Antibodies and methods of use

Abstract

To provide antibodies directed against SLAM family member 6 (SLAMF6) also known as NTB-A or CD352, as well as uses thereof.SOLUTION: Disclosed is an antibody that binds to SLAMF6 and has a specific sequence, or an antigen-binding fragment thereof. Also disclosed are other therapeutic proteins, nucleic acids encoding such antibodies and therapeutic proteins, methods for producing the antibodies and therapeutic proteins, and methods for treating diseases such as cancer using the antibody and the other therapeutic proteins directed against SLAMF6.SELECTED DRAWING: Figure 20

Description

[Technical field]

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

[0002] Introduction Embodiments of the present invention include SLAM family members, also known as NTB-A or CD352. Antibodies and other therapeutic proteins against SLAMF6, such antibodies and Nucleic acids encoding therapeutic proteins, methods for preparing antibodies and other therapeutic proteins, and the use of antibodies and other therapeutic proteins against SLAMF6 to treat diseases such as cancer. The present invention includes methods for treating the disease. [Background technology]

[0003] The target antigen, SLAMF6, is a single-pass type I membrane protein and is a target of immunoglobulin S It is a member of the Parr family and the CD2 subfamily (J Exp Med. 2001 Aug 6; 194(3):235-46). Its activity is mediated by a small cytoplasmic adaptor protein, SH2D1A / SA. This protein is regulated by the presence or absence of P and / or SH2D1B / EAT-2. The cells express a high surface density of natural cytotoxic receptors (J. Exp. Med. 194:235-246(2001)). It induces cytolytic activity only in natural killer (NK) cells that are involved in the production of IL-1. Positive signaling in NK cells implies phosphorylation of VAV1. NK cell activation is mediated by SH2D1B It appears to be dependent on SH2D1A and not on SH2D1A. We investigated the positive selection of thymocyte natural killer T (NKT) cell lineage and its subsequent It regulates the transition between proliferation and differentiation.

[0004] SLAMF6 also upregulates the Th17 phenotype of T helper cells, leading to increased IL-17 secretion. and its costimulatory activity is mediated by SH2D1A (J. Immunol. 177:3170-3177(2006 )) is required. In addition, IL-17 promoter (J. Biol. Chem. 287:38168-38177(2012) ) promotes recruitment of RORC to the endothelial cells. SLAMF1 and CD84 / SLAM Together with F5, SLAMF6 may be a negative regulator of humoral immune responses. In the absence of 2D1A / SAP, SLAMF6 is CD4 + T cells and NKT cells It can transmit negative signals and inhibit T cell:B cell adhesion. This function is likely mediated by SH2D1A / SAP. These results suggest that the association with PTPN6 / SHP-1 is increased via ITSM in the absence of There are.

[0005] WO2008 / 027739 describes an anti-NTB-A antibody and a pharmaceutical composition containing the antibody. The present invention also discloses a method for the detection of erythropoietin-binding protein (NTB-A) that binds to NTB-A and is characterized by the expression of NTB-A. Methods of using the antibodies to treat diseases such as liquid malignancies are also described.

[0006] WO2014 / 100740 and WO2017 / 004330 are specific for NTB-A. and the use of these to test the activity of NTB-A expressing cells. The present invention also discloses methods for the expression or regulation of NTB-A in cells expressing NTB-A. and methods for treating diseases such as multiple myeloma, non-Hodgkin's lymphoma, and acute myeloid leukemia. is also disclosed.

[0007] WO2015 / 104711 describes improved ex vivo and in vivo T Compositions and methods for cell regulation and for the treatment of cancer and other pathologies are described. More specifically, embodiments of the present invention are directed to the treatment of cancer patients, the prevention and treatment of blood cells in susceptible patients, and the prevention and treatment of cancer. -PROTEIN FOR THE PREVENTION AND TREATMENT OF HYPERCYTOSIS AND FOR EX VIVO PREPARATION OF IMPROVED CELL COMPOSITIONS - Patent application Use of water-soluble NTB-A polypeptides or agonists thereof is contemplated. Summary of the Invention

[0008] Aspects of the present invention include specific antibodies against SLAMF6, SLAMF6 and tumor-associated antigens Bispecific antibodies against the The present invention relates to a method for preparing an antibody of the present invention, and a method for treating small cell lung cancer, non-small cell lung cancer ... Cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC) , colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, renal cancer, and hepatocellular carcinoma Liver cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach (stom ach) cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, Hematologic malignancies, including but not limited to sarcomas, GISTs, neuroendocrine tumors, and hematological malignancies. This includes methods for treating diseases such as cancer in humans.

[0009] The antibodies described herein provide antibodies that bind to SLAMF6 (SEQ ID NO:11). Preferably, the antibody binds to the extracellular domain of SLAMF6 (SEQ ID NO: 12). An embodiment of the present invention is the SLAMF6 protein recognized by the antibodies described herein. or binds to an epitope on the clone for binding to an antibody described herein. cross-compete, preferably with an antibody as described herein. at least about 80%, at least about 85% of the binding affinity of the human 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%, An antibody or antibody thereof that retains at least about 98% or at least about 99% of its antigen-binding fragment. In some embodiments, the antibody is an isolated antibody.

[0010] Embodiments of the invention include antibodies or antigen-binding fragments thereof that bind to SLAMF6. The antibody comprises a heavy chain variable region, the heavy chain variable region comprising a CDR- H1 sequence, CDR-H2 sequence comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 15, and SEQ ID NO: and in some embodiments, the antibody or antigen-binding domain comprises a CDR-H3 sequence comprising the sequence of SEQ ID NO:7. The combination fragment comprises a CDR- L1, CDR-L2 comprising any one of the sequences of SEQ ID NO: 9 or SEQ ID NO: 17, and At least one CDR selected from the group consisting of CDR-L3 comprising the sequence of SEQ ID NO: 10 It further comprises a light chain variable region comprising the R sequence.

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

[0012] [Table 1]

[0013] In some preferred embodiments, an antibody or antigen-binding fragment thereof that binds to SLAMF6. The fragment comprises CDR-H1 comprising SEQ ID NO:5, CDR-H2 comprising SEQ ID NO:6, and A heavy chain variable region comprising CDR-H3 comprising SEQ ID NO:7 and CDR-L1 comprising SEQ ID NO:8; A light chain variable region comprising a CDR-L2 comprising SEQ ID NO:9 and a CDR-L3 comprising SEQ ID NO:10. Includes the area.

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

[0015] In a further embodiment, the antibody or antigen-binding fragment thereof of the present invention is The present invention therefore relates to variants of the parent antibody. and providing a variant antibody or antigen-binding fragment thereof comprising a variable region of the parent antibody or The 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, and a CDR-H3 sequence comprising the sequence of SEQ ID NO:7. A variable region, a CDR-L1 comprising the sequence of SEQ ID NO: 16, and a CDR comprising the sequence of SEQ ID NO: 17. -L2, and CDR-L3 comprising the sequence of SEQ ID NO: 10, In one embodiment, the mutated antibody or antigen-binding fragment thereof is selected from 1 to 5 or CDR-H1 with 1 to 4 or 1 to 3 substitutions, additions, or deletions , CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 set In the example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 are collectively 1, 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 3, 4, 5 or 6 amino acid substitutions, additions, and / or deletions, or an antigen-binding fragment thereof retains specific binding to SLAMF6. The variations are substitutions, preferably conservative substitutions or amino acid substitutions in the variable region. The substitution is to revert the amino acid to the corresponding amino acid from the human germline. In one embodiment, the modified antibody or antigen-binding fragment thereof of the present invention has the sequence of SEQ ID NO:5. a CDR-H1 sequence comprising the sequence of SEQ ID NO: 15; and a CDR-H2 sequence comprising the sequence of SEQ ID NO: 7; and CDR-L1 comprising the sequence of SEQ ID NO: 16; CDR-L2 comprising the sequence of sequence number 17; and CDR-L3 comprising the sequence of SEQ ID NO: 10. and one or more of said CDR sequences are identical to the corresponding parent CDR sequences listed above. R sequence and approximately 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, and 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical It will be modified.

[0016] In some embodiments, the heavy chain variable region, or a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 13, about 80%, 85%, 90%, 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical and / or a light chain variable region as set forth in SEQ ID NO: 2 or SEQ ID NO: 14. or about 80%, 85%, 90%, 91%, 92% of SEQ ID NO: 2 or SEQ ID NO: 14 , 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to or an antigen-binding fragment thereof. In a further embodiment, SEQ ID NO: 1 or or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 1 compared to SEQ ID NO: 13 A heavy chain variable region containing two amino acid substitutions, additions and / or deletions, and / or sequences 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 compared to number 2 or SEQ ID NO: 14 An antibody comprising a light chain variable region containing 1 or 12 amino acid substitutions and / or deletions, or Antigen-binding fragments thereof are provided. Preferably, the variants are substitutional, more preferably conserved. Contains optional substitutions.

[0017] Amino acid substitutions, additions and / or deletions may occur within the framework regions and / or CD It will be further evident that in R

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

[0019] In one embodiment, a heavy chain variable region comprising the sequence of SEQ ID NO: 13 and a heavy chain variable region comprising the sequence of SEQ ID NO: 14.

[0023] An antibody or antigen-binding fragment thereof is provided that comprises a light chain variable region comprising:

[0020] In one embodiment, the antibody comprises a heavy chain sequence comprising SEQ ID NO:18 and a light chain sequence comprising SEQ ID NO:19. Full length antibodies comprising the polypeptides are provided.

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

[0022] SEQ ID NOs: 13 to 19 are humanized antibody sequences based on the sequences of SEQ ID NOs: 1 and 2. The supplier claims that SEQ ID NOs: 18-19 represent full-length functional antibodies, i.e., the constant and Fc regions. full-length heavy and light chain sequences including regions other than the variable region necessary to produce Those skilled in the art will readily appreciate that the amino acid sequence from the variable region of the organism in which the antibody is to be produced is with amino acids from the human germline sequence, and administering them to a human subject. It will be understood that the sequences are humanized to minimize the immunogenic effects of the antibodies when administered to humans. The majority of amino acid substitutions will occur in framework regions, but at non-critical positions. A number of amino acids from the CDRs may be substituted, and such substitutions are preferably Conserved in the human germline or amino acids at specific positions are In this case, the amino acids from the replaced CDRs are converted to amino acids using a structural model: Identification by distinguishing between paratopic and non-paratopic residues in the CDR regions This allows antibodies to be humanized to a greater extent than simple CDR grafting. .

[0023] In the present invention, SEQ ID NO:13 has a similar structure to CDR2 (SEQ ID NO:6) of SEQ ID NO:1. In the case of There is a KQ substitution at position 16 of SEQ ID NO:6 and a DG substitution at position 17 of SEQ ID NO:6.

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

[0025] These humanized sequences do not represent distinct, selective antibodies when compared to the parent antibody. Instead, we used structural modeling to more closely resemble the human germline to minimize immunogenicity. It is understood that the present invention relates to the same antibody having the same properties, but modified to correspond to the , as will be understood by those skilled in the art.

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

[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 comprises a framework sequence. In an embodiment, at least a portion of the framework sequence is a human consensus framework. In some embodiments, the light chain variable region comprises a framework sequence. In some embodiments, at least a portion of the framework sequences is It contains consensus framework sequences.

[0028] Also included are substitutions of residues in the antibody lower hinge, such as L234A and L235A (LALA). Alternative strategies to mitigate antibody effector functions have also been employed, including These residues form part of the Fc-γ receptor binding site on the CH2 domain, and The exchange between antibody isotypes identifies their importance in ADCC, and a greater or had smaller effector functions. Alanine substitutions at these positions were Although these substitutions are effective in reducing ADCC in both human and mouse antibodies, is less effective at 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 The marker is an Fc mutant engineered to reduce binding to the Fc gamma receptor. , thereby resulting in reduced effector function and ADCC activity.

[0029] In some embodiments, the antibody, or antigen-binding fragment, is Fc silenced. Engineered IgG1 antibodies that have been modified to reduce binding to one or more Fc receptors. In another embodiment, the antibody is an antigen-binding fragment having either I or no I. It is a gG4 antibody.

[0030] In some embodiments, the antibody, or antigen-binding fragment, is a T cell cytotoxic Bispecific antibodies or antigen-binding fragments mediating cytotoxicity and / or NK cytotoxicity - Patents.com In some embodiments, the antibody or antigen-binding fragment is In one embodiment, immune cells are preferably In another embodiment, the immune cells are preferably NK cells. The terms induction and / or enhancement refer to induction and / or enhancement of cytokine release by immune cells. and / or induce and / or enhance the proliferation of said immune cells, and / or or inducing and / or enhancing cell killing activity. As used in this context, the term "induce" or "induction" It is believed that the antibody or antigen-binding fragment may cause activation of immune cells or inhibit the inactivation of the antibody or antigen-binding fragment. This means increasing immune cell activation above the activation levels seen in the presence of It will be readily apparent to one of ordinary skill in the art that "enhancing" as used in this context means The term refers to increasing the activation level of an already activated immune cell.

