WO2022204529A1 - Anticorps anti-klrg1 - Google Patents

Anticorps anti-klrg1 Download PDF

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Publication number
WO2022204529A1
WO2022204529A1 PCT/US2022/021972 US2022021972W WO2022204529A1 WO 2022204529 A1 WO2022204529 A1 WO 2022204529A1 US 2022021972 W US2022021972 W US 2022021972W WO 2022204529 A1 WO2022204529 A1 WO 2022204529A1
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seq
cdr
antibody
antigen
binding fragment
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PCT/US2022/021972
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English (en)
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Stefano V. GULLA
Kenneth Evan THOMPSON
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Abcuro, Inc.
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Publication of WO2022204529A1 publication Critical patent/WO2022204529A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2851—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the lectin superfamily, e.g. CD23, CD72
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the technical field relates to inhibition of the lymphocyte co-inhibitory receptor Killer Cell Lectin-Like Receptor Subfamily G Member 1.
  • Lymphocyte co-inhibitory receptors modulate the action of the adaptive immune system (see e.g. W02020/060781).
  • the action of co-inhibitory receptors is generally carried out by binding of a ligand to the extracellular domain of the co-inhibitory receptor followed by recruitment of intracellular phosphatases by an immunoreceptor tyrosine-based inhibition motif (ITIM) located in the intracellular domain of the co-inhibitory receptor.
  • ITIM immunoreceptor tyrosine-based inhibition motif located in the intracellular domain of the co-inhibitory receptor.
  • the action of co- inhibitory receptors is generally to dampen the immune response of T-cell receptor (TCR) engagement.
  • TCR T-cell receptor
  • the present disclosure relates to an antibody, or antigen-binding fragment thereof, which comprises: (a) a heavy chain comprising three heavy chain complementarity determining regions comprising a CDR-H1 selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 16, SEQ ID NO:22, SEQ ID NO:28, and SEQ ID NO:34; a CDR-H2 selected from the group consisting of SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO:23, SEQ ID NO:29, and SEQ ID NO:35; and a CDR-H3 selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO:24, SEQ ID NO:30, and SEQ ID NO:36; and (b) a light chain comprising three light chain complementary determining regions comprising a CRD-Ll selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 19, SEQ ID NO:25, SEQ ID NO:31, and S
  • the CDR-L1, CDR-L2, and CDR-L3 may be 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; and (b) the 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
  • the antibody, or antigen-binding fragment thereof may comprise: (a) a heavy chain variable region comprising SEQ ID NO:6 or a sequence having at least 90% sequence identity to SEQ ID NO:6 and comprising three heavy chain complementarity determining regions comprising, respectively, SEQ ID NO: 10 (CDR-H1), SEQ ID NO: 11 (CDR-H2), and SEQ ID NO: 12 (CDR-H3); and (b) a light chain variable region comprising SEQ ID NO:7 or a sequence having at least 90% sequence identity to SEQ ID NO:7 and comprising three light chain complementary determining regions comprising, respectively, SEQ ID NO: 13 (CRD-Ll), SEQ ID NO: 14 (CDR-L2), and SEQ ID NO: 15 (CDR-L3).
  • the heavy chain variable region sequence may have at least 95% identity to SEQ ID NO: 6 and the light chain variable region sequence may have at least 95% identity to SEQ ID NO:7.
  • the heavy chain variable region may have at least 90% sequence identity to SEQ ID NO: 6, and the heavy chain variable region maintains the sequences of CDR-H1, CDR-H2, and CDR- H3.
  • the heavy chain variable region may comprise SEQ ID NO: 6 and the light chain may comprise CDR-L1 (SEQ ID NO: 13), CDR-L2 (SEQ ID NO: 14) and CDR-L3 (SEQ ID NO: 15).
  • the heavy chain may have at least 90% sequence identity to SEQ ID NO: 4, wherein the heavy chain maintains the sequences of CDR-H1, CDR-H2, and CDR-H3.
  • the light chain may comprise CDR-L1 (SEQ ID NO: 13), CDR-L2 (SEQ ID NO: 14) and CDR-L3 (SEQ ID NO: 15).
  • the light chain variable region may have at least 90% sequence identity to SEQ ID NO: 7, wherein the light chain variable region maintains the sequences of CDR-L1, CDR-L2, and CDR- L3.
  • the heavy chain may comprise CDR-H1 (SEQ ID NO: 10), CDR-H2 (SEQ ID NO: 11) and CDR-H3 (SEQ ID NO: 12).
  • the light chain may have at least 90% sequence identity to SEQ ID NO: 5, wherein the light chain maintains the sequences of CDR-L1, CDR-L2, and CDR-L3.
  • the heavy chain may comprise CDR-H1 (SEQ ID NO: 10), CDR-H2 (SEQ ID NO: 11) and CDR-H3 (SEQ ID NO: 12).
  • the light chain may have at least 90% sequence identity to SEQ ID NO: 5, wherein the light chain maintains the sequences of CDR-L1, CDR-L2, and CDR-L3; and the heavy chain may have at least 90%, sequence identity to SEQ ID NO: 4, wherein the heavy chain maintains the sequences of CDR-H, CDR-2, and CDR-H3.
  • 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.
  • the disclosure provides a method of treating cancer, comprising administering to a cancer patient the antibody or antigen-binding fragment thereof disclosed herein.
  • the method may further include administering a checkpoint inhibitor to said cancer patient.
  • the checkpoint inhibitor may be administered at the same time as administering the antibody or antigen-binding fragment thereof.
  • the checkpoint inhibitor may be administered within one day, one week, or one month of the antibody or antigen-binding fragment thereof.
  • the cancer may be one or more of breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia,
  • the disclosure relates to an adjunct therapy, comprising administering to a subject who is undergoing treatment with a checkpoint inhibitor an antibody or antigen-binding fragment disclosed herein, thereby to perform the adjunct therapy.
