Anti-KLRG1 antibodies and uses thereof

Abstract

The present disclosure provides antibodies and polypeptides that specifically bind to killer cell lectin-like receptor G1 (KLRG1). Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating subjects using these antibodies.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to antibodies specific for human killer cell lectin-like receptor Gl (KLRG1) and methods of use thereof. BACKGROUND

[0002] Killer cell lectin-like receptor Gl (KLRG1) is a C-type lectin inhibitory receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) motif in its cytoplasmic domain. It is expressed primarily on natural killer (NK) cells and late-differentiated effector and effector memory CD8+ T cells. KLRG1 has two main ligands, the cell adhesion molecules E-cadherin and N-cadherin, and also recognizes R-cadherin. Cadherin levels have been observed to be higher in several different human cancers, including melanoma, prostate cancer, breast cancer, hepatocellular carcinoma, and renal cancer, among others. Engagement of KLRG1 inhibits NK cytotoxicity, IFN-g and TNF-a production, proliferation, and telomerase activity. KLRG1 expression has been shown to be upregulated in response to various cancer therapies, potentially contributing to acquired resistance, and suggests that KLRG1 blockade can be effective in cancer therapy.

[0003] Accordingly, there is a need for therapies that target KLRG1. SUMMARY

[0004] The present disclosure provides antibodies and polypeptides that specifically bind to KLRG1 (e.g., human KLRG1). Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of using these antibodies to treat subjects. The antibodies disclosed herein are particularly advantageous because, to the best of the present applicant's knowledge, each has a significantly lower IC50 (for inhibiting KLRG1 / E-cadherin interaction) than other antagonistic KLRG1 antibodies currently being developed for therapeutic purposes. The present applicant believes that this lower IC50 will translate into superior efficacy in vivo.

[0005] In one aspect, the present disclosure provides an antibody that specifically binds to human KLRG1, the antibody comprising a VH and a VL, the VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 16, 19, or 48; the VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 17, 31, or 49.

[0006] In one aspect, the present disclosure provides an antibody that specifically binds to human KLRG1, the antibody comprising a VH and a VL, the VH comprising CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence set forth in SEQ ID NO: 1, 11, 19, 32, or 42; the VL comprising CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence set forth in SEQ ID NO: 2, 12, 20, 29, 33, or 43.

[0007] In one embodiment, the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences set forth in SEQ ID NO: 16 and 17, 19 and 31, or 48 and 49, respectively.

[0008] In one embodiment, the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences set forth in SEQ ID NO: 1 and 10, 11 and 12, 19 and 20, 19 and 29, 32 and 33, or 42 and 43, respectively.

[0009] In one embodiment, the antibody comprises CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; or 34, 35, and 50, respectively.

[0010] In one embodiment, the antibody comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 18, and 8; 24, 25, and 26; or 37, 51, and 39, respectively.

[0011] In one embodiment, the antibody comprises CDRH1, CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; 34, 35, and 36; or 34, 35, and 44, respectively.

[0012] In one embodiment, the antibody comprises CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 6, 7, and 8; 6, 13, and 8; 24, 25, and 26; 37, 38, and 39; or 37, 45, and 39, respectively.

[0013] In an embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 18, and 8; 21, 22, 23, 24, 25, and 26; or 34, 35, 50, 37, 51, and 39, respectively.

[0014] In an embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences set forth in SEQ ID NO: 3, 4, 5, 6, 7, and 8; 3, 4, 5, 6, 13, and 8; 21, 22, 23, 24, 25, and 26; 34, 35, 36, 37, 38, and 39; or 34, 35, 44, 37, 45, and 39, respectively.

[0015] In an embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO: 16, 19, or 48.

[0016] In an embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO: 1, 11, 19, 32, or 42.

[0017] In an embodiment, the antibody comprises a heavy chain constant region, optionally selected from the group consisting of human IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0018] In an embodiment, the antibody comprises a heavy chain constant region that is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to an FcγR with an affinity that is lower than the affinity with which the wild-type heavy chain constant region binds to the FcγR.

[0019] In an embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 52, 53, 54, or 55.

[0020] In an embodiment, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59, or 60.

[0021] In an embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO: 17, 31, or 49.

