KLRB1 binder and its application method

Abstract

KLRB1 binders with increased human characteristics (particularly anti-KLRB1 antibodies and their antigen-binding moieties) and combinations thereof, and treatments using these agents, for example, depleted or inhibited or activated cells (particularly in vivo Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, peTh2, ILC2, ILC3, NK cells and / or neoplastic T cells or NK cells), to treat autoimmune diseases, allergic diseases, graft rejection, hematologic malignancies and cancers.

Description

Priority requirements

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 601,664, filed November 21, 2023. The entire foregoing is hereby incorporated by reference.

[0002] Statement Regarding Federal Government-Sponsored Research This invention was carried out with government support under approval HL119145 granted by the National Institutes of Health. The government owns certain rights to this invention. Technical Field

[0003] This disclosure generally relates to KLRB1 binders, particularly anti-KLRB1 antibodies, and treatments using these agents for autoimmune diseases, allergic diseases, graft rejection, hematologic malignancies, and cancers.

[0004] sequence list This application contains a sequence list that has been submitted electronically as an XML file named “29618-0484WO1 _ST26_SL.XML”. This XML file was created on November 19, 2024, and is 135,544 bytes in size. The material in the XML file is hereby incorporated in its entirety by reference. Background Technology

[0005] The expression of cytotoxic lectin-like receptor B subfamily member 1 (KLRB1; also known as CD161) defines a unique population of immune cells 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 are associated with psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (Yang et al., 2014), and other autoimmune diseases. Monoclonal antibody therapeutics targeting IL-17 or stimulating the upstream cytokine IL-23 of IL-17-producing cells have been approved by the FDA and marketed for psoriasis (e.g., secukinumab and ixekizumab) and ankylosing spondylitis (e.g., secukinumab). However, the development of therapeutics targeting these molecules remains limited, and new therapeutics utilizing these targets are still needed for a wide range of indications, including autoimmune diseases, allergic diseases, inflammatory diseases, and cancer. Summary of the Invention

[0006] This article describes KLRB1-binding antibodies (and their antigen-binding fragments) with increased humanity and / or reduced potential deamidation and / or isomerization sites, possessing multiple uses (including therapeutic and diagnostic applications). For example, these antibodies can be used to treat and, in some cases, prevent various diseases associated with KLRB1-expressing cells (i.e., by depleting KLRB1-expressing cells), for example, to treat and, in some cases, prevent the following diseases (i.e., reduce the risk of developing): autoimmune diseases, including psoriasis, psoriatic arthritis, ankylosing spondylitis, palmoplantar pustulosis, hidradenitis suppurativa, and inflammatory bowel disease; allergic diseases, including asthma and atopic dermatitis; graft rejection; hematologic malignancies and cancer.

[0007] This article provides an antibody or antigen-binding moiety thereof that specifically binds to a member of the cytotoxic lectin-like receptor B subfamily 1 (KLRB1; optionally SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3), wherein the antibody or antigen-binding moiety thereof comprises or consists of at least one of the following: (a) a heavy chain variable region (VH) comprising a VH complementarity-determining region (CDR) 1, the VH complementarity-determining region (CDR) 1 comprising a sequence that is at least 95% identical to the amino acid sequence of VH CDR1 shown in Tables 1 to 17, preferably Tables 10, 14 or 4; VH CDR2, the VH CDR2 comprising a sequence that is at least 95% identical to the amino acid sequence of VH CDR2 shown in Tables 1 to 17, preferably Tables 10, 14 or 4; and VH CDR3, the VH CDR3 comprising a sequence that is at least 95% identical to the amino acid sequence of VH CDR2 shown in Tables 1 to 17, preferably Tables 10, 14 or 4; and VH CDR3, the VH CDR3 comprising a sequence that is at least 95% identical to the amino acid sequence of VH CDR2 shown in Tables 1 to 17, preferably Tables 10, 14 or 4. (a) a CDR3 amino acid sequence that is at least 95% identical to the CDR3 amino acid sequence; and (b) a light chain variable region (VL) comprising VL CDR1, which contains a sequence that is at least 95% identical to the VL CDR1 amino acid sequence shown in Tables 1 to 17, preferably Tables 10, 14 or 4; VLCDR2, which contains a sequence that is at least 95% identical to the VL CDR2 amino acid sequence shown in Tables 1 to 17, preferably Tables 10, 14 or 4; and VL CDR3, which contains a sequence that is at least 95% identical to the VLCDR3 amino acid sequence shown in Tables 1 to 17, preferably Tables 10, 14 or 4.

[0008] In some embodiments, this document describes an antibody or antigen-binding fragment thereof that specifically binds to cytotoxic lectin-like receptor B subfamily member 1 (KLRB1), the antibody or antigen-binding fragment thereof comprising: a. A heavy chain variable region (VH) comprising three VH complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3); and b. Light chain variable region (VL), which contains three VL complement-determining regions (CDR-L1, CDR-L2, and CDR-L3). CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are selected from one of the following: i. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55, and 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27, and 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 17-21; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or v. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49 or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49, or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or viii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 58-60; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ix. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or x. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 60 or 66; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 69 or 70; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xiii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28, 30 or 76; and CDR-L3 has the amino acid sequence of SEQ ID NO: 77 or 78; or xiv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 84 or 85; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvii. CDR-H1 has an amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has an amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has an amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has an amino acid sequence of any one of SEQ ID NO: 87-89; CDR-L2 has an amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has an amino acid sequence of SEQ ID NO: 81 or 82.

[0009] In some embodiments, the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are selected from the same antibody numbering rule. In some embodiments, the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 are selected from the Kabat antibody numbering rule.

[0010] In some embodiments, CDR-H1 has the amino acid sequence of SEQ ID NO: 14, CDR-H2 has the amino acid sequence of SEQ ID NO: 37, CDR-H3 has the amino acid sequence of SEQ ID NO: 22, CDR-L1 has the amino acid sequence of SEQ ID NO: 64, CDR-L2 has the amino acid sequence of SEQ ID NO: 28, and CDR-L3 has the amino acid sequence of SEQ ID NO: 31.

[0011] In some embodiments, VH has an amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62, or 68, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62, or 68 and having the CDR-H1, CDR-H2, or CDR-H3 amino acid sequences shown herein (e.g., above in i–xvii). In some embodiments, VH comprises an amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62, or 68. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 68.

[0012] In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86, or 90, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86, or 90 and having the CDR-L1, CDR-L2, CDR-L3 amino acid sequences shown herein (e.g., above in i–xvii). In some embodiments, VL comprises the amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86, or 90. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 65.

[0013] In some implementations, VL and VH are selected from one of the following: a. VH contains SEQ ID NO: 68; and VL contains any one of SEQ ID NO: 65, 71, 75, 79, 80, 83, 86 or 90; b. VH contains any one of SEQ ID NO: 33, 42 or 48; and VL contains SEQ ID NO: 34; c. VH contains any one of SEQ ID NO: 51, 53 or 57; and VL contains SEQ ID NO: 52; d. VH contains SEQ ID NO: 62; and VL contains SEQ ID NO: 63 or SEQ ID NO: 65; or e. VH contains SEQ ID NO: 57; and VL contains SEQ ID NO: 67.

[0014] In some embodiments, VH comprises SEQ ID NO:68; and VL comprises SEQ ID NO:65. In some embodiments, VL is part of a light chain comprising the amino acid sequence of any one of SEQ ID NO:99-110, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO:99-110. In some embodiments, the light chain comprises the amino acid sequence of any one of SEQ ID NO:99-110. In some embodiments, the light chain comprises the amino acid sequence of SEQ ID NO:102.

[0015] In some embodiments, this document discloses antibodies or antibody fragments thereof comprising an Fc region that bind to an Fc γ receptor (FcγR) and induce antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or bind to C1q and induce complement-dependent cytotoxicity (CDC). In some embodiments, the Fc region is unfucosylated. In some embodiments, the antibody is an immunoglobulin G (IgG) isoform IgG1 antibody, IgG2 antibody, or IgG4 antibody.

[0016] In some embodiments, VH is part of the heavy chain, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NO: 91-98, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 91-98. In some embodiments, the heavy chain comprises the amino acid sequence of any one of SEQ ID NO: 91-98. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 98.

[0017] In some implementations, the heavy chain (HC) and the light chain (LC) are selected from one of the following: a. HC contains SEQ ID NO: 98; and LC contains SEQ ID NO: 102 or any one of 104-110; b. HC contains any one of SEQ ID NO: 91-93; and LC contains SEQ ID NO: 99; c. HC contains any one of SEQ ID NO: 94-96; and LC contains SEQ ID NO: 100; d. HC contains SEQ ID NO: 97; and LC contains SEQ ID NO: 101 or SEQ ID NO: 102; or e. HC contains SEQ ID NO: 96; and LC contains SEQ ID NO: 103.

[0018] In some implementations, HC contains SEQ ID NO:98 and LC contains SEQ ID NO:102.

[0019] In some implementations, the antibody or an antibody fragment thereof is conjugated to a cytotoxic agent.

[0020] This article also discloses a polynucleotide set, which includes: a. A first nucleic acid sequence encoding the VH or heavy chain of an antibody or its antigen-binding moiety as disclosed herein (e.g., any of the embodiments above); and b. A second nucleic acid sequence encoding the VL or light chain of an antibody or its antigen-binding portion as described herein (e.g., any of the embodiments above).

[0021] In some implementations, each of the first and second nucleic acid sequences is operatively linked to a promoter. Vectors containing a set of polynucleotides on the same vector are also disclosed herein. A collection of vectors comprising: a. A first nucleic acid sequence comprising a VH or heavy chain encoding an antibody or its antigen-binding moiety as disclosed herein (e.g., any of the embodiments above); and b. A second vector comprising a second nucleic acid sequence encoding an antibody or its antigen-binding portion as described herein (e.g., any of the embodiments above).

[0022] This article also describes host cells that contain the polynucleotide set described herein, or the vector described herein, or the vector set described herein, and optionally express the antibody described herein or its antigen-binding portion.

[0023] This article also describes a method for preparing any of the antibodies or antigen-binding moieties described herein, the method comprising: a. Culture the host cells described herein under conditions sufficient to express the antibody or its antigen-binding moiety; and b. Separate the antibody or its antigen-binding portion.

[0024] In some embodiments, the method further includes formulating the antibody into a pharmaceutical composition. In some embodiments, VH comprises or consists of the VH amino acid sequences shown in Tables 10, 14, or 4. In some embodiments, VL comprises or consists of the VL amino acid sequences shown in Tables 10, 14, or 4. In some embodiments, VH comprises or consists of the VH amino acid sequences shown in Table 14, and VL comprises or consists of the VH amino acid sequences shown in Table 14. In some embodiments, VH comprises or consists of the VH amino acid sequences shown in Table 10, and VL comprises or consists of the VH amino acid sequences shown in Table 10. In some embodiments, VH comprises or consists of the VH amino acid sequences shown in Table 4, and VL comprises or consists of the VH amino acid sequences shown in Table 4. In some embodiments, VH comprises or consists of an amino acid sequence having at least 95% sequence identity with the VH amino acid sequences shown in Table C, and VL comprises or consists of an amino acid sequence having at least 95% sequence identity with the VL amino acid sequences shown in Table C. In some embodiments, VH comprises an amino acid sequence having at least 95% sequence identity with the VH amino acid sequence shown in one of Tables 10, 14 or 4, or consists of an amino acid sequence having at least 95% sequence identity with the VH amino acid sequence shown in one of Tables 10, 14 or 4, and VL comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in one of Tables 10, 14 or 4, or consists of an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in one of Tables 10, 14 or 4, preferably wherein VH and VL are from the same table.

[0025] In some embodiments, the antibody or its antigen-binding portion 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 Tables A.

[0026] In some embodiments, the antibody or its antigen-binding portion 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 Tables D.

[0027] In addition, this article provides an antibody or antigen-binding moiety thereof that specifically binds to a member of the human cytotoxic lectin-like receptor B subfamily 1 (KLRB1; for example, SEQ ID NO: 1), the antibody or antigen-binding moiety thereof comprising or consisting of: a variable region heavy chain consisting of the VH amino acid sequences shown in Table 14 and a variable region light chain consisting of the VL amino acid sequences shown in Table 14, and an optional constant region that optionally contains at least 95% identical sequences to those shown in Table A.

[0028] In addition, this article provides an antibody or antigen-binding moiety thereof that specifically binds to a member of the human cytotoxic lectin-like receptor B subfamily 1 (KLRB1; for example, SEQ ID NO: 1), the antibody or antigen-binding moiety thereof comprising or consisting of: a variable region heavy chain consisting of the VH amino acid sequences shown in Table 10 and a variable region light chain consisting of the VL amino acid sequences shown in Table 10, and an optional constant region that optionally contains at least 95% identical sequences to those shown in Table A.

[0029] In addition, this article provides an antibody or antigen-binding moiety thereof that specifically binds to a member of the human cytotoxic lectin-like receptor B subfamily 1 (KLRB1; for example, SEQ ID NO: 1), the antibody or antigen-binding moiety thereof comprising or consisting of: a variable region heavy chain consisting of the VH amino acid sequences shown in Table 4 and a variable region light chain consisting of the VL amino acid sequences shown in Table 4, and an optional constant region that optionally contains at least 95% identical sequences to those shown in Table A.

[0030] In some implementations, the constant region contains or consists of sequences as shown in Table A.

[0031] In some embodiments, the antibody comprises a heavy chain variable sequence and / or a light chain variable sequence that is at least 95% identical to the sequence shown in Table B, or is composed 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 B.

[0032] In some embodiments, the antibody comprises or consists of a complete heavy chain and / or light chain sequence that is at least 95% identical to the sequence shown in Table C.

[0033] This document also provides antibodies or antigen-binding moieties thereof that specifically bind to human KLRB1, the antibodies or antigen-binding moieties thereof comprising CDRs from different tables herein, or heavy / light chain pairs from different tables herein. In some embodiments, the antibodies or antigen-binding moieties thereof comprise a heavy chain constant region hinge region and an Fc domain.

[0034] In some embodiments, the antibody or its antigen-binding portion is an antibody comprising a heavy chain constant region containing 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 A.

[0035] In some implementations, the antibody or its antigen-binding portion is a monoclonal antibody.

[0036] In some embodiments, the antibody or its antigen-binding portion is chimeric, humanized, or human antibody, and / or contains one or more mutations (e.g., in CDR) that remove the Asn(N)-glycosylation site or remove Cys, Asp, Met, Trp, or Lys.

[0037] In some implementations, the antibody or its antigen-binding portion is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody, or IgG4 antibody.

[0038] In some embodiments, the antibody or its antigen-binding portion is an antibody containing an Fc region (preferably human IgG1) that binds to the Fc γ 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).

[0039] In some implementations, the antibody or its antigen-binding portion is conjugated to a cytotoxic agent.

[0040] In some implementations, the antibody or its antigen-binding portion includes an unfucosylated Fc region.

[0041] Because CD161 is an inhibitory receptor when it binds to its ligand CLEC2D (LLT1), regulation of this interaction can affect immune cell activity. Therefore, inhibiting the CD161 / CLEC2D interaction enhances T cell function, a typical mechanism of action for this type of immuno-oncology therapeutic, while enhancing the CD161 / CLEC2D interaction can be used for immunosuppression in autoimmune and allergic diseases. In some embodiments, the antibodies disclosed herein block the CD161 / CLEC2D interaction, thereby activating T or NK cells to attack tumor cells in various cancers, a hallmark of immuno-oncology checkpoint inhibition mechanisms.

