WO2024091919A2 - Agents de liaison à klrb1 et leurs méthodes d'utilisation - Google Patents

Agents de liaison à klrb1 et leurs méthodes d'utilisation Download PDF

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Publication number
WO2024091919A2
WO2024091919A2 PCT/US2023/077607 US2023077607W WO2024091919A2 WO 2024091919 A2 WO2024091919 A2 WO 2024091919A2 US 2023077607 W US2023077607 W US 2023077607W WO 2024091919 A2 WO2024091919 A2 WO 2024091919A2
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Prior art keywords
antibody
klrb1
amino acid
acid sequence
set forth
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WO2024091919A3 (fr
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Steven A. Greenberg
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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Priority to JP2025523864A priority Critical patent/JP2026502764A/ja
Priority to AU2023367648A priority patent/AU2023367648A1/en
Priority to EP23883632.4A priority patent/EP4608858A2/fr
Priority to CN202380087151.2A priority patent/CN120569402A/zh
Publication of WO2024091919A2 publication Critical patent/WO2024091919A2/fr
Publication of WO2024091919A3 publication Critical patent/WO2024091919A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • KLRB1 binding agents in particular anti-KLRB1- antibodies as well as therapeutic methods of using the agents for autoimmune disease, allergic diseases, transplant rejection, hematologic malignancies, and cancer.
  • KLRB1 killer cell lectin-like receptor subfamily B, member 1
  • CD161 killer cell lectin-like receptor subfamily B, member 1
  • T-helper IL-17 secreting (Th17) cells express KLRB1 (Maggi et al.2010).
  • Th17 cells and the cytokine IL-17 they produce, are implicated in psoriasis, psoriatic arthritis, ankylosing spondylitis, and inflammatory bowel disease (Yang et al. 2014), among other autoimmune diseases.
  • KLRB1-binding antibodies and antigen-binding fragments thereof that have numerous uses, including therapeutic and diagnostic uses.
  • the antibodies can be used for treating, and in some cases preventing, various diseases associated with KLRB1 expressing cells (i.e., by depleting the KLRB1 expressing cells), e.g., for treating, and in some cases preventing (i.e., reducing 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; transplant rejection; hematologic malignancies, and cancer.
  • various diseases associated with KLRB1 expressing cells i.e., by depleting the KLRB1 expressing cells
  • autoimmune diseases including psoriasis, psoriatic arthritis, ankylosing spondylitis, palmoplantar pustulosis, hidradenitis suppurativa, and inflammatory bowel disease
  • allergic diseases
  • the VH comprises or consists of an VH amino acid sequence set forth in Table 9, 8, or 18.
  • the VL comprises or consists of an VL amino acid sequence set forth in Table 9, 8, or 18.
  • the VH comprises or consists of the VH amino acid sequence set forth in Table 3, and the VL comprises or consists of the VH amino acid sequence set forth in Table 3.
  • the VH comprises or consists of the VH amino acid sequence set forth in Table 5, and the VL comprises or consists of the VH amino acid sequence set forth in Table 5.
  • the VH comprises or consists of the VH amino acid sequence set forth in Table 8, and the VL comprises or consists of the VH amino acid sequence set forth in Table 8.
  • the VH comprises or consists of the VH amino acid sequence set forth in Table 9, and the VL comprises or consists of the VH amino acid sequence set forth in Table 9.
  • the VH comprises or consists of the VH amino acid sequence set forth in Table 18, and the VL comprises or consists of the VH amino acid sequence set forth in Table 18.
  • the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence set forth in Table C
  • the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the VL amino acid sequence set forth in Table C.
  • the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence set forth in one of Tables 9, 8, or 18, and the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in one of Tables 9, 8, or 18, preferably wherein the VH and VL are from the same Table.
  • the antibodies or antigen binding portions thereof comprise a constant region heavy chain and light chain, wherein the constant region heavy chain and/or light chain comprises or consists of an amino acid sequence set forth in one of Table A.
  • antibodies or antigen binding portions thereof that specifically bind to human killer cell lectin-like receptor subfamily B, member 1, (KLRB1; e.g., SEQ ID NO:1), comprising or consisting of a variable region heavy chain consisting of the VH amino acid sequence set forth in Table 3 and a variable region light chain consisting of the VL amino acid sequence set forth in Table 3, and optionally a constant region, optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table A.
  • KLRB1 e.g., SEQ ID NO:1
  • antibodies or antigen binding portions thereof that specifically bind to human killer cell lectin-like receptor subfamily B, member 1, (KLRB1; e.g., SEQ ID NOs:1-3), comprising or consisting of a variable region heavy chain consisting of the VH amino acid sequence set forth in Table 5 and a variable region light chain consisting of the VL amino acid sequence set forth in Table 5, and optionally a constant region, optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table A.
  • antibodies or antigen binding portions thereof that specifically bind to human killer cell lectin-like receptor subfamily B, member 1, (KLRB1; e.g., SEQ ID NO:1), comprising or consisting of a variable region heavy chain consisting of the VH amino acid sequence set forth in Table 8, and a variable region light chain consisting of the VL amino acid sequence set forth in Table 8, and optionally a constant region, optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table A.
  • KLRB1 e.g., SEQ ID NO:1
  • antibodies or antigen binding portions thereof that specifically bind to human killer cell lectin-like receptor subfamily B, member 1, (KLRB1; e.g., SEQ ID NO:1), comprising or consisting of a variable region heavy chain consisting of the VH amino acid sequence set forth in Table 9, and a variable region light chain consisting of the VL amino acid sequence set forth in Table 9, and optionally a constant region, optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table A.
  • KLRB1 e.g., SEQ ID NO:1
  • antibodies or antigen binding portions thereof that specifically bind to human killer cell lectin-like receptor subfamily B, member 1, (KLRB1; e.g., SEQ ID NO:1), comprising or consisting of a variable region heavy chain consisting of the VH amino acid sequence set forth in Table 18, and a variable region light chain consisting of the VL amino acid sequence set forth in Table 18, and optionally a constant region, optionally comprising a sequence that is at least 95% identical to a sequence set forth in Table A.
  • the constant region comprises or consists of a sequence as set forth in Table A.
  • the antibodies comprise or consist of a heavy chain variable and/or light chain variable sequence that is at least 95% identical to a sequence set forth in Table B. In some embodiments, the antibodies comprise or consist of a complete heavy chain and/or light chain sequence that is at least 95% identical to a sequence set forth in Table C. Also provided herein are antibodies or antigen binding portions thereof that specifically bind to human KLRB1, comprising CDRs from different tables herein, or heavy/light chain pairs from different tables herein. In some embodiments, the antibodies or antigen binding portions thereof comprise a heavy chain constant region hinge region and Fc domain.
  • the antibody or antigen binding portion thereof is an antibody that comprises a heavy chain constant region comprising an amino acid sequence having at least 80%, 90%, 95%, or 97% sequence identity to a heavy chain constant region amino acid sequence set forth in Table A.
  • the antibody or antigen binding portion thereof is a monoclonal antibody.
  • the antibody or antigen binding portion thereof is a chimeric, humanized, or human antibody, and/or comprises one or more mutations (e.g., in a CDR) that remove Asn (N)-glycosylation sites or remove Cys, Asp, Met, Trp or Lys.
  • the antibody or antigen binding portion thereof is an immunoglobulin G (IgG) subtype IgG1 antibody, an IgG2 antibody, or an IgG4 antibody.
  • the antibody or antigen binding portion thereof is an antibody that comprises an Fc region, preferably human IgG1, that binds to Fc gamma receptors (Fc ⁇ Rs) and induces antibody dependent cell-mediated cytotoxicity (ADCC) to deplete cells expressing KLRB1, or that binds to C1q and induce complement dependent cytotoxicity (CDC).
  • the antibody or antigen binding portion thereof is conjugated to a cytotoxic agent.
  • the antibody or antigen binding portion thereof comprises an Fc region that is afucosylated.
  • CD161 is an inhibitory receptor when bound to ligand CLEC2D (LLT1), modulation of this interaction can therefore affect immune cell activity.
  • LLT1 ligand CLEC2D
  • inhibiting the CD161/CLEC2D interaction could enhance T cell function, a typical mechanism of action of the class of immuno-oncology therapeutics, while augmenting the CD161/CLEC2D interaction could act as immunosuppression for autoimmune and allergic diseases.
  • antibodies disclosed herein block CD161/CLEC2D interaction thereby activating T or NK cells to attack tumor cells in a variety of cancers, typical of an immuno-oncology checkpoint inhibition mechanism.
  • antibodies disclosed herein augment (e.g., increase) CD161/CLEC2D interaction thereby suppressing T or NK cells providing immunosuppression applicable for treating a variety of autoimmune, allergic, and inflammatory diseases.
  • a KLRB1-binding antibody disclosed herein achieves better immune cell activation, for example demonstrates improvements in any one or more of the activities of activating T cells to produce cytokines, activating T cells to kill tumor cells, activating NK cells (e.g., increased expression of CD107a), activating NK cells to produce cytokines or cytotoxic molecules such as granzymes, and activating NK cells to kill tumor cells when compared to the immune cell activation of one or more of 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 JNH25
  • an antibody disclosed herein that has an N-terminal glutamine or glutamate may be post-translationally modified at such N-terminus to form a pyroglutamate (or pyrrolidone carboxylic acid).
  • polynucleotides comprising a nucleic acid sequence encoding an antibody or antigen binding portion thereof as described herein.
  • the nucleic acid sequence is operably linked to a promoter.
  • vectors comprising the polynucleotides described herein, as well as host cells comprising the polynucleotides or the vectors, and optionally expressing an antibody or antigen binding portion thereof as described herein.
  • kits for making the antibodies or antigen binding portions thereof as described herein can include culturing the host cells under conditions sufficient to express the antibody or antigen binding portion thereof and isolating the antibody or antigen binding portion thereof.
  • the methods include formulating the antibody as a pharmaceutical composition.
  • pharmaceutical compositions that comprise an antibody or antigen binding portion thereof of as described herein (e.g., comprises or consists of the antibody or antigen binding portion thereof as an active agent), and a pharmaceutically acceptable carrier or diluent.
  • the antibody or antigen binding portion thereof is not B199.2 (Invitrogen), HP-3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), DX1 (Thermo Fisher), DX12 (BD Biosciences), 191B8 (Miltenyi Biotec), Ab9 (PCT publication WO2023028501A1), KW1.2.1 (United States patent publication US20210122826A1), KW7.3.7 (United States patent publication US20210122826A1), or JNH25G2G22 (Creative Diagnostics).
  • an antibody that binds KLRB1 preferably an antibody or antigen binding portion thereof, polynucleotide, vector, pharmaceutical composition, or host cell that expresses the antibody or antigen binding portion thereof, as described herein, for use in a method of treating one or more of an autoimmune disease, an allergic disease, a transplant rejection, and a hematologic malignancy in a subject in need thereof.
  • the autoimmune disease is rheumatoid arthritis, Sjogren’s syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, or hidradenitis suppurativa.
  • the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyposis, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.
  • the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ transplant.
  • the hematological malignancy is a leukemia, e.g., T cell leukemia, NK cell leukemia, T cell prolymphocytic leukemia (T-PLL), or large granular lymphocytic leukemia (LGLL).
  • the hematological malignancy is a lymphoma, e.g., hepatosplenic T cell lymphoma, NK/T cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), or peripheral T cell lymphoma not otherwise specified (PTCL-NOS).
  • a lymphoma e.g., hepatosplenic T cell lymphoma, NK/T cell lymphoma, mycosis fungoides, Sezary syndrome, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), or peripheral T cell lymphoma not otherwise specified (PTCL-NOS).
  • the antibody or antigen binding portion thereof, the polynucleotide, the vector, the antibody binds to and depletes Th17, Th17.1, ex-Th17, Tc17, mucosal-associated invariant T cells (MAIT), invariant NK-T cells (iNKT), innate lymphoid cells types 2 and 3 (ILC2, and ILC3), pathogenic effector Th2 (peTh2) cells, and/or NK cells expressing KLRB1.
  • the subject is human.
  • the antibodies disclosed herein will demonstrate superiority to previously described antibodies, e.g., commercially available antibodies, in one or more of the following activities: - increased binding potency (lower binding EC 50 to cell expressed KLRB1 or to soluble KLRB1 extracellular domain); - increased ADCC mediated depletion potency (lower EC50 for depletion of CHO- KLRB1+ cells or lower EC50 for reporter cell line ADCC assay); - increased binding affinity kinetics (lower KD or lower Koff); - increased blocking potency (lower IC 50 for inhibition of CLEC2D binding to cell expressed KLRB1); and/or - increased augmentation potency (lower EC 50 for augmenting (increasing) CLEC2D binding to cell expressed KLRB1).
  • - increased binding potency lower binding EC 50 to cell expressed KLRB1 or to soluble KLRB1 extracellular domain
  • ADCC mediated depletion potency lower EC50 for depletion of CHO- KLRB1
  • the antibodies disclosed herein have a higher production yield, lower immunogenicity (due to the presence of humanized variable regions and/or human Fc sequences), and/or improved biophysical parameters (e.g., higher melting temperature, greater freeze-thaw stability, lower isomerization, reduced or absence of deamidation, and/or less susceptibility to oxidation) when compared to previously described antibodies, e.g., commercially available antibodies.
  • 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.
  • FIG.1 shows KLRB1 expression marks Th17, Th17.1, ex-Th17, Tc17, iNKTs, IL2, ILC3, peTh2, and a subset of NK cells.
  • FIG.2 shows body-wide KLRB1 expression profiling data indicating that KLRB1 has no significant expression on any cell type other than immune cells
  • FIG.3 shows KLRB1 is overexpressed in rheumatoid arthritis synovium compared to osteoarthritis and normal synovium.
  • FIG.4 shows KLRB1 is overexpressed in rheumatoid arthritis synovium compared to osteoarthritis and crystal-induced arthritis synovium.
  • FIG.5 shows KLRB1 is overexpressed in advanced and moderate Sjogren’s syndrome salivary gland compared to normal.
  • FIG.6 shows KLRB1 is overexpressed in Sjogren’s syndrome parotid gland compared to normal.
  • FIG.7 shows KLRB1 is overexpressed in muscle tissue from patients with inclusion body myositis.
  • FIG.8 shows KLRB1 is overexpressed in skin tissue from patients with discoid lupus.
  • FIG.9 shows KLRB1 is overexpressed in skin tissue from patients with psoriasis.
  • FIG.10 shows KLRB1 is overexpressed in lung tissue from patients with idiopathic pulmonary fibrosis.
  • FIG.11 shows KLRB1 is overexpressed in pancreas tissue from patients with diabetes.
  • FIG.12 shows KLRB1 is overexpressed in scalp tissue from patients with alopecia universalis.
  • FIG.13 shows KLRB1 is overexpressed in liver tissue from patients with primary biliary cholangitis.
