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] This invention relates to an antibody capable of binding to the SLAMF6 protein, and to the use thereof. ru.

[0002] Introduction Aspects of the present invention include the SLAM family, also known as NTB-A or CD352. Antibodies against member 6 (SLAMF6) and other therapeutic proteins, such antibodies and Methods for preparing nucleic acids encoding therapeutic proteins, antibodies, and other therapeutic proteins, Furthermore, using antibodies against SLAMF6 and other therapeutic proteins can help treat diseases such as cancer. This includes treatment methods for the patient. [Background technology]

[0003] The target antigen SLAMF6 is a single-pass type I membrane protein, and immunoglobulin... It is a member of the Per family and the CD2 subfamily (J Exp Med. 2001 Aug 6; 194(3):235-46). Its activity is attributed to the small cytoplasmic adapter protein, SH2D1A / SA This tamper is controlled by the presence or absence of P and / or SH2D1B / EAT-2. The compound expresses a high surface density of a natural cytotoxic receptor (J. Exp. Med. 194:235-246(2001)). It induces cell-lytic activity only in natural killer (NK) cells. Positive signaling implies phosphorylation of VAV1. NK cell activation is SH2D1B It appears to depend on SLAMF1 and not on SH2D1A. This involves positive selection of thymocyte natural killer T (NKT) cell lineages and subsequent It controls the transition between proliferation and differentiation.

[0004] SLAMF6 also promotes T cell differentiation into helper T cell Th17 phenotypes that lead to increased IL-17 secretion, and costimulatory activity requires SH2D1A (J. Immunol. 177:3170-3177(2006 )). Furthermore, it promotes the recruitment of RORC to an IL-17 promoter (J. Biol. Chem. 287:38168-38177(2012) ). Along with SLAMF1 and CD84 / SLAM F5, SLAMF6 may be a negative regulator of the humoral immune response. When SH 2D1A / SAP is absent, SLAMF6 can transmit negative signals to CD4 T cells and NKT cells , , , . It also negatively regulates germinal center formation by inhibiting T cell:B cell adhesion, and this function likely involves increased association with PTPN6 / SHP-1 via ITSM when SH2D1A / SAP is absent . . .

[0005] WO2008 / 027739 discloses anti-NTB-A antibodies and pharmaceutical compositions containing the above antibodies. Also described is a method of using the above antibodies to treat diseases such as hematological malignancies characterized by NTB-A expression . .

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

[0007] WO2015 / 104711 describes compositions and methods for improved T cell regulation ex vivo and in vivo, and for the treatment of cancer and other pathologies. More specifically, embodiments of the invention are directed to the use of water-soluble NTB-A polypeptides or agonists thereof for the treatment of cancer patients, the prevention and treatment of cytopenia in susceptible patients, and the ex vivo preparation of improved cell compositions. Summary of the Invention Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and tumor-associated antigens, nucleic acids encoding such antibodies of the invention, host cells encoding the antibodies of the invention, methods for preparing the antibodies of the invention, and methods for treating diseases such as cancers of humans including, but not limited to, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, GIST, neuroendocrine tumor, and hematological malignancies. 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). Aspects of the invention include SLAMF6 protein recognized by the antibodies described herein. Brief Description of the Drawings Detailed Description of the Invention

[0008] Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and tumor-associated antigens, nucleic acids encoding such antibodies of the invention, host cells encoding the antibodies of the invention, methods for preparing the antibodies of the invention, and methods for treating diseases such as cancers of humans including, but not limited to, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, GIST, neuroendocrine tumor, and hematological malignancies. 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). Aspects of the invention include SLAMF6 protein recognized by the antibodies described herein. Brief Description of the Drawings Detailed Description of the Invention Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and tumor-associated antigens, nucleic acids encoding such antibodies of the invention, host cells encoding the antibodies of the invention, methods for preparing the antibodies of the invention, and methods for treating diseases such as cancers of humans including, but not limited to, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, GIST, neuroendocrine tumor, and hematological malignancies. 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). Aspects of the invention include SLAMF6 protein recognized by the antibodies described herein. Brief Description of the Drawings 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). Aspects of the invention include SLAMF6 protein recognized by the antibodies described herein. Detailed Description of the Invention Aspects of the invention include specific antibodies against SLAMF6, bispecific antibodies against SLAMF6 and tumor-associated antigens, nucleic acids encoding such antibodies of the invention, host cells encoding the antibodies of the invention, methods for preparing the antibodies of the invention, and methods for treating diseases such as cancers of humans including, but not limited to, small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, GIST, neuroendocrine tumor, and hematological malignancies.

[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). Aspects of the invention include SLAMF6 protein recognized by the antibodies described herein. Brief Description of the Drawings Detailed Description of the Invention It binds to an epitope on the chromosome, or, in relation to binding with the antibodies described herein, it binds to the chromosome. Cross-compete, preferably with the antimicrobial agents described herein. At least approximately 80%, at least approximately 85%, of the body's binding affinity to human SLAMF6. At least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, At least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, An antibody that retains at least approximately 98% or at least approximately 99% of its antigen-binding fraction, or its antigen-binding fraction. It contains a component. In some embodiments, the antibody is an isolated antibody.

[0010] Aspects of the present invention include an antibody that binds to SLAMF6 or an antigen-binding fragment thereof. The antibody includes a heavy chain variable region, and the heavy chain variable region includes the sequence of SEQ ID NO: 5. H1 sequence, CDR-H2 sequence containing sequence number 6 or sequence number 15, and sequence number The CDR-H3 sequence includes sequence 7, and in some embodiments, an antibody or antigen is included. The combined fragment contains either the sequence of sequence number 8 or sequence number 16 in the CDR- CDR-L2 containing one of the sequences L1, sequence number 9, or sequence number 17, and At least one CD selected from the group consisting of CDR-L3 containing the sequence of sequence number 10 It further includes a light chain variable region containing the R sequence.

[0011] In some embodiments, an antibody or antigen-binding flag that binds to SLAMF6 The ment consists of one of eight combinations of heavy and light chain CDRs, as shown in Table 1. It includes heavy chain variable regions and light chain variable regions.

[0012] [Table 1]

[0013] In some preferred embodiments, an antibody or antigen-binding agent that binds to SLAMF6 The fragments include CDR-H1 containing SEQ ID NO: 5, CDR-H2 containing SEQ ID NO: 6, and A heavy chain variable region including CDR-H3 containing SEQ ID NO: 7, and CDR-L1 containing SEQ ID NO: 8, Light chain variable CDR-L2 containing SEQ ID NO: 9, and CDR-L3 containing SEQ ID NO: 10. Includes the region.

[0014] In another preferred embodiment, an antibody that binds to SLAMF6 or its antigen-binding fragment The sequence includes CDR-H1 containing sequence number 5, CDR-H2 containing sequence number 15, and sequence A heavy chain variable region including CDR-H3 containing number 7, and CDR-L1 containing sequence number 16, Includes CDR-L2 containing column number 17, and CDR-L3 containing sequence number 10.

[0015] In a further embodiment, the antibody or antigen-binding fragment of the present invention is described herein. It contains a variable CDR compared to the parent antibody described. Therefore, the present invention is a variant of the parent antibody. This provides a mutant antibody containing a variable region or its antigen-binding fragment, and the parent antibody or The antigen-binding fragment contains the CDR-H1 sequence, which includes the sequence of SEQ ID NO: 5, and the sequence of SEQ ID NO: 15. Heavy chain containing the CDR-H2 sequence including the column and the CDR-H3 sequence including the sequence of sequence number 7. Variable region and CDR-L1 containing the sequence of sequence number 16, CDR containing the sequence of sequence number 17 -Includes a light chain variable region including L2 and CDR-L3 which includes the sequence of sequence number 10, In one embodiment, the mutant antibody or its antigen-binding fragment is used for 1 to 5 specific applications. Alternatively, CDR-H1 having 1 to 4 substitutions, additions, or deletions. , CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 set In the case of the t, collectively, there are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 A In one or more substitutions, additions, and / or deletions of a mino acid, 1, 2, It has substitutions, additions, and / or deletions of 3, 4, 5, or 6 amino acids, and antibodies Alternatively, the antigen-binding fragment retains a specific binding to SLAMF6. Preferably, Variations are substitutions, preferably substitutions are conservative substitutions or ami in the variable region. This is a substitution to return a no acid to the corresponding amino acid from the human germ cell line. Further implementations In this state, the modified antibody of the present invention or its antigen-binding fragment is the sequence of SEQ ID NO: 5 CDR-H1 sequence containing; CDR-H2 sequence containing sequence of sequence number 15; and sequence number CDR-H3 sequence containing sequence 7; and CDR-L1 sequence containing sequence number 16; CDR-L2 containing the sequence at column number 17; and CDR-L3 containing the sequence at sequence number 10. Including a light chain variable region, one or more of the CDR sequences are the corresponding parent CDs listed above. R sequence and approximately 70%, 75%, 80%, 85%, 87%, 88%, 89%, 90%, 91% 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are identical. It will be altered.

[0016] In some embodiments, the heavy chain variable described in Sequence ID No. 1 or Sequence ID No. 13 The region, or the sequence described in SEQ ID NO: 1 or SEQ ID NO: 13, is approximately 80%, 85%, 9%. 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% are the same. The sequence and / or the light chain variable region described in SEQ ID NO: 2 or SEQ ID NO: 14 , or with sequence number 2 or sequence number 14, approximately 80%, 85%, 90%, 91%, 92% Antibodies containing sequences that are identical by 93%, 94%, 95%, 96%, 97%, 98%, and 99%. or the antigen-binding fragment thereof is provided. In a further embodiment, if SEQ ID NO: 1 Compared to sequence number 13, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 1 A heavy chain variable region and / or sequence containing two amino acid substitutions, additions, and / or deletions. Compared to number 2 or sequence number 14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 Antibodies or light chain variable regions containing 1 or 12 amino acid substitutions and / or deletions The antigen-binding fragment is provided. Preferably, the variant is substituted, more preferably retained. Includes existential substitution.

[0017] Amino acid substitutions, additions, and / or deletions are within the framework region and / or CD. It will become even clearer that this is possible within R.

[0018] In one embodiment, a heavy chain variable region including the sequence of sequence number 1, and the sequence of sequence number 2 are included. An antibody or its antigen-binding fragment containing a light chain variable region is provided.

[0019] In one embodiment, a heavy chain variable region including the sequence of sequence number 13, and the sequence of sequence number 14. An antibody or its antigen-binding fragment containing a light chain variable region is provided.

[0020] In one embodiment, the heavy chain sequence includes sequence number 18 and the light chain sequence includes sequence number 19. Full-length antibodies are provided.

[0021] In yet another embodiment of the present invention, three heavy chain CDRs of SEQ ID NO: 1 or SEQ ID NO: 13 and a SLAMF6-specific compound containing three light chain CDRs of SEQ ID NO: 2 or SEQ ID NO: 14. An antibody or antigen-binding fragment that binds to it is provided, and the CDR is by Kabat. It is defined by [the system name] or by the Chothia numbering system. Alternatively, the antigen-binding fragment that specifically binds to the antibody or SLAMF6. This is a sequence number defined by the Kabat or Chothia numbering system. It includes three heavy chain CDRs of sequence number 13 and three light chain CDRs of sequence number 14.

[0022] Sequence IDs 13-19 are humanized antibody sequences based on the sequences of Sequence IDs 1 and 2. The manufacturer states that sequence numbers 18-19 are full-length functional antibodies, i.e., the constant region and Fc region. It is a full-length heavy chain and light chain sequence that includes regions other than the variable region necessary for production. Those skilled in the art will easily understand this. Those skilled in the art will also understand that the amino acids derived from the variable region of the organism from which the antibody is produced are amino acids. By replacing these with amino acids derived from human germline sequences, these can be administered to human subjects. To minimize the immunogenicity of the antibody in that case, it is important to understand that the sequence is humanized. Most amino acid substitutions occur in the framework region, but not in noncritical positions. Many amino acids from the CDR may be substituted, and such substitutions are preferably in the properties. They are either conserved or have amino acids at specific positions present in the corresponding human germ cell system. Return to amino acids. In this case, the amino acids from the substituted CDR are obtained using a structural model. Identification by distinguishing between paratope surface residues and non-paratopic residues in the CDR region. This allows for a higher degree of humanization of the antibody than with a simple CDR graft. .

[0023] In the present invention, Sequence ID 13 is compared with Sequence ID 1's CDR2 (Sequence ID 6). The compound contains two amino acid substitutions in CDR2 (SEQ ID NO: 15). Specifically, There is a KQ substitution at position 16 of sequence number 6, and a DG substitution at position 17 of sequence number 6.

[0024] In the present invention, Sequence ID 14 is compared with Sequence ID 2's CDR1 (Sequence ID 8). The compound contains three amino acid substitutions in CDR1 (SEQ ID NO: 16). Specifically, SQ substitution at position 1 of SEQ ID NO: 8; SQ substitution at position 4 of SEQ ID NO: 8; and position 5 of SEQ ID NO: 8 There is an SD substitution at position 14. Sequence ID 14 is compared with the CDR2 of sequence ID 2 (sequence ID 9). This further includes one amino acid substitution in CDR2 (SEQ ID NO: 17). Specifically, An ST substitution exists at position 7 of sequence number 9.

[0025] These humanized sequences do not represent different, selective antibodies compared to the parent antibody. Instead, structural modeling is used to more closely study human germ cell lines in order to minimize immunogenicity. This concerns the same antibody with the same properties, but modified to correspond to the same characteristics. This will be understood by those skilled in the art.

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

[0027] In some embodiments, the antibody or antigen-binding fragment is a monoclonal antibody. It is a body. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the heavy chain variable region includes a framework sequence. In one embodiment, at least a portion of the framework array is a human consensus frame Includes work array. In some embodiments, the light chain variable region is the framework array. This includes. In some embodiments, at least a portion of the framework array is a single co Includes consensus framework array.

[0028] Furthermore, this includes substitutions of residues within the antibody low hinge, such as L234A and L235A (LALA). Hmm, alternative strategies to mitigate the antibody effector function are also being employed. These residues form part of the Fc-γ receptor binding site on the CH2 domain, and these residues The exchange between antibody isotypes identifies their importance in ADCC, Or it had a smaller effector function. Alanine substitution at these positions is human Both the mouse antibody and the replacement are effective in reducing ADCC, but these substitutions It is not very effective in reducing CDC activity (Lo M et al, J Biol Chem. 2017). Mar 3;292(9):3900-3908). In some embodiments, antibody or antigen-binding fragments are used. The nucleotide is an Fc variant that has been engineered to reduce its binding to the FC gamma receptor. This results in reduced effector functionality and ADCC activity.

[0029] In some embodiments, the antibody or antigen-binding fragment is Fc silence Modified IgG1 antibodies, or antibodies with reduced binding to one or more Fc receptors. An antigen-binding fragment that has or does not have any antigen-binding fragment. In another embodiment, the antibody is I This is a gG4 antibody.

[0030] In some embodiments, antibodies or antigen-binding fragments cause T cell cytotoxicity. Bispecific antibodies or antigen-binding fragments that mediate sex and / or NK cell cytotoxicity In some embodiments, the antibody or antigen-binding fragment is used on immune cells. Activation can be induced and / or enhanced. In one embodiment, immune cells are preferred In another embodiment, the immune cell is preferably an NK cell. Those skilled in the art will know The terms induction and / or enhancement refer to the induction and / or enhancement of cytokine release by immune cells. or enhance and / or induce and / or enhance the proliferation of the immune cells, and / It is important to understand that this can also refer to inducing and / or enhancing cytotoxic activity. It will be. The term "to guide" or "to guide" as used in this context. This involves causing the activation of immune cells, or the absence of antibodies or antigen-binding fragments. This means increasing the activation of immune cells to a level exceeding the activation level observed under normal circumstances. This will be readily apparent to those skilled in the art. The use of "enhance" in this context is The term refers to increasing the activation level of already activated immune cells.