[0031] In some embodiments, the antibody or antigen-binding fragment is binds to Pak (e.g., SEQ ID NO: 11) and binds to one or more additional binding targets; A bispecific or multispecific antibody or antigen-binding fragment, preferably The additional binding target is one or more tumor antigens. The above further binding targets are immunomodulatory molecules. In one embodiment, the additional binding target is P D-L1. In one embodiment, the antibody or antigen-binding fragment thereof is bivalent. In another embodiment, the antibody or antigen-binding fragment thereof is tetravalent. In the present invention, the antibody or antigen-binding fragment thereof is trivalent.

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

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

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

[0035] Aspects of the invention include methods of making an antibody, or an antigen-binding fragment thereof, comprising the steps of: The method comprises incubating a host cell under conditions such that the antibody, or antigen-binding fragment, is expressed in the host cell. and optionally isolating the antibody or antigen-binding fragment. .

[0036] An aspect of the invention is a method for the treatment of cancer, comprising administering to a patient an antibody or antigen-binding fragment as described herein, and administering to a patient a therapeutically effective amount of the antibody or antigen-binding fragment. and a physiologically acceptable carrier. In certain embodiments, the pharmaceutical composition or medicament further comprises an effective amount of a second therapeutic agent.

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

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

[0039] In one embodiment, the antibody or antigen-binding fragment comprises a CDR-H1 comprising SEQ ID NO:5. , CDR-H2 comprising SEQ ID NO:6 or SEQ ID NO:15, and CDR- A heavy chain variable region comprising H3 and 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. and a light chain variable region comprising

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

[0041] Preferably, the light chain variable region comprises CDR-L1 comprising SEQ ID NO: 16, SEQ ID NO: 17 CDR-L2, and CDR-L3 comprising SEQ ID NO:10.

[0042] In some embodiments, a method of treating cancer is provided, wherein a person in need thereof The antibody or antigen-binding fragment of the invention is administered to a patient suffering from said disease, The antigen-binding fragment may be used to induce an immune response, such as a cytotoxic T cell response and / or a NK cell response. Inducing and / or enhancing a cellular response.

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

[0044] In some embodiments, the cancer is small cell lung cancer, non-small cell lung cancer (squamous cell carcinoma and adenocarcinoma). cancer), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric ( ric) cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, cancer Glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, sarcoma, GIST, neuroendocrine secretory tumors, and hematological malignancies.

[0045] According to a further aspect of the invention, there is provided an antibody or a composition according to the invention for use in prophylaxis or therapy. Antigen-binding fragments are provided.

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

[0047] According to a further aspect of the invention, an antibody according to the invention in the manufacture of a medicament for the treatment of cancer. Uses of the antibody or antigen-binding fragment thereof are provided. In some embodiments, the cancer according to the previous aspects is small cell lung cancer, non-small cell lung cancer. (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), Colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, renal cancer, liver cancer including hepatocellular carcinoma visceral cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric (stomac) h) Cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, meat The tumor is selected from the group consisting of a gastrointestinal tumour, a gastrointestinal tumour, a neuroendocrine tumour and a hematological malignancy. [Brief description of the drawings]

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

[0049] Embodiments of the invention include antibodies against SLAMF6 and nucleic acids encoding such antibodies. Host cells containing such nucleic acids encoding the antibodies of the present application, and methods for preparing anti-SLAMF6 antibodies are also provided. and methods for treating 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, uterine cancer, prostate cancer, renal cancer, liver cancer including hepatocellular carcinoma visceral cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric (stomac) h) Cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, meat Hematologic malignancies, including but not limited to, gastrointestinal tumours, gastrointestinal stromal tumors, neuroendocrine tumours and hematological malignancies. and methods for treating diseases such as cancer in humans.

[0050] As used in this specification and the appended claims, the singular forms "a," "an ... "An" and "the" are used interchangeably unless the context clearly indicates otherwise. Please note that the claims may include multiple references. Note further that it can be drafted to exclude primes. This statement may be used in conjunction with the recitation of claim elements, such as "sole," "only," or the like. as a predicate basis for the use of exclusive terminology or "negative" limitations It is intended to be helpful.

[0051] As will be apparent to those skilled in the art upon reading this disclosure, the Each of the individual embodiments may be combined with any of the other embodiments without departing from the scope or spirit of the invention. The features of any of the embodiments may be easily separated or combined into separate components and Any of the recited methods have the following characteristics: It may be performed in any other order possible.

[0052] definition For the purposes of interpreting this specification, the following definitions shall apply and, whenever appropriate, shall be used in the singular: Terms used also include the plural and vice versa.

[0053] The term "SLAMF6" as used herein means a primate mammals, such as mammals (e.g., humans, primates, and rodents (e.g., mice and rats) Refers to any naturally occurring SLAMF6 protein from any vertebrate source, including mammals. SLAM The F6 protein is also called the SLAMF6-like protein. The sequence is provided in SEQ ID NO:11 herein.

[0054] The term "SLAMF6" refers to the "full-length" unprocessed SLAMF6 as well as the intracellular protease This term also encompasses any form of SLAMF6 resulting from sessing. Naturally occurring variants of 6, such as splice variants, allelic variants and isoforms The term also encompasses naturally occurring truncated or unsaturated forms of the SLAMF6 polypeptide. or secreted (e.g., extracellular domain sequences). The polypeptides may be isolated from a variety of sources, such as human tissue types or from another source. The native sequence SL can be prepared by recombinant or synthetic methods. "AMF6 polypeptide" refers to a polypeptide that has the same amino acid sequence as the corresponding naturally occurring SLAMF6 polypeptide. Such native sequence SLAMF6 polypeptides include polypeptides having the following sequence: It can be isolated from nature or produced by recombinant or synthetic means. As used herein, the term "SLAMF6 epitope" refers to any of the SLAMF6 epitopes described herein. and / or is bound by an antibody that contains at least one or more of the CDR sequences. The epitope is exemplified by the binding profile of the anti-SLAMF6 antibody as shown in the Examples. Refers to taupe.

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

[0056] In other embodiments, the term "antibody" as used herein includes, but is not limited to, Nano Includes body®, Unibody® and scFv fragments , it will be understood to refer to a structure that does not include the six CDRs.

[0057] "Anti-SLAMF6 antibody", "SLAMF6 antibody", or "Antibody that binds to SLAMF6" The term "antibody" refers to a compound useful as a diagnostic and / or therapeutic agent that targets SLAMF6. The term "antibody" refers to an antibody that can bind to SLAMF6 with sufficient affinity such that the antibody can bind to SLAMF6 with sufficient affinity to In this study, anti-SLAMF6 antibodies were used to identify SLs that are conserved among SLAMF6 from different species. Binds to an epitope on AMF6.

[0058] An "isolated antibody" is one that has been identified and separated from a component of its environment, and / or Contaminants in the environment are materials that may interfere with the therapeutic use of the antibody. and can contain enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Cut.

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

[0060] The term "antagonist" is used in the broadest sense and refers to a naturally occurring SLAMF6 polypeptide. Any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of a Suitable antagonist molecules include, in particular, antagonist antibodies or antibody fragments. Segments, native SLAMF6 polypeptides, peptides, antisense oligonucleotides , fragments or amino acid sequence variants of small organic molecules. Methods for identifying antagonists of MF6 polypeptide include the steps of: The polypeptide is contacted with a candidate antagonist molecule, and one that is normally associated with the SLAMF6 polypeptide is detected. The method may include measuring a detectable change in the above biological activity.

[0061] The term "agonist" is used in the broadest sense and refers to any naturally occurring SLAMF6 polypeptide. Suitable agonist molecules include any molecule that enhances the biological activity of a stimulant. F6 ligand polypeptides, peptides, antisense oligonucleotides, small organic molecules Agonist antibodies or antibody fragments, fragments or amino acid sequence variants, such as antibodies Specifically included are variants. Methods for identifying agonists of SLAMF6 polypeptides The method comprises contacting the SLAMF6 polypeptide with a candidate agonist molecule, and The method may include measuring a detectable change in one or more biological activities normally associated with the peptide. .

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

[0063] As used herein, the terms "predictive" and "prognostic" also refer to a predictive or prognostic variable. The method for determining whether or not the subject is a person who performs the method includes performing a treatment with an anticancer drug containing an anti-SLAMF6 antibody. Patients who are deemed more likely to respond to treatment (usually, but not necessarily, prior to treatment) are interchangeable in cases where it means to be able to choose .

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

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

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

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

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

[0069] The amino-terminal portion of each antibody chain contains approximately 100 to 110 or more amino acids that are primarily involved in antigen recognition. The variable regions contain amino acid variable regions for each of the heavy and light chain V domains. The three loops are assembled to form the antigen-binding site. Each loop is a complementarity determining region (hereafter, The most notable variation in amino acid sequence is called "CDR". This means that certain segments of the variable region differ significantly in sequence among antibodies. The variability in V regions is not evenly distributed. Instead, V regions are made up of 15–30 amino acids. Each of these regions consists of relatively constant extension regions called framework regions (FRs), each of which has nine A short region of extreme variability called a "hypervariable region" consisting of ~15 amino acids So they are separated.

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

[0071] The hypervariable region generally consists of 24-34 CDRs (CDR-L1; "L" stands for light chain variable region) in the light chain. heavy chain variable region), 50-56 (CDR-L2) and 89-97 (CDR-L3) Approximately 31-35B (CDR-H1; "H" is the heavy chain, 50-65 (CDR-H2) and amino acid residues from 95-102 (CDR-H3), Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Ins. titutes of Health, Bethesda, Md. (1991) and / or hypervariable loops (e.g., light chain Residues 26-32 (CDR-L1), 50-52 (CDR-L2) in the variable region and 91-96 (CDR-L3), and 26-32 (CDR-H1) in the heavy chain variable region , 53-55 (CDR-H2) and 96-101 (CDR-H3) residues, Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. Typical CDRs are listed below.

[0072] Throughout this specification, the Kabat numbering system is used to number the residues in the variable domain ( Approximately residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region) It is commonly used when appropriate (see, for example, Kabat et al., (1991), supra).

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

[0074] The IgG subclass of immunoglobulins contains several immunoglobulin domains in the heavy chain. As used herein, an "immunoglobulin (Ig) domain" refers to an immunoglobulin having a distinct tertiary structure. Of interest in the present invention is the constant heavy chain (CH) In the context of IgG antibodies, the heavy chain domain is the I domain, which includes the I domain and the hinge domain. Each gG isotype has three CH regions.

[0075] Another type of Ig domain in the heavy chain is the hinge region. or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" A flexible polynucleotide comprising amino acids between the first and second constant domains of an antibody. It means a peptide.

[0076] Of particular interest in the present invention is the Fc region. or "Fc region" or "Fc domain" refers to the first constant region immunoglobulin domain. and a polypeptide comprising the constant region of an antibody, optionally excluding a portion of the hinge. Thus, Fc is the last two constant region immunoglobulins of IgA, IgD, and IgG. the last three constant region immunoglobulin domains of IgE and IgM; and the flexible hinge N-terminal to these domains. IgA and IgM For IgG, the Fc domain can include the J chain. Globulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and Cγ1 (Cγ1) The boundaries of an Fc region may vary, but may also include the lower hinge region between Cγ2 and Cγ3. The human IgG heavy chain Fc region typically comprises residues C226 or P23 towards its carboxyl terminus. 0, where the numbering is defined as the EU index as in Kabat. In some embodiments, the amino acid modification is, for example, in one or more FcγR Modifications have been made to the Fc region to alter binding to the FcRn or FcRn receptors. can be.

[0077] In some embodiments, the antibody is full length. As used herein, a "full length antibody" is The present invention relates to a method for the production of a medicament ... By "antibody" is meant the structures which constitute the natural biological formation of an antibody, including

[0078] Alternatively, the antibody may be an antigen-binding fragment, a monoclonal antibody, a bispecific antibody, a mitochondria, or a mitogen-activated antibody. Nibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics") antibodies), chimeric antibodies, humanized antibodies, antibody melts (also called "antibody conjugates") and various antigen-binding fragments thereof, structures that depend on the use of a set of CDRs are within the definition of "antibody." Included.

[0079] In one embodiment, the antibody is an antigen-binding fragment. The fragments include (i) a Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) a single (iv) an Fv fragment consisting of the VL and VH domains of a single antibody; dAb fragments (Ward et al., 1989, Nature 34, incorporated by reference in its entirety) 1:544-546), (v) isolated CDR regions, (vi) F(ab')2 fragments, (vii) a bivalent fragment comprising two linked Fab fragments, (vii) a single chain Fv molecule ( scFv), in which the VH domain and the VL domain are bound to each other to form an antibody linked by a peptide linker that allows the formation of an original binding site (see Bird et al., 1988, Science 242:423-426, Huston et al., 1 988, Proc. Natl. Acad. Sci. USA 85:5879-5883), (viii) Bispecific single strand Fv (WO 03 / 11161, which is incorporated by reference in its entirety) and (ix) "Diabodies" or "Triabodies", multivalent or Multispecific fragments (Tomlinson et al., incorporated by reference in their entirety). al., 2000, Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc . Natl. Acad. Sci. USA 90:6444-6448).

[0080] Chimeric and Humanized Antibodies In some embodiments, the antibodies are of different species, e.g., chimeric antibodies and / or That is, in the present invention, the CDR set may be a mixture of humanized antibodies. With framework and constant regions other than those specifically described by the sequences in the specification. can be used for.

