Anti-KLRG1 Antibody

Abstract

The present disclosure relates to antibodies, or antigen-binding fragments thereof, that specifically bind to killer cell lectin-like receptor G1 (KLRG1). Such antibodies, or antigen-binding fragments thereof, are useful for a variety of therapeutic or diagnostic purposes, including the treatment of cancer, and are useful for increasing the efficacy of vaccines.

Description

[Technical field]

[0001] The technical field relates to inhibition of lymphocyte co-inhibitory receptor killer cell lectin-like receptor subfamily G member 1.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 166,663, filed March 26, 2021, and U.S. Provisional Patent Application No. 63 / 294,436, filed December 29, 2021, the contents of each of which are incorporated by reference in their entirety herein.

[0003] Sequence Listing Reference This application contains a sequence listing in computer readable form, which is incorporated herein by reference. The ASCII copy, created on March 1, 2022, is named 770808_000043_SL.txt and is 32,100 bytes in size. [Background technology]

[0004] Lymphocyte co-inhibitory receptors regulate the action of the adaptive immune system (see, for example, WO2020 / 060781). The action of co-inhibitory receptors is generally carried out by the binding of a ligand to the extracellular domain of the co-inhibitory receptor, followed by the recruitment of intracellular phosphatases by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular domain of the co-inhibitory receptor. The action of co-inhibitory receptors is generally to dampen T cell receptor (TCR)-mediated immune responses. In recent years, it has been shown that drugs that block the activity of co-inhibitory receptors can be used as effective treatments for cancer and infectious diseases.

[0005] Summary of the Invention In one aspect, the disclosure relates to an antibody, or antigen-binding fragment thereof, that specifically binds to the extracellular domain of KLRG1, comprising a heavy chain variable region comprising SEQ ID NO: 4. The antibody, or antigen-binding fragment thereof, may further comprise a light chain comprising a CDR-L1 selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 31, and SEQ ID NO: 37, a CDR-L2 selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 32, and SEQ ID NO: 38, and a CDR-L3 selected from the group consisting of SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 27, SEQ ID NO: 33, and SEQ ID NO: 39. The CDR-L1, CDR-L2, and CDR-L3 may be SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21, respectively, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 33, respectively, or SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively.

[0006] In another aspect, the disclosure relates to an antibody, or antigen-binding fragment thereof, that specifically binds to the extracellular domain of KLRG1, comprising a light chain variable region comprising SEQ ID NO: 5. The antibody, or antigen-binding fragment thereof, of claim 4 may further comprise a heavy chain comprising CDR-L1 selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, CDR-L2 selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, and CDR-L3 selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36. CDR-H1, CDR-H2, and CDR-H3 may be SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24, respectively, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30, respectively, or SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, respectively.

[0007] In one aspect, the disclosure relates to an antibody, or antigen-binding fragment thereof, that specifically binds to the extracellular domain of KLRG1, the antibody, or antigen-binding fragment thereof, comprising: (a) a heavy chain variable region comprising SEQ ID NO: 4; and (b) a light chain variable region comprising SEQ ID NO: 5. The antibody, or antigen-binding fragment thereof, may comprise a heavy chain comprising SEQ ID NO: 8. The antibody, or antigen-binding fragment thereof, may comprise a light chain comprising SEQ ID NO: 9. The antibody, or antigen-binding fragment thereof, may comprise: (a) a heavy chain comprising SEQ ID NO: 8; and (b) a light chain comprising SEQ ID NO: 9.

[0008] The antigen-binding fragment may be a F(ab) fragment. The antigen-binding fragment may be a F(ab2) fragment. The antibody, or antigen-binding fragment thereof, may be bispecific.

[0009] In one embodiment, the present disclosure relates to a method for treating cancer, comprising administering to a cancer patient an antibody or antigen-binding fragment disclosed herein. The method may also comprise administering to a cancer patient a checkpoint inhibitor. The checkpoint inhibitor may be administered simultaneously with the administration of the antibody, or antigen-binding fragment thereof. The checkpoint inhibitor may be administered within a day, a week, or a month of the antibody, or antigen-binding fragment thereof. Cancers 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 cancer, pancreatic cancer, gastric cancer, colon carcinoma, prostate carcinoma, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, malignant insulinoma, malignant pancreatic cancer, The cancer may be one or more of carcinoid carcinoma, 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, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

[0010] The present disclosure also relates to adjunctive therapy, comprising administering an antibody, or antigen-binding fragment thereof, disclosed herein to a subject undergoing treatment with a checkpoint inhibitor, thereby effecting the adjunctive therapy.

[0011] The present disclosure will become more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0012] [Figure 1] 1 is a graph showing PA015 binding to CHOK1 cells expressing human KLRG1. [Diagram 2] 1 is a graph showing PA015 inhibition of E-cadherin binding to CHOK1 cells expressing KLRG1 in a relative binding assay. [Diagram 3] 1 is a graph showing PA015 inhibition of E-cadherin binding to CHOK1 cells expressing KLRG1 compared to HG1N01 and HG1N02 in a relative binding assay. [Figure 4] 1 shows a graph of PA015 binding to human CD8+ T cells from two donors compared to HG1N02 in a FACS binding assay. [Diagram 5] 1 is a graph showing serum concentrations of PA015 over time in a cynomolgus monkey pharmacokinetic assay as described in Example 9. [Figure 6] 1 is a graph showing serum concentration of HG1N01 over time in a cynomolgus monkey pharmacokinetic assay as described in Example 9. [Figure 7] 1 shows a graph of deamidation at specific light chain (LC) and heavy chain (HC) residues of PA015 compared to HG1N02. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Certain exemplary embodiments are 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.

[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 extracellular portion of KLRG1 contains a C-type lectin domain whose known ligands are cadherins. The intracellular portion of KLRG1 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain that is involved in the co-inhibition of T cell receptor (TCR)-mediated signaling. KLRG1 ligands include E-cadherin, N-cadherin, R-cadherin, and combinations thereof.

[0015] In humans, KLRG1 expression is generally restricted to cells of the immune system, specifically CD8+ T cells, NK cells, and to a lesser extent CD4+ T cells. KLRG1 expression is associated with a late differentiation phenotype. As antigen-specific T cells differentiate, they may acquire increased expression of cytotoxic molecules, thus increasing their cytotoxic potential. The biological function of KLRG1 is to inhibit the cytotoxicity and proliferation of these T cells under certain circumstances. In mouse cancer models, blocking the interaction of KLRG1 with its ligands has been shown to be beneficial in controlling tumor growth or metastasis.

[0016] 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 terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure and any invention(s) described herein or otherwise provided.

[0017] Amino acids are referred to herein by either the one-letter or three-letter codes, in accordance with 37 C.FR §1.822 and established usage.

[0018] All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for any teachings relevant to the referenced sentence and / or the paragraph in which the reference is presented.

[0019] definition 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.

[0020] 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").

[0021] As used herein, the terms "about" and "approximately," when referring to a measurable value such as an amount of a polypeptide, a dosage, time, temperature, an 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.

[0022] The transitional phrase "consisting essentially of" means that the claim scope is to be construed to include those specified materials or steps recited in the claim that do not "materially affect the basic and novel characteristic(s)" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ 461,463 (CCPA 1976) (emphasis in original); see also MPEP § 2111.03.

[0023] The term "consists essentially of" (and grammatical variations) as applied to polynucleotide or polypeptide sequences of the present disclosure 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 to 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.

[0024] As used herein, "effective amount" refers to the amount that provides the desired effect. The term "effective amount" refers to the dosage or amount that is sufficient to reduce the activity of KLRG1, leading to the improvement of the patient's condition, or to achieve the 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.