[0022] In an embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33, or 43.

[0023] In one embodiment, the antibody comprises a light chain containing an amino acid sequence of SEQ ID NO: 10, 15, 28, 30, 41, or 47.

[0024] In one embodiment, the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31 or 48 and 49.

[0025] In one embodiment, the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33 or 42 and 43.

[0026] In one embodiment, the heavy chain and the light chain respectively comprise the amino acid sequences shown in SEQ ID NO: 9 and 10, 56 and 10, 14 and 15, 57 and 15, 27 and 28, 58 and 28, 27 and 30, 58 and 30, 40 and 41, 59 and 41, 46 and 47 or 60 and 47.

[0027] In one aspect, this disclosure provides a polypeptide comprising a VH, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, 19, or 48. In one embodiment, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; or 34, 35, and 50. In another embodiment, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; 34, 35, and 36; or 34, 35, and 44. In yet another embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 16, 19, or 48. In yet another embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 1, 11, 19, 32, or 42. In one embodiment, the polypeptide comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59, or 60.

[0028] In one embodiment, this disclosure provides a polypeptide comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, 19, or 48. In one embodiment, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; or 34, 35, and 50. In another embodiment, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 3, 4, and 5; 21, 22, and 23; 34, 35, and 36; or 34, 35, and 44. In yet another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 16, 19, or 48. In yet another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1, 11, 19, 32, or 42. In one embodiment, the polypeptide comprises an amino acid sequence of SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59 or 60.

[0029] In one aspect, this disclosure provides a polypeptide comprising a VL, wherein the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, 31, or 49. In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NO: 6, 18, and 8; 24, 25, and 26; or 37, 51, and 39, respectively. In another embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NO: 6, 7, and 8; 6, 13, and 8; 24, 25, and 26; 37, 38, and 39; or 37, 45, and 39, respectively. In one embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 17, 31, or 49. In another embodiment, the VL comprises the amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33, or 43. In one embodiment, the polypeptide comprises a light chain containing an amino acid sequence of SEQ ID NO: 10, 15, 28, 30, 41, or 47.

[0030] In one embodiment, this disclosure provides a polypeptide comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, 31, or 49. In one embodiment, the polypeptide comprises the amino acid sequences shown in SEQ ID NO: 6, 18, and 8; 24, 25, and 26; or 37, 51, and 39, respectively. In another embodiment, the polypeptide comprises the amino acid sequences shown in SEQ ID NO: 6, 7, and 8; 6, 13, and 8; 24, 25, and 26; 37, 38, and 39; or 37, 45, and 39, respectively. In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 17, 31, or 49. In another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33, or 43. In yet another embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 10, 15, 28, 30, 41, or 47.

[0031] On one hand, this disclosure provides an antibody that specifically binds to human KLRG1, the antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 57 or 65. In one embodiment, the VH comprises up to four cumulative amino acid substitutions from CDRH1, CDRH2, and CDRH3, and the VL comprises up to two cumulative amino acid substitutions from CDRL1, CDRL2, and CDRL3. In another embodiment, the VH and / or VL comprises up to fifteen amino acid substitutions outside the CDR region.

[0032] In one embodiment, the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, respectively.

[0033] In one embodiment, the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 58, 59 and 60; or 66, 67 and 68.

[0034] In one embodiment, the antibodies comprise the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 61, 62, and 63; or 69, 70, and 71, respectively.

[0035] In one embodiment, the antibody comprises the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 58, 59, 60, 61, 62, and 63; or SEQ ID NO: 66, 67, 68, 69, 70, and 71.

[0036] In one embodiment, the antibody comprises the VH amino acid sequence of SEQ ID NO: 56 or 64.

[0037] In one embodiment, the antibody comprises the VL amino acid sequence of SEQ ID NO: 57 or 65.

[0038] In one embodiment, the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65.

[0039] On one hand, this disclosure provides a polypeptide comprising VH, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64. In one embodiment, the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 58, 59, and 60; or 61, 62, and 63, respectively.

[0040] On one hand, this disclosure provides a polypeptide comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64. In one embodiment, the polypeptide comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences shown in SEQ ID NO: 58, 59, and 60; or 61, 62, and 63, respectively.