  • FIG.14 shows KLRB1 is overexpressed in brain tissue from patients with multiple sclerosis.
  • FIG.15 shows KLRB1 is overexpressed in colon tissue from patients with lymphocytic colitis.
  • FIG.16 shows KLRB1 is overexpressed in kidney tissue from patients with renal transplants.
  • FIG.17 shows KLRB1 is overexpressed in broncheoalveolar lavage fluid from patients with lung transplants.
  • FIG.18 shows KLRB1 is overexpressed in skin tissue from patients with atopic dermatitis.
  • FIG.19 shows KLRB1 is overexpressed in skin tissue from patients with palmoplantar pustulosis.
  • FIG.20 shows KLRB1 is overexpressed in skin tissue from patients with hidradenitis suppurativa.
  • FIG.21 shows KLRB1 is overexpressed in airway brushings from patients with asthma.
  • FIGs. 22A-D shows KLRB1 is expressed in various T and NK cell lymphomas and leukemias.
  • FIG.22A shows increased expression of KLRB1 in tumor cells from 4/4 patients with hepatosplenic T cell lymphoma (HSTCL), 7/19 patients with NK/T cell lymphoma (NKTCL), and 2/2 patients with mycosis fungoides.
  • HTCL hepatosplenic T cell lymphoma
  • NKTCL NK/T cell lymphoma
  • FIG.22B shows expression of KLRB1 in various forms of peripheral T cell lymphoma (PTCL) including angioimmunoblastic T cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL; both ALK-positive and ALK- negative), NK/T cell lymphoma (NKTCL), peripheral T cell lymphoma not otherwise specified (PTCL-NOS), and T cell prolymphocytic leukemia (T-PLL).
  • FIG.22C shows KLRB1 increased expression in spleen tumor cells compared to healthy spleen and from tumor cells compared to healthy CD4 T cells in 4 patients each with HSTCL.
  • FIG.22D shows increased expression of KLRB1 an HSTCL cell line (DERL-2) and from aggressive NK cell leukemia (ANKL) cell lines (KHYG-1 and NKL).
  • FIGs. 23A-C shows binding of the murine antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, 5A11D10, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3 against human KLRB1 by ELISA.
  • FIG.23A shows binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, and 5A11D10.
  • FIG. 23B shows binding of 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2.
  • FIG. 23C shows binding of 22F10G3.
  • 24A-E shows binding of the murine antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3 to CHO-K1 cells expressing human or cynomolgus KLRB1 (CHO-hum-KLRB1 or CHO-cyno-KLRB1). Binding was detected by FACS. Secondary anti- mouse IgG was used to detect antibodies bound to CHO-hum-KLRB1 or CHO-cyno-KLRB1.
  • 24A shows binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO- hum-KLRB1.
  • FIG. 24B shows binding of 9C10G11, 57E2E4, 75B10F12, 7B6C7, and 52G9B12B5 to CHO-cyno-KLRB1.
  • FIG.24C shows binding of 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, and 92E12F2 to CHO-hum-KLRB1.
  • FIG. 24D shows binding of 22F10G3 to CHO-hum-KLRB1.
  • FIG.24E shows binding of 22F10G35 to CHO-cyno-KLRB1.
  • FIGs. 25A-B shows an antibody dependent cell-mediated cytotoxicity (ADCC) assay measuring target cell lysis of CHO-K1 target cells expressing human KLRB1 (CHO-hum- KLRB1) incubated with human peripheral blood mononuclear cells (PBMCs) and the following antibodies: (25A) mouse/human IgG1-kappa (wild-type) chimeric antibodies 57E2E4-Chimeric, 7B6C7-Chimeric, 52G9B12B5-Chimeric, 5B1B11-Chimeric, 50A8F2-Chimeric, 47A5H2- Chimeric; 52G9B12B5-Chimeric-EN (a mouse/human chimeric KLRB1-binding antibody with 52G9B12B5 variable regions and human IgG1-kappa mutant L234A/L235A/G237A mutation; used as
  • FIGs. 26A-E show that some anti-KLRB1 binding antibodies may compete with the natural KLRB1 ligand CLEC2D for binding to KLRB1 on CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) and thereby block the ability of CLEC2D to bind to KLRB1.
  • CHO-humKLRB1 cells were incubated with a CLEC2D-Fc-biotin fusion protein and with increasing concentrations of antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 22F10G3, and control mouse IgG. Binding of CLEC2D was determined by addition of streptavidin-AF488. The graphs show mean fluorescent intensity (MFI) over the indicated concentration range.
  • MFI mean fluorescent intensity
  • FIG.26A shows competition of 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, and mouse IgG with CLEC2D.
  • FIG. 26B shows competition of 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, and mouse IgG with CLEC2D.
  • FIG.26C shows competition of 50A8F2 and mouse IgG with CLEC2D, demonstrating that 50A8F2 enhances binding of CLEC2D to KLRB1.
  • FIG.26D shows competition in a second repeated experiment with a greater range of concentrations of 50A8F2, 47A5H2, and mouse IgG with CLEC2D, demonstrating in a second experiment that 50A8F2 enhances binding of CLEC2D to KLRB1.
  • FIG. 26E shows competition of 22F10G3 and mouse IgG with CLEC2D.
  • FIGs. 27A-F show the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies to human KLRB1 extracellular protein by ELISA.
  • FIG. 27A shows binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04.
  • FIG. 27B shows binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08.
  • FIG. 27C shows binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12.
  • FIG. 27D shows binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimeric.
  • FIG.27E shows binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7.
  • FIG.27F shows binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8.
  • FIG. 28A-F show the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies to cynomolgus KLRB1 extracellular protein by ELISA.
  • FIG. 28A shows binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04.
  • FIG. 28B shows binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08.
  • FIG. 28C shows binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12.
  • FIG. 28D shows binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimeric.
  • FIG.28E shows binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7. Hu50A8F2-04 did not bind.
  • FIG.28F shows binding of Hu47A5H2-11, HP-3G10, and DX12. 191B8 did not bind.
  • FIGs. 29A-F show the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies to CHO-hum-KLRB1 cells measured by FACS.
  • FIG.29A shows binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04.
  • FIG. 29B shows binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08.
  • FIG. 29C shows binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12.
  • FIG. 29D shows binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimeric.
  • FIG.29E shows binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7.
  • FIG.29F shows binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8.
  • FIGs. 30A-F show the binding of 47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies to CHO-cyno-KLRB1 cells measured by FACS.
  • FIG.30A shows binding of Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04.
  • FIG. 30B shows binding of Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08.
  • FIG. 30C shows binding of Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2-11, and Hu47A5H2-12.
  • FIG. 30D shows binding of Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimeric.
  • FIG.30E shows binding of Hu47A5H2-11, Hu50A8F2-04, Ab9, KW1.2.1, and KW7.3.7.
  • FIG.30F shows binding of Hu47A5H2-11, HP-3G10, DX12, and 191B8.
  • FIG.31A-F show the blocking by 47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies of CLEC2D binding to CHOK1-human KLRB1 cells measured by FACS.
  • FIG.31A shows blocking by Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47A5H2-04.
  • FIG.31B shows blocking by Hu47A5H2-05, Hu47A5H2-06, Hu47A5H2-07, and Hu47A5H2-08.
  • FIG.31C shows blocking by Hu47A5H2-09, Hu47A5H2-10, Hu47A5H2- 11, and Hu47A5H2-12.
  • FIG.31A shows blocking by Hu47A5H2 chimeric and humanized antibodies and other KLRB1 binding antibodies of CLEC2D binding to CHOK1-human KLRB1 cells measured by FACS.
  • FIG.31A shows blocking by Hu47A5H2-01, Hu47A5H2-02, Hu47A5H2-03, and Hu47
  • FIG. 31D shows blocking by Hu47A5H2-13, Hu47A5H2-14, Hu47A5H2-15, Hu47A5H2-16, and Hu47A5H2-chimeric.
  • FIG.31E shows blocking by Hu47A5H2-11, Ab9, KW1.2.1, and KW7.3.7.
  • FIG.31F shows blocking by Hu47A5H2-11, HP- 3G10, DX12, and 191B8.
  • FIGs. 32A-D show the binding of 50A8F2 chimeric and humanized antibodies to human KLRB1 extracellular protein by ELISA.
  • FIG. 32A shows binding of Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04.
  • FIG.32B shows binding of Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08.
  • FIG.32C shows binding of Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12.
  • FIG.32D shows binding of Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2-chimeric.
  • FIGs. 33A-E show the binding of 50A8F2 chimeric and humanized antibodies to CHO-hum-KLRB1 cells and absence of binding to CHO-cyno-KLRB1 cells measured by FACS.
  • FIG.33A shows binding to CHO-hum-KLRB1 of Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2- 03, and Hu50A8F2-04.
  • FIG. 33B shows binding to CHO-hum-KLRB1 of Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08.
  • FIG.33C shows binding of Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12.
  • FIG.33D shows binding to CHO-hum- KLRB1 of Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2- chimeric.
  • FIG.33E shows absence of binding to CHO-cyno-KLRB1 of the 16 humanized 50A8F2 antibodies Hu50A8F2-01 through Hu50A8F2-16 and the 50A8F2 chimeric, with binding (as a positive control) of Hu47A5H2 chimeric.
  • FIGs. 34A-D show that 50A8F2 chimeric and humanized antibodies augmented CLEC2D binding to CHO-hum-KLRB1 cells measured by FACS.
  • FIG. 34A shows augmentation of CLEC2D/CHO-hum-KLRB1 binding by Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04.
  • FIG. 34A shows augmentation of CLEC2D/CHO-hum-KLRB1 binding by Hu50A8F2-01, Hu50A8F2-02, Hu50A8F2-03, and Hu50A8F2-04.
  • FIG. 34B shows augmentation of CLEC2D/CHO-hum- KLRB1 binding by Hu50A8F2-05, Hu50A8F2-06, Hu50A8F2-07, and Hu50A8F2-08.
  • FIG. 34C shows augmentation of CLEC2D/CHO-hum-KLRB1 binding by Hu50A8F2-09, Hu50A8F2-10, Hu50A8F2-11, and Hu50A8F2-12.
  • FIG.34D shows augmentation of CLEC2D/CHO-hum-KLRB1 binding by Hu50A8F2-13, Hu50A8F2-14, Hu50A8F2-15, Hu50A8F2-16, and Hu50A8F2 chimeric.
  • FIG. 35 shows that 50A8F2 chimeric and humanized antibodies Hu50A8F2-01, Hu50A8F2-03, Hu50A8F2-04, and Hu50A8F2-06 augmented CLEC2D binding to CHOK1- human KLRB1 cells measured by FACS.
  • human IgG1 had no effect on CLEC2D binding to CHOK1-human KLRB1 cells and KW1.2.1, KW7.3.7, and 47A5H2 chimeric antibodies inhibited CLEC2D binding to CHOK1-human KLRB1 cells.
  • FIG.36A shows NK cell CD107a expression was decreased with K562-CLEC2D compared to K562 cells, and then increased with 47A5H2 chimeric (47A5H2-chi) antibody.
  • FIG.36B shows NK cell IFN-gamma (IFNg) expression was decreased with K562-CLEC2D compared to K562 cells, and then increased with 47A5H2 chimeric antibody.
  • FIGs. 37A-B show NK cell activation was suppressed by CLEC2D and anti-KLRB1 blocking antibodies activated NK cells in the presence of CLEC2D suppression.
  • NK cells showed less activation with K562-CLEC2D single cell clone 1D1 than with K562 cells.
  • Hu47A5H2-11 and other anti-KLRB1 antibodies enhanced NK cell activation in assays in which NK cells targeted K562-CLEC2D but not assays in which NK cells targeted K562 cells alone.
  • FIG.37A shows NK cell activation measured as CD107a expression.
  • FIG.37B shows NK cell activation measured as IFNg expression.
  • FIGs. 38A-B show Hu47A5H2-11 enhanced NK cell activation measured by CD107a expression by FACS, with NK cells targeting K562-CLEC2D clone 1D1 cells.
  • Hu47A5H2-11 had superior potency (EC500.039 nM) compared to other tested antibodies.
  • FIG.38A shows Hu47A5H2-11 in comparison to Ab9 (EC500.118 nM), KW1.2.1 (EC504.946 nM), and KW7.3.7 (EC5036.29 nM).
  • FIG. 34B shows Hu47A5H2-11 in comparison to HP-3G10 (EC50 1.071 nM), DX12 (EC506.137 nM), and 191B8 (EC500.085 nM).
  • FIG. 39A-B show Hu47A5H2-11 enhanced NK cell activation measured by IFN- gamma expression by FACS, with NK cells targeting K562-CLEC2D clone 1D1 cells.
  • Hu47A5H2-11 had superior potency (EC500.131 nM) compared to other tested antibodies.
  • FIG. 39A shows Hu47A5H2-11 in comparison to Ab9 (EC500.436 nM), and KW1.2.1 and KW7.3.7 (neither of which activated NK cell IFNg production).
  • FIG.39B shows Hu47A5H2-11 in comparison to HP-3G10 (EC502.510 nM), DX12 (EC507.956 nM), and 191B8 (EC500.2475 nM; more correctly, 0.383 nM given NK cell toxicity at 40 nM).
  • FIG. 40 shows that 47A5H2 chimeric and humanized antibodies enhanced NK cell killing in a primary NK cell vs Raji target cell assay, with superior potency to KW1.2.1 and KW7.3.7.
  • FIG. 40 shows that 47A5H2 chimeric and humanized antibodies enhanced NK cell killing in a primary NK cell vs Raji target cell assay, with superior potency to KW1.2.1 and KW7.3.7.
  • FIG. 41A-B shows that Hu47A5H2-11 enhanced NK cell killing in a primary NK cell vs Raji target cell assay, with superior potency to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8.
  • FIG. 41A shows Hu47A5H2-11 (EC500.036 nM) in comparison to Ab9 (EC50 0.055 nM), KW1.2.1 (EC500.112 nM), and KW7.3.7 (EC500.440 nM).
  • FIG.41B shows Hu47A5H2-11 (EC500.036 nM) in comparison to HP-3G10 (EC500.325 nM), DX12 (no effect), and 191B8 (EC500.064 nM).
  • FIG. 42 shows that 47A5H2-chimeric antibody enhanced T cell activation in Jurkat- NFAT-1G4TCR-KLRB1 vs K562-CLEC2D-HLA loaded with 0.005 ug/ml NY-ESO-1 peptide assay.47A5H2-chimeric, but not 50A8F2 chimeric, increased T cell activation.
  • FIG. 42 shows that 47A5H2-chimeric antibody enhanced T cell activation in Jurkat- NFAT-1G4TCR-KLRB1 vs K562-CLEC2D-HLA loaded with 0.005 ug/ml NY-ESO-1 peptide assay.47A5H2-chimeric, but not 50A8F2 chimeric, increased T cell activation.
