BINDING AGENTS AND METHODS OF USING SAME - Patent application

Abstract

The present invention provides binding agents that specifically bind to CD8+KIR+T regulatory cells, and their use in treating diseases or disorders, such as inflammatory diseases, autoimmune diseases, cancer or infectious diseases.The binding agents and the methods of their use are provided herein for modulating the activity of CD8+KIR+ regulatory T cells (Treg).The binding agents are bispecific or multispecific, and specifically bind to the antigen expressed on the surface of CD8+KIR+Treg.

Description

[Technical field]

[0001] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference herein. The name of the text file containing this sequence listing is 670151_403WO_Sequence_Listing.txt. This text file is 85KB, was created on January 20, 2022, and has been submitted electronically via EFS-Web. [Background technology]

[0002] background The immune system includes the innate and adaptive immune systems. The adaptive immune system has several cell subtypes, including T cell subsets and B cell subsets. The T cell subsets include various types of T cells, including naive and effector T lymphocytes, e.g., cytotoxic and helper T cells, and regulatory T cells. The activity of these T cell types is achieved by a balance between the activity of effector T cells and regulation by regulatory T cells. It is generally believed that effector T cells promote inflammation, while regulatory T cells control it. Thus, Tregs play a key role in autoimmune pathogenesis by maintaining self-tolerance, limiting autoimmunity, and controlling the expansion and activation of autoreactive CD4+ T effector cells. Disruption of the balance between effector and regulatory T cells can result in inappropriate activation or suppression of immune responses, loss of self-tolerance, autoimmune disorders, and cancer. The mechanisms for maintaining the balance of the immune system and the regulation of regulatory T cells are only beginning to be understood. Summary of the Invention [Means for solving the problem]

[0003] A quick overview Provided herein are binding agents and methods of their use for modulating the activity of CD8+KIR+ regulatory T cells (Tregs). The binding agents are bispecific or multispecific and specifically bind to antigens expressed on the surface of CD8+KIR+Tregs. In some embodiments, the CD8+KIR+Tregs are MHC class I restricted. In some embodiments, the CD8+KIR+Tregs are not MHC Qa-1 restricted. Methods of using the binding agents for the treatment of autoimmune diseases, infectious diseases and cancers are also provided.

[0004] In some embodiments, a binding agent is provided that comprises a first binding domain that specifically binds to a first antigen, where the first antigen is selected from an antigen expressed on CD8+KIR+ T regulatory cells (Tregs) other than a KIR protein; and a second binding domain that specifically binds to an inhibitory KIR protein expressed on the surface of CD8+KIR+ Tregs, wherein the binding agent binds to CD8+KIR+ Tregs.

[0005] In some embodiments, the first antigen is selected from the group consisting of CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first antigen is selected from the group consisting of CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the first antigen is selected from the group consisting of CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first antigen is selected from CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first antigen is selected from LAG-3 / CD223, TIM-3, PD-1, S1000A8 / 9, and TLT2. In some embodiments, the first antigen is selected from CD103(ITGAE), CD166, CD177, CXCR3, and S1000A8 / 9. In some embodiments, the first antigen is selected from CCR7, CXCR3, and CXCR5. In some embodiments, the first antigen is selected from PD-1, CXCR3, and ICOS. In some embodiments, the first antigen is selected from CD3, CD5, and CD8. In some embodiments, the first antigen is selected from CD3 and CD8.

[0006] In some embodiments, the binding agent is a bispecific antibody, a diabody, an antibody-Fc fusion, scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-F c, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, BiTe, DART, crossmab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, one-armed tandem scFv-Fc or DART-Fc. In some embodiments, either the first or second binding domain of the binding agent is selected from an antibody or an antigen-binding portion thereof, and the other binding domain is an antibody fragment. In some embodiments, the antigen-binding portion is a Fab, Fab', F(ab')2, Fv, scFv or single domain antibody (also called VHH, VNAR, sdAb or nanobody). In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region.

[0007] In some embodiments, the first binding domain specifically binds to CD3 or a subunit of CD3, optionally CD3 epsilon. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are a. SEQ ID NO:1 and SEQ ID NO:2, respectively; b. SEQ ID NO:9 and SEQ ID NO:10, respectively; c. SEQ ID NO:17 and SEQ ID NO:18, respectively; d. SEQ ID NO:25 and SEQ ID NO:26, respectively; e. SEQ ID NO:33 and SEQ ID NO:34, respectively; f. SEQ ID NO:41 and SEQ ID NO:34, respectively; g. SEQ ID NO:45 and SEQ ID NO:34, respectively; h. SEQ ID NO:49 and SEQ ID NO:50, respectively; i. SEQ ID NO:57 and SEQ ID NO:58, respectively; j. SEQ ID NO:65 and SEQ ID NO:66, respectively; and k. SEQ ID NO:65 and SEQ ID NO:166, respectively The amino acid sequence is selected from the pair of amino acid sequences shown in the group consisting of:

[0008] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, and the CDRs are a. SEQ ID NO:3 to SEQ ID NO:8, respectively; b. SEQ ID NO:11 to SEQ ID NO:16, respectively; c. SEQ ID NO:19 to SEQ ID NO:24, respectively; d. SEQ ID NO:27 to SEQ ID NO:32, respectively; e. SEQ ID NO:35 to SEQ ID NO:40, respectively; f. SEQ ID NO:42 to SEQ ID NO:44 and SEQ ID NO:38 to SEQ ID NO:40, respectively; g. SEQ ID NO:46 to SEQ ID NO:48 and SEQ ID NO:38 to SEQ ID NO:40, respectively; h. SEQ ID NO:51 to SEQ ID NO:56, respectively; i. SEQ ID NO:59 to SEQ ID NO:64, respectively; j. SEQ ID NO: 67 to SEQ ID NO: 72, respectively; and k. SEQ ID NOs: 67-69 and 167-169, respectively The amino acid sequence is selected from the set of amino acid sequences shown in the group consisting of:

[0009] In some embodiments, the first binding domain specifically binds to CD8 or a subunit of CD8, optionally CD8 alpha. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions are a. SEQ ID NO:73 and SEQ ID NO:74, respectively; and b. SEQ ID NO:81 and SEQ ID NO:82, respectively having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: or said first binding domain comprises a VHH chain, said VHH chain comprising: c. SEQ ID NO:89; d. SEQ ID NO:93; and e. SEQ ID NO:97 The amino acid sequence is selected from the amino acid sequences shown in the group consisting of:

[0010] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, and the amino acid sequences of the CDRs are as follows: a. SEQ ID NO: 75 to SEQ ID NO: 80, respectively; or b. SEQ ID NO: 83 to SEQ ID NO: 88, respectively selected from the amino acid sequences set forth in the group consisting of: or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, the amino acid sequences of the CDRs of the VHH being as follows: c. SEQ ID NO: 90 to SEQ ID NO: 92, respectively; d. SEQ ID NO:94 to SEQ ID NO:96, respectively; and e. SEQ ID NO: 98 to SEQ ID NO: 100, respectively The amino acid sequence is selected from the group consisting of:

[0011] In some embodiments, the first binding domain specifically binds to ICOS or a subunit of ICOS. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL have the amino acid sequences of SEQ ID NO: 170 and SEQ ID NO: 171, respectively.

[0012] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs: 172-174, respectively, and lCDR1, lCDR2 and lCDR3 having the amino acid sequences of SEQ ID NOs: 175-177, respectively.

[0013] In some embodiments, the first binding domain specifically binds to PD-1 or a subunit of PD-1. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL have the amino acid sequences of SEQ ID NO: 178 and SEQ ID NO: 179, respectively.

[0014] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs: 180 to 182, respectively, and lCDR1, lCDR2 and lCDR3 having the amino acid sequences of SEQ ID NOs: 183 to 185, respectively.

[0015] In some embodiments, the first binding domain specifically binds to CXCR3 or a subunit of CXCR3. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL have the amino acid sequences of SEQ ID NO: 186 and SEQ ID NO: 187, respectively.

[0016] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs: 188 to 190, respectively, and lCDR1, lCDR2 and lCDR3 having the amino acid sequences of SEQ ID NOs: 191 to 193, respectively.

[0017] In some embodiments, the first binding domain specifically binds to CD5 or a subunit of CD5. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL have the amino acid sequences of SEQ ID NO: 194 and SEQ ID NO: 195, respectively.

[0018] In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs: 196-198, respectively, and lCDR1, lCDR2 and lCDR3 having the amino acid sequences of SEQ ID NOs: 199-201, respectively.

[0019] In some embodiments, the second binding domain specifically binds to an inhibitory KIR protein selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 and KIR2DL3 or a combination thereof. In some embodiments, the second binding domain specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2. In some embodiments, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are a. SEQ ID NO:101 and SEQ ID NO:102, respectively; b. SEQ ID NO:109 and SEQ ID NO:110, respectively; c. SEQ ID NO:117 and SEQ ID NO:118, respectively; d. SEQ ID NO:125 and SEQ ID NO:126, respectively; e. SEQ ID NO:133 and SEQ ID NO:134, respectively; f. SEQ ID NO:141 and SEQ ID NO:142, respectively; g. SEQ ID NO:149 and SEQ ID NO:150, respectively; and h. SEQ ID NO:157 and SEQ ID NO:158, respectively The amino acid sequence is selected from the pair of amino acid sequences shown in the group consisting of:

[0020] In some embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, and the CDRs are a. SEQ ID NO:103 to SEQ ID NO:108, respectively; b. SEQ ID NO:111 to SEQ ID NO:116, respectively; c. SEQ ID NO:119 to SEQ ID NO:124, respectively; d. SEQ ID NO:127 to SEQ ID NO:132, respectively; e. SEQ ID NO:135 to SEQ ID NO:140, respectively; f. SEQ ID NO:143 to SEQ ID NO:148, respectively; g. SEQ ID NO: 151 to SEQ ID NO: 156, respectively; and h. SEQ ID NO:159 and SEQ ID NO:164, respectively The amino acid sequence is selected from the set of amino acid sequences shown in the group consisting of:

[0021] In some embodiments, the binding agent does not contain an Fc domain. In some embodiments, the binding agent comprises an Fc domain. In some embodiments, the Fc domain is selected from an IgG1 and an IgG4 Fc domain. In some embodiments, the binding agent has substantially no effector function activity. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc null.

[0022] In some embodiments, the binding agent is bivalent or tetravalent, hi some embodiments, the binding agent is bispecific.

[0023] Also provided is a pharmaceutical composition comprising a binding agent of any of the embodiments described herein and a pharma- ceutically acceptable carrier.

[0024] Also provided is a nucleic acid encoding any of the binding agent embodiments described herein.Further provided is a vector comprising any of the nucleic acid embodiments described herein.Further provided is a cell line comprising any of the nucleic acid or vector embodiments described herein.

[0025] In some embodiments, methods are provided for treating an autoimmune disease, comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to reduce the number or activity of pathogenic immune cells in the subject, thereby alleviating a symptom of the autoimmune disease.

[0026] In some embodiments, a method of suppressing an immune response mediated by pathogenic immune cells is provided, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), thereby reducing the number or activity of the pathogenic immune cells.

[0027] In some embodiments, a method of suppressing an immune response to an autoantigen is provided, comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to the autoantigen.

[0028] In some embodiments, a method of suppressing an immune response to an antigen is provided, comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to the antigen.

[0029] In some embodiments of these methods of treating an autoimmune disease or suppressing an immune response, the CD8+KIR+Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+Tregs are contacted with the binding agent ex vivo. In some embodiments, the activated CD8+KIR+Tregs are administered to a subject in need thereof in an effective amount. In some embodiments, the pathogenic immune cells are autoreactive CD4 T cells, autoantibody-producing B cells, or autoantigen-presenting dendritic cells. In some embodiments, the pathogenic immune cells are autoantigen-presenting cells. In some embodiments, the titer of autoantibodies is reduced in the subject.

[0030] In some embodiments, the subject has an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE) and ulcerative colitis.

[0031] In some embodiments of the method of treating an autoimmune disease or suppressing an immune response, the binding agent specifically binds to CD8 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD5 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to PD-1 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to ICOS and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CXCR3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the CD8+KIR+Treg is MHC class I restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E(Qa-1b) restricted.

[0032] In some embodiments of the methods of treating an autoimmune disease or suppressing an immune response, the methods further comprise administering an immunosuppressant to the subject. In some embodiments, administering the binding agent to the subject results in an improved treatment outcome in the subject. In some embodiments, the improved treatment outcome is a reduction in the frequency or severity of disease flares, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with the autoimmune disease.

[0033] In some embodiments of the method of treating an autoimmune disease or suppressing an immune response, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the binding agent has substantially no effector function activity.

[0034] In some embodiments, the use of any of the embodiments of the binding agent or pharmaceutical composition described herein is provided for the treatment of autoimmune disease in a subject by activating or stimulating CD8+KIR+Treg.In some embodiments, the use of any of the embodiments of the binding agent or pharmaceutical composition described herein is provided for the reduction of immune response by pathogenic immune cells by activating or stimulating CD8+KIR+Treg.In some embodiments, the use of any of the embodiments of the binding agent or pharmaceutical composition described herein is provided for the reduction of autoantibody titers in a subject by activating or stimulating CD8+KIR+Treg.

[0035] In some embodiments, a method of treating cancer is provided comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ T regulatory cells (Tregs), thereby alleviating a symptom of the cancer, wherein the binding agent is substantially free of effector function activity.

[0036] In some embodiments, a method of stimulating an immune response to an antigen associated with cancer (cancer antigen) is provided, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), wherein the binding agent is substantially free of effector function activity, thereby increasing the immune response to the cancer antigen.

[0037] In some embodiments, a method of treating cancer is provided comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+ T regulatory cells (Tregs), thereby alleviating a symptom of the cancer, wherein the binding agent has effector function activity including at least ADCC.

[0038] In some embodiments, a method of stimulating an immune response to an antigen associated with cancer (cancer antigen) is provided, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC, thereby increasing the immune response to the cancer antigen.

[0039] In some embodiments of the method of treating cancer or stimulating an immune response to an antigen associated with cancer, the CD8+KIR+Treg is contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+Treg is contacted with the binding agent ex vivo. In some embodiments, the activated CD8+KIR+Treg is administered to a subject in need thereof in an effective amount. In some embodiments, the increase in immune response includes reduction of cancer cells or depletion of immunosuppressive immune cells. In some embodiments, the number of cancer cells in the subject is reduced.

[0040] In some embodiments, the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma and leukemia. In some embodiments, the cancer is selected from the group consisting of solid tumors, such as breast, cervical, ovarian, lung, colorectal (CRC) (and other cancers of the intestine), skin, esophageal, adenocarcinoma, bladder and prostate cancer; and lymphoma.

[0041] In some embodiments, the binding agent specifically binds to CD8 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD5 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to PD-1 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CXCR3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to ICOS and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the CD8+KIR+Treg is MHC class I restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E(Qa-1b) restricted.

[0042] In some embodiments, the methods further include administering a chemotherapeutic agent to the subject. In some embodiments, the methods further include administering an immunotherapy to the subject. In some embodiments, the methods further include administering an immunotherapy, e.g., a checkpoint inhibitor, to the subject. In some embodiments, administering the binding agent to the subject results in an improved treatment outcome in the subject. In some embodiments, the improved treatment outcome is a partial response or a complete response. In some embodiments, the improved treatment outcome is a remission.

[0043] In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg.

[0044] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for the treatment of cancer in a subject by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially free of effector function activity.

[0045] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for reducing immunosuppression by immunoinhibitory immune cells by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially free of effector function activity.

[0046] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for reducing tumor burden in a subject by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially free of effector function activity.

[0047] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for the treatment of cancer in a subject by depleting CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC.

[0048] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for depletion of CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC.

[0049] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for reducing tumor burden in a subject by depleting CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC.

[0050] In some embodiments, a method of treating an infectious disease is provided, comprising administering to a subject in need thereof any of the embodiments of the binding agent or pharmaceutical composition described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby alleviating a symptom of the infectious disease.

[0051] In some embodiments, a method is provided for stimulating an immune response against infected cells caused by an infectious disease, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), thereby increasing the immune response against the infected cells.

[0052] In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. In some embodiments, the activated CD8+KIR+ Tregs are administered to a subject in need thereof in an effective amount.

[0053] In some embodiments of treating an infectious disease or stimulating an immune response against an infected cell, the immune response includes a reduction in infected cells or immunosuppressive immune cells selected from CD4 T regulatory cells and tolerant DCs. In some embodiments, the number of infected cells in the subject is reduced. In some embodiments, the infectious disease is selected from bacterial disease, systemic fungal disease, rickettsial disease, parasitic disease and viral disease. In some embodiments, the infectious disease is selected from the group consisting of HIV infection, Hepatitis C virus, (HCV) infection, human papillomavirus (HPV) infection, Epstein-Barr virus (EBV) infection, coronavirus infection, e.g., SARS-COV2 infection (Covid-19), cytomegalovirus (CMV) infection and influenza virus infection.

[0054] In some embodiments, the binding agent specifically binds to CD8 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD5 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to PD-1 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CXCR3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to ICOS and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the CD8+KIR+Treg is MHC class I restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E(Qa-1b) restricted.

[0055] In some embodiments, the methods further include administering an antimicrobial or antiviral agent to the subject. In some embodiments, administering the binding agent to the subject results in an improved treatment outcome in the subject. In some embodiments, the improved treatment outcome is a reduction in infection. In some embodiments, the improved treatment outcome is a reduction in infected cells. In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the binding agent has substantially no effector function activity.

[0056] In some embodiments, there is provided a use of any of the embodiments of the binding agent or pharmaceutical composition described herein for the treatment of an infection in a subject by activating or stimulating CD8+KIR+ Tregs.

[0057] In some embodiments, there is provided a use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for stimulating an immune response by activating or stimulating CD8+KIR+ Tregs, thereby suppressing immunosuppressive immune cells.

[0058] In some embodiments, there is provided a use of any of the embodiments of the binding agent or pharmaceutical composition described herein for reducing an infection in a subject by activating or stimulating CD8+KIR+ Tregs.

[0059] In some embodiments, a method for reducing or preventing the onset of post-transplant graft-versus-host disease (GVHD) is provided, comprising administering to a subject in need thereof any of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby reducing or alleviating at least one symptom of GVHD. In some embodiments, the binding agent has substantially no effector function activity.

[0060] In some embodiments, provided herein is a method of treating a transplanted subject, comprising contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), thereby reducing or suppressing GVHD. In some embodiments, the binding agent has substantially no effector function activity.

[0061] In some embodiments, a method of treating a subject receiving a transplant is provided, comprising administering to a subject in need thereof any of the binding agents or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Tregs, thereby alleviating symptoms of GVHD. In some embodiments, the binding agent has effector function activity including at least ADCC.

[0062] In some embodiments, a method of suppressing GVHD upon transplantation is provided, comprising contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Tregs, thereby reducing GVHD or symptoms thereof. In some embodiments, the binding agent has effector function activity including at least ADCC.

[0063] In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. In some embodiments, the activated CD8+KIR+ Tregs are administered to a subject in need thereof in an effective amount.

[0064] In some embodiments, the reduced or decreased GVHD comprises a reduction in CD4+ T cells active in GVHD. In some embodiments, the transplant is selected from the group consisting of organ transplant, hematopoietic stem cell transplant, umbilical cord blood stem cell transplant, transplant of precursor or differentiated cells derived from induced pluripotent stem cells, and bone marrow transplant. In some embodiments, the transplant is hematopoietic stem cell transplant, umbilical cord blood stem cell transplant, transplant of precursor or differentiated cells derived from induced pluripotent stem cells, or bone marrow transplant. In some embodiments, the transplant is allogeneic.

[0065] In some embodiments, the binding agent specifically binds to CD8 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CD5 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to PD-1 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to CXCR3 and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the binding agent specifically binds to ICOS and inhibitory KIR protein on CD8+KIR+Treg. In some embodiments, the CD8+KIR+Treg is MHC class I restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E(Qa-1b) restricted.

[0066] In some embodiments, an immunosuppressant is also administered to the subject.

[0067] In some embodiments, administering the binding agent to a subject results in an improved treatment outcome in the subject.In some embodiments, the improved treatment outcome is a reduction in symptoms associated with GVHD, a reduction in systemic inflammatory cytokines, a reduction in lesions in tissues affected by GVHD, a reduction in self-reported symptoms associated with immune responses associated with adverse effects on host tissues, an improvement or prolongation of graft survival, a reduction in one or more symptoms, and / or prevention, delay or slowing down the onset or progression of rejection of said graft, or a prolongation of graft survival by reducing the use of broad-spectrum immunosuppressants, such as corticosteroids.

[0068] In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously.

[0069] In some embodiments, the use of any of the binding agents or pharmaceutical compositions described herein is provided for treating transplant-associated GVHD in a subject.In some embodiments, the binding agent has substantially no effector function activity.

[0070] In some embodiments, the use of any of the binding agents or pharmaceutical compositions described herein is provided for treating transplant-associated GVHD in a subject by activating or stimulating CD8+KIR+Treg.In some embodiments, the binding agent has substantially no effector function activity.

[0071] In some embodiments, the use of any of the binding agents or pharmaceutical compositions described herein is provided for reducing transplant-associated GVHD by activating or stimulating CD8+KIR+Treg.In some embodiments, the binding agent has substantially no effector function activity.

[0072] In some embodiments, the present invention provides any of the binders or pharmaceutical compositions described herein for reducing GVHD in transplantation.In some embodiments, the binder has substantially no effector function activity.

[0073] In some embodiments, the use of any of the binding agent or pharmaceutical composition described herein is provided for treating transplant-associated GVHD in a subject by depleting CD8+KIR+Treg.In some embodiments, the binding agent has effector function activity, including at least ADCC.

[0074] In some embodiments, the use of any of the binding agents or pharmaceutical compositions described herein for depleting CD8+KIR+Treg is provided. In some embodiments, the binding agent has effector function activity, including at least ADCC.

[0075] In some embodiments, the use of any of the binding agents or pharmaceutical compositions described herein is provided for depleting CD8+KIR+Treg in transplant recipients to reduce GVHD.In some embodiments, the binding agent has effector function activity, including at least ADCC.

[0076] These and other aspects of the present invention may be more fully understood with reference to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief description of the drawings]

[0077] [Figure 1] FIG. 1 shows various formats of IgG-scFv bispecific antibodies.

[0078] [Diagram 2] FIG. 2 shows certain bispecific antibodies in various formats.

[0079] [Diagram 3] FIG. 3 shows additional bispecific antibodies in various formats.

[0080] [Figure 4A-4D] 4A-4D show the effect of Ly49 blockade on CD8+KIR+ T regulatory cells.

[0081] [Diagram 5] FIG. 5 shows the experimental design for evaluating the effects of Ly49 blockade in a mouse experimental autoimmune encephalomyelitis (EAE) model.

[0082] [Figure 6] FIG. 6 shows disease severity (as measured by clinical score) 7 to 27 days after immunization with MOG alone, MOG+SP, and MOG+Ly49 blockade.

[0083] [Figure 7A-7C]Figures 7A-7C show the predominant T cell profile in celiac patients. Celiac patients have an increased prevalence of CD8+KIR+ T cells (Figure 7A); an increased percentage of CD8+ T cells with intracellular IFN-gamma and perforin (Figure 7B); and an increased percentage of CD8+ T cells with intracellular granzyme B (Figure 7C).

[0084] [Figure 8A-8B] 8A-8B show that celiac patients have more CD8+KIR+ T cells (FIG. 8A) and CD8+KIR+ICOS+ T cells (FIG. 8B) compared to healthy controls.

[0085] [Figure 9A-9B] Figures 9A-9B show that gluten peptide restimulation of CD8+KIR+ T cells from celiac patients increases degranulation (Figure 9A, left) and granzyme B levels (Figure 9A, right) compared to unstimulated cells or cells stimulated with a control influenza peptide. Gluten peptide restimulation also results in a reduction in reactive CD4+ T cells compared to unstimulated cells or cells restimulated with a control influenza peptide (Figure 9B).

[0086] [Figure 10A-10B] 10A-10B show that KIR blockade of CD8+ Tregs ("KIR block") results in increased intracellular granzyme B levels (FIG. 10A) and increased degranulation (CD107) (FIG. 10B).

[0087] [Figure 11] FIG. 11 shows increased CD8+ T cell cytolytic activity, decreased CD4+ T cell activation, and increased CD4+ T cell death in PBMC from celiac patients treated with KIR blockade and gluten restimulation.

[0088] [Figure 12]FIG. 12 shows that KIR blockade of CD8+CD16+ T cells reduced CD4 T cell activation and proliferation (CD69) in samples from three celiac patients.

[0089] [Figure 13] FIG. 13 shows the presence of CD8+KIR+ Treg cells (upper panel) and CD8+CD39+ Treg cells (lower panel) in samples from patients with celiac disease, Crohn's disease, multiple sclerosis (MS), systemic lupus erythematosus (SLE), ulcerative colitis (UC) or type 1 diabetes (T1D) or in healthy subjects.

[0090] [Figure 14] FIG. 14 shows the expression of surface markers CD39, KLRG1, NKG2D, NKG2C, KLRB, CXCR3 and CD122 on CD8+KIR+ Treg cells isolated from celiac patient PBMC samples.

[0091] [Figure 15] FIG. 15 shows granzyme B, perforin and IL-10 production by CD8+ Treg cells isolated from celiac patient PBMC samples.

