Binders and methods for using them

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] Statement regarding sequence listings The sequence listing relating to this application is provided in text format instead of on paper and is incorporated herein by reference. The name of the text file containing this sequence listing is 670151_403WO_Sequence_Listing.txt. This text file is 85KB in size, was created on January 20, 2022, and submitted electronically via EFS-Web. [Background technology]

[0002] background The immune system includes innate and adaptive immunity. Adaptive immunity has several cell subtypes, including T cell subsets and B cell subsets. T cell subsets include various types of T cells, such as naive T lymphocytes and effector T lymphocytes, e.g., cytotoxic T cells and helper T cells, as well as regulatory T cells. The activity of these T cell types is achieved by a balance between the activity of effector T cells and the regulation by regulatory T cells. Effector T cells are generally thought to promote inflammation, while regulatory T cells regulate it. Therefore, Tregs play a crucial role in the development of autoimmune lesions by maintaining self-tolerance, limiting autoimmunity, and controlling the proliferation and activation of autoreactive CD4+ T effector cells. An imbalance between effector T cells and regulatory T cells can lead to inappropriate activation or suppression of the immune response, 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 just beginning to be understood. [Overview of the Initiative] [Means for solving the problem]

[0003] Brief overview Binding agents and methods of using them to modulate the activity of CD8+KIR+ regulatory T cells (Tregs) are provided herein. The binding agents are bispecific or polyspecific and specifically bind to antigens expressed on the surface of CD8+KIR+Tregs. In some embodiments, CD8+KIR+Tregs are MHC class I-restricted. In some embodiments, CD8+KIR+Tregs are not MHC Qa-1-restricted. Methods of using binding agents for the treatment of autoimmune diseases, infectious diseases and cancer are also provided.

[0004] In some embodiments, a binder is provided that binds to CD8+KIR+Tregs, comprising: a first binding domain that specifically binds to a first antigen, wherein the first antigen is selected from antigens expressed on CD8+KIR+T regulatory cells (Tregs) other than KIR proteins; and a second binding domain that specifically binds to an inhibitory KIR protein expressed on the surface of 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 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 binder is a bispecific antibody, diabody, 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 binder is selected from an antibody or its antigen-binding moiety, and the other binding domain is an antibody fragment. In some embodiments, the antigen-binding moiety is Fab, Fab', F(ab')2, Fv, scFv, or a single-domain antibody (also called VHH, VNAR, sdAb, or nanobody). In some embodiments, the first binding domain includes a heavy chain variable region and a light chain variable region. In some embodiments, the second binding domain includes 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, and optionally to CD3 epsilon. In some embodiments, the first binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH and VL are a. Sequence ID 1 and Sequence ID 2, respectively; b. Sequence ID 9 and Sequence ID 10, respectively; c. Sequence ID 17 and Sequence ID 18, respectively; d. Sequence IDs 25 and 26, respectively; e. Sequence ID 33 and Sequence ID 34, respectively; f. respectively, SEQ ID NO: 41 and SEQ ID NO: 34; g. respectively, SEQ ID NO: 45 and SEQ ID NO: 34; h. respectively, SEQ ID NO: 49 and SEQ ID NO: 50; i. respectively, SEQ ID NO: 57 and SEQ ID NO: 58; j. respectively, SEQ ID NO: 65 and SEQ ID NO: 66; and k. respectively, SEQ ID NO: 65 and SEQ ID NO: 166 It has an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of.

[0008] In some embodiments, the first binding domain includes a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions each include hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDRs are a. respectively, SEQ ID NO: 3 to SEQ ID NO: 8; b. respectively, SEQ ID NO: 11 to SEQ ID NO: 16; c. respectively, SEQ ID NO: 19 to SEQ ID NO: 24; d. respectively, SEQ ID NO: 27 to SEQ ID NO: 32; e. respectively, SEQ ID NO: 35 to SEQ ID NO: 40; f. respectively, SEQ ID NO: 42 to SEQ ID NO: 44 and SEQ ID NO: 38 to SEQ ID NO: 40; g. respectively, SEQ ID NO: 46 to SEQ ID NO: 48 and SEQ ID NO: 38 to SEQ ID NO: 40; h. respectively, SEQ ID NO: 51 to SEQ ID NO: 56; i. respectively, SEQ ID NO: 59 to SEQ ID NO: 64; j. respectively, SEQ ID NO: 67 to SEQ ID NO: 72; and k. respectively, SEQ ID NO: 67 to 69 and 167 to 169 It has an amino acid sequence selected from a 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, and optionally to CD8 alpha. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions are a. respectively, SEQ ID NO: 73 and SEQ ID NO: 74; and b. respectively, SEQ ID NO: 81 and SEQ ID NO: 82 having an amino acid sequence selected from the pair of amino acid sequences shown in the group consisting of; or the first binding domain comprises a VHH chain, and the VHH chain is as follows: c. SEQ ID NO: 89; d. SEQ ID NO: 93; and e. SEQ ID NO: 97 having an amino acid sequence 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, and the heavy and light chain variable regions each comprise hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the amino acid sequences of the CDRs are as follows: a. respectively, SEQ ID NO: 75 to SEQ ID NO: 80; or b. respectively, SEQ ID NO: 83 to SEQ ID NO: 88 selected from the amino acid sequences shown in the group consisting of; or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, and the amino acid sequences of the CDRs of the VHH are as follows: c. respectively, SEQ ID NO: 90 to SEQ ID NO: 92; d. respectively, SEQ ID NO: 94 to SEQ ID NO: 96; and e. respectively, SEQ ID NO: 98 to SEQ ID NO: 100 selected from the amino acid sequences shown in 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), the VH and VL having the amino acid sequences of SEQ ID NO: 170 and SEQ ID NO: 171, respectively.

[0012] In some embodiments, the first binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each including hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs. 172-174, 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), the VH and VL having the amino acid sequences of SEQ ID NO: 178 and SEQ ID NO: 179, respectively.

[0014] In some embodiments, the first binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each including hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs. 180-182, and lCDR1, lCDR2 and lCDR3 having the amino acid sequences of SEQ ID NOs. 183-185, 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), where 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 includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each including hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs. 188 to 190, 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 includes a heavy chain variable region (VH) and a light chain variable region (VL), where 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 includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each including hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs. 196-198, 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 includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL are a. Sequence ID 101 and Sequence ID 102, respectively; b. Sequence IDs 109 and 110, respectively; c. Sequence ID 117 and Sequence ID 118, respectively; d. Sequence IDs 125 and 126, respectively; e. Sequence IDs 133 and 134, respectively; f. Sequence ID 141 and Sequence ID 142, respectively; g. Sequence IDs 149 and 150, respectively; and h. Sequence ID 157 and Sequence ID 158, respectively It has an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of the following.

[0020] In some embodiments, the second binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each including hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, respectively, and the CDR is a. Sequence IDs 103 to 108, respectively; b. Sequence numbers 111 to 116, respectively; c. Sequence numbers 119 to 124, respectively; d. Sequence numbers 127 to 132, respectively; e. Sequence numbers 135 to 140, respectively; f. Sequence numbers 143 to 148, respectively; g. Sequence IDs 151 to 156, respectively; and h. Sequence IDs 159 and 164, respectively. It has an amino acid sequence selected from a set of amino acid sequences shown in the group consisting of the following.

[0021] In some embodiments, the binder does not contain an Fc domain. In some embodiments, the binder contains an Fc domain. In some embodiments, the Fc domain is selected from IgG1 and IgG4 Fc domains. In some embodiments, the binder has substantially no effector functional 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 binder is divalent or tetravalent. In some embodiments, the binder is dispecific.

[0023] Pharmaceutical compositions comprising any of the binders and pharmaceutically acceptable carriers described herein are also provided.

[0024] Nucleic acids encoding any of the binders described herein are also provided. Vectors comprising any of the nucleic acid embodiments described herein are further provided. Cell systems comprising any of the nucleic acid or vector embodiments described herein are also further provided.

[0025] In some embodiments, a method is provided for treating an autoimmune disease, comprising the step of administering to a subject in need of it one of the embodiments of the binder or pharmaceutical composition described herein in an amount effective in reducing the number or activity of pathogenic immune cells in the subject and thereby alleviating the symptoms of the autoimmune disease.

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

[0027] In some embodiments, a method is provided for suppressing an immune response to an autoantigen, comprising the step of administering to a subject requiring such suppression in an amount effective to activate or stimulate CD8+KIR+Treg cells, thereby reducing the number or activity of pathogenic immune cells responsive to the autoantigen.

[0028] In some embodiments, a method is provided for suppressing an immune response to an antigen, comprising the step of administering to a subject requiring such suppression in an amount effective to activate or stimulate CD8+KIR+Treg cells, thereby reducing the number or activity of pathogenic immune cells responsive to the antigen.

[0029] In some embodiments of these methods for treating autoimmune diseases or suppressing immune responses, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. In some embodiments, activated CD8+KIR+Treg is administered in an effective dose to a subject requiring it. In some embodiments, pathogenic immune cells are autoreactive CD4 T cells, autoantibody-producing B cells, or autoantigen-presenting dendritic cells. In some embodiments, pathogenic immune cells are autoantigen-presenting cells. In some embodiments, the titer of autoantibodies is reduced in the subject.

[0030] In some embodiments, the subjects have 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 mellitus), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis.

[0031] In some embodiments of methods for treating autoimmune diseases or suppressing immune responses, the binder specifically binds to CD8 and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, the binder specifically binds to CD3 and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, the binder specifically binds to CD5 and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, the binder specifically binds to PD-1 and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, the binder specifically binds to ICOS and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, the binder specifically binds to CXCR3 and inhibitory KIR proteins on CD8+KIR+Treg. In some embodiments, CD8+KIR+Treg is MHC class I constrained. In some embodiments, CD8+KIR+Treg is not MHC HLA E(Qa-1b) constrained.

[0032] In some embodiments of methods for treating autoimmune diseases or suppressing immune responses, these methods further include the step of administering an immunosuppressant to a subject. In some embodiments, administration of a binder to a subject results in an improvement in the treatment outcome in the subject. In some embodiments, the improvement in the treatment outcome is a reduction in the frequency or severity of disease relapses, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with the autoimmune disease.

[0033] In some embodiments of methods for treating autoimmune diseases or suppressing immune responses, the binder is administered intravenously. In some embodiments, the binder is administered subcutaneously. In some embodiments, the binder is administered in doses ranging from about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the binder has substantially no effector functional activity.

[0034] In some embodiments, the use of any embodiment of the binder or pharmaceutical composition described herein is provided for the treatment of autoimmune diseases in a subject by activating or stimulating CD8+KIR+Treg. In some embodiments, the use of any embodiment of the binder or pharmaceutical composition described herein is provided for the reduction of the immune response by pathogenic immune cells by activating or stimulating CD8+KIR+Treg. In some embodiments, the use of any embodiment of the binder 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 for treating cancer is provided, comprising the step of administering one of the embodiments of the binder or pharmaceutical composition described herein to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+T regulatory cells (Treg) and thereby alleviate the symptoms of cancer, wherein the binder substantially lacks effector functional activity.

[0036] In some embodiments, a method is provided for stimulating an immune response to a cancer-associated antigen (cancer antigen), comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with one of the embodiments of a binder or pharmaceutical composition described herein in an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs), wherein the binder has substantially no effector functional activity, thereby increasing the immune response to the cancer antigen.

[0037] In some embodiments, a method for treating cancer is provided, comprising the step of administering one of the embodiments of the binder or pharmaceutical composition described herein to a subject in need thereof in an amount effective to deplete CD8+KIR+T regulatory cells (Tregs) and thereby alleviate the symptoms of cancer, wherein the binder has effector functional activity comprising at least ADCC.

[0038] In some embodiments, a method is provided for stimulating an immune response to a cancer-associated antigen (cancer antigen), comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with one of the embodiments of a binder or pharmaceutical composition described herein in an amount effective to deplete the CD8+KIR+Tregs, wherein the binder has effector functional activity comprising at least ADCC, thereby increasing the immune response to the cancer antigen.

[0039] In some embodiments of methods for treating cancer or stimulating an immune response to cancer-related antigens, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. In some embodiments, activated CD8+KIR+Treg is administered in an effective dose to a subject requiring it. In some embodiments, the increased immune response includes a reduction in 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, cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, and leukemia. In some embodiments, cancer is selected from the group consisting of solid tumors, such as cancers of the breast, cervix, ovary, lung, colorectal (CRC) (and other cancers of the intestine), skin, esophagus, adenocarcinoma, bladder, and prostate; as well as lymphoma.

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

[0042] In some embodiments, these methods further include the step of administering a chemotherapeutic agent to a target. In some embodiments, these methods further include the step of administering an immunotherapy to a target. In some embodiments, these methods further include the step of administering an immunotherapy, such as a checkpoint inhibitor, to a target. In some embodiments, administration of a binder to a target results in an improvement in the treatment outcome in the target. In some embodiments, the improvement in the treatment outcome is a partial response or a complete response. In some embodiments, the improvement in the treatment outcome is remission.

[0043] In some embodiments, the binder is administered intravenously. In some embodiments, the binder is administered subcutaneously. In some embodiments, the binder is administered in doses ranging from approximately 0.01 mg / kg to approximately 20 mg / kg.

[0044] In some embodiments, a use is provided for the treatment of cancer in a subject by activating or stimulating CD8+KIR+Treg, wherein the binder has substantially no effector functional activity.

[0045] In some embodiments, the use of any of the embodiments of the binders or pharmaceutical compositions described herein is provided for reducing immunosuppression by immunosuppressive immune cells by activating or stimulating CD8+KIR+Treg, wherein the binder has substantially no effector functional activity.

[0046] In some embodiments, the use of any of the embodiments of the binders or pharmaceutical compositions described herein is provided for reducing tumor burden in a subject by activating or stimulating CD8+KIR+Treg, wherein the binder has substantially no effector functional activity.

[0047] In some embodiments, the use of any embodiment of the binder or pharmaceutical composition described herein is provided for the treatment of cancer in a subject by depleting CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC.

[0048] In some embodiments, a use is provided for the depletion of CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC.

[0049] In some embodiments, the use of any embodiment of the binder or pharmaceutical composition described herein is provided for reducing tumor burden in a subject by depleting CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC.

[0050] In some embodiments, a method is provided for treating an infection, comprising the step of administering one of the embodiments of the binder or pharmaceutical composition described herein to a subject in need of it in an amount effective to activate or stimulate CD8+KIR+Treg, thereby alleviating the symptoms of the infection.

[0051] In some embodiments, a method is provided for stimulating an immune response to infected cells caused by an infection, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with one of the embodiments of the binder or pharmaceutical composition described herein in an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs), thereby increasing the immune response to the infected cells.

[0052] In some embodiments, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. In some embodiments, activated CD8+KIR+Treg is administered in an effective dose to a subject requiring it.

[0053] In some embodiments for treating an infection or stimulating an immune response against infected cells, 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 infection is selected from bacterial diseases, systemic fungal diseases, rickettsial diseases, parasitic diseases and viral diseases. In some embodiments, the infection is selected from the group consisting of HIV infection, hepatitis C virus (HCV) infection, human papillomavirus (HPV) infection, Epstein-Barr virus (EBV) infection, coronavirus infections, e.g., SARS-CoV-2 infection (Covid-19), cytomegalovirus (CMV) infection and influenza virus infection.

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

[0055] In some embodiments, these methods further include the step of administering an antimicrobial or antiviral agent to a subject. In some embodiments, administration of the binder to the subject results in an improvement in the treatment outcome in the subject. In some embodiments, the improvement in the treatment outcome is a reduction in infection. In some embodiments, the improvement in the treatment outcome is a reduction in infected cells. In some embodiments, the binder is administered intravenously. In some embodiments, the binder is administered subcutaneously. In some embodiments, the binder is administered in doses ranging from about 0.01 mg / kg to about 20 mg / kg. In some embodiments, the binder has substantially no effector functional activity.

[0056] In some embodiments, the use of any of the embodiments of the binders or pharmaceutical compositions described herein is provided for the treatment of infections in a subject by activating or stimulating CD8+KIR+Treg.

[0057] In some embodiments, the use of any embodiment of the binder or pharmaceutical composition described herein is provided for activating or stimulating CD8+KIR+Treg, thereby stimulating an immune response by suppressing immunosuppressive immune cells.

[0058] In some embodiments, the use of any of the embodiments of the binders or pharmaceutical compositions described herein is provided for reducing infection in a subject by activating or stimulating CD8+KIR+Treg.

[0059] In some embodiments, a method is provided for reducing or preventing the development of graft-versus-host disease (GVHD) after transplantation, comprising the step of administering one of the binders or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Treg, thereby reducing or alleviating at least one symptom of GVHD. In some embodiments, the binder is substantially devoid of effector functional activity.

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

[0061] In some embodiments, a method is provided for treating a transplanted subject, comprising the step of administering to a subject in need of either a binder or a pharmaceutical composition described herein in an amount effective to deplete CD8+KIR+Treg and thereby alleviate the symptoms of GVHD. In some embodiments, the binder has effector functional activity comprising at least ADCC.

[0062] In some embodiments, a method is provided for suppressing GVHD in transplantation, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with any of the conjugates or pharmaceutical compositions described herein in an amount effective to deplete the CD8+KIR+Tregs, thereby reducing GVHD or its symptoms. In some embodiments, the conjugate has effector functional activity comprising at least ADCC.

[0063] In some embodiments, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. In some embodiments, activated CD8+KIR+Treg is administered in an effective dose to a subject requiring it.

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

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

[0066] In some embodiments, immunosuppressants are also administered to the subjects.

[0067] In some embodiments, administration of a binder to the subject results in an improvement in the treatment outcome. In some embodiments, the improvement in treatment outcome includes a reduction in GVHD-related symptoms, a reduction in systemic inflammatory cytokines, a reduction in lesions in GVHD-affected tissues, a reduction in self-reported symptoms related to immune responses associated with adverse effects on host tissues, and transplantation. thing To improve or prolong graft survival, alleviate one or more symptoms, and / or transplantation. thing Prevention, delay, or slowing of the onset or progression of rejection, or widening Regional spectrum Immunosuppressants (broad spectrum immunosuppressive agent) For example, transplants due to a decrease in the use of corticosteroids. thing This is an extension of the process of rooting.

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

[0069] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein is provided for the treatment of transplant-related GVHD in a subject. In some embodiments, the binders are substantially devoid of effector functional activity.

[0070] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein is provided for the treatment of transplant-related GVHD in a subject by activating or stimulating CD8+KIR+Treg. In some embodiments, the binder is substantially devoid of effector functional activity.

[0071] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein is provided for reducing transplant-related GVHD by activating or stimulating CD8+KIR+Treg. In some embodiments, the binders are substantially devoid of effector functional activity.

[0072] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein for reducing GVHD in transplants is provided herein. In some embodiments, the binders are substantially devoid of effector functional activity.

[0073] In some embodiments, the use of any of the conjugates or pharmaceutical compositions described herein is provided for the treatment of transplant-related GVHD in a subject by depleting CD8+KIR+Treg. In some embodiments, the conjugate has effector functional activity comprising at least ADCC.

[0074] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein is provided for the depletion of CD8+KIR+Treg. In some embodiments, the binder has effector functional activity comprising at least ADCC.

[0075] In some embodiments, the use of any of the binders or pharmaceutical compositions described herein is provided for CD8+KIR+Treg depletion in transplanted subjects to reduce GVHD. In some embodiments, the binder has effector functional activity comprising at least ADCC.

[0076] These and other aspects of the present invention can be better understood by referring to the following detailed description, non-limiting examples of specific embodiments, and the accompanying drawings. [Brief explanation of the drawing]

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

[0078] [Figure 2] Figure 2 shows various forms of a specific bispecific antibody.

[0079] [Figure 3] Figure 3 shows various forms of additional bispecific antibodies.

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

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

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

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

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

[0085] [Figure 9A-9B] Figures 9A–9B show that gluten peptide restimulation of CD8+ KIR+ T cells derived 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 of reactive CD4+ T cells compared to unstimulated cells or cells restimulated with a control influenza peptide (Figure 9B).

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

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

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

[0089] [Figure 13] Figure 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 mellitus (T1D), or in healthy subjects.

[0090] [Figure 14] Figure 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] Figure 15 shows the production of granzyme B, perforin, and IL-10 by CD8+ Treg cells isolated from celiac patient PBMC samples.

