Killer cell lectin-like receptor subfamily G member 1 (KLRG1)-depleting antibodies

Abstract

The present invention relates to antibodies or fragments thereof that specifically bind to KLRG1, methods for depleting T cells and / or NK cells expressing killer cell lectin-like receptor G1 (KLRG1) in a subject in need thereof, methods for treating a disorder associated with an excess of KLRG1-expressing T cells in a subject in need thereof, methods for treating cancer in a subject, adjunctive therapies for the treatment of cancer in a subject, methods for depleting KLRG1-expressing cells in a mixed population of cells, methods for selectively depleting KLRG1-expressing CDS effector T cells, pharmaceutical compositions of anti-KLRG1 antibodies, and kits for anti-KLRG1 antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 225,828, filed July 26, 2021, and U.S. Provisional Patent Application No. 63 / 308,222, filed February 9, 2022, the entire contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Cytotoxic T cells or NK cells can cause disease through inappropriate cell damage (e.g., autoimmunity) or through uncontrolled proliferation (e.g., certain leukemias and lymphomas involving cytotoxic T cells or NK cells). Damage to tissues by cytotoxic T cells is involved in autoimmune diseases, type 1 diabetes, solid organ transplant rejection, and graft-versus-host disease. Uncontrolled proliferation of cytotoxic T cells or NK cells is also involved in certain T cell leukemias and lymphomas, such as hepatosplenic T cell lymphoma and NK / T cell lymphoma. Killer cell lectin-like receptor G1 (KLRG1), a cell surface marker known to be present on mature cytotoxic T cells, has been demonstrated to be present on cytotoxic T cells with high killing potential (WO2018053264). Thus, there is a need for compositions and methods for depleting KLRG1-expressing T cells and / or NK cells in subjects in need of such depletion.

[0003] Summary of the Invention Among many different embodiments, disclosed herein are antibodies and fragments thereof that specifically bind to the extracellular domain of KLRG1, as well as methods of using such antibodies and fragments to deplete KLRG1-expressing T cells and / or NK cells in subjects in need of depletion treatment.

[0004] In one aspect, the disclosure relates to an antibody or fragment thereof that specifically binds to the extracellular domain of KLRG1, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4, and a light chain variable region comprising three complementarity determining regions comprising the amino acid sequences of SEQ ID NO:11 (CDR-L1), SEQ ID NO:12 (CDR-L2), and SEQ ID NO:13 (CDR-L3). In some embodiments, the antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO:5. In some embodiments, the antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6. In some embodiments, the antibody or fragment thereof comprises a light chain comprising SEQ ID NO:7. In some embodiments, the antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6 and a light chain comprising SEQ ID NO:7. In some embodiments, the antibody or fragment thereof specifically binds the epitope PLNFSRI (SEQ ID NO:14) or a fragment thereof comprising at least 5 consecutive amino acids. The antibody or fragment thereof may be a monoclonal antibody, or a fragment or derivative thereof. The antibody or fragment thereof may be a humanized antibody, or a fragment thereof. The KLRG1 may be human KLRG1 or cynomolgus KLRG1.

[0005] In some aspects, the present disclosure relates to a method for depleting KLRG1-expressing T cells and / or NK cells in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or fragment thereof disclosed herein, thereby depleting KLRG1-expressing T cells and / or NK cells in the subject.

[0006] Another aspect of the present disclosure relates to a method of treating a disorder associated with an excess of KLRG1-expressing T cells in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of an antibody or fragment thereof disclosed herein, thereby depleting KLRG1-expressing T cells. The disorder can be a transplantation disorder. The disorder can be an autoimmune disease. The disorder can be inclusion body myositis.

[0007] In one aspect, the disclosure relates to a method of treating cancer in a subject, wherein the cancer comprises cancer cells that express KLRG1, the method comprising delivering to the subject a therapeutically effective amount of an antibody or fragment thereof disclosed herein, wherein said delivery to the subject depletes the cancer cells that express KLRG1.

[0008] In another aspect, the disclosure relates to the treatment of cancer in a subject with adjunctive therapy, wherein the subject is receiving checkpoint therapy (regardless of whether the cancer expresses KLRG1), and wherein the adjunctive therapy comprises delivering to the subject a therapeutically effective amount of an antibody or fragment thereof disclosed herein, wherein the delivery depletes KLRG1-expressing pathogenic T cells and / or NK cells that attack autologous tissue in the subject.

[0009] In one aspect, the disclosure relates to a method of depleting KLRG1-expressing cells in a mixed population of cells, the KLRG1-expressing cells comprising one or more cells selected from the group consisting of T cells, NK cells, cancer cells, and combinations thereof, comprising delivering to the mixed population of cells a therapeutically effective amount of an antibody or fragment thereof disclosed herein in an amount effective to deplete the KLRG1-expressing T cells, NK cells, cancer cells, or combinations thereof in the mixed population of cells.

[0010] Another aspect of the present disclosure relates to a method of selectively depleting KLRG1-expressing CD8 effector T cells, while relatively sparing naive T cells and / or regulatory T cells, comprising delivering to a subject a therapeutically effective amount of an antibody or fragment thereof disclosed herein, thereby selectively depleting KLRG1-expressing CD8 effector T cells.

[0011] Another aspect of the present disclosure relates to a pharmaceutical composition comprising at least one antibody or fragment thereof disclosed herein and a pharma- ceutically acceptable carrier.

[0012] The present disclosure also relates to kits comprising at least one antibody or fragment thereof disclosed herein and instructions for use. [Brief description of the drawings]

[0013] [Figure 1] 1 shows gene expression of human KLRG1 by hepatosplenic T-cell lymphoma (HSTCL) neoplasms compared to expression in normal spleen. [Diagram 2] 1 shows gene expression of human KLRG1 by hepatosplenic T-cell lymphoma (HSTCL) neoplasms compared to NK cell lines and GD T cell lines. [Diagram 3] 1 shows gene expression of human KLRG1 by hepatosplenic T-cell lymphoma (HSTCL) neoplasms compared to peripheral T-cell lymphoma (PTCL). [Figure 4] 1 shows gene expression of human KLRG1 by NK / T cell lymphoma (NKTCL) neoplasms. [Diagram 5] 1 shows gene expression of human KLRG1 in mycosis fungoides. [Figure 6] 1 shows gene expression of human KLRG1 in mycosis fungoides compared to healthy cells. [Figure 7] FIG. 1 shows gene expression of human KLRG1 by T cell and NK cell lymphoma and leukemia cell lines, including KARPAS-384 (γ-δ T cell line), KHYG-1 (progressive NK cell leukemia), and MTA (progressive NK cell leukemia). [Figure 8] FIG. 1 shows gene expression of human KLRG1 across T-cell prolymphocytic leukemia (T-PLL) neoplasms. [Figure 9] 1 shows the effect of antibody ABC008 on the KLRG1-positive CD8-positive T cell population in cynomolgus monkeys. [Figure 10] FIG. 1 shows the effect of antibody ABC008 on KLRG1+CD8+ T cell populations in three subjects with inclusion body myositis (IBM). [Figure 11]FIG. 1 shows the effect of antibody ABC008 on the CD3+CD57+ large granular lymphocyte (LGL) T-cell population in three subjects with inclusion body myositis (IBM). [Figure 12] 1 shows the effect of different doses and dosing schedules of antibody ABC008 on KLRG1-positive CD8-positive T cell populations in cynomolgus monkeys. [Figure 13] 1 shows the baseline CD8 KLRG1 positive / KLRG1 negative T cell ratios in nine subjects (three cohorts of three subjects) with IBM. [Figure 14] FIG. 14 shows depletion of CD8+KLRG1+ T cells in the three cohorts compared to baseline values ​​shown in FIG. [Figure 15] FIG. 1 shows the effect of antibody ABC008 on regulatory T cells (Tregs) in three subjects with IBM. [Figure 16] Figure 1 shows the effect of antibody ABC008 on Tregs in 9 subjects (3 cohorts of 3 subjects) with IBM. Comparative data for alemtuzumab are also shown. [Figure 17] 1 shows the effect of antibody ABC008 on central memory T cells in 9 subjects with IBM (3 cohorts of 3 subjects each). Comparative data for alemtuzumab are also shown. [Figure 18] Pharmacokinetic data for ABC008 is shown. [Figure 19] Pharmacokinetic data for ABC008 in 9 subjects (3 cohorts of 3 subjects each) who received 0.1 mg / kg, 0.5 mg / kg, or 2.0 mg / kg of ABC008 are shown. [Figure 20A] FIG. 1 shows sporadic inclusion body myositis functional assessment (sIFA) scores over time for three subjects with IBM who received ABC008. [Figure 20B] FIG. 1 shows modified Timed Up and Go (mTUG) scores over time for three subjects with IBM who received ABC008. [Figure 20C]Changes in sIFA, Inclusion Body Myositis Functional Scale (IBMFRS), mTUG, and Manual Muscle Testing (MMT12) scores after 56 days are summarized for three subjects with IBM who received ABC008. [Figure 21] In vitro potency data for ABC008, fucosylated ABC008 (ABC108), and isotype control against CD8+CD57+ LGL are shown. [Figure 22] 1 shows baseline CD8 KLRG1 positive / KLRG1 negative T cell ratios in 11 subjects with IBM. [Diagram 23] FIG. 14 shows depletion of CD8+KLRG1+ T cells in 11 subjects compared to baseline values ​​shown in FIG. [Figure 24] FIG. 1 shows the effect of antibody ABC008 on the CD3+CD57+ large granular lymphocyte (LGL) T-cell population in a first cohort of three subjects suffering from inclusion body myositis (IBM). [Diagram 25] FIG. 1 shows the effect of antibody ABC008 on the CD3+CD57+ large granular lymphocyte (LGL) T-cell population in a second cohort of three subjects suffering from inclusion body myositis (IBM). [Figure 26] FIG. 1 shows the effect of antibody ABC008 on the CD3+CD57+ large granular lymphocyte (LGL) T-cell population in a third cohort of 5 subjects suffering from inclusion body myositis (IBM). [Figure 27] FIG. 1 shows the effect of antibody ABC008 on naive CD8 T cells in three cohorts of subjects with IBM. Comparative data for alemtuzumab are also shown. [Figure 28] Figure 1 shows the effect of antibody ABC008 on CD8 central memory T cells in three cohorts of subjects with IBM. Comparative data for alemtuzumab are also shown. Legend: circle, cohort 1; diamond, cohort 2; inverted triangle, cohort 3; square, alemtuzumab. [Figure 29]Figure 1 shows the effect of antibody ABC008 on CD8 effector memory T cells (TEM) in three cohorts of subjects with IBM. Comparative data for alemtuzumab are also shown. Legend: circle, cohort 1; diamond, cohort 2; inverted triangle, cohort 3; square, alemtuzumab. [Diagram 30] Figure 1 shows the effect of antibody ABC008 on CD8 terminally differentiated effector memory T cells (TEMRA) in three cohorts of subjects with IBM. Comparative data for alemtuzumab is also shown. [Figure 31A] FIG. 1 shows Inclusion Body Myositis Functional Scale (IBMFRS) scores over time for subjects with IBM who received ABC008. [Figure 31B] FIG. 1 shows manual muscle testing (MMT) scores over time for subjects with IBM who received ABC008. [Figure 31C] FIG. 1 shows modified Timed Up and Go (mTUG) scores over time for subjects with IBM receiving ABC008. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane protein that can function as a co-inhibitory receptor by regulating the activity of T cells and NK cells. The intracellular portion of KLRG1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain that is involved in co-inhibition of T cell receptor (TCR)-mediated signaling. The extracellular portion of KLRG1 contains a C-type lectin domain whose known ligands are cadherins. KLRG1 ligands include E-cadherin, N-cadherin, R-cadherin, and combinations thereof.

