KLRB1 binding agents and methods of use thereof

Abstract

KLRB1 binding agents, particularly anti-KLRB1 antibodies and antigen binding portions thereof, and compositions thereof, and methods of using such agents, such as depleted or suppressor or activated cells, particularly Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, peTh2, ILC2, ILC3, NK cells and / or neoplastic T cells or NK cells in vivo, for the treatment of autoimmune diseases, allergic diseases, graft rejection, hematological malignancies, and cancers.

Description

[0001] Priority claim

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 419,218, filed on October 25, 2022, and U.S. Provisional Application Serial No. 63 / 343,434, filed on May 18, 2022. The entire contents of the foregoing are incorporated herein by reference.

[0003] Statement Regarding Federally Sponsored Research

[0004] This invention was made with government support under Grant HL119145 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present disclosure relates generally to KLRB1 binding agents, particularly anti-KLRB1 antibodies, and therapeutic methods using the agents for autoimmune diseases, allergic diseases, transplant rejection, hematological malignancies, and cancer. Background Art

[0006] Expression of killer cell lectin-like receptor subfamily B member 1 (KLRB1; also known as CD161) defines a unique immune cell population associated with a variety of 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 have been implicated in psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (Yang et al., 2014), and other autoimmune diseases. Monoclonal antibody therapeutics targeting IL-17 or the upstream cytokine IL-23 that stimulates IL-17-producing cells have been approved by the FDA and marketed for the treatment of psoriasis (e.g., secukinumab and ixekizumab) and ankylosing spondylitis (e.g., secukinumab). However, the development of therapeutics directly targeting these molecules remains limited, and new therapeutics that reduce the activity of these cytokines remain needed for a variety of indications, including autoimmune diseases. Summary of the invention

[0007] The present invention provides a solution to the above problems by providing antibodies and antigen-binding fragments thereof that bind to cells expressing KLRB1 on the cell surface and target those bound cells for depletion. Those targeted cells directly produce or cause other cells to produce the above cytokines. By depleting those cells, the production of cytokines is reduced, resulting in therapeutic benefit.

[0008] KLRB1 binding antibodies (and antigen-binding fragments thereof) having a variety of uses, including therapeutic and diagnostic uses, are described herein. For example, the antibodies and antigen-binding fragments thereof can be used to treat, and in some cases prevent, various diseases associated with KLRB1 expressing cells (i.e., by depleting KLRB1 expressing cells), such as for treating, and in some cases preventing (i.e., reducing the risk of developing) the following diseases: autoimmune diseases, including psoriasis, psoriatic arthritis, ankylosing spondylitis, palmoplantar pustulosis, hidradenitis suppurativa, and inflammatory bowel disease; allergic diseases, including asthma and atopic dermatitis; transplant rejection; hematological malignancies and cancers. Antigen-binding fragments (also referred to herein as antibody fragments) include those described herein, such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies (e.g., scFv).

[0009] Provided herein is an antibody or antigen-binding portion thereof that specifically binds to KLRB1 (KLRB1; optionally SEQ ID NO: 1 or SEQ ID NO: 2), wherein the antibody or antigen-binding portion thereof comprises at least one of the following or consists of at least one of the following: (a) a heavy chain variable region (VH), the heavy chain variable region (VH) comprising a VH complementarity determining region (CDR) 1, the VH complementarity determining region (CDR) 1 comprising a sequence at least 95% identical to a VH CDR1 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31 or 32; a VH CDR2 comprising a sequence at least 95% identical to a VH CDR2 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and a VH CDR3, the VH CDR3 comprises a sequence at least 95% identical to a VH CDR3 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and (b) a light chain variable region (VL) comprising a VLCDR1, the VL CDR1 comprising a sequence at least 95% identical to a VL CDR1 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; a VL CDR2, the VL CDR2 comprising a sequence at least 95% identical to a VL CDR2 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and a VL CDR3, the VL CDR3 comprising a sequence at least 95% identical to a VL CDR3 amino acid sequence shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53.

[0010] In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 1, 30, 31, 32, or 45 to 53. In some embodiments, VL comprises or consists of a VL amino acid sequence shown in Table 1, 30, 31, 32, or 45 to 53. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 1, and VL comprises or consists of a VH amino acid sequence shown in Table 1. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 30, and VL comprises or consists of a VH amino acid sequence shown in Table 30. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 31, and VL comprises or consists of a VH amino acid sequence shown in Table 31. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 32, and VL comprises or consists of a VH amino acid sequence shown in Table 32. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 45, and VL comprises or consists of a VH amino acid sequence shown in Table 45. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 46, and VL comprises or consists of a VH amino acid sequence shown in Table 46. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 47, and VL comprises or consists of a VH amino acid sequence shown in Table 47. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 48, and VL comprises or consists of a VH amino acid sequence shown in Table 48. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 49, and VL comprises or consists of a VH amino acid sequence shown in Table 49.In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 50, and VL comprises or consists of a VH amino acid sequence shown in Table 50. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 51, and VL comprises or consists of a VH amino acid sequence shown in Table 51. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 52, and VL comprises or consists of a VH amino acid sequence shown in Table 52. In some embodiments, VH comprises or consists of a VH amino acid sequence shown in Table 53, and VL comprises or consists of a VH amino acid sequence shown in Table 53. In some embodiments, VH comprises an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence shown in Table 1, or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence shown in Table 1, and VL comprises an amino acid sequence having at least 95% sequence identity to the VL amino acid sequence shown in Table 1, 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, VH comprises or consists of an amino acid sequence having at least 95% sequence identity to a VH amino acid sequence shown in one of Tables 30, 31, 32, or 45 to 53, and VL comprises or consists of an amino acid sequence having at least 95% sequence identity to an amino acid sequence shown in one of Tables 30, 31, 32, or 45 to 53, preferably wherein VH and VL are from the same table.

[0011] In some embodiments, the antibody or antigen binding portion thereof comprises a constant region heavy chain and a light chain, wherein the constant region heavy chain and / or light chain comprises or consists of an amino acid sequence shown in one of Table 58.

[0012] In addition, the present invention provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or the antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or the antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0013] Also provided herein is an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0014] In addition, the present invention provides an antibody or an 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), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0015] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0016] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0017] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0018] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0019] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: a variable region heavy chain consisting of the HC amino acid sequence shown in Table 48 and a variable region light chain consisting of the LC amino acid sequence shown in Table 48, and optionally a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0020] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0021] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0022] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0023] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or the antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or the antigen-binding portion thereof comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0024] Further, the present invention also provides an antibody or an antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; for example, SEQ ID NO: 1 or 2), the antibody or its antigen-binding portion comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

[0025] In some embodiments, the constant region comprises or consists of a sequence as shown in Table 58.

[0026] In some embodiments, the antibody comprises or consists of a heavy chain variable sequence and / or a light chain variable sequence that is at least 95% identical to the sequence shown in Table 56.

[0027] In some embodiments, the antibody comprises or consists of a complete heavy chain sequence and / or light chain sequence that is at least 95% identical to the sequence shown in Table 57.

[0028] Also provided herein are antibodies or antigen binding portions thereof that specifically bind to human KLRB1, the antibodies or antigen binding portions thereof comprising CDRs from the various tables herein, or heavy / light chain pairs from the various tables herein. Preferably, the antibodies or antigen binding portions thereof bind to KLRB1 expressing cells and target them for depletion, for example, by the presence of an Fc domain for ADCC or CDC-dependent depletion or conjugation to a cytotoxic agent.

[0029] In some embodiments, the antibody, or antigen binding portion thereof, comprises a heavy chain constant region hinge region and an Fc domain.

[0030] In some embodiments, the antibody, or antigen binding portion thereof, is an antibody comprising a heavy chain constant region comprising an amino acid sequence having at least 80%, 90%, 95% or 97% sequence identity to the heavy chain constant region amino acid sequence shown in Table 58.

[0031] In some embodiments, the antibody, or antigen-binding portion thereof, is a monoclonal antibody.

[0032] In some embodiments, the antibody, or antigen-binding portion thereof, is a chimeric, humanized, or human antibody and / or comprises one or more mutations (e.g., in a CDR) that remove an Asn(N)-glycosylation site or remove Cys, Asp, Met, Trp, or Lys.

[0033] In some embodiments, the antibody, or antigen-binding portion thereof, is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody, or IgG4 antibody.

[0034] In some embodiments, the antibody or antigen-binding portion thereof is an antibody comprising an Fc region (preferably human IgG1), which binds to Fcγ receptors (FcγRs) and induces antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or binds to C1q and induces complement-dependent cytotoxicity (CDC).

[0035] In some embodiments, the antibody or antigen-binding portion thereof is an antigen-binding fragment (also referred to herein as an antibody fragment), including those described herein, such as Fab, Fab', F(ab')2, Fv, and single-chain antibodies (eg, scFv).

[0036] In some embodiments, the antibody, or antigen binding portion thereof, is attached to a second molecule (e.g., 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 cells expressing KLRB1 or promote depletion of cells expressing KLRB1.

[0037] In some embodiments, the antibody, or antigen binding portion thereof, comprises an afucosylated Fc region.

[0038] Also provided herein are polynucleotides comprising a nucleic acid sequence encoding an antibody or antigen binding portion thereof as described herein. In some embodiments, the nucleic acid sequence is operably linked to a promoter.

[0039] Additionally provided are vectors comprising the polynucleotides described herein, and host cells comprising the polynucleotides or the vectors and optionally expressing antibodies or antigen binding portions thereof as described herein. Also provided herein are methods for preparing antibodies or antigen binding portions thereof as described herein. The method may include culturing host cells 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.

[0040] Further, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding portion thereof as described herein (e.g., comprising the antibody or antigen-binding portion thereof as an active agent or consisting of the antibody or antigen-binding portion thereof as an active agent) and a pharmaceutically acceptable carrier or diluent.

[0041] 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 US20210122826 (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).

[0042] Also provided herein is a method for treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, 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 expressing the antibody or an antigen-binding portion thereof as described herein. Also provided is an antibody that binds to KLRB1, preferably an antibody or an antigen-binding portion thereof or a pharmaceutical composition as described herein, for use in a method for treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, and a hematological malignancy in a subject in need thereof. Also provided is a polynucleotide, a vector, or a host cell expressing the antibody or an antigen-binding portion thereof, for example, for use in cell or gene therapy. In some embodiments, the autoimmune disease is autoimmune hepatitis, rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or suppurative hidradenitis. In some embodiments, the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders or high eosinophilic syndrome. In some embodiments, transplant rejection can be rejection of kidney, lung, heart, liver, limb, skin or multi-organ transplants. In some embodiments, the hematological malignancy is a leukemia, such as T cell leukemia, NK cell leukemia, T cell prolymphocytic leukemia (T-PLL) or large granular lymphocytic leukemia (LGLL). In some embodiments, the hematological malignancy is a lymphoma, such as hepatosplenic T cell lymphoma, NK / T cell lymphoma, mycosis fungoides, Sezary syndrome (Sezary syndrome), peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL) or peripheral T cell lymphoma not otherwise specified (PTCL-NOS). In some embodiments, the hematological malignancy expresses KLRB1.

[0043] In some embodiments, the antibody, or antigen binding portion thereof, binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), innate lymphoid cells type 2 and type 3 (ILC2 and ILC3), pathogenic effector Th2 (peTh2) cells, and / or NK cells expressing KLRB1. In some embodiments, the antibody, or antigen binding portion thereof, is conjugated to a cytotoxic agent or moiety.

[0044] In some embodiments, the antibody or antigen-binding portion thereof used in the treatment methods 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). Diagnostics) or the antibodies described in US20210122826 (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).

[0045] In some examples of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the subject is a human.

[0046] Without being bound by theory, the inventors believe that the antibodies disclosed herein will exhibit advantages over previously described antibodies (e.g., commercially available antibodies) in one or more of the following activities:

[0047] - Increased binding potency (binding EC to cell-expressed KLRB1 or soluble KLRB1 extracellular domain 50 lower);

[0048] - Increased potency of ADCC-mediated depletion (lower EC50 for depletion in CHO-KLRB1+ cells or EC50 for ADCC assay in reporter cell lines) 50 lower);

[0049] - Increased binding affinity kinetics (lower KD or K off lower);

[0050] - Increased blocking potency (IC for inhibition of CLEC2D binding to cell-expressed KLRB1) 50 lower); and / or

[0051] - Potentiated (enhanced) the binding of CLEC2D to KLRB1 expressed in cells 50 lower).

[0052] 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, lower isomerization, reduced or absent deamidation, and / or lower oxidative sensitivity) compared to previously described antibodies (e.g., commercially available antibodies).

[0053] When aspects or embodiments of the present disclosure are described in terms of Markush groups or other alternative groupings, the present disclosure encompasses not only the entire group listed as a whole, but also each individual member of the group and all possible subgroups of the main group, as well as the main group lacking one or more of the group members. The present disclosure also contemplates the explicit exclusion of one or more of any group members from the claimed disclosure.

[0054] These and other advantages of the present technology will be apparent with reference to the drawings and description that follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 KLRB1 expression was shown to be a marker for Th17, Th17.1, ex-Th17, Tc17, iNKT, IL2, ILC3, peTh2, and NK cell subsets.

[0056] Figure 2 Whole body KLRB1 expression profiling data are shown, which indicate that KLRB1 is not significantly expressed on any cell type except immune cells.

[0057] Figure 3 showed that KLRB1 was overexpressed in rheumatoid arthritis synovium compared with osteoarthritis and normal synovium.

[0058] Figure 4 showed that KLRB1 was overexpressed in rheumatoid arthritis synovium compared with osteoarthritis and crystal-induced arthritis synovium.

[0059] Figure 5 showed that KLRB1 was overexpressed in salivary glands of advanced and moderate Sjögren's syndrome compared with normal controls.

[0060] Figure 6 showed that KLRB1 was overexpressed in the parotid glands of Sjögren's syndrome compared with normal ones.

[0061] Figure 7 showed that KLRB1 is overexpressed in muscle tissue from patients with inclusion body myositis.

[0062] Figure 8showed that KLRB1 is overexpressed in skin tissue from patients with discoid lupus.

[0063] Fig. 9 showed that KLRB1 is overexpressed in skin tissue from patients with psoriasis.

[0064] Fig.10 showed that KLRB1 is overexpressed in lung tissue from patients with idiopathic pulmonary fibrosis.

[0065] Fig.11 showed that KLRB1 is overexpressed in pancreatic tissue from patients with diabetes.

[0066] Fig.12 KLRB1 was shown to be overexpressed in scalp tissue from patients with alopecia universalis.

[0067] Fig.13 showed that KLRB1 is overexpressed in liver tissue from patients with primary biliary cholangitis.

[0068] Fig.14 showed that KLRB1 is overexpressed in brain tissue from patients with multiple sclerosis.

[0069] Fig.15 showed that KLRB1 is overexpressed in colon tissue from patients with lymphocytic colitis.

[0070] Fig.16 showed that KLRB1 was overexpressed in kidney tissue from patients with kidney transplantation.

[0071] Fig.17 showed that KLRB1 was overexpressed in bronchoalveolar lavage fluid from patients with lung transplants.

[0072] Fig.18 showed that KLRB1 is overexpressed in skin tissue from patients with atopic dermatitis.

[0073] Fig.19 showed that KLRB1 is overexpressed in skin tissue from patients with palmoplantar pustulosis.

[0074] Fig. 20 showed that KLRB1 was overexpressed in skin tissue from patients with hidradenitis suppurativa.

[0075] Fig.21 KLRB1 was shown to be overexpressed in airway brushings from patients with asthma.