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

[0093] [Figure 17] Figure 17 shows the decrease in IL-17A and IL-23 production by CD4+ T cells co-cultured with CD8+KIR+Treg cells after gliadin stimulation.

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

[0095] [Figure 19] Figure 19 shows the 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] Figure 20 shows the increased CD8+ Treg cell response to repeated antigen exposure.

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

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

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

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

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

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

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

[0104] [Figure 28] Figure 28 shows examples of bispecific KIR binders and their expected effects on activation signal intensity.

[0105] [Figures 29A-29C] Figure 29A shows a bispecific antibody having a CD8-binding domain and a binding domain that targets KIR2DL1 / 2 / 3. Figure 29B shows degranulation 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), and Figure 29C shows the granzyme B levels.

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

[0107] [Figure 31] Figure 31 shows CD4+ T cell death 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).

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

[0109] [Figure 33] Figure 33 shows the preferential binding of a bispecific blocker (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells compared to NK cells.

[0110] [Figure 34] Figure 34 shows the preferential binding of a bispecific blocker (anti-KIR2DL1 / 2 / 3 and anti-CD8) to CD8+ T cells compared to NK cells and CD4+ T cells. [Modes for carrying out the invention]

[0111] Detailed explanation definition For convenience, certain terms in the specification, examples, and claims are defined herein. Unless otherwise noted or implicitly indicated in the context, the following terms and phrases have the meanings provided below. Since the scope of the invention is limited solely by the claims, these definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention pertains.

[0112] Where used herein, unless otherwise indicated, the terms “a” and “an” shall be interpreted as meaning “one,” “at least one,” or “one or more.” Where otherwise required by context, singular terms herein shall include plural forms, and plural terms herein shall include singular forms.

[0113] Unless the context clearly requires otherwise, throughout the description and claims, the words “comprise,” “comprising,” etc., are interpreted in an inclusive sense, as opposed to an exclusive or thorough sense; that is, they are interpreted as “includes but not limited to.”

[0114] The terms “reduce,” “decrease,” “reduced,” “decrease,” “decrease,” and “inhibit” are all used herein in general to mean a statistically significant reduction compared to a reference.

[0115] The terms “increased,” “boosting,” “enhancing,” or “activating” are all used herein to generally mean an increase of a statically significant amount compared to the reference.

[0116] The terms “isolated” or “partially purified,” as used herein, mean, in the case of nucleic acids, polypeptides, or proteins, a nucleic acid, polypeptide, or protein that has been isolated from at least one other component (e.g., nucleic acid, polypeptide, or protein) that is present with the nucleic acid, polypeptide, or protein found in its natural source, and / or expressed by cells, or secreted in the case of secreted polypeptides and proteins. Chemically synthesized nucleic acids, polypeptides, or proteins, or those synthesized using in vitro transcription / translation, are considered “isolated.” The terms “purified” or “substantially purified” mean, for example, an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight of the nucleic acid, polypeptide, or protein of interest, containing at least 96%, at least 97%, at least 98%, at least 99%, or more.

[0117] As used herein, the terms “protein” and “polypeptide” are interchangeable to specify a set of amino acid residues, each linked to the others 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, glycated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. While “protein” and “polypeptide” are often used to refer to relatively large polypeptides, while the term “peptide” is often used to refer to smaller polypeptides, the usage of these terms in this art is often overlapping. The terms “protein” and “polypeptide” are interchangeable to use herein when referring to the encoded gene product and its fragments. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologues, paralogs, fragments, and other equivalents, variants, fragments, and analogs of those described 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 thus incorporated herein by reference.

[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 thus incorporated herein by reference.

[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 thus incorporated herein by reference.

[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 thus incorporated herein by reference.

[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. Examples of KIR3DL2 polypeptides, but not limited to those listed above, include those having the amino acid sequences shown in NP_006728.2 and NP_001229796.1; these sequences are thus incorporated herein by reference.

[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. Examples of KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_055033.2; this sequence is thus incorporated herein by reference.

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

[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. Examples of KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_056952.2; this sequence is thus incorporated herein by reference.

[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 shown in NP_001233.2; this sequence is thus incorporated herein by reference.

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

[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 thus incorporated herein by reference.

[0129] CD40L, or CD40 ligand, is also known as 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 thus incorporated herein by reference.

[0130] CD45 is also known as the 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. Examples of CD45R0 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-4. Examples of CD45RA polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-8. Examples of CD45RB polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-9. See the UniProtKB database. These sequences are thus incorporated herein by reference.

[0131] CD73 is also known 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; their amino acid sequences are thus incorporated herein by reference.

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

[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 amino acid sequences disclosed in NP_001333152.1, NP_001333151.1, and NP_000869.1; these amino acid sequences are thus incorporated herein by reference.

[0134] CD166, or activated leukocyte adhesion molecule (ALCAM), is also known as MMD. 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 thus incorporated herein by reference.

[0135] CD177 is also known 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 shown in NP_065139.2; this amino acid sequence is thus incorporated herein by reference.

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

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

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

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

[0140] ICOS, or inducible T cell costimulation, is also known as AILIM, CD278, and CVID1. Examples of ICOS polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_036224.1; this amino acid sequence is thus incorporated herein by reference.

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

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

[0143] PD-1 is also known as programmed cell death protein 1. Examples of PD-1 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_005009.2; this amino acid sequence is thus incorporated herein by reference.

[0144] S1000A8 / 9, or S100A8 and S100A9 respectively, are Ca components belonging to the S100 family. 2+These are binding proteins. S100A8, or S100-A8, is also known as 60B8AG, CAGA, CFAG, CGLA, CP-10, L1Ag, MA387, MIF, MRP8, NIF, and P8. Examples of S100A8 polypeptides include, but are not limited to, those having the amino acid sequences shown in NP_001306125.1, NP_001306126.1, NP_001306127.1, NP_001306130.1, and NP_002955.2. S100A9, or S100-A9, is also known as 60B8AG, CAGB, CFAG, CGLB, L1AG, LIAG, MAC387, MIF, MRP14, NIF, and P14. Examples of S100A9 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_002956.1. These amino acid sequences are thus incorporated herein by reference.

[0145] TIM-3, also known as hepatitis A virus cell receptor 2 (HAVCR2), is also known as CD366, HAVcr-2, KIM-3, SPTCL, TIM3, TIMD-3, and TIMD3. Examples of TIM-3 polypeptides, but not limited to those having the amino acid sequence shown in NP_116171.3, are incorporated herein by reference.

[0146] TLT-2, or trigger receptor-like 2 (TREML2) expressed on myeloid cells, is also known as C6orf76 or dJ238O23.1. Examples of TLT-2 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP_079083.2; this amino acid sequence is thus incorporated herein by reference.

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

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

[0149] As used herein, “epitope” refers to amino acids typically bound by immunoglobulin VH / VL pairs, e.g., antibodies and other binders described herein. Epitopes can be formed on polypeptides from consecutive amino acids or discontinuous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from consecutive amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in their own spatial conformation. Epitopes define the minimum binding site for an antibody or other binder and thus indicate the target of the antibody, its antigen-binding portion, or other immunoglobulin-based binder's specificity. In the case of a single-domain antibody, the epitope indicates a structural unit bound sequestered by a variable domain.

[0150] As used herein, "specifically binds" means that KD is 10 -5 M (10000 nM) or less, for example, 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 Refers to the ability of a binder (e.g., an antibody or an antigen-binding portion thereof) described herein to bind to a target at M or less. Specific binding can be affected, for example, by the affinity and binding strength of the antibody or other binder and the concentration of the target polypeptide. One of ordinary skill in the art can determine the appropriate conditions under which the antibodies and other binders described herein bind selectively to the target antigen using any suitable method, e.g., titration of the binder in a suitable cell-binding assay. A binder that binds specifically to a target cannot be replaced by non-similar competing factors. In certain embodiments, a binder, e.g., an antibody or an antigen-binding portion thereof, is said to bind specifically to its target if it preferentially recognizes its target antigen in a complex mixture of proteins and / or macromolecules.

[0151] In some embodiments, a binder described herein, e.g., an antibody or an antigen-binding portion thereof or other binder, is 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 Specifically binds to a target polypeptide with a dissociation constant (KD) of M or less. In some embodiments, an antibody or an antigen-binding portion thereof or other binder described herein specifically binds to a target polypeptide with a dissociation constant (KD) of about 10 -5 M to 10 -6 M. In some embodiments, an antibody or an antigen-binding portion thereof or other binder described herein specifically binds to a target polypeptide with a dissociation constant (KD) of about 10 -6 M to 10 -7 M. In some embodiments, an antibody or an antigen-binding portion thereof or other binder described herein specifically binds to a target polypeptide with a dissociation constant (KD) of about 10 -7 M to 10 -8It specifically binds to the target polypeptide with a dissociation constant (KD) of M. In some embodiments, the antibody or its antigen-binding moiety or other binder described herein is approximately 10 -8 M~10 -9 It specifically binds to the target polypeptide with a dissociation constant (KD) of M. In some embodiments, the antibody or its antigen-binding moiety or other binder described herein is approximately 10 -9 M~10 -10 It specifically binds to the target polypeptide with a dissociation constant (KD) of M. In some embodiments, the antibody or its antigen-binding moiety or other binder described herein is approximately 10 -10 M~10 -11 It specifically binds to the target polypeptide with a dissociation constant (KD) of M. In some embodiments, the antibody or its antigen-binding moiety or other binder described herein is approximately 10 -11 M~10 -12 It specifically binds to the target polypeptide with a dissociation constant (KD) of M. In some embodiments, the antibody or its antigen-binding moiety or other binder described herein is 10 -12 It has a dissociation constant (KD) of less than M and specifically binds to the target polypeptide.

[0152] As used herein, the term "essentially derived from" refers to an element required of a given embodiment. The term allows for the presence of elements that do not substantially affect the basic, novel, or functional features of that embodiment.

[0153] The term "consists of" refers to the compositions, methods, and their respective components described herein, excluding all elements not enumerated in the description of the embodiments.

[0154] Unless otherwise indicated in the examples or elsewhere, all numbers representing the quantities of components or reaction conditions used herein should be understood to be modified in all cases by the term “approximately.” When used in conjunction with percentages, the term “approximately” may mean + / - 1%.

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

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

[0157] Modulation of CD8+KIR+ regulatory T cells Binding agents comprising a binding domain that specifically binds to antigens expressed on CD8+KIR+ regulatory T cells (Tregs) are provided herein. In some embodiments, the CD8+KIR+Tregs are MHC class I-restricted. In some embodiments, the CD8+KIR+Tregs are not MHC Qa-1 (HLA-E)-restricted. Methods of using the binding agents for the treatment of autoimmune diseases, infectious diseases, and cancer are also provided.

[0158] The binding agent comprises a first binding domain that specifically binds to T cell antigens expressed on CD8+KIR+Tregs other than KIR proteins, and a second binding domain that specifically binds to inhibitory KIR proteins expressed on CD8+KIR+Tregs. In some embodiments, the first binding domain specifically binds to antigens 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 antigens 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 subunits of antigens 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 capable of activating 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 to reduce 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 an anchoring portion to enhance the specificity of the binder to CD8 KIR+Treg. 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 an anchoring portion for enhancing tissue specificity. In some embodiments, such antigens are, 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. In 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 binder specifically binds to an inhibitory KIR protein (killer cell immunoglobulin-like receptor protein). The inhibitory KIR protein may be, for example, KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, or KIR2DL3, or a combination thereof, and specifically binds to, for example, KIR2DL1 / 2 / 3 or KIR2DL1 / 2 proteins. 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 proteins. In some embodiments, the second binding domain is a KIR protein antagonist that blocks the interaction between the KIR protein and its binding partner.

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

[0168] The binding domain of the binder may be derived from an antibody or a non-antibody form. In some embodiments, the binding domain is derived from an antibody or its antigen-binding portion (i.e., an antibody fragment). In some embodiments, the antibody fragment is Fab, Fab', F(ab')2, Fv, scFv, or a single-domain antibody (also called VHH, VNAR, sdAb, or nanobody). In some embodiments, the binding domain is or is derived from antikalin, afibody, avimer, DARPin, or adonectin.

[0169] In some embodiments, the binder is a bispecific antibody, diabody, 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, crossumab, antikalin, afibody, avimer, DARPin, adnectin, scFv-Fc, one-arm tandem scFv-Fc, or DART-Fc (see, for example, Figures 2 and 3). In some embodiments, IgG-scFv is 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 binder includes a first binding domain comprising a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain and light chain variable regions of the first binding domain specifically bind to antigens 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 antigens expressed on CD8+KIR+Treg, 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 antigens expressed on CD8+KIR+Treg, such as subunits of CD3, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8 / 9, and 41BB / CD137.

[0171] In some embodiments, the first antigen is selected from functional agonists capable of activating 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 to reduce 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 an anchoring portion to enhance the specificity of the binder to CD8 KIR+Treg. 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 an anchoring portion for enhancing tissue specificity. In some embodiments, such antigens are, 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. In 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] The antibodies for use in the binding domain described herein are known in the art.

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

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

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

[0182] Antibodies against CD27 are described, for example, in U.S. Patent Application Publications No. 20210009706, 20200247898, and 20200131272.

[0183] Antibodies against CD38 are described, for example, in U.S. Patent Application Publications No. 20200408765, 20200399391, 20090304710, and 20050158305.

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

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

[0186] Antibodies against CD45RA, CD45RB, and CD45RO are described, for example, in U.S. Patent Application Publications 20030232009 and 20020168362, and are available from commercial sources.

[0187] Antibodies against CD73 are described, for example, in U.S. Patent Application Publications No. 20200148781, 20200071404, 20190256598, and 20160145350.

[0188] An antibody against CD103(ITGAE) is described, for example, in U.S. Patent Application Publication No. 20050266001.

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

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

[0191] An antibody against CD177 is described, for example, in U.S. Patent Application Publication No. 20190125832.

[0192] Antibodies against CCR7 are described, for example, in U.S. Patent Application Publications No. 20200216548, 20180237529, and 20150344580.

[0193] Antibodies against CXCR3 are described, for example, in U.S. Patent Application Publications No. 20190119391, 20190008955, and 20130251733.

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

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

[0196] Antibodies against ICOS are described, for example, in U.S. Patent Application Publications No. 20160304610 and 20110243929.

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

[0198] Antibodies against OX40 are described, for example, in U.S. Patent Application Publications No. 20140377284, 20140308276, and 20100196359.

[0199] Antibodies against PD-1 are described, for example, in U.S. Patent Application Publications No. 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 Publications No. 20180072804, No. 20170306016, and No. 20150086574.

[0202] An antibody against TLT-2 is described, for example, in U.S. 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 Publications 20170198050 and 20200347144.

[0205] In some embodiments, the first binding domain specifically binds to CD3 epsilon, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs: 1 and 2; SEQ ID NOs: 9 and 10; SEQ ID NOs: 17 and 18; SEQ ID NOs: 25 and 26; SEQ ID NOs: 33 and 34; SEQ ID NOs: 41 and 34; SEQ ID NOs: 45 and 34; SEQ ID NOs: 49 and 50; SEQ ID NOs: 57 and 58; SEQ ID NOs: 65 and 66; or SEQ ID NOs: 65 and 166, respectively.

[0206] In some embodiments, the first binding domain specifically binds to CD3 epsilon, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively; SEQ ID NOs: 9 and 10, respectively; SEQ ID NOs: 17 and 18, respectively; SEQ ID NOs: 25 and 26, respectively; SEQ ID NOs: 33 and 34, respectively; SEQ ID NOs: 41 and 34, respectively; SEQ ID NOs: 45 and 34, respectively; SEQ ID NOs: 49 and 50, respectively; SEQ ID NOs: 57 and 58, respectively; or SEQ ID NOs: 65 and 66, respectively; or SEQ ID NOs: 65 and 166, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is not modified.

[0207] In some embodiments, the first binding domain includes one or more amino acid sequences described in 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 chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs. 73 and 74, respectively; or SEQ ID NOs. 81 and 82, respectively; or the binding domain includes a VHH chain having the amino acid sequence shown in SEQ ID NOs. 89, 93, or 97.

[0209] In some embodiments, the first binding domain specifically binds to CD8 alpha, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs. 73 and 74, respectively; or SEQ ID NOs. 81 and 82, respectively; or the binding domain includes a VHH chain having the amino acid sequence shown in SEQ ID NOs. 89, 93, or 97, respectively; the framework regions of the heavy chain and light chain variable regions or the VHH chain are modified as necessary by 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region or the VHH chain is not modified.

[0210] In some embodiments, the first binding domain includes one or more amino acid sequences described in any of sequence numbers 73 to 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, and the light chain variable region has lCDR1, lCDR2, and lCDR3, and the amino acid sequences of the heavy chain and light chain variable region CDRs are shown in SEQ ID NOs: 3 to 8; SEQ ID NOs: 11 to 16; SEQ ID NOs: 19 to 24; SEQ ID NOs: 27 to 32; SEQ ID NOs: 35 to 40; SEQ ID NOs: 42 to 44 and 38 to 40; SEQ ID NOs: 46 to 48 and 38 to 40; SEQ ID NOs: 51 to 56; SEQ ID NOs: 59 to 64; or SEQ ID NOs: 67 to 72. In some embodiments, the first binding domain specifically binds to CD3 epsilon and includes light chain variable regions lCDR1, lCDR2, and lCDR3 having amino acid sequences shown in SEQ ID NOs. 167, 168, and 169, respectively.

[0212] In some embodiments, the first binding domain specifically binds to CD8 alpha and has a heavy chain variable region having complementarity-determining regions hCDR1, hCDR2, and hCDR3, and the light chain variable region has lCDR1, lCDR2, and lCDR3, and the amino acid sequences of the heavy chain and light chain variable region CDRs are shown in SEQ ID NOs. 75 to 80, respectively; or SEQ ID NOs. 83 to 88, respectively; or the first binding domain comprises a VHH chain having hCDR1, hCDR2, and hCDR3, and the amino acid sequences of the CDRs of the VHH are shown in SEQ ID NOs. 90 to 92, respectively; SEQ ID NOs. 94 to 96, respectively; or SEQ ID NOs. 98 to 100, respectively.

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

[0214] In some embodiments, the first binding domain specifically binds to ICOS, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 170 and SEQ ID NO: 171, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, while the CDR of the heavy chain or light chain variable region is not modified.

[0215] In some embodiments, the first binding domain includes one or more amino acid sequences described in any of sequence numbers 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 chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively.

[0217] In some embodiments, the first binding domain specifically binds to PD-1, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 178 and SEQ ID NO: 179, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is not modified.

[0218] In some embodiments, the first binding domain includes one or more amino acid sequences described in any of sequence numbers 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 chain and light chain variable regions have the amino acid sequences shown 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 chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 186 and SEQ ID NO: 187, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is left unmodified.

[0221] In some embodiments, the first binding domain includes one or more amino acid sequences described in any of sequence numbers 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 chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 194 and SEQ ID NO: 195, respectively.

[0223] In some embodiments, the first binding domain specifically binds to CD5, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 194 and SEQ ID NO: 195, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, while the CDR of the heavy chain or light chain variable region is not modified.

[0224] In some embodiments, the first binding domain includes one or more amino acid sequences described in any of sequence numbers 194-201 (e.g., VH, VL, hCDR1, hCDR1, hCDR3, lCDR1, lCDR2 and / or lCDR3).

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

[0226] Antibodies against inhibitory KIR proteins are well known in this field.

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

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

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

[0230] In some embodiments, the second binding domain specifically binds to KIR3DL1, and the heavy chain and light chain variable regions have amino acid sequences shown in SEQ ID NOs: 133 and 134, respectively; SEQ ID NOs: 141 and 142, respectively; or SEQ ID NOs: 149 and 150, respectively.

[0231] In some embodiments, the first binding domain specifically binds to KIR3DL1, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs: 133 and 134, respectively; SEQ ID NOs: 141 and 142, respectively; or SEQ ID NOs: 149 and 150, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is not modified.

[0232] In some embodiments, the first binding domain specifically binds to KIR3DL2, and the heavy chain and light chain variable regions have amino acid sequences shown in the amino acid sequences shown 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 chain and light chain variable regions have the amino acid sequences shown in SEQ ID NO: 157 and SEQ ID NO: 158, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is left unmodified.

