KLRB1 binding agents and methods of use thereof

Abstract

KLRB1-binding agents (particularly anti-KLRB1 antibodies and antigen-binding portions thereof) and compositions thereof, and therapeutic methods using such agents, for example, to deplete or inhibit cells or activate cells (particularly Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, peTh2, ILC2, ILC3, NK cells, and / or neoplastic T or NK cells in vivo) for the treatment of autoimmune diseases, allergic diseases, transplant rejection, malignant hematological diseases, and cancer.

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 63 / 419,201, filed October 25, 2022, and U.S. Provisional Application No. 63 / 419,186, filed May 25, 2022, the entire contents of which are incorporated herein by reference.

[0002] Statement on Federally Sponsored Research This invention was made with government support under HL119145 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field The present disclosure relates generally to KLRB1 binding agents, particularly anti-KLRB1 antibodies, and methods of treatment using such agents for autoimmune diseases, allergic diseases, transplant rejection, hematological malignancies, and cancer. [Background technology]

[0004] Expression of killer cell lectin-like receptor subfamily B, member 1 (KLRB1, also known as CD161) defines a unique population of immune cells involved in various autoimmune and allergic diseases. For example, T helper IL-17-secreting (Th17) cells express KLRB1 (Maggi et al. 2010). Th17 cells and the cytokine they produce, IL-17, are involved in psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease, among other autoimmune diseases (Yang et al. 2014). Monoclonal antibody therapeutics targeting IL-17 or the upstream cytokine IL-23 that stimulates IL-17-producing cells are FDA-approved and commercially available for use in psoriasis (e.g., secukinumab and ixekizumab) and ankylosing spondylitis (e.g., secukinumab). However, the development of therapies based on targeting these molecules remains limited, and there remains a need for new therapeutic agents utilizing these targets for a variety of indications, including autoimmune diseases, allergic diseases, inflammatory diseases, and cancer. Summary of the Invention

[0005] Described herein are KLRB1-binding antibodies (and antigen-binding fragments thereof) that have many uses, including therapeutic and diagnostic uses. For example, the antibodies can be used to treat, and in some cases prevent (i.e., by depleting KLRB1-expressing cells) various diseases associated with KLRB1-expressing cells, such as autoimmune diseases including psoriasis, psoriatic arthritis, ankylosing spondylitis, palmoplantar pustulosis, hidradenitis suppurativa, and inflammatory bowel disease; allergic diseases including asthma and atopic dermatitis; transplant rejection; hematological malignancies; and cancer, and in some cases prevent (i.e., reduce the risk of developing) the following:

[0006] Provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to Killer Cell Lectin-Like Receptor Subfamily B, Member 1 (KLRB1; optionally SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3), the antibody, or antigen-binding portion thereof, comprising: (a) a heavy chain variable region (VH) comprising: a VH complementarity-Determining Region (CDR)1 comprising a sequence that is at least 95% identical to a VH CDR1 amino acid sequence set forth in one of Tables 1-18, preferably Tables 9, 8, or 18; a VH CDR2 comprising a sequence that is at least 95% identical to a VH CDR2 amino acid sequence set forth in one of Tables 1-18, preferably Tables 9, 8, or 18; and a VH CDR3 comprising a sequence that is at least 95% identical to a VH CDR3 amino acid sequence set forth in one of Tables 1-18, preferably Tables 9, 8, or 18; and (b) a light chain variable region (VL) comprising: a VL complementarity-Determining Region (CDR)1 comprising a sequence that is at least 95% identical to a VH CDR1 amino acid sequence set forth in one of Tables 1-18, preferably Tables 9, 8, or 18. and a VL CDR3 comprising a sequence at least 95% identical to a VL CDR3 amino acid sequence shown in one of Tables 1-18, preferably Tables 9, 8, or 18.

[0007] In some embodiments, the VH comprises or consists of a VH amino acid sequence shown in Table 9, 8, or 18. In some embodiments, the VL comprises or consists of a VL amino acid sequence shown in Table 9, 8, or 18. In some embodiments, the VH comprises or consists of a VH amino acid sequence shown in Table 3, and the VL comprises or consists of a VH amino acid sequence shown in Table 3. In some embodiments, the VH comprises or consists of a VH amino acid sequence shown in Table 5, and the VL comprises or consists of a VH amino acid sequence shown in Table 5. In some embodiments, the VH comprises or consists of a VH amino acid sequence shown in Table 8, and the VL comprises or consists of a VH amino acid sequence shown in Table 8. In some embodiments, the VH comprises or consists of a VH amino acid sequence shown in Table 9, and the VL comprises or consists of a VH amino acid sequence shown in Table 9. In some embodiments, the VH comprises or consists of a VH amino acid sequence set forth in Table 18, and the VL comprises or consists of a VH amino acid sequence set forth in Table 18. In some embodiments, the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to a VH amino acid sequence set forth in Table C, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to a VL amino acid sequence set forth in Table C. In some embodiments, the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to a VH amino acid sequence set forth in one of Tables 9, 8, or 18, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to an amino acid sequence set forth in one of Tables 9, 8, or 18, preferably the VH and VL are from the same table.

[0008] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy and light chain constant region, wherein the heavy and / or light chain constant region comprises or consists of an amino acid sequence set forth in one of Table A.

[0009] Further provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; e.g., SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 3, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 3, and optionally a constant region, optionally comprising a sequence at least 95% identical to a sequence set forth in Table A.

[0010] Also provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; e.g., SEQ ID NOS: 1-3), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 5, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 5, and optionally a constant region, optionally comprising a sequence at least 95% identical to a sequence set forth in Table A.

[0011] Further provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; e.g., SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 8, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 8, and optionally a constant region, optionally comprising a sequence at least 95% identical to a sequence set forth in Table A.

[0012] Further provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; e.g., SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 9, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 9, and optionally a constant region, optionally comprising a sequence at least 95% identical to a sequence set forth in Table A.

[0013] Further provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; e.g., SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 18, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 18, and optionally a constant region, optionally comprising a sequence at least 95% identical to a sequence set forth in Table A.

[0014] In some embodiments, the constant region comprises or consists of a sequence shown in Table A.

[0015] In some embodiments, the antibody comprises or consists of a heavy chain variable sequence and / or a light chain variable sequence that is at least 95% identical to a sequence shown in Table B.

[0016] In some embodiments, the antibody comprises or consists of a complete heavy and / or light chain variable sequence that is at least 95% identical to a sequence shown in Table C.

[0017] Also provided herein is an antibody, or antigen-binding portion thereof, that specifically binds to human KLRB1, comprising a CDR from a different table herein, or a heavy chain / light chain pair from a different table herein. In some embodiments, the antibody, or antigen-binding portion thereof, comprises the hinge region and Fc domain of the heavy chain constant region.

[0018] In some embodiments, the antibody or antigen-binding portion thereof is an antibody comprising a heavy chain constant region comprising an amino acid sequence having at least 80%, 90%, 95%, or 97% sequence identity to the amino acid sequence of the heavy chain constant region shown in Table A.

[0019] In some embodiments, the antibody or antigen-binding portion thereof is a monoclonal antibody.

[0020] In some embodiments, the antibody or antigen-binding portion thereof is a chimeric antibody, a humanized antibody, or a human antibody, and / or contains one or more mutations (e.g., in a CDR) that remove an Asn(N)-glycosylation site or that remove Cys, Asp, Met, Trp, or Lys.

[0021] In some embodiments, the antibody or antigen-binding portion is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody, or IgG4 antibody.

[0022] In some embodiments, the antibody or antigen-binding portion thereof is an antibody comprising an Fc region, preferably a human IgG1, that binds to an Fc gamma receptor (FcγR) and induces antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or that binds to C1q and induces complement-dependent cytotoxicity (CDC).

[0023] In some embodiments, the antibody or antigen-binding portion thereof is conjugated to a cytotoxic agent.

[0024] In some embodiments, the antibody or antigen-binding portion thereof comprises a defucosylated Fc region.

[0025] Because CD161 is an inhibitory receptor when bound to its ligand CLEC2D (LLT1), modulation of this interaction can affect immune cell activity. Thus, inhibition of the CD161 / CLEC2D interaction can enhance T cell function, a typical mechanism of action for a class of immuno-oncology therapeutics, while enhancement of the CD161 / CLEC2D interaction can act as immunosuppressant for autoimmune and allergic diseases. In some embodiments, the antibodies disclosed herein block the CD161 / CLEC2D interaction, thereby activating T cells or NK cells to attack tumor cells in various cancers, which is typical of an immuno-oncology checkpoint inhibition mechanism.

[0026] In some embodiments, the antibodies disclosed herein suppress T or NK cells by enhancing (e.g., increasing) CD161 / CLEC2D interaction, providing immunosuppression applicable to the treatment of various autoimmune, allergic, and inflammatory diseases.

[0027] In some embodiments, the KLRB1-binding antibodies disclosed herein achieve better immune cell activation when compared to the immune cell activation of one or more preceding antibodies (e.g., B199.2, HP-3G10, OTI1D8, 14F1F11, 702228, B-D51, 2F3, EP7169, DX1, DX12, 191B8, Ab9, KW1.2.1, KW7.3.7, or JNH25G2G22), e.g., exhibit improved activity in any one or more of the following: activating T cells to produce cytokines; activating T cells to kill tumor cells; activating NK cells (e.g., increased expression of CD107a); activating NK cells to produce cytokines or cytotoxic molecules such as granzymes; and activating NK cells to kill tumor cells.

[0028] In some embodiments, antibodies disclosed herein having an N-terminal glutamine or glutamic acid can be post-translationally modified at such N-terminus to form pyroglutamic acid (or pyrrolidone carboxylic acid).

[0029] Also provided herein is a polynucleotide comprising a nucleic acid sequence encoding an antibody or antigen-binding portion thereof described herein. In some embodiments, the nucleic acid sequence is operably linked to a promoter.

[0030] Further provided are vectors comprising the polynucleotides described herein, as well as host cells comprising the polynucleotides or vectors and, optionally, expressing the antibodies or antigen-binding portions thereof described herein. Also provided herein are methods for making the antibodies or antigen-binding portions thereof described herein. The methods may include culturing the host cells under conditions sufficient to express the antibodies or antigen-binding portions thereof, and isolating the antibodies or antigen-binding portions thereof. In some embodiments, the methods include formulating the antibodies as pharmaceutical compositions.

[0031] Further provided herein are pharmaceutical compositions comprising an antibody or antigen-binding portion thereof described herein (e.g., comprising or consisting of an antibody or antigen-binding portion thereof as an active agent) and a pharmaceutically acceptable carrier or diluent.

[0032] In some embodiments, the antibody or antigen-binding portion thereof is not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), DX1 (Thermo Fisher), DX12 (BD Biosciences), 191B8 (Miltenyi Biotec), Ab9 (PCT Publication No. WO2023028501A1), KW1.2.1 (U.S. Patent Publication No. US20210122826A1), KW7.3.7 (U.S. Patent Publication No. US20210122826A1), or JNH25G2G22 (Creative Diagnostics).

[0033] Also provided herein are methods for treating one or more of an autoimmune disease, an allergic disease, transplant rejection, and a malignant hematological disease in a subject in need thereof, the methods comprising administering to the subject an antibody that binds to KLRB1, preferably an antibody or an antigen-binding portion thereof, a polynucleotide, a vector, a pharmaceutical composition, or a host cell expressing the antibody or antigen-binding portion thereof (as described herein). Also provided are antibodies that bind to KLRB1, preferably an antibody or an antigen-binding portion thereof, a polynucleotide, a vector, a pharmaceutical composition, or a host cell expressing the antibody or antigen-binding portion thereof (as described herein), for use in treating one or more of an autoimmune disease, an allergic disease, transplant rejection, and a malignant hematological disease in a subject in need thereof. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa. In some embodiments, the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multiorgan transplant. In some embodiments, the hematological malignancy is leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL). In some embodiments, the hematological malignancy is a lymphoma, e.g., hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, mycosis fungoides, Sézary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), or peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

[0034] In some embodiments, the antibody or antigen-binding portion thereof, polynucleotide, vector, or antibody binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), innate lymphoid cell types 2 and 3 (ILC2 and ILC3), pathogenic effector Th2 (peTh2) cells, and / or NK cells expressing KLRB1.

[0035] In some embodiments of each of the above aspects and embodiments, as well as other aspects and embodiments described herein, the subject is a human.

[0036] Without wishing to be bound by theory, Applicants believe that the antibodies disclosed herein exhibit superiority over previously described antibodies, e.g., commercially available antibodies, in one or more of the following activities: - Increased binding potency (binding EC2 for cell-expressed KLRB1 or soluble KLRB1 extracellular domain) 50 decline); - Increased ADCC-mediated depletion potency (decreased EC50 for depletion of CHO-KLRB1+ cells or EC50 for reporter cell line ADCC assay) 50 decline); - Increased binding affinity kinetics (decreased KD or K off decline); - Increased blocking potency (IC for inhibition of CLEC2D binding to KLRB1-expressing cells) 50 a decrease in -Increased potentiation effect (EC 200 for enhanced (increased) CLEC2D binding to cell-expressed KLRB1) 50 (decrease in

[0037] In some embodiments, the antibodies disclosed herein have higher production yields, lower immunogenicity (due to the presence of humanized variable regions and / or human Fc sequences), and / or improved biophysical parameters (e.g., higher melting temperatures, higher freeze-thaw stability, reduced isomerization, reduced or absent deamidation, and / or reduced susceptibility to oxidation) compared to previously described antibodies, e.g., commercially available antibodies. Examples of previously described antibodies include B199.2, HP-3G10, OTI1D8, 14F1F11, 702228, B-D51, 2F3, EP7169, DX1, DX12, 191B8, Ab9, KW1.2.1, KW7.3.7, or JNH25G2G22.

[0038] When aspects or embodiments of the present disclosure are described in terms of Markush groups or other alternative groupings, the present disclosure includes not only the entire group listed together, but also each member of the group individually, as well as all possible subgroups of the main group and the main group lacking one or more of its members. The present disclosure also contemplates the explicit exclusion of any one or more of the group members in the claimed disclosure.

[0039] These and other advantages of the present technology will become apparent when reference is made to the accompanying drawings and the following description. [Brief explanation of the drawings]