[0076] Fig.22A-D shows that KLRB1 is expressed in various T- and NK-cell lymphomas and leukemias. Fig.22A KLRB1 expression was shown to be increased in tumor cells from 4 / 4 patients with hepatosplenic T-cell lymphoma (HSTCL), 7 / 19 patients with NK / T-cell lymphoma (NKTCL), and 2 / 2 patients with mycosis fungoides. Fig. 22B KLRB1 expression was shown in various forms of peripheral T-cell lymphoma (PTCL), including angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL; ALK-positive and ALK-negative), NK / T-cell lymphoma (NKTCL), peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), and T-cell prolymphocytic leukemia (T-PLL). Fig. 22C showed that in four patients, each with HSTCL, KLRB1 expression was increased in spleen tumor cells compared with healthy spleens, and in tumor cells compared with healthy CD4 T cells. Fig.22D showed increased expression of KLRB1 in HSTCL cell lines (DERL-2) and aggressive NK cell leukemia (ANKL) cell lines (KHYG-1 and NKL).

[0077] Fig.23 Binding of murine antibody 10A3D6 to human KLRB1 by ELISA is shown. Human KLRB1 was coated on an ELISA plate and incubated with 10A3D6. 10A3D6 bound to KLRB1 on the ELISA plate was detected using a secondary anti-mouse IgG.

[0078] Fig.24A -B shows the interaction of humanized antibodies 10A3D6hum2.2 and 10A3D6hum2.2-afuc (10A3D6hum2.2 without fucosylation) with CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) ( Fig.24A ) and CHO-K1 cells expressing cynomolgus KLRB1 (CHO-cyno-KLRB1) ( Fig. 24B ). The controls used were human IgG and PBS (negative control). Binding was detected by FACS. Antibodies bound to CHO-hum-KLRB1 or CHO-cyno-KLRB1 were detected using a secondary anti-human IgG.

[0079] Fig.25A-B shows a luminescent antibody-dependent cell-mediated cytotoxicity (ADCC) assay using increasing concentrations of Rituxan (positive control), test antibody, and human IgG1 antibody (negative control), and using target cells + a reporter cell line GS-J2 expressing CD16A (GS-J2 / CD16A; Genscript, Inc.). Fig.25A The cell luminescence of 10A3D6-hIgG1k (a mouse / human chimeric KLRB1 binding antibody having the variable region of 10A3D6 and the constant region of human IgG1-κ) is shown, while Fig.25B Shown is a comparison of 10A3D6-hlgG1k to the negative control antibody 52G9B12B5-hlgG1kEN (a mouse / human chimeric KLRB1 binding antibody having the 52G9B12B5 variable region and the human IgG1-κ mutant L234A / L235A / G237A mutations).

[0080] Fig.26A -B shows an antibody-dependent cell-mediated cytotoxicity (ADCC) assay measuring target cell lysis of CHO-K1 target cells expressing human KLRB1 (CHO-hum-KLRB1) incubated with human peripheral blood mononuclear cells (PBMCs) and the indicated antibodies 10A3D6-hIgG1k (a mouse / human chimeric antibody with 10A3D6 variable regions and human IgG1-κ constant regions) and human IgG1 (negative control) in an ADCC cell lysis assay. The assay utilizes LDH release and formazan detection (Genscript, SC1544). Fig.26A The percentage of target cell lysis at various effector to target (E:T) ratios as indicated for each test antibody and control antibody is shown, while Fig.26B Shown are the percentage of target cell lysis for each antibody tested at the various concentrations indicated.

[0081] Fig. 27 Shown is an antibody-dependent cell-mediated cytotoxicity (ADCC) assay measuring target cell death of CHO-K1 target cells expressing human KLRB1 (CHO-hum-KLRB1) incubated with human natural killer (NK) cells and the indicated antibodies 10A3D6hum2.2, 10A3D6hum2.2-afucosylated, and negative control antibody 52G9B12B5-hIgG1kEN (a mouse / human chimeric KLRB1 binding antibody with 52G9B12B5 variable regions and human IgG1-κ mutant L234A / L235A / G237A mutations. The assay uses flow cytometry to detect dead CHO-hum-KLRB1 cells.

[0082] Fig.28A-G shows the indicated antibodies (10A3D6hum1.2; 10A3D6hum1.5; and 10A3D6hum2.2; and human IgG1 control) and various immune cells (i.e., CD3+ cells ( Fig.28A )、CD4+T cells( Fig.28B ), CD8+T cells ( Fig.28C ), CD3-CD56+ cells ( Fig.28D ), CD14++ monocytes ( Fig.28E )、CD14+ neutrophils ( Fig.28F ) and CD19+ cells ( Figure 28G )) of the binding expressed as mean fluorescence intensity.

[0083] Fig.29A -B shows the competition data of 103AD6hum1.2 antibody and commercially available antibodies against cells expressing KLRB1. CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) ( Fig.29A ) and CHO-K1 cells expressing cynomolgus KLRB1 (CHO-cyno-KLRB1) ( Fig.29B ) were incubated with the fluorescently labeled antibody 10A3D6hum1.2 and then with increasing concentrations of the commercial antibodies HP-3G10, DX12, 191B8 and negative controls mouse IgG and human IgG. Fig.29A The graph in -B shows the mean fluorescence intensity (MFI) (1000s) over the indicated concentration range (nM).

[0084] Fig. 30A -C shows a differential scanning calorimetry (DSC) graph indicating that 10A3D6hum1.2 ( Fig. 30A )、10A3D6hum1.5( Fig. 30B ) and 10A3D6hum2.2( Fig. 30C ) Cp (kcal / mol / K) over the indicated temperature range.

[0085] Fig.31 Depletion of KLRB1+ T cells is shown in cynomolgus monkeys administered 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2) at 3 mg / kg and 10 mg / kg.

[0086] Fig.32A-C shows depletion of KLRB1+ T cells in cynomolgus monkeys by 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg and 3 mg / kg of 10A3D6hum2.2-afuc (fucosylated 10A3D6hum2.2). These studies showed that the KLRB1+CD8+ T cell population was almost completely depleted at 8 hours post-dose, 1, 3, 7 and 35 days ( Fig.32A and Fig.32B ). Gene expression of KLRB1 transcripts by quantitative PCR (qPCR) also showed depletion of KLRB1 transcripts ( Fig.32C ).

[0087] Fig.33 Shown are the pharmacokinetic parameters of 10A3D6hum2.2-afuc in cynomolgus monkeys.

[0088] Fig.34 Shown is the reduction in Th17 pathway gene expression based on RNAseq (heat map representation).

[0089] Fig.35 Shown is the reduction in Th17 pathway gene expression by RNAseq (graphical representation).

[0090] Fig.36 Shown is the reduction in peTh2 pathway gene expression by RNAseq (heat map representation).

[0091] Fig.37 Shown is the reduction in peTh2 pathway gene expression by RNAseq (graphical representation).

[0092] Fig.38 Shown is the reduction in Th1 and CD8 effector pathway gene expression by RNAseq (heat map representation).

[0093] Fig.39 Shown is a reduction in Th1 and CD8 effector pathway gene expression by RNAseq (graphical representation). DETAILED DESCRIPTION

[0094] KLRB1 expression is a marker for a unique set of immune system cells associated with a variety of autoimmune diseases. These include Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, and / or NK cells.

[0095] KLRB1 is also expressed by neoplastic cells in a variety of T-cell and NK-cell malignancies. These include various peripheral T-cell and NK-cell lymphomas and leukemias.

[0096] The KLRB1 binding antibodies described herein can be used to preferentially target Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells and / or neoplastic T cells or NK cells for depletion. The immune cell population expressing KLRB1 expresses IL-17 more abundantly than the total CD4 or CD8 T cell population, and has a higher specificity for Th17 and Tc17 T cells than CD4 or CD8, respectively. Therefore, a method for treating a subject in the following manner is described herein: administering an effective amount of a cell-depleting lectin-like receptor B1 (KLRB1) binding agent having cell depletion activity to a subject in need, thereby depleting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells and / or neoplastic T cells or NK cells in the body. These methods can be used to treat autoimmune diseases, allergic diseases, transplant rejection, or hematological malignancies, for example, as described herein.

[0097] In addition, in some embodiments, since regulating KLRB1 has an inhibitory or stimulatory effect on T cells and NK cells, the present invention describes a method for treating a subject by administering an effective amount of a cell-killing lectin-like receptor B1 (KLRB1) binder having KLRB1 receptor blocking activity (wherein blocking of LLT1 binding to KLRB1 is achieved) to a subject in need, for example, an antibody as described herein having an effector null Fc mutation (e.g., as described in Liu et al., Antibodies (Basel). 2020 Dec; 9(4): 64), thereby activating or inhibiting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2 and / or in vivo. These methods can be used to activate T cells and / or NK cells to enhance their activity against tumor cells, thereby treating cancer.

[0098] In some embodiments, the antibody has antibody-dependent cellular cytotoxicity (ADCC) effector activity or complement-dependent cytotoxicity (CDC) effector activity. Administration of an effective amount of an anti-KLRB1 antibody having ADCC or CDC effector function or connected to a cytotoxic agent to a subject in need can be used 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 present disclosure provides a cell-killing lectin-like receptor B1 (KLRB1) binding agent as described herein that has ADCC or CDC activity or is conjugated to a cytotoxic agent. In various aspects, the present disclosure provides mRNA or cDNA encoding a binding agent. In various aspects, the present disclosure provides a pharmaceutical composition comprising an effective amount of an adhesive.

[0099] Various features of the present disclosure, including KLRB1 and its ligands, anti-KLRB1 antibodies or antigen-binding portions thereof, pharmaceutical compositions, treatments and administrations, and illustrative examples are discussed in turn below. The following sections contain definitions of terms used in the present disclosure. Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. For the purpose of interpreting this specification, the following descriptions of terms will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any description of a term set forth conflicts with any document incorporated by reference herein, the description of the term set forth below shall prevail.

[0100] Killer cell lectin-like receptor B1 (KLRB1)

[0101] 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 a stimulatory receptor (Fergusson et al., 2014) or an inhibitory receptor (Aldemir et al., 2005, Mathewson et al., 2021) for different types of T cells and NK cells. It is present on the surface of a variety of immune cells. The ligand of KLRB1 is LLT1 (also known as CLEC2D) (Aldemir et al., 2005).

[0102] KLRB1 expression in T cells is restricted to cells that have the ability to respond to IL-12 and IL-18 (Fergusson et al., 2014). In CD4+ T helper cells, KLRB1 expression is a unique marker for 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, which are KLRB1 negative, from ex-Th17 IFNG-producing cells, which are KLRB1+ (Basdeo et al., 2017). In CD8+ T cytotoxic cells, KLRB1 expression is a unique marker for Tc17 cells. Therefore, KLRB1 is a marker for T cells that have the ability to produce IL-17, or in the case of ex-Th17 cells, interferon-γ (IFNG). In the context of autoimmune diseases, the production of this cytokine is undesirable.

[0103] Antibodies and antigen-binding fragments thereof that bind to KLRB1 (preferably human KLRB1) are described herein. An exemplary sequence of human KLRB1 is provided as SEQ ID NO: 1:

[0104] MDQQAIYAELNLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIQKSSIEKCSVDIQQSRNKTTERPGLLNCPIYWQQLREKCLLFSHTVN PWNNSLADCSTKESSLLLIRDKDELIHTQNLIRDKAILFWIGLNFSLSEKNWKWINGSFLNSNDLEIRGDAKENSCISISQTSVYSEYCSTEIRWICQKELTPVRNKVYPDS.

[0105] In some embodiments, KLRB1 is cynomolgus monkey KLRB1; an exemplary sequence is provided as SEQ ID NO: 2:

[0106] MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWQQVQEKCLLFSHTVNP WNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0107] Preferably, the antibody binds to cells (e.g., Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T cells or NK cells) that express KLRB1 on their surface and targets such cells for depletion, e.g., by ADCC or CDC (e.g., for full-length antibodies comprising an Fc region) or a cytotoxic agent or portion (e.g., for an antigen-binding fragment lacking an Fc region having ADCC or CDC capability).

[0108] KLRB1 binding antibodies

[0109] Antibodies and antigen-binding fragments thereof that bind to KLRB1 are described herein.The term "antibody" refers to an immunoglobulin molecule or an immunologically active portion thereof, ie, an antigen-binding portion.

[0110] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical, except for possible naturally occurring mutations that may be present in trace amounts. The antibody may be a monoclonal antibody. The antibody may be a human antibody or a humanized antibody. The term "monoclonal antibody" encompasses complete and full-length monoclonal antibodies and antibody fragments (e.g., Fab, Fab', F(ab')2, Fv and single-chain antibodies (e.g., scFv)), fusion proteins comprising antibody fragments, and any other modified immunoglobulin molecules comprising at least one antigen binding site. In addition, "monoclonal antibody" refers to such antibodies made by a variety of techniques including but not limited to hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0111] The term "chimeric antibody" refers to an antibody wherein a portion of the heavy and / or light chains are derived from a first source or species, while the remainder of the heavy and / or light chains are derived from a different source or species.

[0112] The term "humanized antibody" as used herein refers to an antibody comprising a human heavy chain variable region and a light chain variable region, wherein the native CDR residues are replaced by residues of the corresponding CDR from a non-human antibody (e.g., mouse, rat, rabbit, or non-human primate), wherein the non-human antibody has the desired specificity, affinity, and / or activity. In some embodiments, one or more framework region residues of a human heavy chain or light chain variable region are replaced by corresponding residues from a non-human antibody. In addition, a humanized antibody may be included in a residue not seen in a human antibody or in a non-human antibody. In some embodiments, these modifications are in order to further improve and / or optimize antibody properties. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc) of a human immunoglobulin.

[0113] The term "human antibody" as used herein refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or an antibody prepared using any technique known to those skilled in the art for preparing 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.

[0114] "Antibody fragments" or "antigen binding fragments" 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; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen binding fragment is attached to a second molecule, such as a cytotoxic agent or moiety (e.g., a cytotoxic agent or moiety that promotes the 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.

[0115] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the part of an antigen or target that can be recognized and bound by a specific antibody. When the antigen or target is a polypeptide, the epitope can be formed by continuous amino acids and non-continuous amino acids juxtaposed by the tertiary folding of the protein. The epitope formed by continuous amino acids (also referred to as a linear epitope) is usually retained after protein denaturation, while the epitope formed by the tertiary folding (also referred to as a conformational epitope) is usually lost after protein denaturation. The epitope usually comprises at least 3 amino acids in a unique spatial conformation, more usually at least 5, 6, 7 or 8-10 amino acids. Any of a large number of software bioinformatics tools available on the Internet can be used to predict epitopes. X-ray crystallography can be used to characterize epitopes on target proteins by analyzing the interactions of amino acid residues of antigen / antibody complexes.

[0116] "Fv" includes the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight non-covalent association. It is in this configuration of interaction of the three CDRs of each variable domain that the antigen binding site on the surface of the VH-VL dimer is defined. The six CDRs together confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of a complete binding site. The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between the Fab fragment and the Fab' fragment is that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab' in which one or more cysteine ​​residues of the constant domain have a free thiol group is referred to herein as Fab'-SH. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical conjugates of antibody fragments are also known.

[0117] Immunoglobulins can be assigned to different classes based on the amino acid sequence of their heavy chain constant domains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, several of which can be further divided into subclasses (isotypes), such as IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains so that the sFv can form a structure required for antigen binding.

[0118] In various embodiments, the antibody or its antigen-binding fragment comprises a human or humanized antibody. The humanized form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, an immunoglobulin chain, or a fragment thereof (such as Fv, scFv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include such human immunoglobulins (acceptor antibodies), wherein the residues from the complementary determining regions (CDRs) of the acceptor are replaced by residues from the CDRs of non-human species (donor antibodies) such as mice, rats, or rabbits with the desired specificity, affinity, and capacity. In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies can also be included in residues that do not exist in the acceptor antibody or the CDR or framework sequences imported. Typically, the humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein 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.

[0119] The KLRB1 antibodies described herein can be affinity matured, for example, using selection and / or mutagenesis methods known in the art. Typically, an "affinity matured" antibody is an antibody having one or more changes in one or more hypervariable regions that result in an increase in the affinity of the antibody for the antigen compared to a parent antibody that does not have the one or more changes. 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 times, more preferably 10 times, even more preferably 20 or 30 times higher than the starting antibody (usually murine, humanized or human) from which the mature antibody is prepared.