[0092] [Figure 16] FIG. 16 shows the production of RANTES and TNFβ by CD8+KIR+ Treg cells isolated from celiac patient PBMC samples following stimulation with anti-CD3 antibody.

[0093] [Figure 17] FIG. 17 shows reduced IL-17A and IL-23 production by CD4+ T cells co-cultured with CD8+KIR+ Treg cells following gliadin stimulation.

[0094] [Figure 18] FIG. 18 shows changes in activation and IFNγ by CD4+ T cells co-cultured with CD8+KIR+ Treg cells and stimulated with gluten.

[0095] [Figure 19] Figure 19 shows an increase in anti-inflammatory cytokines in CD4+ T cells co-cultured with CD8+KIR+ Treg cells and stimulated with gliadin. IL-10 produced by CD4+ T cells is shown using intracellular cytokine staining.

[0096] [Figure 20] FIG. 20 shows the increase in CD8+ Treg cell responses upon repeated antigen exposure.

[0097] [Figure 21] FIG. 21 shows selective expression of inhibitory KIR proteins KIR2DL1 / 2 / 3 and KIR3DL1 by CD8+ Treg cells derived from celiac patient PBMC.

[0098] [Figure 22] FIG. 22 shows an increase in CD8+KIR+ T cells in celiac patients.

[0099] [Diagram 23] FIG. 23 shows the presence of CD8+KIR+ T cells in intestinal tissue of celiac patients.

[0100] [Figure 24] Figure 24 shows the interaction of granzyme positive CD8+ T cells with CD4+ T cells in the intestinal tissue of a celiac patient. Granzyme B is shown in white, CD8+ T cells in green, CD4+ T cells in ocher, and the interaction between CD8+ T cells and CD4+ T cells in yellow.

[0101] [Diagram 25] FIG. 25 shows the increase in CD8+ Treg cells in peripheral blood 6 days (6D) after gluten challenge.

[0102] [Figure 26]FIG. 26 shows the increase in CD8+ Treg cells in tissue biopsies 14 days (14D) after gluten challenge.

[0103] [Figure 27] FIG. 27 shows CD4+ T cell numbers, costimulatory molecule expression, and proliferation in celiac patient tissue samples 14 days after gluten challenge (compared to equivalent patient tissue before gluten challenge), as well as KIR2DL expressing T cells.

[0104] [Figure 28] FIG. 28 shows examples of bispecific KIR binders and their predicted effects on activation signal strength.

[0105] [Figures 29A-29C] Figure 29A shows a bispecific antibody with a CD8 binding domain and a binding domain targeting KIR2DL1 / 2 / 3. Figure 29B shows degranulation and Figure 29C shows granzyme B levels after administration of anti-CD8 scFv / KIR FAB-Fc (at doses of 10 μg / mL, 1 μg / mL or 0.1 μg / mL) or monoclonal KIR blockade (at 20 μg).

[0106] [Diagram 30] FIG. 30 shows a dose-dependent reduction in pro-inflammatory cytokines following administration of anti-CD8 scFv / KIR FAB-Fc (at doses of 10 μg / mL or 1 μg / mL).

[0107] [Diagram 31] FIG. 31 shows CD4+ T cell death following administration of anti-CD8 scFv / KIR FAB-Fc (at doses of 10 μg / mL, 1 μg / mL or 0.1 μg / mL) or monoclonal KIR blockade (at 20 μg).

[0108] [Diagram 32]FIG. 32 shows CD4+ T cell death following administration of anti-CD8 scFv / KIR FAB-Fc (at doses of 10 μg / mL, 1 μg / mL or 0.1 μg / mL).

[0109] [Diagram 33] FIG. 33 shows preferential binding of bispecific blocking agents (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells compared to NK cells.

[0110] [Diagram 34] FIG. 34 shows preferential binding of bispecific blocking agents (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells compared to NK cells and CD4+ T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] Detailed Description definition For convenience, certain terms in the specification, examples and claims are defined here. Unless otherwise specified or implied from the context, the following terms and phrases have the meanings provided below. Since the scope of the present invention is limited only by the claims, these definitions are provided to help describe certain embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0112] As used herein, unless otherwise indicated, the terms "a" and "an" shall be construed to mean "one," "at least one," or "one or more." Unless otherwise required by context, singular terms as used herein shall include pluralities and plural terms shall include the singular.

[0113] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is, in the sense of "including, but not limited to."

[0114] The terms "reduce," "reduce," "reduced," "reduction," "decrease," and "inhibit" are all used generally herein to mean a statistically significant amount of reduction as compared to a reference.

[0115] The terms "increased," "increase," "enhance," or "activate" are all used herein to generally mean an increase in a statically significant amount compared to a reference.

[0116] The term "isolated" or "partially purified" as used herein refers to a nucleic acid, polypeptide, or protein that, in the case of a nucleic acid, polypeptide, or protein, is separated from at least one other component (e.g., a nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide, or protein found in its natural source and / or is present with the nucleic acid, polypeptide, or protein when expressed by a cell, or when secreted, in the case of secreted polypeptides and proteins, when secreted. Chemically synthesized nucleic acids, polypeptides, or proteins, or those synthesized using in vitro transcription / translation, are considered "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight of the subject nucleic acid, polypeptide, or protein, including, for example, at least 96%, at least 97%, at least 98%, at least 99% or more.

[0117] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues each connected to each other by a peptide bond between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to polymers of protein amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. Although "protein" and "polypeptide" are often used in reference to relatively large polypeptides, while the term "peptide" is often used in reference to small polypeptides, the usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to encoded gene products and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the above.

[0118] CD3 epsilon is a protein expressed on T cells, including regulatory T cells. CD3 epsilon polypeptides include, but are not limited to, those having the amino acid sequence shown in NP000724.1; this sequence is hereby incorporated by reference herein.

[0119] CD5 is a protein expressed on T cells and B cells. CD5 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_055022.2 and NP_001333385.1; these sequences are hereby incorporated by reference herein.

[0120] CD8 alpha is a protein expressed on T cells, including regulatory T cells. CD8 alpha polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_001759.3, NP001139345.1, NP_741969.1, NP_001369627.1, NP_757362.1, NP_001171571.1, NP_742100.1, NP_742099.1 and NP_004922; these sequences are hereby incorporated by reference herein.

[0121] KIR3DL1 is a protein expressed on NK cells and some T cells. It is also known as CD158E1, KIR, KIR2DL5B, KIR3DL1 / S1, NKAT-3, NKAT3, NKB1 and NKB1B. KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_037421.2 and NP_001309097.1; these sequences are hereby incorporated by reference herein.

[0122] KIR3DL2 is a protein expressed on NK cells and some T cells.It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4, NKAT4B and p140.KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_006728.2 and NP_001229796.1; these sequences are hereby incorporated by reference herein.

[0123] KIR2DL1 is a protein expressed on NK cells and some T cells.It is also known as CD158A, KIR-K64, KIR221, KIR2DL3, NKAT, NKAT-1, NKAT1 and p58.1.KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_055033.2; this sequence is hereby incorporated by reference herein.

[0124] KIR2DL2 is a protein that is expressed on NK cells and some T cells.It is also known as CD158B1, CD158b, NKAT-6, NKAT6 and p58.2.KIR2DL2 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_055034.2; this sequence is hereby incorporated by reference herein.

[0125] KIR2DL3 is a protein expressed on NK cells and some T cells.It is also known as CD158B2, CD158b, GL183, KIR-023GB, KIR-K7b, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT, NKAT2, NKAT2A, NKAT2B and p58.KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_056952.2; this sequence is hereby incorporated by reference herein.

[0126] CD27 is also known as TNF receptor superfamily member 7, S152, LPFS2, T14, TNFRSF7, and Tp55. CD27 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_001233.2; this sequence is hereby incorporated by reference herein.

[0127] CD38 is also referred to as ADP-ribosyl cyclase / cyclic ADP-ribose hydrolase 1, ADPRC1, and ADPRC 1. CD38 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_001766.2; this amino acid sequence is hereby incorporated by reference herein.

[0128] CD39 is also known as ectonucleoside triphosphate diphosphohydrolase 1, SPG64 ATPDase and NTPDase-1. It encodes a cell membrane protein that hydrolyzes extracellular ATP and ADP to AMP. CD39 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in NP_001307845.1, NP_001157651.1, NP_001157650.1, NP_001091645.1, NP_001767.3, NP_001299583.1, NP_001157655.1, NP_001157654.1 and NP_001157653.1; these amino acid sequences are hereby incorporated by reference herein.

[0129] CD40L, or CD40 ligand, is also called CD154, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39 and hCD40L. It is expressed on the surface of T cells. CD40L polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_000065.1; this sequence is hereby incorporated by reference herein.

[0130] CD45 is referred to as protein tyrosine phosphatase receptor type C, B220, CD45R, GP180, L-CA, LCA, LY5 and T200. It has many isoforms including CD45RA, CD45Rb and CD45RO. CD45RA and CD45Rb are expressed on naive T cells. CD45RO is expressed on memory T cells. CD45R0 polypeptides include, but are not limited to, those having the amino acid sequence disclosed in P08575-4. CD45RA polypeptides include, but are not limited to, those having the amino acid sequence disclosed in P08575-8. CD45RB polypeptides include, but are not limited to, those having the amino acid sequence disclosed in P08575-9. See the UniProtKB database. These sequences are hereby incorporated by reference herein.

[0131] CD73 is also referred to as 5' nucleotidase ecto, CALJA, CD73, E5NT, NT, NT5, NTE, eN and eNT. CD73 polypeptides include, but are not limited to, those disclosed in NP_001191742.1 and NP_002517.1; these amino acid sequences are hereby incorporated by reference herein.

[0132] CD103, or integrin subunit alpha E (ITGAE), is also referred to as HUMINAE. CD103 polypeptides include, but are not limited to, those having the amino acid sequence disclosed in NP_002199.3; this amino acid sequence is hereby incorporated by reference herein.

[0133] CD122, or interleukin 2 receptor subunit beta, is also known as IL15RB, IMD63, and P70-75. CD122 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in NP_001333152.1, NP_001333151.1, and NP_000869.1; these amino acid sequences are hereby incorporated by reference herein.

[0134] CD166, or activated leukocyte cell adhesion molecule (ALCAM), is also called MEMD. CD166 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_001618.2, NP_001230209.1, NP_001230210.1 and NP_001230212.1; these amino acid sequences are hereby incorporated by reference herein.

[0135] CD177 is also referred to as HNA-2a, HNA2A, NB1, NB1 GP, PRV-1 and PRV1. CD177 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_065139.2; this amino acid sequence is hereby incorporated by reference herein.

[0136] CCR7, or CC motif chemokine receptor 7, is also referred to as BLR2, CC-CKR-7, CCR-7, CD197, CDw197, CMKBR7, and EBI1. CCR7 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001829.1, NP_001288643.1, NP_001288645.1, NP_001288646.1, and NP_001288647.1; these amino acid sequences are hereby incorporated by reference herein.

[0137] CXCR3, or CXC motif chemokine receptor 3, is also called GPR9, MigR, CD182, CD183, Mig-R, CKR-L2, CMKAR3 and IP10-R. CXCR3 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_001495.1 and NP_001136269.1; these amino acid sequences are hereby incorporated by reference herein.

[0138] CXCR5, or CXC motif chemokine 5, is also called BLR1, CD185, and MDR15. CXCR5 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_001707.1 and NP_116743.1; these amino acid sequences are hereby incorporated by reference herein.

[0139] HLA-DR is a class II histocompatibility antigen composed of two chains.HLA-DR alpha chain polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_061984.2.HLA-DR beta chain polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_002116.2, NP_072049.2, NP_001346123.1 and NP_001346122.1.These amino acid sequences are hereby incorporated by reference herein.

[0140] ICOS, or inducible T cell costimulatory, is also referred to as AILIM, CD278, and CVID1. ICOS polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_036224.1, which is hereby incorporated by reference herein.

[0141] LAG-3, or CD223, is also referred to as lymphocyte activation 3. LAG-3 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_002277.4; this amino acid sequence is hereby incorporated by reference herein.

[0142] OX-40 is also known as TNF receptor superfamily member 4 or TNFRSF4, ACT35, CD134, IMD16 and TXGP1L. OX-40 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_003318.1; this amino acid sequence is hereby incorporated by reference herein.

[0143] PD-1 is also called programmed cell death protein 1. PD-1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_005009.2, which is hereby incorporated by reference herein.

[0144] S1000A8 / 9, or S100A8 and S100A9, respectively, are Ca2+ receptors that belong to the S100 family. 2+It is a binding protein.S100A8, or S100-A8, is also called 60B8AG, CAGA, CFAG, CGLA, CP-10, L1Ag, MA387, MIF, MRP8, NIF and P8.S100A8 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_001306125.1, NP_001306126.1, NP_001306127.1, NP_001306130.1 and NP_002955.2.S100A9, or S100-A9, is also called 60B8AG, CAGB, CFAG, CGLB, L1AG, LIAG, MAC387, MIF, MRP14, NIF and P14.S100A9 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_002956.1. These amino acid sequences are hereby incorporated by reference herein.

[0145] TIM-3, also called Hepatitis A Virus Cellular Receptor 2 (HAVCR2), is also known as CD366, HAVcr-2, KIM-3, SPTCL, TIM3, TIMD-3 and TIMD3. TIM-3 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_116171.3; this amino acid sequence is hereby incorporated by reference herein.

[0146] TLT-2, or triggering receptor expressed on myeloid cells like 2 (TREML2), is also called C6orf76 or dJ238O23.1. TLT-2 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_079083.2; this amino acid sequence is hereby incorporated by reference herein.

[0147] 2B4, or CD244, is also referred to as NAIL, NKR2B4, Nmrk, and SLAMF4. 2B4 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP_057466.1, NP_001160135.1, or NP_001160136.1; these amino acid sequences are hereby incorporated by reference herein.

[0148] 41BB, or TNF receptor superfamily member 9 (TNFSF9), is also referred to as ILA, 4-1BB, CD137, and CDw137. 41BB polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP_001552.2; this amino acid sequence is hereby incorporated by reference herein.

[0149] As used herein, "epitope" refers to amino acids that are normally bound by an immunoglobulin VH / VL pair, e.g., the antibodies and other binding agents described herein. Epitopes can be formed on a polypeptide from contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. An epitope typically comprises at least three, more usually at least five, about nine or about eight to ten amino acids in a unique spatial conformation. An epitope defines a minimal binding site for an antibody or other binding agent, and thus represents the target of specificity of the antibody, its antigen-binding portion or other immunoglobulin-based binding agent. In the case of a single domain antibody, an epitope represents a unit of structure that is bound in isolation by the variable domains.

[0150] As used herein, "specifically binds" means a KD of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Specific binding refers to the ability of the binding agent described herein (e.g., an antibody or an antigen-binding portion thereof) to bind to a target at or below M. Specific binding can be influenced, for example, by the affinity and avidity of the antibody or other binding agent and the concentration of the target polypeptide. Those skilled in the art can determine the appropriate conditions under which the antibodies and other binding agents described herein selectively bind to a target antigen using any suitable method, for example, titration of the binding agent in a suitable cell binding assay. A binding agent that specifically binds to a target is not displaced by a dissimilar competitor. In certain embodiments, a binding agent, for example, an antibody or an antigen-binding portion thereof, is said to specifically bind to a target when it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0151] In some embodiments, a binding agent described herein, e.g., an antibody or antigen-binding portion thereof, or other binding agent, is administered in a manner similar to that described herein, for example, in a concentration of 10 -5 M (10000 nM) or less, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 M or less. -5 M~10 -6 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -6 M~10 -7 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -7 M~10 -8In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -8 M~10 -9 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -9 M~10 -10 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -10 M~10 -11 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of about 10 -11 M~10 -12 In some embodiments, the antibodies or antigen-binding portions thereof or other binding agents described herein specifically bind to a target polypeptide with a dissociation constant (KD) of 10 -12 It specifically binds to a target polypeptide with a dissociation constant (KD) less than M.

[0152] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

[0153] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding all elements not recited in that description of an embodiment.

[0154] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," which when used in conjunction with percentages can mean + / - 1%.

[0155] The terms "statistically significant" or "significantly" refer to statistical significance, generally meaning a difference of two standard deviations (2SD) above or below a reference value.

[0156] Other terms are defined herein within the description of various aspects of the invention.

[0157] Modulation of CD8+KIR+ regulatory T cells Provided herein is a binding agent that comprises a binding domain that specifically binds to an antigen expressed on CD8+KIR+ regulatory T cells (Treg). In some embodiments, the CD8+KIR+Treg is MHC class I restricted. In some embodiments, the CD8+KIR+Treg is not MHC Qa-1 (HLA-E) restricted. Also provided is a method of using the binding agent for the treatment of autoimmune disease, infectious disease and cancer.

[0158] The binding agent comprises a first binding domain that specifically binds to a T cell antigen expressed on CD8+KIR+Treg other than a KIR protein, and a second binding domain that specifically binds to an inhibitory KIR protein expressed on CD8+KIR+Treg. In some embodiments, the first binding domain specifically binds to an antigen selected from CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, CD5, and 41BB. In some embodiments, the first binding domain specifically binds to an antigen selected from CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the first binding domain specifically binds to a subunit of an antigen selected from CD3, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8 / 9, and 41BB / CD137.

[0159] In some embodiments, the first antigen is selected from functional agonists that can activate CD8 KIR+Treg. In some embodiments, such antigens are, for example, CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR. In some embodiments, such antigens are, for example, CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR. In some embodiments, the first binding domain has agonist activity when bound to such antigen.

[0160] In some embodiments, the first antigen is selected from functional antagonists for reducing the functional inhibition of CD8 KIR+Treg. In some embodiments, such antigens are, for example, LAG-3 / CD223, TIM-3, PD-1, S1000A8 / 9 and TLT2. In some embodiments, the first binding domain has antagonist activity (e.g., blocking activity) when bound to such antigen.

[0161] In some embodiments, the first antigen is a tethering moiety to enhance the specificity of the binding agent for CD8 KIR+ Tregs. In some embodiments, such antigens are, for example, CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, such antigens are, for example, CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the first binding domain specifically binds to such antigens.

[0162] In some embodiments, the first antigen is a tethering moiety for enhancing tissue specificity.In some embodiments, such antigen is, for example, CD103 (ITGAE), CD166, CD177, CXCR3 and S1000A8 / 9.In some embodiments, the first binding domain specifically binds to such antigen.

[0163] In some embodiments, the first antigen is an agonist for enhancing CD8 KIR+ Treg cell migration.In some embodiments, such antigen is, for example, CCR7, CXCR3 or CXCR5.In some embodiments, the first binding domain specifically binds to such antigen.

[0164] In some embodiments, the first antigen is selected from PD-1, ICOS, and CXCR3, hi some embodiments, the first binding domain specifically binds to such antigen.

[0165] In some embodiments, the first antigen is selected from CD3 or CD8. In some embodiments, the first antigen is selected from CD3, CD5, or CD8. In some embodiments, the first antigen is selected from a subunit of CD3 or CD8. In some embodiments, the first antigen is CD3 epsilon. In some embodiments, the first antigen is CD8 alpha.

[0166] The second binding domain of the binding agent specifically binds to inhibitory KIR protein (killer cell immunoglobulin-like receptor protein). The inhibitory KIR protein can be, for example, KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 or KIR2DL3 or a combination thereof, for example, specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2 protein. In some embodiments, the KIR protein is selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 or KIR2DL3 or a combination thereof, for example, KIR2DL1 / 2 / 3 or KIR2DL1 / 2 protein. In some embodiments, the second binding domain is a KIR protein antagonist that blocks the interaction between KIR protein and its binding partner.

[0167] The binding agent may be any suitable agent that contains binding domains for both antigens. In some embodiments, the binding agent is bispecific (i.e., has binding domains for two different antigens). In some embodiments, the binding agent is bivalent (i.e., has two binding domains). In some embodiments, the binding agent is tetravalent (i.e., has four binding domains).

[0168] The binding domain of the binding agent may be derived from an antibody or a non-antibody format. In some embodiments, the binding domain is derived from an antibody or an antigen-binding portion thereof (i.e., an antibody fragment). In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, Fv, scFv, or a single domain antibody (also called a VHH, VNAR, sdAb, or nanobody). In some embodiments, the binding domain is or is derived from an anticalin, an affibody, an avimer, a DARPin, or an adnectin.

[0169] In some embodiments, the binding agent is a bispecific antibody, a diabody, an antibody-Fc fusion, scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, BiTe, DART, crossmab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, one-arm tandem scFv-Fc or DART-Fc (see, e.g., Figures 2 and 3). In some embodiments, the IgG-scFv is an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, 2scFV-IgG or IgG-2scFv (as shown in Figure 1).

[0170] In some embodiments, the binding agent comprises a first binding domain comprising a heavy chain variable region and a light chain variable region. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to an antigen expressed on CD8+KIR+Treg, such as CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to an antigen expressed on CD8+KIR+ Tregs, such as CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to an antigen expressed on CD8+KIR+Tregs, such as CD3, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8 / 9, and 41BB / CD137 subunits. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to an antigen expressed on CD8+KIR+Tregs, such as CD3, CD5, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8 / 9, and 41BB / CD137 subunits.

[0171] In some embodiments, the first antigen is selected from functional agonists that can activate CD8 KIR+Treg. In some embodiments, such antigens are, for example, CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR. In some embodiments, such antigens are, for example, CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR. In some embodiments, the first binding domain has agonist activity when bound to such antigen.

[0172] In some embodiments, the first antigen is selected from functional antagonists for reducing the functional inhibition of CD8 KIR+Treg. In some embodiments, such antigens are, for example, LAG-3 / CD223, TIM-3, PD-1, S1000A8 / 9 and TLT2. In some embodiments, the first binding domain has antagonist activity (e.g., blocking activity) when bound to such antigen.

[0173] In some embodiments, the first antigen is a tethering moiety to enhance the specificity of the binding agent for CD8 KIR+ Tregs. In some embodiments, such antigens are, for example, CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, such antigens are, for example, CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the first binding domain specifically binds to such antigens.

[0174] In some embodiments, the first antigen is a tethering moiety for enhancing tissue specificity.In some embodiments, such antigen is, for example, CD103 (ITGAE), CD166, CD177, CXCR3 and S1000A8 / 9.In some embodiments, the first binding domain specifically binds to such antigen.

[0175] In some embodiments, the first antigen is an agonist for enhancing CD8 KIR+ Treg cell migration.In some embodiments, such antigen is, for example, CCR7, CXCR3 or CXCR5.In some embodiments, the first binding domain specifically binds to such antigen.

[0176] In some embodiments, the first antigen is selected from PD-1, ICOS, and CXCR3, hi some embodiments, the first binding domain specifically binds to such antigen.

[0177] In some embodiments, the first antigen is selected from CD3 or CD8. In some embodiments, the first antigen is selected from CD3, CD5, or CD8. In some embodiments, the first antigen is selected from a subunit of CD3 or CD8. In some embodiments, the first antigen is CD3 epsilon. In some embodiments, the first antigen is CD8 alpha.

[0178] Antibodies for use in the binding domains described herein are known in the art.

[0179] Antibodies to CD3 are described, for example, in U.S. Pat. Nos. 5,929,212; 5,885,573; and 8,551,478, as well as International Patent Publication WO2018223004.

[0180] Antibodies to CD8 are described, for example, in U.S. Patent Application Publication Nos. 20190382488 and 20190071500, and International Patent Publications WO2014164553 and WO2017134306.

[0181] Antibodies against CD5 are described, for example, in U.S. Patent Application Publication Nos. 2018 / 0104308, 2011 / 0250203, and 2008 / 0254027.

[0182] Antibodies against CD27 are described, for example, in U.S. Patent Application Publication Nos. 20210009706, 20200247898, and 20200131272.

[0183] Antibodies against CD38 are described, for example, in U.S. Patent Application Publication Nos. 20200408765, 20200399391, 20090304710, and 20050158305.

[0184] Antibodies to CD39 are described, for example, in U.S. Patent Application Publication Nos. 20190062448, 20130273062, and 20100303828.

[0185] Antibodies against CD40L are described, for example, in U.S. Patent Application Publication Nos. 20190092868, 20100092482, 20030031668, and 20010018041.

[0186] Antibodies to CD45RA, CD45RB and CD45RO are described, for example, in US Patent Application Publication Nos. 20030232009 and 20020168362, and are available from commercial sources.

[0187] Antibodies against CD73 are described, for example, in U.S. Patent Application Publication Nos. 20200148781, 20200071404, 20190256598, and 20160145350.

[0188] Antibodies against CD103 (ITGAE) are described, for example, in US Patent Application Publication No. 20050266001.

[0189] Antibodies against CD122 are described, for example, in U.S. Patent Application Publication Nos. 20180362655 and 20110250213.

[0190] Antibodies against CD166 are described, for example, in U.S. Patent Application Publication Nos. 20160355587 and 20090269787.

[0191] Antibodies against CD177 are described, for example, in US Patent Application Publication No. 20190125832.

[0192] Antibodies against CCR7 are described, for example, in U.S. Patent Application Publication Nos. 20200216548, 20180237529, and 20150344580.

[0193] Antibodies against CXCR3 are described, for example, in U.S. Patent Application Publication Nos. 20190119391, 20190008955, and 20130251733.