[0042] In some implementations, the antibodies disclosed herein enhance (e.g., increase) CD161 / CLEC2D interaction, thereby suppressing T or NK cells and providing immunosuppression suitable for the treatment of a variety of autoimmune, allergic, and inflammatory diseases.

[0043] In some embodiments, the KLRB1 binding antibody disclosed herein achieves better immune cell activation, for example, by exhibiting improvements in any one or more activities compared to immune cell activation by one or more prior antibodies (e.g., B199.2, HP-3G10, OTI1D8, 14F1F11, 702228, B-D51, 2F3, EP7169, DX1, DX12, 191B8, Ab9, KW1.2.1, KW7.3.7, or JNH25G2G22), such as activation of T cells to produce cytokines, activation of T cells to kill tumor cells, activation of NK cells (e.g., increased expression of CD107a), activation of NK cells to produce cytokines or cytotoxic molecules (such as granzymes), and activation of NK cells to kill tumor cells.

[0044] In some embodiments, the antibodies disclosed herein with N-terminal glutamine or glutamate can be post-translational modified at such N-terminus to form pyroglutamic acid (or pyrrolidone carboxylic acid).

[0045] This document also provides polynucleotides comprising nucleic acid sequences encoding antibodies or their antigen-binding moieties as described herein. In some embodiments, this nucleic acid sequence is operatively linked to a promoter.

[0046] Additionally, a vector comprising the polynucleotide described herein is provided, as well as a host cell comprising the polynucleotide or the vector and optionally expressing an antibody or its antigen-binding moiety as described herein. Methods for preparing antibodies or their antigen-binding moieties as described herein are also provided herein. These methods may include culturing host cells under conditions sufficient to express the antibody or its antigen-binding moieties and isolating the antibody or its antigen-binding moieties. In some embodiments, the method includes formulating the antibody into a pharmaceutical composition.

[0047] Furthermore, this document provides pharmaceutical compositions comprising an antibody or its antigen-binding moiety as described herein (e.g., comprising the antibody or its antigen-binding moiety as an active agent or consisting of the antibody or its antigen-binding moiety as an active agent) and a pharmaceutically acceptable carrier or diluent.

[0048] In some embodiments, the antibody or its antigen-binding moiety is not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), DX1 (Thermo Fisher), DX12 (BD Biosciences), 191B8 (Miltenyi Biotec), Ab9 (PCT publication WO2023028501A1), KW1.2.1 (US Patent publication US20210122826A1), KW7.3.7 (US Patent publication US20210122826A1), or JNH25G2G22 (Creative Diagnostics).

[0049] This document also provides a method for treating one or more of autoimmune diseases, allergic diseases, graft rejection, and hematologic malignancies in subjects of need, comprising administering to the subject an effective amount of an antibody or antigen-binding moiety of KLRB1, preferably an antibody or antigen-binding moiety of KLRB1 as described herein, a polynucleotide, a carrier, a pharmaceutical composition, or a host cell expressing the antibody or antigen-binding moiety of KLRB1. An antibody binding to KLRB1, preferably an antibody or antigen-binding moiety of KLRB1 as described herein, a polynucleotide, a carrier, a pharmaceutical composition, or a host cell expressing the antibody or antigen-binding moiety of KLRB1, is also provided for use in a method for treating one or more of autoimmune diseases, allergic diseases, graft rejection, and hematologic malignancies in subjects of need. In some embodiments, the autoimmune disease is rheumatoid arthritis, Sjogren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes mellitus, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa. In some embodiments, the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, graft rejection can be rejection of kidney, lung, heart, liver, limb, skin, or multi-organ grafts. In some embodiments, the hematologic malignancy is leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell prolymphoblastic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL). In some embodiments, the hematologic malignancy is lymphoma, such as 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 (PTCL-NOS) unless otherwise specified.

[0050] In some embodiments, the antibody or its antigen-binding moiety, polynucleotide, vector, antibody binding and depletion of Th17, Th17.1, ex-Th17, Tc17, mucosa-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 and / or NK cells expressing KLRB1.

[0051] In some implementations of each of the aspects and implementation schemes mentioned above, as well as in other aspects and implementation schemes described herein, the subject is a human being.

[0052] Unbound by theory, the applicant believes that the antibody disclosed herein will exhibit superiority over previously described antibodies (e.g., commercially available antibodies) in one or more of the following activities: - Increased binding potency (binding to cellularly expressed KLRB1 or the extracellular domain of soluble KLRB1 EC) 50 (lower); - ADCC-mediated increased exhaustion potency (lower EC50 in CHO-KLRB1+ cells or EC50 as measured by ADCC in reporter gene cell lines) 50 (lower); - Increased binding affinity kinetics (lower KD or K off (lower); - Increased blocking efficacy (IC50) of CLEC2D inhibiting the binding of CLEC2D to cell-expressed KLRB1 50 (lower); and / or - Enhanced potency (enhanced binding of CLEC2D to KLRB1 expressed in EC cells) 50 (Lower).

[0053] In some embodiments, the antibodies disclosed herein exhibit 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, and / or lower oxidative sensitivity) compared to previously described antibodies (e.g., commercially available antibodies). Examples of previously described antibodies include B199.2, HP-3G10, OTI1D8, 14F1F11, 702228, B-D51, 2F3, EP7169, DX1, DX12, 191B8, Ab9, KW1.2.1, KW7.3.7, or JNH25G2G22.

[0054] When aspects or embodiments of this disclosure are described in the form of Markush groups or other alternative groupings, this disclosure covers not only the entire group listed as a whole, but also each individual member of that group and all possible subgroups of the main group, as well as the main group lacking one or more members. This disclosure also contemplates the explicit exclusion of one or more members from any of the claimed disclosures.

[0055] These and other advantages of this technology will become apparent from the accompanying drawings and the following description. Attached Figure Description

[0056] Figure 1KLRB1 expression is shown to be a marker for Th17, Th17.1, ex-Th17, Tc17, iNKT, IL2, ILC3, peTh2, and subsets of NK cells.

[0057] Figure 2 The data shown are whole-body KLRB1 expression profiling data, which indicate that KLRB1 is not significantly expressed in any cell type other than immune cells.

[0058] Figure 3 The results showed that KLRB1 was overexpressed in the synovium of rheumatoid arthritis compared with osteoarthritis and normal synovium.

[0059] Figure 4 The results showed that KLRB1 was overexpressed in the synovium of rheumatoid arthritis compared to the synovium of osteoarthritis and crystal-induced arthritis.

[0060] Figure 5 The results showed that KLRB1 was overexpressed in the salivary glands of late and moderate Sjögren's syndrome compared to normal conditions.

[0061] Figure 6 The results showed that KLRB1 was overexpressed in the parotid glands of Sjögren's syndrome compared to normal conditions.

[0062] Figure 7 The study showed that KLRB1 was overexpressed in muscle tissue from patients with inclusion body myositis.

[0063] Figure 8 The study showed that KLRB1 was overexpressed in skin tissue from patients with discoid lupus.

[0064] Figure 9 The study showed that KLRB1 was overexpressed in skin tissue from patients with psoriasis.

[0065] Figure 10 The study showed that KLRB1 was overexpressed in lung tissue from patients with idiopathic pulmonary fibrosis.

[0066] Figure 11 The study showed that KLRB1 was overexpressed in pancreatic tissue from patients with diabetes.

[0067] Figure 12 The study showed that KLRB1 was overexpressed in scalp tissue from patients with alopecia universalis.

[0068] Figure 13 The study showed that KLRB1 was overexpressed in liver tissue from patients with primary biliary cholangitis.

[0069] Figure 14The study showed that KLRB1 was overexpressed in brain tissue from patients with multiple sclerosis.

[0070] Figure 15 The study showed that KLRB1 was overexpressed in colon tissue from patients with lymphocytic colitis.

[0071] Figure 16 The study showed that KLRB1 was overexpressed in kidney tissue from patients with kidney transplants.

[0072] Figure 17 KLRB1 was found to be overexpressed in bronchoalveolar lavage fluid from patients with lung transplants.

[0073] Figure 18 The study showed that KLRB1 was overexpressed in skin tissue from patients with atopic dermatitis.

[0074] Figure 19 The study showed that KLRB1 was overexpressed in skin tissue from patients with palmoplantar pustulosis.

[0075] Figure 20 The study showed that KLRB1 was overexpressed in skin tissue from patients with hidradenitis suppurativa.

[0076] Figure 21 The study showed that KLRB1 was overexpressed in airway brushings from patients with asthma.

[0077] Figure 22A -D indicates that KLRB1 is expressed in various T and NK cell lymphomas and leukemias. Figure 22A Increased KLRB1 expression was observed in tumor cells from 4 / 4 of patients with hepatosplenic T-cell lymphoma (HSTCL), 7 / 19 of patients with NK / T-cell lymphoma (NKTCL), and 2 / 2 of patients with mycosis fungoides. Figure 22B This study demonstrates KLRB1 expression 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 (PTCL-NOS) without further specification, and T-cell prolymphocytic leukemia (T-PLL). Figure 22C The results showed that in four patients with HSTCL, KLRB1 expression was increased in splenic tumor cells compared to healthy spleens, and KLRB1 expression from tumor cells was increased compared to healthy CD4 T cells. Figure 22DIncreased KLRB1 expression was observed in the HSTCL cell line (DERL-2) and the aggressive NK cell leukemia (ANKL) cell lines (KHYG-1 and NKL).

[0078] Figure 23 This assay demonstrates the measurement of target cell lysis in antibody-dependent cell-mediated cytotoxicity (ADCC) lysis of CHO-K1 target cells (CHO-hum-KLRB1) expressing human KLRB1, incubated with human peripheral blood mononuclear cells (PBMCs) and indicated antibodies 12.0, 13.2, 2.2, 11, and human IgG1 (negative control). The percentage of target cell lysis for each test antibody at the indicated concentrations is shown. The assay utilizes LDH release and formazan salt detection (Genscript, SC1544). Detailed Implementation

[0079] KLRB1 expression is a marker of 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.

[0080] KLRB1 is also expressed by neoplastic cells in various T-cell and NK-cell malignancies. These include various peripheral T-cell and NK-cell lymphomas and leukemias.

[0081] The KLRB1 binding antibody described herein can be used to preferentially target and deplete Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T cells or NK cells. Immune cell populations expressing KLRB1 express IL-17 more abundantly than total CD4 or CD8 T cell populations, and exhibit higher specificity for Th17 and Tc17 T cells than for CD4 or CD8, respectively. Therefore, this article describes a method for treating subjects by administering an effective amount of a cytotoxic lectin-like receptor B1 (KLRB1) binder with cell-depleting activity to subjects in need, thereby depleting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells in the body. These methods can be used to treat autoimmune diseases, allergic diseases, graft rejection, or hematologic malignancies, as described in this article.

[0082] Furthermore, since the regulation of KLRB1 has both inhibitory and stimulatory effects on T and NK cells, this article describes a method for treating subjects by administering an effective amount of a cytotoxic lectin-like receptor B1 (KLRB1) binder with KLRB1 receptor blocking activity (which achieves blockade of CLEC2D (LLT1) binding to KLRB1) to the subject in need, thereby activating or inhibiting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, and / or peTh2 in vivo. These methods can be used to activate T and / or NK cells to enhance their activity against tumor cells, thereby for the treatment of cancer.

[0083] In some embodiments, the antibody has antibody-dependent cytotoxic (ADCC) effector activity or complement-dependent cytotoxic (CDC) effector activity. Administration of an effective amount of an anti-KLRB1 antibody having ADCC or CDC effector function or conjugated to a cytotoxic agent to a subject in need can eliminate or reduce the number of Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells. In some embodiments, this disclosure provides a cytotoxic lectin-like receptor B1 (KLRB1) binder as described herein, having ADCC or CDC activity or conjugated to a cytotoxic agent. In various aspects, this disclosure provides mRNA or cDNA encoding the binder. In various aspects, this disclosure provides a pharmaceutical composition comprising an effective amount of the binder.

[0084] The various features of this disclosure are discussed below, including KLRB1 and its ligands, anti-KLRB1 antibodies or their antigen-binding moieties, pharmaceutical compositions, treatment and administration, and illustrative examples. The following sections contain definitions of terms used in this disclosure. Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. For the purpose of interpreting this specification, the following description of terminology will apply, and where appropriate, terms used in the singular will also include the plural, and vice versa. If any description of the terms set forth herein conflicts with any document incorporated herein by reference, the description of the terms set forth below shall prevail.

[0085] Killer cell lectin-like receptor B1 (KLRB1) Killer cell lectin-like receptor B1 (KLRB1), also known as CD161, is a type II transmembrane protein. It is a receptor that regulates the activity of T cells and NK cells and can act as either a stimulatory 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 various immune cells. The ligand for KLRB1 is LLT1 (also known as CLEC2D) (Aldemir et al., 2005).

[0086] KLRB1 expression in T cells is limited to cells capable of responding to IL-12 and IL-18 (Fergusson et al., 2014). In CD4+ T helper cells, KLRB1 expression is a unique marker of 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 of Tc17 cells. Therefore, KLRB1 is a marker of T cells capable of producing IL-17, or, in the case of ex-Th17 cells, T cells capable of producing interferon-γ (IFNG). In the context of autoimmune diseases, the production of this cytokine is undesirable.

[0087] This article describes antibodies that bind to KLRB1 (preferably human KLRB1) and their antigen-binding fragments. An exemplary sequence of human KLRB1 is provided as SEQ ID NO: 1: MDQQAIYAELNLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIQKSSIEKCSVDIQQSRNKTTERPGLLNCPIYWQQLREKCLLFSHTVNPWNNSLADCSTKESSLLLIRDKDELIHTQNLIRDKAILFWIGLNFSLSEKNWKWINGSFLNSNDLEIRGDAKENSCISISQTSVYSEYCSTEIRWICQKELTPVRNKVYPDS.

[0088] In some implementations, KLRB1 is cynomolgus monkey KLRB1 (e.g., AOA2K5WYI1 from UniParc UPI0003ABB264); an exemplary sequence is provided as SEQ ID NO: 2: MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWQQVQEKCLLFSHTVNPWNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0089] In some implementations, KLRB1 is cynomolgus monkey KLRB1 (e.g., UniProt A0A7N9D796); an exemplary sequence is provided as SEQ ID NO: 3: MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSLSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWKQVQEKCLLFSHTVNPWNNSLADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKITGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.

[0090] KLRB1 binding antibody This article describes antibodies that bind to KLRB1 and their antigen-binding fragments. The term "antibody" refers to an immunoglobulin molecule or its immunoactive portion, i.e., the antigen-binding part.

[0091] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous population of antibodies, for example, the individual antibodies comprising that population are identical, except for possible naturally occurring mutations that may be present in trace amounts. Antibodies can be monoclonal antibodies. Antibodies can be human antibodies or humanized antibodies. The term "monoclonal antibody" encompasses full-length and complete monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab', F(ab')2, Fv), single-chain antibodies (e.g., scFv), fusion proteins containing antibody fragments, and any other modified immunoglobulin molecules containing at least one antigen-binding site. Furthermore, "monoclonal antibody" refers to antibodies prepared using a variety of techniques, including but not limited to hybridoma production, phage library display, recombinant expression, and transgenic animals.

[0092] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a primary source or species, while the remaining heavy chain and / or light chain is derived from a different source or species.