[0015] The receptor KLRG1 is expressed on the cell surface of T cells and NK cells, which binds to ligands on epithelial and mesenchymal cells. In humans, KLRG1 expression is generally restricted to cells of the immune system, specifically CD8 positive T cells, NK cells, and if not, CD4 positive T cells. KLRG1 expression is associated with late differentiation phenotype. As antigen-specific T cells differentiate, they can acquire high expression of cytotoxic molecules, and thus have high cytotoxicity.

[0016] Therefore, KLRG1-expressing (or KLRG1-positive) T cells and / or NK cells can be pathogenic and therefore are advantageous targets for cell depletion therapy. For example, administering an effective amount of a KLRG1 depleting agent (e.g., a KLRG1-expressing cell depleting agent) having antibody-dependent cellular cytotoxicity (ADCC) effector function to a subject in need thereof can eliminate or reduce the number of cytotoxic T cells and / or NK cells that damage healthy cells, for example, in the case of autoimmune disease and transplantation disorders. Methods that include administering a KLRG1-expressing cell depleting agent, such as the antibodies and fragments thereof disclosed herein, are also advantageous in treating patients with cancer cells that express KLRG1.

[0017] Depletion of KLRG1 positive cells may be desirable in diseases with abnormal accumulation of KLRG1 cells or KLRG1 positive cells in tissue samples, including certain lymphomas and leukemias of mature T and NK cells, particularly NK / T cell lymphoma (NKTCL), aggressive NK cell leukemia (ANKL), hepatosplenic T cell lymphoma (HSTCL), gamma delta T cell lymphoma (GDTCL), NK / T cell lymphoma (NKTCL), aggressive NK cell leukemia (ANKL), T cell prolymphocytic leukemia (T-PLL), mature T cell leukemia / lymphoma (ATLL), and other diseases. LL), angioimmunoblastic T-cell lymphoma (AITL), subacute panniculitis-like T-cell lymphoma (SPTCL), enteropathic T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), cutaneous T-cell peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), T-cell lymphoma not otherwise specified (TCL-UN), and T-cell large granular lymphocytic leukemia (T-LGLL). In addition, for certain autoimmune diseases, particularly inclusion body myositis (IBM), primary biliary cholangitis, primary sclerosing cholangitis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, oral lichen planus, vitiligo, Sjogren's syndrome, pure red cell aplasia, aplastic anemia, type 1 diabetes, lupus, lupus nephritis, alopecia areata, and Addison's disease, it may be desirable to deplete KLRG1 positive cells.

[0018] Certain exemplary embodiments will now be described to provide a general understanding of the principles of the structure, function, manufacture, and use of the antibodies, fragments thereof, compositions of matter, kits, and related methods and therapies disclosed herein. Those skilled in the art will understand that the disclosures described herein are exemplary embodiments, and that the scope of the disclosure is defined only by the claims. Features shown or described in connection with one exemplary embodiment may be combined with features of other embodiments. Such modifications and variations are intended to be encompassed within the scope of the disclosure.

[0019] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms used in the description of the disclosure herein are for the purpose of describing specific embodiments only and are not intended to be limiting of the disclosure and any invention(s) described herein or otherwise provided.

[0020] Amino acids are represented herein by either the one-letter or three-letter symbols, in accordance with established usage.

[0021] All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0022] As used in the description of this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0023] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives ("or").

[0024] As used herein, the terms "about" and "approximately" when referring to a measurable value such as an amount of a polypeptide, dosage, time, temperature, enzymatic activity, or other biological activity, are meant to encompass variations of ±20%, ±10%, ±5%, ±1%, +0.5%, or even ±0.1% of the specified amount.

[0025] The transitional phrase "consisting essentially of" means that the claim scope is to be construed to include the specified materials or steps recited in the claim, and which do not "materially affect the basic and novel characteristic(s)" of the claimed invention.

[0026] When applied to a polynucleotide or polypeptide sequence of the present disclosure, the term "consists essentially of" (and grammatical variants) means a polynucleotide or polypeptide that consists of both a recited sequence (e.g., SEQ ID NO:) and a total of 10 or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminus and / or C-terminus of the recited sequence, such that the function of the polypeptide is not substantially altered. A total of 10 or less additional amino acids can include the total number of additional amino acids at both ends added together.

[0027] As used herein, an "effective amount" is an amount that provides a desired effect. The term "effective amount" refers to the dosage or amount of an antibody or antigen-binding fragment that is sufficient to reduce the activity of KLRG1, resulting in the improvement of a patient's condition, or to achieve a desired biological outcome, such as reducing the activity of KLRG1, modulating lymphocyte co-inhibitory response, increasing or decreasing the activation of cytotoxic T cells and NK cells, or increasing or decreasing the release of IFNγ by cytotoxic T cells or NK cells.

[0028] As used herein, "therapeutically effective amount" refers to an amount that provides some clinical improvement or benefit to the subject.In other words, "therapeutically effective" amount refers to an amount that causes some relief, alleviation, or reduction in at least one clinical symptom in the subject.Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some benefit is provided to the subject.

[0029] The terms "treat", "treating", or "treatment of" intend that the severity of the subject's condition is reduced or at least partially ameliorated or corrected, and that some alleviation, relief, or reduction of at least one clinical symptom is achieved.

[0030] The term "deplete" as used herein with respect to T cells and / or NK cells and / or KLRG1-expressing cancer cells refers to a measurable reduction in the number of such cells in a subject or in a sample. The reduction can be at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In certain embodiments, the term refers to a reduction in the number of T cells and / or NK cells and / or KLRG1-expressing cancer cells in a subject or in a sample to an amount below detectable limits.

[0031] The term "autoimmune disorder" refers to any disorder associated with an autoimmune response, including, but not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, lupus, and inflammatory bowel disease.

[0032] The term "cancer" as used herein refers to any malignant abnormal growth of cells. Examples include breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain tumor, primary brain tumor, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head and neck carcinoma, breast carcinoma, ovarian carcinoma, lung cancer, small cell lung cancer, Wilms' tumor, cervical cancer, testicular cancer, bladder carcinoma, pancreatic carcinoma, stomach cancer, colon carcinoma, prostate carcinoma, renal urinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, malignant pancreatic insulinoma, The cancer includes, but is not limited to, malignant carcinoid cancer, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteosarcoma, primary macroglobulinemia, and retinoblastoma. In some embodiments, the cancer is selected from the group of tumor-forming cancers. Those skilled in the art will recognize which cancers are within the group.

[0033] Another example of cancer is myelodysplastic syndrome (MDS).

[0034] As used herein, the term "transplant" refers to a section of tissue or a complete organ that is removed from its original native site or host and transferred to a new location in the same human or a different individual. The method for treating a transplant recipient relates to a method for inhibiting organ or tissue transplant rejection, particularly in a mammal. More specifically, the present disclosure relates to a method for inhibiting transplant rejection in a mammal in need thereof, the method comprising administering to such mammal a transplant rejection inhibiting amount of an anti-KLRG1 binding agent, including antibodies and fragments thereof that specifically bind KLRG1.

[0035] The term "isolated" as used herein refers to a polypeptide that is substantially free of cellular material, viral material, and / or culture medium (if produced by recombinant DNA techniques), or chemical precursors or other chemicals (if chemically synthesized). Additionally, an "isolated fragment" is a fragment of a polypeptide that does not naturally occur as a fragment and is not found in the natural state. "Isolated" does not mean that the preparation is technically pure (homogeneous), but rather that the polypeptide or nucleic acid is sufficiently pure to provide in a form that can be used for the intended purpose. Thus, the term "isolated" refers to a molecule that is substantially free of its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cellular or tissue source from which it is derived. The term "isolated" also refers to a preparation in which the isolated protein is sufficiently pure to be administered as a pharmaceutical composition or is about at least 70-80% (w / w) pure, more preferably about at least 80-90% (w / w) pure, even more preferably about 90-95% pure, and most preferably about at least 95%, about at least 96%, about at least 97%, about at least 98%, about at least 99%, or about at least 100% (w / w) pure.

[0036] The term "fragment" as used herein when applied to a polypeptide refers to an amino acid sequence that is shorter in length compared to a reference polypeptide or amino acid sequence and that comprises, consists essentially of, and / or consists of an amino acid sequence that is identical or nearly identical (e.g., approximately 90%, approximately 92%, approximately 95%, approximately 98%, approximately 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the present disclosure may be included within the larger polypeptide of which they are constituent, where appropriate. In some embodiments, such fragments may comprise, consist essentially of, and / or consist of a peptide having a length of at least about 4, about 5, about 6, about 8, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 100, about 150, about 200, or more contiguous amino acids of a polypeptide or amino acid sequence according to the present disclosure.

[0037] As used herein, the terms "protein" and "polypeptide" are used interchangeably, unless otherwise indicated, and include both peptides and proteins.

[0038] As used herein, a fusion protein refers to a polypeptide produced when two heterologous nucleotide sequences, or fragments thereof, encoding two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame. Exemplary fusion polypeptides include fusions of a polypeptide of the present disclosure (or fragments thereof) to all or a portion of glutathione-transferase, maltose binding protein, or a reporter protein (e.g., green fluorescent protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-Myc, the FLAG epitope, etc.

[0039] The term "antibody" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Antibodies can be monoclonal or polyclonal and can be of any species of origin, including, for example, mouse, rat, rabbit, horse, goat, sheep, camel, human, humanized, or chimeric antibodies. Antibodies can be recombinant monoclonal antibodies, produced by the methods disclosed in, for example, U.S. Pat. No. 4,474,893 or U.S. Pat. No. 4,816,567. Antibodies can also be chemically constructed, for example, by the methods disclosed in U.S. Pat. No. 4,676,980.

