KLRB1 binder and method of using the same

Abstract

For the treatment of autoimmune diseases, allergic diseases, graft rejection, malignant blood diseases and cancer, for example, to deplete cells, or inhibit cells, or activate cells (specifically, Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, peTh2, ILC2, ILC3, NK cells, and / or tumorous T or NK cells, in vivo), KLRB1 binders (in particular, anti-KLRB1 antibodies and antigen-binding portions thereof) and compositions thereof, as well as a treatment method using such agents.

Description

Technical Field

[0001] Claims of Priority This application claims the benefit of U.S. Provisional Application No. 63 / 419,218, filed Oct. 25, 2022, and U.S. Provisional Application No. 63 / 343,434, filed May 18, 2022. The entire contents of the foregoing are incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. HL119145 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field This disclosure generally relates to KLRB1 binders, particularly anti-KLRB1 antibodies, and methods of treatment using such agents for autoimmune diseases, allergic diseases, transplant rejection, hematologic malignancies, and cancer.

Background Art

[0004] Expression of killer cell lectin-like receptor subfamily B, member 1 (KLRB1, also known as CD161) defines a distinct 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 IL-17 they produce, 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, have been FDA-approved and are commercially available for use in psoriasis (e.g., secukinumab and ixekizumab) and ankylosing spondylitis (e.g., secukinumab). However, development of therapies based on directly targeting these molecules has remained limited, and new therapeutics that reduce the activity of these cytokines for various indications, including autoimmune diseases, are still needed.

SUMMARY OF THE INVENTION

[0005] The present invention provides a solution to the above problems by providing an antibody and an antigen-binding fragment thereof that bind to cells expressing KLRB1 on the cell surface and target the bound cells for depletion. These target cells directly produce the above-mentioned cytokines or cause other cells to produce them. By depleting those cells, cytokine production is reduced, providing a therapeutic benefit.

[0006] This specification describes KLRB1-binding antibodies (and antigen-binding fragments thereof) having many uses including therapeutic and diagnostic uses. For example, the antibodies and antigen-binding fragments thereof can be used to treat, and in some cases prevent (i.e., by depleting KLRB1-expressing cells), various diseases associated with KLRB1-expressing cells, for example, for treatment and in some cases, autoimmune diseases such as psoriasis, psoriatic arthritis, ankylosing spondylitis, palmoplantar pustulosis, hidradenitis suppurativa, and inflammatory bowel disease; allergic diseases such as asthma and atopic dermatitis; graft rejection; malignant blood diseases, and cancer (i.e., reducing the risk of developing). Antigen-binding fragments (also referred to herein as antibody fragments) include those described herein, for example, Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies (e.g., scFv).

[0007] Antibodies or antigen-binding portions thereof that specifically bind to killer cell lectin-like receptor subfamily B, member 1 (KLRB1; optionally SEQ ID NO: 1 or SEQ ID NO: 2) are provided herein, and the antibody or antigen-binding portion thereof comprises: (a) a heavy chain variable region (VH) comprising a VH complementarity-determining region (CDR) 1 having a sequence that is at least 95% identical to the VH CDR1 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, or 32; a VH CDR2 having a sequence that is at least 95% identical to the VH CDR2 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, 32, or 45-53; and a VH CDR3 having a sequence that is at least 95% identical to the VH CDR3 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, 32, or 45-53; and (b) a light chain variable region (VL) comprising at least one or consisting of a VL CDR1 having a sequence that is at least 95% identical to the VL CDR1 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, 32, or 45-53; a VL CDR2 having a sequence that is at least 95% identical to the VL CDR2 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, 32, or 45-53; and a VL CDR3 having a sequence that is at least 95% identical to the VL CDR3 amino acid sequence shown in any one of Tables 1-53, preferably Table 1, 30, 31, 32, or 45-53.

[0008] In some embodiments, this VH comprises or consists of the VH amino acid sequences shown in Tables 1, 30, 31, 32, or 45 - 53. In some embodiments, this VL comprises or consists of the VL amino acid sequences shown in Tables 1, 30, 31, 32, or 45 - 53. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 1, and this VL comprises or consists of the VH amino acid sequence shown in Table 1. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 30, and this VL comprises or consists of the VH amino acid sequence shown in Table 30. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 31, and this VL comprises or consists of the VH amino acid sequence shown in Table 31. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 32, and this VL comprises or consists of the VH amino acid sequence shown in Table 32. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 45, and this VL comprises or consists of the VH amino acid sequence shown in Table 45. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 46, and this VL comprises or consists of the VH amino acid sequence shown in Table 46. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 47, and this VL comprises or consists of the VH amino acid sequence shown in Table 47. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 48, and this VL comprises or consists of the VH amino acid sequence shown in Table 48. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 49, and this VL comprises or consists of the VH amino acid sequence shown in Table 49. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 50, and this VL comprises or consists of the VH amino acid sequence shown in Table 50.In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 51, and this VL comprises or consists of the VH amino acid sequence shown in Table 51. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 52, and this VL comprises or consists of the VH amino acid sequence shown in Table 52. In some embodiments, this VH comprises or consists of the VH amino acid sequence shown in Table 53, and this VL comprises or consists of the VH amino acid sequence shown in Table 53. In some embodiments, this VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence shown in Table 1, and this VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the VL amino acid sequence shown in Table 1. In some embodiments, this VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence shown in one of Tables 30, 31, 32, or 45 - 53, and this VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in one of Tables 30, 31, 32, or 45 - 53, preferably, VH and VL are from the same table.

[0009] In some embodiments, the antibody or antigen - binding portion thereof comprises the heavy and light chains of the constant region, and the heavy and / or light chains of this constant region comprise or consist of the amino acid sequence shown in one of Table 58.

[0010] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 1, and a variable region light chain consisting of the LC amino acid sequence shown in Table 1, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0011] 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; SEQ ID NO:), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 30, and a variable region light chain consisting of the LC amino acid sequence shown in Table 30, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0012] Furthermore, 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), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 31, and a variable region light chain consisting of the LC amino acid sequence shown in Table 31, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0013] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 32, and a variable region light chain consisting of the LC amino acid sequence shown in Table 32, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0014] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 45, and a variable region light chain consisting of the LC amino acid sequence shown in Table 45, and optionally comprises or consists of a constant region, and optionally this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0015] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 46, and a variable region light chain consisting of the LC amino acid sequence shown in Table 46, and optionally comprises or consists of a constant region, and optionally this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0016] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 47, and a variable region light chain consisting of the LC amino acid sequence shown in Table 47, and optionally comprises or consists of a constant region, and optionally, the antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0017] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 48, and a variable region heavy chain consisting of the LC amino acid sequence shown in Table 48, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0018] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 49, and a variable region light chain consisting of the LC amino acid sequence shown in Table 49, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0019] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 50, and a variable region light chain consisting of the LC amino acid sequence shown in Table 50, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0020] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 51, and a variable region light chain consisting of the LC amino acid sequence shown in Table 51, and optionally comprises or consists of a constant region, and optionally, the antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0021] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 52, and a variable region light chain consisting of the LC amino acid sequence shown in Table 52, and optionally comprises or consists of a constant region, and optionally this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0022] Furthermore, 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 or 2), which comprises a variable region heavy chain consisting of the HC amino acid sequence shown in Table 53, and a variable region light chain consisting of the LC amino acid sequence shown in Table 53, and optionally comprises or consists of a constant region, and optionally, this antibody or antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0023] In some embodiments, this constant region comprises or consists of the sequence shown in Table 58.

[0024] In some embodiments, this antibody comprises or consists of a heavy chain variable sequence and / or a light chain variable sequence that is at least 95% identical to the sequence shown in Table 56.

[0025] In some embodiments, this antibody comprises or consists of a heavy chain variable sequence and / or a light chain variable sequence that is at least 95% identical to the sequence shown in Table 57.

[0026] Also provided herein is an antibody or antigen-binding portion thereof that specifically binds to human KLRB1, comprising CDRs from different tables herein, or heavy chain / light chain pairs from different tables herein. Preferably, this antibody or antigen-binding portion thereof binds to KLRB1-expressing cells and targets them for depletion (e.g., via the presence of an Fc domain for ADCC-dependent or CDC-dependent depletion or conjugation to a cytotoxic agent).

[0027] In some embodiments, this antibody or antigen-binding portion thereof comprises the hinge region and Fc domain of the heavy chain constant region.

[0028] In some embodiments, this 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 58.

[0029] In some embodiments, this antibody or antigen-binding portion thereof is a monoclonal antibody.

[0030] In some embodiments, this antibody or antigen-binding portion thereof is a chimeric antibody, a humanized antibody, or a human antibody and / or comprises one or more mutations (e.g., within a CDR) that remove an Asn (N)-glycosylation site or remove Cys, Asp, Met, Trp, or Lys.

[0031] In some embodiments, this antibody or antigen-binding site is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody, or IgG4 antibody.

[0032] In some embodiments, this antibody or antigen-binding portion thereof is an antibody comprising an Fc region that binds to an Fc gamma receptor (FcγR), induces antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or binds to C1q to induce complement-dependent cytotoxicity (CDC), preferably human IgG1.

[0033] In some embodiments, this antibody or antigen-binding portion thereof is an antigen-binding fragment (also referred to herein as an antibody fragment) comprising those described herein, e.g., Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies (e.g., scFv).

[0034] In some embodiments, the antibody or antigen-binding portion thereof binds to a second molecule, such as a cytotoxic agent or moiety, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection. In some embodiments, the antibody or antigen-binding portion thereof is conjugated to a cytotoxic agent or moiety to deplete or facilitate the depletion of cells expressing KLRB1.

[0035] In some embodiments, the antibody or antigen-binding portion thereof comprises a defucosylated Fc region.

[0036] Also provided herein is a polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen-binding portion thereof described herein. In some embodiments, the nucleic acid sequence is operably linked to a promoter.

[0037] Further provided are vectors comprising the polynucleotides described herein, and host cells comprising the polynucleotide or vector and optionally expressing the antibody or antigen-binding portion thereof described herein. Also provided herein is a method for producing the antibody or antigen-binding portion thereof described herein. The method can include culturing the host cell under conditions sufficient to express the antibody or antigen-binding portion thereof, and isolating the antibody or antigen-binding portion thereof. In some embodiments, the method includes formulating the antibody as a pharmaceutical composition.

[0038] Further provided herein is a pharmaceutical composition comprising the antibody or antigen-binding portion thereof described herein (e.g., comprising or consisting of the antibody or antigen-binding portion thereof as an active agent) and a pharmaceutically acceptable carrier or diluent.

[0039] In some embodiments, the antibody or antigen-binding portion thereof 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), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent Application Publication No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7).

[0040] Also provided herein is a method of treating one or more of an autoimmune disease, an allergic disease, a graft rejection reaction, and a hematological malignancy in a subject in need thereof, the method 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 that expresses the antibody or an antigen-binding portion thereof (described herein). Also provided is an antibody that binds to KLRB1, preferably an antibody or an antigen-binding portion thereof or a pharmaceutical composition described herein, for use in a method of treating one or more of an autoimmune disease, an allergic disease, a graft rejection reaction, and a hematological malignancy in a subject in need thereof. Also provided are a polynucleotide, a vector, or a host cell that expresses an antibody or an antigen-binding portion thereof, for use, for example, in cell or gene therapy. In some embodiments, the autoimmune disease is autoimmune hepatitis, 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. 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 graft rejection reaction can be a rejection reaction against a kidney, lung, heart, liver, limb, skin, or multi-organ 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 lymphoma, such as 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, unspecified (PTCL-NOS). In some embodiments, the hematological malignancy expresses KLRB1.

[0041] In some embodiments, this antibody or its antigen-binding portion binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), natural lymphocyte-like cell types 2 and 3 (ILC2 and ILC3), pathogenic effector Th2 (peTh2) cells, and / or NK cells expressing KLRB1. In some embodiments, this antibody or its antigen-binding portion is conjugated to a cytotoxic agent or cytotoxic moiety.

[0042] In some embodiments, this antibody or its antigen-binding portion used in the methods of treatment described herein is or 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), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent Application Publication No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7).

[0043] In some embodiments of each of the above aspects and embodiments, and in other aspects and embodiments described herein, the subject is human.

[0044] Without wishing to be bound by theory, the inventors believe that the antibodies disclosed herein are superior to the aforementioned antibodies, e.g., commercially available antibodies, in one or more of the following activities. - Increased binding efficacy (decrease in binding EC for cell-expressed KLRB1 or soluble KLRB1 extracellular domain 50 ); - Increased ADCC-mediated depletion efficacy (decrease in EC50 for depletion of CHO-KLRB1+ cells, or decrease in EC 50 for reporter cell line ADCC assay); - Increased kinetics of binding affinity (decrease in KD or K off ); - Increased blocking efficacy (decrease in IC for inhibition of CLEC2D binding to KLRB1-expressing cells 50 ); and / or - Increased enhancement effect (decrease in EC for enhancement (increase) of CLEC2D binding to cell-expressed KLRB1 50 ).