[0234] In some embodiments, the first binding domain specifically binds to KIR2DL1 / 2 / 3, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs: 101 and 102, respectively; SEQ ID NOs: 109 and 110, respectively; SEQ ID NOs: 117 and 118, respectively; or SEQ ID NOs: 125 and 126, respectively.

[0235] In some embodiments, the first binding domain specifically binds to KIR2DL1 / 2 / 3, and the heavy chain and light chain variable regions have the amino acid sequences shown in SEQ ID NOs: 101 and 102, respectively; SEQ ID NOs: 109 and 110, respectively; SEQ ID NOs: 117 and 118, respectively; or SEQ ID NOs: 125 and 126, respectively; the framework regions of the heavy chain and light chain variable regions are modified as necessary with 1-8, 1-6, 1-4, or 1-2 amino acid substitutions, deletions, or insertions, and the CDR of the heavy chain or light chain variable region is not modified.

[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, and the light chain variable region has lCDR1, lCDR2, and lCDR3, and the amino acid sequences of the heavy chain and light chain variable region CDRs are the amino acid sequences shown in SEQ ID NOs. 135 to 140; SEQ ID NOs. 143 to 148; 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, and the light chain variable region has lCDR1, lCDR2, and lCDR3, and the amino acid sequences of the heavy chain and light chain variable region CDRs are shown in the amino acid sequences of SEQ ID NOs. 159 to 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 shown in SEQ ID NO: 103 to SEQ ID NO: 108; SEQ ID NO: 111 to SEQ ID NO: 116; SEQ ID NO: 119 to SEQ ID NO: 124; or SEQ ID NO: 127 to SEQ ID NO: 132, respectively.

[0239] Binding agent The binding agent can be any suitable agent comprising at least a first binding domain and a second binding domain. The first binding domain specifically binds to a first antigen selected from antigens expressed on CD8+KIR+ regulatory T cells (Tregs) other than the 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 domains of the binding agent can be derived from an antibody or 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 single-domain antibody (also referred to as VHH, VNAR, sdAb, or nanobody). In some embodiments, the binding domain is derived from an anticalin, affibody, avimer, DARPin, adnectin, or receptor ectodomain Fc fusion protein.

[0242] In some embodiments, the binder is a bispecific antibody, diabody, 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, crossumab, antikalin, afibody, avimer, DARPin, adnectin, scFv-Fc, one-arm tandem scFv-Fc, or DART-Fc. In some embodiments, IgG-scFv is 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 an immunoglobulin molecule and a molecule containing an immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site (antigen-binding portion) that specifically binds to a target antigen. This term generally refers to an antibody composed of a full-length antibody (having heavy and light chain constant regions) and two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including its antigen-binding moiety; examples include intact monoclonal antibodies, Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAb), diabodies, polyspecific antibodies, bispecific antibodies, dispecific antibodies, and monochain antibodies (see, for example, Huston et al., Proc. Natl. Acad. Sci. USA, 85, 5879-5883 (1988) and Bird et al., Science 242, 423-426 (1988), which are thus incorporated herein by reference).

[0244] In antibodies, each heavy chain consists 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 called an "Fc domain." As used herein, if a conjugate contains an Fc domain, the conjugate may contain one or more Fc domains, or an entire Fc region, unless otherwise specified by the context. Each light chain consists of a variable region (abbreviated as VL) and a constant region or constant domain. The light chain constant region is the CL domain. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with conserved regions called framework regions (FRs). Thus, each VH and VL region consists of three CDRs and four FRs aligned from the N-terminus to the 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 moiety” of an antibody refers to the portion of the antibody described herein that has VH and VL sequences or heavy and light chain variable regions CDR. Examples of antigen-binding moieties, according to the term “antigen-binding moiety” of an antibody, include Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single-domain antibodies (dAb), diabodies, and single-chain antibodies. As used herein, the terms Fab, F(ab')2, and Fv refer to: (i) a Fab fragment, i.e., a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked to each other in a hinge region via disulfide crosslinks; and (iii) an Fv fragment consisting of the VL and VH domains of an antibody. The two domains of the Fv fragment, namely VL and VH, are encoded by separate coding regions, but these can be further linked together using a synthetic linker, for example, a poly-G4S amino acid sequence (disclosed as SEQ ID NO: 165, "(G4S)n", where n=1-5), allowing them to be prepared as a single protein chain in which the VL and VH regions are combined 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 the VH and VL domains are expressed on a single polypeptide chain, but the chain is too short for the two domains on the same chain to combine. A linker is used to connect the VH and VL domains, thereby forcing the VH and VL domains to pair with complementary domains (VL and VH, respectively) on different chains, forming two antigen-binding sites (see, e.g., Holliger, R, et al. (1993) Proc. Natl. Acad. Sci. USA 90:64446448; Poljak, R. J, et al. (1994) Structure 2:1121-1123).

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

[0248] In some embodiments, the binder lacks an Fc region or domain. In some embodiments, the binder has an entire Fc region or Fc domain. In some embodiments, the binder has an Fc region or Fc domain of the IgG1 isotype. In some embodiments, the binder has an Fc region or Fc domain of the IgG2 isotype. In some embodiments, the binder has an Fc region or Fc domain of the IgG3 isotype. In some embodiments, the binder has an Fc region or Fc domain of the IgG4 isotype. In some embodiments, the Fc domain may have a hybrid isotype containing constant regions derived 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 the Fc domain (e.g., heavy chain) may be a complete C-terminus ending with the amino acid residue PGK. In some embodiments, the C-terminus of the Fc domain may also be a shortened C-terminus with one or two of the C-terminal amino acid residues removed. In some embodiments, the C-terminus of the Fc domain is a shortened C-terminus ending with PG. In some embodiments, the binder containing a heavy chain with a C-terminal CH3 domain includes a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In some embodiments, the binder containing a heavy chain with a C-terminal CH3 domain includes a C-terminal glycine residue (G446, numbered according to the Kabat EU index).

[0250] The binders described herein are multispecific, typically bispecific, binders. In some embodiments, the binder is a multispecific antibody or antibody-like molecule, such as a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites or antigens. The binders described herein typically have binding specificity to different antigens. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Examples of bispecific and polyspecific 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, IgG-scFv is 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 producing multispecific antibodies include, but are not limited to, the 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 the "knob-in-hole" operation (see, for example, U.S. Patent No. 5,731,168). Polyspecific antibodies are produced by manipulating the electrostatic steering effect to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1); crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent 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" techniques to produce 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., See 152:5368 (1994); and it can also be prepared by preparing trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147: 60 (1991).

[0252] Manipulated antibodies having three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, for example, U.S. Patent Application Publication 2006 / 0025576A1).

[0253] The conjugates described herein (e.g., antibodies or antigen-binding fragments) also include "dual-acting FAb" or "DAF" containing antigen-binding sites that bind to two different antigens (see, for example, U.S. Patent Application Publication 2008 / 0069820 and Bostrom et al., 2009, Science 323:1610-14). "Crosumab" antibodies are also included herein (see, for example, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254 and WO2013 / 026833).

[0254] In some embodiments, the binder contains different antigen-binding sites fused to one or the other of two subunits of the Fc domain; thus, the two subunits of the Fc domain may be contained within two non-identical polypeptide chains. Recombinant co-expression of these polypeptides, followed by dimerization, results in several possible recombinations of the two polypeptides. Therefore, to improve the yield and purity of bispecific molecules in recombinant production, it is beneficial to introduce modifications into the Fc domain of the binder that promote the association of the desired polypeptides.

[0255] Accordingly, in certain embodiments, the binder comprises an Fc domain consisting of (a) at least a first binding domain, (b) a second binding domain, and (c) a first and second subunit capable of stable association, wherein the Fc domain includes a modification that facilitates the association of the first and second subunits of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the human IgG Fc domain is located in the CH3 domain of the Fc domain. Accordingly, in one embodiment, the modification is located in the CH3 domain of the Fc domain.

[0256] In specific embodiments, the Fc modification is a so-called "knob-into-hole" modification, which includes 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 certain embodiments, the first subunit of the Fc domain includes amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain includes amino acid substitutions Y349C, T366S, and Y407V (numbering according to the Kabat EU index).

[0257] The knob-into-hole technique is described, for example, in U.S. Patent No. 5,731,168; No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001). Generally, this method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, so that the protrusion can be positioned in a cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a smaller 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 a larger amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine).

[0258] Accordingly, in some embodiments, in the CH3 domain of the Fc domain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a projection within the CH3 domain that can be placed in a cavity within the CH3 domain of the second Fc domain, and in the CH3 domain of the second Fc domain, an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second Fc domain in which the projection within the CH3 domain of the first Fc domain can be placed. The projection and cavity can be created, for example, by modifying the nucleic acid encoding the polypeptide by site-directed mutagenesis or by peptide synthesis. In specific embodiments, 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, the threonine residue at position 366 is further replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A).

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

[0260] In some embodiments, the modifications that promote the association of the first Fc domain and the second Fc domain include, for example, modifications that mediate an electrostatic steering effect, as described in PCT Publication WO2009 / 089004. Generally, this method involves replacing one or more amino acid residues at the interface of the two Fc domains with charged amino acid residues such that homodimer formation is electrostatically unfavorable while heterodimerization is electrostatically favorable.

[0261] In some embodiments, the binder comprises one or more scFvs or "single-chain variable fragments". An scFv is a fusion protein 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 VH to the C-terminus of VL or vice versa. This protein retains the specificity of the original antibody despite removal of the constant regions and introduction of the linker. scFv antibodies are described, for example, in Houston, J. S., Methods in Enzymol. 203 (1991) 46-96. Methods for making scFv molecules and designing appropriate peptide linkers are described, for example, in U.S. Patent No. 4,704,692; U.S. Patent No. 4,946,778; Raag and Whitlow, FASEB 9:73-80 (1995) and Bird and Walker, TIBTECH, 9: 132-137 (1991).

[0262] The binder that is 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, e.g., WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains aligned on a single polypeptide. For example, a single polypeptide chain may comprise two single-chain Fv (scFv) fragments, each having a variable heavy chain (VH) domain and a variable light chain (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. This 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 to different proteins so that both proteins are bound by BiTE.

[0264] Since it is 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., the CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be required to separate the monomeric bispecific T cell engager from other multimer species that may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a sodium chloride concentration gradient. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimer species. In some embodiments, the conjugate, which is a bispecific antibody, consists of a single polypeptide chain containing two single-chain FV fragments (scFVs) fused to each other by a peptide linker.

[0265] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies may originate from variable domains of antibody heavy chains derived from camelid animals (e.g., nanobodies or VHH fragments). Furthermore, the term single-domain antibody includes autonomous human heavy chain variable domains (aVHs) or VNAR fragments derived from sharks (see, for example, Hasler et al., Mol. Immunol. 75:28-37, 2016).

[0266] Techniques for producing single-domain antibodies (DABs or VHHs) 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 from, for example, camels, alpacas, or llamas by standard immunoassay techniques (see, e.g., 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). VHH may possess potent antigen-binding ability 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 (HCAb) (see, e.g., Maass et al., 2007). Alpacas can be immunized with antigens, and VHH that binds to and neutralizes target antigens can be isolated (see, e.g., Maass et al., 2007). PCR primers for amplifying alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH phage display libraries that can be used for antibody fragment isolation by standard biopanning techniques well known in the field (see, e.g., Maass et al., 2007).

[0267] In some embodiments, the binder is IgG-scFV. Examples of IgG-scFv forms include IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, svFc-(L)IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody forms, as well as methods for producing 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 bivariable 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, as well as in WO08 / 024188 and WO07 / 024715. Triomabs are described by Chelius et al., MAbs 2(3):309-319, 2010. Two-in-one IgG (2-in-1-IgG) is described by Kontermann et al., Drug Discovery Today 20(7):838-847, 2015. Tandem antibodies, or TandAb, are described by Kontermann et al., ibid. The scFv-HSA-scFv antibody has also been described by Kontermann et al. (ibid.).

[0269] In some embodiments, the binding agent is a scaffold antigen-binding protein that has been used as an alternative scaffold for the antigen-binding domain, such as fibronectin and engineered ankyrin repeat protein (DARPin). 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 lipocalin (anticalin), molecules derived from protein A, e.g., the Z-domain (affibody), A-domain (avimer / maxibody) of protein A, serum transferrin (trans-body); designed ankyrin repeat protein (DARPin), fibronectin (adonectin), C-type lectin domain (tetranectin); variable domain (VNAR fragment) of novel antigen receptor beta-lactamase, human gamma-crystallin or ubiquitin (affilin molecule); Knitz-type domain of human protease inhibitor, and microbodies, e.g., proteins from the Nottin family, peptide aptamers, and fibronectin (adonectin).

[0270] Lipokalins are a family of extracellular proteins that transport small hydrophobic molecules, such as steroids, bilines, retinoids, and lipids. They have a rigid beta-sheet secondary structure with several loops at the open end of a conical structure that can be manipulated to bind to different target antigens. Antikarins are derived from lipokalins and are between 160 and 180 amino acids in size. 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] The engineered ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the binding of endogenous membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and one beta-turn. These can be engineered to bind to different target antigens by randomizing the residues in the first alpha-helix and beta-turn of each repeat. Their binding interfaces can be increased by increasing the number of modules (affinity maturation method). 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), as well as U.S. Patent Application Publication No. 20040132028A1.

[0272] Fc domain modification to change effect pedal functions 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 shows 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” means weak binding to the selected Fc gamma receptor(s). In some embodiments, “substantially no binding” means a reduction in binding affinity to the Fc gamma receptor (e.g., an increase in Kd) of at least 1 / 1000. In some embodiments, the Fc domain or region is Fc null. As used herein, “Fc null” refers to an Fc region or domain that exhibits weak binding to any of the Fc gamma receptors or no binding. In some embodiments, the Fc null domain or region exhibits a reduction in binding affinity to the Fc gamma receptor (e.g., an increase in Kd) by at least 1 / 1000.

[0273] In some embodiments, the Fc domain has reduced effector functional activity or substantially no effector functional activity. As used herein, “effector functional activity” refers to antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cell-mediated 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 a reduction in effector functional activity of at least one-thousandth compared to a wild-type Fc domain.

[0274] In some embodiments, the Fc domain has reduced ADCC activity or no ADCC activity. As used herein, reduced ADCC activity or no ADCC activity refers to a reduction 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 CDC activity or no CDC activity. As used herein, reduced CDC activity or no CDC activity means a reduction 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 times.

[0276] In vitro and / or in vivo cytotoxicity assays may be performed to confirm reduced / depleted ADCC and / or CDC activity. For example, Fc receptor (FcR) binding assays may be performed to ensure that an antibody lacks the Fc gamma receptor (and therefore may lack ADCC activity). NK cells, the primary cells for mediating ADCC, express only Fc gamma RIII, while 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 for evaluating the ADCC activity of a target molecule are described in U.S. Patent 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); and U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

[0277] C1q binding assays may also be performed to confirm that an antibody or Fc domain or region is unable to bind to C1q and therefore lacks or has reduced CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC assays may also be performed to assess 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 ADCP activity or no ADCP activity. As used herein, reduced ADCP activity or no ADCP activity means 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 times.

[0279] ADCP binding assays may also be performed to confirm that an antibody or Fc domain or region lacks ADCP activity or has reduced ADCP activity. See, for example, U.S. Patent Application Publication 20190079077 and U.S. Patent Application Publication 20190048078 and the references disclosed therein.

[0280] Antibodies with reduced effector activity include those having one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (see U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297, and Fc variants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327 (see U.S. Patent No. 7,332,581). Certain antibody variants with attenuated 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 binder comprises an Fc domain or region having one or more amino acid substitutions that attenuate Fc gamma-R binding, e.g., 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 the human IgG1 Fc region. In some embodiments, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from the 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 the human IgG1 Fc region.

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

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

[0284] For the sake of conservation substitution, amino acids can be grouped according to the similarity in the properties of their side chains (AL 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) Non-charged: 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 conserved substitutions, naturally occurring residues can be divided into groups based on common 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 affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions inevitably involve replacing one or more members of these classes.

[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, the conservatively modified variant of the binding domain is 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 greater identical to the reference VH or VL sequence, and the CDRs of the VH and VL sequences are not modified. The degree of homology (percent identity) between the reference sequence and the modified sequence can be determined, for example, by comparing the two sequences using a freely available computer program commonly used for this purpose on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0288] Modification of a 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 having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used to provide a modified nucleotide sequence having 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. Patents Nos. 4,518,584 and 4,737,462, which are thus incorporated herein by reference in their entirety.

[0289] CD8+KIR+regulatory T cells Regulatory T cells are characterized by a phenotype that is CD8+KIR+ and are typically MHC class I-restricted. In humans, CD8+Kir+ regulatory T cells express inhibitory KIR proteins. In some embodiments, the KIR proteins expressed by the cells may include one or more inhibitory KIR proteins, such as KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2; specifically, one or more of KIR2DL2, KIR2DL3, and KIR3DL1 may be mentioned. In some embodiments, CD8+KIR+ regulatory T cells are not HLA E(Qa-1b) constrained (see, for example, Lohwasser et al., International Immunology 13:321-327 (2001) and Sarantopoulos et al., J. Clin. Invest. 114(9):1218-1221 (2004) for a general description of mouse Qa-1b and human HLA E constraints). In some embodiments, CD8+KIR+ regulatory T cells may also be characterized as CD44+, CD122+ and not HLA E(Qa-1b) constrained. 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+Treg expresses the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103, and inhibitory KIR proteins.

[0291] In some embodiments, the CD8+KIR+Treg expresses 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, the CD8+KIR+Treg expresses 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, the CD8+KIR+Treg expresses one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR3, and CD122.

[0292] Production of binders In various embodiments, the binders may be produced in cell lines derived from humans, mice, or other animals. Recombinant DNA expression may be used to produce the binders. This allows for the production of antibodies, as well as a range of antigen-binding moieties and other binders (including fusion proteins) in selected host species. The production of antibodies, their antigen-binding moieties, and other binders in bacteria, yeast, transgenic animals, and chicken eggs is also an alternative to cell-based production systems. The main advantage of transgenic animals is the potential high yield from a renewable source.

[0293] As used herein, the terms “nucleic acid,” “nucleic acid sequence,” “polynucleotide sequence,” or “nucleotide” refer to polymeric molecules that incorporate units of ribonucleic acid, deoxyribonucleic acid, or their analogues. Nucleic acids can be single-stranded or double-stranded. Single-stranded nucleic acids can be the nucleic acid of one strand of denatured double-stranded DNA. In some embodiments, nucleic acids can be cDNA, for example, nucleic acids lacking introns.

[0294] Nucleic acid molecules encoding amino acid sequences of antibodies or their antigen-binding moieties and other binders can be prepared by various methods known in the art. These methods include, but are not limited to, the preparation of synthetic nucleotide sequences encoding antibodies, antigen-binding moieties, or other binders. Furthermore, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis may be used to prepare nucleotide sequences encoding antibodies or antigen-binding moieties and other binders. Nucleic acid sequences encoding at least the antibodies, their antigen-binding moieties, binders, or polypeptides described herein can be recombined with vector DNA according to conventional techniques, such as blunt-end or staggered-ended termination for ligation, restriction enzyme digestion to provide suitable termination, cohesive end filling-in where appropriate, alkaline phosphatase treatment to avoid undesirable conjugation, and ligation with appropriate ligase. Techniques for such operations are disclosed, for example, in 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 an antibody or its antigen-binding moiety or its VH and / or VL polypeptide. Where the binder contains an antibody or its antigen-binding moiety, 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, both the VH and VL polypeptides are encoded by a single nucleic acid.

[0295] A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences containing transcriptional and translational regulatory information, and such sequences are "operably linked" to nucleotide sequences encoding a polypeptide. An operable linkage is a linkage in which a regulatory DNA sequence and a DNA sequence to be expressed (e.g., an antibody or its antigen-binding portion) are connected in such a manner that it enables the gene expression of a recoverable amount of polypeptide(s) or antigen-binding portion. The exact nature of the regulatory regions required for gene expression can vary from organism to organism, as is well known in related fields. See, for example, Sambrook et al., 1989; Ausubel et al., 1987–1993.