[0120] An antibody that "binds to" a particular polypeptide or an epitope on a particular polypeptide, "binds specifically to" a particular polypeptide or an epitope on a particular polypeptide, or "is specific for a particular polypeptide or an epitope on a particular polypeptide" is an antibody that binds to a particular polypeptide or an epitope on a particular polypeptide and does not substantially bind to any other polypeptide or polypeptide epitope. As used herein, the term "specific binding" refers to the interaction of a KLRB1 agent (e.g., an anti-KLRB1 antibody) with a particular antigen, epitope, protein, or target molecule that is more frequent, more rapid, longer lasting, with greater affinity, or some combination of the above, than with an alternative substance. 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 skilled in the art. Therefore, functional equivalents of the specific anti-KLRB1 antibodies are described herein. In some cases, KLRB1 antibodies may cross-react with various similar KLRB1 proteins (e.g., having the highest affinity for one KLRB1 protein, such as human KLRB1, and a lower affinity for other KLRB1 proteins, such as mouse KLRB1). A binding agent that specifically binds to an antigen has a higher binding affinity for the target antigen than it has for a different antigen. The different antigen may be a related antigen. In some embodiments, a binding agent that specifically binds to an antigen binds to the target antigen with an affinity that is at least 20 times higher than its affinity for a different antigen, for example, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 60 times higher, at least 70 times higher, at least 80 times higher, at least 90 times higher, or at least 100 times higher than its affinity for a different antigen. In some embodiments, a binding agent that specifically binds to a particular antigen binds to a different antigen with such a low affinity that the binding cannot be detected using other assays described herein or known in the art. In some embodiments, affinity is measured using SPR technology, for example, in a Biacore system or other systems known to those of skill in the art.

[0121] The term "identical" or "percent identity" in the context of two or more polypeptides (e.g., two anti-KLRB1 polypeptides) refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues when compared and aligned (introducing gaps if necessary) for maximum correspondence, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.

[0122] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / number of total positions × 100), taking into account the number of spaces and the length of each space that need to be introduced to achieve optimal alignment of the two sequences. Mathematical algorithms can be used to complete the comparison of sequences and the determination of the percent identity between two sequences. Such homology is well embodied in the art by local alignment tools and / or algorithms, and may include pairwise alignments, multiple sequence alignment methods, structural alignment methods, and / or phylogenetic analysis methods. When sequences differ in conservative substitutions, the percent identity of the sequence may (but not necessarily) be adjusted upward to correct for the conservative nature of the substitution. Methods for making such adjustments 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 percent identity of the sequence. Thus, for example, when the same amino acid is given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score between 0 and 1.

[0123] In some embodiments, two polypeptides of the present disclosure (e.g., antibodies or antibody domains thereof (e.g., VL, CL, VH, CH1, CH2, CH3 domains)) are substantially identical, meaning that when compared and aligned for maximum correspondence, they have 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% amino acid residue identity, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, the percent identity exists over a region of at least about 10, at least about 20, at least about 20-40, at least about 40-60 amino acid residues, at least about 60-80 nucleotides or amino acid residues in length, or any integer value therebetween. In some embodiments, the percent identity exists over a region longer than 60-80 amino acid residues (such as at least about 80-100 amino acid residues), and in some embodiments, the sequences are substantially identical over the entire length of the compared sequences (e.g., amino acid sequences).

[0124] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences need to be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences to obtain optimal alignment, and non-homologous sequences can be ignored for comparison purposes). In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the two molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the 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 need to be introduced to achieve optimal alignment of the two sequences.

[0125] Mathematical algorithms can be used to complete the comparison of sequences and the determination of the percent identity between two sequences. 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 in the GCG software package (available on the World Wide Web at gcg.com) using default parameters (e.g., the Blossum 62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4 and a frameshift gap penalty of 5).

[0126] As used herein, the term "conservative sequence modification" or "conservative substitution" may refer to an amino acid modification of a target epitope or an antibody of the present disclosure and an antigen-binding portion thereof that does not significantly affect or alter the binding properties of the anti-KLRB1 antibody. As used herein, "conservative substitution" refers to a substitution in which one amino acid residue is replaced by 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, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar 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, a substitution of tyrosine with phenylalanine is considered a conservative substitution. Methods for identifying conservative substitutions of amino acids that do not eliminate binding are well known in the art.

[0127] In various embodiments, the antibody is a blocker or antagonist binder. "Blocking" or "antagonist" means that the agent (e.g., an antibody or binding fragment thereof) is an agent that inhibits or reduces the biological activity of the antigen to which it binds. Certain blockers or antagonists substantially or completely inhibit the biological activity of the antigen. For example, a KLRB1 binder as described herein with an effector null Fc mutation can block KLRB1 signaling (e.g., thereby disrupting KLRB1 signaling and regulating Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells and / or neoplastic T cells or NK cells).

[0128] In some embodiments, the KLRB1 binding agent is an antibody comprising: a. a full-length antibody that binds to KLRB1 and comprises an Fc domain that can bind to an Fcγ receptor having an effector function for triggering antibody-dependent cell-mediated cytotoxicity (ADCC); b. an antibody that binds to KLRB1 and comprises an Fc domain that can bind to complement protein 1q (C1q) having an effector function for triggering complement-dependent cytotoxicity (CDC); c. an antibody conjugate that binds to KLRB1 and comprises a cytotoxic agent, such as an antibody-drug conjugate (ADC); or d. a multispecific antibody (e.g., a bispecific or trispecific antibody), wherein the antibody binds to KLRB1 and another antigen.

[0129] In some embodiments, the KLRB1 antibodies described herein bind to the extracellular domain of human KLRB1; in some embodiments, the antibody cross-reacts with (binds to) the extracellular domains of human and cynomolgus monkey KLRB1. In some embodiments, the antibody binds to an epitope of the extracellular domain of KLRB1, wherein the epitope is at least 90% identical in humans and cynomolgus monkeys. Preferably, the antibody or antigen-binding fragment thereof comprises or is conjugated to an Fc region, a cytotoxic agent, or a second antigen-binding domain that results in depletion of KLRB1-expressing cells.

[0130] In some embodiments, the antibody binds to KLRB1 and is not a mouse antibody.

[0131] 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 (CellSciences), 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 US20210122826 (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).

[0132] In some embodiments, the KLRB1 binder is an antibody, such as 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, IgD, IgE, IgG, 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.

[0133] 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 bond-linked scFv. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In one embodiment, the antibody is a monovalent antibody. In one embodiment, the antibody is a monospecific antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is separated. 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 animals (e.g., rabbits, rats, mice, goats, donkeys) with an antigen of interest (e.g., purified peptide fragments, recombinant proteins, or fusion proteins) using multiple subcutaneous or intraperitoneal injections. In some embodiments, the antigen is conjugated to a carrier (such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor). The antigen (with or without a carrier protein) is diluted in sterile saline and is usually combined with an adjuvant (e.g., complete or incomplete Freund's adjuvant) to form a stable emulsion. After a period of time, the polyclonal antibodies are recovered from the immunized animal (e.g., from blood or ascites). In some embodiments, 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.

[0134] In some embodiments, the KLRB1 binding agent is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, monoclonal antibodies are prepared using hybridoma methods known to those skilled in the art. For example, mice, rats, rabbits, hamsters or other suitable host animals are immunized as described above using hybridoma methods. 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.

[0135] After immunization, lymphocytes are separated and fused with suitable myeloma cell lines using, for example, polyethylene glycol. Hybridoma cells are selected using specialized culture medium as known in the art, and unfused lymphocytes and myeloma cells cannot survive in the selection process. Hybridomas producing monoclonal antibodies specifically for selected antigens can be identified by a variety of 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 desired specificity, affinity and / or activity are identified, clones can be subcloned by limiting dilution techniques. Hybridomas can be bred in vitro culture using standard methods, or they can be bred as ascites tumors in animals. Monoclonal antibodies can be purified from culture medium or ascites according to the standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis and dialysis.

[0136] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques as known to those skilled in the art. For example, polynucleotides encoding the antibody are isolated from mature B cells or hybridoma cells using RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using standard techniques. Subsequently, the isolated polynucleotides encoding the heavy and light chains are cloned into suitable expression vectors, which produce monoclonal antibodies when being transfected into cells such as Escherichia coli cells, ape COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not generate immunoglobulins in addition.

[0137] In some embodiments, recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of the desired species. Screening of phage libraries can be accomplished by various techniques known in the art.

[0138] In some embodiments, monoclonal antibodies are modified using recombinant DNA technology to produce alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of mouse monoclonal antibodies are replaced with the constant regions of human antibodies to produce chimeric antibodies. In some embodiments, the constant regions are truncated or removed to produce the desired antibody fragments of monoclonal antibodies. In some embodiments, site-directed or high-density mutagenesis of one or more variable regions is used to optimize the specificity and affinity of monoclonal antibodies.

[0139] In some embodiments, the KLRB1 binding agent is a humanized antibody. A variety of methods for producing humanized antibodies are known in the art. In some embodiments, a humanized antibody comprises one or more amino acid residues introduced therein from a non-human source. In some embodiments, humanization is performed by replacing one or more non-human CDR sequences with corresponding CDR sequences of a human antibody. In some embodiments, a humanized antibody is constructed by replacing all six CDRs of a non-human antibody (e.g., a mouse antibody) with corresponding CDRs of a human antibody.

[0140] Can be based on various factors and by various methods known in the art to select which kind of people's heavy chain variable region and / or light chain variable region to generate humanized antibodies.In some embodiments, use " best fit " method, wherein the whole library of known people's variable region sequence is screened for the sequence of the variable region of non-human (for example, rodent) antibody.Select the human sequence most similar to the non-human (for example, rodent) sequence as the human variable region framework of humanized antibody.In some embodiments, select the specific variable region framework of the consensus sequence of all human antibodies derived from specific light chain or heavy chain subgroup as variable region framework.In some embodiments, variable region framework sequence is derived from the consensus sequence of the most abundant people's subclass.In some embodiments, people's germline gene is used as the source of variable region framework sequence.

[0141] Other methods for humanization include, but are not limited to, a method called "superhumanization" which is described as transferring CDRs directly to a human germline framework, a method called human string content (HSC) which is based on the metric of "antibody humanity", methods based on generating large libraries of humanized variants (including phage, ribosome, and yeast display libraries), and methods based on framework region shuffling.

[0142] In some embodiments, the KLRB1 binding agent is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are produced by immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are produced by lymphocytes isolated from immunized individuals. In any case, cells that produce antibodies against target antigens can be generated and isolated. In some embodiments, human antibodies are selected from a phage library, wherein the phage library expresses human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable region gene libraries from unimmunized donors. The generation and use of antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art (including but not limited to chain shuffling and site-directed mutagenesis) can be used to generate higher affinity human antibodies. In some embodiments, human antibodies are produced in transgenic mice containing human immunoglobulin loci. After immunization, these mice are able to produce a complete human antibody library in the absence of endogenous immunoglobulin production.

[0143] In some embodiments, the KLRB1 binding agent is an scFv antibody. ScFv is a molecule comprising a variable heavy chain region and a variable light chain region connected to form a single polypeptide. ScFv can be produced using recombinant techniques known in the art. In some embodiments, scFv comprises a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, scFv comprises (from N-terminal to C-terminal) (i) heavy chain variable region, (ii) linker, and (iii) orientation of the light chain variable region. In some embodiments, scFv comprises (from N-terminal to C-terminal) (i) light chain variable region, (ii) linker, and (iii) orientation of the heavy chain variable region. In some embodiments, scFv is a disulfide-linked scFv (dsscFv), which is an scFv comprising an engineered disulfide bond between the light chain variable region and the heavy chain variable region of the scFv. In some embodiments, the scFv (e.g., dsscFv) is attached (directly or indirectly) to a half-life extending moiety, such as, for example, an Fc molecule, a 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), polysialic acid, N-(2-hydroxypropyl) methacrylamide or dextran, or modified by, for example, hyperglycosylation, to extend the half-life of the scFv (e.g., dsscFv). In some embodiments, the scFv is attached to a second molecule (e.g., a cytotoxic agent or moiety), or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0144] A variety of suitable joints are known to those skilled in the art and are not limited by any specific sequence disclosed herein. In some embodiments, the polypeptide joint is composed of naturally or non-naturally occurring amino acids. In some embodiments, the joint comprises amino acids that allow flexibility. In some embodiments, the joint comprises amino acids that allow suitable solubility. In some embodiments, the joint comprises glycine amino acids. In some embodiments, the joint comprises glycine and serine amino acids. In certain embodiments, the joint comprises one or more glycine / serine repeat sequence sets. In some embodiments, the polypeptide joint is selected from the group consisting of: (GGGGS)n (where n=1-4) (SEQ ID NO: 85), GGGGS (SEQ ID NO: 86), GGGGSGGGGS (SEQ ID NO: 87), GG GGSGGGGSGGGGS (SEQ ID NO: 88), GGGGSGGGGSGGGGSGG GGS (SEQ ID NO: 89) and (GGGGA)n (where n=1-4) (SEQ ID NO: 90). In some embodiments, the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 91).

[0145] In some embodiments, the KLRB1 binding agent is an Fv. The Fv comprises a heavy chain variable region and a light chain variable region. In some embodiments, the Fv is attached (directly or indirectly) to a portion that extends the half-life, such as, for example, an Fc molecule, an IgG CH3 domain (e.g., IgG1 CH3), PEG or PEG mimetics, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl) methacrylamide or dextran, or modified, for example, by hyperglycosylation to extend the half-life of the Fv. In some embodiments, the Fv is attached to a second molecule (e.g., a cytotoxic agent or moiety), or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0146] In some embodiments, the KLRB1 binding agent is Fab. Fab is one of the molecules produced by papain digestion of immunoglobulin antibodies. Fab is a monovalent molecule comprising a light chain, a heavy chain variable region, a CH1 region, and an optional heavy chain constant region hinge region or a portion thereof. Fab can be produced using recombinant techniques known in the art. In some embodiments, Fab comprises a polypeptide linker between a light chain constant region and a heavy chain variable region. In some embodiments, Fab comprises a polypeptide linker between a heavy chain constant region and a light chain variable region. A variety of suitable linkers are known to those skilled in the art and are not limited to any specific sequence disclosed herein. In certain embodiments, the linker is a linker described herein. In some embodiments, Fab is attached (directly or indirectly) to a portion that extends the half-life, such as, for example, an Fc molecule, an IgG CH3 domain (e.g., IgG1 CH3), PEG or PEG mimetics, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl) methacrylamide or dextran, or, for example, modified by hyperglycosylation to extend the half-life of Fab. In some embodiments, the Fab is attached to a second molecule (eg, a cytotoxic agent or moiety), or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0147] In some embodiments, Fab comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region. In some embodiments, Fab comprises a disulfide bond that increases the stability of the Fab molecule. In some embodiments, Fab comprises a disulfide bond that increases the thermal stability of the Fab molecule.

[0148] In some embodiments, the KLRB1 binder is F(ab')2. F(ab')2 is one of the molecules produced by digesting an immunoglobulin antibody with pepsin. F(ab')2 is a bivalent molecule comprising a first light chain associated with a first polypeptide comprising a first heavy chain variable region, a first CH1, and a first hinge region; and a second light chain associated with a second polypeptide comprising a second heavy chain variable region, a second CH1, and a second hinge region, wherein the first hinge region is connected 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 attached (directly or indirectly) to a half-life extending portion, such as, for example, the CH3 domain of IgG (e.g., CH3 of IgG1), PEG or PEG mimetics, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl) methacrylamide, or dextran, or modified, for example, by hyperglycosylation, to extend the half-life of F(ab')2. In some embodiments, the F(ab')2 is attached to a second molecule (eg, a cytotoxic agent or moiety), or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0149] 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.