[0040] [Figure 1] KLRB1 expression marks Th17, Th17.1, ex-Th17, Tc17, iNKTs, IL2, ILC3, peTh2, and NK cell subsets. [Figure 2] 1 shows whole-body KLRB1 expression profiling data showing that KLRB1 has no significant expression on any cell type other than immune cells. [Figure 3]1 shows that KLRB1 is overexpressed in rheumatoid arthritis synovium compared with osteoarthritis and normal synovium. [Figure 4] We show that KLRB1 is overexpressed in rheumatoid arthritis synovium compared with osteoarthritis and crystal-induced arthritis synovium. [Figure 5] We show that KLRB1 is overexpressed in salivary glands of patients with advanced and moderate Sjögren's syndrome compared to normal. [Figure 6] We show that KLRB1 is overexpressed in the parotid glands of patients with Sjögren's syndrome compared with normal. [Figure 7] We show that KLRB1 is overexpressed in muscle tissue from patients with inclusion body myositis. [Figure 8] We show that KLRB1 is overexpressed in skin tissue from patients with discoid lupus. [Figure 9] We show that KLRB1 is overexpressed in skin tissue from psoriasis patients. [Figure 10] We show that KLRB1 is overexpressed in lung tissue from patients with idiopathic pulmonary fibrosis. [Figure 11] We show that KLRB1 is overexpressed in pancreatic tissue from diabetic patients. [Figure 12] We show that KLRB1 is overexpressed in scalp tissue from patients with alopecia universalis. [Figure 13] We show that KLRB1 is overexpressed in liver tissue from patients with primary biliary cholangitis. [Figure 14] We show that KLRB1 is overexpressed in brain tissue from patients with multiple sclerosis. [Figure 15] We show that KLRB1 is overexpressed in colonic tissue from patients with lymphocytic colitis. [Figure 16] We show that KLRB1 is overexpressed in kidney tissue from kidney transplant patients. [Figure 17] We show that KLRB1 is overexpressed in broncheoalveolar lavage fluid from lung transplant patients. [Figure 18] We show that KLRB1 is overexpressed in skin tissues from patients with atopic dermatitis. [Figure 19] We show that KLRB1 is overexpressed in skin tissue from patients with palmoplantar pustulosis. [Figure 20] We show that KLRB1 is overexpressed in skin tissue from patients with hidradenitis suppurativa. [Figure 21] We show that KLRB1 is overexpressed in airway brushings from asthmatic patients. [Figure 22A] We show that KLRB1 is expressed in various T- and NK-cell lymphomas and leukemias. We show increased expression of KLRB1 in tumor cells from 4 / 4 patients with hepatosplenic T-cell lymphoma (HSTCL), 7 / 19 patients with NK / T-cell lymphoma (NKTCL), and 2 / 2 patients with mycosis fungoides. [Figure 22B] We show that KLRB1 is expressed in various T- and NK-cell lymphomas and leukemias. We show KLRB1 expression in various forms of peripheral T-cell lymphoma (PTCL), including angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL; both ALK-positive and ALK-negative), NK / T-cell lymphoma (NKTCL), peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), and T-cell prolymphocytic leukemia (T-PLL). [Figure 22C] Figure 1 shows that KLRB1 is expressed in various T and NK cell lymphomas and leukemias. In four patients each with HSTCL, increased KLRB1 expression is observed in splenic tumor cells compared to healthy spleens, and in tumor cells compared to healthy CD4 T cells. [Figure 22D] Figure 1 shows that KLRB1 is expressed in various T and NK cell lymphomas and leukemias. Increased expression of KLRB1 is shown from an HSTCL cell line (DERL-2) and aggressive NK cell leukemia (ANKL) cell lines (KHYG-1 and NKL). [Figure 23A]Figure 1 shows the binding of mouse antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, and 5A11D10 to human KLRB1 by ELISA. Human KLRB1 was coated onto an ELISA plate and incubated with the indicated antibodies. Secondary anti-mouse IgG was used to detect antibody binding to KLRB1 on the ELISA plate. Figure 2 shows the binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, and 5A11D10. [Figure 23B] Figure 1 shows the binding of mouse antibodies 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2 to human KLRB1 by ELISA. Human KLRB1 was coated onto an ELISA plate and incubated with the indicated antibodies. Secondary anti-mouse IgG was used to detect antibody binding to KLRB1 on the ELISA plate. Figure 2 shows the binding of 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2. [Figure 23C] Figure 1 shows the binding of mouse antibody 22F10G3 to human KLRB1 by ELISA. Human KLRB1 was coated onto an ELISA plate and incubated with the indicated antibodies. Secondary anti-mouse IgG was used to detect antibody bound to KLRB1 on the ELISA plate. Binding of 22F10G3 is shown. [Figure 24A] Figure 1 shows the binding of mouse antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1). Binding was detected by FACS. Antibody bound to CHO-hum-KLRB1 was detected using a secondary anti-mouse IgG. Figure 1 shows the binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO-hum-KLRB1. [Figure 24B]Figure 1 shows the binding of mouse antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO-K1 cells expressing cynomolgus monkey KLRB1 (CHO-cyno-KLRB1). Binding was detected by FACS. Antibody bound to CHO-cyno-KLRB1 was detected using a secondary anti-mouse IgG. Figure 1 shows the binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO-cyno-KLRB1. [Figure 24C] Figure 1 shows the binding of mouse antibodies 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2 to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1). Binding was detected by FACS. Antibody binding to CHO-hum-KLRB1 was detected using a secondary anti-mouse IgG. Figure 1 shows the binding of 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2 to CHO-hum-KLRB1. [Figure 24D] Figure 1 shows the binding of mouse antibody 22F10G3 to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1). Binding was detected by FACS. Antibody bound to CHO-hum-KLRB1 was detected using a secondary anti-mouse IgG. Figure 2 shows the binding of 22F10G3 to CHO-hum-KLRB1. [Figure 24E] Binding of mouse antibody 22F10G3 to CHO-K1 cells expressing cynomolgus monkey KLRB1 (CHO-cyno-KLRB1) is shown. Binding was detected by FACS. Antibody bound to CHO-cyno-KLRB1 was detected using secondary anti-mouse IgG. Binding of 22F10G35 to CHO-cyno-KLRB1 is shown. [Figure 25A]This figure shows an antibody-dependent cell-mediated cytotoxicity (ADCC) assay measuring target cell lysis of CHO-K1 target cells expressing human KLRB1 (CHO-hum-KLRB1) incubated with human peripheral blood mononuclear cells (PBMCs) and the following antibodies: mouse / human IgG1-kappa (wild-type) chimeric antibodies 57E2E4-chimera, 7B6C7-chimera, 52G9B12B5-chimera, 5B1B11-chimera, 50A8F2-chimera, and 47A5H2-chimera; 52G9B12B5-chimera EN (a mouse / human chimeric KLRB1-binding antibody with the 52G9B12B5 variable region and human IgG1-kappa mutations L234A / L235A / G237A; used as a negative control); and human IgG1 (negative control). The assay used detection of LDH release and formazan salt (Genscript, SC1544). The figure shows the % target cell lysis for each antibody at the various concentrations indicated. [Figure 25B] Figure 1 shows an antibody-dependent cell-mediated cytotoxicity (ADCC) assay measuring target cell lysis of CHO-K1 target cells expressing human KLRB1 (CHO-hum-KLRB1) incubated with human peripheral blood mononuclear cells (PBMCs) and the following antibodies in an ADCC cytolysis assay: mouse / human IgG1-kappa (wild-type) chimeric antibody 22F10G3-chimera, and human IgG1 (negative control). The assay used detection of LDH release and formazan salt (Genscript, SC1544). The figure shows the % target cell lysis for each antibody at the various concentrations indicated. [Figure 26A]This figure shows that some anti-KLRB1-binding antibodies compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1), potentially blocking CLEC2D's ability to bind to KLRB1. CHO-humKLRB1 cells were incubated with CLEC2D-Fc-biotin fusion protein and increasing concentrations of antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, and control mouse IgG. CLEC2D binding was determined by the addition of streptavidin-AF488. The graph shows the mean fluorescence intensity (MFI) over the indicated concentration range. Competition between 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, and mouse IgG and CLEC2D is shown. [Figure 26B] This figure shows that some anti-KLRB1-binding antibodies compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1), potentially blocking CLEC2D's ability to bind to KLRB1. CHO-humKLRB1 cells were incubated with CLEC2D-Fc-biotin fusion proteins and increasing concentrations of antibodies 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, and control mouse IgG. CLEC2D binding was determined by the addition of streptavidin-AF488. The graph shows the mean fluorescence intensity (MFI) over the indicated concentration range. Competition between 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, and mouse IgG and CLEC2D is shown. [Figure 26C]This figure shows that some anti-KLRB1-binding antibodies compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1), potentially blocking the ability of CLEC2D to bind to KLRB1. CHO-humKLRB1 cells were incubated with CLEC2D-Fc-biotin fusion protein and increasing concentrations of antibody 50A8F2 and control mouse IgG. CLEC2D binding was determined by the addition of streptavidin-AF488. The graph shows mean fluorescence intensity (MFI) across the indicated concentration range. Competition between 50A8F2 and mouse IgG with CLEC2D is shown, demonstrating that 50A8F2 enhances CLEC2D binding to KLRB1. [Figure 26D] This figure shows that some anti-KLRB1-binding antibodies compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1), potentially blocking CLEC2D's ability to bind to KLRB1. CHO-humKLRB1 cells were incubated with CLEC2D-Fc-biotin fusion protein and increasing concentrations of antibodies 50A8F2, 47A5H2, and control mouse IgG. CLEC2D binding was determined by the addition of streptavidin-AF488. The graph shows mean fluorescence intensity (MFI) across the indicated concentration range. A second replicate experiment shows competition between CLEC2D and a larger concentration range of 50A8F2, 47A5H2, and mouse IgG, demonstrating that 50A8F2 enhances CLEC2D binding to KLRB1. [Figure 26E]This figure shows that some anti-KLRB1-binding antibodies compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1), potentially blocking the ability of CLEC2D to bind to KLRB1. CHO-humKLRB1 cells were incubated with CLEC2D-Fc-biotin fusion protein and increasing concentrations of antibody 22F10G3 and control mouse IgG. CLEC2D binding was determined by the addition of streptavidin-AF488. The graph shows mean fluorescence intensity (MFI) across the indicated concentration range. Competition between 22F10G3 and mouse IgG with CLEC2D is shown. [Figure 27A] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04 is shown. [Figure 27B] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08 is shown. [Figure 27C] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12 is shown. [Figure 27D] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimera is shown. [Figure 27E]1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7 is shown. [Figure 27F] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8 is shown. [Figure 28A] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular proteins by ELISA. Binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04 is shown. [Figure 28B] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular proteins by ELISA. Binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08 is shown. [Figure 28C] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular proteins by ELISA. Binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12 is shown. [Figure 28D] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular proteins by ELISA. Binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimera is shown. [Figure 28E]

[0033] Figure 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular proteins by ELISA. Binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7 is shown. Hu50A8F2-04 did not bind. [Figure 28F] Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to cynomolgus monkey KLRB1 extracellular protein by ELISA is shown. Binding of Hu47A5H2-11, HP-3G10, and DX12 is shown. 191B8 did not bind. [Figure 29A] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04 is shown. [Figure 29B] Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS is shown. Binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08 is shown. [Figure 29C] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12 is shown. [Figure 29D] Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS is shown. Binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimera is shown. [Figure 29E]Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS is shown. Binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7 is shown. [Figure 29F] Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-hum-KLRB1 cells as measured by FACS is shown. Binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8 is shown. [Figure 30A] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04 is shown. [Figure 30B] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08 is shown. [Figure 30C] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12 is shown. [Figure 30D] Binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS is shown. Binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimera is shown. [Figure 30E]1 shows binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7 is shown. [Figure 30F] 1 shows the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies to CHO-cyno-KLRB1 cells as measured by FACS. Binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8 is shown. [Figure 31A] Figure 1 shows blocking of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, as measured by FACS. Blocking by Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04 is shown. [Figure 31B] Figure 1 shows blocking of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, as measured by FACS. Blocking by Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08 is shown. [Figure 31C] Figure 1 shows blocking of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, as measured by FACS. Blocking of binding by Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12 is shown. [Figure 31D] Figure 1 shows blocking of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, as measured by FACS. Blocking by Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimera is shown. [Figure 31E]Blockade of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, as measured by FACS, is shown, along with Hu47A5H2-11, Ab9, KW1.2.1, and KW7.3.7. [Figure 31F] Blockade of CLEC2D binding to CHOK1-human KLRB1 cells by 47A5H2 chimeric and humanized antibodies and other KLRB1-binding antibodies, Hu47A5H2-11, HP-3G10, DX12, and 191B8, as measured by FACS, is shown. [Figure 32A] 1 shows the binding of 50A8F2 chimeric and humanized antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04 is shown. [Figure 32B] 1 shows the binding of 50A8F2 chimeric and humanized antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08 is shown. [Figure 32C] 1 shows the binding of 50A8F2 chimeric and humanized antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12 is shown. [Figure 32D] 1 shows the binding of 50A8F2 chimeric and humanized antibodies to human KLRB1 extracellular protein by ELISA. Binding of Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2-chimera is shown. [Figure 33A] Figure 1 shows the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells, and the lack of binding to CHO-cyno-KLRB1 cells, as measured by FACS. Figure 2 shows the binding of Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04 to CHO-hum-KLRB1. [Figure 33B] Figure 1 shows the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells, and the lack of binding to CHO-cyno-KLRB1 cells, as measured by FACS. Figure 2 shows the binding of Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08 to CHO-hum-KLRB1. [Figure 33C] Figure 1 shows the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells, and the lack of binding to CHO-cyno-KLRB1 cells, as measured by FACS. Binding of Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12 is shown. [Figure 33D] Figure 1 shows the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells, and the lack of binding to CHO-cyno-KLRB1 cells, as measured by FACS. Figure 2 shows the binding of Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2-chimera to CHO-hum-KLRB1. [Figure 33E] Figure 1 shows the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells, and the lack of binding to CHO-cyno-KLRB1 cells, as measured by FACS. The lack of binding of 16 humanized 50A8F2 antibodies Hu50A8F2-01 to Hu50A8F2-16 and the 50A8F2 chimera to CHO-cyno-KLRB1 is shown, along with the binding of Hu47A5H2 chimera (as a positive control). [Figure 34A] 1 shows that the 50A8F2 chimeric and humanized antibodies enhanced CLEC2D binding to CHO-hum-KLRB1 cells as measured by FACS. 1 shows enhancement of CLEC2D / CHO-hum-KLRB1 binding by Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04. [Figure 34B]1 shows that the 50A8F2 chimeric and humanized antibodies enhanced CLEC2D binding to CHO-hum-KLRB1 cells as measured by FACS. 1 shows enhancement of CLEC2D / CHO-hum-KLRB1 binding by Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08. [Figure 34C] 1 shows that the 50A8F2 chimeric and humanized antibodies enhanced CLEC2D binding to CHO-hum-KLRB1 cells as measured by FACS. 1 shows enhancement of CLEC2D / CHO-hum-KLRB1 binding by Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12. [Figure 34D] Figure 1 shows that the 50A8F2 chimeric and humanized antibodies enhanced CLEC2D binding to CHO-hum-KLRB1 cells as measured by FACS. Figure 2 shows enhancement of CLEC2D / CHO-hum-KLRB1 binding by Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2 chimera. Hu47A5H2 inhibited, rather than enhanced, CLEC2D / CHO-hum-KLRB1 binding. [Figure 35] Figure 1 shows that the 50A8F2 chimeric and humanized antibodies Hu50A8F2-01, Hu50A8F2-03, Hu50A8F2-04, and Hu50A8F2-06 enhanced CLEC2D binding to CHOK1-human KLRB1 cells as measured by FACS. In comparison, human IgG1 had no effect on CLEC2D binding to CHOK1-human KLRB1 cells, and the KW1.2.1, KW7.3.7, and 47A5H2 chimeric antibodies inhibited CLEC2D binding to CHOK1-human KLRB1 cells. [Figure 36]Figures A and B show that the 47A5H2 chimeric antibody increases NK cell activation as measured by FACS. Figure A shows that CD107a expression on NK cells was reduced by K562-CLEC2D compared to K562 cells, and then increased by 47A5H2 chimeric (47A5H2-chi) antibody. Figure B shows that IFN-gamma (IFNg) expression on NK cells was reduced by K562-CLEC2D compared to K562 cells, and then increased by 47A5H2 chimeric antibody. [Figure 37A] These figures show that NK cell activation was inhibited by CLEC2D, and that anti-KLRB1 blocking antibodies activated NK cells in the presence of CLEC2D inhibition. Primary NK cells were less activated by K562-CLEC2D single-cell clone 1D1 than by K562 cells. Hu47A5H2-11 and other anti-KLRB1 antibodies enhanced NK cell activation in assays targeting NK cells to K562-CLEC2D, but not in assays targeting NK cells alone to K562 cells. NK cell activation measured as CD107a expression is shown. [Figure 37B] These results show that NK cell activation was inhibited by CLEC2D, and that anti-KLRB1 blocking antibodies activated NK cells in the presence of CLEC2D inhibition. Primary NK cells were less activated with K562-CLEC2D single-cell clone 1D1 than with K562 cells. Hu47A5H2-11 and other anti-KLRB1 antibodies enhanced NK cell activation in assays targeting NK cells to K562-CLEC2D, but not in assays targeting NK cells alone to K562 cells. NK cell activation measured as IFNg expression is shown. [Figure 38]Figures A-B show that Hu47A5H2-11 enhanced NK cell activation as measured by CD107a expression by FACS, targeting K562-CLEC2D clone 1D1 cells. Hu47A5H2-11 had superior potency (EC50 0.039 nM) compared to other tested antibodies. Figure A shows Hu47A5H2-11 compared to Ab9 (EC50 0.118 nM), KW1.2.1 (EC50 4.946 nM), and KW7.3.7 (EC50 36.29 nM). Figure B shows Hu47A5H2-11 compared to HP-3G10 (EC50 1.071 nM), DX12 (EC50 6.137 nM), and 191B8 (EC50 0.085 nM). [Figure 39] (A-B) show that Hu47A5H2-11 enhanced NK cell activation, as measured by IFN-gamma expression by FACS, targeting K562-CLEC2D clone 1D1 cells. Hu47A5H2-11 had superior potency (EC50 0.131 nM) compared to other tested antibodies. (A) shows Hu47A5H2-11 compared to Ab9 (EC50 0.436 nM), as well as KW1.2.1 and KW7.3.7 (neither of which activated NK cell IFNg production). B shows Hu47A5H2-11 compared to HP-3G10 (EC50 2.510 nM), DX12 (EC50 7.956 nM), and 191B8 (EC50 0.2475 nM; more precisely 0.383 nM taking into account NK cytotoxicity at 40 nM). [Figure 40] 4 shows that the 47A5H2 chimeric and humanized antibody enhances NK cell killing in a primary NK cell versus Raji target cell assay and has superior potency than KW1.2.1 and KW7.3.7. [Figure 41](A-B) Hu47A5H2-11 enhances NK cell killing in a primary NK cell versus Raji target cell assay and has superior potency to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8. (A) Hu47A5H2-11 (EC50 0.036 nM) compared to Ab9 (EC50 0.055 nM), KW1.2.1 (EC50 0.112 nM), and KW7.3.7 (EC50 0.440 nM). B shows Hu47A5H2-11 (EC50 0.036 nM) compared to HP-3G10 (EC50 0.325 nM), DX12 (no effect), and 191B8 (EC50 0.064 nM). [Figure 42] Figure 1 shows that 47A5H2-chimera antibody enhanced T cell activation in a Jurkat-NFAT-1G4TCR-KLRB1 vs. K562-CLEC2D-HLA assay loaded with 0.005 μg / ml of NY-ESO-1 peptide. 47A5H2-chimera, but not 50A8F2, increased T cell activation. [Figure 43] (A-B) show that Hu47A5H2-11 enhances T cell activation in a Jurkat-NFAT-1G4TCR-KLRB1 vs. K562-CLEC2D-HLA assay loaded with 0.005 μg / ml of NY-ESO-1 peptide, with superior potency to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8. (A) shows Hu47A5H2-11 (EC50 0.379 nM) compared to Ab9 (EC50 0.675 nM), KW1.2.1 (not activating), KW7.3.7 (not activating), and Hu50A8F2-04 (not activating). B shows Hu47A5H2-11 (EC50 0.379 nM) compared to HP-3G10 (EC50 12.56 nM), DX12 (EC50 0.179 nM), and 191B8 (EC50 0.392 nM). DETAILED DESCRIPTION OF THE INVENTION

[0041] KLRB1 expression marks a unique set of immune system cells involved in various autoimmune diseases, including Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, and / or NK cells.

[0042] KLRB1 is also expressed by tumor cells present in many T and NK cell malignancies, including a variety of peripheral T cell and NK cell lymphomas and leukemias.

[0043] The KLRB1-binding antibodies described herein can be used to preferentially target Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells for depletion. Populations of KLRB1-expressing immune cells express IL-17 more abundantly than total CD4 or CD8 T cell populations, and are more specific for Th17 and Tc17 T cells than CD4 or CD8, respectively. Accordingly, disclosed herein are methods of treating a subject in need thereof by administering to the subject an effective amount of a killer cell lectin-like receptor B1 (KLRB1)-binding agent with cell-depleting activity, thereby depleting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells in vivo. These methods can be used to treat autoimmune diseases, allergic diseases, transplant rejection, or hematological malignancies, for example, as described herein.