[0045] In some embodiments, the antibodies disclosed herein have a higher production yield, lower immunogenicity (due to the presence of humanized variable regions and / or human Fc sequences), and / or improved biophysical parameters (e.g., higher melting temperature, higher freeze-thaw stability, reduced isomerization, reduced or absent deamidation, and / or reduced susceptibility to oxidation) compared to previously described antibodies, such as commercially available antibodies.

[0046] 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 recited group collectively, but also each individual component of that group, as well as every possible subgroup of the major group and the major group with one or more of the components of that group missing from the major group. In the present disclosure, it is also contemplated that one or more of the components of the group may be explicitly excluded in the claimed disclosure.

[0047] These and other advantages of the present technology will become apparent when referring to the accompanying drawings and the following description.

Brief Description of the Drawings

[0048]

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Mode for Carrying Out the Invention

[0049] The expression of KLRB1 characterizes a unique set of immune system cells associated with various autoimmune diseases. These include Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, and / or NK cells.

[0050] KLRB1 is also expressed by tumor cells present in many T and NK cell malignancies. These include lymphomas and leukemias of various peripheral T cells and NK cells.

[0051] Using the KLRB1-binding antibodies described herein, for depletion, Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells can be preferentially targeted. The population of KLRB1-expressing immune cells expresses more IL-17 than the population of total CD4 or CD8 T cells and is specific for Th17 and Tc17 T cells rather than CD4 or CD8, respectively. Thus, 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) binder having cell depletion 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 malignant blood diseases, such as those described herein.

[0052] Furthermore, in some embodiments, modulation of KLRB1 has an inhibitory or stimulatory effect on T cells and NK cells. Thus, disclosed herein are methods of activating or inhibiting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, and / or in vivo in a subject in need thereof by administering to the subject an effective amount of a killer cell lectin-like receptor B1 (KLRB1) binder having KLRB1 receptor blocking activity (blocking of the binding of LLT1 to KLRB1 is achieved), such as an antibody described herein having an effector null Fc mutation (e.g., Liu et al., Antibodies (D) Basel. 2020 Dec;9(4):64). Using these methods, T cells and / or NK cells can be activated and their activity against tumor cells that can be used for the treatment of cancer can be enhanced.

[0053] In some embodiments, the antibody has antibody-dependent cell-mediated cytotoxicity (ADCC) effector activity or complement-dependent cytotoxicity (CDC) effector activity. An effective amount of an anti-KLRB1 antibody that has ADCC or CDC effector function, or is conjugated to a cytotoxic agent, is administered to a subject in need thereof to 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 disclosure provides a killer cell lectin-like receptor B1 (KLRB1) binder as described herein that has ADCC or CDC activity, or is conjugated to a cytotoxic agent. In various aspects, the disclosure provides an mRNA or cDNA encoding the binder. In various aspects, the disclosure provides a pharmaceutical composition comprising an effective amount of the binder.

[0054] Various features of the disclosure, including KLRB1 and its ligands, anti-KLRB1 antibodies or antigen-binding portions thereof, pharmaceutical compositions, treatments and administrations, and exemplary examples, are considered in order below. The following sections include definitions of terms used in the disclosure. Unless otherwise defined herein, technical and scientific terms used in the description of the invention have the meanings commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following explanations of terms will apply, and where appropriate, terms used in the singular will include the plural, and vice versa. If any explanation of a term shown conflicts with any document incorporated herein by reference, the following explanation of the term will prevail.

[0055] 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 is a receptor that 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 for 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).

[0056] KLRB1 expression among T cells is limited to cells that have the ability to respond 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 with the ability to produce interleukin-17 or, in the case of ex-Th17 cells, T cells with the ability to produce interferon-gamma (IFNG). This cytokine production is undesirable in the case of autoimmune diseases.

[0057] This specification describes 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.

[0058] In some embodiments, KLRB1 is cynomolgus monkey KLRB1; an exemplary sequence is provided as SEQ ID NO:2: MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWQQVQEKCLLFSHTVNPWNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0059] Preferably, this antibody binds to cells expressing KLRB1 on their surface (e.g., Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or tumorous T or NK cells) and targets such cells for depletion, for example, via ADCC or CDC (e.g., in the case of a full-length antibody containing an Fc region) or a cytotoxic agent or moiety (e.g., an antigen-binding fragment lacking an ADCC or CDC competent Fc).

[0060] KLRB1-binding antibody This specification describes 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., the antigen-binding portion.

[0061] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., each antibody in the population is identical except for possible naturally occurring mutations that may be present in minor amounts. An antibody can be monoclonal. An antibody can be human or humanized. The term "monoclonal antibody" includes intact monoclonal antibodies and full-length monoclonal antibodies, as well as antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv, and single-chain antibodies (e.g., scFv)), fusion proteins containing antibody fragments, and any other modified immunoglobulin molecule containing at least one antigen-binding site. Further, "monoclonal antibody" refers to antibodies produced by a number of techniques, including but not limited to production by hybridomas, phage library display, recombinant expression, and transgenic animals.

[0062] The term "chimeric antibody" refers to an antibody in which a portion of its heavy chain and / or light chain is derived from a first source or species, while the remainder of its heavy chain and / or light chain is derived from a different source or species.

[0063] As used herein, the term "humanized antibody" refers to an antibody that includes human heavy and light chain variable regions, wherein the native CDR residues thereof are replaced with residues derived from the corresponding CDRs of a non-human antibody (e.g., an antibody of a mouse, rat, rabbit, or non-human primate), and wherein 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 with the corresponding residues derived from the non-human antibody. Further, a humanized antibody may include residues not found in human or non-human antibodies. In some embodiments, these modifications are made for the purpose of further improving and / or optimizing the properties of the antibody. In some embodiments, the humanized antibody includes an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically at least a portion of the constant region of a human immunoglobulin.

[0064] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human, and / or an antibody produced using any technique known to those skilled in the art as being for the production of human antibodies. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B cell hybridoma technology.

[0065] "Antibody fragment" or "antigen-binding fragment" may include a portion of an intact antibody, preferably the antigen-binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; bispecific antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding fragment binds to a second molecule, such as a cytotoxic agent or moiety, e.g., a molecule that promotes depletion of KLRB1+ cells to which the antigen-binding fragment binds, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0066] The terms "epitope" and "antigenic determinant" are used synonymously herein and refer to the portion of an antigen or target to which a particular antibody can recognize and bind. Where 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 its tertiary structure. Epitopes formed from contiguous amino acids (also called linear epitopes) are typically retained even when the protein is denatured, whereas epitopes formed by folding into the tertiary structure (also called conformational epitopes) are typically lost upon denaturation of the protein. Epitopes typically contain at least 3 amino acids, more commonly at least 5, 6, 7, or 8-10 amino acids, in a unique spatial conformation. Epitopes can be predicted using any one of a number of software bioinformatics tools available on the Internet. Epitopes on a target protein may be characterized by analyzing the amino acid residue interactions of the antigen / antibody complex using X-ray crystallography.

[0067] "Fv" contains the smallest antibody fragments that contain the complete antigen recognition and antigen-binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain in a tight non-covalent association. It is in this arrangement that the three CDRs of each variable domain interact to define the 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 the Fv containing only three CDRs specific for the antigen), although with a lower affinity than the full binding site, has the ability to recognize and bind the antigen. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of several residues to the carboxy terminus of the heavy chain CH1 domain that includes 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 domain carry free thiol groups. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments that have a hinge cysteine in between. Other chemical couplings of antibody fragments are also known.

[0068] Depending on the amino acid sequence of their heavy chain constant domains, immunoglobulins can be assigned to different classes. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), for example, IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. The "single-chain Fv" or "sFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further contains a polypeptide linker between the VH and VL domains, enabling the sFv to form the structure desired for antigen binding.

[0069] In various embodiments, the antibody or antigen-binding fragment thereof includes a human or humanized antibody. A humanized form of a non-human (e.g., mouse) antibody is an immunoglobulin chain or fragment thereof (e.g., Fv, Fab, Fab′, F(ab′)2, or other antigen-binding subsequence of an antibody) that includes a minimal sequence derived from a chimeric immunoglobulin, non-human immunoglobulin. A humanized antibody is a human immunoglobulin (recipient antibody) in which the residues of the recipient's complementarity-determining regions (CDRs) are replaced with the residues of the CDRs of a non-human species such as a mouse, rat, or rabbit (donor antibody) having the desired specificity, affinity, and capacity. Optionally, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human residues. A humanized antibody may also include residues not found in the recipient antibody or in the imported CDR or framework sequences. In general, a humanized antibody includes substantially all of at least one, typically two variable domains, where 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.

[0070] 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 within one or more of its hypervariable regions, and these modifications improve the affinity of the antibody for the antigen as compared to the parental antibody that does not have these modification(s). In one embodiment, the affinity matured antibody has a nanomolar or even picomolar affinity for the target antigen. Preferred affinity matured antibodies have an affinity that is 5-fold, more preferably 10-fold, even more preferably 20 or 30-fold better than the starting antibody (generally mouse, humanized, or human) from which the mature antibody is prepared.

[0071] An antibody that "binds to", "specifically binds to", or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is one that binds to the particular polypeptide or the epitope on the particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. 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, faster rate, longer duration, higher affinity, or some combination thereof than with other substances. A binding agent (e.g., an antibody) that specifically binds to an antigen can be identified, for example, by immunoassay, ELISA, surface plasmon resonance (SPR) assay (e.g., Biacore), or other techniques known to those of skill in the art. Accordingly, functional equivalents of the particular anti-KLRB1 antibodies described herein are described herein. In some cases, a KLRB1 antibody can be cross-reactive with various similar KLRB1 proteins (e.g., having the highest affinity for a protein such as human KLRB1 and a lower affinity for other proteins such as mouse KLRB1). A binding agent that specifically binds to an antigen binds to its target antigen with a higher affinity than its affinity for different antigens. The different antigens can be related antigens. In some embodiments, a binding agent that specifically binds to an antigen binds to its target antigen with an affinity that is at least 20-fold higher, e.g., at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, or at least 100-fold higher than its affinity for different antigens. In some embodiments, a binding agent that specifically binds to a particular antigen binds to different antigens with such a low affinity that binding cannot be detected using the assays described herein or other assays known in the art. In some embodiments, affinity is measured using SPR techniques known to those of skill in the art, e.g., a Biacore system or other systems.

[0072] For two or more polypeptides (e.g., two anti-KLRB1 antibodies), the term "identical" or "percent identity" refers to sequences or subsequences in which, as part of the sequence identity, when compared and aligned to maximize match (introducing gaps if necessary) without considering any conservative amino acid substitutions, two or more amino acid residues are the same, or two or more sequences or subsequences in which the same amino acid residues are a given percentage. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to perform amino acid sequence or nucleotide sequence alignments are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.

[0073] 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). Comparison of sequences and determination of the percent identity between two sequences can be achieved using mathematical algorithms. Such homology is well known in the art by local alignment tools and / or algorithms, and can include pairwise alignment methods, multiple sequence alignment methods, structure alignment methods, and / or phylogenetic analysis methods. If the sequences differ by conservative substitutions, the percent sequence identity may be upregulated to correct for the conservative nature of the substitution, but this is not necessary. 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 mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if a score of 1 is given to identical amino acids and a score of 0 is given to non-conservative substitutions, a score of 0-1 is given to conservative substitutions.

[0074] In some embodiments, for two polypeptides of the present disclosure (e.g., an antibody or an antibody domain thereof (e.g., VL, CL, VH, CH1, CH2, CH3 domain)) to be substantially identical, when compared and aligned to maximize match and measured using a sequence comparison algorithm or by visual inspection, the amino acid residue identity is 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%. In some embodiments, the percent identity is present 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 nucleotide lengths or amino acid residue lengths, or any integer value therebetween. In some embodiments, the percent identity is seen over a region of 60 - 80 amino acid residues, e.g., longer than at least about 80 - 100 amino acid residues, and in some embodiments, the sequence is substantially identical over the entire length of the sequences being compared, e.g., the amino acid sequence.

[0075] 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., for optimal alignment, gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be disregarded 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 the corresponding amino acid positions or nucleotide positions are then compared. If 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 equal 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.

[0076] Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the 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 using a Blossum 62 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 at gcg.com on the world wide web).