[0093] As used herein, the term "humanized antibody" refers to an antibody comprising a human heavy chain variable region and a light chain variable region, wherein 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 possesses the desired specificity, affinity, and / or activity. In some embodiments, one or more framework region residues of the human heavy chain or light chain variable region are replaced by corresponding residues from the non-human antibody. Additionally, humanized antibodies may contain residues not found in human antibodies or in non-human antibodies. In some embodiments, these modifications are intended to further refine and / or optimize antibody properties. In some embodiments, humanized antibodies comprise at least a portion of the immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically at least a portion of the immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc) of human immunoglobulins. Exemplary constant regions include those shown in Table A.

[0094] Table A. Sequences of Exemplary Constant Regions

[0095] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies produced by humans and / or an antibody prepared using any technique known to those skilled in the art for the preparation of human antibodies. These techniques include, but are not limited to, phage display libraries, yeast display libraries, transgenic animals, recombinant protein production, and B-cell hybridoma techniques.

[0096] An "antibody fragment" may include a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0097] The terms “epitope” and “antigenic determinant” are used interchangeably in this document, referring to the portion of an antigen or target that can be recognized and bound by a specific antibody. When the antigen or target is a polypeptide, an epitope can be formed from consecutive amino acids and discontinuous amino acids juxtaposed through the ternary folding of a protein. Epitopes formed from consecutive amino acids (also known as linear epitopes) are generally retained after protein denaturation, while epitopes formed from ternary folding (also known as conformational epitopes) are generally lost after protein denaturation. Epitopes typically contain at least 3 amino acids in a unique spatial conformation, more commonly at least 5, 6, 7, or 8–10 amino acids. Epitopes can be predicted using any of the numerous software bioinformatics tools available on the internet. X-ray crystallography can be used to characterize epitopes on target proteins by analyzing the amino acid residue interactions of antigen / antibody complexes.

[0098] "Fv" comprises the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly non-covalently associated heavy chain variable domain and a light chain variable domain. The antigen binding site on the surface of the VH-VL dimer is defined by this configuration of three interacting CDRs in each variable domain. The six CDRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only the three antigen-specific CDRs) can recognize and bind to the antigen, although with less affinity than the complete binding site. The Fab fragment also contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. In this paper, Fab' where one or more cysteine ​​residues in the constant domain have free thiol groups is referred to as Fab'-SH. The F(ab')2 antibody fragment is initially generated as a Fab' fragment pair with a hinge cysteine ​​residue between the Fab' fragments. Other chemical conjugates of the antibody fragment are also known.

[0099] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. A "single-chain Fv" or "sFv" antibody fragment contains the VH and VL domains of the antibody, where these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further includes a polypeptide linker between the VH and VL domains, enabling the sFv to form the structure required for antigen binding.

[0100] In various embodiments, the antibody or its antigen-binding fragment comprises a human or humanized antibody. Humanized forms of non-human (e.g., mouse) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies comprise human immunoglobulins (recipient antibodies) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from a CDR of a non-human species such as a mouse, rat, or rabbit (donor antibody) having 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 may also contain residues not present in either the recipient antibody or the introduced CDR or framework sequence. Typically, the humanized antibody will contain at least one and usually two substantially all of the variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of non-human immunoglobulins and all or substantially all of the FR regions are those FR regions of human immunoglobulin consensus sequences. Methods for humanizing non-human antibodies are well known in the art.

[0101] The affinity maturation of the KLRB1 antibody described herein can be achieved, for example, using selection and / or mutagenesis methods known in the art. Typically, an "affinity-matured" antibody is an antibody with one or more alterations in one or more hypervariable regions, which result in an increased affinity of the antibody for the antigen compared to a parent antibody without such alterations. In one embodiment, the affinity-matured antibody has nanomolar or even picomolar affinity for the target antigen. Preferred affinity-matured antibodies have an affinity 5-fold, more preferably 10-fold, and even more preferably 20 or 30-fold higher than that of the starting antibody from which the mature antibody is prepared (typically murine, humanized, or human).

[0102] An antibody that “binds to” a specific polypeptide or an epitope on a specific polypeptide, “specifically binds to” a specific polypeptide or an epitope on a specific polypeptide, or “is specific to a specific polypeptide or an epitope on a specific polypeptide” is an antibody that binds to a specific polypeptide or an epitope on a specific polypeptide and substantially does not bind to any other polypeptide or polypeptide epitope. As used herein, the term “specific binding” refers to a KLRB1 agent (e.g., an anti-KLRB1 antibody) interacting with a specific antigen, epitope, protein, or target molecule more frequently, more rapidly, for a longer duration, with greater affinity, or a 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 antibody are described herein. In some cases, KLRB1 antibodies can cross-react with various similar KLRB1 proteins (e.g., exhibiting the highest affinity for one KLRB1 protein, such as human KLRB1, and a lower affinity for other KLRB1 proteins, such as mouse KLRB1). A binder that specifically binds to an antigen has a higher affinity for the target antigen than for different antigens. These different antigens can be related antigens. In some embodiments, the binder that specifically binds to an antigen has an affinity for the target antigen at least 20-fold higher than its affinity for different antigens, for example, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher. In some embodiments, the binder that specifically binds to a particular antigen binds to different antigens with such low affinity that the binding is undetectable using any other assay described herein or known in the art. In some implementations, SPR technology is used to measure affinity, for example, in the Biacore system or other systems known to those skilled in the art.

[0103] In the context of two or more peptides (e.g., two anti-KLRB1 peptides), the term "identity" or "identity percentage" 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 (with vacancies introduced if necessary) to obtain maximum correspondence, regardless of any conserved amino acid substitutions as part of sequence identity. The identity percentage can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software available for obtaining alignments of amino acid or nucleotide sequences are well known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof.

[0104] The percentage of identity between two sequences is a function of the number of common positions shared by the sequences (i.e., % homology = number of common positions / total number of positions × 100), taking into account the number of vacancies and the length of each vacancy required to achieve optimal alignment of the two sequences. Mathematical algorithms can be used to compare sequences and determine the percentage of identity between them. This homology is well represented in the art through local alignment tools and / or algorithms, and can include pairwise alignment, multiple sequence alignment methods, structural alignment methods, and / or phylogenetic analysis methods. When sequences differ in terms of conserved substitutions, the percentage of sequence identity can be (but not necessarily) adjusted upwards to correct for the conservatism of the substitutions. Methods for making such adjustments are well known to those skilled in the art. Typically, but not necessarily, this involves classifying conserved substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage of sequence identity. Thus, for example, conserved substitutions are assigned a score between 0 and 1 when the same amino acid is assigned a score of 1 instead of a conserved substitution being assigned a score of 0.

[0105] In some embodiments, the two polypeptides of this disclosure (e.g., their antibody or antibody domains (e.g., VL, CL, VH, CH1, CH2, CH3 domains)) are substantially identical, meaning that when compared and aligned to obtain 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 sequence comparison algorithms or by visual inspection. In some embodiments, the identity percentage is present in regions of sequence lengths 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, or any integer value between these. In some embodiments, the identity percentage is present in regions longer than 60-80 amino acid residues (e.g., at least about 80-100 amino acid residues), and in some embodiments, the sequences are substantially identical across the full length of the compared sequences (e.g., amino acid sequences).

[0106] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences need to be aligned for optimal comparison (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences to achieve optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments at least 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid 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 percentage of identity between the two sequences is a function of the number of shared positions in the sequences, taking into account the number of vacancies that need to be introduced for optimal alignment of the two sequences and the length of each vacancy.

[0107] Mathematical algorithms can be used to compare sequences and determine the percentage of identity between two sequences. For example, the percentage of identity between two amino acid sequences can be determined using Needleman and Wunsch's (1970) GAP program, which has been incorporated into the GCG software package (available at gcg.com on the World Wide Web). J. Mol. Biol. The algorithm (48:444-453) uses default parameters (e.g., Blossum 62 rating matrix, vacancy penalty of 12, vacancy extension penalty of 4, and shifted vacancy penalty of 5) to determine the parameters.

[0108] As used herein, the terms "conserved sequence modification" or "conserved substitution" can refer to amino acid modifications to the target epitope or the antibody and its antigen-binding moiety disclosed herein, which do not significantly affect or alter the binding properties of the anti-KLRB1 antibody. As used herein, "conserved substitution" means the substitution of one amino acid residue for another amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are 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), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of tyrosine with phenylalanine is considered a conserved substitution. Methods for identifying conserved substitutions of amino acids that do not eliminate the binding are well known in the art.

[0109] In various implementations, the antibody is a blocking or antagonistic binding agent. "Blocking" or "antagonistic" means that the agent (e.g., an antibody or its binding fragment) is an agent that inhibits or reduces the biological activity of the antigen it binds to. Some blocking or antagonistic agents substantially or completely inhibit the biological activity of the antigen. For example, KLRB1 binders can block KLRB1 signaling (e.g., thereby disrupting KLRB1 signaling and modulating Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and / or neoplastic T or NK cells).

[0110] In some embodiments, the KLRB1 binder is an antibody comprising: a. a full-length antibody that binds to KLRB1 and contains an Fc domain of an Fcγ receptor having an effector function for triggering antibody-dependent cell-mediated cytotoxicity (ADCC); b. an antibody that binds to KLRB1 and contains an Fc domain of complement protein 1q (C1q) having an effector function for triggering complement-dependent cytotoxicity (CDC); c. an antibody conjugate that binds to KLRB1 and contains a cytotoxic agent, such as an antibody-drug conjugate (ADC); or d. a multispecific antibody (e.g., a bispecific or trispecific antibody) wherein the antibody binds to KLRB1 and another antigen.

[0111] In some embodiments, the KLRB1 antibody described herein binds to the extracellular domain of human KLRB1; in some embodiments, the antibody cross-reacts with the extracellular domain of human and cynomolgus monkey KLRB1 (binding to both). In some embodiments, the antibody binds to an epitope of the extracellular domain of KLRB1, wherein the epitope is at least 90% identical in both humans and cynomolgus monkeys.

[0112] In some implementations, the antibody binds to KLRB1 and is not a mouse antibody.

[0113] In some implementations, the antibodies described herein are not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), 191B8 (Miltenyi), DX12 (BD Biosciences), or JNH25G2G22 (Creative Diagnostics).

[0114] In some embodiments, the KLRB1 binder is an antibody, such as a full-length antibody comprising an Fc domain containing 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.

[0115] In some embodiments, the antibody is an antibody fragment containing an antigen-binding site. In some embodiments, the antibody is an scFv. In some embodiments, the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a bispecific 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 isolated. In some embodiments, the antibody is substantially pure. In some embodiments, the KLRB1 binder is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, the polyclonal antibody is generated by immunizing an animal (e.g., rabbit, rat, mouse, goat, donkey) with the antigen of interest (e.g., a purified peptide fragment, recombinant protein, or fusion protein) via multiple subcutaneous or intraperitoneal injections. In some embodiments, the antigen is conjugated to a vector (such as keyhole hare hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor). The antigen (with or without a carrier protein) is diluted in sterile saline and typically combined with an adjuvant (e.g., complete or incomplete Freund's adjuvant) to form a stable emulsion. After a period of time, the polyclonal antibody is recovered from the immunized animal (e.g., from blood or ascites). In some embodiments, the polyclonal antibody is 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.

[0116] In some embodiments, the KLRB1 binder is a monoclonal antibody. Monoclonal antibodies can be prepared by any method known to those skilled in the art. In some embodiments, the monoclonal antibody is prepared using a hybridoma method known to those skilled in the art. For example, using a hybridoma method, mice, rats, rabbits, hamsters, or other suitable host animals are immunized as described above. In some embodiments, immunization is performed in vitro against lymphocytes. In some embodiments, the immunoantigen is a human protein or a fragment thereof. In some embodiments, the immunoantigen is a mouse protein or a fragment thereof.

[0117] Following immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol. Hybridoma cells are selected using specialized culture media as known in the art, and unfused lymphocytes and myeloma cells cannot survive the selection process. Hybridomas producing monoclonal antibodies specifically against selected antigens can be identified by a variety of methods, including but not limited to immunoprecipitation, Western blotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay). Once hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones can be subcloned using limiting dilution techniques. Hybridomas can be propagated in vitro using standard methods or in vivo as ascites tumors. Monoclonal antibodies can be purified from culture media or ascites according to standard methods in the art, including but not limited to affinity chromatography, ion exchange chromatography, gel electrophoresis, and dialysis.

[0118] In some embodiments, monoclonal antibodies are prepared using recombinant DNA techniques known to those skilled in the art. For example, polynucleotides encoding the antibody may be isolated from mature B cells or hybridoma cells using RT-PCR with oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences may be determined using standard techniques. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which, when transfected into cells such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not additionally produce immunoglobulins, generates monoclonal antibodies.

[0119] In some implementations, recombinant monoclonal antibodies are isolated from phage display libraries expressing the variable domains or CDRs of the desired species. Screening of the phage library can be performed using various techniques known in the art.

[0120] In some embodiments, monoclonal antibodies are modified using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of mouse monoclonal antibodies are replaced with constant regions of human antibodies to generate chimeric antibodies. In some embodiments, constant regions are truncated or removed to generate 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 the monoclonal antibody.

[0121] In some embodiments, the KLRB1 binder is a humanized antibody. Various methods for generating humanized antibodies are known in the art. In some embodiments, the humanized antibody comprises one or more amino acid residues introduced therefrom from a non-human source. In some embodiments, humanization is performed by replacing one or more non-human CDR sequences with the corresponding CDR sequences of a human antibody. In some embodiments, the humanized antibody is constructed by replacing all six CDRs of a non-human antibody (e.g., a mouse antibody) with the corresponding CDRs of a human antibody.

[0122] The choice of which human heavy chain variable region and / or light chain variable region to use for generating humanized antibodies can be based on a variety of factors and through various methods known in the art. In some embodiments, a "best-fit" approach is used, where sequences of variable regions for non-human (e.g., rodent) antibodies are screened against an entire library of known human variable region sequences. The human sequence most similar to the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody. In some embodiments, a specific variable region framework derived from a consensus sequence of all human antibodies from a particular light chain or heavy chain subgroup is selected as the variable region framework. In some embodiments, the variable region framework sequence is derived from a consensus sequence of the most abundant human subclass. In some embodiments, human germline genes are used as the source of the variable region framework sequence.

[0123] Other methods for humanization include, but are not limited to, methods described as “hyperhumanization” that directly transfer CDRs to human germline frameworks, methods based on “antibody humanization” metrics called human string content (HSC), methods based on generating large libraries of humanized variants (including phage, ribosome, and yeast display libraries), and methods based on framework region shuffling.

[0124] Humanity can be defined by an OASis score computed as part of the BioPhi platform. BioPhi is an open-source platform featuring novel methods for humanization (Sapiens) and humanity assessment (OASis). Sapiens is a deep learning-based humanization method trained on the Observed Antibody Space (OAS) using language modeling. Based on a computer-based humanization benchmark of 177 antibodies, Sapiens generates sequences on a large scale while achieving results comparable to human experts. OASis is a finely detailed, interpretable, and diverse humanity score based on 9-peptide retrieval in OAS. OASis distinguishes between human and non-human sequences with high accuracy and is correlated with clinical immunogenicity (Prihoda D et al. 2022). Humanity assessment settings can be selected, such as Kabat numbering, Kabat CDR definition, and relaxed OASis prevalence thresholds.