[0025] As used herein, a "therapeutically effective" amount is an amount that provides some degree of improvement or benefit to a subject. Alternatively, a "therapeutically effective" amount is an amount that provides some degree of relief, alleviation, or reduction of at least one clinical symptom in a subject. Those skilled in the art will understand that the therapeutic effect does not need to be complete or curative, as long as some degree of benefit is provided to the subject.

[0026] By the terms "treat," "treating," or "treatment of," it is intended that the severity of the subject's condition is reduced, or at least partially ameliorated or corrected, and some alleviation, relief, or reduction of at least one clinical symptom is achieved.

[0027] The term "cancer" as used herein refers to any benign or 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 cancer, pancreatic carcinoma, stomach cancer, colon carcinoma, prostate carcinoma, genitourinary cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, malignant insulin ... The cancer includes, but is not limited to, 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, osteogenic sarcoma, 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 scope of the group.

[0028] The term "isolated" can refer 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 would not be 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 its 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 cell 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 at least about 70-80% (w / w) pure, more preferably at least about 80-90% (w / w) pure, even more preferably about 90-95% pure, and most preferably at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% (w / w) pure.

[0029] The term "fragment" as applied to a polypeptide is understood to mean 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 of contiguous amino acids that are identical or nearly identical (e.g., about 90%, about 92%, about 95%, about 98%, about 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the present disclosure may be optionally included in a larger polypeptide of which it is a component. In some embodiments, such a fragment may comprise, consist essentially of, and / or consist of a peptide having a length of at least about 4, 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 75, about 100, about 150, about 200, or more contiguous amino acids of a polypeptide or amino acid sequence according to the present disclosure.

[0030] As used herein, the terms "protein" and "polypeptide" are used interchangeably, unless otherwise indicated, and include both peptides and proteins.

[0031] 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, or human, or can be chimeric antibodies. Antibodies can be recombinant monoclonal antibodies produced according to 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, according to the methods disclosed in U.S. Pat. No. 4,676,980.

[0032] The terms "antigen-binding domain", "antigen-binding fragment" and "binding fragment" refer to the part 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 part of the antigen. The part of the antigen molecule that is responsible for the specific interaction with the antigen-binding domain is called the "epitope" or "antigenic determinant".

[0033] An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily comprise both. For example, an Fd antibody fragment consists only of a VH domain but still retains some antigen-binding function of an intact antibody.

[0034] The anti-KLRG1 antibody comprises an antibody constant region or a part thereof. For example, the VL domain may have an antibody light chain constant domain attached to its C-terminus, including a human Cκ or Cλ chain. Similarly, a specific antigen-binding domain based on a VH domain may have all or part of an immunoglobulin heavy chain attached 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 of the C-terminal fragments of are well known in the art.

[0035] 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 about 10 8 M -1A specific binding is considered to be specific when it is higher than 0.01. 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.

[0036] In certain embodiments, the antibody can specifically bind to an epitope within the extracellular domain (ECD) of human 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, and the amino acid sequence of human E-cadherin is set forth in SEQ ID NO:3 below.

[0037] Antibodies and Compositions The present disclosure describes antibodies and fragments thereof that specifically bind to the extracellular domain of KLGR1 on cells by interfering with the binding of E-cadherin, R-cadherin, and / or N-cadherin to the extracellular domain of KLRG1. By interfering with the binding of E-cadherin, R-cadherin, and / or N-cadherin ligands to KLRG1, such antibodies prolong the cytotoxicity of T cells and NK cells that have KLRG1 on their surface. The antibodies described herein can be used as effective therapeutic agents for the treatment of cancer, either as monotherapy or in combination with other immunotherapeutic agents (such as checkpoint inhibitors, e.g., anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies), or in combination with chemotherapeutic agents, or cancer vaccines. Such antibodies, or fragments thereof, can be used as adjunctive therapy for the treatment of cancer, regardless of whether the cancer cells express KLRG1.

[0038] 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 closest 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 scaffold three regions of hypervariable sequence called complementarity determining regions (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, and the CDR components of the light chain are called L1, L2, and L3. CDR3, and especially H3, is the greatest source of molecular diversity within the antigen-binding domain. H3 can be as short as, for example, two amino acid residues or longer than 26.

[0039] Fab fragments (fragment antigen binding), or F(ab), consist of or contain 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 (50,000 daltons) 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, so-called single chain (sc) Fv fragments (scFv) can be constructed. In scFvs, a flexible and sufficiently long polypeptide links 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: 42) can be used as a linker, but other linkers are known in the art.

[0040] Antibody diversity is the result of the combinatorial assembly of multiple germline genes encoding the variable regions and various somatic events. Somatic events can include rearrangement of variable gene segments with diverse (D) and joining (J) gene segments to create a complete VH region, and rearrangement of variable and joining gene segments to create a complete VL region. The rearrangement process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junction. 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.

[0041] Fundamental Immunology, 3 rd Based on the estimated number of germline gene segments, the random rearrangements of these segments, and the random VH-VL pairings, the maximum number of germline gene segments is approximately 1.6 × 10 7 They can produce different antibodies. Immunoglobulin Genes, 2 nd Taking into account other processes that contribute to antibody diversity (such as somatic mutations), as supported by the 1995 American Journal of Clinical Chemistry (2019) 11:139–140, 2015, ed., Jonio et al., Academic Press, San Diego, Calif., 1995, the antibody diversity is 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.

[0042] The present disclosure provides novel antibody variable heavy and light chain regions (VH and VL) and antibody heavy and light chains derived from a human immunoglobulin gene library that are effective in blocking KLRG1 signaling in cells expressing cell surface KLGR1. These heavy and light chains and their variable regions provide a scaffold structure for carrying CDRs. The CDRs can generally be, but are not limited to, the heavy or light chains 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. [Table 1]

[0043] Antibody binding specificity It is contemplated that the antibodies of the present disclosure may also bind to other proteins, including, for example, recombinant proteins that include all or a portion of KLRG1.

[0044] The KLRG1 antibody may also be a multispecific antibody, such as a bispecific or tetraspecific antibody, a diabody, or a similar molecule (for a description of diabodies, see, e.g., PNAS USA 90(14), 6444-8 (1993)). Indeed, the bispecific antibodies, diabodies, etc. provided by the present invention may bind to any suitable target in addition to a portion of KLRG1. For example, in one embodiment, the bispecific antibody may bind to KLRG1 and HER2, thereby activating and redirecting KLRG1+ T cells and NK cells to HER2+ cancer cells. In another embodiment, the bispecific antibody may bind to KLRG1 and PD-1, thereby activating T cells and NK cells by blocking two checkpoint receptors (KLRG1 and PD-1).

[0045] The present disclosure also provides multispecific antibody fusion proteins that include an antibody portion, e.g., a Fab or Fab' fragment, with a first specificity for an antigen of interest and further include at least one single domain antibody with specificity for a second antigen of interest. The multispecific antibody fusion will be able to selectively bind to two or more antigens of interest.

[0046] In one embodiment, the first and second antigens are the same antigen.