  • FIG. 1 is a graph showing GA015 binding to CHOK1 cells expressing human KLRG1 .
  • FIG. 2 is a graph showing GA015 inhibition of E-cadherin binding to CHOK1 cells expressing KLRG1 in a relative binding assay.
  • FIG. 3 is a graph showing GAO 15 inhibits melanoma growth in a humanized mouse patient derived xenograft model.
  • FIGS. 4A AND 4B are graphs showing GAO 15 enhances NK cell cytotoxicity (FIG. 4A) and enhances NK CD 107a degranulation (FIG. 4B).
  • FIG. 5 is a graph showing GA015 inhibition of E-cadherin binding to CHOK1 cells expressing KLRGl, relative to HG1N01 and HG1N02, in a relative binding assay.
  • FIG. 6 is a graph showing serum concentration of GAO 15 over time in a cynomolgus monkey pharmacokinetic assay described in Example 7.
  • FIG. 7 is a graph showing serum concentration of HG1N01 over time in the cynomolgus monkey pharmacokinetic assay described in Example 7.
  • KLRGl Killer cell lectin-like receptor G1
  • KLRGl is a type II transmembrane protein which can function as a co-inhibitory receptor by modulating the activity of T and NK cells.
  • the extracellular portion of KLRGl contains a C-type lectin domain whose known ligands are cadherins.
  • the intracellular portion of KLRGl contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain responsible for co-inhibition of T cell receptor (TCR) mediated signaling.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • TCR T cell receptor
  • KLRGl ligands include E-cadherin, N-cadherin, R-cadherin, and combinations thereof.
  • KLRGl expression is generally confined to cells of the immune system, and specifically to CD8 positive T cells, NK cells, and, to a lesser extent, CD4 positive T cells.
  • KLRGl expression has been associated with the late differentiated phenotype. As antigen specific T cells differentiate, they can acquire increased expression of cytotoxic molecules, and therefore can have increased cytotoxic potential.
  • the biological function of KLRGl is to inhibit cytotoxicity and proliferation of these T cells under certain circumstances. In murine cancer models, the blockade of KLRGl’s interaction with its ligands has been shown to be beneficial to controlling tumor growth or metastases.
  • Amino acids are represented herein by either the one-letter code, or the three- letter code, both in accordance with 37 C.F.R. ⁇ 1.822 and established usage.
  • the total of ten or less additional amino acids can include the total number of additional amino acids on both ends added together.
  • an "effective amount" as used herein is an amount that provides a desired effect.
  • the term “effective amount” refers to a dosage or amount that is sufficient to reduce the activity of KLRG1 to result in amelioration of symptoms in a patient or to achieve a desired biological outcome, e.g., reduced activity of KLRG1, modulation of lymphocyte co-inhibition response, increased or decreased activation of cytotoxic T and NK cells, or increased or decreased release oflFNy by cytotoxic T cells or NK cells.
  • a “therapeutically effective” amount as used herein is an amount that provides some improvement or benefit to the subject.
  • a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject.
  • the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • treat By the terms “treat,” “treating,” or “treatment of” it is intended that the severity of the condition of the subject is reduced, or at least partially improved or modified, and that some alleviation, mitigation, or decrease in at least one clinical symptom is achieved.
  • cancer refers to any benign or malignant abnormal growth of cells. Examples include, without limitation, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hyper
  • isolated can refer to a polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an "isolated fragment” is a fragment of a polypeptide that is not naturally occurring as a fragment and would not be found in the natural state.
  • isolated does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • isolated refers to a molecule that is substantially free of its natural environment.
  • an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived.
  • isolated also refers to preparations where the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or approximately at least 70-80% (w/w) pure, more preferably, approximately at least 80-90% (w/w) pure, even more preferably, approximately 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.
  • polypeptide fragment as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or nearly identical ( e.g ., approximately 90%, approximately 92%, approximately 95%, approximately 98%, approximately 99% identical) to the reference polypeptide or amino acid sequence.
  • a polypeptide fragment according to the present disclosure may be, where appropriate, included in a larger polypeptide of which it is a constituent.
  • such fragments can comprise, consist essentially of, and/or consist of peptides 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 consecutive amino acids of a polypeptide or amino acid sequence according to the present disclosure.
  • protein and “polypeptide” are used interchangeably and encompass both peptides and proteins, unless indicated otherwise.
  • antibody or " antibodies " as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibody 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 a chimeric antibody.
  • the antibodies can be recombinant monoclonal antibodies produced according to the methods disclosed, for example, in U.S. Patent No. 4,474,893 or U.S. Patent No. 4,816,567.
  • the antibodies can also be chemically constructed, for example, according to the methods disclosed in U.S. Patent No. 4,676,980.
  • antigen-binding domain refers to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as " epitope " or " antigenic determinant
  • an antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), it does not necessarily have to comprise both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a Fd antibody fragment consists only of a VH domain, but still retains some antigen-binding function of the intact antibody.
  • Anti-KLRGl antibodies comprise antibody constant regions or parts thereof.
  • a VL domain may have attached, at its C terminus, antibody light chain constant domains including human CK or CX chains.
  • a specific antigen-binding domain based on a VH domain may have attached all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g ., IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4.
  • immunoglobulin heavy chain derived from any antibody isotope, e.g ., IgG, IgA, IgE, and IgM and any of the isotope sub-classes, which include but are not limited to, IgGl and IgG4.
  • the DNA and amino acid sequences for the C- terminal fragment of are well known in the art.
  • binding refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding can be characterized by a high affinity and a low to moderate capacity, as distinguished from non specific binding, which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than approximately 10 6 M _1 , or more preferably higher than approximately 10 8 M _1 . If necessary, non-specific binding can be reduced without substantially affecting specific binding, for example, by varying the binding conditions.
  • the appropriate binding conditions such as concentration of antibodies, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g, serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.