[0044] Furthermore, because modulation of KLRB1 has inhibitory or stimulatory effects on T cells and NK cells, methods are described herein for treating a subject in need thereof by administering to the subject an effective amount of a killer cell lectin-like receptor B1 (KLRB1)-binding agent having KLRB1 receptor-blocking activity (achieved by blocking the binding of CLEC2D (LLT1) to KLRB1), thereby activating or inhibiting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, and / or peTh2 in vivo. These methods can be used to activate T cells and / or NK cells and enhance their activity against tumor cells used in the treatment of cancer.

[0045] In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) effector activity or complement-dependent cytotoxicity (CDC) effector activity. Administration of an effective amount of an anti-KLRB1 antibody having ADCC or CDC effector function or linked to a cytotoxic agent to a subject in need thereof can eliminate or reduce the number of Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells. In some embodiments, the present disclosure provides a killer cell lectin-like receptor B1 (KLRB1)-binding agent described herein that has ADCC or CDC activity or is conjugated to a cytotoxic agent. In various aspects, the present disclosure provides mRNA or cDNA encoding the binding agent. In various aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of the binding agent.

[0046] Various features of the present disclosure, including KLRB1 and its ligands, anti-KLRB1 antibodies or antigen-binding portions thereof, pharmaceutical compositions, treatments and administration, and illustrative examples, are discussed in order below. The following section contains definitions of terms used in this disclosure. Unless otherwise defined herein, technical and scientific terms used in describing the present invention have the meanings commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following terminology shall apply, and where appropriate, terms used in the singular shall include the plural and terms used in the plural shall also include the singular. In the event that any provided terminology conflicts with any document incorporated herein by reference, the following terminology shall control.

[0047] Killer cell lectin-like receptor B1 (KLRB1) Killer cell lectin-like receptor B1 (KLRB1), also known as CD161, is a type II transmembrane protein. It regulates the activity of T cells and NK cells and can act as either a stimulatory (Fergusson et al. 2014) or inhibitory (Aldemir et al. 2005, Mathewson et al. 2021) receptor on different types of T cells and NK cells. This receptor is present on the surface of various immune cells. The ligand for KLRB1 is LLT1 (also known as CLEC2D) (Aldemir et al. 2005).

[0048] KLRB1 expression among T cells is restricted to cells capable of responding to IL-12 and IL-18 (Fergusson et al. 2014). Among CD4+ T helper cells, KLRB1 expression uniquely characterizes Th17, Th17.1, and ex-Th17 cells (cells that no longer produce IL-17 but produce IFN-γ). KLRB1 expression distinguishes Th1 IFNG-producing cells (KLRB1-negative) from ex-Th17 IFNG-producing cells (which are KLRB1+) (Basdeo et al. 2017). Among CD8+ cytotoxic T cells, KLRB1 expression uniquely characterizes Tc17 cells. Thus, KLRB1 characterizes T cells capable of IL-17 production or, in the case of ex-Th17 cells, interferon-gamma (IFNG) production. This cytokine production is undesirable in autoimmune diseases.

[0049] Described herein are antibodies and antigen-binding fragments thereof that bind to KLRB1, preferably human KLRB1. An exemplary sequence of human KLRB1 is provided as SEQ ID NO: 1:MDQQAIYAELNLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIQKSSIEKCSVDIQQSRNKTTERPGLLNCPIYWQQLREKCLLFSHTVNPWNNSLADCSTKESSLLLIRDKDELIHTQNLIRDKAILFWIGLNFSLSEKNWKWINGSFLNSNDLEIRGDAKENSCISISQTSVYSEYCSTEIRWICQKELTPVRNKVYPDS.

[0050] In some embodiments, the KLRB1 is cynomolgus monkey KLRB1 (e.g., AOA2K5WYI1 from UniParc UPI0003ABB264); an exemplary sequence is provided as SEQ ID NO: 2:MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWQQVQEKCLLFSHTVNPWNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0051] In some embodiments, the KLRB1 is cynomolgus monkey KLRB1 (e.g., UniProt A0A7N9D796); an exemplary sequence is provided as SEQ ID NO: 3:MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWKQVQEKCLLFSHTVNPWNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0052] KLRB1 binding antibody Described herein are antibodies and antigen-binding fragments thereof that bind to KLRB1.The term "antibody" refers to an immunoglobulin molecule or an immunologically active portion thereof, i.e., an antigen-binding portion.

[0053] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for the possibility of naturally occurring mutations that may be present in minor amounts. The antibody may be monoclonal. The antibody may be human or humanized. The term "monoclonal antibody" includes intact, full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (e.g., scFv), fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecule comprising at least one antigen-binding site. Furthermore, "monoclonal antibody" refers to such antibodies produced by a number of techniques, including, but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0054] The term "chimeric antibody" refers to an antibody in which a portion of its heavy and / or light chain is derived from a first source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0055] The term "humanized antibody," as used herein, refers to an antibody comprising human heavy and light chain variable regions in which native CDR residues are replaced by residues from the corresponding CDR of a non-human antibody (e.g., a mouse, rat, rabbit, or non-human primate antibody), such that the non-human antibody has the desired specificity, affinity, and / or activity. In some embodiments, one or more framework region amino acid residues of the human heavy or light chain variable region are replaced by the corresponding residue from the non-human antibody. Furthermore, humanized antibodies may contain residues that are not found in human or non-human antibodies. In some embodiments, these modifications are made to further refine and / or optimize the antibody's properties. In some embodiments, the humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically that of a human immunoglobulin. Exemplary constant regions include those set forth in Table A. [Table 1-1] [Table 1-2]

[0056] The term "human antibody," as used herein, refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or an antibody made using any technique known to those of skill in the art for making human antibodies, including, but not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma technology.

[0057] An "antibody fragment" can include a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0058] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the portion of an antigen or target that can be recognized and bound by a specific antibody. When the antigen or target is a polypeptide, an epitope can be formed from both contiguous amino acids and non-contiguous amino acids juxtaposed by the folding of the protein into a tertiary structure. Epitopes formed from contiguous amino acids (also called linear epitopes) typically persist upon denaturation of the protein, whereas epitopes formed by folding into a tertiary structure (also called conformational epitopes) typically are lost upon denaturation of the protein. Epitopes typically contain at least three, and more commonly at least five, six, seven, or eight to ten, amino acids in a unique spatial conformation. Epitopes can be predicted using any one of numerous software bioinformatics tools available online. X-ray crystallography may be used to characterize epitopes on target proteins by analyzing the interactions of amino acid residues in an antigen / antibody complex.

[0059] "Fv" comprises the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, noncovalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site. Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy-chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0060] Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0061] In various embodiments, the antibody or antigen-binding fragment thereof comprises a human or humanized antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. Methods for humanizing non-human antibodies are well known in the art.

[0062] The KLRB1 antibodies described herein can be affinity matured, for example, using selection and / or mutagenesis methods known in the art. Generally, an "affinity matured" antibody has one or more modifications in one or more hypervariable regions thereof that improve the affinity of the antibody for an antigen compared to a parent antibody lacking those modification(s). In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Preferred affinity matured antibodies have 5-fold, more preferably 10-fold, and even more preferably 20- or 30-fold better affinity than the starting antibody (typically murine, humanized, or human) from which the matured antibody is prepared.

[0063] An antibody that "binds," "specifically binds to," or is "specific" for a particular polypeptide or epitope on a particular polypeptide is one that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptides or polypeptide epitopes. The term "specifically binds," as used herein, refers to a KLRB1 agent (e.g., an anti-KLRB1 antibody) that interacts with a particular antigen, epitope, protein, or target molecule with a higher frequency, a faster rate, a longer duration, or with higher affinity than other substances, or some combination thereof. Binding agents (e.g., antibodies) that specifically bind to an antigen can be identified, for example, by immunoassays, ELISA, surface plasmon resonance (SPR) assays (e.g., Biacore), or other techniques known to those skilled in the art. Accordingly, functional equivalents of the specific anti-KLRB1 antibodies described herein are described. In some cases, KLRB1 antibodies may be cross-reactive with various similar KLRB1 proteins (e.g., having the highest affinity for proteins such as human KLRB1 and lower affinity for other proteins such as mouse KLRB1). A binding agent that specifically binds to an antigen binds to its target antigen with higher affinity than to a different antigen, which may be a related antigen. In some embodiments, a binding agent that specifically binds to an antigen binds to its target antigen with at least 20-fold higher affinity than to a different antigen, e.g., at least 30-fold higher affinity, at least 40-fold higher affinity, at least 50-fold higher affinity, at least 60-fold higher affinity, at least 70-fold higher affinity, at least 80-fold higher affinity, at least 90-fold higher affinity, or at least 100-fold higher affinity than to a different antigen. In some embodiments, a binding agent that specifically binds to a particular antigen binds to a different antigen with such low affinity that binding is not detectable using the assays described herein or otherwise known in the art. In some embodiments, affinity is measured using SPR techniques known to those of skill in the art, such as a Biacore system or other systems.

[0064] With respect to two or more polypeptides (e.g., two anti-KLRB1 antibodies), the term "identical" or percent "identity" refers to two or more sequences or subsequences that have the same amino acid residues or a specified percentage of the same amino acid residues when compared and aligned for maximum correspondence (introducing gaps, if necessary), without considering any conservative amino acid substitutions as part of sequence identity. Percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.

[0065] The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions × 100). Sequence comparison and determination of percent identity between two sequences can be achieved using mathematical algorithms. Such homology can be determined using local alignment tools and / or algorithms well known in the art, and can include pairwise alignment, multiple sequence alignment, structural alignment, and / or phylogenetic analysis. When sequences differ by conservative substitutions, the percent sequence identity may, but need not, be adjusted upward to compensate for the conservative nature of the substitution. Means for making this adjustment are well known to those skilled in the art. Typically, but not necessarily, this involves scoring conservative substitutions as partial rather than full mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, then conservative substitutions are given a score of 0-1.

[0066] In some embodiments, two polypeptides of the disclosure (e.g., antibodies or antibody domains thereof (e.g., VL, CL, VH, CH1, CH2, CH3 domains)) are substantially identical means that the amino acid residues are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99%, identical when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the percent identity exists over a region of the sequence that is at least about 10, at least about 20, at least about 20-40, at least about 40-60 amino acid residues, at least about 60-80 nucleotides or amino acid residues in length, or any integer value therebetween. In some embodiments, the percent identity is over a region longer than 60-80 amino acid residues, e.g., at least about 80-100 amino acid residues, and in some embodiments, the sequences are substantially identical over the entire length of the sequences being compared, e.g., the amino acid sequences.

[0067] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences.

[0068] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program of the GCG software package (available on the World Wide Web at gcg.com) using a Blossum62 scoring matrix with default parameters, e.g., a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5 (available on the World Wide Web at gcg.com).

[0069] As used herein, the term "conservative sequence modifications" or "conservative substitutions" may refer to amino acid modifications to the target epitopes or antibodies and antigen-binding portions thereof of the present disclosure that do not significantly affect or alter the binding characteristics of the anti-KLRB1 antibodies. The phrase "conservative amino acid substitutions," as used herein, refers to substitutions in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have generally been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of tyrosine with phenylalanine is considered a conservative substitution. Methods for identifying conservative amino acid substitutions that do not abolish binding are well known in the art.

[0070] In various embodiments, the antibody is a blocking or antagonist binding agent. "Blocking" or "antagonist" refers to an agent (e.g., an antibody or binding fragment thereof) that inhibits or reduces the biological activity of the antigen to which it binds. Particular blocking or antagonist agents substantially or completely inhibit the biological activity of the antigen. For example, a KLRB1-binding agent can block KLRB1 signaling (e.g., thereby interfering with KLRB1 signaling and modulating Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or tumor T cells or NK cells).

[0071] In some embodiments, the KLRB1-binding agent is an antibody comprising: a. a full-length antibody comprising an Fc domain that binds to KLRB1 and has an effector function capable of binding to an Fc gamma receptor and inducing antibody-dependent cell-mediated cytotoxicity (ADCC); b. an antibody comprising an Fc domain that binds to KLRB1 and has an effector function capable of binding to complement protein 1q (C1q) and inducing complement-dependent cytotoxicity (CDC); c. an antibody conjugate, e.g., an antibody-drug conjugate (ADC), that binds to KLRB1 and comprises a cytotoxic drug; or d. a multispecific antibody (e.g., a bispecific or trispecific antibody) that binds to KLRB1 and another antigen.

[0072] In some embodiments, the KLRB1 antibodies described herein bind to the extracellular domain of human KLRB1. In some embodiments, the antibodies cross-react with (bind to) both the extracellular domains of human and cynomolgus KLRB1. In some embodiments, the antibodies bind to an epitope in the extracellular domain of KLRB1 that is at least 90% identical in humans and cynomolgus monkeys.

[0073] In some embodiments, the antibody binds to KLRB1 and is not a murine antibody.

[0074] In some embodiments, the antibody described herein is not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), 191B8 (Miltenyi), DX12 (BD Biosciences), or JNH25G2G22 (Creative Diagnostics).

[0075] In some embodiments, the KLRB1-binding agent is an antibody, e.g., a full-length antibody comprising an Fc domain including at least one heavy chain. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is an IgA antibody, an IgD antibody, an IgE antibody, an IgG antibody, or an IgM antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG2 antibody. In some embodiments, the antibody is an IgG3 antibody. In some embodiments, the antibody is an IgG4 antibody.

[0076] In some embodiments, the antibody is an antibody fragment comprising an antigen-binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a bispecific or multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure. In some embodiments, the KLRB1 binding agent is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those of skill in the art. In some embodiments, polyclonal antibodies are produced by immunizing animals (e.g., rabbits, rats, mice, goats, donkeys) with an antigen of interest (e.g., purified peptide fragments, recombinant proteins, or fusion proteins) using multiple subcutaneous or intraperitoneal injections. In some embodiments, the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. The antigen (with or without a carrier protein) is diluted in sterile saline and typically combined with an adjuvant (e.g., complete or incomplete Freund's adjuvant) to form a stable emulsion. After a period of time, polyclonal antibodies are recovered from the immunized animal (e.g., from the blood or ascites fluid). In some embodiments, the polyclonal antibodies are purified from the serum or ascites fluid using standard methods in the art, including, but not limited to, affinity chromatography, ion exchange chromatography, gel electrophoresis, and / or dialysis.

[0077] In some embodiments, the KLRB1 binding agent is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known to those of skill in the art. In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those of skill in the art. For example, using the hybridoma method, a mouse, rat, rabbit, hamster, or other suitable host animal is immunized as described above. In some embodiments, lymphocytes are immunized in vitro. In some embodiments, the immunizing antigen is a human protein or a fragment thereof. In some embodiments, the immunizing antigen is a mouse protein or a fragment thereof.

[0078] After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, e.g., using polyethylene glycol. The hybridoma cells are selected using specialized media, as known in the art, and unfused lymphocytes and myeloma cells do not survive the selection process. Hybridomas producing monoclonal antibodies specifically directed against a given antigen can be identified by various methods, including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution techniques. The hybridomas can be grown in vitro in culture using standard methods or in vivo as ascites tumors in animals. Monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art, including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.

[0079] In some embodiments, monoclonal antibodies are produced using recombinant DNA techniques known to those of skill in the art. For example, polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the antibody heavy and light chains, and the sequences of the heavy and light chains are determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector that produces the monoclonal antibody when transfected into host cells (such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin protein unless transfected with an expression vector).

[0080] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of the desired species. Screening of phage libraries can be carried out by various techniques known in the art.

[0081] In some embodiments, monoclonal antibodies are modified using recombinant DNA technology to generate alternative antibodies. In some embodiments, the light and heavy chain constant domains of a murine monoclonal antibody are replaced with constant regions from a human antibody to generate chimeric antibodies. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. In some embodiments, site-directed or high-density mutagenesis of the variable region(s) is used to optimize the specificity and affinity of the monoclonal antibody.

[0082] In some embodiments, the KLRB1 binding agent is a humanized antibody. Various methods for producing humanized antibodies are known in the art. In some embodiments, a humanized antibody contains one or more amino acid residues from a non-human source introduced into its sequence. In some embodiments, humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody. In some embodiments, a humanized antibody is constructed by substituting all six CDRs of a non-human antibody (e.g., a murine antibody) with the corresponding CDRs of a human antibody.

[0083] The selection of human heavy and / or light chain variable regions to use in generating a humanized antibody can be based on a variety of factors and can be performed by a variety of methods known in the art. In some embodiments, a "best-fit" approach is used, in which the sequence of a variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence most similar to the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody. In some embodiments, a particular variable region framework is selected as the variable region framework, derived from the consensus sequence of all human antibodies with a particular subgroup of light or heavy chains. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most commonly observed human subclass. In some embodiments, human germline genes are used as the source of the variable region framework sequences.

[0084] Other humanization methods include, but are not limited to, a method called "hyperhumanization," which has been described as directly transferring CDRs into human germline frameworks; a method named human string content (HSC), which is based on a metric called "antibody humanness"; methods based on generating large libraries of humanized variants (including phage, ribosomal, and yeast display libraries); and methods based on framework region shuffling.

[0085] In some embodiments, the KLRB1-binding agent is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced from lymphocytes isolated from immunized individuals. In either case, cells producing antibodies against the target antigen can be generated and isolated. In some embodiments, human antibodies are selected from phage libraries, where the phage library expresses human antibodies. Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable region gene repertoires derived from unimmunized donors. Techniques for generating and using antibody phage libraries are well known in the art. Once an antibody is identified, affinity maturation methods known in the art, including, but not limited to, chain shuffling and site-directed mutagenesis, can be used to generate human antibodies with higher affinity. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. These mice are capable, upon immunization, of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production.