[0031] In some embodiments, the antibody or antigen-binding fragment is SLAMF6 tannin. It binds to PAC (e.g., SEQ ID NO: 11) and to one or more additional binding targets, A bispecific or multispecific antibody or antigen-binding fragment, preferably the aforementioned The additional binding targets are one or more tumor antigens. In a further embodiment, one The further binding targets mentioned above are immunomodulatory molecules. In one embodiment, the additional binding target is P It is D-L1. In one embodiment, the antibody or its antigen-binding fragment is divalent. In another embodiment, the antibody or its antigen-binding fragment is quadrivalent. The antibody or its antigen-binding fragment is trivalent.

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

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

[0034] In another embodiment, a vector comprising one or more nucleic acids of the present invention is provided. In another aspect of the present invention, the heavy chain and / or light chain, or both, of the antibody of the present invention are used. A host cell containing one or more nucleic acids is provided. In some embodiments, The host cells are grown under conditions in which nucleic acids are expressed. In other embodiments, the present invention A method for recovering antibodies is provided.

[0035] Aspects of the present invention include a method for producing an antibody or an antigen-binding fragment thereof. The method involves the host cell being expressed under conditions in which the antibody or antigen-binding fragment is expressed in the host cell. This includes culturing the antibody or antigen-binding fragment, and optionally isolating the antibody or antigen-binding fragment. .

[0036] Aspects of the present invention include an antibody or antigen-binding fragment as described herein, and a drug The pharmaceutical composition includes a scientifically acceptable carrier. In one embodiment, the pharmaceutical composition or pharmaceutical further comprises an effective amount of a second therapeutic agent.

[0037] In a further embodiment of the present invention, a method for treating a disorder is provided, the method which does not require In patients requiring treatment, the antibody or antigen conjugate of the present invention to SLAMF6 (SEQ ID NO: 11) This includes administering a fragment. In one embodiment, the disorder is cancer.

[0038] In a further embodiment, an effective amount of the antibody or antigen-binding fragment of the present invention is used. A method for treating cancer is provided, which includes administering the drug to the target in need.

[0039] In one embodiment, the antibody or antigen-binding fragment is CDR-H1 containing SEQ ID NO: 5 , CDR-H2 containing SEQ ID NO: 6 or SEQ ID NO: 15, and CDR-H2 containing SEQ ID NO: 7 A heavy chain variable region containing H3, and CDR-L1 containing sequence number 8 or sequence number 16, sequence number CDR-L2 containing code number 9 or sequence number 17, and CDR-L3 containing sequence number 10. Includes a light chain variable region.

[0040] Preferably, the heavy chain variable region is CDR-H1 containing SEQ ID NO: 5, and C containing SEQ ID NO: 15. Includes DR-H2 and CDR-3, which includes sequence number 7.

[0041] Preferably, the light chain variable region includes CDR-L1, which includes SEQ ID NO: 16, and SEQ ID NO: 17. Includes CDR-L2 and CDR-L3, which includes sequence number 10.

[0042] In some embodiments, a method for treating cancer is provided, and here it is necessary The antibody or antigen-binding fragment of the present invention is administered to the patient, and the antibody or antigen-binding fragment of the present invention Antigen-binding fragments trigger immune responses, such as cytotoxic T cell responses and / or NK cells. It induces and / or enhances cellular responses.

[0043] In a further embodiment, the antibody or its antigen-binding fragment is SLAMF6( SEQ ID NO: 11) and bispecific or multispecific antibodies that bind to tumor-specific antigens or It contains the antigen-binding fragment.

[0044] In some embodiments, cancer is small cell lung cancer, non-small cell lung cancer (squamous cell carcinoma and glandular cancer). Cancer (including melanoma), skin cancer, breast cancer (including TNBC), colorectal cancer, stomach cancer 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, neurological The group is selected from secretory tumors and hematological malignancies.

[0045] According to a further aspect of the present invention, the antibody or An antigen-binding fragment is provided.

[0046] Preferably, the antibody or antigen-binding fragment is used for the prevention or treatment of cancer. It belongs to them.

[0047] According to a further aspect of the present invention, the present invention relates to the manufacture of a pharmaceutical product for the treatment of cancer. The use of a body or antigen-binding fragment is provided. In some embodiments, the cancers described in the previous model are small cell lung cancer and 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, and 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, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, muscle The group is selected from tumors, GISTs, neuroendocrine tumors, and hematological malignancies. [Brief explanation of the drawing]

[0048] [Figure 1] Figure 1a shows the amino acid sequence of the variable region of the heavy chain of the parental mouse 1B3 antibody (SEQ ID NO: 1). Figure 1b shows the amino acid sequence of the variable region of the light chain of the parental mouse 1B3 antibody (SEQ ID NO: 2). [Figure 2] Figure 2a shows the amino acid sequence of the variable region of the heavy chain of the humanized antibody Hu_1B3 (SEQ ID NO: 13). Figure 2b shows the amino acid sequence of the variable region of the light chain of the humanized antibody Hu_1B3 (SEQ ID NO: 14). [Figure 3] Figure 3a shows the sequence alignment of the heavy chain variable region of the 1B3 parental mouse antibody sequence and the humanized heavy chain variable region 1B3 sequence. Figure 3b shows the sequence alignment of the light chain variable region of the 1B3 parental mouse antibody sequence and the humanized light chain variable region Hu_1B3 sequence. [Figure 4] Figure 4 shows the specific dose-dependent binding of antibody 1B3 to 1B3 expressing Raji cells. [Figure 5] Figure 5 shows that antibody Hu_1B3 enhanced the ability to mediate IFNγ production upon T cell activation compared to SLAMF6 antibodies at another clinical stage. [Figure 6]Figure 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. This assay demonstrates enhanced activity of antibody 1B3 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. This assay demonstrates enhanced activity of antibody 1B3 compared to pembrolizumab. [Figure 9] Figure 9 shows that activating SLAMF6 present on T cells isolated using the antibody Hu_1B3 induces proliferation of CD8+ T cells. [Figure 10] Figure 10 shows an MLR assay, demonstrating that the antibody Hu_1B3 can induce DC-mediated T cell activation, as highlighted by increased IFNy release. [Figure 11] Figure 11 shows that the anti-SLAMF6 antibody Hu_1B3 can induce activated T cells and produce granzyme B in a dose-dependent manner. [Figure 12] Figure 12 shows that the antibody Hu_1B3 dose-dependently upregulates perforin expression on CD8+ T cells. [Figure 13] Figure 13 shows that the Hu_1B3 antibody is blocked from binding to receptors on the surface of PBMCs in the presence of the human SLAMF6 ECD-mIgG2a Fc fusion protein. [Figure 14] Figure 14 shows the incorporation of the humanized antibody Hu_1B3 into SLAMF6-expressing cells at a significantly lower rate than that of the anti-SLAMF6 antibody produced by Seattle Genetics. [Figure 15]Figure 15 shows that the addition of Hu_1B3 enhances lymphocytotoxicity against SKBR-3 cells when used in conjunction with an anti-her2 anti-CD3Bi specific antibody. [Figure 16] Figure 16 shows that the addition of Hu_1B3 enhances lymphocytotoxicity against SKBR-3 cells when used in conjunction with an anti-her2 anti-CD3Bi specific antibody. [Figure 17] Figure 17 shows that the addition of Hu_1B3 enhances lymphocyte cytotoxicity against HCT116 cells when used in conjunction with an anti-her2 anti-CD3 bispecific antibody. [Figure 18] Figure 18 shows that the addition of Hu_1B3 enhances lymphocytotoxicity against MDA-MB-231 cells when used in conjunction with an anti-her2 anti-CD3 bispecific antibody. [Figure 19] Figure 19 shows that after activating lymphocytes with various antibodies for 96 hours, the antibody HU_1B3 resulted in significantly more SKBR-3 cell death than either urelumab (anti-CD137) or its isotype. [Figure 20] Figure 20 shows that when used with a fixed-concentration anti-Her2 / anti-CD3 bispecific antibody, the addition of Hu_1B3 dose-dependently enhances lymphocyte cytotoxicity against SKBR-3 cells. [Figure 21] Figures 21a and 21b show that the addition of Hu_1B3 in the absence of enabling bispecific antibodies also enhances lymphocytotoxicity against SKBR-3 cells. [Modes for carrying out the invention]

[0049] Embodiments of the present invention include an antibody against SLAMF6, and a nucleic acid encoding such an antibody. Using host cells containing such nucleic acids encoding the antibody of this invention, an anti-SLAMF6 antibody is prepared. Methods for doing so, and 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, kidney cancer, and 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, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, muscle This includes, but is not limited to, tumors, GISTs, neuroendocrine tumors, and hematological malignancies. This includes treatment methods for diseases such as cancer.

[0050] As used herein and in the appended claims, the singular "one (a)" "An" and "the" are used unless the context explicitly indicates otherwise. Please note that this includes multiple references. Thus, the claims include any and all necessary references. It should be noted that we can draft it in a way that excludes primes. In that way, This description is related to the description of elements of the claims, such as "only" or "only" As a prerequisite for the use of exclusive terminology or the restrictive use of "negative" It is intended to be helpful.

[0051] As will be apparent to those skilled in the art by reading this disclosure, the information described and illustrated herein is Each of these individual embodiments may, without departing from the scope or spirit of the present invention, describe several other embodiments. Individual components that can be easily separated or combined from any of the features of the embodiment It has the following characteristics. In any of the cited methods, the cited events are in the order or logically. It can be executed in any other possible order.

[0052] definition For the purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, the singular form The terms used also include plural forms, and vice versa.

[0053] The term "SLAMF6" as used herein, unless otherwise specified, refers to primates. Pups of species such as humans, primates, and rodents (e.g., mice and rats) Refers to any natural SLAMF6 protein of any vertebrate source, including dairy. The F6 protein is also called a SLAMF6-like protein. The amino acid composition of human SLAMF6 is... The column is provided herein in Sequence ID No. 11.

[0054] The term "SLAMF6" refers to both "full-length" untreated SLAMF6 and the intracellular progenitor. This term encompasses any form of SLAMF6 arising from cessation. 6 naturally occurring variants, such as splice variants, allele variants, and iso This term encompasses the naturally occurring cleavage forms of the SLAMF6 polypeptide. This includes secretory types (e.g., extracellular domain sequences). SLAMF6 as described herein Polypeptides can be isolated from various sources, such as human tissue types or other sources. It can be prepared by recombinant or synthetic methods. The AMF6 polypeptide is the same amino acid as the naturally derived SLAMF6 polypeptide. It contains polypeptides having an acid sequence. Such a natural sequence SLAMF6 polypeptide is It can be isolated from nature, or produced by recombinant or synthetic means. The term "SLAMF6 epitope" as used herein refers to the term used herein. Conjugated by an antibody containing at least one or more CDR sequences, and / or Epi, as illustrated by the binding profile of the anti-SLAMF6 antibody shown in the examples. It refers to taupe.

[0055] The term "antibody" is used in its broadest sense, for example, one anti-SLAMF6 monoantibody. Clonal antibodies (agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies) A composition of anti-SLAMF6 antibody having polyepitopy specificity (including a polychloro antibody), polychloro A multispecific antibody formed from a single antibody, a multivalent antibody, and at least two intact antibodies. (For example, a bispecific antibody that exhibits the desired biological activity), single-chain anti-SLAMF6 antibody The body, and Fab, Fab', F(ab')2 and Fv and FV-TCR fragments Antigen-binding fragments, diabodies, and single-domain antibodies containing anti-SLAMF6 antibodies are also included. (sdAb) specifically include insofar as they exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" as used herein. The antibodies may be chimeric, human, humanized, and / or affinity-matured. In some embodiments, at least the antibody is six as defined herein. It will be understood by those skilled in the art that the set of CDRs includes conventional antibodies (monoc (Containing both ronal and polyclonal antibodies), humanized, human and / or chimeric Antibodies, antibody fragments, and modified antibodies (for example, amino acid-modified antibodies outlined below) (with embellishments), multispecific antibodies (including bispecific antibodies), and known in the art, This includes, but is not limited to, other analogues discussed in the specification.

[0056] In other embodiments, the term “antibody” as used herein is not limited to, but may also refer to Nano Includes body(registered trademark), Unibody(registered trademark), and scFv fragments. It will be understood that this refers to a structure that does not include the six CDRs.

[0057] "Anti-SLAMF6 antibody," "SLAMF6 antibody," or "Anti-SLAMF6 binding antibody" The term "body" is useful as a diagnostic and / or therapeutic agent targeting SLAMF6. This refers to an antibody that can bind to SLAMF6 with sufficient affinity. In this state, anti-SLAMF6 antibodies are conserved among SLAMF6 from different species. It binds to the AMF6 epitope.

[0058] "Isolated antibodies" are identified and isolated from the components of that environment, and / or It was recovered. The contaminants in that environment are materials that would interfere with the therapeutic use of the antibody. It may contain enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Cut.

[0059] Regarding the binding of antibodies to target molecules, "specific binding" refers to a specific polypeptide or specific "Specifically binds" or "specifically" to epitopes on specific polypeptide targets. The term refers to a binding that is measurably different from a nonspecific interaction. Specific binding is, For example, compare the binding of a control molecule (generally a molecule with a similar structure that does not possess binding activity). This can be measured by determining the molecular bonding.

[0060] The term "antagonist" is used in its broadest sense, referring to natural SLAMF6 polypeptide Contains any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of [the substance]. Hmm. Suitable antagonist molecules include, specifically, antagonist antibodies or antibody flashes. Ingredients, natural SLAMF6 polypeptide, peptides, antisense oligonucleotides Examples include fragments or amino acid sequence variants of small organic molecules. A method for identifying MF6 polypeptide antagonists is SLAMF6 polypeptide The drug is brought into contact with a candidate antagonist molecule, and one that is typically associated with the SLAMF6 polypeptide. This may include measuring the detectable changes in the above biological activities.

[0061] The term "agonist" is used in its broadest sense to refer to natural SLAMF6 polypeptides. Contains any molecule that enhances the biological activity of the cytoplasm. Suitable agonist molecules include SLAM. F6 ligand polypeptide, peptide, antisense oligonucleotide, low molecular weight organic compound Agonist antibodies or antibody fragments, fragments or amino acid sequence modifications, such as children. The variant is specifically included. A method for identifying agonists of the SLAMF6 polypeptide. This involves contacting the SLAMF6 polypeptide with a candidate agonist molecule, and then the SLAMF6 polypeptide This may include measuring a detectable change in one or more biological activities typically associated with tides. .

[0062] As used herein, "tumor" refers to all neoplasms, whether malignant or benign. This refers to the growth and proliferation of cells, as well as all precancerous and cancerous cells and tissues.

[0063] As used herein, the terms “predictive” and “prognostic” also mean “predictive or prognostic.” The method for determination is that the person performing this method is using anticancer drugs containing anti-SLAMF6 antibodies. Patients who are considered more likely to respond to treatment (usually before treatment, but not necessarily) Interchangeable in the sense that it allows for the selection of (otherwise) .