[0194] Antibodies against CXCR5 are described, for example, in U.S. Patent Application Publication Nos. 20190169283, 20160053014, and 20130236476.

[0195] Antibodies against HLA-DR are described, for example, in U.S. Patent Application Publication Nos. 20180355043 and 20190071503.

[0196] Antibodies against ICOS are described, for example, in U.S. Patent Application Publication Nos. 20160304610 and 20110243929.

[0197] Antibodies against LAG-3 / CD223 are described, for example, in U.S. Patent Application Publication Nos. 20210009687, 20200277372, 20200071403, and 20190276538.

[0198] Antibodies to OX40 are described, for example, in U.S. Patent Application Publication Nos. 20140377284, 20140308276, and 20100196359.

[0199] Antibodies against PD-1 are described, for example, in U.S. Patent Application Publication Nos. 20190322749, 20190309069, 20170313774, and 20110171215.

[0200] Antibodies against S1000A8 / 9 are described, for example, in U.S. Patent Application Publication Nos. 20180256710 and 20200023045.

[0201] Antibodies against TIM-3 are described, for example, in U.S. Patent Application Publication Nos. 20180072804, 20170306016 and 20150086574.

[0202] Antibodies against TLT-2 are described, for example, in US Patent Application Publication No. 20130216540.

[0203] Antibodies against 2B4 are available, for example, from commercial suppliers.

[0204] Antibodies against 41BB are described, for example, in U.S. Patent Application Publication Nos. 20170198050 and 20200347144.

[0205] In some embodiments, the first binding domain specifically binds to CD3 epsilon, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; SEQ ID NO:17 and SEQ ID NO:18, respectively; SEQ ID NO:25 and SEQ ID NO:26, respectively; SEQ ID NO:33 and SEQ ID NO:34, respectively; SEQ ID NO:41 and SEQ ID NO:34, respectively; SEQ ID NO:45 and SEQ ID NO:34, respectively; SEQ ID NO:49 and SEQ ID NO:50, respectively; SEQ ID NO:57 and SEQ ID NO:58, respectively; SEQ ID NO:65 and SEQ ID NO:66, respectively; or SEQ ID NO:65 and SEQ ID NO:166, respectively.

[0206] In some embodiments, the first binding domain specifically binds CD3 epsilon, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively; SEQ ID NO:9 and SEQ ID NO:10, respectively; SEQ ID NO:17 and SEQ ID NO:18, respectively; SEQ ID NO:25 and SEQ ID NO:26, respectively; SEQ ID NO:33 and SEQ ID NO:34, respectively; SEQ ID NO:41 and SEQ ID NO:34, respectively; SEQ ID NO:45 and SEQ ID NO:34, respectively; SEQ ID NO:49 and SEQ ID NO:50, respectively; SEQ ID NO:57 and SEQ ID NO:58, respectively; or SEQ ID NO:65 and SEQ ID NO:66, respectively; or SEQ ID NO:65 and SEQ ID NO:166, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0207] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 1-72 and 166-169 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0208] In some embodiments, the first binding domain specifically binds to CD8 alpha, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:73 and SEQ ID NO:74, respectively; or SEQ ID NO:81 and SEQ ID NO:82, respectively; or the binding domain comprises a VHH chain having the amino acid sequence set forth in SEQ ID NO:89, SEQ ID NO:93 or SEQ ID NO:97.

[0209] In some embodiments, the first binding domain specifically binds CD8 alpha, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 73 and SEQ ID NO: 74, respectively; or SEQ ID NO: 81 and SEQ ID NO: 82, respectively; or the binding domain comprises a VHH chain having the amino acid sequence set forth in SEQ ID NO: 89, SEQ ID NO: 93 or SEQ ID NO: 97, respectively; the heavy and light chain variable regions or framework regions of the VHH chain are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions or VHH chain are unmodified.

[0210] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 73-100 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0211] In some embodiments, the first binding domain specifically binds to CD3 epsilon, the heavy chain variable region has complementarity determining regions hCDR1, hCDR2 and hCDR3, the light chain variable region has lCDR1, lCDR2 and lCDR3, and the amino acid sequences of the heavy and light chain variable region CDRs are set forth in SEQ ID NO:3 to SEQ ID NO:8, respectively; SEQ ID NO:11 to SEQ ID NO:16, respectively; SEQ ID NO:19 to SEQ ID NO:24, respectively; SEQ ID NO:27 to SEQ ID NO:32, respectively; SEQ ID NO:35 to SEQ ID NO:40, respectively; SEQ ID NO:42 to SEQ ID NO:44 and SEQ ID NO:38 to SEQ ID NO:40, respectively; SEQ ID NO:46 to SEQ ID NO:48 and SEQ ID NO:38 to SEQ ID NO:40, respectively; SEQ ID NO:51 to SEQ ID NO:56, respectively; SEQ ID NO:59 to SEQ ID NO:64, respectively; or SEQ ID NO:67 to SEQ ID NO:72, respectively. In some embodiments, the first binding domain specifically binds to CD3 epsilon and comprises light chain variable regions lCDR1, lCDR2 and lCDR3 having the amino acid sequences set forth in SEQ ID NOs: 167, 168 and 169, respectively.

[0212] In some embodiments, the first binding domain specifically binds to CD8 alpha and comprises a heavy chain variable region having complementarity determining regions hCDR1, hCDR2 and hCDR3, and a light chain variable region having lCDR1, lCDR2 and lCDR3, the amino acid sequences of the heavy and light chain variable region CDRs being set forth in SEQ ID NO:75 to SEQ ID NO:80, respectively; or SEQ ID NO:83 to SEQ ID NO:88, respectively; or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, the amino acid sequences of the VHH CDRs being set forth in SEQ ID NO:90 to SEQ ID NO:92, respectively; SEQ ID NO:94 to SEQ ID NO:96, respectively; or SEQ ID NO:98 to SEQ ID NO:100, respectively.

[0213] In some embodiments, the first binding domain specifically binds to ICOS, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively.

[0214] In some embodiments, the first binding domain specifically binds ICOS, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 170 and SEQ ID NO: 171, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0215] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 170-177 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0216] In some embodiments, the first binding domain specifically binds to PD-1, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:178 and SEQ ID NO:179, respectively.

[0217] In some embodiments, the first binding domain specifically binds PD-1, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:178 and SEQ ID NO:179, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0218] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 178-185 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0219] In some embodiments, the first binding domain specifically binds to CXCR3, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:186 and SEQ ID NO:187, respectively.

[0220] In some embodiments, the first binding domain specifically binds to CXCR3, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 186 and SEQ ID NO: 187, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0221] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 186-193 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0222] In some embodiments, the first binding domain specifically binds to CD5, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:194 and SEQ ID NO:195, respectively.

[0223] In some embodiments, the first binding domain specifically binds CD5, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 194 and SEQ ID NO: 195, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0224] In some embodiments, the first binding domain comprises an amino acid sequence set forth in any one or more of SEQ ID NOs: 194-201 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

[0225] In some embodiments, the binding agent comprises a second binding domain that comprises a heavy chain variable region and a light chain variable region.The second binding domain of the binding agent specifically binds to inhibitory KIR protein (killer cell immunoglobulin-like receptor protein).The inhibitory KIR protein can be KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 or KIR2DL3 or combinations thereof, for example, specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2 protein.

[0226] Antibodies to inhibitory KIR proteins are known in the art.

[0227] Antibodies against KIR3DL1 are described, for example, in U.S. Pat. No. 5,770,387 and International Patent Publication WO2018148223.

[0228] Antibodies against KIR3DL2 are described, for example, in U.S. Patent Application Publication Nos. 20200199228 and 20150232556.

[0229] Antibodies against KIR2DL1, KIR2DL2, KIR2DL3, and combinations thereof are described, for example, in U.S. Pat. Nos. 10,668,180 and 10,253,095, International Patent Publication WO2006003179, U.S. Patent Application Publication Nos. 20150290316 and 20130251711, and European Patent No. 3072522.

[0230] In some embodiments, the second binding domain specifically binds to KIR3DL1, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 133 and SEQ ID NO: 134, respectively; SEQ ID NO: 141 and SEQ ID NO: 142, respectively; or SEQ ID NO: 149 and SEQ ID NO: 150, respectively.

[0231] In some embodiments, the first binding domain specifically binds to KIR3DL1, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 133 and SEQ ID NO: 134, respectively; SEQ ID NO: 141 and SEQ ID NO: 142, respectively; or SEQ ID NO: 149 and SEQ ID NO: 150, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0232] In some embodiments, the first binding domain specifically binds to KIR3DL2, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 157 and SEQ ID NO: 158, respectively.

[0233] In some embodiments, the first binding domain specifically binds to KIR3DL2, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO: 157 and SEQ ID NO: 158, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0234] In some embodiments, the first binding domain specifically binds to KIR2DL1 / 2 / 3, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:101 and SEQ ID NO:102, respectively; SEQ ID NO:109 and SEQ ID NO:110, respectively; SEQ ID NO:117 and SEQ ID NO:118, respectively; or SEQ ID NO:125 and SEQ ID NO:126, respectively.

[0235] In some embodiments, the first binding domain specifically binds to KIR2DL1 / 2 / 3, and the heavy and light chain variable regions have the amino acid sequences set forth in SEQ ID NO:101 and SEQ ID NO:102, respectively; SEQ ID NO:109 and SEQ ID NO:110, respectively; SEQ ID NO:117 and SEQ ID NO:118, respectively; or SEQ ID NO:125 and SEQ ID NO:126, respectively; the framework regions of the heavy and light chain variable regions are optionally modified with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and the CDRs of the heavy or light chain variable regions are unmodified.

[0236] In some embodiments, the first binding domain specifically binds to KIR3DL1, the heavy chain variable region has complementarity determining regions (CDRs) hCDR1, hCDR2 and hCDR3, the light chain variable region has lCDR1, lCDR2 and lCDR3, and the amino acid sequences of the heavy and light chain variable region CDRs have the amino acid sequences set forth in SEQ ID NOs: 135 to 140, respectively; SEQ ID NOs: 143 to 148, respectively; or SEQ ID NOs: 151 to 156, respectively.

[0237] In some embodiments, the first binding domain specifically binds to KIR3DL2, the heavy chain variable region has complementarity determining regions hCDR1, hCDR2 and hCDR3, the light chain variable region has lCDR1, lCDR2 and lCDR3, and the amino acid sequences of the heavy and light chain variable region CDRs are set forth in the amino acid sequences of SEQ ID NO: 159 to SEQ ID NO: 164, respectively.

[0238] In some embodiments, the first binding domain specifically binds to KIR2DL1 / 2 / 3, the heavy chain variable region has complementarity determining regions hCDR1, hCDR2 and hCDR3, the light chain variable region has lCDR1, lCDR2 and lCDR3, and the amino acid sequences of the heavy and light chain variable region CDRs are set forth in SEQ ID NO: 103 to SEQ ID NO: 108, respectively; SEQ ID NO: 111 to SEQ ID NO: 116, respectively; SEQ ID NO: 119 to SEQ ID NO: 124, respectively; or SEQ ID NO: 127 to SEQ ID NO: 132, respectively.

[0239] Binder The binding agent may be any suitable agent comprising at least a first binding domain and a second binding domain, wherein the first binding domain specifically binds to a first antigen selected from an antigen expressed on CD8+KIR+T regulatory cells (Tregs) other than a KIR protein; the second binding domain specifically binds to an inhibitory KIR protein, and the binding agent binds to CD8+KIR+Tregs.

[0240] In some embodiments, the binding agent is bispecific (i.e., has binding domains for two different antigens). In some embodiments, the binding agent is bivalent (i.e., has two binding domains). In some embodiments, the binding agent is tetravalent (i.e., has four binding domains). In some embodiments, the binding agent is trivalent, hexavalent, or octavalent.

[0241] The binding domain of the binding agent may be derived from an antibody or a non-antibody format. In some embodiments, the binding domain is derived from an antibody or an antigen-binding portion thereof (i.e., an antigen-binding antibody fragment). In some embodiments, the antibody fragment is a Fab, Fab', F(ab')2, Fv, scFv, or a single domain antibody (also called a VHH, VNAR, sdAb, or nanobody). In some embodiments, the binding domain is derived from an anticalin, an affibody, an avimer, a DARPin, an adnectin, or a receptor ectodomain Fc fusion protein.

[0242] In some embodiments, the binding agent is a bispecific antibody, a diabody, an antibody-Fc fusion, scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, BiTe, DART, crossmab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, one-arm tandem scFv-Fc or DART-Fc. In some embodiments, the IgG-scFv is an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, 2scFV-IgG or IgG-2scFv (as shown in Figure 1).

[0243] As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site (antigen-binding portion) that specifically binds to a target antigen. The term generally refers to antibodies composed of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including a full-length antibody (having heavy and light chain constant regions) and the antigen-binding portion thereof; for example, intact monoclonal antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAbs), diabodies, multispecific antibodies, bispecific antibodies, diabodies, and single chain antibodies (see, e.g., Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are hereby incorporated by reference).

[0244] In antibodies, each heavy chain is composed of a variable region (abbreviated as VH) and a constant region. The heavy chain constant region may contain three domains CH1, CH2 and CH3, and optionally a fourth domain CH4. Each of these domains is referred to as an "Fc domain". As used herein, if a binding agent contains an Fc domain, the binding agent may contain one or more Fc domains, or the entire Fc region, unless otherwise specified by the context. Each light chain is composed of a variable region (abbreviated as VL) and a constant region or domain. The light chain constant region is a CL domain. The VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDRs), interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region is composed of three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. This structure is well known to those skilled in the art.

[0245] As used herein, the "antigen-binding portion" of an antibody refers to the portion of an antibody described herein that has VH and VL sequences or heavy and light chain variable region CDRs. According to the term "antigen-binding portion" of an antibody, examples of antigen-binding portions include Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibody (dAb), diabody and single chain antibody. As used herein, the terms Fab, F(ab')2 and Fv refer to: (i) Fab fragment, i.e., a monovalent fragment that is composed of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, i.e., a bivalent fragment that comprises two Fab fragments linked together at the hinge region via disulfide bridges; and (iii) Fv fragment that is composed of the VL and VH domains of an antibody. The two domains of the Fv fragment, VL and VH, are encoded by separate coding regions, but they can be further linked to each other using a synthetic linker, such as a poly-G4S amino acid sequence ("(G4S)n", where n=1-5, as disclosed as SEQ ID NO: 165), allowing them to be prepared as a single protein chain in which the VL and VH regions combine to form a monovalent molecule (also known as single-chain Fv (ScFv)). The term "antigen-binding portion" of an antibody is intended to include such single-chain antibodies.

[0246] Other forms of single chain antibodies, such as "diabodies", are also included herein. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but use a linker connecting VH and VL domains that is too short to allow the two domains on the same chain to combine, thereby forcing the VH and VL domains to pair with complementary domains (VL and VH, respectively) of different chains and form two antigen binding sites (see, for example, Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, R. J, et al. (1994) Structure 2:1121-1123).

[0247] Immunoglobulin constant region, or Fc region, refers to heavy or light chain constant region. The amino acid sequences of human heavy and light chain constant regions are known in the art. The constant region can be of any suitable type, which can be selected from immunoglobulin classes, IgA, IgD, IgE, IgG and IgM. Some immunoglobulin classes can be further divided into isotypes, for example, IgG1, IgG2, IgG3, IgG4, or IgAl and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins can be α, δ, ε, γ and μ, respectively. The light chain can be one of either kappa (or κ) and lambda (or λ).

[0248] In some embodiments, the binding agent lacks its Fc region or domain. In some embodiments, the binding agent has its entire Fc region or Fc domain. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG1 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG2 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG3 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG4 isotype. In some embodiments, the Fc domain may have a hybrid isotype that includes constant regions from two or more isotypes. In some embodiments, the Fc region or Fc domain may be an IgG1 or IgG4 constant region.

[0249] In some embodiments, the C-terminus of an Fc domain (e.g., heavy chain) may be a complete C-terminus ending in amino acid residue PGK. In some embodiments, the C-terminus of an Fc domain may also be a truncated C-terminus, with one or two of the C-terminal amino acid residues removed. In some embodiments, the C-terminus of an Fc domain is a truncated C-terminus ending in PG. In some embodiments, a binding agent comprising a heavy chain comprising a C-terminal CH3 domain comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In some embodiments, a binding agent comprising a heavy chain comprising a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, numbering according to Kabat EU index).

[0250] The binding agent described herein is a multispecific, typically a bispecific binding agent.In some embodiments, the binding agent is a multispecific antibody or antibody-like molecule, such as a bispecific antibody.A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites or antigens.The binding agent described herein typically has binding specificity for different antigens.A bispecific antibody can be prepared as a full-length antibody or an antibody fragment. Bispecific and multispecific antibodies include: scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, scFv-Fc, one-arm tandem scFv-Fc and DART-Fc. In some embodiments, the IgG-scFv is an IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG or IgG-2scFv (as shown in Figure 1).

[0251] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO93 / 08829 and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 1999). 152:5368 (1994)); and by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol. 147: 60 (1991).

[0252] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576A1).

[0253] The binding agents (e.g., antibodies or antigen-binding fragments) herein also include "Dual Acting FAbs" or "DAFs" that contain antigen-binding sites that bind to two different antigens (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820 and Bostrom et al., 2009, Science 323:1610-14). "Crossmab" antibodies are also included herein (see, e.g., WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254 and WO2013 / 026833).

[0254] In some embodiments, the binding agent comprises different antigen binding sites fused to one or the other of the two subunits of the Fc domain; thus, the two subunits of the Fc domain can be contained in two non-identical polypeptide chains. The recombinant co-expression of these polypeptides and subsequent dimerization leads to some possible recombination of the two polypeptides. Therefore, in order to improve the yield and purity of the bispecific molecule in recombinant production, it is beneficial to introduce modifications in the Fc domain of the binding agent that promote the association of the desired polypeptides.

[0255] Thus, in a particular embodiment, the binding agent comprises (a) at least a first binding domain, (b) a second binding domain, and (c) an Fc domain composed of a first subunit and a second subunit capable of stable association, wherein the Fc domain comprises a modification that promotes the association of the first subunit with the second subunit of the Fc domain. The most extensive protein-protein interaction site between the two subunits of the human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment, the modification is in the CH3 domain of the Fc domain.

[0256] In a specific embodiment, the Fc modification is a so-called "knob-into-hole" modification, which comprises a "knob" modification in one of the two subunits of the Fc domain and a "hole" modification in the other of the two subunits of the Fc domain. In a particular embodiment, the first subunit of the Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0257] Knobs-into-holes technology is described, for example, in U.S. Patent Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). In general, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") at the interface of a second polypeptide, so that the protrusion can be positioned in the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protrusion is created at the interface of the second polypeptide by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0258] Thus, in some embodiments, in the CH3 domain of the Fc domain, amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby generating a protuberance in the CH3 domain that can be placed in the cavity in the CH3 domain of the second Fc domain, and in the CH3 domain of the second Fc domain, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity in the CH3 domain of the second Fc domain, into which the protuberance in the CH3 domain of the first Fc domain can be placed. The protuberance and the cavity can be created by modifying the nucleic acid encoding the polypeptide, for example, by site-directed mutagenesis or by peptide synthesis. In a specific embodiment, in the CH3 domain of the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In another embodiment, in the second Fc domain, furthermore, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).

[0259] In yet a further aspect, in the first Fc domain, the serine residue at position 354 is further replaced with a cysteine ​​residue (S354C), and in the second Fc domain, the tyrosine residue at position 349 is further replaced with a cysteine ​​residue (Y349C). The introduction of these two cysteine ​​residues results in the formation of a disulfide bridge between the two Fc domains, which further stabilizes the dimer (Carter (2001), J Immunol Methods 248, 7-15). In some embodiments, the first Fc domain comprises the amino acid substitutions S354C and T366W (EU numbering), and the second Fc domain comprises the amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0260] In some embodiments, the modification that promotes the association of a first Fc domain with a second Fc domain comprises a modification that mediates an electrostatic steering effect, e.g., as described in PCT Publication WO2009 / 089004. In general, this method involves replacing one or more amino acid residues at the interface of the two Fc domains with a charged amino acid residue, such that homodimer formation is electrostatically disfavored, but heterodimerization is electrostatically favored.

[0261] In some embodiments, the binding agent comprises one or more scFvs or "single chain variable fragments". scFvs are fusion proteins of the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, connected using a short linker peptide of 10 to about 25 amino acids. The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility, and can connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The protein retains the specificity of the original antibody despite the removal of the constant region and the introduction of the linker. scFv antibodies are described, for example, in Houston, JS, Methods in Enzymol. 203 (1991) 46-96. Methods for making scFv molecules and designing suitable peptide linkers are described, for example, in U.S. Pat. No. 4,704,692; U.S. Pat. No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137 (1991).

[0262] A binding agent that is an scFv-Fc is described by Sokolowska-Wedzina et al., Mol. Cancer Res. 15(8):1040-1050, 2017.

[0263] In some embodiments, the binding agent is a "bispecific T cell engager," or BiTE (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, the single polypeptide chain may contain two single chain Fv (scFv) fragments, each having a variable heavy (VH) domain and a variable light (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. The single polypeptide further comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different proteins, such that both proteins are bound by the BiTE.

[0264] Being a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., CHO cell system. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, the solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using a gradient of imidazole concentration. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a gradient of sodium chloride concentration. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species. In some embodiments, the bispecific antibody binder is composed of a single polypeptide chain comprising two single chain FV fragments (scFV) fused to each other by a peptide linker.

[0265] A single domain antibody is an antibody fragment that consists of a single monomeric variable antibody domain. A single domain antibody can be derived from the variable domain of an antibody heavy chain from a camelid (e.g., nanobody or VHH fragment). In addition, the term single domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragment from a shark (see, e.g., Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0266] Techniques for producing single domain antibodies (DAB or VHH) are known in the art, for example, as disclosed in Cossins et al. (2006, Prot Express Purif 51: 253-259 and Li et al., Immunol. Lett. 188: 89-95, 2017). Single domain antibodies can be obtained, for example, from camels, alpacas or llamas by standard immunization techniques (see, for example, Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003; and Maass et al., J Immunol Methods 324:13-25, 2007). VHHs can have strong antigen-binding capacity and can interact with novel epitopes inaccessible to conventional VH-VL pairs (see, e.g., Muyldermans et al., 2001). Alpaca serum IgG contains approximately 50% camelid heavy chain only IgG antibodies (HCAbs) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens and VHHs that bind and neutralize the target antigen can be isolated (see, e.g., Maass et al., 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct an alpaca VHH phage display library that can be used to isolate antibody fragments by standard biopanning techniques well known in the art (see, e.g., Maass et al., 2007).

[0267] In some embodiments, the binding agent is an IgG-scFv. IgG-scFv formats include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods for making them are described, for example, in Brinkmann and Kontermann, MAbs 9(2):182-212 (2017); Wang et al., Antibodies, 2019, 8, 43; Dong et al., 2011, MAbs 3:273-88; Natsume et al., J. Biochem. 140(3):359-368, 2006; Cheal et al., Mol. Cancer Ther. 13(7):1803-1812, 2014; and Bates and Power, Antibodies, 2019, 8, 28.

[0268] Igg-like dual variable domain antibodies (DVD-Ig) are described by Wu et al., 2007, Nat Biotechnol 25:1290-97; Hasler et al., Mol. Immunol. 75:28-37, 2016, and in WO08 / 024188 and WO07 / 024715. Triomabs are described by Chelius et al., MAbs 2(3):309-319, 2010. 2-in-1-IgGs are described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tandem antibodies, or TandAbs, are described by Kontermann et al., supra. An scFv-HSA-scFv antibody has also been described by Kontermann et al. (supra).

[0269] In some embodiments, the binding agent is a scaffold antigen-binding protein, such as fibronectin and designed ankyrin repeat proteins (DARPins), which have been used as alternative scaffolds for antigen-binding domains.See, for example, Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In some embodiments, the scaffold antigen binding protein is selected from the group consisting of lipocalins (anticalins), molecules derived from Protein A, such as the Z-domain of Protein A (affibody), A-domain (avimer / maxibody), serum transferrin (trans-body); designed ankyrin repeat proteins (DARPins), fibronectins (adnectins), C-type lectin domains (tetranectins); variable domains of the novel antigen receptor beta-lactamase (VNAR fragments), human gamma-crystallin or ubiquitin (Affilin molecules); Kunitz-type domains of human protease inhibitors, and microbodies, such as proteins from the knottin family, peptide aptamers and fibronectins (adnectins).

[0270] Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with several loops at the open end of a cone structure that can be engineered to bind different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482: 337-350 (2000), U.S. Patent No. 7,250,297B1 and U.S. Patent Application Publication No. 20070224633.

[0271] Designed ankyrin repeat proteins (DARPins) are derived from ankyrin, a family of proteins that mediate the binding of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and one beta-turn. They can be engineered to bind different target antigens by randomizing the residues in the first alpha-helix and beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007), and US Patent Publication No. 20040132028A1.