[0081] In general, "chimeric antibodies" and "humanized antibodies" refer to antibodies that combine regions from more than one species. For example, "chimeric antibodies" are typically derived from mice (or rats, as the case may be). A "humanized antibody" generally refers to an antibody that contains variable regions from a human antibody and constant regions from a human. A non-human antibody having variable domain framework regions exchanged for sequences found in human antibodies. Generally, in a humanized antibody, the entire antibody except for the CDRs is made from a polynucleotide of human origin. or identical to such an antibody except within its CDRs. The CDRs are encoded in part or in whole by nucleic acid derived from a non-human organism. The antibody is then grafted onto the beta-sheet framework of a human antibody variable region to produce an antibody. The specificity is determined by the grafted CDR. The production of such antibodies is described, for example, in WO 9 2 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:15 34-1536, all of which are incorporated by reference in their entirety. In this regard, the antibodies of the present invention may be multispecific antibodies, particularly bispecific antibodies, and are sometimes referred to as "diabodies." These are called "cell bodies." These are cells that react with two (or more) different antigens, or with the same antigen. Diabodies are antibodies that bind to different epitopes on the same antigen. Various methods (see Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449) The nucleophilic substance can be produced by, for example, chemical or hybridization. It can be produced from a lid hybridoma.

[0082] In one embodiment, the antibody is a minibody. Minimize antibody-like proteins, including bound scFvs. Hue et al., et al., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv is linked to the Fc region. The minibody may include a portion or the entire hinge region. Although they do not have a complete set of DRs, they are included within the definition of an "antibody." Please note.

[0083] The antibodies of the present invention are generally isolated or recombinant. In some embodiments, the antibodies of the invention are recombinant proteins, isolated proteins or A substantially pure protein. An "isolated" protein is one that is normally present in its native state. free of at least some of the associated substances, e.g., less than 10% of the total protein in a given sample Constitutes at least about 5% by weight, or at least about 50% by weight. It is understood that, depending on the circumstances, the total protein content may comprise 5-99.9% by weight. For example, proteins can be expressed using inducible or high expression promoters. Thus, it can be produced at a much higher concentration, so that the protein is produced at the increased concentration levels. In the case of a recombinant protein, the definition is that it is produced in the art where it is not naturally produced. This includes the production of antibodies in a wide variety of organisms and / or host cells known in the art. The isolated polypeptide is prepared by at least one purification step. "Antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, The isolated antibodies that specifically bind to SLAMF6 are not specific for antigens other than SLAMF6. Substantially free of bound antibody.

[0084] Isolated monoclonal antibodies with different specificities are available in well-defined compositions. Thus, for example, the antibodies of the invention may be combined with any of the antibodies discussed further below. As described above, the active ingredient may be optionally and individually included or excluded from the formulation.

[0085] Specific binding to a particular antigen or epitope is, for example, at least about 10 -4 M , at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, less About 10 -8 M, at least about 10 -9 M, or at least about 10 -10 M, small At least about 10 -11 M, at least about 10 -12 For antigens or epitopes of M or more K D In one embodiment, the antibody has the formula: D specific antibody-antigen interactions Typically, an antibody that specifically binds to an antigen has a higher dissociation rate compared to a control molecule. 20-, 50-, 100-, 500-, 1000-, 50 for antigen or epitope 00-, 10,000-fold lower K D would have.

[0086] Specific binding to a particular antigen or epitope can also be achieved by, for example, A or K a but When referring to the association rate of a particular antibody-antigen interaction, it refers to a decrease in the binding of an antibody to an epitope compared to a control. At least 20-, 50-, 100-, 500-, 1000-, 5000-, 10,000 -fold or greater K for an antigen or epitope A or K a By showing by the antibody having can be done.

[0087] The standard assay to assess the binding ability of an antibody to SLAMF6 is protein immunoassay. or at the cellular level, e.g., ELISA, Western blot, RIA , Octet®, BIAcore® Assays and Flow Cytometry Suitable assays include, but are not limited to, the Tree analysis and are known in the art. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of the antibodies can also be determined using Biacore® or According to standard assays known in the art, such as by Octet® system analysis, can be evaluated more precisely.

[0088] SLAMF6 antibody The present invention provides SLAMF6 antibodies that bind to SLAMF6 polypeptides or portions thereof. An example of an SLAMF6 amino acid sequence is provided in SEQ ID NO: 11. The AMF6 antibody inhibits immune cell activation, e.g., T These can induce or enhance cell activation and / or NK cell activation. The antibody is referred to herein as an "anti-SLAMF6" antibody, or, for ease of exposition, as an "S It is called "LAMF6 antibody."

[0089] In some embodiments, the subject SLAMF6 antibodies bind to T cells, particularly to SLAs on their surface. Contact with MF6-expressing CD4+ or CD8+ T cells induces cytokine release or In this context, cytokine release or or T cell proliferation can be measured in several ways. In one embodiment, The SLAMF6 antibodies of the invention can be used to detect the activity of activated T cells using standard assays such as ELISA. In a further embodiment, the subject SLAMF6 antibody is contacted with and killing can be induced and / or enhanced.

[0090] In one embodiment, the antibody is an antibody that comprises the following CDRs; In addition, these CDR sequences can also be modified with a limited number of amino acid variants, as previously described. May contain: [Table 2]

[0091] In some embodiments, the antibody is selected from the group consisting of SEQ ID NOs: 5, 15, 7, 16, 17 and 10. In some embodiments, the amino acid sequence of at least one of the CDR sequences provided in In some embodiments, the antibody comprises the CDRs provided in SEQ ID NOs: 5, 15, 7, 16, 17 and 10. At least about 75%, 80%, 85%, 90%, It comprises an amino acid sequence that is 95%, 96%, 97%, 98% or 99% identical.

[0092] Provided herein are variable heavy and light chains comprising the CDR sets of the invention, as well as full length heavy and light chains. The light and light chains (e.g., including constant regions) are also disclosed. The disclosed CDR set may comprise a murine, humanized or human constant region (including framework regions). An embodiment of the present invention can be incorporated into the heavy chain variable region sequences disclosed herein. 13) and at least about 80%, 85%, Heavy chain variable regions and light chain variable regions that are 90%, 95%, 96%, 97%, 98% or 99% identical It contains the chain variable region.

[0093] In some embodiments, the present invention provides a method for the treatment of a SLAMF6 of the invention described herein. Monoclonal antibodies (i.e., the monoclonal antibodies of the invention described herein and S As an antibody that has the ability to cross-compete for binding to the LAMF6 protein, human S To provide antibodies that bind to or cross-compete with the same epitope on LAMF6. For antibodies that may cross-compete, blocking of the reference antibody may not be complete. It will be understood that the present invention does not necessarily block the The binding of the antibody is at least about 10, 20, 30, 40, 50, 60, 70, 75, 80, Decreased by 85, 90, 95, 97, 98 or 99%.

[0094] antibody modification The present invention further relates to mutant antibodies, sometimes referred to as "antibody derivatives" or "antibody analogs." That is, amino acid modification in the CDR (affinity maturation), Amino acid modifications, glycosylation variants, and other types of covalent modifications (e.g., drug The antibodies of the present invention may be subjected to various methods, including, but not limited to, methods for the binding of conjugates, etc. There are many modifications that can be made.

[0095] A "variant" is a polypeptide that differs from the sequence of a parent polypeptide by at least one amino acid modification. In some embodiments, the parent polypeptide refers to a polypeptide sequence comprising the sequence Any of the full length variable heavy or light chains listed in columns 1 or 2, 13 or 14 or any of SEQ ID NOs: 5 to 10, 15, 16 or 17. In some embodiments, the amino acid modification is one or more of the following: Substitutions, insertions and / or deletions may be included, the former being often preferred. In some embodiments, the substitutions may be conservative substitutions.

[0096] Generally, the variants will retain the functionality of the antibody as described herein. For example, the antibody may be specific for human SLAMF6. Similarly, if amino acid variants are made in the Fc region, e.g. The variant antibody should maintain the receptor binding function necessary for the particular use or indication of the antibody. be.

[0097] A "variant" of the subject antibody, as described herein, is any one of the CDR sequences listed. one or more, one or more framework regions, or one or more defined regions of an antibody Having amino acid mutations in any of the constant regions (e.g., the Fc region) can be done.

[0098] In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, Nine or ten amino acid modifications are commonly utilized, with the goal being to produce a product that functions with the minimum number of modifications. In some embodiments, the modification is one to five (1, 2, 3 1, 4 or 5) modifications (e.g., individual amino acid substitutions, insertions and / or deletions). Also, 1-2, 1-3 and 1-4 will find use in many embodiments. For example, in some embodiments, one or more of the CDR sequences of an antibody of the invention comprises one or more of: For example, 1, 2, 3, 4 or 5 amino acid modifications, preferably 1-4, 1-3, 1 or Each of the modifications may contain two modifications. Generally, no more than 4, 5, 6, 7, 8, 9, or 10 modifications. The changes are made within the set of CDRs.

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

[0100] As used herein, an "amino acid substitution" or "substitution" refers to an amino acid that is introduced at a particular position in a parent polypeptide sequence. As used herein, "amino acid" refers to the replacement of an amino acid at one position with another amino acid. "Amino acid insertion" or "insertion" refers to the addition of an amino acid at a particular position in a parent polypeptide sequence. As used herein, an "amino acid deletion" or "deletion" refers to a deletion of a parent polypeptide. It refers to the removal of an amino acid at a particular position in a peptide sequence.

[0101] As used herein, "parent polypeptide," "parent protein," and "precursor polypeptide" refer to or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. Generally, the parent polypeptide as used herein refers to the 1B3 polypeptide. Chid, for example 1B3 V H Chain or V l Alternatively, it may refer to the CDR sequence. Thus, as used herein, a "parent antibody" refers to an antibody that has been modified to generate a variant antibody. The antibody is a marker for the antibody that is to be labeled with the antibody.

[0102] As used herein, "wild type" or "WT" or "naive" refers to an allele that is WT means an amino acid sequence or a nucleic acid sequence found in nature, including genetic variations. Pak, polypeptide, antibody, immunoglobulin, IgG, etc., not intentionally modified It has an amino acid sequence or a nucleic acid sequence.

[0103] As used herein, a "mutant (mutant) Fc region" refers to a region that has at least one amino acid modification. An Fc variant refers to an Fc sequence that differs from the wild-type Fc sequence due to a mutation. It may refer to the polypeptide itself, a composition comprising the Fc variant polypeptide, or an amino acid sequence.

[0104] In some embodiments, the anti-SLAMF6 antibodies of the invention comprise a variant Fc domain. As is known in the art, the Fc region of an antibody is composed of multiple Fc receptors. They interact with targets and ligands and exert an array of important functional capabilities called effector functions. Suitable modifications may be made at one or more positions to reduce or enhance binding, particularly to Fc receptors. can be made to silence specific amino acid substitutions.

[0105] In addition to the modifications outlined above, other modifications can be made. For example, the molecule can be modified to and VL domains (see references). (Reiter et al., 1996, Nature Biotech. 14:1239-1245) .

[0106] Additionally, modifications at cysteines may be used in antibody-drug conjugates (ADCs), as described further below. In some embodiments, the constant region of the antibody is a One that is specifically "thiol-reactive" to allow for more specific and controlled placement. The polypeptides can be engineered to contain one or more cysteines, see, for example, the entire disclosure of which is incorporated herein by reference. See U.S. Pat. No. 7,521,541, which is incorporated herein by reference.

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

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

[0109] bispecific molecule In another aspect, the invention includes an anti-SLAMF6 antibody or fragment thereof of the invention. The antibodies or antigen-binding fragments thereof of the present invention include bispecific and multispecific molecules. The fusion protein may be a functional molecule, such as a peptide or protein (e.g., a receptor) that binds to another molecule. derivatized or linked to one or more of the following molecules (antibodies or ligands) to form two distinct binding sites or target molecules: In some embodiments, bispecific molecules can be generated that bind to The disclosed antibody, or antigen-binding fragment thereof, comprises at least two functional molecules, e.g., Induced by other peptides or proteins (e.g., other antibodies or ligands for the receptor) Multispecific molecules that bind to at least three different binding sites or target molecules when combined or linked together To generate bispecific or multispecific molecules of the invention, To this end, the antibodies of the present invention may be functionally (e.g., by chemical coupling, genetic fusion, non-covalent bonding) or otherwise) to generate a bispecific or multispecific molecule. Linked to one or more other binding molecules, such as antibody fragments, peptides, or binding mimetics. It can be concluded.

[0110] Thus, the present invention provides a method for the production of a first target epitope (i.e., SLAMF6) At least one first binding domain and a second binding domain for a second target epitope. The second target epitope is bound by the first binding specificity. The second target epitope may be present on the same target protein as the one bound, or may be may be present on a target protein different from that bound by the first binding specificity. The second target epitope is located in the same cellular compartment as the first target epitope (i.e., SLAMF6). The second target epitope may be present on a cell that exhibits the first target epitope. As used herein, the term "binding "Specificity" refers to a moiety that comprises at least one antibody variable domain.