  • the antibodies can specifically bind an epitope within the extracellular domain (ECD) of human or monkey KLRG1, with an affinity, as expressed in KD, of at least about 2 nM, about lnm, about 100 pM, about 10 pM, or about 5 pM.
  • ECDs of human and cynomolgus KLRG1 are set out in SEQ ID NO:l and SEQ ID NO:2, and the amino acid sequence of human E-Cadherin is set forth in SEQ ID NO: 3, below.
  • the present disclosure describes antibodies and fragments thereof that specifically bind to the extracellular domain of KLGR1 of cells by interfering with the binding of E-cadherin, R-cadherin, and/or N-cadherin to the extracellular domain of KLRG1.
  • E-cadherin, R-cadherin, and/or N-cadherin ligand to KLRG1
  • such antibodies prolong the cytotoxicity of T- and NK-cells which bear KLRG1 on their surface.
  • the antibodies described herein can be used as effective therapeutic agents for treatment of cancer either as a monotherapy, or in combination with other immunotherapy agents (such as checkpoint inhibitors, e.g., anti-PD-1 antibodies, anti-PD-Ll antibodies, or anti-CTLA4 antibodies), or in combination with chemotherapy agents, or cancer vaccines.
  • immunotherapy agents such as checkpoint inhibitors, e.g., anti-PD-1 antibodies, anti-PD-Ll antibodies, or anti-CTLA4 antibodies
  • Such antibodies, or fragments thereof can be used as an adjunct therapy for cancer treatment, regardless of whether the cancer cells express KLRG1.
  • each light chain can be composed of an N- terminal variable domain (VL) and a constant domain (CL).
  • Each heavy chain can be composed of an N-terminal variable domain (VH), three or four constant domains (CH), and a hinge region.
  • the CH domain most proximal to VH is designated as CHI .
  • the VH and VL domains consist or comprise of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for 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.
  • CDR1, CDR2, and CDR3 The three CDRs are referred to as CDR1, CDR2, and CDR3.
  • CDR constituents on the heavy chain are referred to as HI, H2, and H3, while CDR constituents on the light chain are referred to as LI, L2, and L3, accordingly.
  • CDR3 and particularly H3, are the greatest source of molecular diversity within the antigen-binding domain.
  • H3, for example, can be as short as two amino acid residues or greater than 26.
  • the Fab fragment (Fragment antigen-binding), or F(ab), consists of or comprises the VH-CHl and VL-CL domains covalently linked by a disulfide bond between the constant regions.
  • a Fab 50,000 daltons
  • IgG and IgM consisting or comprising of the VH, CHI and VL, CL regions, linked by an intramolecular disulfide bond.
  • a flexible and adequately long polypeptide links either the C-terminus of the VH to the N-terminus of the VL, or the C-terminus of the VL to the N-terminus of the VH.
  • a 15-residue (Gly4Ser)3 peptide can be used as a linker, but other linkers are also known in the art.
  • Antibody diversity is a result of combinatorial assembly of multiple germline genes encoding variable regions and a variety of somatic events.
  • the somatic events can include recombination of variable gene segments with diversity (D) and joining (J) gene segments to make a complete VH region and the recombination of variable and joining gene segments to make a complete VL region.
  • D diversity
  • J joining
  • the recombination process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions.
  • the present disclosure provides novel antibody variable heavy and light chain regions (VH and VL) and antibody heavy and light chains derived from human immunoglobulin gene libraries, which are effective in blocking KLRG1 signaling in cells expressing cell surface KLGR1 .
  • VH and VL antibody variable heavy and light chain regions
  • VL antibody heavy and light chain regions
  • These heavy and light chains and their variable regions provide a scaffold structure for carrying CDRs.
  • a CDR can generally be, though is not limited to, an antibody heavy or light chain, or a portion thereof, in which the CDR is located at a location corresponding to the CDR of naturally occurring VH and VL.
  • the structures and locations of immunoglobulin variable domains may be determined, for example, as described in Rabat et ah, Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.
  • antibodies of the present disclosure may also bind with other proteins, including, for example, recombinant proteins comprising all or a portion of KLRG1 .
  • a KLRG1 antibody may also be a multispecific antibody, such as a bispecific or tetraspecific antibody, diabody, or similar molecule (see for instance PNAS USA 90(14), 6444-8 (1993) for a description of diabodies).
  • bispecific antibodies, diabodies, and the like, provided by the present invention may bind any suitable target in addition to a portion of KLRG1.
  • a bispecific antibody may bind KLRG1 and HER2, which would activate and redirect KLRG1+ T and NK cells to HER2+ cancer cells.
  • a bispecific antibody binds KLRG1 and PD-1, which would activate T and NK cells by blocking two checkpoint receptors (KLRG1 and PD-1).
  • the present disclosure also provides for multispecific antibody fusion proteins which comprise an antibody moiety, for example a Fab or Fab' fragment, with a first specificity for an antigen of interest, and further comprise at least one single domain antibody with specificity for a second antigen of interest.
  • a multispecific antibody fusion will be capable of selectively binding to two or more antigens of interest.
  • the first and second antigen are the same antigen.
  • an antigen of interest bound by the antibody or antigen binding fragment thereof may be a cell-associated protein, for example a cell surface protein on cells such as bacterial cells, yeast cells, T-cells, endothelial cells or tumor cells, or it may be a soluble protein.
  • Antigens of interest may also be any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands.
  • cell surface proteins include adhesion molecules, for example integrins such as 131 integrins e.g.
  • VLA-4 E-selectin, P selectin or L-selectin
  • 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 for example respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins, such as IgE, interferons such as interferon a, interferon p or interferon y, tumor necrosis factor-a, tumor necrosis factor-13, colony stimulating factors such as G-CSF or GM-CSF, and platelet derived growth factors such as PDGF-a, and PDGF-I3 and where appropriate receptors thereof.
  • 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.
  • the antigens of interest include IL-2, soluble IL-15 or membrane- bound IL-15Ra/IL-15 complex, and IL-12.