[0125] In some embodiments, the KLRB1 binder is a human antibody. Human antibodies can be prepared using various techniques known in the art. In some embodiments, the human antibody is generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, the human antibody is generated from lymphocytes isolated from an immunized individual. In any case, cells that produce antibodies against the target antigen can be generated and isolated. In some embodiments, the human antibody is selected from a phage library expressing human antibodies. Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from an immunoglobulin variable region gene library from an unimmunized donor. Techniques for generating and using 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 human antibodies with higher affinity. In some embodiments, the human antibody is generated in transgenic mice containing human immunoglobulin loci. After immunization, these mice are able to generate a complete human antibody library in the absence of endogenous immunoglobulin production.

[0126] In some embodiments, the KLRB1 binder is an scFv antibody. ScFv is a molecule comprising a variable heavy chain region and a variable light chain region linked to form a single polypeptide. ScFv can be produced using recombinant techniques known in the art. In some embodiments, the scFv comprises a polypeptide linker between the heavy chain variable region and the light chain variable region. In some embodiments, the scFv comprises (from N-terminus to C-terminus) the orientation of (i) the heavy chain variable region, (ii) the linker, and (iii) the light chain variable region. In some embodiments, the scFv comprises (from N-terminus to C-terminus) the orientation of (i) the light chain variable region, (ii) the linker, and (iii) the heavy chain variable region. In some embodiments, the scFv is a disulfide-linked scFv (dsscFv), which is an scFv containing an engineered disulfide bond between the light chain variable region and the heavy chain variable region. In some embodiments, scFv (e.g., dsscFv) is attached (directly or indirectly) to a portion that extends half-life, such as, for example, an Fc molecule, the CH3 domain of an immunoglobulin (e.g., CH3 of IgG1), polyethylene glycol (PEG) or a PEG mimic, 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 scFv (e.g., dsscFv).

[0127] Various suitable linkers are known to those skilled in the art and are not limited to any particular sequence disclosed herein. In some embodiments, the peptide linker is composed of amino acids that are naturally or non-naturally present. In some embodiments, the linker contains amino acids that allow for flexibility. In some embodiments, the linker contains amino acids that allow for suitable solubility. In some embodiments, the linker contains glycine amino acids. In some embodiments, the linker contains both glycine and serine amino acids. In some embodiments, the linker contains one or more sets of glycine / serine repeat sequences. In some embodiments, the peptide linker is selected from the group consisting of: (GGGGS)n, where n = 1-4 (SEQ ID NO: 116); GGGGS (SEQ ID NO: 117); GGGGSGGGGS (SEQ ID NO: 118); GGGGSGGGGSGGGGS (SEQ ID NO: 119); GGGGSGGGGSGGGGSGGGS (SEQ ID NO: 120); and (GGGGA)n, where n = 1-4 (SEQ ID NO: 121). In some implementations, the connector contains GGGGSGGGGSGGGGS (SEQ ID NO:119).

[0128] In some embodiments, the KLRB1 binder is Fv. Fv comprises a heavy chain variable region and a light chain variable region. In some embodiments, Fv is attached (directly or indirectly) to a portion that extends half-life, such as, for example, an Fc molecule, the CH3 domain of IgG (e.g., the CH3 domain of IgG1), PEG or a PEG mimic, 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 Fv.

[0129] In some embodiments, the KLRB1 binder is Fab. Fab is one of the molecules produced by digesting immunoglobulin antibodies with papain. Fab is a monovalent molecule comprising a light chain, a heavy chain variable region, a CH1 region, and optionally a heavy chain constant region hinge region or a portion thereof. Fab can be produced using recombinant techniques known in the art. In some embodiments, Fab comprises a polypeptide linker between the light chain constant region and the heavy chain variable region. In some embodiments, Fab comprises a polypeptide linker between the heavy chain constant region and the light chain variable region. A variety of suitable linkers are known to those skilled in the art and are not limited to any particular sequence disclosed herein. In some embodiments, the linker is the linker described herein. In some embodiments, Fab is attached (directly or indirectly) to a portion that extends half-life, such as, for example, an Fc molecule, the CH3 domain of IgG (e.g., CH3 of IgG1), PEG or a PEG mimic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or modified, for example, by high glycosylation, to extend the half-life of Fab.

[0130] In some embodiments, Fab comprises disulfide bonds formed between the heavy chain variable region and the light chain variable region. In some embodiments, Fab comprises disulfide bonds that increase the stability of the Fab molecule. In some embodiments, Fab comprises disulfide bonds that increase the thermal stability of the Fab molecule.

[0131] In some embodiments, the KLRB1 binder is F(ab′)2. F(ab′)2 is one of the molecules produced by digesting immunoglobulin antibodies with pepsin. F(ab′)2 is a divalent molecule comprising a first light chain associated with a first polypeptide, the 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, the second polypeptide comprising a second heavy chain variable region, a second CH1, and a second hinge region, wherein the first hinge region is linked to the second hinge region via at least one disulfide bond. F(ab′)2 can be produced using recombinant techniques known in the art. In some embodiments, F(ab′)2 is attached (directly or indirectly) to a portion that extends half-life, such as, for example, the CH3 domain of IgG (e.g., the CH3 of IgG1), PEG or a PEG mimic, XTEN, serum albumin (e.g., human serum albumin), polysialic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or modified, for example, by high glycosylation, to extend the half-life of F(ab′)2.

[0132] In some embodiments, F(ab′)₂ contains disulfide bonds formed between the heavy chain variable region and the light chain variable region. In some embodiments, F(ab′)₂ contains disulfide bonds that increase the stability of the F(ab′)₂ molecule. In some embodiments, F(ab′)₂ contains disulfide bonds that increase the thermal stability of the F(ab′)₂ molecule.

[0133] 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 containing 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 moiety, such as, for example, an Fc molecule, the CH3 domain of IgG (e.g., the CH3 domain of IgG1), PEG or a PEG mimic, 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′).

[0134] In some embodiments, F(ab′) comprises disulfide bonds formed between the heavy chain variable region and the light chain variable region. In some embodiments, F(ab′) comprises disulfide bonds that increase the stability of the F(ab′) molecule. In some embodiments, F(ab′) comprises disulfide bonds that increase the thermal stability of the F(ab′) molecule.

[0135] In some embodiments, the KLRB1 binder is a bispecific antibody. A bispecific antibody is capable of recognizing and binding to 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., one epitope on KLRB1 and one epitope on a different target). In some embodiments, the bispecific antibody has enhanced potency compared to a single antibody or a combination of more than one antibody. In some embodiments, the bispecific antibody has reduced toxicity compared to a single antibody or a combination of more than one antibody. Those skilled in the art know that any therapeutic agent may have a unique pharmacokinetic (PK) profile (e.g., circulating half-life). In some embodiments, the bispecific antibody has the ability to synchronize the PK of two active binders, wherein the two individual binders have different PK profiles. In some embodiments, the bispecific antibody has the ability to concentrate the effects of the two agents on a common region (e.g., tissue) of the subject (e.g., human). In some embodiments, the bispecific antibody has the ability to concentrate the effects of the 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.

[0136] 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 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 as a single agent.

[0137] Several techniques for manufacturing bispecific antibodies are known to those skilled in the art. In some embodiments, the bispecific antibody comprises a heavy chain constant region that is modified in an amino acid that is part of the interface between two heavy chains. These modifications are intended to enhance heterodimer formation and typically reduce or eliminate homodimer formation. In some embodiments, the bispecific antibody is generated using a knob-into-hole (KIH) strategy. In some embodiments, the bispecific antibody comprises a modified hinge region that prevents the formation of disulfide bonds between identical heavy chains (e.g., reducing homodimer formation). In some embodiments, the bispecific antibody comprises a heavy chain in an amino acid having variations that alter electrostatic interactions. In some embodiments, the bispecific antibody comprises a heavy chain in an amino acid having variations that alter hydrophobic / hydrophilic interactions.

[0138] Bispecific antibodies can be complete antibodies or antibody fragments containing antigen-binding sites.

[0139] KLRB1 binders with more than two valence states were also considered. In some implementations, trispecific or tetraspecific antibodies were generated.

[0140] In some embodiments, the KLRB1 binder 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 11.12, for example, as shown in Table 1, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.12, for example, as shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.12, for example, as shown in Table 1 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.12, for example, as shown in Table 1. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.27, for example, as shown in Table 2, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.27, for example, as shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.27, for example, as shown in Table 2 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.27, for example, as shown in Table 2. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.29, for example, as shown in Table 3, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.29, for example, as shown in Table 3. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.29, for example, as shown in Table 3 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.29, for example, as shown in Table 3. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.41.1, for example, as shown in Table 4, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.41.1, for example, as shown in Table 4. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.41.1, for example, as shown in Table 4 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.41.1, for example, as shown in Table 4. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.44.1, for example, as shown in Table 5, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.44.1, for example, as shown in Table 5.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.44.1, for example, as shown in Table 5 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.44.1, for example, as shown in Table 5. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.45, for example, as shown in Table 6, and / or (ii) three light chain CDRs from antibody 11.45, for example, as shown in Table 6. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.45, for example, as shown in Table 6 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.45, for example, as shown in Table 6. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.54, for example, as shown in Table 7, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.54, for example, as shown in Table 7. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.54, for example, as shown in Table 7 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.54, for example, as shown in Table 7. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.55, for example, as shown in Table 8, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.55, for example, as shown in Table 8. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.55, for example, as shown in Table 8 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.55, for example, as shown in Table 8. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 11.57, for example, as shown in Table 9, and / or (ii) one, two, and / or three light chain CDRs from antibody 11.57, for example, as shown in Table 9. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 11.57, for example, as shown in Table 9 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 11.57, for example, as shown in Table 9. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 12.0, for example, as shown in Table 10, and / or (ii) one, two, and / or three light chain CDRs from antibody 12.0, for example, as shown in Table 10.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 12.0, for example, as shown in Table 10 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 12.0, for example, as shown in Table 10. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 12.1.3, for example, as shown in Table 11, and / or (ii) one, two, and / or three light chain CDRs from antibody 12.1.3, for example, as shown in Table 11. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 12.1.3, for example, as shown in Table 11 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 12.1.3, for example, as shown in Table 11. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 12.2, for example, as shown in Table 12, and / or (ii) one, two, and / or three light chain CDRs from antibody 12.2, for example, as shown in Table 12. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 12.2, for example, as shown in Table 12 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 12.2, for example, as shown in Table 12. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 12.3, for example, as shown in Table 13, and / or (ii) one, two, and / or three light chain CDRs from antibody 12.3, for example, as shown in Table 13. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 12.3, for example, as shown in Table 13 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 12.3, for example, as shown in Table 13. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 13.2, for example, as shown in Table 14, and / or (ii) one, two, and / or three light chain CDRs from antibody 13.2, for example, as shown in Table 14. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13.2, for example, as shown in Table 14 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 13.2, for example, as shown in Table 14. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 13.4, for example, as shown in Table 15, and / or (ii) one, two, and / or three light chain CDRs from antibody 13.4, for example, as shown in Table 15.In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13.4, for example, as shown in Table 15 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 13.4, for example, as shown in Table 15. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 13.5, for example, as shown in Table 16, and / or (ii) one, two, and / or three light chain CDRs from antibody 13.5, for example, as shown in Table 16. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13.5, for example, as shown in Table 16 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 13.5, for example, as shown in Table 16. In some embodiments, the anti-KLRB1 antibody comprises (i) one, two, and / or three heavy chain CDRs from antibody 13.6, for example, as shown in Table 17, and / or (ii) one, two, and / or three light chain CDRs from antibody 13.6, for example, as shown in Table 17. In some embodiments, the anti-KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13.6, for example, as shown in Table 17 (i.e., CDR1, CDR2, and CDR3), and (ii) three light chain CDRs from antibody 13.6, for example, as shown in Table 17.

[0141] In some embodiments, the KLRB1 binder is a humanized version of an anti-KLRB1 antibody comprising (i) one, two, and / or three heavy chain CDRs, and / or (ii) one, two, and / or three light chain CDRs from any of Tables 1-17. In some embodiments, the KLRB1 binder is a humanized version of an anti-KLRB1 antibody comprising (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) and (ii) three light chain CDRs from any of Tables 1-17, i.e., where the heavy chain CDRs and light chain CDRs are from the same table.

[0142] Table 1: Antibody 11.12 sequence

[0143] Table 2: Antibody 11.27 sequence

[0144] Table 3: Antibody 11.29 sequence

[0145] Table 4: Antibody 11.41.1 sequence

[0146] Table 5: Antibody 11.44.1 sequence

[0147] Table 6: Antibody 11.45 sequence

[0148] Table 7: Antibody 11.54 Sequence

[0149] Table 8: Antibody 11.55 Sequence

[0150] Table 9: Antibody 11.57 Sequence

[0151] Table 10: Antibody 12.0 Sequence

[0152] Table 11: Antibody 12.1.3 Sequence

[0153] Table 12: Antibody 12.2 Sequence

[0154] Table 13: Antibody 12.3 Sequence

[0155] Table 14: Antibody 13.2 Sequence

[0156] Table 15: Antibody 13.4 Sequence

[0157] Table 16: Antibody 13.5 Sequence

[0158] Table 17: Antibody 13.6 Sequence

[0159] In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from the antibodies described herein. In some embodiments, the KLRB1 binder comprises a humanized version or humanized variant of the antibodies described herein. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.12 (Table 1) or a humanized version thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.27 (Table 2) or a humanized version thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.29 (Table 3) or a humanized version thereof. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.41.1 (Table 4) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.44.1 (Table 5) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.45 (Table 6) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.54 (Table 7) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.55 (Table 8) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 11.57 (Table 9) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 12.0 (Table 10) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 12.1.3 (Table 11) or its humanized version.In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 12.2 (Table 12) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 12.3 (Table 13) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 13.2 (Table 14) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 13.4 (Table 15) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 13.5 (Table 16) or its humanized version. In some embodiments, the KLRB1 binder comprises heavy chain CDR1, CDR2, and CDR3 and / or light chain CDR1, CDR2, and CDR3 from antibody 13.6 (Table 17) or its humanized version.

[0160] In some embodiments, the KLRB1 binder comprises a more humanized version or humanized variant of the antibody described herein. In some embodiments, the KLRB1 binder comprises the heavy chain CDR1, CDR2, and CDR3 and / or the light chain CDR1, CDR2, CDR3, or more humanized versions thereof, as shown in Tables 1-17.

[0161] Those skilled in the art define CDRs using various methods / systems. These systems and / or definitions have been developed and refined over many years and include Kabat, Chothia, IMGT, AbM, and Contact. The Kabat definition is based on sequence variability and is generally the most commonly used. The Chothia definition is based on the location of structural loop regions. The IMGT system is based on sequence variability and location within the structure of variable domains. 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 antibody sequence analysis and CDR determination, 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.

[0162] The specific CDR sequences defined herein are generally based on the Kabat definition. However, it should be understood that references to one or more heavy chain CDRs and / or one or more light chain CDRs for a particular antibody will encompass all CDR definitions known to those skilled in the art, such as those shown in the tables herein. In some embodiments, all CDR sequences used will be identified using the same definition, i.e., all will be Chothia, all will be Kabat, all will be IMGT, etc. The term "antibody numbering rule" is defined as the various rules used to define CDR regions such as Kabat, Chothia, AbM, Contact, and IMGT.