  • FIG. 42 shows that 47A5H2-chimeric antibody enhanced T cell activation in Jurkat- NFAT-1G4TCR-KLRB1 vs K562-CLEC2D-HLA loaded with 0.005 ug/ml NY
  • FIG. 43A-B shows that Hu47A5H2-11 enhanced T cell activation in Jurkat-NFAT- 1G4TCR-KLRB1 vs K562-CLEC2D-HLA loaded with 0.005 ug/ml NY-ESO-1 peptide assay, with superior potency to Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8.
  • FIG. 43A shows Hu47A5H2-11 (EC500.379 nM) in comparison to Ab9 (EC500.675 nM), KW1.2.1 (did not activate), KW7.3.7 (did not activate), and Hu50A8F2-04 (did not activate).
  • FIG.43B shows Hu47A5H2-11 (EC500.379 nM) in comparison to HP-3G10 (EC5012.56 nM), DX12 (EC50 0.179 nM), and 191B8 (EC500.392 nM).
  • DETAILED DESCRIPTION KLRB1 expression marks a unique set of immune system cells implicated in a variety of autoimmune diseases. These include Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, and/or NK cells. KLRB1 is also expressed by neoplastic cells present in a number of T and NK cell malignancies. These include various peripheral T cell and NK cell lymphomas and leukemias.
  • the KLRB1-binding antibodies described herein can be used to preferentially target Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and/or neoplastic T or NK cells for depletion.
  • the population of KLRB1 expressing immune cells more abundantly expresses IL-17 than the population of total CD4 or CD8 T cells, and are more specific to Th17 and Tc17 T cells than CD4 or CD8, respectively.
  • KLRB1 killer cell lectin- like receptor B1
  • methods for treating a subject by administering to a subject in need thereof an effective amount of a killer cell lectin- like receptor B1 (KLRB1) binding agent with cell depleting activity, thereby depleting Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and/or neoplastic T or NK cells in vivo.
  • KLRB1 killer cell lectin- like receptor B1
  • KLRB1 killer cell lectin-like receptor B1
  • KLRB1 receptor blocking activity in which blocking of binding of CLEC2D (LLT1) to KLRB1 is achieved
  • Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, and/or peTh2 in vivo.
  • the antibodies have antibody dependent cellular cytotoxicity (ADCC) effector activity or complement dependent cytotoxicity (CDC) effector activity.
  • ADCC antibody dependent cellular cytotoxicity
  • CDC complement dependent cytotoxicity
  • Administering to a subject in need thereof an effective amount of an anti-KLRB1 antibody that has ADCC or CDC effector function, or that is linked to a cytotoxic agent, can be used to eliminate or reduce the number of Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, ILC2, ILC3, peTh2, NK cells, and/or neoplastic T or NK cells.
  • the disclosure provides a killer cell lectin-like receptor B1 (KLRB1) binding agent as described herein that has ADCC or CDC activity or is conjugated to a cytotoxic agent.
  • KLRB1 killer cell lectin-like receptor B1
  • the disclosure provides an mRNA or cDNA encoding the binding agent.
  • the disclosure provides a pharmaceutical composition comprising an effective amount of the binding agent.
  • Killer Cell Lectin-Like Receptor B1 (KLRB1)
  • KLRB1 Killer cell lectin-like receptor B1 (KLRB1), also known as CD161
  • CD161 is a type II transmembrane protein. It is a receptor modulating the activity of T and NK cells and can act as either a stimulatory (Fergusson et al.2014) or inhibitory (Aldemir et al.2005, Mathewson et al. 2021) receptor for different types of T and NK cells. It is present on the surface of a variety of immune cells.
  • KLRB1 The ligand for KLRB1 is LLT1 (also called CLEC2D) (Aldemir et al.2005). KLRB1 expression among T cells is limited to those with an ability to respond to IL-12 and IL-18 (Fergusson et al. 2014). Among CD4+ T helper cells, KLRB1 expression uniquely marks Th17, Th17.1, and ex-Th17 cells (cells that no longer produce IL-17 but produce IFN- ⁇ ). KLRB1 expression distinguishes Th1 IFNG producing cells (which are KLRB1-negative) from ex-Th17 IFNG producing cells (which are KLRB1+) (Basdeo et al.2017).
  • KLRB1 expression uniquely marks Tc17 cells.
  • KLRB1 marks T cells with capacity for IL-17 production or, in the case of ex-Th17 cells, interferon-gamma (IFNG) production.
  • IFNG interferon-gamma
  • KLRB1 An exemplary sequence of human KLRB1 is provided as SEQ ID NO:1: MDQQAIYAELNLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSV SVTSLIQKSSIEKCSVDIQQSRNKTTERPGLLNCPIYWQQLREKCLLFSHTVNPWNNSLA DCSTKESSLLLIRDKDELIHTQNLIRDKAILFWIGLNFSLSEKNWKWINGSFLNSNDLEIR GDAKENSCISISQTSVYSEYCSTEIRWICQKELTPVRNKVYPDS.
  • the KLRB1 is cynomolgus KLRB1(e.g., AOA2K5WYI1 from UniParc UPI0003ABB264); an exemplary sequence is provided as SEQ ID NO:2: MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLS LSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWQQVQEKCLLFSHTVNPWNNSL ADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKI TGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.
  • the KLRB1 is cynomolgus KLRB1 (e.g., UniProt A0A7N9D796); an exemplary sequence is provided as SEQ ID NO:3: MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLS LSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWKQVQEKCLLFSHTVNPWNNSL ADCSTKESSLLLIQDKDELTRTQNLIHDKAISFWIGLNFSLSEKNWKWINGSFLSSNDLKI TGDAKENSCVYISQTSVYSEYCSTEMKWICQKELTLVRNKVSPDSWL.
  • SEQ ID NO:3 MDQQMMYAELTLPKDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLS LSVASLLQKPSIGKCSVDIQQNRTKTTERPDLLNCPIYWKQVQEKCLLFSH
  • KLRB1-Binding Antibodies Described herein are antibodies and antigen binding fragments thereof that bind to KLRB1.
  • the term “antibody” refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. An antibody can be monoclonal. An antibody can be a human or humanized antibody.
  • monoclonal antibody encompasses intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab’, F(ab’)2, Fv), single chain antibodies (e.g., scFv), fusion proteins comprising an antibody fragment, and any other modified immunoglobulin molecule comprising at least one antigen-binding site.
  • monoclonal antibody refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage library display, recombinant expression, and transgenic animals.
  • chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a first source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.
  • humanized antibody refers to an antibody that comprises a human heavy chain variable region and a light chain variable region wherein the native CDR residues are replaced by residues from corresponding CDRs from a nonhuman antibody (e.g., mouse, rat, rabbit, or nonhuman primate), wherein the nonhuman antibody has the desired specificity, affinity, and/or activity.
  • one or more framework region residues of the human heavy chain or light chain variable regions are replaced by corresponding residues from nonhuman antibody.
  • humanized antibodies can comprise residues that are not found in the human antibody or in the nonhuman antibody. In some embodiments, these modifications are made to further refine and/or optimize antibody characteristics.
  • the humanized antibody comprises at least a portion of an immunoglobulin constant region (e.g., CH1, CH2, CH3, Fc), typically that of a human immunoglobulin. Exemplary constant regions include those shown in Table A. Table A. Sequences of Exemplary Constant Regions
  • human antibody refers to an antibody that possesses an amino acid sequence that corresponds to an antibody produced by a human and/or an antibody that has been made using any of the techniques that are known to those of skill in the art for making human antibodies.
  • antibody fragments can include a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecif ⁇ c antibodies formed from antibody fragments.
  • epipe and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen or target capable of being recognized and bound by a particular antibody.
  • epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of the protein.
  • Epitopes formed from contiguous amino acids also referred to as linear epitopes
  • epitopes formed by tertiary folding also referred to as conformational epitopes
  • An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation. Epitopes can be predicted using any one of a large number of software bioinformatic tools available on the internet.
  • X-ray crystallography may be used to characterize an epitope on a target protein by analyzing the amino acid residue interactions of an antigen/antibody complex.
  • Fv includes the minimum antibody fragment which contains a complete antigen- recognition and binding site. This region consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them.
  • immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgB1, IgG2, IgG3, IgG4, IgA, and IgA2. “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • the antibody or antigen binding fragment thereof comprises a human or humanized antibody.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • donor antibody non- human species
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • Methods for humanizing non-human antibodies are well known in the art.
  • the KLRB1 antibodies described herein can be affinity matured, for example using selection and/or mutagenesis methods known in the art.
  • an “affinity matured” antibody is one with one or more alterations in one or more hyper variable regions thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s).
  • an affinity matured antibody has nanomolar or even picomolar affinities for the target antigen.
  • Preferred affinity matured antibodies have an affinity that is five times, more preferably 10 times, even more preferably 20 or 30 times greater than the starting antibody (generally murine, humanized or human) from which the matured antibody is prepared.
  • An antibody that “binds to,” “specifically binds to,” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
  • KLRB1 agent e.g., an anti-KLRB1 antibody
  • a binding agent e.g. antibody
  • SPR Surface Plasmon Resonance
  • the KLRB1 antibody may be cross reactive with various similar KLRB1 proteins (e.g., with highest affinity for one, such as human KLRB1, and lower affinity for others, such as mouse KLRB1).
  • a binding agent that specifically binds an antigen binds the target antigen with a higher affinity than its affinity for a different antigen.
  • the different antigen can be a related antigen.
  • a binding agent that specifically binds an antigen binds the target antigen with an affinity that is at least 20 times greater than its affinity for a different antigen, e.g., at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different antigen.
  • a binding agent that specifically binds a particular antigen binds a different antigen at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art.
  • affinity is measured using SPR technology, e.g., in a Biacore system or other system known to those of skill in the art.
  • identity or “percent identity” in the context of two or more polypeptides (e.g., two anti-KLRB1 antibodies), refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity.
  • the percent identity may be measured using sequence comparison software or algorithms or by visual inspection.
  • Various algorithms and software that may be used to obtain 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 variants thereof.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • sequences differ in conservative substitutions may be, but not necessarily is, adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically, but not necessarily, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1.
  • two polypeptides e.g., antibodies or antibody domains (e.g., VL, CL, VH, CH1, CH2, CH3 domains) thereof) of the disclosure are substantially identical, meaning 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, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection.
  • antibodies or antibody domains e.g., VL, CL, VH, CH1, CH2, CH3 domains
  • percent identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 20-40, at least about 40-60 amino acid residues, at least about 60-80 nucleotides or amino acid residues in length or any integral value there between. In some embodiments, percent identity exists over a longer region than 60-80 amino acid residues, such as at least about 80-100 amino acid residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, an amino acid sequence.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can determined using the Needleman and Wunsch ((1970) J. Mol. Biol.
  • conservative sequence modifications or “conservative substitutions” as used herein may refer to amino acid modifications to a target epitope or antibodies and antigen- binding portions thereof of the disclosure that does not significantly affect or alter the binding characteristics of the anti-KLRB1 antibodies. “Conservative substitution” as used herein refers to a substitution in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been generally defined in the art, 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic
  • the antibody is a blocking or antagonist binding agent.
  • “Blocking” or “antagonist” means the agent (e.g., antibody or binding fragment thereof) is one that inhibits or reduces biological activity of the antigen it binds. Certain blocking agents or antagonist agents substantially or completely inhibit the biological activity of the antigen.
  • a KLRB1 binding agent 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 cells).
  • the KLRB1 binding agent is an antibody that comprises: a. a full length antibody that binds KLRB1 and comprises an Fc domain that can bind Fcgamma receptors with effector function to trigger antibody-dependent cell-mediated cytotoxicity (ADCC); b.
  • the KLRB1 antibodies described herein bind to the extracellular domain of human KLRB1; in some embodiments, the antibody cross-reacts with (binds to both of) the extracellular domains of human and cynomolgus KLRB1.
  • the antibody binds to an epitope of the extracellular domain of KLRB1, wherein the epitope is at least 90% identical in human and cynomolgus. In some embodiments, the antibody binds to KLRB1 and is not a mouse antibody.
  • the antibody described herein is not B199.2 (Invitrogen), HP- 3G10 (Invitrogen), OTI1D8 (OriGene), 14F1F11 (OriGene), 702228 (R&D Systems), B-D51 (Cell Sciences), 2F3 (Novus Biologics), EP7169 (Abcam), 191B8 (Miltenyi), DX12 (BD Biosciences), or JNH25G2G22 (Creative Diagnostics).
  • a KLRB1-binding agent is an antibody, e.g., a full length antibody comprising an Fc domain including at least one heavy chain.
  • the antibody is a recombinant antibody.
  • 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. In some embodiments, the antibody is an antibody fragment comprising an antigen- binding site. In some embodiments, the antibody is a scFv.
  • the antibody is a disulfide-linked scFv. In some embodiments, the antibody is a bispecific antibody or a multispecific antibody. In some embodiments, the antibody is a monovalent antibody. In some embodiments, the antibody is a monospecific antibody. In some embodiments, the antibody is a bivalent antibody. In some embodiments, the antibody is isolated. In some embodiments, the antibody is substantially pure. In some embodiments, a KLRB1-binding agent is a polyclonal antibody. Polyclonal antibodies can be prepared by any method known to those of skill in the art.
  • polyclonal antibodies are produced by immunizing an animal (e.g., a rabbit, rat, mouse, goat, donkey) with an antigen of interest (e.g., a purified peptide fragment, a recombinant protein, or a fusion protein) using multiple subcutaneous or intraperitoneal injections.
  • an antigen of interest e.g., a purified peptide fragment, a recombinant protein, or a fusion protein
  • the antigen is conjugated to a carrier such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
  • the antigen (with or without a carrier protein) is diluted in sterile saline and usually combined with an adjuvant (e.g., Complete or Incomplete Freund's Adjuvant) to form a stable emulsion.
  • an adjuvant e.g., Complete or Incomplete Freund's Adjuvant
  • polyclonal antibodies are recovered from the immunized animal (e.g., from blood or ascites).
  • the polyclonal antibodies are purified from serum or ascites according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and/or dialysis.
  • a KLRB1-binding agent is a monoclonal antibody.
  • Monoclonal antibodies can be prepared by any method known to those of skill in the art.
  • monoclonal antibodies are prepared using hybridoma methods known to one of skill in the art. For example, using a hybridoma method, a mouse, rat, rabbit, hamster, or other appropriate host animal, is immunized as described above.
  • lymphocytes are immunized in vitro.
  • the immunizing antigen is a human protein or a fragment thereof.
  • the immunizing antigen is a mouse protein or a fragment thereof.
  • lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol.
  • hybridoma cells are selected using specialized media as known in the art and unfused lymphocytes and myeloma cells do not survive the selection process.
  • Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen can be identified by a variety of methods including, but not limited to, immunoprecipitation, immunoblotting, and in vitro binding assays (e.g., flow cytometry, FACS, ELISA, SPR (e.g., Biacore), and radioimmunoassay).
  • the clones may be subcloned by limiting dilution techniques.
  • the hybridomas can be propagated either in in vitro culture using standard methods or in vivo as ascites tumors in an animal.
  • the monoclonal antibodies can be purified from the culture medium or ascites fluid according to standard methods in the art including, but not limited to, affinity chromatography, ion-exchange chromatography, gel electrophoresis, and dialysis.
  • monoclonal antibodies are made using recombinant DNA techniques as known to one skilled in the art.
  • the polynucleotides encoding an antibody are isolated from mature B-cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using standard techniques.
  • the isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors which produce the monoclonal antibodies when transfected into host cells such as E. coli, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins.
  • recombinant monoclonal antibodies are isolated from phage display libraries expressing variable domains or CDRs of a desired species. Screening of phage libraries can be accomplished by various techniques known in the art.
  • a monoclonal antibody is modified by using recombinant DNA technology to generate alternative antibodies.
  • the constant domains of the light chain and heavy chain of a mouse monoclonal antibody are substituted for constant regions of a human antibody to generate a chimeric antibody.
  • the constant regions are truncated or removed to generate a desired antibody fragment of a monoclonal antibody.
  • a KLRB1-binding agent is a humanized antibody.
  • a humanized antibody comprises one or more amino acid residues that have been introduced into it from a source that is non-human.
  • humanization is performed by substituting one or more non-human CDR sequences for the corresponding CDR sequences of a human antibody.
  • the humanized antibodies are constructed by substituting all six CDRs of a non-human antibody (e.g., a mouse antibody) for the corresponding CDRs of a human antibody.
  • a non-human antibody e.g., a mouse antibody
  • the choice of which human heavy chain variable region and/or light chain variable region is used for generating humanized antibodies can be made based on a variety of factors and by a variety of methods known in the art.
  • the “best-fit” method is used where the sequence of the variable region of a non-human (e.g., rodent) antibody is screened against the entire library of known human variable region sequences. The human sequence that is most similar to that of the non-human (e.g., rodent) sequence is selected as the human variable region framework for the humanized antibody.
  • variable region framework sequence is selected as the variable region framework.
  • variable region framework sequence is derived from the consensus sequences of the most abundant human subclasses.
  • human germline genes are used as the source of the variable region framework sequences.
  • a KLRB1-binding agent is a human antibody.
  • Human antibodies can be prepared using various techniques known in the art. In some embodiments, human antibodies are generated from immortalized human B lymphocytes immunized in vitro. In some embodiments, human antibodies are generated from lymphocytes isolated from an immunized individual.
  • a human antibody is selected from a phage library, where that phage library expresses human antibodies.
  • phage display technology may be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable region gene repertoires from unimmunized donors. Techniques for the generation and use of antibody phage libraries are well known in the art. Once antibodies are identified, affinity maturation strategies known in the art, including but not limited to, chain shuffling and site-directed mutagenesis, may be employed to generate higher affinity human antibodies.
  • human antibodies are produced in transgenic mice that contain human immunoglobulin loci.
  • a KLRB1-binding agent is a scFv antibody.
  • ScFvs are molecules that comprise a variable heavy chain region and a variable light chain region linked to form a single polypeptide. ScFvs can be produced using recombinant technologies known in the art.
  • a scFv comprises a polypeptide linker between the heavy chain variable region and the light chain variable region.
  • the scFv comprises an orientation of (from N- to C-terminus) (i) heavy chain variable region, (ii) linker, and (iii) light chain variable region.
  • the scFv comprises an orientation (from N- to C- terminus) of (i) light chain variable region, (ii) linker, and (iii) heavy chain variable region.
  • the scFv is a disulfide-linked scFv (dsscFv), which is a scFv comprising an engineered disulfide bond between the light chain variable region and heavy chain variable region of the scFv.
  • the scFv (e.g., dsscFv) is attached (either directly or indirectly) to a half-life extending moiety such as, e.g., an Fc molecule, a CH3 domain of an immunoglobulin (e.g., CH3 of IgG1), polyethylene glycol (PEG) or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysicalic acid, N-(2- hydroxypropyl)methacrylamide, or dextran, or is modified by, e.g., hyperglycosylation, to extend the half-life of the scFv (e.g., dsscFv).
  • a half-life extending moiety such as, e.g., an Fc molecule, a CH3 domain of an immunoglobulin (e.g., CH3 of IgG1), polyethylene glycol (PEG) or a PEG mimetic, X
  • the polypeptide linker is comprised of naturally, or non-naturally, occurring amino acids.
  • the linker comprises amino acids that allow for flexibility.
  • the linker comprises amino acids that allow for suitable solubility.
  • the linker comprises glycine amino acids.
  • the linker comprises glycine and serine amino acids.
  • the linker comprises one or more sets of glycine/serine repeats.
  • the linker comprises GGGGSGGGGSGGGGS (SEQ ID NO:401).
  • a KLRB1-binding agent is a Fv.
  • a Fv comprises a heavy chain variable region and a light chain variable region.
  • the Fv is attached (either directly or indirectly) to a half-life extending moiety such as, e.g., an Fc molecule, a CH3 domain of an IgG (e.g., CH3 of IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysicalic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, e.g., by hyperglycosylation, to extend the half-life of the Fv.
  • a KLRB1-binding agent is a Fab.
  • a Fab is one of the molecules that result from digestion of an immunoglobulin antibody with papain.
  • Fabs are monovalent molecules that comprise a light chain, a heavy chain variable region, a CH1 region, and, optionally, a heavy chain constant region hinge region or a portion thereof. Fabs can be produced using recombinant technologies known in the art.
  • a Fab comprises a polypeptide linker between the light chain constant region and the heavy chain variable region.
  • a Fab comprises a polypeptide linker between the heavy chain constant region and the light chain variable region.
  • suitable linkers are known to those of skill in the art and are not limited by any specific sequences disclosed herein.
  • the linker is a linker described herein.
  • the Fab is attached (either directly or indirectly) to a half-life extending moiety such as, e.g., an Fc molecule, a CH3 domain of an IgG (e.g., CH3 of IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysicalic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, e.g., by hyperglycosylation, to extend the half-life of the Fab.
  • a Fab comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region.
  • a Fab comprises a disulfide bond that increases stability of the Fab molecule. In some embodiments, a Fab comprises a disulfide bond that increases thermostability of the Fab molecule.
  • a KLRB1-binding agent is a F(ab′)2.
  • a F(ab′)2 is one of the molecules that results from digestion of an immunoglobulin antibody with pepsin.
  • a F(ab′)2 is a divalent molecule that comprises a first light chain in association with a first polypeptide comprising a first heavy chain variable region, a first CH1, and a first hinge region, and a second light chain in association with a second polypeptide comprising a second heavy chain variable region, a second CH1, and a second hinge region, wherein the first hinge region is linked to the second hinge region via at least one disulfide bond.
  • F(ab′)2s can be produced using recombinant technologies known in the art.
  • the F(ab′)2 is attached (either directly or indirectly) to a half-life extending moiety such as, e.g., a CH3 domain of an IgG (e.g., CH3 of IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysicalic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, e.g., by hyperglycosylation, to extend the half-life of the F(ab′) 2 .
  • a F(ab′) 2 comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region.
  • a F(ab′)2 comprises a disulfide bond that increases stability of the F(ab′)2 molecule. In some embodiments, a F(ab′)2 comprises a disulfide bond that increases thermostability of the F(ab′)2 molecule.
  • a KLRB1-binding agent is a F(ab′).
  • a F(ab′) is a molecule that results from treatment of a F(ab′) 2 with beta-mercaptoethanol.
  • a F(ab′) is a monovalent molecule that comprises a light chain in association with a polypeptide comprising a heavy chain variable region, a CH1, and a hinge region.
  • the F(ab′) is attached (either directly or indirectly) to a half-life extending moiety such as, e.g., an Fc molecule, a CH3 domain of an IgG (e.g., CH3 of IgG1), PEG or a PEG mimetic, XTEN, serum albumin (e.g., human serum albumin), polysicalic acid, N-(2-hydroxypropyl)methacrylamide, or dextran, or is modified, e.g., by hyperglycosylation, to extend the half-life of the F(ab′).
  • a F(ab′) comprises a disulfide bond formed between the heavy chain variable region and the light chain variable region.
  • a F(ab′) comprises a disulfide bond that increases stability of the F(ab′) molecule. In some embodiments, a F(ab′) comprises a disulfide bond that increases thermostability of the F(ab′) molecule.
  • a KLRB1-binding agent is a bispecific antibody. Bispecific antibodies are capable of recognizing and binding at least two different antigens or epitopes. The different epitopes can either be 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).
  • a bispecific antibody has enhanced potency as compared to an individual antibody or to a combination of more than one antibody. In some embodiments, a bispecific antibody has reduced toxicity as compared to an individual antibody or to a combination of more than one antibody. It is known to those of skill in the art that any therapeutic agent may have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific antibody has the ability to synchronize the PK of two active binding agents wherein the two individual binding agents have different PK profiles. In some embodiments, a bispecific antibody has the ability to concentrate the actions of two agents in a common area (e.g., tissue) in a subject (e.g., a human).
  • a common area e.g., tissue
  • a subject e.g., a human
  • a bispecific antibody has the ability to concentrate the actions of two agents to a common target (e.g., a specific cell type). In some embodiments, a bispecific antibody has the ability to target the actions of two agents to more than one biological pathway or function. In some embodiments, a bispecific antibody has the ability to target two different cells and bring them closer together. In some embodiments, a bispecific antibody has decreased toxicity and/or side effects. In some embodiments, a bispecific antibody has decreased toxicity and/or side effects as compared to a mixture of the two individual antibodies or the antibodies as single agents. In some embodiments, a bispecific antibody has an increased therapeutic index.
  • a bispecific antibody has an increased therapeutic index as compared to a mixture of the two individual antibodies or the antibodies as single agents.
  • the bispecific antibodies comprise heavy chain constant regions with modifications in the amino acids that are part of the interface between the two heavy chains. These modifications are made to enhance heterodimer formation and generally reduce or eliminate homodimer formation.
  • the bispecific antibodies are generated using a knobs-into-holes (KIH) strategy.
  • the bispecific antibodies comprise variant hinge regions incapable of forming disulfide linkages between identical heavy chains (e.g., reduce homodimer formation).
  • the bispecific antibodies comprise heavy chains with changes in amino acids that result in altered electrostatic interactions. In some embodiments, the bispecific antibodies comprise heavy chains with changes in amino acids that result in altered hydrophobic/hydrophilic interactions. Bispecific antibodies can be intact antibodies or antibody fragments comprising antigen-binding sites. KLRB1-binding agents with more than two valencies are also contemplated. In some embodiments, trispecific or tetraspecific antibodies are generated. In some embodiments, a KLRB1-binding agent is an anti-KLRB1 antibody that comprises one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 18G11F11, e.g., as shown in Table 1 and/or (ii) one, two, and/or three light chain CDRs from antibody 18G11F11, e.g., as shown in Table 1.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 18G11F11, e.g., as shown in Table 1 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 18G11F11, e.g., as shown in Table 1.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 9C10G11, e.g., as shown in Table 2, and/or (ii) one, two, and/or three light chain CDRs from antibody 9C10G11, e.g., as shown in Table 2.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 9C10G11, e.g., as shown in Table 2 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 9C10G11, e.g., as shown in Table 2.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 57E2E4, e.g., as shown in Table 3, and/or (ii) one, two, and/or three light chain CDRs from antibody 57E2E4, e.g., as shown in Table 3.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 57E2E4, e.g., as shown in Table 3 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 57E2E4, e.g., as shown in Table 3.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 75B10F12, e.g., as shown in Table 4, and/or (ii) one, two, and/or three light chain CDRs from antibody 75B10F12, e.g., as shown in Table 4.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 75B10F12, e.g., as shown in Table 4 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 75B10F12, e.g., as shown in Table 4.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 7B6C7, e.g., as shown in Table 5 and/or (ii) one, two, and/or three light chain CDRs from antibody 7B6C7, e.g., as shown in Table 5.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 7B6C7, e.g., as shown in Table 5 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 7B6C7, e.g., as shown in Table 5.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 52G9B12B5, e.g., as shown in Table 6 and/or (ii) one, two, and/or three light chain CDRs from antibody 52G9B12B5, e.g., as shown in Table 6.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 52G9B12B5, e.g., as shown in Table 6 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 52G9B12B5, e.g., as shown in Table 6.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 5B1B11, e.g., as shown in Table 7 and/or (ii) one, two, and/or three light chain CDRs from antibody 5B1B11, e.g., as shown in Table 7.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 5B1B11, e.g., as shown in Table 7 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 5B1B11, e.g., as shown in Table 7.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 50A8F2, e.g., as shown in Table 8 and/or (ii) one, two, and/or three light chain CDRs from antibody 50A8F2, e.g., as shown in Table 8.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 50A8F2, e.g., as shown in Table 8 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 50A8F2, e.g., as shown in Table 8.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 47A5H2, e.g., as shown in Table 9 and/or (ii) one, two, and/or three light chain CDRs from antibody 47A5H2, e.g., as shown in Table 9.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 47A5H2, e.g., as shown in Table 9 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 47A5H2, e.g., as shown in Table 9.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 66G9C11, e.g., as shown in Table 10 and/or (ii) one, two, and/or three light chain CDRs from antibody 66G9C11, e.g., as shown in Table 10.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 66G9C11, e.g., as shown in Table 10 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 66G9C11, e.g., as shown in Table 10.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 33F11A6, e.g., as shown in Table 11 and/or (ii) one, two, and/or three light chain CDRs from antibody 33F11A6, e.g., as shown in Table 11.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 33F11A6, e.g., as shown in Table 11 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 33F11A6, e.g., as shown in Table 11.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 37G6A7, e.g., as shown in Table 12 and/or (ii) one, two, and/or three light chain CDRs from antibody 37G6A7, e.g., as shown in Table 12.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 37G6A7, e.g., as shown in Table 12 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 37G6A7, e.g., as shown in Table 12.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 39C5D6, e.g., as shown in Table 13 and/or (ii) one, two, and/or three light chain CDRs from antibody 39C5D6, e.g., as shown in Table 13.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 39C5D6, e.g., as shown in Table 13 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 39C5D6 e.g., as shown in Table 13.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 62H1D6, e.g., as shown in Table 14 and/or (ii) one, two, and/or three light chain CDRs from antibody 62H1D6, e.g., as shown in Table 14.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 62H1D6, e.g., as shown in Table 14 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 62H1D6, e.g., as shown in Table 14.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 92E12F2, e.g., as shown in Table 15 and/or (ii) one, two, and/or three light chain CDRs from antibody 92E12F2, e.g., as shown in Table 15.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 92E12F2, e.g., as shown in Table 15 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 92E12F2, e.g., as shown in Table 15.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 13H2D11, e.g., as shown in Table 16 and/or (ii) one, two, and/or three light chain CDRs from antibody 13H2D11, e.g., as shown in Table 16.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 13H2D11, e.g., as shown in Table 16 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 13H2D11, e.g., as shown in Table 16.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 5A11D10, e.g., as shown in Table 17 and/or (ii) one, two, and/or three light chain CDRs from antibody 5A11D10, e.g., as shown in Table 17.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 5A11D10, e.g., as shown in Table 17 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 5A11D10, e.g., as shown in Table 17.