  • the antibodies of this present disclosure may be used to detect, measure, and inhibit proteins that differ somewhat from KLRG1.
  • the antibodies can be expected to retain the specificity of binding so long as the target protein comprises a sequence which 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 contiguous amino acids in the sequence set forth SEQ ID NO: 1 or SEQ ID NO:2.
  • the percent identity is determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol.
  • BLAST Basic Local Alignment Tool
  • epitope mapping see, e.g ., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996) and secondary and tertiary structure analyses can be carried out to identify 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, N. Y.).
  • CDRs complementarity-determining regions
  • VH and VL identified in the Examples below
  • variants of these sequences that retain the ability to specifically bind KLRG1.
  • Such variants may be derived from the sequences listed in the Examples by a skilled artisan using techniques well known in the art.
  • amino acid substitutions, deletions, or additions can be made in the framework regions (FRs) and/or in CDRs. While changes in the FRs can usually be designed to improve stability and immunogenicity of the antibody, changes in the CDRs can typically be designed to increase affinity of the antibody for its target.
  • Changes to FRs include, but are not limited to, humanizing a non-human derived antibody, or engineering certain framework residues that are important for antigen contact or for stabilizing the binding site, e.g ., changing the class or subclass of the constant region, changing specific amino acid residues which might alter the effector function such as Fc receptor binding, e.g, as described in U.S. Patent 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.
  • Variants of FRs also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes may be determined empirically by routine techniques that involve altering the CDR 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 alterations can be made according to the methods described, for example, in Antibody Engineering, 2 nd ed., Oxford University Press, ed. Borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a “silent” change.
  • the nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine, and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • any native residue in the polypeptide may 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).
  • substantially as set out means that the relevant CDR, VH, or VL domain of the disclosure will be either identical to, or have only insubstantial differences in the specified regions (e.g., a CDR) from the sequence of which is set out. Insubstantial differences include minor amino acid changes, such as substitutions of one (1) or two (2) out of any five (5) amino acids in the sequence of a specified region.
  • KLRG1 activity refers to one or more lymphocyte co-inhibitory activities associated with KLRG1.
  • KLRG1 activity may mean modulation of cytotoxic T and NK cell activation.
  • modulate refers to a reduction or an increase in the activity of KLRG1 associated with activation of T cells and NK cells due to its interaction with an anti-KLRGl antibody, wherein the reduction or increase is relative to the activity of KLRG1 in the absence of the same antibody.
  • a reduction or an 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.
  • percent (%) sequence identity refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ClustalW, Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • 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.
  • F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (see Huse et al, Science 1989 Dec 8;246(4935): 1275-1281).
  • 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 (such as Fv, Fab, Fabl, F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a 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.
  • CDR complementarity determining region
  • Fv framework residues of the human immunoglobulin can be replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody, nor in the imported CDR or framework sequences.
  • the humanized antibody can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions (i.e., the sequences between the CDR regions) are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the VH and/or VL domains may be germlined, i.e., the framework regions (FRs) of these domains are mutated using conventional molecular biology techniques to match those produced by the germline cells.
  • the framework sequences remain diverged from the consensus germline sequences.
  • Monoclonal antibodies used to carry out the present disclosure can be produced in a hybridoma cell line according to the technique of Kohler and Milstein, Nature 265:495 (1975).
  • a solution containing the appropriate antigen can be injected into a mouse and, after a sufficient time, the mouse sacrificed, and spleen cells obtained.
  • the spleen cells can then be immortalized by fusing them, for example with myeloma cells or with 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 screened for monoclonal antibodies having 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 the target polypeptide can also be obtained by phage display techniques known in the art.
  • Various immunoassays can be used for screening to identify antibodies having the desired specificity for the extracellular domain of KLRG1.
  • Numerous protocols for competitive binding or immunoradiometric assays using monoclonal antibodies with established specificity are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between an antigen and its specific antibody (e.g ., antigen/antibody complex formation).
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on the polypeptides or peptides of this disclosure can be used as well as a competitive binding assay.
  • anti-KLRGl antibodies described herein can be conjugated to a solid support (e.g., beads, plates, slides, or wells formed from materials such as latex or polystyrene) in accordance with known techniques.
  • Anti-KLRGl antibodies described herein can likewise be conjugated to detectable groups such as radiolabels e.g ., 35 S, 125 I, 131 I or 99m Tc, which may also be attached to antibodies using conventional chemistry), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescence labels (e.g, fluorescein) in accordance with known techniques.
  • 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 an antibody/antigen complex in the methods of this disclosure can be by detection of, for example, precipitation, agglutination, flocculation, radioactivity, color development or change, fluorescence, luminescence, etc., and such methods are well known in the art.
  • anti-KLRGl antibodies described herein can be linked to another functional molecule, e.g, another peptide or protein (albumin, another antibody, etc.), toxin, radioisotope, cytotoxic or cytostatic agents.
  • Antibodies as described herein can be conjugated to toxins.
  • 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.
  • the antibodies can be linked by chemical cross-linking or by recombinant methods.
  • the antibodies may also be linked to one of a variety of nonproteinaceous polymers, e.g, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent No. 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 conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods to attach them are also shown in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546.
  • an anti-KLRGl antibody comprises a combination of any six CDRs of any one of the antibodies described herein.
  • an anti-KLRGl antibody comprises (i) three heavy chain CDRs from Table 2 (i.e., a CDR1, a CDR2, and a CDR3), and (ii) three light chain CDRs from Table 2.
  • the IMGT unique numbering for the V-REGION has allowed to redefine the limits of the Complementarity Determining Regions and Framework Regions, designated as CDR-IMGT and FR-IMGT, respectively in IMGT.
  • the IMGT unique numbering for V- DOMAIN provides a standardized numbering for the CDR3-IMGT and FR4-IMGT of rearranged V-J-REGION and V-D-J-REGION.
  • CDR-IMGT Complementarity Determining Region
  • V-DOMAIN Variable (V) Domain
  • IMGT unique numbering for 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.