[0064] SLAMF6 protein According to UNIPROT, SLAMF6 is part of the immunoglobulin superfamily and It is a single-pass type I membrane protein of the CD2 subfamily. The protein is sequence number 11 extracellular domains between amino acids 22-226, and one between amino acids 227-247 It consists of a transmembrane region and a single cytoplasmic region between amino acids 248 and 331.

[0065] In some embodiments, the antibody of the present invention binds to human SLAMF6. The terms "human SLAMF6" or "human SLAMF6 protein" used in this document refer to the information herein. This refers to the protein with sequence number 11 as defined in [the relevant documentation].

[0066] The antibodies according to embodiments of the present invention may, in some cases, be derived from non-human species of SLAMF6 tannin. It may cross-react with Pak. For example, to facilitate preclinical and toxicity tests, the present invention The antibody may cross-react with the mouse or primate SLAMF6 protein. Alternatively, In certain embodiments, the antibody may be specific to the human SLAMF6 protein, and also It does not need to exhibit other types of non-human cross-reactivity.

[0067] antibody Aspects of the present invention, as described herein, include anti-SLAMF6 antibodies, generally used in therapeutic anti- This includes antibodies for body and / or diagnostic use. The antibodies found to be used in the methods of the present invention are specified herein. As further described, traditional antibodies and antibody derivatives, antigen-binding fragments This includes any of the numerous formats described herein, including imitations. This is possible. In some embodiments, the antibody is one of the six CDRs defined herein. One or one selected from the set (including a small number of amino acid changes described herein) It has multiple CDRs. As reviewed above, the term "antibody" as used herein This refers to various structures.

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

[0069] The amino-terminus of each antibody chain consists of approximately 100-110 or more amino acids, which are primarily involved in antigen recognition. It includes the variable region of the mino acid. In the variable region, the V domains of the heavy chain and light chain are respectively The three loops then converge to form an antigen-binding site. Each loop is a complementarity-determining region (hereinafter, It is called "CDR" (Cardiomeric Variability), and the most pronounced mutations are in the amino acid sequence. "Variable" means This means that specific segments of the variable region have significantly different sequences between antibodies. The variability is not evenly distributed. Instead, the V region consists of 15 to 30 amino acids. It consists of relatively invariant extension areas called framework areas (FRs), each of which is 9 A short region with extreme variability called the "hypervariable region," consisting of approximately 15 amino acids. Therefore, they are separated.

[0070] Each VH and VL has three hypervariable regions ("complementary decision regions," "CDRs") and four Composed of two FRs, from the amino terminus to the carboxyl terminus, FR1-CDR1-FR2- They are arranged in the order CDR2-FR3-CDR3-FR4.

[0071] The ultra-variable region is generally the 24-34 (CDR-L1; "L" stands for light) range in the light chain variable region. Chain), 50-56 (CDR-L2) and 89-97 (CDR-L3), and heavy chain variable region Approximately 31-35B (CDR-H1; "H" indicates heavy chain), 50-65 (CDR-H2) in the region. and amino acid residues derived from 95-102 (CDR-H3), Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Ins. Titles of Health, Bethesda, Md. (1991) and / or hypervariable loops (e.g., light chains) 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. These form 53-55 (CDR-H2) and 96-101 (CDR-H3). The residues include those described in Chothia and Lesk (1987) J. Mol. Biol. 196:901-917. The embodiment of the present invention The following are examples of CDRs.

[0072] Throughout this specification, the Kabat numbering system refers to residues in variable domains ( Refer to residues 1-107 of the light chain variable region and residues 1-113 of the heavy chain variable region. It is commonly used in such cases (for example, Kabat et al., (1991)).

[0073] CDRs contribute to the formation of antigen-binding sites on antibodies, more specifically, epitope-binding sites. A single antigen can have multiple epitopes.

[0074] The IgG subclass of immunoglobulins has several immunoglobulin domains in its heavy chain. Yes. In this specification, the "immunoglobulin (Ig) domain" has a distinct tertiary structure. This refers to the region of immunoglobulins. What is interesting in this invention is the constant heavy chain (CH) It is a heavy chain domain containing the domain and hinge domain. In relation to IgG antibodies, I Each gG isotype has three CH regions.

[0075] Another type of Ig domain in the heavy chain is the hinge region. In this specification, "hinge" refers to... What are "hinge regions," "antibody hinge regions," or "immunoglobulin hinge regions"? , flexible poly containing amino acids between the first and second constant domains of the antibody It means peptide.

[0076] Of particular interest in this invention is the Fc region. The term "Fc" as used herein refers to... The term "Fc region" or "Fc domain" refers to the first constant region immunoglobulin domain. And in some cases, this means a polypeptide that includes the constant region of the antibody, excluding part of the hinge. Therefore, Fc is the last two constant-region immunoglobulins of IgA, IgD, and IgG. Immunoglobulin domains: the last three constant regions of IgE and IgM, and the flexible hinge N-terminus to these domains. IgA and IgM Regarding Fc, it can contain a J chain. Regarding IgG, the Fc domain is immune Globulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3), and Cγ1 (Cγ1) It includes the lower hinge region between Cγ2(Cγ2). The boundary of the Fc region can vary, The human IgG heavy chain Fc region is typically the residue C226 or P23 at its carboxyl terminus. Defined to include 0, where the numbering is EU indexed as in Kabat. It follows the rules. In some embodiments, amino acid modifications include, for example, one or more FcγR To modify the binding to the receptor or FcRn receptor, the Fc region is subjected to It can be done.

[0077] In some embodiments, the antibody is full length. The term "full-length antibody" as used herein and This includes a variable region and a constant region which optionally include one or more modifications as outlined herein. This refers to the structures that constitute the natural biological formation of antibodies.

[0078] Alternatively, antibodies can be antigen-binding fragments, monoclonal antibodies, bispecific antibodies, and more. Nibodies, domain antibodies, synthetic antibodies (sometimes referred to as "antibody mimics" in this specification) ), chimeric antibodies, humanized antibodies, antibody fusions (sometimes called "antibody conjugates") A variety of antigen-binding fragments, including, but not limited to, these, and their respective antigen-binding fragments. A structure that can be formed and depends on the use of a set of CDRs falls within the definition of an "antibody". It is included.

[0079] In one embodiment, the antibody is an antigen-binding fragment. Specific antigen-binding antibody fragment The fragment consists of (i) VL, VH, CL, and CH1 domains. (ii) Fd fragment consisting of VH and CH1 domains, (iii) (iv) From a single variable region, an Fv fragment consisting of the VL and VH domains of one antibody, (iv) from a single variable region This is a dAb fragment (the whole is incorporated by reference Ward et al., 1989, Nature 34). 1:544-546), (v) isolated CDR region, (vi) F(ab')2 fragment, 2 A bivalent fragment containing two linked Fab fragments, (vii) a single-chain Fv molecule ( scFv) and the VH domain and VL domain are formed when the two domains are joined together to protect Linked by peptide linkers that enable the formation of a primary binding site (see reference) The whole is incorporated by 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 (International Publication 03 / 11161, which is incorporated in its entirety by reference), and (ix) "Diabody" or "Triabody", a polyvalent or constructed by gene fusion Multiple specific fragments (which are incorporated by reference throughout their entirety, as shown by Tomlinson et al.) al., 2000, Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc This includes, but is not limited to, the following (e.g., Natl. Acad. Sci. USA 90:6444-6448).

[0080] Chimeric and humanized antibodies In some embodiments, the antibody is of a different species, e.g., a chimeric antibody and / or It may be a mixture from humanized antibodies. That is, in the present invention, the CDR set is the present invention Frameworks and constant domains other than those specifically described by the arrangement in the details are also included. It can be used for this purpose.

[0081] Generally, "chimeric antibodies" and "humanized antibodies" are antibodies that combine regions from multiple species. Yes, for example, "chimeric antibodies" are typically from mice (or rats, in some cases) It includes a variable region from and a constant region from humans. "Humanized antibody" generally refers to a human antibody Non-human antibodies with a variable domain framework region that has been exchanged for sequences found in the body It refers to the body. Generally, in humanized antibodies, the entire antibody, excluding CDRs, is of human origin polynucleonucleotides. Encoded by leotide, or identical to such antibody except within its CDR Yes, CDRs are encoded by nucleic acids, some or all of which originate from non-human organisms. Therefore, the beta sheet framework of the human antibody variable region is transplanted to produce antibodies, and Specificity is determined by the engrafted CDR. Such antibody production is, for example, WO 9 2 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:15 This is described in 34-1536 and is fully incorporated by reference. In one embodiment, In this context, the antibody of the present invention may be a multispecific antibody, particularly a bispecific antibody, and sometimes "dia Also called "bodies." These are two (or more) different antigens, or the same anti- It is an antibody that binds to different epitopes on the original surface. Diabody is known in the art. Various methods (Holliger and Winter, 1993, Current Opinion Biotechnol. 4:446-449, see reference) It can be manufactured by (being fully integrated by illumination), for example, chemically or by hive It can be manufactured from Lid Hybridoma.

[0082] In one embodiment, the antibody is a minibody. The minibody has a CH3 domain. Minimize the antibody-like protein containing the bound scFv. (Fully referenced by Hu e) t al., 1996, Cancer Res. 56:3055-3061. In some cases, scFv is linked to the Fc region. It can be joined and may include part or all of the hinge area. The mini body is C Despite not having a complete set of DRs, it is included within the definition of "antibody". Please take note.

[0083] The antibodies of the present invention are generally isolated or recombinant. In some embodiments, the antibody of the present invention is a recombinant protein, an isolated protein, or It is essentially a pure protein. An "isolated" protein is one that is normally present in its natural state. Without at least some of the associated substances, for example, the total protein in a given sample It constitutes at least approximately 5% by weight, or at least approximately 50% by weight. Isolated protein It is understood that, depending on the circumstances, it can constitute 5 to 99.9% by weight of the total protein content. For example, proteins use either an inducible promoter or a high-expression promoter. Therefore, it can be produced at a considerably high concentration, and as a result, the protein is produced at the increased concentration level. It is produced. In the case of recombinant proteins, the definition is that it is not produced naturally in the relevant technical field. It includes the production of antibodies in a wide range of organisms and / or host cells known in the art. Usually, the isolated polypeptide is prepared by at least one purification step. An "isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities. For example, an isolated antibody that specifically binds to SLAMF6 substantially does not contain antibodies that specifically bind to antigens other than SLAMF6.

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

[0085] Specific binding to a particular antigen or epitope can be demonstrated, for example, by an antibody having a K -4 of at least about 10 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 1M, at least about 10 -9 M, or at least about 10 -10 M, at least [[ID=4"]]0]at least about 10 -11 M, at least about 10 -12 M or more for an antigen or epitope, where K is the dissociation rate of a particular antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen will have a K D that is 20-, 50-, 100-, 500-, 1000-, 50 D 00-, 10,000-fold or more lower for the antigen or epitope compared to a control molecule. D ​​​​​​​

[0086] Furthermore, specific binding to a particular antigen or epitope is, for example, K A or K a but When referring to the association rate of a specific antibody-antigen interaction, a smaller value relative to the epitope compared to the control is used. Even without that, 20, 50, 100, 500, 1000, 5000, 10,000 - K against antigens or epitopes with a ratio of - times or more A or K a This is demonstrated by antibodies that possess It is possible.

[0087] A standard assay to evaluate the binding ability of antibodies to SLAMF6 is the protein Alternatively, it can be done at the cellular level, for example, by ELISA, Western blot, or RIA. Octet® assay and flow cytometry This includes tree analysis, which is known in the art. Appropriate assays are described in detail in the examples. The binding kinetics of antibodies (e.g., binding affinity) are also known as Biacore® or Through Octet® system analysis and other methods, the standard assay known in this field is applied. It can be evaluated more highly.

[0088] SLAMF6 antibody This invention provides a SLAMF6 antibody that binds to the SLAMF6 polypeptide or a portion thereof. Provided. An example of the SLAMF6 amino acid sequence is provided in SEQ ID NO: 11. Subject SL AMF6 antibodies enhance the immune response in tumors by activating immune cells, for example, T These can induce or enhance cell activation and / or NK cell activation. In this specification, the antibody is referred to as the "anti-SLAMF6" antibody, or, for ease of explanation, as "S It is called the "LAMF6 antibody."

[0089] In some aspects, the subject SLAMF6 antibody attacks T cells, particularly those on their surface, which are SLA When MF6 is in contact with CD4+ or CD8+ T cells expressing MF6, cytokine release or Proliferation can be induced and / or enhanced. Cytokine release in this situation T cell proliferation can be measured in several ways. In one embodiment, The SLAMF6 antibody of the present invention can be used in standard assays such as ELISA to detect activated T cells. It comes into contact with. In a further embodiment, the target SLAMF6 antibody activates NK cells. And it can induce and / or enhance killing.

[0090] In one embodiment, the antibody is an antibody containing the following CDR; in addition, as discussed below Furthermore, these CDR sequences also have a limited number of amino acid variants, as previously described. It can contain: [Table 2]

[0091] In some embodiments, the antibody is sequence numbers 5, 15, 7, 16, 17 and 10. It includes the amino acid sequence of at least one CDR sequence provided to. Several implementations In this state, the antibody is provided in the CDRs of SEQ ID NOs. 5, 15, 7, 16, 17 and 10. One or more amino acid sequences of the sequence and at least about 75%, 80%, 85%, 90%, Contains amino acid sequences that are 95%, 96%, 97%, 98%, or 99% identical.

[0092] This specification includes variable heavy and light chains, as well as full-length heavy chains, including the CDR set of the present invention. The light chain (including, for example, the steady-state region) is also disclosed. As those skilled in the art will understand, Akira's CDR set includes mouse, humanized, or human constant regions (including framework regions). It can be incorporated into. An aspect of the present invention is a heavy chain variable region array (arrangement) disclosed herein. Column number 13) and light chain variable region sequence (sequence number 14) and at least about 80%, 85%, Heavy and light chain variable regions are 90%, 95%, 96%, 97%, 98%, or 99% identical. Includes a variable chain region.

[0093] In some embodiments, the present invention is described herein as the SLAMF6 Monoclonal antibody (i.e., the monoclonal antibody of the present invention as described herein and S As an antibody that has the ability to cross-compete for binding to the LAMF6 protein, human S We provide antibodies that bind to the same epitope on LAMF6 or cross-compete with it. For antibodies that are likely to cross-compete, block binding of the reference antibody is not necessarily required to be complete. It will be understood that this does not block it. In some embodiments, refer Antibody binding occurs at least approximately 10, 20, 30, 40, 50, 60, 70, 75, 80 times. It decreases by 85, 90, 95, 97, 98, or 99%.

[0094] antibody modification The present invention further includes mutant antibodies, sometimes referred to as "antibody derivatives" or "antibody analogs." It provides the body. That is, amino acid modification (affinity maturation) in the CDR, in the Fc region Amino acid modifications, glycosylation variants, and other types of covalent modifications (e.g., drug This includes, but is not limited to, the application of the antibody of the present invention to the antibody (for purposes such as binding of conjugates). There are many possible modifications.

[0095] A "mutant" is a molecule whose sequence differs from that of the parent polypeptide due to a modification of at least one amino acid. This means a polypeptide sequence. In some embodiments, the parent polypeptide is a polypeptide sequence. Either a variable-length heavy chain or light chain listed in row number 1 or 2, 13 or 14. C is either or is disclosed in any of SEQ ID NOs. 5-10, 15, 16, or 17. It is one or more DR sequences. In some embodiments, the amino acid modification is This can include substitution, insertion, and / or deletion, with the former being preferred in many cases. In that embodiment, the substitution may be a conservative substitution.