[0040] As used herein, the terms "antigen-binding domain", "antigen-binding fragment" and "binding fragment" refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. In cases where the antigen is large, the antigen-binding domain may only bind to a portion of the antigen. The portion of the antigen molecule that is responsible for the specific interaction with the antigen-binding domain is referred to as the "epitope" or "antigenic determinant".

[0041] The phrase "KLRG1-associated disorder" as used herein refers to any disease, disorder, or condition in which KLRG1 protein and / or expression of KLRG1 plays a role in the cause, pathology, side effects, symptoms, or other aspects of the disease, disorder, or condition. Examples of such disorders include, but are not limited to, autoimmune disorders (e.g., inclusion body myositis (IBM), multiple sclerosis, and rheumatoid arthritis), transplant disorders, type 1 diabetes, and cancer (e.g., melanoma, prostate cancer, and certain leukemias and lymphomas, such as mature T and NK cell lymphomas and T cell large granular lymphocytic leukemia (T-LGLL)). Other examples of KLRG1-associated disorders include, but are not limited to, infections caused by microorganisms (e.g., bacteria), viruses (e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or shingles), or parasites.

[0042] Another example of a disorder associated with KLRG1 is myelodysplastic syndrome (MDS).

[0043] As used herein, the term "KLRG1 activity" refers to one or more lymphocyte co-inhibitory activities associated with KLRG1. For example, KLRG1 activity can refer to the regulation of cytotoxic T cell and NK cell activation.

[0044] The term "modulate" and its cognates refer to a reduction or increase in the activity of KLRG1 associated with T cell and NK cell activation resulting from interaction with an anti-KLRG1 antibody, which reduction or increase is compared to the activity of KLRG1 in the absence of the same antibody. The reduction or increase in activity is preferably at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more. When KLRG1 activity is reduced, the terms "modulatory" and "modulating" are interchangeable with the terms "inhibitory" and "inhibiting". When KLRG1 activity is increased, the terms "modulatory" and "modulating" are interchangeable with the terms "activating" and "activating".

[0045] Antibodies and Compositions Antibodies, such as those described herein, exhibit at least two functions in the immune system: they bind antigens, e.g., KLRG1, and eliminate these antigens, including cells expressing the antigens, through immunoglobulin effector functions, including, but not limited to, activation of the complement system or interaction with cellular receptors (Fc receptors) on phagocytes, such as macrophages, and / or cellular receptors (Fc receptors) on other immune cells, such as NK cells, leukocytes, platelets, and placental trophoblasts. Disclosed herein are antibodies or fragments thereof that specifically bind to the extracellular domain of KLRG1 but do not compete with the binding of E-cadherin, N-cadherin, or R-cadherin to KLRG1, and that deplete cells expressing KLRG1 when administered to a subject.

[0046] Antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) are three well-known antibody-mediated mechanisms for killing, and thus depleting, target cells.

[0047] Without wishing to be bound by mechanism or theory, binding of an antibody to a target cell via the antigen-binding region (variable domain) of the antibody can provide binding of the target cell to an immune effector via the Fc region(s) of the antibody's constant region. In ADCC, typically, the Fc region of an antibody binds to an FcγRIIIa receptor on an immune effector cell, such as an NK cell, and can then kill the target cell. In ADCP, typically, the Fc region of an antibody binds to an FcγRIIa receptor on an immune effector cell, such as a macrophage cell, and can then phagocytose and kill the target cell. Binding of immune complex C1q to the Fc region of an antibody bound to a target cell induces CDC, triggering the formation of a membrane attack complex that opens a hole into the surface of the target cell.

[0048] Thus, the constant region of the antibody mediates effector functions including activation of complement and interaction with Fc receptors, enabling effects such as ADCC, ADCP, or CDC. Neither the CH1 domain nor the Cκ or Cλ domains mediate effector functions, which is why Fabs do not exhibit ADCC, ADCP, or CDC.

[0049] There are three classes of Fcγ receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). Only FcγRI can bind IgG in a monomeric form, and the affinity of the FcγRI receptor is higher than that of the immunoglobulin receptors FcγRII and FcγRIII. The high affinity receptor FcγRl is constitutively expressed on monocytes, macrophages, and dendritic cells, and expression can be induced on neutrophils and eosinophils. Thus, these cells can be recruited to target cells through antibodies or antibody fragments thereof that contain the Fc region and that bind to the target cells.

[0050] FcγRIIa receptors are found on macrophages, monocytes, and neutrophils, and FcγRIIb receptors are found on B cells, macrophages, mast cells, and eosinophils. FcγRIIIa receptors are found on NK cells, macrophages, eosinophils, monocytes, and T cells, and FcγRIIIb receptors are expressed at high levels on neutrophils. Again, these various cell types can be recruited to a target cell by an antibody that binds to the target cell through an antibody or antibody fragment thereof that contains an fc region that binds to the target cell.

[0051] Thus, in some embodiments, the KLRG1 binding molecules, including the antibodies and antigen-binding fragments thereof disclosed herein, comprise a KLRG1 antigen-binding site together with an antibody constant domain or fragment thereof, which can function to mediate effector functions, including but not limited to ADCC, ADCP, or CDC. In some embodiments, the KLRG1 binding molecules consist of or comprise an antibody antigen-binding site and a peptide-binding Fc-effector molecule, as described in International Patent Application Publication No. WO02 / 44215.

[0052] Intact antibodies, also known as immunoglobulins, are typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kilodaltons (kDa) each and two heavy (H) chains of approximately 50 kDa each. An exemplary carbohydrate moiety on which antibodies may be glycosylated is the fucose moiety. Two types of light chains, designated lambda and kappa, are found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes, namely A, D, E, G, and M, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0053] The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. Briefly, each light chain can be composed of an N-terminal variable domain (VL) and a constant domain (CL). Each heavy chain can be composed of an N-terminal variable domain (VH), three or four constant domains (CH), and a hinge region. The CH domain most proximal to the VH is called CH1. The VH and VL domains consist of or are composed of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of highly variable sequence called CDRs. The CDRs can contain most of the residues responsible for specific interactions with the antigen. The three CDRs are called CDR1, CDR2, and CDR3. The CDR components of the heavy chain are called H1, H2, and H3, while the CDR components of the light chain are called L1, L2, and L3, as appropriate. CDR3, and especially H3, are the greatest source of molecular diversity within the antigen-binding domain. H3 can be as short as, for example, two or more amino acid residues.

[0054] Examples of antibody fragments included within the scope of the present disclosure include Fab, Fab', F(ab')2, and Fv fragments; domain antibodies, diabodies, bispecific antibodies; vaccibodies, linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Such fragments can be produced by known techniques. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be generated by reducing disulfide bridges of F(ab')2 fragments. Alternatively, Fab expression libraries can be constructed to allow for rapid and easy identification of monoclonal Fab fragments with the desired specificity (see Huse et al, Science 1989 Dec 8;246(4935):1275-1281).

[0055] Fab fragment (antigen-binding fragment), or Fab, consists of or comprises the VH-CH1 and VL-CL domains covalently linked by disulfide bonds between the constant regions. As known to those skilled in the art, Fab (approximately 50 kDa in size) is a monovalent fragment produced from IgG and IgM, consisting of or composed of the VH, CH1 and VL, CL domains linked by intramolecular disulfide bonds. To overcome the tendency of the non-covalently linked VH and VL domains in Fv to dissociate when co-expressed in a host cell, single chain (sc) Fv fragments (scFv) can be constructed. In scFv, a flexible and sufficiently long polypeptide is linked, either the C-terminus of VH to the N-terminus of VL, or the C-terminus of VL to the N-terminus of VH. Most commonly, a 15-residue (Gly4Ser)3 peptide (SEQ ID NO: 15) can be used as a linker, although other linkers are known in the art.

[0056] Antibody diversity is the result of the combinatorial assembly of multiple germline genes encoding variable regions and various somatic events. Somatic events can include recombination of variable gene segments with diverse (D) and joining (J) gene segments to create a complete VH region, and recombination of variable and joining gene segments to create a complete VL region. The recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions. These mechanisms of diversity occur in developing B cells prior to antigen exposure. After antigen stimulation, antibody genes expressed in B cells can undergo somatic mutation.

[0057] According to Fundamental Immunology, 3rd ed., ed. Paul, Raven Press, New York, NY, 1993, based on the estimated number of germline gene segments, random recombination of these segments, and random VH-VL pairing, the maximum number of VH-VL pairs is approximately 1.6 × 10 7different antibodies can be produced. Taking into account other processes that contribute to antibody diversity (such as somatic mutations), as supported by Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, Calif., 1995, approximately 1 × 10 10 It is believed that one or more different antibodies could potentially be generated. Because of the many processes involved in antibody diversity, it is highly unlikely that independently generated antibodies will have identical amino acid sequences in the CDRs and heavy and light chain variable regions.

[0058] The present disclosure provides novel variable heavy chain and complete heavy chain sequences that are effective in depleting cells expressing cell surface KLRG1. The scaffolding structure for carrying the CDRs can generally be, but is not limited to, the heavy or light chain of an antibody or a portion thereof, and the CDRs are located at positions corresponding to the CDRs of naturally occurring VH and VL. The structure and position of immunoglobulin variable domains can be determined, for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.

[0059] In certain embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, and a light chain variable region comprising the three light chain CDRs of SEQ ID NO:11 (CDR-L1), SEQ ID NO:12 (CDR-L2), and SEQ ID NO:13 (CDR-L3). In some aspects of these embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, and a light chain variable region comprising SEQ ID NO:5. In some aspects of these embodiments, the antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6. Alternatively, or in addition, the antibody or fragment thereof comprises a light chain comprising SEQ ID NO:7. In more specific aspects of these embodiments, the antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6, and a light chain comprising SEQ ID NO:7. [Table 1] [Table 2]

[0060] In some embodiments, the KLRG1 to which the disclosed antibody or fragment thereof binds can be human KLRG1 or cynomolgus KLRG1. In some embodiments, the antibody or fragment thereof can specifically bind the epitope PLNFSRI (SEQ ID NO: 14) or a fragment thereof that includes at least 5 consecutive amino acids.

[0061] In some embodiments, an antibody can be expected to retain binding specificity and / or ability to be a KLRG1-expressing cell depletion agent so long as the antibody amino acid sequence comprises a sequence that is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical to SEQ ID NO:4, 5, 6, or 7. In some aspects of these embodiments, the antibody or fragment thereof retains the heavy chain CDRs and light chain CDRs of SEQ ID NO:8-13. In each of these aspects, the heavy chain variable region comprises SEQ ID NO:4, and the light chain CDRs of SEQ ID NO:11-13. The percentage of identity can be determined, for example, by standard alignment algorithms such as the Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215:403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48:444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4:11-17. Appl. Biosci., 4:11-17.

