINPGSGGTNYNEKFKD 7 1B3_VH_CDR3_aa RGWDYFDY 8 1B3_VL_CDR1_aa SASSSVSYIY 9 1B3_VL_CDR2_aa RTSNLAS 10 1B3_VL_CDR3_aa QQWDNNPYT 11 SLAMF6 (Q96DU3) MLWLFQSLLFVFCFGPGNVVSQSSLTPLMVNGILGESVTLPLEFPAGEKVNFITWLFNETSLAFIVPHETKSPEIHVTNPQGKRLNFTQSYSLQLSNLKMEDTGSYRAQISTKTSAKLSYTLRILRQLRNIQTNHLQLFADHLEDFR EALGNTLSSQPNLTVSWDPRICEQDYTCIAENAVSNLSFSVSAQKLCEDVKIQYTDTKMILFMVSGICIVFGFIILLLLVLRKRRDSSLSTQRTQGPAESARNLEYVSPTNNTVYASVTHSNRETEIWTPRENDTIYSTINHSKESKPTFSRATALDNVVVV 12 SLAMF6 ECD (서열번호 11의 aa22 - 226) QSSLTPLMVNGILGESVLTLPLEFPAGEKVNFITWLFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQSYSLQLSNLKMEDTGSYRAQISTKTSAKLSSYT LRILRQLRNIQVTNHSQLFQNMTCELHLTCSVEDADDNVSFRWEALGNTLSSQPNLTVSWDPRISSEQDYTCIAENAVSNLSFSVSAQKLCEDVKIQYTDTKM 13 1B3_VHBhu aa QVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWIGV INPGSGGTNY NEKFQGRVTL TADKSISTAY MELSRLRSDD TAVYYCARRG WDYFDYWGQG TLVTVSS 14 1B3VLBhu aa DIQLTQSPSS LSASVGDRVT ITCQASQDVS YIYWYQQKPG KAPKPWIYRT SNLATGVPSR FSGSGSGTDY TFTISSLQPE DIATYYCQQW DNNPYTFGQG TKLEIK 15 1B3_VHBhu_CDR2 VINPGSGGTNYNEKFQG 16 1B3_VLBhu_CDR1 QASQDVSYIY 17 1B3_VLBhu_CDR2 RTSNLAT 18 1B3_VHBhu_Fc_aa QVQLVQSGAE VKKPGASVKV SCKASGYAFT NYLIEWVRQA PGQGLEWIGV INPGSGGTNY NEKFQGRVTL TADKSISTAY MELSRLRSDD TAVYYCARRG WDYFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS SSLGTQTYIC NVNHKPSNTK VDKKVEPKSC DKTHTCPPCP APEAAGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKSLSLSPGK 19 1B3_VLBhu_Fc_aa DIQLTQSPSS LSASVGDRVT ITCQASQDVS YIYWYQQKPG KAPKPWIYRT SNLATGVPSR FSGSGSGTDY TFTISSLQPE DIATYYCQQW DNNPYTFGQG TKLEIKRTVA APSVFIFPPS DEQLKSGTAS VVCLLNNFYP REAKVQWKVD NALQSGNSQE SVTEQDSKDS TYSLSSTLTL SKADYEKHKV YACEVTHQGL SSPVTKSFNR GEC 20 1B3_VHBhu_nt CAAGTGCAACTGGTGCAATCTGGTGCTGAAGTCAAGAAGCCTGGTGCTTCCGTCAAGGTTTCTTGTAAGGCATCTGGTTACGCATTCACCAACTATCTCATTGAATGGGTTAGGCAAGCACCTGGACAAGGACTGGAGTGGATCGGAGTGATCAACCCAGGTTCTGGAGGCACAAACTACAACGAGAAGTTCCAAGGTCGCGTCACACTCACTGCAGACAAATCCATTTCTACAGCCTACATGGAGCTGTCTCGCCTCCGCTCCGATGACACTGCTGTGTACTACTGCGCTCGCAGAGGTTGGGACTACTTCGACTACTGGGGTCAAGGTACCCTCGTTACAGTGTCCAGC 21 1B3_VLBhu_nt GACATCCAACTGACTCAATCTCCATCTAGCCTGTCTGCATCCGTTGGTGATAGGGTCACTATCACATGCCAAGCATCTCAAGACGTGAGCTACATCTATTGGTATCAACAGAAACCCGGTAAGGCTCCTAAACCTTGGATCTACAGGACATCTAATCTGGCCACTGGTGTTCCTTCTCGCTTCTCTGGCAGCGGTAGCGGAACCGACTACACTTTCACCATCAGCTCTCTCCAACCTGAAGACATTGCTACCTACTACTGTCAGCAATGGGATAACAACCCATACACCTTTGGACAAGGTACCAAGCTGGAGATCAAG 