  • an anti- KLRB1 antibody comprises (i) one, two, and/or three heavy chain CDRs from antibody 22F10G3, e.g., as shown in Table 18 and/or (ii) one, two, and/or three light chain CDRs from antibody 22F10G3, e.g., as shown in Table 18.
  • an anti- KLRB1 antibody comprises (i) three heavy chain CDRs from antibody 22F10G3, e.g., as shown in Table 18 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from antibody 22F10G3, e.g., as shown in Table 18.
  • a KLRB1-binding agent is a humanized version of an anti- KLRB1 antibody that comprises (i) one, two, and/or three heavy chain CDRs, and/or (ii) one, two, and/or three light chain CDRs from any one of Tables 1-18.
  • a KLRB1-binding agent is a humanized version of an anti-KLRB1 antibody that comprises (i) three heavy chain CDRs (i.e., a CDR1, a CDR2, and a CDR3) and (ii) three light chain CDRs from any one of Tables 1-18, i.e., wherein the heavy chain CDRs and light chain CDRs are from the same table.
  • Table 1 Antibody 18G11F11 Sequences Table 2: Antibody 9C10G11 Sequences Table 3: Antibody 57E2E4 Sequences Table 4: Antibody 75B10F12 Sequences Table 5: Antibody 7B6C7 Sequences Table 6: Antibody 52G9B12B5 Sequences Table 7: Antibody 5B1B11 Sequences Table 8: Antibody 50A8F2 Sequences with Humanized Sequence Variants Table 9: Antibody 47A5H2 Sequences with Humanized Sequence Variants Table 10: Antibody 66G9C11 Sequences Table 11: Antibody 33F11A6 Sequences Table 12: Antibody 37G6A7 Sequences Table 13: Antibody 39C5D6 Sequences Table 14: Antibody 62H1D6 Sequences Table 15: Antibody 92E12F2 Sequences Table 16: Antibody 13H2D11 Sequences Table 17: Antibody 5A11D10 Sequences Table 18: Antibody 22F10G3 Sequences and
  • a KLRB1-binding agent comprises a humanized version or humanized variant of an antibody described herein.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 18G11F11 (Table 1) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 9C10G11 (Table 2) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 57E2E4 (Table 3) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 75B10F12 (Table 4) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 7B6C7 (Table 5) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 52G9B12B5 (Table 6) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 5B1B11 (Table 7) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 50A8F2 (Table 8) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 47A5H2 (Table 9) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 66G9C11 (Table 10) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 33F11A6 (Table 11) or a humanized version thereof. In some embodiments, a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 37G6A7 (Table 12) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 39C5D6 (Table 13) or a humanized version thereof. In some embodiments, a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 62H1D6 (Table 14) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 92E12F2 (Table 15) or a humanized version thereof. In some embodiments, a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 13H2D11 (Table 16) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 5A11D10 (Table 17) or a humanized version thereof.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 from antibody 22F10G3 (Table 18) or a humanized version thereof.
  • a KLRB1-binding agent comprises a humanized version or humanized variant of an antibody described herein.
  • a KLRB1-binding agent comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain CDR1, CDR2, CDR3 shown in Tables 1-18 or a humanized version thereof.
  • CDRs are defined by a variety of methods/systems by those skilled in the art. These systems and/or definitions have been developed and refined over a number of years and include Kabat, Chothia, IMGT, AbM, and Contact.
  • the Kabat definition is based on sequence variability and generally is the most commonly used.
  • the Chothia definition is based on the location of the structural loop regions.
  • the IMGT system is based on sequence variability and location within the structure of the variable domain.
  • the AbM definition is a compromise between Kabat and Chothia.
  • the Contact definition is based on analyses of the available antibody crystal structures.
  • An Exemplary system is a combination of Kabat and Chothia.
  • Software programs e.g., abYsis (bioinf.org.uk/abysis/sequence_input/key_annotation/ key_annotation.cgi)
  • abYsis bioinf.org.uk/abysis/sequence_input/key_annotation/ key_annotation.cgi
  • the specific CDR sequences defined herein are generally based on Kabat definitions. However, it will be understood that reference to a heavy chain CDR or CDRs and/or a light chain CDR or CDRs of a specific antibody will encompass all CDR definitions as known to those of skill in the art, e.g., as shown in the Tables herein.
  • CDR sequences used will all be identified using the same definitions, i.e., will all be Chothia, all Kabat, all IMGT, and so on.
  • a KLRB1-binding agent is a variant of an agent described herein.
  • a KLRB1-binding agent (e.g., an antibody) comprises (a) a heavy chain CDR1, CDR2, CDR3 from a VH sequence presented herein (e.g., in Tables 1-18, Table B, or Table C), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions; and/or a light chain CDR1, CDR2, and/or CDR3 from a VL sequence presented herein (e.g., in Tables 1-18, Table B, or Table C), or a variant thereof comprising 1, 2, 3, or 4 amino acid substitutions.
  • the amino acid substitutions are conservative substitutions.
  • a CDR comprises one conservative amino acid substitution.
  • a CDR comprises two conservative amino acid substitutions. In some embodiments, a CDR comprises three conservative amino acid substitutions. In some embodiments, a CDR comprises four conservative amino acid substitutions. In some embodiments, the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain CDR2. In some embodiments, the CDR is a heavy chain CDR3. In some embodiments, the CDR is a light chain CDR1. In some embodiments, the CDR is a light chain CDR2. In some embodiments, the CDR is a light chain CDR3. In some embodiments, the substitutions are made as part of a humanization process. In some embodiments, the substitutions are made as part of a germline humanization process.
  • a KLRB1-binding agent e.g., an antibody
  • a KLRB1-binding agent comprises one or more heavy chain or light chain CDRs that have been modified, e.g., to reduce deamidation within the CDR sequence, to remove Asn (N)-glycosylation sites, to remove cysteines, or to remove Asp to reduce isomerization sites, to remove Met/Trp or Lys, e.g., to reduce the likelihood within a CDR sequence of asparagine (N)-glycosylation, cysteinylation, asparagine (Asn) deamidation, aspartate (Asp) isomerization, methionine/tryptophan (Met/Trp) oxidation, and non-enzymatic lysine (Lys) glycation (see, e.g., Haberger et
  • Deamidation is a chemical reaction in which an amide functional group in the side chain of the amino acids asparagine (N) or glutamine (Q) is removed or converted to another functional group.
  • asparagine is converted to aspartic acid or isoaspartic acid and glutamine is converted to glutamic acid or polyglutamic acid.
  • deamidation may change the structure, function, and/or stability of a polypeptide, potentially resulting in decreased biological activity.
  • Exemplary human heavy chain variable region and light chain variable region sequences are provided in Table B. Table B.
  • a KLRB1-binding agent comprises a heavy chain variable region comprising heavy chain CDRs 1, 2, and 3, and a light chain variable region comprising light chain CDRs 1, 2, and 3 as shown in Tables 1-18, Table B, or Table C.
  • a KLRB1-binding agent (e.g., an antibody) comprises a heavy chain variable region or sequence having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a heavy chain variable region sequence presented herein (e.g., in Tables 1-18, Table B, or Table C), and/or a light chain variable region having at least 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a light chain variable region sequence presented herein (e.g., in Tables 1-18, Table B, or Table C).
  • Consensus sequence refers to a composite or genericized VL or VH sequence defined based on information as to which amino acid residues within the VL or VH chain are amenable to modification without detriment to antigen binding.
  • certain amino acid positions are occupied by one of multiple possible amino acid residues at that position. For example, if an arginine (R) or a serine (S) occur at a particular position, then that particular position within the consensus sequence can be either arginine or serine (R or S).
  • Consensus sequences for VH and VL chain can be defined, for example, by in vitro affinity maturation (e.g., randomizing every amino acid position in a certain CDR using degenerate coding primers), by scanning mutagenesis (e.g., alanine scanning mutagenesis) of amino acid residues within the antibody CDRs, or any other methods known in the art, followed by evaluation of the binding of the mutants to the antigen to determine whether the mutated amino acid position affects antigen binding.
  • mutations are introduced in the CDR regions.
  • mutations are introduced in framework regions.
  • mutations are introduced in CDR and framework regions.
  • a KLRB1-binding agent described herein (e.g., an antibody) comprises one or more constant heavy domains (e.g., CH1, CH2 and/or CH3 regions).
  • the KLRB1-binding agent comprises a constant heavy domain 1 (CH1) having an amino acid sequence set forth herein, e.g., in Tables B or C.
  • the KLRB1- binding agent comprises a constant heavy domain 2 (CH2) comprising an amino acid sequence set forth herein, e.g., in Tables B or C.
  • the KLRB1-binding agent comprises a constant heavy domain 3 (CH3) comprising an amino acid sequence set forth herein, e.g., in Tables B or C.
  • the KLRB1-binding agent comprises a heavy chain constant region comprising an amino acid sequence having at least 80, 85%, 90%, 95%, 97%, or 99% sequence identity to an amino acid sequence set forth herein, e.g., in Tables B or C.
  • the one or more constant regions of the KLRB1-binding agent has/have been modified.
  • the antibodies 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).
  • the heavy chain constant region of the modified antibodies comprises at least one human constant region. In some embodiments, the heavy chain constant region of the modified antibodies comprises more than one human constant region. In some embodiments, modifications to the constant region comprise additions, deletions, or substitutions of one or more amino acids in one or more regions. In some embodiments, one or more regions are partially or entirely deleted from the constant regions of the modified antibodies. In some embodiments, the entire CH2 domain has been removed from an antibody ( ⁇ CH2 constructs). In some embodiments, a deleted constant region is replaced by a short amino acid spacer that provides some of the molecular flexibility typically imparted by the absent constant region. In some embodiments, a modified antibody comprises a CH3 domain directly fused to the hinge region of the antibody.
  • a modified antibody comprises a peptide spacer inserted between the hinge region and modified CH2 and/or CH3 domains.
  • the constant region(s) of an antibody mediates several effector functions and these effector functions can vary depending on the isotype of the antibody.
  • the Fc region of an antibody can bind a cell expressing a Fc receptor (FcR).
  • Fc receptors There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).
  • an antibody comprises a variant Fc region.
  • the amino acid sequences of the Fc region of human IgG1, IgG2, IgG3, and IgG4 are known to those of ordinary skill in the art (e.g., a representative human IgG1 Fc region is shown in Lobner et al., Immunol Rev.2016 Mar; 270(1): 113–131; see, e.g., Table A, Table C).
  • Fc regions with amino acid variations have been identified in native antibodies.
  • a variant Fc region is engineered with substitutions at specific amino acid positions as compared to a native Fc region.
  • the Fc region is mutated to alter (reduce) antibody dependent cell- mediated cytotoxicity (ADCC), antibody induced complement dependent cytotoxicity (CDC), and/or antibody dependent cell-mediated phagocytosis (ADCP) (see, e.g., Kang and Jung, Experimental & Molecular Medicine.2019. 51:1–9; Wang et al., Antibody Therapeutics, January 2021.4 (1):45–54; Lobner et al., Immunol Rev.2016 Mar; 270(1): 113–131).
  • ADCC antibody dependent cell- mediated cytotoxicity
  • CDC antibody induced complement dependent cytotoxicity
  • ADCP antibody dependent cell-mediated phagocytosis
  • the Fc region is afucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs.2009 May-Jun; 1(3): 230–236, which describes methods for production of therapeutic antibodies with controlled levels of fucosylation of Fc region N-glycans).
  • the modified antibodies e.g., modified Fc region
  • the deletion or inactivation (through point mutations or other means) of a constant region enhances Fc receptor binding of the modified antibody as it circulates.
  • the constant region modifications increase the serum half-life of the antibody.
  • the constant region modifications reduce the serum half-life of the antibody. In some embodiments, the constant region modifications increase or enhance ADCC and/or complement dependent cytotoxicity (CDC) of the antibody. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moieties. In some embodiments, the constant region is modified to add/substitute one or more amino acids to provide one or more cytotoxin, oligosaccharide, or carbohydrate attachment sites. Modifications to the constant region of antibodies described herein may be made using well known biochemical or molecular engineering techniques.
  • antibody variants are prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Using these antibody variants, it may be possible to enhance the activity or effector function provided by a specific sequence or region while substantially maintaining the structure, binding activity, and other desired characteristics of the modified antibody.
  • HC Heavy Chain
  • LC Light Chain
  • a KLRB1-binding agent e.g., an antibody
  • a heavy chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a HC sequence set forth in Table C, and/or a light chain region having at least about 80% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to a LC sequence set forth in Table C.
  • the VH comprises or consists of an amino acid sequence having at least 95% sequence identity to the VH amino acid sequence set forth in Table C
  • the VL comprises or consists of an amino acid sequence having at least 95% sequence identity to the VL amino acid sequence set forth in Table C.
  • the present disclosure further embraces additional variants and equivalents that are substantially homologous to the recombinant, monoclonal, chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. In some embodiments, it is desirable to improve the binding affinity of the antibody.
  • it is desirable to modulate biological properties of the antibody including but not limited to, specificity, thermostability, expression level, effector function(s), glycosylation, immunogenicity, or solubility.
  • amino acid changes may alter post- translational processes of an antibody, such as changing the number or position of glycosylation sites or altering membrane anchoring characteristics. Variations may be a substitution, deletion, or insertion of one or more nucleotides encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide sequence.
  • amino acid substitutions are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, e.g., conservative amino acid replacements.
  • Insertions or deletions may optionally be in the range of about 1 to 5 amino acids.
  • the substitution, deletion, or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the parent molecule.
  • variants may include addition of amino acid residues at the amino- and/or carboxyl-terminal end of the antibody or polypeptide.
  • the length of additional amino acids residues may range from one residue to a hundred or more residues.
  • a variant comprises an N-terminal methionyl residue.
  • the variant comprises an additional polypeptide/protein, i.e., a fusion protein.
  • a variant is engineered to be detectable and may comprise a detectable label and/or protein (e.g., an enzyme).
  • a cysteine residue not involved in maintaining the proper conformation of an antibody may be substituted or deleted to modulate the antibody’s characteristics, for example, to improve oxidative stability and/or prevent aberrant disulfide crosslinking.