[0047] In one embodiment, the antigen of interest bound by the antibody, or antigen-binding fragment thereof, can be a cell-associated protein, e.g., a cell surface protein on cells such as bacterial cells, yeast cells, T cells, endothelial cells, or tumor cells, or it can be a soluble protein. The antigen of interest can also be any medically relevant protein, such as those proteins that are upregulated during disease or infection, e.g., receptors and / or their corresponding ligands. Particular examples of cell surface proteins include adhesion molecules, e.g., integrins such as integrin 131, e.g., VLA-4, E-selectin, P-selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD134(0X40), ICOS, BCMP7, CD137, CD27L, CDCP1, DPCR1, dudul in2, FLJ20584, FLJ40787, HEK2, KIAA0634, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2, nectin-like 2, NKCCI, PTK7, RAIG1, TCAM1, SC6, BCMP101, BCMP84, BCMP11, DTD, carcinoembryonic antigen (CEA), human milk fat globulin (HMFG1 and 2), MHC class I and MHC class II antigens, and VEGF, and, where appropriate, their receptors. Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-16, or IL-17, viral antigens, e.g., respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins such as IgE, interferons such as interferon a, interferon p, or interferon y, colony stimulating factors such as tumor necrosis factor-a, tumor necrosis factor-I3, G-CSF, or GM-CSF, and platelet-derived growth factors such as PDGF-a, and PDGF-I3, and optionally their receptors.Other antigens include bacterial cell surface antigens, bacterial toxins, viruses such as influenza, EBV, HepA, B, and C, bioterrorism agents, radionuclides and heavy metals, and snake and spider venoms and toxins. In certain embodiments, antigens of interest include IL-2, soluble IL-15, or membrane-bound IL-15Ra / IL-15 complex, and IL-12.

[0048] One of skill in the art will recognize that the antibodies of the present disclosure may be used to detect, measure, and inhibit proteins somewhat different from KLRG1. The antibodies may be expected to retain binding specificity so long as the target protein comprises a sequence that is at least about 60%, about 70%, about 80%, about 90%, about 95% or more identical to any sequence of at least about 130, about 100, about 80, about 60, about 40, or about 20 consecutive amino acids in the sequence shown in SEQ ID NO:1 or SEQ ID NO:2. Percent identity is determined, for example, by standard sequence 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.

[0049] In addition to sequence homology analysis, epitope mapping (see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996), as well as secondary and tertiary structure analysis, can be performed to identify the specific 3D structures assumed by the disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0050] Antibody variants The present disclosure also provides a method for obtaining an antibody specific to KLRG1. The complementarity determining regions (CDRs) in such antibodies are not limited to the specific sequences of VH and VL specified in the following examples, but may include variants of these sequences that retain the ability to specifically bind to KLRG1. Such variants may be derived from the sequences listed in the examples 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.

[0051] Modifications to the FR include, but are not limited to, humanization of antibodies of non-human origin, or manipulation of certain framework residues important for antigen contact or stabilization of the binding site, e.g., changing the class or subclass of the constant region, e.g., changing specific amino acid residues that may alter effector function such as Fc receptor binding, e.g., as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, and Lund et al. (1991) J. Immun. 147:2657-2662 and Morgan et al. (1995) Immunology 86:319-324, or changing the species from which the constant region is derived.

[0052] Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes can be empirically determined by routine techniques that involve modifying the CDRs and testing the affinity antibody for its target. For example, conservative amino acid substitutions can be made within any one of the disclosed CDRs. Various modifications can be made, for example, as described in Antibody Engineering, 2 nded., 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. Positively charged (basic) amino acids include arginine, lysine, histidine. 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. 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).

[0053] The phrase "substantially depicted" means that the relevant CDR, VH, or VL domain of the present disclosure is identical to the particular region (e.g., CDR) whose sequence is depicted, or has only insubstantial differences, including minor amino acid changes, such as substitutions of one or two of any five amino acids in the sequence of the particular region.

[0054] 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 activation of cytotoxic T cells and NK cells.

[0055] The term "modulate" and similar terms refer to a decrease or increase in the activity of KLRG1 associated with T cell and NK cell activation resulting from interaction with an anti-KLRG1 antibody, the decrease or increase being compared to the activity of KLRG1 in the absence of the same antibody. The decrease 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 decreased, 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 "activation" and "activating".

[0056] The term "percent (%) sequence identity" as used herein refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in a reference nucleic acid sequence, after aligning sequences to achieve maximum sequence identity percentage and introducing gaps as necessary.Alignment to determine sequence identity percentage can be achieved using various methods within the skill of the art, for example, publicly available computer software such as ClustalW, Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software.

[0057] Antibody fragments included within the scope of the present disclosure include, for example, Fab, Fab', F(ab')2, and Fv fragments; domain antibodies, diabodies; vaccibodies, linear antibodies; single-chain antibody (ScFv) molecules; and multispecific (e.g., bispecific, tetraspecific) 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).

[0058] The antibodies of the present disclosure are humanized forms of non-human (e.g., murine) antibodies and may include 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 include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin can be replaced by corresponding non-human residues. Humanized antibodies may also include 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 can also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0059] 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.

[0060] Methods for humanizing non-human antibodies are well known in the art. The present disclosure, and any invention(s) provided herein, is not limited to any particular source, species of origin, or method of production.

[0061] The monoclonal antibodies used to carry out the present disclosure can be produced in hybridoma cell lines according to the technique of Kohler and Milstein, Nature 265:495 (1975). For example, a solution containing an 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 a suitable medium, and the supernatant is 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 known in the art.

[0062] Various immunoassays can be used for screening to identify antibodies with desired specificity for the extracellular domain of KLRG1. Numerous protocols for competitive binding assays or immunoradiometric assays using monoclonal antibodies with established specificity are well known in the art. Such immunoassays typically involve measuring the complex formation (e.g., antigen / antibody complex formation) between an antigen and its specific antibody. Two-site monoclonal-based immunoassays utilizing monoclonal antibodies that react with two non-interfering epitopes on the polypeptide or peptide of the present disclosure, and competitive binding assays can be used.

[0063] In addition to the above conjugates, the anti-KLRG1 antibodies described herein can be conjugated to a solid support (e.g., a bead, plate, slide, or well formed from a material such as latex or polystyrene) in accordance with known techniques. The anti-KLRG1 antibodies described herein can also be conjugated to a radiolabel (e.g., a radiolabel that can be bound to the antibody using conventional chemical methods) in accordance with known techniques. 35 S, 125 I, 131I, or 99m The antibody / antigen complex may be conjugated to a detectable group such as .Tc), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein). Detectable labels further include chemical moieties such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin. Determination of the formation of the antibody / antigen complex in the methods of the present disclosure may be by detection of, for example, precipitation, aggregation, flocculation, radioactivity, color development or change, fluorescence, luminescence, and the like, such methods being well known in the art.

[0064] As mentioned above, the anti-KLRG1 antibody described herein can be linked to another functional molecule, for example, another peptide or protein (such as albumin, another antibody, etc.), a toxin, a radioisotope, a cytotoxic agent, or a cytostatic agent. The antibody described herein can be conjugated to a toxin. Antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) are three well-known antibody-mediated mechanisms for killing target cells.

[0065] For example, the antibodies can be linked by chemical cross-linking or recombinant methods. The antibodies can also be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. The antibodies can be chemically modified by covalent attachment to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching them are also shown in U.S. Pat. Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546.

[0066] PA015 composition In some embodiments, the anti-KLRG1 antibody comprises a combination of any six CDRs of any one of the antibodies described herein. In some embodiments, the anti-KLRG1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3) from Table 2, and (ii) three light chain CDRs from Table 2. [Table 2]

[0067] Kabat (Wu and Kabat,1970, J Exp Med. (1970)132:211-50), Kabat EA, Te Wu T, Foeller C, Perry HM, Gottesman KS. Sequences of Proteins of Immunological Interest. Diane Publishing Company (1992)), Chothia (1987, Chothia C, Lesk a M. Canonical structures for the hypervariable regions of Immunol There are multiple antibody variable chain numbering schemes, including those proposed by Today (1997) 18:509, Lefranc et al., 2005 IMGT-ONTOLOGY for immunogenetics and immunoinformatics. In Silico Biol. (2004) 4:17-29, and Honegger (Honegger and Pluckthun, 2001, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J Mol Biol. (2001) 309:657-70), which adds to the complexity.