[0077] As used herein, the terms "conservative sequence modification" or "conservative substitution" can refer to amino acid modifications to the target epitopes, antibodies, and antigen-binding portions thereof of the present disclosure that do not significantly affect and do not change the binding characteristics of the anti-KLRB1 antibody. As used herein, the phrase "conservative amino acid substitution" refers to a substitution 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 been generally defined in the art and include 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, the substitution of tyrosine with phenylalanine is considered a conservative substitution. Methods for identifying amino acid conservative substitutions that do not abolish binding are well known in the art.

[0078] In various embodiments, the antibody is a blocking or antagonist binding agent. "Block," "blocking," or "antagonist" means an agent (e.g., an antibody or a binding fragment thereof) that inhibits or reduces the biological activity of the antigen to which it binds. Certain blocking or antagonist agents substantially or completely inhibit the biological activity of the antigen. For example, the KLRB1 binding agents described herein with effector null Fc mutations can block the signaling of KLRB1 (e.g., thereby interfering with the signaling of KLRB1 and modulating Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or tumorigenic T cells or NK cells).

[0079] In some embodiments, this KLRB1 binder is an antibody, including: a. a full-length antibody that binds to KLRB1 and contains an Fc domain that binds to the Fc gamma receptor with effector function to induce antibody-dependent cell-mediated cytotoxicity (ADCC); b. an antibody that binds to KLRB1 and contains an Fc domain that binds to complement protein 1q (C1q) using effector function to induce complement-dependent cytotoxicity (CDC); c. an antibody conjugate that binds to KLRB1 and contains a cytotoxic agent, such as an antibody-drug conjugate (ADC); or d. a multispecific antibody (e.g., a bispecific or trispecific antibody) that binds to KLRB1 and another antigen.

[0080] In some embodiments, the KLRB1 antibody described herein binds to the extracellular domain of human KLRB1. In some embodiments, this antibody cross-reacts with (binds to both) the extracellular domains of human and cynomolgus monkey KLRB1. In some embodiments, the antibody binds to an epitope of the extracellular domain of KLRB1, and this epitope is at least 90% identical in humans and cynomolgus monkeys. Preferably, the antibody or its antigen-binding fragment contains or is conjugated to an Fc region, a cytotoxic agent, or a second antigen-binding domain that causes depletion of KLRB1-expressing cells.

[0081] In some embodiments, this antibody binds to KLRB1 and is not a mouse antibody.

[0082] In some embodiments, the antibodies described herein are not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent Application Publication No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7).

[0083] In some embodiments, the KLRB1 binder is an antibody, e.g., a full-length antibody comprising an Fc domain comprising 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, IgD antibody, IgE antibody, IgG antibody or 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.

[0084] In some embodiments, the antibody is an antibody fragment comprising an antigen-binding site. In some embodiments, the antibody is a 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 binder is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, polyclonal antibodies are produced by immunizing an animal (e.g., rabbit, rat, mouse, goat, donkey) with the antigen of interest (e.g., purified peptide fragment, recombinant protein or fusion protein) 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 the carrier protein) is diluted in sterile saline and is usually 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 blood or ascites). In some embodiments, the polyclonal antibodies are purified from serum or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis and / or dialysis.

[0085] In some embodiments, the KLRB1 binder is a monoclonal antibody. The monoclonal antibody can be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using the hybridoma method known to those skilled 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.

[0086] After immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line, for example using polyethylene glycol. The hybridoma cells are selected using a special medium as known in the art, and unfused lymphocytes and myeloma cells are excluded in the selection process. Hybridomas producing monoclonal antibodies specifically related to a given antigen can be identified by various methods, including but not limited to immunoprecipitation, immunoblotting, and in vitro binding assays (such as flow cytometry, FACS, ELISA, SPR (such as Biacore) and radioimmunoassay). Once hybridoma cells producing antibodies with 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 using standard methods, or in vivo as ascites tumors in animals. Monoclonal antibodies can be purified from the culture medium or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis and dialysis.

[0087] In some embodiments, monoclonal antibodies are produced using recombinant DNA techniques known to those skilled in the art. For example, the polynucleotide encoding the antibody is isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, 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 monoclonal antibodies, and transfected into host cells (such as E. coli cells that do not produce immunoglobulin proteins unless transfected with the expression vector, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells).

[0088] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries that express the variable domains or CDRs of the desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

[0089] In some embodiments, monoclonal antibodies are modified to produce alternative antibodies by using recombinant DNA techniques. In some embodiments, the constant domains of the light and heavy chains of a murine monoclonal antibody are replaced with the constant regions of a human antibody to produce a chimeric antibody. In some embodiments, the constant region is shortened or removed to produce the desired antibody fragment of the monoclonal antibody. In some embodiments, site-directed mutagenesis or high-density mutagenesis of the variable region(s) is used to optimize the specificity and affinity of the monoclonal antibody.

[0090] In some embodiments, the KLRB1 binder is a humanized antibody. Various methods for making humanized antibodies are known in the art. In some embodiments, the humanized antibody contains one or more amino acid residues derived from non-human sources introduced into its sequence. In some embodiments, humanization is performed by replacing the corresponding CDR sequences of a human antibody with one or more non-human CDR sequences. In some embodiments, the humanized antibody is constructed by replacing all six CDRs of a non-human antibody (e.g., a mouse antibody) with the corresponding CDRs of a human antibody.

[0091] The selection of the human heavy chain variable region and / or human light chain variable region used to make the humanized antibody can be performed by various methods known in the art based on various factors. In some embodiments, a "best fit" method is used in which the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against an entire library of known human variable region sequences. The human sequence that is most similar to the non-human (e.g., rodent) sequence is selected as the human variable region framework of the humanized antibody. In some embodiments, a specific variable region framework derived from the consensus sequence of all human antibodies having a particular subgroup of light or heavy chains is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from the consensus sequence of the most prevalent human subclass. In some embodiments, human germline genes are used as the source of the variable region framework sequence.

[0092] Other humanization methods include, but are not limited to, a method called "hyperhumanization" described as directly transferring CDRs into a human germline framework, a method named human string content (HSC) based on a measure of "humanity" of an antibody, methods based on the generation of large libraries of humanized variants (including phage libraries, ribosome libraries, and yeast display libraries), and methods based on framework region shuffling.

[0093] In some embodiments, the KLRB1 binder is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some embodiments, the human antibody is made from immortalized human B lymphocytes immunized in vitro. In some embodiments, the human antibody is made from lymphocytes isolated from an immunized individual. In either case, cells producing antibodies against the target antigen may be made and isolated. In some embodiments, the human antibody is selected from a phage library, in which case the phage library expresses human antibodies. Alternatively, human antibodies and antibody fragments may be produced in vitro from an immunoglobulin variable region gene repertoire derived from non-immunized donors using phage display technology. Techniques for making and using antibody phage libraries are well known in the art. Once an antibody is identified, more highly affinity human antibodies may be made using affinity maturation methods known in the art, including but not limited to chain shuffling and site-directed mutagenesis. In some embodiments, the human antibody is produced in transgenic mice containing the human immunoglobulin locus. These mice, when immunized, can produce a complete repertoire of human antibodies without producing endogenous immunoglobulins.

[0094] In some embodiments, the KLRB1 binder is a scFv antibody. An scFv is a molecule that contains a variable heavy chain region and a variable light chain region linked to form a single polypeptide. The scFv can be produced using recombinant techniques known in the art. In some embodiments, the scFv contains a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, this scFv contains (i) a heavy chain variable region, (ii) a linker, and (iii) a light chain variable region in the direction (from N-terminus to C-terminus). In some embodiments, this scFv contains (i) a light chain variable region, (ii) a linker, and (iii) a heavy chain variable region in the direction (from N-terminus to C-terminus). In some embodiments, this scFv is a disulfide-linked scFv (dsscFv), which is an scFv that contains a disulfide bond engineered between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, this scFv (e.g., dsscFv) is bound (either directly or indirectly) to a moiety that extends its half-life, such as, for example, an Fc molecule, the CH3 domain of an immunoglobulin (e.g., CH3 of IgG1), polyethylene glycol (PEG) or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polyanionic phosphate, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, for example, by hyperglycosylation, to extend the lifespan of the scFv (e.g., dsscFv). In some embodiments, this scFv binds to a second molecule, such as a cytotoxic agent or moiety, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0095] A variety of suitable linkers are known to those skilled in the art and are not limited by any specific sequences disclosed herein. In some embodiments, the polypeptide linker is composed of naturally occurring amino acids or non-naturally occurring amino acids. In some embodiments, the linker contains amino acids that allow for plasticity. In some embodiments, the linker contains amino acids that allow for solubility. In some embodiments, the linker contains glycine amino acids. In some embodiments, the linker contains glycine and serine amino acids. In certain embodiments, the linker contains 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 to 4) (SEQ ID NO: 85), GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), GGGGSGGGGSGGGGS (SEQ ID NO: 88), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 89), and (GGGGA)n (where n = 1 to 4) (SEQ ID NO: 90). In some embodiments, the linker contains GGGGSGGGGSGGGGS (SEQ ID NO: 91).

[0096] In some embodiments, the KLRB1 binder is an Fv. The Fv contains 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 extending moiety such as, for example, an Fc molecule, the CH3 domain of IgG (e.g., 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. In some embodiments, the Fv binds to a second molecule, such as a cytotoxic agent or moiety, or a detectable substance or molecule that allows the antibody to be used for diagnosis and / or detection.

[0097] In some embodiments, the KLRB1 binder is a Fab. A Fab is one of the molecules resulting from the digestion of an immunoglobulin antibody by papain. A Fab is a monovalent molecule that includes a light chain, a heavy chain variable region, a CH1 region, and optionally a heavy chain constant region hinge region or a portion thereof. A Fab can be produced using recombinant techniques known in the art. In some embodiments, the Fab includes a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, the Fab includes a polypeptide linker between the heavy chain constant region and the light chain variable region. Various suitable linkers are known to those of skill in the art and are not limited by any particular sequence disclosed herein. In certain embodiments, the linker is the linker described herein. In some embodiments, this Fab is conjugated (either directly or indirectly) to a half-life extending moiety such as, for example, an Fc molecule, a CH3 domain of IgG (e.g., 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 Fab. In some embodiments, this Fab binds to a second molecule, such as a cytotoxic agent or moiety, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0098] In some embodiments, the Fab includes a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, the Fab includes a disulfide bond that increases the stability of the Fab molecule. In some embodiments, the Fab includes a disulfide bond that increases the thermal stability of the Fab molecule.

[0099] In some embodiments, the KLRB1 binder is F(ab’)2. F(ab′)2 is one of the molecules resulting from the digestion of an immunoglobulin antibody by pepsin. AF(ab’)2 is 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, F(ab′)2 is bound (either directly or indirectly) to a moiety that extends its half-life, such as, for example, the CH3 domain of IgG (e.g., 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 F(ab′)2. In some embodiments, this F(ab’)2 is bound to a second molecule, such as a cytotoxic agent or moiety, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0100] In some embodiments, F(ab’)2 comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, F(ab’)2 comprises a disulfide bond that increases the stability of the F(ab’)2 molecule. In some embodiments, F(ab’)2 comprises a disulfide bond that increases the thermal stability of the F(ab’)2 molecule.

[0101] In some embodiments, the KLRB1 binder is F(ab’). F(ab’) is a molecule resulting from the treatment of F(ab’)2 with beta-mercaptoethanol. F(ab’) is a monovalent molecule comprising a light chain associated with a polypeptide containing a heavy chain variable region, CH1, and a hinge region. In some embodiments, this F(ab’) is bound (either directly or indirectly) to a moiety that extends the half-life, such as, for example, an Fc molecule, the CH3 domain of IgG (e.g., 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 F(ab’). In some embodiments, this F(ab’) binds to a second molecule, such as, for example, a cytotoxic agent or moiety, or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0102] In some embodiments, F(ab’) comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, F(ab’) comprises a disulfide bond that increases the stability of the F(ab’) molecule. In some embodiments, F(ab’) comprises a disulfide bond that increases the thermal stability of the F(ab’) molecule.

[0103] In some embodiments, the KLRB1 binder is a bispecific antibody. A bispecific antibody can recognize and bind to at least two different antigens or epitopes. Those different epitopes may be epitopes within the same molecule (e.g., two epitopes on KLRB1), or epitopes of different molecules (e.g., one epitope is on KLRB1 and one epitope is on a different target). In some embodiments, the bispecific antibody has enhanced potency compared to individual antibodies or a combination of two or more antibodies. In some embodiments, the bispecific antibody has reduced toxicity compared to individual antibodies or a combination of two or more antibodies. It is known to those skilled in the art that any therapeutic agent can have its own pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, the bispecific antibody has the ability to synchronize the PK of two active binders, and the two individual binders have different PK profiles. In some embodiments, the bispecific antibody has the ability to concentrate the actions of two drugs in a common region (e.g., tissue) in a subject (e.g., a human). In some embodiments, the bispecific antibody has the ability to concentrate the actions of two drugs on a common target (e.g., a given type of cell). In some embodiments, the bispecific antibody has the ability to act on two or more biological pathways or functions. In some embodiments, the bispecific antibody has the ability to target two different cells and bring them closer together.