[0150] In some embodiments, the KLRB1 binder is F(ab'). F(ab') is a molecule produced by treating F(ab')2 with β-mercaptoethanol. F(ab') is a monovalent molecule comprising a light chain associated with a polypeptide comprising a heavy chain variable region, CH1, and a hinge region. In some embodiments, F(ab') is attached (directly or indirectly) to a half-life extending portion, such as, for example, an Fc molecule, a CH3 domain of IgG (e.g., CH3 of IgG1), PEG or PEG mimetics, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl) methacrylamide or dextran, or modified, for example, by hyperglycosylation to extend the half-life of F(ab'). In some embodiments, the F(ab') is attached to a second molecule (e.g., a cytotoxic agent or moiety), or a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection.

[0151] 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.

[0152] In some embodiments, the KLRB1 binding agent is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding at least two different antigens or epitopes. The different epitopes may be located within the same molecule (e.g., two epitopes on KLRB1) or on different molecules (e.g., an epitope on KLRB1 and an epitope on different targets). In some embodiments, bispecific antibodies have enhanced efficacy compared to a single antibody or a combination of more than one antibody. In some embodiments, bispecific antibodies have reduced toxicity compared to a single antibody or a combination of more than one antibody. It is known to those skilled in the art that any therapeutic agent may have a unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, bispecific antibodies have the ability to synchronize the PK of two active binding agents, wherein the two separate binding agents have different PK profiles. In some embodiments, bispecific antibodies have the ability to concentrate the effects of two agents in a common area (e.g., tissue) of a subject (e.g., a human). In some embodiments, bispecific antibodies have the ability to concentrate the effects of two agents on a common target (e.g., a specific cell type). In some embodiments, bispecific antibodies have the ability to target the effects of two agents to more than one biological pathway or function. In some embodiments, bispecific antibodies have the ability to target two different cells and bring them closer together.

[0153] 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 a mixture of two individual antibodies or an antibody as a single agent. In some embodiments, the bispecific antibody has an increased therapeutic index. In some embodiments, the bispecific antibody has an increased therapeutic index compared to a mixture of two individual antibodies or an antibody as a single agent.

[0154] Several technologies for making bispecific antibodies are known to those skilled in the art. In some embodiments, the bispecific antibody comprises such a heavy chain constant region, which has a modification in the amino acid as a part of the interface between the two heavy chains. These modifications are to enhance the formation of heterodimers, and generally reduce or eliminate the formation of homodimers. In some embodiments, the bispecific antibody is generated using a knob-into-hole (KIH) strategy. In some embodiments, the bispecific antibody comprises a variant hinge region (for example, reducing the formation of homodimers) that can not form a disulfide bond between the same heavy chain. In some embodiments, the bispecific antibody is included in an amino acid with a heavy chain that causes a change in electrostatic interaction. In some embodiments, the bispecific antibody is included in an amino acid with a heavy chain that causes a change in hydrophobic / hydrophilic interaction.

[0155] Bispecific antibodies can be complete antibodies or antibody fragments that contain the antigen-binding site.

[0156] KLRB1 binding agents having more than two valencies are also contemplated.In some embodiments, trispecific or tetraspecific antibodies are produced.

[0157] In some embodiments, the KLRB1 binding agent is an anti-KLRB1 antibody comprising one, two, three, four, five and / or six CDRs of any 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, e.g., as shown in Table 1, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6, e.g., as shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6, e.g., as shown in Table 1 (i.e., CDR1, CDR2 and CDR3), and (ii) three light chain CDRs from antibody 10A3D6, e.g., as shown in Table 1. In some embodiments, an anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6C109S, e.g., as shown in Table 2, and / or (ii) one, two, and / or three light chain CDRs from antibody 10A3D6C109S, e.g., as shown in Table 2. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6C109S, e.g., as shown in Table 2 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6C109S, e.g., as shown in Table 2. In some embodiments, an anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6C109F, e.g., as shown in Table 3, and / or (ii) one, two, and / or three light chain CDRs from antibody 10A3D6C109F, e.g., as shown in Table 3. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6C109F, e.g., as shown in Table 3 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6C109F, e.g., as shown in Table 3. In some embodiments, an anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 10A3D6C109V, e.g., as shown in Table 4, and / or (ii) one, two, and / or three light chain CDRs from antibody 10A3D6humC109V, e.g., as shown in Table 4. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6humC109V, e.g., as shown in Table 4 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6humC109V, e.g., as shown in Table 4.

[0158] In some embodiments, the KLRB1 binding agent is a humanized anti-KLRB1 antibody comprising: (i) one, two and / or three heavy chain CDRs from any one of Tables 1-53 or Tables 5-53, and / or (ii) one, two and / or three light chain CDRs. In some embodiments, the KLRB1 binding agent is a humanized anti-KLRB1 antibody comprising: (i) three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3) from any one of Tables 1-53 or 5-53 and (ii) three light chain CDRs, i.e., wherein the heavy chain CDRs and the light chain CDRs are from the same table. In some embodiments, the heavy chain and light chain are from different tables, or the CDRs include CDRs from different tables.

[0159] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum1.2, e.g., as shown in Table 30, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum1.2, e.g., as shown in Table 30. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum1.2, e.g., as shown in Table 30 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum1.2, e.g., as shown in Table 30.

[0160] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum1.5, e.g., as shown in Table 31, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum1.5, e.g., as shown in Table 31. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum1.5, e.g., as shown in Table 31 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum1.5, e.g., as shown in Table 31.

[0161] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum2.2, e.g., as shown in Table 32, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum2.2, e.g., as shown in Table 32. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum2.2, e.g., as shown in Table 32 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum2.2, e.g., as shown in Table 32.

[0162] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.1, e.g., as shown in Table 45, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.1, e.g., as shown in Table 45. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.1, e.g., as shown in Table 45 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.1, e.g., as shown in Table 45.

[0163] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.2, e.g., as shown in Table 46, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.2, e.g., as shown in Table 46. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.2, e.g., as shown in Table 46 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.2, e.g., as shown in Table 46.

[0164] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.3, e.g., as shown in Table 47, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.3, e.g., as shown in Table 47. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.3, e.g., as shown in Table 47 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.3, e.g., as shown in Table 47.

[0165] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.4, e.g., as shown in Table 48, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.4, e.g., as shown in Table 48. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.4, e.g., as shown in Table 48 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.4, e.g., as shown in Table 48.

[0166] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.5, e.g., as shown in Table 49, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.5, e.g., as shown in Table 49. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.5, e.g., as shown in Table 49 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.5, e.g., as shown in Table 49.

[0167] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.6, e.g., as shown in Table 50, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.6, e.g., as shown in Table 50. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.6, e.g., as shown in Table 50 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.6, e.g., as shown in Table 50.

[0168] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.7, e.g., as shown in Table 51, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.7, e.g., as shown in Table 51. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.7, e.g., as shown in Table 51 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.7, e.g., as shown in Table 51.

[0169] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.8, e.g., as shown in Table 52, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.8, e.g., as shown in Table 52. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.8, e.g., as shown in Table 52 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.8, e.g., as shown in Table 52.

[0170] In some embodiments, an anti-KLRB1 antibody comprises (i) one, two and / or three heavy chain CDRs from antibody 10A3D6hum8.9, e.g., as shown in Table 53, and / or (ii) one, two and / or three light chain CDRs from antibody 10A3D6hum8.9, e.g., as shown in Table 53. In some embodiments, an anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 10A3D6hum8.9, e.g., as shown in Table 53 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 10A3D6hum8.9, e.g., as shown in Table 53.

[0171] Table 1: Antibody 10A3D6 sequence

[0172]

[0173] Table 2: Antibody 10A3D6C109S sequence

[0174]

[0175]

[0176] Table 3: Antibody 10A3D6C109F sequence

[0177]

[0178] Table 4: Antibody 10A3D6C109V sequence

[0179]

[0180] Table 5: Antibody 10A3D6C109Chum1.1 sequence

[0181]

[0182]

[0183] Table 6: Antibody 10A3D6C109Chum1.2 sequence

[0184]

[0185] Table 7: Antibody 10A3D6C109Chum1.3 sequence

[0186]

[0187]

[0188] Table 8: Antibody 10A3D6C109Chum1.4 sequence

[0189]

[0190] Table 9: Antibody 10A3D6C109Chum1.5 sequence

[0191]

[0192] Table 10: Antibody 10A3D6C109Chum2.1 sequence

[0193]

[0194]

[0195] Table 11: Antibody 10A3D6C109Chum2.2 sequence

[0196]

[0197] Table 12: Antibody 10A3D6C109Chum2.3 sequence

[0198]

[0199]

[0200] Table 13: Antibody 10A3D6C109Chum2.4 sequence

[0201]

[0202] Table 14: Antibody 10A3D6C109Chum2.5 sequence

[0203]

[0204]

[0205] Table 15: Antibody 10A3D6C109Chum3.1 sequence

[0206]

[0207] Table 16: Antibody 10A3D6C109Chum3.2 sequence

[0208]

[0209]

[0210] Table 17: Antibody 10A3D6C109Chum3.3 sequence

[0211]

[0212]

[0213] Table 18: Antibody 10A3D6C109Chum3.4 sequence

[0214]

[0215] Table 19: Antibody 10A3D6C109Chum3.5 sequence

[0216]

[0217]

[0218] Table 20: Antibody 10A3D6C109Chum4.1 sequence

[0219]

[0220] Table 21: Antibody 10A3D6C109Chum4.2 sequence

[0221]

[0222]

[0223] Table 22: Antibody 10A3D6C109Chum4.3 sequence

[0224]

[0225] Table 23: Antibody 10A3D6C109Chum4.4 sequence

[0226]

[0227]

[0228] Table 24: Antibody 10A3D6C109Chum4.5 sequence

[0229]

[0230] Table 25: Antibody 10A3D6C109Chum5.1 sequence

[0231]

[0232] Table 26: Antibody 10A3D6C109Chum5.2 sequence

[0233]

[0234]

[0235] Table 27: Antibody 10A3D6C109Chum5.3 sequence

[0236]

[0237] Table 28: Antibody 10A3D6C109Chum5.4 sequence

[0238]

[0239]

[0240] Table 29: Antibody 10A3D6C109Chum5.5 sequence

[0241]

[0242] Table 30: Antibody 10A3D6hum1.2 sequence

[0243]

[0244]

[0245] Table 31: Antibody 10A3D6hum1.5 sequence

[0246]

[0247] Table 32: Antibody 10A3D6hum2.2 sequence

[0248]

[0249] Table 33: Antibody 10A3D6hum6.1 sequence

[0250]

[0251]

[0252] Table 34: Antibody 10A3D6hum6.2 sequence

[0253]

[0254] Table 35: Antibody 10A3D6hum6.3 sequence

[0255]

[0256]

[0257] Table 36: Antibody 10A3D6hum6.4 sequence

[0258]

[0259] Table 37: Antibody 10A3D6hum6.5 sequence

[0260]

[0261]

[0262] Table 38: Antibody 10A3D6hum6.6 sequence

[0263]

[0264] Table 39: Antibody 10A3D6hum7.1 sequence

[0265]

[0266] Table 40: Antibody 10A3D6hum7.2 sequence

[0267]

[0268]

[0269] Table 41: Antibody 10A3D6hum7.3 sequence

[0270]

[0271] Table 42: Antibody 10A3D6hum7.4 sequence

[0272]

[0273]

[0274] Table 43: Antibody 10A3D6hum7.5 sequence

[0275]

[0276] Table 44: Antibody 10A3D6hum7.6 sequence

[0277]

[0278]

[0279] Table 45: Antibody 10A3D6hum8.1 sequence

[0280]

[0281] Table 46: Antibody 10A3D6hum8.2 sequence

[0282]

[0283] Table 47: Antibody 10A3D6hum8.3 sequence

[0284]

[0285]

[0286] Table 48: Antibody 10A3D6hum8.4 sequence

[0287]

[0288] Table 49: Antibody 10A3D6hum8.5 sequence

[0289]

[0290]

[0291] Table 50: Antibody 10A3D6hum8.6 sequence

[0292]

[0293] Table 51: Antibody 10A3D6hum8.7 sequence

[0294]

[0295]

[0296] Table 52: Antibody 10A3D6hum8.8 sequence

[0297]

[0298] Table 53: Antibody 10A3D6hum8.9 sequence

[0299]

[0300]

[0301] In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and / or a light chain CDR1, CDR2, CDR3 from an antibody described herein. In some embodiments, the KLRB1 binding agent comprises a humanized version or humanized variant of an antibody described herein. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and / or a light chain C DR1, CDR2, CDR3 from antibody 10A3D6 (Table 1) or a humanized version thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and / or a light chain C DR1, CDR2, CDR3 from antibody 10A3D6C109S (Table 2) or a humanized version thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and / or light chain CDR1, CDR2, CDR3 from antibody 10A3D6C109F (Table 3) or a humanized version thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and / or light chain CDR1, CDR2, CDR3 from antibody 10A3D6C109V (Table 4) or a humanized version thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and / or light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum1.2 (Table 30) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and / or a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum1.5 (Table 31) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum2.2 (Table 32) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.1 (Table 33) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.2 (Table 34) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.3 (Table 35) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.4 (Table 36) or a variant thereof.In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.5 (Table 37) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 38) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 39) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 40) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 41) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 42) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 43) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 44) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 45) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 46) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 47) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 48) or a variant thereof.In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 49) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 50) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 51) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 52) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 53) or a variant thereof. In some embodiments, the KLRB1 binding agent comprises the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2, CDR3 from antibody 10A3D6hum6.6 (Table 53) or a variant thereof. 3, 10A3D6hum8.4, 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.

[0302] In some embodiments, the KLRB1 binding agent comprises a humanized version or humanized variant of an antibody described herein. In some embodiments, the KLRB1 binding agent comprises a heavy chain CDR1, CDR2 and CDR3 shown in Tables 1-4 and / or a light chain CDR1, CDR2, C DR3 or a humanized version thereof, e.g., as shown in Tables 5-53 or 56-57.

[0303] Those skilled in the art define CDR by a variety of methods / systems. These systems and / or definitions are developed and improved over many years, and include Kabat, Chothia, IMGT, Ab M and Contact. The Kabat definition is based on sequence variability, and is usually the most commonly used. The Chothia definition is based on the position of the structural loop region. The IMGT system is based on sequence variability and position 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. For the analysis of antibody sequences and the determination of CDR, software programs (e.g., abYsis (bioinf.org.uk / abysis / sequence_input / key_annotation / key_annotation.cgi)) are available and known to those skilled in the art.

[0304] The specific CDR sequences defined herein are generally based on the Kabat definition. However, it will be appreciated that reference to a heavy chain CDR or multiple heavy chain CDRs and / or a light chain CDR or multiple light chain CDRs of a particular antibody will encompass all CDR definitions as known to those skilled in the art, e.g., as shown in the tables herein. In some embodiments, the CDR sequences used will all be identified using the same definition, i.e., will all be Chothia, all Kabat, all IMGT, etc.

[0305] In some embodiments, the KLRB1 binding agent is a variant of an agent described herein. In some embodiments, the KLRB1 binding agent (e.g., an antibody) comprises (a) a heavy chain CDR1, CDR2, CDR3 from a VH sequence presented herein (e.g., in Tables 1-53 or 56-57), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and / or a light chain CDR1, CDR2, and / or CDR3 from a VL sequence presented herein (e.g., in Tables 1-53 or 56-57), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitution is a conservative substitution. In some embodiments, the CDR comprises one conservative amino acid substitution. In some embodiments, the CDR comprises two conservative amino acid substitutions. In some embodiments, the CDR comprises three conservative amino acid substitutions. In some embodiments, the CDR comprises four conservative amino acid substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain CDR2. In some embodiments, the CDR is a heavy chain CDR3. In some embodiments, the CDR is a light chain CDR1. In some embodiments, the CDR is a light chain CDR2. In some embodiments, the CDR is a light chain CDR3. In some embodiments, the substitution is performed as part of a humanization process. In some embodiments, the substitution is performed as part of a germline humanization process. In some embodiments, the substitution is performed as part of an affinity maturation process. In some embodiments, the substitution is performed as part of an optimization process.