[0068] Chothia and his colleagues defined CDRs based on structure, ie, loop location (Chothia and Lesk, 1987, supra; Al-Lazikani et al., 1997, supra).

[0069] Kabat and Wu first defined CDRs in the early 1970s by amino acid sequence variation analysis (Wu and Kabat, 1970, supra; Kabat and Wu, 1971, supra). Since then, detailed genetic and three-dimensional structural analyses have more clearly defined the structural basis of antigen binding sites from the HVL / CDR perspective, leading to the definition of contact residues (MacCallum et al., 1996, Antibody-antigen interactions: contact analysis and binding site topography. J Mol Biol. (1996) 262: 732-745; Martin and Allen, 2007, Bioinformatics tools for antibody engineering. In: Dubel S, editor. Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag GmbH (2008). p. 95-117).

[0070] The AbM definition of CDRs, which was a compromise between the Kabat and Chothia definitions, was used in the AbM modeling software (Martin and Allen, supra, 2007). Martin and his colleagues also based their definition of CDRs on the actual paratope, i.e., those residues that actually contact the antigen (Martin and Allen, supra, 2007), and developed and tested an automated application called "Abnum" for variable chain numbering and CDR determination based on a correction of the Chothia approach (Abhinandan and Martin, 2008, Analysis and prediction of VH / VL packing in antibodies. Protein Eng Des Sel. (2010) 23:689-97).

[0071] The Contact definition of CDRs is based on the observation that only 20-33% of the amino acids in a CDR are in direct contact with the antigen (Padlan EA. Anatomy of the antibody molecule. Mol Immunol. (1994) 31:169-217). These residues, called "specificity-determining residues (SDRs)", were first described by Padlan et al. (Padlan EA, Abergel C, Tipper JP. Identification of specificity-determining residues in antibodies. FASEB J OffPubl Fed Am Soc Exp Biol. (1995) 9:133-9). Their results show that these SDRs are involved in the interaction with the antigen and in most cases coincide with the most variable positions present in the CDRs. Using this concept of SDRs, MacCallum and colleagues proposed a new way to define CDRs and renamed SDRs "contact residues". They also suggested that contact residues are more frequently located in the center of the paratope and, as Chothia noted above, non-contact residues play a role in shaping the conformation of the CDR loops, thus optimally orienting the contact residues for efficient and specific antigen binding.

[0072] The V-REGION IMGT-specific numbering allowed for redefining the limits of the complementarity determining regions and framework regions, designated in the IMGT as CDR-IMGT and FR-IMGT, respectively. The V-DOMAIN IMGT-specific numbering provided a standardized numbering of the CDR3-IMGT and FR4-IMGT of the rearranged VJ-REGION and VDJ-REGION.

[0073] Complementarity determining regions (CDR-IMGT) are the loop regions of the V-DOMAIN (variable (V) domain) delimited according to the IMGT unique numbering of the V domain (Lefranc M. et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Dev Comp Immunol 27:55-77). In the V-DOMAIN, there are three CDR-IMGT: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C''), and CDR3-IMGT (loop FG).

[0074] In the V-DOMAIN (immunoglobulin (IG) or V-domain of antibodies and T-cell receptors (TR)), the amino acids of the CDR-IMGT bind to the antigen (epitope) and confer specificity to the IG and TR (Paratope, IMGT-ONTOLOGY, SpecificityType). The first two CDR-IMGT are part of the V-REGION (encoded by the variable (V) genes), whereas the CDR3-IMGT corresponds to the junction and results from a rearrangement between the V gene and the joining (J) gene (VJ rearrangement) or between the V gene, the diversity (D) gene, and the J gene (VDJ rearrangement) (immunoglobulin synthesis).

[0075] PA015 sequence One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, and (ii) a light chain comprising CDR-L1 (e.g., SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37), CDR-L2 (e.g., SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38), and CDR-L3 (e.g., SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39). In more specific embodiments, the heavy chain variable region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 in regions other than the complementarity determining regions (CDR) regions, including but not limited to the framework (FR) regions.

[0076] One embodiment is an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, comprising: (i) a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, and in regions other than the complementarity determining region (CDR) regions, including, but not limited to, framework (FR) regions; and (ii) a light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, CDR-L2, and CDR-L3 are SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.

[0077] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4, and (ii) a light chain comprising CDR-L1 (SEQ ID NO:13), CDR-L2 (SEQ ID NO:14), and CDR-L3 (SEQ ID NO:15). In a more specific embodiment, a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0078] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8, and (ii) a light chain comprising CDR-L1 (e.g., SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37), CDR-L2 (e.g., SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38), and CDR-L3 (e.g., SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39). In more specific embodiments, a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0079] One embodiment is an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, comprising: (i) a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions, with the amino acid sequence of SEQ ID NO:8; and or (ii) a light chain comprising CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, CDR-L2, and CDR-L3 have SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively, or SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively.

[0080] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, and (ii) a heavy chain comprising CDR-H1 (e.g., SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34), CDR-H2 (e.g., SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35), and CDR-H3 (e.g., SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36). In more specific embodiments, the light chain variable region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5 in regions other than the complementarity determining regions (CDR) regions, including but not limited to the framework (FR) regions.

[0081] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, and (ii) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; or SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

[0082] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5, and (ii) a heavy chain comprising CDR-H1 (SEQ ID NO:10), CDR-H2 (SEQ ID NO:11), and CDR-H3 (SEQ ID NO:12). In more specific embodiments, the light chain variable region has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:5 in regions other than the complementarity determining regions (CDR) regions, including but not limited to the framework (FR) regions.

[0083] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and (ii) a heavy chain comprising CDR-H1 (e.g., SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34), CDR-H2 (e.g., SEQ ID NO:11, SEQ ID NO:17, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35), and CDR-H3 (e.g., SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36). In more specific embodiments, the light chain has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0084] Certain embodiments include an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, comprising: (i) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, respectively, in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions; and (ii) a heavy chain comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1, CDR-H2, and CDR-H3 have SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, or SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively.

[0085] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9, and (ii) a heavy chain comprising CDR-H1 (SEQ ID NO: 10), CDR-H2 (SEQ ID NO: 11), and CDR-H3 (SEQ ID NO: 12). In more specific embodiments, the light chain has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0086] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:9, and (ii) a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:8. In a more specific embodiment, the light chain has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions, and the heavy chain has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0087] One embodiment is an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:9 in regions other than the complementarity determining regions (CDR) regions, including but not limited to the framework (FR) regions. and (ii) a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in regions other than the complementarity determining region (CDR) regions, including, but not limited to, the framework (FR) regions, with the amino acid sequence of SEQ ID NO:8.

[0088] One embodiment is an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, comprising: (i) a light chain variable region containing CDRs (CDR-L1, CDR-L2, CDR-L3) having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (ii) a heavy chain variable region containing CDRs (CDR-H1, CDR-H2, CDR-H3) having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; and (iii) a heavy chain variable region containing CDRs having at least 50%, 60%, or more identical to the amino acid sequence of SEQ ID NO: 6. 7. In one embodiment, the present invention provides a method for the preparation of a human medicament ...