[0062] In some embodiments, the antibody or fragment thereof comprises a monoclonal antibody. In some embodiments, the monoclonal antibody or fragment thereof comprises a chimeric antibody or fragment thereof. In some embodiments, the monoclonal antibody or fragment thereof comprises a humanized antibody or fragment thereof.

[0063] In some embodiments, the monoclonal antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, and a light chain variable region comprising the three light chain CDRs of SEQ ID NO:11 (CDR-L1), SEQ ID NO:12 (CDR-L2), and SEQ ID NO:13 (CDR-L3). In some aspects of these embodiments, the monoclonal antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, and a light chain variable region comprising SEQ ID NO:5. In some aspects of these embodiments, the monoclonal antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6. In some aspects of these embodiments, the monoclonal antibody or fragment thereof comprises a light chain comprising SEQ ID NO:7. In some more specific aspects of these embodiments, the monoclonal antibody or fragment thereof comprises a heavy chain comprising SEQ ID NO:6, and a light chain comprising SEQ ID NO:7.

[0064] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, a heavy chain comprising the amino acid sequence of SEQ ID NO:6, or a sequence having approximately 90% sequence identity thereto, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99% identical sequence identity thereto, and a light chain comprising light chain CDRs SEQ ID NO:11 (CDR-L1), SEQ ID NO:12 (CDR-L2), and SEQ ID NO:13 (CDR-L3). In some embodiments, the antibody or fragment thereof comprises a heavy chain comprising at least 50 contiguous amino acids of the amino acid sequence of SEQ ID NO:6, or a sequence approximately at least 90% identical to said contiguous amino acids, e.g., at least about 100 or about 150 or about 200 or more contiguous amino acids.

[0065] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, a light chain comprising the amino acid sequence of SEQ ID NO:7 or a sequence having approximately 90% sequence identity thereto, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99% identical thereto. In some embodiments, the antibody or fragment thereof comprises a light chain comprising at least 50 contiguous amino acids of the amino acid sequence of SEQ ID NO:7, or a sequence approximately at least 90% identical thereto, e.g., at least about 100 or about 150 or about 200 or more contiguous amino acids.

[0066] In some embodiments, the antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:4, a heavy chain comprising the amino acid sequence of SEQ ID NO:6, or a sequence having approximately at least 90% sequence identity thereto, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99% identical thereto, and a light chain comprising the amino acid sequence of SEQ ID NO:7, or a sequence having approximately at least 90% sequence identity thereto, e.g., at least about 95%, about 96%, about 97%, about 98%, or about 99% identical thereto.

[0067] Depletion of KLRG1-expressing cells Disclosed herein is a method for depleting KLRG1-expressing T cells and / or NK cells in a subject in need of treatment. The method provided herein includes delivering to a subject in need of treatment an effective amount of a KLRG1 depleting agent, such as an antibody or fragment thereof, such as those disclosed herein, that specifically binds to the extracellular domain of KLRG1, thereby depleting KLRG1-expressing T cells in the subject.

[0068] Also disclosed herein is a method of treating a disorder associated with excess and / or unwanted KLRG1-expressing T cells in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of a KLRG1-depleting agent (e.g., an anti-KLRG1 antibody disclosed above). The KLRG1-depleting agent can deplete KLRG1-expressing T cells, and delivery to the subject depletes the excess or unwanted KLRG1-expressing T cells.

[0069] In some embodiments of the method, the disorder can be a transplant-associated disorder, and delivery to the subject depletes KLRG1-expressing pathogenic T cells and / or NK cells that attack transplanted tissue in the subject. In some embodiments of the method, the disorder can be an autoimmune disease (e.g., inclusion body myositis), and delivery to the subject depletes KLRG1-expressing pathogenic T cells and / or NK cells that attack autologous tissue in the subject.

[0070] Also disclosed herein is a method of treating cancer in a subject, the cancer comprising cancer cells expressing KLRG1. The method can include delivering to the subject a therapeutically effective amount of a KLRG1 depleting agent, such as an antibody or fragment thereof disclosed herein, that specifically binds to the extracellular domain of KLRG1.

[0071] KLRG1, a cell surface marker known to be present on mature cytotoxic T cells and NK cells, is also present on certain mature T cell and NK cell lymphomas and leukemias (see Examples 1-8). The methods of treatment described herein relate to the discovery that KLRG1 depleting agents may be useful in treating aggressive NK cell leukemia (ANKL), NK-T cell lymphoma (NKTCL), hepatosplenic T cell lymphoma (HSTCK), gamma-delta T cell lymphoma, among other certain mature T cell and NK cell lymphomas / leukemias. For example, the methods of treatment can include administering to a subject in need thereof an effective amount of a KLRG1 depleting agent (e.g., a KLRG1 expressing cell depleting agent) comprising an antibody or fragment thereof described herein. In addition, antibody drug conjugates (ADCs) of the antibodies or fragments thereof described herein can eliminate or reduce the number of neoplastic T cells or NK cells. The ADCs disclosed herein can include a toxic agent or another therapeutic agent, as described below.

[0072] In some embodiments, certain leukemias and lymphomas that involve proliferation of cells expressing KLRG1 are treated. KLRG1 is usually expressed in T cells and NK cells, particularly in subpopulations of mature T cells and NK cells. Since some hematological neoplasms involve proliferation of mature T cells and NK cells, these tumor cells often express KLRG1. Exemplary leukemias and lymphomas that may be treated include T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, chronic lymphoproliferative dysplasia of NK cells, aggressive NK cell leukemia, systemic EBV-positive T-cell lymphoma of childhood, variola vaccinia-like lymphoproliferative dysplasia, adult T-cell leukemia / lymphoma, NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma, chronic gastrointestinal T-cell lymphoproliferative dysplasia with smoldering and no adverse prognostic factors, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous CD30-positive T-cell lymphoma, and primary cutaneous CD30-positive T-cell lymphoma. These include follicular lymphoproliferative dysplasia, lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma, primary cutaneous gamma-delta T-cell lymphoma, primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acral CD8-positive T-cell lymphoma, primary cutaneous CD4-positive small / medium T-cell lymphoproliferative dysplasia, peripheral T-cell lymphoma unspecified type, angioimmunoblastic T-cell lymphoma, follicular lymphoma, nodal peripheral T-cell lymphoma with TFH phenotype, ALK1-positive anaplastic large cell lymphoma, ALK1-negative anaplastic large cell lymphoma, and breast implant-associated anaplastic large cell lymphoma.

[0073] In particular, aggressive NK cell leukemia (ANKL) and NK / T cell lymphoma (NKTCL) are related disorders that may constitute a spectrum of diseases and may be treated by administration of the antibodies disclosed herein. As some hepatosplenic T cell lymphomas (HSTCL) express gamma / delta T cell receptors, HSTCL and gamma-delta T cell lymphomas (GDTCL) overlapping with HSTCL may also be treated using the antibodies and methods disclosed herein.

[0074] Disclosed herein is an adjuvant therapy for the treatment of cancer in a subject, such as a subject undergoing checkpoint therapy. The adjuvant therapy can be performed regardless of whether the cancer expresses KLRG1. The adjuvant therapy can include delivering a therapeutically effective amount of a KLRG1 depleting agent, such as an antibody or a fragment thereof, that specifically binds to the extracellular domain of KLRG1 to the subject. The delivery to the subject can modulate KLRG1 activity by depleting KLRG1-expressing pathogenic T cells and / or NK cells that attack self-tissue in the subject.

[0075] Disclosed herein is a method for depleting KLRG1-expressing cells in a mixed population of cells. The KLRG1-expressing cells can include one or more cells selected from the group consisting of T cells and / or NK cells and / or cancer cells. The method can include delivering an effective amount of a KLRG1 depleting agent, such as an antibody or a fragment thereof, that specifically binds to KLRG1 and depletes KLRG1-expressing T cells and / or NK cells and / or cancer cells to the mixed population of cells, thereby depleting KLRG1-expressing T cells and / or NK cells and / or cancer cells in the mixed population of cells.

[0076] Disclosed herein is a method for selectively depleting KLRG1-expressing CD8 effector T cells while relatively sparing naive T cells and / or regulatory T cells. The method can include delivering to a subject a therapeutically effective amount of an antibody or fragment thereof that specifically binds to the extracellular domain of KLRG1, thereby selectively depleting KLRG1-expressing CD8 effector T cells.

[0077] Disclosed herein is a method for selectively depleting KLRG1-expressing T cells and / or NK cells in a subject in need thereof, the method comprising delivering to the subject a therapeutically effective amount of an antibody or fragment thereof that specifically binds to the extracellular domain of KLRG1, thereby depleting KLRG1-expressing T cells and / or NK cells in the subject.

[0078] Immunotoxins as a tool for depletion of KLRG1-expressing cells In some embodiments, the antibodies and / or antigen-binding fragments thereof provided by the present disclosure are conjugated to a toxic drug and thus do not necessarily rely on endogenous effector cells in ADCC, ADCP, or CDC to deplete target cells, e.g., pathogenic cells and / or cancer cells expressing cell surface KLRG1.

[0079] Immunoconjugates containing one or more cytotoxins are referred to as "immunotoxins." Antibodies conjugated to cytotoxic agents, drugs, or the like are also known as antibody-drug conjugates (ADCs). Immunoconjugates may have a half-life sufficient for the antibody-drug conjugate to be internalized and degraded, and induce cell killing by the released toxin. Cytotoxins or cytotoxic drugs may include any agent that is detrimental to cells (e.g., kills cells). Suitable cytotoxic agents for forming the immunoconjugates of the present disclosure include taxol, tubulysin, duostatin, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, maytansine or an analogue or derivative thereof, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; calicheamicin or or its analogs or derivatives, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives such as carboplatin;and duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin), or an analog or derivative of CC-1065), dolastatins, auristatins, pyrrolo[2,1-c][1,4]benzodiazepines (PDB), indolinobenzodiazepines (IGN), or analogs thereof, antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria Antimitotics (e.g., tubulin targeting agents), such as toxins and related molecules (such as diphtheria A chain and its active fragments and conjugated molecules); ricin toxins (such as ricin A or deglycosylated ricin A chain toxins), cholera toxin, Shiga-like toxins (SLT-I, SLT-II, SLT-IIV), LT toxins, C3 toxins, Shiga toxins, pertussis toxins, tetanus toxins, soybean Bowman-Birk protease inhibitors, Pseudomonas exotoxins, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, and enomycin toxins. Other suitable binding molecules include antibacterial / lytic peptides such as CLIP, magainin 2, melittin, cecropin, and P18, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas exotoxin.