[0306] In some embodiments, a KLRB1 binding agent (e.g., an antibody) comprises one or more heavy or light chain CDRs that have been modified, for example, to reduce deamidation within the CDR sequence, to remove Asn(N)-glycosylation sites, to remove cysteine, or to remove Asp to reduce isomerization sites, remove Met / Trp or Lys, for example, to reduce the likelihood of asparagine (N)-glycosylation, cysteinization, asparagine (Asn) deamidation, aspartate (Asp) isomerization, methionine / tryptophan (Met / Trp) oxidation, and non-enzymatic lysine (Lys) glycosylation within the CDR sequence (see, e.g., Haberger et al., MAbs. 2014 Mar 1; 6(2): 327–339; Lu et al., MAbs. 2019 Jan; 11(1): 45-57). Deamidation is a chemical reaction in which the amide functional group in the side chain of the amino acids asparagine (N) or glutamine (Q) is removed or converted to another functional group. Typically, asparagine is converted to aspartic acid or isoaspartic acid, while glutamine is converted to glutamic acid or polyglutamic acid. In some cases, deamidation may alter the structure, function, and / or stability of a polypeptide, which may result in reduced biological activity.

[0307] In certain embodiments, a KLRB1 binding agent comprises a heavy chain variable region comprising heavy chain CDRs 1, 2, and 3 as shown in Tables 1-53 or 56-57 and a light chain variable region comprising light chain CDRs 1, 2, and 3 as shown in Tables 1-53 or 56-57.

[0308] In some embodiments, a KLRB1 binding agent (e.g., an antibody) comprises a heavy chain variable region or sequence having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a heavy chain variable region sequence presented herein (e.g., in Tables 1-53 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 a light chain variable region sequence presented herein (e.g., in Tables 1-53 or 56-57).

[0309] The term "consensus sequence" as used herein for light chain (VL) and heavy chain (VH) variable regions refers to a composite or universal VL or VH sequence defined based on information about which amino acid residues within a VL or VH chain are susceptible to modification without impairing antigen binding. Thus, in a "consensus sequence" of a VL or VH chain, certain amino acid positions are occupied by one of a plurality of possible amino acid residues at that position. For example, if arginine (R) or serine (S) appears at a particular position, then that particular position within the consensus sequence can be arginine or serine (R or S). The consensus sequence of the VH and VL chains can be determined, for example, by in vitro affinity maturation (e.g., randomizing each amino acid position in a CDR using degenerate coding primers), by scanning mutagenesis of amino acid residues within an antibody CDR (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by evaluation of the binding of the mutant to the antigen to determine whether the mutated amino acid position affects antigen binding. In some aspects, mutations are introduced in the CDR region. In other aspects, mutations are introduced in the framework region. In some other aspects, mutations are introduced in the CDR and framework regions.The consensus sequence can be determined by software such as EMBOSS Cons, which is available at: ebi.ac.uk / Tools / msa / embos s_cons / .

[0310] In some embodiments, the KLRB1 binding agents (e.g., antibodies) described herein comprise one or more constant heavy domains (e.g., CH1, CH2, and / or CH3 regions). In some embodiments, the KLRB1 binding agent comprises a constant heavy domain 1 (CH1) having an amino acid sequence as shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binding agent comprises a constant heavy domain 2 (CH2) comprising an amino acid sequence as shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binding agent comprises a constant heavy domain 3 (CH3) comprising an amino acid sequence as shown herein, e.g., in Table 57 or 58. In some embodiments, the KLRB1 binding agent comprises a heavy chain constant region comprising an amino acid sequence having at least 80, 85%, 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence as shown herein, e.g., in Table 57 or 58. In some embodiments, one or more constant regions of the KLRB1 binding agent have been modified. In some embodiments, the antibody may include modifications to one or more of the three heavy chain constant regions (CH1, CH2 or CH3) and / or to the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody includes at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody includes more than one human constant region. In some embodiments, the modification of the constant region includes adding, deleting or replacing one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or completely deleted from the constant region of the modified antibody. In some embodiments, the entire CH2 domain has been removed from the antibody (ΔCH2 construct). In some embodiments, the deleted constant region is replaced by a short amino acid spacer, which provides some molecular flexibility in the molecular flexibility usually conferred by the deleted constant region. In some embodiments, the modified antibody includes a CH3 domain fused directly to the hinge region of the antibody. In some embodiments, the modified antibody includes a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0311] One or more constant regions of antibodies known in the art mediate several effector functions and these effector functions can vary according to the isotype of the antibody. In addition, the Fc region of the antibody can be combined with cells expressing Fc receptors (FcR). There are many Fc receptors that are specific to antibodies of different classes, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). The combination of antibodies and Fc receptors on the cell surface can trigger many important and diverse biological responses, including the phagocytosis and destruction of antibody-coated particles, the removal of immune complexes, killing cells to the cracking of antibody-coated target cells (called antibody-dependent cellular toxicity or ADCC), cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), the release of inflammatory mediators, placental transfer and the control 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 of ordinary skill in the art (e.g., a representative human IgG1 Fc region is shown in Lobner et al., Immunol Rev. 2016 Mar; 270(1): 113–131; see, e.g., Table 58). In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, variant Fc regions are engineered to have substitutions at specific amino acid positions compared to native Fc regions. In some embodiments, the Fc region is mutated to alter (reduce) antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-induced complement-dependent cytotoxicity (CDC), and / or antibody-dependent cell-mediated phagocytosis (ADCP) (see, e.g., Kang and Jung, Experimental & Molecular Medicine. 2019. 51: 1–9; Wang et al., Antibody Therapeutics, January 2021. 4(1): 45–54; Lobner et al., Immunol Rev. 2016 Mar; 270(1): 113–131). In some embodiments, the Fc region is afucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. 2009 May-June; 1(3): 230–236, which describes a method for producing therapeutic antibodies with controlled fucosylation levels of Fc region N-glycans).

[0312] In some embodiments, modified antibodies (e.g., modified Fc regions) provide altered effector functions, which in turn affect the biological spectrum of the antibody. For example, in some embodiments, mutations in the constant region enhance the Fc receptor binding of the modified antibody when it circulates. In some embodiments, constant region modifications increase the serum half-life of the antibody. In some embodiments, constant region modifications reduce the serum half-life of the antibody. In some embodiments, constant region modifications increase or enhance the ADCC and / or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, the constant region is modified to eliminate disulfide bonds or oligosaccharide moieties. In some embodiments, the constant region is modified to add / replace one or more amino acids, thereby providing one or more cytotoxins, oligosaccharides, or carbohydrate attachment sites.

[0313] The constant regions of the antibodies described herein can be modified using well-known biochemical or molecular engineering techniques. In some embodiments, antibody variants are prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesizing the desired antibody or polypeptide. Using these antibody variants, the activity or effector function provided by a specific sequence or region can be enhanced while substantially maintaining the structure, binding activity and other desired properties of the modified antibody.

[0314] The present disclosure further includes additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized and human antibodies described herein, or their antibody fragments. 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, one or more effector functions, glycosylation, immunogenicity or solubility. It will be appreciated by those skilled in the art that amino acid changes may alter the post-translational processes of the antibody, such as changing the number or position of glycosylation sites or changing membrane anchoring properties.

[0315] Variation can be the replacement, deletion or insertion of one or more nucleotides encoding antibodies or polypeptides, which causes amino acid sequences to change compared to native antibodies or polypeptide sequences. In some embodiments, amino acid substitution is to replace a kind of amino acid with another amino acid with similar structure and / or chemical properties, such as leucine is replaced with serine, for example, the result of conservative amino acid substitution. Insertion or deletion can be optionally located in the range of about 1 to 5 amino acids. In some embodiments, relative to the parent molecule, replacement, deletion or insertion include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions. In some embodiments, by systematically inserting, deleting or replacing in sequence, and testing the activity of the obtained variant protein compared with the parent antibody to determine the change in biologically useful and / or relevant amino acid sequence.

[0316] In some embodiments, variants can include adding amino acid residues at the aminoterminal and / or carboxylterminal of an antibody or polypeptide. The length of the additional amino acid residues can be in the range of one residue to one hundred or more residues. In some embodiments, variants include N-terminal methionyl residues. In some embodiments, variants include additional polypeptides / proteins, i.e., fusion proteins. In some embodiments, variants are engineered to be detectable and can include detectable markers and / or proteins (e.g., enzymes).

[0317] In some embodiments, cysteine ​​residues that are not involved in maintaining the correct conformation of the antibody can be substituted or deleted to adjust the properties of the antibody, for example, to improve oxidative stability and / or prevent abnormal disulfide cross-linking. Conversely, in some embodiments, one or more cysteine ​​residues can be added to create one or more disulfide bonds to improve stability.

[0318] Variant antibodies or polypeptides described herein can be produced using methods known in the art including, but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0319] In some embodiments, the KLRB1 binding agents described herein are chemically modified. In some embodiments, the KLRB1 binding agent 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 attachment to cellular ligands or other proteins. Any of a number of chemical modifications can be performed by known techniques.

[0320] The present disclosure encompasses KLRB1 binding agents constructed on non-immunoglobulin backbones, wherein the agent binds to the same epitope or substantially the same epitope as the anti-KLRB1 antibodies disclosed herein. In some embodiments, the non-immunoglobulin-based binding agent is an agent that competes with the anti-KLRB1 antibodies described herein in a competitive binding assay. In some embodiments, the alternative KLRB1 binding agent comprises a scaffold protein. Generally, based on the architecture of the backbone of the scaffold protein, the scaffold protein can be assigned to one of the following three groups: (1) scaffolds composed of α-helices; (2) small scaffolds with a small amount of secondary structure, or an irregular architecture of α-helices and β-sheets; and (3) scaffolds composed primarily of β-sheets. Scaffold proteins include, but are not limited to, anticalins, which are based on the lipocalin scaffold; adnectins, which are based on the 10th domain of human nectin type 3; affimers, which are based on the B-domain in the Ig binding region of Staphylococcus aureus protein A; darpins, which are based on ankyrin repeat domain proteins; fynomers, which are based on the SH3 domain of human Fyn protein kinase; affitins, which are based on Sac7d from Sulfolobus acidocaldarius; affilins, which are based on human γ-B crystallin or human ubiquitin; aVimers, which are based on the A domain of membrane receptor proteins; knottins (cysteine ​​knot miniproteins), which are based on a stable 30 amino acid antiparallel β-strand protein fold; and Kunitz domain inhibitor scaffolds, which are based on a structure containing three disulfide bonds and three loops. In some embodiments, the KLRB1 binding agent comprises an engineered scaffold protein comprising the heavy chain CDR1, CDR2 and CDR3 and the light chain CDR1, CDR2 and CDR3 shown in any one of Tables 1 to 54, e.g., Tables 1, 31, 32 or 33. In some embodiments, these agents are conjugated to a cytotoxic agent or moiety that causes depletion of KLR B1+ cells.

[0321] Typically, antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. When describing the strength of an antigen-antibody complex, the terms affinity and / or avidity are often used. The binding of an antibody to its antigen is a reversible process, and the affinity of the binding is usually expressed in terms of the equilibrium dissociation constant (K D ) to report. D is the antibody dissociation rate (k off ) (how quickly the antibody dissociates from its antigen) and the antibody association rate (k onIn some embodiments, the k of a particular antibody / antigen interaction is measured by measuring the k of the antibody to its antigen. on and k off rate, and then use the ratio of these values ​​to calculate K D The value of K D Value. K D The K value can be used to evaluate and rank the strength of individual antibody / antigen interactions. D The lower the affinity of the antibody to its target, the higher the affinity of the antibody to its target. In some embodiments, the SPR technology in the Biacore system is used to measure affinity. Avidity gives a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: (i) the affinity of the antibody to the target, (ii) the valence of both the antibody and the antigen, and (iii) the structural arrangement of the interacting parts.

[0322] In some embodiments, a KLRB1 binding agent (e.g., an antibody) has a dissociation constant (K) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 0.1 nM or less, 50 pM or less, 10 pM or less, or 1 pM or less. D ) binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 20 nM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 10 nM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 1 nM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 0.5 nM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 0.1 nM or less. D Binds to KLRB1 (e.g., human KLR B1). In some embodiments, the KLRB1 binding agent has a K of about 50 pM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 25 pM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 10 pM or less. D Binds to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binding agent has a K of about 1 pM or less.D Binding to KLRB1 (e.g., human KLRB1). In some embodiments, the dissociation constant of the binding agent (e.g., antibody) to KLRB1 is a dissociation constant determined using a KLRB1 protein immobilized on a Biacore chip and the binding agent flowing through the chip. In some embodiments, the dissociation constant of the binding agent (e.g., antibody) to KLRB1 is a dissociation constant determined using a binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble KLRB1 flowing through the chip.

[0323] In some embodiments, a KLRB1 binding agent (e.g., an antibody) binds to KLRB1 (e.g., human KLRB1) at a half-maximal effective concentration (EC50) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a KLRB1 binding agent binds to human KLRB1 at an EC50 of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a KLRB1 binding agent binds to cynomolgus monkey KLRB1 and / or human KLRB1 at 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.

[0324] Without being bound by theory, the antibodies disclosed herein exhibit superiority over commercial antibodies in one or more of the following activities:

[0325] Increased binding potency (lower EC50 for binding to cell-expressed KLRB1 or soluble KLRB1 extracellular domain);

[0326] Increased potency of ADCC-mediated depletion (lower EC50 for depletion in CHO-KLRB1+ cells or lower EC50 for ADCC assay in reporter cell lines);

[0327] Increased binding affinity kinetics (lower KD or lower Koff);

[0328] Increased blocking potency (lower IC50 for inhibition of CLEC2D binding to cell-expressed KLRB1); and

[0329] • Increased potency of potentiation (lower EC50 for potentiation (increase) of CLEC2D binding to cell-expressed KLRB1).

[0330] In some embodiments, the antibody is in combination with a commercial antibody (e.g., B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (CellSciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative The antibodies disclosed herein have higher production yields, lower immunogenicity (due to the presence of humanized variable regions and / or human Fc sequences), and / or improved biophysical parameters (e.g., higher melting temperatures, higher freeze-thaw stability, lower isomerization, reduced or absent deamidation, or lower sensitivity to oxidation) compared to the antibodies described in US Pat.

[0331] The KLRB1 binding agents (e.g., antibodies) described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding polypeptide sequences and expressing these sequences in a suitable host. In some embodiments, recombinant technology is used to construct DNA sequences by isolating or synthesizing DNA sequences encoding wild-type proteins of interest. Optionally, the sequence can be mutated by site-specific mutagenesis to provide functional variants thereof. In some embodiments, a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons that are favored in the host cell that will produce the recombinant polypeptide of interest. Standard methods can be used to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, the complete amino acid sequence can be used to construct a reverse-translated gene. Further, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then linked. A single oligonucleotide typically contains a 5′ or 3′ overhang for complementary assembly.

[0332] Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence encoding the polypeptide of particular interest can be inserted into an expression vector and can be operably linked to expression control sequences suitable for expressing the protein in the desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping and / or expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of the transfected gene in the host, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0333] In some embodiments, a recombinant expression vector is used to amplify and express DNA encoding an antibody or fragment thereof against human KLRB1. For example, a recombinant expression vector can be a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of a KLRB1 binder (such as an anti-KLRB1 antibody or antigen-binding fragment thereof), which is operably linked to a suitable transcription and / or translation regulatory element derived from a mammalian, microbial, viral or insect gene. A transcription unit generally comprises the following assembly: (1) one or more genetic elements that have a regulatory effect on gene expression, such as a transcription promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein, and (3) appropriate transcription and translation start and stop sequences. The regulatory element may include an operator sequence for controlling transcription. The ability to replicate in a host, which is usually conferred by an origin of replication, and a selection gene for facilitating the recognition of transformants may also be additionally incorporated. DNA regions are "operably linked" when they are functionally related to each other. For example, if the DNA of a signal peptide (secretion leader) is expressed as a precursor that participates in the secretion of a polypeptide, the DNA of the signal peptide is operably linked to the DNA of the polypeptide; if a promoter controls the transcription of a coding sequence, the promoter is operably linked to the sequence; or, if a ribosome binding site is positioned to allow translation, the ribosome binding site is operably linked to the coding sequence. In some embodiments, the structural elements intended for use in a yeast expression system include a leader sequence that enables a host cell to secrete translated proteins to the extracellular space. In some embodiments, in the case where a recombinant protein is expressed without a leader sequence or a transport sequence, the polypeptide may include an N-terminal methionine residue. This residue may then be optionally cleaved from the expressed recombinant protein to provide a final product.