  • CDR1-IMGT loop BC
  • CDR2-IMGT loop C'C
  • CDR3-IMGT loop a loop region of a V-DOMAIN
  • V-DOMAIN V domain of the immunoglobulins (IG) or antibodies and T cell receptors (TR)
  • the amino acids of the CDR-IMGT bind to an antigen (Epitope) and confer the specificity to the IG and TR (Paratope, IMGT-ONTOLOGY, SpecificityType).
  • the first two CDR-IMGT are part of the V-REGION (encoded by a variable (V) gene), whereas the CDR3- IMGT corresponds to the junction and results from the rearrangement between a V gene and a joining (J) gene (V-J rearrangement) or between a V gene, a diversity (D) gene, and a J gene (V- D-J rearrangement) (Immunoglobulin Synthesis).
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain variable region with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 6 and (ii) a light chain comprising a CDR-Ll (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).
  • CDR-Ll e
  • 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: 6 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain variable region with 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 such as including, but not limited to, framework (FR) regions, with amino acid sequences of SEQ ID NO: 6 and (ii) a light chain comprising a CDR-L1, a CDR-L2, and a CDR-L3, wherein the CDR-L1, CDR-L2 and CDR-L3 are SEQ ID N0:13, SEQ ID N0:14 and SEQ ID N0:15, respectively; SEQ ID NO: 19, SEQ ID NO:20 and SEQ ID N0:21, respectively; SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain variable region with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 6 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).
  • 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: 6 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 4 and (ii) a light chain comprising a 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).
  • CDR-L1 e.g.,
  • 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 SEQ ID NO:4 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain with 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 such as including, but not limited to, framework (FR) regions, with amino acid sequences of SEQ ID NO: 4 and (ii) a light chain comprising a CDR-L1, a CDR-L2, and a CDR-L3, wherein the 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 N0
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a heavy chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences 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).
  • 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 SEQ ID NO:4 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain variable region with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 7 and (ii) a heavy chain comprising a 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).
  • CDR-H1 e
  • 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: 7 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain variable region with 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 such as including, but not limited to, framework (FR) regions, with amino acid sequences of SEQ ID NO: 7 and (ii) a heavy chain comprising a CDR-H1, a CDR-H2, and a CDR-H3, wherein the 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
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain variable region with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 7 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).
  • 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: 7 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 5 and (ii) a heavy chain comprising a 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).
  • CDR-H1 e.g., S
  • 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: 5 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions.
  • CDR complementarity-determining region
  • a particular embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain with 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 such as including, but not limited to, framework (FR) regions, with amino acid sequences of SEQ ID NO: 5 and (ii) a heavy chain comprising a CDR-H1, a CDR-H2, and a CDR-H3, wherein the 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,
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences 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).
  • the light chain has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof having (i) a light chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 5 and (ii) a heavy chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with amino acid sequences of SEQ ID NO: 4.
  • 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: 5 in regions other than the complementarity-determining region (CDR) regions such as including, but not limited to, framework (FR) regions; and the heavy chain has at least 50%, 60%, 70%, 75%, 80%, 85%,
  • CDR complementarity-determining region
  • One embodiment provides an antibody, preferably a monoclonal antibody, or an antigen binding fragment thereof, having (i) a light chain variable region with at least 50%,
  • One embodiment provides an antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has (i) a light chain variable region containing CDRs (CDR-L1, CDR-L2, CDR-L3) having amino acid sequences SEQ ID Nos: 13, 14 and 15, respectively; (ii) a heavy chain variable region containing CDRs (CDR-H1, CDR-H2, CDR-H3) having amino acid sequences SEQ ID NOs: 10, 11 and 12, respectively; (iii) a heavy chain constant region with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
  • One embodiment provides an antibody, preferably a monoclonal antibody, or antigen binding fragment thereof that has (i) a light chain variable region containing CDRs (CDR-L1, CDR-L2, CDR-L3) having amino acid sequences SEQ ID NOs:13, 14 and 15, respectively; (ii) a heavy chain variable region containing CDRs (CDR-H1, CDR-H2, CDR-H3) having amino acid sequences SEQ ID Nos: 10, 11 and 12, respectively; (iii) a heavy chain with at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the antibody, or an antigen binding fragment thereof comprises (i) a heavy chain variable region that includes at least one, two or three CDR (e.g., CDR-H1, CDR-H2, or CDR-H3), or a portion thereof, having the amino acid sequence of SEQ ID Nos: 10, 11, or 12, respectively, and (ii) a light chain variable region that includes at least one, two or three CDR (e.g., CDR-L1, CDR-L2, or CDR-L3), or a portion thereof, having the amino acid sequence of SEQ ID Nos: 13, 14, or 15, respectively.
  • CDR e.g., CDR-H1, CDR-H2, or CDR-H3
  • a light chain variable region that includes at least one, two or three CDR (e.g., CDR-L1, CDR-L2, or CDR-L3), or a portion thereof, having the amino acid sequence of SEQ ID Nos: 13, 14, or 15, respectively.
  • the antibodies and/or antigen binding fragments thereof provided for by the instant disclosure are conjugated to a toxic agent.
  • Immunoconjugates which include one or more cytotoxins are referred to as "immunotoxins.”
  • Antibodies conjugated to a cytotoxic agent, drug, or the like are also known as antibody-drug conjugates (ADC).
  • ADC antibody-drug conjugates
  • An immunoconjugate may have a half-life of sufficient periods of time for the antibody-drug conjugate to be internalized, degraded, and induce cell killing by the released toxin.
  • a cytotoxin or cytotoxic agent can include any agent that is detrimental to ( e.g ., kills) cells.