[0163] In some embodiments, the KLRB1 binder is a variant of the agent described herein. In some embodiments, the KLRB1 binder (e.g., an antibody) comprises (a) heavy chain CDR1, CDR2, CDR3 of the VH sequence presented herein (e.g., in Tables 1-17, Table B, or Table C), or variants thereof containing 1, 2, 3, or 4 amino acid substitutions; and / or light chain CDR1, CDR2, and / or CDR3 of the VL sequence presented herein (e.g., in Tables 1-17, Table B, or Table C), or variants thereof containing 1, 2, 3, or 4 amino acid substitutions. In some embodiments, the amino acid substitutions are conserved substitutions. In some embodiments, the CDR contains one conserved amino acid substitution. In some embodiments, the CDR contains two conserved amino acid substitutions. In some embodiments, the CDR contains three conserved amino acid substitutions. In some embodiments, the CDR contains four conserved amino acid substitutions. In some embodiments, the CDR is heavy chain CDR1. In some embodiments, the CDR is 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, substitution is performed as part of a humanization process. In some embodiments, substitution is performed as part of a germline humanization process. In some embodiments, substitution is performed as part of an affinity maturation process. In some embodiments, substitution is performed as part of an optimization process.

[0164] In some implementations, the KLRB1 binder (e.g., an antibody) comprises one or more such 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, to remove Met / Trp, or Lys, for example, to reduce the likelihood of asparagine(N)-glycosylation, cysteine ​​glycosylation, asparagine (Asn) deamidation, aspartic acid (Asp) isomerization, methionine / tryptophan (Met / Trp) oxidation, and non-enzymatic lysine (Lys) glycosylation within the CDR sequence (see, for example, Haberger et al., MAbs. 1 Mar 2014; 6(2): 327–339; Lu et al., MAbs. 1 Jan 2019; 11(1): 45–57). Deamidation is a chemical reaction in which the amide group in the side chain of the amino acid 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 the peptide, potentially leading to reduced biological activity.

[0165] Exemplary sequences of human heavy chain variable regions and light chain variable regions are provided in Table B.

[0166] Table B. Sequences of Exemplary Antibody Variant Sequences

[0167] In some embodiments, the KLRB1 binder comprises a heavy chain variable region containing heavy chain CDRs 1, 2 and 3 as shown in Tables 1-17, B or C, and a light chain variable region containing light chain CDRs 1, 2 and 3 as shown in Tables 1-17, B or C.

[0168] In some embodiments, the KLRB1 binder (e.g., an antibody) comprises a heavy chain variable region or sequence having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the heavy chain variable region sequence presented herein (e.g., in Tables 1-17, Table B, or Table C), and / or a light chain variable region having at least 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the light chain variable region sequence presented herein (e.g., in Tables 1-17, Table B, or Table C). The term “consensus sequence” as used herein with respect to light chain (VL) and heavy chain (VH) variable regions refers to a composite or generic VL or VH sequence defined based on information about which amino acid residues within the VL or VH chain are readily modifiable without impairing antigen binding. Therefore, in the “consensus sequence” of the VL or VH chain, certain amino acid positions are occupied by one of several possible amino acid residues at that position. For example, if arginine (R) or serine (S) is present at a specific position, then that specific position within the consensus sequence can be either arginine or serine (R or S). The consensus sequences 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 given CDR using degenerate-encoding primers), by scanning mutagenesis of amino acid residues within the antibody CDR (e.g., alanine scanning mutagenesis), or any other method known in the art, followed by evaluation of the mutant's binding 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 frame region. In some other aspects, mutations are introduced in both the CDR and frame regions. The consensus sequence can be determined using software such as EMBOSS Cons, which is available at ebi.ac.uk / Tools / msa / emboss_cons / .

[0169] In some embodiments, the KLRB1 binder (e.g., an antibody) described herein comprises one or more constant restructured domains (e.g., CH1, CH2, and / or CH3 regions). In some embodiments, the KLRB1 binder comprises a constant restructured domain 1 (CH1) having the amino acid sequence shown herein (e.g., in Table B or C). In some embodiments, the KLRB1 binder comprises a constant restructured domain 2 (CH2) having the amino acid sequence shown herein (e.g., in Table B or C). In some embodiments, the KLRB1 binder comprises a constant restructured domain 3 (CH3) having the amino acid sequence shown herein (e.g., in Table B or C). In some embodiments, the KLRB1 binder comprises a heavy chain constant region containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence shown herein (e.g., in Table B or C). In some embodiments, one or more constant regions of the KLRB1 binder have been modified. In some embodiments, the antibody may comprise modifications to one or more of the three heavy chain constant regions (CH1, CH2, or CH3) and / or to the light chain constant region (CL). In some embodiments, the heavy chain constant region of the modified antibody comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibody comprises more than one human constant region. In some embodiments, modifications to the constant region include adding, deleting, or substituting 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 that provides some of the molecular flexibility typically conferred by the missing constant region. In some embodiments, the modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody. In some embodiments, the modified antibody comprises a peptide spacer inserted between the hinge region and the modified CH2 and / or CH3 domains.

[0170] It is known in the art that one or more constant regions of an antibody mediate several effector functions, and these effector functions can vary depending on the antibody isotype. Furthermore, the Fc region of an antibody can bind to cells expressing Fc receptors (FcRs). Many Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (ε receptor), IgA (α receptor), and IgM (μ receptor). Binding of an antibody to Fc receptors on the cell surface triggers many important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by cytotoxic cells (referred to as antibody-dependent cytotoxicity or ADCC), cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), release of inflammatory mediators, placental transfer, and 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 skilled 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, for example, Tables A and C). In some cases, Fc regions with amino acid variations have been identified in native antibodies. In some embodiments, the variant Fc regions are engineered to have substitutions at specific amino acid positions compared to the 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, Jan. 2021. 4(1):45–54; Lobner et al., Immunol Rev. Mar. 2016; 270(1): 113–131). In some embodiments, the Fc region is unfucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs. Mar. 2009; 1(3):230–236, which describes a method for producing therapeutic antibodies with controlled levels of fucosylation of Fc region N-glycans).

[0171] In some embodiments, the modified antibody (e.g., a modified Fc region) provides altered effector functions, which in turn affect the antibody's biological profile. For example, in some embodiments, the deletion or inactivation of the constant region (through point mutation or other means) enhances the Fc receptor binding of the modified antibody during its circulation. In some embodiments, constant region modification increases the serum half-life of the antibody. In some embodiments, constant region modification decreases the serum half-life of the antibody. In some embodiments, constant region modification increases or enhances the antibody's ADCC and / or complement-dependent cytotoxicity (CDC). 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 cytotoxic, oligosaccharide, or carbohydrate attachment sites.

[0172] 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 coding DNA and / or by synthesizing the desired antibody or peptide. 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 characteristics of the modified antibody.

[0173] Exemplary full-length heavy chain (HC) and light chain (LC) sequences are provided in Table C.

[0174] Table C. Exemplary sequences of complete heavy chain (HC) and light chain (LC) regions

[0175] In some embodiments, the KLRB1 binder (e.g., an antibody) as described herein comprises a heavy chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the HC sequence shown in Table C, and / or a light chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity with the LC sequence shown in Table C. In some embodiments, VH comprises an amino acid sequence having at least 95% sequence identity with the VH amino acid sequence shown in Table C, or is composed of an amino acid sequence having at least 95% sequence identity with the VH amino acid sequence shown in Table C, and VL comprises an amino acid sequence having at least 95% sequence identity with the VL amino acid sequence shown in Table C, or is composed of an amino acid sequence having at least 95% sequence identity with the VL amino acid sequence shown in Table C.

[0176] This disclosure further includes additional variants and equivalents substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to enhance the binding affinity of the antibody. In some embodiments, it is desirable to modulate the biological properties of the antibody, including but not limited to specificity, thermostability, expression level, one or more effector functions, glycosylation, immunogenicity, or solubility. Those skilled in the art will understand that amino acid changes may alter the post-translational processes of the antibody, such as changing the number or location of glycosylation sites or altering membrane anchoring properties.

[0177] Mutations can be substitutions, deletions, or insertions of one or more nucleotides encoding an antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native antibody or polypeptide sequence. In some embodiments, an amino acid substitution is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, such as replacing leucine with serine, for example, a conserved amino acid substitution. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. In some embodiments, substitutions, deletions, or insertions relative to the parent molecule include substitutions of fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids. In some embodiments, changes in the amino acid sequence that are biologically useful and / or relevant are determined by systematically performing insertions, deletions, or substitutions in the sequence and testing the activity of the resulting variant protein compared to the parent antibody.

[0178] In some embodiments, the variant may include the addition of amino acid residues to the amino terminus and / or carboxyl terminus of the antibody or peptide. The length of the additional amino acid residues may range from one residue to one hundred or more residues. In some embodiments, the variant contains an N-terminal methionyl residue. In some embodiments, the variant contains an additional peptide / protein, i.e., a fusion protein. In some embodiments, the variant is engineered to be detectable and may contain a detectable label and / or protein (e.g., an enzyme).

[0179] In some embodiments, cysteine ​​residues that do not participate in maintaining the correct conformation of the antibody can be replaced or deleted to modulate antibody properties, for example, to improve oxidative stability and / or prevent abnormal disulfide crosslinking. Conversely, in some embodiments, one or more cysteine ​​residues can be added to create one or more disulfide bonds to improve stability.

[0180] The variant antibodies or peptides described herein can be generated using methods known in the art, including but not limited to site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.

[0181] In some embodiments, the KLRB1 binder described herein is chemically modified. In some embodiments, the KLRB1 binder is an anti-KLRB1 antibody that has been chemically modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, and / or linking to cellular ligands or other proteins. Any of many chemical modifications can be performed using known techniques.

[0182] This disclosure covers KLRB1 binding agents constructed on a non-immunoglobulin backbone, wherein the agent binds to the same epitope or substantially the same epitope as the anti-KLRB1 antibody disclosed herein. In some embodiments, the non-immunoglobulin-based binding agent is an agent that competes with the anti-KLRB1 antibody described herein in a competitive binding assay. In some embodiments, alternative KLRB1 binding agents comprise scaffold proteins. Generally, based on the architecture of the scaffold protein backbone, scaffold proteins can be assigned to one of 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, anticarrier proteins based on lipid carrier scaffolds; adnectin based on the 10th domain of human adhesion protein type 3; and affinity proteins based on Staphylococcus aureus (S. aureus). Staphylococcus aureusThe B domain in the Ig-binding region of protein A; darpin, which is based on the ankyrin repeat domain protein; fynomer, which is based on the SH3 domain of human Fyn protein kinase; affitin, which is based on the acidophilic thermosulfur leaf fungus ( Sulfolobus acidocaldarius The KLRB1 binder comprises Sac7d; affilin, based on human γ-β lens protein or human ubiquitin; aVimer, based on the A domain of a membrane receptor protein; knottin (cysteine ​​knottin), based on a 30-amino acid-stabilizing anti-parallel β-chain protein folding; and a Kunitz domain inhibitor scaffold, based on a structure containing three disulfide bonds and three rings. In some embodiments, the KLRB1 binder comprises engineered scaffold proteins comprising, for example, the heavy chains CDR1, CDR2, and CDR3 and the light chains CDR1, CDR2, and CDR3 shown in any of Tables 1 to 17, such as Tables 14, 4, or 10.

[0183] Typically, antigen-antibody interactions are nonvalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic forces, and van der Waals forces. The terms affinity and / or cohesive force are often used to describe the strength of antigen-antibody complexes. The binding of an antibody to its antigen is a reversible process, and the affinity of binding is usually expressed as an equilibrium dissociation constant (…). KD (Report to K) D It is the antibody dissociation rate (k off (How antibodies rapidly dissociate from their antigens) and antibody association rate (k) on The ratio of how rapidly an antibody binds to its antigen. In some implementations, this is achieved by measuring the k-value of a specific antibody / antigen interaction. on and k off Rate, and then use the ratio of these values ​​to calculate K. D Value to determine K D Value. K D Values ​​can be used to evaluate and rate the strength of individual antibody / antigen interactions. The lower the KD of an antibody, the higher its affinity for its target. In some implementations, SPR technology in the Biacore system is used to measure affinity. Affinity provides a measure of the overall strength of the antibody-antigen complex. It depends on three main parameters: (i) the antibody's affinity for the target, (ii) the valence state of both the antibody and the antigen, and (iii) the structural arrangement of the interacting parts.

[0184] In some embodiments, the KLRB1 binder (e.g., antibody) has a dissociation constant (KL) 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 (e.g., human KLRB1) binds to KLRB1. In some embodiments, the KLRB1 binder is at about 20 nM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 10 nM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 1 nM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 0.5 nM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 0.1 nM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 50 pM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 25 pM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 10 pM or lower K D Bound to KLRB1 (e.g., human KLRB1). In some embodiments, the KLRB1 binder is at about 1 pM or lower K D The conjugate binds to KLRB1 (e.g., human KLRB1). In some embodiments, the dissociation constant of the conjugate (e.g., antibody) to KLRB1 is determined using the KLRB1 protein immobilized on a Biacore chip and the conjugate flowing through the chip. In some embodiments, the dissociation constant of the conjugate (e.g., antibody) to KLRB1 is determined using the conjugate captured by an anti-human IgG antibody on a Biacore chip and soluble KLRB1 flowing through the chip.

[0185] In some embodiments, a KLRB1 binder (e.g., an antibody) binds to KLRB1 (e.g., human KLRB1) at a half-maximum 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 binder 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, the KLRB1 binder binds 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.

[0186] The KLRB1 binders (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 those sequences in a suitable host. In some embodiments, DNA sequences are constructed using recombinant techniques by isolating or synthesizing DNA sequences encoding wild-type proteins of interest. Optionally, the sequences can be mutagenized by site-specific mutagenesis to provide functional variants. In some embodiments, DNA sequences encoding polypeptides of interest are 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 preferred in the host cell from which the recombinant polypeptide of interest will be produced. Standard methods can be used to synthesize polynucleotide sequences encoding isolated polypeptides of interest. For example, complete amino acid sequences can be used to construct reverse-translated genes. Further, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then linked together. Individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.

[0187] Once assembled (by synthesis, site-directed mutagenesis, or other methods), the polynucleotide sequence encoding a specific polypeptide of interest can be inserted into an expression vector and operatively ligated to expression control sequences suitable for protein expression in the desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and / or expression of the bioactive polypeptide in a suitable host. As is well known in the art, to achieve high expression levels of the transfected gene in a host, the gene must be operatively ligated to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0188] In some implementations, recombinant expression vectors are used to amplify and express DNA encoding an antibody against human KLRB1 or a fragment thereof. For example, the recombinant expression vector may be a reproducible DNA construct having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain encoding a KLRB1 binding agent (such as an anti-KLRB1 antibody or an antigen-binding fragment thereof), operatively linked to a suitable transcriptional and / or translational regulatory element derived from a mammalian, microbial, viral, or insect gene. A transcriptional unit typically comprises an assembly of: (1) one or more genetic elements that regulate gene expression, such as a transcription promoter or enhancer; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) appropriate transcriptional and translational initiation and termination sequences. Regulatory elements may include operon sequences for controlling transcription. Additional inclusions may also include the ability to replicate in the host, typically conferred by the origin of replication, and selection genes for facilitating the recognition of transformants. DNA regions are “operatively 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 involved in the secretion of the polypeptide, then the DNA of the signal peptide is operatively linked to the DNA of the polypeptide; if a promoter controls transcription of a coding sequence, then the promoter is operatively linked to that sequence; or, if a ribosome binding site is localized to allow translation, then the ribosome binding site is operatively linked to the coding sequence. In some embodiments, the structural element intended for use in a yeast expression system comprises a leader sequence that enables the host cell to secrete the translated protein extracellularly. In some embodiments, when the recombinant protein is expressed without a leader sequence or transport sequence, the polypeptide may contain an N-terminal methionine residue. This residue may then optionally be cleaved from the expressed recombinant protein to provide the final product.