[0096] In general, the variant still retains the function of the antibody as described herein. As long as it is specific to human SLAMF6, any number of modifications can be included. For example, the antibody may be specific to human SLAMF6. They should bind. Similarly, if amino acid variants are generated within the Fc region, for example, Mutant antibodies should maintain the receptor binding function necessary for the specific use or labeling of the antibody. be.

[0097] A "mutant" of the antibody in question is one of the listed CDR sequences, as described herein. One or more, one or more framework regions, or one or more antibodies To have an amino acid mutation in any of the normal regions (e.g., the Fc region). It is possible.

[0098] In some embodiments, compared to the parent array, 1, 2, 3, 4, 5, 6, 7, 8, Modifications of 9 or 10 amino acids are commonly used, with the aim of achieving function with the minimum number of modifications. is often to be modified. In some embodiments, there are 1 to 5 (1, 2, 3 , 4 or 5) modifications (e.g., individual amino acid substitutions, insertions and / or deletions), and are also found to be used in many embodiments from 1 to 2, 1 to 3 and 1 to 4. For example, in some embodiments, one or more of the CDR sequences of the antibody of the present invention may each individually contain, for example, 1, 2, 3, 4 or 5 amino acid modifications, preferably 1-4, 1-3, 1 or 2 modifications. Generally, 4, 5, 6, 7, 8, 9, or 10 or fewer changes are made within the set of CDRs. It should be noted that the number of amino acid modifications is, for example, desirable to have 1 to 5 modifications in the Fc region of the wild-type or genetically engineered protein, and, for example, 1 to 5 modifications in the Fv region. The mutant polypeptide sequence preferably has at least about 75%, 80%, 85%, 90%, 91%, 92

[0099] %, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the parent sequence (e.g., variable region sequence, constant region sequence, and / or, for example, the CDRs of antibody 1B3 and / or the heavy and light chain sequences). As used herein, "amino acid substitution" or "substitution" means substituting an amino acid at a specific position in the parent polypeptide sequence with another amino acid, and "amino acid insertion" or "insertion" as used herein means adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" as used herein means removing an amino acid at a specific position in the parent poly peptide sequence.

[0100] As used herein, "amino acid substitution" or "substitution" means substituting an amino acid at a specific position in the parent polypeptide sequence with another amino acid, and "amino acid insertion" or "insertion" as used herein means adding an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" as used herein means removing an amino acid at a specific position in the parent poly peptide sequence. As used herein, "amino acid deletion" or "deletion" means removing an amino acid at a specific position in the parent polypeptide sequence.

[0101] ​​ The terms "parent polypeptide," "parent protein," and "precursor polypeptide" as used herein A "precursor protein" is an unmodified polymer that is subsequently modified to generate a mutant. This refers to peptides. Generally, the parent polypeptide used herein is 1B3 peptide. Chido, for example, 1B3 V H Chain or V l Alternatively, it may refer to the CDR sequence. Therefore, as used herein, "parent antibody" means a modified antibody used to produce a mutant antibody. It means an antibody that is decorated.

[0102] In this specification, "wild type," "WT," or "naive (natural type)" refers to the opposite of the opposite. This refers to naturally occurring amino acid sequences or nucleic acid sequences, including genetic mutations. WT Tan Proteins, polypeptides, antibodies, immunoglobulins, IgG, etc., are not intentionally modified. It has an amino acid sequence or a nucleic acid sequence.

[0103] In this specification, "mutant Fc region" refers to a modified Fc region containing at least one amino acid. This means that the Fc sequence is different from the wild-type Fc sequence. Fc mutants have an Fc polypeptide The term "do" itself can refer to a composition containing an Fc variant polypeptide, or to an amino acid sequence.

[0104] In some embodiments, the anti-SLAMF6 antibody of the present invention has a mutant Fc domain or It is composed of the following. As is known in the art, the Fc region of an antibody is composed of numerous Fc receptors It interacts with receptors and ligands, forming an array of important functional capabilities called effector functions. The appropriate modification reduces binding to the Fc receptor at one or more positions. This can be done for specific amino acid substitutions that require silence.

[0105] In addition to the changes outlined above, other changes can be made. For example, the molecule may be VH Furthermore, it can be stabilized by the incorporation of disulfide bridges linking the VL domains (see reference). Completely incorporated by illumination (Reiter et al., 1996, Nature Biotech. 14:1239-1245) .

[0106] Furthermore, the modification with cysteine ​​is further described below for antibody-drug conjugates (ADCs). It is particularly useful in application. In some embodiments, the constant region of the antibody is the drug portion. To enable more specific and controlled arrangements, one in particular is "thiol-reactive". The above cysteine ​​can be incorporated by means of the whole. See U.S. Patent No. 7,521,541, which is incorporated herein by reference.

[0107] Furthermore, various covalent modifications can be made to antibodies, as outlined below. Covalent modification of antibodies is included within the scope of this invention and is generally performed post-translation, This is not always done. For example, a specific amino acid residue of an antibody is used in a selected side chain. Alternatively, react with an organic derivatizing agent that can react with the N- or C-terminal residue. This process introduces several types of covalent modifications to the antibody molecule.

[0108] In addition, as will be understood by those skilled in the art, labeling (including fluorescent, enzyme, magnetic, radioactive, etc.) These can all be added to antibodies (as well as other compositions of the present invention).

[0109] bispecific molecule In another aspect, the invention includes bispecific and multispecific molecules comprising an anti-SLAMF6 antibody or fragment thereof of the invention. The antibody or antigen-binding fragment thereof of the invention is derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds to two different binding sites or target molecules . In some embodiments, the antibody of the invention, or an antigen-binding fragment thereof, is derivatized or linked to at least two functional molecules, such as other peptides or proteins (e.g., other antibodies or ligands for a receptor), to generate a multispecific molecule that binds to at least three different binding sites or target molecules . To create the bispecific or multispecific molecules of the invention, the antibody of the invention is functionally (e.g., by chemical coupling, genetic fusion, non-covalent binding or otherwise) linked to one or more other binding molecules, such as another antibody, an antibody fragment, a peptide or a binding mimic, such that a bispecific or multispecific molecule results . Thus, the invention includes bispecific molecules comprising at least one first binding domain for a first target epitope (i.e., SLAMF6) and a second binding domain for a second target epitope . The second target epitope may be present on the same target protein as that bound by the first binding specificity, or the second target epitope may be present on a different target protein than that bound by the first binding specificity . The second target epitope is on the same cell as the first target epitope (i.e., SLAMF6) . To create the bispecific or multispecific molecules of the invention, the antibody of the invention is functionally (e.g., by chemical coupling, genetic fusion, non-covalent binding or otherwise) linked to one or more other binding molecules, such as another antibody, an antibody fragment, a peptide or a binding mimic, such that a bispecific or multispecific molecule results

[0110] <00,00979>Thus, the invention includes bispecific molecules comprising at least one first binding domain for a first target epitope (i.e., SLAMF6) and a second binding domain for a second target epitope . The second target epitope may be present on the same target protein as that bound by the first binding specificity, or the second target epitope may be present on a different target protein than that bound by the first binding specificity . The second target epitope may be present on the same target protein as that bound by the first binding specificity, or the second target epitope may be present on a different target protein than that bound by the first binding specificity[[ID=D37]] . The second target epitope is on the same cell They may be present on the cell, or the second target epitope may be a cell showing the first target epitope. They may be present on targets not indicated by cells. The term "binding" is used herein. "Specificity" refers to the portion that contains at least one antibody variable domain.

[0111] In another embodiment of the present invention, the second target epitope is located on tumor cells. Therefore, an aspect of the present invention is SLAMF6-expressing effector cells (for example, SLAMF6-expressing cells) Binding to both cytotoxic T cells and tumor cells expressing a second target epitope. It contains a bispecific molecule that can perform this function.

[0112] In one embodiment, the bispecific antibody of the present invention comprises a total of two or three antibody-variables. It can have a domain, where the first part of the bispecific antibody is human immunoeffect Human immunotherapy is achieved by specifically binding to effector antigens located on effector cells. It can mobilize the activity of effector cells, and the effector antigen is SLAMF6, Part 1 consists of at least one antibody variable domain, and is the second part of the bispecific antibody. It can specifically bind to target antigens other than effector antigens, and the target antigens are human Located on target cells other than immune effector cells, the second portion contains at least one antibody Includes variable domains.

[0113] In embodiments of the present invention in which the binding protein is highly specific, the molecule is antitumor, binding specific In addition to sex and anti-SLAMF6 binding specificity, a third binding specificity can be further included. In one embodiment, the third binding specificity is an anti-enhancing factor (EF) moiety, for example, It binds to surface proteins involved in cytotoxic activity, thereby triggering an immune response against target cells. It is a molecule that increases [something]. The "anti-enhancing factor moiety" is an antibody, a functional antibody fragment, and a predetermined [something]. It binds to molecules, such as antigens or receptors, thereby determining binding to target cell antigens. It may be a ligand that enhances the effect of the base. The "anti-enhancing factor moiety" is applied to the target cell antigen. It can bind. Alternatively, the anti-enhancing factor portion is the entity to which the first and second binding specificities bind. It can bind to entities different from (the entity). For example, the anti-enhancing factor portion can Cytotoxic T cells (e.g., CD2, CD2, which result in an increased immune response against target cells) 3. Via CD8, CD28, CD4, CD40, ICAM-1 or other immune cells) It can be combined with this.

[0114] In one embodiment, the bispecific protein of the present invention has, as binding specificity, at least Both are one antibody, or its antigen-binding fragment (e.g., Fab, Fab', F(a) b')2, Fv, FVTCR, Fd, dAb or single-stranded Fv) are included. The antibodies are Also, light chain or heavy chain dimer, or any smallest fragment thereof, for example, U.S. Patent It may also be an Fv or single-chain structure as described in No. 4,946,778. The amount is explicitly incorporated by reference.

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

[0116] The binding of bispecific molecules to specific targets can be performed, for example, by enzyme immunosorbent assay (ELISA), and Geoimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), Alternatively, this can be confirmed by a Western blot assay. Each generally uses a reagent (e.g., an antibody) that is specifically labeled for the target complex. This method detects the presence of the target protein-antibody complex.

[0117] In one embodiment of the present invention, the bispecific antibody contains four antigen-binding regions. It is a tetravalent antibody. In a preferred embodiment, the antibody has two Fabs that target the first antigen. It contains domains, each consisting of a heavy chain and a light chain Fab region. These are natural IgG It is sequenced in the same conformation as Fabs. The antibody further has two that target the second antigen. It contains a chimeric Fab domain, and each of these is a chimeric " containing a variable heavy chain domain It consists of two chimeric polypeptide domains containing a "heavy chain," which, via its C-terminus, The N-terminus of the constant region of the alpha or beta chain of the T cell receptor (TCR), and It is linked to a chimeric "light" chain containing a modified light chain domain, which, via its C-terminus, controls the TCR. They are ligated to the N-terminus of the constant region of the beta or alpha chain, respectively. The chimera's "heavy chain" and "light chain" are located in the chimera Fab domain, and these are the chimera The C-terminus of the constant region of the alpha or beta chain of the TCR of the "heavy chain" is natural FabD It is linked to the natural Fab domain by binding to the N-terminus of the variable region of the main heavy chain. Therefore, the overall symmetrical structure is such that each of the two different antigens is bivalent. The formula generates a bispecific antibody that targets the first antigen, and therefore, a natural F The ab domain and the chimeric Fab domain targeting the second antigen are such tetravalent antibodies It is located on both arms of the body.

[0118] In another embodiment, the chimeric Fab domain is located proximal to the Fc domain, and as a result, The C-terminus of the α-chain or β-chain of the constant region of the TCR containing the chimeric "heavy" chain is native to hindrance. The natural Fab domain binds to the N-terminus of the Fc domain, and as a result, the natural Fab domain is located at the tip of the Fc domain. The C-terminus of the CH1 domain of the heavy chain containing the natural Fab domain may contain a chimeric Fab domain. It binds to the N-terminus of the multiplicative chain.

[0119] In another embodiment, an asymmetric trivalent format consisting of two different antibody arms is Used, as a result, one arm has a single Fab domain (natural or chimeric) The second arm of the bispecific antibody is the Fab domain (natural and chimeric) as described above. It has both. Heterodimization of two different arms is possible in the art (e.g., Nobu-I). As described in the section on knobs-into-wholes (electrostatic steering, etc.) This is made possible through antibody engineering in the Fc domain.

[0120] In yet another embodiment, an asymmetric bivalent formal composed of two different antibody arms Using a tactic, one arm has a single Fab domain, either natural or chimeric, while the other... Even if each arm has a single Fab-binding domain, a chimeric or native domain Good. Heterodimerization of two different arms is well described in the art. Antibody engineering in domains (e.g., knob-into-wholes) This is possible via (such as electrostatic steering).

[0121] Glycosylation Another type of modification by covalent bonding is a change in glycosylation. For example, Lycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Glycosylation is a process that, for example, alters an antibody to increase its affinity for an antigen. This can be achieved. Such carbohydrate modifications include, for example, one or more parts of glycosylation within an antibody sequence. This can be achieved by changing the position. For example, one or more variable region frames This results in the removal of the glycosylation site, thereby eliminating glycosylation at that site. One or more amino acid substitutions can be removed. Such glycosylation can lead to antigen This could potentially increase the affinity of antibodies against it. Such an approach was proposed by Co et al. Further details are provided in U.S. Patent Nos. 5,714,350 and 6,350,861. This can be achieved by removing asparagine at position 297.

[0122] Another type of covalent modification of antibodies, for example, is fully incorporated by reference. NektarTherapeutics 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 As described in issues 70,417, 4,791,192, or 4,179,337 In the manner described, polyethylene glycol, polypropylene glycol or polyoxyal Various non-protein polyols, including but not limited to kylenes. This includes binding an antibody to the mer. Furthermore, as is known in the art, amino acid placement The exchange can occur at various locations within the antibody to facilitate the addition of polymers such as PEG. For example, U.S. Patent Application Publication No. 2005 / 0114, which is incorporated as a whole by reference. See issue 037.

[0123] In further embodiments, for example, use of the antibodies of the present invention for diagnostic or detection purposes. In this specification, the antibody may include a label. In this specification, "labeled" means that the compound is As described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland To enable the detection of compounds such as the one in question, at least one bonded part, element, and It means having a tectonic plate or chemical compound.

[0124] The present invention's method for producing antibodies The present invention further provides a method for producing the disclosed anti-SLAMF6 antibody. These methods involve culturing host cells containing isolated nucleic acids encoding the antibodies of the present invention. To include. As those skilled in the art will understand, this can be done in various ways depending on the properties of the antibody. It is possible to do so. In some embodiments, the antibody of the present invention is a full-length conventional antibody. In some cases, for example, host cells can be cultured under conditions such that antibodies can be produced and isolated. It contains nucleic acids that encode heavy chain variable regions and light chain variable regions.