[0334] The selection of expression control sequences and expression vectors usually depends on the selection of the host. A variety of expression host / vector combinations can be adopted. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli (including pCR1, pBR322, pMB9 and derivatives thereof), and plasmids of wider host range, such as M13 and other filamentous single-stranded DNA phages.

[0335] The KLRB1 binding agents (e.g., antibodies) disclosed herein can be expressed by 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.

[0336] Suitable host cells for expressing KLRB1 binding agents (e.g., antibodies) or KLRB1 proteins or fragments thereof for use as antigens or immunogens include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative organisms or gram-positive organisms, such as Escherichia coli or Bacillus. Higher eukaryotic cells include established mammalian-derived cell lines as described herein. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors and protein production methods (including antibody production) for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art.

[0337] Various mammalian culture systems can be used to express recombinant polypeptides. The expression of recombinant proteins in mammalian cells may be desirable because these proteins are usually folded correctly, appropriately modified and have biological functions. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney derived), L-929 (mouse fibroblast derived), C127 (mouse breast tumor derived), 3T3 (mouse fibroblast derived), CHO (Chinese hamster ovary derived), HeLa (human cervical cancer derived), BHK (hamster kidney fibroblast derived), HEK-293 (human embryonic kidney derived) cell lines and their variants. Mammalian expression vectors may include non-transcriptional elements, such as replication origins, suitable promoters and enhancers connected to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences, and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences.

[0338] The expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also provides a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for producing heterologous proteins in insect cells are well known to those skilled in the art. Therefore, the present disclosure provides cells comprising KLRB1 binding agents described herein. In some embodiments, the cells produce KLRB1 binding agents described herein. In some embodiments, the cells produce antibodies. In some embodiments, the cells produce antibodies that bind to human KLRB1. In some embodiments, the cells produce antibodies that bind to cynomolgus monkey KLRB1. In some embodiments, the cells produce antibodies that bind to human KLRB1 and cynomolgus monkey K LRB1. In some embodiments, the cell produces an antibody designated as 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 cell produces an scFv version of the antibody 10A3D6, 10A3D6hum1.2, 10A3D6hum1.5, 10A3D6hum2.2, 10A3D6hum6.1, 10A3D6hum6.2, 10A3D6hum6.3, 10A3D6hum6.4, 10A3D6hum6.5, or 10A3D6hum6.6. In some embodiments, the cell is a prokaryotic cell (e.g., Escherichia coli). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hybridoma cell. The protein produced by the host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility or any other standard techniques for protein purification. Affinity tags (such as hexa-histidine, maltose binding domain, influenza capsid sequence and glutathione-S-transferase) can be attached to the protein to allow easy purification by passing through from an appropriate affinity column. Affinity chromatography for purifying immunoglobulins can include protein A chromatography, protein G chromatography and protein L chromatography. Separated 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, a commercially available protein concentration filter (eg. or Millipore Ultrafiltration device) concentrates the supernatant from the expression system that secretes the recombinant protein into the culture medium. After the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin is used, such as a matrix or substrate with a side chain diethylaminoethyl (DEAE) group. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. In some embodiments, a cation exchange step is used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. In some embodiments, hydroxyapatite media are used, including but not limited to ceramic hydroxyapatite (CHT). In some embodiments, one or more reverse phase HPLC steps are used to further purify the recombinant protein, and the reverse phase HPLC step uses a hydrophobic RP-HPLC medium (e.g., silica gel with side chain methyl or other aliphatic groups). In some embodiments, hydrophobic interaction chromatography (HIC) is used to separate recombinant proteins based on the hydrophobicity of the recombinant protein. HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other technologies. Some or all of the foregoing purification steps in various combinations may be used to provide a homogeneous recombinant protein.

[0339] The physical / chemical properties and / or biological activities of the anti-KLRB1 antibodies of the present disclosure can be analyzed by various assays known in the art. In some embodiments, the ability of the anti-KLRB1 antibodies to bind to KLRB1 (e.g., human KLRB1 and / or cynomolgus monkey KLRB1) is tested. Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS. In some embodiments, the ability of the anti-KLRB1 antibodies to induce ADCC, ADCP, and / or CDC, as well as the ability of the antibodies to kill KLRB1 target cells (cell depletion), are tested. Assays include, but are not limited to, ADCC cell lysis assays, such as those using LDH release and formazan detection. In addition, the solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency of the antibodies can also be evaluated.

[0340] In some embodiments, the purified anti-KLRB1 antibodies are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0341] Antibody conjugates

[0342] The present disclosure also provides a conjugate comprising an anti-KLRB1 antibody or antigen-binding fragment thereof as described herein. In some embodiments, the antibody or antigen-binding fragment thereof is attached 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 promotes the depletion of KLRB1-expressing cells to which the antibody or antigen-binding fragment is bound. 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, doxorubicin / doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines, (PBD) or other intercalating agents. In some embodiments, the cytotoxic agent is a microtubule inhibitor, including but not limited to auristatins, maytansinoids (eg, DM1 and DM4), and tubulysin. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant or animal origin, or a fragment thereof, including but not limited to diphtheria toxin A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and tricothecene. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as calicheamicin, maytansinoids, tricothecenes and CC1065. Derivatives of any of these toxins may be used so long as the derivative retains the cytotoxic activity of the parent molecule.

[0343] Any suitable method known in the art can be used to prepare conjugates comprising an anti-KLRB1 antibody or antigen-binding fragment thereof described herein. In some embodiments, conjugates are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0344] In some embodiments, the anti-KLRB1 antibodies or antigen-binding fragments thereof described herein are conjugated to detectable substances or molecules that enable the antibodies to be used for diagnosis and / or detection. Detectable substances can include, but are not limited to: enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; prosthetic groups such as biotin and one or more riboflavins; fluorescent materials such as umbelliferone, luciferin, luciferin isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials 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, 186Re, 188 Re, 105 Rh, 97 Such as 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.

[0345] The anti-KLRB1 antibodies or antigen-binding fragments thereof described herein may also be conjugated to a second antibody to form an antibody heteroconjugate.

[0346] The anti-KLRB1 antibodies or antigen-binding fragments thereof as described herein can be attached 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 antibodies are used in immunoassays. In some embodiments, the immobilized anti-KLRB1 antibodies are used in the purification of the target antigen.

[0347] Polynucleotides / methods of making binding agents

[0348] Also provided herein are nucleic acids encoding polypeptides described herein and vectors containing nucleic acids encoding 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 attached, and may include plasmids, cosmids, or viral vectors. The vector is capable of autonomous replication or may be integrated into host DNA. Viral vectors include, for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses.

[0349] The vector may include a nucleic acid in a form suitable for expressing the nucleic acid in a host cell. Preferably, the recombinant expression vector includes 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 nucleotide sequences, 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 the host cell to be transformed, the expression level of the desired protein, and the like. The expression vector of the present invention may be introduced into a host cell to thereby produce a protein or polypeptide encoded by a nucleic acid as described herein encoding a KLRB1 binding agent as described herein, including a fusion protein or polypeptide.

[0350] The recombinant expression vector of the present invention can be designed to express KLRB1 binder protein in prokaryotic cells. Preferably, 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 control function of the expression vector is often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40.

[0351] Vector DNA can be introduced into host cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to various art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection or electroporation.

[0352] The host cell can be used to produce (i.e., express) a KLRB1 binding agent protein. Therefore, the present invention further provides a method for producing a KLRB1 binding agent protein using the host cell of the present invention. In one embodiment, the method comprises culturing a host cell of the present invention (in which a recombinant expression vector encoding a KLRB1 binding agent protein has been introduced) in a suitable culture medium, thereby producing a KLRB1 binding agent protein. In another embodiment, the method further comprises isolating the KLRB1 binding agent protein from the culture medium or the host cell. In some embodiments, the Fc region is afucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. 2009 May-June; 1(3):230–236, which describes a method for producing a therapeutic antibody with a controlled level of fucosylation of the Fc region N-glycans).

[0353] Pharmaceutical composition

[0354] Also provided herein are pharmaceutical compositions comprising a KLRB1 binding agent described herein as an active ingredient. In various embodiments, the KLRB1 binding agent is prepared as a pharmaceutical composition, such as a pharmaceutical composition for use as a medicament. In various embodiments, the pharmaceutical composition is used as a medicament for treating a disease as described herein (optionally an autoimmune disease, an allergic disease, a transplant rejection, or a hematological malignancy) in a subject in need thereof. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, suppurative hidradenitis, 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, transplant rejection can be rejection of kidney, lung, heart, liver, limb, skin or multi-organ transplants. In some embodiments, hematological malignancies are leukemias, such as T-cell leukemias, NK-cell leukemias, T-cell lymphomas, T-cell prolymphocytic leukemias (T-PLL) or large granular lymphocytic leukemias (LGLL). In some embodiments, lymphomas are hepatosplenic T-cell lymphomas (HSTCL), NK / T-cell lymphomas (NKTCL), extranodal NK / T-cell lymphomas (ENKL), aggressive NK-cell leukemias (ANKL), mycosis fungoides, Sezary syndrome, peripheral T-cell lymphomas, angioimmunoblastic T-cell lymphomas (AITL) and peripheral T-cell lymphomas not otherwise specified (PTCL-NOS). In some embodiments, leukemias are aggressive NK-cell leukemias or T-cell prolymphocytic leukemias.

[0355] Those skilled in the art can formulate the KLRB1 binding agent into a pharmaceutical composition according to known methods.

[0356] The pharmaceutical composition may include a carrier. As used herein, a "carrier" may include a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic (or relatively nontoxic) to cells or subjects exposed thereto at the doses and concentrations employed. A physiologically acceptable carrier is often an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, 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 including glucose, mannose, or dextrins; 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 TM , polyethylene glycol (PEG) and PLURONICS TM .

[0357] In various embodiments, the KLRB1 binding agent is contained in an injectable formulation, such as a subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection formulation. The injectable formulation can be an aqueous solution, such as an aqueous solution in a physiologically compatible buffer, such as Hanks' solution, Ringer's solution or physiological saline buffer. The injectable formulation can contain a formulation agent such as a suspending agent, a stabilizer and / or a dispersing agent. Alternatively, the KLRB1 binding agent can be in a dry form or powder form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to use.

[0358] The binding agents of the present disclosure may be formulated in any suitable form for delivery to target cells / tissues. In some embodiments, the KLRB1 binding agent may be formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nanoparticle, nanocapsule or macroemulsion. In some embodiments, the pharmaceutical preparation comprises an agent of the present disclosure complexed with a liposome. Methods for producing liposomes are known to those skilled in the art. For example, some liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE).

[0359] In some embodiments, the KLRB1 binding agent is formulated as a sustained release preparation. Suitable examples of sustained release preparations include a semipermeable matrix of a solid hydrophobic polymer containing the agent, wherein the matrix is ​​in the form of a shaped article (e.g., a film or microcapsule). Sustained release matrices include, but are not limited to, polyesters, hydrogels (such as poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides, 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 TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0360] Treatment and administration

[0361] The present disclosure provides a method comprising administering a KLRB1 binding agent as described herein, or a pharmaceutical composition comprising a KLRB1 binding agent as described herein, to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the method is performed in vivo (e.g., as opposed to ex vivo). As used herein, "treatment" refers to therapeutic treatment (treatment of a subject with a disease); the method can also be used for preventive or preventive measures, wherein the purpose is to prevent or mitigate (mitigate) the disease in a subject who does not suffer from the targeted pathological disorder or condition. Those in need of treatment may include those already suffering from the disorder, those susceptible to the disorder, or those in need of prevention of the disorder (as used herein, "prevention" is intended to reduce the risk of illness).

[0362] In various aspects and embodiments, the present disclosure provides methods for treating autoimmune diseases, allergic diseases, transplant rejection or hematological malignancies in subjects in need. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, Crohn's disease, ulcerative colitis or celiac disease. In some embodiments, allergic diseases are asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders or high eosinophilic syndrome. In some embodiments, transplant rejection can be the rejection of kidney, lung, heart, liver, limbs, skin or multi-organ transplants. In some embodiments, the hematological malignancy is a leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL). In some embodiments, the lymphoma is hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), extranodal NK / T-cell lymphoma (ENKL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sezary 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 associated with the pathogenesis of these diseases; therefore, in some embodiments, the hematological malignancy expresses KLRB1. According to the present disclosure, depletion of such KLRB1 expressing cells may provide therapeutic benefit.

[0363] In various aspects and embodiments, the present disclosure provides methods for treating or preventing transplant rejection.The transplant rejection can be, for example, kidney rejection.

[0364] "Administering" and "treating" as they apply to animals, humans, experimental subjects, cells, tissues, organs or biological fluids can include contacting an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, human, subject, cell, tissue, organ or biological fluid. "Administering" and "treating" include in vivo treatment, and in some embodiments in vitro or ex vivo treatment.

[0365] Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the subject or population being treated, whether to induce regression of the one or more symptoms or to inhibit the progression of the one or more symptoms to any clinically measurable extent. The amount of therapeutic agent effective to alleviate any particular disease symptom may vary according to factors such as the disease state, age and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether disease symptoms have been alleviated can be assessed by any clinical measurement commonly used by a physician or other skilled healthcare provider to assess the severity or progression of the symptom.

[0366] Thus, in various embodiments, the term "effective amount" or "therapeutically effective amount" is the concentration or amount of a KLRB1 binding agent that results in achieving a particular stated purpose (e.g., alleviation of one or more symptoms of a disease described herein). An "effective amount" of a KLRB1 binding agent can be determined empirically. In addition, a "therapeutically effective amount" is a concentration or amount of a KLRB1 binding agent that is effective to achieve a stated therapeutic effect. This amount can also be determined empirically.

[0367] In some embodiments, treatment with a KLRB1 binding agent of the disclosure can kill at least about 20%, such as at least about 30%, 40%, 50%, 60%, 70%, or 80% of KLRB1 expressing cells involved in the pathogenesis of a disease disclosed herein.

[0368] The term "subject" refers to any animal (eg, mammal), including, but not limited to, humans and non-human veterinary subjects (including primates).

[0369] As used herein, reference to "about" or "approximately" a value or parameter includes (and describes) embodiments for that value or parameter. For example, a description referring to "about X" includes a description of "X." As used herein, "about" means plus or minus 10%.

[0370] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0371] It should be understood that whenever an embodiment is described herein with the term "comprising", other similar embodiments described with "consisting of" and / or "consisting essentially of" are also provided. It should be understood that whenever an embodiment is described herein with the phrase "consisting essentially of", other similar embodiments described with "consisting of" are also provided.

[0372] The term "and / or" as used herein in phrases such as "A and / or B" is meant to include: both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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 (alone); B (alone); and C (alone).

[0373] In various embodiments, a KLRB1 binding agent can be administered by providing an mRNA encoding the binding agent to a subject.

[0374] The following examples are for illustrative purposes only and are not limiting. After reading this disclosure, those skilled in the art will be aware of many variations of the technology. Therefore, the scope of the technology should not be determined with reference to the embodiments, but should be determined with reference to the full scope of the appended claims and their equivalents.

[0375] Example

[0376] Example 1: KLRB1 gene expression is a marker of a unique immune cell population and is restricted to immune cells

[0377] KLRB1 expression spans traditional lymphocyte classifications and is expressed in Th17, Th17.1, ex-Th17, Tc17, iNKT, ILC2, ILC3, peTh2, and NK cell subsets ( Figure 1 )'s marker.