[0296] Therefore, the expression of antibodies or their antigen-binding moieties or other binders described herein may occur in either prokaryotic or eukaryotic cells. Suitable hosts include bacterial or eukaryotic hosts, or host cells of mammalian, insect, bird, or yeast origin, including yeast, insect, fungal, avian, and mammalian cells, either in vivo or in situ. Mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, cattle, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cells may be used. Furthermore, for example, in vivo synthesis of ubiquitin-transmembrane polypeptide fusion proteins can be achieved by using a yeast ubiquitin hydrolase system. The fusion proteins thus produced may be processed in vivo or purified and processed in vitro to enable the synthesis of antibodies or their antigen-binding moieties described herein having the specified amino-terminal sequence. Furthermore, problems associated with the retention of methionine residues derived from start codons 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 the yeast gene expression systems incorporating promoters and termination elements derived from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich medium, can be used to obtain recombinant antibodies or their antigen-binding moieties or other binders. Known glycolytic genes can also provide highly efficient transcriptional regulatory signals. For example, promoter and terminator signals from phosphoglycerate kinase genes can be used.

[0297] The production of antibodies or their antigen-binding moieties and other binders in insects can be achieved, for example, by infecting an insect host with a baculovirus engineered to express polypeptides by methods known to those skilled in the art. See Ausubel et al., 1987–1993.

[0298] In some embodiments, the introduced nucleic acid sequence (encoding an antibody or its antigen-binding portion or its polypeptide or other binder) is incorporated into a plasmid or viral vector capable of autonomous replication in recipient host cells. Any of the broad range of vectors can be used for this purpose, are known to those skilled in the art, and are available. 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 not containing the vector; the desired number of copies of the vector in a particular host; and whether it is desirable that the vector can be "shuttle" between different species of host cells.

[0299] Examples of prokaryotic vectors known in this field include plasmids, for example, those that can replicate in E. coli. Other gene expression elements useful for the expression of DNA encoding antibodies or their antigen-binding portions and other binders include, but are not limited to, (a) viral transcription promoters and their enhancer elements, for example, 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 mouse leukemia virus LTR (Grosschedl et al., 41 Cell 885 (1985)); (b) splice regions and polyadenylation sites, for example, those derived from the SV40 late region (Okayarea et al., 1983); and (c) polyadenylation sites, for example, those in SV40 (Okayama et al., 1983). Immunoglobulin-coding DNA genes can be expressed using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, the SV40 late region mRNA splicing, rabbit S-globin intercalated sequences, immunoglobulin and rabbit S-globin polyadenylation sites, and SV40 polyadenylation elements as expression elements, as described in Liu et al., hereafter and Weidle et al., 51 Gene 21 (1987).

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

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

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

[0303] In some embodiments, nucleic acids containing a coding region encoding an antibody or its antigen-binding portion are assembled in separate expression vectors subsequently 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, the first selectable gene designed for selection in a bacterial system and the second selectable gene designed for selection in a eukaryotic system, with each vector having one set of coding regions. This strategy first directs the production of a nucleotide sequence in a bacterial system, resulting in a vector that enables its amplification. The DNA vector thus produced and amplified in the bacterial host is subsequently used to co-transfect eukaryotic cells, enabling the selection of co-transfected cells possessing the desired transfected nucleic acid (e.g., encoding antibody heavy and light chains). Non-limiting examples of selectable genes for use in a bacterial system include genes conferring resistance to ampicillin and genes conferring resistance to chloramphenicol. Selectable genes for use in eukaryotic transformants include the xanthine guanine phosphoribosyltransferase gene (referred to as gpt) and the Tn5-derived phosphotransferase gene (referred to as 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 their antigen-binding moieties or other binders, the recipient cell line may be a Chinese hamster ovary cell line (e.g., DG44) or myeloma cells. Myeloma cells can synthesize, assemble, and secrete immunoglobulins encoded by the transfected immunoglobulin gene and possess the mechanism for immunoglobulin glycosylation. For example, in some embodiments, the recipient cells are recombinant Ig-producing myeloma cells SP2 / 0. SP2 / 0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells may be grown in culture or in the peritoneal cavity of mice, and in the peritoneal cavity of mice, secreted immunoglobulins may be obtained from ascites fluid.

[0305] Expression vectors encoding antibodies or their antigen-binding moieties or other binders 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, as well as mechanical means such as electroporation, direct microinjection, and particle guns. (Johnston et al., 240 Science 1538 (1988), known to those skilled in the art.)

[0306] Yeast offers certain advantages over bacteria in the production of immunoglobulin heavy and light chains. Yeast performs post-translational peptide modifications, including glycosylation. Several recombinant DNA strategies exist that utilize strong promoter sequences and high copy number plasmids that can be used to produce desired proteins in yeast. Yeast recognizes the leader sequence of a cloned mammalian gene product and secretes a polypeptide (i.e., prepolypeptide) that possesses the leader sequence. See, for example, Hitzman et al., 11th Intl. Conf. Yeast, Genetics & Molec. Biol. (Montpelier, France, 1982).

[0307] Yeast gene expression systems can be conventionally evaluated for the level of production, secretion, and stability of antibodies and assembled antibodies and their antigen-binding moieties. Various yeast gene expression systems can be utilized that incorporate promoters and termination elements derived from actively expressed genes encoding glycolytic enzymes, which are produced in large quantities when yeast is grown in glucose-rich medium. Known glycolytic genes can also provide highly efficient transcriptional regulatory 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 immunoglobulin expression in yeast. See II DNA Cloning 45, (Glover, ed., IRL Press, 1985) and, for example, U.S. Patent Application Publication 2006 / 0270045A1.

[0308] Bacterial strains can also be used as hosts for the production of antibody molecules or their antigen-binding moieties or other binders described herein. E. coli K12 strain, e.g., E. coli W3110, Bacillus species, Enterobacteriaceae, e.g., Salmonella typhimurium or Serratia marcescens, and various Pseudomonas species may be used. Plasmid vectors containing replicons and regulatory sequences derived from host cell-compatible species are used in conjunction with these bacterial hosts. The vectors contain replication sites and specific genes capable of providing phenotypic selection in transformed cells. Several approaches can be taken to evaluate expression plasmids for antibody and antigen-binding moiety production 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 antibody molecules, and secretion of functional antibodies and / or their antigen-binding moieties.

[0310] Mammalian cells that may be useful as hosts for antibody protein production include, in addition to the lymphoid cells mentioned above, fibroblast-derived cells, such as Vero or CHO-K1 cells. 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, specific eukaryotic host cells are selected based on their ability to perform desired post-translational modifications to the 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 their antigen-binding moieties or other binders may be produced in vivo in animals manipulated or transfected with one or more nucleic acid molecules encoding polypeptides, according to any suitable method.

[0312] In some embodiments, the antibody or its antigen-binding moiety 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, the VH and VL chains, along with their associated constant regions as needed, can be co-expressed in the same cell to achieve intracellular association and ligation of the VH and VL chains, resulting in a complete tetrameric H2L2 antibody or its antigen-binding moiety. Co-expression can occur in the same host by using either the same or different plasmids. The nucleic acids encoding the VH and VL chains or their antigen-binding moieties may be placed in the same plasmid, which is then transfected into cells to directly select cells that express both chains. Alternatively, cells may be first transfected with a plasmid encoding one chain, e.g., the VL chain, and then the resulting cell line may be transfected with a VH chain plasmid containing a second selectable marker. Cell lines that produce antibodies, their antigen-binding moieties, or other binders via any of these pathways may be transfected with plasmids encoding additional copies of peptide, VH, VL, or VH-plus-VL chains, along with additional selectable markers, to generate cell lines with enhanced properties, such as higher production of assembled antibodies or their antigen-binding moieties, or enhanced stability of the transfected cell line.

[0314] Furthermore, plants are emerging as a simple, safe, and economical alternative expression system for recombinant antibody production based on large-scale cultures of microbial or animal cells. Antibodies or antigen-binding moieties can be expressed in plant cell cultures or in normally grown plants. Expression in plants may be systemic, limited to intracellular plastids, or limited to seeds (endosperm). See, for example, U.S. Patent Application Publication 2003 / 0167531; U.S. Patent No. 6,080,560; U.S. 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 ligated to at least a portion of the immunoglobulin constant region (Fc), typically that of human immunoglobulin. Human constant region DNA sequences can be isolated from various human cells, e.g., immortalized B cells, by well-known procedures (see, e.g., WO87 / 02671; this is thus incorporated herein by reference in its entirety). Antibodies may contain both light chain and heavy chain constant regions. The heavy chain constant region may include the CH1, hinge, CH2, CH3, and sometimes the CH4 region. In some embodiments, the CH2 domain may be deleted or omitted.

[0316] Alternatively, the described techniques for the production of single-chain antibodies (see, for example, U.S. Patent 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); these are incorporated herein by reference in their entirety) may be adapted to produce single-chain antibodies that specifically bind to a desired antigen. Single-chain antibodies are formed by linking the heavy chain and light chain variable regions of the Fv region via amino acid crosslinking to produce a single-chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (see, for example, Skerra et al., Science 242:1038-1041 (1988); this is incorporated herein by reference in its entirety). Methods for preparing other binders are described above.

[0317] Intact (e.g., whole) antibodies, their dimers, individual light and heavy chains, or their antigen-binding moieties can be recovered and purified by known techniques, e.g., immunoadsorption or immunoaffinity chromatography, chromatography methods, e.g., HPLC (high-performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination thereof. See, for general information, Scopes, Protein Purification (Springer-Verlag, NY, 1982). Particularly beneficial for pharmaceutical use are antibodies with 98%–99% or greater homogeneity; therefore, substantially pure antibodies or their antigen-binding moieties with at least approximately 90%–95% homogeneity are desirable. Once partially or homogeneously purified as desired, the intact antibodies or their antigen-binding moieties can then be used therapeutically or in the development and implementation of assay procedures, immunofluorescence staining, etc. For general information, please refer to Vols. I & II Immunol. Meth. (Lefkovits & Pernis, eds., Acad. Press, NY, 1979 and 1981).

[0318] Pharmaceutical preparations In some embodiments, the binder relates to a composition comprising an active ingredient (i.e., a nucleic acid encoding the binder described herein or an antibody or its antigen-binding moiety or other binder described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term “pharmaceutical composition” means an active agent combined with a pharmaceutically acceptable carrier permitted for use in the pharmaceutical industry. The term “pharmaceutically acceptable” is used herein to mean a compound, material, composition and / or dosage form that is appropriate for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications, and that is balanced by a reasonable benefit / risk ratio, within the bounds of reasonable medical judgment.

[0319] The preparation of pharmacological compositions containing an active ingredient dissolved or dispersed therein is well understood in the art and does not need to be limited to any particular formulation. Typically, such compositions are prepared as injectables, either as liquids or suspensions; however, solid forms suitable for rehydration or suspension in liquid before use may also be prepared. Preparations may also be emulsified or presented as liposome compositions. Antibodies or their antigen-binding moieties or other binders may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. Furthermore, if desired, the pharmaceutical composition may contain trace amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., that enhance or maintain the efficacy of the active ingredient (e.g., antibody or its antigen-binding moiety or other binder). The pharmaceutical compositions described herein may contain pharmaceutically acceptable salts of the constituent components. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or with organic acids such as acetic acid, tartaric acid, or mandelic acid (formed with the free amino group of the polypeptide). Salts formed with the free carboxyl group may 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, or procaine. Physiologically tolerable carriers are well known in the art. An exemplary liquid carrier is a sterile aqueous solution containing an active ingredient (e.g., an antibody and / or its antigen-binding moiety or other binder) and water, which may contain a buffer, such as sodium phosphate, physiological saline, or both at physiological pH, such as phosphate-buffered saline. Furthermore, aqueous carriers may contain more than one buffer salt, as well as salts, such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. In addition to water, and in addition to the removal of water, the liquid composition may also contain a liquid phase.Examples of such additional liquid phases include glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active agent effective in treating a particular disorder or condition depends 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 its antigen-binding moiety or other binder described herein, or a nucleic acid encoding an antibody or its antigen-binding moiety or other binder, may be a lyophilized product.

[0321] In some embodiments, a syringe containing a therapeutically effective amount of the binder or pharmaceutical composition described herein is provided.

[0322] Treatment of inflammatory and autoimmune diseases In some embodiments, the binders described herein may be used in a method comprising the step of administering the binder or pharmaceutical composition described herein to a subject having an inflammatory disease. In some embodiments, the binders described herein may be used in a method comprising the step of administering the binder or pharmaceutical composition described herein to a subject having an autoimmune disease or an immune response to an autoantigen or 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 requires treatment for an autoimmune disease. In some embodiments, a method is provided for treating an autoimmune disease, comprising the step of administering either the binder or pharmaceutical composition described herein to a subject in need, in an amount effective in reducing 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 the step of contacting CD8+KIR+T regulatory cells (Tregs) with either a binder or a pharmaceutical composition described herein in an amount effective to activate or stimulate the 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 to an autoantigen, comprising the step of administering either a binder or a pharmaceutical composition described herein to a subject requiring such action in an amount effective to activate or stimulate the 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 to an autoantigen that causes or results in the development of an autoimmune disorder, comprising the step of administering either a binder or a pharmaceutical composition described herein to a subject in need of such use in an amount effective to activate or stimulate CD8+KIR+Treg, 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 autoantigen that causes or results in the development of an autoimmune disorder, comprising the step of administering either a binder or a pharmaceutical composition described herein to a subject in need of such use in an amount effective to activate or stimulate CD8+KIR+Treg, 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 the step of administering to a subject in need of either a binder or a pharmaceutical composition described herein in an amount effective to activate or stimulate CD8+KIR+Treg, thereby reducing or suppressing the immune response to the antigen or autoantigen in the subject.

[0323] As used herein, the term “autoantigen” means an antigen (e.g., a cell surface protein or other antigen) that is normally recognized as self by the immune system (e.g., the healthy human immune system). Antigens or autoantigens are targets of humoral or cell-mediated immune responses, or induce an excessive immune response, in autoimmune diseases or conditions (collectively, “autoimmune diseases”). As used herein, “activating or stimulating” CD8+KIR+Treg means that activated CD8+KIR+Tregs increase the regulatory T cell function of such cells, e.g., the ability to suppress immune responses, particularly immune responses to autoantigens, or to suppress immune responses to autoantigens that cause or result in the development of autoimmune disorders. Activation or stimulation of CD8+KIR+Tregs may include the removal of their inhibitory effects on such cells in order to restore the CD8+KIR+Tregs (e.g., to restore balance to the immune system or restore balanced immune activity in a subject or in a subject requiring treatment before the onset of disease). Activation or stimulation of CD8+KIR+Treg may also include the removal of such cells, such as CD4+ cells, B cells, or other cells that mediate the immune response, by cell lysis.

[0324] As used herein, pathogenic immune cells refer to immune cells that are reactive to or induce a response to autoantigens. Examples of such pathogenic immune cells include, as is well known in the art, autoreactive CD4+ T cells, autoantibody-producing B cells, autoantigen-presenting dendritic cells, and other autoantigen-presenting cells.

[0325] In some embodiments, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. The activated CD8+KIR+Treg can then be administered in an effective dose to a subject requiring it.

[0326] In some embodiments, 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, 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, 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, activated CD8+KIR+Treg modulates the pathogenic effect of pathogenic immune cells and reduces the titer of autoantibodies in the target. In some embodiments, activated CD8+KIR+Treg reduces the titer of autoantibodies in the target.

[0327] In some embodiments, the binder is selected from any of the binders described herein, each having reduced effector activity or substantially no effector activity. In some embodiments, reduced effector activity means reduced ADCC, ADCP, or CDC effector activity, or the absence of ADCC, ADCP, or CDC effector activity. In some embodiments, substantially no effector activity means substantially no ADCC, ADCP, and CDC effector activity. In some embodiments, the binder lacks an Fc domain or region and has reduced effector activity or substantially no effector activity. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region, e.g., an Fc null substitution. In some embodiments, the binder is either lacking an Fc domain or region, or having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder is lacking an Fc domain or region. In some embodiments, the binder is either having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder has an Fc domain or region having reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0328] While not intended to be bound by any particular theory, the reduction or absence of effector functional activity by the binder may limit the interaction between the binder and other cell types (i.e., non-CD8+KIR+Treg) and / or limit the depletion of CD8+KIR+Treg bound to the binder.

[0329] In some embodiments, the subjects requiring treatment have autoimmune diseases. In some embodiments, the subjects requiring treatment have autoimmune diseases 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, Behçet's disease, avian lung, bronchial asthma, Kaplan syndrome, cardiomyopathy, celiac disease, Chagas disease, chronic glomerulonephritis, chronic graft-versus-host disease, Cogan syndrome, cold agglutinin disease, CREST syndrome, Crohn's disease, cryoglobulinemia, Cushing's syndrome, dermatomyositis, discoid lupus erythematosus, and Dressier's syndrome. Eaton-Lambert syndrome, encephalomyelitis, endocrine ophthalmopathy, lupus erythematosus, Evans syndrome, Felty syndrome, fibromyalgia, Fuchs cyclitis, gastric atrophy, gastrointestinal allergy, giant cell arteritis, glomerulonephritis, Goodpasture syndrome, graft-versus-host disease, Graves' disease, Guillain-Barré 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 1), juvenile arthritis, juvenile idiopathic arthritis, juvenile diabetes mellitus (type 1), Lambert-Eaton syndrome Laminitis, lichen planus, lupoid hepatitis, lupus, lupus nephritis, lymphopenia, macroglobulinemia, Meniere's disease, mixed connective tissue disease, monoclonal gammaglobulinemia of unknown origin, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus / bullous pemphigoid, 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, Schulmann syndrome, Sjögren's syndromeThey may have conditions such as Stiffman syndrome, sympathetic ophthalmitis, systemic lupus erythematosus, Takayasu's arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyrotoxicosis, toxic epidermal necrolysis, type B insulin resistance, type 1 diabetes mellitus, ulcerative colitis, uveitis, leukoplakia, Waldenström macroglobulinemia and / or Wegener's granulomatosis.

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

[0332] The methods described herein include the step of administering a therapeutically effective dose of a binder to a subject having an autoimmune disease. As used herein, the terms “therapeutic dose,” “effective dose,” “effective amount,” and “effective dosage” may mean the amount of the binder described herein that provides a therapeutic benefit in treating an autoimmune disease, managing an autoimmune disease, preventing recurrence of an autoimmune disease, delaying the onset of an autoimmune disease, or preventing the onset of an autoimmune disease, for example, the amount that provides a statistically significant reduction in at least one symptom, sign, or marker of an autoimmune disease. Determining the therapeutically effective dose is well within the capabilities of those skilled in the art. Generally, the therapeutically effective dose may vary depending on the subject’s medical history, age, condition, and sex, as well as the severity and type of the medical condition in the subject, and the administration of other pharmaceutically active agents.

[0333] The methods described herein are intended to reduce symptoms, lesions, disease progression, or disease relapse in subjects. As used herein, “subject” refers to a human or an animal. Typically, animals are vertebrates, e.g., primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, e.g., domestic cats, canids, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient,” “individual,” and “subject” are used interchangeably herein.

[0334] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be usefully used, for example, as subjects to present animal models of various autoimmune diseases. Furthermore, the methods described herein may be used to treat domesticated animals and / or pets. The subject may be male or female. In certain embodiments, the subject is human.

[0335] A subject may be one who has been previously diagnosed with or identified as having an autoimmune disease and requires treatment, but does not need to have already received treatment for the autoimmune disease. Alternatively, a subject may also be one who requires treatment but has not been previously diagnosed with an autoimmune disease. A subject may exhibit one or more risk factors for a condition or one or more complications associated with an autoimmune disease, or may not exhibit any risk factors. A subject “requiring treatment” for an autoimmune disease may be one who has the disease or one who has been diagnosed with the disease. In some embodiments, a subject requires treatment if there is a risk that the subject will develop an immune response to an antigen or autoantigen that can cause or may cause the development of an autoimmune disorder. In other embodiments, a subject “at risk of developing” an autoimmune disease refers to one who has been diagnosed as being at risk of developing the disease or condition.

[0336] As used herein, the terms “to treat,” “treatment,” “to treat,” or “to alleviate,” when used in reference to a disease, disorder, or medical condition (e.g., autoimmune disease), refer to a therapeutic treatment of the condition, the purpose of which is to reverse, reduce, alleviate, inhibit, slow, or halt the progression or severity of the symptoms or condition. The term “to treat” includes reducing or mitigating 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 interrupted. That is, “treatment” may include not only improvement of symptoms or markers but also cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of the treatment. Beneficial or desired clinical outcomes include, but are not limited to, reduction of one or more symptoms, reduction of disease relapse in the subject, reduction of one or more symptoms, attenuation of the degree of defect, stabilization (i.e., non-worsening) of an autoimmune disease, or prevention, delay, or slowing of the onset or progression of an autoimmune disease. As used herein, the term “administer” means bringing a binder described herein or the nucleic acid encoding a binder described herein into contact (e.g., by administration to a subject) by a method or route that results in the binding of the binder to CD8+KIR+Treg. Similarly, pharmaceutical compositions disclosed herein, comprising a binder described herein or the nucleic acid encoding a binder described herein, may be administered by any suitable route that results in an effective treatment in a subject.