  • Suitable cytotoxic agents for forming immunoconjugates of the present disclosure include taxol, tubulysins, duostatins, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, maytansine or an analog or derivative thereof, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin; calicheamicin or analogs or derivatives thereof, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine
  • rachelmycin or analogs or derivatives of CC-1065
  • dolastatin auristatin
  • pyrrolo[2,l-c][l,4]benzodiazepins PDBs
  • indolinobenzodiazepine IGNs or analogues thereof
  • antibiotics such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)
  • anti-mitotic agents e.g., tubulin-targeting agents
  • diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules
  • ricin toxin such as ricin A or a deglycosylated ricin A chain toxin
  • cholera toxin a Shiga-like toxin
  • conjugated molecules include antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and PI 8; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.
  • antimicrobial/lytic peptides such as CLIP, Magainin 2, mellitin, Cecropin, and PI 8
  • RNase ribonuclease
  • DNase I DNase I
  • Staphylococcal enterotoxin-A Staphylococcal enterotoxin-A
  • pokeweed antiviral protein diphtheria toxin
  • Pseudomonas endotoxin Pseudomonas endotoxin.
  • the antibodies of the present disclosure can optionally be delivered to a patient in conjunction with other therapeutic agents.
  • the additional therapeutic agents can be delivered before, concurrently with, or after the antibodies of the present disclosure.
  • concurrently means sufficiently close in time to produce a combined effect (that is, concurrently can be simultaneously, or it can be two or more events occurring within a short time period before or after each other).
  • the antibodies of the present disclosure can be administered in conjunction with anti-cancer agents, such as: 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-alfa); 6) platinum coordinating complexes (e.g, cisplatin and carboplatin); 7) anthracenediones (e.g, mitoxantrone); 8) substituted ureas (e.g, hydroxyurea);
  • anti-cancer agents such as:
  • methylhydrazine derivatives e.g, procarbazine (N-methylhydrazine; MTH)); 10) adrenocortical suppressants (e.g, mitotane (o,r'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g, prednisone); 12) progestins (e.g, hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g, diethylstilbestrol and ethinyl estradiol); 14) 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).
  • the antibodies of the present disclosure can be administered in conjunction with anti -angiogenesis agents, such as antibodies to VEGF (e.g, bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g, bFGF, angiopoietin-1), antibodies to alpha-v/beta-3 vascular integrin (e.g, VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide (SEQ ID NO: 43) TNF alpha conjugate, cyclophosphamide, combretastatin A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzastaurin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation (Abraxane), soy isoflavone (Genistein), tamoxifen citrate,
  • the antibodies of the present disclosure can be administered in conjunction with immunosuppressive agents including, for example, cyclosporine A, rapamycin, glucocorticoids, azathioprine, mizoribine, aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamide and FK506 (tacrolimus).
  • immunosuppressive agents including, for example, cyclosporine A, rapamycin, glucocorticoids, azathioprine, mizoribine, aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamide and FK506 (tacrolimus).
  • the present disclosure further provides isolated nucleic acids encoding the disclosed antibodies.
  • the nucleic acids may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.
  • the nucleic acids provided herein comprise a coding sequence for a CDR, a VH domain, and/or a VL domain disclosed herein.
  • the present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes which comprise at least one nucleic acid encoding a CDR, a VH domain, and/or a VL domain disclosed here.
  • the disclosure further provides a host cell which comprises one or more constructs as above.
  • the coding sequence for the heavy chain is the nucleotide sequence of SEQ ID NO:40.
  • the coding sequence for the light chain is the nucleotide sequence of SEQ ID NO:41.
  • Suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems.
  • Mammalian cell lines available in the art for expression of a heterologous polypeptide 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 invention may be used to produce the disclosed antibodies.
  • Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.
  • Suitable vectors can be chosen or constructed, so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids or viral, e.g., phage, or phagemid, as appropriate.
  • phage e.g., phage, or phagemid
  • a further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed here.
  • a still further aspect provides a method comprising introducing such nucleic acid into a host cell.
  • the introduction may employ any available technique.
  • suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus.
  • suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.
  • the introduction of the nucleic acid into the cells may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.
  • the disclosed anti-KLRGl antibodies are capable of modulating the KLRG1- associated immune responses.
  • the activation of cytotoxic T and NK cells is mediated by modulation of KLRG1 signaling.
  • the disclosed antibodies can act as either agonists or antagonists of KLRG1, depending on the method of their use.
  • the antibodies can be used to prevent, diagnose, or treat medical disorders in mammals, especially, in humans.
  • Antibodies of the disclosure can also be used for isolating KLRG1 or KLRG1 -expressing cells.
  • the antibodies can be used to treat a subject at risk of, or susceptible to, a disorder, or having a disorder associated with aberrant KLRG1 expression or function.
  • Antibodies of the disclosure can be used in circumstances where modulation of cytotoxic T and NK cell activation may be desirable, for example, in certain types of cancers and infectious diseases.
  • the disorders being treated or prevented by the disclosed methods include, but are not limited to, infections with microbes (e.g. bacteria), viruses (e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or shingles), or parasites; and cancer (e.g., melanoma and prostate cancers).
  • microbes e.g. bacteria
  • viruses e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or shingles
  • cancer e.g., melanoma and prostate cancers.
  • Cytotoxic T and NK cell activation with the anti-KLRGl antibodies disclosed herein enhance T and NK cell responses.
  • antibodies act as antagonists of KLRG1.
  • the antibodies can be used to inhibit or reduce the downregulatory activity associated with KLRG1, i.e., the activity associated with downregulation of cytotoxic T and NK cell activation.
  • the antibodies inhibit binding of E- cadherin 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.
  • the antibodies or antibody compositions of the present disclosure are administered in therapeutically effective amounts.
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the severity of the medical condition of the subject.
  • a therapeutically effective amount of 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 observed effects of the treatment.
  • the appropriate dose is chosen based on clinical indications by a treating physician.
  • the antibodies may be given as a bolus dose, to maximize the circulating levels of antibodies for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
  • Immune cells can also be isolated from a patient and incubated ex vivo with antibodies of the disclosure.