[0089] One embodiment is an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, comprising: (i) a light chain variable region comprising CDRs (CDR-L1, CDR-L2, CDR-L3) having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (ii) a heavy chain variable region comprising CDRs (CDR-H1, CDR-H2, CDR-H3) having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; (iii) a CDR having an amino acid sequence of SEQ ID NO: 8 and at least 50 amino acids therein; 9, and (iv) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9.

[0090] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises (i) a heavy chain variable region comprising at least one, two, or three CDRs (e.g., CDR-H1, CDR-H2, or CDR-H3) having the amino acid sequence of SEQ ID NO: 10, 11, or 12, respectively, or a portion thereof, and (ii) a light chain variable region comprising at least one, two, or three CDRs (e.g., CDR-L1, CDR-L2, or CDR-L3) having the amino acid sequence of SEQ ID NO: 13, 14, or 15, respectively, or a portion thereof.

[0091] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5 in regions other than the complementarity determining region (CDR) regions, including but not limited to the framework (FR) regions.

[0092] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 in regions other than the CDR regions, including but not limited to the FR regions.

[0093] One embodiment provides an antibody, preferably a monoclonal antibody, or antigen-binding fragment thereof, having (i) a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:5 in regions other than the CDR regions, including but not limited to the framework (FR) regions, and (ii) a heavy chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4 in regions other than the CDR regions, including but not limited to the FR regions.

[0094] One embodiment is an antibody, preferably a monoclonal antibody, or an antigen-binding fragment thereof, comprising: (i) a light chain variable region containing CDRs (CDR1, CDR2, CDR3) having the amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively; (ii) a heavy chain variable region containing CDRs (CDR1, CDR2, CDR3) having the amino acid sequences of SEQ ID NOs: 10, 11, and 12, respectively; (iii) a heavy chain variable region containing CDRs (CDR1, CDR2, CDR3) having at least 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 600%, 700%, 750%, 800%, 900%, 950%, 900%, 1000%, 1000%, 1000%, 1100%, 1200%, 1300%, 1400%, 150 , 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:7, and (iv) a light chain constant region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:7.

[0095] In certain aspects of all of the foregoing embodiments, when a light or heavy chain, variable region, or constant region is said to have a certain percentage of sequence identity to a reference SEQ ID NO:, such identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0096] immunotoxin In some embodiments, the antibodies and / or antigen-binding fragments thereof provided by the present disclosure are conjugated to a toxic agent. Immunoconjugates containing one or more cytotoxins are referred to as "immunotoxins." Antibodies conjugated to cytotoxic agents, drugs, and the like are also known as antibody-drug conjugates (ADCs). The immunoconjugates may have a half-life of sufficient duration for the antibody-drug conjugate to be internalized, degraded, and induce cell killing by the released toxin. Cytotoxins or cytotoxic agents may include any agent that is harmful 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 analog or derivative thereof, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; calicheamatidine, tetracaine, lidocaine, propranolol, and puromycin; isine or their analogs or derivatives, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (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 their analogs, antibiotics (dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitom ... antimitotic agents (e.g., thuringian targeting agents), such as xanthrone, plicamycin, anthramycin (AMC), etc.), diphtheria toxin and related molecules (e.g., diphtheria A chain and its active fragments and hybrid molecules); ricin toxins (e.g., 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 Exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins. Other suitable conjugate molecules include antibacterial / lytic peptides such as CLIP, Magainin2, melitin, Cecropin, and P18, ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.

[0097] Complementary Therapeutic Agents The antibodies of the present disclosure, including fragments and conjugates thereof, can optionally be delivered to a patient in conjunction with other therapeutic agents. The additional therapeutic agents can be delivered before, simultaneously, or after the antibodies of the present disclosure. As used herein, the word "concurrently" means close enough in time to produce a combined effect (i.e., concurrently can be simultaneously, or it can be two or more events occurring within a short period of time before or after each other).

[0098] In some embodiments, the antibodies of the disclosure are directed to or comprised within a class of compounds that are capable of binding to or inhibiting 1) vinca alkaloids (e.g., vinblastine, vincristine), 2) epipodophyllotoxins (e.g., etoposide and teniposide), 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)), 4) enzymes (e.g., L-asparaginase), 5) biological response modifiers (e.g., interferon-alpha), 6) platinum coordination complexes (e.g., cisplatin and carboplatin), 7) anthracenediones (e.g., mitoxantrone), 8) substituted ureas (e.g., hydroxyurea), 9) methylhydrazine derivatives (e.g., procal, It may be administered in conjunction with anti-cancer drugs such as vasodilator (N-methylhydrazine, MIH), 10) adrenal cortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide), 11) adrenal cortical steroids (e.g., prednisone), 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate), 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol), 14) and antiestrogens (e.g., tamoxifen), 15) androgens (e.g., testosterone propionate and fluoxymesterone), 16) antiandrogens (e.g., flutamide), and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).In some embodiments, the antibodies of the disclosure are used in combination with antibodies 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 (SEQ ID NO: 43) TNF alpha complex, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, and antiangiogenic agents such as rifabutin, enzastaurin, paclitaxel, paclitaxel albumin stabilized nanoparticle formulation (Abraxane), soy isoflavone (genistein), tamoxifen citrate, thalidomide, ADH-1 (EXHERIN), 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.

[0099] In some embodiments, antibodies of the disclosure may be administered in conjunction with immunosuppressants including, for example, cyclosporine A, rapamycin, glucocorticoids, azathioprine, mizoribine, aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamide, and FK506 (tacrolimus).

[0100] 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.

[0101] The nucleic acids provided herein comprise coding sequences for the CDRs, VH domains, and / or VL domains disclosed herein. The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription, or expression cassettes that comprise at least one nucleic acid encoding the CDRs, VH domains, and / or VL domains disclosed herein. The present disclosure further provides host cells that comprise one or more of the above constructs.

[0102] In some embodiments, the coding sequence for the heavy chain is the nucleotide sequence of SEQ ID NO:40.

[0103] In some embodiments, the coding sequence for the light chain is the nucleotide sequence of SEQ ID NO:41.

[0104] 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 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.

[0105] 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. Vectors can be plasmids or viruses, such as phages or phagemids, as needed. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. 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.

[0106] A further aspect of the disclosure provides a host cell comprising a nucleic acid disclosed herein. Yet a further aspect 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 baculovirus for insect cells. 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.

[0107] Treatment methods The disclosed anti-KLRG1 antibodies can regulate KLRG1-related immune responses. In certain embodiments, the activation of cytotoxic T cells and NK cells is mediated by the regulation of KLRG1 signaling. The disclosed antibodies can act as either agonists or antagonists of KLRG1 depending on how they are used. The antibodies can be used to prevent, diagnose, or treat medical disorders in mammals, particularly humans. The disclosed antibodies can also be used to isolate KLRG1 or KLRG1-expressing cells. Furthermore, the antibodies can be used to treat subjects at risk or susceptible to disorders or having disorders associated with abnormal KLRG1 expression or function.

[0108] The antibodies of the disclosure can be used in situations where modulation of cytotoxic T cell and NK cell activation may be desirable, for example, in certain types of cancer and infectious diseases.

[0109] In certain circumstances, it may be desirable to induce or enhance a patient's immune response to treat cancer or infectious diseases. Disorders treated or prevented by the disclosed methods 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, as well as cancers (e.g., melanoma and prostate cancer). Activation of cytotoxic T cells and NK cells by the anti-KLRG1 antibodies disclosed herein enhances the response of T cells and NK cells. In such cases, the antibodies act as antagonists of KLRG1. Thus, in some embodiments, the antibodies can be used to inhibit or reduce downregulation activities associated with KLRG1, i.e., activities associated with downregulation of the activation of cytotoxic T cells and NK cells.