[0104] In some embodiments, the bispecific antibody has reduced toxicity and / or side effects. In some embodiments, the bispecific antibody has reduced toxicity and / or side effects compared to two individual antibodies or a mixture of antibodies as a single agent. In some embodiments, the bispecific antibody has an improved therapeutic index. In some embodiments, the bispecific antibody has an improved therapeutic index compared to two individual antibodies or a mixture of antibodies as a single agent.

[0105] Several techniques for generating bispecific antibodies are known to those skilled in the art. In some embodiments, the bispecific antibody comprises a heavy chain constant region with modifications to the 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 antibody is generated using a knobs-into-holes (KIH) strategy. In some embodiments, the bispecific antibody comprises a variant hinge region that cannot form disulfide bonds between identical heavy chains (e.g., reduces homodimer formation). In some embodiments, the bispecific antibody comprises a heavy chain with amino acid changes that modify electrostatic interactions. In some embodiments, the bispecific antibody comprises a heavy chain with amino acid changes that modify hydrophobic / hydrophilic interactions.

[0106] A bispecific antibody may be an intact antibody or an antibody fragment that includes an antigen-binding site.

[0107] Trivalent or higher KLRB1 binders are also contemplated. In some embodiments, a trispecific or tetravalent antibody is generated.

[0108] In some embodiments, the KLRB1 binder is an anti-KLRB1 antibody comprising one, two, three, four, five, and / or six of any one of the CDRs of the antibodies described herein. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6, such as those shown in Table 1, and / or (ii) one, two, and / or three light chain CDRs from antibody 10A3D6 as shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) from antibody 10A3D6 as shown in Table 1, and (ii) three light chain CDRs from antibody 10A3D6 as shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6C109S, such as those shown in Table 2, and / or (ii) one, two, and / or three light chain CDRs from antibody 10A3D6C109S as shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) from antibody 10A3D6C109S as shown in Table 2, and (ii) three light chain CDRs from antibody 10A3D6C109S as shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6C109F, such as those shown in Table 3, and / or (ii) three light chain CDRs from antibody 10A3D6C109F as shown in Table 3. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) from antibody 10A3D6C109F as shown in Table 3, and (ii) three light chain CDRs from antibody 10A3D6C109F as shown in Table 3.In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6C109V, such as those shown in Table 4, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6humC109V, such as those shown in Table 4. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6humC109V, such as those shown in Table 4, and (ii) three light chain CDRs derived from antibody 10A3D6humC109V, such as those shown in Table 4.

[0109] In some embodiments, the KLRB1 binder is a humanized 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 derived from any one of Tables 1-53 or Tables 5-53. In some embodiments, the KLRB1 binder is a humanized anti-KLRB1 antibody that comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) and (ii) three light chain CDRs derived from any one of Tables 1-53 or Tables 5-53, wherein the heavy chain CDRs and the light chain CDRs are from the same table. In some embodiments, the heavy chain and the light chain are from different tables, or the CDRs comprise CDRs from different tables.

[0110] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum1.2, such as those shown in Table 30, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum1.2, such as those shown in Table 30. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum1.2, such as those shown in Table 30, and (ii) three light chain CDRs derived from antibody 10A3D6hum1.2, such as those shown in Table 30.

[0111] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum1.5, such as those shown in Table 31, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum1.5, such as those shown in Table 31. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum1.5, such as those shown in Table 31, and (ii) three light chain CDRs derived from antibody 10A3D6hum1.5, such as those shown in Table 31.

[0112] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum2.2, such as those shown in Table 32, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum2.2, such as those shown in Table 32. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs derived from antibody 10A3D6hum2.2, such as those shown in Table 32, and (ii) three light chain CDRs derived from antibody 10A3D6hum2.2, such as those shown in Table 32.

[0113] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.1, such as those shown in Table 45, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.1, such as those shown in Table 45. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.1, such as those shown in Table 45, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.1, such as those shown in Table 45.

[0114] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.2, such as those shown in Table 46, and / or (ii) one, two and / or three light chain CDRs derived from antibody 10A3D6hum8.2, such as those shown in Table 46. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.2, such as those shown in Table 46, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.2, such as those shown in Table 46.

[0115] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.3, such as those shown in Table 47, and / or (ii) one, two and / or three light chain CDRs derived from antibody 10A3D6hum8.3, such as those shown in Table 47. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.3, such as those shown in Table 47, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.3, such as those shown in Table 47.

[0116] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.4, such as those shown in Table 48, and / or (ii) one, two and / or three light chain CDRs derived from antibody 10A3D6hum8.4, such as those shown in Table 48. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.4, such as those shown in Table 48, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.4, such as those shown in Table 48.

[0117] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.5, such as those shown in Table 49, for example, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.5, such as those shown in Table 49, for example. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.5, such as those shown in Table 49, for example, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.5, such as those shown in Table 49, for example.

[0118] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.6, such as those shown in Table 50, for example, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.6, such as those shown in Table 50, for example. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.6, such as those shown in Table 50, for example, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.6, such as those shown in Table 50, for example.

[0119] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.7, such as those shown in Table 51, for example, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.7, such as those shown in Table 51, for example. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.7, such as those shown in Table 51, for example, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.7, such as those shown in Table 51, for example.

[0120] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.8, such as those shown in Table 52, for example, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.8, such as those shown in Table 52, for example. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.8, such as those shown in Table 52, for example, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.8, such as those shown in Table 52, for example.

[0121] In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs derived from antibody 10A3D6hum8.9, such as those shown in Table 53, for example, and / or (ii) one, two, and / or three light chain CDRs derived from antibody 10A3D6hum8.9, such as those shown in Table 53, for example. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) derived from antibody 10A3D6hum8.9, such as those shown in Table 53, for example, and (ii) three light chain CDRs derived from antibody 10A3D6hum8.9, such as those shown in Table 53, for example.

Table 1

Table 2

Table 3

Table 4

Table 5

Table 6

Table 7

Table 8

Table 9

Table 10

Table 11

Table 12

Table 13

Table 14

Table 15

Table 16

Table 17

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Table 25

Table 26

Table 27

Table 28

Table 29

Table 30

Table 31

Table 32

Table 33

Table 34

Table 35

Table 36

Table 37

Table 38

Table 39

Table 40

Table 41

Table 42

Table 43

Table 44

Table 45

Table 46

Table 47

Table 48

Table 49

Table 50

Table 51

Table 52

Table 53

[0122] In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from the antibodies described herein. In some embodiments, the KLRB1 binder comprises a humanized form or a humanized variant of the antibodies described herein. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6 (Table 1) or its humanized form. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6C109S (Table 2) or its humanized form. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6C109F (Table 3), or its humanized form. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6C109V (Table 4), or its humanized form. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum1.2 (Table 30), or its variant. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum1.5 (Table 31), or its variant. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum2.2 (Table 32), or its variant. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.1 (Table 33), or its variant.In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.2 (Table 34), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.3 (Table 35), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.4 (Table 36), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.5 (Table 37), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 38), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 39), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 40), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 41), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 42), or a variant thereof. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3, and the light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 43), or a variant thereof.In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 44), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 45), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 46), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 47), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 48), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 49), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 50), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 51), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 52), or a variant thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3 derived from antibody 10A3D6hum6.6 (Table 53), or a variant thereof.In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, CDR3, derived from antibody 10A3D6hum1.2, 10A3D6hum1.5, 10A3D6hum2.2, 10A3D6hum6.1, 10A3D6hum6.2, 10A3D6hum6.3, 10A3D6hum6.4, 10A3D6hum6.5, 10A3D6hum6.6, 10A3D6hum7.1, 10A3D6hum7.2, 10A3D6hum7.3, 10A3D6hum7.4, 10A3D6hum7.5, 10A3D6hum7.6, 10A3D6hum8.1, 10A3D6hum8.2, 10A3D6hum8.3, 10A3D6hum8.4, 10A3D6hum8.5, 10A3D6hum8.6, 10A3D6hum8.7, 10A3D6hum8.8, or 10A3D6hum8.9, or variants thereof.

[0123] In some embodiments, the KLRB1 binder comprises a humanized form or a humanized variant of an antibody described herein. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, CDR3 shown in Tables 1-4, or humanized forms thereof such as those shown in, for example, Tables 5-53 or 56-57.

[0124] CDRs are defined in various ways / systems by those skilled in the art. 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 position of structural loop regions. The IMGT system is based on the diversity and position of sequences within the structure of the variable domain. The AbM definition is a compromise between Kabat and Chothia. The Contact definition is based on the analysis of available antibody crystal structures. An exemplary system is a combination of Kabat and Chothia. Software programs (e.g., abYsis (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi)) are available and are known to those skilled in the art for the analysis and determination of CDR antibody sequences.

[0125] The specific CDR sequences defined herein are generally based on the Kabat definition. However, when referring to the heavy chain CDR(s) and / or light chain CDR(s) of a specific antibody, it will be understood that all CDR definitions known to those skilled in the art are included, for example, as shown in the tables herein. In some embodiments, all of the CDR sequences used are identified using the same definition, i.e., all Chothia, all Kabat, all IMGT, etc.

[0126] In some embodiments, the KLRB1 binder is a variant of the agents described herein. In some embodiments, the KLRB1 binder (e.g., an antibody) comprises (a) a heavy chain CDR1, CDR2, CDR3, or a variant thereof containing 1, 2, 3, or 4 amino acid substitutions, derived from the VH sequences presented herein (e.g., Tables 1-53 or 56-57); and / or a light chain CDR1, CDR2, and / or CDR3, or a variant thereof containing 1, 2, 3, or 4 amino acid substitutions, derived from the VL sequences presented herein (e.g., Tables 1-53 or 56-57). In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the CDR contains one conservative amino acid substitution. In some embodiments, the CDR contains two conservative amino acid substitutions. In some embodiments, the CDR contains three conservative amino acid substitutions. In some embodiments, the CDR contains four conservative amino acid substitutions. In some embodiments, the CDR is the heavy chain CDR1. In some embodiments, the CDR is the heavy chain CDR2. In some embodiments, the CDR is the heavy chain CDR3. In some embodiments, the CDR is the light chain CDR1. In some embodiments, the CDR is the light chain CDR2. In some embodiments, the CDR is the light chain CDR3. In some embodiments, the substitutions are made as part of the humanization process. In some embodiments, the substitutions are made as part of the germline humanization process. In some embodiments, the substitutions are made as part of the affinity maturation process. In some embodiments, the substitutions are made as part of the optimization process.

[0127] In some embodiments, the KLRB1 binder (e.g., an antibody) comprises one or more heavy or light chain CDRs modified to reduce, for example, deamidation within the CDR sequence, remove Asn (N)-glycosylation sites, remove cysteines, or remove Asp to reduce isomerization sites, or remove Met / Trp or Lys, e.g., to reduce the likelihoods within the CDR sequence of asparagine (N)-glycosylation, cysteinylation, asparagine (Asn) deamidation, aspartic acid (Asp) isomerization, methionine / tryptophan (Met / Trp) oxidation, and non-enzymatic lysine (Lys) glycation (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 of the side chain of the amino acids 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 change the structure, function, and / or stability of the polypeptide and potentially reduce biological activity.

[0128] In certain embodiments, the KLRB1 binder comprises a heavy chain variable region comprising heavy chain CDR1, 2, and 3, and a light chain variable region comprising light chain CDR1, 2, and 3, as set forth in Tables 1-53 or 56-57.

[0129] In some embodiments, the KLRB1 binder (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 the heavy chain variable region sequences presented herein (e.g., Tables 1-53 or 56-57), and / or a light chain variable region having at least 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the light chain variable region sequences presented herein (e.g., Tables 1-53 or 56-57).