[0125] The variable heavy and light chains of the antibody of the present invention are disclosed herein (protein and nucleic acid sequences) Both); as understood in the art, these can be easily enhanced to create full-length heavy chains and A light chain can be generated. That is, as outlined herein, V H and V If you provide DNA fragments that encode an L segment, these DNA fragments For example, the variable region gene is used in the full-length antibody chain gene, and the Fab fragment gene is used in the Fab fragment gene. Alternatively, further manipulation using standard recombinant DNA techniques to convert to the scFv gene. This is possible. In these operations, VL or V H It contains nucleic acids that encode The DNA fragment then connects to another protein, such as the antibody's constant region or a flexible linker. It is operablely (operatically) ligated to another DNA fragment that codes. The term "operably linked" as used means that two DNA fragments are linked together. The two DNA fragments remain in frame so that the amino acid sequence being coded remains in frame. This is intended to mean that they are linked together.

[0126] V H The isolated DNA encoding the region is V H The heavy chain constant region of the DNA encoding Another DNA molecule that codes (C H 1, C H 2 and C H 3) Connecting to operable Therefore, it can be converted to a full-length heavy chain gene. The sequence of the rat heavy chain constant region gene is It is publicly known in the technical field (e.g., Kabat, EA, et al. (1991) Sequences of Proteins) of Immunological Interest, Fifth Edition, US Department of Health and Human Serv (See 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 a constant region of IgG3, IgG4, IgA, IgE, IgM, or IgD. In a preferred embodiment, the heavy chain constant region is the IgG1 or IgG4 constant region. In the case of a b-fragment heavy chain gene, V H The DNA that codes for this is heavy chain C H Only the steady-state region It can be operablely linked to another DNA molecule that codes for it.

[0127] The isolated DNA encoding the VL region is V L The light chain constant region of the DNA encoding Another DNA molecule that codes, C L By operably linking to, full-length light chain genes (and Fab light chain gene) can be converted. Sequence of rat light chain constant region gene This is publicly known in the art (for example, Kabat, EA, et al. (1991) Sequences of Prote ins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, see NIH Publication No. 91-3242), DNA flags encompassing these regions The ment can be obtained by standard PCR amplification. In one preferred embodiment, The light chain steady region is the kappa or lambda steady region.

[0128] To construct the polynucleotide sequence encoding the scFv antibody fragment, V H - and the DNA fragments encoding VL are VL and V H The array is a flexibility linker VL and V linked by - H It is expressed as a continuous single-chain protein containing a region. Another fragment encoding the flexibility linker, for example, an amino acid sequence, may be used. (for example) is operablely concatenated to another fragment that codes for (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 present invention include nucleic acids that encode the antibody of the present invention. Such polynucleotides This codes for both the variable and constant regions of the heavy and light chains, respectively, but as specified herein Other combinations are also intended by the present invention according to the compositions described. Aspects of the present invention are disclosed. Oligonucleotide fragments derived from polynucleotides, and these poly It contains nucleic acid sequences complementary to nucleotides.

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

[0131] In some embodiments, the nucleic acid encoding the antibody of the present invention is extrachromosomal or Alternatively, it can be an expression vector designed to be incorporated into the genome of the host cell into which it is introduced. - It is incorporated into the expression vector. The expression vector contains any number of appropriate regulatory sequences (transcriptional and translational regulatory sequences). This includes columns, promoters, ribosome binding sites, enhancers, origins of replication, etc. It may contain, but is not limited to, other components (such as select genes), and these Everything is operably connected as is well known in the art. In some cases, two Nucleic acids are used, each with a different expression vector (for example, the weight in the first expression vector) The chain (light chain in the second expression vector) can be inserted into the second expression vector, or they can be inserted into the same expression vector. It can be placed inside. Designing an expression vector, including the selection of regulatory sequences, is a host cell selection process. Those skilled in the art will understand that this may depend on factors such as the desired protein expression level. .

[0132] Generally, nucleic acids and / or expression are introduced into selected host cells in any manner as appropriate (e.g., Recombinant host using transformation, transfection, electroporation, or infection It can be introduced into host cells as needed to create cells, and as a result, nucleic acid molecules are 1 or more The expression regulatory elements above (for example, constructs created by intracellular processing in a vector) In this process, it is operably linked to (which is incorporated into the host cell genome). Chief cells are expressed under conditions suitable for expression (for example, in the presence of an inducer, in a suitable non-human animal, and in a suitable environment). (In a suitable culture medium supplemented with salt, growth factors, antibiotics, nutritional supplements, etc.) It can be maintained, thereby producing the encoded polypeptide. In some embodiments, the heavy and light chains are produced in the same host cell. In this morphology, the heavy chain is produced in one host cell, and the light chain is produced in another host cell. It is produced there.

[0133] Mammalian cell lines that can be used as hosts for expression are well known in the art, and American Many are available from an Type Culture Collection (ATCC), Manassas, and VA. This includes immortalized cell lines, including Chinese hamster ovary (CHO) cells, HEK 293 cells, FS293, Expi293, NSO cells, HeLa cells, baby hamster - Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2) This includes, but is not limited to, bacteria, yeast, insects, and many other cell lines. Non-mammalian cells, including but not limited to plants, also express recombinant antibodies. It can be used for cattle or chickens. In some embodiments, the antibody is used for cattle or chickens. It can be produced in such transgenic animals.

[0134] Common methods for antibody molecular biology, expression, purification, and screening include, for example, U.S. Patent Nos. 4,816,567, 4,816,397, and 6,331,415 and Nos. 7,923,221, and 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 See also Morrison, S. (1985) Science 229:1202.

[0135] Pharmaceutical composition One or more embodiments of the present invention are formulated with a pharmaceutically acceptable carrier. A composition containing an antibody (or a combination thereof) or its antigen-binding fragment, for example For example, a pharmaceutical composition. Such a composition is one of the antibodies or bispecific molecules of the present invention. Alternatively, it may include a combination (for example, two or more different ones). For example, the present invention The pharmaceutical composition binds to different epitopes on the target antigen or has complementary activity. It can include a combination of antibodies.

[0136] The pharmaceutical composition of the present invention can also be used in combination therapy, i.e., administered in combination with other drugs. Yes, it is possible. For example, combination therapy may involve at least one other antitumor agent, or an anti-inflammatory agent. The present invention may include antibodies combined with immunosuppressants. Used in combination therapy. Examples of therapeutic agents obtained are described in more detail below in the section relating to the use of the antibodies of the present invention. It will be published.

[0137] As used herein, “pharmaceutically acceptable carrier” means a physiologically compatible carrier. All solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and It contains absorption retarders and the like. Preferably, the carrier is delivered intravenously, intramuscularly, subcutaneously, parenterally, or via the spinal cord. It is suitable for epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the activity Compounds, i.e., antibodies or antibody fragments, are inactivated by acids and other substances. To protect the compound from its natural state of operation, it can be coated into the material.

[0138] The pharmaceutical compounds of the present invention may contain one or more pharmaceutically acceptable salts. A "scientifically acceptable salt" retains the desired biological activity of the parent compound and any undesirable properties. This refers to salts that do not impart any toxicological effects (e.g., Berge, SM, et al. (1977) J. Pharm.). (Sci. 66:1-19). The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants. Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include: Water, ethanol, polyol (glycerin, propylene glycol, polyethylene glycol) (such as ethanol), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectionable Examples of suitable organic esters (such as ethyl oleate) are used. Appropriate fluidity is, for example, By using coating substances such as citin, the required particle size can be achieved in the case of dispersants. This is maintained by and by using surfactants.

[0139] These compositions also include adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Good. Prevention of the presence of microorganisms can be achieved through the sterilization procedures described above, along with various antimicrobial and antifungal agents, for example. This is ensured by both the inclusion of parabens, chlorobutanol, phenolsorbic acid, etc. It is possible to include isotonic agents such as sugar, sodium chloride, and similar substances in the composition. Furthermore, drugs that delay absorption, such as aluminum monostearate and gelatin. By incorporating the agent, long-term absorption of the injectable pharmaceutical form can be achieved.

[0140] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile injection solutions or fractions. Includes sterile powder for immediate preparation of powder. The above medium and for pharmaceutically active substances. The use of any conventional medium or drug is known in the art. Unless otherwise found to be incompatible with the compound, its use in the pharmaceutical composition of the present invention is intended. (Supplementary Information) The active compounds can also be incorporated into the composition.

[0141] The administration plan is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a bolus can be administered as a single dose, or it can be administered in several doses over a period of time, or Depending on the urgency of the treatment situation, the dose may be proportionally reduced or increased. For ease of administration and uniformity of dosage, parenteral compositions are formulated in dosage unit form. This is particularly advantageous. As used herein, “dosage unit form” refers to the treatment of This refers to physically distinct units suitable as a single dose for elephants, each unit being the required medicine. It contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in conjunction with the carrier. The description of the dosage unit form of the present invention is (a) the unique characteristics and achievements of the active compound. (b) specific therapeutic effects to be expected, and (b) such activation combinations for the treatment of individual sensitivity It is shaped by and directly dependent on the inherent limitations of the technology used to formulate substances.

[0142] For antibody administration, the dosage is approximately 0.0001 to 100 mg / kg of host body weight, approximately 0.001~50mg / kg, approx. 0.001~10mg / kg, approx. 0.01~10mg / The dose may be in the range of kg, and more commonly, 0.01 to 5 mg / kg. For example, administration The amounts are 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 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 within the range of 0.1 to 5 mg / kg or 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 a month, once every 3 months, or once every 3 to 6 months The administration is as follows. The preferred dosing regimen for the anti-SLAMF6 antibody of the present invention is intravenous administration. It contains 1 mg / kg body weight, 3 mg / kg, 5 mg / kg, or 10 mg / kg body weight. The antibodies are administered using one of the following dosing plans: (i) weekly for six doses. After that, (ii) monthly; (iii) weekly; (iii) 3 mg / kg body weight once, followed by 1 mg / weekly. Weight in kg.

[0143] In some methods, two or more monoclonal antibodies with different binding specificities are simultaneously It is administered, and in that case, the dose of each antibody administered falls within the indicated range. In this embodiment, the antibody is administered multiple times. The interval between single doses is, for example, weeks. The interval can be monthly, every three months, or annually. The blood concentration of the antibody may be irregular, as can be shown by measuring the concentration of the antibody in the blood. In several embodiments, the dosage is such that a plasma antibody concentration of approximately 1 to 1000 μg / ml is achieved. It is adjusted to be approximately 25-300 μg / ml in some cases.

[0144] In some embodiments, the antibody can be administered as a sustained-release formulation, in place In general, administration is not necessary. The dosage and frequency should be determined based on the antibody levels in the patient. It varies depending on the half-life. Generally, human antibodies have the longest half-lives, followed by humanized antibodies. Chimeric antibodies and non-human antibodies follow. Dosage and frequency of administration depend on whether the treatment is prophylactic. It can vary depending on whether it is therapeutic or not. For prophylactic use, relatively low doses are used over a long period of time. It is administered at relatively infrequent intervals. Some patients may continue to receive treatment for life. In some cases, this may occur. In therapeutic applications, the patient may continue treatment until the progression of the disease decreases or ends, preferably until the patient is fully recovered. A relatively high dose at relatively short intervals until the patient shows partial or complete improvement in the symptoms of the disease. It may be necessary at times. Afterward, the patient may be administered a prophylactic regimen.

[0145] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is not toxic to the patient. Effective in achieving the desired therapeutic response for specific patients, compositions, and modes of administration. The amount of the active ingredient may be modified. The selected dosage level is used in the present invention. The activity, administration route, administration time, and usage of a specific composition, or its ester, salt, or amide. The excretion rate of the specific compound used, the duration of treatment, and the combination with the specific composition used. Other drugs, compounds, and / or materials used, age, sex, and weight of the patient being treated, Condition, overall health and medical history, as well as various drugs, including similar factors well known in the medical field. It depends on dynamic factors.

[0146] The "therapeutably effective dose" of the antibody of the present invention is preferably a dose that reduces the severity of disease symptoms. Increased frequency and duration of asymptomatic periods, or functional or physical impairment due to disease-related pain. It provides protection against harm. For example, a "therapeutably effective dose" is preferably for untreated subjects. Compared to, it is preferred by at least about 10%, at least about 20%, and at least about 30%. Or at least about 40%, at least about 50%, and more preferably at least about 6 0%, at least about 70%, more preferably at least about 80%, at least about 9 It inhibits cell growth or tumor growth by 0%, or at least about 95%. The ability of these compounds is evaluated in animal model systems that predict efficacy in human tumors. This can be done. Alternatively, this property of the composition can be used to test the ability of a compound to inhibit cell proliferation. This can be evaluated by assays known to those skilled in the art. It can be measured in vitro. A therapeutically effective amount of the therapeutic compound is measured in the subject. This can reduce the size of the tumor or improve symptoms in other ways. , the size of the subject, the severity of the subject's symptoms, and the selected specific composition or route of administration Such quantities can be determined based on factors like these.

[0147] The compositions of the present invention are obtained by using one or more of the various methods known in the art, one or more It can be administered via the route of administration. As will be understood by those skilled in the art, the route of administration and The method and / or form may vary depending on the desired result. Preferred administration cycle for the antibody of the present invention The pathways include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other routes, for example, by injection or infusion. This includes parenteral administration routes. The term "parenteral administration" as used herein is usually understood to mean parenteral administration. This refers to, but is not limited to, methods of administration other than enteral and local administration, such as injection, but also includes static administration. Intravascular, intramuscular, intraarterial, subarachnoid, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, skin This includes injections and infusions of the subarticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal regions.

[0148] Alternatively, the antibody of the present invention may be transmitted via parenteral pathways such as local, epidermal, or mucosal pathways. For example, it can be administered intranasally, orally, vaginally, rectally, sublingually, or topically.

[0149] The active compound is delivered via implants, transdermal patches, and microencapsulated delivery systems. It can be prepared using a carrier that protects the compound from rapid release, such as a controlled-release formulation. It can be made from ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polio By using biodegradable, biocompatible polymers such as luteoesters and polylactic acid. This can be done. Many methods for preparing the above formulations are patented or are known to those skilled in the art. (Sustained and Controlled Release Drug Delivery Systems) (See JR Robinson, ed., Marcel Dekker, Inc., NY, 1978).

[0150] In a particular embodiment, the monoclonal antibody of the present invention provides appropriate distribution in vivo. It can be formulated to ensure effectiveness. For example, the blood-brain barrier (BBB) ​​is composed of many hydrophilic water molecules. To eliminate highly effective compounds. To ensure that the therapeutic compound of the present invention can pass through the blood-brain barrier (BBB). Therefore, (if desired), it can be formulated, for example, in liposomes. For information on the manufacturing method, see, for example, U.S. Patent No. 4,522,811; No. 5,374,5 See issues 48 and 5,399,331. Liposomes are found in certain cells or It may contain one or more parts that are selectively transported to organs and thus enhance targeted drug delivery. (See, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary target area The minutes contain folic acid or biotin (see, for example, U.S. Patent No. 5,416,016); manno Sid (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 present invention include a wide range of applications, including the diagnosis and treatment of immune-mediated disorders. It has in vitro and in vivo diagnostic and therapeutic utility.

[0152] In some embodiments, these molecules treat, prevent and / or diagnose various disorders. To diagnose, in culture, in vitro or ex vivo, in cells, or in human subjects, for example It can be administered in vivo. As used herein, the term "subject" refers to human subjects. This is intended to include non-human animals. Non-human animals include all vertebrates, such as mammals. This includes animals, such as non-human primates and non-mammals. Preferred subjects include human patients. This includes the present invention. When administering the antibody of this invention together with another drug, the order of the two is determined. They can be administered simultaneously.