[0110] As shown in the Examples, blocking KLRG1 / E-cadherin interactions with antagonistic anti-KLRG1 antibodies results in enhanced T cell proliferative responses and IFNy secretion by cytotoxic T cells and NK cells, consistent with a downregulatory role of the KLRG1 pathway in the activation of these cells. In various embodiments, the antibodies inhibit E-cadherin binding to KLRG1 with an IC50 of less than about 50 nM, and more preferably less than about 40, 30, 20, 10, or 5 nM. Inhibition of E-cadherin binding can be measured using techniques known in the art.

[0111] The antibody or antibody composition of the present disclosure is administered in a therapeutically effective amount. In general, the therapeutically effective amount may vary depending on the age, condition, and sex of the subject, as well as the severity of the subject's condition. The therapeutically effective amount of the antibody ranges from about 0.001 to about 30 mg / kg body weight, preferably from about 0.01 to about 25 mg / kg body weight, from about 0.1 to about 20 mg / kg body weight, or from about 1 to about 10 mg / kg body weight. The dosage may be adjusted as necessary to suit the observed effects of the treatment.

[0112] The appropriate dose is selected by the treating physician based on clinical indication. The antibody may be given as a bolus dose to maximize the circulating levels of the antibody for the longest period of time after administration. Continuous infusion may also be used after the bolus dose.

[0113] Immune cells (e.g., T cells or NK cells) can also be isolated from a patient and incubated ex vivo with the antibodies of the present disclosure. In some embodiments, activation of T cells and NK cells can be modulated by removing immune cells from a subject and contacting the immune cells in vitro with an anti-KLRG1 antibody of the present disclosure. In such embodiments, anti-KLRG1 antibodies can be used in a multivalent form such that KLRG1 molecules on the surface of immune cells are "cross-linked" upon binding to such antibodies. For example, anti-KLRG1 antibodies can be bound to a solid support, e.g., beads, or cross-linked via a secondary antibody. Immune cells can then be isolated using methods known in the art and re-implanted into the patient.

[0114] The antibody of the present disclosure can also be used to detect the presence of KLRG1 in biological samples.The amount of KLRG1 detected can be correlated with the expression level of KLRG1, which then correlates with the activation state of immune cells (e.g., activated T cells or NK cells) in subjects.

[0115] Detection methods using antibodies are well known in the art, and include, for example, ELISA, radioimmunoassay, immunoblot, Western blot, immunofluorescence, immunoprecipitation.Antibody can be provided in a diagnostic kit that incorporates one or more of these techniques to detect KLRG1.Such kits can contain other components, packaging, instructions, or other materials to assist in the detection of protein.

[0116] If the antibodies are for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope, or an enzyme). If necessary, the antibodies of the present disclosure may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands with specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment quantifiable by a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, as well as the numerous receptor-ligand pairs known in the art. Other permutations and possibilities will be readily apparent to those of skill in the art and are considered equivalents within the scope of the present disclosure.

[0117] The antibodies of the present disclosure can be used in screening methods to identify inhibitors of the KLRG1 pathway that are effective as therapeutic agents. In such screening assays, a first binding mixture is formed by combining KLRG1 with an antibody of the present disclosure, and the amount of binding in the first binding mixture (MO) is measured. A second binding mixture is also formed by combining KLRG1, an antibody, and the compound or agent to be screened, and the amount of binding in the second binding mixture (M1) is measured. The compound to be tested may be another anti-KLRG1 antibody, as shown in the examples. The amount of binding in the first and second binding mixtures is then compared, for example, by calculating the M1 / M0 ratio. It is considered that the compound or agent can modulate KLRG1-related downregulation of immune responses if a decrease in binding is observed in the second binding mixture compared to the first binding mixture.

[0118] Formulation and optimization of the binding mixture is within the level of the ordinary skilled artisan, and such binding mixture may also contain buffers and salts necessary to enhance or optimize binding, and additional control assays may be included in the screening assays of the present disclosure. Thus, compounds found to reduce KLRG1 antibody binding by at least about 10% (i.e., M1 / M0<0.9), preferably more than about 30%, may be identified and then, if necessary, screened secondarily for their ability to ameliorate the disorder in other assays or animal models described below. The strength of binding between KLRG1 and the antibody may be measured, for example, using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance-based technology (e.g., Biacore), all of which are techniques well known in the art.

[0119] Compounds may then be tested in vitro as shown in the examples or animal models. For example, preliminary doses determined according to animal tests and dosage scaling for human administration are performed according to accepted practices in the art. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Data obtained from cell culture assays or animal studies can be used to formulate dosage ranges for use in humans.

[0120] Therapeutically effective dosages achieved in one animal model can be converted for use in other animals, including humans, using conversion factors known in the art (see, e.g., Freireich et al. (1966) Cancer Chemother. Reports, 50(4):219-244).

[0121] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising anti-KLRG1 antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions typically comprise one or more antibodies of the present disclosure and a pharma- ceutically acceptable excipient. The phrase "pharma- ceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, etc., 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 supplementary, additional, or enhanced therapeutic functions. The pharmaceutical compositions may be contained in a container, pack, or dispenser together with instructions for administration.

[0122] The pharmaceutical composition of the present disclosure is formulated to be compatible with its intended route of administration.Methods for achieving administration are known to those skilled in the art.Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.It may also be possible to obtain a composition that may be topically or orally administered, or that may be capable of being transmitted across mucous membranes.

[0123] The solution or suspension used for intradermal or subcutaneous application typically includes one or more of the following components: sterile diluent such as water for injection, saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antibacterial agent such as benzyl alcohol or methylparaben; antioxidant such as ascorbic acid or sodium hydrogen sulfate; chelating agent such as ethylenediaminetetraacetic acid; buffer such as acetate, citrate, or phosphate; and agent for adjusting osmotic pressure such as sodium chloride or dextrose. pH can be adjusted using acid or base such as hydrochloric acid or sodium hydroxide. Such formulations may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0124] 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). In all cases, the composition must be sterile and fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, a polyalcohol, such as sugar, 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.

[0125] Administration and Dosage Methods for accomplishing administration are known to those of skill in the art. Administration can be, for example, parenteral (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous).

[0126] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is 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, by using lozenges, nasal sprays, inhalers, or suppositories. For example, in the case of antibodies that contain an Fc portion, the composition may be capable of being 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 into ointments, salves, gels, or creams, as 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 containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[0127] In certain embodiments, the antibody of the present disclosure is prepared with a carrier that protects the compound from rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Liposomal suspensions containing the antibody disclosed herein can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0128] It may be advantageous to formulate parenteral compositions into dosage unit form for ease of administration and uniformity of dosage.The term "dosage unit form" as used herein refers to physically discrete units suitable for use 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.

[0129] 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 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50.

[0130] Compositions that exhibit large therapeutic indices are preferred. For any composition used in the present disclosure, the therapeutically effective dose can be initially estimated from cell culture assays. Examples of suitable bioassays include 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. 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 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 on the dosage form used and the route of administration used.

[0131] The following examples are not intended to limit the scope of the present disclosure in any way. Those skilled in the art will recognize numerous modifications and variations that can be made without changing 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 incorporated herein by reference.