[0378] Whole-body KLRB1 expression profiling data showed that KLRB1 was not significantly expressed in any cell type except immune cells ( Figure 2 ).

[0379] Example 2: KLRB1 expression is enhanced under various immune conditions

[0380] Rheumatoid arthritis: Analysis of expression data from synovial biopsies from patients with rheumatoid arthritis compared to patients with crystal-induced arthritis (GSE1919) showed that KLRB1 expression was increased (10-fold ratio) ( Figure 3 ). Rheumatoid arthritis is therefore a particularly attractive target for therapies according to the present disclosure.

[0381] Analysis of expression data from synovial biopsies from patients with rheumatoid arthritis compared to normal patients (GSE36700) showed that the expression of KLRB1 was increased (3.3-fold ratio) ( Figure 4 ). Rheumatoid arthritis is therefore a particularly attractive target for therapies according to the present disclosure.

[0382] Sjögren's syndrome: Analysis of expression data from salivary gland biopsies from patients with Sjögren's syndrome compared to normal controls (GSE23117) showed increased expression of KLRB1 in late (13.1-fold ratio), moderate (5.3-fold ratio), and early (1.6-fold ratio) stages of the disease. Figure 5 ). Therefore, Sjögren's syndrome is a particularly attractive target for therapies according to the present disclosure.

[0383] Analysis of expression data from parotid gland biopsies from patients with Sjögren's syndrome compared to normal controls (GSE40611) showed that KLRB1 expression was increased (2.6-fold ratio) ( Figure 6 ). Therefore, Sjögren's syndrome is a particularly attractive target for therapies according to the present disclosure.

[0384] Inclusion body myositis: Analysis of expression data from muscle biopsies from patients with inclusion body myositis compared to normal subjects (GSE38454) showed increased expression of KLRB1 (2.9-fold ratio) ( Figure 7 ). Therefore, inclusion body myositis is a particularly attractive target for therapy according to the present disclosure.

[0385] Discoid lupus: Analysis of expression data from skin biopsies from patients with discoid lupus compared to normal subjects (GSE52471) showed increased expression of KLRB1 (7.2-fold ratio) ( Figure 8 ). Discoid lupus is therefore a particularly attractive target for therapies according to the present disclosure.

[0386] Psoriasis: Analysis of expression data from skin biopsies from patients with psoriasis compared to normal subjects (GSE52471) showed that the expression of KLRB1 was increased (11.2-fold ratio) ( Fig. 9 ). Discoid lupus is therefore a particularly attractive target for therapies according to the present disclosure.

[0387] Idiopathic Pulmonary Fibrosis: Analysis of expression data from lung biopsies from patients with idiopathic pulmonary fibrosis compared to normal subjects (GSE53845) showed that the expression of KLRB1 was increased (1.5-fold ratio) ( Fig.10 ). Idiopathic pulmonary fibrosis is therefore a particularly attractive target for therapies according to the present disclosure.

[0388] Diabetes: Analysis of expression data from pancreatic biopsies from patients with diabetes compared to normal subjects (GSE72492) showed that the expression of KLRB1 was increased (3.7-fold) ( Fig.11 ). Therefore, diabetes is a particularly attractive target for therapies according to the present disclosure.

[0389] Alopecia universalis: Analysis of expression data from scalp biopsies from patients with idiopathic pulmonary fibrosis compared to normal subjects (GSE74761) showed increased expression of KLRB1 (4.3-fold ratio) ( Fig.12 ). Therefore, alopecia universalis is a particularly attractive target for therapies according to the present disclosure.

[0390] Primary biliary cholangitis: Analysis of expression data from liver biopsies from patients with primary biliary cholangitis who ultimately required liver transplantation (GSE79850) showed increased expression of KLRB1 (5.6-fold ratio) compared to normal controls ( Fig.13 ). Therefore, primary biliary cholangitis is a particularly attractive target for therapy according to the present disclosure.

[0391] Multiple Sclerosis: Analysis of expression data from brain biopsies of patients with multiple sclerosis (GSE5839) showed that expression of KLRB1 was elevated (2.5-fold) compared to control brains ( Fig.14 ). Multiple sclerosis is therefore a particularly attractive target for therapies according to the present disclosure.

[0392] Lymphocytic colitis: Analysis of expression data from colon biopsies of 4 patients with lymphocytic colitis compared with 4 healthy subjects (GSE65107) showed that the expression of KLRB1 was increased (3.2-fold ratio) ( Fig.15 ). Therefore, lymphocytic colitis is a particularly attractive target for therapy according to the present disclosure.

[0393] Renal transplant rejection: Analysis of expression data from renal biopsies of 7 patients with acute renal rejection compared to 9 healthy subjects (GSE1563) showed that KLRB1 expression was increased (2.1-fold ratio) ( Fig.16 ). Therefore, renal transplant rejection is a particularly attractive target for therapies according to the present disclosure.

[0394] Lung transplantation: Analysis of expression data from bronchoalveolar lavage (BAL) fluid from 7 patients with lung transplant rejection compared to 27 patients with lung transplants that did not experience rejection (GSE65107) showed increased expression of KLRB1 (3.6-fold ratio) ( Fig.17 ). Therefore, lung transplant rejection is a particularly attractive target for therapy according to the present disclosure.

[0395] Atopic dermatitis: Analysis of expression data from skin biopsies from 5 patients with atopic dermatitis compared to 5 healthy subjects (GSE65107) showed that the expression of KLRB1 was increased (1.9-fold ratio) ( Fig.18). Therefore, atopic dermatitis is a particularly attractive target for therapy according to the present disclosure.

[0396] Palmoplantar pustulosis: Analysis of expression data from skin biopsies from three patients with palmoplantar pustulosis lesional skin and eight patients with palmoplantar pustulosis non-lesional skin compared with seven healthy subjects (GSE185856) showed that KLRB1 expression was increased (the ratio of the lesional group to the healthy group was 11.0 times; the ratio of the non-lesional group to the healthy group was 5.9 times) ( Fig.19 ). Palmoplantar pustulosis is therefore a particularly attractive target for therapy according to the present disclosure.

[0397] Hidradenitis suppurativa: Analysis of skin biopsies from 18 patients with lesional skin of hidradenitis suppurativa and 7 patients with non-lesional skin of hidradenitis suppurativa compared with 8 healthy subjects (GSE148027) showed increased expression of KLRB1 (8.0-fold in lesional group vs. healthy group; 3.0-fold in non-lesional group vs. healthy group) ( Fig. 20 ). Thus, hidradenitis suppurativa is a particularly attractive target for therapy according to the present disclosure.

[0398] Asthma: Analysis of expression data from lung airway brushings from 10 patients with severe asthma, 37 patients with moderate asthma, and 44 patients with mild asthma compared to 47 healthy subjects (GSE41861) showed increased expression of KLRB1 (1.9-fold in severe asthma, 1.5-fold in moderate asthma, and 1.3-fold in mild asthma) compared to healthy subjects ( Fig.21 ). Therefore, atopic dermatitis is a particularly attractive target for therapy according to the present disclosure.

[0399] Example 3: KLRB1 expression is enhanced in various T-cell and NK-cell lymphomas and leukemias

[0400] Analysis of expression data from tumor cells from patients with various T and NK cell lymphomas and leukemias compared to normal NK cell expression (GSE19067) for example showed increased expression of KLRB1 (e.g., hepatosplenic T cell lymphomas had a 17-fold ratio compared to normal NK cell lines) (Figure 22). Therefore, various T cell and NK cell lymphomas are particularly attractive targets for therapies according to the present disclosure.

[0401] In particular, some 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, Sezary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) had increased KLRB1 expression or KLRB1 expression similar to that of CD4+ T cells ( FIG. 22 ). Thus, hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), extranodal NK / T-cell lymphoma (ENKL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Sezary 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 therapies according to the present disclosure.

[0402] Example 4: Monoclonal anti-KLRB1 antibody 10A3D6 binds to human KLRB1

[0403] Mouse hybridomas were generated by immunizing mice (5 balb / c and 5 SJL) with the extracellular domain (ECD) of the human KLRB1 protein (Uniprot ID Q12918, amino acids 67-225) to produce monoclonal antibodies against human KLRB1. The parent hybridomas were subsequently screened by ELISA for binding to the human KLRB1 ECD and by FACS for binding to a stable CHO-K1 cell line expressing human KLRB1 (SEQ ID NO: 1) and separately expressing cynomolgus monkey KLRB1 (SEQ ID NO: 2). 20 hybridomas were selected for subcloning based on binding to human and cynomolgus monkey CHO-K1 cells expressing KLRB1, resulting in 91 subclones. These 91 subclones were subjected to repeated FACS binding studies against CHO-K1 human-KLRB1 and CHO-K1 cynomolgus monkey KLRB1. The binding ability of one of these mAbs (designated 10A3D6) was tested in an ELISA assay. Human KLRB1 ECD was coated on an ELISA plate and the plate was incubated with 10A3D6 antibody. 10A3D6 antibody bound to the ELISA plate was detected using a secondary goat anti-mouse peroxidase-labeled IgG (see Fig.23 ).

[0404] Example 5: Monoclonal anti-KLRB1 chimeric and humanized antibodies bind to human KLRB1 and cynomolgus monkey KLRB1

[0405] The chimeric antibody 10A3D6-hIgG1k (a mouse / human chimeric antibody having a 10A3D6 variable region and a human IgG1-κ constant region), chimeric mutant variants (10A3D6C109S, 10A3D6C109F, and 10A3D6C109V), and 10A3D6 humanized variants generated by CDR grafting (Almagro et al., 2008) were determined by surface plasmon resonance (SPR) for binding to the human KLRB1 extracellular domain (ECD). The results are shown in Table 54.

[0406] In separate assays, humanized antibodies 10A3D6hum2.2 and 10A3D6hum2.2-afuc (10A3D6hum2.2 produced without fucosylation) were assayed by FACS for binding to CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) and CHO-K1 cells expressing cynomolgus monkey KLRB1 (CHO-cyno-KLRB1) at a concentration range up to 90 nM. Antibody binding to CHO-hum-KLRB1 or CHO-cyno-KLRB1 was detected using a secondary goat anti-mouse peroxidase-labeled IgG. 10A3D6hum2.2 and 10A3D6hum2.2-afuc had similar binding EC50s (see FIG. 24 ).

[0407] Table 54: SPR binding kinetics for 10A3D6, 10A3D6-chimeric variants, and 10A3D6-humanized variants

[0408]

[0409] Example 6: Anti-KLRB1 antibodies can induce antibody-dependent cell-mediated cytotoxicity

[0410] To determine whether the 10A3D6 antibody would effectively kill KLRB1 positive cells, the ability of these antibodies to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) was tested using a reporter gene assay (Genscript ADCC assay using the CD16A expressing reporter cell line GS-J2). CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) were incubated with luminescent reporter cells GS-J2 / CD16A and various antibodies. In contrast to the negative control IgG1, Rituxan (positive control) and the chimeric antibody 10A3D6-hIgG1k (a mouse / human chimeric antibody with the 10A3D6 variable region and the human IgG1-κ constant region) showed activation of the luminescent reporter cells ( Fig.25AIn contrast to the negative control antibody 52G9B12B5-hIgG1kEN (a mouse / human chimeric KLRB1 binding antibody with the 52G9B12B5 variable region and the human IgG1-κ mutant L234A / L235A / G237A mutations), antibody 10A3D6-hIgG1k activated luminescent reporter gene cells ( Fig.25B ).

[0411] In a separate assay, ADCC elicited by the anti-KLRB1 antibodies of the present disclosure was evaluated using a cell lysis assay involving LDH release and formazan detection (Genscript, SC1544). CHO-hum-KLRB1 was incubated with human peripheral blood mononuclear cells (PBMCs) and various antibodies. In contrast to the negative control antibody 52G9B12B5 with effector null mutations, antibody 10A3D6-hIgG1k resulted in target cell lysis ( Fig.26A -B). This confirmed that the ADCC activity of the anti-KLRB1 antibody is mediated through the Fc region of the antibody.

[0412] In a separate assay, a non-fucosylated version of 10A3D6hum2.2 (10A3D6hum2.2-afuc) was generated using FUT8 knockout cells (Satoh et al., 2006). ADCC elicited by 10A3D6hum2.2 and non-fucosylated 10A3D6hum2.2 was determined by incubating the antibody with CHO-K1 cells expressing KLRB1 (CHO-hum-KLRB1) and human NK cells, and target cell death was detected using flow cytometry. 10A3D6hum2.2-afuc had enhanced target cell killing (EC50 0.010 nM) compared to 10A3D6hum2.2 (EC50 0.052) ( Fig. 27 ).

[0413] Example 6: Humanized monoclonal anti-KLRB1 antibody binds to specific immune cells in whole blood

[0414] To determine whether the anti-KLRB1 antibodies of the present disclosure can effectively bind to immune cells, FACS binding assays were performed using blood from three donors (experiments were performed at iQ Biosciences). Fresh whole human blood was incubated with a fixable viability dye, treated with TruStain Fc Blocker (BioLegend) in FACS buffer, and then incubated with 10ug / ml monoclonal humanized antibodies 10A3D6hum1.2, 10A3D6hum1.5, and 10A3D6hum2.2 and a human IgG1 control. All three monoclonal humanized anti-KLRB1 antibodies were found to bind to CD3+ cells at levels significantly higher than the control antibody ( Fig.28A )、CD4+T cells( Fig.28B ), CD8+T cells ( Fig.28C ) and CD3-CD56+NK cells ( Fig.28D Overall, the tested antibodies were positive for CD14++ monocytes ( Fig.28E )、CD14+ neutrophils ( Fig.28F ) or CD19+B cells ( Figure 28G These results indicate that the humanized monoclonal anti-KLRB1 antibodies of the present disclosure bind to specific immune cells (particularly CD8+ and CD4+ T cells and NK cells) in whole blood.

[0415] Example 7: Anti-KLRB1 Antibodies Have Improved Binding in Competition Assays with Commercially Available Anti-KLRB1 Antibodies

[0416] 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 ( Fig.29A ) and CHO-K1 cells expressing cynomolgus macaque KLRB1 ( Fig.29B ) were incubated with the fluorescently labeled antibody 10A3D6hum1.2 and then with increasing concentrations of the commercial antibodies HP-3G10, DX12, and 191B8. The results showed that 10A3D6 bound to a specific epitope more tightly than any other antibody did.

[0417] Example 8: Biophysical properties of anti-KLRB1 antibodies

[0418] 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 (melting temperature).

[0419] 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 are depicted in Fig. 30A -C.

[0420] Table 55: DSC test results of 10A3D6hum1.2, 10A3D6hum1.5 and 10A3D6hum2.2

[0421] Clone ID Tonset Tm1 Tm2 10A3D6hum1.2 64.96℃ 74.10℃ 84.31℃ 10A3D6hum1.5 64.14℃ 73.33℃ 84.57℃ 10A3D6hum2.2 65.09℃ 73.47℃ 78.99℃

[0422] Table 56. Sequences of exemplary antibody variable sequences

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429] Table 57. Sequences of exemplary complete HC and LC regions

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444] Table 58. Sequences of exemplary constant regions

[0445]

[0446]

[0447] Example 9. Depletion of KLRB1+ T cells in cynomolgus monkeys by 3 mg / kg and 10 mg / kg of 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2).

[0448] The antibody 10A3D6hum2.2-afuc was administered intravenously to two cynomolgus monkeys, one at a single dose of 3 mg / kg and the other at a single dose of 10 mg / kg. The blood was analyzed by FACS to detect changes in immune cell populations, using the anti-KLRB1 (anti-CD161) antibody 57E2E4 to detect KLRB1+ cells. These studies showed that the KLRB1+CD8+T cell population was almost completely depleted on days 1, 3, and 7 ( Fig.31 ).

[0449] Example 10. Depletion of KLRB1+ T cells in cynomolgus monkeys by 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg and 3 mg / kg of 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2).