[0337] The dosage range for the binder depends on the efficacy and includes a sufficiently large amount to produce the desired effect, e.g., reduction of one or more symptoms, reduction of disease relapse in the subject, alleviation of one or more symptoms, reduction of the degree of defect, stabilization (i.e., non-exacerbating) of the autoimmune disease, or prevention, delay, or slowing of the onset or progression of the autoimmune disease. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary depending on the age, condition, and sex of the subject and may be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if there are any complications. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, the dosage ranges from about 0.5 mg / kg body weight to about 15 mg / kg body weight. In some embodiments, the dose range is about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be dose-set 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 conjugates 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 conjugates described herein, for example, for the prevention of an immune response that may result in an autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the conjugates 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 conjugates described herein, for example, for the prevention of an immune response that may result in 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 over several weeks, months, or years. The duration of treatment depends on the clinical progression of the subject and their response to the treatment.

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

[0340] A pharmaceutical composition containing any of the binders described herein may be administered in unit doses. When used in reference to a pharmaceutical composition, the term “unit dose” refers to a physically distinct unit appropriate as a unit dosage for a subject, each unit containing a predetermined amount of active material (e.g., a binder) calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., a carrier or vehicle.

[0341] In some embodiments, administration of any of the binders described herein may result in improved treatment outcomes, such as a reduction in systemic inflammatory cytokines, a reduction in lesions in disease-affected tissues, a reduced frequency and / or severity of relapses, a reduction in self-reported disease-related symptoms, a reduction in one or more symptoms, and / or prevention, delay, or slowing of the onset or progression of autoimmune disease.

[0342] In some embodiments, the binders or any of the pharmaceutical compositions of the binders described herein are administered in conjunction with immunotherapy. As used herein, “immunotherapy” refers to a therapeutic strategy designed to modulate the subject’s own immune system. Examples of immunotherapies, but not limited to, include antibodies, e.g., checkpoint inhibitors and modulators, and immunosuppressants, e.g., cyclosporine, cyclosporine A, mycophenolate mofetil, sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate, cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab, abatacept, beratacept, and adalimumab.

[0343] Treatment of transplant complications In some embodiments, methods are provided for treating transplant complications associated with graft-versus-host disease, or GVHD. In some embodiments, methods are provided for reducing GVHD complications in a subject, comprising the step of administering a conjugate or pharmaceutical composition described herein to a transplanted subject to suppress the host immune response to the transplant. In some embodiments, methods are provided for reducing GVHD complications in a subject, comprising the step of administering a conjugate or pharmaceutical composition described herein to a transplanted subject to suppress the immune response associated with the transplant. In some embodiments, methods are provided for reducing GVHD, comprising the step of administering a conjugate or pharmaceutical composition described herein to a transplanted subject currently experiencing GVHD, thereby reducing the symptoms of GVHD. In some embodiments, methods are provided for suppressing GVHD, comprising the step of administering a conjugate or pharmaceutical composition described herein to a transplanted subject currently experiencing GVHD, thereby suppressing or reducing GVHD. In some embodiments, a method is provided for suppressing or reducing GVHD, comprising the step of contacting CD8+KIR+T regulatory cells (Treg) derived from a transplanted subject with a binder or pharmaceutical composition described herein, thereby activating the CD8+KIR+T regulatory cells (Treg). In some embodiments, a method is provided for suppressing or reducing GVHD, comprising the step of contacting CD8+KIR+T regulatory cells (Treg) derived from a transplanted subject, thereby depleting the transplanted CD8+KIR+T regulatory cells (Treg). In some embodiments, a method is provided for suppressing or reducing GVHD, comprising the step of contacting CD8+KIR+T regulatory cells (Treg) derived from a transplanted subject with a binder or pharmaceutical composition described herein, thereby depleting the CD8+KIR+T regulatory cells (Treg).

[0344] In some embodiments, subjects have undergone hematopoietic stem cell transplantation, umbilical cord blood stem cell transplantation, transplantation of induced pluripotent stem cell precursors or differentiated cells, bone marrow transplantation, or solid organ transplantation. The transplantation is typically allogeneic transplantation. In some embodiments, subjects have undergone hematopoietic stem cell transplantation, umbilical cord blood stem cell transplantation, transplantation of induced pluripotent stem cell precursors or differentiated cells, bone marrow transplantation, or solid organ transplantation and are currently experiencing GVHD. In some embodiments, subjects have undergone hematopoietic stem cell transplantation, umbilical cord blood stem cell transplantation, transplantation of induced pluripotent stem cell precursors or differentiated cells, bone marrow transplantation, or solid organ transplantation and are at risk of experiencing GVHD. In some embodiments, subjects have undergone hematopoietic stem cell transplantation and are currently experiencing GVHD. In some embodiments, subjects have undergone hematopoietic stem cell transplantation and are at risk of experiencing GVHD. In some embodiments, subjects have undergone umbilical cord blood stem cell transplantation and are currently experiencing GVHD. In some embodiments, subjects have undergone umbilical cord blood stem cell transplantation and are at risk of experiencing GVHD. In some embodiments, the subjects have received transplantation of induced pluripotent stem cell precursors or differentiated cells and are currently experiencing GVHD. In some embodiments, the subjects have received transplantation of induced pluripotent stem cell precursors or differentiated cells and are at risk of experiencing GVHD. In some embodiments, the subjects have received bone marrow transplantation and are currently experiencing GVHD. In some embodiments, the subjects have received bone marrow transplantation and are at risk of experiencing GVHD. In some embodiments, the subjects have received solid organ transplantation and are currently experiencing GVHD. In some embodiments, the subjects have received solid organ transplantation and are at risk of experiencing GVHD.

[0345] In some embodiments, GVHD is suppressed or reduced by depletion of pathogenic immune cells, such as CD4 T cells. In some embodiments, GVHD is suppressed or reduced by depletion of pathogenic immune cells, such as transplant-derived CD8+KIR+Tregs. In some embodiments, CD8+KIR+Tregs are contacted with a binder in vivo. In some embodiments, CD8+KIR+Tregs are contacted with a binder ex vivo. The activated CD8+KIR+Tregs can then be administered in an effective dose to a target requiring them.

[0346] In some embodiments, the binder is selected from any of the binders described herein, each having reduced effector activity or substantially no effector activity. In some embodiments, reduced effector activity means reduced ADCC, ADCP, or CDC effector activity, or the absence of ADCC, ADCP, or CDC effector activity. In some embodiments, substantially no effector activity means substantially no ADCC, ADCP, and CDC effector activity. In some embodiments, the binder lacks an Fc domain or region and has reduced effector activity or substantially no effector activity. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region, such as an Fc null substitution. In some embodiments, the binder is either lacking an Fc domain or region, or having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder is lacking an Fc domain or region. In some embodiments, the binder is either having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder has an Fc domain or region having 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 binder is selected from any of the binders described herein, each having effector functional activity comprising at least ADCC. In some embodiments, the effector functional activity is ADCC combined with ADCP and / or CDC effector functional activity. In some embodiments, having effector functional activity means having ADCC, ADCP, and CDC effector functional activity. In various embodiments, such binders have an Fc domain, or have an Fc domain that binds to one or more Fc gamma receptors.

[0348] While not intended to be bound by any particular theory, the reduction or absence of effector functional activity by the binder may limit the interaction between the binder and other cell types (i.e., non-CD8+KIR+Treg) and / or limit the depletion of CD8+KIR+Treg. In contrast, the presence of effector functional activity by the binder is thought to bias the immune response toward the depletion of CD8+KIR+Treg.

[0349] In some embodiments, transplantation is a stem cell transplant, a bone marrow transplant, or a solid organ transplant. As used herein, the term “transplantation” means an organ, tissue, or cell that has been transplanted from one subject to another subject, or within the same subject (e.g., to different regions within the subject). Organs, e.g., liver, kidney, heart, or lung, or other body parts, e.g., bone or skeletal matrix, e.g., bone marrow, tissues, e.g., 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 induced pluripotent stem cell-derived precursors or differentiated cells are all examples of transplantation. In some embodiments, solid organ transplantation is a liver, kidney, lung, pancreas, and / or heart transplant. The term transplantation includes grafts. A transplant may be an allograft (or allogeneic graft) or a xenograft. The term "allograft" refers to a graft between two genetically non-identical members of the same 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 precursors or differentiated cells.

[0351] In some embodiments, the subjects have graft-versus-host disease, or GVHD. In some embodiments, the subjects are at risk of having GVHD.

[0352] The methods described herein include the step of administering a therapeutically effective dose of a binder to a subject currently undergoing transplantation. As used herein, the terms “therapeutic dose,” “effective dose,” “effective amount,” and “effective dosage” may refer to the amount of the binder described herein that provides a therapeutic benefit in the management of transplant-related GVHD. In some embodiments, the therapeutic benefit is the delay 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 the therapeutic dose is well within the capabilities of those skilled in the art. Generally, the therapeutic dose may vary depending on the subject’s medical history, age, condition, and sex, as well as the severity and type of the subject’s medical condition, and the administration of other pharmaceutically active agents.

[0353] The methods of the present invention are intended to reduce, mitigate, or prevent GVHD in transplanted subjects. As used herein, “subject” refers to a human or an animal. Typically, animals are vertebrates, e.g., primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, e.g., domestic cats, canines, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient,” “individual,” and “subject” are used interchangeably in this specification.

[0354] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be usefully used, for example, as subjects to present animal models of various autoimmune diseases. Furthermore, the methods described herein may be used to treat domesticated animals and / or pets. The subject may be male or female. In certain embodiments, the subject is human.

[0355] The subjects may be those who will receive a transplant. Alternatively, the subjects may be those who have already received a transplant. The subjects may be those who will receive a transplant and are at risk of GVHD. The subjects may be those who have received a transplant and are at risk of having GVHD. The subjects may be those who have received a transplant and have GVHD.

[0356] As used herein, the terms “to treat,” “treatment,” “to treat,” or “relieve,” when used in reference to a disease, disorder, or medical condition (e.g., GVHD), refer to a therapeutic treatment of the condition whose purpose is to reverse, reduce, alleviate, inhibit, slow, or halt the progression or severity of the symptoms or condition of GVHD. The term “to treat” includes reducing or mitigating 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 interrupted. That is, “treatment” may include not only improvement of symptoms or markers but also 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 “administer” means bringing a binder described herein or the nucleic acid encoding a binder described herein into contact (e.g., by administration to a subject) by a method or route that results in the binding of the binder to CD8+KIR+Treg. Similarly, a pharmaceutical composition comprising a binder described herein or the nucleic acid encoding a binder described herein, as disclosed herein, may be administered by any suitable route that results in an effective treatment in a subject.

[0358] The dosage range for the binder depends on the efficacy and includes a sufficiently large amount to produce the desired effect, e.g., reduction of one or more symptoms of GVHD or reduction or prevention of GVHD. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage may vary depending on the age, condition, and sex of the subject and may be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if there are any complications. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, the dosage ranges from about 0.5 mg / kg body weight to about 15 mg / kg body weight. In some embodiments, the dose range is about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be dose-set 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 conjugates described herein, for example, for the treatment of acute GVHD after transplantation. In some embodiments, the subject receives repeated doses of any of the conjugates described herein, for example, for the treatment of chronic GVHD. In some embodiments, the dose is administered weekly, every two weeks, every three weeks, monthly, every two months, or every six months over several weeks, months, or years. The duration of treatment depends on the clinical progression of the subject and their response to treatment.

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

[0361] A pharmaceutical composition containing any of the binders described herein may be administered in unit doses. When used in reference to a pharmaceutical composition, the term “unit dose” refers to a physically distinct unit appropriate as a unit dosage for a subject, each unit containing a predetermined amount of active material (e.g., a binder) calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., a carrier or vehicle.

[0362] In some embodiments, administration of any of the binders described herein improves treatment outcomes, such as reducing systemic inflammatory cytokines, reducing lesions in disease-affected tissues, reducing self-reported symptoms related to immune responses associated with adverse effects on host tissues, and transplantation. thing To improve or prolong graft survival, alleviate one or more symptoms, and / or transplantation. thing Prevention, delay, or slowing of the onset or progression of rejection, or widening Regional spectrum Reduced use of immunosuppressants, such as corticosteroids. accompanied transplant thing This can result in an extended period of engraftment.

[0363] In some embodiments, the binders described herein or any of the binder pharmaceutical compositions are administered together with an immunosuppressant, such as a corticosteroid(s).

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

[0365] In some embodiments of the method for treating an infection, CD8+KIR+Treg is contacted in vivo with one of the binders described herein. In some embodiments, CD8+KIR+Treg is contacted ex vivo with one of the binders described herein. The activated CD8+KIR+Treg is subsequently administered in an effective dose to a subject requiring it. In some embodiments, the immune response includes a reduction in immunosuppressive immune cells. When used herein, immunosuppressive immune cells include CD4 Treg and tolerant DCs. In some embodiments, the number of infected cells in the subject is reduced.

[0366] In some embodiments, the binder is selected from any of the binders described herein, each having reduced effector activity or substantially no effector activity. In some embodiments, reduced effector activity means reduced ADCC, ADCP, or CDC effector activity, or the absence of ADCC, ADCP, or CDC effector activity. In some embodiments, substantially no effector activity means substantially no ADCC, ADCP, and CDC effector activity. In some embodiments, the binder lacks an Fc domain or region and has reduced effector activity or substantially no effector activity. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region, such as an Fc null substitution. In some embodiments, the binder is either lacking an Fc domain or region, or having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder is lacking an Fc domain or region. In some embodiments, the binder is either having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder has an Fc domain or region having reduced binding to one or more Fc gamma receptors due to amino acid substitutions in the Fc domain or region.

[0367] While not intended to be bound by any particular theory, the reduction or absence of effector functional activity by the binder may limit the interaction between the binder and other cell types (i.e., non-CD8+KIR+Treg) and / or limit the depletion of CD8+KIR+Treg bound to the binder.

[0368] In some embodiments, the subject is someone who requires treatment for an infectious disease or is infected with an infectious agent, such as a bacterial disease, systemic fungal disease, rickettsial disease, parasitic disease, or viral disease. In some embodiments, the infectious disease is a bacterial disease, such as diphtheria, pertussis, latent bacteremia, urinary tract infection, gastroenteritis, cellulitis, epiglottitis, tracheitis, adenoid hypertrophy, retropharyngeal abscess, impetigo, pustule, pneumonia, endocarditis, suppurative arthritis, pneumococcal pneumonia, peritonitis, bacteremia, meningitis, acute suppurative meningitis, urethritis, cervicitis, proctitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, anthrax, nocardiosis, salmonella, typhoid fever, dysentery, conjunctivitis, sinusitis, or brucellosis. Tularemia, cholera, bubonic plague, tetanus, necrotizing enterocolitis, actinomycosis, mixed anaerobic infections, syphilis, relapsing fever, leptospirosis, Lyme disease, rat-bite fever, tuberculosis including Mycobacterium tuberculosis, lymphadenitis, leprosy, chlamydia, chlamydial pneumonia, trachoma or inclusion conjunctivitis; systemic fungal diseases, e.g., histoplasmosis, coccidioidomycosis, blastomycosis, sporotrichumosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, mycomatosis or melanomycosis; rickettsial diseases, e.g., typhus, Rocky Mountain spotted fever, ehrlichiosis, Eastern tick-borne rickettsial diseases. Rickettsioses, rickettsial pox, Q fever or bartonellosis; parasitic diseases, e.g., malaria, babesiosis, African sleeping sickness, Chagas disease, leishmaniasis, dum-dum fever, toxoplasmosis; meningoencephalitis, keratitis, dinuclear amebiasis, giardiasis, cryptosporidiosis, isosporiasis, cyclosporasis, microsporidiasis, ascariasis, whipworm infection, hookworm infection, strongyloidiasis, ocular larval migrans, trichinellosis, dung worm disease, lymphangiofilariasis, loa filariasis, river blindness, heartworm infection, schistosomiasis, cercariasis (swimmer's itch), Oriental lung fluke, Oriental liver fluke Fluke infections, hepatitis, hypertrophic fluke infections, opisthochiosis, tapeworm infections, hydatid infections, and pulmonary hydatid infections;Furthermore, viral diseases such as measles, subacute sclerosing panencephalitis, common cold, mumps, rubella, roseola, erythema infectiosum, chickenpox, coronavirus infection, Covid-19, polynuclear respiratory virus infection, croup, bronchiolitis, infectious mononucleosis, poliomyelitis, herpangina, hand-foot-and-mouth disease, Bornholm's disease, genital herpes, genital warts, aseptic meningitis, myocarditis, echovirus infection, 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, and viral "walking" pneumonia. These include 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, giant cell inclusion disease, rabies, progressive multifocal leukoencephalopathy, kuru, fatal familial insomnia, Creutzfeldt-Jakob disease, Gerstmann-Sfraussler-Scheinker disease, tropical spastic paraplegia, 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 infection is a viral infection. In some embodiments, the infection 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-CoV-2 infection (Covid-19) and influenza virus infection (influenza). In some embodiments, the infection is caused by an infectious agent, such as coronavirus, diphtheria, Ebola, influenza (flu), HIV, human papillomavirus (HPV), hepatitis A, hepatitis B, hepatitis C, measles virus, polynuclear respiratory virus, rotavirus and herpesvirus.

[0370] The methods described herein include the step of administering a therapeutically effective dose of a binder to a subject having an infection or a subject having cells infected with an infectious agent. As used herein, the terms “therapeutic dose,” “effective dose,” “effective dose,” or “effective amount” may mean any amount of any of the binders or pharmaceutical compositions described herein that provides a therapeutic benefit in the treatment of an infectious disease, the management of an infectious disease, or the prevention of recurrence of an infectious disease, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of the infection. Determining the therapeutically effective dose is well within the capabilities of those skilled in the art. Generally, the therapeutically effective dose may vary depending on the subject’s medical history, age, condition, and sex, as well as the severity and type of the medical condition in the subject, and the administration of other pharmaceutically active agents.

[0371] As used herein, “subject” refers to a human or an animal. Typically, animals are vertebrates, e.g., primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, e.g., domestic cats, canids, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient,” “individual,” and “subject” are used interchangeably herein.

[0372] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be usefully used, for example, as subjects for presenting animal models of various infectious diseases. Furthermore, the methods described herein may be used to treat domesticated animals and / or pets. The subject may be male or female. In certain embodiments, the subject is human.

[0373] A subject may be one who has been previously diagnosed with or identified as having an infectious disease and requires treatment, but does not need to have already received treatment for the infectious disease. Alternatively, a subject may also be one who has not been previously diagnosed with an infectious disease but requires treatment. A subject may exhibit one or more risk factors for an infectious disease-related condition or one or more complications, or may not exhibit such risk factors. A subject “requiring treatment for infectious disease” may be one who has the infectious disease or one who has been diagnosed with the infectious disease. In other embodiments, a subject “at risk of developing” an infectious disease refers to one who has been diagnosed as being at risk of developing an infectious disease.

[0374] As used herein, the terms “to treat,” “treatment,” “to treat,” or “to alleviate,” when used in reference to a disease, disorder, or medical condition (e.g., an infection), refer to a therapeutic treatment of the condition whose purpose is to reverse, reduce, alleviate, inhibit, prevent, slow, or halt the progression or severity of the symptoms or condition. The term “to treat” includes reducing or mitigating 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 interrupted. That is, “treatment” includes not only improvement of symptoms or markers but also cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of the treatment. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in infected cells in the subject, a reduction in one or more symptoms, a reduction in the degree of defect, a stabilized (i.e., non-worsening) state of the infection, or a delay or slowing of the infection or its progression, compared to what would be expected in the absence of the treatment. As used herein, the term “administer” means bringing a binder described herein or the nucleic acid encoding a binder described herein into contact (e.g., by administration to a subject) by a method or route that results in the binding of the binder to CD8+KIR+Treg. Similarly, pharmaceutical compositions disclosed herein, comprising a binder described herein or the nucleic acid encoding a binder described herein, may be administered by any suitable route that results in an effective treatment in a subject.