  • one or more cysteine residues may be added to create disulfide bond(s) to improve stability.
  • the variant antibodies or polypeptides described herein may be generated using methods known in the art, including but not limited to, site-directed mutagenesis, alanine scanning mutagenesis, and PCR mutagenesis.
  • KLRB1-binding agents described herein are chemically modified.
  • the KLRB1-binding agents are anti- KLRB1 antibodies that have been chemically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, and/or linkage to a cellular ligand or other protein. Any of numerous chemical modifications may be carried out by known techniques.
  • the present disclosure encompasses KLRB1-binding agents built upon non- immunoglobulin backbones, wherein the agents bind to the same epitope or essentially the same epitope as an anti- KLRB1 antibody disclosed herein.
  • a non- immunoglobulin-based binding agent is an agent that competes with an anti- KLRB1 antibody described herein in a competitive binding assay.
  • alternative KLRB1- binding agents comprise a scaffold protein.
  • scaffold proteins can be assigned to one of three groups based on the architecture of their backbone (1) scaffolds consisting of ⁇ -helices; (2) small scaffolds with few secondary structures or an irregular architecture of ⁇ -helices and ⁇ - sheets; and (3) scaffolds consisting of predominantly ⁇ -sheets.
  • Scaffold proteins include, but are not limited to, anticalins, which are based upon the lipocalin scaffold; adnectins, which are based on the 10 th domain of human fibronectin type 3; affibodies, which are based on the B-domain in the Ig-binding region of Staphylococcus aureus protein A; darpins, which are based on ankyrin repeat domain proteins; fynomers, which are based on the SH3 domain of the human Fyn protein kinase; affitins, which are based on Sac7d from Sulfolobus acidocaldarius; affilins, which are based on human ⁇ -B-crystallin or human ubiquitin; avimers, which are based on the A-domains of membrane receptor proteins; knottins (cysteine knot miniproteins), which are based upon a stable 30-amino acid anti-parallel ⁇ -strand protein fold; and Kunitz domain
  • a KLRB1-binding agent comprises an engineered scaffold protein comprising a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3 shown in any one of Table 1 to 18, e.g., in Table 8, 9, or 18.
  • antigen-antibody interactions are non-covalent and reversible, formed by a combination of hydrogen bonds, hydrophobic interactions, electrostatic and van der Waals forces.
  • affinity and/or avidity are commonly used mentioned.
  • the binding of an antibody to its antigen is a reversible process, and the affinity of the binding is typically reported as an equilibrium dissociation constant (KD).
  • KD is the ratio of an antibody dissociation rate (koff) (how quickly it dissociates from its antigen) to the antibody association rate (kon) (how quickly it binds to its antigen).
  • KD values are determined by measuring the kon and koff rates of a specific antibody/antigen interaction and then using a ratio of these values to calculate the KD value. KD values may be used to evaluate and rank order the strength of individual antibody/antigen interactions. The lower the K D of an antibody, the higher the affinity of the antibody for its target.
  • affinity is measured using SPR technology in a Biacore system. Avidity gives a measure of the overall strength of an antibody-antigen complex.
  • a KLRB1-binding agent binds KLRB1 (e.g., an antibody) with a dissociation constant (KD) of about 1 ⁇ M 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.
  • KD dissociation constant
  • a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a K D of about 20 nM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a KD of about 10 nM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a KD of about 1 nM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a KD of about 0.5 nM or less.
  • a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a KD of about 0.1 nM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a K D of about 50 pM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a K D of about 25 pM or less. In some embodiments, a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a K D of about 10 pM or less.
  • a KLRB1-binding agent binds KLRB1 (e.g., human KLRB1) with a KD of about 1 pM or less.
  • the dissociation constant of the binding agent (e.g., an antibody) for KLRB1 is the dissociation constant determined using a KLRB1 protein immobilized on a Biacore chip and the binding agent flowed over the chip.
  • the dissociation constant of the binding agent (e.g., an antibody) for KLRB1 is the dissociation constant determined using the binding agent captured by an anti-human IgG antibody on a Biacore chip and soluble KLRB1 flowed over the chip.
  • a KLRB1-binding agent binds KLRB1 (e.g., an antibody) with a half maximal effective concentration (EC50) of about 1 ⁇ M 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.
  • EC50 half maximal effective concentration
  • a KLRB1-binding agent binds to human KLRB1 with an EC50 of about 1 ⁇ M or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a KLRB1-binding agent binds cyno KLRB1 and/or human KLRB1with 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.
  • the KLRB1-binding agents (e.g., antibodies) described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host.
  • a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest.
  • the sequence can be mutagenized by site-specific mutagenesis to provide functional variants thereof.
  • a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer.
  • Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated.
  • the individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.
  • the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host.
  • the gene in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.
  • recombinant expression vectors are used to amplify and express DNA encoding antibodies, or fragments thereof, against human KLRB1.
  • recombinant expression vectors can be replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of a KLRB1-binding agent, such as an anti-KLRB1 antibody, or antigen-binding fragment thereof, operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes.
  • a transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are “operatively linked” when they are functionally related to each other.
  • DNA for a signal peptide is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation.
  • structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell.
  • a polypeptide may include an N-terminal methionine residue.
  • This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.
  • the choice of an expression control sequence and an expression vector generally depends upon the choice of host. A wide variety of expression host/vector combinations can be employed.
  • Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus.
  • Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E.
  • the KLRB1-binding agents (e.g., antibodies) of the present disclosure can be expressed from one or more vectors.
  • a heavy chain polypeptide is expressed by one vector and a light chain polypeptide is expressed by a second vector.
  • a heavy chain polypeptide and a light chain polypeptide are expressed by one vector.
  • Suitable host cells for expression of a KLRB1-binding agent (e.g., an antibody) or a KLRB1 protein or fragment thereof to use as an antigen or immunogen include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters.
  • Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus.
  • Higher eukaryotic cells include established cell lines of mammalian origin as described herein. Cell-free translation systems may also be employed.
  • Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts, as well as methods of protein production, including antibody production are well known in the art.
  • mammalian culture systems may be used to express recombinant polypeptides.
  • Expression of recombinant proteins in mammalian cells may be desirable because these proteins are generally correctly folded, appropriately modified, and biologically functional.
  • suitable mammalian host cell lines include, but are not limited to, COS-7 (monkey kidney- derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), HEK-293 (human embryonic kidney- derived) cell lines and variants thereof.
  • Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non- translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.
  • Expression of recombinant proteins in insect cell culture systems e.g., baculovirus
  • Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.
  • the present disclosure provides cells comprising the KLRB1- binding agents described herein.
  • the cells produce the KLRB1-binding agents described herein. In some embodiments, the cells produce an antibody. In some embodiments, the cells produce an antibody that binds human KLRB1. In some embodiments, the cells produce an antibody that binds cyno KLRB1. In some embodiments, the cells produce an antibody that binds human KLRB1 and cyno KLRB1.
  • the cells produce an antibody designated 18G11F11, 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 22F10G3, or variants thereof.
  • the cells produce a scFv version of antibody 18G11F11, 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, or 22F10G3.
  • the cell is a prokaryotic cell (e.g., E. coli).
  • the cell is an eukaryotic cell.
  • the cell is a mammalian cell.
  • the cell is a hybridoma cell.
  • Proteins produced by a 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 by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Affinity chromatography used for purifying immunoglobulins can include Protein A, Protein G, and Protein L chromatography.
  • Isolated proteins can be physically characterized using such techniques 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.
  • SEC size exclusion chromatography
  • MS mass spectrometry
  • NMR nuclear magnetic resonance
  • IEF isoelectric focusing
  • HPLC high performance liquid chromatography
  • x-ray crystallography The purity of isolated proteins can be determined using techniques known to those of skill in the art, including but not limited to, SDS-PAGE, SEC, capillary gel electrophoresis, IEF, and capillary isoelectric focusing (cIEF).
  • supernatants from expression systems which secrete recombinant protein into culture media are first concentrated using a commercially available protein concentration filter, for example, an Amicon® or Millipore Pellicon® ultrafiltration unit.
  • the concentrate can be applied to a suitable purification matrix.
  • an anion exchange resin is employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
  • the matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification.
  • a cation exchange step is employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups.
  • a hydroxyapatite media is employed, including but not limited to, ceramic hydroxyapatite (CHT).
  • one or more reverse- phase HPLC steps employing hydrophobic RP-HPLC media are employed to further purify a recombinant protein.
  • hydrophobic interaction chromatography HIC is used to separate recombinant proteins based on their hydrophobicity.
  • HIC is a useful separation technique for purifying proteins while maintaining biological activity due to the use of conditions and matrices that operate under less denaturing conditions than some other techniques.
  • Anti-KLRB1 antibodies of the present disclosure may be analyzed for their physical/chemical properties and/or biological activities by various assays known in the art.
  • an anti- KLRB1 antibody is tested for its ability to bind KLRB1 (e.g., human KLRB1 and/or cyno KLRB1). Binding assays include, but are not limited to, SPR (e.g., Biacore), ELISA, and FACS.
  • an anti- KLRB1 antibody is tested for its ability to induce ADCC, ADCP, and/or CDC, as well as the ability of the antibody to kill KLRB1 target cells (cell depletion).
  • Assays include, but are not limited to, ADCC cell lysis assays, e.g., that use LDH release and detection of formazan salt.
  • antibodies may be evaluated for solubility, stability, thermostability, viscosity, expression levels, expression quality, and/or purification efficiency.
  • purified anti-KLRB1 antibodies are characterized by assays including, but not limited to, N-terminal sequencing, amino acid analysis, high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.
  • Antibody Conjugates The present disclosure also provides conjugates comprising an anti-KLRB1 antibody or antigen-binding fragment thereof described herein.
  • the antibody or antigen-binding fragment thereof is attached to a second molecule. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent or moiety. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent to form an ADC (antibody-drug conjugate).
  • the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamycin/doxorubicin, melphalan, mitomycin C, chlorambucil, duocarmycin, daunorubicin, pyrrolobenzodiazepines (PBDs), or other intercalating agents.
  • the cytotoxic agent is a microtubule inhibitor including, but not limited to, auristatins, maytansinoids (e.g., DM1 and DM4), and tubulysins.
  • the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tric
  • an antibody is conjugated to one or more small molecule toxins, such as calicheamicins, maytansinoids, trichothenes, and CC1065.
  • a derivative of any one of these toxins may be used as long as the derivative retains the cytotoxic activity of the parent molecule.
  • Conjugates comprising an anti-KLRB1 antibody or antigen-binding fragment thereof described herein can be made using any suitable method known in the art.
  • conjugates are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p- diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis- active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
  • SPDP N-succinimidyl-3-(2-pyridyidithi
  • an anti-KLRB1 antibody or antigen-binding fragment thereof described herein is conjugated to a detectable substance or molecule that allows the antibody to be used for diagnosis and/or detection.
  • a detectable substance can include but is not limited to, enzymes, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase; prosthetic groups, such as biotin and flavine(s); fluorescent materials, such as, umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylrhodamine isothiocyanate (TRITC), dichlorotriazinylamine fluorescein, dansyl chloride, cyanine (Cy3), and phycoerythrin; bioluminescent materials, such as luciferase; radioactive materials, such as 212 Bi, 14 C, 57 Co, 51 Cr, 67 Cu,
  • An anti-KLRB1 antibody or antigen-binding fragment thereof described herein can also be conjugated to a second antibody to form an antibody heteroconjugate.
  • An anti-KLRB1 antibody or antigen-binding fragment thereof as described herein may be attached to a solid support.
  • Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.
  • immobilized anti-KLRB1 antibodies are used in immunoassays.
  • immobilized anti-KLRB1 antibodies are used in purification of the target antigen.
  • nucleic acids encoding a polypeptide described herein and vectors, preferably expression vectors, containing the nucleic acid encoding a polypeptide described herein.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked and can include a plasmid, cosmid or viral vector.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
  • a vector can include a nucleic acid in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vector includes 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 those which direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences.
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein that encode a KLRB1 binding agent as described herein.
  • the recombinant expression vectors of the invention can be designed for expression of KLRB1 binding agent proteins in prokaryotic cells.
  • the KLRB1 binding agents can be expressed in mammalian cells, preferably human cells. See, e.g., Frenzel et al., Front Immunol.2013; 4: 217.
  • the expression vector's control functions are often provided by viral regulatory elements.
  • Vector DNA can be introduced into host cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • a host cell can be used to produce (i.e., express) a KLRB1 binding agent protein.
  • the invention further provides methods for producing a KLRB1 binding agent protein using the host cells of the invention.
  • the method includes culturing the host cell of the invention (into which a recombinant expression vector encoding a KLRB1 binding agent protein has been introduced) in a suitable medium such that a KLRB1 binding agent protein is produced.
  • the method further includes isolating a KLRB1 binding agent protein from the medium or the host cell.
  • the Fc region is afucosylated (see, e.g., Yamane-Ohnuki and Satoh, MAbs.2009 May-Jun; 1(3): 230– 236, which describes methods for production of therapeutic antibodies with controlled levels of fucosylation of Fc region N-glycans).
  • Pharmaceutical compositions comprising a KLRB1-binding agent as described herein as an active ingredient.
  • the KLRB1 binding agent is prepared as a pharmaceutical composition, for example as a pharmaceutical composition for use as a medicament.
  • the pharmaceutical composition is for use as a medicament for treating a disease as described herein, optionally an autoimmune disease, an allergic disease, a transplant rejection, or a hematologic malignancy, in a subject in need thereof.
  • the autoimmune disease is rheumatoid arthritis, Sjögren’s syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn’s disease, ulcerative colitis, or celiac disease.
  • the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyposis, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.
  • the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ transplant.
  • the hematological malignancy is a leukemia, e.g., T cell leukemia, NK cell leukemia, T cell lymphoma, or large granular lymphocytic leukemia (LGLL).
  • the lymphoma is hepatosplenic T cell lymphoma (HSTCL), NK/T cell lymphomas (NKTCL), extranodal NK/T cell lymphomas (ENKL), aggressive NK cell leukemia (ANKL), mycosis fungoides, Sezary syndrome, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), and peripheral T cell lymphoma not otherwise specified (PTCL-NOS).
  • the leukemia is aggressive NK cell leukemia or T cell prolymphocytic leukemia.
  • KLRB1-binding agent as a pharmaceutical composition according to known methods.
  • compositions can include a carrier.
  • Carriers as used herein can include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic (or relatively non-toxic) to the cell or subject being exposed thereto at the dosages and concentrations employed.
  • the physiologically acceptable carrier is an aqueous pH buffered solution.
  • physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • buffers such as phosphate, citrate, and other organic acids
  • antioxidants including ascorbic acid
  • proteins such as serum albumin
  • the KLRB1 binding agent is comprised in an injectable formulation, for example, a subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection formulation.
  • injectable formulations can be aqueous solutions, for example in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer.