[0132] Recombinant proteins were produced by standard molecular cloning and expression protocols and include human KLRG1 ECD (SEQ ID NO: 1), cynomolgus KLRG1 ECD (SEQ ID NO: 2), and human E-cadherin (SEQ ID NO: 3), the amino acid sequences of which are well known in the art and disclosed in PCT Publication No. WO2020060781, the entire contents of which are incorporated herein by reference. Recombinant proteins were produced as FC-fusion or HIS-tagged versions by cloning the respective cDNAs into pCDNA4 vectors (Invitrogen) and transiently transfecting in mammalian HEK293. Purification of the expressed proteins was performed by chromatography using Protein A affinity resin for the FC-fusion versions and nickel-NTA resin for the HIS-tagged proteins. All purified proteins were characterized by SDS-PAGE electrophoresis to confirm purity and molecular weight. Exemplary Sequences PA015 heavy chain variable region sequence >PA015_VH (SEQ ID NO: 4) QVTLRESGPALVKPTQTLTLTCTVSGFSLSTFGMGVGWIRQPPGKALEWLAHIWWDDDKWYELALKSRLTISKDTSKNQVVLTITNMDPVDTATYYCARVIYYGSRSAYYSMDYWGQGTTVTVSS PA015 light chain variable region sequence >PA015_VL (SEQ ID NO: 5) DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSNAHTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPVTFGQGTKLEIKRTV PA015 heavy chain constant region sequence >PA015_Heavy chain constant region (SEQ ID NO: 6) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PA015 light chain constant region sequence >PA015_Light chain constant region (SEQ ID NO:7) AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC PA015 heavy chain sequence >PA015_HC (SEQ ID NO: 8) QVTLRESGPALVKPTQTLTLTCTVSGFSLSTFGMGVGWIRQPPGKALEWLAHIWWDDDKWYELALKSRLTISKDTSKNQVVLTITNMDPVDTATYYCARVIYYGSRSAYYSMD YWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK PA015 light chain sequence >PA015_LC (SEQ ID NO: 9) DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSNAHTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPVTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC >PA015_VH_CDR1 (SEQ ID NO: 10) - Chothia format GFSLSTFGM >PA015_VH_CDR2 (SEQ ID NO: 11) - Chothia format WWDDD >PA015_VH_CDR3 (SEQ ID NO: 12) - Chothia format VIYYGSRSAYYSMDY >PA015_VL_CDR1 (SEQ ID NO: 13) - Chothia format KSSQSIVHSNAHTYLE >PA015_VL_CDR2 (SEQ ID NO: 14) - Chothia format KVSNRFS >PA015_VL_CDR3 (SEQ ID NO: 15) - Chothia format FQGSHVPVT >PA015_HC (SEQ ID NO: 40) >PA015_LC(SEQ ID NO: 41) ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCGGCGCTCATTCTGATGTGGTCATGACACAGACCCCTCTGAGCCTGTCTGTGACACCTGGACAGCCTGCCTCCATCTCCTGCAAGTCCTCTCAGTCCATCGTGCACAGCAACGCCCACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACAAGGTGTCCAACAGATTCTCTGGCGTGCCCGACAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTTTCCAAGGCTCTCACGTGCCCGTGACCTTTGGCCAGGGAACAAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCTTCCGTGTTCATCTTTCCACCTTCCGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACTCTCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGCTGATGA

Example

[0133] Example 1: Generation of Anti-KLRG1 Antibody The PA015 antibody is a humanized IgG1 antibody against the extracellular domain of KLRG1. Mouse monoclonal antibodies (MAB) against human KLRG1 were generated by standard immunization of female B ALB / c and SJL mice with human and cynomolgus KLRG1 and subsequent screening of hybridomas. Several immunization strategies have been employed to generate a diverse number of antibody hits. Briefly, SJL and Balb / c mice were repeatedly immunized with either cDNA, recombinant antigen, or CHO cells expressing the antigen of interest. Antigen-specific antibody titers were monitored periodically by ELISA, and animals were sacrificed when an appropriate titer was reached, usually a dilution factor of 1:1000 to 1:10000. Spleen cells from sacrificed mice were fused to mouse myeloma cells to produce hybridoma cells, which were then cultured and subcloned into single cells. Stable clones were scaled up, and conditioned media was collected and tested for expression of anti-KLRG1 antibodies by ELISA and FACS.

[0134] Example 2: PA015 binding to CHOK1 cells expressing human KLRG1. Binding of anti-KLRG1 monoclonal antibodies to cells expressing human and cynomolgus-KLRG1 is performed by FACS. Chinese hamster ovary (CHO) cells are stably transfected to express full-length human KLRG1 (CHOK1-hKLRG1). CHOK1-hKLRG1 expressing cells were cultured and incubated with various concentrations of either PA015 or control human IgG1. The cells were then centrifuged, the supernatant was discarded, and washed with incubation buffer (HBSS+2%FBS+10mM Ca2+). The cells were resuspended, incubated with anti-human IgG-Alexa488, washed, resuspended, and FACS tested. The mean fluorescence intensity (MFI) of cells incubated with PA015 and control human IgG1 is shown in Figure 1. The EC50 of PA015 was 1.33nM.

[0135] Example 3: PA015 inhibition of E-cadherin binding to CHOK1 cells expressing KLRG1 in a relative binding assay. The ability of monoclonal antibodies to inhibit E-cadherin / KLRG1 binding is measured by FACS.

[0136] CHOK1-hKLRG1 expressing cells were cultured and incubated with various concentrations of either PA015 or control human IgG1. 0.3 μM of recombinant human E-cadherin-ECD-FC-biotin was then added and incubated. The cells were then centrifuged, the supernatant discarded, and washed with incubation buffer (HBSS+2% FBS+10 mM Ca2+). The cells were resuspended, incubated with streptavidin-Alexa488, washed, resuspended, and FACS tested. The mean fluorescence intensity (MFI) of cells incubated with PA015 and control human IgG1 is shown in Figure 2. IC50 values ​​are determined by monitoring the loss of E-cadherin binding as a function of varying concentrations of monoclonal antibody from 0.1 to 100 nM. The IC50 for PA015 inhibition of E-cadherin binding was 8.05 nM.

[0137] Example 4: Superior Purity of PA015 Compared to HG1N02 Examples 4-6 refer to the antibody PA015 described herein and an antibody designated HG1N02, which is an anti-KLRG1 antibody described in WO2020 / 060781.

[0138] PA015 and HG1N02 were produced at 1 L scale using the ExpiCho Expression System (Thermo Fisher). Reducing and non-reducing capillary electrophoresis (rCE) were performed to measure the percentage of the major peak in three independent experiments. The results show superior purity of PA015 during CHO cell production compared to HG1N02 (Table 3). [Table 3]

[0139] Example 5: Superior thermal properties (melting and coagulation temperatures) of PA015 compared to HG1N02 PA015 and HG1N02 were produced at 1 L scale using the ExpiCho Expression System (Thermo Fisher). Melting temperatures (Tm) were measured by full spectrum fluorescence, and small molecule aggregate formation (Tagg266) and large molecule aggregate formation (Tagg473) were measured by static light scattering (SLS) using UNCLE (Unchained Labs, Inc.) at 4 hours, 7 days, and 28 days in three independent experiments. Results show superior thermal properties (higher melting point and aggregation temperature) for PA015 compared to HG1N02 (Table 4). [Table 4]

[0140] Example 6: Superior thermal properties (melting and coagulation temperatures) after stress testing of PA015 compared to HG1N02 PA015 and HG1N02 were subjected to stress testing with heat stress, low pH, and high pH stress for 4 hours, 7 days, and 28 days. Melting temperature (Tm) was measured by full spectrum fluorescence, and small molecule aggregate formation (Tagg266) and large molecule aggregate formation (Tagg473) were measured by static light scattering (SLS) using UNCLE (Unchained Labs, Inc.) at 4 hours, 7 days, and 28 days. The results show superior stability of thermal properties (higher melting point and aggregation temperature) after stress testing for PA015 compared to HG1N02 (Tables 5-7). [Table 5] [Table 6] [Table 7]