22 1B3_VHB_Fc_nt CAAGTGCAACTGGTGCAATCTGGTGCTGAAGTCAAGAAGCCTGGTGCTTCCGTCAAGGTTTCTTGTAAGGCATCTGGTTACGCATTCACCAACTATCTCATTGAATGGGTTAGGCAAGCACCTGGACAAGGACTGGAGTGGATCGGAGTGATCAACCCAGGTTCTGGAGGCACAAACTACAACGAGAAGTTCCAAGGTCGCGTCACACTCACTGCAGACAAATCCATTTCTACAGCCTACATGGAGCTGTCTCGCCTCCGCTCCGATGACACTGCTGTGTACTACTGCGCTCGCAGAGGTTGGGACTACTTCGACTACTGGGGTCAAGGTACCCTCGTTACAGTGTCCAGCGCTAGCACCAAGGGCCCATCCGTTTTCCCTCTGGCTCCTAGCTCCAAATCAACCAGCGGTGGCACAGCAGCCCTGGGATGTCTCGTGAAGGACTACTTCCCCGAGCCCGTCACCGTCTCCTGGAACTCCGGCGCACTCACCTCCGGCGTCCACACCTTTCCCGCCGTTCTGCAGAGTTCTGGCCTGTACAGTCTGAGTTCCGTGGTGACCGTCCCATCCTCCTCCCTCGGGACCCAGACCTACATTTGTAATGTTAATCACAAGCCATCAAACACCAAAGTGGATAAGAAGGTCGAACCTAAAAGCTGCGACAAGACTCACACCTGCCCACCCTGCCCCGCACCAGAAGCTGCAGGTGGCCCCTCAGTTTTCCTGTTCCCACCAAAGCCCAAAGATACCCTCATGATCTCAAGAACCCCAGAGGTCACCTGCGTCGTCGTCGACGTGTCACACGAAGATCCCGAAGTCAAGTTTAATTGGTATGTTGATGGGGTCGAAGTGCATAACGCCAAAACAAAACCCCGCGAAGAGCAGTATAACAGCACTTACAGAGTTGTTTCCGTTCTGACAGTGCTCCACCAGGATTGGCTGAATGGTAAGGAGTACAAATGCAAGGTGTCTAACAAGGCTCTGCCAGCCCCTATTGAGAAAACCATAAGCAAGGCCAAGGGTCAGCCCAGGGAGCCACAGGTGTATACCCTCCCACCTTCACGGGATGAGCTGACCAAGAACCAAGTGAGTCTCACCTGTCTGGTGAAGGGCTTCTACCCAAGCGATATTGCTGTGGAATGGGAATCTAACGGGCAGCCTGAAAATAACTACAAGACCACACCACCAGTGCTCGATTCCGACGGTAGCTTCTTTCTGTATTCCAAACTGACCGTGGACAAAAGCAGATGGCAGCAGGGAAATGTGTTCAGTTGTAGCGTGATGCATGAGGCCCTCCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCCGGTAAG 23 1B3_VLB_Fc_nt GACATCCAACTGACTCAATCTCCATCTAGCCTGTCTGCATCCGTTGGTGATAGGGTCACTATCACATGCCAAGCATCTCAAGACGTGAGCTACATCTATTGGTATCAACAGAAACCCGGTAAGGCTCCTAAACCTTGGATCTACAGGACATCTAATCTGGCCACTGGTGTTCCTTCTCGCTTCTCTGGCAGCGGTAGCGGAACCGACTACACTTTCACCATCAGCTCTCTCCAACCTGAAGACATTGCTACCTACTACTGTCAGCAATGGGATAACAACCCATACACCTTTGGACAAGGTACCAAGCTGGAGATCAAGAGAACAGTGGCTGCACCTAGTGTGTTCATCTTCCCTCCTTCCGATGAGCAACTGAAGAGCGGAACCGCCAGTGTTGTCTGTCTGCTGAACAACTTCTACCCTCGGGAAGCCAAAGTTCAGTGGAAAGTCGACAACGCTCTGCAATCCGGCAACTCCCAGGAGAGTGTCACAGAGCAAGATTCCAAGGACTCCACATATAGTCTGTCCTCTACTCTGACTCTGAGCAAGGCTGACTACGAGAAGCACAAAGTGTACGCTTGCGAAGTGACACATCAAGGCCTGTCCAGTCCCGTTACCAAGAGCTTCAATAGAGGAGAATGT