[0080] Complementary Therapeutic Agents The antibodies of the present disclosure, including fragments and conjugates thereof, can be optionally delivered to a patient in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered in parallel with the antibodies of the present disclosure. As used herein, the term "in parallel" means close enough in time to produce a combined effect (i.e., in parallel can be at the same time, or in parallel can be two or more events occurring within a short period of time before or after each other). In certain embodiments, other therapeutic agents can be conjugated to the antibodies or fragments disclosed herein to form ADCs.

[0081] In some embodiments, the antibodies of the disclosure are directed to compounds that are capable of binding to, or are capable of binding to, vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide and teniposide), antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)), enzymes (e.g., L-asparaginase), biological response modifiers (e.g., interferon-α), platinum coordination complexes (e.g., cisplatin and carboplatin), anthracenediones (e.g., mitoxantrone), substituted ureas, (e.g., hydroxyurea), methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine, MH)), adrenal cortical suppressants (e.g., mitotane (o,P'-DDD) and aminoglutethimide), corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), estrogens (e.g., diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone propionate and fluoxymesterone), antiandrogens (e.g., flutamide), and gonadotropin-releasing hormone analogs (e.g., leuprolide).In some embodiments, the antibodies of the disclosure are directed against VEGF (e.g., bevacizumab (Avastin), ranibizumab (Lucentis)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin 1), antibodies against alpha-v / beta-3 vascular integrin (e.g., vitaxin), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide TNF alpha complex ("CN" disclosed as SEQ ID NO: 16), GRC"), cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetate, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin stabilized nanoparticle formulation (Abraxane), soy isoflavone (genistein), tamoxifen citrate, thalidomide, ADH-1 (Exhelin), AG-013736, AMG-706, AZD2171, sorafenib tosylate, BMS-582664, CHIR-265, pazopanib, PI-88, vatalanib, everolimus, suramin, sunitinib malate, XL184, ZD6474, ATN-161, cilengitide, and celecoxib.

[0082] In some embodiments, including but not limited to, the treatment of autoimmune diseases or in transplant treatments, the antibodies of the present disclosure can be administered in conjunction with immunosuppressants, including, for example, cyclosporine A, rapamycin, glucocorticoids, azathioprine, mizoribine, aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamide, and FK506 (tacrolimus). In certain embodiments, immunosuppressants can be conjugated to the antibodies or fragments disclosed herein to form ADCs.

[0083] The anti-KLRG1 antibody may optionally include an antibody constant region or a portion thereof. For example, the VL domain may be bound to an antibody light chain constant domain, including a human Cκ or Cλ chain, at its C-terminus. Similarly, a specific antigen-binding domain based on a VH domain may be bound to all or a portion of an immunoglobulin heavy chain from any antibody isotope, such as IgG, IgA, IgE, and IgM, as well as any of the isotope subclasses, including but not limited to IgG1 and IgG4. The DNA and amino acid sequences for the C-terminal fragments of are well known in the art.

[0084] The term "repertoire" refers to a genetically diverse collection of nucleotides derived in whole or in part from sequences encoding expressed immunoglobulins. The sequences can be generated by in vivo rearrangement of, for example, V, D, and J segments for H chains, and, for example, V and J segments for L chains. Alternatively, the sequences can be generated from cell lines in response to an in vitro stimulus in which rearrangement occurs. Alternatively, parts or all of the sequences can be obtained by, for example, combining unrearranged V segments with D and J segments, by nucleotide synthesis, random mutagenesis, and other methods, such as those disclosed in, for example, U.S. Pat. No. 5,565,332.

[0085] The terms "specific interaction" and "specific binding" refer to two molecules that form a complex that is relatively stable under physiological conditions. Specific binding can be characterized by high affinity and low to moderate capacity, as distinguished from nonspecific binding, which usually has low affinity with moderate to high capacity. Typically, binding occurs with an affinity constant KA of approximately 10 6 M -1 or more preferably approximately 10 8 M -1When the binding affinity is higher than 0.01, it is considered specific. If necessary, non-specific binding can be reduced, for example, by changing the binding conditions, without substantially affecting the specific binding. Suitable binding conditions, such as the concentration of the antibody, the ionic strength of the solution, the temperature, the time allowed for binding, the concentration of the blocking agent (e.g., serum albumin, milk casein), etc., can be optimized by those skilled in the art using routine techniques.

[0086] In certain embodiments, the antibody can specifically bind an epitope within the extracellular domain (ECD) of human or mouse or monkey KLRG1 with an affinity represented by a KD of at least about 2 nM, about 1 nm, about 100 pM, about 10 pM, or about 5 pM. The amino acid sequences of the ECD of human and cynomolgus KLRG1 are shown in SEQ ID NO:1 and SEQ ID NO:2, as listed in Table 1.

[0087] Methods for Producing Antibodies and Fragments Thereof The present disclosure also provides a method for obtaining an antibody specific to KLRG1. The CDRs in such an antibody are not limited to the specific sequences of VH and VL disclosed herein, but may include variants of these sequences. Such variants may be obtained from the sequences provided herein by those skilled in the art using techniques well known in the art. For example, amino acid substitutions, deletions, or additions may be made in the framework regions (FRs) and / or in the CDRs. FR changes may typically be designed to improve the stability and immunogenicity of the antibody, while CDR changes may typically be designed to increase the affinity of the antibody to its target.

[0088] Modifications to the FRs include, but are not limited to, humanizing non-human origins or manipulating certain framework residues important for antigen contact or for stabilizing the binding site, e.g., by changing specific amino acid residues that may alter effector function such as Fc receptor activity, as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, as well as Lund et al. (1991) J. Immun. 147:2657-2662 and Morgan et al. (1995) Immunology 86:319-324.

[0089] Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-enhancing changes can be empirically determined by routine techniques that include modifying CDRs and testing the affinity of the antibody against its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various modifications can be made according to the methods described, for example, in Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that are modified by substitution of different codons that code for functionally equivalent amino acid residues within the sequence, thus resulting in "silent" changes. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (i.e., nonpolar, polar neutral, basic, or acidic).

[0090] In one embodiment, the substitutions may be selected from the exemplary conservative substitutions listed in Table 3. [Table 3]

[0091] Additionally, any naturally occurring residues in the polypeptide can also be substituted with alanine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; ​​Sasaki et al. (1998) Adv. Biophys. 35:1-24).

[0092] Antibodies of the present disclosure may be modified or mutated for compatibility with species other than the one in which the antibody was raised. For example, antibodies may be humanized or camelized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab, Fab1, F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies comprise a human immunoglobulin (recipient antibody) in which residues from the recipient CDRs are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin can be replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody can 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 framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also can comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0093] In certain embodiments, the VH and / or VL domains may be germlined, i.e., the framework regions (FR) of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells, while in other embodiments, the framework sequences remain divergent from the consensus germline sequences.

[0094] Methods for humanizing non-human antibodies are well known in the art. The present disclosure, and any invention(s) provided herein, are not limited to any particular source, species of origin, or method of production. In general, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues because they are typically taken from an "import" variable domain. Humanization can be performed essentially by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534 (1988)). Such humanized antibodies thus are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially none of the non-intact non-human variable domains have been substituted by the corresponding sequences from the non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues (e.g., all or part of the CDRs) and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0095] Human antibodies can also be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al, J. Mol Biol 222:581 (1991)). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147:86 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed that closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 9,434,782, 9,253,965, 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in the following scientific publications:Lee, E-Chiang et al. “Complete humanization of the mouse immunoglobulin loci enables efficient therapeutic antibody discovery”Nature Biotechnology volume 32, pages 356-363(2014), Marks et ah,Bio / Technology 10:779(1992), Lonberg et l,Nature 368:856 (1994), Morrison, Nature 368:812 (1994), Fishwild et ah, Nature Biotechnol.14:M5(1996), Neuberger, Nature Biotech 14:826(1 96), Lonberg and Huszar, Intern, Rev. Immunol 13:65(1995).

[0096] The monoclonal antibodies used to carry out the present disclosure can be produced in hybridoma cell lines by the technique of Kohler and Milstein, Nature 265:495 (1975). For example, a solution containing the appropriate antigen can be injected into a mouse, and after a sufficient time, the mouse can be sacrificed and spleen cells can be obtained. The spleen cells can then be immortalized by, for example, fusing with myeloma or lymphoma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells. The hybridoma cells can then be grown in an appropriate medium, and the supernatant can be screened for monoclonal antibodies with the desired specificity. Monoclonal Fab fragments can be produced in E. coli by recombinant techniques known to those skilled in the art. Antibodies specific to target polypeptides can also be obtained by phage display techniques, as is well known in the art.

[0097] Various immunoassays can be used for screening to identify antibodies with desired specificity for the extracellular domain of KLRG1. Many protocols for competitive binding or immunoradiometric analysis using monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve measuring the complex formation between an antigen and its specific antibody (e.g., antigen / antibody complex formation). Two-site monoclonal-based immunoassays that utilize monoclonal antibodies that react with two non-interfering epitopes on the polypeptide or peptide of the present disclosure can be used as well as competitive binding assays.

[0098] The anti-KLRG1 antibodies described herein can be bound to a solid support (e.g., beads, plates, slides, or wells formed from materials such as latex or polystyrene) in accordance with known techniques. The anti-KLRG1 antibodies described herein can also be bound to a radiolabel (e.g., a radiolabel that can be attached to the antibody using conventional chemical properties) in accordance with known techniques. 35 S, 125 I, 131 I or 99 mTc), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein). Detectable labels further include chemical moieties such as biotin, which can be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin. Determination of the formation of an antibody / antigen complex in the methods of the present disclosure can be by detection of, for example, precipitation, aggregation, flocculation, radioactivity, color development or change, fluorescence, luminescence, and the like, and is well known in the art.

[0099] As mentioned above, the anti-KLRG1 antibody described herein can be linked to another functional molecule, such as another peptide or protein (such as albumin, another antibody, etc.), a toxin, a radioisotope, a cytotoxic agent, or a cytostatic agent. For example, the antibody can be linked to such other functional molecules by chemical crosslinking or by recombinant methods. The antibody can also be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. The antibody can be chemically modified by covalent attachment to a polymer, for example, to extend their circulating half-life. Exemplary polymers and methods of attaching the same are also set forth in US Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546.