[0041] On one hand, this disclosure provides a polypeptide comprising a VL, wherein the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequences shown in SEQ ID NO: 57 and 65. In one embodiment, the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NO: 61, 62, and 63; or the CDRL1, CDRL2, and CDRL3 amino acid sequences shown in SEQ ID NO: 69, 70, and 71, respectively.

[0042] On one hand, this disclosure provides a polypeptide comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequences shown in SEQ ID NO: 57 and 65. In one embodiment, the polypeptide comprises the amino acid sequences shown in SEQ ID NO: 61, 62, and 63; or 69, 70, and 71, respectively. In one embodiment, the antibody or polypeptide disclosed herein is conjugated with a cytotoxic agent, cell inhibitor, toxin, radionuclide, or detectable label.

[0043] On one hand, this disclosure provides a polynucleotide that encodes: the VH, VL, heavy chain and / or light chain of the antibody disclosed herein; or the polypeptide disclosed herein.

[0044] On the one hand, this disclosure provides a vector comprising the polynucleotides disclosed herein.

[0045] On one hand, this disclosure provides a recombinant host cell, the recombinant host cell comprising: (a) The polynucleotides disclosed herein; (b) The medium disclosed herein; (c) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes a heavy chain variable region or heavy chain of the antibody disclosed herein, and the second polynucleotide encodes a light chain variable region or light chain of the antibody disclosed herein. (d) A first carrier and a second carrier, the first carrier comprising a first polynucleotide encoding a heavy chain variable region or heavy chain of the antibody disclosed herein, and the second carrier comprising a second polynucleotide encoding a light chain variable region or light chain of the antibody disclosed herein.

[0046] On one hand, this disclosure provides a pharmaceutical composition comprising the antibody, peptide, polynucleotide, vector, host cell disclosed herein, and pharmaceutically acceptable carrier or excipient disclosed herein.

[0047] On one hand, this disclosure provides a method for generating antibodies, the method comprising culturing the host cells disclosed herein under suitable conditions to express the polynucleotide and generate the antibody.

[0048] On the other hand, this disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody, peptide, polynucleotide, vector, host cell, or pharmaceutical composition disclosed herein.

[0049] On the one hand, this disclosure provides the use of the antibodies, peptides, polynucleotides, vectors, host cells, or pharmaceutical compositions disclosed herein in the preparation of a medicament for treating cancer in a subject in need.

[0050] On the one hand, this disclosure provides antibodies, peptides, polynucleotides, vectors, host cells, or pharmaceutical compositions disclosed herein for use in medicine.

[0051] On the one hand, this disclosure provides antibodies, peptides, polynucleotides, vectors, host cells, or pharmaceutical compositions disclosed herein for the treatment of cancer in subjects in need.

[0052] On one hand, this disclosure provides a method for treating cancer, the method comprising administering to a subject suffering from cancer a therapeutically effective amount of: (a) an antibody or peptide disclosed herein; and (b) a bispecific T-cell connective. In one embodiment, the bispecific T-cell connective specifically binds to: (a) a tumor-associated antigen; and (b) a molecule on an effector cell. In one embodiment, the tumor-associated antigen is Her2. In one embodiment, the tumor-associated antigen is CD19. In one embodiment, the molecule on the effector cell is CD3. In one embodiment, the tumor-associated antigen is Her2, and the molecule on the effector cell is CD3. In one embodiment, the tumor-associated antigen is CD19, and the molecule on the effector cell is CD3. In one embodiment, the bispecific T-cell connective comprises trastuzumab and a CD3 binder. In one embodiment, the bispecific T-cell connective is blinatumomab.

[0053] In one aspect, this disclosure provides a method comprising administering to a subject a therapeutically effective amount of: (a) an antibody or peptide disclosed herein; and (b) a PD-1 and / or PD-L1 agent. In one embodiment, the PD-1 or PD-L1 agent is or comprises an antibody. In one embodiment, the antibody is an antagonist antibody. Attached Figure Description

[0054] Figure 1 This diagram illustrates the binding of the KLRG1 antibody to CHO cells expressing human and cynomolgus monkey KLRG1. The binding is shown as a function of mean fluorescence intensity (MFI) versus antibody concentration in nM.