[0086] In some embodiments, the KLRB1-binding agent is an scFv antibody. An ScFv is a molecule comprising a variable heavy chain region and a variable light chain region linked to form a single polypeptide. ScFvs can be produced using recombinant techniques known in the art. In some embodiments, the scFv comprises a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, the scFv comprises, in N-terminal to C-terminal direction, (i) a heavy chain variable region, (ii) a linker, and (iii) a light chain variable region. In some embodiments, the scFv comprises, in N-terminal to C-terminal direction, (i) a light chain variable region, (ii) a linker, and (iii) a heavy chain variable region. In some embodiments, the scFv is a disulfide-linked scFv (dsscFv), which is an scFv that comprises an engineered disulfide bond between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, the scFv (e.g., dsscFv) is conjugated (either directly or indirectly) to a half-life extending moiety, such as, for example, an Fc molecule, an immunoglobulin CH3 domain (e.g., IgG1 CH3), polyethylene glycol (PEG) or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polydiphosphate, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, e.g., by hyperglycosylation, to extend the half-life of the scFv (e.g., dsscFv).

[0087] A variety of suitable linkers are known to those of skill in the art and are not limited by any of the specific sequences disclosed herein. In some embodiments, the polypeptide linker is composed of naturally occurring or non-naturally occurring amino acids. In some embodiments, the linker includes amino acids that allow for flexibility. In some embodiments, the linker includes amino acids that allow for solubility. In some embodiments, the linker includes glycine amino acids. In some embodiments, the linker includes glycine and serine amino acids. In certain embodiments, the linker includes one or more sets of glycine / serine repeats. In some embodiments, the polypeptide linker is selected from the group consisting of (GGGGS)n (where n=1-4) (SEQ ID NO:399), GGGGS (SEQ ID NO:399), GGGGSGGGGS (SEQ ID NO:400), GGGGSGGGGSGGGGS (SEQ ID NO:401), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:402), and (GGGGA)n (where n=1-4) (SEQ ID NO:403). In some embodiments, the linker comprises GGGGSGGGSGGGGS (SEQ ID NO: 401).

[0088] In some embodiments, the KLRB1-binding agent is an Fv. The Fv comprises a heavy chain variable region and a light chain variable region. In some embodiments, the Fv is conjugated (either directly or indirectly) to a half-life-enhancing moiety, such as, for example, an Fc molecule, an IgG CH3 domain (e.g., the CH3 of IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, for example, by hyperglycosylation, to extend the half-life of the Fv.

[0089] In some embodiments, the KLRB1-binding agent is a Fab. Fab is one of the molecules resulting from digestion of an immunoglobulin antibody with papain. Fab is a monovalent molecule comprising a light chain, a heavy chain variable region, a CH1 region, and optionally a heavy chain constant region hinge region or a portion thereof. Fab can be produced using recombinant techniques known in the art. In some embodiments, the Fab comprises a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, the Fab comprises a polypeptide linker between the heavy chain constant region and the light chain variable region. A variety of suitable linkers are known to those skilled in the art and are not limited by any particular sequence disclosed herein. In certain embodiments, the linker is a linker described herein. In some embodiments, the Fab is conjugated (either directly or indirectly) to a half-life extending moiety, such as, for example, an Fc molecule, a CH3 domain of an IgG (e.g., the CH3 of an IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, for example, by hyperglycosylation, to extend the half-life of the Fab.

[0090] In some embodiments, a Fab comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, a Fab comprises a disulfide bond that increases the stability of the Fab molecule. In some embodiments, a Fab comprises a disulfide bond that increases the thermal stability of the Fab molecule.

[0091] In some embodiments, the KLRB1 binding agent is F(ab')2. F(ab')2 is one of the molecules resulting from digestion of an immunoglobulin antibody with pepsin. F(ab')2 is a bivalent molecule comprising a first light chain associated with a first polypeptide comprising a first heavy chain variable region, a first CH1, and a first hinge region, and a second light chain associated with a second polypeptide comprising a second heavy chain variable region, a second CH1, and a second hinge region, wherein the first hinge region is linked to the second hinge region via at least one disulfide bond. F(ab')2 can be produced using recombinant techniques known in the art. In some embodiments, the F(ab')2 is conjugated (either directly or indirectly) to a half-life extending moiety, such as, for example, the CH3 domain of an IgG (e.g., the CH3 of an IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, for example, by hyperglycosylation, to extend the half-life of the F(ab')2.

[0092] In some embodiments, the F(ab')2 comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, the F(ab')2 comprises a disulfide bond that increases the stability of the F(ab')2 molecule. In some embodiments, the F(ab')2 comprises a disulfide bond that increases the thermal stability of the F(ab')2 molecule.

[0093] In some embodiments, the KLRB1-binding agent is a F(ab'). An F(ab') is a molecule resulting from treatment of an F(ab') with beta-mercaptoethanol. An F(ab') is a monovalent molecule comprising a light chain associated with a polypeptide comprising a heavy chain variable region, a CH1, and a hinge region. In some embodiments, the F(ab') is conjugated (either directly or indirectly) to a half-life-extending moiety, such as, for example, an Fc molecule, an IgG CH3 domain (e.g., the CH3 of an IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, for example, by hyperglycosylation, to extend the half-life of the F(ab').

[0094] In some embodiments, the F(ab') comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, the F(ab') comprises a disulfide bond that increases the stability of the F(ab') molecule. In some embodiments, the F(ab') comprises a disulfide bond that increases the thermal stability of the F(ab') molecule.

[0095] In some embodiments, the KLRB1-binding agent is a bispecific antibody. A bispecific antibody can recognize and bind to at least two different antigens or epitopes. These different epitopes may be epitopes within the same molecule (e.g., two epitopes on KLRB1) or epitopes on different molecules (e.g., one epitope on KLRB1 and one epitope on a different target). In some embodiments, a bispecific antibody has enhanced efficacy compared to an individual antibody or a combination of two or more antibodies. In some embodiments, a bispecific antibody has reduced toxicity compared to an individual antibody or a combination of two or more antibodies. Those skilled in the art know that any therapeutic agent can have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents, where the two individual binding agents have different PK profiles. In some embodiments, bispecific antibodies are capable of focusing the action of two agents on a common area (e.g., tissue) in a subject (e.g., a human). In some embodiments, bispecific antibodies are capable of focusing the action of two agents on a common target (e.g., a given type of cell). In some embodiments, bispecific antibodies are capable of directing the action of two agents on more than one biological pathway or function. In some embodiments, bispecific antibodies are capable of targeting two different cells and bringing them into proximity with each other.

[0096] In some embodiments, bispecific antibodies have reduced toxicity and / or side effects. In some embodiments, bispecific antibodies have reduced toxicity and / or side effects compared to two individual antibodies or a mixture of antibodies as a single agent. In some embodiments, bispecific antibodies have an improved therapeutic index. In some embodiments, bispecific antibodies have an improved therapeutic index compared to two individual antibodies or a mixture of antibodies as a single agent.

[0097] Several techniques for generating bispecific antibodies are known to those skilled in the art. In some embodiments, bispecific antibodies comprise heavy chain constant regions with modifications to amino acids at the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation. In some embodiments, the bispecific antibodies are generated using the knobs-into-holes (KIH) method. In some embodiments, the bispecific antibodies comprise variant hinge regions that prevent disulfide bond formation between identical heavy chains (e.g., reducing homodimer formation). In some embodiments, the bispecific antibodies comprise heavy chains with amino acid modifications that alter electrostatic interactions. In some embodiments, the bispecific antibodies comprise heavy chains with amino acid modifications that alter hydrophobic / hydrophilic interactions.

[0098] Bispecific antibodies may be intact antibodies or antibody fragments that contain the antigen-binding site.

[0099] Trivalent or higher valent KLRB1 binding agents are also contemplated. In some embodiments, trispecific or tetraspecific antibodies are generated.

[0100] In some embodiments, the KLRB1-binding agent is an anti-KLRB1 antibody that comprises one, two, three, four, five, and / or six CDRs of any one of the antibodies described herein. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 18G11F11, e.g., those shown in Table 1, and / or (ii) one, two, and / or three light chain CDRs from antibody 18G11F11, e.g., those shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 18G11F11, e.g., those shown in Table 1 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 18G11F11, e.g., those shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 9C10G11, e.g., those shown in Table 2, and / or (ii) one, two, and / or three light chain CDRs from antibody 9C10G11, e.g., those shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 9C10G11, e.g., those shown in Table 2 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 9C10G11, e.g., those shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 57E2E4, e.g., those shown in Table 3, and / or (ii) one, two, and / or three light chain CDRs from antibody 57E2E4, e.g., those shown in Table 3. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 57E2E4, e.g., those shown in Table 3 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 57E2E4, e.g., those shown in Table 3. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 75B10F12, e.g., those shown in Table 4, and / or (ii) one, two, and / or three light chain CDRs from antibody 75B10F12, e.g., those shown in Table 4.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 75B10F12, e.g., those shown in Table 4 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 75B10F12, e.g., those shown in Table 4. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 7B6C7, e.g., those shown in Table 5, and / or (ii) one, two, and / or three light chain CDRs from antibody 7B6C7, e.g., those shown in Table 5. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 7B6C7, e.g., those shown in Table 5 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 7B6C7, e.g., those shown in Table 5. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 52G9B12B5, e.g., those shown in Table 6, and / or (ii) one, two, and / or three light chain CDRs from antibody 52G9B12B5, e.g., those shown in Table 6. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 52G9B12B5, e.g., those shown in Table 6 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 52G9B12B5, e.g., those shown in Table 6. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 5B1B11, e.g., those shown in Table 7, and / or (ii) one, two, and / or three light chain CDRs from antibody 5B1B11, e.g., those shown in Table 7. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 5B1B11, e.g., those shown in Table 7 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chains from antibody 5B1B11, e.g., those shown in Table 7.In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 50A8F2, e.g., those shown in Table 8, and / or (ii) one, two, and / or three light chain CDRs from antibody 50A8F2, e.g., those shown in Table 8. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 50A8F2, e.g., those shown in Table 8 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 50A8F2, e.g., those shown in Table 8. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 47A5H2, e.g., those shown in Table 9, and / or (ii) one, two, and / or three light chain CDRs from antibody 47A5H2, e.g., those shown in Table 9. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 47A5H2, e.g., those shown in Table 9 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 47A5H2, e.g., those shown in Table 9. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 66G9C11, e.g., those shown in Table 10, and / or (ii) one, two, and / or three light chain CDRs from antibody 66G9C11, e.g., those shown in Table 10. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 66G9C11, e.g., those shown in Table 10 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 66G9C11, e.g., those shown in Table 10. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 33F11A6, e.g., those shown in Table 11, and / or (ii) one, two, and / or three light chain CDRs from antibody 33F11A6, e.g., those shown in Table 11.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 33F11A6, e.g., those shown in Table 11 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 33F11A6, e.g., those shown in Table 11. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 37G6A7, e.g., those shown in Table 12, and / or (ii) one, two, and / or three light chain CDRs from antibody 37G6A7, e.g., those shown in Table 12. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 37G6A7, e.g., those shown in Table 12 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 37G6A7, e.g., those shown in Table 12. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 39C5D6, e.g., those shown in Table 13, and / or (ii) one, two, and / or three light chain CDRs from antibody 39C5D6, e.g., those shown in Table 13. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 39C5D6, e.g., those shown in Table 13 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 39C5D6, e.g., those shown in Table 13. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 62H1D6, e.g., those shown in Table 14, and / or (ii) one, two, and / or three light chain CDRs from antibody 62H1D6, e.g., those shown in Table 14. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 62H1D6, e.g., those shown in Table 14 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 62H1D6, e.g., those shown in Table 14.In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 92E12F2, e.g., those shown in Table 15, and / or (ii) one, two, and / or three light chain CDRs from antibody 92E12F2, e.g., those shown in Table 15. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 92E12F2, e.g., those shown in Table 15 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 92E12F2, e.g., those shown in Table 15. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 13H2D11, e.g., those shown in Table 16, and / or (ii) one, two, and / or three light chain CDRs from antibody 13H2D11, e.g., those shown in Table 16. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13H2D11, e.g., those shown in Table 16 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 13H2D11, e.g., those shown in Table 16. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 5A11D10, e.g., those shown in Table 17, and / or (ii) one, two, and / or three light chain CDRs from antibody 5A11D10, e.g., those shown in Table 17. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 5A11D10, e.g., those shown in Table 17 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 5A11D10, e.g., those shown in Table 17. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 22F10G3, e.g., those shown in Table 18, and / or (ii) one, two, and / or three light chain CDRs from antibody 22F10G3, e.g., those shown in Table 18.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 22F10G3, e.g., those shown in Table 18 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 22F10G3, e.g., those shown in Table 18.

[0101] In some embodiments, the KLRB1 binding agent is a humanized version of an anti-KLRB1 antibody that comprises (i) one, two, and / or three heavy chain CDRs and / or (ii) one, two, and / or three light chain CDRs from any one of Tables 1 to 18. In some embodiments, the KLRB1 binding agent is a humanized version of an anti-KLRB1 antibody that comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) and (ii) three light chain CDRs from any one of Tables 1 to 18, wherein the heavy chain CDRs and light chain CDRs are from the same table. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9-1] [Table 9-2] Table 10-1 Table 10-2 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Table 18 Table 19-1 Table 19-2

[0102] In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from an antibody described herein. In some embodiments, the KLRB1-binding agent comprises a humanized version or humanized variant of an antibody described herein. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 18G11F11 (Table 1) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 9C10G11 (Table 2) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, derived from antibody 57E2E4 (Table 3) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, derived from antibody 75B10F12 (Table 4) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, derived from antibody 7B6C7 (Table 5) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, derived from antibody 52G9B12B5 (Table 6) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, CDR3, from antibody 5B1B11 (Table 7) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, CDR3, from antibody 50A8F2 (Table 8) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, CDR3, from antibody 47A5H2 (Table 9) or a humanized version thereof.In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 66G9C11 (Table 10) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 33F11A6 (Table 11) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 37G6A7 (Table 12) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 39C5D6 (Table 13) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 62H1D6 (Table 14) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 92E12F2 (Table 15) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 13H2D11 (Table 16) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, from antibody 5A11D10 (Table 17) or a humanized version thereof. In some embodiments, the KLRB1-binding agent comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, CDR3 from antibody 22F10G3 (Table 18) or a humanized version thereof.

[0103] In some embodiments, the KLRB1-binding agent comprises a humanized version or humanized variant of an antibody described herein. In some embodiments, the KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3, and / or a light chain CDR1, CDR2, CDR3, or a humanized version thereof, as set forth in Tables 1-18.

[0104] CDRs are defined by those skilled in the art using a variety of methods / systems. These systems and / or definitions have been developed and refined over the years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence diversity and is generally the most widely used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence diversity and location within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs, such as abYsis (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi), are available and known to those skilled in the art for analyzing antibody sequences and determining CDRs.

[0105] The CDR sequences defined herein are generally based on the Kabat definitions. However, it will be understood that when referring to the heavy chain CDR(s) and / or light chain CDR(s) of a given antibody, all CDR definitions known to those of skill in the art are included, e.g., as set forth in the tables herein. In some embodiments, all CDR sequences used are identified using the same definition, i.e., all Chothia, all Kabat, all IMGT, etc.

[0106] In some embodiments, the KLRB1-binding agent is a variant of an agent described herein. In some embodiments, the KLRB1-binding agent (e.g., an antibody) comprises (a) a heavy chain CDR1, CDR2, CDR3, or variants thereof comprising one, two, three, or four amino acid substitutions from a VH sequence presented herein (e.g., in Tables 1-18, Table B, or Table C); and / or a light chain CDR1, CDR2, and / or CDR3, or variants thereof comprising one, two, three, or four amino acid substitutions from a VL sequence presented herein (e.g., in Tables 1-18, Table B, or Table C). In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, a CDR comprises one conservative amino acid substitution. In some embodiments, a CDR comprises two conservative amino acid substitutions. In some embodiments, a CDR comprises three conservative amino acid substitutions. In some embodiments, a CDR comprises four conservative amino acid substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain CDR2. In some embodiments, the CDR is a heavy chain CDR3. In some embodiments, the CDR is a light chain CDR1. In some embodiments, the CDR is a light chain CDR2. In some embodiments, the CDR is a light chain CDR3. In some embodiments, the substitution is made as part of a humanization process. In some embodiments, the substitution is made as part of a germline humanization process. In some embodiments, the substitution is made as part of an affinity maturation process. In some embodiments, the substitution is made as part of an optimization process.

[0107] In some embodiments, a KLRB1-binding agent (e.g., an antibody) comprises one or more heavy or light chain CDRs that have been modified to reduce the potential for asparagine (N)-glycosylation, cysteinylation, asparagine (Asn) deamidation, aspartic acid (Asp) isomerization, methionine / tryptophan (Met / Trp) oxidation, and non-enzymatic lysine (Lys) glycosylation within the CDR sequences, e.g., to reduce deamidation within the CDR sequences, remove Asn(N)-glycosylation sites, remove cysteines, or remove Asps to reduce isomerization sites, or remove Met / Trp or Lys (see, e.g., Haberger et al., MAbs. 2014 Mar 1;6(2):327-339; Lu et al., MAb. 2019 Jan;11(1):45-57). Deamidation is a chemical reaction in which the amide functional group in the side chain of the amino acid asparagine (N) or glutamine (Q) is removed or converted to another functional group. Generally, asparagine is converted to aspartic acid or isoaspartic acid, and glutamine is converted to glutamic acid or polyglutamic acid. In some cases, deamidation can alter the structure, function, and / or stability of a polypeptide, potentially resulting in a loss of biological activity.