[0100] The anti-KLRG1 antibodies described herein may also be modified to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties may be deleted and / or one or more glycosylation sites may be added to the original antibody. Addition of glycosylation sites to the antibodies of the present disclosure may be accomplished by altering the amino acid sequence to contain a glycosylation site consensus sequence known in the art. Such methods are described in International Patent Application Publication No. WO87 / 05330, and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22:259-306. Reduction of glycosylation may be accomplished by removing a glycosylation site by altering one or two amino acids that make up such a site. Removal of any carbohydrate moieties from the antibody may also be accomplished chemically or enzymatically, for example, as described by Hakimuddin et al. (1987) Arch. Biochem. Biohys., 259:52, and Edge et al. (1981). Biochem, 118:131, and by Thotakura et al.

[0101] In one embodiment, the antibody or fragment thereof may contain one or more fucosylated amino acid residues. In another embodiment, the antibody or fragment thereof may lack any fucosylated amino acid residues and / or lack any other glycosylated amino acid residues. The lack of fucosylation may result from the manufacturing conditions of the antibody or fragment thereof or may be affected by techniques known to those skilled in the art.

[0102] In certain embodiments, the monoclonal antibody or fragment thereof may be a chimeric or humanized antibody. In further embodiments, the chimeric or humanized antibody comprises at least a portion of the CDRs of a monoclonal antibody. As used herein, a "portion" of a CDR is defined as one or more of the three loops (e.g., from CDRs 1-6) from each of the light and heavy chains that make up the CDR, or one or more portions of the loops that comprise, consist essentially of, or consist of at least three consecutive amino acids. For example, a chimeric or humanized antibody may comprise 1, 2, 3, 4, 5, or 6 CDR loops, portions of 1, 2, 3, 4, 5, or 6 CDR loops, or mixtures thereof, in any combination.

[0103] Nucleic Acids, Cloning, and Expression Systems The present disclosure further provides the isolated nucleic acid that encodes the disclosed antibody.Nucleic acid may comprise DNA or RNA, and may be wholly or partially synthetic or recombinant.Reference to the nucleotide sequence shown herein includes the DNA molecule with the specified sequence, and includes the RNA molecule with the specified sequence in which T is replaced by U, unless otherwise required by the context.

[0104] The nucleic acids provided herein can include coding sequences for the CDRs, VH and / or VL domains, full-length heavy chains, and / or full-length light chains disclosed herein. The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes that can include at least one nucleic acid encoding the CDRs, VH and / or VL domains, full-length heavy chains, and / or full-length light chains disclosed herein. The present disclosure further provides host cells that can include one or more constructs as described above.

[0105] Nucleic acids encoding any of the CDRs (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3), VH and / or VL domains, heavy and / or light chains, and methods of making the encoded products are also provided. The methods may include expressing the encoded product from the encoding nucleic acid. Expression may be achieved, for example, by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression of the VH or VL domain, the specific binding member may be isolated and / or purified using any suitable technique and then used as appropriate and as would be understood by the skilled artisan.

[0106] Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. For suitable cells for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, as well as yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the present invention may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals as described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0107] Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed.Vector can be plasmid or virus, such as phage or phagemid, as needed.For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, 2012.For example, many known techniques and protocols for the manipulation of nucleic acids in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds.Ausubel et al., John Wiley & Sons, 1992.

[0108] A further aspect of the disclosure provides a host cell comprising a nucleic acid disclosed herein or otherwise derivable therefrom. A still further aspect of the disclosure provides a method comprising introducing such a nucleic acid into a host cell. The introduction can use any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or for insect cells, baculovirus. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following introduction of the nucleic acid into the cell, expression from the nucleic acid can be caused or allowed, for example, by culturing the host cell under conditions for expression of the gene.

[0109] Pharmaceutical Compositions and Methods of Administration The present disclosure provides compositions as described herein, comprising at least one KLRG1 depleting agent, anti-KLRG1 antibody, and / or fragment thereof, and / or conjugate and / or fusion protein thereof as described herein. The compositions of the present disclosure may optionally include medicinal agents, pharmaceutical agents, carriers, pharma- ceutically acceptable carriers, adjuvants, dispersing agents, diluents, and the like. Such compositions may be suitable for pharmaceutical use and administration to patients. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects, such as toxicity. The compositions typically include one or more antibodies of the present disclosure and a pharma- ceutical acceptable excipient. The phrase "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical active substances is well known in the art. The compositions may also contain other active compounds that provide supplemental, additional, or enhanced therapeutic functions. The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0110] Those skilled in the art, with the benefit of this disclosure as a whole, will understand that a variety of pharma- ceutically acceptable carriers can be used, including, but not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; surfactants; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; histidine buffers; and other non-toxic compatible materials employed in pharmaceutical formulations. Pharmaceutically acceptable carriers can include, for example, materials that are "Generally Regarded as Safe" (GRAS) as designated by the FDA.

[0111] The composition of the present disclosure can be formulated for administration in pharmaceutical carrier according to known technology.In the manufacture of pharmaceutical preparations according to the present disclosure, the compound (including its physiologically acceptable salt) can typically be mixed with, inter alia, an acceptable carrier.The carrier can be solid or liquid, or both, and the compound can be formulated with the compound as a unit dose preparation, for example, a tablet, which can contain approximately 0.1% by weight or approximately 0.5% by weight to approximately 95% by weight or approximately 99% by weight or 99% by volume of the compound.One or more compounds can be incorporated into the preparation of the present disclosure, which can be prepared by any of the techniques of pharmacy known to those skilled in the art.

[0112] The pharmaceutical composition of the present disclosure can be formulated to be compatible with its intended route of administration. Methods for achieving administration are known to those skilled in the art. Administration can be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, and / or transdermal. It may also be possible to obtain compositions that can be administered in other ways, including topically or orally, or that can be delivered transmucosally.

[0113] The solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: sterile diluents such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Such formulations can be enclosed in ampoules, disposable syringes, or multiple dose vials, for example made of glass or plastic.

[0114] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). Typically, the composition should be sterile and fluid to the extent that easy injectable conditions exist. The composition should be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, including, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and / or polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and / or by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent which prolongs absorption, for example, aluminum monostearate and gelatin.

[0115] Oral compositions generally include an inert diluent or an edible carrier. Oral compositions may be enclosed in gelatin capsules or may be compressed into tablets. For oral administration, the antibody may be combined with an excipient and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents and / or adjuvant materials may be included as part of the composition. The tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0116] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate for the barrier to be permeated can be used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved, for example, using lozenges, nasal sprays, inhalants, or suppositories. For example, in the case of antibodies that contain Fc portions, the composition can be delivered across the mucous membranes of the intestine, mouth, or lungs (e.g., via the FcRn receptor-mediated pathway described in U.S. Pat. No. 6,030,613). For transdermal administration, the active compound can be formulated, for example, into ointments, gels, or creams that are generally known in the art. For administration by inhalation, the antibody can be delivered in the form of an aerosol spray from a pressurized container or dispenser that can contain a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[0117] In certain embodiments, the antibody of the present disclosure can be prepared with a carrier configured to protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and / or polylactic acid can be used. Methods for the preparation of such formulations are apparent to those skilled in the art. Liposomal suspensions containing the antibody disclosed herein can also be used as pharma- ceutically acceptable carriers. The liposomal suspensions can be prepared by methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0118] Oral or parenteral compositions can be advantageously formulated into unit dosage form for ease of administration and uniformity of dosage.The term "unit dosage form" as used herein refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0119] Toxicity and therapeutic efficacy of the compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to approximately 50% of the population) and the ED50 (the dose therapeutically effective in approximately 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are typically preferred.

[0120] For any composition used in or obtainable from the present disclosure, the therapeutically effective amount can be estimated initially from cell culture assays. Examples of suitable bioassays include, but are not limited to, DNA replication assays, cytokine release assays, transcription-based assays, KLRG1 / cadherin binding assays, immunological assays, and other assays, such as those described in the Examples below. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody that achieves half-maximal inhibition of symptoms). Circulating levels in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by a suitable bioassay. Dosages are preferably within a range of circulating concentrations with little or no toxicity. Dosages can vary depending at least in part on the dosage form employed and the route of administration utilized. Alternatively, the antibodies of the present disclosure or obtainable from the present disclosure can be administered in a local rather than systemic manner, for example, in a depot or sustained release formulation.

[0121] The pharmaceutical composition of any of the antibodies described herein may be in any form suitable for intravenous administration.

[0122] Further aspects of the present disclosure relate to kits for use in the methods provided herein or that may be otherwise obtained in light of the present disclosure. The kits may include at least one or more antibodies or fragments thereof of the present disclosure and / or one or more antibodies obtainable from the present disclosure in a form suitable for administration to a subject and / or in a form suitable for incorporation into a formulation. The kits may further comprise other components, such as therapeutic agents, carriers, buffers, containers, devices for administration, etc. The kits may be designed for therapeutic, diagnostic, and / or research use, and the additional components may be appropriate for the intended use. Those of skill in the art will recognize various such components suitable for inclusion in kits of this nature. The kits may further comprise a label and / or instructions, for example, for treatment of a disorder. Such labeling and / or instructions may include, for example, information regarding the amount, frequency, and method of administration of the antibody. In light of the present disclosure, those of skill in the art will recognize the types of instructions that may be included in combination as part of the kit. The instructions may be provided herein or may be obtained by the skilled artisan in light of the present disclosure. EXAMPLES

[0123] Example 1: Hepatosplenic T-cell lymphoma (HSTCL) Gene expression analysis was performed on expression data (GSE57520) from tumor biopsies from four patients suffering from hepatosplenic T-cell lymphoma (HSTCL), and expression compared to normal spleen from three patients showed elevated expression of KLRG1 (13.0-fold ratio) (Figure 1). Datasets were obtained from the Gene Expression Omnibus database of the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, hepatosplenic T-cell lymphoma (HSTCL) is a particularly attractive target for therapy according to the present invention.

[0124] Example 2: Hepatosplenic T-cell lymphoma (HSTCL) Gene expression analysis was performed on expression data (GSE19067) from tumor biopsies from four patients suffering from hepatosplenic T-cell lymphoma (HSTCL) and expression was compared with NK cell lines from 11 patients and gamma-delta T-cell lines from three patients, showing elevated expression of KLRG1 (10.0-fold ratio compared to NK cell lines) (Figure 2). Data sets were obtained from the Gene Expression omnibus database at the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, hepatosplenic T-cell lymphoma (HSTCL) is a particularly attractive target for therapy according to the present invention.