[0375] The dosage range for the binder depends on the efficacy and includes a sufficiently large amount to produce the desired effect, such as stimulating an immune response against infected cells, reducing the number of infected cells, or slowing or preventing the progression of infection. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage may vary depending on the age, condition, and sex of the subject and may be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if there are any complications. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage ranges from about 0.1 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage ranges from 0.5 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage range is about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be set 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 binders or pharmaceutical compositions described herein, for example, for the treatment of an acute infection. In some embodiments, the subject receives a single dose of any of the binders or pharmaceutical compositions described herein, for example, for the prevention of an immune response to an infection that may result in an autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binders or pharmaceutical compositions described herein, for example, for the treatment of a chronic infection. In some embodiments, the dose is administered weekly, every two weeks, every three weeks, monthly, every two months, or every six months over several weeks, months, or years. The duration of treatment depends on the clinical progression of the subject and their response to the treatment.

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

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

[0379] Pharmaceutical compositions containing a binder may be administered in unit doses. When the term “unit dose” is used in reference to a pharmaceutical composition, it refers to a physically distinct unit appropriate as a unit dosage for the subject, each unit containing a predetermined amount of the active material (e.g., binder) calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., a carrier or vehicle.

[0380] In some embodiments, administration of any of the binders described herein may result in improved treatment outcomes, such as relief of clinical symptoms, reduction of viral load, or elimination or reduction of pathogens or a reduction of infected cells.

[0381] In some embodiments, any of the binders or pharmaceutical compositions of the embodiments described herein are administered together with infectious disease control agents, such as antibacterial agents, antifungal agents, or antiviral agents.Antibacterial gents include, for example, lactam antibiotics such as penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bacampicillin, azulocillin, carbenicillin, mezlocillin, piperacillin, or ticalcillin; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netylmycin, streptomycin, or tobramycin; and macrolides such as azithromycin, clarithromycin. Erythromycin, lincomycin, or clindamycin; tetracyclines, e.g., demeclocycline, doxycycline, minocycline, oxytetracycline, or tetracycline; quinolones, e.g., cinoxacin or nalidixic acid; fluoroquinolones, e.g., ciprofloxacin, enoxacin, glepafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin, or trovafloxacin; polypeptides, e.g., bacitracin, colisti or polymyxin B; sulfonamides, e.g., sulfisoxazole, sulfamethoxazole, sulfadiazine, sulfamethazole or sulfacetamide; and other antibacterial agents, e.g., trimethoprim, sulfamethazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dalfopristin, rifampin, spectinomycin or nitrofurantoin; and antiviral agents, e.g., general antiviral agents, e.g., idoxuridiol These may include vidarabine, trifluridine, acyclovir, famciclovir, penciclovir, valacyclovir, ganciclovir, foscarnet, ribavirin, amantadine, rimantadine, or cidofovir; antisense oligonucleotides; immunoglobulins; interferons; and other drugs, such as zidovudine, didanosine, zalcitabine, stabudine, lamivudine, nevirapine, delavirudine, saquinavir, oseltamivir, and peramivir, ritonavir, indinavir, or nelfinavir.

[0382] Cancer treatment In some embodiments, the binders or pharmaceutical compositions described herein may be used in methods for treating cancer, comprising the step of administering the binders or pharmaceutical compositions described herein to a subject requiring such administration. In some embodiments, a method for treating cancer is provided, comprising the step of administering one of the binders or pharmaceutical compositions described herein to a subject requiring such administration in an amount effective to activate or stimulate CD8+KIR+Tregs, thereby alleviating the symptoms of cancer, wherein the binders substantially lack effector functional activity. In some embodiments, a method for stimulating an immune response to a cancer-related antigen (cancer antigen; for example, an antigen expressed on cancer cells) is provided, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with one of the binders or pharmaceutical compositions described herein in an amount effective to activate or stimulate CD8+KIR+Tregs (activated Tregs), wherein the binders substantially lack effector functional activity, thereby increasing the immune response to the cancer antigen.

[0383] In some embodiments, the binder is selected from any of the binders described herein, each having reduced effector activity or substantially no effector activity. In some embodiments, reduced effector activity means reduced ADCC, ADCP, or CDC effector activity, or the absence of ADCC, ADCP, or CDC effector activity. In some embodiments, substantially no effector activity means substantially no ADCC, ADCP, and CDC effector activity. In some embodiments, the binder lacks an Fc domain or region and has reduced effector activity or substantially no effector activity. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region. In some embodiments, the binder has an Fc domain or region having reduced effector activity or substantially no effector activity due to amino acid substitutions in the Fc domain or region, such as an Fc null substitution. In some embodiments, the binder is either lacking an Fc domain or region, or having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder is lacking an Fc domain or region. In some embodiments, the binder is either having an Fc domain or region having reduced binding to one or more Fc gamma receptors, or an Fc null domain. In some embodiments, the binder has an Fc domain or region having 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 for treating cancer is provided, comprising the step of administering to a subject in need of it one of the binders or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Treg and thereby alleviate the symptoms of cancer, wherein the binder has effector functional activity comprising at least ADCC. In some embodiments, a method for stimulating an immune response against cancer is provided, comprising the step of contacting CD8+KIR+T regulatory cells (Treg) with one of the binders or pharmaceutical compositions described herein in an amount effective to deplete CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC and thereby increases the immune response against cancer. In some embodiments, a method is provided for stimulating an immune response to a cancer-associated antigen (cancer antigen), comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with any of the binders described herein or any of the pharmaceutical compositions described herein in an amount effective to deplete the CD8+KIR+Tregs, wherein the binder has effector functional activity comprising at least ADCC, thereby increasing the immune response to the cancer antigen.

[0385] In some embodiments, the binder is selected from any of the binders described herein, each having effector functional activity comprising at least ADCC. In some embodiments, the effector functional activity is ADCC combined with ADCP and / or CDC effector functional activity. In some embodiments, having effector functional activity means having ADCC, ADCP, and CDC effector functional activity. In various embodiments, such binders have an Fc domain, or have an Fc domain that binds to one or more Fc gamma receptors.

[0386] In some embodiments, CD8+KIR+Treg is contacted with a binder in vivo. In some embodiments, CD8+KIR+Treg is contacted with a binder ex vivo. In some embodiments, activated CD8+KIR+Treg is administered in an effective dose to a subject requiring it.

[0387] While not intended to be bound by any particular theory, the reduction or absence of effector functional activity by the binder may limit the interaction between the binder and other cell types (i.e., non-CD8+KIR+Treg) and / or limit the depletion of CD8+KIR+Treg. In contrast, the presence of effector functional activity by the binder is thought to bias the immune response toward the depletion of CD8+KIR+Treg.

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

[0389] The terms “cancer” and “malignant disease” refer to the uncontrolled growth of cells that interfere with the normal functioning of organs and systems of the body. Cancer or malignant disease can be primary or metastatic; that is, cancer or malignant disease can be invasive and cause tumor growth in tissues distant from the original tumor site. “Tumor” refers to the uncontrolled growth of cells that interfere with the normal functioning of organs and systems of the body. As used herein, the term cancer includes malignant disease and tumors unless otherwise indicated by the context. An object having cancer is an object that has objectively measurable cancer cells present throughout the object's body. Benign tumors and malignant cancers, as well as potentially dormant tumors and micrometastases, are included in this definition. Cancer that migrates from its original site and disseminates to other vital organs can ultimately lead to the death of the object through functional deterioration of the affected organ. Hematological malignancies (hematopoietic cancers), such as leukemia and lymphoma, can overwhelm normal hematopoietic compartments in a patient, thereby leading to hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia) that ultimately result in death.

[0390] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia. More specific examples of such cancers 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; cholangiocarcinoma, choriocarcinoma, chondrosarcoma, colorectal cancer, connective tissue cancer, gastrointestinal cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer and stomach cancer), glioblastoma (GBM), liver cancer, hepatoma, carcinoma in situ, kidney or renal cancer. (e.g., clear cell kidney cancer or non-clear cell kidney 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 and non-Hodgkin lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, respiratory cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, severe uterine cancer (uterine Severe carcinoma, cancers of the urinary tract, vulvar cancer; and other cancers and sarcomas, as well as B-cell lymphomas (including low-grade / follicular non-Hodgkin 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 non-incisional cell NHL, giant tumor NHL, mantle cell lymphoma, AIDS-associated lymphoma and Waldenström macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia and post-transplant lymphoproliferative disorders (PTLD), as well as abnormal angiogenesis, edema (e.g., associated with brain tumors) and Meigs syndrome associated with nevus disorders; and myeloma, e.g., multiple myeloma.

[0391] In some embodiments, the cancer is selected from solid tumors including, but not limited to, hepatocellular carcinoma, lung cancer, such as 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 methods described herein include the step of administering a therapeutically effective amount of the binder or pharmaceutical composition described herein to a subject having cancer or malignancy. Where used herein, the terms “therapeutic effective amount,” “effective amount,” “effective quantity,” or “effective dose” may mean an amount of the binder or pharmaceutical composition described herein that provides a therapeutic benefit in the treatment of cancer or malignancy, the management of cancer or malignancy, or the prevention of recurrence of cancer or malignancy, for example, an amount that provides a statistically significant reduction in at least one symptom, sign, or marker of cancer, tumor, or malignancy. Determining the therapeutically effective amount is well within the capabilities of those skilled in the art. Generally, the therapeutically effective amount may vary depending on the subject’s medical history, age, condition, and sex, as well as the severity and type of the medical condition in the subject, and the administration of other pharmaceutically 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, these methods reduce tumor size by 10%, 20%, 30%, or more. In various embodiments, these 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, animals are vertebrates, e.g., primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, e.g., rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, felines, e.g., domestic cats, canids, e.g., dogs, foxes, wolves, birds, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms “patient,” “individual,” and “subject” are used interchangeably herein.

[0395] Preferably, the subject is a mammal. The mammal may be, but is not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be usefully used, for example, as subjects for presenting animal models of various cancers. Furthermore, the methods described herein may be used to treat domesticated animals and / or pets. The subject may be male or female. In certain embodiments, the subject is human.

[0396] A subject may be one who has been previously diagnosed with or identified as having cancer and requires treatment, but does not need to have already received treatment for cancer. Alternatively, a subject may also be one who requires treatment but has not been previously diagnosed with cancer. A subject may exhibit one or more risk factors for a cancer-related condition or one or more complications, or may not exhibit any risk factors. A subject “requiring treatment” for a particular cancer may be one who has that condition or has been diagnosed with that condition. In other embodiments, a subject “at risk of developing” a condition refers to one who has been diagnosed as being at risk of developing the condition.

[0397] As used herein, the terms “to treat,” “treatment,” “to treat,” or “to alleviate,” when used in reference to a disease, disorder, or medical condition (e.g., cancer), refer to a therapeutic treatment of the condition whose purpose is to reverse, reduce, alleviate, inhibit, slow, or halt the progression or severity of the symptoms or condition. The term “to treat” includes reducing or mitigating 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 interrupted. That is, “treatment” includes not only improvement of symptoms or markers but also cessation or at least slowing of the progression or worsening of symptoms that would be expected in the absence of the treatment. Beneficial or desired clinical outcomes include, but are not limited to, a reduction in cancer cells in the subject, a reduction in one or more symptoms, a reduction in the degree of defect, a stabilized (i.e., non-worsening) state of cancer or malignancy, a delay or slowing of tumor growth and / or metastasis, and an increase in life expectancy, compared to what would be expected in the absence of the treatment. As used herein, the term “administer” means bringing a binder or pharmaceutical composition or nucleic acid encoding a binder described herein into contact (e.g., by administration to a subject) by a method or route that results in the binding of the binder to CD8+KIR+Treg. Similarly, a pharmaceutical composition comprising a binder or nucleic acid encoding a binder disclosed herein may be administered by any suitable route that results in an effective treatment in a subject.

[0398] The dosage range for the binder depends on the efficacy and includes a sufficiently large amount to produce the desired effect, e.g., slowing of tumor growth or reduction in tumor size. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage varies depending on the age, condition, and sex of the subject and can be determined by those skilled in the art. The dosage may also be adjusted by the individual physician if there are any complications. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 20 mg / kg body weight. In some embodiments, the dosage ranges from about 0.01 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage ranges from about 0.1 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage ranges from 0.5 mg / kg body weight to about 10 mg / kg body weight. In some embodiments, the dosage range is about 0.5 mg / kg body weight to about 5 mg / kg body weight. Alternatively, the dose range may be set to maintain serum levels between 1 ug / mL and 1000 ug / mL.

[0399] The doses cited above may be repeated. In preferred embodiments, the doses cited above are administered weekly, every two weeks, every three weeks, or monthly over several weeks or months. The duration of treatment depends on the clinical progress of the subject and their response to the treatment.

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

[0401] Pharmaceutical compositions containing a binder may be administered in unit doses. When the term “unit dose” is used in reference to a pharmaceutical composition, it refers to a physically distinct unit appropriate as a unit dosage for the subject, each unit containing a predetermined amount of the active material (e.g., binder) calculated to produce the desired therapeutic effect in relation to the required physiologically acceptable diluent, i.e., a carrier or vehicle.

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

[0403] In some embodiments, any of the binders or pharmaceutical compositions of the embodiments described herein are administered in conjunction with immunotherapy or chemotherapy. As used herein, “immunotherapy” refers to a therapeutic strategy designed to induce or enhance the subject’s own immune system to fight cancer or malignant disease. Examples of immunotherapies, but not limited to, include antibodies, such as checkpoint inhibitors. In some embodiments, chemotherapy includes, for example, alkylating agents, such as nitrogen mustard, such as cyclophosphamide, ifosfamide, trophosfamide, or chlorambucil; nitrosoureas, such as carmustine (BCNU) and lomustine (CCNU); alkyl sulfonates, 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; epipodophilin, for example For example, etoposide, teniposide, topotecan, 9-aminocamptothecin, camptothecin, exatecan, and cristatol; mitomycin, e.g., mitomycin C; antimetabolites, e.g., folate antimetabolites, e.g., DHFR inhibitors, e.g., methotrexate and trimethrexate; IMP dehydrogenase inhibitors, e.g., mycophenolic acid, thiazophrine, ribavirin, and EICAR; ribonucleotide (Ribonuclotide) reductase inhibitors, e.g., hydroxyurea and deferoxamine; and pyrimidine analogs, e.g., uracil analogs, e.g., 5-fluorouracil, phloxuridine, doxifluridine, and ratitrexed; cytosine analogs, e.g., cytarabine (ara) C) Cytosine arabinoside and fludarabine; purine analogs, e.g., mercaptopurine and thioguanine; hormone therapy, e.g., receptor antagonists, e.g., anti-estrogen drugs, e.g., tamoxifen, raloxifene and megestrol;LHRH agonists, e.g., goscrclin and leuprolide acetate; antiandrogens, e.g., flutamide and bicalutamide; retinoids / deltoids; vitamin D3 analogs, e.g., EB 1089, CB 1093 and KH 1060; photodynamic therapy, e.g., verteporfin (BPD-MA), phthalocyanines, photosensitizers Pc4 and demethoxy-hypocrelin A. A) and (2BA-2-DMHA); cytokines, e.g., interferon-alpha and interferon-gamma; tumor necrosis factor; and others, e.g., isoprenylation inhibitors, e.g., lovastatin; dopaminergic neurotoxins, e.g., 1-methyl-4-phenylpyridinium ion; cell cycle inhibitors, e.g., staurosporine; actinomycins, e.g., actinomycin D and dactinomycin; bleomycins, e.g., bleomycin A2, bleomycin B2 and peplomycin; anthracyclines, e.g., daunorabicin, doxorubicin (adriamycin), idarubicin, epirubicin, pirarubicin, zorabicin and mitoxantrone; MDR inhibitors, e.g., verapamil and Ca2+ ATPase inhibitors, e.g., thapsigargin.