[0108] Exemplary human heavy and light chain variable region sequences are shown in Table B. [Table 20-1] [Table 20-2] [Table 20-3] [Table 20-4] [Table 20-5] [Table 20-6] [Table 20-7] [Table 20-8] [Table 20-9] [Table 20-10] [Table 20-11] [Table 20-12] [Table 20-13] [Table 20-14] [Table 20-15]

[0109] In certain embodiments, the KLRB1-binding agent comprises a heavy chain variable region comprising heavy chain CDRs 1, 2, and 3, and a light chain variable region comprising light chain CDRs 1, 2, and 3, as shown in Tables 1-18, Table B, or Table C.

[0110] In some embodiments, a KLRB1-binding agent (e.g., an antibody) comprises a heavy chain variable region or sequence having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a heavy chain variable region sequence presented herein (e.g., in Tables 1-18, Table B, or Table C), and / or a light chain variable region having at least 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a light chain variable region sequence presented herein (e.g., in Tables 1-18, Table B, or Table C). The term "consensus sequence," as used herein with respect to the light chain (VL) and heavy chain (VH) variable regions, refers to a composite or generalized VL or VH sequence that is defined based on information such that amino acid residues within the VL or VH chain can be modified without adversely affecting antigen binding. Thus, in a VL or VH chain "consensus sequence," a particular amino acid position is occupied by one of multiple possible amino acid residues at that position. For example, if arginine (R) or serine (S) is present at a particular position, that particular position in the consensus sequence can be either arginine or serine (R or S). Consensus sequences for VH and VL chains can be defined, for example, by in vitro affinity maturation (e.g., randomizing every amino acid position in a particular CDR using degenerate-coded primers), by scanning mutagenesis of amino acid residues within antibody CDRs (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by assessing binding of the mutants to the antigen to determine whether the mutated amino acid position affects antigen binding. In some embodiments, mutations are introduced in the CDR regions. In other embodiments, mutations are introduced in the framework regions. In some other embodiments, mutations are introduced in the CDRs and framework regions. Consensus sequences can be determined by software such as EMBOSS Cons, available at ebi.ac.uk / Tools / msa / emboss_cons / .

[0111] In some embodiments, the KLRB1-binding agents (e.g., antibodies) described herein comprise one or more constant heavy chain domains (e.g., CH1, CH2, and / or CH3 regions). In some embodiments, the KLRB1-binding agents comprise constant heavy chain domain 1 (CH1) having an amino acid sequence described herein, e.g., in Table B or C. In some embodiments, the KLRB1-binding agents comprise constant heavy chain domain 2 (CH2) comprising an amino acid sequence described herein, e.g., in Table B or C. In some embodiments, the KLRB1-binding agents comprise constant heavy chain domain 3 (CH3) comprising an amino acid sequence described herein, e.g., in Table B or C. In some embodiments, the KLRB1-binding agents comprise a heavy chain constant region comprising an amino acid sequence having at least 80, 85%, 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence described herein, e.g., in Table B or C. In some embodiments, one or more constant regions of the KLRB1-binding agents are modified. In some embodiments, an antibody may comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody comprises two or more human constant regions. In some embodiments, modifications to the constant region comprise the addition, deletion, or substitution of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain is removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced with a short amino acid spacer, which provides some of the flexibility of the molecule normally provided by the deleted constant region. In some embodiments, the modified antibody comprises a CH3 domain fused directly to the hinge region of the antibody. In some embodiments, the modified antibodies comprise a peptide spacer inserted between the hinge region and the modified CH2 domain and / or the modified CH3 domain.

[0112] It is known in the art that the constant region(s) of an antibody mediate several effector functions, and these effector functions can vary depending on the antibody isotype. Additionally, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). There are numerous Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors), and IgM (mu receptors). When an antibody binds to an Fc receptor on a cell surface, many important and diverse biological responses are elicited, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cellular cytotoxicity, or ADCC), cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. In some embodiments, an antibody comprises a variant Fc region. The amino acid sequences of the Fc regions of human IgG1, IgG2, IgG3, and IgG4 are known to those of skill in the art (e.g., a representative human IgG1 Fc region is shown in Lobner et al., Immunol Rev. 2016 Mar;270(1):113-131; see, e.g., Tables A and C). In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, variant Fc regions have been engineered to have substitutions at predetermined amino acid positions compared to native Fc regions. In some embodiments, the Fc region is mutated to alter (reduce) antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-induced complement-dependent cytotoxicity (CDC), and / or antibody-dependent cell-mediated phagocytosis (ADCP) (see, e.g., Kang and Jung, Experimental & Molecular Medicine. 2019. 51:1-9; Wang et al., Antibody Therapeutics, January 2021. 4(1):45-54; Lobner et al., Immunol Rev. 2016 Mar; 270(1):113-131).In some embodiments, the Fc region is defucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. 2009 May-Jun;1(3):230-236, which describes methods for producing therapeutic antibodies with controlled levels of Fc region N-glycan fucosylation).

[0113] In some embodiments, an engineered antibody (e.g., an engineered Fc region) alters effector function, which in turn affects the biological profile of the antibody. For example, in some embodiments, the constant region is deleted or inactivated (through point mutation or other means) to enhance Fc receptor binding of the engineered antibody when it is in circulation. In some embodiments, the constant region is modified to extend the serum half-life of the antibody. In some embodiments, the constant region is modified to shorten the serum half-life of the antibody. In some embodiments, the constant region is modified to increase or enhance ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or carbohydrate moieties. In some embodiments, the constant region is modified to add / substitute one or more amino acids to provide one or more cytotoxins, carbohydrates, or carbohydrate binding sites.

[0114] Modifications to the constant regions of the antibodies described herein may be made using well-known biochemical or molecular engineering techniques. In some embodiments, antibody variants are prepared by introducing appropriate nucleotide changes into the encoding DNA and / or by synthesizing the desired antibody or polypeptide. These antibody variants may be used to enhance the activity or effector function conferred by a particular sequence or region, while substantially retaining the structure, binding activity, and other desired characteristics of the modified antibody.

[0115] Exemplary complete heavy chain (HC) and light chain (LC) sequences are shown in Table C. Table 21-1 Table 21-2 Table 21-3 Table 21-4 Table 21-5 Table 21-6 Table 21-7 Table 21-8 Table 21-9 Table 21-10 Table 21-11 Table 21-12 Table 21-13 Table 21-14 Table 21-15 Table 21-16 Table 21-17 Table 21-18 Table 21-19 Table 21-20 Table 21-21 Table 21-22 Table 21-23 Table 21-24 Table 21-25 Table 21-26 Table 21-27

[0116] In some embodiments, a KLRB1-binding agent (e.g., an antibody) described herein comprises a heavy chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to an HC sequence set forth in Table C, and / or a light chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to an LC sequence set forth in Table C. In some embodiments, the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to a VH amino acid sequence set forth in Table C, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to a VL amino acid sequence set forth in Table C.

[0117] The present disclosure further includes additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including, but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, or solubility. It will be apparent to those skilled in the art that amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0118] Variations may be substitutions, deletions, or insertions of one or more nucleotides encoding the antibody or polypeptide that alter the amino acid sequence compared to that of the native antibody or polypeptide. In some embodiments, amino acid substitutions result from replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., conservative amino acid substitutions, such as replacing leucine with serine. Insertions or deletions may optionally range from about 1 to 5 amino acids. In some embodiments, the substitutions, deletions, or insertions include fewer than 25, 20, 15, 10, 5, 4, 3, or 2 amino acid substitutions relative to the parent molecule. In some embodiments, biologically useful and / or relevant variations in amino acid sequence are determined by systematically making insertions, deletions, or substitutions in the sequence and testing the activity of the resulting variant proteins relative to the parent protein.

[0119] In some embodiments, variants may include additional amino acid residues at the amino and / or carboxyl termini of the antibody or polypeptide. The additional amino acid residues may range in length from one residue to 100 or more residues. In some embodiments, variants include an N-terminal methionyl residue. In some embodiments, variants include an additional polypeptide / protein, i.e., a fusion protein. In some embodiments, variants may be engineered to be detectable and include a detectable label and / or protein (e.g., an enzyme).

[0120] In some embodiments, cysteine ​​residues that are not involved in maintaining the proper conformation of the antibody may be substituted or deleted to adjust antibody characteristics, e.g., to improve oxidative stability and / or prevent aberrant disulfide bridges. Conversely, in some embodiments, one or more cysteine ​​residues may be added to form disulfide bond(s) to improve stability.

[0121] The variant antibodies or variant polypeptides described herein may be made using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0122] In some embodiments, the KLRB1-binding agents described herein are chemically modified. In some embodiments, the KLRB1-binding agents are anti-KLRB1 antibodies that have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linkage to a cellular ligand or other protein. Any of a number of chemical modifications may be performed by known techniques.

[0123] The present disclosure includes KLRB1-binding agents constructed on a non-immunoglobulin scaffold, which bind to the same or essentially the same epitope as the anti-KLRB1 antibodies disclosed herein. In some embodiments, the non-immunoglobulin-based binding agent is an agent that competes with the anti-KLRB1 antibodies described herein in a competitive binding assay. In some embodiments, alternative KLRB1-binding agents include scaffold proteins. Generally, scaffold proteins can be assigned to one of three groups based on their backbone configuration: (1) scaffolds composed of α-helices, (2) small scaffolds with little secondary structure or a disordered configuration of α-helices and β-sheets, and (3) scaffolds composed primarily of β-sheets. Scaffold proteins include, but are not limited to, anticalins, which are based on lipocalin scaffolds; adnectins, which are based on the 10th domain of human fibronectin type 3; affibodies, which are based on the B domain of the Ig-binding region of Staphylococcus aureus protein A; darpins, which are based on ankyrin repeat domain proteins; phinomers, which are based on the SH3 domain of human Fyn protein kinase; affitins, which are based on Sac7d from Sulfolobus acidocaldarius; affilins, which are based on human gamma-B-crystallin or human ubiquitin; avimers, which are based on the A domain of membrane receptor proteins; knottins (cysteine ​​knot miniproteins), which are based on a stable 30-amino acid antiparallel beta-strand protein fold; and Kunitz domain inhibitor scaffolds, which are based on a structure containing three disulfide bonds and three loops. In some embodiments, the KLRB1-binding agent comprises an engineered scaffold protein comprising a heavy chain CDR1, CDR2, and CDR3, and a light chain CDR1, CDR2, and CDR3, as shown in any one of Tables 1-18, e.g., Table 8, 9, or 18.

[0124] Antigen-antibody interactions are generally non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. When describing the strength of an antigen-antibody complex, the terms affinity and / or avidity are commonly used. The binding of an antibody to its antigen is a reversible process, and the affinity of that binding is typically measured by the equilibrium dissociation constant (K D ) and report it as K D is the antibody association rate (k on ) (the rate at which an antibody binds to its antigen) relative to the antibody dissociation rate (k off ) (the rate at which an antibody dissociates from its antigen). In some embodiments, K D The k value for a particular antibody / antigen interaction on and k off The velocity is measured and then the ratio of these values ​​is used to calculate K D It is found by calculating the value of K D The K value may be used to assess and rank order the strength of individual antibody / antigen interactions. D The lower the affinity, the higher the affinity of the antibody for the target. In some embodiments, affinity is measured using SPR technology in a Biacore system. Avidity provides a measure of the overall strength of the antibody-antigen complex. Avidity is determined by three main parameters: (i) the affinity of the antibody for the target, (ii) the valency of both the antibody and the antigen, and (iii) the structural organization of the interacting moieties.

[0125] In some embodiments, the KLRB1 binding agent (e.g., an antibody) has a dissociation constant (K) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) at a K of about 20 nM or less. DIn some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 10 nM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 1 nM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 0.5 nM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 0.1 nM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 50 pM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 25 pM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 10 pM or less. D In some embodiments, the KLRB1 binding agent binds to KLRB1 (e.g., human KLRB1) with a K of about 1 pM or less. D In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) for KLRB1 is determined using KLRB1 protein immobilized on a Biacore chip and the binding agent loaded onto the chip. In some embodiments, the dissociation constant of a binding agent (e.g., an antibody) for KLRB1 is determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble KLRB1 loaded onto the chip.

[0126] In some embodiments, a KLRB1-binding agent (e.g., an antibody) binds to KLRB1 (e.g., human KLRB1) with a half-maximal effective concentration (EC50) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a KLRB1-binding agent binds to human KLRB1 with an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a KLRB1-binding agent binds to cyno KLRB1 and / or human KLRB1 with an EC50 of about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

[0127] The KLRB1-binding agents (e.g., antibodies) described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis to constructing DNA sequences encoding the polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest using recombinant techniques. Optionally, the sequence can be mutated by site-directed mutagenesis to create functional variants thereof. In some embodiments, a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the codon selection that will be favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding a particular isolated polypeptide can be synthesized. For example, several short oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0128] Once a polynucleotide sequence encoding a particular polypeptide of interest has been constructed (by synthesis, site-directed mutagenesis, or otherwise), it can be inserted into an expression vector and operably linked to appropriate expression control sequences to express the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and / or expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0129] In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies against human KLRB1 or fragments thereof. For example, a recombinant expression vector can be a replicable DNA construct containing synthetic or cDNA-derived DNA fragments encoding the polypeptide chain of a KLRB1-binding agent, such as an anti-KLRB1 antibody, operably linked to suitable transcriptional and / or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises an assembly of: (1) genetic element(s) that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structural sequence, i.e., a coding sequence, that is transcribed into mRNA and translated into protein; and (3) appropriate start and stop sequences for transcription and translation. Regulatory elements can include operator sequences to control transcription. The ability to replicate in a host (usually conferred by an origin of replication) and a selection gene to facilitate recognition of transformants may also be incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of that polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of that coding sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of the translated protein by the host cell. In some embodiments, in situations where a recombinant protein is expressed without a leader or transport sequence, the polypeptide may include an N-terminal methionine residue, which can optionally be later cleaved from the expressed recombinant protein to provide the final product.

[0130] The choice of expression control sequences and expression vectors is generally determined by the choice of host. A wide variety of expression host / vector combinations can be used. Expression vectors useful for eukaryotic hosts include, for example, vectors containing expression control sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids such as those derived from E. coli, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host-range plasmids such as M13 and other filamentous single-stranded DNA phages.

[0131] KLRB1-binding agents (e.g., antibodies) of the present disclosure can be expressed from one or more vectors. For example, in some embodiments, a heavy chain polypeptide is expressed by one vector and a light chain polypeptide is expressed by a second vector. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed by a single vector.

[0132] Suitable host cells for expressing KLRB1-binding agents (e.g., antibodies) or KLRB1 proteins or fragments thereof for use as antigens or immunogens include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive bacteria, such as E. coli or Bacillus. Higher eukaryotic cells include mammalian cell lines, as described herein. Cell-free translation systems may also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as methods for protein production, including antibody production, are well known in the art.

[0133] Various mammalian culture systems may be used to express recombinant polypeptides. It may be desirable to express recombinant proteins in mammalian cells because these proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney) cell lines, L-929 (mouse fibroblast) cell lines, C127 (mouse mammary carcinoma) cell lines, 3T3 (mouse fibroblast) cell lines, CHO (Chinese hamster ovary) cell lines, HeLa (human cervical carcinoma) cell lines, BHK (hamster kidney fibroblast) cell lines, HEK-293 (human embryonic kidney) cell lines, and variants thereof. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, as well as other 5' or 3' flanking non-transcribed sequences and 5' or 3' untranslated sequences, such as essential ribosome binding sites, polyadenylation sites, splice donor sites, splice acceptor sites, and transcription termination sequences.

[0134] Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a powerful method for producing correctly folded, biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those of skill in the art. Accordingly, the present disclosure provides cells comprising a KLRB1-binding agent described herein. In some embodiments, the cell produces a KLRB1-binding agent described herein. In some embodiments, the cell produces an antibody. In some embodiments, the cell produces an antibody that binds to human KLRB1. In some embodiments, the cell produces an antibody that binds to cyno KLRB1. In some embodiments, the cell produces an antibody that binds to human KLRB1 and cyno KLRB1. In some embodiments, the cells produce antibodies designated 18G11F11, 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 22F10G3, or variants thereof. In some embodiments, the cell produces an scFv version of antibody 18G11F11, 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, or 22F10G3. In some embodiments, the cell is a prokaryotic cell (e.g., E. coli). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hybridoma cell. Proteins produced by the host cell can be purified according to any suitable method.Standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, and glutathione-S-transferase, can be attached to proteins to facilitate purification by loading them onto an appropriate affinity column. Affinity chromatography used to purify immunoglobulins includes protein A chromatography, protein G chromatography, and protein L chromatography. Isolated proteins can be physically characterized using techniques such as proteolysis, size exclusion chromatography (SEC), mass spectrometry (MS), nuclear magnetic resonance (NMR), isoelectric focusing (IEF), high-performance liquid chromatography (HPLC), and X-ray crystallography. The purity of isolated proteins can be determined using techniques known to those skilled in the art, including, but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF). In some embodiments, supernatants from expression systems that secrete recombinant protein into the culture medium are first concentrated using a commercially available protein concentration filter, such as an Amicon® or Millipore Pellicon® ultrafiltration device. After the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin is used, such as a matrix or substrate with pendant diethylaminoethyl (DEAE) groups. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. In some embodiments, a cation exchange step is used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups.In some embodiments, hydroxyapatite media is used, including, but not limited to, ceramic hydroxyapatite (CHT). In some embodiments, the recombinant protein is further purified using one or more reverse-phase HPLC steps using a hydrophobic RP-HPLC media, such as silica gel with pendant methyl or other aliphatic groups. In some embodiments, hydrophobic interaction chromatography (HIC) is used to separate recombinant proteins based on their hydrophobicity. HIC is a separation technique useful for purifying proteins while preserving biological activity through the use of conditions and matrices that operate in a less denaturing manner than some other techniques. Some or all of the above purification steps can be used in various combinations to yield homogeneous recombinant proteins.