[0125] Example 3: Hepatosplenic T-cell lymphoma (HSTCL) Gene expression analysis was performed on expression data (GSE11946) from tumor biopsies from five patients suffering from hepatosplenic T-cell lymphoma (HSTCL), and expression was compared with tumor biopsies from 11 patients suffering from other types of peripheral T-cell lymphoma (PTCL), showing elevated expression of KLRG1 (2.1-fold ratio) (Figure 3). Datasets were obtained from the National Center for Bioinformatics' Gene Expression Omnibus database and analyzed for KLRG1 expression. Therefore, hepatosplenic T-cell lymphoma (HSTCL) is a particularly attractive target for therapy according to the present invention.

[0126] Example 4: NK / T cell lymphoma (NKTCL). Gene expression analysis was performed on expression data (GSE19067) from tumor biopsies from 19 patients suffering from NK / T cell lymphoma (NKTCL) and expression was compared with NK cell lines from 11 patients and gamma-delta T cell lines from 3 patients, showing elevated expression of KLRG1 (2.2-fold ratio compared to NK cell lines) (Figure 4). The data set was obtained from the Gene Expression omnibus database of the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, NK / T cell lymphoma (NKTCL) is a particularly attractive target for therapy according to the present invention.

[0127] Example 5: Mycosis Fungoides Gene expression analysis was performed on expression data (GSE19067) from tumor biopsies from two patients suffering from mycosis fungoides, comparing expression with NK cell lines from 11 patients and gamma-delta T cell lines from three patients, showing elevated expression of KLRG1 (9.1-fold ratio compared to NK cell lines) (Figure 5). The data set was obtained from the Gene Expression omnibus database at the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, mycosis fungoides is a particularly attractive target for therapy according to the present invention.

[0128] Example 6: Mycosis Fungoides Gene expression analysis was performed on expression data (GSE39041) from tumor biopsies from six patients suffering from mycosis fungoides, and expression was compared with healthy skin CD4 positive T cells from three patients, showing elevated expression of KLRG1 (3.1-fold ratio) (Figure 6). Data sets were obtained from the Gene Expression omnibus database at the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, mycosis fungoides is a particularly attractive target for therapy according to the present invention.

[0129] Example 7: T-cell and NK-cell lymphoma cell lines Gene expression analysis of expression data from T-cell and NK-cell lymphoma and leukemia cell lines (GSE114085) was performed and showed elevated expression of KLRG1 in certain cell lines including KARPAS-384 (γ-δ T-cell line), KHYG-1 (progressive NK cell leukemia), and MTA (progressive NK cell leukemia) (Figure 7). Data sets were obtained from the National Center for Bioinformatics' Gene Expression omnibus database and analyzed for KLRG1 expression. Therefore, γ-δ T-cell lymphoma and aggressive NK cell leukemia are particularly attractive targets for therapy according to the present invention.

[0130] Example 8: T-cell prolymphocytic leukemia (T-PLL) Gene expression analysis was performed on expression data (GSE5788) from tumor biopsies from six patients suffering from T-cell prolymphocytic leukemia (T-PLL), comparing expression with healthy donor T cells from eight patients, showing elevated expression of KLRG1 (1.4-fold ratio) (Figure 8). Datasets were obtained from the Gene Expression Omnibus database of the National Center for Bioinformatics and analyzed for KLRG1 expression. Therefore, T-cell prolymphocytic leukemia is a particularly attractive target for therapy according to the present invention.

[0131] Example 9: Superior Purity of Fucosylated ABC008 Compared to HG1D03 Examples 9-12 refer to antibodies designated ABC108 and HG1D03. ABC008 is an antibody characterized by the amino acid sequence shown in SEQ ID NO:4-SEQ ID NO:13 of Table 2, the antibody being further defucosylated. ABC108 has the same amino acid sequence as ABC008 and retains wild-type fucosylation. HG1D03 is a humanized antibody that exerts a depleting effect on KLRG1 positive T cells. HG1D03 is described in more detail, including the heavy and kappa chain variable region sequences, in U.S. Pat. No. 11,180,561.

[0132] ABC108 and HG1D03 were produced using the ExpiCho Expression System (Thermo Fisher) at 1 L scale. Reduced capillary electrophoresis (rCE), non-reduced capillary electrophoresis (nrCE), and capillary isoelectric focusing (cIEF) were performed to measure the percentage of the main peak in 4 hour, 7 day, and 28 day stress experiments (stress experiments described in Examples 11-12) in three independent experiments. The results show superior purity during CHO cell production of ABC108 compared to HG1D03 (Table 4). [Table 4]

[0133] Example 10. Superior thermal properties (melting point and aggregation temperature) of ABC108 compared to HG1D03 ABC108 and HG1D03 were produced using the ExpiCho Expression System (Thermo Fisher) at 1 L scale. Melting temperature (Tm) was measured by full spectrum fluorescence, and small molecule aggregate formation (Tagg 266) and large molecule aggregate formation (Tagg 473) were measured by static light scattering (SLS) using UNCLE (Unchained Labs, Inc.). Data were collected in 4 hour, 7 day, and 28 day stress experiments (stress experiments described in Examples 11-12) in three independent experiments. Results show superior thermal properties (higher melting point and aggregation temperature) for ABC108 compared to HG1D03 (Table 5). [Table 5]

[0134] Example 11: Superior stability of ABC108 after 40°C stress test compared to HG1D03 ABC108 and HG1D03 were subjected to thermal stability stress testing at 1 mg / mL in PBS by exposure to 40° C. for 4 hours, 7 days, and 28 days. Non-reduced capillary electrophoresis (nrCE) and capillary isoelectric focusing (cIEF) were performed to measure the percentage of the main peak. Tm1, Tagg 266, and Tagg 473 parameters were measured using the UNCLE platform. Temperature scans were performed from 25° C. to 95° C. at a scan rate of 0.3° C. per minute. Tm values ​​were calculated by analysis of the centroid mean method (BCM). Purity results (Table 6) show similar rCE-measured purity, with nrCE-measured purity (e.g., 85.5% vs. 79.3% at 28 days) and cIEF-measured purity (e.g., 78.0% vs. 41.6% at 7 days) being improved for ABC108 vs. HG1D03. The thermal properties results (Table 7) show similar or slightly better thermal properties for ABC108 versus HG1D03. [Table 6] [Table 7]

[0135] Example 12: Superior stability of ABC108 after low and high pH stress tests compared to HG1D03 ABC108 and HG1D03 were subjected to low pH (3.5) and high pH (8.5) stress tests at 1 mg / mL in PBS for 4 hours, 7 days, and 28 days. Purity results (Table 8) show improved purity as measured by sensitive capillary isoelectric focusing (cIEF) for ABC108 versus HG1D03. Thermal properties results (Tables 9-10) show similar or slightly better thermal properties for ABC108 versus HG1D03. [Table 8] [Table 9] [Table 10]

[0136] Example 13: Depletion of KLRG1-positive blood cells by ABC008 in cynomolgus monkeys Cynomolgus monkeys were subcutaneously administered vehicle control or ABC008 at 5 mg / kg, 25 mg / kg, or 50 mg / kg. Blood immune cell populations were monitored by FACS. ABC008 caused almost complete depletion of KLRG1-positive CD8-positive T cell populations (Figure 9).

[0137] In another experiment, cynomolgus monkeys were subcutaneously administered multiple doses of vehicle control or ABC008 at 0.1 mg / kg, 0.3 mg / kg, 10 mg / kg, or 30 mg / kg according to the dosing schedule shown in Figure 12. Blood immune cell populations were monitored by FACS. 0.1 mg / kg ABC008 caused approximately >50% reduction in the KLRG1+CD8+ T cell population. When 0.3 mg / kg or more of ABC008 was administered, depletion of the KLRG1+CD8+ T cell population was nearly complete (Figure 12).

[0138] Example 14: Depletion of KLRG1-positive blood cells by ABC008 in patients with IBM Three patients suffering from IBM were administered a single dose of ABC008 (0.1 mg / kg subcutaneously) in clinical trial NCT04659031. Blood immune cell populations were monitored by FACS. At baseline, KLRG1-positive % of CD8 T cells ranged from 50 to 88%. ABC008 resulted in a peak depletion of 46 to 96% of the KLRG1-positive CD8-positive T cell population (Figure 10).

[0139] The first three patients constituted the first cohort. Two additional cohorts, each of three patients with IBM, received a single dose of ABC008 subcutaneously in clinical trial NCT04659031. The doses were: Cohort 1: 0.1 mg / kg, Cohort 2: 0.5 mg / kg, and Cohort 3: 2.0 mg / kg.

[0140] Blood immune cell populations were monitored by FACS. As shown in Figure 13, at baseline, the percentage of KLRG1-positive CD8 T cells ranged from 46 to 88%. As shown in Figure 14, administration of ABC008 resulted in a peak depletion of approximately 70% of the KLRG1-positive CD8-positive T cell population in cohort 1, and approximately 95% in both cohort 2 and cohort 3.

[0141] Two additional patients met the requirements for inclusion in Cohort 3 and have been anonymized as C3P4 and C3P5. As shown in Figure 22, at baseline, the percentage of KLRG1-positive CD8 T cells in these two patients ranged from 54-60%.

[0142] Figure 23 complements Figure 14 by following Cohort 3 for 112 days. As shown in Figure 23, across all three cohorts, the KLRG1 positive CD8 positive T cell population for Cohort 1 and Cohort 2 remained approximately 50-70% below baseline from day 84 to day 168 post-dose. The KLRG1 positive CD8 positive T cell population for Cohort 3 was approximately 40% depleted after 112 days of dosing.

[0143] Example 15. Depletion of Large Granular Lymphocytes (LGL) by ABC008 in Patients with IBM Three patients suffering from IBM received a single dose of ABC008 0.1 mg / kg subcutaneously in clinical trial NCT04659031. Blood immune cell populations were monitored by FACS. At baseline, the KLRG1 positive % of large granular lymphocyte (CD3+CD57+LGL) T cells ranged from 64-98%. Administration of ABC008 resulted in a peak depletion of the CD3+CD57+LGL T cell population of 40-100% (Figure 11).

[0144] Patients were followed for 168 days. Two showed persistent depletion of the CD3+CD57+ LGL T cell population (FIG. 24).

[0145] In two additional cohorts, 1 in 3 patients and 1 in 5 patients received a single dose of ABC008 as described for Cohort 2 and Cohort 3 in Example 14, and blood immune cell populations were monitored by FACS for 168 days (Cohort 2) or 28 to 112 days (Cohort 3).

[0146] For cohort 2, baseline KLRG1 positivity of large granular lymphocyte (CD3+CD57+LGL) T cells was 69-97%. Administration of ABC008 resulted in a peak depletion of approximately 90-100% of the CD3+CD57+LGL T cell population, and sustained depletion of approximately 40-80% over the monitoring period (Figure 25).