[0404] Exemplary Embodiments The present 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 the first antigen is selected from antigens other than KIR proteins that are expressed on CD8+KIR+T regulatory cells (Treg); and Second binding domain that specifically binds to inhibitory KIR proteins A binder containing the following, which binds to CD8+KIR+Treg. Embodiment 2. The binder according to the preceding embodiment, wherein 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. Embodiment 3. The first antigen is 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 A binder as described in a prior embodiment, selected from the group consisting of -1, S1000A8 / 9, TIM-3, TLT-2, 2B4, and 41BB. Embodiment 4. The first antigen is 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 A combined domain as described in any of the prior embodiments, selected from the above. Embodiment 5. The first antigen is 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 A combined domain as described in any of the prior embodiments, selected from the above. Embodiment 6. A binder according to any of the preceding embodiments, which is a bispecific antibody, diabody, 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, antikalin, afibody, avimer, DARPin, or adonectin. Embodiment 7. A binder according to any of the preceding embodiments, wherein either the first or second binding domain is selected from an antibody or its antigen-binding portion, and the other binding domain is an antibody fragment. Embodiment 8. The binder according to Embodiment 7, wherein the antigen-binding portion is Fab, Fab', F(ab')2, Fv, scFv, or a single-domain antibody, such as VHH, VNAR, sdAb, or a nanobody. Embodiment 9. A binder according to any of the prior embodiments, wherein the first binding domain includes a heavy chain variable region and a light chain variable region. Embodiment 10. The binder according to any of the preceding embodiments, wherein the second binding domain includes a heavy chain variable region and a light chain variable region. Embodiment 11. A binder according to any of the preceding embodiments, wherein the first binding domain specifically binds to CD3 or a subunit of CD3, and optionally to 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 VH and VL are a. Sequence ID 1 and Sequence ID 2, respectively; b. Sequence ID 9 and Sequence ID 10, respectively; c. Sequence ID 17 and Sequence ID 18, respectively; d. Sequence IDs 25 and 26, respectively; e. Sequence ID 33 and Sequence ID 34, respectively; f. Sequence ID 41 and Sequence ID 34, respectively; g. Sequence ID 45 and Sequence ID 34, respectively; h. Sequence IDs 49 and 50, respectively; i. Sequence ID 57 and Sequence ID 58, respectively; j. Sequence IDs 65 and 66, respectively; and k. Sequence IDs 65 and 166, respectively. A binder according to Embodiment 11, selected from a pair of amino acid sequences represented by the group consisting of the following. Embodiment 13. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 1 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 2. Embodiment 14. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 10. Embodiment 15. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 17 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 18. Embodiment 16. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 25 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 26. Embodiment 17. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 33 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 34. Embodiment 18. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 41 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 34. Embodiment 19. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 45 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 34. Embodiment 20. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 49 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 50. Embodiment 21. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 57 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 58. Embodiment 22. The first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region having the amino acid sequence shown in SEQ ID NO: 66. A binder according to Embodiment 11, including a region (VL). Embodiment 23. The binder according to Embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 65 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 166. Embodiment 24. The first binding domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain and light chain variable regions each include hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDR is a. Sequence IDs 3 through 8, respectively; b. Sequence IDs 11 to 16, respectively; c. Sequence numbers 19 to 24, respectively; d. Sequence numbers 27 to 32, respectively; e. Sequence numbers 35 to 40, respectively; f. Sequence IDs 42-44 and 38-40, respectively; g. Sequence IDs 46-48 and 38-40, respectively; h. Sequence IDs 51 to 56, respectively; i. Sequence numbers 59 to 64, respectively; j. Sequence IDs 67 to 72, respectively; and k. Sequence numbers 67-69 and 167-169, respectively. The binder according to Embodiment 11, having an amino acid sequence selected from a set of amino acid sequences represented by the group consisting of the following. Embodiment 25. The binder according to any one of Embodiments 1 to 10, wherein the first binding domain specifically binds to CD8 or a subunit of CD8, and optionally to CD8 alpha. Embodiment 26. The first binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions are a. Sequence IDs 73 and 74, respectively; and b. Sequence ID 81 and Sequence ID 82, respectively It has an amino acid sequence selected from a pair of amino acid sequences shown in the group consisting of; Alternatively, the first binding domain includes a VHH chain, and the VHH chain is c. Sequence ID 89; d. Sequence ID 93; and e. Sequence ID 97 The binder according to Embodiment 25, having an amino acid sequence selected from the group of amino acid sequences represented by the following. Embodiment 27. The binder according to Embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 73 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 74. Embodiment 28. The binder according to Embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 81 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 82. Embodiment 29. The binder according to Embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID NO: 89. Embodiment 30. The binder according to Embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID NO: 93. Embodiment 31. The binder according to Embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID NO: 97. Embodiment 32. The first binding domain includes a heavy chain variable region and a light chain variable region, wherein the heavy chain and light chain variable regions each include hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDR is a. Sequence numbers 75 to 80, respectively; or b. Sequence IDs 83 to 88, respectively It has an amino acid sequence selected from the set of amino acid sequences shown in the group consisting of; Alternatively, the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, and the amino acid sequence of the VHH CDR is c. Sequence numbers 90 to 92, respectively; d. Sequence numbers 94 to 96, respectively; and e. Sequence IDs 98 to 100, respectively A binder according to Embodiment 25, selected from a set of amino acid sequences represented by the group consisting of the following. Embodiment 33. The binder according to any one of Embodiments 1 to 10, wherein the first binding domain specifically binds to ICOS. Embodiment 34. The binder according to 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 binder according to Embodiment 33, wherein the first binding domain includes a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions each include the hCDR1, hCDR1 and hCDR3 amino acid sequences described in SEQ ID NOs. 172, 173 and 174, and the lCDR1, lCDR2 and lCDR3 amino acid sequences described in SEQ ID NOs. 175, 176 and 177, respectively. Embodiment 36. The binder according to any one of Embodiments 1 to 10, wherein the first binding domain specifically binds to PD-1. Embodiment 37. The binder according to 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 binder according to Embodiment 36, wherein the first binding domain includes a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions each include the hCDR1, hCDR1 and hCDR3 amino acid sequences described in SEQ ID NOs. 180, 181 and 182, and the lCDR1, lCDR2 and lCDR3 amino acid sequences described in SEQ ID NOs. 183, 184 and 185, respectively. Embodiment 39. The binder according to any one of Embodiments 1 to 10, wherein the first binding domain specifically binds to CXCR3. Embodiment 40. The binder according to 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 binder according to Embodiment 39, wherein the first binding domain includes a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions each include the hCDR1, hCDR1 and hCDR3 amino acid sequences described in SEQ ID NOs. 188, 189 and 190, and the lCDR1, lCDR2 and lCDR3 amino acid sequences described in SEQ ID NOs. 191, 192 and 193, respectively. Embodiment 42. The binder according to any one of Embodiments 1 to 10, wherein the first binding domain specifically binds to CD5. Embodiment 43. The binder according to 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 binder according to Embodiment 42, wherein the first binding domain includes a heavy chain variable region and a light chain variable region, and the heavy chain and light chain variable regions each include the hCDR1, hCDR1 and hCDR3 amino acid sequences described in SEQ ID NOs: 196, 197 and 198, and the lCDR1, lCDR2 and lCDR3 amino acid sequences described in SEQ ID NOs: 199, 200 and 201, respectively. Embodiment 45. The inhibitory KIR protein is KIR3DL1, KIR3DL2, K A binder according to any of the prior embodiments, selected from IR2DL1, KIR2DL2, and KIR2DL3 or a combination thereof. Embodiment 46. The binder according to Embodiment 45, wherein the second binding domain specifically binds to KIR2DL1 / 2 / 3 or KIR2DL1 / 2. Embodiment 47. The second binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions are a. Sequence ID 101 and Sequence ID 102, respectively; b. Sequence IDs 109 and 110, respectively; c. Sequence ID 117 and Sequence ID 118, respectively; d. Sequence IDs 125 and 126, respectively; e. Sequence IDs 133 and 134, respectively; f. Sequence ID 141 and Sequence ID 142, respectively; g. Sequence IDs 149 and 150, respectively; and h. Sequence ID 157 and Sequence ID 158, respectively A binder according to any of the prior embodiments, having an amino acid sequence selected from a pair of amino acid sequences represented by the group consisting of the following. Embodiment 48. The binder according to any of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 101 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 102. Embodiment 49. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 109 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 110. Embodiment 50. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 117 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 118. Embodiment 51. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 125 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 126. Embodiment 52. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 133 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 134. Embodiment 53. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 141 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 142. Embodiment 54. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 149 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 150. Embodiment 55. The binder according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 157 and a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 158. Embodiment 56. The second binding domain includes a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable regions each include hCDR1, hCDR1 and hCDR3, and lCDR1, lCDR2 and lCDR3, and the CDR is a. Sequence IDs 103 to 108, respectively; b. Sequence numbers 111 to 116, respectively; c. Sequence numbers 119 to 124, respectively; d. Sequence numbers 127 to 132, respectively; e. Sequence numbers 135 to 140, respectively; f. Sequence numbers 143 to 148, respectively; g. Sequence IDs 151 to 156, respectively; and h. Sequence IDs 159 and 164, respectively. A binder according to any one of claims 1 to 47, having an amino acid sequence selected from a set of amino acid sequences shown in the group consisting of CDRs having an amino acid sequence selected from a set of amino acid sequences shown in the group consisting of CDRs. Embodiment 57. A binder according to any of the prior embodiments, which does not contain an Fc domain. Embodiment 58. A binder according to any one of Embodiments 1 to 56, further comprising an Fc domain. Embodiment 59. The binder according to Embodiment 58, wherein the Fc domain is selected from IgG1 and IgG4 Fc domains. Embodiment 60. The binder according to Embodiment 59, which substantially does not have effector function activity. Embodiment 61. The binder according to any one of Embodiments 58 to 60, wherein the Fc domain is an IgG1 Fc domain. Embodiment 62. The binder according to any one of Embodiments 58 to 61, wherein the Fc domain is IgG1 Fc null. Embodiment 63. A binder according to any of the prior embodiments, which is divalent or tetravalent. Embodiment 64. A binder according to any of the prior embodiments, which is two-specific. Embodiment 65. A pharmaceutical composition comprising a binder and a pharmaceutically acceptable carrier as described in any of Embodiments 1 to 64. Embodiment 66. A nucleic acid encoding the binder described in any one of Embodiments 1 to 64. Embodiment 67. A vector comprising the nucleic acid described in Embodiment 66. Embodiment 68. A cell system comprising the vector described in Embodiment 67. Embodiment 69. A method for treating an autoimmune disease, comprising the step of administering to a subject in need of the binder described in any of Embodiments 1 to 64 or the pharmaceutical composition described in Embodiment 65 in an amount effective in reducing the number or activity of pathogenic immune cells in the subject, thereby alleviating the symptoms of the autoimmune disease. Embodiment 70. A method for suppressing an immune response mediated by pathogenic immune cells, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs) with a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65, 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 the step of administering to a subject requiring such action an amount effective to activate or stimulate CD8+KIR+Treg, thereby reducing the number or activity of pathogenic immune cells responsive to the antigen or autoantigen. Embodiment 72. The method according to Embodiment 70, wherein the CD8+KIR+Treg comes into contact with the binder in vivo. Embodiment 73. The method according to Embodiment 70, wherein the CD8+KIR+Treg is in ex vivo contact with the binder. Embodiment 74. The method according to Embodiment 73, wherein the activated CD8+KIR+Treg is administered in an effective amount to a subject requiring administration. Embodiment 75. The method according to any one 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 according to any one of Embodiments 69 to 74, wherein the pathogenic immune cells are autoantigen-presenting cells. Embodiment 77. The method according to Embodiment 71, wherein the titer of the autoantibody is reduced in the subject. Embodiment 78. The subject is celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory The method according to any one of embodiments 69 and 74 to 77, wherein the patient has an autoimmune disease selected from the group consisting of intestinal disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes mellitus), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), and ulcerative colitis. Embodiment 79. The method according to 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 mellitus), 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 according to any one of Embodiments 69 to 79, wherein the binder specifically binds to CD8 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 81. The method according to any one of Embodiments 69 to 79, wherein the binder specifically binds to CD3 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 82. The method according to any one of Embodiments 69 to 79, wherein the binder specifically binds to CD5 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 83. The method according to any one of Embodiments 69 to 79, wherein the binder specifically binds to PD-1 and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 84. The method according to any one of Embodiments 69 to 79, wherein the binder specifically binds to ICOS and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 85. The method according to any one of Embodiments 69 to 79, wherein the binder specifically binds to CXCR3 on CD8+KIR+Treg and to the inhibitory KIR protein. Embodiment 86. The method according to any one of embodiments 69 to 85, wherein the CD8+KIR+Treg is MHC class I constrained. Embodiment 87. The method according to any one of embodiments 69 to 86, wherein the CD8+KIR+Treg is not constrained by MHC HLA E(Qa-1b). Embodiment 88. The method according to any one of Embodiments 69 to 87, further comprising the step of administering an immunosuppressant to the subject. Embodiment 89. The method according to any one of Embodiments 69 to 88, wherein the administration of the binder to the subject results in an improvement in the treatment outcome in the subject. Embodiment 90. The method according to Embodiment 89, wherein the improvement in the treatment outcome is a reduction in the frequency or severity of disease relapses, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with the autoimmune disease. Embodiment 91. The method according to any one of Embodiments 69 to 90, wherein the binder is administered intravenously. Embodiment 92. The method according to any one of Embodiments 69 to 91, wherein the binder is administered subcutaneously. Embodiment 93. The method according to any one of Embodiments 69 to 92, wherein the binder is administered in a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 94. The method according to any one of Embodiments 69 to 93, wherein the binder substantially does not have effector functional activity. Embodiment 95. Use of a binder 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+Treg. Embodiment 96. Use of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for reducing the immune response by pathogenic immune cells by activating or stimulating CD8+KIR+Treg. Embodiment 97. Use of a binder 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+Treg. Embodiment 98. A method for treating cancer, comprising the step of administering a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Treg and thereby alleviate the symptoms of the cancer, wherein the binder substantially lacks effector functional activity. Embodiment 99. A method for stimulating an immune response to a cancer-related antigen (cancer antigen), comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs) of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65, wherein the binder substantially lacks effector functional activity, thereby increasing the immune response to the cancer antigen. Embodiment 100. A method for treating cancer, comprising the step of administering to a subject in need of the binder described in any of Embodiments 1 to 64 or the pharmaceutical composition described in Embodiment 65 in an amount effective to deplete CD8+KIR+Treg and thereby alleviate the symptoms of the cancer, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 101. A method for stimulating an immune response to a cancer-related antigen (cancer antigen), comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 having effector functional activity comprising at least ADCC, in an amount effective to deplete the CD8+KIR+Tregs, thereby increasing the immune response to the cancer antigen. Embodiment 102. The method according to Embodiment 99 or Embodiment 101, wherein the CD8+KIR+Treg is in contact with the binder in vivo. Embodiment 103. The method according to Embodiment 99, wherein the CD8+KIR+Treg is in ex vivo contact with the binder. Embodiment 104. The method according to Embodiment 103, wherein the activated CD8+KIR+Treg is administered in an effective amount to a subject requiring administration. Embodiment 105. The method according to any one of embodiments 99 and 101 to 104, wherein the increase in the immune response includes a reduction in cancer cells or depletion of immunosuppressive immune cells. Embodiment 106. The method according to any one of Embodiments 98 to 105, wherein the cancer cells in the subject are reduced. Embodiment 107. The method according to any one 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 according to any one 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 according to any one of Embodiments 98 to 108, wherein the binder specifically binds to CD8 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 110. The method according to any one of Embodiments 98 to 108, wherein the binder specifically binds to CD3 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 111. The method according to any one of Embodiments 98 to 108, wherein the binder specifically binds to CD5 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 112. The binder is PD-1 on CD8+KIR+Treg and the inhibitor The method according to any one of embodiments 98 to 108, which specifically binds to harmful KIR proteins. Embodiment 113. The method according to any one of Embodiments 98 to 108, wherein the binder specifically binds to ICOS and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 114. The method according to any one of Embodiments 98 to 108, wherein the binder specifically binds to CXCR3 on CD8+KIR+Treg and to the inhibitory KIR protein. Embodiment 115. The method according to any one of embodiments 98 to 114, wherein the CD8+KIR+Treg is MHC class I constrained. Embodiment 116. The method according to any one of embodiments 98 to 115, wherein the CD8+KIR+Treg is not constrained by MHC HLA E(Qa-1b). Embodiment 117. The method according to any one of Embodiments 98 to 116, further comprising the step of administering an immunotherapy, such as a chemotherapeutic agent or a checkpoint inhibitor, to the subject. Embodiment 118. The method according to any one of Embodiments 98 to 117, wherein the administration of the binder to the subject results in an improvement in the treatment outcome in the subject. Embodiment 119. The method according to Embodiment 118, wherein the improvement in the treatment outcome is partial response or complete response. Embodiment 120. The method according to Embodiment 118, wherein the improvement in the treatment outcome is remission. Embodiment 121. The method according to any one of Embodiments 98 to 120, wherein the binder is administered intravenously. Embodiment 122. The method according to any one of Embodiments 98 to 120, wherein the binder is administered subcutaneously. Embodiment 123. The method according to any one of Embodiments 98 to 122, wherein the binder is administered in a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 124. Use of a binder according to any one 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+Treg, wherein the binder substantially does not have effector functional activity. Embodiment 125. Use of a binder according to any one 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+Treg, wherein the binder substantially does not have effector functional activity. Embodiment 126. Use of a binder according to any one 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+Treg, wherein the binder substantially does not have effector functional activity. Embodiment 127. Use of a binder according to any one 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+Treg, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 128. Use of a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the depletion of CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 129. Use of a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for reducing tumor burden in a subject, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 130. A method for treating an infection, comprising the step of administering to a subject in need an amount effective in activating or stimulating CD8+KIR+Treg and thereby alleviating the symptoms of the infection, the binder described in any of Embodiments 1 to 64 or the pharmaceutical composition described in Embodiment 65. Embodiment 131. A method for stimulating an immune response to infected cells caused by an infectious disease, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 in an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs), thereby stimulating the immune response to the infected cells. Embodiment 132. The method according to Embodiment 131, wherein the CD8+KIR+Treg is in contact with the binder in vivo. Embodiment 133. The method according to Embodiment 131, wherein the CD8+KIR+Treg is in ex vivo contact with the binder. Embodiment 134. The method according to Embodiment 133, wherein the activated CD8+KIR+Treg is administered in an effective amount to a subject requiring administration. Embodiment 135. The method according to any one 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 according to Embodiment 135, wherein the number of infected cells in the subject is reduced. Embodiment 137. The method according to any one of Embodiments 130 to 136, wherein the infectious disease is selected from bacterial diseases, systemic fungal diseases, rickettsial diseases, parasitic diseases, and viral diseases. Embodiment 138. The method according to Embodiment 137, wherein 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, for example, SARS-CoV-2 infection (Covid-19), cytomegalovirus (CMV) infection, and influenza virus infection. Embodiment 139. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to CD8 and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 140. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to CD3 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 141. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to CD5 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 142. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to PD-1 and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 143. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to ICOS and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 144. The method according to any one of Embodiments 130 to 138, wherein the binder specifically binds to CXCR3 on CD8+KIR+Treg and to the inhibitory KIR protein. Embodiment 145. The method according to any one of Embodiments 130 to 144, wherein the CD8+KIR+Treg is MHC class I constrained. Embodiment 146. The method according to any one of Embodiments 130 to 145, wherein the CD8+KIR+Treg is not constrained by MHC HLA E(Qa-1b). Embodiment 147. The method according to any one of Embodiments 130 to 146, further comprising the step of administering an antimicrobial agent or an antiviral agent to the subject. Embodiment 148. The method according to any one of Embodiments 130 to 147, wherein the administration of the binder to the subject results in an improvement in the treatment outcome in the subject. Embodiment 149. The method according to Embodiment 148, wherein the improvement in the treatment outcome is a reduction in infection or infected cells. Embodiment 150. The method according to any one of Embodiments 130 to 149, wherein the binder is administered intravenously. Embodiment 151. The method according to any one of Embodiments 130 to 149, wherein the binder is administered subcutaneously. Embodiment 152. The method according to any one of Embodiments 130 to 151, wherein the binder is administered in a dose of about 0.01 mg / kg to about 20 mg / kg. Embodiment 153. The method according to any one of Embodiments 130 to 149, wherein the binder substantially does not have effector functional activity. Embodiment 154. Use of a binder 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+Treg. Embodiment 155. Use of a binder 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+Treg, thereby suppressing immunosuppressive immune cells. Embodiment 156. Use of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the reduction of infection or infected cells in a subject by activating or stimulating CD8+KIR+Treg. Embodiment 157. Transplantation thing A method for reducing or preventing the development of subsequent graft-versus-host disease (GVHD), comprising the step of administering a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Treg, thereby reducing or alleviating at least one symptom of GVHD, wherein the binder substantially lacks effector functional activity. Embodiment 158. Transplantation thing A method for treating a subject that has been affected by GVHD, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 in an amount effective to activate or stimulate the CD8+KIR+Tregs (activated Tregs), wherein the binder has substantially no effector functional activity, thereby reducing or suppressing GVHD. Embodiment 159. Transplantation thingA method for treating a subject that has received a disease, comprising the step of administering to a subject in need of the binder described in any of Embodiments 1 to 64 or the pharmaceutical composition described in Embodiment 65 in an amount effective to deplete CD8+KIR+Treg and thereby alleviate the symptoms of GVHD, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 160. Transplantation thing A method for suppressing GVHD in a person, comprising the step of contacting CD8+KIR+T regulatory cells (Tregs) with a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 in an amount effective to deplete the CD8+KIR+Tregs, thereby reducing GVHD or its symptoms, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 161. The method according to Embodiment 158 ​​or 160, wherein the CD8+KIR+Treg is in contact with the binder in vivo. Embodiment 162. The method according to Embodiment 158, wherein the CD8+KIR+Treg is in ex vivo contact with the binder. Embodiment 163. The method according to Embodiment 162, wherein the activated CD8+KIR+Treg is administered in an effective amount to a subject requiring administration. Embodiment 164. The reduced or diminished GVHD is described in any of Embodiments 157 and 160 to 163, including a reduction in CD4+ T cells active in GVHD. Method of loading. Embodiment 165. The transplant thing However, organ transplantation thing , hematopoietic stem cell transplantation thing umbilical cord blood stem cell transplantation thing Transplantation of precursor or differentiated cells derived from induced pluripotent stem cells. thing , and bone marrow transplantation thing A method according to any one of embodiments 157 to 164, selected from the group consisting of the following. Embodiment 166. The transplant thing However, hematopoietic stem cell transplantation thing umbilical cord blood stem cell transplantation thingTransplantation of precursor or differentiated cells derived from induced pluripotent stem cells. thing , or bone marrow transplant thing The method according to any of Embodiments 165. Embodiment 167. The transplant thing The method according to any one of embodiments 157 to 166, wherein the two are of the same type but different in nature. Embodiment 168. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to CD8 and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 169. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to CD3 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 170. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to CD5 on CD8+KIR+Treg and the inhibitory KIR protein. Embodiment 171. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to PD-1 and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 172. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to ICOS and the inhibitory KIR protein on CD8+KIR+Treg. Embodiment 173. The method according to any one of Embodiments 157 to 167, wherein the binder specifically binds to CXCR3 on CD8+KIR+Treg and to the inhibitory KIR protein. Embodiment 174. The method according to any one of embodiments 157 to 173, wherein the CD8+KIR+Treg is MHC class I constrained. Embodiment 175. The method according to any one of Embodiments 157 to 174, wherein the CD8+KIR+Treg is not constrained by MHC HLA E(Qa-1b). Embodiment 176. The method according to any one of Embodiments 157 to 175, further comprising the step of administering an immunosuppressant to the subject. Embodiment 177. The method according to any one of Embodiments 157 to 176, wherein the administration of the binder to the subject results in an improvement in the treatment outcome in the subject. Embodiment 178. The improvement in the treatment outcome is due to a reduction in GVHD-related symptoms, a reduction in systemic inflammatory cytokines, a reduction in lesions in tissues affected by GVHD, a reduction in self-reported symptoms related to immune responses associated with adverse effects on host tissues, and transplantation. thing To improve or prolong graft survival, alleviate one or more symptoms, and / or transplantation. thing Prevention, delay, or slowing of the onset or progression of rejection, or widening Regional spectrum Transplantation due to reduced use of immunosuppressants, such as corticosteroids. thing The method according to Embodiment 177, which is an extension of the engraftment process. Embodiment 179. The method according to any one of Embodiments 157 to 178, wherein the binder is administered intravenously. Embodiment 180. The method according to any one of Embodiments 157 to 178, wherein the binder is administered subcutaneously. Embodiment 181. Transplantation in the subject thing Use of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the treatment of GVHD related thereto, wherein the binder has substantially no effector functional activity. Embodiment 182. Transplantation in subjects by activating or stimulating CD8+KIR+Treg thing For the treatment of GVHD related to any of embodiments 1 to 64 Use of a binder or the pharmaceutical composition described in Embodiment 65, wherein the binder has substantially no effector functional activity. Embodiment 183. Implantation by activating or stimulating CD8+KIR+Treg thing Use of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the reduction of GVHD related thereto, wherein the binder has substantially no effector functional activity. Embodiment 184. Transplantation thingUse of a binder according to any one of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for reducing GVHD, wherein the binder substantially does not have effector functional activity. Embodiment 185. Transplantation in subjects by depleting CD8+KIR+Treg thing Use of a binder according to any one of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the treatment of GVHD related thereto, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 186. Use of a binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 for the depletion of CD8+KIR+Treg, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 187. Transplantation to reduce GVHD thing Use of a binder according to any one of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65 for the depletion of CD8+KIR+Treg in a subject that has received a certain treatment, wherein the binder has effector functional activity comprising at least ADCC. Embodiment 188. Use of a binder described in any of Embodiments 1 to 64 or a pharmaceutical composition described in 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 binder according to any of Embodiments 1 to 64 or a pharmaceutical composition according to Embodiment 65 in the manufacture of a pharmaceutical 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 embodiments of this disclosure is not intended to be exhaustive, nor is it intended to limit this disclosure to the exact form disclosed. Specific embodiments and examples of this disclosure are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art. 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. The aspects of this disclosure may be modified, if necessary, to use the composition, function and concepts of the above-mentioned references and applications to provide even further embodiments of this disclosure. These and other modifications may be made to this disclosure with regard to the detailed description.