[0153] Considering the specific binding of the antibody of the present invention to SLAMF6, the antibody of the present invention is immunosuppressive. It can be used to specifically detect SLAMF6 expression on the surface of cells, and furthermore, It can be used to purify SLAMF6 via immunoaffinity purification.

[0154] Furthermore, considering the expression of SLAMF6 on immune cells, the antibody and antibody composition of the present invention The term and method refer to tumorigenetic diseases, such as small cell lung cancer, non-small cell lung cancer (squamous cell carcinoma and glandular cancer). Cancer (including melanoma), skin cancer, breast cancer (including TNBC), colorectal cancer, stomach cancer 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, neurological Treating patients with diseases characterized by the presence of tumor cells, such as secretory tumors and hematological malignancies. It can be used for medical treatment.

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

[0156] In further embodiments, the antibodies of the present invention are used, for example, to treat small cell lung cancer and non-small cell lung cancer (tonsil cancer). Skin cancers including squamous cell carcinoma and adenocarcinoma, melanoma, breast cancer (including TNBC), colon cancer Rectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, and 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, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, For the manufacture of pharmaceuticals for the treatment of cancers such as GIST, neuroendocrine tumors, and hematological malignancies. It will be used.

[0157] In one embodiment, the antibody of the present invention (e.g., monoclonal antibody, antibody fragmentation) (e.g., Nanobody™, multispecific and bispecific molecules and compositions) This refers to the amount (level) of SLAMF6, or immune cells that contain SLAMF6 on their membrane surface. It can be used to detect the amount of these quantities, which are then used for specific diagnostic purposes. It can be linked to disease symptoms.

[0158] In another embodiment, the antibody of the present invention (e.g., monoclonal antibody, multispecific and bi) (Heavily specific molecules and compositions) are primarily related to therapeutic or diagnostic use in vitro. The binding activity can be tested. For example, the composition of the present invention can be tested in the following examples. It can be tested using the flow cytometry assay described in [reference].

[0159] In some embodiments, the antibodies of the present invention (e.g., monoclonal antibodies, multispecific antibodies) These (sexual and bispecific molecules and compositions) are of further usefulness in the treatment and diagnosis of diseases. It has the following properties. 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. To: To induce and / or enhance the activation of immune cells; to express SLAMF6 Mediating cellular phagocytosis or ADCC in the presence of toeffector cells. or blocking the binding of the SLAMF6 ligand to SLAMF6.

[0160] In certain embodiments, antibodies (e.g., monoclonal antibodies, multispecific and bispecific) Sex molecules and compositions are used in vivo to treat, prevent, or diagnose various diseases. Examples of related diseases include, in particular, small cell lung cancer and non-small cell lung cancer (squamous cell lung cancer). Skin cancers (including cutaneous cancer and adenocarcinoma), melanoma, breast cancer (including TNBC), colorectal cancer Cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer visceral cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial cancers, stomach cancer, Glioma, glioblastoma, testis, thyroid, bone, gallbladder and bile duct, uterus, adrenal cancer, sarcoma, GI Examples include human cancerous tissues such as ST, neuroendocrine tumors, and hematological malignancies.

[0161] The antibody composition of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) Suitable routes for administering the (and compositions) in vivo and in vitro are well known to those skilled in the art. The method can be selected by those skilled in the art. For example, the antibody composition may be administered by injection (e.g., intravenously). It can be administered intravenously or subcutaneously. The appropriate dosage of the molecule used is determined by the target It depends on the age and weight of the individual, as well as the concentration and / or formulation of the antibody composition.

[0162] As described above, the antibody of the present invention can be used with one or more additional therapeutic agents, such as immunostimulants, cells Antibodies can be administered concurrently with toxic agents, radiotoxic agents, or immunosuppressants. It can be attached to the active substance (as a combination) or administered separately from the active substance. In the case of patients (separate administration), antibodies can be administered before, after, or simultaneously with the drug. or can be administered concurrently with other known treatments, such as anti-cancer therapy, such as radiation therapy. Yes, it is possible. Such therapeutic drugs include, in particular, doxorubicin (adriamycin), and Suplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphate This includes antitumor drugs such as famide hydroxyurea, which themselves affect the child. It is effective only at levels that are toxic or subtoxic. Suitable for co-administration with the antibody of the present invention. Other drugs include other drugs used to treat cancer, such as Avastin® and 5-FU. and gemcitabine. The anti-SLAMF6 antibody of the present invention or its antigen-binding flag. The simultaneous administration of menthol and chemotherapy agents produces different cytotoxic effects on human tumor cells. It provides two anticancer drugs that act through a mechanism. Such simultaneous administration of drugs This can solve problems caused by the development of resistance or changes in the antigenicity of tumor cells.

[0163] Target-specific effector cells, for example, the composition of the present invention (e.g., monoclonal antibodies) Effector cells linked to multispecific and bispecific molecules are also used as therapeutic agents. It can be used. Effector cells for targeting include macrophages, neutrophils, and These can be human white blood cells such as monocytes. Other cells include eosinophils, natural killer cells, and This includes cells with other IgG or IgA receptors. If necessary, Effector cells can be obtained from the target being treated. Target-specific effector cells are viable. It can be administered as a suspension of cells in a phytoacidically acceptable solution. (Controlled cells) The number is 10 8 ~10 9 This order is fine, but it will vary depending on the treatment objective.

[0164] Therapies using target-specific effector cells can be performed in combination with other technologies. (Example) For example, the compositions of the present invention (e.g., monoclonal antibodies, multispecific and bispecific molecules) Antitumor therapies using effector cells equipped with ) and / or these compositions are, It can be used in combination with chemotherapy. Furthermore, combination immunotherapy involves two different types of cell damage. It can be used to direct a population of harmful effectors toward tumor cell rejection.

[0165] The bispecific and multispecific molecules of the present invention also capsulate receptors on the cell surface. By adding or removing FcγR, the amount of FcγR on effector cells is regulated. It can be used for this purpose. A mixture of anti-Fc receptors can also be used for this purpose. It is possible.

[0166] Aspects of the present invention include the antibody composition of the present invention (for example, monoclonal antibody, bispecificity or Kits containing multispecific molecules, and their use in, for example, cancer treatment. The kit also includes instructions for use. The kit further includes immunosuppressant reagents, cytotoxic agents, or radiotoxic agents, etc. One or more additional reagents, or one or more additional antibodies of the present invention (e.g., the first antibody and It contains an antibody that has complementary activity to bind to an epitope in another SLAMF6 antigen. It is possible.

[0167] Therefore, patients treated with the antibody composition of the present invention will have enhanced or increased therapeutic effects of the antibody. In addition, other therapeutic agents such as cytotoxic or radiotoxic agents may be administered. (Before, simultaneously with, or after administration of the antibody of the present invention.)

[0168] In other embodiments, the subject is treated, for example, with cytokines. Drugs that regulate, for example, enhance or inhibit the expression or activity of Fcγ or Fcγ receptors. It can be further treated with a drug. It is preferable to administer a multispecific molecule to cytokines during treatment. This includes granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor ( GM-CSF, interferon-γ (IFN-γ), and tumor necrosis factor (TNF) It includes.

[0169] The compositions of the present invention (e.g., antibodies, multispecific and bispecific molecules) can also be, for example, To label such cells, we target cells that express FcγR or SLAMF6. It can be used for that purpose. For such use, the binder is linked to the molecules that can be detected. This is possible. Therefore, the present invention relates to Fc receptors such as FcγR or SLAMF6. This provides a method for localizing ex vivo or in vitro cells that express the expression. Possible labels may be, for example, radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors.

[0170] In certain embodiments, the present invention relates to a compound 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 for body formation, and The sample and control sample are subjected to a monoclonal antibody that specifically binds to SLAMF6 or A method for measuring the amount of SLAMF6 antigen, including contact with an antigen-binding fragment. This provides the following: Next, the formation of a complex is detected, and here, the complex between the samples compared with the control sample is provided. Differences in agglomeration indicate the presence of SLAMF6 antigen in the sample.

[0171] In other embodiments, the present invention relates to immune inflammatory diseases in the subject, such as human cancer. Small cell lung cancer, non-small cell lung cancer (including squamous cell carcinoma and adenocarcinoma), skin cancer including melanoma, Breast cancer (including TNBC), colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, 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, testis, thyroid, bone, gallbladder, and It treats bile duct, uterine, and adrenal cancers, sarcomas, GISTs, neuroendocrine tumors, and hematological malignancies. To provide a method for doing so.

[0172] All references cited herein are considered in their entirety in accordance with this specification. This includes all papers, publications, patents, patent applications, presentations, and textbooks incorporated by the system. Reports, manuscripts, brochures, books, internet submissions, journal articles, periodicals This includes, but is not limited to, products, product fact sheets, etc. References in this specification include: The discussion is intended to simply summarize the claims made by those authors, however The references cited by the applicant do not constitute prior art, and the applicant's cited references are not considered to be prior art. This does not grant the right to object to the accuracy and appropriateness of the contributions.

[0173] The aforementioned invention is described in several details as examples and illustrations to clarify its understanding. However, without departing from the intent or scope of the dependent claims, certain changes and modifications may be made. It is readily apparent to those skilled in the art, in light of the teachings of this invention, that positive values ​​can be added to these. It will be easy.

[0174] The present invention will be further described by the following embodiments, which will not limit it further. It should not be interpreted as such. The following examples, sequences, and figures are intended to aid in understanding the present invention. Provided, the true scope thereof is described in the attached claims. Deviating from the spirit of the present invention. It is understood that changes can be made to the described procedures without having to deviate from them.

[0175] Examples: Example 1: Antibody generation and screening. Hybridoma formation Recombinant ECD protein was used in SLAMF6 EC at Alere, San Diego.-SLAMF6-hum. It was used for immunizing mice to generate mouse Fab for D (SEQ ID NO: 12). Using ECD spleen cells derived from infected mice, live fabrication of a fab using industrial standard techniques. I created a rally car.

[0176] Secondary screening The Fab supernatant contains SLAMF6 protein and activated cells expressed on the surface of Raji cells. Binding to PBMCs was tested. The supernatant was diluted to 1 / 10 with FACS buffer.

[0177] Example 2: Structural characteristics of a monoclonal antibody against SLAMF6 The cDNA sequences encoding the heavy and light chain variable regions of monoclonal antibodies are standard The DNA was obtained using PCR technology and sequenced using standard DNA sequencing techniques. The heavy and light chain variable regions of 1B3 selected from the above can be seen in Figure 1.

[0178] The nucleotide and amino acid sequences of the heavy chain variable region of 1B3 are respectively represented by Sequence ID No. 3. As shown in 1.

[0179] The nucleotide and amino acid sequences of the light chain variable region of 1B3 are respectively represented by Sequence ID No. 4. This is shown in part 2.

[0180] Further analysis of the 1B3 VH sequence using the Kabat system for CDR region determination is needed. Heavy-chain CDR1, CDR2, and CDR3 as shown in columns 5, 6, and 7, respectively. This led to the visualization of the region. Figure 1a shows CDR1, CDR2, and CDR3 enclosed in boxes. This shows the 1B3 VH sequence.

[0181] Further analysis of the 1B3 VL sequence using the Kabat system for CDR region determination is needed. As shown in columns 8, 9, and 10, respectively, the light chains CDR1, CDR2, and CD This resulted in visualization of the R3 region. Figure 1b shows CDR1, CDR2, and CDR3 boxed together. The 1B3 VL sequence enclosed in brackets is shown.

[0182] Example 3: Fab supernatant for SLAMF6 measured by flow cytometry analysis Specificity of noclonal antibodies 5 x 10 6 Raji cells were placed in each well of a 96-well plate and FACS buffer was added. The cells were washed once with DPBS, 2% FBS. The cells were spun at 1200 rpm for 5 minutes. This process resulted in pelletization. The pellets were then 1% FACS buffer (DPBS, 2% FBS). It is washed once and then pelletized by spinning again at 1200 rpm for 5 minutes, FA The antibody was resuspended in CS buffer. The test antibody was diluted to 30 nM / l in FACS buffer and shown in Figure 4. As shown, the increase was added to each well and incubated on ice for 30 minutes. Next Then, the cells are washed once with FACS buffer (DPBS, 2% FBS), pelletized, and washed again. Then, it was resuspended in FACS buffer. The secondary goat anti-mouse antibody was diluted to 1 μg / ml and each was used. Add 100 μl to the gel, incubate the plate on ice for 30 minutes, then FAC The cells were washed once with S buffer (DPBS, 2% FBS). The cells were pelleted and packed into 200 µl containers. The sample was resuspended in FACS buffer. - Read using a cytometer, and the results are from Guava Cytosoft software. The analysis was performed using a suite.

[0183] As can be seen in Figure 4, antibody 1B3 is dose-specific to SLAMF6-expressing Raji cells. It shows a dependent coupling.

[0184] Example 4: Ability of anti-SLAMF6 antibody to activate T cells and stimulate IFNγ production. In a 96-well non-tissue culture plate, 250 ng / ml of OKT3 was added overnight at 4°C. Plates were coated with different anti-SLAMF6 antibody / isotype concentrations. Wash twice with S, then in R10 medium (10% FBS, 1% L-glutamine, 1% penicillin Blocked for 30 minutes with RPMI (Rapid Mitigation with streptomycin). After blocking, 100,000 The T cells were resuspended in 100 μl of R10 medium and added to the plate (according to the manufacturer's instructions). According to the instructions, use PBMC with Miltenyi kit code: 130-096-535 or (T cells isolated from the plate). Incubate the plate at 37°C for 72 hours, collect the supernatant, and I Diluted for use in the FNγ ELISA assay.

[0185] IFNγ is used in the IFN-Gamma DuoSet ELISA kit (manufactured by R&D Systems). Measurements were taken using part number DY285B) in accordance with the manufacturer's instructions. result

[0186] Humanized antibody Hu_1B3 is a SLAMF described in WO2017 / 004330. Compared to the Seattle Gnenetics antibody against 6, IFN at lower antibody concentrations This demonstrated increased activity in OKT3 pre-activated T cells, reflected in increased γ production (Figure 5). This induction of IFNγ production occurs when antibodies directed at SLAMF6 inhibit the immune system. To demonstrate that it has a therapeutic effect in patients.

[0187] Example 5: Humanization of antibody 1B3. Humanization of mouse 1B3 monoclonal antibody was performed using CDR transplantation technology. Guide the process and preserve the parent mouse residues, or use them in human germline counterparts. To help decide whether to substitute, the homologous molecule of Fv of the 1B3 mouse monoclonal antibody is used. I built Dell.

[0188] The definition of CDR is based on Kabat nomenclature. The 1B3 rat CDR region is transplanted. The selection of human framework receptor regions facilitates the analysis of immunoglobulin V region sequences. To do this, we used IgBLAST, developed at NCBI, to analyze the 1B3 mouse variable region sequence. This can be achieved by searching the IMGT mouse and human V gene databases as input. The strategy applied was to identify specific somatic mutations observed in individual human antibody sequences. The goal was to use a human germline sequence, which is a natural human sequence free of foreign elements.

[0189] Heavy Chain Design Amino acid sequence of VH isolated from mouse 1B3 hybridoma (Kabat number) The CDR area under the ping scheme (shown in bold) is shown below. [ka]

[0190] Selection of the human framework receptor VH region IgBLAST was used with the mouse VH region amino acid sequence as input to perform IMGT human VH gene generation. By searching the gene database, the 1B3 mouse CDR region is transplanted into human follicles. We achieved selection of the VH region of the germline receptor. Sequence sequence of the parental antibody against human germline cells. Based on the entry, the closest matching entry was identified. Optimal human life as a receptor. Identification of proliferative cell lines involves sequence identity between frameworks, as defined by Kabat. , and / or identity between interchain interface residues and supporting loops and the standard conformation of the parent CDR Based on and / or ordered criteria of compatibility. Human germline IGHV1-2 *02 was selected as the most appropriate heavy chain.