[0135] KLRB1 antibodies of the present disclosure may be analyzed for their physical / chemical properties and / or biological activity by various methods known in the art. In some embodiments, anti-KLRB1 antibodies are tested for their ability to bind to KLRB1 (e.g., human KLRB1 and / or cyno KLRB1). Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS. In some embodiments, anti-KLRB1 antibodies are tested for their ability to induce ADCC, ADCP, and / or CDC, as well as their ability to kill KLRB1 target cells (cell depletion). Assays include, but are not limited to, ADCC cytolysis assays using, for example, LDH release and formazan salt detection. In addition, antibodies may be evaluated for solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency.

[0136] In some embodiments, purified antibodies are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0137] antibody conjugates The present disclosure also provides conjugates comprising the anti-KLRB1 antibodies or binding fragments described herein. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a second molecule. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic drug or moiety. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic drug to form an ADC (antibody-drug conjugate). In some embodiments, the cytotoxic drug is a chemotherapeutic agent, including but not limited to, methotrexate, adriamycin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents. In some embodiments, the cytotoxic drug is a microtubule inhibitor, including but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), and tubulysin. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and a trichothecene. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothecenes, and CC1065. Derivatives of any one of these toxins may be used, provided they retain the cytotoxic activity of the parent molecule.

[0138] Conjugates comprising the KLRB1 antibodies or antigen-binding fragments thereof described herein may be prepared using any suitable method known in the art. In some embodiments, the conjugates are prepared using various bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0139] In some embodiments, the KLRB1 antibodies or antigen-binding fragments thereof described herein are conjugated to a detectable substance or molecule, allowing the antibody to be used for diagnosis and / or detection. Detectable substances include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and flavin(s); fluorescent substances such as umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent substances such as luciferase; radioactive substances such as 212 Bi, 14 C. 57 Co, 51 Cr, 67 Cu, 18 F, 68 Ga, 67 Ga, 153 Gd, 159 Gd, 68 Ge,3 H, 166 Ho, 131 I, 125 I, 123 I, 121 I, 115 In, 113 In, 112 In, 111 In, 140 La, 177 Lu, 54 Mn, 99 Mo, 32 P, 103 Pd, 149 Pm, 142 Pr, 186 Re, 188 Re, 105 Rh, 97 Ru, 35 S, 47 Sc, 75 Se, 153 Sm, 113 Sn, 117 Sn, 85 Sr, 99m Tc, 201 Ti, 133 Xe, 90 Y, 69 Yb, 175 Yb, 65 Zn; positron-emitting metals; and magnetic metal ions.

[0140] The anti-KLRB1 antibodies or antigen-binding fragments thereof described herein may also be conjugated to a second antibody to form an antibody heteroconjugate.

[0141] The anti-KLRB1 antibody or antigen-binding fragment thereof described herein can be bound to a solid support. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, the immobilized anti-KLRB1 antibody is used in immunoassays. In some embodiments, the immobilized anti-KLRB1 antibody is used to purify target antigens.

[0142] Methods for making polynucleotide / binding agents Also provided herein are nucleic acids encoding the polypeptides described herein, and vectors, preferably expression vectors, comprising nucleic acids encoding the polypeptides described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked, and may include a plasmid, cosmid, or viral vector. Vectors may be capable of autonomous replication or may integrate into host DNA. Viral vectors include, for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses.

[0143] A vector can contain a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably, a recombinant expression vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory sequences and / or inducible sequences. The design of an expression vector can depend on factors such as the choice of host cell to be transformed and the desired protein expression level. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides encoded by the nucleic acids described herein that encode the KLRB1-binding agents described herein.

[0144] The recombinant expression vector of the present invention may be designed for expression of a KLRB1-binding agent protein in prokaryotic cells. Preferably, the KLRB1-binding agent can be expressed in mammalian cells, preferably human cells. See, for example, Frenzel et al., Front Immunol. 2013;4:217. When used in mammalian cells, the control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, or simian virus 40.

[0145] Vector DNA may be introduced into host cells via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, and electroporation.

[0146] The host cells may be used to produce (i.e., express) the KLRB1-binding agent protein. Accordingly, the invention further provides methods for producing a KLRB1-binding agent protein using the host cells of the invention. In one embodiment, the method comprises culturing a host cell of the invention (introduced with a recombinant expression vector encoding the KLRB1-binding agent protein) in an appropriate medium such that the KLRB1-binding agent protein is produced. In another embodiment, the method further comprises isolating the KLRB1-binding agent protein from the medium or the host cell. In some embodiments, the Fc region is defucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. 2009 May-Jun;1(3):230-236, which describes methods for producing therapeutic antibodies with controlled levels of Fc-region N-glycan fucosylation).

[0147] Pharmaceutical Composition Also provided herein are pharmaceutical compositions comprising the KLRB1-binding agents described herein as an active ingredient. In various embodiments, the KLRB1-binding agents are prepared as pharmaceutical compositions, e.g., as pharmaceutical compositions for use as a medicament. In various embodiments, the pharmaceutical composition is for use as a medicament for treating a disease described herein, optionally an autoimmune disease, an allergic disease, a transplant rejection, or a malignant hematological disease, in a subject in need thereof. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn's disease, ulcerative colitis, or celiac disease. In some embodiments, the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ transplant. In some embodiments, the hematological malignancy is a leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell lymphoma, or large granular lymphocytic leukemia (LGLL). In some embodiments, the lymphoma is hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), extranodal NK / T-cell lymphoma (ENKL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sézary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). In some embodiments, the leukemia is aggressive NK-cell leukemia or T-cell prolymphocytic leukemia.

[0148] Those skilled in the art can formulate the KLRB1 binding agent as a pharmaceutical composition according to known methods.

[0149] The pharmaceutical composition may include a carrier. As used herein, a "carrier" may include a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic (or generally non-toxic) to a cell or subject exposed thereto, at the dosage and concentration employed. Often, the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, for example, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0150] In various embodiments, the KLRB1-binding agent is contained in an injectable formulation, for example, a subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection formulation. The injection formulation may be, for example, an aqueous solution in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. The injection formulation may contain formulatory agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the KLRB1-binding agent may be in dry or powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0151] The binding agents of the present disclosure may be formulated in any suitable form for delivery to target cells / tissues. In some embodiments, the KLRB1-binding agent may be formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nanoparticle, nanocapsule, or macroemulsion. In some embodiments, the pharmaceutical formulation comprises an agent of the present disclosure complexed with a liposome. Methods for making liposomes are known to those skilled in the art. For example, some liposomes can be made by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).

[0152] In some embodiments, the KLRB1 binding agent is formulated as a sustained-release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the drug, which matrices are in the form of shaped articles (e.g., films or microcapsules). Sustained-release matrices include, but are not limited to, polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactide, copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0153] Treatment and Administration The present disclosure provides methods comprising administering a KLRB1-binding agent described herein, or a pharmaceutical composition comprising a KLRB1-binding agent described herein, to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the method is performed in vivo (e.g., as opposed to ex vivo). As used herein, "treatment" refers to therapeutic treatment (treating a subject with a disease); the method may also be used for prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) the targeted pathological condition or disorder in a subject who does not have the disease. Thus, those in need of treatment include those already suffering from the disorder, those susceptible to the disorder, or those in whom the disorder is to be prevented (as used herein, "prevent" means reducing the risk of developing).

[0154] In various aspects and embodiments, the present disclosure provides methods for treating an autoimmune disease, an allergic disease, transplant rejection, or a malignant hematological disease in a subject in need thereof. As described herein, KLRB1-expressing cells are involved in the pathogenesis of these diseases. According to the present disclosure, depletion of such KLRB1-expressing cells provides a therapeutic benefit. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn's disease, ulcerative colitis, or celiac disease. In some embodiments, the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multiorgan transplant. In some embodiments, the hematological malignancy is a leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL). In some embodiments, the lymphoma is hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), extranodal NK / T-cell lymphoma (ENKL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sézary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). In some embodiments, the leukemia is aggressive NK cell leukemia or T cell prolymphocytic leukemia.

[0155] In various aspects and embodiments, the present disclosure provides methods for treating or preventing transplant rejection, which can be, for example, kidney rejection.

[0156] "Administration" and "treatment," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, can include contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" include in vivo treatments, and in some embodiments, in vitro or ex vivo treatments.

[0157] Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population by inducing regression of such symptom(s) or inhibiting the progression of the symptom to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular disease symptom may vary depending on factors such as the patient's disease state, age, and weight, as well as the ability of the agent to induce a desired response in the subject. Whether the disease symptom has been alleviated can be assessed by any clinical measurement commonly used by a physician or other skilled medical provider to assess the severity or progression of the symptom.

[0158] Thus, in various embodiments, the term "effective amount" or therapeutically effective amount refers to a concentration or amount of a KLRB1-binding agent that achieves a particular stated purpose, e.g., alleviation of one or more symptoms of a disease described herein. An "effective amount" of a KLRB1-binding agent may be empirically determined. Furthermore, a "therapeutically effective amount" refers to a concentration or amount of a KLRB1-binding agent that is effective to achieve a stated therapeutic effect. This amount may also be empirically determined.

[0159] In some embodiments, treatment with a KLRB1 binding agent of the present disclosure may kill at least about 20%, e.g., at least about 30%, 40%, 50%, 60%, 70%, or 80% of KLRB1-expressing cells involved in the pathogenesis of a disease disclosed herein.

[0160] The term "subject" refers to any animal (eg, a mammal), including, but not limited to, humans and non-human veterinary subjects, including non-human primates.

[0161] As used herein, a reference to "about" or "approximately" a value or parameter includes (describes) embodiments that are relevant to that value or parameter. For example, a statement that refers to "about X" includes the statement "X." As used herein, "about" means plus or minus 10 percent.

[0162] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0163] Any time an embodiment is described herein with the term "comprising," it is understood that other similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also included. Also, any time an embodiment is described herein with the phrase "consisting essentially of," it is understood that other similar embodiments described with the term "consisting of" are also included.

[0164] As used herein, the term "and / or" when used in phrases such as "A and / or B" is intended to include both A and B; A or B; A (A only); and B (B only). Similarly, the term "and / or" when used in phrases such as "A, B and / or C" is intended to include the following embodiments, respectively: A, B, and C; A, B, or C; A or C; A or B; B, or C; A and C; A and B; B and C; A (A only); B (B only); and C (C only).

[0165] In various embodiments, the KLRB1-binding agent can be administered by providing to the subject mRNA encoding the binding agent.

[0166] The following examples are illustrative, not limiting. Many variations of the present technology will become apparent to those skilled in the art upon review of this disclosure. Accordingly, the scope of the invention should be determined not with reference to the examples, but instead with reference to the appended claims, along with their full scope of equivalents. [Example]

[0167] Example 1: Gene expression of KLRB1 is characterized by and restricted to a unique population of immune cells. KLRB1 expression spans traditional lymphocyte classifications and characterizes subsets of Th17, Th17.1, ex-Th17, Tc17, iNKTs, ILC2, ILC3, peTh2, and NK cells (Figure 1).

[0168] Whole-body KLRB1 expression profiling data indicates that KLRB1 has no significant expression on any cell type other than immune cells (FIG. 2).

[0169] Example 2: KLRB1 expression is enhanced in various immune states Rheumatoid arthritis: Analysis of expression data (GSE1919) from synovial biopsies from rheumatoid arthritis patients compared to normal patients shows increased expression of KLRB1 (10-fold ratio) (Figure 3). Rheumatoid arthritis is therefore a particularly attractive target for treatment according to the present disclosure.

[0170] Analysis of expression data (GSE36700) from synovial biopsies from rheumatoid arthritis patients compared to patients with crystal-induced arthritis shows increased expression of KLRB1 (3.3-fold ratio) (Figure 4). Rheumatoid arthritis is therefore a particularly attractive target for treatment according to the present disclosure.

[0171] Sjogren's syndrome: Analysis of expression data (GSE23117) from salivary gland biopsies of Sjogren's syndrome patients shows increased expression of KLRB1 in advanced (13.1-fold ratio), intermediate (5.3-fold ratio), and early (1.6-fold ratio) stages compared to normal (Figure 5). Thus, Sjogren's syndrome is a particularly attractive target for treatment according to the present disclosure.

[0172] Analysis of expression data (GSE40611) from parotid gland biopsies from Sjogren's syndrome patients shows increased expression of KLRB1 (2.6-fold ratio) compared to normal ( FIG. 6 ). Thus, Sjogren's syndrome is a particularly attractive target for treatment according to the present disclosure.

[0173] Inclusion body myositis: Analysis of expression data from muscle biopsies from inclusion body myositis patients (GSE38454) shows increased expression of KLRB1 (2.9-fold ratio) compared to normal (Figure 7). Thus, inclusion body myositis is a particularly attractive target for treatment according to the present disclosure.

[0174] Discoid lupus: Analysis of expression data from skin biopsies of discoid lupus patients (GSE52471) shows increased expression of KLRB1 (7.2-fold ratio) compared to normal (Figure 8). Thus, discoid lupus is a particularly attractive target for treatment according to the present disclosure.

[0175] Psoriasis: Analysis of expression data from skin biopsies from patients with psoriasis (GSE52471) shows increased expression of KLRB1 (11.2-fold ratio) compared to normal (Figure 9). Thus, discoid lupus is a particularly attractive target for treatment according to the present disclosure.

[0176] Idiopathic pulmonary fibrosis: Analysis of expression data (GSE53845) derived from lung biopsies from idiopathic pulmonary fibrosis patients shows increased expression of KLRB1 (1.5-fold ratio) compared to normal (Figure 10). Idiopathic pulmonary fibrosis is therefore a particularly attractive target for treatment according to the present disclosure.

[0177] Diabetes: Analysis of expression data from pancreatic biopsies from diabetic patients (GSE72492) shows increased expression of KLRB1 (3.7-fold ratio) compared to normal (Figure 11). Thus, diabetes is a particularly attractive target for treatment according to the present disclosure.

[0178] Alopecia Universalis: Analysis of expression data from scalp biopsies of patients with idiopathic pulmonary fibrosis (GSE74761) shows increased expression of KLRB1 (4.3-fold ratio) compared to normal (Figure 12). Thus, alopecia universalis is a particularly attractive target for treatment according to the present disclosure.

[0179] Primary biliary cholangitis: Analysis of expression data (GSE79850) from liver biopsies from patients with primary biliary cholangitis who ultimately required liver transplantation shows increased expression of KLRB1 (5.6-fold ratio) compared to normal (Figure 13). Thus, primary biliary cholangitis is a particularly attractive target for treatment according to the present disclosure.

[0180] Multiple sclerosis: Analysis of expression data (GSE5839) derived from brain biopsies of multiple sclerosis patients shows elevated KLRB1 expression (2.5-fold) compared to control brains (Figure 14). Thus, multiple sclerosis is a particularly attractive target for treatment according to the present disclosure.

[0181] Lymphocytic colitis: Analysis of expression data from colon biopsies of four patients with lymphocytic colitis (GSE65107) shows increased expression of KLRB1 (3.2-fold ratio) compared to four healthy controls (Figure 15). Thus, lymphocytic colitis is a particularly attractive target for treatment according to the present disclosure.

[0182] Kidney transplant rejection: Analysis of expression data (GSE1563) from kidney biopsies from seven patients with acute kidney rejection shows increased expression of KLRB1 (2.1-fold ratio) compared to nine healthy controls (Figure 16). Thus, kidney transplant rejection is a particularly attractive target for treatment according to the present disclosure.

[0183] Lung Transplantation: Analysis of expression data (GSE65107) from lung bronchoalveolar lavage (BAL) fluid from seven patients who underwent lung transplant rejection shows increased expression of KLRB1 (3.6-fold ratio) compared to 27 patients with lung transplants who did not experience rejection (Figure 17). Thus, lung transplant rejection is a particularly attractive target for treatment according to the present disclosure.

[0184] Atopic dermatitis: Analysis of expression data (GSE65107) from five patients with atopic dermatitis shows increased expression of KLRB1 (1.9-fold ratio) compared to five healthy controls (Figure 18). Thus, atopic dermatitis is a particularly attractive target for treatment according to the present disclosure.

[0185] Palmoplantar pustulosis: Analysis of expression data (GSE185856) from skin biopsies from three patients with palmoplantar pustulosis lesional skin and eight patients with palmoplantar pustulosis non-lesional skin shows increased expression of KLRB1 compared to seven healthy controls (lesional vs. healthy ratio 11.0-fold; non-lesional vs. healthy ratio 5.9-fold) (Figure 19). Thus, palmoplantar pustulosis is a particularly attractive target for treatment according to the present disclosure.

[0186] Hidradenitis Suppurativa: Analysis of expression data (GSE148027) from skin biopsies from 18 patients with hidradenitis suppurativa lesional skin and 7 patients with hidradenitis suppurativa non-lesional skin shows increased expression of KLRB1 compared to 8 healthy subjects (8.0-fold lesional to healthy, 3.0-fold non-lesional to healthy) (Figure 20). Thus, hidradenitis suppurativa is a particularly attractive target for treatment according to the present disclosure.

[0187] Asthma: Analysis of expression data (GSE41861) from lung airway brushings of 10 patients with severe asthma, 37 patients with moderate asthma, and 44 patients with mild asthma shows increased expression of KLRB1 compared to 47 healthy controls (1.9-fold ratio for severe asthma vs. healthy controls; 1.5-fold ratio for moderate asthma vs. healthy controls; and 1.3-fold ratio for mild asthma vs. healthy controls) (Figure 21). Thus, atopic dermatitis is a particularly attractive target for treatment according to the present disclosure.