[0272] Fc domain engineering to alter effector function In some embodiments, the Fc region or Fc domain has substantially no binding to at least one Fc receptor selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a) and FcyRIIIB (CD16b). In some embodiments, the Fc region or domain exhibits substantially no binding to any of the Fc receptors selected from FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b), FcyRIIIA (CD16a) and FcyRIIIB (CD16b). As used herein, "substantially no binding" refers to weak to no binding to the selected Fc gamma receptor(s). In some embodiments, "substantially no binding" refers to a reduction in binding affinity (e.g., an increase in Kd) to the Fc gamma receptor by at least 1000-fold. In some embodiments, the Fc domain or region is Fc null. As used herein, "Fc null" refers to an Fc region or Fc domain that exhibits weak to no binding to any of the Fc gamma receptors. In some embodiments, the Fc null domain or region exhibits at least a 1000-fold reduction in binding affinity (e.g., increased Kd) to the Fc gamma receptor.

[0273] In some embodiments, the Fc domain has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP or CDC activity compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits reductions in ADCC, ADCP and CDC compared to a wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP or CDC). As used herein, "substantially no effector function" refers to at least a 1000-fold reduction in effector function activity compared to a wild-type Fc domain.

[0274] In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to a decrease in the ADCC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500 fold.

[0275] In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or no CDC activity refers to a decrease in the CDC activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500 fold.

[0276] In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks Fc gamma receptors (and therefore may lack ADCC activity). NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, whereas monocytes express Fc gamma RI, Fc gamma RII and Fc gamma RIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96™ Non-Radioactive Cytotoxicity Assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0277] C1q binding assay can also be carried out to confirm that antibody or Fc domain or region cannot bind C1q and therefore lacks or has reduced CDC activity.See, for example, the C1q and C3c binding ELISA in WO2006 / 029879 and WO2005 / 100402.CDC assay can be carried out to evaluate complement activation (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)).

[0278] In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, reduced or no ADCP activity refers to a reduction in the ADCP activity of the Fc domain by at least 10, at least 20, at least 30, at least 50, at least 100, or at least 500 fold.

[0279] ADCP binding assays can also be performed to confirm that an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity.See, for example, US Patent Application Publication No. 20190079077 and US Patent Application Publication No. 20190048078 and references disclosed therein.

[0280] Antibodies with reduced effector function activity include those with one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (see U.S. Patent No. 6,737,056). Such Fc variants include those with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (see U.S. Patent No. 7,332,581). Certain antibody variants with reduced binding to FcR are also known (see, for example, U.S. Patent No. 6,737,056; WO2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0281] In certain embodiments, the binding agent comprises an Fc domain or region having one or more amino acid substitutions that attenuate Fc gamma R binding, for example, substitutions at positions 234 and 235 (EU numbering of residues) of the Fc region. In some embodiments, the substitutions are L234A and L235A (LALA). In some embodiments, the Fc domain further comprises D265A and / or P329G in the Fc region derived from a human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from a human IgG1 Fc region (see, for example, WO2012 / 130831). In some embodiments, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from a human IgG1 Fc region.

[0282] In some embodiments, modifications are made in the Fc region that result in altered (i.e., alternatively attenuated) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0283] Binding domain engineering In some embodiments, the binding domain may be modified by conservative substitution(s).For conservative amino acid substitution, a given amino acid may be replaced by a residue with similar physicochemical characteristics, for example, one aliphatic residue is substituted for another aliphatic residue (e.g., Ile, Val, Leu or Ala instead of each other), or one polar residue is used instead of another polar residue (e.g., between Lys and Arg; between Glu and Asp; or between Gln and Asn).Other such conservative amino acid substitutions, for example, the substitution of entire regions with similar hydrophobic characteristics, are well known.Polypeptides containing conservative amino acid substitutions may be tested in any one of the assays described herein to confirm that the desired activity, for example, antigen binding activity and specificity, of native or reference polypeptide is retained.

[0284] For conservative substitutions, amino acids can be grouped according to similarities in the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) nonpolar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M); (2) uncharged polar: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q); (3) acidic: Asp(D), Glu(E); and (4) basic: Lys(K), Arg(R), His(H).

[0285] Alternatively, for conservative substitutions, naturally occurring residues can be divided into groups based on shared side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that affect chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions entail exchanging members of one of these classes or another.

[0286] Specific conservative substitutions include, for example, Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile or Leu.

[0287] In some embodiments, conservatively modified variants of binding domains are preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to a reference VH or VL sequence, and the CDRs of VH and VL are unmodified. The degree of homology (percent identity) between a reference sequence and a modified sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn using default settings).

[0288] Modification of the native (or reference) amino acid sequence can be achieved by any of several techniques known to those skilled in the art. Mutations can be introduced at specific sites, for example, by synthesizing oligonucleotides containing the desired mutant sequence flanked by restriction sites that allow ligation to a fragment of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide altered nucleotide sequences with specific codons altered according to the desired substitution, deletion or insertion. Techniques for making such modifications are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are hereby incorporated by reference in their entireties.

[0289] CD8+KIR+regulatory T cells Regulatory T cells are characterized by the phenotype of CD8+KIR+ and are typically MHC class I restricted.In humans, CD8+Kir+ regulatory T cells express inhibitory KIR protein.In some embodiments, the KIR protein expressed by cells can include one or more of inhibitory KIR proteins, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1 and KIR3DL2; specifically, can include one or more of KIR2DL2, KIR2DL3 and KIR3DL1. In some embodiments, CD8+KIR+ regulatory T cells are not HLA E(Qa-1b) restricted (e.g., for a general description of mouse Qa-1b and human HLA E restriction, see Lohwasser et al., International Immunology 13:321-327 (2001) and Sarantopoulos et al., J. Clin. Invest. 114(9):1218-1221 (2004)). In some embodiments, CD8+KIR+ regulatory T cells may also be characterized as CD44+, CD122+, and are not HLA E(Qa-1b) restricted. In some embodiments, CD8+KIR+ regulatory T cells may also be characterized as CD28-. In some embodiments, CD8+KIR+ regulatory T cells may also be characterized as CD28-, CD44+, and CD122+. In some embodiments, CD8+KIR+ regulatory T cells may also be characterized as CD28-, CD44+ and CD122+, and are not HLA E(Qa-1b) restricted.

[0290] In some embodiments, CD8+KIR+ Tregs express the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103 and inhibitory KIR proteins.

[0291] In some embodiments, the CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD3, CD5, CD16, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, KLRB1, KLRG1, LAG-3 / CD223, NKG2C, NKG2D, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, CD8+KIR+ Tregs express one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR3, and CD122.

[0292] Production of binders In various embodiments, the binding agent can be produced in cell lines derived from human, mouse or other animals. Recombinant DNA expression can be used to produce the binding agent. This allows the production of antibodies and a range of antigen-binding moieties and other binding agents, including fusion proteins, in selected host species. The production of antibodies, their antigen-binding moieties and other binding agents in bacteria, yeast, transgenic animals and chicken eggs are also alternatives to cell-based production systems. The main advantage of transgenic animals is the potential high yield from renewable sources.

[0293] As used herein, the term "nucleic acid" or "nucleic acid sequence" or "polynucleotide sequence" or "nucleotide" refers to a polymeric molecule incorporating units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of a denatured double-stranded DNA. In some embodiments, a nucleic acid can be a cDNA, e.g., a nucleic acid lacking introns.

[0294] Nucleic acid molecules encoding the amino acid sequence of an antibody or antigen-binding portion thereof and other binding agents can be prepared by various methods known in the art. These methods include, but are not limited to, preparation of synthetic nucleotide sequences encoding an antibody, antigen-binding portion or other binding agent(s). In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding antibodies or antigen-binding portions and other binding agents. Nucleic acid sequences encoding at least the antibodies, antigen-binding portions thereof, binding agents, or polypeptides thereof described herein can be recombined with vector DNA according to conventional techniques, such as blunt or staggered-ended ends for ligation, restriction enzyme digestion to provide suitable termini, filling in cohesive ends where appropriate, alkaline phosphatase treatment to avoid undesired junctions, and ligation with a suitable ligase. Techniques for such manipulations are disclosed, for example, by Maniatis et al., Molecular Cloning, Lab. Manual (Cold Spring Harbor Lab. Press, NY, 1982 and 1989) and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and can be used to construct nucleic acid sequences and vectors encoding antibodies or antigen-binding portions thereof or their VH and / or VL polypeptides. When the binding agent comprises an antibody or antigen-binding portion thereof, in some embodiments, the VH polypeptide is encoded by a first nucleic acid. In some embodiments, the VL polypeptide is encoded by a second nucleic acid. In some embodiments, the VH and VL polypeptides are encoded by one nucleic acid.

[0295] A nucleic acid molecule, e.g., DNA, is said to be "capable of expressing" a polypeptide when it contains a nucleotide sequence containing transcriptional and translational regulatory information, and such a sequence is "operably linked" to a nucleotide sequence encoding the polypeptide. An operable link is a linkage in which the regulatory DNA sequence and the DNA sequence desired to be expressed (e.g., an antibody or antigen-binding portion thereof) are connected in such a way as to allow gene expression of the polypeptide(s) or antigen-binding portion in recoverable amounts. The exact nature of the regulatory regions required for gene expression may vary from organism to organism, as is well known in the art. See, e.g., Sambrook et al., 1989; Ausubel et al., 1987-1993.

[0296] Thus, expression of the antibody or antigen-binding portion thereof or other binding agent described herein can occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian and mammalian cells, either in vivo or in situ, or host cells of mammalian, insect, avian or yeast origin. Mammalian cells or tissues can be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine or feline origin, although any other mammalian cells can be used. Additionally, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved, for example, by use of the yeast ubiquitin hydrolase system. The fusion proteins so produced can be processed in vivo or purified and processed in vitro to allow synthesis of the antibody or antigen-binding portion thereof described herein with a specified amino-terminal sequence. Furthermore, problems associated with retention of the methionine residue from the start codon in direct yeast (or bacterial) expression can be avoided (see, e.g., Sabin et al., 7 Bio / Technol. 705 (1989); Miller et al., 7 Bio / Technol. 698 (1989)). Any of a range of yeast gene expression systems incorporating promoter and termination elements from actively expressed genes encoding glycolytic enzymes that are produced in large quantities when yeast is grown in glucose-rich medium can be utilized to obtain recombinant antibodies or antigen-binding portions thereof or other binding agents. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase gene can be utilized.

[0297] Production of antibodies or antigen-binding portions thereof and other binding agents in insects can be accomplished, for example, by infecting an insect host with a baculovirus engineered to express the polypeptide by methods known to those of skill in the art, see Ausubel et al., 1987-1993.

[0298] In some embodiments, the introduced nucleic acid sequence (encoding the antibody or its antigen-binding portion, or its polypeptide or other binding agent) is incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host cell. Any of a wide variety of vectors can be used for this purpose, and are known and available to those skilled in the art. See, for example, Ausubel et al., 1987-1993. Important factors in selecting a particular plasmid or viral vector include: the ease with which recipient cells containing the vector can be recognized and selected from recipient cells that do not contain the vector; the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.

[0299] Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replicating in E. coli.Other gene expression elements useful for expressing DNA encoding antibodies or antigen-binding portions thereof and other binding agents include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early promoter (Okayama et al., 3 Mol. Cell. Biol. 280 (1983)), Rous sarcoma virus LTR (Gorman et al., 79 PNAS 6777 (1982)) and Moloney murine leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985));(b) splice regions and polyadenylation sites, such as those derived from the SV40 late region (Okayarea et al., 1983), and (c) polyadenylation sites, such as those in SV40 (Okayama et al., 1983). Immunoglobulin-encoding DNA genes can be expressed using the SV40 early promoter and enhancer, the mouse immunoglobulin heavy chain promoter enhancer, the SV40 late region mRNA splicing, the rabbit S-globin intervening sequence, the immunoglobulin and rabbit S-globin polyadenylation sites, and the SV40 polyadenylation elements as expression elements, as described in Liu et al., infra, and Weidle et al., 51 Gene 21 (1987).

[0300] For immunoglobulin-encoding nucleotide sequences, the transcription promoter can be, for example, human cytomegalovirus and the promoter enhancer can be cytomegalovirus and mouse / human immunoglobulin.

[0301] In some embodiments, for expression of the DNA coding region in rodent cells, the transcription promoter can be a viral LTR sequence, the transcription promoter enhancer can be either or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, as well as polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the expression elements listed above to achieve expression of the protein in mammalian cells.

[0302] Each coding region or gene fusion is assembled in or inserted into an expression vector. Recipient cells capable of expressing the variable region(s) or antigen-binding portion thereof are then transfected alone with nucleotides encoding the antibody or antibody polypeptide or antigen-binding portion thereof, or co-transfected with polynucleotides encoding the VH and VL chain coding regions. The transfected recipient cells are cultured under conditions that allow expression of the incorporated coding regions, and the expressed antibody chains or intact antibodies or antigen-binding portions are recovered from the culture.

[0303] In some embodiments, nucleic acids containing coding regions encoding antibodies or antigen-binding portions thereof are assembled in separate expression vectors that are then used to co-transfect recipient host cells. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, a first selectable gene designed for selection in a bacterial system and a second selectable gene designed for selection in a eukaryotic system, with each vector having one set of coding regions. This strategy results in a vector that first directs the production of a nucleotide sequence in a bacterial system and allows its amplification. The DNA vector so produced and amplified in a bacterial host is subsequently used to co-transfect eukaryotic cells, allowing the selection of co-transfected cells carrying the desired transfected nucleic acid (e.g., encoding antibody heavy and light chains). Non-limiting examples of selectable genes for use in bacterial systems are genes that confer resistance to ampicillin and genes that confer resistance to chloramphenicol. Selectable genes for use in eukaryotic transformants include the xanthine guanine phosphoribosyltransferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, fused nucleotide sequences encoding the VH and VL chains can be assembled on the same expression vector.

[0304] For transfection of expression vectors and production of antibodies or antigen-binding portions thereof or other binding agents, the recipient cell line can be a Chinese hamster ovary cell line (e.g., DG44) or a myeloma cell. The myeloma cell can synthesize, assemble and secrete immunoglobulins encoded by the transfected immunoglobulin genes and possesses the machinery for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is a recombinant Ig-producing myeloma cell SP2 / 0. The SP2 / 0 cell produces only immunoglobulins encoded by the transfected genes. The myeloma cell can be grown in culture or in the peritoneal cavity of a mouse, where secreted immunoglobulins can be obtained from ascites.

[0305] Expression vectors encoding antibodies or antigen-binding portions thereof or other binding agents can be introduced into suitable host cells by any of a variety of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, and application of polycations such as diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and particle bombardment. Johnston et al., 240 Science 1538 (1988), known to those of skill in the art.

[0306] Yeast offers certain advantages over bacteria for the production of immunoglobulin heavy and light chains. Yeast carries out post-translational peptide modifications, including glycosylation. There are several recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids that can be used for the production of desired proteins in yeast. Yeast recognizes leader sequences in cloned mammalian gene products and secretes polypeptides (i.e., pre-polypeptides) that bear the leader sequence. See, e.g., Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0307] Yeast gene expression systems can be routinely evaluated for the levels of production, secretion and stability of antibodies and assembled antibodies and their antigen-binding portions. Various yeast gene expression systems can be utilized that incorporate promoter and termination elements from actively expressed genes that code for glycolytic enzymes that are produced in large amounts when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional control signals. For example, the promoter and terminator signals of the phosphoglycerate kinase (PGK) gene can be utilized. Another example is the translation elongation factor 1 alpha promoter. Several approaches can be taken to evaluate the optimal expression plasmid for the expression of immunoglobulins in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, for example, U.S. Patent Application Publication No. 2006 / 0270045A1.

[0308] Bacterial strains can also be utilized as hosts for the production of the antibody molecules or antigen-binding portions thereof or other binding agents described herein. E. coli K12 strains, such as E. coli W3110, Bacillus species, enterobacteria, such as Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species can be used. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these bacterial hosts. The vector carries a replication site and specific genes that can provide phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for the production of antibodies and their antigen-binding portions in bacteria (see Glover, 1985; Ausubel, 1987, 1993; Sambrook, 1989; Colligan, 1992-1996).

[0309] Host mammalian cells can be grown in vitro or in vivo. Mammalian cells provide post-translational modifications to immunoglobulin molecules including leader peptide removal, folding and assembly of VH and VL chains, glycosylation of the antibody molecule, and secretion of functional antibodies and / or antigen-binding portions thereof.

[0310] Mammalian cells that may be useful as hosts for the production of antibody proteins include cells of fibroblast origin, such as Vero or CHO-K1 cells, in addition to the above-mentioned cells of lymphoid origin.Exemplary eukaryotic cells that may be used to express immunoglobulin polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1 and DG44 cells; PERC6™ cells (Crucell); and NSO cells.In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to heavy and / or light chains.For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells.

[0311] In some embodiments, one or more antibodies or antigen-binding portions thereof or other binding agents may be produced in vivo in an animal engineered or transfected with one or more nucleic acid molecules encoding the polypeptide, according to any suitable method.

[0312] In some embodiments, the antibody or antigen-binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems are described, for example, in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); and Endo et al., Biotechnol. Adv. 21: 695-713 (2003).

[0313] Many vector systems are available for the expression of VH and VL chains in mammalian cells (see Glover, 1985). Intact antibodies can be obtained according to various approaches. As discussed above, it is possible to co-express VH and VL chains, and optionally associated constant regions, in the same cell to achieve intracellular association and linkage of the VH and VL chains to a complete tetrameric H2L2 antibody or antigen-binding portion thereof. Co-expression can occur by using either the same or different plasmids in the same host. Nucleic acids encoding the VH and VL chains or antigen-binding portions thereof can be placed in the same plasmid, which is then transfected into cells, thereby directly selecting for cells expressing both chains. Alternatively, cells can be first transfected with a plasmid encoding one chain, e.g., the VL chain, and the resulting cell line can then be transfected with a VH chain plasmid containing a second selectable marker. Cell lines producing antibodies, antigen-binding portions thereof, or other binding agents via either route can be transfected with additional copies of plasmids encoding the peptide, VH, VL, or VH plus VL chains, along with additional selectable markers, to generate cell lines with enhanced properties, e.g., higher production of assembled antibody or antigen-binding portions thereof, or enhanced stability of the transfected cell line.

[0314] Furthermore, plants have emerged as a convenient, safe and economical alternative expression system for recombinant antibody production based on large-scale culture of microorganisms or animal cells. Antibodies or antigen-binding portions can be expressed in plant cell cultures or in normally grown plants. Expression in plants can be systemic, intracellular plastid-limited, or seed (endosperm)-limited. See, for example, US Patent Application Publication No. 2003 / 0167531; US ​​Patent No. 6,080,560; US Patent No. 6,512,162; and WO0129242. Several plant-derived antibodies have reached advanced stages of development, including clinical trials (see, for example, Biolex, NC).

[0315] For intact antibodies, the variable regions (VH and VL) of the antibody are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, such as immortalized B cells, according to well-known procedures (see, e.g., WO87 / 02671, which is hereby incorporated by reference in its entirety). The antibody can contain both light and heavy chain constant regions. The heavy chain constant region can include CH1, hinge, CH2, CH3, and sometimes CH4 regions. In some embodiments, the CH2 domain can be deleted or omitted.

[0316] Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989); which are hereby incorporated by reference in their entireties) can be adapted to produce single chain antibodies that specifically bind to a desired antigen. Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge to produce a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (see, e.g., Skerra et al., Science 242:1038-1041 (1988); which are hereby incorporated by reference in their entireties). Methods for making other binders are described above.

[0317] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or antigen-binding portions thereof can be recovered and purified by known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982). Substantially pure antibodies or antigen-binding portions thereof of at least about 90%-95% homogeneity are beneficial, with 98%-99% or greater homogeneity being particularly beneficial for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the intact antibodies or antigen-binding portions thereof can then be used therapeutically or in developing and performing assay procedures, immunofluorescence staining, and the like. See generally, Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0318] Pharmaceutical preparations In some aspects, the binding agent refers to a composition comprising an active ingredient (i.e., a binding agent described herein or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier approved for use in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment.

[0319] The preparation of a pharmacological composition containing an active ingredient dissolved or dispersed therein is well understood in the art and need not be limited based on any particular formulation. Typically, such compositions are prepared as injections, either as liquid solutions or suspensions; however, solid forms suitable for rehydration or suspension in liquid prior to use may also be prepared. The preparation may also be emulsified or presented as a liposomal composition. The antibody or antigen-binding portion thereof or other binding agent may be mixed with an excipient that is pharmaceutically acceptable and compatible with the active ingredient, in an amount appropriate for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, if desired, the pharmaceutical composition may contain minor amounts of auxiliary substances that enhance or maintain the effectiveness of the active ingredient (e.g., the antibody or antigen-binding portion thereof or other binding agent), such as wetting or emulsifying agents, pH buffering agents, and the like. The pharmaceutical compositions described herein may include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of polypeptides) formed with inorganic acids, such as hydrochloric or phosphoric acid, or organic acids, such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide, and organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing active ingredients (e.g., antibodies and / or antigen-binding portions thereof or other binding agents) and water, and may contain buffers, such as sodium phosphate, saline, or both, such as phosphate-buffered saline, at physiological pH values. Still further, aqueous carriers may contain more than one buffer salt, as well as salts, such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions may also contain liquid phases in addition to, and to the exclusion of, water.Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0320] In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding portion thereof or other binding agent described herein, or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent, may be a lyophilizate.

[0321] In some embodiments, a syringe is provided comprising a therapeutically effective amount of a binding agent or pharmaceutical composition described herein.

[0322] Treatment of Inflammatory and Autoimmune Diseases In some aspects, the binding agents described herein may be used in a method(s) comprising administering the binding agents or pharmaceutical compositions described herein to a subject with an inflammatory disease. In some aspects, the binding agents described herein may be used in a method(s) comprising administering the binding agents or pharmaceutical compositions described herein to a subject with an autoimmune disease or a subject with an immune response to an autoantigen or a subject with an immune response to an antigen that causes or results in the development of an autoimmune disorder (which may be collectively referred to herein as an autoimmune disease). In some embodiments, the subject is in need of treatment for an autoimmune disease. In some embodiments, a method of treating an autoimmune disease is provided, comprising administering any of the binding agents described herein or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to reduce the number or activity of pathogenic immune cells in the subject, thereby alleviating the symptoms of the autoimmune disease. In some embodiments, a method is provided for suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents described herein or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), thereby reducing the number or activity of pathogenic immune cells. In some embodiments, a method is provided for suppressing an immune response against an autoantigen, comprising administering to a subject in need thereof any of the binding agents described herein or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to the autoantigen.In some embodiments, a method is provided for preventing an immune response against an autoantigen that causes or results in the development of an autoimmune disorder, comprising administering to a subject in need thereof any of the binding agents described herein or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to the autoantigen, or reducing the titer of autoantibodies in the subject. In some embodiments, a method is provided for suppressing an immune response against an autoantigen that causes or results in the development of an autoimmune disorder, comprising administering to a subject in need thereof any of the binding agents described herein or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to the autoantigen, or reducing the titer of autoantibodies in the subject. In some embodiments, a method is provided for suppressing an immune response to an antigen or autoantigen that causes or results in the development of an autoimmune disorder, comprising administering to a subject in need thereof any of the binding agents described herein or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+ Tregs, whereby the immune response to the antigen or autoantigen is reduced or suppressed in the subject.

[0323] As used herein, the term "autoantigen" refers to an antigen (e.g., a cell surface protein or other antigen) that is normally recognized as self by the immune system (e.g., a healthy human immune system). An antigen or autoantigen is a target of humoral or cell-mediated immune response or induces an excessive immune response in an autoimmune disease or condition (collectively "autoimmune disease"). As used herein, "activating or stimulating" CD8+KIR+Treg or activated CD8+KIR+Treg refers to the regulatory T cell function of such cells, e.g., increasing the ability to suppress immune responses, particularly immune responses against autoantigens, or suppress immune responses against autoantigens that cause or result in the development of an autoimmune disorder. Activating or stimulating CD8+KIR+Treg can include removing the suppressive effect on such cells to restore CD8+KIR+Treg (e.g., to restore balance of the immune system or restore balanced immune activity in a subject or in a subject in need of treatment prior to the onset of disease). Activation or stimulation of CD8+KIR+ Tregs may also include removal of such cells, for example, removal of CD4+ cells, B cells, or other cells mediating the immune response, such as by cell lysis.

[0324] As used herein, pathogenic immune cells refer to immune cells that react with self-antigens or induce responses to self-antigens.Examples of such pathogenic immune cells include autoreactive CD4+ T cells, autoantibody-producing B cells, autoantigen-presenting dendritic cells and other autoantigen-presenting cells, as known in the art.

[0325] In some embodiments, the CD8+KIR+Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+Tregs are contacted with the binding agent ex vivo. The activated CD8+KIR+Tregs can then be administered in an effective amount to a subject in need thereof.

[0326] In some embodiments, the activated CD8+KIR+Treg exerts an inhibitory effect on pathogenic immune cells, such as autoreactive CD4+T cells, autoantibody-producing B cells, autoantigen-presenting dendritic cells, or autoantigen-presenting cells. In some embodiments, the activated CD8+KIR+Treg exerts an inhibitory effect on pathogenic immune cells, such as autoreactive CD4+T cells, autoantibody-producing B cells, and autoantigen-presenting dendritic cells. In some embodiments, the activated CD8+KIR+Treg depletes pathogenic immune cells, such as autoreactive CD4+T cells, autoantibody-producing B cells, and autoantigen-presenting dendritic cells. In some embodiments, the activated CD8+KIR+Treg modulates the pathogenic effect of pathogenic immune cells and reduces the titer of autoantibodies in the subject. In some embodiments, the activated CD8+KIR+Treg reduces the titer of autoantibodies in the subject.