[0055] Figure 2 This diagram illustrates the binding of the KLRG1 antibody to human CD8+ T cells from a healthy donor. The binding is shown as a function of MFI and antibody concentration in nM.

[0056] Figure 3 This graph illustrates the ability of the KLRG1 antibody to block the interaction between human KLRG1 and cells expressing E-cadherin. The blocking is shown as a function of the MFI of KLRG1 tetramer-PE versus antibody concentration (nM).

[0057] Figures 4A-4F This is a series of figures illustrating the ability of the KRLG1 antibody to block the interaction between human KRLG1 and N-cadherin on A375 cells expressing endogenous N-cadherin: Figure 4A Excluding KLRG1 tetramer, Figure 4B Includes only KLRG1 tetramer, Figure 4C Including KLRG1 tetramer and human IgG4 isotype control antibodies, Figure 4D Including KLRG1 tetramer and 16F5 antibody, Figure 4E Including KLRG1 tetramer and 5P13 antibody, and Figure 4F A diagram illustrating the interaction between KLRG1 tetramer and N-cadherin is provided, along with a representation relative to the mode normalization. Figure 4A -E commonality map. KLRG1 binding to cells was analyzed by flow cytometry.

[0058] Figure 5 This is a graph showing the ability of the KLRG1 antibody to reverse the inhibition of CD8+ T cell activation by E-cadherin, measured by IFN-γ secretion levels in pg / mL.

[0059] Figure 6 This graph illustrates the ability of the KLRG1 antibody to enhance CD8+ T cell activation by increasing the expression of IFN-γ and TNF-α in CD8+ T cells from healthy donors. The levels of IFN-γ and TNF-α were measured by ELISA and are shown as a function of antibody concentration in pg / mL.

[0060] Figure 7 This is a graph illustrating the ability of the KLRG1 antibody to enhance the activation of KLRG1-enriched CD8+ T cells isolated from PBMCs of three different cancer patients. IFN-γ levels were measured by ELISA and are shown in pg / mL.

[0061] Figure 8This figure illustrates the ability of the 1D21v1 KLRG1 antibody alone and in combination with an anti-PD-1 antibody to induce activation of CD8+ tumor-infiltrating lymphocytes from dissociated tumor cells in tumor tissue samples. CD8+ T cell activation was analyzed using IFN-γ (IFNG) intracellular staining and measured by flow cytometry.

[0062] Figure 9 This is a graph illustrating the ability of the 1D21v1 KLRG1 antibody to enhance the cytotoxicity of the CD3 x CD19 bispecific T cell connector (BiTE) in HEK cells engineered to express CD19 and E-cadherin. HEK cell viability (%) was measured by flow cytometry and is shown as a function of BiTE concentration.

[0063] Figure 10 This figure illustrates the ability of the 1D21v1 KLRG1 antibody to enhance the cytotoxicity of CD3 x HER2 BiTE in HCC2935 cells expressing endogenous HER2 and E-cadherin. HCC2935 cell viability was measured by flow cytometry and is expressed as a function of BiTE concentration. Detailed Implementation

[0064] This disclosure provides anti-KLRG1 antibodies and peptides. Pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for preparing these antibodies, and methods for treating subjects using these antibodies are also provided. The antibodies disclosed herein are particularly useful for treating cancer in subjects.

[0065] definition As used herein, the expression “KLRG1” refers to cytotoxic lectin-like receptor G1. The amino acid sequence of human cytotoxic lectin-like receptor G1 can be found, for example, in NP_001316028.1, NP_001316030.1, NP_001316031.1, NP_001316032.1, and NP_005801.3. KLRG1 is a type C lectin inhibitory receptor expressed on NK and T cells, containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. All references to proteins, peptides, and protein fragments herein are intended to refer to the human version of the corresponding protein, peptide, or protein fragment unless explicitly stated to be from a non-human species. Therefore, the expression “KLRG1” means human KLRG1 unless specified as from a non-human species, such as “mouse KLRG1,” “monkey KLRG1,” etc.

[0066] As used herein, the terms “antibody” and “antibodies” include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDR, VH region, and / or VL region. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugated antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affinity molecules, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies or their classes (e.g., human IgG1 or IgG4) or subclasses. In some embodiments, the antibodies are humanized monoclonal antibodies. In another specific embodiment, the antibody is a human monoclonal antibody.