[0189] The choice of expression control sequences and expression vectors is often host-dependent. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as those from *E. coli* (including pCR1, pBR322, pMB9, and their derivatives), as well as plasmids with a broader host range, such as M13 and other filamentous single-stranded DNA bacteriophages.

[0190] The KLRB1 binding agent (e.g., antibody) disclosed herein can be expressed by one or more vectors. For example, in some embodiments, the heavy chain polypeptide is expressed by one vector while 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 a single vector.

[0191] Suitable host cells for expressing KLRB1 binding agents (e.g., antibodies) or KLRB1 protein or fragments thereof for use as antigens or immunogens include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include Gram-negative or Gram-positive 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 used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts, as well as protein production methods (including antibody production), are well known in the art.

[0192] Various mammalian culture systems can be used to express recombinant peptides. Expression of recombinant proteins in mammalian cells is often desirable because these proteins are typically correctly folded, appropriately modified, and possess biological function. Examples of suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney), L-929 (mouse fibroblast), C127 (mouse mammary tumor), 3T3 (mouse fibroblast), CHO (Chinese hamster ovary), HeLa (human cervical cancer), BHK (hamster kidney fibroblast), HEK-293 (human embryonic kidney), and their variants. Mammalian expression vectors may contain non-transcriptional elements such as origin of replication, appropriate promoters and enhancers linked to the gene to be expressed, as well as other 5′ or 3′ flanked non-transcriptional sequences and 5′ or 3′ untranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splicing donor and acceptor sites, and transcription termination sequences.

[0193] Expression of recombinant proteins in insect cell culture systems (e.g., baculoviruses) 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, this disclosure provides cells comprising the KLRB1 binder described herein. In some embodiments, the cells produce the KLRB1 binder described herein. In some embodiments, the cells produce antibodies. In some embodiments, the cells produce antibodies binding to human KLRB1. In some embodiments, the cells produce antibodies binding to cynomolgus monkey KLRB1. In some embodiments, the cells produce antibodies binding to both human KLRB1 and cynomolgus monkey KLRB1. In some embodiments, the cell produces antibodies or variants thereof named 11.12, 11.27, 11.29, 11.41.1, 11.44.1, 11.45, 11.54, 11.55, 11.57, 12.0, 12.1.3, 12.2, 12.3, 13.2, 13.4, 13.5, or 13.6. In some embodiments, the cell produces scFv versions of antibodies 11.12, 11.27, 11.29, 11.41.1, 11.44.1, 11.45, 11.54, 11.55, 11.57, 12.0, 12.1.3, 12.2, 12.3, 13.2, 13.4, 13.5, or 13.6. In some embodiments, the cell is a prokaryotic cell (e.g., *E. coli*). In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a hybridoma cell. Proteins produced by the host cell can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags (such as hexahistine, maltose-binding domains, influenza capsid sequences, and glutathione S-transferases) can be attached to proteins to allow for easy purification by passing through a suitable affinity column. Affinity chromatography for purifying immunoglobulins can include protein A chromatography, protein G chromatography, and protein L chromatography. The 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 proteins can be determined using techniques known to those skilled in the art, including but not limited to SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).In some embodiments, the supernatant from the expression system that secretes the recombinant protein into the culture medium is first concentrated using a commercially available protein concentrator filter (e.g., Amicon® or Millipore Pellicon® ultrafiltration device). Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, anion exchange resins are used, such as matrices or substrates having diethylaminoethyl (DEAE) side chains. This matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. In some embodiments, a cation exchange step is employed. 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 reversed-phase HPLC steps are used to further purify the recombinant protein using 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 for the hydrophobic separation of the recombinant protein based on its recombinant protein composition. HIC is a useful separation technique for purifying proteins while maintaining their biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques. Some or all of the aforementioned purification steps, in various combinations, can be used to provide homogeneous recombinant proteins.

[0194] The physical / chemical properties and / or biological activity of the anti-KLRB1 antibody disclosed herein can be analyzed using various assays known in the art. In some embodiments, the ability of the anti-KLRB1 antibody 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 antibody to induce ADCC, ADCP, and / or CDC, as well as the ability of the antibody to kill KLRB1 target cells (cell exhaustion), is tested. Assays include, but are not limited to, ADCC cell lysis assays using, for example, LDH release and formazan salt detection. Furthermore, the antibody's solubility, stability, thermal stability, viscosity, expression level, expression quality, and / or purification efficiency can also be evaluated.

[0195] In some implementations, the purified anti-KLRB1 antibody is characterized by methods including, but not limited to, N-terminal sequencing, amino acid analysis, high-performance liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion assays.

[0196] Antibody conjugates This disclosure also provides conjugates comprising the anti-KLRB1 antibody or its antigen-binding fragment described herein. In some embodiments, the antibody or its antigen-binding fragment is attached to a second molecule. In some embodiments, the antibody or its antigen-binding fragment is conjugated to or partially conjugated to a cytotoxic agent. In some embodiments, the antibody or its antigen-binding fragment 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, docalimcin, daunorubicin, pyrrolobenzodiazepine (PBD), or other intercalating agents. In some embodiments, the cytotoxic agent is a microtubule inhibitor, including but not limited to oliguria guanylate, maytansine alkaloids (e.g., DM1 and DM4), and tubulolysin. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including but not limited to diphtheria toxin A chain, the non-conjugated active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, absinthecin A chain, modeccin A chain, alpha-sarcin, and tung oil (…). Aleurites fordii Protein, caryophyllin, American pokeweed Phytolaca americana Proteins (PAPI, PAPII, and PAP-S), ornamental bitter gourd (Momordica charantia) Inhibitors, curcin, crotin, soapwort (Sapaonaria officinalis) Inhibitors, white tree toxins, mitogellin, aspergillin, phenolmycin, enoxamycin, and trichothecene. In some embodiments, the antibody is conjugated to one or more small molecule toxins, such as galicariin, maytansine alkaloids, trichothecene, and CC1065. Derivatives of any of these toxins may be used, as long as the derivative retains the cytotoxic activity of the parent molecule.

[0197] Conjugates comprising the anti-KLRB1 antibody or its antigen-binding fragment described herein can be prepared using any suitable method known in the art. In some embodiments, conjugates are prepared using a variety of bifunctional protein conjugates such as N-succinimide-3-(2-pyridyldithiol)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (such as dimethyl adipamide ester HCl), active esters (such as disuccinimide-octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), diazide derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bifunctional fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

[0198] In some embodiments, the anti-KLRB1 antibody described herein, or its antigen-binding fragment, is conjugated to a detectable substance or molecule that enables the antibody to be used for diagnosis and / or detection. Detectable substances may include, but are not limited to: enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, and acetylcholinesterase; cofactors such as biotin and one or more riboflavins; fluorescent materials such as umbelliferone, luciferin, luciferin isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazineamine luciferin, dansyl chloride, cyanin (Cy3), and phycoerythrin; bioluminescent materials such as luciferase; and 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 Ru、 35 S, 47 Sc、 75 Se、 153 Sm、 113 Sn、 117 Sn、 85 Sr、 99m Tc, 201 Ti、 133 Xe, 90 Y、 69 Yb、 175 Yb、 65 Zn; a positron-emitting metal; and a magnetic metal ion.

[0199] The anti-KLRB1 antibody or its antigen-binding fragment described in this article can also be conjugated with a second antibody to form an antibody heteroconjugate.

[0200] Anti-KLRB1 antibodies or their antigen-binding fragments as described herein can be attached to solid supports. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. In some embodiments, immobilized anti-KLRB1 antibodies are used in immunoassays. In some embodiments, immobilized anti-KLRB1 antibodies are used in the purification of target antigens.

[0201] Polynucleotides / Methods for preparing binding agents This document also provides nucleic acids encoding the polypeptides described herein and vectors, preferably expression vectors, containing nucleic acids encoding the polypeptides described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto, and may include plasmids, granules, or viral vectors. The vector is capable of autonomous replication or may integrate into the host DNA. Viral vectors include, for example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses.

[0202] Vectors may include nucleic acids in a form suitable for expression in host cells. Preferably, recombinant expression vectors include one or more regulatory sequences operatively 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 expression vectors may depend on factors such as the choice of host cells to be transformed, the desired protein expression level, etc. The expression vectors of the present invention may be introduced into host cells to produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein that encode a KLRB1 binder as described herein.

[0203] The recombinant expression vector of the present invention can be designed for expression of the KLRB1 binding protein in prokaryotic cells. Preferably, the KLRB1 binding protein 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 polyomaviruses, adenovirus 2, cytomegaloviruses, and simian viruses 40.

[0204] Vector DNA can be introduced into host cells using conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to various art-recognized techniques used to introduce foreign nucleic acids (e.g., DNA) into host cells, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipid transfection, or electroporation.

[0205] Host cells can be used to produce (i.e., express) KLRB1 binding protein. Therefore, the present invention further provides a method for producing KLRB1 binding protein using the host cells of the present invention. In one embodiment, the method comprises culturing the host cells of the present invention in a suitable culture medium (in which a recombinant expression vector encoding the KLRB1 binding protein has been introduced) to produce the KLRB1 binding protein. In another embodiment, the method further comprises isolating the KLRB1 binding protein from the culture medium or the host cells. In some embodiments, the Fc region is fucosylated-free (see, for example, Yamane-Ohnuki and Satoh, MAbs. May-June 2009; 1(3): 230–236, which describes a method for producing a therapeutic antibody having a controlled level of fucosylation of Fc region N-glycans).

[0206] Pharmaceutical Composition This document also provides pharmaceutical compositions comprising the KLRB1 binder described herein as an active ingredient. In various embodiments, the KLRB1 binder is prepared as a pharmaceutical composition, for example, a pharmaceutical composition intended for use as a medicine. In various embodiments, the pharmaceutical composition is intended for use as a medicine for treating a subject in need of autoimmune diseases, allergic diseases, graft rejection, or hematologic malignancies. In some embodiments, the autoimmune diseases are rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn's disease, ulcerative colitis, or celiac disease. In some embodiments, the allergic diseases are asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some implementations, graft rejection can be rejection of kidney, lung, heart, liver, limb, skin, or multi-organ transplants. In some implementations, the hematologic malignancy is leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell lymphoma, or large granular lymphoblastic leukemia (LGLL). In some implementations, 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, Cezari syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphoma (PTCL-NOS) not otherwise specified. In some implementations, the leukemia is aggressive NK-cell leukemia or T-cell prolymphoblastic leukemia.

[0207] Those skilled in the art can formulate KLRB1 binders into pharmaceutical compositions using known methods.

[0208] Pharmaceutical compositions may include carriers. As used herein, “carrier” may include pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic (or relatively non-toxic) to cells or subjects exposed thereto at the doses and concentrations employed. Physiologically acceptable carriers are often aqueous pH buffer solutions. 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) peptides; 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0209] In various embodiments, the KLRB1 binder is contained in an injectable formulation, such as a subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection formulation. The injectable formulation may be an aqueous solution, such as an aqueous solution of a physiologically compatible buffer, like Hanks' solution, Ringer's solution, or physiological saline buffer. The injectable formulation may contain a formulatory agent such as a suspending agent, stabilizer, and / or dispersant. Alternatively, the KLRB1 binder may be in a dry or powder form for reconstitution with a suitable medium (e.g., sterile, pyrogen-free water) prior to use.

[0210] The binders of this disclosure can be formulated in any suitable form for delivery to target cells / tissues. In some embodiments, the KLRB1 binder can be formulated as liposomes, microparticles, microcapsules, albumin microspheres, microemulsions, nanoparticles, nanocapsules, or macroemulsions. In some embodiments, the pharmaceutical formulation comprises an agent of this 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-derived phosphatidylethanolamine (PEG-PE).

[0211] In some embodiments, the KLRB1 binder is formulated as a sustained-release preparation. Suitable examples of sustained-release preparations include a semi-permeable matrix of a solid hydrophobic polymer containing the agent, wherein the matrix is ​​in the form of a molded article (e.g., a membrane 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-glutamic acid esters, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0212] Treatment and application This disclosure provides a method comprising administering to a subject in need a KLRB1 binder as described herein, or a pharmaceutical composition comprising a KLRB1 binder as described herein. In some embodiments, the subject is a human being. In some embodiments, the method is performed in vivo (e.g., as opposed to ex vivo). As used herein, “treatment” refers to therapeutic treatment (treating a subject with a disease); the method can also be used for preventative or preventative measures, wherein the aim is to prevent or alleviate (reduce) the disease in subjects who do not have the targeted pathological disease or condition. Those in need of treatment may include those who already have the condition, those who are susceptible to the condition, or those who need to prevent the condition (as used herein, “prevention” is intended to reduce the risk of developing the disease).

[0213] In various aspects and embodiments, this disclosure provides methods for treating autoimmune diseases, allergic diseases, graft rejection, or hematologic malignancies in subjects of need. As described herein, KLRB1-expressing cells are associated with the pathogenesis of these diseases. According to this disclosure, depletion of such KLRB1-expressing cells provides therapeutic benefit. In some embodiments, autoimmune diseases are rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn's disease, ulcerative colitis, or celiac disease. In some embodiments, allergic diseases are asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome. In some embodiments, graft rejection can be rejection of kidney, lung, heart, liver, limb, skin, or multi-organ grafts. In some implementations, the hematologic malignancy is leukemia, such as T-cell leukemia, NK-cell leukemia, T-cell lymphoma, or large granular lymphoblastic leukemia (LGLL). In some implementations, 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, Cezari syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL), and peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). In some implementations, the leukemia is aggressive NK-cell leukemia or T-cell prolymphoblastic leukemia.

[0214] In various aspects and implementations, this disclosure provides methods for treating or preventing graft rejection. Graft rejection can be, for example, kidney rejection.

[0215] "Administration" and "treatment," when applicable 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. "Administration" and "treatment" include in vivo treatment, as well as in vitro or ex vivo treatment in some embodiments.

[0216] Typically, the agent is administered in an amount that effectively alleviates one or more symptoms of a disease in the treated subject or population, either inducing the resolution of these symptoms or inhibiting their progression to any clinically measurable extent. The amount of a therapeutic agent that effectively alleviates any particular disease symptom can vary depending on factors such as the patient's disease state, age, and weight, as well as the drug's ability to elicit the desired response in the subject. The extent of symptom relief can be assessed using any clinical measurement commonly used by a physician or other skilled healthcare provider to evaluate the severity or progression of the symptom.

[0217] Therefore, in various embodiments, the term "effective amount" or "therapeutic effective amount" refers to the concentration or amount of KLRB1 binder that results in achieving the stated purpose (e.g., relief of one or more symptoms of the disease described herein). The "effective amount" of the KLRB1 binder can be determined empirically. Furthermore, the "therapeutic effective amount" is the concentration or amount of KLRB1 binder that effectively achieves the stated therapeutic effect. This amount can also be determined empirically.

[0218] In some embodiments, treatment with the KLRB1 binder of this disclosure can kill at least about 20%, for example at least about 30%, 40%, 50%, 60%, 70%, or 80% of KLRB1-expressing cells associated with the pathogenesis of the diseases disclosed herein.

[0219] The term “subject” refers to any animal (e.g., mammal), including but not limited to human and non-human veterinary subjects (including non-human primates).

[0220] As used herein, references to “about” or “approximate” values ​​or parameters include (and describe) implementations for that value or parameter. For example, a reference to “about X” includes a description of “X”. As used herein, “about” means plus or minus 10%.

[0221] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” used in this disclosure and claims include the plural forms.