  • the injectable formulation can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the KLRB1 binding agent can be in a dried or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the binding agents of the present disclosure can be formulated in any suitable form for delivery to a target cell/tissue.
  • a KLRB1-binding agent can be formulated as a liposome, microparticle, microcapsule, albumin microsphere, microemulsion, nano-particle, nanocapsule, or macroemulsion.
  • the pharmaceutical formulation includes an agent of the present disclosure complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE).
  • PEG-PE PEG-derivatized phosphatidylethanolamine
  • a KLRB1-binding agent is formulated as a sustained-release preparation.
  • sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing an agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules).
  • Sustained-release matrices include but are not limited to polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic
  • treatment and Administration provides methods comprising administering a KLRB1 binding agent as described herein, or a pharmaceutical composition comprising a KLRB1 binding agent as described herein, to a subject in need thereof.
  • the subject is a human.
  • the methods are carried out in vivo (e.g., as opposed to ex vivo).
  • treatment refers to therapeutic treatment (treating a subject who has a disease); the methods can also be used for prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder, in a subject who does not have the disease.
  • Those in need of treatment can include those already with the disorder, those prone to have the disorder, or those in whom the disorder is to be prevented (as used herein, “prevent” means to reduce the risk of developing).
  • the disclosure provides methods for treating an autoimmune disease, an allergic disease, a transplant rejection, or a hematologic malignancy, in a subject in need thereof.
  • KLRB1 expressing cells are implicated in the pathogenesis of these diseases. In accordance with the present disclosure, depletion of such KLRB1 expressing cells provides a therapeutic benefit.
  • the autoimmune disease is rheumatoid arthritis, Sjögren’s syndrome, inclusion body myositis (IBM), discoid lupus, psoriasis, idiopathic pulmonary fibrosis, diabetes, alopecia universalis, primary biliary cholangitis, multiple sclerosis, lymphocytic colitis, palmoplantar pustulosis, hidradenitis suppurativa, Crohn’s disease, ulcerative colitis, or celiac disease.
  • IBM inclusion body myositis
  • the allergic disease is asthma, allergic eosinophilic asthma, allergy, atopic dermatitis, nasal polyposis, eosinophilic gastrointestinal disorder, or hypereosinophilic syndrome.
  • the transplant rejection can be a rejection of a kidney, lung, heart, liver, limb, skin, or multi-organ transplant.
  • the hematological malignancy is a leukemia, e.g., T cell leukemia, NK cell leukemia, T cell lymphoma, T cell prolymphocytic leukemia (T- PLL), or large granular lymphocytic leukemia (LGLL).
  • the lymphoma is hepatosplenic T cell lymphoma (HSTCL), NK/T cell lymphomas (NKTCL), extranodal NK/T cell lymphomas (ENKL), aggressive NK cell leukemia (ANKL), mycosis fungoides, Sezary syndrome, peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), and peripheral T cell lymphoma not otherwise specified (PTCL-NOS).
  • the leukemia is aggressive NK cell leukemia or T cell prolymphocytic leukemia.
  • the disclosure provides methods for treating or preventing transplant rejection.
  • the transplant rejection can be, for example, kidney rejection.
  • administering can include contacting an exogenous pharmaceutical, therapeutic agent, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid.
  • administering and “treatment” include in vivo, as well as in some embodiments in vitro or ex vivo treatments.
  • the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree.
  • the amount of a therapeutic agent that is effective to alleviate any particular disease symptom can vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.
  • the term “effective amount” or therapeutically effective amount” is a concentration or amount of the KLRB1 binding agent that results in achieving a particular stated purpose, e.g., reduction in one or more symptoms of a disease described herein.
  • An “effective amount” of a KLRB1 binding agent can be determined empirically.
  • a “therapeutically effective amount” is a concentration or amount of a KLRB1 binding agent which is effective for achieving a stated therapeutic effect. This amount can also be determined empirically.
  • treatment with the KLRB1-binding agents of the disclosure can kill at least about 20%, e.g., at least about 30%, 40%, 50%, 60%, 70%, or 80% of the KLRB1 expressing cells that are implicated in the pathogenesis of the diseases disclosed herein.
  • subject refers to any animal (e.g., a mammal), including, but not limited to, humans and non-human veterinary subjects including non-human primates.
  • the KLRB1 binding agent can be administered by providing an mRNA encoding the binding agent to the subject.
  • the following examples are illustrative and not restrictive. Many variations of the technology will become apparent to those of skill in the art upon review of this disclosure. The scope of the technology should, therefore, be determined not with reference to the examples, but instead should be determined with reference to the appended claims along with their full scope of equivalents.
  • Body-wide KLRB1 expression profiling data indicates KLRB1 has no significant expression on any cell type other than immune cells (FIG. 2).
  • Example 2 KLRB1 expression is enhanced in various immune conditions
  • Rheumatoid arthritis Analysis of expression data (GSE1919) from synovium biopsies from patients with rheumatoid arthritis compared to normal patients shows increased expression of KLRB1 (10 fold ratio) (FIG.3). Accordingly, rheumatoid arthritis is a particularly attractive target for therapies according to the present disclosure.
  • Sjögren’s syndrome Analysis of expression data (GSE23117) from salivary gland biopsies from patients with Sjogren’s syndrome compared to normal shows increased expression of KLRB1 in advanced stage (13.1 fold ratio), moderate stage (5.3 fold ratio), and early stage (1.6 fold ratio) (FIG.5). Accordingly, Sjogren’s syndrome is a particularly attractive target for therapies according to the present disclosure.
  • Discoid lupus Analysis of expression data (GSE52471) from skin biopsies from patients with discoid lupus compared to normal shows increased expression of KLRB1 (7.2 fold ratio) (FIG.8). Accordingly, discoid lupus is a particularly attractive target for therapies according to the present disclosure.
  • Psoriasis Analysis of expression data (GSE52471) from skin biopsies from patients with psoriasis compared to normal shows increased expression of KLRB1 (11.2 fold ratio) (FIG. 9). Accordingly, discoid lupus is a particularly attractive target for therapies according to the present disclosure.
  • Idiopathic pulmonary fibrosis Analysis of expression data (GSE53845) from lung biopsies from patients with idiopathic pulmonary fibrosis compared to normal shows increased expression of KLRB1 (1.5 fold ratio) (FIG. 10). Accordingly, idiopathic pulmonary fibrosis is a particularly attractive target for therapies according to the present disclosure.
  • Diabetes Analysis of expression data (GSE72492) from pancreas biopsies from patients with diabetes compared to normal shows increased expression of KLRB1 (3.7 fold ratio) (FIG.11). Accordingly, diabetes is a particularly attractive target for therapies according to the present disclosure.
  • Alopecia universalis Analysis of expression data (GSE74761) from scalp biopsies from patients with idiopathic pulmonary fibrosis compared to normal shows increased expression of KLRB1 (4.3 fold ratio) (FIG. 12). Accordingly, alopecia universalis is a particularly attractive target for therapies according to the present disclosure.
  • Primary biliary cholangitis Analysis of expression data (GSE79850) from liver biopsies from patients with primary biliary cholangitis eventually requiring liver transplantation compared to normal shows increased expression of KLRB1 (5.6 fold ratio) (FIG. 13). Accordingly, primary biliary cholangitis is a particularly attractive target for therapies according to the present disclosure.
  • Multiple Sclerosis Analysis of expression data (GSE5839) from brain biopsies from patients with multiple sclerosis show elevated expression of KLRB1 compared to control brain (2.5-fold) (FIG. 14). Accordingly, multiple sclerosis is a particularly attractive target for therapies according to the present disclosure.
  • Lymphocytic colitis Analysis of expression data (GSE65107) from colon biopsies from 4 patients with lymphocytic colitis compared to 4 healthy persons shows increased expression of KLRB1 (3.2 fold ratio) (FIG. 15). Accordingly, lymphocytic colitis is a particularly attractive target for therapies according to the present disclosure.
  • Kidney transplant rejection Analysis of expression data (GSE1563) from kidney biopsies from 7 patients with acute kidney rejection compared to 9 healthy persons shows increased expression of KLRB1 (2.1 fold ratio) (FIG.16). Accordingly, kidney transplant rejection is a particularly attractive target for therapies according to the present disclosure.
  • Lung transplant Analysis of expression data (GSE65107) from lung broncheoalveolar lavage (BAL) fluid from 7 patients with lung transplant rejection compared to 27 patients with lung transplants not undergoing rejection shows increased expression of KLRB1 (3.6 fold ratio) (FIG.17). Accordingly, lung transplant rejection is a particularly attractive target for therapies according to the present disclosure.
  • Atopic dermatitis Analysis of expression data (GSE65107) from skin biopsies from 5 patients with atopic dermatitis compared to 5 healthy persons shows increased expression of KLRB1 (1.9 fold ratio) (FIG.18). Accordingly, atopic dermatitis is a particularly attractive target for therapies according to the present disclosure.
  • Palmoplantar pustulosis Analysis of expression data (GSE185856) from skin biopsies from 3 patients with palmoplantar pustulosis lesional skin and 8 patients palmoplantar pustulosis non-lesional skin compared to 7 healthy persons shows increased expression of KLRB1 (11.0 fold ratio lesional to healthy; 5.9 fold ratio non-lesional to healthy) (FIG. 19).
  • Hidradenitis suppurativa Analysis of expression data (GSE148027) from skin biopsies from 18 patients with hidradenitis suppurativa lesional skin and 7 patients hidradenitis suppurativa non-lesional skin compared to 8 healthy persons shows increased expression of KLRB1 (8.0 fold ratio lesional to healthy; 3.0 fold ratio non-lesional to healthy) (FIG. 20). Accordingly, hidradenitis suppurativa is a particularly attractive target for therapies according to the present disclosure.
  • Asthma Analysis of expression data (GSE41861) from lung airway brushings from 10 patients with severe asthma, 37 patients with moderate asthma, and 44 patients with mild asthma compared to 47 healthy persons shows increased expression of KLRB1 (1.9 fold ratio severe asthma to healthy; 1.5 fold ratio moderate asthma to healthy; and 1.3 fold ratio mild asthma to healthy) (FIG.21). Accordingly, atopic dermatitis is a particularly attractive target for therapies according to the present disclosure.
  • Example 3 KLRB1 expression is enhanced in various T and NK cell lymphomas and leukemias Analysis of expression data (GSE19067) from tumor cells from patients with various T and NK cell lymphomas and leukemias compared to normal NK cell expression shows increased expression of KLRB1 (e.g., hepatosplenic T cell lymphoma has 17 fold ratio compared to normal NK cell line) (FIG. 22). Accordingly, various T and NK cell lymphomas are particularly attractive targets for therapies according to the present disclosure.
  • HSCL hepatosplenic T cell lymphoma
  • NKTCL NK/T cell lymphomas
  • ANKL aggressive NK cell leukemia
  • mycosis fungoides Sezary syndrome
  • PTCL-NOS peripheral T cell lymphoma not otherwise specified
  • T-PLL T cell prolymphocytic leukemia
  • PTCL peripheral T cell lymphoma
  • HTCL hepatosplenic T cell lymphoma
  • NKTCL NK/T cell lymphomas
  • ANKL aggressive NK cell leukemia
  • mycosis fungoides Sezary syndrome
  • PTCL-NOS peripheral T cell lymphoma not otherwise specified
  • T-PLL T cell prolymphocytic leukemia
  • PTCL peripheral T cell lymphoma
  • Example 4 Monoclonal murine anti-KLRB1 antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 18G11F11, and 22F10G3 and various derived chimeric antibodies bind to human KLRB1 Monoclonal antibodies were generated against human KLRB1 by immunizing mice (5 balb/c and 5 SJL) with human KLRB1 protein extracellular domain (ECD) (Uniprot ID Q12918, amino acids 67-225) to create mouse hybridomas.
  • ECD human KLRB1 protein extracellular domain
  • Parental hybridoma clones were subsequently screened for binding by ELISA against human KLRB1 ECD and by FACS against stable CHO- K1 cell lines expressing human KLRB1 (SEQ ID NO:1) and separately expressing cynomolgus KLRB1 (SEQ ID NO:2). Twenty hybridomas were selected for subcloning based on binding to both human and cynomolgus CHO-K1 expressing KLRB1 cells, resulting in 91 subclones. These 91 subclones underwent repeat FACS binding studies to CHO-K1 human-KLRB1 and CHO-K1 cynomolgus KLRB1.
  • a number of these mAbs designated 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 13H2D11, 5A11D10, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3 were tested for binding ability in an ELISA assay.
  • Human KLRB1 ECD was coated on ELISA plates and the plates were incubated with these antibodies.
  • Secondary goat anti-mouse peroxidase labelled IgG was used to detect antibodies bound to the ELISA plate (see FIGs.23A-C).
  • Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, and 22F10G3 were also tested in dose-dependent FACS binding assays against CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1). Secondary goat anti-mouse peroxidase labelled IgG was used to detect antibodies bound to CHO-hum-KLRB1 (see FIG.24A, FIG.24C, and FIG.24D).
  • Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, and 22F10G3 were also tested in dose-dependent FACS binding assays against CHO-K1 cells expressing cynomolgus KLRB1 (CHO-cyno-KLRB1). Secondary goat anti-mouse peroxidase labelled IgG was used to detect antibodies bound to CHO-cyno-KLRB1 (see FIG.24B and FIG.24E).
  • Antibodies 9C10G11, 57E2E4, 75B10F12, 7B6C7, 52G9B12B5, 5B1B11, 50A8F2, 47A5H2, 66G9C11, 33F11A6, 37G6A7, 39C5D6, 62H1D6, 92E12F2, 13H2D11, 5A11D10, 18G11F11, and 22F10G3 were also tested in single-concentration FACS binding assays against both CHO-hum-KLRB1 and CHO-cyno-KLRB1, with untransfected CHO-K1 cells as control.
  • Table 19 Single concentration FACS binding for murine antibodies
  • Table 20 Binding kinetics by SPR for murine and mouse/human chimeric antibodies
  • Table 21A Binding kinetics by SPR for 22F10G3 and 22F10G3 humanized variants
  • Table 21B Binding kinetics by SPR for 22F10G3 and 22F10G3-2.3 humanized variants
  • Example 5 Monoclonal anti-KLRB1 antibodies can elicit antibody-dependent cell- mediated cytotoxicity To determine whether the anti-KLRB1 antibodies will be effective in killing KLRB1 positive cells, the ability of anti-KLRB1 antibodies to elicit antibody-dependent cell-mediated cytotoxicity (ADCC) was tested.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC elicited by was evaluated using a cell lysis assay involving LDH release and detection of formazan salt (Genscript, SC1544).
  • CHO-hum-KLRB1 were incubated with human peripheral blood mononuclear cells (PBMCs) and various antibodies.