[0067] A "multispecific antibody" is an antibody that specifically binds to two or more different antigens or two or more different regions of the same antigen (e.g., a bispecific antibody). Multispecific antibodies include bispecific antibodies containing two different antigen-binding sites (excluding the Fc region). Multispecific antibodies can include, for example, recombinant antibodies, human antibodies, humanized antibodies, surface-repaired antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramer antibodies containing two heavy chains and two light chains, antibody light chain monomers, heteroconjugated antibodies, linked single-chain antibodies or linked single-chain Fv (scFv), camelized antibodies, affinity molecules, linked Fab fragments, F(ab')2 fragments, chemically linked Fv, and disulfide-linked Fv (sdFv). Multispecific antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the multispecific antibodies described herein are IgG antibodies or their classes (e.g., human IgG1, IgG2, or IgG4) or subclasses.

[0068] As used herein, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site present in the variable regions of heavy and light chain polypeptides. These specific regions have been described by, for example, the following literature: Kabat et al., *Journal of Biol. Chem.* 252, 6609-6616 (1977); and Kabat et al., *Sequences of protein of immunological interest* (1991); Chothia et al., *Journal of Molecular Biology* 196:901-917 (1987); and MacCallum et al., *Journal of Molecular Biology* 262:732-745 (1996), all of which are incorporated herein by reference in their entirety, wherein, when compared with each other, the definition includes overlaps or subgroups of amino acid residues. In some implementations, the term "CDR" is as defined by the following literature: MacCallum et al., *Journal of Molecular Biology* 262:732-745 (1996); and Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," *Antibody Engineering*. AntibodyEngineering (See *Molecular Biology Journal*, edited by Kontermann and Dübel, Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)). In some embodiments, the term "CDR" is the CDR as defined by the following literature: Kabat et al., *Molecular Biology Journal* 252, 6609-6616 (1977); and Kabat et al., *Protein Sequences of Immunological Significance* (1991). In some embodiments, the heavy chain CDR and light chain CDR of the antibody are defined using different conventions. In some embodiments, the heavy chain CDR and / or light chain CDR are defined by structural analysis of the antibody and identification of residues in the variable region predicted to contact the epitope region of the target molecule (e.g., human KLRG1). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0069] As used herein, the terms “variable region” and “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids from the amino terminus in the mature heavy chain and about 90 to 115 amino acids in the mature light chain. These regions vary considerably in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in regions known as complementarity-determining regions (CDRs), while more conserved regions within the variable region are called frame regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of both the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region includes rodent or mouse CDRs and human frame regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some implementations, the variable region includes the rodent or mouse CDR and the primate (e.g., non-human primate) frame region (FR).

[0070] As used herein, the terms “VH” and “VL” refer to the variable regions of the antibody heavy and light chains, respectively, as described in Kabat et al., (1991) “Sequences of Immunologically Significant Proteins” (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0071] As used herein, the term "constant region" is common in the art. A constant region is an antibody moiety, such as the carboxyl-terminal portion of the light chain and / or heavy chain, which is not directly involved in the binding of the antibody to the antigen but can exhibit various effector functions, such as interaction with Fc receptors (e.g., Fcγ receptors).

[0072] As used herein, based on the amino acid sequence of the constant region, the term “heavy chain” when used in reference antibody can refer to any different type, such as α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG such as IgG1, IgG2, IgG3, and IgG4.

[0073] As used herein, based on the amino acid sequence of the constant region, the term "light chain" when used with reference antibodies can refer to any different type, such as κ (kappa) or λ (lambda). Light chain amino acid sequences are well known in the art. In this specific embodiment, the light chain is the human light chain.

[0074] As used herein, the term "specific binding" refers to the specificity of a binding molecule (e.g., an antibody) for an antigen, as understood by those skilled in the art. Binding molecules that specifically bind to an antigen typically have a binding density of less than 1 × 10⁻⁶. -6 The equilibrium dissociation constant (KD) of M is bound to the antigen, as measured by, for example, an ELISA assay, surface plasmon resonance, or other suitable assays known in the art. Those skilled in the art will understand that in some embodiments, the binding molecule can specifically bind to different antigens, such as different antigens sharing a common epitope recognized by the binding molecule.