[0111] In another embodiment of the invention, the second target epitope is present on a tumor cell. Thus, an embodiment of the invention provides an SLAMF6-expressing effector cell (e.g., an SLAMF6-expressing cell Binding to both cytotoxic T lymphocytes and tumor cells expressing a second target epitope The present invention includes bispecific molecules that can

[0112] In one embodiment, a bispecific antibody of the invention comprises a total of two or three antibody variable domains. The bispecific antibody can have a human immune effector domain, wherein the first portion of the bispecific antibody is a human immune effector domain. It specifically binds to effector antigens located on effector cells to produce human immune effects. The effector antigen is SLAMF6, which can recruit the activity of effector cells. The first part consists of at least one antibody variable domain and the second part of the bispecific antibody is capable of specifically binding to a target antigen other than an effector antigen, the target antigen being a human located on a target cell other than an immune effector cell, and the second portion is linked to at least one antibody Contains a variable domain.

[0113] In embodiments of the invention in which the binding protein is multispecific, the molecule has anti-tumor, binding specificity In addition to the anti- and anti-SLAMF6 binding specificities, the antibody may further comprise a third binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) moiety, e.g., Binding to surface proteins involved in cytotoxic activity, thereby inducing an immune response against target cells An "anti-enhancement factor moiety" is an antibody, a functional antibody fragment, a , which binds to a molecule, such as an antigen or receptor, thereby determining the binding to the target cell antigen. The "anti-enhancement factor moiety" may be a ligand that provides an enhancement of the effect of a group that binds to a target cell antigen. Alternatively, the anti-enhancement factor portion may bind to an entity to which the first and second binding specificities bind. (entity) can be bound to a different entity. For example, the anti-enhancement factor moiety can be Cytotoxic T cells (e.g., CD2, CD3, CD4, CD5, CD6, CD8, CD9, CD10, CD11, CD12, CD13, CD14, CD15, CD16, CD17, CD18, CD19, CD20, CD21, CD22, CD19, 3. via CD8, CD28, CD4, CD40, ICAM-1 or other immune cells) can be combined with

[0114] In one embodiment, a bispecific protein of the invention has at least one binding specificity Each of the antibodies, or antigen-binding fragments thereof (e.g., Fab, Fab', F(a b') 2, including Fv, FVTCR, Fd, dAb or single chain Fv. Also, light or heavy chain dimers, or any minimal fragments thereof, such as those described in U.S. Pat. It may be an Fv or single chain construct as described in US Pat. No. 4,946,778. , the contents of which are expressly incorporated by reference.

[0115] In some embodiments, antibodies that can be used in the bispecific molecules of the invention include The monoclonal antibody may be a rat, mouse, human, chimeric or humanized monoclonal antibody.

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

[0117] In one embodiment of the invention, the bispecific antibody contains four antigen-binding regions. In a preferred embodiment, the antibody is a tetravalent antibody. The IgG1 domains each consist of a heavy chain and a light chain Fab region. Antibodies are arranged in the same conformation as Fabs. They also contain two fragments that target a second antigen. These comprise chimeric Fab domains, each of which comprises a chimeric " It consists of two chimeric polypeptide domains including a "heavy" chain, which are connected via their C-terminus to The N-terminus of the constant region of the alpha or beta chain of the T cell receptor (TCR), and possibly The chimeric "light" chain contains a variable light domain, which is linked via its C-terminus to the TCR. They are linked to the N-terminus of the beta or alpha chain constant region, respectively. The chimeric "heavy" and "light" chains are arranged in a chimeric Fab domain. The C-terminus of the constant region of the TCR alpha or beta chain of the heavy chain is It is linked to the native Fab domain by binding to the N-terminus of the main heavy chain variable region. Thus, the overall symmetric structure allows each of the two different antigens to be bound in a bivalent manner. Thus, a bispecific antibody is generated that targets a natural F that targets a first antigen. The ab domain and a chimeric Fab domain targeting a second antigen are used to produce such tetravalent antibodies. Present on both arms of the body.

[0118] In another embodiment, the chimeric Fab domain is located proximal to the Fc domain, such that The C-terminus of the constant region of the α or β chain of the TCR containing the chimeric "heavy" chain is the natural Hin The native Fab domain is located at the tip of the Fc domain, resulting in The C-terminus of the CH1 domain of a heavy chain containing a natural Fab domain may contain a chimeric Fab domain. It binds to the N-terminus of the variable heavy chain.

[0119] In another embodiment, an asymmetric trivalent format composed of two different antibody arms is Thus, one arm has a single Fab domain (natural or chimeric). The second arm of the bispecific antibody is a Fab domain (natural and chimeric) as described above. Heterodimerization of two different arms is known in the art (e.g., knob-in As described in (knobs-into-wholes, electrostatic steering, etc.) In particular, this is made possible through antibody engineering in the Fc domain.

[0120] In yet another embodiment, an asymmetric bivalent form of the antibody is formed from two different antibody arms. Using a kit, one arm has a single Fab domain, natural or chimeric, and the other Each arm may have a single Fab binding domain, chimeric or native. Heterodimerization of two different arms is well described in the art. Domain-specific antibody engineering (e.g., knobs-into-holes, This is possible via electrostatic steering, etc.

[0121] Glycosylation Another type of covalent modification is altered glycosylation. A glycosylated antibody can be made (ie, the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be, for example, at least one site of glycosylation within the antibody sequence. This can be achieved by varying the position of one or more variable region frames. This results in the removal of the murk glycosylation site, thereby preventing glycosylation at that site. One or more amino acid substitutions can be made to remove glycosylation. This approach may increase the affinity of the antibody for the target protein. and is described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861. This can be achieved by removing the asparagine at position 297.

[0122] Other types of covalent modifications of antibodies include, for example, those described in the literature, all of which are fully incorporated by reference. Nektar Therapeutics' 2005-2006 PEG Catalog (available on the Nektar website), U.S. Patent No. 4,640,835, No. 4,496,689, No. 4,301,144, No. 4,6 70,417, 4,791,192, or 4,179,337 The method is characterized in that polyethylene glycol, polypropylene glycol or polyoxyethylene glycol is used. Various non-proteinaceous polyols, including, but not limited to, various polyols such as xylene. In addition, as is known in the art, the amino acid substitutions may be modified to modify the antibody. Conversions can be performed at various positions within the antibody to facilitate addition of polymers such as PEG. See, for example, U.S. Patent Application Publication No. 2005 / 0114, which is incorporated by reference in its entirety. See issue 037.

[0123] In further embodiments, the use of the antibodies of the invention, e.g., for diagnostic or detection purposes. In the present specification, the antibody may include a label. As described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland At least one moiety, element, or analogue is attached to enable detection of such a compound. It means having a position or chemical compound.

[0124] Methods for producing the antibodies of the present invention The present invention further provides methods for producing the disclosed anti-SLAMF6 antibodies. These methods involve culturing host cells containing isolated nucleic acid encoding an antibody of the invention. As will be appreciated by those skilled in the art, this can be accomplished in a variety of ways, depending on the nature of the antibody. In some embodiments, the antibodies of the invention are full-length traditional antibodies. In such cases, for example, the host cells can be cultured under conditions such that the antibody can be produced and isolated. , containing nucleic acids encoding a heavy chain variable region and a light chain variable region.

[0125] The variable heavy and light chains of the antibodies of the invention are disclosed herein (protein and nucleic acid sequences). as will be appreciated in the art, these can be readily augmented to produce full length heavy chains and That is, as outlined herein, the V H and V When DNA fragments encoding the L segment are provided, these DNA fragments For example, the variable region genes are converted to full-length antibody chain genes, Fab fragment genes, or further engineered by standard recombinant DNA techniques to convert it into an scFv gene. In these operations, VL or V H Contains a nucleic acid encoding The DNA fragment can then be used to encode another protein, such as an antibody constant region or a flexible linker. It is operably linked to another DNA fragment that encodes The term "operably linked" as used herein means that the linker is a DNA fragment. The two DNA fragments are aligned so that the amino acid sequences being loaded remain in frame. are concatenated.

[0126] V H The isolated DNA encoding the region is H The DNA encoding the heavy chain constant region Another DNA molecule (C H 1. C H 2 and C H 3) operatively connected to Thus, it can be converted into a full-length heavy chain gene. The sequence of the rat heavy chain constant region gene is As known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Serv ices, NIH Publication No. 91-3242), DNA fragments encompassing these regions, The heavy chain constant region can be obtained by standard PCR amplification. It may be an IgG3, IgG4, IgA, IgE, IgM or IgD constant region. In a preferred embodiment, the heavy chain constant region is an IgG1 or IgG4 constant region. For the b-fragment heavy chain gene, V H The DNA encoding the heavy chain C H 1 constant region only It can be operably linked to another DNA molecule encoding the

[0127] The isolated DNA encoding the VL region is L The DNA encoding the light chain constant region Another DNA molecule that encodes C L The full-length light chain gene is operably linked to (and Fab light chain gene). The sequence of the rat light chain constant region gene are known in the art (see, e.g., Kabat, EA, et al. (1991) Sequences of Proteins ins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), DNA flags encompassing these regions The fragment can be obtained by standard PCR amplification. In one preferred embodiment, The light chain constant region is a kappa or lambda constant region.

[0128] To generate a polynucleotide sequence encoding an scFv antibody fragment, H - and VL coding DNA fragments are VL and V H The sequence is a flexible linker VL and VL connected by H Expressed as a continuous single-chain protein with Another fragment encoding a flexible linker, e.g., the amino acid sequence (Gly4-Ser)3 , Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0129] Aspects of the invention include nucleic acids that encode the antibodies of the invention. The sequences encode both the variable and constant regions of each of the heavy and light chains, but are not limited to those described herein. Other combinations are contemplated by the present invention in accordance with the compositions described. and oligonucleotide fragments derived from the polynucleotides selected from these polynucleotides. It includes a nucleic acid sequence complementary to the nucleotide sequence.

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

[0131] In some embodiments, the nucleic acid encoding an antibody of the invention is extrachromosomal or or an expression vector that can be designed to be integrated into the genome of a host cell into which it is introduced. Expression vectors can include any number of suitable regulatory sequences, including transcriptional and translational control sequences. These include sequences, promoters, ribosome binding sites, enhancers, and replication origins. These may contain, but are not limited to, the following: All are operably linked as is known in the art. Nucleic acids are used, each in a different expression vector (e.g., a duplicate in a first expression vector) the light chain in a second expression vector) or The design of the expression vector, including the selection of regulatory sequences, can be influenced by the choice of host cell. It will be appreciated by those skilled in the art that the amount of protein expression that can be achieved may depend on factors such as the level of expression of the desired protein. .

[0132] Generally, the nucleic acid and / or expression is carried out in any manner appropriate to the selected host cell (e.g., transformation, transfection, electroporation, infection) to produce recombinant hosts The nucleic acid molecule can be introduced into a suitable host cell to produce a cell, such that the nucleic acid molecule is The above expression control elements (e.g., in a vector, a construct generated by processing in a cell) The resulting recombinant host cell is operably linked to a vector that is integrated into the host cell genome. The subject cells are cultured in a suitable non-human animal under conditions suitable for expression (e.g., in the presence of an inducer). in appropriate culture medium supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc. The polypeptide can be maintained in a culture medium containing the desired nucleotide sequence, thereby producing the encoded polypeptide. In embodiments, the heavy and light chains are produced in the same host cell. In an embodiment, the heavy chain is produced in one host cell and the light chain is produced in another host cell. It is produced by

[0133] Mammalian cell lines available as hosts for expression are known in the art and are available from American Many available from the American Type Culture Collection (ATCC), Manassas, VA. These include immortalized cell lines such as Chinese hamster ovary (CHO) cells, HEK 293 cells, FS293, Expi293, NSO cells, HeLa cells, baby hamster cells - Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. HepG2 ), and many other cell lines, including but not limited to bacteria, yeast, insect, and non-mammalian cells, including but not limited to plants, for expressing recombinant antibodies. In some embodiments, the antibodies can be derived from bovine or chicken The gene can be produced in such transgenic animals.

[0134] General methods for antibody molecular biology, expression, purification, and screening can be found, for example, in , U.S. Patent Nos. 4,816,567, 4,816,397, and 6,331,415 and No. 7,923,221, as well as Antibody Engineering, edited by Konterma nn & Dubel, Springer, Heidelberg, 2001 and 2010 Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76 ; and Morrison, S. (1985) Science 229:1202.

[0135] Pharmaceutical Compositions An embodiment of the invention is one or more of the compounds of the invention formulated together with a pharma- ceutically acceptable carrier. or a combination thereof), or compositions containing antigen-binding fragments thereof, e.g. Such compositions include, for example, pharmaceutical compositions. or a combination (e.g., two or more different). The pharmaceutical compositions bind to different epitopes on the target antigen or have complementary activities. It may include a combination of antibodies.

[0136] The pharmaceutical compositions of the present invention may also be administered in combination therapy, i.e., in combination with other drugs. For example, the combination therapy may include at least one other anti-tumor agent, or an anti-inflammatory or The antibody of the present invention may be combined with an immunosuppressant. Examples of therapeutic agents that may be used are described in more detail below in the section on uses of the antibodies of the invention. It will be published.

[0137] As used herein, a "pharmaceutical acceptable carrier" refers to any physiologically compatible Any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and Preferably, the carrier is administered intravenously, intramuscularly, subcutaneously, parenterally, spinally or intramuscularly. Depending on the route of administration, the active ingredient may be The compound, i.e., the antibody or antibody fragment, is protected from acids and other agents that may inactivate the compound. To protect the compound from the action of its natural state, it may be coated into a material.