  • PBMCs peripheral blood mononuclear cells
  • Antibodies tested were mouse/human IgG1-kappa (wild-type) chimeric antibodies 57E2E4-Chimeric, 7B6C7-Chimeric, 52G9B12B5-Chimeric, 5B1B11-Chimeric, 50A8F2- Chimeric, 47A5H2-Chimeric; 52G9B12B5-Chimeric-EN (a mouse/human chimeric KLRB1- binding antibody with 52G9B12B5 variable regions and human IgG1-kappa mutant L234A/L235A/G237A mutation; used as a negative control); and human IgG1 (negative control) (FIG.
  • Example 6 Monoclonal anti-KLRB1 antibodies can block binding of CLEC2D to KLRB1 CLEC2D is the natural ligand for KLRB1.
  • a fusion protein of CLEC2D-Fc-biotin was produced through transfection of CHO cells with an expression construct.
  • Example 7 Monoclonal anti-KLRB1 antibody 50A8F2 enhances CLEC2D binding to KLRB1
  • CLEC2D is the natural ligand for KLRB1.
  • a fusion protein of CLEC2D-Fc-biotin was produced through transfection of CHO cells with an expression construct.
  • Monoclonal antibody 50A8F2 ability to compete with CLEC2D-Fc-biotin against CHO-K1 cells expressing human KLRB1 (CHO-hum-KLRB1) was tested using streptavidin-AF488 to detect presence or absence of CLEC2D bound to these cells.50A8F2 enhanced CLEC2D binding in two independent experiments (FIG. 26C and FIG. 26D).
  • Example 8 Humanization of 47A5H2 Humanized sequence variants of 47A5H2 were produced for both heavy and light chains by CDR grafting (Almagro et al.2008) of the murine antibody 47A5H2 onto human frameworks IGHV1-46*01/JH1, IGKV4-1*01/JK2, and IGKV3-11*01/JK2. These are shown in Table 22. The sequence variants were combined and expressed as human IgG1 kappa antibodies with IgG1 L234A/L235A/G237A mutations, to produce 16 antibodies as shown in Table 23. Table 22. 47A5H2 humanized sequence variants for individual heavy and light chains Table 23.
  • Example 11 Monoclonal anti-KLRB147A5H2 chimeric and humanized antibodies bind to human KLRB1 and cynomolgus KLRB1 by ELISA Humanized variants of 47A5H2 created by CDR grafting (Almagro et al.2008) were assayed for binding against human and cynomolgus KLRB1 protein extracellular domain (ECD) via ELISA.
  • Human KLRB1 extracellular domain was amino acids 67-225 from human KLRB1 protein (SEQ ID NO:1).
  • Cynomolgus extracellular domain was amino acids 67-227 from cynomolgus KLRB1 protein (SEQ ID NO:3).
  • Monoclonal anti-KLRB147A5H2 chimeric and humanized antibodies inhibit binding of CLEC2D to human KLRB1 expressed on CHO-K1 cells
  • Competition assays of humanized variants of 47A5H2 with a biotin labeled CLEC2D fusion protein were performed by FACS.
  • Antibodies starting at 200 nM were 5-fold serial diluted into wells along with 50 ul biotin-CLEC2D (final concentration 60 nM) with 2.0E+05 CHO-hum-KLRB1 or CHO-cyno-KLRB1 (SEQ ID NO:2) cells per well, at 4 degrees Celsius for 1 hour.
  • FIG.31A-D Blocking of CLEC2D binding by these humanized antibodies is shown in FIG.31A-D and Table 31. Comparison experiments of Hu47A5H2-11 with Hu50A8F2-04 and other antibodies showed that Hu47A5H2-11 demonstrated a lower IC50 than other antibodies tested. The results are shown in FIGs.31E-F and summarized in Tables 28 and 29 (above). Table 31.
  • Example 14 Monoclonal anti-KLRB147A5H2 and 22F10G3 chimeric and humanized antibodies binding kinetics against human KLRB1. 47A5H2 chimeric and humanized antibodies and other anti-KLRB1 antibodies were assayed for binding against human KLRB1 extracellular domain protein (ECD) via surface plasma resonance (SPR). Human KLRB1 extracellular domain was amino acids 67-225 from human KLRB1 protein (SEQ ID NO:1).
  • a Biacore 8K instrument was used with series S sensor CM5 chips, multiple cycle kinetics and affinity capture in 96-well microplates, and full range antibody concentrations of 0.2 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM. 12.5 nM, and 25 nM.
  • Antibody was fixed to the chip with anti-human-Fc antibody and analyte of human KLRB1 extracellular domain (ECD) was flowed over the surface at 30 ul/min, with contact time 180 seconds and dissociation time 400 seconds.
  • ECD extracellular domain
  • Table 32A Binding kinetics of 47A5H2 chimeric and humanized antibodies and other anti- KLRB1 antibodies against human KLRB1 extracellular domain (ECD).
  • ECD extracellular domain protein
  • 22F10G3 chimeric and humanized antibodies were assayed for binding against human KLRB1 extracellular domain protein (ECD) via surface plasma resonance (SPR).
  • Human KLRB1 extracellular domain was amino acids 67-225 from human KLRB1 protein (SEQ ID NO:1).
  • a Biacore 8K instrument was used with multiple cycle kinetics and affinity capture in 96-well microplates, and single antibody concentration of 20 nM.
  • Antibody was fixed to the chip with anti-human-Fc antibody and analyte of cynomolgus KLRB1 extracellular domain (ECD) was flowed over the surface at 30 ul/min, with contact time 180 seconds and dissociation time 400 seconds. Comparison experiments were done with other antibodies: Ab9, KW7.3.7, HP-3G10, DX12, and 191B8. The results are shown in Table 33. Table 33. Binding kinetics of 47A5H2 chimeric and humanized antibodies and other anti- KLRB1 antibodies against cynomolgus KLRB1 extracellular domain (ECD).
  • Example 16 Monoclonal anti-KLRB150A8F2 chimeric and humanized antibodies bind to human KLRB1 by ELISA Humanized variants of 50A8F2 created by CDR grafting (Almagro et al.2008) were assayed for binding against human KLRB1 protein extracellular domain (ECD) via ELISA. ELISA plates were coated with KLRB1 ECD at 1 ⁇ g/ml, 4 ⁇ , overnight prior to adding primary antibody (3 nM starting concentration, and 3-fold dilution) at 37 ⁇ for 1 hour and then secondary anti-human IgG antibody 1:5000 at 37 ⁇ for 45 minutes. The results are shown in FIGs.32A-D and Table 34. Table 34.
  • Antibodies starting at 200 nM were 5-fold serial diluted into wells along with 50 ul biotin-CLEC2D (final concentration 60 nM) with 2.0E+05 CHO- hum-KLRB1 cells per well, at 4 degrees Celsius for 1 hour. Secondary streptavidin-Alexa 467 (Thermo, CAT#S21374, 1:1000) and LIVE/DEAD dye (Invitrogen, CAT#L34964A, 1:1000) were used for FACS detection. Augmentation of (increased) CLEC2D binding by these humanized antibodies is shown in FIGs. 34A-D and Table 36.
  • Example 19 Monoclonal anti-KLRB150A8F2 chimeric and humanized antibodies binding kinetics against human KLRB1. Humanized variants of 50A8F2 and other antibodies were assayed for binding against human KLRB1 extracellular domain protein (ECD) via surface plasma resonance (SPR).
  • ECD extracellular domain protein
  • SPR surface plasma resonance
  • a Biacore 8K instrument was used with series S sensor CM5 chips, multiple cycle kinetics and affinity capture in 96-well microplates, and full range antibody concentrations of 0.2 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.13 nM, 6.25 nM. 12.5 nM, and 25 nM.
  • Antibody was fixed to the chip with anti-human-Fc antibody and analyte of human KLRB1 extracellular domain (ECD) was flowed over the surface at 30 ul/min, with contact time 180 seconds and dissociation time 400 seconds. The results are shown in Table 38. Table 38. Binding kinetics of 50A8F2 chimeric and humanized and other antibodies to human KLRB1. Example 20.
  • NK cell activation assay was developed as follows: K562 cells were stably transfected with human CLEC2D to create pools and single clones of K562-CLEC2D target cells, with clone 1D1 selected for subsequent single clone experiments.
  • Primary NK cells were isolated from human PBMCs with the human NK Cell Enrichment Kit (Stemcell, Cat #19055).
  • Primary NK cells 1.0E+06 were cultured in RPMI 1640 plus 10% FBS with 100 ng/mL rhIL-2 (Peprotech, Cat #200-02) overnight.
  • NK cells were washed and resuspended with assay buffer (RPMI 1640 plus 10% FBS).50 ul of NK cells (1.0E+05 cells/well) were incubated with various antibodies (50 ul/well) at 37°C for 0.5h. K562- CLEC2D target cells were confirmed by FACS to express human CLEC2D using goat anti- human CLEC2D polyclonal antibody (Invitrogen, Cat# PA5-47496) and Alexa 488 anti-goat IgG (Invitrogen, Cat# A11055).
  • K562 or K562-CLEC2D target cells were incubated with RPMI 1640 plus 10% FBS and 100 ng/mL rhIL-2 at 37°C for 4 hours.
  • Anti-CD107a-APC BioLegend, Cat#328620 was added at the start of the incubation.
  • Bredeldin A BioLegend, Cat# 420601
  • monensin BioLegend, Cat #420701
  • Flow detection antibodies Live/dead dye, Invitrogen, Cat# L34964; anti-CD3-PE- Cy7, BioLegend, Cat# 317334; anti-CD56-FITC, BioLegend, Cat# 304604. were added and incubated at 4°C for 30 minutes in the dark. Cells were then washed twice, suspended in 100 ul of Cytofix/Cytoperm solution (BD, Cat# 554722) for 20 minutes at 4°C, washed again and stained with IFN-gamma-PE (BioLegend, Cat# 502509) at 4°C for 30 minutes in the dark, washed again, and analyzed by FACS.
  • Cytofix/Cytoperm solution BD, Cat# 554722
  • Results are shown in FIGs.36-39 and summarized in Tables 39 and 40 below, and demonstrated that 47A5H2 chimeric and humanized antibodies increased NK cell activation using primary NK cells. These further demonstrated that NK cell activation is suppressed by CLEC2D (K562-CLEC2D cells cause less NK activation than K562 cells, FIGs. 36 and 37) and that Hu47A5H2-11 antibody increased NK cell activation with target cells K562-CLEC2D single clone 1D1.
  • the EC50s for Hu47A5H2-11 NK cell activation for both CD107a expression and IFNg expression were superior to that of antibodies Ab9, KW1.2.1, and KW7.3.7 (FIGs.38A, 39A).
  • Antibodies KW1.2.1, and KW7.3.7 did not activate NK cell IFNg expression (FIG.39A).
  • Antibody 191B8 at 40 nM and at 200 nM caused NK cell toxicity and had a decreased effect on NK cell activation (FIGs.37A, 37B, 38B, and 39B).
  • Antibody Hu50A8F2-04 did not activate NK cell CD107a or IFNg expression (FIGs.38A, 39A).
  • NK cell killing assay was developed as follows: Donors with high expression of KLRB1 (CD161) on NK cells, detected by FACS using PE-anti-CD161 antibody (BioLegend, Cat#339903), were selected for further studies. Primary NK cells were isolated from human PBMCs with the human NK cell enrichment Kit (Stemcell, Cat #19055). Primary NK cells 1.0E+06 were cultured in RPMI 1640 plus 10% FBS with 100 ng/mL rhIL-2 (Peprotech, Cat #200-02) overnight.
  • NK cells were washed and resuspended with assay buffer (RPMI 1640 plus 10% FBS).100 ul of NK cells (4.0E+04 cells/well) were incubated with various antibodies (50 ul/well) at various concentrations at 37°C for 0.5h.
  • Raji target cells were confirmed by FACS to express human CLEC2D using goat anti- human CLEC2D polyclonal antibody (Invitrogen, Cat# PA5-47496) and Alexa 488 anti-goat IgG (Invitrogen, Cat# A11055, 1:1000). Raji target cells were stained with CellTrace Violet (CTV, Invitrogen, Cat# C34557).
  • T cell activation assay was developed as follows: Jurkat-NFAT cells were stably transfected with 1G4-TCR and human CD161 (KLRB1) to create pools and single clones of Jurkat-NFAT-1G4TCR-CD161 effector cells. Expression was confirmed by FACS using anti-HA tag on TCR alpha (BioLegend, Cat#682404), anti-PC tag on TCR beta (Genscript, Cat# A01774-100), and anti-CD161 Hu47A5H2-7 antibody.
  • K562 cells were stably transfected with human CLEC2D and HLA A*02:01 and E2m to create pools and single clones of K562-CLEC2D-HLA target cells.
  • Jurkat-NFAT-1G4TCR-CD161 cells were cultured with K562-CLEC2D-HLA cells pulsed with NY-ESO-1 peptide (SLLMWITQC) under a variety of conditions.
  • SLLMWITQC NY-ESO-1 peptide
  • various concentrations of NY-ESO-1 peptide were added to assay medium (RPMI 1640 plus 10% FBS), and 25 ul of assay medium and 25 ul target K562-CLEC2D-HLA cells were incubated at 37°C for 120 minutes.
  • Jurkat-NFAT-1G4TCR-CD161 effector cells 25 ul with 25 ul of various antibodies were added and incubated at 37°C for 5 hours.
  • ONE-Glo Promega, Cat#E6120 100 ul was added and incubated at room temperature for 3 minutes.
  • Well luminescence was then measured with a Varioskan Lux plate reader.
  • the results of one experiment (Experiment 22.1) using Jurkat cell pools and K562- CLEC2D-HLA cell pools are shown in FIG.42, and demonstrated that 47A5H2 chimeric antibody but not 50A8F2 chimeric antibody, increased T cell activation.
  • Hu47A5H2-11 had superior potency (smaller EC50) compared with Ab9, KW1.2.1, KW7.3.7, HP-3G10, DX12, and 191B8.
  • CD161 is a marker of all human IL-17-producing T-cell subsets and is induced by RORC.

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Abstract

Des agents de liaison à KLRB1 (en particulier des anticorps anti-KLRB1 et une partie de liaison à l'antigène de ceux-ci) et des compositions de ceux-ci, ainsi que des procédés thérapeutiques d'utilisation des agents, par exemple, pour appauvrir des cellules ou inhiber des cellules ou activer des cellules (en particulier, Th17, Th17.1, ex-Th17, Tc17, MAIT, iNKT, peTh2, ILC2, ILC3, cellules NK, et/ou des lymphocytes T ou NK néoplasiques in vivo), pour le traitement d'une maladie auto-immune, de maladies allergiques, d'un rejet de greffe, de malignités hématologiques et d'un cancer.
PCT/US2023/077607 2022-10-25 2023-10-24 Agents de liaison à klrb1 et leurs méthodes d'utilisation Ceased WO2024091919A2 (fr)

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