[0327] In some embodiments, the binding agent is selected from any of the binding agents described herein, in each case with reduced or substantially no effector function activity. In some embodiments, reduced effector function activity is reduced ADCC, ADCP or CDC effector function activity, or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or region and has reduced or substantially no effector function. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region, for example, Fc null substitution. In some embodiments, the binding agent lacks an Fc domain or region, or has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent lacks an Fc domain or region.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0328] Without intending to be bound by any particular theory, the reduced or absent effector function activity by the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-CD8+KIR+ Tregs) and / or limit the depletion of CD8+KIR+ Tregs bound by the binding agent.

[0329] In some embodiments, the subject in need of treatment has an autoimmune disease. In some embodiments, the subject in need of treatment has an autoimmune disease, such as autoimmune induced hepatitis, Addison's disease, alopecia areata, Alport syndrome, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis, atopic allergy, atopic dermatitis, atopic rhinitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myositis, Behcet's disease, bird breeder's lung, bronchial asthma, Kaplan's syndrome, cardiomyopathy, celiac disease, Chagas' disease, chronic glomerulonephritis, chronic graft-versus-host disease, Cogan's syndrome, cold agglutinin disease, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus erythematosus, Dressler's syndrome, Syndrome), Eaton-Lambert syndrome, encephalomyelitis, endocrine ophthalmopathy, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs's cyclitis, gastrointestinal atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture's syndrome, graft-versus-host disease, Graves' disease, Guillain-Barre disease (syndrome), Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, hyperviscosity syndrome, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy, inflammatory bowel disease (syndrome), insulin-dependent diabetes mellitus (IDDM or type I), juvenile arthritis, juvenile idiopathic arthritis, juvenile diabetes mellitus (type I), Lambert-Eaton syndrome Laminitis, Lichen planus, Lupoid hepatitis, Lupus, Lupus nephritis, Lymphocytopenia, Macroglobulinemia, Meniere's disease, Mixed connective tissue disease, Monoclonal gammopathy of unknown origin, Multiple sclerosis, Myasthenia gravis, Myocarditis, Pemphigus / Pemphoid, Pernicious anemia, POEMS syndrome, Polyglandular syndrome, Polyarteritis nodosa, Polymyositis, Presenile dementia, Primary agammaglobulinemia, Primary biliary cirrhosis / cholangitis, Psoriasis, Psoriatic arthritis, Raynaud's phenomenon, Reiter's syndrome, Rheumatic fever, Rheumatoid arthritis, Sampter's syndrome, Schmidt's syndrome, Scleroderma / systemic sclerosis, Schulman's syndrome, Sjögren's syndromeSyndrome), stiff man syndrome, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type I diabetes mellitus, ulcerative colitis, uveitis, leukoplakia, Waldenström's macroglobulinemia and / or Wegener's granulomatosis.

[0330] In some embodiments, the autoimmune disease is autoimmune hepatitis, celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, primary biliary cirrhosis / cholangitis, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE) or ulcerative colitis.

[0331] In some embodiments, the autoimmune disease is selected from autoimmune hepatitis, celiac disease, Crohn's disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or ulcerative colitis.

[0332] The method described herein comprises administering a therapeutically effective amount of a binding agent to a subject having an autoimmune disease. As used herein, the phrases "therapeutically effective amount", "effective amount", "effective amount" and "effective dose" can refer to the amount of a binding agent described herein that provides a therapeutic benefit in treating an autoimmune disease, managing an autoimmune disease, preventing the recurrence of an autoimmune disease, delaying the onset of an autoimmune disease, or preventing the onset of an autoimmune disease, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of an autoimmune disease. The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art. In general, a therapeutically effective amount can vary depending on the subject's medical history, age, condition, and sex, as well as the severity and type of medical condition in the subject, and the administration of other pharmacologic active agents.

[0333] The method herein is intended to reduce symptoms, lesions, disease progression or disease relapse in a subject. As used herein, "subject" refers to a human or animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient", "individual" and "subject" are used interchangeably herein.

[0334] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow. Non-human mammals can be, for example, beneficially used as subjects that present animal models of various autoimmune diseases. Furthermore, the methods described herein can be used to treat domestic animals and / or pets. The subject can be male or female. In certain embodiments, the subject is a human.

[0335] The subject may be a subject who has been previously diagnosed with an autoimmune disease or previously identified as suffering from an autoimmune disease and needs treatment, but does not need to have already been treated for the autoimmune disease. Alternatively, the subject may also be a subject who has not been previously diagnosed with an autoimmune disease and needs treatment. The subject may be a subject who shows one or more risk factors for a condition or one or more complications associated with an autoimmune disease, or a subject who does not show risk factors. A "subject in need" of treatment for an autoimmune disease may be a subject who has the disease or a subject who has been diagnosed with the disease. In some embodiments, a subject needs treatment if the subject is at risk of developing an immune response to an antigen or autoantigen that may cause or result in the development of an autoimmune disorder. In other embodiments, a subject who is "at risk of developing" an autoimmune disease refers to a subject who has been diagnosed as at risk of developing a disease or condition.

[0336] As used herein, the terms "treat", "treatment", "treating" or "alleviation", when used in reference to a disease, disorder or medical condition (e.g., an autoimmune disease), refer to therapeutic treatment for the condition, the purpose of which is to reverse, alleviate, relieve, inhibit, slow down or stop the progression or severity of the symptoms or condition. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of the condition or disease. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" can include not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in one or more symptoms, a reduction in disease flares in a subject, a relief of one or more symptoms, a reduction in the degree of the defect, a stabilized (i.e., not worsening) state of the autoimmune disease, or prevention, delay or slowing of the onset or progression of the autoimmune disease. As used herein, the term "administering" refers to contacting (e.g., by administration to a subject) a binding agent described herein or a nucleic acid encoding a binding agent described herein by a method or route that results in binding of the binding agent to CD8+KIR+Treg. Similarly, the pharmaceutical compositions disclosed herein that include a binding agent described herein or a nucleic acid encoding a binding agent described herein can be administered by any suitable route that results in an effective treatment in a subject.

[0337] Dosage ranges for binding agents depend on efficacy and include amounts large enough to produce the desired effect, e.g., reduction of one or more symptoms, reduction of disease flares in a subject, alleviation of one or more symptoms, attenuation of the degree of the defect, stabilized (i.e., not worsening) state of an autoimmune disease, or prevention, delay or slowing of the onset or progression of an autoimmune disease. Dosages should not be so large as to cause unacceptable adverse side effects. In general, dosages vary according to the age, condition and sex of the subject and can be determined by one of skill in the art. Dosages can also be adjusted by the individual physician in the event of any complications. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, dosages range from about 0.5 mg / kg body weight to about 15 mg / kg body weight. In some embodiments, dosage ranges from about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, dosage ranges can be titrated to maintain serum levels between 1 ug / mL and 1000 ug / mL.

[0338] In some embodiments, the subject receives a single dose of any of the binding agents described herein, for example, for the treatment of an acute autoimmune disease or condition. In some embodiments, the subject receives a single dose of any of the binding agents described herein, for example, for the prevention of immune responses that may lead to an autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binding agents described herein, for example, for the treatment of a chronic autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binding agents described herein, for example, for the prevention of immune responses that may lead to an autoimmune disease or condition. In some embodiments, the dose is administered weekly, every two weeks, every three weeks, monthly, every two months, or every six months for several weeks, months, or years. The duration of treatment depends on the subject's clinical progression and responsiveness to treatment.

[0339] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.

[0340] The pharmaceutical composition containing any of the binding agents described herein can be administered in unit doses.The term "unit dose", when used in reference to pharmaceutical compositions, refers to a physically separate unit suitable as a unitary dosage for subjects, each unit containing a predetermined amount of active material (e.g., binding agent) calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0341] In some embodiments, administration of any of the binding agents described herein may result in improved treatment outcomes, such as reduced systemic inflammatory cytokines, reduced lesions in tissues affected by the disease, reduced relapse frequency and / or severity, reduced self-reported symptoms associated with the disease, alleviation of one or more symptoms, and / or prevention, delay or slowing of the onset or progression of an autoimmune disease.

[0342] In some embodiments, the binding agent or any pharmaceutical composition of the binding agent described herein is administered together with immunotherapy.As used herein, "immunotherapy" refers to a therapeutic strategy designed to modulate the subject's own immune system.Examples of immunotherapy include, but are not limited to, antibodies, such as checkpoint inhibitors and modulators, and immunosuppressants, such as cyclosporine, cyclosporine A, mycophenylate mofetil, sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab, abatacept, belatacept and adalimumab.

[0343] Treatment of transplant complications In some embodiments, a method for treating transplant complications associated with graft-versus-host disease, or GVHD, is provided. In some embodiments, a method for reducing complications of GVHD in a subject is provided, comprising administering a binder or pharmaceutical composition described herein to a subject who has received a transplant to suppress host immune responses to the transplant. In some embodiments, a method for reducing complications of GVHD in a subject is provided, comprising administering a binder or pharmaceutical composition described herein to a subject who has received a transplant to suppress immune responses associated with the transplant. In some embodiments, a method for reducing GVHD is provided, comprising administering a binder or pharmaceutical composition described herein to a subject who has received a transplant and is currently experiencing GVHD, thereby reducing symptoms of GVHD. In some embodiments, a method for suppressing GVHD is provided, comprising administering a binder or pharmaceutical composition described herein to a subject who has received a transplant and is currently experiencing GVHD, thereby suppressing or reducing GVHD. In some embodiments, a method of suppressing or reducing GVHD is provided, comprising contacting a binding agent or pharmaceutical composition described herein with CD8+KIR+T regulatory cells (Treg) from a subject receiving a transplant, thereby activating the CD8+KIR+T regulatory cells (Treg). In some embodiments, a method of suppressing or reducing GVHD is provided, comprising contacting a binding agent or pharmaceutical composition described herein with CD8+KIR+T regulatory cells (Treg) from a subject receiving a transplant, thereby depleting the CD8+KIR+T regulatory cells (Treg) of the transplant. In some embodiments, a method of suppressing or reducing GVHD is provided, comprising contacting a binding agent or pharmaceutical composition described herein with CD8+KIR+T regulatory cells (Treg) from a subject receiving a transplant, thereby depleting the CD8+KIR+T regulatory cells (Treg).

[0344] In some embodiments, the subject has undergone hematopoietic stem cell transplantation, cord blood stem cell transplantation, transplantation of induced pluripotent stem cell derived precursor or differentiated cells, bone marrow transplantation or solid organ transplantation. The transplantation is typically an allogeneic transplantation. In some embodiments, the subject has undergone hematopoietic stem cell transplantation, cord blood stem cell transplantation, transplantation of induced pluripotent stem cell derived precursor or differentiated cells, bone marrow transplantation or solid organ transplantation and is currently experiencing GVHD. In some embodiments, the subject has undergone hematopoietic stem cell transplantation, cord blood stem cell transplantation, transplantation of induced pluripotent stem cell derived precursor or differentiated cells, bone marrow transplantation or solid organ transplantation and is at risk of experiencing GVHD. In some embodiments, the subject has undergone hematopoietic stem cell transplantation and is currently experiencing GVHD. In some embodiments, the subject has undergone hematopoietic stem cell transplantation and is at risk of experiencing GVHD. In some embodiments, the subject has undergone umbilical cord blood stem cell transplantation and is currently experiencing GVHD. In some embodiments, the subject has undergone umbilical cord blood stem cell transplantation and is at risk of experiencing GVHD. In some embodiments, the subject has received a transplant of induced pluripotent stem cell derived progenitors or differentiated cells and is currently experiencing GVHD. In some embodiments, the subject has received a transplant of induced pluripotent stem cell derived progenitors or differentiated cells and is at risk of experiencing GVHD. In some embodiments, the subject has received a bone marrow transplant and is currently experiencing GVHD. In some embodiments, the subject has received a bone marrow transplant and is at risk of experiencing GVHD. In some embodiments, the subject has received a solid organ transplant and is currently experiencing GVHD. In some embodiments, the subject has received a solid organ transplant and is at risk of experiencing GVHD.

[0345] In some embodiments, GVHD is suppressed or reduced by depletion of pathogenic immune cells, for example, CD4 T cells. In some embodiments, GVHD is suppressed or reduced by depletion of pathogenic immune cells, for example, transplant-derived CD8+KIR+Treg. In some embodiments, CD8+KIR+Treg is contacted with the binding agent in vivo. In some embodiments, CD8+KIR+Treg is contacted with the binding agent ex vivo. The activated CD8+KIR+Treg can then be administered to a subject in need thereof in an effective amount.

[0346] In some embodiments, the binding agent is selected from any of the binding agents described herein, in each case with reduced or substantially no effector function activity. In some embodiments, reduced effector function activity is reduced ADCC, ADCP or CDC effector function activity, or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or region, has reduced or substantially no effector function. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region, for example, Fc null substitution. In some embodiments, the binding agent lacks an Fc domain or region, or has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent lacks an Fc domain or region.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0347] In some embodiments, the binding agent is selected from any of the binding agents described herein, each having effector function activity including at least ADCC.In some embodiments, the effector function activity is ADCC combined with ADCP and / or CDC effector function activity.In some embodiments, having effector function activity means having ADCC, ADCP and CDC effector function activity.In various embodiments, such binding agent has Fc domain or has Fc domain that binds to one or more Fc gamma receptors.

[0348] Without intending to be bound by any particular theory, it is believed that the reduced or absent effector function activity by the binder may limit the interaction of the binder with other cell types (i.e., non-CD8+KIR+Tregs) and / or limit the depletion of CD8+KIR+Tregs. In contrast, the presence of effector function activity by the binder is believed to bias the immune response toward the depletion of CD8+KIR+Tregs.

[0349] In some embodiments, the transplant is a stem cell transplant, a bone marrow transplant or a solid organ transplant. As used herein, the term "transplant" refers to an organ, tissue or cell transplanted from one subject to a different subject or transplanted within the same subject (e.g., to a different region within the subject). Organs, such as liver, kidney, heart or lung, or other body parts, such as bone or skeletal matrix, such as bone marrow, tissues, such as skin, cornea, intestine, endocrine gland, or stem cells or various types or hematopoietic cells, including hematopoietic stem cells and progenitor cells, umbilical cord blood stem cells and precursor or differentiated cells derived from induced pluripotent stem cells, are all examples of transplants. In some embodiments, the solid organ transplant is a liver, kidney, lung, pancreas and / or heart transplant. The term transplant includes grafts. Transplants can be allografts (or allogeneic grafts) or xenografts. The term "allograft" refers to a graft between two genetically non-identical members of one species. The term "xenograft" refers to a graft between members of different species.

[0350] In some embodiments, the transplant is a bone marrow transplant. In some embodiments, the transplant is a hematopoietic stem cell transplant. In some embodiments, the transplant is an umbilical cord blood stem cell transplant. In some embodiments, the transplant is a transplant of induced pluripotent stem cell derived precursors or differentiated cells.

[0351] In some embodiments, the subject has graft-versus-host disease, or GVHD. In some embodiments, the subject is at risk of having GVHD.

[0352] The methods described herein include administering a therapeutically effective amount of a binding agent to a subject currently undergoing a transplant. As used herein, the phrases "therapeutically effective amount", "effective amount", "effective amount" and "effective dose" may refer to an amount of a binding agent described herein that provides a therapeutic benefit in managing transplant-associated GVHD. In some embodiments, the therapeutic benefit is a delay in the onset or prevention of the onset of GVHD. In some embodiments, the therapeutic benefit is a statistically significant reduction in at least one symptom, sign or marker of GVHD. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. In general, a therapeutically effective amount may vary depending on the subject's medical history, age, condition and sex, as well as the severity and type of medical condition in the subject, and the administration of other pharmacologic active agents.

[0353] The method of the present invention is intended to reduce symptoms of GVHD, reduce GVHD, or prevent GVHD in transplanted subjects. As used herein, "subject" refers to human or animal. Usually, animals are vertebrates, such as primates, rodents, domestic animals, or game animals. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

[0354] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow. Non-human mammals can be, for example, beneficially used as subjects that present animal models of various autoimmune diseases. Furthermore, the methods described herein can be used to treat domestic animals and / or pets. The subject can be male or female. In certain embodiments, the subject is a human.

[0355] The subject may be a subject who is about to receive a transplant. Alternatively, the subject may be a subject who has already received a transplant. The subject may be a subject who is about to receive a transplant and is at risk for GVHD. The subject may be a subject who has received a transplant and is at risk for having GVHD. The subject may be a subject who has received a transplant and has GVHD.

[0356] As used herein, the terms "treat", "treatment", "treating" or "alleviation", when used in reference to a disease, disorder or medical condition (e.g., GVHD), refer to therapeutic treatment for the condition, the purpose of which is to reverse, alleviate, relieve, inhibit, slow down or stop the progression or severity of a symptom or condition of GVHD. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of the condition or disease. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" can include not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms associated with GVHD.

[0357] As used herein, the term "administering" refers to contacting (e.g., by administration to a subject) a binding agent described herein or a nucleic acid encoding a binding agent described herein by a method or route that results in binding of the binding agent to CD8+KIR+Treg. Similarly, a pharmaceutical composition comprising a binding agent described herein or a nucleic acid encoding a binding agent described herein disclosed herein can be administered by any suitable route that results in an effective treatment in a subject.

[0358] Dosage ranges for binding agents depend on efficacy and include amounts large enough to produce the desired effect, e.g., reduction of one or more symptoms of GVHD or reduction or prevention of GVHD. Dosages should not be so large as to cause unacceptable adverse side effects. In general, dosages vary according to the age, condition, and sex of the subject and can be determined by one of skill in the art. Dosages can also be adjusted by the individual physician in the event of any complications. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, dosages range from about 0.5 mg / kg body weight to about 15 mg / kg body weight. In some embodiments, dosage ranges from about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, dosage ranges can be titrated to maintain serum levels between 1 ug / mL and 1000 ug / mL.

[0359] In some embodiments, the subject receives a single dose of any of the binders described herein, for example, for the treatment of acute GVHD after transplantation.In some embodiments, the subject receives repeated doses of any of the binders described herein, for example, for the treatment of chronic GVHD.In some embodiments, the dose is administered every week, every two weeks, every three weeks, every month, every two months, or every six months for several weeks, months, or years.The duration of treatment depends on the subject's clinical progression and responsiveness to treatment.

[0360] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.

[0361] The pharmaceutical composition containing any of the binding agents described herein can be administered in unit doses.The term "unit dose", when used in reference to pharmaceutical compositions, refers to a physically separate unit suitable as a unitary dosage for subjects, each unit containing a predetermined amount of active material (e.g., binding agent) calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0362] In some embodiments, administration of any of the binding agents described herein may result in improved treatment outcomes, e.g., reduction in systemic inflammatory cytokines, reduction in lesions in tissues affected by the disease, reduction in self-reported symptoms associated with immune responses associated with adverse effects on host tissues, improved or prolonged graft survival, alleviation of one or more symptoms, and / or prevention, delay or slowing of the onset or progression of graft rejection, or prolonged graft survival due to reduced use of broad-spectrum immunosuppressants, e.g., corticosteroids.

[0363] In some embodiments, the binding agent or any pharmaceutical composition of the binding agent described herein is administered with an immunosuppressant, for example, a corticosteroid(s).

[0364] Treatment of infectious diseases In some aspects, the binding agents described herein may be used in a method or methods comprising administering the binding agents or pharmaceutical compositions described herein to a subject having an infectious disease. In some embodiments, the subject is in need of treatment for an infectious disease. In some embodiments, a method is provided for treating an infectious disease by administering any of the binding agents or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby alleviating the symptoms of the infectious disease. In some embodiments, the method comprises stimulating an immune response against the infectious agent by contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), thereby stimulating an immune response against the infectious agent. In some embodiments, the method includes stimulating an immune response against cells infected with an infectious agent by contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), thereby depleting the infected cells. In some embodiments, the method includes stimulating an immune response against cells infected with an infectious agent by contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), thereby stimulating an immune response against the infected cells.

[0365] In some embodiments of the method of treating an infectious disease, CD8+KIR+Treg is contacted in vivo with any of the binding agents described herein. In some embodiments, CD8+KIR+Treg is contacted ex vivo with any of the binding agents described herein. The activated CD8+KIR+Treg is then administered to a subject in need thereof in an effective amount. In some embodiments, the immune response comprises a reduction in immunosuppressive immune cells. As used herein, immunosuppressive immune cells include CD4 Treg and tolerant DC. In some embodiments, the number of infected cells in the subject is reduced.

[0366] In some embodiments, the binding agent is selected from any of the binding agents described herein, in each case with reduced or substantially no effector function activity. In some embodiments, reduced effector function activity is reduced ADCC, ADCP or CDC effector function activity, or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or region, has reduced or substantially no effector function. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region, for example, Fc null substitution. In some embodiments, the binding agent lacks an Fc domain or region, or has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent lacks an Fc domain or region.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0367] Without intending to be bound by any particular theory, the reduced or absent effector function activity by the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-CD8+KIR+ Tregs) and / or limit the depletion of CD8+KIR+ Tregs bound by the binding agent.

[0368] In some embodiments, the subject is in need of treatment for an infectious disease or is infected with an infectious agent, such as a bacterial disease, a systemic fungal disease, a rickettsial disease, a parasitic disease, or a viral disease, etc. In some embodiments, the infectious disease is a bacterial disease, such as diphtheria, whooping cough, occult bacteremia, urinary tract infection, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid hypertrophy, retropharyngeal abscess, impetigo, ecthyma, pneumonia, endocarditis, septic arthritis, pneumococcal pneumonia, peritonitis, bacteremia, meningitis, acute purulent meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella, typhoid, dysentery, conjunctivitis, sinusitis, brucellosis. Tularemia, cholera, bubonic plague, tetanus, necrotizing enteritis, actinomycosis, mixed anaerobic infections, syphilis, relapsing fever, leptospirosis, Lyme disease, rat-bite fever, tuberculosis including mycobacterial tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial pneumonia, trachoma or inclusion conjunctivitis; systemic fungal diseases such as histoplasmosis, coccidioidomycosis, blastomycosis, sporotrichosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycetoma or chromomycosis; rickettsial diseases such as typhoid, Rocky Mountain spotted fever, ehrlichiosis, Eastern Tick-Borne rickettsioses rickettsioses, rickettsialpox, Q fever or bartonellosis; parasitic diseases such as malaria, babesiosis, sleeping sickness, Chagas disease, leishmaniasis, dum-dum fever, toxoplasmosis meningoencephalitis, keratitis, dientamebiasis, giardiasis, cryptosporidiosis, isosporiasis, cyclosporiasis, microsporidiosis, ascariasis, whipworm infection, hookworm infection, strongyloides infection, ocular larva migrans, trichinosis, dracunculiasis, lymphatic filariasis, loiasis, river blindness, heartworm infection, schistosomiasis, cercarial dermatitis (swimmer's itch), oriental lung fluke, oriental liver fluke Fluke), Fascioliasis, Fascioliasis, Opisthorchiasis, Tapeworm Infection, Hydatidosis and Alveolar Hydatidosis;and viral diseases, such as measles, subacute sclerosing panencephalitis, common cold, mumps, rubella, roseola, erythema infectiosum, chickenpox, coronavirus infections, Covid19 disease, respiratory syncytial virus infections, croup, bronchiolitis, infectious mononucleosis, poliomyelitis, herpangina, hand, foot and mouth disease, Bornholm disease, genital herpes, genital warts, aseptic meningitis, myocarditis, echovirus infections, Epstein-Barr virus pericarditis, gastroenteritis, hepatitis A infection, hepatitis B infection, hepatitis C infection, HIV infection, human papillomavirus (HPV) infection, Reye's syndrome, Kawasaki syndrome, influenza, bronchitis, viral "walking" pneumonia. pneumonia, acute febrile respiratory disease, acute pharyngoconjunctival fever, epidemic keratoconjunctivitis, herpes simplex virus 1 (HSV-1) infection, herpes simplex virus 2 (HSV-2) infection, herpes zoster, cytomegalic inclusion disease, rabies, progressive multifocal leukoencephalopathy, kuru, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstmann-Sfraussler-Scheinker disease, tropical spastic paraparesis, western equine encephalitis, California encephalitis, St. Louis encephalitis, yellow fever, dengue fever, lymphocytic choriomeningitis, Lassa fever, hemorrhagic fever, Hantavirus pulmonary syndrome, Marburg virus infection, Ebola virus infection, and smallpox;

[0369] In some embodiments, the infectious disease is a viral infection. In some embodiments, the infectious disease is a viral infection, such as HIV infection, hepatitis A infection, hepatitis B infection, hepatitis C infection, Epstein-Barr virus infection, coronavirus infection, such as SARS-COV2 infection (Covid-19) and influenza virus infection (influenza). In some embodiments, the infectious disease is caused by an infectious agent, such as coronavirus, diphtheria, Ebola, influenza (flu), HIV, human papilloma virus (HPV), hepatitis A, hepatitis B, hepatitis C, measles virus, respiratory syncytial virus, rotavirus and herpes virus.