[0138] The pharmaceutical compounds of the present invention may include one or more pharma- ceutically acceptable salts. "Pharmfully acceptable salts" are those which retain the desired biological activity of the parent compound and do not remove any undesirable It refers to salts that do not impart any significant toxicological effects (e.g., Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). The pharmaceutical compositions of the present invention may also include a pharma- ceutically acceptable antioxidant. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include: Water, ethanol, polyol (glycerin, propylene glycol, polyethylene glycol) oils (such as olive oil), and suitable mixtures thereof; Suitable fluidity may include, for example, ethyl oleate. By using coating substances such as cytin, the required particle size can be achieved in the case of dispersions. and by using surfactants.

[0139] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be achieved by the sterilization procedures described above and the use of various antibacterial and antifungal agents, e.g. Ensured by the inclusion of parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, into the compositions. In addition, drugs that delay absorption, such as aluminum monostearate and gelatin, may be used. By including an agent, prolonged absorption of the injectable pharmaceutical form can be brought about.

[0140] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile injectable solutions or dispersions. Sterile powders for the extemporaneous preparation of dispersions are included. The use of such media and agents is known in the art. Any conventional media or agent may be used to activate the Except insofar as the compound is incompatible with the composition, its use in the pharmaceutical compositions of the present invention is contemplated. Active compounds can also be incorporated into the compositions.

[0141] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, the bolus may be administered as a single dose, or in several doses administered over time, or The dosage may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Parenteral compositions are formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, a "dosage unit form" refers to a single unit dose of "means physically discrete units suitable as single doses for an adult, each unit containing the required medicinal Contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a carrier. The description of the dosage unit forms of the present invention is based on (a) the unique characteristics of the active compound and the function to be achieved. (b) the specific therapeutic effect to be achieved; and It is dictated by and directly dependent on the limitations inherent in the technology of compounding materials.

[0142] For administration of the antibody, the dosage is about 0.0001 to 100 mg / kg of the host body weight, about 0.001~50mg / kg, approx. 0.001~10mg / kg, approx. 0.01~10mg / kg, and more usually in the range of 0.01 to 5 mg / kg. The doses were 0.1mg / kg, 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, and 0 .5mg / kg, 0.75mg / kg body weight, 1mg / kg body weight, 3mg / kg body weight, 4m g / kg body weight, 5 mg / kg body weight, 7.5 mg / kg body weight or 10 mg / kg body weight, Alternatively, it may be in the range of 0.1 to 5 mg / kg or in the range of 1 to 10 mg / kg. Examples of treatment regimens include daily, every other day, twice a week, once a week, and once every two weeks. , once every 3 weeks, once every 4 weeks, once every month, once every 3 months, or once every 3-6 months A preferred dosing regimen for the anti-SLAMF6 antibodies of the present invention is intravenous administration. Includes 1mg / kg body weight, 3mg / kg, 5mg / kg or 10mg / kg body weight The antibody is given using one of the following dosing regimens: (i) weekly for six doses; (ii) weekly; (iii) 3 mg / kg body weight once, followed by 1 mg / kg weekly Body weight in kg.

[0143] In some methods, two or more monoclonal antibodies with different binding specificities are simultaneously , where the dose of each antibody administered falls within the ranges indicated. In this embodiment, the antibody is administered multiple times. The interval between single doses can be, for example, weekly, The intervals can be monthly, quarterly, or yearly. The intervals depend on the patient's response to the target antigen. The patient may be irritable, as indicated by measuring blood levels of antibodies that In some embodiments, the dosage is sufficient to achieve a plasma antibody concentration of about 1-1000 μg / ml. In some methods, the concentration is adjusted to about 25-300 μg / ml.

[0144] In some embodiments, the antibody can be administered as a sustained release formulation, In such cases, less frequent administration is necessary. The dosage and frequency of administration should be determined based on the antibody titer in the patient. The half-life varies depending on the antibody. Generally, human antibodies show the longest half-life, followed by humanized antibodies, followed by chimeric antibodies, and non-human antibodies. The dosage and frequency of administration will depend on whether the treatment is prophylactic or not. In prophylactic applications, relatively low doses may be used over a long period of time. It is administered at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, the patient is preferably treated with the drug until progression of the disease is reduced or terminated. Relatively high doses are administered at relatively short intervals until the patient shows partial or complete improvement of the symptoms of the disease. Thereafter, the patient may be administered a prophylactic regimen, if necessary.

[0145] The practical dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be adjusted to be as high as possible without being toxic to the patient. and an effective activity to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The dosage level selected may be varied to obtain the desired amount of active ingredient. The activity, route of administration, time of administration, use of a particular composition or its esters, salts or amides In combination with the excretion rate of the particular compound used, the duration of treatment, and the particular composition used Other drugs, compounds and / or materials used, the age, sex, weight of the patient to be treated, Various medications, including the condition, general health and medical history, and similar factors well known in the medical arts. Depends on kinetic factors.

[0146] A "therapeutically effective dose" of an antibody of the invention preferably results in a reduction in the severity of disease symptoms, Increased frequency and duration of disease-free periods, or functional impairment or disability due to disease distress For example, a "therapeutically effective dose" preferably provides a therapeutically effective dose for At least about 10%, at least about 20%, at least about 30%, more preferably or at least about 40%, at least about 50%, and even more preferably at least about 6 0%, at least about 70%, even more preferably at least about 80%, at least about 9 Inhibits cell growth or tumor growth by at least about 95%. The ability of compounds to inhibit IL-19 expression should be evaluated in animal model systems that are predictive of efficacy in human tumors. Alternatively, this property of the composition can be used to test the ability of a compound to inhibit cell proliferation. Such inhibition can be assessed by assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound can be measured in a subject by in vitro assay. The treatment may reduce the size of tumors that develop during therapy, or otherwise ameliorate symptoms. , the size of the subject, the severity of the subject's symptoms, and the particular composition or route of administration selected. Such amounts can be determined based on such factors.

[0147] The compositions of the present invention can be prepared by one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route of administration and The administration route and / or mode will vary depending on the desired results. The route may be, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other, by injection or infusion. The term "parenteral administration" as used herein includes the parenteral route of administration of the It means modes of administration other than enteral and topical administration, including but not limited to intravenous administration. Intravenous, intramuscular, intraarterial, subarachnoid, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, intradermal This includes subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injections and infusions.

[0148] Alternatively, the antibodies of the invention can be administered parenterally, such as via topical, epidermal or mucosal routes. For example, administration can be intranasally, orally, intravaginally, rectally, sublingually or topically.

[0149] The active compound is delivered via implants, transdermal patches, and microencapsulated delivery systems. The compound can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation containing Ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyol By using biodegradable, biocompatible polymers such as poly(lactic acid), poly(vinyl esters), and poly(lactic acid), Many methods for the preparation of such formulations are patented or available to those skilled in the art. Sustained and Controlled Release Drug Delivery Systems ( 1978) J. R. Robinson, ed., Marcel Dekker, Inc., NY).

[0150] In certain embodiments, the monoclonal antibodies of the invention have suitable distribution in vivo. For example, the blood-brain barrier (BBB) ​​is made up of many hydrophilic To ensure that the therapeutic compounds of the present invention cross the BBB For example, the compound can be formulated in liposomes, if desired. For methods of preparation, see, for example, U.S. Patent Nos. 4,522,811; 48; and 5,399,331. Liposomes can be used to deliver specific cell or may contain one or more moieties that are selectively transported to an organ, thus enhancing targeted drug delivery. (See, e.g., VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary Targeting Moieties The components include folic acid or biotin (see, e.g., U.S. Pat. No. 5,416,016); Cid (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. A gents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 26 9:9090);K. Keinanen;ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; I See also J. Fidler (1994) Immunomethods 4:273.

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

[0152] In some embodiments, these molecules are useful for treating, preventing, and / or diagnosing various disorders. To isolate a gene, the gene may be introduced into cells in culture, in vitro or ex vivo, or into a human subject, e.g. As used herein, the term "subject" refers to a human or Non-human animals are intended to include all vertebrates, e.g., mammals, mammalian Animals include, for example, non-human primates, and non-mammals. Preferred subjects include human patients. When the antibody of the invention is administered together with another agent, the two may be administered in any order. or simultaneously.

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

[0154] Furthermore, in consideration of the expression of SLAMF6 on immune cells, the antibody, antibody composition and The method includes the steps of: treating tumorigenic diseases, such as small cell lung cancer, non-small cell lung cancer (squamous cell carcinoma and adenocarcinoma); cancer), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric ( ric) cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, cancer Glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, sarcoma, GIST, neuroendocrine The present invention relates to treating subjects having diseases characterized by the presence of tumor cells, such as secretory tumors and hematological malignancies. It can be used to treat

[0155] In one embodiment, the antibody of the present invention is used to treat cancer, such as small cell lung cancer, non-small cell lung cancer ( cancer, including squamous cell carcinoma and adenocarcinoma; skin cancer, including melanoma; breast cancer, including TNBC; Colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma cancer, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, gastric (stomach) ) Cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterine, adrenal cancer, sarcoma It is used to treat GIST, neuroendocrine tumors, and hematological malignancies.

[0156] In further embodiments, the antibodies of the invention are used to treat, for example, small cell lung cancer, non-small cell lung cancer (NSCLC), cancer, including squamous cell carcinoma and adenocarcinoma; skin cancer, including melanoma; breast cancer, including TNBC; colon Rectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma , pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach (stomach) Cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, For the manufacture of medicines for the treatment of cancers such as GIST, neuroendocrine tumors, and hematological malignancies. Used.

[0157] In one embodiment, an antibody of the invention (e.g., a monoclonal antibody, an antibody fragment (e.g., Nanobodies, multispecific and bispecific molecules and compositions) are the levels of SLAMF6 or immune cells that contain SLAMF6 on their membrane surface. These amounts can then be used to detect specific It can be associated with disease symptoms.

[0158] In another embodiment, the antibodies of the invention (e.g., monoclonal, multispecific and bispecific antibodies) are The polyspecific molecules and compositions are initially associated with in vitro therapeutic or diagnostic uses. For example, the compositions of the invention can be tested for binding activity to the The antibodies can be tested using the flow cytometry assay described in.

[0159] In some embodiments, the antibodies of the invention (e.g., monoclonal antibodies, multispecific antibodies) The present invention provides a method for the treatment and diagnosis of diseases using ... For example, monoclonal antibodies, multispecific or bispecific molecules have the following properties: It can be used to induce one or more biological activities in vivo or in vitro. Induce and / or enhance the activation of immune cells; Mediating phagocytosis or ADCC of cells in the presence of effector cells , or blocking the binding of an SLAMF6 ligand to SLAMF6.

[0160] In certain embodiments, antibodies (e.g., monoclonal antibodies, multispecific and bispecific antibodies) The therapeutic molecules and compositions can be used in vivo to treat, prevent or diagnose various diseases. Examples of relevant diseases include small cell lung cancer, non-small cell lung cancer (squamous cell carcinoma), and skin cancer, including melanoma, breast cancer (including TNBC), colorectal Cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic visceral cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, Glioma, Glioblastoma, Testicular, Thyroid, Bone, Gallbladder and Bile Duct, Uterine, Adrenal Cancer, Sarcoma, GI Human cancer tissues include those represented by STs, neuroendocrine tumors, and hematological malignancies.

[0161] The antibody compositions of the invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) Suitable routes of administration of the compounds (and compositions) in vivo and in vitro are well known to those of skill in the art. For example, the antibody composition may be administered by injection (e.g., intravenously). The appropriate dosage of the molecule to be used may be determined by the subject. The amount of antibody that can be administered will depend on the age and weight of the recipient, as well as the concentration and / or formulation of the antibody composition.

[0162] As mentioned above, the antibodies of the present invention may be administered in combination with one or more additional therapeutic agents, e.g., immunostimulants, cells. The antibody may be administered simultaneously with a toxic, radiotoxic or immunosuppressive agent. The compound may be linked to the agent (as a conjugate) or may be administered separately from the agent. In the case of patients (separate administration), the antibody can be administered before, after, or simultaneously with the agent. or may be co-administered with other known therapies, e.g., anti-cancer therapies, e.g., radiation therapy. Such therapeutic agents include, among others, doxorubicin (adriamycin), Suprathreshold bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide These include antitumor agents such as famidohydroxyurea, which may have their own adverse effects on patients. These antibodies are effective only at levels that are toxic or subtoxic to humans. Other drugs include other drugs used in the treatment of cancer, such as Avastin®, 5FU and gemcitabine. The anti-SLAMF6 antibody or its antigen-binding fragment thereof of the present invention is Coadministration of cefotaxime and chemotherapeutic agents results in different cytotoxic effects on human tumor cells. This provides two anticancer agents that act via a mechanism that is consistent with the drug. This can overcome problems due to the development of resistance to or changes in the antigenicity of tumor cells.

[0163] Target-specific effector cells, such as compositions of the invention (e.g., monoclonal antibodies Effector cells linked to IgG1, IgG2, and IgG3 (multispecific and bispecific molecules) can also be used as therapeutic agents. Effector cells for targeting can be macrophages, neutrophils or The cells may be human white blood cells such as monocytes. Other cells include eosinophils, natural killer cells and and other IgG or IgA receptor-bearing cells. Target-specific effector cells can be obtained from the subject to be treated. The cells may be administered as a suspension of cells in a physiologically acceptable solution. The number of 8 ~10 9 The order of magnitude may be sufficient, but varies depending on the purpose of treatment.