[0406] Specific elements of any of the embodiments described above can be combined or used in place of elements in other embodiments. Furthermore, while advantages related to certain embodiments of this disclosure have been described in light of those embodiments, other embodiments may also demonstrate such advantages, and not all embodiments are necessarily required to demonstrate such advantages in order to fall within the scope of this disclosure.

[0407] All identified patents and other publications are expressly incorporated herein by reference, for example, for the purpose of describing and disclosing methodologies described in such publications that may be used in conjunction with the present invention. These publications are provided exclusively for their disclosures prior to the filing date of this application. In this regard, nothing should be construed as an acknowledgment that the inventors are not entitled to precede such disclosures, either on the grounds of prior invention or for any other reason. All statements regarding dates or representations regarding the contents of these documents are based on information available to the applicant and do not constitute any acknowledgment of the accuracy of the dates or contents of these documents. [Examples]

[0408] (Example 1) Testing of 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 the functional restoration of CD8 KIR+ Treg cell activation and cytotoxicity-mediated elimination of pathogenic immune cells, e.g., autoreactive CD4 T cells, primary CD8+ KIR+ T cells are incubated with escalating concentrations of CD3 agonist antibodies, which mimic peptide / MHC binding to the CD8 KIR+ T cell receptor in the presence of escalating concentrations of inhibitory KIR blockers. Blocking inhibitory KIR signaling can be predicted 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 CD8+ KIR+ Treg cell activation is confirmed by increased secretion of certain cytokines (e.g., IFN-gamma, IL-10, TNF-alpha, IL-35, or their subunits), increased expression markers associated with activation (e.g., CD69, CD25, CD62L, CD44, CD45), and increased proliferation.

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

[0410] (Example 2) Testing of 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) will be tested for functional restoration of CD8 KIR+ Treg cell activation, as well as for the direct and indirect removal of pathogen-infected cells from a cytotoxicity-mediated state. Human CMV-specific T cells (Cellero) will be cultured with an increasing dose of a virus-derived dominant epitope (pp65) that binds to the MHC class I molecule HLA-A2. The peptide will be loaded onto HLA-A2-expressing lymphoma cell line T2. To test the function of specific inhibitory KIR molecules, either an MHC I-deficient lymphoma T1 cell line or a K562 cell line will be transfected with an appropriate congeneral MHC class I molecule (e.g., HLA-C2 if blocking KIR2DL1). CD8 Treg cell activation, cytotoxicity, cytokine production, and proliferation will be tested to confirm that KIR blockade specifically and effectively re-establishes the removal of pathogen-infected cells. The degree of target cell removal and apoptosis will also be determined using Annexin V staining and proliferation. As controls, the inventors use, for example, unrelated 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 higher sensitivity, the inventors transfect Jurkat cells, for example, with SHP1 / 2 or NFAT reporters to show enhanced activation upon inhibitory KIR blockade and / or agonist binding, in order to prioritize molecules for use.

[0411] (Example 3) Testing of 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 using high antigen loading in the presence of a panel of KIR bispecific molecules. The KIR bispecific molecules are tested alone, in combination with other KIR bispecific molecules, and in combination with other immune checkpoint inhibitors. As an example, the study tests dose escalation of anti-CD3 agonist antibodies in combination with 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). Haplotype dependence on the response is determined, for example, using primary NY-ESO-1 specific T cells (Cellero) versus NY-ESO1 peptide-pulsed T2 cells (HLA-A2-restricted cell line) and / or HLA-A2 K562 cells. To assess whether a subset of HLA expression (e.g., HLA-B binding KIR3DL1 or KIR2DL1 / 2 / 3 binding HLA-C) is required for the optimal response by targeting tumor cells, appropriate HLA molecules are overexpressed in either K562 or T1 cells and pulsed with the appropriate dominant epitope (https: / / antibodies.cancer.gov / detail / MajorHistocompatibilityComplexClassICPeptide1).

[0412] (Example 4) Testing of monospecific and bispecific molecules for CD8+KIR+Treg cell activation and cytotoxicity in GVHD transplantation models. After hematopoietic stem cell transplantation and other transplantation procedures, serious and life-threatening complications can occur, in which donor-derived cells recognize allogeneic host tissue as foreign, become activated, and destroy healthy cells in the recipient, a condition known as graft-versus-host disease (GVHD). Alloreactive GVHD causes transplant-related pathological conditions in up to 50% of transplant recipients and accounts for approximately 20% of post-transplant mortality. If transplanted cells destroy healthy tissue and recognize it as foreign, KIR blockade on CD8+KIR+Treg may reduce the severity of graft-versus-host disease. To test the effect of KIR blockade on GVHD severity, a well-characterized GVHD model was used, in which human immune cells were injected into NOD / SCID / gamma chain (NSG)-deficient mice, and subsequent multi-organ acute lesions were observed as a result of human cell activation and destruction of mouse tissue. KIR-blocking monospecific and bispecific molecules were injected every 72 hours over the duration of the study, which lasted 30–45 days. Endpoint analysis included serum pro-inflammatory cytokine expression, 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 usefulness of KIR blockade as a method to reduce the severity of GVHD while maintaining graft survival, but also determines the effects of KIR blockade on systemic diseases 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+ Treg cells was confirmed in vitro. Briefly, cells were isolated from the spleen 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+ cells were isolated using magnetic separation and stimulated with CD3 / CD28 in the absence of the blocking antibody (control) or in the presence of the blocking antibody LY49 C / l F(ab')2 fragment (clone 5e6; lacking the Fc portion of the antibody) (anti-Ly49), and cultured 1:1 with CD4 T cells.

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

[0415] The supernatant from the cells (described above) was collected 48 hours after the start of co-culture and analyzed for various analytes (cytokines) using the Bioplex assay.

[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 peptides 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 peptides compared to mice treated with MOG peptide (data not shown). Levels of the anti-inflammatory cytokines IL-22 and MCP-3 were reduced in samples from mice treated with MOG and suppressor peptides 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 / l (clone 5E6; lacking the Fc portion of the antibody) (anti-Ly49 or "Ly49 blockade") (Figure 5). Ly49 blockade delayed disease onset and reduced disease severity (Figure 6), suggesting that CD8+ Treg recruitment at the time of autoimmune triggering contributed to disease control.

[0418] (Example 6) CD8 KIR+ T cells have a higher cytolytic potential than KIR-negative CD8 T cells in celiac patients. Peripheral blood mononuclear cells (PBMCs) were obtained from celiac patients and healthy donors. PBMCs were enriched with 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 evaluated for intracellular granzyme, perforin, and IFN gamma levels.

[0419] PBMCs derived from celiac patients had a higher percentage of CD8+KIR+Treg cells (Figure 7A). KIR+CD8+ T cells had a higher percentage of cells containing 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 (6 individuals) 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 also 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+Treg cells, as well as CD4+ T cell loss. To determine the effects of gluten restimulation on CD8+KIR+ T cells, PMBCs derived from celiac patients were enriched with gluten peptides for 12 days in the presence of IL-7 and IL-15, enriching both CD4-responsive cells and CD8+ Treg cells. CD8 Treg and CD4 T cells were then selected and combined 1:1 with autologous APCs, either without peptide pulses or pulsed with influenza peptides or gluten peptides. After 48 hours, the cells were analyzed by flow cytometry.

[0422] Restimulation with gluten peptides increased degranulation (Figure 9A, left) and granzyme B levels (Figure 9A, right), as measured by CD107, compared to cells restimulated with control influenza peptides or unstimulated cells (598 refers to PBMCs from patient 598). Restimulation with gluten peptides also resulted in a reduction in the percentage of viable CD4+ cells, which was not caused by restimulation with influenza peptides (Figure 9B). These results indicate that the antigen response by CD8+ KIR+ Tregs is specific, rapid, and persistent, consistent with the source of Tregs from celiac patients. These results suggest 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 in a 1:1 ratio with CD4+ T cells and 1 ug / ml anti-CD3 agonist antibody (clone OKT3) in or without 100 ug / ml of KIR2DL1 / 2 / 3 and KIR3DL1 antagonist antibodies (50 ug 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 effects of KIR blockade were evaluated in vitro in another experiment using PBMCs derived from celiac patients. Briefly, CD8+ Tregs were enriched from celiac patient PBMCs and 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 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, decreased CD4+ T cell activation, and increased CD4+ T cell death (Figure 11). These results were consistently observed across several patient samples during 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 in a 1:1 ratio with CD4+ T cells and 1ug / ml anti-CD3 agonist antibody (clone OKT3) in or without 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) The relationship between selective KIR protein 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 for CD8 T cells, the percentage positivity of cells for KIR ligands and HLA haplotypes was determined (HLA and KIR classification was performed in collaboration with Cisco Genetics).

[0427] Patient-derived CD8 T cells expressed KIRs as follows: in peripheral blood, all three expressed KIR2DL, and two expressed KIR3DL. HLA ligands for selected KIRs were overpresented in celiac patient samples. Nine out of ten patients had at least one copy of HLA-C 07:01:01, and all ten patients had at least one copy of HLA-B 08:01:01.

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

[0429] KIR2L1 / 2 / 3-CD8 alpha-closumab was tested for co-binding to KIR2DL1 or KIR2DL3 and CD8 alpha by biolayer interferometry using an Octet instrument. For co-binding studies, the closumab was captured in an AHC (anti-human Fc) biosensor using 2-fold dilutions ranging from 0.3125 ug / ml to 20 ug / ml. The analytes (KIR2DL1, KIR2DL3, and CD8 alpha) were maintained at a constant 100 nM. Post-capture analyte co-binding was analyzed in two ways: first, association of KIR2DL1 or KIR2DL3, followed immediately by association of CD8 alpha, or association of CD8 alpha, followed by direct association of KIRDL1 or KIR2DL3. KIR2DL1, KIR2DL3, and CD8 alpha were tagged with hexahistidine peptide. Crosumab was able to co-bind to the target KIR2DL1 or KIR2DL3 and CD8 alpha.

[0430] Using an Octet instrument, the affinity of crossumab to KIR2DL1, KIR2DL3, and CD8 alpha ligands was measured and compared with anti-CD8 alpha and anti-KIR2DL1 / L2 / L3 parental antibodies. For kinetic analysis, crossumab was captured using an AHC (anti-human Fc) biosensor at a loading concentration of 1.25 ug / ml. The concentrations of each analyte (KIR2DL1, KIR2DL3, and CD8 alpha) ranged from 6.25 nM to 200 nM. After capture, analyte binding was analyzed first for the association of KIR2DL1, KIR2DL3, or CD8 alpha, and then independently for the dissociation of each analyte. This allowed for the acquisition of ka (on-rate), kd (off-rate), and KD values, ensuring that they could be directly compared with parental antibodies. Kinetic analysis revealed that crossumab retained affinity for the target 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 separately captured on an AHC (anti-human Fc) biosensor using a loading concentration of 1.25 ug / ml. For IPH2102 IgG1r mAb, the KIR2DL1 or KIR2DL3 analytes ranged from 6.25 nM to 200 nM. The analyte binding after capture was analyzed first for the association of KIR2DL1 or KIR2DL3, and then independently for the dissociation of each analyte. Similarly, for Mb1b IgG1r mAb, the CD8 alpha analytes ranged from 6.25 nM to 200 nM. The analyte binding after capture was analyzed first for the association of CD8 alpha, and then independently for dissociation. [Table 1]

[0432] (Example 13) Analysis of PBMC samples from patients diagnosed with other autoimmune diseases. PBMCs derived from patients with lupus, ulcerative colitis, Crohn's disease, multiple sclerosis, and type 1 diabetes were analyzed using flow cytometry and bioplex assays. CD8+KIR+Treg cells were identified in these patient samples using a cocktail of antibodies against a subset of inhibitory KIR surface receptors, KIR2DL1 / 2 / 3 and KIR3DL1 (Figure 13). Treg cells were found to express CXCR3, CD39, and other cell surface markers, consistent with CD8+Treg cells derived from celiac disease patients (data not shown). CD8+Treg cells were found to produce soluble analytes associated with CD8+Treg cell function, including IFN gamma and IL-22 (data not shown).

[0433] (Example 14) Phenotypic and functional characterization of CD8+KIR+Treg cells PBMCs derived from individuals with celiac disease and the HLA DQ2.5 haplotype were analyzed using flow cytometry and bioplex assays to detect soluble analytes in the supernatant. CD8+KIR+Treg cells were identified in these patient samples and were found to express the surface markers CD39, KLRG1, NKG2D, NKG2C, KLRB, CXCR3, and CD122 (Figure 14).

[0434] When cultured under optimized conditions with autologous CD4 T cells pulsed with gliadin peptide and antigen-presenting cells, CD8+KIR+Treg cells produced soluble analytes related to CD8+Treg cell function, including the cell lysis markers granzyme B, perforin, and CD107a, the intracellular anti-inflammatory cytokines IL-10, IFNγ, and TNFα, and secreted cytokines (see, for example, Figure 15). Stimulation of isolated CD8+KIR+Treg cells with escalating doses of anti-CD3 antibody pr...

Claims

1. A first binding domain that specifically binds to CD8 or a subunit of CD8, comprising the amino acid sequences hCDR1, hCDR2, hCDR3, lCDR1, lCDR2, and lCDR3 according to SEQ ID NOs. 83 to 88, respectively; and A second binding domain that specifically binds to the inhibitory KIR protein, comprising the amino acid sequences hCDR1, hCDR2, hCDR3, lCDR1, lCDR2, and lCDR3, respectively, according to SEQ ID NOs. 103 to 108. A binder containing a compound that binds to CD8 + KIR + Treg.

2. Bispecific antibody, diabody, antibody Fc fusion, scFv1-ScFv2, scFv1 ２ -Fc-scFv2 ２ IgG-scFv, DVD-IgG, Triomab / Quadroma, Two-in-One IgG, scFv ２ -Fc, TandAb, scFv-HSA-scFv, Fab-scFv-Fc, dAb-IgG, IgG-VHH, tandem scFv-Fc, (scFv1) ２ -Fc-(VHH) ２ The binder according to claim 1, which is BiTe, DART, crossumab, scFv-Fc, one-arm tandem scFv-Fc, or DART-Fc.

3. The binder according to claim 2, wherein each of the first and second binding domains comprises an antibody fragment, and the antibody fragment is Fab, Fab', F(ab')2, Fv, or scFv.

4. The binder according to claim 1, wherein the first binding domain comprises scFv and the second binding domain comprises Fab.

5. The binder according to any one of claims 1 to 4, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL regions each have the amino acid sequences shown in SEQ ID NO: 81 and SEQ ID NO: 82, respectively.

6. The binder according to any one of claims 1 to 5, wherein the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL each have the amino acid sequences shown in SEQ ID NO: 101 and SEQ ID NO: 102, respectively.

7. A binder according to any one of claims 1 to 6, which does not contain an Fc domain.

8. A binder according to any one of claims 1 to 6, further comprising an Fc domain.

9. The binder according to claim 8, wherein the Fc domain is an IgG Fc domain.

10. The binder according to claim 9, wherein the Fc domain is selected from IgG1 and IgG4 Fc domains.

11. A pharmaceutical composition comprising a binder according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A nucleic acid encoding the binder according to any one of claims 1 to 10.

13. A vector comprising the nucleic acid according to claim 12.

14. A cell system comprising the vector according to claim 13.

15. A pharmaceutical composition for use in a method for treating an autoimmune disease, the method comprising the step of administering the pharmaceutical composition to a subject in need of it in an amount effective to reduce the number or activity of pathogenic immune cells in the subject and thereby alleviate the symptoms of the autoimmune disease.

16. The pharmaceutical composition for use according to claim 15, wherein the autoimmune disease is celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes mellitus), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis.

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

18. The pharmaceutical composition for use according to any one of claims 15 to 17, wherein the pathogenic immune cells are autoreactive CD4+ T cells, autoantibody-producing B cells, autoantigen-presenting cells, or autoantigen-presenting dendritic cells.

19. A pharmaceutical composition for use according to any one of claims 15 to 18, wherein the subject is celiac disease, Crohn's disease, juvenile idiopathic arthritis, inflammatory bowel disease (IBD), insulin-dependent diabetes mellitus (IDDM or type 1 diabetes mellitus), lupus nephritis, myasthenia gravis, myocarditis, multiple sclerosis (MS), pemphigus / bullous pemphigoid, rheumatoid arthritis (RA), scleroderma / systemic sclerosis, Sjögren's syndrome (SjS), systemic lupus erythematosus (SLE), or ulcerative colitis.

20. The pharmaceutical composition for use according to any one of claims 15 to 19, further comprising the step of administering an immunosuppressant to the subject.

21. A pharmaceutical composition for use according to any one of claims 15 to 20, wherein the administration of the pharmaceutical composition results in a reduction in the frequency or severity of disease relapses, a reduction in systemic inflammatory cytokines, or a reduction in self-reported symptoms associated with the autoimmune disease.

22. A pharmaceutical composition for use in a method for reducing or preventing the development of graft-versus-host disease (GVHD) after transplantation, the method comprising the step of administering the pharmaceutical composition to a subject in need thereof in an amount effective to activate or stimulate CD8+KIR+Treg, thereby reducing or alleviating at least one symptom of GVHD, wherein the binder substantially has no effector functional activity.

23. The pharmaceutical composition for use according to claim 22, wherein the administration of the pharmaceutical composition to the subject results in a reduction of symptoms associated with GVHD, a reduction of systemic inflammatory cytokines, a reduction of lesions in tissues affected by GVHD, a reduction in self-reporting of symptoms associated with immune responses related to adverse effects on host tissues, improvement or extension of graft survival, alleviation of one or more symptoms, and / or prevention, delay or slowing of the onset or progression of graft rejection, or extension of graft survival accompanied by a reduction in the use of broad-spectrum immunosuppressants.

24. A pharmaceutical composition for use according to any one of claims 17 to 23, characterized in that the CD8+KIR+Treg is in vivo in contact with the binder.

25. A pharmaceutical composition for use according to any one of claims 15 to 24, characterized in that the binder is to be administered intravenously or subcutaneously; and / or the binder is to be administered in a dose of 0.01 mg / kg to 20 mg / kg.

26. A pharmaceutical composition for use according to claim 17 or claim 18, characterized in that the CD8+KIR+Treg is in ex vivo contact with the binder.

27. The pharmaceutical composition for use according to claim 26, characterized in that the activated CD8+KIR+Treg is administered in an effective amount to a subject requiring administration.

28. A pharmaceutical composition for use according to any one of claims 17 to 27, wherein the CD8+KIR+Treg is MHC class I constrained and / or not MHC HLA E(Qa-1b) constrained.

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