[0147] For cohort 3, baseline KLRG1 positivity of large granular lymphocyte (CD3+CD57+LGL) T cells was 47-85%. Administration of ABC008 resulted in peak depletion of approximately 50-100% of the CD3+CD57+LGL T cell population, with sustained depletion of approximately 25-55% for patient 1 (monitored for 112 days) and >90% for patient 3 (monitored for 84 days) (Figure 26).

[0148] Example 16. Depletion of large granular lymphocytes (LGL) by ABC008 in patients with T-cell large granular lymphocytic leukemia (T-LGLL). Three patients suffering from T-cell large granular lymphocytic leukemia (T-LGLL) are administered ABC008 at 0.25 mg / kg subcutaneously on day 1 and weeks 1, 12, 24, and 36 in a clinical trial. Blood immune cell populations are monitored by FACS. The group receiving ABC008 shows depletion of LGL cells.

[0149] Example 17: Regulatory T cells (Tregs) are preserved by ABC008 in patients with IBM. Three patients suffering from IBM were administered a single dose of ABC008 (0.1 mg / kg) subcutaneously in clinical trial NCT04659031. Blood immune cell populations were monitored by FACS. ABC008 resulted in negligible depletion of Treg cell populations (less than about 20% on days 21-84 after administration) (Figure 15).

[0150] Three cohorts, each containing three IBM patients, were subcutaneously administered ABC008 as shown in Example 14. Treg cell populations were monitored by FACS and compared with historical data from alemtuzumab trials for multiple sclerosis. Administration of ABC008 resulted in negligible depletion of Tregs (peak depletion about less than 10%) over 180 days (Figure 16). In contrast, alemtuzumab depleted about 75% of Tregs over the same period (Figure 16).

[0151] Example 18: Central memory T cells are preserved by ABC008 in patients with IBM. Three cohorts, each containing three IBM patients, were subcutaneously administered ABC008 as shown in Example 14. Central memory T cell populations were monitored by FACS and compared with historical data from alemtuzumab trials for multiple sclerosis. Administration of ABC008 resulted in negligible depletion of central memory T cells (peak depletion less than about 25%) over 180 days (Figure 17). In contrast, alemtuzumab depleted more than about 80% of central memory T cells over the same period (Figure 17).

[0152] Three cohorts, two of which included three IBM patients and the third of which included five patients, were administered ABC008 subcutaneously as shown in Example 14. Central memory T cell populations were monitored by FACS and compared with historical data from alemtuzumab trials for multiple sclerosis. Administration of ABC008 resulted in less than about 60% peak depletion of central memory T cells over 180 days (Figure 28). In contrast, alemtuzumab depleted more than about 80% of central memory T cells over the same period (Figure 28).

[0153] Example 19: Pharmacokinetics of ABC008 in patients with IBM. Patients suffering from IBM were administered a single dose of ABC008 (0.1 mg / kg) subcutaneously in clinical trial NCT04659031. As shown in Figure 18, ABC008 demonstrated prolonged absorption and slow clearance typical of monoclonal antibody therapy.

[0154] Three cohorts, each containing three patients with IBM, were administered ABC008 subcutaneously as described in Example 14. As shown in Figure 19, ABC008 demonstrated prolonged absorption and slow clearance typical of monoclonal antibody therapy.

[0155] Example 20: Effect of ABC008 on disease severity in patients with IBM. Disease severity in three patients with IBM was assessed in clinical trial NCT04659031 using the Sporadic Inclusion Body Myositis Functional Assessment (sIFA), Inclusion Body Myositis Functional Rating Scale (IBMFRS), modified Timed Up and Go (mTUG), and manual muscle testing (MMT12) followed by a single dose of ABC008 (0.1 mg / kg) subcutaneously administered, followed by the same assessments over a 56-day period. Figure 20A shows the absolute sIFA scores at various time points after ABC008 administration. Figure 20B shows the percentage of improvement in mTUG at various time points. Figure 20C summarizes the changes in all four assessments after 56 days. The most significant improvement was seen in patient #2, who had the highest IBM disease severity at baseline.

[0156] Figures 31A-C show trends toward functional stability or improvement in IBMFRS, MMT, and mTUG by at least day 56 for combined functional readouts across three cohorts of IBM patients receiving single ascending subcutaneous doses of ABC008 (0.1, 0.5, and 2.0 mg / kg) in the same clinical trial.

[0157] Example 21: In vitro depletion of human CD8+CD57+ LGLs with ABC008 or ABC108. Purified human CD8+CD57+ LGLs were incubated with ABC008, ABC108 (a fucosylated form of ABC008), or an isotype control at concentrations ranging from about 0.1 nM to about 1 mM. ABC008 showed higher potency than ABC108 against CD8+CD57+ LGLs (Figure 21).

[0158] Example 22: Naive T cells are preserved by ABC008 in patients with IBM. Three cohorts, two containing three IBM-afflicted patients and the third containing five patients, were subcutaneously administered ABC008 as shown in Example 14. Naive T cell populations were monitored by FACS and compared with historical data from alemtuzumab trials for multiple sclerosis. Administration of ABC008 resulted in less than about 40% peak depletion of naive T cells over 180 days (Figure 27). In contrast, alemtuzumab depleted more than about 80% of naive T cells over the same period (Figure 27).

[0159] Example 23: CD8 effector memory T cells are depleted by ABC008 in patients with IBM. Three cohorts, two containing three IBM-affected patients and the third containing five patients, were administered ABC008 subcutaneously as shown in Example 14. CD8 effector memory T (TEM) cell populations were monitored by FACS and compared to historical data from trials of alemtuzumab for multiple sclerosis. Administration of ABC008 resulted in a peak depletion of about 50-75% of TEM cells, with sustained depletion of about 30-50% over 112-180 days (Figure 29). In comparison, alemtuzumab depleted more than about 80% of TEM cells over the same period (Figure 29).

[0160] Example 24: CD8 terminally differentiated effector memory T cells are depleted by ABC008 in patients with IBM. Three cohorts, two containing three IBM-affected patients and the third containing five patients, were administered ABC008 subcutaneously as shown in Example 14. CD8 terminally differentiated effector memory T (TEMRA) cell populations were monitored by FACS and compared to historical data from trials of alemtuzumab for multiple sclerosis. Administration of ABC008 resulted in a peak depletion of TEMRA cells of about 55-95%, with sustained depletion of about 20-70% over 112-180 days (Figure 30). In comparison, alemtuzumab depleted more than about 80% of TEMRA cells over the same period (Figure 29).

[0161] Those skilled in the art will recognize numerous modifications and variations that may be made without altering the spirit or scope of the present disclosure. Such modifications and variations are encompassed within the scope of the present disclosure. The entire contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference.

[0162] References Swerdlow SH,et al.The 2016 revision of the World Health Organization classification of lymphoid neoplasms.Blood 2016;127(20):2375-90.

[0163] Takata K, et al.Primary cutaneous NK / T-cell lymphoma,nasal type and CD56-positive peripheral T-cell lymphoma:a cellular lineage and clinicopathologic study of 60 patients from Asia.The American journal of surgical pathology 2015;39(1):1-12.

[0164] Zing NPC,et al.Peripheral T-Cell Lymphomas:Incorporating New Developments in Diagnostics,Prognostication,and Treatment Into Clinical Practice-PART 2:ENKTL,EATL,Indolent T-Cell LDP of the GI Tract,ATLL,and Hepatosplenic T-Cell Lymphoma.Oncology(Williston Park)2018;32(8):e83-e9。

Claims

1. An antibody or fragment thereof that specifically binds to the extracellular domain of KLRG1, comprising a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 4 and a light chain variable region containing light chain complementarity-determining regions (CDRs) with the amino acid sequences of SEQ ID NO: 11 (CDR-L1), SEQ ID NO: 12 (CDR-L2), and SEQ ID NO: 13 (CDR-L3).

2. The antibody or fragment thereof according to claim 1, wherein the light chain variable region contains the amino acid sequence of SEQ ID NO:

5.

3. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO:

6.

4. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a light chain containing the amino acid sequence of SEQ ID NO:

7.

5. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 6 and a light chain containing the amino acid sequence of SEQ ID NO:

7.

6. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof specifically binds to the epitope PLNFSRI (SEQ ID NO: 14) or a fragment thereof containing at least 5 consecutive amino acids.

7. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a monoclonal antibody or a fragment or derivative thereof.

8. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment thereof comprises a humanized antibody or a fragment thereof.

9. The antibody or fragment thereof according to claim 1, wherein the KLRG1 comprises human KLRG1 or cynomolgus monkey KLRG1.

10. A method of depleting KLRG1-expressing T cells and / or NK cells in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of the antibody or fragment thereof according to claim 1, thereby depleting KLRG1-expressing T cells and / or NK cells in the subject.

11. A method of treating a disorder associated with excessive KLRG1-expressing T cells in a subject in need thereof, comprising delivering to the subject a therapeutically effective amount of the antibody or fragment thereof according to claim 1, thereby reducing the excessive KLRG1-expressing T cells in the subject.

12.

13. The method according to claim 11, wherein the disorder is a transplantation disorder.

14. ​ The method according to claim 11, wherein the disorder is an autoimmune disease.

14. The method according to claim 11, wherein the disorder is inclusion body myositis (IBM).

15. A method of treating cancer in a subject, wherein the cancer comprises cancer cells expressing KLRG1, comprising delivering to the subject a therapeutically effective amount of the antibody or fragment thereof according to claim 1, wherein the delivery to the subject depletes cancer cells expressing KLRG1.

16. An adjuvant therapy for treating cancer in a subject, the adjuvant therapy comprising delivering to the subject a therapeutically effective amount of the antibody or fragment thereof according to claim 1, wherein the delivery depletes KLRG1-expressing pathogenic T cells and / or NK cells that attack self-tissues in the subject, regardless of whether the cancer expresses KLRG1. wherein the subject is receiving checkpoint therapy.

17. A method of depleting KLRG1-expressing cells in a mixed population of cells, wherein the KLRG1-expressing cells comprise one or more cells selected from the group consisting of T cells, NK cells, cancer cells, and combinations thereof, comprising delivering to the mixed population of cells a therapeutically effective amount of the antibody or fragment thereof according to claim 1, wherein the delivery depletes KLRG1-expressing T cells, NK cells, cancer cells, and combinations thereof in the mixed population of cells.

18. A method of selectively depleting KLRG1-expressing CD8 effector T cells while relatively sparing naive T cells and / or regulatory T cells, comprising delivering to a subject a therapeutically effective amount of the antibody or fragment thereof according to claim 1, thereby selectively depleting KLRG1-expressing CD8 effector T cells.

19. A pharmaceutical composition comprising at least one antibody or fragment thereof according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

20. A kit comprising at least one antibody or fragment thereof according to any one of claims 1 to 9 and instructions for use.

Images