Claims

Claim 1 An antibody or an antigen-binding fragment thereof that binds to SLAMF6, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising a CDR-H1 sequence including SEQ ID NO. 5; a CDR-H2 sequence including SEQ ID NO. 15; and a CDR-H3 sequence including SEQ ID NO. 7; and a CDR-L1 sequence including SEQ ID NO. 16; a CDR-L2 sequence including SEQ ID NO. 17; and a light chain variable region comprising a CDR-L3 sequence including SEQ ID NO.

10. Claim 2 An antibody or an antigen-binding fragment thereof that binds to SLAMF6, wherein the antibody or antigen-binding fragment comprises: a heavy chain variable region comprising a CDR-H1 sequence including SEQ ID NO. 5; a CDR-H2 sequence including SEQ ID NO. 6; and a CDR-H3 sequence including SEQ ID NO. 7; and a light chain variable region comprising a CDR-L1 sequence including SEQ ID NO. 8; a CDR-L2 sequence including SEQ ID NO. 9; and a CDR-L3 sequence including SEQ ID NO.

10. Claim 3 An antibody or its antigen-binding fragment that binds to SLAMF6, comprising i) three heavy chain CDRs of SEQ ID NO. 1 and three light chain CDRs of SEQ ID NO. 2, or ii) three heavy chain CDRs of SEQ ID NO. 13 and three light chain CDRs of SEQ ID NO. 14, wherein the CDRs are defined by the Kabat or Chothia numbering system. Claim 4 An antibody or an antigen-binding fragment thereof comprising, in claim 1, a heavy chain variable region having a sequence identical to SEQ ID NO. 13 by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, and a light chain variable region having a sequence identical to SEQ ID NO. 14 by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Claim 5 An antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region including SEQ ID NO. 13 and a light chain variable region including SEQ ID NO.

14. Claim 6 An antibody or an antigen-binding fragment thereof, wherein, in any one of claims 1 to 5, the antibody or antigen-binding fragment is a monoclonal antibody. Claim 7 An antibody or its antigen-binding fragment, wherein the antibody or antigen-binding fragment is a chimeric, humanized, bispecific, multispecific, or human antibody or antigen-binding fragment. Claim 8 In claim 7, the antibody or antigen-binding fragment thereof is a bispecific or multispecific antibody that binds to one or more additional binding targets including CD3 or PD-L1. Claim 9 An antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody or its antigen-binding fragment is a silenced engineered IgG1 antibody or antigen-binding fragment having reduced or no binding to one or more Fc receptors. Claim 10 An antibody or an antigen-binding fragment thereof, wherein, in any one of claims 1 to 5, the antibody or the antigen-binding fragment thereof is capable of inducing and / or enhancing T-cell cytotoxicity. Claim 11 An antibody or an antigen-binding fragment thereof, wherein, in any one of claims 1 to 5, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)2, F(ab')2, Fv, FV-TCR fragment, scFv, and single-domain antibodies. Claim 12 i) a heavy chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 5; and / or ii) one or more polynucleotides encoding a light chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 5. Claim 13 An expression vector comprising one or more polynucleotides according to paragraph 12. Claim 14 i. an expression vector comprising a polynucleotide according to claim 12; or ii. a first expression vector comprising a polynucleotide encoding a heavy chain variable region of the antibody of claim 12 or its antigen-binding fragment, and a second expression vector comprising a polynucleotide encoding a light chain variable region of the antibody of claim 12 or its antigen-binding fragment. Claim 15 A method for producing an antibody or an antigen-binding fragment thereof, comprising the step of culturing a host cell according to claim 14 under conditions in which the antibody or the antigen-binding fragment thereof is expressed in the host cell. Claim 16 A method according to claim 15, further comprising the step of isolating the antibody or antigen-binding fragment. Claim 17 A pharmaceutical composition for treating cancer comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier. Claim 18 A pharmaceutical composition for treating cancer comprising an effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5. Claim 19 A composition according to claim 18, wherein the cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, skin cancer including melanoma, breast cancer, colorectal cancer, stomach cancer, ovarian cancer, cervical cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, gastric cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and cholangiocarcinoma, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumor and hematological malignancy. Claim 20 A composition according to claim 19, wherein the non-small cell lung cancer is squamous cell carcinoma or adenocarcinoma, and the breast cancer is TNBC. Claim 21 A composition comprising a second therapeutic agent in addition to the one described in paragraph 18. Claim 22 An antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5, for use in therapy or as a pharmaceutical agent. Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete

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