[0130] As used herein with respect to the variable regions of the light chain (VL) and heavy chain (VH), the term "consensus sequence" refers to a composite or generalized VL or VH sequence defined on the basis of information such that amino acid residues within the VL or VH chain can be modified without having a deleterious effect on antigen binding. Thus, in the "consensus sequence" of a VL or VH chain, a particular amino acid position is occupied by one of a plurality of possible amino acid residues at that position. For example, if arginine (R) or serine (S) is present at a particular position, that particular position within the consensus sequence may be either arginine or serine (R or S). The consensus sequences of the 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 coding primers), by scanning mutagenesis of amino acid residues within the antibody CDR (e.g., alanine scan mutagenesis), or by any other method known in the art, followed by evaluating the binding of the variants to the antigen to determine whether the mutated amino acid position affects antigen binding. In some embodiments, the mutations are introduced into the CDR regions. In other embodiments, the mutations are introduced into the framework regions. In some other embodiments, the mutations are introduced into both the CDR and framework regions. The consensus sequence can be determined by software such as EMBOSS Cons available at ebi.ac.uk / Tools / msa / emboss_cons / .

[0131] In some embodiments, the KLRB1 binder (e.g., an antibody) described herein comprises one or more constant heavy chain domains (e.g., CH1, CH2, and / or CH3 regions). In some embodiments, the KLRB1 binder comprises a constant heavy chain domain 1 (CH1) having an amino acid sequence shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binder comprises a constant heavy chain domain 2 (CH2) having an amino acid sequence shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binder comprises a constant heavy chain domain 3 (CH3) having an amino acid sequence shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binder comprises a heavy chain constant region comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to the amino acid sequence shown herein, e.g., in Table 57 or 58. In some embodiments, one or more constant regions of the KLRB1 binder are modified. In some embodiments, the 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, the modifications to the constant region comprise 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 wholly 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 that provides some degree of molecular plasticity normally conferred by the deleted constant region. In some embodiments, the modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, the modified antibody comprises a peptide spacer inserted between the hinge region and the modified CH2 domain and / or the modified CH3 domain.

[0132] 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 isotype of the antibody. In addition, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). There are a number of Fc receptors specific for different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). When an antibody binds to an Fc receptor on the cell surface, many important and diverse biological responses are induced, including phagocytosis and destruction of antibody-coated particles, removal of immune complexes, lysis of antibody-coated target cells by killer cells (referred to as antibody-dependent cell-mediated cytotoxicity, or ADCC), cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. In some embodiments, the 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 skilled in the art (for example, a representative human IgG1 Fc region is shown in Lobner et al., Immunol Rev. 2016 Mar;270(1):113-131; see, for example, Table 58). In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the variant Fc region is engineered to have substitutions at predetermined amino acid positions compared to the native Fc region. 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, for example, 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, this Fc region is defucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. 2009 May-Jun;1(3):230-236, which describes a method for producing a therapeutic antibody in which the level of fucosylation of the Fc region N-glycan is controlled).

[0133] In some embodiments, a modified antibody (e.g., a modified Fc region) modifies effector function, which in turn affects the biological profile of the antibody due to the modification. For example, in some embodiments, a mutation in the constant region enhances the binding of the modified antibody to Fc receptors when the modified antibody is circulating. In some embodiments, modification of the constant region extends the serum half-life of the antibody. In some embodiments, modification of the constant region shortens the serum half-life of the antibody. In some embodiments, modification of the constant region increases or enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. In some embodiments, the constant region is modified to exclude disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified by adding / substituting one or more amino acids to provide one or more cytotoxins, oligosaccharides, or carbohydrate-binding sites.

[0134] Modifications to the constant region of the antibodies described herein may be made using well-known biochemical or molecular manipulation techniques. In some embodiments, antibody variants are prepared by introducing appropriate changes in nucleotides into the coding DNA and / or synthesizing the desired antibody or polypeptide. It may be possible to enhance the activity or effector function provided by a given sequence or region using these antibody variants while substantially retaining the structure, binding activity, and other desired characteristics of the modified antibody.

[0135] The present disclosure further includes additional variants and equivalents that are substantially homologous to the recombinant antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies described herein, or antibody fragments thereof. 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, thermal stability, expression level, effector function(s), glycosylation, immunogenicity, or solubility. It will be apparent to those skilled in the art that amino acid changes can alter post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchor properties.

[0136] A variation is a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide, which may be a substitution, deletion, or insertion that changes the amino acid sequence compared to the sequence of the native antibody or polypeptide. In some embodiments, the amino acid substitution is the result of replacing one amino acid with another having similar structural and / or chemical properties, such as replacing leucine with serine, for example, as a result of a conservative amino acid substitution. The insertion or deletion may be optional and may range from about 1 to 5 amino acids. In some embodiments, the substitution, deletion, or insertion includes fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions with respect to the parent molecule. In some embodiments, among the variations in the amino acid sequence, biologically useful and / or relevant variations are determined by systematically inserting, deleting, or substituting in the sequence and testing the activity of the resulting variant protein compared to the parent protein.

[0137] In some embodiments, variants can include those having amino acid residues added to the amino terminus and / or carboxyl terminus of the antibody or polypeptide. The length of the additional amino acid residues can range from 1 residue to over 100 residues. In some embodiments, the variant includes an N-terminal methionyl residue. In some embodiments, the variant includes an additional polypeptide / protein, i.e., a fusion protein. In some embodiments, the variant is engineered to be detectable and may include a detectable label and / or a protein (e.g., an enzyme).

[0138] In some embodiments, to modulate the properties of the antibody, cysteine residues not involved in maintaining the proper higher-order structure of the antibody may be substituted or deleted, for example, to improve oxidative stability and / or prevent abnormal disulfide bridging. Conversely, in some embodiments, one or more cysteine residues may be added to form disulfide bond(s) to improve stability.

[0139] 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.

[0140] In some embodiments, the KLRB1 binders described herein are chemically modified. In some embodiments, the KLRB1 binder is an anti-KLRB1 antibody that has been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or conjugation to a cell ligand or other protein. Any number of chemical modifications can be performed by known techniques.

[0141] The present disclosure includes KLRB1 binders constructed on a non-immunoglobulin backbone, which bind to the same epitope or substantially the same epitope as the anti-KLRB1 antibodies disclosed herein. In some embodiments, the non-immunoglobulin-based binder is an agent that competes with the anti-KLRB1 antibodies described herein in a competitive binding assay. In some embodiments, alternative KLRB1 binders include a scaffold protein. Generally, scaffold proteins can be assigned to one of three groups based on the composition of their backbone: (1) scaffolds consisting of α-helices, (2) small scaffolds with little secondary structure or an irregular composition of α-helices and β-sheets, and (3) scaffolds consisting mainly of β-sheets. Scaffold proteins include, but are not limited to, anticalins based on lipocalin scaffolds; adnectins based on the tenth domain of human fibronectin type 3; affibodies based on the B domain in the Ig-binding region of Staphylococcus aureus protein A; darpins based on ankyrin repeat domain proteins; finomers based on the SH3 domain of human Fyn protein kinase; affitins based on Sac7d derived from Sulfolobus acidocaldarius; affilins based on human γ-B-crystallin or human ubiquitin; avimers based on the A domain of membrane receptor proteins; knottins (cysteine knot miniproteins) based on a stable 30-amino acid antiparallel β-strand protein fold; and Kunitz domain inhibitor scaffolds based on a structure containing three disulfide bonds and three loops. In some embodiments, the KLRB1 binder includes an engineered scaffold protein that includes heavy chain CDR1, CDR2, and CDR3, and light chain CDR1, CDR2, and CDR3, shown in any one of Tables 1-54, for example, Tables 1, 31, 32, or 33. In some embodiments, these agents are conjugated to a cytotoxic agent or moiety that results in depletion of KLRB1+ cells.

[0142] Generally, antigen-antibody interactions are non-covalent and reversible, formed by the combination of hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. When explaining the strength of the antigen-antibody complex, the terms affinity and / or binding activity (avidity) are commonly used. The binding of an antibody to its antigen is a reversible process, and the affinity of this binding is typically reported as the equilibrium dissociation constant (K D ). K D is the ratio of the antibody dissociation rate (k on ) (the rate at which the antibody dissociates from its antigen) to the antibody association rate (k off ) (the rate at which the antibody binds to its antigen). In some embodiments, the K D value is determined by measuring k on and k off for a given antibody / antigen interaction and then calculating the K D value using the ratio of these values. The K D value is used to evaluate and rank the strength of individual antibody / antigen interactions. The lower the K D of an antibody, the higher its affinity for its target. In some embodiments, the affinity is measured using the SPR technology of the Biacore system. The binding activity (avidity) provides a measure of the overall strength of the antibody-antigen complex. The binding activity (avidity) depends on three main parameters: (i) the affinity of the antibody for its target, (ii) the valency of both the antibody and the antigen, and (iii) the structural composition of the interacting parts.

[0143] In some embodiments, a KLRB1 binder (e.g., an antibody) binds to KLRB1 (e.g., human KLRB1) with a dissociation constant (K D ) 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. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K Dbinds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 1 nM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 0.5 nM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 0.1 nM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 50 pM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 25 pM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 10 pM or less D binds. In some embodiments, the KLRB1 binder binds to KLRB1 (e.g., human KLRB1) with a K of about 1 pM or less D binds. In some embodiments, the dissociation constant of the binder (e.g., antibody) for KLRB1 is the dissociation constant determined using the KLRB1 protein immobilized on a Biacore chip and the binder flowed over the chip. In some embodiments, the dissociation constant of the binder (e.g., antibody) for KLRB1 is the dissociation constant determined using the binder captured by an anti-human IgG antibody on a Biacore chip and the soluble KLRB1 flowed over the chip.

[0144] In some embodiments, the KLRB1 binder (e.g., an antibody) binds to KLRB1 (e.g., human KLRB1) with a maximum 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, the KLRB1 binder 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, the KLRB1 binder 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.

[0145] Without being bound by theory, the antibodies disclosed herein exhibit superiority over commercially available antibodies in one or more of the following activities: ● Increased binding potency (decrease in binding EC50 to cell-expressed KLRB1 or soluble KLRB1 extracellular domain); ● Increased ADCC-mediated depletion potency (decrease in EC50 for depletion of CHO-KLRB1+ cells or decrease in EC50 for reporter cell line ADCC assay); ● Increased kinetics of binding affinity (decrease in KD or decrease in Koff); ● Increased blocking potency (decrease in IC50 for inhibition of CLEC2D binding to KLRB1-expressing cells); and ● Increased enhancement effect (decrease in EC50 for enhancement (increase) of CLEC2D binding to cell-expressed KLRB1).

[0146] 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 temperature, higher freeze-thaw stability, low isomerization, reduced or absent deamidation, or reduced susceptibility to oxidation) compared to commercially available antibodies (e.g., B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent Application Publication No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7).

[0147] The KLRB1 binders (e.g., antibodies) described in this specification can be prepared by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, recombinant techniques are used to construct the DNA sequence by isolating or synthesizing the DNA sequence encoding the wild-type protein of interest. Optionally, mutations can be induced in the sequence by site-directed mutagenesis to obtain its functional variants. In some embodiments, the DNA sequence encoding the polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. The oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the codon selection that is advantageous in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be applied to synthesize the polynucleotide sequence encoding the target isolated polypeptide. For example, a gene can be constructed by reverse translation using the complete amino acid sequence. Additionally, DNA oligomers containing the nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, several short oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0148] Once the polynucleotide sequence encoding the specific polypeptide of interest has been constructed (by synthesis, site-directed mutagenesis, or another method), it can be inserted into an expression vector and operably linked to expression control sequences appropriate for expressing the protein in the desired host. Appropriate 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, for high expression levels 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.

[0149] In some embodiments, a recombinant expression vector is used to amplify and express DNA encoding an antibody against human KLRB1, or a fragment thereof. For example, a recombinant expression vector is a replicable DNA construct having a synthetic DNA fragment or a DNA fragment derived from cDNA encoding a polypeptide chain of a KLRB1 binder such as an anti-KLRB1 antibody, which may be a replicable DNA construct operably linked to suitable transcriptional regulatory elements and / or translational regulatory elements derived from mammalian genes, microbial genes, viral genes or insect genes. A transcription unit generally includes an assembly of (1) genetic element(s) (plural) having a regulatory role in gene expression, such as a transcription promoter or a transcription enhancer, (2) a structural sequence that is transcribed into mRNA and translated into a protein, i.e., a coding sequence, and (3) appropriate start and stop sequences for transcription and translation. The regulatory element may include an operator sequence to control transcription. A gene for replication ability in a host (usually conferred by an origin of replication) and a selectable gene to facilitate recognition of the transformant may also be added and incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, the DNA of a signal peptide (secretory leader) is operably linked to the DNA of that polypeptide when expressed as a precursor involved in the secretion of the polypeptide; a promoter is operably linked to the coding sequence when it controls the transcription of the coding sequence, or a ribosome binding site is operably linked to the coding sequence when it is in a position to enable translation. In some embodiments, the structural elements intended for use in a yeast expression system include a leader sequence that causes the host cell to secrete the translated protein extracellularly. In some embodiments, in situations where a recombinant protein is expressed without a leader sequence or transport sequence, the polypeptide may include an N-terminal methionine residue. This residue may optionally be cleaved later from the expressed recombinant protein to yield the final product.