  • T cell and NK cell activation can be modulated by removing immune cells from a subject, contacting the immune cells in vitro with an anti-KLRGl antibody of the disclosure.
  • the anti- KLRG1 antibody may be used in a multivalent form such that KLRG1 molecules on the surface of an immune cell become "crosslinked" upon binding to such antibodies.
  • the anti- KLRGl antibodies can be bound to solid support, such as beads, or crosslinked via a secondary antibody.
  • the immune cells may be then isolated using methods known in the art and reimplanted into the patient.
  • the antibodies of the disclosure may also be used to detect the presence of KLRG1 in biological samples.
  • the amount of KLRG1 detected may be correlated with the expression level of KLRG1, which, in turn, is correlated with the activation status of immune cells (e.g., activated T cells or NK cells) in the subject.
  • immune cells e.g., activated T cells or NK cells
  • Detection methods that employ antibodies are well known in the art and include, for example, ELISA, radioimmunoassay, immunoblot, Western blot, immunofluorescence, immunoprecipitation.
  • the antibodies may be provided in a diagnostic kit that incorporates one or more of these techniques to detect KLRG1.
  • Such a kit may contain other components, packaging, instructions, or other material to aid in the detection of the protein.
  • the antibodies are intended 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).
  • a detectable label such as a fluorescent group, a radioisotope or an enzyme.
  • the antibodies of the disclosure may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having 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 with a spectrophotometer.
  • TMB tetramethylbenzidine
  • binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant disclosure.
  • Antibodies of the disclosure can be used in screening methods to identify inhibitors of the KLRG1 pathway effective as therapeutics.
  • a first binding mixture is formed by combining KLRG1 and an antibody of the disclosure; and the amount of binding in the first binding mixture (MO) is measured.
  • a second binding mixture is also formed by combining KLRG1, the antibody, and the compound or agent to be screened, and the amount of binding in the second binding mixture (Ml) is measured.
  • a compound to be tested may be another anti-KLRGl antibody, as illustrated in the Examples.
  • the amounts of binding in the first and second binding mixtures are then compared, for example, by calculating the M1/M0 ratio.
  • the compound or agent is considered to be capable of modulating a KLRG1 -associated downregulation of immune responses if a decrease in binding in the second binding mixture as compared to the first binding mixture is observed.
  • binding mixtures may also contain buffers and salts necessary to enhance, or to optimize, binding, and additional control assays may be included in the screening assay of the disclosure.
  • Compounds found to reduce the KLRG1 -antibody binding by at least about 10% (i.e., M1/MCK0.9), preferably greater than about 30% may thus be identified and then, if desired, secondarily screened for the ability to ameliorate a disorder in other assays or animal models as described below.
  • the strength of the binding between KLRG1 and an antibody can be measured using, for example, an enzyme-linked immunoadsorption assay (ELISA), radio-immunoassay (RIA), surface plasmon resonance-based technology (e.g., Biacore), all of which are techniques well known in the art.
  • ELISA enzyme-linked immunoadsorption assay
  • RIA radio-immunoassay
  • Biacore surface plasmon resonance-based technology
  • the compound may then be tested in vitro as described in the Examples or in an animal model.
  • Preliminary doses as, for example, determined according to animal tests, and the scaling of dosages for human administration is performed according to art-accepted practices.
  • Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from the cell culture assays or animal studies can be used in formulating a range of dosages for use in humans.
  • Therapeutically effective dosages achieved in one animal model can be converted for use in another animal, including humans, using conversion factors known in the art (see, e.g., Freireich et al. (1966) Cancer Chemother. Reports, 50(4): 219-244).
  • compositions comprising anti-KLRGl 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 pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, coatings, antibacterial agents and antifungal agents, isotonic agents, and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
  • the pharmaceutical compositions may also be included in a container, pack, or dispenser together with instructions for administration.
  • a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art.
  • the administration may, for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal. It may also be possible to obtain compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be 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.
  • 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.
  • the 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 dispersion and/or by the use of surfactants.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate, and gelatin.
  • the administration may, for example, be parental administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous).
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration may be accomplished, for example, through the use of lozenges, nasal sprays, inhalers, or suppositories;
  • compositions may be capable of transmission across mucous membranes in intestine, mouth, or lungs (e.g., via the FcRn receptor- mediated pathway as described in U.S. Pat.
  • the active compounds may be formulated into ointments, salves, gels, or creams as generally known in the art.
  • the antibodies may be delivered in the form of an aerosol spray from pressured container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • the presently disclosed antibodies are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • Liposomal suspensions containing the presently disclosed antibodies 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.
  • parenteral compositions in a dosage unit form for ease of administration and uniformity of dosage.
  • dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Toxicity and therapeutic efficacy of the composition of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining 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 and it can be expressed as the ratio LD50/ED50.
  • Compositions that exhibit large therapeutic indices are preferred.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • bioassays include DNA replication assays, cytokine release assays, transcription-based assays, KLRGl/cadherin binding assays, immunological assays other assays as, for example, described in the Examples.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the antibody which achieves a half-maximal inhibition of symptoms). Circulating levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay.
  • the dosage lies preferably within a range of circulating concentrations with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • Recombinant proteins were produced by standard molecular cloning and expression protocols include human KLRG1 ECD (SEQ ID NO:l), cynomolgus KLRG1 ECD (SEQ ID NO:2), and human E-cadherin (SEQ ID NO:3), whose amino acid sequences are known in art and are disclosed in the PCT publication, W02020060781, which is hereby by incorporated by reference in its entirety.
  • Recombinant proteins were produced as FC fusion or as HIS tagged versions by cloning the respective cDNA into pCDNA4 vector (Invitrogen) and transient transfection in mammalian HEK293.