[0075] As used herein, the term "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can typically be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0076] As used herein, the term “EU numbering system” refers to the EU numbering convention for the constant regions of antibodies, as described in the following references: Edelman, GM, et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); and Kabat, et al., Protein sequences of immunological significance, US Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0077] As used herein, the terms “treat,” “treating,” and “treatment” refer to the therapeutic or preventative measures described herein. A “treatment” method involves administering antibodies to a subject who has or is susceptible to a disease or condition, in order to prevent, cure, delay, relapse, reduce the severity of, or improve one or more symptoms of the disease or condition, or extend the subject’s survival beyond what would be expected in the absence of such treatment.

[0078] As used herein, in the context of administering therapy to a subject, the term “effective amount” refers to the amount of treatment that achieves the desired preventive or therapeutic effect.

[0079] As used herein, the term "subject" includes any human or non-human mammal. In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is a human.

[0080] As used herein with respect to antibodies or polynucleotides, the term "isolated" means an antibody or polynucleotide isolated from one or more contaminants (e.g., peptides, polynucleotides, lipids, or carbohydrates) present in the natural source of the antibody or polynucleotide. All instances of "isolated antibody" described herein are also considered to be, but not necessarily, isolated antibodies. All instances of "isolated polynucleotide" described herein are also considered to be, but not necessarily, isolated polynucleotides. All instances of "antibody" described herein are also considered to be, but not necessarily, isolated antibodies. All instances of "polynucleotide" described herein are also considered to be, but not necessarily, isolated polynucleotides.

[0081] The determination of the “percentage of identity” between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using mathematical algorithms. Specific, non-limiting examples of mathematical algorithms used for comparing two sequences are those in the following literature: Karlin S and Altschul SF (1990), *Proceedings of the National Academy of Sciences* 87: 2264-2268, which is modified in Karlin S and Altschul SF (1993), *Proceedings of the National Academy of Sciences* 90: 5873-5877, each of which is incorporated herein by reference in its entirety. Such algorithms are also incorporated in the NBLAST and XBLAST procedures of Altschul SF et al. (1990), *Journal of Molecular Biology* 215: 403, which is also incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide procedure parameter set (e.g., fraction = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST procedure parameter set (e.g., fraction = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For vacancy-based alignments for comparative purposes, vacancy-based BLAST can be utilized, as described in: Altschul SF et al., (1997) Nucleic Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, PSI BLAST can be used to perform iterative searches to detect distance relationships between molecules (ibid.). When using BLAST, BLAST with vacancies, and PSI Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (see, for example, the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm for sequence alignment is the algorithm of Myers and Miller, 1988, *Computers in the Biological Sciences (CABIOS)* 4:11-17, which is incorporated herein by reference in its entirety. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When comparing amino acid sequences using the ALIGN program, the PAM120 weighted residue table, vacancy length penalty of 12, and vacancy penalty of 4 can be used.

[0082] Whether gaps are allowed or not, techniques similar to those described above can be used to determine the percentage of identity between two sequences. When calculating the percentage of identity, typically only exact matches are counted.

[0083] Anti-KLRG1 antibody On the one hand, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1). The amino acid sequences of exemplary antibodies are shown in Table 1.

[0084] Table 1. Amino acid sequences of exemplary anti-KLRG1 antibodies. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VH domain, the VH domain comprising one, two, or all three CDRs of the VH domains shown in Table 1. In some embodiments, the antibody comprises CDRH1 of the VH domains shown in Table 1. In some embodiments, the antibody comprises CDRH2 of the VH domains shown in Table 1. In some embodiments, the antibody comprises CDRH3 of the VH domains shown in Table 1.

[0085] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VL domain comprising one, two, or all three CDRs of the VL domains disclosed in Table 1. In some embodiments, the antibody comprises CDRL1 of the VL domains shown in Table 1. In some embodiments, the antibody comprises CDRL2 of the VL domains shown in Table 1. In some embodiments, the antibody comprises CDRL3 of the VL domains shown in Table 1.