[0150] The selection of the expression control sequence and the expression vector generally depends on the selection of the host. A wide variety of combinations of expression hosts / vectors may be used. Examples of expression vectors useful for eukaryotic hosts include vectors containing expression control sequences derived from, for example, SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of expression vectors useful for bacterial hosts include known bacterial plasmids such as plasmids derived from E. coli, including pCR1, pBR322, pMB9, and their derivatives, as well as more extensive host plasmids such as M13 and other filamentous single-stranded DNA phages.

[0151] The KLRB1 binder (e.g., antibody) of the present disclosure can be expressed from one or more vectors. For example, in some embodiments, the heavy chain polypeptide is expressed by one vector and the light chain polypeptide is expressed by a second vector. In some embodiments, the heavy chain polypeptide and the light chain polypeptide are expressed by one vector.

[0152] Suitable host cells for expressing a KLRB1 binder (e.g., an antibody) or a KLRB1 protein or a fragment thereof for use as an antigen or immunogen include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Examples of prokaryotes include Gram-negative or Gram-positive bacteria, such as E. coli or Bacillus. Examples of higher eukaryotic cells include established cell lines derived from mammals as described herein. A cell-free translation system may also be used. Appropriate cloning vectors 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.

[0153] Recombinant polypeptides may be expressed using a variety of mammalian culture systems. In some cases, 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, the COS-7 (derived from monkey kidney) cell line, the L-929 (derived from mouse fibroblasts) cell line, the C127 (derived from mouse mammary carcinoma) cell line, the 3T3 (derived from mouse fibroblasts) cell line, the CHO (derived from Chinese hamster ovary) cell line, the HeLa (derived from human cervical carcinoma) cell line, the BHK (derived from hamster kidney fibroblasts) cell line, the HEK-293 (derived from human fetal kidney) cell line, and variants thereof. Mammalian expression vectors may contain non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' untranslated sequences such as an essential ribosome binding site, a polyadenylation site, a splice donor site and acceptor site, and a transcription termination sequence.

[0154] Even in the expression of recombinant proteins in insect cell culture systems (e.g., baculovirus), a powerful method can be obtained to produce correctly folded, biologically functional proteins. The baculovirus system for producing heterologous proteins in insect cells is well known to those skilled in the art. Accordingly, the present disclosure provides cells comprising the KLRB1 binders described herein. In some embodiments, the cells produce the KLRB1 binders described herein. In some embodiments, the cells produce an antibody. In some embodiments, the cells produce an antibody that binds to human KLRB1. In some embodiments, the cells produce an antibody that binds to cyno KLRB1. In some embodiments, the cells produce an antibody that binds to both human KLRB1 and cyno KLRB1. In some embodiments, the cells produce an antibody called 10A3D6, 10A3D6hum1.2, 10A3D6hum1.5, 10A3D6hum2.2, 10A3D6hum6.1, 10A3D6hum6.2, 10A3D6hum6.3, 10A3D6hum6.4, 10A3D6hum6.5, 10A3D6hum6.6, or a variant thereof. In some embodiments, the cells produce the scFv version of the antibodies 10A3D6, 10A3D6hum1.2, 10A3D6hum1.5, 10A3D6hum2.2, 10A3D6hum6.1, 10A3D6hum6.2, 10A3D6hum6.3, 10A3D6hum6.4, 10A3D6hum6.5, or 10A3D6hum6.6. In some embodiments, the cells are prokaryotic cells (e.g., E. coli). In some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are hybridoma cells. The proteins produced by the host cells can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, solubility differences, 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 the protein and easily purified by applying it to a suitable 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 the isolated protein 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, the supernatant from an expression system that secretes the recombinant protein into the culture medium is first concentrated using a commercially available protein concentration filter, such as an Amicon® or Millipore Pellicon® ultrafiltration device. After the concentration step, the concentrate may be added to a suitable purification matrix. In some embodiments, an anion exchange resin is used, such as a matrix or substrate having a pendant diethylaminoethyl (DEAE) group. The matrix may 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 a sulfopropyl group or a carboxymethyl group. In some embodiments, a hydroxyapatite medium 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 medium, such as silica gel having a pendant methyl or other aliphatic group.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 maintaining biological activity due to the use of conditions and matrices that act in a less denaturing state than several other techniques. Some or all of the above purification steps can be used in various combinations to yield a homogeneous recombinant protein.

[0155] The KLRB1 antibodies of the present disclosure can be analyzed by various methods known in the art for their physical / chemical properties and / or biological activities. 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 the ability of the antibody to kill KLRB1 target cells (cell depletion). Assays include, but are not limited to, for example, an ADCC cell lysis assay using release of LDH and detection of formazan salts. Additionally, the antibody may be evaluated for solubility, stability, thermal stability, viscosity, expression level, quality of expression, and / or purification efficiency.

[0156] In some embodiments, the purified antibody is characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0157] Antibody conjugate 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 agent or moiety that, for example, causes or facilitates depletion of KLRB1-expressing cells to which the antibody or antigen-binding fragment binds. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate). In some embodiments, the cytotoxic agent is a chemotherapeutic agent, including but not limited to methotrexate, adriamycin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepine (PBD) or other intercalating agents. In some embodiments, the cytotoxic agent is a microtubule inhibitor, including but not limited to auristatin, maytansinoid (e.g., DM1 and DM4), and tubulysin. In some embodiments, the cytotoxic agent is an enzymatically active toxin or fragment thereof derived from bacteria, fungi, plants or animals, including but not limited to diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecene. In some embodiments, the antibody is conjugated to one or more small molecule toxins such as calicheamicin, maytansinoid, trichothecene and CC1065. Derivatives of any one of these toxins may be used if the derivative retains the cytotoxic activity of its parent molecule.

[0158] The conjugate containing the KLRB1 antibody or its antigen-binding fragment described herein may be prepared using any suitable method known in the art. In some embodiments, the conjugate is prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0159] In some embodiments, the KLRB1 antibody or its antigen-binding fragment described herein is conjugated to a detectable substance or molecule that enables the use of the antibody for diagnosis and / or detection. Detectable substances include, but are not limited to, enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; cofactor families 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.

[0160] The anti-KLRB1 antibodies or antigen-binding fragments thereof described herein may also be conjugated to a second antibody to form an antibody heteroconjugate.

[0161] The anti-KLRB1 antibodies or antigen-binding fragments 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 an immunoassay. In some embodiments, the immobilized anti-KLRB1 antibody is used for the purification of the target antigen.

[0162] Method for Producing Polynucleotide / Binder Also provided herein are nucleic acids encoding the polypeptides described herein, and vectors comprising nucleic acids encoding the polypeptides described herein, preferably expression vectors. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound, and may include plasmids, cosmids or viral vectors. The vector may be capable of autonomous replication or may be integrated into the host DNA. Examples of viral vectors include replication-defective retroviruses, adenoviruses and adeno-associated viruses.

[0163] The vector may contain the nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably, the 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 the expression vector may depend on factors such as the choice of host cell to be transformed and the expression level of the desired protein. The expression vector of the present invention may be introduced into a host cell to thereby produce a protein or polypeptide comprising a fusion protein or polypeptide encoded by the nucleic acid described herein that encodes the KLRB1 binder described herein.

[0164] The recombinant expression vector of the present invention may be designed for expression of the KLRB1 binder protein in prokaryotic cells. Preferably, the KLRB1 binder 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 regulatory functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, or simian virus 40.

[0165] The vector DNA may be introduced into the host cell via conventional transformation or transfection techniques. The terms "transformation" and "transfection" refer to various art-recognized techniques for introducing foreign nucleic acids (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation.

[0166] A host cell may be used to produce (i.e., express) the KLRB1 binder protein. Accordingly, the present invention further provides a method for producing a KLRB1 binder protein using the host cell of the present invention. In one embodiment, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector encoding the KLRB1 binder protein has been introduced) in a suitable medium such that the KLRB1 binder protein is produced. In another embodiment, the method further comprises isolating the KLRB1 binder 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. This describes a method for producing a therapeutic antibody in which the level of fucosylation of the Fc region N-glycan is controlled).

[0167] Pharmaceutical composition Also provided herein is a pharmaceutical composition comprising the KLRB1 binder described herein as an active ingredient. In various embodiments, the KLRB1 binder is formulated as a pharmaceutical composition, for example, as a pharmaceutical composition for use as a medicine. In various embodiments, the pharmaceutical composition is for use as a medicine for treating the diseases described herein, optionally an autoimmune disease, an allergic disease, a graft rejection reaction, or a hematological malignancy, in a subject in need thereof. In some embodiments, 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, 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 graft rejection reaction can be a rejection reaction against kidney, lung, heart, liver, limb, skin, or multi-organ transplantation. In some embodiments, the hematological malignancy is leukemia, for example, T cell leukemia, NK cell leukemia, T cell lymphoma, T cell prolymphocytic leukemia (T-PLL), or large granular lymphocyte 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.

[0168] One of ordinary skill in the art can formulate the KLRB1 binder as a pharmaceutical composition according to known methods.

[0169] The pharmaceutical composition may contain a carrier. As used herein, "carrier" may include a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic (or generally non-toxic) to the cells or subject to which it is exposed, at the dosages and concentrations used. In many cases, the physiologically acceptable carrier is a pH buffered aqueous 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 such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS™.

[0170] In various embodiments, the KLRB1 binder is included in an injectable formulation, such as a subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection formulation. The injectable formulation may be, for example, an aqueous solution in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline buffer. This injectable formulation may include formulation agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the KLRB1 binder may be in a dry form or in a powder form for constitution prior to use with a suitable vehicle, such as sterile pyrogen-free water.

[0171] The binder of the present disclosure can be formulated in any suitable form for delivery to target cells / tissues. In some embodiments, the KLRB1 binder can be formulated as liposomes, microparticles, microcapsules, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or macroemulsions. In some embodiments, the pharmaceutical formulation includes a complex of the agent of the present disclosure with liposomes. 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 containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).

[0172] In some embodiments, the KLRB1 binder is formulated as a sustained-release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the drug, and the matrix is in the form of a molded article (e.g., a film or microcapsule). Examples of 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 a lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0173] Treatment and Administration The present disclosure provides a method comprising administering to a subject in need thereof a KLRB1 binder described herein, or a pharmaceutical composition comprising a KLRB1 binder described herein. In some embodiments, the subject is human. In some embodiments, the method is performed in vivo (e.g., as contrasted with ex vivo). As used herein, "treatment" refers to therapeutic treatment (treating a subject having a disease); the method may also be used for prophylactic or preventive means, the purpose of which is to prevent or delay (mitigate) the target pathological condition or disorder in a subject not having the disease. Thus, those in need of treatment include those already suffering from the disorder, those prone to suffering from the disorder, or those in whom the disorder should be prevented (as used herein, "prevent" means reducing the risk of onset).

[0174] In various aspects and embodiments, the present disclosure provides methods for treating autoimmune diseases, allergic diseases, graft rejection, or hematological malignancies in a subject in need thereof. In some embodiments, autoimmune diseases include 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, Crohn's disease, ulcerative colitis, or celiac disease. In some embodiments, allergic diseases include asthma, allergic eosinophilic asthma, allergies, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, the graft rejection can be rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ transplant. In some embodiments, hematological malignancies include 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, lymphoma includes 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. As described herein, KLRB1-expressing cells are involved in the etiology of these diseases; thus, in some embodiments, hematological malignancies express KLRB1. According to the present disclosure, depletion of such KLRB1-expressing cells provides a therapeutic benefit.

[0175] In various aspects and embodiments, the present disclosure provides methods for treating or preventing graft rejection. The graft rejection can be, for example, kidney rejection.

[0176] As used herein, "administering" and "treating" can include contacting an animal, human, subject, cell, tissue, organ, or biological fluid with an exogenous pharmaceutical, therapeutic, diagnostic, or composition when applied to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can include in vivo treatment, as well as in some embodiments in vitro or ex vivo treatment.

[0177] Typically, the agent is administered in an amount effective to reduce one or more symptoms of a disease in the subject or population being treated by inducing regression of such symptom(s) or inhibiting progression of the symptom(s) to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any particular disease symptom can vary depending on factors such as the subject's disease state, age, and weight, as well as the ability of the agent to elicit the desired response in the subject. Whether a disease symptom has been alleviated can be evaluated by any clinical measurement commonly used by a physician or other skilled healthcare provider to assess the severity or progression of that symptom.