[0191] Design using IGHV1-2*02 human germline as the framework receptor region. Humanized version Move the mouse CDR (bold), defined by Kabat nomenclature, to IGHV1-2*02. The following detailed sequences were obtained. Many of the residues are framework mouse residues (CDR residues). (External) and, that is, they are conserved from the 1B3 VH sequence of the parent mouse, and they are anti It is preserved because it may be structurally important for maintaining the body's full activity. . [ka]

[0192] Humanized version using IGHV1-2*02 human germ cell line (OBT577-12 -86.7% identity of VHB (85 identical residues out of a total of 98 residues in the V gene).

[0193] Lightweight chain design The amino acid sequence of mouse 1B3 VL (the CDR region as defined by Kabat nomenclature) The following shows the emphasis. [ka]

[0194] Selection of the human framework receptor VL region IgBLAST was used with the mouse VL region amino acid sequence as input to analyze the IMGT human VL gene By searching the gene database, the 1B3 VL mouse CDR region is transplanted. Selection of the VL region of the framework receptor was achieved. Based on column alignment, the closest matching entry was identified. Optimal as a receptor. Identification of human germline cells involves the arrangement of frameworks as defined by Kabat. Identity, as well as the identity between the interchain interface residues and supporting loops and the standard conformation of the parent CDR. Based on and / or ordered criteria for compatibility (interchangeability). From this analysis, humans Germline cells, IGKV1-33*01 is the best choice as a human framework receptor region. It was thought that this human germline was used to design a humanized version. It was used.

[0195] Design using IGKV1-33*01 human germline as the framework receptor region. Humanized version The mouse CDR defined by Kabat numbering was transplanted to IGKV1-33*01. And the following detailed sequence was obtained. Numerous structurally important components for maintaining the full activity of the antibody. The residues are retained. This results in a humanized version with 86.3% identity (9 5 (82 amino acid residues) were obtained. [ka] 86.3% identity between the humanized version and the IGKV1-33*01 human germline (8 2 / 95)

[0196] Example 6: ELISPOT with tumor-infiltrating lymphocytes Primary tumors derived from NSCLC (Figure 6), breast cancer (Figure 7), or CRC (Figure 8) tumors. Infiltrating lymphocytes (TILs) were treated with mouse 1B3 or pembrolizumab at a dose of 10 μg / ml. KT3 was diluted to 1 μg / ml in complete IMDM culture medium and stimulated for 96 hours. After stimulation, T The ILs were collected, counted, and placed on an IFNγ ELISPOT plate (Mabtech) for 10 minutes. Cells were plated (cultured on a plate) at 0000 cells / well. The plates were incubated at 37°C for 24 hours. Incubated and then developed according to the manufacturer's instructions. The number of spots is Immu The readings were taken using the noSpot(registered trademark) Series 5 ELISPOT analyzer. The data was analyzed using GraphPad Prism software.

[0197] Figure 6 shows that antibody 1B3 produces significantly higher IFNγ than the isotype antibody, reflecting this. This demonstrates the activation of TILs derived from SCLC.

[0198] Figure 7 shows that antibody 1B3 activates a significantly larger number of breast cancer-derived TILs than pembrolizumab. This demonstrates that it produces IFNγ.

[0199] Figure 8 shows that antibody 1B3 significantly increased the number of colorectal cancer-derived TILs compared to pembrolizumab. This demonstrates that it activates and produces IFNγ.

[0200] Example 7 Binding affinity of humanized 1B3 antibody Binding affinity experiments for Biacore T-200 were conducted at 25°C. Flow of CM5 chip Cells 2, 3, and 4 were coated with the maximum amount of goat anti-human IgG, 500 RU. The test antibody was then applied. The data was captured in flow cells 2, 3, and 4. Flow cell 1 was left blank and used for reference subtraction. The antigen was then poured onto the chip. The binding of the antigen to the antibody was monitored in real time. Observed k on and k off Based on that, the KD (Kick-Draw) decision was made.

[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) with anti-human CD3 (eBioscience) (USA) Antibody 250 ng / ml alone or humanized anti-SLAMF6 antibody (Hu_1B3) The concentrations of the isotype control antibody (0, 0.2, 0.4, 0.6, 0.9, 1.2 ug / ml) are shown. After coating with a certain temperature and incubating overnight at 4°C, the plates were washed the following day and AIM was applied. Blocked with V medium. T cells isolated from PBMCs produced from healthy donors were used in the cell Stain with growth dye eFluor® 670 (eBioscience, USA), wash, and FCS. Penicillin-streptomycin-containing AIM V (Thermofisher Scientific, USA) culture Seeds were seeded into antibody-coated plates containing ground (100,000 cells / well in 100 μl), and tissue samples were taken. The cells were cultured in a culture incubator at 37°C for 72 hours. Cells were collected on the third day and treated with anti-human C2. FITC labeled with D8, Brilliant Viole labeled with anti-human CD4 t 711 (trademark), anti-human CD69 (Biolegend, USA) labeled PE, and fixed The sample was stained with the viable dye eFluor(trademark) 506 (eBioscience, USA). Analysis was performed using a Tune NxT flow cytometer (Thermofisher Scientific, USA), and Fl The data was analyzed using owJo software (TreeStar, USA).

[0203] Figure 9 shows the presence of anti-SLAMF6 antibodies on T cells isolated using anti-SLAMF6 antibodies in the presence of CD3. Stimulation of SLAMF6 leads to T cell proliferation compared to isotype or CD3 alone. It indicates an increase in intent.

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

[0205] Frozen PBMCs from the second donor (Donor 2) were cultured in culture medium at a rate of 2E6 cells / ml. The samples were treated with mitomycin C at a density of 50 ug / ml. They were then washed with culture medium and treated with mitomycin. Before removing C, the cells were treated at 37°C for 90 minutes.

[0206] Next, 100,000 cells from donor 1 were placed in a 96-well, U-bottom plate. It was combined with 100,000 mitomycin C-treated cells from step 2. Cells were treated in solutions of various concentrations of the humanized antibody Hu_1B3 and a control solution for 6 days. The cells were cultured in 100 µl of medium / well. Anti-CD13 was used as a negative control for the isotype. It was used in the same way as the 7mAb and included in the comparison.

[0207] The culture supernatant was collected on day 6 and subjected to ELISA (R&D Systems: DY285B). The concentration of IFN-γ was analyzed according to the manufacturer's instructions. Figure 10 shows that the anti-SLAMF6 antibody Hu_1B3 increases cytokine release in a dose-dependent manner. This indicates that T cells are activated by the presence of antibodies. Furthermore, the activation of SLAMF6... Sexualization is associated with higher cytokine release from isolated T cells than with CD137-mediated activation. This indicates that it will bring about.

[0208] Example 10: SLAMF6-mediated release of granzyme B and perforin from T cells PBMC isolation As the first step in T cell isolation, PBMCs are buffy coat (Stanford Blood Center or It is isolated from Leucopak. Blood is mixed in PBS in a 1:4 ratio (10 ml blood + PBS). Dilute the diluted blood (30 ml) with 15 ml of Ficoll-Hypaque (GE-Healthcar). Place carefully on top of (e cat.No.17-1440-03). The tube is 400g (1400rpm), The monocytes are centrifuged in a Sorvall centrifuge for 30 minutes with the brake off, under RT (Restoration Time). They are separated. Approximately 10 ml of the white fraction of cells from all tubes is transferred to a single 50 ml tube. Pool in a container, wash, and count using a Cellometer Auto 2000. Buffy coat or The PBMCs generated are used for T cell isolation.

[0209] T cell isolation Pan T cell isolation from PBMCs, except for CD4 and CD8 T cells, leaves all others A subset of immune cells was labeled with a biotin-conjugated antibody and subjected to streptavidin in a high magnetic field. This is a negative selection process captured by microbeads coated with a special material. Wash the PBMCs isolated from the buffy coat and add FACS sorting buffer (0.1 PBS). Resuspend in %BSA at a concentration of 2.5e7 cells / mL, and Pan T cell biotin antibody Add the cocktail to the cells, mix with the antibody cell suspension using a 1 ml pipette, and leave on ice for 5 minutes. Incubate for a while. Add the Pan T cell microbead isolation cocktail to the mixture. Incubate on ice for another 10 minutes. MACS isolation method isolates untreated T cells. Used for the purpose of biotin-labeled microbead mixture of PBMCs, high-field MACS The sample was passed through a separator on an LS column (Miltneyi Biotec, Cat#130-042-401) and used on non-T cells. The depleted flow-through is collected, washed once with FACS sorting buffer, and 5 Resuspend in AIMV complete medium with %FBS.

[0210] Granzyme B ELISA For the granzyme B functional assay, non-tissue culture-treated 96-well plates were used. 50 ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific Cat#) Combined with 16-0031-85), coat with human Hu_1B3 or isotype control antibody. Start with 4.2 μg / ml of each test antibody and administer 10 infusions in 100 μl of PBS. Coat three times with a 2:3 dilution for settling. Seal the plate and leave the ink overnight at 4°C. To devise, on the day of the Granzyme B functional assay setup, wash the plate and 5 Block in AIMV complete medium containing %FCS for 20 minutes to reduce nonspecific binding.

[0211] Granzyme B immunoassay is performed using the Human Granzyme BD R&D system (Cat# DY2906-05). Set up using the uoSet ELISA kit. Nunc-immunoassay plate Coated with granzyme B capture antibody diluted 1:60 in PBS, and the plate was... Incubate at 4°C overnight. The next day, wash the plate in ELISA washing buffer (PBS+). Wash with 0.05% tween20) and block in PBS with 1% BSA. After blocking 100 μl of the sample diluted 1:60 in the buffer solution, each of the following steps was performed. Add the standard solution to the bottle and incubate overnight at 4°C. The next day, develop the plate and Ver. OD values ​​were captured using the saMax tunable microplate reader. Granzyme B The emission is quantified and plotted using Graphpad Prism 8 software.

[0212] conclusion Granzyme-mediated apoptosis is a process in which cytotoxic lymphocytes eliminate transformed cells. This is one of the main mechanisms used for tumor suppression. This data indicates that the antibody Hu_1B3 is important for tumor suppression. This demonstrates that it can induce cytotoxic functions. The antibody Hu_1B3 is released from activated T cells. Lanzyme B was dose-dependently increased (Figure 11), and EC 50 It is 0.51 μg / ml. .

[0213] Perforin intracellular assay Perforin intracellular quantitative assay setup: Non-tissue culture treated 96-well plate, 250 ng / ml anti-human CD3 (OKT3 cloned) (Thermofisher Scientific Ca Combined with t#16-0031-85), coat with human Hu_1B3 or isotype control antibody ( Coat the samples. Start with 4.2 μg / ml of each test antibody and add 10 samples in 100 μl of PBS. Coat with a 2:3 dilution for titration. Seal the plate and incubate overnight at 4°C. On the day of the Granzyme B functional assay setup, wash the plate and rinse with 5% FCS. Blocking for 20 minutes in AIMV complete medium containing reduces nonspecific binding. For the purpose, use 20 per well in 100 μl of AIMV culture medium containing 5% FBS. 0,000 pan-T cells were added and cultured in a tissue culture incubator at 37°C for 3 days. On the day of the assay, use a protein transport inhibitor cocktail (Thermofisher Scientific, Cat#). Add 00-4980-93) to all wells to prevent perforin transport into the extracellular space. Incubate for 4 hours. Harvest the cells and add T cell surface markers CD3, CD4, and CD8. After staining, use the FIX&PERM (trademark) cell permeabilization kit (Thermofisher Science) Intracellular perforin staining is performed using tific (Cat# GAS-004). Cells are then transferred to Attune N The data was analyzed using an xT FACS analyzer (Thermofisher Scientific, MD), and the data was then flown. The analysis was performed using wJo software (BD Biosciences, San Jose).

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

[0215] Example 11: Competitive binding assay to evaluate the binding epitope of antibody 1B3. Frozen human PBMCs were thawed and placed in FACS buffer (DPBS containing 2% FCS). The cells are washed once with a suspension, and then pelletized by centrifuging at 1200 rpm for 5 minutes. The supernatant was then discarded (using the same method as used for subsequent washing). The cells were then placed in FACS buffer. Inside, 100,000 cells per well are dispensed into a 96-well assay plate, and then, Washed again. Next, SLAM was performed using 100 μl of FACS buffer on ice for 1 hour before washing. Regarding F6 receptor blocking, cells are treated with SLAMF6 ECD-mIgG2a Fc The fusion protein was blocked at 100 nM or 300 nM for 1 hour. Humanized Hu_1B 3 antibodies or human IgG1 isotype control, at a maximum concentration of 10 nM, in a 1 / 3 truncated set. Titrate with a subsequent dilution, then add to the blocked PBMC and infuse the cells on ice for 1 hour. It was evaporated and then washed twice. Next, it was treated with a solution of 1 μg / mL FACS buffer. The next antibody, goat anti-human-IgG-RPE (Southern Biotech, Ref: 2040-05), was tested on ice for 3 minutes. The treatment was applied to the treated cells for 0 minutes. One of the untreated wells containing cells was also stained with the secondary antibody. The other sample was left unstained to act as a control for secondary antibody binding. After this final incubation, the cells are washed twice more, and the final cell pellet is processed using FACS. The antibody was resuspended in buffer. The average fluorescence intensity of the secondary antibody was 96W according to industry standard protocols. Attune NxT Flow Cytometer (ThermoF) in a 3D format Measurements were taken for each sample using (ischer Scientific), and the raw data was recorded using FlowJo The analysis was performed using analytical software.

[0216] As shown in Figure 13, the Hu_1B3 antibody is human SLAMF6 ECD-mIgG2 In the presence of the Fc fusion protein, binding to receptors on the surface of PBMCs is blocked, H This shows that u_1B3 competitively binds to human SLAMF6 ECD. Therefore Hu_1B3 binds to the homodimerized epitope of SLAMF6 and acts as an agonist antibody. It is thought that this enhances cytotoxic T cell function via the SAP-mediated activation pathway. .

[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 In No. 10-041-CM, Mediatech, Manassas VA, 10% fetal bovine serum (HyClone* Cosmic Calf Serum, Cat No. SH30087-03, Thermo Scientific, Waltham, MA) and 1% Pillbital Sodium phosphate (Cellgro, Cat. # 25-000-Cl) is added using industry-standard sterile techniques. RAJI cells were cultured in 24-well glass-bottom plates at a rate of 5 × 10⁴ cells per well. 5 The cells were plated by density and grown in growth medium at 37°C for 48 hours. The wells were as follows: The sample was examined at 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, and 24 hours. Prepared: Secondary antibody only control, human IgG isotype control, antibody Hu_1B3, Seattle Genetics-derived clinical anti-SLAMF6 antibody (positive control). For fluorescence control, only secondary fluorescence is used. Control wells and antibody control wells were not used.