[0141] Example 7: PA015 has superior blocking of E-cadherin compared to HG1N01 and HG1N02 on KLRG1-expressing CHOK1 cells in a relative binding assay. CHOK1-hKLRG1 expressing cells were cultured and incubated with various concentrations of PA015, HG1N01, HG1N02 or control human IgG1. Then, 0.3 μM of recombinant human E-cadherin-ECD-FC-biotin was added and incubated. The cells were centrifuged, the supernatant was discarded, and washed with incubation buffer (HBSS + 2% FBS + 10 mM Ca2+). The cells were resuspended, incubated with streptavidin-Alexa488, washed, resuspended, and FACS tested. The mean fluorescence intensity (MFI) of cells incubated with the antibodies is shown in Figure 3. The IC50 was 5.3 nM for PA015, 8.3 nM for HG1N01, and 7.9 nM for HG1N02.

[0142] Example 8: PA015 has superior binding to human CD8+ T cells compared to HG1N02 in a FACS binding assay. Human CD8+ T cells were isolated by magnetic separation from three healthy donors, incubated with various concentrations of PA015, HG1N02, or control human IgG1, and detected with a fluorescently labeled anti-human IgG secondary antibody. The percentage of positive cells and EC50 values ​​are shown in Figure 4.

[0143] Example 9: PA015 has superior pharmacokinetic properties compared to HG1N01 in non-human primates. PA015 and HG1N01 (3 mg / kg) were each administered intravenously as a single dose to two cynomolgus monkeys. Serum concentrations of each antibody were measured over 42 days (Figures 5 and 6). PA015 compared to HG1N01 demonstrates superior clearance (6.99 vs. 38.6 mL / day / kg), area under the curve (AUC; 429 vs. 77.6 days*ug / mL), and beta half-life (6.23 vs. 4.7 days) (Tables 8 and 9). [Table 8] [Table 9]

[0144] Example 10: PA015 has improved deamidation properties compared to HG1N02. HG1N02 has a theoretical deamidation risk (NG at light chain residue N33). However, in a study of 131 clinically approved therapeutic mAbs (Lu Y et al. MAbs. 2019 Jan;11(1):45-57), 48% (13 / 27) of the CDR NG sites actually had no deamidation under stress conditions, so the sequence does not actually predict the occurrence of deamidation. Tryptic peptide mapping of unstressed PA015 and HG1N02 showed that PA015 had less deamidation than HG1N02 at the key N33 light chain CDR residue (Figure 7). Cited by Reference

[0145] The disclosures of any and all U.S. and foreign patents and pending patent applications and publications referred to herein are specifically incorporated herein by reference in their entirety, as are the contents of the sequence listing and figures. The teachings of PCT / US2019 / 050110 (published as WO2020060781) are incorporated herein by reference in their entirety.

[0146] Equivalent Those skilled in the art will understand that the disclosure described herein and shown in the accompanying drawings are non-limiting exemplary embodiments, and 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. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be covered by the following claims. Any combination of the embodiments disclosed in any of the dependent claims or examples is contemplated to be within the scope of the disclosure.

[0147] Other embodiments From the above description, it will be apparent that variations and modifications can be made to the invention described herein to adapt it to a variety of applications and conditions, including the use of different signal sequences and nucleic acid sequences for expressing the antibodies of the invention having the same or similar protein sequences, and are considered to be within the scope of the invention. Other embodiments according to the invention are within the scope of the following claims.

[0148] The recitation of a list of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0149] All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains.

Claims

1. A heavy chain variable region comprising SEQ ID NO: 4 and a light chain variable region, wherein CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are selected from the following (a) to (e): (a) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (b) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; (c) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (d) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or (e) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1, which is any one of the following:

2. A light chain variable region comprising SEQ ID NO:5, and a heavy chain variable region, wherein CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are selected from the following (a) to (e): (a) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; (b) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; (c) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; (d) the CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; or (e) the CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1, which is any one of the following:

3. An antibody, or an antigen-binding fragment thereof, that specifically binds to the extracellular domain of KLRG1, comprising a heavy chain variable region comprising SEQ ID NO:4 and a light chain variable region comprising SEQ ID NO:

5.

4. A heavy chain that specifically binds to the extracellular domain of KLRG1 and includes SEQ ID NO: 8, and a light chain, wherein CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region are selected from the following (a) to (e): (a) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively; (b) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21, respectively; (c) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively; (d) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively; or (e) the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; 1. An antibody, or an antigen-binding fragment thereof,

5. The antibody specifically binds to the extracellular domain of KLRG1 and comprises a light chain having SEQ ID NO: 9, and a heavy chain, wherein CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region are selected from the following (a) to (e): (a) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; (b) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively; (c) the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; (d) the CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; or (e) the CDR-H1, CDR-H2, and CDR-H3 are SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively; 1. An antibody, or an antigen-binding fragment thereof,

6. An antibody, or an antigen-binding fragment thereof, that specifically binds to the extracellular domain of KLRG1 and comprises a heavy chain comprising SEQ ID NO:8 and a light chain comprising SEQ ID NO:

9.

7. The antibody or antigen-binding fragment of any one of claims 1 to 3, wherein the antigen-binding fragment is an F(ab) fragment.

8. The antigen-binding fragment is F(ab ２ The antibody or antigen-binding fragment of any one of claims 1 to 3, which is a .alpha.-amino acid fragment.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof is bispecific.

10. 10. A pharmaceutical composition for use in a method of treating cancer, the pharmaceutical composition comprising an antibody, or antigen-binding fragment thereof, according to any one of claims 1 to 6, the method comprising administering the antibody, or antigen-binding fragment thereof, to a cancer patient.

11. The pharmaceutical composition of claim 10, wherein the method further comprises administering a checkpoint inhibitor to the cancer patient.

12. The pharmaceutical composition of claim 11, wherein the method comprises administering the checkpoint inhibitor simultaneously with administration of the antibody, or antigen-binding fragment thereof.

13. The pharmaceutical composition of claim 11, wherein the method comprises administering the checkpoint inhibitor within one day, within one week, or within one month of administration of the antibody, or antigen-binding fragment thereof.

14. The cancer is selected from the group consisting of 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 cancer, pancreatic carcinoma, gastric cancer, colon carcinoma, prostate carcinoma, urogenital cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical carcinoma, malignant pancreatic insulinoma, and malignant carcinoid.

11. The pharmaceutical composition of claim 10, wherein the cancer is one or more of: carcinoma, 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, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

15. The pharmaceutical composition of claim 10, wherein the patient is undergoing treatment with a checkpoint inhibitor.

16. An isolated nucleic acid encoding the antibody, or antigen-binding fragment thereof, of any one of claims 1 to 6.

17. 17. The isolated nucleic acid of claim 16, comprising SEQ ID NO:

40.

18. 17. The isolated nucleic acid of claim 16, comprising SEQ ID NO:

41.

19. An expression vector comprising the nucleic acid of claim 16.

20. An expression vector comprising the nucleic acid described in claim 17.

21. An expression vector comprising the nucleic acid described in claim 18.

22. A host cell comprising the expression vector of claim 19.

23. A host cell comprising the expression vector described in claim 20.

24. A host cell comprising the expression vector described in claim 21.

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