[0178] Accordingly, in various embodiments, the term "effective amount" or therapeutically effective amount is the concentration or amount of a KLRB1 binder that achieves a particular stated purpose, e.g., reduction of one or more symptoms of a disease described herein. The "effective amount" of a KLRB1 binder can be determined empirically. Further, a "therapeutically effective amount" is the concentration or amount of a KLRB1 binder effective to achieve the described therapeutic effect. This amount can also be determined empirically.

[0179] In some embodiments, treatment with a KLRB1 binder of the present disclosure can kill at least about 20%, such as at least about 30%, 40%, 50%, 60%, 70%, or 80% of KLRB1-expressing cells associated with the etiology of a disease disclosed herein.

[0180] The term "subject" refers to any animal (e.g., a mammal) including, but not limited to, non-human veterinary subjects including humans and primates.

[0181] As used herein, the recitation of "about" or "approximately" for a value or parameter includes (and describes) embodiments that are for that value or parameter. For example, a recitation of "about X" includes a recitation of "X". As used herein, "about" means plus or minus 10 percent.

[0182] As used in this disclosure and the claims, the singular forms "a", "an", and "the" include the plural forms unless the context clearly dictates otherwise.

[0183] It is understood that whenever embodiments are described in terms of the term "comprising", otherwise similar embodiments described in terms of the terms "consisting of" and / or "consisting essentially of" are also shown. Also, whenever embodiments are described in terms of the phrase "consisting essentially of", it is understood that otherwise similar embodiments described in terms of the term "consisting of" are also shown.

[0184] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B; A or B; A only (A); and B only (B). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to include the embodiments of 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 only (A); B only (B); and C only (C).

[0185] In various embodiments, the KLRB1 binder can be administered by providing mRNA encoding the binder to a subject.

[0186] The following examples are illustrative and not limiting. Many variations of the present technology will be apparent to those skilled in the art upon review of the present disclosure. Accordingly, the scope of the present invention should be determined with reference to the appended claims, together with their full scope of equivalents, rather than with reference to the examples.

Example

[0187] Example 1: Gene expression of KLRB1 is characteristic of and limited to immune cells, which have a unique repertoire. Expression of KLRB1 transcends conventional lymphocyte classification and characterizes subsets of Th17, Th17.1, ex-Th17, Tc17, iNKTs, ILC2, ILC3, peTh2, and NK cells (Figure 1).

[0188] Whole-body KLRB1 expression profiling data shows that KLRB1 does not have significant expression in any cell type other than immune cells (Figure 2).

[0189] 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 patients with crystal-induced arthritis shows an increase in KLRB1 expression (10-fold) (Figure 3). Thus, rheumatoid arthritis is a particularly attractive target for treatment according to the present disclosure.

[0190] Analysis of expression data (GSE36700) from synovial biopsies from rheumatoid arthritis patients compared to normal patients shows an increase in KLRB1 expression (3.3-fold) (Figure 4). Thus, rheumatoid arthritis is a particularly attractive target for treatment according to the present disclosure.

[0191] Sjögren's syndrome: Analysis of expression data (GSE23117) from salivary gland biopsies of patients with Sjögren's syndrome shows that the expression of KLRB1 increases at the advanced stage (13.1-fold), moderate stage (5.3-fold), and early stage (1.6-fold) compared to normal (Figure 5). Therefore, Sjögren's syndrome is a particularly attractive target for the treatment according to the present disclosure.

[0192] Analysis of expression data (GSE40611) from parotid gland biopsies of patients with Sjögren's syndrome shows an increase in the expression of KLRB1 (2.6-fold) compared to normal (Figure 6). Therefore, Sjögren's syndrome is a particularly attractive target for the treatment according to the present disclosure.

[0193] Inclusion body myositis: Analysis of expression data (GSE38454) from muscle biopsies of patients with inclusion body myositis shows an increase in the expression of KLRB1 (2.9-fold) compared to normal (Figure 7). Therefore, inclusion body myositis is a particularly attractive target for the treatment according to the present disclosure.

[0194] Discoid lupus: Analysis of expression data (GSE52471) from skin biopsies of patients with discoid lupus shows an increase in the expression of KLRB1 (7.2-fold) compared to normal (Figure 8). Therefore, discoid lupus is a particularly attractive target for the treatment according to the present disclosure.

[0195] Psoriasis: Analysis of expression data (GSE52471) from skin biopsies of patients with psoriasis shows an increase in the expression of KLRB1 (11.2-fold) compared to normal (Figure 9). Therefore, discoid lupus is a particularly attractive target for the treatment according to the present disclosure.

[0196] Idiopathic pulmonary fibrosis: Analysis of expression data (GSE53845) from lung biopsies of patients with idiopathic pulmonary fibrosis shows an increase in the expression of KLRB1 (1.5-fold) compared to normal (Figure 10). Therefore, idiopathic pulmonary fibrosis is a particularly attractive target for the treatment according to the present disclosure.

[0197] Diabetes: Analysis of expression data (GSE72492) from pancreatic biopsies from diabetic patients showed an increase in the expression of KLRB1 (3.7-fold) compared to normal (Figure 11). Thus, diabetes is a particularly attractive target for the treatment according to the present disclosure.

[0198] Alopecia universalis: Analysis of expression data (GSE74761) from scalp biopsies from patients with idiopathic pulmonary fibrosis showed an increase in the expression of KLRB1 (4.3-fold) compared to normal (Figure 12). Thus, alopecia universalis is a particularly attractive target for the treatment according to the present disclosure.

[0199] Primary biliary cholangitis: Analysis of expression data (GSE79850) from liver biopsies from patients with primary biliary cholangitis who ultimately required liver transplantation showed an increase in the expression of KLRB1 (5.6-fold) compared to normal (Figure 13). Thus, primary biliary cholangitis is a particularly attractive target for the treatment according to the present disclosure.

[0200] Multiple sclerosis: Analysis of expression data (GSE5839) from brain biopsies from patients with multiple sclerosis showed an increase in KLRB1 expression (2.5-fold) compared to control brains (Figure 14). Thus, multiple sclerosis is a particularly attractive target for the treatment according to the present disclosure.

[0201] Lymphocytic colitis: Analysis of expression data (GSE65107) from colonic biopsies from 4 patients with lymphocytic colitis showed an increase in the expression of KLRB1 (3.2-fold) compared to 4 healthy individuals (Figure 15). Thus, lymphocytic colitis is a particularly attractive target for the treatment according to the present disclosure.

[0202] Kidney transplant rejection: Analysis of expression data (GSE1563) from kidney biopsies from 7 patients with acute kidney rejection showed an increase in the expression of KLRB1 (2.1-fold) compared to 9 healthy individuals (Figure 16). Thus, kidney transplant rejection is a particularly attractive target for the treatment according to the present disclosure.

[0203] Lung transplantation: Analysis of expression data (GSE65107) from bronchoalveolar lavage (BAL) fluid of the lungs of 7 patients who developed lung allograft rejection showed an increase in the expression of KLRB1 (a ratio of 3.6-fold) compared to 27 patients with lung transplants who did not experience rejection (Figure 17). Thus, lung allograft rejection is a particularly attractive target for the treatment according to the present disclosure.

[0204] Atopic dermatitis: Analysis of expression data (GSE65107) from 5 patients with atopic dermatitis showed an increase in the expression of KLRB1 (a ratio of 1.9-fold) compared to 5 healthy individuals (Figure 18). Thus, atopic dermatitis is a particularly attractive target for the treatment according to the present disclosure.

[0205] Palmoplantar pustulosis: Analysis of expression data (GSE185856) from skin biopsies of 3 patients with palmoplantar pustulosis lesions and 8 patients with non-lesional palmoplantar pustulosis skin showed an increase in the expression of KLRB1 compared to 7 healthy individuals (lesional vs. healthy ratio of 11.0-fold; non-lesional vs. healthy ratio of 5.9-fold) (Figure 19). Thus, palmoplantar pustulosis is a particularly attractive target for the treatment according to the present disclosure.

[0206] Hidradenitis suppurativa: Analysis of expression data (GSE148027) from skin biopsies of 18 patients with hidradenitis suppurativa lesions and 7 patients with non-lesional hidradenitis suppurativa skin showed an increase in the expression of KLRB1 compared to 8 healthy individuals (lesional vs. healthy ratio of 8.0-fold, non-lesional vs. healthy ratio of 3.0-fold) (Figure 20). Thus, hidradenitis suppurativa is a particularly attractive target for the treatment according to the present disclosure.

[0207] 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 showed an increase in the expression of KLRB1 compared to 47 healthy individuals (severe asthma vs. healthy individuals ratio of 1.9-fold; moderate asthma vs. healthy individuals ratio of 1.5-fold; and mild asthma vs. healthy individuals ratio of 1.3-fold) (Figure 21). Thus, atopic dermatitis is a particularly attractive target for the treatment according to the present disclosure.

[0208] Example 3: KLRB1 expression is enhanced in various T and NK cell lymphomas and leukemias Analysis of expression data (e.g., GSE19067) from tumor cells derived from patients with various T and NK cell lymphomas and leukemias compared to normal NK cell expression (e.g., shows an increase in the expression of KLRB1 (e.g., hepatosplenic T cell lymphoma has a 17-fold ratio compared to a normal NK cell line) (Figure 22). Thus, various T cell and NK cell lymphomas are particularly attractive targets for treatment according to the present disclosure.

[0209] In particular, in patients with 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), there may be an increase in the expression of KLRB1 or a similar expression of KLRB1 with respect to CD4+ T cells (Figure 22). Therefore, 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) are particularly attractive targets for treatment according to the present disclosure.

[0210] Example 4: Monoclonal anti-KLRB1 antibody 10A3D6 binds to human KLRB1 Monoclonal antibodies were generated against human KLRB1 by immunizing mice (5balb / c and 5SJL) with the extracellular domain (ECD) of human KLRB1 protein (Uniprot ID Q12918, amino acids 67 - 225) to create mouse hybridomas. Subsequently, parental hybridoma clones were successively screened by ELISA for binding to human KLRB1 ECD and by FACS for binding to stable CHO - K1 cell lines that express human KLRB1 (SEQ ID NO: 1) and cynomolgus KLRB1 (SEQ ID NO: 2) separately. Based on binding to both human and cynomolgus CHO - K1 expressed KLRB1 cells, 20 hybridomas were selected for subcloning and 91 subclones were obtained. FACS binding studies against CHO - K1 human - KLRB1 and CHO - K1 cynomolgus KLRB1 were repeated for these 91 subclones. One of these mAbs, named 10A3D6, was tested for binding ability in an ELISA assay. Human KLRB1 ECD was coated on an ELISA plate and the plate was incubated with the 10A3D6 antibody. A secondary goat anti - mouse peroxidase - labeled IgG was used to detect the 10A3D6 antibody bound to the ELISA plate (see Figure 23).

[0211] Example 5: Monoclonal anti - KLRB1 chimeric and humanized antibodies bind to human KLRB1 and cynomolgus KLRB1 The chimeric antibody 10A3D6 - hIgG1k (a mouse / human chimeric antibody having the 10A3D6 variable region and the human IgG1 - kappa constant region), chimeric mutant variants (10A3D6C109S, 10A3D6C109F, and 10A3D6C109V), and a humanized variant of 10A3D6 created by CDR grafting (Almagro et al. 2008) were assayed for binding to the extracellular domain (ECD) of human KLRB1 via surface plasmon resonance (SPR). The results are shown in Table 54.

[0212] In another assay, the humanized antibodies 10A3D6hum2.2 and 10A3D6hum2.2-afuc (which produces defucosylated 10A3D6hum2.2) were assayed for binding to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) and CHO-K1 cells expressing cynomolgus KLRB1 (CHO-cyno-KLRB1) by FACS in a concentration range up to 90 nM. Secondary goat anti-mouse peroxidase-labeled IgG was used to detect the antibodies bound to CHO-hum-KLRB1 or CHO-cyno-KLRB1. 10A3D6hum2.2 and 10A3D6hum2.2-afuc had similar binding EC50s (see Figure 24).

Table 54

[0213] Example 6: Anti-KLRB1 antibodies can induce antibody-dependent cell-mediated cytotoxicity To determine whether the 10A3D6 antibodies are effective in killing KLRB1-positive cells, their ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) was tested using a reporter assay (Genscript ADCC assay using the reporter cell line GS-J2 expressing CD16A). CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) were incubated with the luminescent reporter cells GS-J2 / CD16A and various antibodies. Rituxan (positive control) and the chimeric antibody 10A3D6-hIgG1k (a mouse / human chimeric antibody of the 10A3D6 variable region and the human IgG1-kappa constant region) showed activation of the luminescent reporter cells, in contrast to the negative control IgG1 (Figure 25A). The antibody 10A3D6-hIgG1k activated the luminescent reporter cells, in contrast to the negative control antibody 52G9B12B5-hIgG1kEN (a mouse / human chimeric KLRB1-binding antibody with the 52G9B12B5 variable region and the human IgG1-kappa mutant L234A / L235A / G237A mutation) (Figure 25B).