[0188] Example 3: KLRB1 expression is enhanced in various T and NK cell lymphomas and leukemias Analysis of expression data (GSE19067) from tumor cells derived from patients with various T and NK cell lymphomas and leukemias compared to normal NK cell expression shows increased expression of KLRB1 (e.g., hepatosplenic T cell lymphomas have a 17-fold ratio compared to normal NK cell lines) (Figure 22). Thus, various T cell and NK cell lymphomas are particularly attractive targets for treatment according to the present disclosure.

[0189] In particular, some patients with hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sézary syndrome, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), T-cell prolymphocytic leukemia (T-PLL), and peripheral T-cell lymphoma (PTCL) have increased or similar KLRB1 expression in CD4+ T cells (Figure 22). Thus, hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sézary syndrome, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), T-cell prolymphocytic leukemia (T-PLL), and peripheral T-cell lymphoma (PTCL) are particularly attractive targets for treatment according to the present disclosure.

[0190] Example 4: Monoclonal mouse anti-KLRB1 antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 18G11F11, and 22F10G3 and various derived chimeric antibodies bind to human KLRB1 Monoclonal antibodies against human KLRB1 were generated by immunizing five balb / c and five SJL mice with the human KLRB1 protein extracellular domain (ECD) (Uniprot ID Q12918, amino acids 67-225) to generate mouse hybridomas. The parental hybridoma clones were then screened by ELISA for binding to the human KLRB1 ECD and by FACS for binding to stable CHO-K1 cell lines expressing human KLRB1 (SEQ ID NO: 1) and cynomolgus monkey KLRB1 (SEQ ID NO: 2), respectively. Based on binding to both human and cynomolgus monkey CHO-K1-expressing KLRB1 cells, 20 hybridomas were selected for subcloning, resulting in 91 subclones. These 91 subclones were repeatedly subjected to FACS binding studies against CHO-K1 human-KLRB1 and CHO-K1 cynomolgus monkey KLRB1. A number of these mAbs, designated 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, 5A11D10, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3, were tested for binding ability in an ELISA assay. Human KLRB1 ECD was coated onto an ELISA plate, and the plate was incubated with these antibodies. Antibody binding to the ELISA plate was detected using a secondary goat anti-mouse peroxidase-labeled IgG (see Figures 23A-C).

[0191] Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3 were also tested in a dose-dependent FACS assay against CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1). Antibody binding to CHO-hum-KLRB1 was detected using a secondary goat anti-mouse peroxidase-labeled IgG (see Figures 24A, 24C, and 24D).

[0192] Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, and 22F10G3 were also tested in a dose-dependent FACS assay against CHO-K1 cells expressing cynomolgus monkey KLRB1 (CHO-cyno-KLRB1). Secondary goat anti-mouse peroxidase-labeled IgG was used to detect antibody binding to CHO-cyno-KLRB1 (see Figures 24B and 24E).

[0193] Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 18G11F11, and 22F10G3 were also tested in single-concentration FACS binding assays against both CHO-hum-KLRB1 and CHO-cyno-KLRB1, using untransduced CHO-K1 cells as a control. Secondary goat anti-mouse peroxidase-labeled IgG was used to detect antibody binding to CHO-hum-KLRB1 or CHO-cyno-KLRB1. All antibodies bound to CHO-hum-KLRB1, and all except 50A8F2 bound to CHO-cyno-KLRB1. The results are shown in Table 19.

[0194] Various murine and mouse / human chimeric antibodies to human IgG1-kappa were assayed for binding to the human KLRB1 extracellular domain (ECD) by surface plasma resonance (SPR), and the results are shown in Tables 20 and 21A-B. [Table 22] [Table 23] [Table 24] [Table 25]

[0195] Example 5: Monoclonal anti-KLRB1 antibodies can induce antibody-dependent cell-mediated cytotoxicity To determine whether anti-KLRB1 antibodies are effective in killing KLRB1-positive cells, we tested their ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Induced ADCC was assessed using a cytolytic assay involving LDH release and detection of formazan salt (Genscript, SC1544). CHO-hum-KLRB1 cells were incubated with human peripheral blood mononuclear cells (PBMCs) and various antibodies. The antibodies tested were mouse / human IgG1-kappa (wild-type) chimeric antibodies 57E2E4-chimera, 7B6C7-chimera, 52G9B12B5-chimera, 5B1B11-chimera, 50A8F2-chimera, 47A5H2-chimera; 52G9B12B5-chimera-EN (a mouse / human chimeric KLRB1-binding antibody with the 52G9B12B5 variable region and human IgG1-kappa mutations L234A / L235A / G237A mutations; used as a negative control), and human IgG1 (negative control) (Figure 25A), or 22F10G3-chimera and human IgG1 (negative control) (Figure 25B).

[0196] Example 6: Monoclonal anti-KLRB1 antibodies can block the binding of CLEC2D to KLRB1 CLEC2D is the natural ligand for KLRB1. A fusion protein of CLEEC2D-Fc-biotin was produced by transfection of CHO cells with the expression construct. The ability of monoclonal antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, and 22F10G3 to compete with CLEC2D-Fc-biotin for CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) was tested using streptavidin-AF488 to detect the presence or absence of CLEC2D bound to these cells. All antibodies except 50A8F2 demonstrated the ability to block CLEC2D binding (Figures 26A, 26B, and 26E).

[0197] Example 7: Monoclonal anti-KLRB1 antibody 50A8F2 enhances CLEC2D binding to KLRB1 CLEC2D is the natural ligand for KLRB1. A CLEEC2D-Fc-biotin fusion protein was generated by transfection of CHO cells with the expression construct. The ability of monoclonal antibody 50A8F2 to compete with CLEC2D-Fc-biotin for CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) was tested using streptavidin-AF488 to detect the presence or absence of CLEC2D binding to these cells. 50A8F2 enhanced CLEC2D binding in two independent experiments (Figures 26C and 26D).

[0198] Example 8. Humanization of 47A5H2 Humanized sequence variants of 47A5H2 were generated by CDR-grafting (Almagro et al. 2008) of the murine antibody 47A5H2 into the human frameworks IGHV1-46*01 / JH1, IGKV4-1*01 / JK2, and IGKV3-11*01 / JK2 for both the heavy and light chains and are shown in Table 22.

[0199] The sequence variants were combined and expressed as human IgG1 kappa antibodies with the IgG1 L234A / L235A / G237A mutations to generate the 16 antibodies shown in Table 23. [Table 26] [Table 27]

[0200] Example 9. Humanization of 50A8F2 Humanized sequence variants of 50A8F2 were generated by CDR grafting (Almagro et al. 2008) of the murine antibody 50A8F2 into the human frameworks IGHV1-46*01 / JH6 and IGKV1-39*01 / JK2 for both the heavy and light chains and are shown in Table 24.

[0201] These sequence variants were combined and expressed as human IgG1 kappa antibodies with the IgG1 L234A / L235A / G237A mutations to generate the 16 antibodies shown in Table 25. [Table 28] [Table 29]

[0202] Example 10: Humanization of 22F10G3 Humanized sequence variants of 22F10G3 were generated by CDR-grafting (Almagro et al. 2008) of the murine antibody 22F10G3 into the human frameworks IGHV4-31*02 / J4*01 and IGKV4-1*01 / J2*01 for both the heavy and light chains and are shown in Table 26A.

[0203] These sequence variants were combined and expressed as human IgG1 kappa antibodies to generate the 16 antibodies shown in Table 26B. [Table 30] [Table 31]

[0204] Example 11: Monoclonal anti-KLRB1 47A5H2 chimeric and humanized antibodies bind to human KLRB1 and cynomolgus monkey KLRB1 by ELISA Humanized variants of 47A5H2, generated by CDR grafting (Almagro et al. 2008), were assayed by ELISA for binding to the human and cynomolgus monkey KLRB1 protein extracellular domains (ECDs). The human KLRB1 extracellular domain consisted of amino acids 67-225 of the human KLRB1 protein (SEQ ID NO: 1). The cynomolgus monkey extracellular domain consisted of amino acids 67-227 of the cynomolgus monkey KLRB1 protein (SEQ ID NO: 3). ELISA plates were coated with 1 μg / ml KLRB1 ECD overnight at 4°C, followed by the addition of primary antibodies (for human, starting concentration 3 nM and 3-fold dilutions; for cynomolgus monkey, starting concentration 10 nM and 5-fold dilutions) for 1 hour at 37°C, followed by the addition of a secondary anti-human IgG antibody at 1:5000 for 45 minutes at 37°C. The results are shown in Figures 27A-D and 28A-D and Table 27.

[0205] Comparison experiments of Hu47A5H2-11 and Hu50A8F2-04 were performed with other antibodies containing the N297A effector null mutation, Ab9, KW1.2.1, and KW7.3.7, as well as HP-3G10, DX12, and 191B8. Ab9 is described in PCT Publication No. WO2023028501A1, and KW1.2.1 and KW7.3.7 are described in U.S. Patent Application Publication No. US20210122826A1. HP-3G10, DX12, and 191B8 are commercially available antibody reagents.

[0206] These experiments showed that Hu47A5H2-11 exhibited a lower EC50 for human KLRB1 binding, and that Hu50A8F2-04 and 191B8 did not bind to cynomolgus monkey KLRB1 protein. The results are shown in Figures 27E-F and 28E-F and summarized in Tables 28 and 29. [Table 32] [Table 33] [Table 34]

[0207] Example 12: Monoclonal anti-KLRB1 47A5H2 chimeric and humanized antibodies bind to human KLRB1 and cynomolgus monkey KLRB1 expressed in CHO-K1 cells The 47A5H2 chimeric and humanized antibodies were assayed by FACS for binding to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) and CHO-K1 cells expressing cynomolgus monkey KLRB1 (SEQ ID NO: 2) (CHO-cyno-KLRB1). Starting at 100 nM for human assays and 200 nM for cynomolgus monkey assays, antibodies were serially diluted 5-fold into wells containing 2.0E+05 CHO-hum-KLRB1 or CHO-cyno-KLRB1 cells per well. Secondary Alexa488 anti-human Fc antibody (Jackson, catalog no. 109-545-098, 1:500) and LIVE / DEAD dye (Invitrogen, catalog no. L34964A, 1:1000) were used for FACS detection. The binding is shown in Figures 29A-D and 30A-D and in Table 30.

[0208] Comparison experiments of Hu47A5H2-11 with Hu50A8F2-04 and other antibodies showed that Hu50A8F2-04 and 191B8 did not bind to the cynomolgus monkey KLRB1 protein. We believe that the 47A5H2 chimeric and humanized antibodies bind to a different epitope than 191B8. The results are shown in Figures 29E-F and 30E-F and summarized in Tables 28 and 29 (above). [Table 35]

[0209] Example 13. Monoclonal anti-KLRB1 47A5H2 chimeric and humanized antibodies inhibit CLEC2D binding to human KLRB1 expressed in CHO-K1 cells A competition assay between the humanized variants of 47A5H2 and biotin-labeled CLEC2D fusion proteins was performed by FACS. Starting at 200 nM, antibodies were serially diluted 5-fold in wells with 2.0E+05 CHO-hum-KLRB1 or CHO-cyno-KLRB1 (SEQ ID NO: 2) cells and 50 μl of biotin-CLEC2D (final concentration 60 nM) per well for 1 hour at 4°C. Secondary streptavidin-Alexa 467 (Thermo, Cat. No. S21374, 1:1000) and LIVE / DEAD dye (Invitrogen, Cat. No. L34964A, 1:1000) were used for FACS detection. Blocking of CLEC2D binding by these humanized antibodies is shown in Figures 31A-D and Table 31.

[0210] Comparison experiments of Hu47A5H2-11 with Hu50A8F2-04 and other antibodies showed that Hu47A5H2-11 exhibited a lower IC50 than the other antibodies tested, the results of which are shown in Figures 31E-F and summarized in Tables 28 and 29 (above). [Table 36]

[0211] Example 14: Binding kinetics of monoclonal anti-KLRB1 47A5H2 and 22F10G3 chimeric and humanized antibodies to human KLRB1. The 47A5H2 chimeric and humanized antibodies, as well as other anti-KLRB1 antibodies, were assayed for binding to the human KLRB1 extracellular domain protein (ECD) by surface plasma resonance (SPR). The human KLRB1 extracellular domain was amino acids 67-225 of the human KLRB1 protein (SEQ ID NO: 1). A Biacore 8K instrument was used with a Series S sensor CM5 chip, multi-cycle kinetics and affinity capture in a 96-well microplate, and a full range of antibody concentrations: 0.2 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, and 25 nM. The antibody was immobilized to the chip with an anti-human Fc antibody, and the human KLRB1 extracellular domain (ECD) analyte was flowed over the surface at 30 μl / min with a contact time of 180 seconds and a dissociation time of 400 seconds. Comparison experiments were performed with other antibodies: Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8, and the results are shown in Table 32A. [Table 37]

[0212] Additionally, the 22F10G3 chimeric and humanized antibodies were assayed for binding to the human KLRB1 extracellular domain protein (ECD) by surface plasma resonance (SPR). The human KLRB1 extracellular domain was amino acids 67-225 of the human KLRB1 protein (SEQ ID NO: 1). A Biacore 8K instrument was used with multi-cycle kinetics and affinity capture in 96-well microplates and a single antibody concentration of 20 nM. The antibody was immobilized on a chip, and the human KLRB1 extracellular domain (ECD) analyte was flowed over the surface at 30 μl / min with a contact time of 120 seconds and a dissociation time of 360 seconds. The results are shown in Table 32B. [Table 38]

[0213] Example 15: Binding kinetics of monoclonal anti-KLRB1 47A5H2 chimeric and humanized antibodies to cynomolgus monkey KLRB1. The 47A5H2 chimeric and humanized antibodies, as well as other anti-KLRB1 antibodies, were assayed for binding to the cynomolgus monkey KLRB1 extracellular domain protein (ECD) by surface plasma resonance (SPR). The cynomolgus monkey extracellular domain was amino acids 67-227 of the cynomolgus monkey KLRB1 protein (SEQ ID NO: 3). A Biacore 8K instrument was used with a Series S sensor CM5 chip, multi-cycle kinetics and affinity capture in a 96-well microplate, and a full range of antibody concentrations: 0.2 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, and 25 nM. The antibody was immobilized to the chip with an anti-human Fc antibody, and the cynomolgus monkey KLRB1 extracellular domain (ECD) analyte was flowed over the surface at 30 μl / min with a contact time of 180 seconds and a dissociation time of 400 seconds. Comparison experiments were conducted with other antibodies: Ab9, KW7.3.7, HP-3G10, DX12, and 191B8, and the results are shown in Table 33. [Table 39]

[0214] Example 16: Monoclonal anti-KLRB1 50A8F2 chimeric and humanized antibodies bind to human KLRB1 by ELISA Humanized variants of 50A8F2, generated by CDR grafting (Almagro et al. 2008), were assayed for binding to the human KLRB1 protein extracellular domain (ECD) by ELISA. ELISA plates were coated with 1 μg / ml KLRB1 ECD overnight at 4°C, followed by the addition of primary antibody (starting concentration 3 nM and 3-fold dilution) for 1 hour at 37°C, followed by the addition of secondary anti-human IgG antibody at 1:5000 for 45 minutes at 37°C. The results are shown in Figures 32A-D and Table 34. [Table 40]

[0215] Example 17: Monoclonal anti-KLRB1 50A8F2 chimeric and humanized antibodies bind to human KLRB1 expressed in CHO-K1 cells but not to cynomolgus monkey KLRB1 expressed in CHO-K1 cells The 50A8F2 chimeric and humanized antibodies were assayed by FACS for binding to CHO-K1 cells expressing human KLRB1 (SEQ ID NO: 1) (CHO-hum-KLRB1) and CHO-K1 cells expressing cynomolgus monkey KLRB1 (SEQ ID NO: 2) (CHO-cyno-KLRB1). Starting at 100 nM for human assays and 200 nM for cynomolgus monkey assays, the antibodies were serially diluted 5-fold into wells containing 2.0E+05 CHO-hum-KLRB1 or CHO-cyno-KLRB1 cells per well. A secondary Alexa488 anti-human Fc antibody (Jackson, catalog no. 109-545-098, 1:500) and LIVE / DEAD dye (Invitrogen, catalog no. L34964A, 1:1000) were used for FACS detection. The binding of the 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 is shown in Figures 33A-D and Table 35. The lack of binding of the 50A8F2 chimeric and humanized antibodies to CHO-cyno-KLRB1 is shown in Figure 33E, along with the presence of binding of the 47A5H2 chimera (also called HU47A5H2 chimera) as a positive control. [Table 41]

[0216] Example 18. Monoclonal anti-KLRB1 50A8F2 chimeric and humanized antibodies enhance CLEC2D binding to human KLRB1 expressed in CHO-K1 cells Competition assays of 50A8F2 chimeric and humanized antibodies with biotin-labeled CLEC2D were performed by FACS. Starting at 200 nM, antibodies were serially diluted 5-fold into wells with 2.0E+05 CHO-hum-KLRB1 cells and 50 μl of biotin-CLEC2D (final concentration 60 nM) per well for 1 hour at 4°C. Secondary streptavidin-Alexa 467 (Thermo, catalog no. S21374, 1:1000) and LIVE / DEAD dye (Invitrogen, catalog no. L34964A, 1:1000) were used for FACS detection. The enhanced (increased) CLEC2D binding by these humanized antibodies is shown in Figures 34A-D and Table 36. In another comparative experiment, the 50A8F2 humanized antibody showed enhanced CLEC2D binding, whereas antibodies KW1.2.1 and KW7.3.7 did not; rather, as previously shown in Figure 31E, these antibodies inhibited CLEC2D binding (Figure 35 and Table 37). [Table 42] [Table 43]

[0217] Example 19: Binding kinetics of monoclonal anti-KLRB1 50A8F2 chimeric and humanized antibodies to human KLRB1. The humanized variant of 50A8F2 and other antibodies were assayed for binding to the human KLRB1 extracellular domain protein (ECD) by surface plasma resonance (SPR). A Biacore 8K instrument was used with a Series S sensor CM5 chip, multi-cycle kinetics and affinity capture in a 96-well microplate, and a full range of antibody concentrations: 0.2 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM, 12.5 nM, and 25 nM. The antibody was immobilized to the chip with an anti-human Fc antibody, and the human KLRB1 extracellular domain (ECD) analyte was flowed over the surface at 30 μl / min with a contact time of 180 seconds and a dissociation time of 400 seconds. The results are shown in Table 38. [Table 44]

[0218] Example 20. Anti-KLRB1 47A5H2 chimeric and humanized antibodies increase activation of primary NK cells An NK cell activation assay was developed as follows: K562 cells were stably transfected with human CLEC2D to generate pools and single clones of K562-CLEC2D target cells, and clone 1D1 was selected for subsequent single clone experiments.