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

[0165] The bispecific and multispecific molecules of the present invention also cap receptors on cell surfaces. Regulating FcγR or the amount of FcγR on effector cells, such as by binding and depletion A mixture of anti-Fc receptors can also be used for this purpose. It is possible.

[0166] An embodiment of the invention is an antibody composition of the invention (e.g., a monoclonal antibody, bispecific or and kits containing the multispecific molecules, and methods for their use, e.g., in the treatment of cancer. The kit may further comprise an immunosuppressant reagent, a cytotoxic agent, or a radiotoxic agent, etc. or one or more additional antibodies of the invention (e.g., the first antibody and contains an antibody having complementary activity that binds to an epitope in another SLAMF6 antigen It is possible.

[0167] Thus, patients treated with the antibody compositions of the present invention may have an enhanced or increased therapeutic effect of the antibody. Another therapeutic agent, such as a cytotoxic or radiotoxic agent, can be administered to enhance the prior to, concurrently with, or following administration of an antibody of the invention.

[0168] In other embodiments, the subject is diagnosed with, e.g., by treating the subject with a cytokine. Agents that modulate, e.g., enhance or inhibit, the expression or activity of Fcγ or Fcγ receptors Preferred cytokines for administration during multispecific molecule therapy include Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor ( GM-CSF), interferon-gamma (IFN-γ), and tumor necrosis factor (TNF) Includes:

[0169] The compositions of the invention (e.g., antibodies, multispecific and bispecific molecules) can also be used, e.g., To label such cells, target cells expressing FcγR or SLAMF6. For such use, the binding agent can be linked to a molecule that can be detected. Thus, the present invention relates to a method for the preparation of a medicament for the treatment of an Fc receptor, such as FcγR or SLAMF6. Methods for localizing ex vivo or in vitro cells expressing detectable The capable label can be, eg, a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

[0170] In a particular embodiment, the present invention relates to a conjugate between an antibody or a portion thereof and SLAMF6. A method for detecting the presence of SLAMF6 antigen in a sample under conditions that allow the formation of a cellular body, The samples and control samples were incubated with a monoclonal antibody or antibodies that specifically bind to SLAMF6. A method for determining the amount of SLAMF6 antigen comprising contacting the antibody with an antigen-binding fragment of The formation of complexes is then detected, where the difference in the complexes between the sample compared to the control sample is Differences in complex formation indicate the presence of SLAMF6 antigen in the sample.

[0171] In other embodiments, the present invention relates to a method for treating an immune-mediated inflammatory disease in a subject, e.g., a human cancer, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer, including melanoma, Breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, Kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer, and others urothelial carcinoma, stomach cancer, glioma, glioblastoma, testicular, thyroid, bone, gallbladder and and bile duct, uterine, adrenal cancer, sarcoma, GIST, neuroendocrine tumors, and hematological malignancies. This provides a method for

[0172] All references cited herein are hereby incorporated by reference in their entirety. All articles, publications, patents, patent applications, presentations, textbooks, , reports, manuscripts, pamphlets, books, internet submissions, journal articles, periodicals This includes, but is not limited to, articles, product fact sheets, and the like. The discussion is intended merely to summarize the arguments made by those authors and does not include any Applicant does not admit that any reference constitutes prior art, and No part of this publication is intended to be a substitute for the right to challenge the accuracy or appropriateness of any contribution.

[0173] The foregoing invention has been described in some detail by way of illustration and example, for clarity of understanding. However, certain modifications and modifications may be made without departing from the spirit or scope of the appended claims. It will be readily apparent to one of ordinary skill in the art in light of the teachings of the present invention that positive and negative values ​​can be added to these. It would be clear.

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

[0175] Working Example: Example 1: Antibody generation and screening. Hybridoma generation Recombinant ECD protein was transformed with SLAMF6 EC D (SEQ ID NO: 12) was used to immunize mice for the generation of mouse Fab. Live Fab fragments were isolated using industry standard techniques using ECD splenocytes from infected mice. Larry created.

[0176] Secondary Screening Fab supernatant was analyzed by immunoprecipitation of SLAMF6 protein expressed on the surface of Raji cells and activated Binding to PBMC was tested: Supernatants were diluted 1:10 in FACS buffer.

[0177] Example 2: Structural characterization of monoclonal antibodies against SLAMF6 The cDNA sequences encoding the heavy and light chain variable regions of the monoclonal antibodies are The clones were obtained using PCR techniques and sequenced using standard DNA sequencing techniques. The heavy and light chain variable regions of 1B3 selected from can be seen in FIG.

[0178] The nucleotide and amino acid sequences of the heavy chain variable region of 1B3 are set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively. The figures are shown in Fig. 1.

[0179] The nucleotide and amino acid sequences of the light chain variable region of 1B3 are set forth in SEQ ID NO:4 and SEQ ID NO:5, respectively. 2 shows the results.

[0180] Further analysis of the 1B3 VH sequence using the Kabat system of CDR region determination revealed that the sequence Heavy chain CDR1, CDR2 and CDR3 as shown in columns 5, 6 and 7, respectively Figure 1a shows the CDR1, CDR2, and CDR3 regions boxed. The 1B3 VH sequence is shown.

[0181] Further analysis of the 1B3 VL sequence using the Kabat system of CDR region determination revealed that the sequence As shown in columns 8, 9 and 10, respectively, the light chain CDR1, CDR2 and CDR3 are Figure 1b shows the boxed CDR1, CDR2, and CDR3 regions. The 1B3 VL sequence is boxed.

[0182] Example 3: Fab Supernatant Model for SLAMF6 Measured by Flow Cytometry Analysis Specificity of monoclonal antibodies 5×10 6 Raji cells were placed in each well of a 96-well plate and treated with FACS buffer. The cells were washed once with DPBS (2% FBS). The cells were spun at 1200 rpm for 5 min. The pellet was pelleted by diluting in FACS buffer (DPBS, 2% FBS) for 1 h. Wash once and pellet again by spinning at 1200 rpm for 5 min. The test antibodies were diluted to 30 nM / l in FACS buffer and shown in FIG. Increasing amounts were added to each well as indicated, which were then incubated on ice for 30 min. , wash cells once with FACS buffer (DPBS, 2% FBS), pellet and wash The secondary goat anti-mouse antibody was diluted to 1 μg.ml and Add 100 μl to the wells, incubate the plate on ice for 30 min, and then FAC The cells were washed once with S buffer (DPBS, 2% FBS). The cells were pelleted and 200 ul The samples were resuspended in FACS buffer and then analyzed using the Guava Easycyte Plus HT Flow Cytometer. - The results are read using a cytometer and analyzed using Guava Cytosoft software The analysis was performed using the suite.

[0183] As can be seen from FIG. 4, antibody 1B3 inhibited the binding of SLAMF6-expressing Raji cells in a dose-specific manner. It shows dependent binding.

[0184] Example 4: Ability of anti-SLAMF6 antibodies to activate T cells and stimulate IFNγ production 96-well non-tissue culture plates were incubated overnight at 4°C with 250 ng / ml OKT3 and Plates were coated with PBMC and different anti-SLAMF6 antibody / isotype concentrations. Wash twice with S, followed by R10 medium (10% FBS, 1% L-glutamine, 1% penicillin The cells were blocked with 100,000 mL of RPMI containing 100% ethanol / streptomycin for 30 minutes. T cells were resuspended in 100 μl of R10 medium and added onto the plate (according to the manufacturer's instructions). PBMC was cultured using the Miltenyi kit code: 130-096-535 according to the Plates were incubated at 37°C for 72 hours, and the supernatants were collected and analyzed by I Diluted for use in FNγ ELISA assay.

[0185] IFNγ was measured using the IFN-gamma DuoSet ELISA kit (R&D Systems, Inc.). Measurements were performed using a fluoroscopy machine (product number DY285B) according to the manufacturer's instructions. result

[0186] The humanized antibody Hu_1B3 was synthesized using the SLAMF IFN at lower antibody concentrations compared to the Seatle Genetics antibody against 6 We showed increased activity in OKT3 preactivated T cells, reflected by increased γ production ( Fig. 5 This induction of IFNγ production was due to the fact that antibodies directed against SLAMF6 suppressed the immune system. It has been shown to have therapeutic efficacy in patients.

[0187] Example 5: Humanization of antibody 1B3. The mouse 1B3 monoclonal antibody was humanized using CDR grafting technology. This guides the process by conserving parent mouse residues or replacing them with their human germline counterparts. To help determine which replacement to use, a homologous molecule model of the Fv of the 1B3 mouse monoclonal antibody was used. Dell was built.

[0188] The CDR definition is based on the Kabat nomenclature. The 1B3 rat CDR region was grafted. The selection of human framework acceptor regions facilitates the analysis of immunoglobulin V-region sequences. The 1B3 mouse variable region sequence was identified using IgBLAST, which was developed by NCBI. As input, this was achieved by searching the IMGT mouse and human V gene databases. The strategy employed was to identify specific somatic mutations found in individual human antibody sequences. The aim of this study was to use human germline sequences, which are naturally occurring human sequences that contain no mutations.

[0189] Heavy Chain Design The amino acid sequence of VH isolated from mouse 1B3 hybridoma (Kabat numbering) The CDR regions are shown in bold in accordance with the coding scheme below: [ka]

[0190] Selection of human framework receptor VH regions Using the mouse VH region amino acid sequence as input, IgBLAST was used to identify the IMGT human VH gene. By searching gene databases, it was possible to identify human fragments into which the 1B3 mouse CDR region could be grafted. Selection of framework receptor VH regions was achieved by sequence alignment of parent antibodies against human germline Based on the objective of the study, the closest matching entry was identified. Germline identification was based on sequence identity between frameworks as defined by Kabat. and / or identity of the interchain interface residues and supporting loops with the canonical conformation of the parent CDRs. and / or based on ordered criteria of compatibility. *02 was selected as the most suitable heavy chain.

[0191] Designing IGHV1-2*02 human germline using the framework acceptor region Humanized version The mouse CDRs (bold) defined by the Kabat nomenclature were transferred to IGHV1-2*02. The following detailed sequence was obtained: Many of the residues are framework murine residues (CDR residues external), i.e., conserved from the parent mouse 1B3 VH sequence, Conserved because they may be structurally important for maintaining full activity in the body . [ka]

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

[0193] Light Chain Design The amino acid sequence of mouse 1B3 VL (CDR regions defined by Kabat nomenclature) (emphasis mine) is shown below: [ka]

[0194] Selection of human framework receptor VL domains Using the mouse VL region amino acid sequence as input, IgBLAST was used to search for the IMGT human VL gene. By searching gene databases, we identified human CDR regions into which the 1B3 VL mouse CDR regions could be grafted. Selection of the framework receptor VL region was achieved. Based on the column alignment, the closest matching entries were identified. Human germline identification was performed using interframework sequences as defined by Kabat. Identity and identity of the interchain interface residues and support loops with the canonical conformation of the parent CDRs and / or based on ordered criteria of compatibility. Germline, IGKV1-33*01 is the best choice for human framework acceptor domain Therefore, this human germline was used to engineer a humanized version. was used.

[0195] Designed using IGKV1-33*01 human germline as the framework acceptor region Humanized version Grafting of mouse CDRs defined by Kabat numbering into IGKV1-33*01 The following detailed sequences were obtained: This resulted in a humanized version with 86.3% identity (9 5) (82 amino acid residues in total) were obtained. [ka] The humanized version shares 86.3% identity with the IGKV1-33*01 human germline (8 2 / 95)

[0196] Example 6 ELISPOT with tumor-infiltrating lymphocytes Tumors derived from primary tumors derived from NSCLC (Figure 6), breast cancer (Figure 7) or CRC (Figure 8) tumors TILs were treated with mouse 1B3 or pembrolizumab at 10 μg / ml, KT3 cells were stimulated for 96 hours at 1 μg / ml in complete IMDM culture medium. ILs were harvested, counted and plated on IFNγ ELISPOT plates (Mabtech) at 10 0,000 cells / well were plated. The plates were incubated at 37°C for 24 hours. Incubation and subsequent development were performed according to the manufacturer's instructions. Spot counts were determined by immunofluorescence. The data were read using a noSpot® Series 5 ELISPOT analyzer. The data was analyzed using GraphPad Prism software.

[0197] FIG. 6 shows that antibody 1B3 significantly increased IFNγ production compared to the isotype antibody. Activation of SCLC-derived TILs.

[0198] Figure 7 shows that antibody 1B3 activated significantly higher numbers of breast cancer-derived TILs than pembrolizumab. The results show that IFNγ is produced by the IFN-γ-binding protein 1 (IFNγ-binding protein 1

[0199] FIG. 8 shows that antibody 1B3 induces significantly higher numbers of colorectal cancer-derived TILs than pembrolizumab. Activation of IFNγ by IFN-γ production.