[0214] In another assay, the ADCC induced by the anti-KLRB1 antibodies of the present disclosure was evaluated using a cell lysis assay that included detection of LDH release and formazan salt (Genscript, SC1544). CHO-hum-KLRB1 was incubated with human peripheral blood mononuclear cells (PBMCs) and various antibodies. Antibody 10A3D6-hIgG1k resulted in target cell lysis, in contrast to the negative control antibody 52G9B12B5 that has an effector null mutation (Figures 26A - B). This confirmed that the ADCC activity of the anti-KLRB1 antibody is mediated through the Fc region of the antibody.

[0215] In another assay, a defucosylated form of 10A3D6hum2.2 (10A3D6hum2.2-afuc) was generated using FUT8 knockout cells (SatOH et al. 2006). The ADCC induced by 10A3D6hum2.2 and by defucosylated 10A3D6hum2.2 was assayed by incubating CHO-K1 cells expressing KLRB1 (CHO-hum-KLRB1) and human NK cells with the antibody, and target cell death was detected using flow cytometry. 10A3D6hum2.2-afuc showed improved killing of target cells compared to 10A3D6hum2.2 (EC50 0.052) (EC50 was 0.010 nM) (Figure 27).

[0216] Example 6: Humanized monoclonal anti-KLRB1 antibody binds to specific immune cells in whole blood To determine whether the anti-KLRB1 antibodies of the present disclosure can effectively bind to immune cells, a FACS binding assay was performed using blood from 3 donors (experiment conducted at iQ Biosciences). Fresh human whole blood was incubated with a fixable viability dye, treated with TruStain Fc Block (BioLegend) in FACS buffer, and then incubated with 10 μg / ml of monoclonal humanized antibodies 10A3D6hum1.2, 10A3D6hum1.5, and 10A3D6hum2.2, as well as a human IgG1 control. All three monoclonal humanized anti-KLRB1 antibodies were found to bind at significantly higher levels to CD3+ cells (Figure 28A), CD4+ T cells (Figure 28B), CD8+ T cells (Figure 28C), and CD3-CD56+ NK cells (Figure 28D) than the control antibody. Overall, there was no increase in binding of the test antibodies to CD14+ monocytes (Figure 28E), CD14+ neutrophils (Figure 28F), or CD19+ B cells (Figure 28G). These results demonstrate that the humanized monoclonal anti-KLRB1 antibodies of the present disclosure bind to specific immune cells in whole blood, particularly CD8+ and CD4+ T cells and NK cells.

[0217] Example 7: The anti-KLRB1 antibody has improved binding in a competition experiment with a commercially available anti-KLRB1 antibody To determine whether the anti-KLRB1 antibodies of the present disclosure have improved binding compared to commercially available anti-KLRB1 antibodies, a competition assay was performed. CHO-K1 cells expressing human KLRB1 (Figure 29A) and CHO-K1 cells expressing cynomolgus KLRB1 (Figure 29B) were incubated with the fluorescently labeled antibody 10A3D6hum1 and then with increasing concentrations of the commercially available antibodies HP-3G10, DX12, and 191B8. The results show that 10A3D6 binds more tightly to a specific epitope than any other antibody that binds to that specific epitope.

[0218] Example 8: Biophysical properties of the anti-KLRB1 antibody To determine the properties of the anti-KLRB1 antibodies of the present disclosure, differential scanning calorimetry (DSC) was performed. DSC measures Tonset (the onset temperature at which melting / unfolding is first detected) and Tm (the melting temperature).

[0219] The DSC test results of the monoclonal humanized anti-KLRB1 antibodies 10A3D6hum1.2, 10A3D6hum1.5, and 10A3D6hum2.2 are shown in Table 55 below and in Figures 30A - C. [Table 55] [Table 56 - 1] [Table 56 - 2] [Table 56 - 3] [Table 56 - 4] [Table 56 - 5] [Table 57 - 1] [Table 57 - 2] [Table 57 - 3] [Table 57 - 4] [Table 57 - 5] [Table 57 - 6] [Table 57 - 7]

Table 57-8

Table 57-9

Table 57-10

Table 57-11

Table 57-12

Table 57-13

Table 58

[0220] Depletion of KLRB1+ T cells in cynomolgus monkeys by 3 mg / kg and 10 mg / kg of Example 9.10A3D6hum2.2-afuc (defucosylated 10A3D6hum2.2). Antibody 10A3D6hum2.2-afuc was intravenously administered to two cynomolgus monkeys at a single dose of 3 mg / kg to one monkey and 10 mg / kg to the other monkey. Blood was analyzed by FACS to detect changes in immune cell populations, and anti-KLRB1 (anti-CD161) antibody 57E2E4 was used to detect KLRB1+ cells. These studies showed that the KLRB1+ CD8+ T cell population was almost completely depleted on days 1, 3, and 7 (Figure 31).

[0221] Depletion of KLRB1+ T cells in cynomolgus monkeys by 0.0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg of 10A3D6hum2.2-afuc (defucosylated 10A3D6hum2.2). Antibody 10A3D6hum2.2-afuc was intravenously administered to four cynomolgus monkeys at a single dose of 0.1 mg / kg to one monkey, 0.3 mg / kg to one monkey, 1 mg / kg to one monkey, and 3 mg / kg to one monkey. Blood was analyzed by FACS to detect changes in immune cell populations, and anti-KLRB1 antibody 57E2E4 was used to detect KLRB1+ cells. These studies showed that the KLRB1+CD8+ T cell population was almost completely depleted at 8 hours, day 1, day 3, day 7, and day 35 after administration (Figures 32A and 32B). Gene expression of KLRB1 transcripts by quantitative PCR (qPCR) also showed depletion of KLRB1 transcripts (Figure 32C).

[0222] Example 11. Pharmacokinetic parameters of 10A3D6hum2.2-afuc in cynomolgus monkeys. Antibody 10A3D6hum2.2-afuc was intravenously administered to four cynomolgus monkeys at a single dose of 0.1 mg / kg to one monkey, 0.3 mg / kg to one monkey, 1 mg / kg to one monkey, and 3 mg / kg to one monkey. The serum concentration of 10A3D6hum2.2-afuc was measured, showing acceptable pharmacokinetic parameters (Figure 33 and Table 59). [Table 59]

[0223] Example 12. Suppression of the Th17 pathway by 10A3D6hum2.2-afuc (defucosylated 10A3D6hum2.2) in cynomolgus monkeys. Antibody 10A3D6hum2.2-afuc was intravenously administered to four cynomolgus monkeys at a single dose of 0.1 mg / kg to one monkey, 0.3 mg / kg to one monkey, 1 mg / kg to one monkey, and 3 mg / kg to one monkey. Blood was analyzed by RNAseq to detect changes in Th17 pathway transcripts. These studies showed significant suppression of the important Th17 transcription factor RORC as well as the major Th17 cell markers IL-23R and CCR6 receptor (Figures 34 and 35).

[0224] Example 13. Suppression of the peTh2 pathway by 10A3D6hum2.2-afuc (defucosylated 10A3D6hum2.2) in cynomolgus monkeys. The antibody 10A3D6hum2.2-afuc was intravenously administered to four cynomolgus monkeys at single doses of 0.1 mg / kg to one monkey, 0.3 mg / kg to one monkey, 1 mg / kg to one monkey, and 3 mg / kg to one monkey. Blood was analyzed by RNAseq to detect changes in pathogenic effector Th2 (peTh2) pathway transcripts. These studies showed significant suppression of the major peTh2 transcription factors GATA3 and RORA, as well as the major peTh2 cell marker CRTH2 (Figures 36 and 37).

[0225] Example 14. Suppression of the Th1 pathway by 10A3D6hum2.2-afuc (defucosylated 10A3D6hum2.2) in cynomolgus monkeys. The antibody 10A3D6hum2.2-afuc was intravenously administered to four cynomolgus monkeys at single doses of 0.1 mg / kg to one monkey, 0.3 mg / kg to one monkey, 1 mg / kg to one monkey, and 3 mg / kg to one monkey. Blood was analyzed by RNAseq to detect changes in Th1 and CD8 effector pathway transcripts. These studies showed significant suppression of the major Th1 and CD8 effector transcription factors Tbet (TBX21) and EOMES (Figures 38 and 39).

[0226] Example 15. KLRB1 ECD binding The antibodies and appropriate controls disclosed herein are assayed by ELISA for binding to the extracellular domain (ECD) of human and cynomolgus macaque KLRB1 protein. ELISA plates are coated overnight at 4°C with KLRB1 ECD at 1 μg / ml, followed by addition of the primary antibody (starting concentration 3 nM and three-fold dilutions thereof for human; starting concentration 10 nM and five-fold dilutions for cynomolgus macaque) at 37°C for 1 hour, and then a labeled secondary anti-human IgG antibody at 1:5000 at 37°C for 45 minutes. The amount of antibody bound to KLRB1 is determined by the intensity of the signal of the bound secondary antibody.

[0227] The antibodies and appropriate controls (e.g., antibodies known in the art or described herein, such as B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent Application Publication No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7)) disclosed herein are assayed for binding to the extracellular domain (ECD) of human or cynomolgus macaque KLRB1 by a similar method such as surface plasmon resonance (SPR) or biolayer interferometry (BLI). SPR is performed using a Biacore, in which binding of a ligand to its receptor is detected by a change in the refractive index of the sensor surface. BLI is performed using an Octet, in which binding of a ligand to its receptor is detected by a change in the optical thickness of the biolayer.

[0228] The antibodies of the present disclosure may have increased binding potency to cell-expressed KLRB1 or the extracellular domain of soluble KLRB1 and a low binding EC50 as compared to the control.

[0229] Example 16. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Activation The antibodies disclosed in this specification and appropriate controls (e.g., antibodies known in the art or described in this specification, such as B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7)) are assayed for antibody-dependent cell-mediated cytotoxicity (ADCC) using a reporter assay (Genscript ADCC assay using the reporter cell line GS-J2 expressing CD16A). CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) are incubated with the luminescent reporter cell GS-J2 / CD16A and various antibodies. In another assay, ADCC induced by the antibody is evaluated using a cell lysis assay involving detection of LDH release and formazan salt (Genscript, SC1544). CHO-hum-KLRB1 is incubated with human peripheral blood mononuclear cells (PBMC) and the antibody. These antibodies show an increase in ADCC-mediated depletion efficacy, a decrease in the EC50 for depletion of CHO-KLRB1+ cells, or a decrease in the EC50 for the reporter cell line ADCC assay, compared to the control.

[0230] Example 17. Further Biochemical and Biophysical Characterization of Antibodies A competition assay of the antibodies disclosed in this specification and appropriate controls (e.g., antibodies known in the art or described in this specification, such as B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), or the antibodies described in U.S. Patent No. 20210122826 (i.e., KW1, KW1.2.1, KW1.3.12, KW7, KW7.2.2, KW7.3.7, KW9, KW9.3.3, KW17, KW17.3.4, KM12, KM12.2.3, KM12.3.2, or KM12.4.7)) with biotin-labeled CLEC2D was performed by FACS. CHO-hum-KLRB1 or CHO-cyno-KLRB1 cells were incubated with biotin-labeled CLEC2D, and the fluorescence signal was detected by FACS. CLEC2D was blocked over a concentration range (e.g., 0 - 200 nM), and the IC50 potency of antibody inhibition or enhancement was measured using the ability of the antibody to cause loss or increase of the fluorescence signal.

[0231] The kinetics of the antibody binding affinity has increased (lower K D or lower Koff ), and the blocking efficacy has increased because the IC50 for inhibition of CLEC2D binding, e.g., binding to cells expressing KLRB1, is lower, and the enhancement efficacy has increased when compared to the control because the EC50 for enhancement (increase) of CLEC2D binding, e.g., binding to KLRB1-expressing cells, is low.

[0232] Differential scanning calorimetry (DSC) was performed, and the antibody T onset (the onset temperature at which melting / unfolding is first detected) and Tm Measure the (melting temperature). These antibodies may have improved biophysical parameters such as an improved melting temperature, improved freeze-thaw stability, reduced isomerization, reduced or non-amidation of deamidation, and / or low sensitivity to oxidation when compared to controls.

[0233] 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.

[0234] 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

[Claim 1] The invention described in the specification.