[0086] The individual CDRs of the antibodies disclosed herein can be determined based on any CDR numbering scheme known in the art.

[0087] In some embodiments, one or more of the CDRs of the antibodies disclosed herein may be determined according to Kabat et al., Journal of Biochemistry 252, 6609-6616 (1977) and Kabat et al., Protein Sequences of Immunological Significance (1991), each of which is incorporated herein by reference in its entirety.

[0088] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of the antibodies disclosed in Table 1 herein, as determined by the Kabat numbering scheme.

[0089] In some embodiments, one or more of the CDRs of the antibodies disclosed herein may be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loop (see, for example, Chothia C and Lesk AM, (1987), Journal of Molecular Biology 196: 901-917; Al-Lazikani B et al., (1997) Journal of Molecular Biology 273: 927-948; Chothia C et al., (1992) Journal of Molecular Biology 227: 799-817; Tramontano A et al., (1990) Journal of Molecular Biology 215(1): 175-82; and U.S. Patent No. 7,709,226, all of which are incorporated herein by reference in their entirety).

[0090] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of the antibodies disclosed in Table 1 herein, as determined by the Chothia numbering system.

[0091] In some embodiments, one or more of the CDRs of the antibodies disclosed herein may be determined according to MacCallum RM et al., (1996) *Journal of Molecular Biology* 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin, A. “Protein Sequence and Structural Analysis of Variable Domains of Antibodies”, *Antibody Engineering*, edited by Kontermann and Dübel, Chapter 31, pp. 422-439, Springer Verlag Berlin (2001), which is incorporated herein by reference in its entirety.

[0092] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of the antibodies disclosed in Table 1 herein, as determined by the MacCallum numbering system.

[0093] In some embodiments, the CDR of the antibodies disclosed herein can be determined according to the IMGT numbering system described in the following references: Lefranc MP, (1999) The Immunologist 7: 132-136; Lefranc MP et al., (1999) Nucleic Acid Research 27: 209-212, each of which is incorporated herein by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acid Research 37: D1006-D1012.

[0094] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of the antibodies disclosed in Table 1 herein, as determined by the IMGT numbering system.

[0095] In some embodiments, the CDR of the antibodies disclosed herein can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable region, representing a compromise between the Kabat CDR and the Chothia structural loop, and is used by the AbM antibody modeling software of Oxford Molecular (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0096] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of an antibody disclosed in Table 1 herein, as determined by the AbM numbering scheme.

[0097] In some embodiments, the CDR of the antibodies disclosed herein may be determined according to the AHo numbering system as described in the following literature: Honegger and Plückthun, A., Journal of Molecular Biology 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0098] In some embodiments, this disclosure provides antibodies that specifically bind to KLRG1 (e.g., human KLRG1) and comprise a CDR of the antibodies disclosed in Table 1 herein, as determined by the AHo numbering system.

[0099] In some embodiments, the individual CDRs of the antibodies disclosed herein are determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that are in contact with the epitope region of KLRG1.

[0100] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, 19, or 48; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, 31, or 49, wherein each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that are in contact with epitope regions of KLRG1 (e.g., human KLRG1).

[0101] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31, or 48 and 49, respectively.

[0102] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 1, 11, 19, 32, or 42; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 2, 12, 20, 29, 33, or 43, wherein each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that are in contact with epitope regions of KLRG1 (e.g., human KLRG1).

[0103] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33, or 42 and 43, respectively.

[0104] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VH, the VH comprising the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 3, 4 and 5; 21, 22 and 23; or 34, 35 and 50.

[0105] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VL containing the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 6, 18, and 8; 24, 25, and 26; or 37, 51, and 39.

[0106] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH, the VH comprising the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 3, 4 and 5; 21, 22 and 23; 34, 35 and 36; or 34, 35 and 44, respectively.

[0107] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VL, the VL comprising the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 6, 7 and 8; 6, 13 and 8; 24, 25 and 26; 37, 38 and 39; or 37, 45 and 39.

[0108] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises VH and VL, wherein the VH comprises CDRH1, CDRH2, and CDRH3 regions; and the VL comprises CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions respectively comprise the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 18, and 8; 21, 22, 23, 24, 25, and 26; or 34, 35, 50, 37, 51, and 39.