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

[0201] [Table 3]

[0202] Example 8 In vitro proliferation assay using anti-SLAMF6 antibody 1B3 method. Non-tissue culture treated 96-well plates (BD Falcon, USA) were pre-treated with anti-human CD3 (eBioscience , USA) antibody 250ng / ml alone or humanized anti-SLAMF6 antibody (Hu_1B3) or are the concentrations of isotype control antibody (0, 0.2, 0.4, 0.6, 0.9, 1.2ug / ml) The next day, the plates were washed and then coated with AIM. T cells isolated from PBMCs generated from healthy donors were then blocked with V medium. The cells were stained with the proliferation dye eFluor™ 670 (eBioscience, USA), washed, and then incubated with FCS. AIM V (Thermofisher Scientific, USA) medium containing penicillin-streptomycin Cells were seeded onto antibody-coated plates (100,000 cells / well in 100 μl) containing tissue samples. The cells were cultured in a culture incubator at 37°C for 72 hours. On the third day, the cells were harvested and stained with anti-human C D8 labeled FITC, anti-human CD4 labeled Brilliant Violet t 711™, PE labeled with anti-human CD69 (Biolegend, USA), and fixed The samples were stained with the viability dye eFluor™ 506 (eBioscience, USA). The results were analyzed using a Tune NxT flow cytometer (Thermofisher Scientific, USA). Data were analyzed using owJo software (TreeStar, USA).

[0203] FIG. 9 shows that SLAMF6 is present on T cells isolated using anti-SLAMF6 antibodies in the presence of CD3. SLAMF6 stimulation enhanced T cell proliferation compared to isotype or CD3 alone This indicates a significant increase.

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

[0205] Cryopreserved PBMCs from a second donor (donor 2) were cultured at 2E6 cells / ml in culture medium. The cells were treated with 50ug / ml mitomycin C at a density of 1.5g / ml. The cells were washed with culture medium and then resuspended in mitomycin C. Cells were treated at 37°C for 90 min before removing C.

[0206] Then, 100,000 cells from donor 1 were transferred to donor 2 in a 96-well, U-bottom plate. The combined cells were then combined with 100,000 mitomycin C-treated cells from The cells were treated with various concentrations of humanized antibody Hu_1B3 and a control solution for 6 days. The cells were cultured in 100 ul of medium per well. Anti-CD13 was used as a negative control for isotype. It was used similarly to 7mAb and was included in the comparison.

[0207] The culture supernatant was collected on the sixth day and subjected to ELISA (R&D Systems: DY285B). The IFN-γ concentration was analyzed according to the manufacturer's instructions. Figure 10 shows that the anti-SLAMF6 antibody Hu_1B3 showed a dose-dependent increase in cytokine release. This indicates that T cells are activated by the presence of antibodies. Activation results in higher cytokine release from isolated T cells than activation via CD137 This shows that it brings about

[0208] Example 10 SLAMF6-Mediated Granzyme B and Perforin Release from T Cells PBMC isolation As a first step in T cell isolation, PBMCs were cultured using buffy coat cultures (obtained from the Stanford Blood Center). The blood was diluted in PBS at a ratio of 1:4 (10 ml blood + PBS). The diluted blood was then diluted with 30 ml of Ficoll-Hypaque (GE Healthcare Carefully place the tube on the 400g (1400 rpm) The monocytes are centrifuged in a Sorvall centrifuge with the brake off for 30 min at RT. Approximately 10 ml of the white cell fraction from all tubes was transferred to a single 50 ml tube. The buffy coat is pooled in a tub, washed and counted using a Cellometer Auto 2000. PBMCs generated from the mice are used for T cell isolation.

[0209] T cell isolation Pan T cell isolation from PBMCs excludes CD4 and CD8 T cells and all remaining All immune cell subsets were labeled with biotin-conjugated antibodies and bound to streptavidin in a high magnetic field. This is a negative selection process in which the target cells are captured by microbeads coated with PBMCs isolated from the buffy coat were washed and sorted in FACS sorting buffer (0.1 in PBS). 100% BSA) at a concentration of 2.5e7 cells / mL and incubated with Pan T cell biotin-antibody Add the cocktail to the cells, mix the antibody-cell suspension with a 1 ml pipette, and incubate on ice for 5 min. Add Pan T Cell MicroBead Isolation Cocktail to the mixture and incubate for 24 hours. Incubate on ice for an additional 10 minutes. The MACS separation method is used to isolate naive T cells. The mixture of PBMCs and biotin-labeled microbeads is then subjected to high-field MACS. In the separator, the non-T cells were run on an LS column (Miltneyi Biotec, Cat#130-042-401). The depleted flow-through was collected, washed once with FACS sorting buffer, and then incubated for 5 min. Resuspend in AIMV complete medium with 100% FBS.

[0210] Granzyme B ELISA For the granzyme B functional assay, non-tissue culture treated 96-well plates were 50ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific Cat# 16-0031-85) and coated with human Hu_1B3 or isotype control antibody. Each test antibody was administered in 100 μl of PBS starting at 4.2 μg / ml. Coat plates in triplicate with a 2:3 dilution for each well. Seal plates and incubate overnight at 4°C. On the day of Granzyme B functional assay setup, wash the plate and incubate for 5 min. Block with AIMV complete medium containing 50% FCS for 20 min to reduce non-specific binding.

[0211] Granzyme B immunoassay was performed using the Human Granzyme BD R&D System (Cat# DY2906-05). Nunc-Immunoassay plates are set up using the uoSet ELISA kit. The plates were coated with Granzyme B capture antibody diluted 1:60 in PBS. Incubate overnight at 4°C. The next day, wash the plate with ELISA wash buffer (PBS+ 0.05% Tween 20) and block in 1% BSA in PBS. After blocking, 100 μl of sample diluted 1:60 in dilution buffer was added to each well. Add the standard solution to the plate and incubate at 4°C overnight. The next day, develop the plate and OD values ​​were captured on a saMax tunable microplate reader. Granzyme B Release is quantified and plotted using Graphpad Prism 8 software.

[0212] conclusion Granzyme-mediated apoptosis is a mechanism by which cytotoxic lymphocytes eliminate transformed cells. This data indicates that the antibody Hu_1B3 is important in tumor suppression. This indicates that the antibody Hu_1B3 can induce cytotoxicity from activated T cells. It increased Ranzyme B in a dose-dependent manner (Figure 11) and EC 50 is 0.51μg / ml .

[0213] Perforin intracellular assay Perforin Intracellular Quantitation Assay Set-up Non-tissue Culture Treated 96-well Plates 250ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific t#16-0031-85) in combination with human Hu_1B3 or isotype control antibody ( Each test antibody was diluted in 100 μl of PBS starting at 4.2 μg / ml. Coat with 2:3 dilutions for titration. Seal plates and incubate overnight at 4°C. On the day of Granzyme B functional assay setup, wash the plate and resuspend in 5% FCS. Block with AIMV complete medium containing 50 mM Tris-HCl for 20 min to reduce non-specific binding. For the assay, 20 cells were cultured per well in 100 μl of AIMV culture medium containing 5% FBS. Add 0,000 pan T cells and incubate at 37°C in a tissue culture incubator for 3 days. On the day of the assay, protein transport inhibitor cocktail (Thermofisher Scientific, Cat# 00-4980-93) was added to all wells to prevent perforin transport into the extracellular space. The cells were then cultured at 4°C for 4 hours. The cells were harvested and analyzed for T cell surface markers CD3, CD4, and CD8. Then, the cells were stained with FIX&PERM™ Cell Permeabilization Kit (Thermofisher Science Intracellular perforin staining was performed using the Attune N The data were analyzed using a Flocculation Scale (FACS Analyzer, Thermofisher Scientific, MD). Analysis was performed with wJo software (BD Biosciences, San Jose).

[0214] conclusion Perforin-mediated necrosis is another major mechanism of killing induced by cytotoxic T lymphocytes. Hu_1B3 has an EC of 0.44μg / ml. 50 In a dose-dependent manner Enhances perforin upregulation in CD8+ T cells (Figure 12)

[0215] Example 11 Competitive Binding Assay to Evaluate the Binding Epitope of Antibody 1B3 Thaw cryopreserved human PBMCs and place them in FACS buffer (DPBS containing 2% FCS). Wash once with suspension, then pellet cells by centrifugation at 1200 rpm for 5 minutes The cells were washed with FACS buffer and the supernatant was discarded (the same method used for the subsequent washes). Dispense cells into 96-well assay plates at 100,000 cells per well in Then, SLAM in 100 μl FACS buffer on ice for 1 hour before washing. For F6 receptor blocking, cells were transfected with SLAMF6 ECD-mIgG2a Fc Blocking was performed with fusion protein at 100 nM or 300 nM for 1 hour. Humanized Hu_1B Three antibodies or a human IgG1 isotype control were assayed in triplicate at a top concentration of 10 nM. The antibodies were titrated in serial dilutions and then added to the blocked PBMCs and incubated on ice for 1 hour. The cells were incubated with 1 μg / mL of 200 mM NaCl in FACS buffer and then washed twice. The secondary antibody, goat anti-human IgG-RPE (Southern Biotech, Ref: 2040-05), was incubated for 3 h on ice. One of the untreated wells containing cells was also stained with the secondary antibody. , and the other was left unstained to act as a control for secondary antibody binding. After this final incubation, the cells were washed again twice and the final cell pellet was subjected to FACS The mean fluorescence intensity of the secondary antibody was measured using 96-well plates according to industry standard protocols. In a single plate format, the Attune NxT Flow Cytometer (ThermoF Each sample was measured using a FlowJoint (Fast Calculator) and the raw data were The data was analyzed using analytical software.

[0216] As shown in FIG. 13, the Hu_1B3 antibody inhibited human SLAMF6 ECD-mIgG2 a In the presence of Fc fusion protein, binding to receptors on the surface of PBMCs is blocked, and H This indicates that u_1B3 competitively binds to human SLAMF6 ECD. Hu_1B3 binds to the homodimerization epitope of SLAMF6 and acts as an agonist antibody. This may enhance cytotoxic T cell function through a SAP-mediated activation pathway that functions in the .

[0217] Example 12 Internalization of Hu_1B3 antibody RAJI, human Burkitt lymphoma cells (Cat No. CCL-86, American Type Culture Collection [ATCC], Manassas VA) and RPMI-1640 medium (Cellgro, Cat No. 10-041-CM, Mediatech, Manassas VA) in 10% fetal bovine serum (HyClone* Cosmic Calf Serum, Cat No SH30087-03, Thermo Scientific, Waltham, MA) and 1% Pilby Sodium phosphate (Cellgro, Cat. # 25-000-Cl) was added using industry standard aseptic technique. RAJI cells were cultured at 5 × 10 cells per well in a 24-well glass-bottom culture plate. 5 Cells were plated at a density of 1000× and allowed to grow in growth medium at 37° C. for 48 hours. Samples were examined at 0, 0.5, 1, 2, 4 and 24 hours. Prepared: secondary antibody only control, human IgG isotype control, antibody Hu_1B3, Seattle Clinical anti-SLAMF6 antibody (positive control) from Biosciences genetics. As a fluorescent control, only the secondary antibody was used. No control wells, antibody control wells were used.

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

[0219] After primary and secondary antibody labeling, human IgG isotype control, secondary antibody only control, and Test and positive controls were treated at 0 min. Cells were washed twice with IF buffer and diluted with 4% paraformaldehyde. Methylaldehyde (4% diluted in DPBS, Cat No 19943, Affymetrix, Santa Clara, CA) a, CA) on ice to stop internalization and cells was fixed.

[0220] The remaining cells were washed twice with IF buffer and 1 mL of warmed growth medium was added to the wells of a 37°C incubator. was added to each well before placing it in the oven. For 24 hours, cells were incubated in paraformaldehyde on ice as described for the control samples. Fixed.

[0221] All cells remained in fixative for at least 15 min on ice. Cells were washed with IF buffer. Then, as a nuclear counterstain, add 2-3 drops of Prolong Gold Anti-Fade reagent + DAPI (Cat A coverslip was added to each well along with 100% PBS (No. P-36931, Invitrogen, Grand Island, NY). The cell images were taken with a Leica microscope (Leica DMI600B) and a Leica monochrome Camera (Leica DFC350FX), filter set for DAPI and Alexa Fluor Acquired using 488 and 63x oil immersion lenses. Images are in TIFF format. The images were saved and analyzed using ImageJ.

[0222] FIG. 14 shows that the humanized antibody Hu_1B3 has significantly less cytotoxicity than the clinical SeattleGenetics antibody. This indicates that SLAMF6 is internalized into SLAMF6-expressing cells when bound to antibody Hu_1B3. The receptor remains on the cell surface for a long time, which induces a more sustained response in T cells. These results suggest that the agonist activity of β-lactamase inhibitors is increased by 100% compared to β-lactamase inhibitors, and therefore may be more effective agonists.

[0223] Example 13 Cytotoxicity Assay SKBr3 HCT116 and MDA-MB-231 were purchased from ATCC. The strains were maintained according to the manufacturer's (ATCC) instructions using standard sterile techniques. The nutrient medium consisted of 2 mM L-glutamine and 25 mM HEPES (Corning), 1% penicillin, and 1% ethanol. Cylindryl / streptomycin (Sigma-Aldrich), and 10% heat-inactivated FCS (HyClon e) were used. The cell lines were maintained in exponential phase. The cells were grown in a 37°C incubator containing 5% CO2. SKBr3 is a high copy number He r2(approx. 5×10 6 HCT116 is a breast cancer cell line that expresses low copy number (100 copies / cell). MDA-MB-231 is a colon cancer cell line that expresses a low copy number of Her2. Her2 expressing breast cancer cell line.