[0218] Next, all antibody incubation and washing steps are performed on ice using ice-cooling reagents. The culture medium was aspirated from the wells, and IF buffer (Dulbeccio phosphate-buffered saline (DP) was added. Wash twice with BS, Thermo Scientific, Waltham VA, Cat. # SH30028-03)) + 2% FBS). The primary antibody (purified OBT humanized Hu_1B3; isotype or positive control antibody) was used. Dilute the body (Seattle Genetics) to 2 ug / ml with IF buffer, and add 200 μg to each well as needed. The solution was applied for 15 minutes. Only the same volume of IF buffer was used for the secondary antibody control. Added to the wells. Secondary antibody (goat anti-human IgG-Alexa Fluor 488, Invitrogen Cat. # Dilute A11013) to a concentration of 2 ug / mL in IF buffer, and then incubate for 15 minutes in a primary incubation chamber. I added it to my vaudeville.

[0219] After primary and secondary antibody labeling, human IgG isotype control, secondary antibody only control, and Tests and positive controls were processed at 0 minutes. Cells were washed twice with IF buffer and 4% paraformaldehyde. Maldehyde (4% diluted by half with DPBS, Cat No. 19943, Affymetrix, Santa Claus) Place the cells in a second 24-well plate containing a, CA) on ice to stop internal migration. It was fixed in place.

[0220] Wash the remaining cells twice with IF buffer, then add 1 mL of warmed growth medium and incubate at 37°C. It was added to each well before placing it in the tank. 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours The cells were then placed on ice in paraformaldehyde for 24 hours, as described for the control sample. It's fixed in place.

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

[0222] Figure 14 shows that the humanized antibody Hu_1B3 is significantly less abundant than the clinical SeattleGenetics antibody. This indicates that it is internalized in SLAMF6-expressing cells. This means that when it binds to the antibody Hu_1B3, Because the receptor remains on the cell surface for a long time, it induces a more sustained response in T cells. This indicates that it will be a more effective agonist.

[0223] Example 13: Cytotoxicity assay SKBr3 HCT116 and MDA-MB-231 were purchased from ATCC. The strains were maintained using standard sterile techniques according to the manufacturer's (ATCC) instructions. The culture medium consists of 2 mM L-glutamine and 25 mM HEPES (Corning), and 1% penicillin. Sylin / Streptomycin (Sigma-Aldrich), and 10% thermally inactivated FCS (HyClon It consisted of RPMI-1640 including e). The cell line was maintained in an exponential phase. It was 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 (copy / cell). It is a colorectal cancer cell line that expresses a low number of Her2 proteins. MDA-MB-231 is a low copy number This is a breast cancer cell line that expresses Her2.

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

[0225] The day before the assay, the frozen PBMCs were thawed in a 37°C water bath and washed in cell culture medium. They were then incubated overnight in a 37°C incubator with 5% CO2. The next day, the survival rate was approximately Cell concentration was measured by dye exclusion. PBMCs were added to target cells in 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 antibody was Her2 and It detects both CD3ε. A Cris7-Her2 bispecific antibody is used. The solution was diluted to 0 ng / ml, and then continuously diluted to 3 times its original concentration, up to 0.0012 ng / ml. Furthermore, Cris7-Her2+ agonist antibodies were tested. To observe the enhanced cytotoxicity, a Cris7-Her2 bispecific antibody and an agonist were used. This included antibody combinations. As a positive control, urelumab (Ur), an antibody against 4-1BB, was used. elumab was used. Isotype was used as a negative control. Hu_1B3 was affected. The test substance was urerumab, isotype, and Hu at a final concentration of 2.5 ug / ml. _1B3 was used. All samples were tested three times. For control, target cells alone or target cells Cells + effector cells were included. Further controls included target cells and effector cells. +Hu_1B3, or including target cells and effector cells + isotype control .

[0227] Cytotoxicity assays were incubated for 48-96 hours depending on the cell line. After the procedure, the viability of the target cells was measured. Viability was based on the quantitative determination of ATP, and A TP serves as a signal indicating the presence of metabolically active cells. This assay is luminescence-based. Cell Titer-Glo® (Promega, Madison, WI) measures living cells. These were the reagents used for the purpose of the assay. For assay conditions, please follow the manufacturer's instructions. All processes were carried out at room temperature. In short, 96-well plates and Cell Ti The ter-Glo® reagent was equilibrated at room temperature for 30 minutes. After 30 minutes, the cell culture medium was used. Remove the residue, wash the target cells with 200 µl of PBS, and repeat the washing step once more. Next, 100 µl of Cell Titer-Glo(registered trademark) reagent was added to all of the The cells were added to the gel. The plate was placed on an orbital shaker and mixed at 250 rpm for 2 minutes to lyse the cells. This was induced. The plate was removed from the orbital shaker and incubated in the dark for 10 minutes. The luminescence signal was stabilized. After 10 minutes, the sample was transferred to a 96-well plate with opaque walls. The light emission was recorded.

[0228] The viability of target cells was proportional to the generated luminescence signal. All samples were repeated three times. The assay was performed, and the average was calculated for each assay condition. The cytotoxicity percentage of the sample was used as a control. In contrast, the normalized control group was the target cell in the presence of effector cells (target + effector). This was the survival rate. 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 remaining non-viable cells, as shown in Figure 15. In SKBr3 cells, either bispecific antibody alone or bispecific antibody + isotype control was used. It exhibits effective cytotoxicity at higher concentrations of 0.8-100 ng / ml. Urelumab When added, the cytotoxicity curve shifts slightly to the left, but it remains below 5%. However, Furthermore, the addition of 2.5 ug / ml of Hu_1B3 increased the cytotoxicity of SKBR-3 cells by 10-40. It increased by %.

[0229] Figure 16 shows that similar results were observed in the second PBMC donor.

[0230] Figure 17 shows the enhanced cytotoxicity of HCT116 cells by Hu_1B3. HCT116 cells, a colon cancer cell line, express low levels of Her2 on their cell surface. The cytotoxicity assay involved PBMCs and a bispecific antibody for T cell engagement, Cris7-Her2. The assay was performed using HCT116 cells. As described above, Hu_1B3 was added to the assay. In addition, it determines whether it enhances the function of T cells or NK cells present in PBMCs. Furthermore, T cells and NK cells both express the co-activation receptor SLAMF6. The T cell engagement bispecific antibody, Cris7-Her2, is dose-dependent. Cytotoxicity is shown at various concentrations tested. More importantly, the data is for Hu_1B3. This indicates that an increase in cytotoxicity was observed when it was added. Cris7-Her2 special The EC50 of a single or bispecific antibody + isotype control is approximately 0.25 ng / ml. In contrast, the EC50 of bispecific antibody + Hu_1B3 is approximately 0.07 ng / ml. This represents an increase of approximately 3.6 times in cytotoxicity.

[0231] Figure 18 shows the enhanced cytotoxicity of MDA-MB-231 cells by Hu_1B3. This shows that the breast cancer cell line MDA-MB-231 cells have low levels of Her2 on their cell surface. It expresses. The cytotoxicity assay is performed on MDA-MB-231 cells as described above, using PBM. The procedure was performed using C and the T cell engagement bispecific antibody, Cris7-Her2. Furthermore, Hu_1B3 is a cytotoxic with Cris7-Her2 bispecificity at various concentrations tested. It enhances the properties. Specifically, compared to the isotype control, Cris7 at 1 ng / mL A 40% increase in cytotoxicity was observed.

[0232] Figure 19 shows that after 96 hours of incubation, the combination of bispecificity and Hu_1B3 was Even at the lowest levels of bispecific antibody concentration, high levels (quantity) of SKBR-3 cell elimination occurred. This indicates that the above was shown. On the other hand, at these levels, bispecificity alone or bispecificity and urea In combination with urelumab, or with an isotype control, either at a very low level ( Since Hu_1B3 caused cell death (in a certain amount) or did not cause cell death, it is determined that Hu_1B3 This suggests that it leads to a powerful activation of lymphocytes.

[0233] Figure 20 shows that in the cytotoxicity assay described above, the concentration of the test antibody increased from 7.5 ug / ml. Under the condition that titration is performed with a 1 / 3 dilution within the range of 0.0034 ug / ml, it exhibits bispecificity. This demonstrates that Cris7-Her2 antibody levels are maintained at a constant level (0.0457 ng / ml). Dose-dependent killing of SKBR-3 cells is observed with urelumab or isotype controls. This was observed in comparison to the ability of Hu_1B3 to activate cytotoxic lymphocytes. did.

[0234] Example 14: Cytotoxicity assay in the absence of Cris7 bispecificity Another cytotoxicity assay was set up as described above, but bispecific antibodies were not used. Cytotoxicity studies using SKBR-3 as a monotherapy include Hu_1B3, isotype controls, and Only urelumab was used. The concentration range used in the assay was 1 / 3 dilution per donor. The levels ranged from 33 ug / ml to 0.13 ug / ml. For the second donor, further testing of the test antibody was performed. It was diluted to 0.0152 ug / ml (Figure 21b).

[0235] Figures 21a and 21b show the absence of the CD3-Her2 bispecific antibody Hu_1B3. This demonstrates that lymphocytes can be activated to induce cell death in SKBR-3 cells. This is in contrast to urelumab or isotype controls, which show little to no cell killing. be.

[0236] Sequence List JPEG0007843824000008.jpg245170JPEG0007843824000009.jpg245170JPEG0007843824000010.jpg245170

Claims

1. A bispecific antibody or multispecific antibody or its antigen-binding fragment that binds to SLAMF6 and one or more additional binding targets, The CDR-H1 sequence containing sequence number 5, A CDR-H2 sequence containing sequence number 6 or sequence number 15, The CDR-H3 sequence containing sequence number 7, Heavy chain variable region including, A CDR-L1 sequence containing sequence number 8 or sequence number 16, A CDR-L2 sequence containing sequence number 9 or sequence number 17, The CDR-L3 sequence containing sequence number 10, Light chain variable region including, A bispecific antibody or a multispecific antibody or its antigen-binding fragment, including the above.

2. The aforementioned heavy chain variable region is CDR-H1 containing the sequence of sequence number 5, CDR-H2 containing the sequence of sequence number 15, CDR-H3 containing the sequence of sequence number 7, A bispecific antibody or multispecific antibody or its antigen-binding fragment according to claim 1, comprising:

3. The light chain variable region is CDR-L1 containing the sequence of sequence number 16, CDR-L2 containing the sequence of sequence number 17, CDR-L3 containing the sequence of sequence number 10, A bispecific antibody or multispecific antibody or its antigen-binding fragment according to claim 1 or claim 2, comprising:

4. A bispecific antibody or multispecific antibody that binds to SLAMF6, or an antigen-binding fragment thereof, CDR-H1 containing Sequence ID 5, CDR-H2 containing sequence number 15, CDR-H3 containing Sequence ID 7, Heavy chain variable region including, CDR-L1 containing sequence number 16, CDR-L2 containing sequence number 17, CDR-L3 containing sequence number 10, Light chain variable region including A bispecific antibody or a multispecific antibody or its antigen-binding fragment, including the above.

5. A bispecific antibody or multispecific antibody that binds to SLAMF6, or an antigen-binding fragment thereof, i) Three heavy chain CDRs of SEQ ID NO: 1 and three light chain CDRs of SEQ ID NO: 2, or ii) Three heavy chain CDRs of SEQ ID NO: 13 and three light chain CDRs of SEQ ID NO: 14 Includes, The CDR is a bispecific antibody or a multispecific antibody or its antigen-binding fragment, as defined by the Kabat or Chothia numbering system.

6. A heavy chain variable region containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 13, A light chain variable region containing a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequence number 14, A bispecific antibody or multispecific antibody or its antigen-binding fragment that binds to SLAMF6, further comprising the above, according to claim 4.

7. A bispecific antibody or a multispecific antibody or its antigen-binding fragment according to claim 6, comprising a heavy chain variable region containing SEQ ID NO: 13 and a light chain variable region containing SEQ ID NO:

14.

8. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein the one or more additional binding targets include PD-L1.

9. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein the one or more additional binding targets include CD3.

10. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein the bispecific antibody or multispecific antibody or its antigen-binding fragment is a monoclonal antibody.

11. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 10, wherein the bispecific antibody or multispecific antibody or its antigen-binding fragment is a chimeric or humanized antibody, or the antigen-binding fragment thereof.

12. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 11, wherein the bispecific antibody or multispecific antibody or its antigen-binding fragment is an Fc-silencing engineered IgG1 antibody in which binding to one or more Fc receptors is reduced or does not occur at all, or is an antigen-binding fragment thereof.

13. The bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 12, wherein the bispecific antibody or multispecific antibody or its antigen-binding fragment can induce and / or enhance T cell cytotoxicity.

14. The antigen-binding fragment is Fab, Fab', F(ab) ２ , F(ab') ２ A bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 13, selected from the group consisting of Fv, FV-TCR fragment, and scFv.

15. i) A heavy chain variable region of a bispecific antibody or multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 14, and / or ii) A light chain variable region of a bispecific antibody or a multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 14, A polynucleotide that codes for [something].

16. An expression vector comprising at least one polynucleotide as described in claim 15.

17. i. An expression vector comprising the polynucleotide described in claim 15, or ii. A first expression vector comprising a polynucleotide encoding the heavy chain variable region of a bispecific antibody or multispecific antibody or its antigen-binding fragment according to claim 15, and a second expression vector comprising a polynucleotide encoding the light chain variable region of a bispecific antibody or multispecific antibody or its antigen-binding fragment according to claim 15, Host cells containing these cells.

18. A method for producing a bispecific antibody or a multispecific antibody or its antigen-binding fragment, comprising culturing the host cells described in claim 17 under conditions in which the bispecific antibody or multispecific antibody or its antigen-binding fragment is expressed in the host cells.

19. The method according to claim 18, further comprising isolating the bispecific antibody or multispecific antibody or its antigen-binding fragment.

20. A pharmaceutical composition comprising a bispecific antibody or a multispecific antibody or its antigen-binding fragment according to any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

21. Use of a bispecific antibody or multispecific antibody or its antigen-binding fragment, or a pharmaceutical composition, according to any one of claims 1 to 14 or 20, in the manufacture of a pharmaceutical product for use in the treatment of cancer.

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

23. The use according to claim 21 or 22, wherein the pharmaceutical composition or drug further comprises an effective amount of a second therapeutic agent.

24. A bispecific antibody or multispecific antibody or its antigen-binding fragment, or a pharmaceutical composition, according to any one of claims 1 to 14 or 20, for use in the treatment of cancer.

25. A bispecific antibody or a multispecific antibody or its antigen-binding fragment, or a pharmaceutical composition for use according to claim 24, wherein the cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer including squamous cell carcinoma and adenocarcinoma, skin cancer including melanoma, breast cancer including TNBC, colorectal cancer, gastric cancer, ovarian cancer, uterine cancer, prostate cancer, kidney cancer, liver cancer including hepatocellular carcinoma, pancreatic cancer, head and neck cancer, nasopharyngeal cancer, esophageal cancer, bladder cancer and other urothelial carcinomas, stomach cancer, glioma, glioblastoma, testicular cancer, thyroid cancer, bone cancer, gallbladder cancer and bile duct cancer, uterine cancer, adrenal cancer, sarcoma, GIST, neuroendocrine tumors and hematological malignancies.

26. A bispecific antibody or a multispecific antibody or its antigen-binding fragment, or a pharmaceutical composition, for use according to any one of claims 24 to 25, wherein the pharmaceutical composition or drug further comprises an effective amount of a second therapeutic agent.

27. A bispecific antibody or multispecific antibody or antigen-binding fragment thereof, or a pharmaceutical composition, according to any one of claims 1 to 14 or 20, for use in treatment or as a pharmaceutical.

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