[0222] It should be understood that whenever an implementation is described herein using the term "comprising," other similar implementations described as "consisting of" and / or "substantially consisting of" are also provided. It should be understood that whenever an implementation is described herein using the phrase "substantially consisting of," other similar implementations described as "consisting of" are also provided.

[0223] As used herein, the term "and / or" such as "A and / or B" means including: both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" such as "A, B, and / or C" is intended to cover 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).

[0224] In various implementation schemes, the KLRB1 binder can be administered by providing the subject with mRNA encoding the binder.

[0225] The following embodiments are for illustrative purposes only and are not intended to be limiting. After reading this disclosure, those skilled in the art will understand many variations of this technology. Therefore, the scope of this technology should not be determined by reference to the embodiments, but rather by reference to the appended claims and their equivalents.

[0226] Example Example 1: KLRB1 gene expression is a marker of a unique aggregate of immune cells, and is limited to immune cells. KLRB1 expression spans traditional lymphocyte classifications and is a marker for Th17, Th17.1, ex-Th17, Tc17, iNKT, ILC2, ILC3, peTh2, and subsets of NK cells. Figure 1 ).

[0227] Whole-body KLRB1 expression profiling data showed that KLRB1 was not significantly expressed in any cell type other than immune cells. Figure 2 ).

[0228] Example 2: Enhanced KLRB1 expression under various immune conditions Rheumatoid Arthritis: Analysis of expression data from synovial biopsies of patients with rheumatoid arthritis (GSE1919) compared to normal patients showed increased KLRB1 expression (10-fold). Figure 3 Therefore, rheumatoid arthritis is a particularly attractive target for the treatments according to this disclosure.

[0229] Analysis of expression data from synovial biopsies of patients with rheumatoid arthritis (GSE36700) compared to patients with crystal-induced arthritis showed increased KLRB1 expression (3.3-fold). Figure 4 Therefore, rheumatoid arthritis is a particularly attractive target for the treatments according to this disclosure.

[0230] Sjögren's syndrome: Analysis of expression data from salivary gland biopsies of patients with Sjögren's syndrome (GSE23117) compared to normal conditions showed increased KLRB1 expression in the late (13.1-fold), intermediate (5.3-fold), and early (1.6-fold) stages. Figure 5 Therefore, Sjögren's syndrome is a particularly attractive target for the treatments according to this disclosure.

[0231] Analysis of expression data from parotid gland biopsies of patients with Sjögren's syndrome (GSE40611) compared to normal conditions showed increased KLRB1 expression (2.6-fold). Figure 6 Therefore, Sjögren's syndrome is a particularly attractive target for the treatments according to this disclosure.

[0232] Inclusion body myositis: Analysis of expression data from muscle biopsies of patients with inclusion body myositis (GSE38454) compared to normal conditions showed increased KLRB1 expression (2.9-fold). Figure 7 Therefore, inclusion body myositis is a particularly attractive target for the treatment according to this disclosure.

[0233] Discoid lupus: Analysis of expression data from skin biopsies of patients with discoid lupus (GSE52471) compared to normal conditions showed increased KLRB1 expression (7.2-fold increase). Figure 8 Therefore, discoid lupus is a particularly attractive target for the treatments according to this disclosure.

[0234] Psoriasis: Analysis of expression data from skin biopsies of patients with psoriasis (GSE52471) compared to normal conditions showed an increase in KLRB1 expression (11.2-fold). Figure 9 Therefore, discoid lupus is a particularly attractive target for the treatments according to this disclosure.

[0235] Idiopathic pulmonary fibrosis: Analysis of expression data from lung biopsies of patients with idiopathic pulmonary fibrosis (GSE53845) compared to normal conditions showed increased KLRB1 expression (1.5-fold). Figure 10 Therefore, idiopathic pulmonary fibrosis is a particularly attractive target for the treatments according to this disclosure.

[0236] Diabetes: Analysis of expression data from pancreatic biopsies of patients with diabetes (GSE72492) compared to normal conditions showed increased KLRB1 expression (3.7-fold). Figure 11 Therefore, diabetes is a particularly attractive target for the treatments according to this disclosure.

[0237] Alopecia universalis: Analysis of expression data from scalp biopsies of patients with idiopathic pulmonary fibrosis (GSE74761) compared to normal conditions showed increased KLRB1 expression (4.3-fold). Figure 12 Therefore, alopecia areata is a particularly attractive target for the treatments disclosed herein.

[0238] Primary biliary cholangitis: Analysis of expression data from liver biopsies of patients with primary biliary cholangitis who ultimately require liver transplantation (GSE79850) compared to normal conditions showed increased KLRB1 expression (5.6-fold increase). Figure 13 Therefore, primary biliary cholangitis is a particularly attractive target for the treatments according to this disclosure.

[0239] Multiple sclerosis: Analysis of expression data from brain biopsies of patients with multiple sclerosis (GSE5839) showed that KLRB1 expression was 2.5-fold higher than in control brains. Figure 14 Therefore, multiple sclerosis is a particularly attractive target for the treatments according to this disclosure.

[0240] Lymphocytic colitis: Analysis of expression data from colon biopsies of four patients with lymphocytic colitis (GSE65107) compared with four healthy individuals showed increased KLRB1 expression (3.2-fold). Figure 15 Therefore, lymphocytic colitis is a particularly attractive target for the treatments according to this disclosure.

[0241] Kidney graft rejection: Analysis of expression data from kidney biopsies of 7 patients with acute kidney rejection (GSE1563) compared to 9 healthy individuals showed increased KLRB1 expression (2.1-fold ratio). Figure 16 Therefore, kidney transplant rejection is a particularly attractive target for the therapy according to this disclosure.

[0242] Lung transplantation: Analysis of expression data from bronchoalveolar lavage (BAL) fluid of 7 patients who experienced lung graft rejection (GSE65107) compared with 27 patients who did not experience rejection showed increased KLRB1 expression (3.6-fold ratio). Figure 17 Therefore, lung graft rejection is a particularly attractive target for the therapies according to this disclosure.

[0243] Atopic dermatitis: Analysis of expression data from skin biopsies of 5 patients with atopic dermatitis (GSE65107) compared with 5 healthy individuals showed increased KLRB1 expression (1.9-fold). Figure 18 Therefore, atopic dermatitis is a particularly attractive target for the treatments according to this disclosure.

[0244] Palmoplantar pustulosis: An analysis of expression data from skin biopsies (GSE185856) from 3 patients with palmoplantar pustulosis lesions and 8 patients with non-lesion palmoplantar pustulosis skin compared to 7 healthy individuals showed increased KLRB1 expression (11.0-fold increase in the lesion group compared to the healthy group; 5.9-fold increase in the non-lesion group compared to the healthy group). Figure 19 Therefore, palmoplantar pustulosis is a particularly attractive target for the treatment according to this disclosure.

[0245] Hidradenitis suppurativa: Analysis of expression data (GSE148027) from skin biopsies of 18 patients with lesions of hidradenitis suppurativa and 7 patients with non-lesions of hidradenitis suppurativa compared to 8 healthy individuals showed increased KLRB1 expression (8.0-fold increase in the lesion group compared to the healthy group; 3.0-fold increase in the non-lesion group compared to the healthy group). Figure 20 Therefore, hidradenitis suppurativa is a particularly attractive target for the treatments according to this disclosure.

[0246] Asthma: Analysis of lung airway brushing samples (GSE41861) from 10 patients with severe asthma, 37 patients with moderate asthma, and 44 patients with mild asthma compared to 47 healthy individuals showed increased KLRB1 expression (1.9-fold increase in severe asthma compared to healthy individuals; 1.5-fold increase in moderate asthma compared to healthy individuals; and 1.3-fold increase in mild asthma compared to healthy individuals). Figure 21 Therefore, atopic dermatitis is a particularly attractive target for the treatments according to this disclosure.

[0247] Example 3: Enhanced KLRB1 expression in various T-cell and NK-cell lymphomas and leukemias Analysis of expression data from tumor cells from patients with various T-cell and NK-cell lymphomas and leukemias (GSE19067) compared to normal NK cell expression showed increased KLRB1 expression (e.g., a 17-fold ratio in hepatocellular and splenic T-cell lymphomas compared to normal NK cell lines) (Figure 22). Therefore, various T-cell and NK-cell lymphomas are particularly attractive targets for therapies according to this disclosure.

[0248] In particular, some patients with hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Cézari syndrome, unspecified peripheral T-cell lymphoma (PTCL-NOS), T-cell prolymphocytic leukemia (T-PLL), and peripheral T-cell lymphoma (PTCL) have increased KLRB1 expression or KLRB1 expression similar to that of CD4+ T cells (Figure 22). Therefore, hepatosplenic T-cell lymphoma (HSTCL), NK / T-cell lymphoma (NKTCL), aggressive NK-cell leukemia (ANKL), mycosis fungoides, Cézari syndrome, unspecified peripheral T-cell lymphoma (PTCL-NOS), T-cell prolymphocytic leukemia (T-PLL), and peripheral T-cell lymphoma (PTCL) are particularly attractive targets for therapies according to this disclosure.

[0249] Example 4: Improved anti-KLRB1 antibody To increase humanization and / or remove potential deamidation and isomerization sites, various anti-KLRB1 variants were derived from the anti-KLRB1 antibodies listed in Table 18. These variants (antibodies 11.12, 11.27, 11.29, 11.41.1, 11.44.1, 11.45, 11.54, 11.55, 11.57, 12.0, 12.1.3, 12.2, 12.3, 13.2, 13.4, 13.5, and 13.6), the novel sequences they contain, and their humanization calculated using the BioPhi Humanization Report are shown in Table 19. The humanization assessment settings were selected as Kabat number, Kabat CDR definition, and relaxed OASis prevalence threshold.

[0250] Table 18. Initial Antibody Variants

[0251] Table 19. Antibodies with improved humanization or removal of deamidation or isomerization sites. Amino acid changes are indicated (e.g., DS-->DY indicates replacement of DS with DY).

[0252] Example 5: Anti-KLRB1 antibodies 10A3D6hum2.2, 10A3D6hum8.2, 10A3D6hum1.2, 11.12, 11.27, 11.29, 11.41.1, 11.44.1, 11.45, 11.54, 11.55, 11.57, 12.0, 12.1.3, 12.2, 12.3, 13.2, 13.4, 13.5, and 13.6 bind to human KLRB1. Various humanized antibodies derived from humanized variants of antibody 10A3D6 containing human IgG1-κ were generated in CHO cells, and their binding to the human KLRB1 extracellular domain (ECD) was then determined by surface plasmon resonance (SPR). The human KLRB1 extracellular domain is amino acids 67-225 from the human KLRB1 protein (SEQ ID NO: 1). A Biacore 8K instrument was used with multi-cycle kinetics and affinity capture in a 96-well microplate and a single antibody concentration of 20 nM. The antibody was immobilized on the chip, and the human KLRB1 extracellular domain (ECD) analyte was flowed through the surface at 30 μL / min for a contact time of 120 s and a dissociation time of 360 s. Results from two independent experiments are shown in Tables 20 and 21. Overall, these results indicate that some variants (e.g., 11.27, 12.0, and 13.2) retained binding capacity, while others (e.g., 12.3 and 13.5) lost binding capacity.

[0253] Table 20: Binding kinetics and production yield of anti-KLRB1 antibody obtained by SPR

[0254] Table 21: Binding kinetics and production yield of anti-KLRB1 antibody obtained by SPR

[0255] Example 6: Anti-KLRB1 antibodies 12.0, 13.2, 10A3D6hum2.2, and 10A3D6hum1.2 can induce antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC induced by anti-KLRB1 antibodies 12.0, 13.2, 10A3D6hum2.2, and 10A3D6hum1.2 was evaluated using a cell lysis assay involving LDH release and formazan detection (Genscript, SC1544). CHO-hum-KLRB1 (a stable CHO cell line expressing human KLRB1 SEQ ID NO: 1) was incubated with human peripheral blood mononuclear cells (PBMCs) and these antibodies, as well as control human IgG1. The EC50 of ADCC was 5.4 pM for antibody 12.0, 8.3 pM for antibody 13.2, 4.8 pM for antibody 10A3D6hum2.2, and 5.8 pM for antibody 10A3D6hum1.2. Results were presented in... Figure 23 As shown in the image.

[0256] Example 7: Other anti-KLRB1 humanized antibody sequences Table D shows the complete HC and LC sequences of various other humanized anti-KLRB1 antibodies. In some embodiments, the antibody or its antigen-binding moiety comprises a constant region heavy chain and a light chain, wherein the constant region heavy chain and / or light chain comprises or consists of the amino acid sequences shown in Table D.

[0257] Table D. Complete heavy chain (HC) and light chain (LC) regions of various other anti-KLRB1 antibodies

[0258] References Exemplary embodiments of the present invention are described in the following enumerated paragraphs: E1. An antibody or an antigen-binding fragment thereof that specifically binds to a member of the cytotoxic lectin-like receptor B subfamily 1 (KLRB1), said antibody or antigen-binding fragment thereof comprising: c. A heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises three VH complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3); and d. A light chain variable region (VL), wherein the light chain variable region (VL) comprises three VL complement-determining regions (CDR-L1, CDR-L2, and CDR-L3). The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 mentioned above are selected from one of the following: i. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55, and 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27, and 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 17-21; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or v. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49 or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49, or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or viii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 58-60; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ix. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or x. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 60 or 66; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 69 or 70; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xiii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28, 30 or 76; and CDR-L3 has the amino acid sequence of SEQ ID NO: 77 or 78; or xiv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 84 or 85; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvii. CDR-H1 has an amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has an amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has an amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has an amino acid sequence of any one of SEQ ID NO: 87-89; CDR-L2 has an amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has an amino acid sequence of SEQ ID NO: 81 or 82.

[0259] E2. The antibody or its antigen-binding fragment as described in embodiment E1, wherein the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are selected from the same antibody numbering rule.

[0260] E3. The antibody or its antigen-binding fragment as described in embodiment E2, wherein the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are selected from the Kabat antibody numbering rules.

[0261] E4. The antibody or antigen-binding fragment thereof as described in embodiment E3, wherein CDR-H1 has the amino acid sequence of SEQ ID NO: 14, CDR-H2 has the amino acid sequence of SEQ ID NO: 37, CDR-H3 has the amino acid sequence of SEQ ID NO: 22, CDR-L1 has the amino acid sequence of SEQ ID NO: 64, CDR-L2 has the amino acid sequence of SEQ ID NO: 28, and CDR-L3 has the amino acid sequence of SEQ ID NO: 31.

[0262] E5. An antibody or antigen-binding fragment thereof as described in any one of embodiments E1-E4, wherein the VH has an amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68 and having the amino acid sequences of CDR-H1, CDR-H2, CDR-H3 shown in embodiment E1.

[0263] E6. The antibody or antigen-binding fragment thereof as described in embodiment E5, wherein the VH comprises the amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68.

[0264] E7. An antibody or antigen-binding fragment thereof as described in embodiment E6, wherein the VH comprises the amino acid sequence of SEQ ID NO:68.

[0265] E8. An antibody or antigen-binding fragment thereof as described in any one of embodiments E1-E8, wherein the VL comprises an amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90 and having the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 shown in embodiment E1.

[0266] E9. An antibody or antigen-binding fragment thereof as described in embodiment E8, wherein the VL comprises an amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90.

[0267] E10. An antibody or antigen-binding fragment thereof as described in embodiment E9, wherein the VL comprises the amino acid sequence of SEQ ID NO:65.