[0370] The methods described herein include administering a therapeutically effective amount of a binding agent to a subject having an infectious disease or a subject having cells infected with an infectious agent. As used herein, the phrases "therapeutically effective amount", "effective amount", "effective amount" or "effective dose" can refer to an amount of any of the binding agents or pharmaceutical compositions described herein that provides a therapeutic benefit in treating an infectious disease, managing an infectious disease, or preventing recurrence of an infectious disease, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of an infectious disease. The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art. In general, a therapeutically effective amount can vary depending on the subject's medical history, age, condition, and sex, as well as the severity and type of medical condition in the subject, and the administration of other pharmacologic active agents.

[0371] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

[0372] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow. Non-human mammals can be, for example, beneficially used as subjects to present animal models of various infectious diseases. Furthermore, the methods described herein can be used to treat domestic animals and / or pets. The subject can be male or female. In certain embodiments, the subject is a human.

[0373] The subject may be a subject who has been previously diagnosed with or previously identified as suffering from an infectious disease and who needs treatment, but does not need to have already been treated for the infectious disease. Alternatively, the subject may also be a subject who has not previously been diagnosed with an infectious disease but who needs treatment. The subject may be a subject who shows one or more risk factors for a condition or one or more complications associated with an infectious disease, or a subject who does not show such risk factors. A "subject in need" of treatment for an infectious disease may be a subject who has the infectious disease or a subject who has been diagnosed with the infectious disease. In other embodiments, a subject who is "at risk of developing" an infectious disease refers to a subject who has been diagnosed as being at risk of developing an infectious disease.

[0374] As used herein, the terms "treat", "treatment", "treating" or "alleviation", when used in reference to a disease, disorder or medical condition (e.g., an infectious disease), refer to a therapeutic treatment for the condition, the purpose of which is to reverse, alleviate, relieve, inhibit, prevent, slow down or stop the progression or severity of the symptoms or condition. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of the condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in infected cells in a subject, a reduction in one or more symptoms, a reduction in the degree of the defect, a stabilized (i.e., not worsening) state of the infection, or a delay or slowing of the infection or the progression of the infection, compared to that expected in the absence of treatment. As used herein, the term "administering" refers to contacting (e.g., by administration to a subject) a binding agent described herein or a nucleic acid encoding a binding agent described herein by a method or route that results in binding of the binding agent to CD8+KIR+Treg. Similarly, the pharmaceutical compositions disclosed herein that include a binding agent described herein or a nucleic acid encoding a binding agent described herein can be administered by any suitable route that results in an effective treatment in a subject.

[0375] Dosage ranges for binding agents depend on efficacy and include amounts large enough to produce the desired effect, e.g., stimulating an immune response against infected cells, reducing the number of infected cells, or slowing or preventing the progression of an infectious disease. Dosages should not be so large as to cause unacceptable adverse side effects. In general, dosages vary with the age, condition, and sex of the subject and can be determined by one of skill in the art. Dosages can also be adjusted by the individual physician in the event of any complications. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosages range from about 0.1 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosages range from 0.5 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosage ranges from about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be titrated to maintain serum levels between 1 ug / mL and 1000 ug / mL.

[0376] In some embodiments, the subject receives a single dose of any of the binding agents or pharmaceutical compositions described herein, for example, for the treatment of acute infection. In some embodiments, the subject receives a single dose of any of the binding agents or pharmaceutical compositions described herein, for example, for the prevention of immune responses to infections that may result in autoimmune diseases or conditions. In some embodiments, the subject receives repeated doses of any of the binding agents or pharmaceutical compositions described herein, for example, for the treatment of chronic infection. In some embodiments, the dose is administered weekly, every two weeks, every three weeks, monthly, every two months, or every six months for several weeks, months, or years. The duration of treatment depends on the subject's clinical progression and responsiveness to treatment.

[0377] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.

[0378] In some embodiments, about 2 to about 10 total doses are administered to the subject. In some embodiments, 4 total doses are administered. In some embodiments, 5 total doses are administered. In some embodiments, 6 total doses are administered. In some embodiments, 7 total doses are administered. In some embodiments, 8 total doses are administered. In some embodiments, 9 total doses are administered. In some embodiments, 10 total doses are administered. In some embodiments, more than 10 total doses are administered.

[0379] The pharmaceutical composition containing the binder can be administered in unit doses. The term "unit dose", when used in reference to pharmaceutical compositions, refers to a physically discrete unit suitable as a unitary dosage for a subject, each unit containing a predetermined amount of active material (e.g., binder) calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0380] In some embodiments, administration of any of the binding agents described herein may result in improved treatment outcomes, for example, alleviation of clinical symptoms, or reduction in viral load, or elimination or reduction of pathogens or reduction in infected cells.

[0381] In some embodiments, the binding agent or pharmaceutical composition of any of the embodiments described herein is administered with an infectious disease control agent, for example, an antibacterial, antifungal, or antiviral agent.Antibacterial gents include, for example, lactam antibiotics, such as penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azlocillin, carbenicillin, mezlocillin, piperacillin or ticarcillin; aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin or tobramycin; macrolides, such as azithromycin, clarithromycin, erythromycin, lincomycin or clindamycin; tetracyclines, such as demeclocycline, doxycycline, minocycline, oxytetracycline or tetracycline; quinolones, such as cinoxacin or nalidixic acid; fluoroquinolones, such as ciprofloxacin, enoxacin, grepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin; polypeptides, such as bacitracin, colistin, sulfonamides, such as sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethizole, or sulfacetamide; and other antibacterial agents, such as trimethoprim, sulfamethazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampin, spectinomycin, or nitrofurantoin; and antiviral agents, such as general antivirals, such as idoxuridine, rifampin, rifampin, spectinomycin, or nitrofurantoin. antisense oligonucleotides; immunoglobulins; interferons; and other drugs such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine, saquinavir, oseltamivir and peramivir, ritonavir, indinavir or nelfinavir.

[0382] Cancer Treatment In some aspects, the binding agent or pharmaceutical composition described herein may be used in a method for the treatment of cancer, comprising administering the binding agent or pharmaceutical composition described herein to a subject in need thereof. In some embodiments, a method of treating cancer is provided, comprising administering any of the binding agents described herein or any of the pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby alleviating symptoms of cancer, wherein the binding agent has substantially no effector function activity. In some embodiments, a method of stimulating an immune response against an antigen associated with cancer (cancer antigen; e.g., an antigen expressed on cancer cells), comprising contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents described herein or any of the pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), wherein the binding agent has substantially no effector function activity, thereby increasing the immune response against the cancer antigen.

[0383] In some embodiments, the binding agent is selected from any of the binding agents described herein, in each case with reduced or substantially no effector function activity. In some embodiments, reduced effector function activity is reduced ADCC, ADCP or CDC effector function activity, or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or region, has reduced or substantially no effector function. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region. In some embodiments, the binding agent has an Fc domain or region with reduced or substantially no effector function due to amino acid substitution in the Fc domain or region, for example, Fc null substitution. In some embodiments, the binding agent lacks an Fc domain or region, or has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent lacks an Fc domain or region.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors, or is an Fc null domain.In some embodiments, the binding agent has an Fc domain or region that has reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0384] In some embodiments, a method of treating cancer is provided, comprising administering to a subject in need thereof any of the binding agents described herein or any of the pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Tregs, thereby alleviating symptoms of the cancer, wherein the binding agent has effector function activity including at least ADCC. In some embodiments, a method of stimulating an immune response against cancer is provided, comprising contacting CD8+KIR+T regulatory cells (Tregs) with any of the binding agents described herein or any of the pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Tregs, wherein the binding agent has effector function activity including at least ADCC, thereby increasing the immune response against the cancer. In some embodiments, a method of stimulating an immune response to an antigen associated with cancer (cancer antigen) is provided, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with any of the binding agents described herein or any of the pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC, thereby increasing the immune response to the cancer antigen.

[0385] In some embodiments, the binding agent is selected from any of the binding agents described herein, each having effector function activity including at least ADCC.In some embodiments, the effector function activity is ADCC combined with ADCP and / or CDC effector function activity.In some embodiments, having effector function activity means having ADCC, ADCP and CDC effector function activity.In various embodiments, such binding agent has Fc domain or has Fc domain that binds to one or more Fc gamma receptors.

[0386] In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent in vivo. In some embodiments, the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. In some embodiments, the activated CD8+KIR+ Tregs are administered to a subject in need thereof in an effective amount.

[0387] Without intending to be bound by any particular theory, it is believed that the reduced or absent effector function activity by the binder may limit the interaction of the binder with other cell types (i.e., non-CD8+KIR+Tregs) and / or limit the depletion of CD8+KIR+Tregs. In contrast, the presence of effector function activity by the binder is believed to bias the immune response toward the depletion of CD8+KIR+Tregs.

[0388] In some embodiments, the increase in immune response comprises the reduction of cancer cells or the depletion of immunosuppressive immune cells.In this context, immunosuppressive immune cells refer to, for example, tumor-associated macrophages, CD4+Tregs and / or tolerant dendritic cells (DCs).In some embodiments, the number of cancer cells in a subject is reduced.In some embodiments, the number of immunosuppressive immune cells in a subject is reduced.

[0389] The terms "cancer" and "malignant disease" refer to the uncontrolled growth of cells that interfere with the normal function of bodily organs and systems. Cancer or malignant disease can be primary or metastatic, i.e., the cancer or malignant disease becomes invasive and causes tumor growth in tissues distant from the original tumor site. "Tumor" refers to the uncontrolled growth of cells that interfere with the normal function of bodily organs and systems. As used herein, the term cancer includes malignant diseases and tumors, unless otherwise indicated by the context. A subject with cancer is one who has objectively measurable cancer cells present in the subject's body. Benign and malignant tumors, as well as potentially dormant tumors and micrometastases, are included in this definition. Cancer that migrates from its original location and disseminates to other vital organs can ultimately result in the death of the subject through the functional deterioration of the affected organ. Hematological malignancies (hematopoietic cancers), such as leukemia and lymphoma, can, for example, overwhelm the normal hematopoietic compartment in a subject, thereby resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) that ultimately causes death.

[0390] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia. More specific examples of such cancer include, but are not limited to, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer (e.g., triple-negative breast cancer), peritoneal cancer, cervical cancer; bile duct cancer, choriocarcinoma, chondrosarcoma, colon and rectal cancer (colorectal cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer and stomach cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney or renal cancer. (e.g., clear cell renal cancer or non-clear cell renal cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), lymphoma including Hodgkin's and non-Hodgkin's lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, respiratory system cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, uterine cancer, serious carcinoma), cancer of the urinary system, cancer of the vulva; as well as other carcinomas and sarcomas, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small noncleaved cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with nevus syndrome, edema (e.g., associated with brain tumors) and Meigs syndrome; and myelomas, e.g., multiple myeloma.

[0391] In some embodiments, the cancer is selected from solid tumors, including but not limited to hepatocellular carcinoma, lung cancer, e.g., small cell lung cancer and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and bladder cancer.

[0392] The method described herein comprises administering a therapeutically effective amount of the binding agent or pharmaceutical composition described herein to a subject having cancer or malignant disease. As used herein, the phrase "therapeutically effective amount", "effective amount", "effective amount", "effective amount", or "effective dose" can refer to an amount of the binding agent or pharmaceutical composition described herein that provides a therapeutic benefit in treating cancer or malignant disease, managing cancer or malignant disease, or preventing recurrence of cancer or malignant disease, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of cancer, tumor, or malignant disease. The determination of a therapeutically effective amount is well within the capabilities of a person skilled in the art. In general, a therapeutically effective amount can vary depending on the subject's medical history, age, condition, and sex, as well as the severity and type of medical condition in the subject, and the administration of other pharmacologic active agents.

[0393] In some embodiments, the methods described herein reduce tumor size or tumor burden in a subject and / or reduce metastasis in a subject. In various embodiments, the tumor size in a subject is reduced by about 25-50%, about 40-70%, or about 50-90% or more. In various embodiments, the methods reduce tumor size by 10%, 20%, 30% or more. In various embodiments, the methods reduce tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0394] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments, the subject is a mammal, such as a primate, such as a human. The terms "patient," "individual," and "subject" are used interchangeably herein.

[0395] Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse or a cow. Non-human mammals can be, for example, beneficially used as subjects to present animal models of various cancers. Furthermore, the methods described herein can be used to treat domestic animals and / or pets. The subject can be male or female. In certain embodiments, the subject is a human.

[0396] The subject may be a subject who has been previously diagnosed with cancer or previously identified as suffering from cancer, and who needs treatment, but does not need to have already been treated for cancer. Alternatively, the subject may also be a subject who has not been previously diagnosed with cancer, who needs treatment. The subject may be a subject who shows one or more risk factors for a condition or one or more complications related to cancer, or a subject who does not show risk factors. A "subject in need" of treatment for a particular cancer may be a subject who has the condition or a subject who has been diagnosed with the condition. In other embodiments, a subject who is "at risk of developing" a condition refers to a subject who has been diagnosed as being at risk for developing the condition.

[0397] As used herein, the terms "treat", "treatment", "treating" or "palliation", when used in reference to a disease, disorder or medical condition (e.g., cancer), refer to therapeutic treatment for a condition, the purpose of which is to reverse, alleviate, relieve, inhibit, slow down or stop the progression or severity of a symptom or condition. The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a condition. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of a condition is reduced or halted. That is, "treatment" includes not only improvement of symptoms or markers, but also cessation or at least slowing of progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of cancer cells in a subject, relief of one or more symptoms, attenuation of the degree of the defect, a stabilized (i.e., not worsening) state of cancer or malignant disease, delay or slowing of tumor growth and / or metastasis, and increased life span, compared to that expected in the absence of treatment. As used herein, the term "administering" refers to contacting (e.g., by administration to a subject) a binding agent or pharmaceutical composition described herein or a nucleic acid encoding a binding agent described herein by a method or route that results in binding of the binding agent to CD8+KIR+Treg. Similarly, a pharmaceutical composition comprising a binding agent described herein or a nucleic acid encoding a binding agent described herein disclosed herein can be administered by any suitable route that results in an effective treatment in a subject.

[0398] Dosage ranges for binding agents depend on efficacy and include amounts large enough to produce the desired effect, e.g., slowing of tumor growth or reduction in tumor size. Dosages should not be so large as to cause unacceptable adverse side effects. In general, dosages vary with the age, condition, and sex of the subject and can be determined by one of skill in the art. Dosages can also be adjusted by the individual physician in the event of any complications. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, dosages range from about 0.01 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosages range from about 0.1 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosages range from 0.5 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, dosage ranges from about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be titrated to maintain serum levels between 1 ug / mL and 1000 ug / mL.

[0399] The administration of the doses cited above can be repeated.In a preferred embodiment, the doses cited above are administered every week, every two weeks, every three weeks or every month for several weeks or months.The duration of treatment depends on the clinical progression of the subject and its responsiveness to treatment.

[0400] In some embodiments, the dose may be administered intravenously. In some embodiments, the intravenous administration may be an infusion administered over a period of about 10 minutes to about 4 hours. In some embodiments, the intravenous administration may be an infusion administered over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.

[0401] The pharmaceutical composition containing the binder can be administered in unit doses. The term "unit dose", when used in reference to pharmaceutical compositions, refers to a physically discrete unit suitable as a unitary dosage for a subject, each unit containing a predetermined amount of active material (e.g., binder) calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier or vehicle.

[0402] In some embodiments, administration of any of the binding agents described herein may result in an improved treatment outcome, such as an objective response selected from stable disease, partial response, or complete response, as determined by standard medical criteria for the cancer being treated. In some embodiments, the improved treatment outcome is reduced tumor burden. In some embodiments, the improved treatment outcome is progression-free survival or disease-free survival.

[0403] In some embodiments, the binding agent or pharmaceutical composition of any of the embodiments described herein is administered together with immunotherapy or chemotherapy.As used herein, "immunotherapy" refers to a therapeutic strategy designed to induce or strengthen the subject's own immune system to fight cancer or malignant disease.Examples of immunotherapy include, but are not limited to, antibodies, such as checkpoint inhibitors. In some embodiments, chemotherapy may include, for example, alkylating agents, such as nitrogen mustards, such as cyclophosphamide, ifosfamide, trofosfamide, or chlorambucil; nitrosoureas, such as carmustine (BCNU) and lomustine (CCNU); alkylsulfonates, such as busulfan and treosulfan; triazenes, such as dacarbazine; platinum-containing compounds, such as cisplatin and carboplatin; plant alkaloids, such as vinca alkaloids, such as vincristine, vinblastine, vindesine, and vinorelbine; taxoids, such as paclitaxel and docetaxol; DNA topoisomerase inhibitors; epipodophyllins, such as for example, etoposide, teniposide, topotecan, 9-aminocamptothecin, camptothecin, exatecan and crisnatol; mitomycins, for example mitomycin C; antimetabolites, for example, antifolates, for example, DHFR inhibitors, for example, methotrexate and trimetrexate; IMP dehydrogenase inhibitors, for example, mycophenolic acid, tiazofurin, ribavirin and EICAR; ribonuclotide reductase inhibitors, for example, hydroxyurea and deferoxamine; and pyrimidine analogs, for example, uracil analogs, for example, 5-fluorouracil, floxuridine, doxifluridine and raltitrexed; cytosine analogs, for example, cytarabine (arabinose), C), cytosine arabinoside and fludarabine; purine analogues, such as mercaptopurine and thioguanine; hormonal therapy, such as receptor antagonists, such as antiestrogens, such as tamoxifen, raloxifene and megestrol;LHRH agonists, such as goserelin and leuprolide acetate; antiandrogens, such as flutamide and bicalutamide; retinoids / deltoids; vitamin D3 analogues, such as EB 1089, CB 1093 and KH 1060; photodynamic therapy, such as verteporfm (BPD-MA), phthalocyanines, photosensitizer Pc4, demethoxy-hypocrellin A (DHA), phthalocyanines, ... A) and (2BA-2-DMHA); cytokines such as interferon-alpha and interferon-gamma; tumor necrosis factors; and others such as isoprenylation inhibitors such as lovastatin; dopaminergic neurotoxins such as 1-methyl-4-phenylpyridinium ion; cell cycle inhibitors such as staurosporine; actinomycins such as actinomycin D and dactinomycin; bleomycins such as bleomycin A2, bleomycin B2 and peplomycin; anthracyclines such as daunorabicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorabicin and mitoxantrone; MDR inhibitors such as verapamil and Ca2+ATPase inhibitors such as thapsigargin;