[0450] The antibody 10A3D6hum2.2-afuc was administered intravenously to four cynomolgus monkeys, one monkey at a single dose of 0.1 mg / kg, one monkey at a single dose of 0.3 mg / kg, one monkey at a single dose of 1 mg / kg, and one monkey at a single dose of 3 mg / kg. Blood was analyzed by FACS to detect changes in immune cell populations, using the anti-KLRB1 antibody 57E2E4 to detect KLRB1+ cells. These studies showed that the KLRB1+CD8+T cell population was almost completely depleted at 8 hours after dosing, on days 1, 3, 7, and 35 ( Fig.32A and Fig.32B ). Gene expression of KLRB1 transcripts by quantitative PCR (qPCR) also showed depletion of KLRB1 transcripts ( Fig.32C ).

[0451] Example 11. Pharmacokinetic parameters of 10A3D6hum2.2-afuc in cynomolgus monkeys

[0452] The antibody 10A3D6hum2.2-afuc was administered intravenously to four cynomolgus monkeys, one monkey at a single dose of 0.1 mg / kg, one monkey at a single dose of 0.3 mg / kg, one monkey at a single dose of 1 mg / kg, and one monkey at a single dose of 3 mg / kg. Serum concentrations of 10A3D6hum2.2-afuc were measured and showed acceptable pharmacokinetic parameters ( Fig.33 and Table 59).

[0453] Table 59. Pharmacokinetic parameters

[0454] PK parameters <![CDATA[βT 1 / 2 ]]> CL <![CDATA[MRT last ]]> <![CDATA[AUC last ]]> Vss unit Number of days mL / day / kg Number of days Day*ug / mL mL / kg 0.1 mg / kg IV 3.51 4.8 4.67 20.8 27.3 0.3 mg / kg IV 5.08 2.97 11.42 100.9 33.9 1.0 mg / kg IV 3.37 3.92 8.71 255.2 34.2 3.0 mg / kg IV 9.63 2.61 15.21 1152.2 39.6

[0455] Example 12. Suppression of the Th17 pathway in cynomolgus monkeys by 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2).

[0456] The antibody 10A3D6hum2.2-afuc was administered intravenously to four cynomolgus monkeys, one monkey at a single dose of 0.1 mg / kg, one monkey at a single dose of 0.3 mg / kg, one monkey at a single dose of 1 mg / kg, and one monkey at a single dose of 3 mg / kg. The blood was analyzed by RNAseq to detect changes in Th17 pathway transcripts. These studies showed that the key Th17 transcription factor RORC and the key Th17 cell markers IL-23R and CCR6 receptors were significantly suppressed ( Fig.34 and 35 ).

[0457] Example 13. Suppression of the peTh2 pathway in cynomolgus monkeys by 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2).

[0458] The antibody 10A3D6hum2.2-afuc was administered intravenously to four cynomolgus monkeys, one at a single dose of 0.1 mg / kg, one at 0.3 mg / kg, one at 1 mg / kg, and one at 3 mg / kg. Blood was analyzed by RNAseq to detect changes in pathogenic effector Th2 (peTh2) pathway transcripts. These studies showed that the key peTh2 transcription factors GATA3 and RORA, as well as the key peTh2 cell marker CRTH2, were significantly suppressed ( Fig.36 and 37 ).

[0459] Example 14. Suppression of the Th1 pathway in cynomolgus monkeys by 10A3D6hum2.2-afuc (afucosylated 10A3D6hum2.2).

[0460] The antibody 10A3D6hum2.2-afuc was administered intravenously to four cynomolgus monkeys, one monkey at a single dose of 0.1 mg / kg, one monkey at a single dose of 0.3 mg / kg, one monkey at a single dose of 1 mg / kg, and one monkey at a single dose of 3 mg / kg. The blood was analyzed by RNAseq to detect changes in Th1 and CD8 effector pathway transcripts. These studies showed that the key Th1 and CD8 effector transcription factors Tbet (TBX21) and EOMES were significantly suppressed ( Fig.38 and 39 ).

[0461] Example 15. KLRB1 ECD Binding

[0462] The antibodies disclosed herein and appropriate controls were determined by ELISA for binding to the extracellular domain (ECD) of human and cynomolgus KLRB1 proteins. The ELISA plate was coated with 1 μg / ml KLRB1 ECD overnight at 4°C, and then the primary antibody (for humans, 3nM starting concentration and its 3-fold dilution; for cynomolgus monkeys, 10nM starting concentration and its 5-fold dilution) was added at 37°C for 1 hour, and then the labeled secondary anti-human IgG antibody was added at 1:5000 at 37°C and kept for 45 minutes. The amount of antibody bound to KLRB1 is determined by the signal intensity of the bound second antibody.

[0463] The antibodies disclosed herein and appropriate controls (e.g., antibodies known in the art or described herein, 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 a similar method, such as bio-layer interferometry (BLI)) are assayed for expression of 5-mercaptoethanol (e.g., 10-mercaptoethanol (10-mercaptoethanol)) in an amount of 100 ng / mL (about 200 ng / mL). Diagnostics) or US20210122826 (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)) to the extracellular domain (ECD) of human or cynomolgus monkey KLRB1. SPR is performed using Biacore, where the binding of a ligand to its receptor is detected by a change in the refractive index on the sensor surface. BLI is performed using Octet, where the binding of a ligand to its receptor is detected by a change in the optical thickness in the biolayer.

[0464] The disclosed antibodies may have increased binding potency and lower EC50 for binding to cell-expressed KLRB1 or soluble KLRB1 extracellular domain compared to a control.

[0465] Example 16. Antibody-dependent cell-mediated cytotoxicity (ADCC) activation

[0466] The antibodies disclosed herein and appropriate controls (e.g., antibodies known in the art or described herein, e.g., B199.2 (Invitrogen), HP-3G10 (Invit rogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics) are assayed using a reporter gene assay (Genscript ADCC assay using the reporter cell line GS-J2 expressing CD16A). cs) or US20210122826 (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)) antibody-dependent cell-mediated cytotoxicity (ADCC). CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) were incubated with luminescent reporter gene cells GS-J2 / CD16A and various antibodies. In a separate assay, ADCC triggered by the antibody was evaluated using a cell lysis assay involving LDH release and formazan detection (Gen script, SC1544). CHO-hum-KLRB1 was incubated with human peripheral blood mononuclear cells (PBMCs) and the antibody. The antibody exhibited increased ADCC-mediated depletion potency and lower EC50 for depletion of CHO-KLRB1+ cells or lower EC50 for reporter cell line ADCC assays compared to controls.

[0467] Example 17. Additional biochemical and biophysical characterization of antibodies

[0468] The antibodies disclosed herein and appropriate controls (e.g., antibodies known in the art or described herein, 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 US20210122826 described antibodies (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) and FACS competition assay. CHO-hum-KLRB1 or CHO-cyno-KLRB1 cells were incubated with biotin-labeled CLEC2D, and the fluorescent signal was detected by FACS. The IC50 potency of the inhibition or enhancement of the antibody was measured using the ability of the antibody to block CLEC2D within a certain concentration range (e.g., 0 to 200 nM) and cause loss or increase of the fluorescent signal.

[0469] When compared to the control group, the antibody had increased binding affinity kinetics (lower K D or lower Koff ), lower IC50 for inhibiting CLEC2D binding, such as binding to cells expressing KLRB1, and thus having increased blocking potency; and lower EC50 for enhancing (increasing) CLEC2D binding, such as binding to cells expressing KLRB1, and thus having increased enhancing potency.

[0470] Differential scanning calorimetry (DSC) was performed to measure antibody T onset (the onset temperature at which melting / unfolding is first detected) and T m (Melting Temperature). The antibody has improved biophysical parameters (such as higher melting temperature, higher freeze-thaw stability, lower isomerization, reduced or absent deamidation, and / or lower oxidative sensitivity) when compared to a control.

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[0475] Fergusson,J.R.,et al.(2014).″CD161 defines a transcriptional andfunctional phenotype across distinct human T cell lineages.″Cell Rep 9(3):1075-1088.

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Claims

1. An antibody or antigen-binding portion thereof that specifically binds to KLRB1 (optionally SEQ ID NO: 1 or SEQ ID NO: 2), wherein the antibody or antigen-binding portion thereof comprises at least one of the following: (a) a heavy chain variable region (VH), comprising a VH complementarity determining region (CDR) 1 comprising a sequence at least 95% identical to a VH CDR1 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; a VH CDR2 comprising a sequence at least 95% identical to a VH CDR2 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and a VH CDR3 comprising a sequence at least 95% identical to a VH CDR3 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and (b) a light chain variable region (VL), comprising a VL CDR1 comprising a sequence that is at least 95% identical to a VL CDR1 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; a VL CDR2 comprising a sequence that is at least 95% identical to a VL CDR2 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53; and a VL CDR3 comprising a sequence that is at least 95% identical to a VL CDR3 amino acid sequence as shown in Tables 1 to 53, preferably one of Tables 1, 30, 31, 32, or 45 to 53.

2. The antibody or antigen binding portion thereof of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Tables 1, 30, 31, 32, or 45 to 53.

3. The antibody or antigen binding portion thereof of claim 1, wherein the VL comprises or consists of a VL amino acid sequence as shown in Tables 1, 30, 31, 32, or 45 to 53.

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

5. The antibody or antigen binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 30, and the VL comprises or consists of the VH amino acid sequence shown in Table 30.

6. The antibody or antigen-binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 31, and the VL comprises or consists of the VH amino acid sequence shown in Table 31.

7. The antibody or antigen binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 32, and the VL comprises or consists of the VH amino acid sequence shown in Table 32.

8. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 45, and the VL comprises or consists of the VH amino acid sequence shown in Table 45.

9. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 46, and the VL comprises or consists of the VH amino acid sequence shown in Table 46.

10. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 47, and the VL comprises or consists of the VH amino acid sequence shown in Table 47.

11. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Table 48, and the VL comprises or consists of a VH amino acid sequence as shown in Table 48.

12. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Table 49, and the VL comprises or consists of a VH amino acid sequence as shown in Table 49.

13. The antibody or antigen binding portion thereof of claim 1, wherein the VH comprises or consists of the VH amino acid sequence shown in Table 50, and the VL comprises or consists of the VH amino acid sequence shown in Table 50.

14. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Table 51, and the VL comprises or consists of a VH amino acid sequence as shown in Table 51.

15. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Table 52, and the VL comprises or consists of a VH amino acid sequence as shown in Table 52.

16. The antibody, or antigen binding portion thereof, of claim 1, wherein the VH comprises or consists of a VH amino acid sequence as shown in Table 53, and the VL comprises or consists of a VH amino acid sequence as shown in Table 53.

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

18. The antibody or antigen binding portion thereof of claim 1, wherein the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to a VH amino acid sequence shown in one of Tables 30, 31, 32, or 45 to 53, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to an amino acid sequence shown in one of Tables 30, 31, 32, or 45 to 53, preferably wherein the VH and VL are from the same table.

19. The antibody or antigen binding portion thereof of claims 1-18, comprising a constant region heavy chain and a light chain, wherein the constant region heavy chain and / or light chain comprises or consists of an amino acid sequence as shown in Table 58.

20. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

21. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

22. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

23. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

24. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

25. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

26. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

27. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: A variable region heavy chain consisting of the HC amino acid sequence shown in Table 48 and a variable region light chain consisting of the LC amino acid sequence shown in Table 48, and optionally a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

28. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

29. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen-binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

30. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

31. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

32. An antibody or antigen-binding portion thereof that specifically binds to human killer cell lectin-like receptor subfamily B member 1 (KLRB1; SEQ ID NO: 1), the antibody or antigen-binding portion thereof comprising or consisting of: 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 a constant region, optionally wherein the antibody or its antigen binding portion comprises a sequence that is at least 95% identical to the sequence shown in Table 57.

33. The antibody or antigen binding portion thereof of claim 1, further comprising a heavy chain constant region hinge region and an Fc domain.

34. The antibody or antigen binding portion thereof of claim 1, wherein the antibody or antigen binding portion thereof is an antibody comprising a heavy chain constant region comprising an amino acid sequence having at least 80%, 90%, 95% or 97% sequence identity with the heavy chain constant region amino acid sequence shown in Table 58.

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

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

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

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

39. The antibody, or antigen binding portion thereof, of claim 1, conjugated to a cytotoxic agent.

40. The antibody, or antigen binding portion thereof, of claim 1, comprising an afucosylated Fc region.

41. A polynucleotide comprising a nucleic acid sequence encoding the antibody or antigen binding portion thereof of claims 1 to 40.

42. The polynucleotide of claim 41, wherein the nucleic acid sequence is operably linked to a promoter.

43. A vector comprising the polynucleotide of any one of claims 41-42.

44. A host cell comprising the polynucleotide of any one of claims 41-42, or the vector of claim 43, and optionally expressing the antibody or antigen binding portion thereof.

45. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof as claimed in any one of the preceding claims, and a pharmaceutically acceptable carrier or diluent.

46. ​​A method of preparing the antibody or antigen binding portion thereof of any of the preceding claims, the method comprising culturing the host cell of claim 44 under conditions sufficient for expression of the antibody or antigen binding portion thereof and isolating the antibody or antigen binding portion thereof.

47. The method of claim 46, further comprising formulating the antibody as a pharmaceutical composition.

48. A method for treating one or more of an autoimmune disease, an allergic disease, a transplant rejection and a hematological malignancy in a subject in need thereof, the method comprising administering to the subject an antibody that binds KLRB1, preferably the antibody of any one of claims 1-41, the polynucleotide of any one of claims 41-42, the vector of claim 43, the pharmaceutical composition of claim 45, or the host cell of claim 44 expressing the antibody or antigen-binding portion thereof.

49. The method of claim 48, wherein the autoimmune disease is autoimmune hepatitis, rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.

50. The method of claim 48, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.

51. The method of claim 48, wherein the transplant rejection can be rejection of a kidney, lung, heart, liver, limb, skin or multi-organ transplant.

52. The method of claim 48, wherein the hematological malignancy is a lymphoma or a leukemia.

53. The method of claim 52, wherein the lymphoma is NK / T cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), or peripheral T cell lymphoma not otherwise specified (PTCL-NOS).

54. The method of claim 52, wherein the leukemia is T-cell leukemia, aggressive NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).

55. The antibody of any one of claims 1-40, the polynucleotide of any one of claims 41-42, the vector of claim 43, or the method of claims 48-54, wherein the antibody binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), type 2 and type 3 innate lymphoid cells (ILC2 and ILC3), pathogenic effector Th2 (peTh2) cells, NK cells and / or neoplastic T or NK cells expressing KLRB1.

56. The antibody of any of claims 1-40, which is not clone B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), or JNH25G2G22 (Creative Diagnostics), B199.2 (Invitrogen), 191B8 (Miltenyi), DX12 (BD Biosciences), JNH25G2G22 (Creative Diagnostics), 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.

57. The antibody of any one of claims 1-40, the polynucleotide of any one of claims 41-42, or the vector of claim 43, for use in a method of treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, and a hematological malignancy in a subject in need thereof.

58. The antibody, polynucleotide or vector for use as claimed in claim 57, wherein the autoimmune disease is autoimmune hepatitis, rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis or hidradenitis suppurativa.

59. The antibody, polynucleotide or vector for use as claimed in claim 57, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyposis, eosinophilic gastrointestinal disorder or hypereosinophilic syndrome.

60. The antibody, polynucleotide or vector for use as claimed in claim 57, wherein the transplant rejection can be rejection of a kidney, lung, heart, liver, limb, skin or multi-organ transplant.

61. The antibody, polynucleotide or vector for use as claimed in claim 57, wherein the hematological malignancy is a lymphoma or a leukemia.

62. The antibody, polynucleotide or vector for use as claimed in claim 61, wherein the lymphoma is hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL) or peripheral T-cell lymphoma not otherwise specified (PTCL-NOS).

63. The antibody, polynucleotide or vector for use according to claim 61, wherein the leukemia is T cell leukemia, NK cell leukemia, T cell prolymphocytic leukemia (T-PLL) or large granular lymphocytic leukemia (LGLL).

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