[0268] E11. The antibody or antigen-binding fragment thereof as described in any one of embodiments E1-E10, wherein the VL and VH are selected from one of the following: f. The VH comprises SEQ ID NO: 68; and the VL comprises any one of SEQ ID NO: 65, 71, 75, 79, 80, 83, 86 or 90; g. The VH comprises any one of SEQ ID NO: 33, 42 or 48; and the VL comprises SEQ ID NO: 34; h. The VH comprises any one of SEQ ID NO: 51, 53 or 57; and the VL comprises SEQ ID NO: 52; i. The VH comprises SEQ ID NO: 62; and the VL comprises SEQ ID NO: 63 or SEQ ID NO: 65; or j. The VH contains SEQ ID NO: 57; and the VL contains SEQ ID NO: 67.

[0269] E12. The antibody or antigen-binding fragment thereof as described in embodiment E11, wherein the VH comprises SEQ ID NO:68; and the VL comprises SEQ ID NO:65.

[0270] E13. An antibody or antigen-binding fragment thereof as described in any one of embodiments E1-E12, wherein the VL is part of a light chain, wherein the light chain comprises an amino acid sequence of any one of SEQ ID NO: 99-110, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 99-110.

[0271] E14. An antibody or antigen-binding fragment thereof as described in embodiment E13, wherein the light chain comprises the amino acid sequence of any one of SEQ ID NO: 99-110.

[0272] E15. An antibody or antigen-binding fragment thereof as described in embodiment E14, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 102.

[0273] E16. An antibody or antigen-binding portion thereof as described in any one of embodiments E1-E15, wherein the antibody or antigen-binding portion thereof is an antibody containing an Fc region, the antibody binding to an Fc γ receptor (FcγR) and inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or binding to C1q and inducing complement-dependent cytotoxicity (CDC).

[0274] E17. An antibody or its antigen-binding portion as described in embodiment E16, wherein the Fc region is unfucosylated.

[0275] E18. An antibody or its antigen-binding portion as described in any one of embodiments E1-E17, wherein the antibody is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody or IgG4 antibody.

[0276] E19. An antibody or antigen-binding fragment thereof as described in embodiment E18, wherein the VH is part of a heavy chain, wherein the heavy chain comprises an amino acid sequence of any one of SEQ ID NO: 91-98, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 91-98.

[0277] E20. An antibody or antigen-binding fragment thereof as described in embodiment E19, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NO: 91-98.

[0278] E21. An antibody or antigen-binding fragment thereof as described in embodiment E20, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 98.

[0279] E22. An antibody or antigen-binding fragment thereof as described in any one of embodiments E1-E21, wherein the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), wherein the heavy chain (HC) and the light chain (LC) are selected from one of the following: f. The HC comprises SEQ ID NO: 98; and the LC comprises SEQ ID NO: 102 or any one of 104-110; g. The HC comprises any one of SEQ ID NO: 91-93; and the LC comprises SEQ ID NO: 99; h. The HC comprises any one of SEQ ID NO: 94-96; and the LC comprises SEQ ID NO: 100; i. The HC contains SEQ ID NO: 97; and the LC contains SEQ ID NO: 101 or SEQ ID NO: 102; or j. The HC contains SEQ ID NO: 96; and the LC contains SEQ ID NO: 103.

[0280] E23. An antibody or antigen-binding fragment thereof as described in embodiment E22, wherein the HC comprises SEQ ID NO:98 and the LC comprises SEQ ID NO:102.

[0281] E24. An antibody or antigen-binding portion thereof as described in any one of embodiments E1-E22, wherein the antibody or antigen-binding portion thereof is conjugated to a cytotoxic agent.

[0282] E25. A collection of polynucleotides, said collection of polynucleotides comprising: c. The first nucleic acid sequence of the VH or heavy chain of the antibody or its antigen-binding moiety as described in embodiments E1 to E22; and d. Encoding a second nucleic acid sequence of the VL or light chain of the antibody or its antigen-binding portion as described in embodiments E1 to E22.

[0283] E26. A collection of polynucleotides as described in embodiment E25, wherein each of the first nucleic acid sequence and the second nucleic acid sequence is operatively linked to a promoter.

[0284] E27. A vector comprising a set of polynucleotides as described in embodiment E25 or 26.

[0285] E28. A collection of carriers, the collection of carriers comprising: c. A first vector comprising the first nucleic acid sequence as described in implementation scheme E25 or 26; and d. A second vector comprising the second nucleic acid sequence as described in implementation scheme E25 or E26.

[0286] E29. A host cell comprising a polynucleotide set as described in embodiment E25 or E26, or a vector as described in embodiment E27, or a vector set as described in embodiment E28, and optionally expressing an antibody or its antigen-binding portion.

[0287] E30. A method for preparing an antibody or its antigen-binding portion according to any one of embodiments E1-E24, the method comprising the following steps: c. Culture the host cells as described in embodiment E29 under conditions sufficient to express the antibody or its antigen-binding moiety; and d. Separate the antibody or its antigen-binding portion.

[0288] E31. The method as described in embodiment E28, the method further comprising formulating the antibody into a pharmaceutical composition.

[0289] E32. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof as described in any one of embodiments E1-E24, and a pharmaceutically acceptable carrier or diluent.

[0290] E33. A method for treating a subject in need of one or more of an autoimmune disease, an allergic disease, graft rejection, and a hematologic malignancy, the method comprising administering to the subject an antibody or its antigen-binding portion, as described in any one of embodiments E1-E24, or a pharmaceutical composition as described in embodiment E32, in an effective therapeutic amount.

[0291] E34. The method as described in implementation scheme E33, wherein the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.

[0292] E35. The method as described in embodiment E33, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome.

[0293] E36. The method as described in embodiment E33, wherein the graft rejection can be rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ graft.

[0294] E37. The method as described in embodiment E33, wherein the hematologic malignancy is lymphoma or leukemia.

[0295] E38. The method as described in embodiment E37, wherein the lymphoma is NK / T-cell lymphoma, mycosis fungoides, Cezari syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL) or peripheral T-cell lymphoma (PTCL-NOS) or mycosis fungoides unless otherwise specified.

[0296] E39. The method as described in embodiment E37, wherein the leukemia is T-cell leukemia, aggressive NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).

[0297] Other implementation plans It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to a member of the cytotoxic lectin-like receptor B subfamily 1 (KLRB1), said antibody or antigen-binding fragment comprising: a. A heavy chain variable region (VH), said heavy chain variable region (VH) comprising three VH complementarity-determining regions (CDR-H1, CDR-H2, and CDR-H3); and b. A light chain variable region (VL), wherein the light chain variable region (VL) comprises three VL complement-determining regions (CDR-L1, CDR-L2, and CDR-L3). The CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 mentioned above are selected from one of the following: i. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55, and 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27, and 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 17-21; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or iv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 40 or 41; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or v. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49, or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 23, 49, or 50; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or vii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 25-27; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or viii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 58-60; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or ix. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 12-16; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or x. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 43-47; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 60 or 66; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 29, 30 or 61; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 59, 69 or 70; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xiii. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 72-74; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28, 30 or 76; and CDR-L3 has the amino acid sequence of SEQ ID NO: 77 or 78; or xiv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 31 or 32; or xv. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 27 or 64; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvi. CDR-H1 has the amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has the amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has the amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has the amino acid sequence of any one of SEQ ID NO: 26, 84 or 85; CDR-L2 has the amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has the amino acid sequence of SEQ ID NO: 81 or 82; or xvii. CDR-H1 has an amino acid sequence of any one of SEQ ID NO: 14, 15, 54, 55 or 56; CDR-H2 has an amino acid sequence of any one of SEQ ID NO: 35-39; CDR-H3 has an amino acid sequence of any one of SEQ ID NO: 22-24; CDR-L1 has an amino acid sequence of any one of SEQ ID NO: 87-89; CDR-L2 has an amino acid sequence of any one of SEQ ID NO: 28-30; and CDR-L3 has an amino acid sequence of SEQ ID NO: 81 or 82.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are selected from the same antibody numbering rule.

3. The antibody or its antigen-binding fragment as described in claim 2, wherein the amino acid sequences of each group of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 are selected from the Kabat antibody numbering rules.

4. The antibody or antigen-binding fragment thereof as claimed in claim 3, wherein CDR-H1 has the amino acid sequence of SEQ ID NO: 14, CDR-H2 has the amino acid sequence of SEQ ID NO: 37, CDR-H3 has the amino acid sequence of SEQ ID NO: 22, CDR-L1 has the amino acid sequence of SEQ ID NO: 64, CDR-L2 has the amino acid sequence of SEQ ID NO: 28, and CDR-L3 has the amino acid sequence of SEQ ID NO:

31.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, wherein the VH has an amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68, or has at least 90% sequence identity with any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68 and has an amino acid sequence of the CDR-H1, CDR-H2, CDR-H3 amino acid sequence as shown in claim 1.

6. The antibody or antigen-binding fragment thereof as claimed in claim 5, wherein the VH comprises the amino acid sequence of any one of SEQ ID NO: 33, 42, 48, 51, 53, 57, 62 or 68.

7. The antibody or antigen-binding fragment thereof as claimed in claim 6, wherein the VH comprises the amino acid sequence of SEQ ID NO:

68.

8. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-7, wherein the VL comprises an amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90 and having the amino acid sequences of CDR-L1, CDR-L2, CDR-L3 as shown in claim 1.

9. The antibody or antigen-binding fragment thereof as claimed in claim 8, wherein the VL comprises the amino acid sequence of any one of SEQ ID NO: 34, 52, 63, 65, 67, 71, 75, 79, 80, 83, 86 or 90.

10. The antibody or antigen-binding fragment thereof as claimed in claim 9, wherein the VL comprises the amino acid sequence of SEQ ID NO:

65.

11. The antibody or antigen-binding fragment thereof as described in any one of claims 1-10, wherein the VL and VH are selected from one of the following: a. The VH comprises SEQ ID NO: 68; and the VL comprises any one of SEQ ID NO: 65, 71, 75, 79, 80, 83, 86 or 90; b. The VH comprises any one of SEQ ID NO: 33, 42 or 48; and the VL comprises SEQ ID NO: 34; c. The VH comprises any one of SEQ ID NO: 51, 53 or 57; and the VL comprises SEQ ID NO: 52; d. The VH comprises SEQ ID NO: 62; and the VL comprises SEQ ID NO: 63 or SEQ ID NO: 65; or e. The VH contains SEQ ID NO: 57; and the VL contains SEQ ID NO:

67.

12. The antibody or antigen-binding fragment thereof as claimed in claim 11, wherein the VH comprises SEQ ID NO: 68; and the VL comprises SEQ ID NO:

65.

13. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-12, wherein the VL is part of a light chain, wherein the light chain comprises an amino acid sequence of any one of SEQ ID NO: 99-110, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 99-110.

14. The antibody or antigen-binding fragment thereof as claimed in claim 13, wherein the light chain comprises the amino acid sequence of any one of SEQ ID NO: 99-110.

15. The antibody or antigen-binding fragment thereof as claimed in claim 14, wherein the light chain comprises the amino acid sequence of SEQ ID NO:

102.

16. The antibody or antigen-binding portion thereof as claimed in any one of claims 1-15, wherein the antibody or antigen-binding portion thereof is an antibody comprising an Fc region, the antibody binding to an Fc γ receptor (FcγR) and inducing antibody-dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or binding to C1q and inducing complement-dependent cytotoxicity (CDC).

17. The antibody or antigen-binding portion thereof as claimed in claim 16, wherein the Fc region is unfucosylated.

18. The antibody or its antigen-binding portion as claimed in any one of claims 1-17, wherein the antibody is an immunoglobulin G (IgG) subtype IgG1 antibody, IgG2 antibody or IgG4 antibody.

19. The antibody or antigen-binding fragment thereof as claimed in claim 18, wherein the VH is part of a heavy chain, wherein the heavy chain comprises an amino acid sequence of any one of SEQ ID NO: 91-98, or an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 91-98.

20. The antibody or antigen-binding fragment thereof as claimed in claim 19, wherein the heavy chain comprises the amino acid sequence of any one of SEQ ID NO: 91-98.

21. The antibody or antigen-binding fragment thereof as claimed in claim 20, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO:

98.

22. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-21, wherein the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), wherein the heavy chain (HC) and the light chain (LC) are selected from one of the following: a. The HC comprises SEQ ID NO: 98; and the LC comprises SEQ ID NO: 102 or any one of 104-110; b. The HC comprises any one of SEQ ID NO: 91-93; and the LC comprises SEQ ID NO: 99; c. The HC comprises any one of SEQ ID NO: 94-96; and the LC comprises SEQ ID NO: 100; d. The HC contains SEQ ID NO: 97; and the LC contains SEQ ID NO: 101 or SEQ ID NO: 102; or e. The HC contains SEQ ID NO: 96; and the LC contains SEQ ID NO:

103.

23. The antibody or antigen-binding fragment thereof as claimed in claim 22, wherein the HC comprises SEQ ID NO: 98 and the LC comprises SEQ ID NO:

102.

24. The antibody or antigen-binding portion thereof as claimed in any one of claims 1-22, wherein the antibody or antigen-binding portion thereof is conjugated to a cytotoxic agent.

25. A collection of polynucleotides, said polynucleotide collection comprising: a. A first nucleic acid sequence encoding the VH or heavy chain of the antibody or its antigen-binding moiety as claimed in claims 1 to 22; and b. A second nucleic acid sequence encoding the VL or light chain of the antibody or its antigen-binding portion as claimed in claims 1 to 22.

26. The polynucleotide set of claim 25, wherein each of the first nucleic acid sequence and the second nucleic acid sequence is operatively linked to a promoter.

27. A vector comprising the polynucleotide set of claim 25 or 26.

28. A collection of carriers, the collection of carriers comprising: a. A first vector comprising the first nucleic acid sequence of claim 25 or 26; and b. A second vector comprising the second nucleic acid sequence as described in claim 25 or 26.

29. A host cell comprising the polynucleotide set of claim 25 or 26, or the vector of claim 27, or the vector set of claim 28, and optionally expressing an antibody or its antigen-binding portion.

30. A method for preparing the antibody or antigen-binding portion thereof according to any one of claims 1-24, the method comprising the following steps: a. Culture the host cells of claim 29 under conditions sufficient to express the antibody or its antigen-binding moiety; and b. Separate the antibody or its antigen-binding portion.

31. The method of claim 28, wherein the method further comprises formulating the antibody into a pharmaceutical composition.

32. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof as described in any one of claims 1-24, and a pharmaceutically acceptable carrier or diluent.

33. A method for treating a subject in need of one or more of an autoimmune disease, an allergic disease, graft rejection, and a hematologic malignancy, the method comprising administering to the subject an antibody or an antigen-binding portion thereof, as described in any one of claims 1-24, or a pharmaceutical composition as described in claim 32, in an effective therapeutic amount.

34. The method of claim 33, wherein the autoimmune disease is rheumatoid arthritis, Sjögren's syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.

35. The method of claim 33, wherein the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyps, eosinophilic gastrointestinal disorders, or hypereosinophilic syndrome.

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

37. The method of claim 33, wherein the hematologic malignancy is lymphoma or leukemia.

38. The method of claim 37, wherein the lymphoma is NK / T-cell lymphoma, mycosis fungoides, Cezari syndrome, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma (AITL) or peripheral T-cell lymphoma (PTCL-NOS) or mycosis fungoides unless otherwise specified.

39. The method of claim 37, wherein the leukemia is T-cell leukemia, aggressive NK-cell leukemia, T-cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).

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