[0404] Exemplary embodiments This invention is further illustrated by the following embodiments, which should not be construed as limiting. Embodiment 1. A first binding domain that specifically binds to a first antigen, wherein said first antigen is selected from an antigen expressed on CD8+KIR+ T regulatory cells (Tregs), other than a KIR protein; and A second binding domain that specifically binds to an inhibitory KIR protein wherein the binding agent binds to CD8+KIR+Treg. Embodiment 2. The binding agent of the preceding embodiment, wherein said first antigen is selected from the group consisting of CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. Embodiment 3. The binding agent of the preceding embodiment, wherein said first antigen is selected from the group consisting of CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4 and 41BB. Embodiment 4. The first antigen is selected from the following group of antigens: a.CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR; b.LAG-3 / CD223, TIM-3, PD-1, S1000A8 / 9 and TLT2; c.CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4 and 41BB; d.CD103(ITGAE), CD166, CD177, CXCR3 and S1000A8 / 9; e. CCR7, CXCR3 and CXCR5; f.PD-1, ICOS and CXCR3; g. CD3, CD5 and CD8; and h.CD3 and CD8 5. The binding domain of any of the preceding embodiments, selected from: Embodiment 5. The first antigen is selected from the following group of antigens: a.CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR; b.LAG-3 / CD223, TIM-3, PD-1, S1000A8 / 9 and TLT2; c.CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103(ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4 and 41BB; d.CD103(ITGAE), CD166, CD177, CXCR3 and S1000A8 / 9; e. CCR7, CXCR3 and CXCR5; and f. CD3 and CD8 5. The binding domain of any of the preceding embodiments, selected from: Embodiment 6. The binding agent of any of the preceding embodiments, which is a bispecific antibody, a diabody, an antibody-Fc fusion, scFv1-ScFv2, scFv12-Fc-scFv22, IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv2-Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1)2-Fc-(VHH)2, BiTe, DART, CrossMab, scFv-Fc, one-arm tandem scFv-Fc, DART-Fc, anticalin, affibody, avimer, DARPin or adnectin. Embodiment 7. The binding agent of any of the preceding embodiments, wherein either the first or second binding domain is selected from an antibody or antigen-binding portion thereof, and the other binding domain is an antibody fragment. Embodiment 8. The binding agent of embodiment 7, wherein the antigen-binding portion is a Fab, Fab', F(ab')2, Fv, scFv or a single domain antibody, such as a VHH, VNAR, sdAb or nanobody. Embodiment 9. The binding agent of any of the preceding embodiments, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region. Embodiment 10. The binding agent of any of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region and a light chain variable region. Embodiment 11. The binding agent of any of the preceding embodiments, wherein said first binding domain specifically binds to CD3 or a subunit of CD3, optionally CD3 epsilon. Embodiment 12. The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the amino acid sequences of the VH and VL are: a. SEQ ID NO:1 and SEQ ID NO:2, respectively; b. SEQ ID NO:9 and SEQ ID NO:10, respectively; c. SEQ ID NO:17 and SEQ ID NO:18, respectively; d. SEQ ID NO:25 and SEQ ID NO:26, respectively; e. SEQ ID NO:33 and SEQ ID NO:34, respectively; f. SEQ ID NO:41 and SEQ ID NO:34, respectively; g. SEQ ID NO:45 and SEQ ID NO:34, respectively; h. SEQ ID NO:49 and SEQ ID NO:50, respectively; i. SEQ ID NO:57 and SEQ ID NO:58, respectively; j. SEQ ID NO:65 and SEQ ID NO:66, respectively; and k. SEQ ID NO:65 and SEQ ID NO:166, respectively 12. The binding agent of embodiment 11, wherein the binding agent is selected from the pair of amino acid sequences shown in the group consisting of: Embodiment 13. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO:1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO:2. Embodiment 14. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 10. Embodiment 15. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 18. Embodiment 16. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 25 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 26. Embodiment 17. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 33 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 34. Embodiment 18. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 41 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 34. Embodiment 19. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 45 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 34. Embodiment 20. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 49 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 50. Embodiment 21. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 57 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 58. Embodiment 22. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 66. Embodiment 23. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 65 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 166. Embodiment 24. The first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions respectively comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDRs are a. SEQ ID NO:3 to SEQ ID NO:8, respectively; b. SEQ ID NO:11 to SEQ ID NO:16, respectively; c. SEQ ID NO:19 to SEQ ID NO:24, respectively; d. SEQ ID NO:27 to SEQ ID NO:32, respectively; e. SEQ ID NO:35 to SEQ ID NO:40, respectively; f. SEQ ID NO:42 to SEQ ID NO:44 and SEQ ID NO:38 to SEQ ID NO:40, respectively; g. SEQ ID NO:46 to SEQ ID NO:48 and SEQ ID NO:38 to SEQ ID NO:40, respectively; h. SEQ ID NO:51 to SEQ ID NO:56, respectively; i. SEQ ID NO:59 to SEQ ID NO:64, respectively; j. SEQ ID NO: 67 to SEQ ID NO: 72, respectively; and k. SEQ ID NOs: 67-69 and 167-169, respectively 12. The binding agent of embodiment 11, having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: Embodiment 25. The binding agent of any of embodiments 1 to 10, wherein the first binding domain specifically binds to CD8 or a subunit of CD8, optionally CD8 alpha. Embodiment 26. The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions are: a. SEQ ID NO:73 and SEQ ID NO:74, respectively; and b. SEQ ID NO:81 and SEQ ID NO:82, respectively having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: or said first binding domain comprises a VHH chain, said VHH chain comprising: c. SEQ ID NO:89; d. SEQ ID NO:93; and e. SEQ ID NO:97 26. The binding agent of embodiment 25, having an amino acid sequence selected from the amino acid sequences set forth in the group consisting of: Embodiment 27. The binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 73 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 74. Embodiment 28. The binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 81 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 82. Embodiment 29. The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence set forth in SEQ ID NO:89. Embodiment 30. The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence set forth in SEQ ID NO: 93. Embodiment 31 The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence set forth in SEQ ID NO:97. Embodiment 32. The first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions respectively comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDRs are a. SEQ ID NO: 75 to SEQ ID NO: 80, respectively; or b. SEQ ID NO: 83 to SEQ ID NO: 88, respectively having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, and the amino acid sequence of the VHH CDRs is c. SEQ ID NO: 90 to SEQ ID NO: 92, respectively; d. SEQ ID NO:94 to SEQ ID NO:96, respectively; and e. SEQ ID NO: 98 to SEQ ID NO: 100, respectively 26. The binding agent of embodiment 25, selected from the set of amino acid sequences set forth in the group consisting of: Embodiment 33. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds to ICOS. Embodiment 34. The binding agent of embodiment 33, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 170 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 171. Embodiment 35. The binding agent of embodiment 33, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising the hCDR1, hCDR1 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 172, 173 and 174, respectively, and the lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 175, 176 and 177, respectively. Embodiment 36 The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds to PD-1. Embodiment 37. The binding agent of embodiment 36, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 178 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 179. Embodiment 38. The binding agent of embodiment 36, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, and the heavy and light chain variable regions comprise the hCDR1, hCDR1 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 180, 181 and 182, respectively, and the lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 183, 184 and 185, respectively. Embodiment 39. The binding agent of any of embodiments 1 to 10, wherein the first binding domain specifically binds to CXCR3. Embodiment 40. The binding agent of embodiment 39, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 186 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 187. Embodiment 41. The binding agent of embodiment 39, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, and the heavy and light chain variable regions comprise the hCDR1, hCDR1 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 188, 189 and 190, respectively, and the lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 191, 192 and 193, respectively. Embodiment 42 The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds to CD5. Embodiment 43. The binding agent of embodiment 42, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 194 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO: 195. Embodiment 44. The binding agent of embodiment 42, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising the hCDR1, hCDR1 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 196, 197 and 198, respectively, and the lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 199, 200 and 201, respectively. Embodiment 45. The binding agent of any of the preceding embodiments, wherein said inhibitory KIR proteins are selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 and KIR2DL3, or a combination thereof. Embodiment 46 The binding agent of embodiment 45, wherein said second binding domain specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2. Embodiment 47. The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL regions are: a. SEQ ID NO:101 and SEQ ID NO:102, respectively; b. SEQ ID NO:109 and SEQ ID NO:110, respectively; c. SEQ ID NO:117 and SEQ ID NO:118, respectively; d. SEQ ID NO:125 and SEQ ID NO:126, respectively; e. SEQ ID NO:133 and SEQ ID NO:134, respectively; f. SEQ ID NO:141 and SEQ ID NO:142, respectively; g. SEQ ID NO:149 and SEQ ID NO:150, respectively; and h. SEQ ID NO:157 and SEQ ID NO:158, respectively 11. The binding agent of any of the preceding embodiments, having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: Embodiment 48. The binding agent of any of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 101 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 102. Embodiment 49. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 109 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 110. Embodiment 50. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 117 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 118. Embodiment 51. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 125 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 126. Embodiment 52. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 133 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 134. Embodiment 53. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 141 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 142. Embodiment 54. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 149 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 150. Embodiment 55. The binding agent of any of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID NO: 157 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID NO: 158. Embodiment 56. The second binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, and the CDRs are a. SEQ ID NO:103 to SEQ ID NO:108, respectively; b. SEQ ID NO:111 to SEQ ID NO:116, respectively; c. SEQ ID NO:119 to SEQ ID NO:124, respectively; d. SEQ ID NO:127 to SEQ ID NO:132, respectively; e. SEQ ID NO:135 to SEQ ID NO:140, respectively; f. SEQ ID NO:143 to SEQ ID NO:148, respectively; g. SEQ ID NO: 151 to SEQ ID NO: 156, respectively; and h. SEQ ID NO:159 and SEQ ID NO:164, respectively 48. The binding agent of any one of claims 1 to 47, having an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of CDRs having an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of: Embodiment 57 A binding agent according to any of the preceding embodiments, which does not contain an Fc domain. Embodiment 58. A binding agent according to any preceding embodiment, further comprising an Fc domain. Embodiment 59. The binding agent of embodiment 58, wherein the Fc domain is selected from an IgG1 and an IgG4 Fc domain. Embodiment 60. The binding agent of embodiment 59, which is substantially devoid of effector function activity. Embodiment 61 The binding agent of any of embodiments 58 to 60, wherein the Fc domain is an IgG1 Fc domain. Embodiment 62. The binding agent of any of embodiments 58 to 61, wherein the Fc domain is IgG1 Fc null. Embodiment 63. The binding agent of any of the preceding embodiments, which is bivalent or tetravalent. Embodiment 64. A binding agent according to any of the preceding embodiments which is bispecific. Embodiment 65. A pharmaceutical composition comprising a binding agent according to any one of embodiments 1 to 64 and a pharma- ceutically acceptable carrier. Embodiment 66. A nucleic acid encoding a binding agent according to any one of embodiments 1 to 64. Embodiment 67. A vector comprising the nucleic acid of embodiment 66. Embodiment 68. A cell line comprising the vector of embodiment 67. Embodiment 69. A method of treating an autoimmune disease, comprising administering to a subject in need thereof a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to reduce the number or activity of pathogenic immune cells in the subject, thereby alleviating a symptom of the autoimmune disease. Embodiment 70. A method for suppressing an immune response mediated by pathogenic immune cells, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), thereby reducing the number or activity of pathogenic immune cells. Embodiment 71. A method for suppressing an immune response to an antigen, such as an autoantigen, comprising administering to a subject in need thereof a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby reducing the number or activity of pathogenic immune cells responsive to said antigen or autoantigen. Embodiment 72. The method of embodiment 70, wherein the CD8+KIR+ Treg is contacted with the binding agent in vivo. Embodiment 73 The method of embodiment 70, wherein the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. Embodiment 74 The method of embodiment 73, wherein the activated CD8+KIR+ Tregs are administered in an effective amount to a subject in need thereof. Embodiment 75. The method of any of embodiments 69 to 74, wherein the pathogenic immune cells are autoreactive CD4 T cells, autoantibody-producing B cells, or autoantigen-presenting dendritic cells. Embodiment 76 The method of any one of embodiments 69 to 74, wherein the pathogenic immune cells are autologous antigen-presenting cells. Embodiment 77 The method of embodiment 71, wherein titers of autoantibodies are reduced in the subject. Embodiment 78. The method of any of embodiments 69 and 74 to 77, wherein the subject has an autoimmune disease selected from the group consisting of celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE) and ulcerative colitis. Embodiment 79. The method of embodiment 78, wherein the autoimmune disease is selected from the group consisting of celiac disease, Crohn's disease, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, multiple sclerosis (MS), rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE) and ulcerative colitis. Embodiment 80. The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to CD8 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 81 The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to CD3 and the inhibitory KIR protein on CD8+KIR+ Tregs. Embodiment 82. The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to CD5 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 83. The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to PD-1 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 84. The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to ICOS on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 85. The method of any of embodiments 69 to 79, wherein the binding agent specifically binds to CXCR3 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 86 The method of any one of embodiments 69 to 85, wherein the CD8+KIR+ Tregs are MHC class I restricted. Embodiment 87. The method of any one of embodiments 69 to 86, wherein the CD8+KIR+ Tregs are not MHC HLA E(Qa-1b) restricted. Embodiment 88 The method of any of embodiments 69 to 87, further comprising administering to the subject an immunosuppressant. Embodiment 89 The method of any of embodiments 69 to 88, wherein said administering to said subject of said binding agent results in an improved treatment outcome in said subject. Embodiment 90. The method of embodiment 89, wherein the improved treatment outcome is a reduction in the frequency or severity of disease flares, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with the autoimmune disease. Embodiment 91 The method of any one of embodiments 69 to 90, wherein the binding agent is administered intravenously. Embodiment 92 The method of any one of embodiments 69 to 91, wherein the binding agent is administered subcutaneously. Embodiment 93. The method of any one of embodiments 69 to 92, wherein the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 94 The method of any one of embodiments 69 to 93, wherein the binding agent has substantially no effector function activity. Embodiment 95. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of an autoimmune disease in a subject by activating or stimulating CD8+KIR+ Tregs. Embodiment 96. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing an immune response by pathogenic immune cells by activating or stimulating CD8+KIR+Tregs. Embodiment 97. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing autoantibody titers in a subject by activating or stimulating CD8+KIR+ Tregs. Embodiment 98. A method of treating cancer, comprising administering to a subject in need thereof a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby alleviating a symptom of said cancer, wherein said binding agent is substantially devoid of effector function activity. Embodiment 99. A method for stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting CD8+KIR+ T regulatory cells (Tregs) with a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), wherein the binding agent is substantially devoid of effector function activity, thereby increasing the immune response against the cancer antigen. Embodiment 100. A method of treating cancer, comprising administering to a subject in need thereof a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 in an amount effective to deplete CD8+KIR+ Tregs, thereby alleviating a symptom of said cancer, wherein said binding agent has effector function activity including at least ADCC. Embodiment 101. A method for stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting CD8+KIR+ T regulatory cells (Tregs) with a binding agent having effector function activity including at least ADCC according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to deplete CD8+KIR+ Tregs, thereby increasing said immune response against said cancer antigen. Embodiment 102. The method of embodiment 99 or embodiment 101, wherein the CD8+KIR+ Treg is contacted with the binding agent in vivo. Embodiment 103. The method of embodiment 99, wherein the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. Embodiment 104. The method of embodiment 103, wherein the activated CD8+KIR+ Tregs are administered in an effective amount to a subject in need thereof. Embodiment 105. The method of any of embodiments 99 and 101 to 104, wherein the increased immune response comprises a reduction in cancer cells or a depletion of immunosuppressive immune cells. Embodiment 106 The method of any of embodiments 98 to 105, wherein the cancer cells in the subject are reduced. Embodiment 107. The method of any of embodiments 98 to 106, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, myeloma and leukemia. Embodiment 108. The method of any of embodiments 98 to 107, wherein the cancer is selected from the group consisting of solid tumors, such as breast, cervical, ovarian, lung, CRC (and other cancers of the intestine), skin, esophageal, adenocarcinoma, bladder and prostate; and lymphoma. Embodiment 109. The method of any of embodiments 98 to 108, wherein the binding agent specifically binds to CD8 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 110. The method of any of embodiments 98 to 108, wherein said binding agent specifically binds to CD3 on CD8+KIR+ Tregs and to said inhibitory KIR protein. Embodiment 111. The method of any of embodiments 98 to 108, wherein the binding agent specifically binds to CD5 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 112. The method of any of embodiments 98 to 108, wherein the binding agent specifically binds to PD-1 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 113. The method of any of embodiments 98 to 108, wherein the binding agent specifically binds to ICOS on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 114 The method of any of embodiments 98 to 108, wherein said binding agent specifically binds to CXCR3 on CD8+KIR+ Tregs and to said inhibitory KIR protein. Embodiment 115. The method of any of embodiments 98 to 114, wherein the CD8+KIR+ Tregs are MHC class I restricted. Embodiment 116. The method of any of embodiments 98 to 115, wherein the CD8+KIR+ Tregs are not MHC HLA E(Qa-1b) restricted. Embodiment 117. The method of any one of embodiments 98 to 116, further comprising administering to the subject a chemotherapeutic agent or an immunotherapy, such as a checkpoint inhibitor. Embodiment 118 The method of any of embodiments 98 to 117, wherein said administering to said subject of said binding agent results in an improved treatment outcome in said subject. Embodiment 119. The method of embodiment 118, wherein the improved treatment outcome is a partial response or a complete response. Embodiment 120. The method of embodiment 118, wherein the improved treatment outcome is remission. Embodiment 121 The method of any one of embodiments 98 to 120, wherein the binding agent is administered intravenously. Embodiment 122 The method of any one of embodiments 98 to 120, wherein the binding agent is administered subcutaneously. Embodiment 123. The method of any of embodiments 98 to 122, wherein the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 124. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of cancer in a subject by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent has substantially no effector function activity. Embodiment 125. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing immunosuppression by immunosuppressive immune cells by activating or stimulating CD8+KIR+Tregs, wherein the binding agent has substantially no effector function activity. Embodiment 126. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing tumor burden in a subject by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially devoid of effector function activity. Embodiment 127. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of cancer in a subject by depleting CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC. Embodiment 128. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for depletion of CD8+KIR+ Tregs, wherein said binding agent has effector function activity including at least ADCC. Embodiment 129. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing tumor burden in a subject, wherein the binding agent has an effector function activity including at least ADCC. Embodiment 130. A method of treating an infectious disease, comprising administering to a subject in need thereof a binding agent described in any of embodiments 1 to 64 or a pharmaceutical composition described in embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby alleviating a symptom of the infectious disease. Embodiment 131. A method for stimulating an immune response against infected cells caused by an infectious disease, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), thereby enhancing said immune response against said infected cells. Embodiment 132. The method of embodiment 131, wherein the CD8+KIR+ Treg is contacted with the binding agent in vivo. Embodiment 133. The method of embodiment 131, wherein the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. Embodiment 134. The method of embodiment 133, wherein the activated CD8+KIR+ Tregs are administered in an effective amount to a subject in need thereof. Embodiment 135. The method of any of embodiments 131 to 134, wherein the immune response comprises a reduction in infected cells, or a reduction in immunosuppressive immune cells selected from CD4+ T regulatory cells and tolerant DCs. Embodiment 136 The method of embodiment 135, wherein the number of infected cells in the subject is reduced. Embodiment 137. The method of any of embodiments 130 to 136, wherein the infectious disease is selected from a bacterial disease, a systemic fungal disease, a rickettsial disease, a parasitic disease and a viral disease. Embodiment 138. The method of embodiment 137, wherein the infection is selected from the group consisting of an HIV infection, a Hepatitis C virus (HCV) infection, a human papillomavirus (HPV) infection, an Epstein-Barr virus (EBV) infection, a coronavirus infection, e.g., a SARS-COV2 infection (Covid-19), a cytomegalovirus (CMV) infection and an influenza virus infection. Embodiment 139. The method of any of embodiments 130 to 138, wherein the binding agent specifically binds to CD8 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 140 The method of any of embodiments 130 to 138, wherein said binding agent specifically binds to CD3 and said inhibitory KIR protein on CD8+KIR+ Tregs. Embodiment 141 The method of any of embodiments 130 to 138, wherein said binding agent specifically binds to CD5 on CD8+KIR+ Tregs and to said inhibitory KIR protein. Embodiment 142. The method of any of embodiments 130 to 138, wherein the binding agent specifically binds to PD-1 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 143 The method of any of embodiments 130 to 138, wherein the binding agent specifically binds to ICOS on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 144 The method of any of embodiments 130 to 138, wherein the binding agent specifically binds to CXCR3 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 145. The method of any of embodiments 130 to 144, wherein the CD8+KIR+ Tregs are MHC class I restricted. Embodiment 146. The method of any of embodiments 130 to 145, wherein the CD8+KIR+ Tregs are not MHC HLA E(Qa-1b) restricted. Embodiment 147. The method of any of embodiments 130 to 146, further comprising administering to the subject an antimicrobial or antiviral agent. Embodiment 148 The method of any of embodiments 130 to 147, wherein said administering to said subject of said binding agent results in an improved treatment outcome in said subject. Embodiment 149 The method of embodiment 148, wherein the improved treatment outcome is a reduction in infection or infected cells. Embodiment 150. The method of any one of embodiments 130 to 149, wherein the binding agent is administered intravenously. Embodiment 151 The method of any one of embodiments 130 to 149, wherein the binding agent is administered subcutaneously. Embodiment 152. The method of any one of embodiments 130 to 151, wherein the binding agent is administered at a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 153 The method of any of embodiments 130 to 149, wherein the binding agent has substantially no effector function activity. Embodiment 154. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of an infection in a subject by activating or stimulating CD8+KIR+ Tregs. Embodiment 155. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for stimulating an immune response by activating or stimulating CD8+KIR+ Tregs, thereby suppressing immunosuppressive immune cells. Embodiment 156. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing infections or infected cells in a subject by activating or stimulating CD8+KIR+ Tregs. Embodiment 157. A method for reducing or preventing the onset of graft-versus-host disease (GVHD) after transplantation, comprising administering to a subject in need thereof a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby reducing or alleviating at least one symptom of GVHD, wherein the binding agent is substantially devoid of effector function activity. Embodiment 158. A method of treating a subject who has received a transplant, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with an effective amount of a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 to activate or stimulate CD8+KIR+ Tregs (activated Tregs), wherein the binding agent has substantially no effector function activity, thereby reducing or suppressing GVHD. Embodiment 159. A method of treating a subject who has received a transplant, comprising administering to a subject in need thereof a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in an amount effective to deplete CD8+KIR+ Tregs, thereby alleviating symptoms of GVHD, wherein the binding agent has effector function activity including at least ADCC. Embodiment 160. A method for suppressing GVHD upon transplantation, comprising contacting CD8+KIR+ T regulatory cells (Tregs) with a binding agent of any of embodiments 1 to 64 or a pharmaceutical composition of embodiment 65 in an amount effective to deplete CD8+KIR+ Tregs, thereby reducing GVHD or a symptom thereof, and wherein the binding agent has effector function activity including at least ADCC. Embodiment 161. The method of embodiment 158 ​​or 160, wherein the CD8+KIR+ Treg is contacted with the binding agent in vivo. Embodiment 162. The method of embodiment 158, wherein the CD8+KIR+ Tregs are contacted with the binding agent ex vivo. Embodiment 163. The method of embodiment 162, wherein the activated CD8+KIR+ Tregs are administered in an effective amount to a subject in need thereof. Embodiment 164. The method of any of embodiments 157 and 160 to 163, wherein the reduced or decreased GVHD comprises a reduction in CD4+ T cells active in GVHD. Embodiment 165. The method of any of embodiments 157 to 164, wherein the transplant is selected from the group consisting of organ transplantation, hematopoietic stem cell transplantation, umbilical cord blood stem cell transplantation, transplantation of precursors or differentiated cells derived from induced pluripotent stem cells, and bone marrow transplantation. Embodiment 166. The method of any of embodiments 165, wherein the transplant is a hematopoietic stem cell transplant, an umbilical cord blood stem cell transplant, a transplant of precursors or differentiated cells derived from induced pluripotent stem cells, or a bone marrow transplant. Embodiment 167. The method of any one of embodiments 157 to 166, wherein the transplant is allogeneic. Embodiment 168. The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to CD8 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 169. The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to CD3 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 170. The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to CD5 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 171. The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to PD-1 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 172. The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to ICOS on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 173 The method of any of embodiments 157 to 167, wherein the binding agent specifically binds to CXCR3 on CD8+KIR+ Tregs and to the inhibitory KIR protein. Embodiment 174. The method of any one of embodiments 157 to 173, wherein the CD8+KIR+ Tregs are MHC class I restricted. Embodiment 175. The method of any of embodiments 157 to 174, wherein the CD8+KIR+ Tregs are not MHC HLA E(Qa-1b) restricted. Embodiment 176 The method of any of embodiments 157 to 175, further comprising administering to the subject an immunosuppressant. Embodiment 177. The method of any of embodiments 157 to 176, wherein said administering to said subject of said binding agent results in an improved treatment outcome in said subject. Embodiment 178. The method of embodiment 177, wherein the improved treatment outcome is a reduction in symptoms associated with GVHD, a reduction in systemic inflammatory cytokines, a reduction in lesions in tissues affected by GVHD, a reduction in self-reported symptoms associated with immune responses associated with adverse effects on host tissues, an improvement or prolongation of graft survival, a reduction in one or more symptoms, and / or prevention, delay or slowing of the onset or progression of rejection of the graft, or a prolongation of graft survival due to a reduction in the use of broad-spectrum immunosuppressants, e.g., corticosteroids. Embodiment 179. The method of any one of embodiments 157 to 178, wherein the binding agent is administered intravenously. Embodiment 180. The method of any one of embodiments 157 to 178, wherein the binding agent is administered subcutaneously. Embodiment 181. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of transplant-associated GVHD in a subject, wherein the binding agent is substantially devoid of effector function activity. Embodiment 182. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of transplant-associated GVHD in a subject by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially devoid of effector function activity. Embodiment 183. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing transplant-associated GVHD by activating or stimulating CD8+KIR+ Tregs, wherein the binding agent is substantially devoid of effector function activity. Embodiment 184. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing GVHD upon transplantation, wherein the binding agent is substantially free of effector function activity. Embodiment 185. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the treatment of transplant-associated GVHD in a subject by depleting CD8+KIR+ Tregs, wherein the binding agent has effector function activity including at least ADCC. Embodiment 186. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for depletion of CD8+KIR+ Tregs, wherein said binding agent has effector function activity including at least ADCC. Embodiment 187. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for depletion of CD8+KIR+ Tregs in a subject who has undergone a transplant to reduce GVHD, wherein the binding agent has effector function activity including at least ADCC. Embodiment 188. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in any of the methods described in embodiments 69 to 94, 98 to 123, 130 to 153 and 157 to 180. Embodiment 189. Use of a binding agent according to any of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 in the manufacture of a medicament for use in any of the methods described in embodiments 69 to 94, 98 to 123, 130 to 153 and 157 to 180.

[0405] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but as one skilled in the art will recognize, various equivalent modifications are possible within the scope of the present disclosure. The teachings of the disclosure provided herein may be applied to other procedures or methods as appropriate. The various embodiments described herein may be combined to provide further embodiments. Aspects of the present disclosure may be modified, if necessary, to use the compositions, functions and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes may be made to the present disclosure in light of the detailed description.

[0406] Specific elements of any of the above-described embodiments can be combined or substituted for elements in other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily need to exhibit such advantages in order to fall within the scope of the present disclosure.

[0407] All patents and other publications identified are hereby expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in conjunction with the present invention. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by reason of prior invention or for any other reason. All statements as to the date or representation as to the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents. EXAMPLES

[0408] Example 1 Testing monospecific and bispecific molecules for CD8+KIR+Treg cell activation and cytotoxicity against pathogenic immune cells in autoimmune disorders To test a panel of monospecific and bispecific molecules (including inhibitory KIR blockers) for functional restoration of CD8 KIR+ Treg cell activation and cytotoxicity-mediated elimination of pathogenic immune cells, such as autoreactive CD4 T cells, primary CD8+ KIR+ T cells are incubated with increasing concentrations of CD3 agonist antibodies, which mimic peptide / MHC binding of CD8 KIR+ T cell receptor in the presence of increasing concentrations of inhibitory KIR blocking molecules. Blockade of inhibitory KIR signals may be expected to reduce the activation threshold of TCR engagement required for CD8 KIR+ Treg cell activation resulting in a specific and increased activation status of CD8 KIR+ T cells. Increased activation of CD8+ KIR+ Treg cells is confirmed by increased secretion of certain cytokines (such as IFN gamma, IL-10, TNF alpha, IL-35 or their subunits), increased expression markers associated with activation (such as CD69, CD25, CD62L, CD44, CD45) and increased proliferation.

[0409] A panel of monospecific and bispecific molecules is tested based on their efficacy by inducing CD8 KIR+Treg cell-mediated functional outcomes, followed by testing using CD8 KIR+Treg cells derived from celiac patient peripheral blood mononuclear cells in response to gluten-restimulated CD4 T cells. Increased activation and enhanced activity against pathogenic immune cells by CD8KIR+Treg cells is confirmed by increased secretion of certain cytokines (such as IFN gamma, IL-10, TNF alpha and / or IL-35 or their subunits), increased expression markers (such as CD69, CD25, CD62L, CD44 and / or CD45), decreased inhibitory molecules (LAG-3, TIM-3 and / or PD-1), increased proliferation, increased inhibition of autoreactive CD4+T cells and other pathogenic immune cells, such as autoantibody-producing B cells, autoantigen-presenting dendritic cells and autoAPC.

[0410] Example 2 Testing monospecific and bispecific molecules for CD8+KIR+Treg cell activation and cytotoxicity in infectious disease models A panel of monospecific and bispecific molecules (including inhibitory KIR blockers) is tested for functional restoration of CD8 KIR+ Treg cell activation and direct and indirect cytotoxicity-mediated elimination of pathogen-infected cells. Human CMV-specific T cells (Cellero) are cultured with increasing doses of a dominant epitope (pp65) derived from the virus that binds to the MHC class I molecule HLA-A2. Peptides are loaded onto the HLA-A2-expressing lymphoma cell line T2. For testing of specific inhibitory KIR molecule function, either MHC I-deficient lymphoma T1 cell line or K562 cell line are transfected with the appropriate cognate MHC class I molecule (e.g., HLA-C2 when blocking KIR2DL1). To confirm that KIR blockade specifically and effectively re-establishes elimination of pathogen-infected cells, CD8 Treg cell activation, cytotoxicity, cytokine production and proliferation are tested. The extent of target cell elimination and apoptosis is also determined using Annexin V staining and proliferation. As controls, we use, for example, an irrelevant influenza hemaglutinin peptide (negative control), CD3 antibody bead activation (positive control), or MHC-deficient lymphoma T1 cell line or parental K562 cells (negative control). To detect activation with greater sensitivity, we transfect, for example, Jurkat cells with SHP1 / 2 or NFAT reporters to show enhanced activation upon inhibitory KIR blockade and / or agonist binding to prioritize molecules for use.

[0411] Example 3 Testing monospecific and bispecific molecules for CD8+KIR+Treg cell activation and cytotoxicity in cancer models To determine whether KIR blockade improves CD8 KIR Treg cell activation and target cell killing, CD8 KIR+Treg cells are tested against a panel of tumor cell lines with high antigen load in the presence of a panel of KIR bispecific molecules.KIR bispecific molecules are tested alone or in combination with other KIR bispecific molecules, and in combination with other immune checkpoint inhibitors.As an example, the study tests the dose escalation of anti-CD3 agonist antibodies together with the dose escalation of KIR molecules in co-culture with tumor cell lines including, for example, A549 (NSCLC), H1229 (NSCLC), A375 (melanoma), SK-Mel 3 (melanoma), Caki-1 (RCC) and / or 786-O (RCC). The haplotype dependency of the response is determined, for example, using primary NY-ESO-1 specific T cells (Cellero) versus NY-ESO1 peptide-pulsed T2 (HLA-A2 restricted cell line) and / or HLA-A2 K562. To assess whether a subset of HLA expression by tumor cell targets (e.g., HLA-B binding KIR3DL1 or KIR2DL1 / 2 / 3 binding HLA-C) is required for optimal response, the appropriate HLA molecule is overexpressed in either K562 or T1 cells and pulsed with the appropriate dominant epitope (https: / / antibodies.cancer.gov / detail / MajorHistocompatibilityComplexClassICPeptide1).