[0224] Target and effector (PBMC) cells were prepared under the following conditions: On the day of infection, target cells are washed with PBS and incubated with 0.25% trypsin. The target cells were resuspended in culture medium. The viability and cell concentration were measured by dye exclusion. 10,000 cells / well were plated in 96-well tissue culture treated plates. Cells were grown overnight in a 37°C incubator with 5% CO2.

[0225] The day before the assay, thaw frozen PBMCs in a 37°C water bath and wash with cell culture medium. The cells were then cultured overnight in a 37°C incubator with 5% CO2. PBMCs were added to target cells at an E:T ratio of 10:1. 10,000 target cells were mixed with 100,000 effector cells.

[0226] The bispecific antibody Cris7-Her2 was used in the cytotoxicity assay. The Cris7-Her2 bispecific antibody detects both CD3ε and CD3ε. Diluted to 0 ng / ml, then serially diluted 3-fold to 0.0012 ng / ml In addition, we tested Cris7-Her2+ agonist antibodies. Cris7-Her2 bispecific antibody and agonist to observe enhanced cytotoxicity The antibody combination was included. As a positive control, urelumab (Urelumab), an antibody against 4-1BB, was used. elumab) was used. Isotype was used as a negative control. Hu_1B3 was the target. The final concentration of urelumab, isotype, and Hu were 2.5ug / ml. All samples were run in triplicate. Controls included target cells alone or Further controls included target and effector cells. + Hu_1B3, or target and effector cells + isotype control .

[0227] Cytotoxicity assays were incubated for 48–96 h depending on the cell line. After transfection, target cell viability was measured. Viability was based on quantification of ATP and A TP signals the presence of metabolically active cells. The assay is luminescence-based. Cell Titer-Glo® (Promega, Madison, WI) measures viable cells The assay conditions were as follows: All steps were performed at room temperature. Briefly, 96-well plates and Cell Ti The ter-Glo® reagent was allowed to equilibrate to room temperature for 30 minutes. After 30 minutes, the cell culture media Remove the supernatant and wash the target cells with 200ul of PBS and repeat the washing step one more time. Next, 100 ul of Cell Titer-Glo® Reagent was added to all The plate was placed on an orbital shaker and mixed at 250 rpm for 2 minutes to allow cell lysis. The plate was removed from the orbital shaker and incubated in the dark for 10 minutes. The luminescence signal was allowed to stabilize. After 10 min, the samples were transferred to an opaque-walled 96-well plate and The luminescence was recorded.

[0228] The viability of the target cells was proportional to the luminescence signal produced. All samples were run in triplicate. The percent cytotoxicity of the samples was compared with that of the controls. Normalized to target cells in the presence of effector cells (target + effector) To calculate the survival rate, the luminescence signal of the sample was divided by the luminescence signal of the control. Cytotoxicity was calculated based on the percentage of non-viable cells remaining. As can be seen in Figure 15 , SKBr3 cells, bispecific antibody alone or bispecific antibody plus isotype control has effective cytotoxicity at higher concentrations of 0.8-100ng / ml. The addition of α-tocopherol shifted the cytotoxicity curve slightly to the left, but by less than 5%. The addition of 2.5ug / ml of Hu_1B3 increased the cytotoxicity of SKBR-3 cells by 10-40%. % increase.

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

[0230] FIG. 17 shows the enhanced cytotoxicity by Hu_1B3 against HCT116 cells. HCT116 cells, a colon cancer cell line, express low levels of Her2 on the cell surface. Cytotoxicity assays were performed using PBMCs and a T cell-engaging bispecific antibody, Cris7-Her2. The assay was performed using HCT116 cells with Hu_1B3 as described above. We added IgG1-dependent IL-1α to PBMCs to determine whether it enhances the function of T cells or NK cells present in PBMCs. T cells and NK cells both express the coactivation receptor SLAMF6. Dose-dependent induction of T cell-engaging bispecific antibody, Cris7-Her2 Cytotoxicity is shown at the various concentrations tested. More importantly, the data show that Hu_1B3 When the Cris7-Her2 antibody was added, increased cytotoxicity was observed. The EC50 for the single or bispecific antibody plus isotype control is approximately 0.25 ng / ml. In contrast, the EC50 of the bispecific antibody + Hu_1B3 is approximately 0.07ng / ml. This represents approximately a 3.6-fold increase in cytotoxicity.

[0231] FIG. 18 shows enhanced cytotoxicity by Hu_1B3 against MDA-MB-231 cells. The breast cancer cell line, MDA-MB-231 cells, express low levels of Her2 on the cell surface. Cytotoxicity assays were performed as described above using MDA-MB-231 cells expressing PBM C, and with the T cell-engaging bispecific antibody, Cris7-Her2. Hu_1B3 inhibited the Cris7-Her2 bispecific cytotoxic activity at various concentrations tested. Specifically, 1 ng / mL of Cris7 increased the expression of A 40% increase in cytotoxicity was observed.

[0232] FIG. 19 shows that after 96 hours of incubation, the combination of bispecific with Hu_1B3 Even the lowest concentrations of bispecific antibodies induced high levels of killing of SKBR-3 cells. On the other hand, at these levels, the bispecific alone or the bispecific and urea Either the urelumab combination or the isotype control showed very low levels ( The results indicate that Hu_1B3 induces cell death in the 100% IL-16 (100% IL-16) or does not induce cell death. It was suggested that this results in strong activation of lymphocytes.

[0233] Figure 20 shows the results of the cytotoxicity assay described above, with the test antibody concentrations ranging from 7.5ug / ml. Bispecific, provided that it is titrated at 1 / 3 dilutions in the range of 0.0034ug / ml The Cris7-Her2 antibody was maintained at a constant level (0.0457ng / ml). Dose-dependent killing of SKBR-3 cells was observed in 116 patients with urelumab or isotype control. This was observed in comparison with Hu_1B3, and further demonstrated the ability of Hu_1B3 to activate cytotoxic lymphocytes. did.

[0234] Example 14 Cytotoxicity Assay in the Absence of Cris7 Bispecific Another cytotoxicity assay was set up as above, but without the use of bispecific antibodies. Single agent SKBR-3 cytotoxicity studies included Hu_1B3, isotype control, and Only urelumab was used. The concentration range used in the assay was 1:3 dilution from one donor. The serum levels of the second donor were 33ug / ml to 0.13ug / ml. The antibody was diluted to 0.0152 ug / ml (Figure 21b).

[0235] Figures 21a and 21b show the results in the absence of the CD3-Her2 bispecific antibody Hu_1B3. , showing that it can activate lymphocytes and induce cell death in SKBR-3 cells. This is in contrast to urelumab or the isotype control, where little cell killing was observed. be.

[0236] Sequence Listing JPEG2025041695000009.jpg245170JPEG2025041695000010.jpg245170JPEG2025041695000011.jpg245170

Claims

1. An antibody or antigen-binding fragment thereof that binds to SLAMF6, a CDR-H1 sequence comprising SEQ ID NO:5; a CDR-H2 sequence comprising SEQ ID NO:6 or SEQ ID NO:15; a CDR-H3 sequence comprising SEQ ID NO:7; An antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising:

2. A CDR-L1 sequence comprising a sequence selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:

16. Columns, A CDR-L2 sequence comprising a sequence selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:

17. and, a CDR-L3 sequence comprising SEQ ID NO: 10; and a light chain variable region comprising at least one CDR sequence selected from the group consisting of:

2. The antibody or antigen-binding fragment of claim 1.

3. The heavy chain variable region CDR-L1 comprising the sequence of SEQ ID NO:5; CDR-L2 comprising the sequence of SEQ ID NO: 15; CDR-L3 comprising the sequence of SEQ ID NO: 7; 3. The antibody or antigen-binding fragment of claim 1 or claim 2, comprising:

4. The light chain variable region comprises: CDR-L1 comprising the sequence of SEQ ID NO: 16; CDR-L2 comprising the sequence of SEQ ID NO: 17; CDR-L3 comprising the sequence of SEQ ID NO: 10; 4. The antibody or antigen-binding fragment of claim 2 or claim 3, comprising:

5. An antibody or antigen-binding fragment thereof that binds to SLAMF6, CDR-H1 comprising SEQ ID NO:5; CDR-H2 comprising SEQ ID NO: 15; and CDR-H3 comprising SEQ ID NO:7; and a heavy chain variable region comprising CDR-L1 comprising SEQ ID NO: 16; and CDR-L2 comprising SEQ ID NO: 17; and CDR-L3 comprising SEQ ID NO: 10; and or a light chain variable region comprising Including variants thereof, The mutant is i) The above CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and in one or more of CDR-L3, independently, 1, 2, 3, 4, 5, or 6 amino acid substitutions, additions and / or deletions, or ii) The above CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L 2, and in the set of CDRs including CDR-L3, 1, 2, 3, 4, 5, Antibodies with 6, 7, 8, 9 or 10 amino acid substitutions, additions and / or deletions. or an antigen-binding fragment.

6. An antibody, or an antigen-binding fragment thereof, comprising: i) the three heavy chain CDRs of SEQ ID NO: 1 and the three light chain CDRs of SEQ ID NO: 2, or Or ii) the three heavy chain CDRs of SEQ ID NO: 13 and the three light chain CDRs of SEQ ID NO: 14 R Including, The CDRs are defined according to the Kabat or Chothia numbering systems. An antibody or antigen-binding fragment thereof.

7. SEQ ID NO: 13 and at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a heavy chain variable region of the present invention; 、 SEQ ID NO: 14 and at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a light chain variable region of the present invention; 、 An antibody or antigen-binding fragment thereof comprising:

8. A heavy chain variable region comprising SEQ ID NO: 13 and a light chain variable region comprising SEQ ID NO:

14. The antibody or antigen-binding fragment thereof according to claim 7.

9. The antibody or antigen-binding fragment of any one of claims 1 to 8 is or any one of claims 1 to 8. An antibody or antigen-binding fragment thereof that cross-competes for binding with the antibody of claim 1. to.

10. A human SLAM antibody or antigen-binding fragment of any one of claims 1 to 8. At least about 80%, at least about 85%, at least about 90% of the binding affinity for F6 %, 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% 9. The antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof retains about 99% or at least about 99% of the total binding of the antibody or antigen-binding fragment thereof. segment.

11. The antibody or antigen-binding fragment of claim 1 is a monoclonal antibody. Any of the antibodies or antigen-binding fragments thereof described herein.

12. The antibody or antigen-binding fragment may be chimeric, humanized, bispecific, or human The antibody or antigen-binding fragment according to any one of claims 1 to 11. is an antigen-binding fragment thereof.

13. The antibody or antigen-binding fragment is an Fc-silenced engineered IgG. or has reduced or complete binding to one or more Fc receptors. The antibody or antigen-binding fragment according to any one of claims 1 to 12, and an antigen-binding fragment thereof.

14. The antibody or antigen-binding fragment induces and / or enhances T cell cytotoxicity. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, nt.

15. The antigen-binding fragment may be Fab, Fab', F(ab) ２ , F(ab′) ２ , F v, Fv-TCR fragment, scFv, and single domain antibody. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14.

16. i) A heavy chain variable region of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15. Areas, and / or ii) The light chain variable region of the antibody or antigen-binding fragment according to any one of claims 1 to 15. area, A polynucleotide encoding the

17. 17. An expression vector comprising at least one polynucleotide according to claim 16.

18. i. an expression vector comprising the polynucleotide of claim 16, or ii. A polypeptide encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof according to claim 16. A first expression vector comprising a polynucleotide encoding the antibody or its derivative according to claim 16. and a polynucleotide encoding the light chain variable region of the antigen-binding fragment of said first polypeptide. The current vector, A host cell comprising:

19. A method for producing an antibody or antigen-binding fragment thereof comprising the steps of: and subjecting the host cell to conditions in which the antibody or antigen-binding fragment is expressed in the host cell. and optionally isolating said antibody or antigen-binding fragment. Includes, a method.

20. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 15 and a pharmaceutical and a physiologically acceptable carrier.

21. A method for treating a subject having cancer comprising administering to said subject an effective amount of any one of claims 1 to 15 or 20. Administering the antibody or antigen-binding fragment thereof or pharmaceutical composition of any one of claims 1 to a subject. The method includes:

22. A method for the manufacture of a pharmaceutical composition for use in the treatment of cancer, comprising administering to said patient a compound according to any one of claims 1 to 15 or 20. Use of the antibody or antigen-binding fragment thereof, or pharmaceutical composition described in any one of claims 1 to 4.

23. An antibody or a polypeptide according to any one of claims 1 to 15 or 20 for use in the treatment of cancer. or an antigen-binding fragment thereof, or a pharmaceutical composition.

24. The cancer includes small cell lung cancer, non-small cell lung cancer including squamous cell carcinoma and adenocarcinoma, melanoma. Skin cancer, breast cancer including TNBC, colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and and other urothelial cancers, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer , gallbladder and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies The method according to any one of claims 21 to 23, wherein the tumor is selected from the group consisting of: use.

25. Claims 21-24, wherein the pharmaceutical composition or medicament further comprises an effective amount of a second therapeutic agent.

2. The method or use according to any one of claims 1 to 11.

26. The compound according to claims 1 to 15 or 20 for use in therapy or as a medicament.

2. The antibody or antigen-binding fragment thereof, or pharmaceutical composition according to any one of claims 1 to 11.

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