[0219] Primary NK cells were isolated from human PBMCs using a human NK cell enrichment kit (Stemcell, catalog no. 19055). Primary NK cells (1.0E+06) were cultured overnight in RPMI 1640 plus 10% FBS containing 100 ng / mL rhIL-2 (Peprotech, catalog no. 200-02). On day 2, NK cells were washed and resuspended in assay buffer (RPMI 1640 plus 10% FBS). 50 μl of NK cells (1.0E+05 cells / well) were incubated with various antibodies (50 μl / well) for 0.5 hours at 37°C. Expression of human CLEC2D by FACS was confirmed on K562-CLEC2D target cells using a goat anti-human CLEC2D polyclonal antibody (Invitrogen, catalog no. PA5-47496) and Alexa488 anti-goat IgG (Invitrogen, catalog no. A11055). 100 μl of cells (5.0E+05 per well) of K562 or K562-CLEC2D target cells (either pooled or single clones), along with various controls, were incubated with RPMI 1640 plus 10% FBS and 100 ng / mL rhIL-2 for 4 hours at 37°C. Anti-CD107a-APC (BioLegend, Catalog No. 328620) was added at the beginning of the incubation. After 1 hour of incubation, Bredeldin A (BioLegend, Catalog No. 420601) and Monensin (BioLegend, Catalog No. 420701) were added. Cells were then washed twice and blocked with human Fc (BD Catalog No. 564220) for 10 minutes at room temperature. Flow detection antibodies (Live / dead dye, Invitrogen, Cat. No. L34964; anti-CD3-PE-Cy7, BioLegend, Cat. No. 317334; anti-CD56-FITC, BioLegend, Cat. No. 304604) were added and incubated for 30 min at 4°C in the dark.Cells were then washed twice, suspended in 100 ul of Cytofix / Cytoperm solution (BD, Cat. No. 554722) for 20 minutes at 4°C, washed again, stained with IFN-gamma-PE (BioLegend, Cat. No. 502509) for 30 minutes at 4°C in the dark, washed again, and analyzed by FACS.

[0220] The results, shown in Figures 36-39 and summarized in Tables 39 and 40 below, demonstrate that the 47A5H2 chimeric and humanized antibodies increased NK cell activation using primary NK cells. These results further demonstrate that NK cell activation is suppressed by CLEC2D (K562-CLEC2D cells result in lower NK activation than K562 cells, Figures 36 and 37), and that the Hu47A5H2-11 antibody increased NK cell activation with the target cell K562-CLEC2D monoclonal clone 1D1. The EC50 values ​​for NK cell activation by Hu47A5H2-11 in terms of both CD107a and IFNg expression were superior to those of Ab9, KW1.2.1, and KW7.3.7 (Figures 38A and 39A). Antibodies KW1.2.1 and KW7.3.7 did not activate IFNg expression in NK cells (Figure 39A). Antibody 191B8 at 40 nM and 200 nM caused NK cell toxicity and had a reduced effect on NK cell activation (Figures 37A, 37B, 38B, and 39B). Antibody Hu50A8F2-04 did not activate CD107a or IFNg expression on NK cells (Figures 38A and 39A).

[0221] Example 21. Anti-KLRB1 47A5H2 humanized variant increases primary NK cell killing An NK cell killing assay was developed as follows: Donors with high expression of KLRB1 (CD161) on NK cells, as detected by FACS using a PE anti-CD161 antibody (BioLegend, catalog no. 339903), were selected for further testing. Primary NK cells were isolated from human PBMCs using a human NK cell enrichment kit (Stemcell, catalog no. 19055). Primary NK cells (1.0E+06) were cultured overnight in RPMI 1640 plus 10% FBS containing 100 ng / mL rhIL-2 (Peprotech, catalog no. 200-02). On day 2, NK cells were washed and resuspended in assay buffer (RPMI 1640 plus 10% FBS). 100 μl of NK cells (4.0E+04 cells / well) were incubated with various concentrations of various antibodies (50 μl / well) at 37°C for 0.5 hours. Raji target cells were confirmed to express human CLEC2D by FACS using goat anti-human CLEC2D polyclonal antibody (Invitrogen, catalog no. PA5-47496) and Alexa488 anti-goat IgG (Invitrogen, catalog no. A11055, 1:1000). Raji target cells were stained with CellTrace Violet (CTV, Invitrogen, catalog no. C34557). Staining was stopped with an equal volume of FBS for 5 minutes at room temperature, washed with sterile FACS buffer, and 50 μl of cells (4.0E+04 per well) were co-incubated with the primary NK cell / antibody mixture for 4 hours at 37°C. Cells were then washed twice with FACS buffer and stained with 100 μl / well of Live / Dead far-red fluorescent dye (Invitrogen, catalog no. L10120) for 0.5 hours at 4°C. Cells were then washed twice with FACS buffer and analyzed by FACS.

[0222] The results of one experiment (Experiment 21.1) are shown in Figure 40 and demonstrate that the 47A5H2-chimeric, Hu47A5H2-07, and Hu47A5H2-11 antibodies increased NK cell killing. Compared to the anti-KLRB1 antibodies KW1.2.1 and KW7.3.7, the 47A5H2 chimeric and humanized antibodies had superior potency (smaller EC50).

[0223] In another experiment (Experiment 21.2) shown in Figure 41, Hu47A5H2-11 had superior potency (smaller EC50) compared to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8. The results are summarized in Tables 39 and 40 below.

[0224] Example 22. Anti-KLRB1 47A5H2 and humanized variants activate Jurkat T cells A T cell activation assay was developed as follows: Jurkat-NFAT cells were stably transfected with 1G4-TCR and human CD161 (KLRB1) to generate pools and single clones of Jurkat-NFAT-1G4TCR-CD161 effector cells. Expression was confirmed by FACS using an anti-HA tag on the TCR alpha (BioLegend, catalog no. 682404), an anti-PC tag on the TCR beta (Genscript, catalog no. A01774-100), and an anti-CD161 Hu47A5H2-7 antibody.

[0225] K562 cells were stably transfected with human CLEC2D and HLA A*02:01 and β2m to generate pools and single clones of K562-CLEC2D-HLA target cells.

[0226] Jurkat-NFAT-1G4TCR-CD161 cells were cultured with K562-CLEC2D-HLA cells pulsed with NY-ESO-1 peptide (SLLMWITQC) under various conditions. In a typical experiment, various concentrations of NY-ESO-1 peptide were added to assay medium (RPMI 1640 plus 10% FBS), and 25 μl of assay medium and 25 μl of target K562-CLEC2D-HLA cells were incubated at 37°C for 120 minutes. 25 μl of various antibodies were added to 25 μl of Jurkat-NFAT-1G4TCR-CD161 effector cells and incubated at 37°C for 5 hours. 100 μl of ONE-Glo (Promega, catalog no. E6120) was added and incubated at room temperature for 3 minutes. Luminescence of the wells was then measured using a Varioskan Lux plate reader.

[0227] The results of one experiment (Experiment 22.1) using Jurkat and K562-CLEC2D-HLA cell pools are shown in Figure 42 and demonstrated that the 47A5H2 chimeric antibody, but not the 50A8F2 chimeric antibody, increased T cell activation.

[0228] In another experiment (Experiment 22.2) using a single clone of Jurkat cells (clone 2F8) and a single clone of K562-CLEC2D-HLA cells (clone 5E8) shown in Figure 43, Hu47A5H2-11 had superior potency (smaller EC50) compared to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8. The results are summarized in Tables 39 and 40 below. [Table 45] [Table 46]

[0229] References Almagro,J.C.,et al.(2008). “Humanization of antibodies.” Front Biosci 13:1619-1633. Aldemir,H.,et al.(2005). 「Cutting edge:lectin-like transcript 1 is a ligand for the CD161 receptor.」J Immunol 175(12):7791-7795. Basdeo,S. A.,et al.(2017). ”Ex-Th17(Nonclassical Th1)Cells Are Functionally Distinct from Classical Th1 and Th17 Cells and Are Not Constrained by Regulatory T Cells.” J Immunol 198(6):2249-2259. Fergusson,J. R.,et al.(2014). ”CD161 defines a transcriptional and functional phenotype across distinct human T cell lineages.” Cell Rep 9(3):1075-1088. Maggi,L.,et al.(2010). ”CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC.” Eur J Immunol 40(8):2174-81. Mathewson,N.D.,et al.(2021). “Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis.” Cell 184(5):1281-1298. Satoh,M.,et al.(2008). “Non-fucosylated therapeutic antibodies as next-generation therapeutic antibodies.” Expert Opin Biol Ther 6(11):1161-1173. Yang,J.,et al.(2014). ”Targeting Th17 cells in autoimmune diseases.” Trends Pharmacol Sci 35(10):493-500.

Claims

1. 1. An antibody or antigen-binding portion thereof that specifically binds to Killer Cell Lectin-Like Receptor Subfamily B, Member 1 (KLRB1; optionally SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3), wherein the antibody or antigen-binding portion thereof comprises: (a) a heavy chain variable region (VH) comprising: a VH complementarity determining region (CDR) 1 comprising a sequence at least 95% identical to a VH CDR1 amino acid sequence set forth in one of Tables 1-18, preferably Table 9, 8, or 18; a VH CDR2 comprising a sequence at least 95% identical to a VH CDR2 amino acid sequence set forth in one of Tables 1-18, preferably Table 9, 8, or 18; and a VH CDR3 comprising a sequence at least 95% identical to a VH CDR3 amino acid sequence set forth in one of Tables 1-18, preferably Table 9, 8, or 18; and (b) the antibody or antigen-binding portion thereof, comprising at least one light chain variable region (VL), wherein the light chain variable region (VL) comprises a VL CDR1 comprising a sequence at least 95% identical to a VL CDR1 amino acid sequence as set forth in one of Tables 1-18, preferably Table 9, 8, or 18; a VL CDR2 comprising a sequence at least 95% identical to a VL CDR2 amino acid sequence as set forth in one of Tables 1-18, preferably Table 9, 8, or 18; and a VL CDR3 comprising a sequence at least 95% identical to a VL CDR3 amino acid sequence as set forth in one of Tables 1-18, preferably Table 9, 8, or 18.

2. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of a VH amino acid sequence shown in Table 9, 8, or 18.

3. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VL comprises or consists of a VL amino acid sequence shown in Table 9, 8, or 18.

4. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 9, and the VL comprises or consists of the VH amino acid sequence shown in Table 9.

5. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 8, and the VL comprises or consists of the VH amino acid sequence shown in Table 8.

6. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 18, and the VL comprises or consists of the VH amino acid sequence shown in Table 18.

7. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 3 or 5, and the VL comprises or consists of the VH amino acid sequence shown in Table 3 or 5.

8. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence shown in Table 9, 8 or 18, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the VL amino acid sequence shown in (the same) Table 9, 8 or 18.

9. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of an amino acid sequence having at least 95% sequence identity with a VH amino acid sequence shown in Table C, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity with an amino acid sequence shown in Table C, preferably wherein the VH and the VL are from the same row of the table.

10. 10. The antibody or antigen-binding portion thereof according to claim 1, comprising a heavy chain and a light chain constant region, wherein the heavy chain and / or the light chain constant region comprises or consists of an amino acid sequence as set forth in Table A.

11. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 9, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 9, and optionally a constant region, and optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table C.

12. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence set forth in Table 8, and a variable region light chain consisting of a VL amino acid sequence set forth in Table 8, and optionally a constant region, and optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table C.

13. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of the HC amino acid sequence set forth in Table 18, and a variable region light chain consisting of the LC amino acid sequence set forth in Table 18, and optionally a constant region, and optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table C.

14. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B, member 1 (KLRB1; SEQ ID NO: 1), comprising or consisting of a variable region heavy chain consisting of a VH amino acid sequence shown in Table 3 or 5, and a variable region light chain consisting of a VL amino acid sequence shown in Table 3 or 5, and optionally a constant region, and optionally comprising a sequence that is at least 95% identical to a sequence shown in Table C.

15. The antibody or antigen-binding portion thereof according to claims 1 to 14, further comprising a hinge region and an Fc domain of the heavy chain constant region.

16. The antibody or antigen-binding portion thereof according to claim 1, which is an antibody comprising a heavy chain constant region comprising an amino acid sequence having at least 80%, 90%, 95%, or 97% sequence identity with the amino acid sequence of the heavy chain constant region shown in Table A.

17. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody is a monoclonal antibody.

18. 2. The antibody or antigen-binding portion thereof of claim 1, wherein the antibody is a chimeric, humanized, or human antibody and / or comprises one or more mutations (optionally within a CDR) that remove an Asn(N)-glycosylation site or that remove Cys, Asp, Met, Trp, or Lys.

19. The antibody or antigen-binding portion thereof of claim 1 , wherein the antibody is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody, or IgG4 antibody.

20. 2. The antibody or antigen-binding portion thereof according to claim 1, wherein the antibody or antigen-binding portion thereof is an antibody comprising an Fc region, preferably a human IgG1, that binds to an Fc gamma receptor (FcγR) and induces antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or that binds to C1q and induces complement-dependent cytotoxicity (CDC).

21. The antibody or antigen-binding portion thereof of claim 1 conjugated to a cytotoxic drug.

22. The antibody or antigen-binding portion thereof of claim 1, comprising a defucosylated Fc region.

23. A polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen-binding portion thereof according to claims 1 to 22.

24. 24. The polynucleotide of claim 23, wherein the nucleic acid sequence is operably linked to a promoter.

25. A vector comprising the polynucleotide according to any one of claims 23 to 24.

26. A host cell comprising the polynucleotide of any one of claims 23 to 24 or the vector of claim 25, and optionally expressing the antibody or antigen-binding portion thereof.

27. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof according to any one of the preceding claims and a pharmaceutically acceptable carrier or diluent.

28. 27. A method of making the antibody or antigen-binding portion thereof of any one of the preceding claims, comprising culturing the host cell of claim 26 under conditions sufficient to express the antibody or antigen-binding portion thereof, and isolating the antibody or antigen-binding portion thereof.

29. 29. The method of claim 28, further comprising formulating the antibody as a pharmaceutical composition.

30. A method for treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, and a malignant hematological disease in a subject in need thereof, the method comprising administering to the subject an antibody that binds to KLRB1, preferably an antibody described in any one of claims 1 to 22, a polynucleotide described in any one of claims 23 to 24, a vector described in claim 25, a pharmaceutical composition described in claim 27, or a host cell described in claim 26 that expresses the antibody or an antigen-binding portion thereof.

31. 31. The method of claim 30, wherein the autoimmune disease is rheumatoid arthritis, Sjogren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.

32. 31. The method of claim 30, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.

33. 31. The method of claim 30, wherein the transplant rejection can be a kidney, lung, heart, liver, limb, skin, or multi-organ transplant rejection.

34. 31. The method of claim 30, wherein the hematological malignancy is lymphoma or leukemia.

35. 35. The method of claim 34, wherein the lymphoma, NK / T-cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), or peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) or mycosis fungoides.

36. 35. The method of claim 34, wherein the leukemia is T-cell leukemia, aggressive NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).

37. The antibody of any one of claims 1 to 22, the polynucleotide of any one of claims 23 to 24, the vector of claim 25, or the method of claims 30 to 36, wherein the antibody binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), innate lymphoid cell types 2 and 3 (ILC2 and ILC3), pathogenic effector Th2 (peTh2) cells, NK cells, and / or neoplastic T or NK cells expressing KLRB1.

38. The antibody of any one of claims 1 to 22, which is not clone B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), DX1, DX12, 191B8, Ab9, KW1.2.1, KW7.3.7, or JNH25G2G22 (Creative Diagnostics).

39. 26. The antibody of any one of claims 1 to 22, the polynucleotide of any one of claims 23 to 24, or the vector of claim 25, for use in a method for treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, and a malignant hematological disease in a subject in need thereof.

40. 40. The antibody, polynucleotide or vector for use according to claim 39, wherein the autoimmune disease is rheumatoid arthritis, Sjogren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.

41. 40. The antibody, polynucleotide or vector for use according to claim 39, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.

42. 40. The antibody, polynucleotide or vector for use according to claim 39, wherein the transplant rejection can be kidney, lung, heart, liver, limb, skin or multi-organ transplant rejection.

43. 40. The antibody, polynucleotide, or vector for use according to claim 39, wherein the hematological malignancy is lymphoma or leukemia.

44. 44. The antibody, polynucleotide, or vector for use according to claim 43, wherein the lymphoma is NK / T-cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), or peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

45. 44. The antibody, polynucleotide, or vector for use according to claim 43, wherein the leukemia is T-cell leukemia, NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).

Images