[0412] Example 4 Testing monospecific and bispecific molecules for CD8+KIR+Treg cell activation and cytotoxicity in a GVHD transplantation model After hematopoietic stem cell transplantation and other transplantation procedures, serious and life-threatening complications can occur when donor-derived cells recognize allogeneic host tissue as foreign and become activated to destroy the recipient's healthy cells, known as graft-versus-host disease (GVHD). Alloreactive GVHD causes transplant-related morbidity in up to 50% of transplant recipients and accounts for approximately 20% of post-transplant deaths. KIR blockade on CD8+KIR+Tregs can reduce the severity of graft-versus-host disease, where transplanted cells destroy healthy tissue and recognize it as foreign. To test the effect of KIR blockade on GVHD severity, a well-characterized GVHD model is used in which human immune cells are injected into NOD / SCID / gamma chain (NSG)-deficient mice, and subsequent multi-organ acute lesions are observed as a result of human cell activation and destruction of mouse tissue. KIR-blocking monospecific and bispecific molecules were injected every 72 h for the duration of the study, 30-45 days, and endpoint analyses included serum pro-inflammatory cytokines, human T cell activation marker expression, disease scoring (including survival time and body weight), and histopathological analysis of intestinal tissue for inflammation and epithelial cell killing. This study not only supports the utility of KIR blockade as a method to reduce the severity of GVHD while preserving graft survival, but also determines the effect of KIR blockade on a systemic disease that may affect multiple organs and tissues.

[0413] Example 5 Ly49 blockade increases the activity of CD8+Ly49+ T regulatory cells The effect of Ly49 blockade on CD8+Ly49+ Tregs was confirmed in vitro. Briefly, cells were isolated from spleens and lymph nodes of C57BL / 6 mice 10 days after EAE induction using a standard MOG peptide protocol on day 10 (see Saligrama et al., Nature 572:481-487 (2019)). CD4+ T cells, CD8+CD28- regulatory T cells and CD8+CD28+ were isolated using magnetic separation and cultured 1:1 with CD4 T cells stimulated with CD3 / CD28 in the absence of blocking antibodies (control) or in the presence of the F(ab')2 fragment of the blocking antibody LY49 C / l (clone 5e6; lacking the Fc portion of the antibody) (anti-Ly49).

[0414] Referring to Figures 4A-4D, in the presence of Ly49 blockade, there was a statistically significant increase in CD8+ Treg activation (Figure 4A), production of immunosuppressive cytokines (Figure 4B), cytolytic activity (granzyme B) (Figure 4C), and an increase in CD4 T cell production of the anti-inflammatory IL-10 cytokine (Figure 4D). Similar results were observed with cells stimulated with CD3 / CD28 in the presence of full-length Ly49 C / I blocking antibody (clone 5E6) (data not shown). These results confirm that CD8+Ly49+ T cells show increased activation with Ly49 / KIR blockade.

[0415] Supernatants from cells (described above) were collected 48 hours after the initiation of co-culture and analyzed for various analytes (cytokines) using Bioplex assays.

[0416] The results of this analysis showed that Ly49 blockade suppressed the following pro-inflammatory cytokines in samples from mice treated with MOG and suppressor peptide compared to mice treated with MOG peptide alone: ​​RANTES, IL-6, IL-18, GM-CSF, TNF-alpha, and IFN-gamma (data not shown). Furthermore, IL-2 and IL-15 levels were reduced in samples from mice treated with MOG and suppressor peptide compared to mice treated with MOG peptide (data not shown). Levels of anti-inflammatory cytokines IL-22 and MCP-3 were reduced in samples from mice treated with MOG and suppressor peptide compared to mice treated with MOG peptide (data not shown).

[0417] The effect of Ly49 blockade was evaluated in vivo in a mouse EAE model. Briefly, EAE was induced in C57BL / 6 mice using a standard MOG injection protocol (see Saligrama et al., Nature 572:481-487 (2019)). Mice were administered MOG alone or in combination with a "surrogate peptide" cocktail ("SP") or the F(ab')2 fragment of the blocking antibody LY49 C / I (clone 5E6; lacking the Fc portion of the antibody) (anti-Ly49 or "Ly49 blockade") (Figure 5). Ly49 blockade delayed disease onset and attenuated disease severity (Figure 6), suggesting that CD8+ Treg recruitment at the time of the autoimmune trigger contributed to disease control.

[0418] Example 6 CD8 KIR+ T cells have a higher cytolytic potential than CD8 T cells negative for KIR expression in celiac patients Peripheral blood mononuclear cells (PBMCs) were obtained from celiac patients and healthy donors. PBMCs were enriched for CD8+ T cells, then stained for several surface markers, including CD8 and a mix of pan-inhibitory KIR-reactive peptides, and sorted to obtain CD8+KIR+ T cells and CD8+KIR- cells. After sorting, PBMCs were stimulated with gluten peptides. Six days after stimulation, CD8 Treg cells were assessed for intracellular granzyme, perforin, and IFN-gamma levels.

[0419] PBMCs from celiac patients had a higher percentage of CD8+KIR+Treg cells (Figure 7A). KIR+CD8+T cells had a higher percentage of cells with perforin, as well as intracellular IFN gamma and granzyme B, compared to CD8+KIR-T cells (Figures 7B and 7C). These results indicate that celiac patients possess CD8+KIR+T cells with higher cytolytic potential than KIR-negative CD8+T cells.

[0420] Example 7 Celiac patients have more KIR+CD8+ T cells and more ICOS expression on KIR+CD8+ T cells than healthy controls PBMCs from celiac patients (n=6) or healthy donors were analyzed by flow cytometry and gated for CD8+ T cells. PBMCs from celiac patients had more CD3+ / pan-KIR+ T cells than PBMCs from healthy donors (Figure 8A). PBMCs from celiac patients had more CD3+ / pan-KIR+ / ICOS+ cells than PBMCs from healthy donors (Figure 8B). These results indicate that celiac patients have more ICOS expression on KIR+CD8+ T cells.

[0421] Example 8 Gluten restimulation increases granzyme B levels and degranulation of CD8+KIR+ Tregs and loss of CD4+ T cells To determine the effect of gluten restimulation on CD8+KIR+T cells, PMBCs from celiac patients were stimulated with gluten peptides in the presence of IL-7 and 15 for 12 days to enrich for both CD4 reactive cells and CD8+Treg cells. CD8 Treg and CD4 T cells were then selected and combined 1:1 with autologous APCs without peptide pulse or pulsed with influenza or gluten peptides. After 48 hours, cells were analyzed by flow cytometry.

[0422] Restimulation with gluten peptides increased degranulation (Figure 9A, left) and granzyme B levels (Figure 9A, right) measured by CD107 compared to cells restimulated with control influenza peptide or unstimulated (598 refers to PBMCs from patient 598). Restimulation with gluten peptides also caused a reduction in the percentage of viable CD4+ cells, whereas restimulation with influenza peptides did not (Figure 9B). These results indicate that antigen responses by CD8+KIR+Tregs are specific, rapid and sustained, consistent with the source of Tregs from celiac patients. These results indicate that CD8+Treg cells are upregulated, CD4+T cell activation is downregulated, and pathogenic CD4+T cells are eliminated.

[0423] Example 9 KIR blockade increases granzyme B content and degranulation in CD8+ T cells CD8+CD16+ T cells were selected from three patients diagnosed with celiac disease and cultured 1:1 with CD4+ T cells and 1ug / ml anti-CD3 agonist antibody (clone OKT3) in the presence or absence of 100ug / ml KIR2DL1 / 2 / 3 and KIR3DL1 antagonist antibodies (50ug each). After 48 hours, CD8+ Treg cells were analyzed using flow cytometry. KIR blockade ("KIR block") increased intracellular granzyme B levels (Figure 10A) and degranulation (CD107) (Figure 10B).

[0424] The effect of KIR blockade was evaluated in vitro in a separate experiment using PBMCs from celiac patients. Briefly, CD8+ Tregs were enriched from celiac patient PBMCs, cultured with autologous CD4 T cells and antigen-presenting cells pulsed with a gliadin peptide cocktail, and analyzed using flow cytometry. Upon restimulation, cells treated with anti-inhibitory KIR antibodies (anti-KIR2DL1 / 2 / 3, anti-KIR3DL1, or a cocktail of anti-KIR2DL1 / 2 / 3 and KIR3DL1) showed induction of CD8+ T cell cytolytic activity, reduced CD4+ T cell activation, and increased CD4+ T cell death (Figure 11). These results were consistently observed across several patient samples upon monospecific and bispecific KIR blockade.

[0425] Example 10 KIR blockade reduces CD4+ T cell activation CD8+CD16+ T cells were selected from three patients diagnosed with celiac disease and cultured 1:1 with CD4+ T cells and 1ug / ml anti-CD3 agonist antibody (clone OKT3) in the presence or absence of 100ug / ml KIR2DL1 / 2 / 3 and KIR3DL1 antagonist antibodies (50ug each). After 48 hours, CD8+ Treg cells were analyzed using flow cytometry. KIR blockade reduced CD4+ T cell activation and proliferation (CD69) in samples from all three patients (Figure 12).

[0426] Example 11 Association between selected KIR proteins and HLA ligand expression in CD8+ Treg cells in celiac disease Celiac patient PBMCs were stained with antibodies against KIR2DL1 / 2 / 3 and KIR3DL1. After gating on CD8 T cells, the percentage positivity of cells for KIR ligands and HLA haplotypes was determined (HLA and KIR typing was performed in collaboration with Scisco Genetics).

[0427] CD8 T cells from patients expressed KIRs as follows: in peripheral blood, 3 out of 3 expressed KIR2DL and 2 out of 3 expressed KIR3DL. HLA ligands for selected KIRs are over-represented in celiac patient samples. 9 out of 10 patients had at least one copy of HLA-C 07:01:01 and 10 out of 10 patients had at least one copy of HLA-B 08:01:01.

[0428] Example 12 Characterization of a bispecific molecule that co-binds to CD8 and KIR2DL CrossMab was prepared using a Fab that binds KIR2L1 / 2 / 3 (prepared from IPH2102 IgG1r mAb (parent antibody VH and VL sequences, SEQ ID NOs: 101 and 102, respectively)) and a scFv that binds CD8 alpha (prepared from Mb1b IgG1r mAb (parent antibody VH and VL sequences, SEQ ID NOs: 81 and 82)). The Fab and scFv were attached to the hinge-CH2-CH3 of IgG1, where the CH3 domain was engineered to contain "knob-into-hole" mutations that force correct association of the two heterodimeric heavy chains. The "knob" heavy chain contained the mutations S354C and T366W. The "hole" heavy chain contained the mutations Y349C, T366S, L368A and Y407V.

[0429] KIR2L1 / 2 / 3-CD8alpha crossmab was tested for co-binding to KIR2DL1 or KIR2DL3 and CD8alpha by biolayer interferometry using an Octet instrument. For co-binding studies, crossmab was captured on AHC (anti-human Fc) biosensor using 2-fold dilutions ranging from 0.3125ug / ml to 20ug / ml. Analytes (KIR2DL1, KIR2DL3 and CD8alpha) were held constant at 100nM. Analyte co-binding after capture was analyzed in two ways: first, KIR2DL1 or KIR2DL3 association followed immediately by CD8alpha association, or CD8alpha association followed by KIRDL1 or KIR2DL3 direct association. KIR2DL1, KIR2DL3 and CD8alpha were tagged with hexahistidine peptides. Crossmab could co-bind to the targets KIR2DL1 or KIR2DL3 and CD8 alpha.

[0430] The affinity of Crossmab for KIR2DL1, KIR2DL3 and CD8 alpha ligands was measured using an Octet instrument and compared to anti-CD8 alpha and anti-KIR2DL1 / L2 / L3 parent antibodies. For kinetic analysis, Crossmab was captured on an AHC (anti-human Fc) biosensor using a loading concentration of 1.25ug / ml. Each analyte (KIR2DL1, KIR2DL3 and CD8 alpha) concentration ranged from 6.25nM to 200nM. Analyte binding after capture was analyzed first for association of KIR2DL1, KIR2DL3 or CD8 alpha and then independently for dissociation of each analyte. This ensured that ka (on rate), kd (off rate) and KD values ​​could be obtained and directly compared to the parent antibodies. Kinetic analysis revealed that Crossmab retained affinity for targets KIR2DL1, KIR2DL3 and CD8 alpha.

[0431] The affinity of the parental antibodies anti-KIR2DL1 / L2 / L3 IPH2102 IgG1r mAb and anti-CD8 alpha Mb1b IgG1r mAb was also analyzed. For kinetic analysis, the parental antibodies were captured separately on an AHC (anti-human Fc) biosensor using a loading concentration of 1.25ug / ml. For IPH2102 IgG1r mAb, the KIR2DL1 or KIR2DL3 analytes ranged from 6.25nM to 200nM. Analyte binding after capture was analyzed first for KIR2DL1 or KIR2DL3 association and then for dissociation of each analyte independently. Similarly, for Mb1b IgG1r mAb, the CD8 alpha analytes ranged from 6.25nM to 200nM. Analyte binding after capture was analyzed first for CD8 alpha association and then for dissociation. [Table 1]

[0432] Example 13 Analysis of PBMC samples from patients diagnosed with other autoimmune diseases PBMCs from patients with lu...

Claims

1. a first binding domain that specifically binds to a first antigen, the first antigen being selected from an antigen expressed on CD8+KIR+ T regulatory cells (Tregs), other than a KIR protein; and A second binding domain that specifically binds to an inhibitory KIR protein wherein the binding agent binds to CD8+KIR+Treg.

2. 2. The binding agent of claim 1, wherein the first antigen is selected from the group consisting of CD8, ICOS, CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, LAG-3 / CD223, OX-40, PD-1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB.

3. Bispecific antibodies, diabodies, antibody-Fc fusions, scFv1-ScFv2, scFv1 ２ -Fc-scFv2 ２ , IgG-scFv, DVD-Ig, triomab / quadroma, two-in-one IgG, scFv ２ -Fc, TandAb, scFv-HSA-scFv, scFv-VHH, Fab-scFv-Fc, Fab-VHH-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1) ２ -Fc-(VHH) ２ , BiTe, DART, crossmab, scFv-Fc, one-arm tandem scFv-Fc, DART-Fc, anticalin, affibody, avimer, DARPin or adnectin.

4. The binding agent of claim 1, wherein the first and second binding domains each comprise an antibody fragment selected from Fab, Fab', F(ab')2, Fv, scFv and a single domain antibody.

5. 5. The binding agent according to any one of claims 1 to 4, wherein the first binding domain specifically binds to CD8 or a subunit of CD8, optionally CD8 alpha.

6. The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions comprising: a. SEQ ID NO:81 and SEQ ID NO:82, respectively; and b. SEQ ID NO:73 and SEQ ID NO:74, respectively having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of: or the first binding domain comprises a VHH chain, the VHH chain comprising: c. SEQ ID NO:89; d. SEQ ID NO:93; and e. SEQ ID NO:97 The binding agent of claim 5, having an amino acid sequence selected from the amino acid sequences set forth in the group consisting of:

7. The first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, the CDRs being a. SEQ ID NO:83 to SEQ ID NO:88, respectively; and b. SEQ ID NO: 75 to SEQ ID NO: 80, respectively having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of: or said first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, wherein the CDRs of said VHH are c. SEQ ID NO:90 to SEQ ID NO:92, respectively; d. SEQ ID NO:94 to SEQ ID NO:96, respectively; and e. SEQ ID NOs: 98 to 100, respectively 6. The binding agent of claim 5, having an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of:

8. 5. The binding agent of claim 1, wherein the first binding domain specifically binds to ICOS.

9. 9. The binding agent of claim 8, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 170 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

171.

10. The binding agent of claim 8, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 172, 173 and 174, respectively, and lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 175, 176 and 177, respectively.

11. 5. The binding agent according to any one of claims 1 to 4, wherein the first binding domain specifically binds to CD3 or a subunit of CD3, optionally CD3 epsilon.

12. The first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the amino acid sequences of the VH and VL are a. SEQ ID NO:1 and SEQ ID NO:2, respectively; b. SEQ ID NO:9 and SEQ ID NO:10, respectively; c. SEQ ID NO:17 and SEQ ID NO:18, respectively; d. SEQ ID NO:25 and SEQ ID NO:26, respectively; e. SEQ ID NO:33 and SEQ ID NO:34, respectively; f. SEQ ID NO:41 and SEQ ID NO:34, respectively; g. SEQ ID NO:45 and SEQ ID NO:34, respectively; h. SEQ ID NO:49 and SEQ ID NO:50, respectively; i. SEQ ID NO:57 and SEQ ID NO:58, respectively; j. SEQ ID NO:65 and SEQ ID NO:66, respectively; and k. SEQ ID NO:65 and SEQ ID NO:166, respectively The binding agent of claim 11, selected from the pair of amino acid sequences shown in the group consisting of:

13. The first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, the CDRs being a. SEQ ID NO:3 to SEQ ID NO:8, respectively; b. SEQ ID NO:11 to SEQ ID NO:16, respectively; c. SEQ ID NO:19 to SEQ ID NO:24, respectively; d. SEQ ID NO:27 to SEQ ID NO:32, respectively; e. SEQ ID NO:35 to SEQ ID NO:40, respectively; f. SEQ ID NO:42 to SEQ ID NO:44 and SEQ ID NO:38 to SEQ ID NO:40, respectively; g. SEQ ID NO:46 to SEQ ID NO:48 and SEQ ID NO:38 to SEQ ID NO:40, respectively; h. SEQ ID NOs:51 to 56, respectively; i. SEQ ID NO:59 to SEQ ID NO:64, respectively; j. SEQ ID NO:67 to SEQ ID NO:72, respectively; and k. SEQ ID NOs: 67-69 and 167-169, respectively 12. The binding agent of claim 11, having an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of:

14. 5. The binding agent of any one of claims 1 to 4, wherein the first binding domain specifically binds to PD-1.

15. 15. The binding agent of claim 14, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 178 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

179.

16. The binding agent of claim 14, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 180, 181 and 182, respectively, and lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 183, 184 and 185, respectively.

17. The binding agent of claim 1 , wherein the first binding domain specifically binds to CXCR3.

18. 18. The binding agent of claim 17, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 186 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

187.

19. The binding agent of claim 17, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 188, 189 and 190, respectively, and lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 191, 192 and 193, respectively.

20. The binding agent according to claim 1 , wherein the first binding domain specifically binds to CD5.

21. 21. The binding agent of claim 20, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID NO: 194 and a light chain variable region (VL) having the amino acid sequence of SEQ ID NO:

195.

22. The binding agent of claim 20, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions comprising hCDR1, hCDR2 and hCDR3 amino acid sequences set forth in SEQ ID NOs: 196, 197 and 198, respectively, and lCDR1, lCDR2 and lCDR3 amino acid sequences set forth in SEQ ID NOs: 199, 200 and 201, respectively.

23. 23. The binding agent of any one of claims 1 to 22, wherein the inhibitory KIR protein is selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 and KIR2DL3 or a combination thereof.

24. 24. The binding agent of claim 23, wherein the second binding domain specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2.

25. The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL comprising: a. SEQ ID NO:101 and SEQ ID NO:102, respectively; b. SEQ ID NO:109 and SEQ ID NO:110, respectively; c. SEQ ID NO:117 and SEQ ID NO:118, respectively; d. SEQ ID NO:125 and SEQ ID NO:126, respectively; e. SEQ ID NO:133 and SEQ ID NO:134, respectively; f. SEQ ID NO:141 and SEQ ID NO:142, respectively; g. SEQ ID NO:149 and SEQ ID NO:150, respectively; and h. SEQ ID NO: 157 and SEQ ID NO: 158, respectively 25. The binding agent of any one of claims 1 to 24, having an amino acid sequence selected from the pair of amino acid sequences set forth in the group consisting of:

26. The second binding domain comprises a heavy chain variable region (VH) and a light chain variable region, the heavy and light chain variable regions comprising hCDR1, hCDR2 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, the CDRs being a. SEQ ID NO:103 to SEQ ID NO:108, respectively; b. SEQ ID NOs:111 to 116, respectively; c. SEQ ID NOs:119 to 124, respectively; d. SEQ ID NOs:127 to 132, respectively; e. SEQ ID NOs:135 to 140, respectively; f. SEQ ID NOs:143 to 148, respectively; g. SEQ ID NO:151 to SEQ ID NO:156, respectively; and h. SEQ ID NO: 159 and SEQ ID NO: 164, respectively 26. The binding agent of any one of claims 1 to 25, having an amino acid sequence selected from the set of amino acid sequences set forth in the group consisting of:

27. 27. The binding agent of any one of claims 1 to 26, which does not contain an Fc domain.

28. 27. The binding agent of any one of claims 1 to 26, further comprising an Fc domain.

29. 29. The binding agent of claim 28, wherein the Fc domain is selected from an IgG1 and an IgG4 Fc domain.

30. 30. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 29 and a pharma- ceutically acceptable carrier.

31. 30. A nucleic acid encoding a binding agent according to any one of claims 1 to 29.

32. A vector comprising the nucleic acid of claim 31.

33. A cell line comprising the vector of claim 32.

34. 31. The pharmaceutical composition of claim 30 for use in a method of treating an autoimmune disease, said method comprising the step of administering said pharmaceutical composition to a subject in need thereof in an amount effective to reduce the number or activity of pathogenic immune cells in said subject, thereby alleviating a symptom of said autoimmune disease.

35. 31. The pharmaceutical composition of claim 30 for use in a method of suppressing an immune response mediated by pathogenic immune cells, said method comprising the step of contacting CD8+KIR+ T regulatory cells (Tregs) with an amount of said pharmaceutical composition effective to activate or stimulate CD8+KIR+ Tregs (activated Tregs), thereby reducing the number or activity of pathogenic immune cells.

36. 36. The pharmaceutical composition for use according to claim 34 or claim 35, wherein the pathogenic immune cell is an autoreactive CD4+ T cell, an autoantibody-producing B cell, an autoantigen-presenting cell, or an autoantigen-presenting dendritic cell.

37. 37. The pharmaceutical composition for use according to any one of claims 34 to 36, wherein the subject has an autoimmune disease selected from the group consisting of celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjogren's syndrome (SjS), systemic lupus erythematosus (SLE) and ulcerative colitis.

38. A pharmaceutical composition for use according to any one of claims 34 to 37, wherein the method further comprises the step of administering an immunosuppressant to the subject.

39. 39. The pharmaceutical composition for use according to any one of claims 34 to 38, wherein said administration of said pharmaceutical composition results in a reduction in the frequency or severity of disease flares, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with said autoimmune disease.

40. 31. The pharmaceutical composition of claim 30 for use in a method of treating cancer or stimulating an immune response to an antigen associated with cancer, said method comprising the step of administering said pharmaceutical composition to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby alleviating a symptom of said cancer, wherein said binding agent is substantially free of effector function activity.

41. 41. The pharmaceutical composition for use according to claim 40, wherein the cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, myeloma and leukemia.

42. 41. The pharmaceutical composition for use according to claim 40, wherein the cancer is selected from the group consisting of solid tumors, e.g. breast, cervical, ovarian, lung, CRC (and other cancers of the intestine), skin, esophageal, adenocarcinoma, bladder and prostate; and lymphoma.

43. 31. The pharmaceutical composition according to claim 30 for use in a method of treating an infectious disease or stimulating an immune response against infected cells caused by an infectious disease, said method comprising the step of administering said pharmaceutical composition to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby alleviating a symptom of said infectious disease.

44. 31. The pharmaceutical composition of claim 30, for use in a method for reducing or preventing the onset of graft-versus-host disease (GVHD) after transplantation, the method comprising administering to a subject in need thereof the pharmaceutical composition in an amount effective to activate or stimulate CD8+KIR+ Tregs, thereby reducing or alleviating at least one symptom of GVHD, wherein the binding agent is substantially free of effector function activity.

45. The pharmaceutical composition for use according to claim 44, wherein administration of the pharmaceutical composition to the subject results in a reduction in symptoms associated with GVHD, a reduction in systemic inflammatory cytokines, a reduction in lesions in tissues affected by GVHD, a reduction in self-reported symptoms associated with immune responses associated with adverse effects on host tissues, improved or prolonged graft survival, alleviation of one or more symptoms, and / or prevention, delay or slowing of the onset or progression of rejection of the graft, or prolonged graft survival accompanied by reduced use of broad-spectrum immunosuppressants, e.g., corticosteroids.

46. 46. ​​The pharmaceutical composition for use according to any one of claims 34 to 45, characterized in that the CD8+KIR+ Tregs are contacted with the binding agent in vivo.

47. The pharmaceutical composition for use according to any one of claims 34 to 46, characterized in that the binding agent is to be administered intravenously or subcutaneously; and / or the binding agent is to be administered at a dose of from about 0.01 mg / kg to about 20 mg / kg.

48. 37. The pharmaceutical composition for use according to claim 35 or claim 36, characterized in that the CD8+KIR+ Tregs are contacted with the binding agent ex vivo.

49. The pharmaceutical composition for use according to claim 48, characterized in that the activated CD8+KIR+ Tregs are administered in an effective amount to a subject in need thereof.

50. 50. The pharmaceutical composition for use according to any one of claims 34 to 49, wherein said CD8+KIR+ Tregs are MHC class I restricted and / or are not MHC HLA E(Qa-1b) restricted.

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