  • VTLKESGPGLVKPTQTLTLTCTF SGF SL S TF GMGV GWIRQPPGK ALEWL AHIW WDDDKWYELALKSRLTISKDS SKNQ VVLTMTNMDP VDTAT YY CARVIYY GSRS AYYSMDYWGPGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VT V SWNSGALT SGVHTFP AVLQ S SGL Y SL S SWT VP S S SLGTQT YICNVNHKP SN TKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVV VD V SHEDPEVKFNW YVDGVEVHNAKTKPREEQ YN ST YRV V S VLT VLHQDWLN GKE YKCK V SNK ALP APIEKTI SK AKGQPREPQ V YTLPP SREEMTKN Q V SLTCL
  • VTLKESGPGLVKPTQTLTLTCTF SGF SL S TF GMGV GWIRQPPGK ALEWL AHIW WDDDKWYELALKSRLTISKDS SKNQ VVLTMTNMDP VDTAT YY CARVIYY GSRS A Y Y SMD YW GPGTT VT V S S
  • the GAO 15 antibodies are humanized IgGl antibodies against the extracellular domain of KLRG1.
  • Mouse monoclonal antibodies (MAB) against human KLRGl was generated by standard immunizations of female B ALB/c mice and SJL mice with human and cynomolgus KLRGl, and subsequent hybridoma screening.
  • 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 periodically monitored by ELISA and animals were sacrificed when appropriate titers were reached, usually between 1 : 1000 and 1 : 10000 dilution factor. Splenocytes from sacrificed mice were fused to mouse myeloma cells to produce hybridoma cells and later cultured and sub-cloned into single cells. Stable clones were scaled up and condition media was harvested and tested for expression of anti-KLRGl antibodies by ELISA and FACS.
  • CHOKl-hKLRGl expressing cells were cultured and incubated with varying concentrations of either GAO 15 or control human IgGl. Cells were then centrifuged, the supernatant discarded, and washed with an incubation buffer (HBSS + 2% FBS + lOmM Ca2+). Cells were resuspended and incubated with anti-human IgG-Alexa 488, washed, resuspended, and underwent FACS study. The mean fluorescent intensity (MFI) of the cells incubated with GAO 15 and with control human IgGl are shown in FIG. 1. The EC50 for GAO 15 was 1.42 nM.
  • Example 3 GAO 15 blocking of E-cadherin to CHOK1 cells expressing KLRG1 in relative binding assay.
  • CHOKl-hKLRGl expressing cells were cultured and incubated with varying concentrations of either GAO 15 or control human IgGl.
  • Recombinant human E-cadherin-ECD- FC-biotin at 0.3 uM was then added and incubated.
  • Cells were then centrifuged, the supernatant discarded, and washed with an incubation buffer (HBSS + 2% FBS + lOmM Ca2+).
  • HBSS + 2% FBS + lOmM Ca2+ Cells were resuspended and incubated with Streptavidin-Alexa 488, washed, resuspended, and underwent FACS study.
  • the mean fluorescent intensity (MFI) of the cells incubated with GAO 15 and with control human IgGl are shown in FIG. 2.
  • the IC50 for GA015 was 9.99 nM.
  • Example 4 Efficacy of GAO 15 in humanized mouse model with a patient-derived xenograft of melanoma.
  • mice through implantation of bone marrow/liver/thymus (Allen TM et al. Nat Immunol 2019 Jul;20(7):770-774).
  • Mice with greater than 25% human CD45+ cell levels were implanted with 2 million patient-derived (PDX) melanoma tumor cells.
  • PDX patient-derived
  • FIG. 3 shows that GAO 15 inhibits melanoma growth in the humanized mouse patient derived xenograft model.
  • Example 5 GAO 15 enhances human NK cell killing and degranulation.
  • Human donor blood underwent NK cell isolation by magnetic bead separation (NK Cell Isolation Kit, Miltenyi).
  • Target cells were K562-Ecad (a K562 cell line transfected with nucleic acid encoding human E-cadherin to produce K562-E-cadherin surface expressing cells).
  • NK cells effectors
  • CD 107a and IL-2 were added and incubated with cells and antibodies for 4 hours at 37°C 5% C02.
  • Example 6 GAO 15 has superior blocking compared with HG1N01 and HG1N02 of E-cadherin to CHOK1 cells expressing KLRG1 in relative binding assay.
  • CHOKl-hKLRGl-expressing cells were cultured and incubated with varying concentrations of GAO 15, HG1N01, HG1N02 or control human IgGl. Recombinant human E- cadherin-ECD-FC-biotin at 0.3 mM was then added and incubated. Cells were centrifuged, the supernatant discarded, and cells washed with an incubation buffer (HBSS + 2% FBS + lOmM Ca2+). Cells were resuspended and incubated with Streptavidin-Alexa 488, washed, resuspended, and underwent FACS study.
  • the mean fluorescent intensity (MFI) of the cells incubated with the antibodies is shown in FIG.5.
  • the IC50 for GA015 was 2.0 nM, for HG1N01, 8.3 nM, and for HG1N02, 7.9 nM.
  • Example 7 GAO 15 has superior pharmacokinetic properties compared to HG1N01 in non human primates.
  • GAO 15 and HG1N01 were administered as a single dose intravenously to two cynomolgus monkeys each. Serum concentration of each antibody was measured over 42 days (GAO 15, FIG. 6; and HG1N01, FIG. 7). GAO 15 compared to HG1N01 has superior clearance (9.84 vs 38.6 mL/day/kg), area under the curve (AUC; 331 vs 77.6 day*ug/mL) and beta halfdife (7.16 vs 4.7 days) (Tables 3 and 4). Table 3. Pharmacokinetic parameters of GA015

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Abstract

La divulgation concerne des anticorps, ou des fragments de liaison à l'antigène de ceux-ci, qui se lient de manière spécifique au récepteur G1 de type lectine des cellules tueuses (KLRG1). De tels anticorps, ou fragments de liaison à l'antigène de ceux-ci, sont utiles à diverses fins thérapeutiques ou diagnostiques, notamment pour le traitement de cancers et pour augmenter l'efficacité de vaccins.
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