[0109] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises VH and VL, wherein the VH comprises CDRH1, CDRH2, and CDRH3 regions; and the VL comprises CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions respectively comprise the amino acid sequences shown in SEQ ID NO: 3, 4, 5, 6, 7, and 8; 3, 4, 5, 6, 13, and 8; 21, 22, 23, 24, 25, and 26; 34, 35, 36, 37, 38, and 39; or 34, 35, 44, 37, 45, and 39.

[0110] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 16, 19, or 48. In some embodiments, the amino acid sequence of said VH consists of the amino acid sequence shown in SEQ ID NO: 16, 19, or 48.

[0111] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 17, 31, or 49. In some embodiments, the amino acid sequence of said VL consists of the amino acid sequence shown in SEQ ID NO: 17, 31, or 49.

[0112] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 1, 11, 19, 32, or 42. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 1, 11, 19, 32, or 42.

[0113] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 2, 12, 20, 29, 33, or 43. In some embodiments, the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO: 2, 12, 20, 29, 33, or 43.

[0114] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 16, 19, or 48; and the VL comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 17, 31, or 49. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 16, 19, or 48; and the VL comprises the amino acid sequence of SEQ ID NO: 17, 31, or 49. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 16, 19, or 48; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO: 17, 31, or 49.

[0115] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising the VH and VL amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31, or 48 and 49, respectively. In some embodiments, the amino acid sequences of VH and VL consist of the amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31, or 48 and 49, respectively.

[0116] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 1, 11, 19, 32, or 42; and the VL comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 2, 12, 20, 29, 33, or 43. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, 11, 19, 32, or 42; and the VL comprises the amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33, or 43. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 1, 11, 19, 32, or 42; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO: 2, 12, 20, 29, 33, or 43.

[0117] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising the VH and VL amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33, or 42 and 43, respectively. In some embodiments, the VH and VL amino acid sequences consist of the amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33, or 42 and 43, respectively.

[0118] In some embodiments, this disclosure provides an antibody that cross-competitively binds to KLRG1 (e.g., human KLRG1), the antibody comprising the VH and VL amino acid sequences shown in SEQ ID NO: SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33 or 42 and 43, respectively.

[0119] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 57 or 65, wherein each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that are in contact with epitope regions of KLRG1 (e.g., human KLRG1).

[0120] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a humanized variant of the antibody comprising VH and VL, the VH comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64; the VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 57 or 65, wherein the humanized variant comprises up to four cumulative substitutions in the CDRs of the VH and up to two cumulative substitutions in the CDRs of the VL, the substitutions not degrading the binding properties of the antibody, and wherein each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that contact the epitope region of KLRG1 (e.g., human KLRG1).

[0121] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises CDRH1, CDRH2, and CDRH3 amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the VH amino acid sequence shown in SEQ ID NO: 56 or 64; the VL comprises the same amino acid sequence as SEQ ID NO: The CDRL1, CDRL2, and CDRL3 amino acid sequences shown in 57 or 65 are at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical VL amino acid sequences, wherein each CDR is determined independently according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of a multispecific molecule, wherein the structural analysis identifies residues in the predicted variable region that are in contact with the epitope region of KLRG1 (e.g., human KLRG1).

[0122] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, respectively.

[0123] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a humanized variant of the antibody, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, having up to four cumulative substitutions in the CDR of the VH and up to two cumulative substitutions in the CDR of the VL, wherein the substitutions in the VH and VL do not degrade the binding properties of the antibody.

[0124] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VH, the VH comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 58, 59, and 60, respectively; or 66, 67, and 68. In some embodiments, this disclosure provides a humanized variant of an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VH, the VH comprising the amino acid sequences of CDRH1, CDRH2, and CDRH3 shown in SEQ ID NO: 58, 59, and 60, respectively; or 66, 67, and 68, having up to four cumulative substitutions in the CDR of the VH, wherein the substitutions in the VH and VL do not degrade the binding properties of the antibody.

[0125] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VL containing the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 61, 62, and 63, respectively; or SEQ ID NO: 69, 70, and 71. In some embodiments, this disclosure provides a humanized variant of an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises a VL containing the amino acid sequences CDRL1, CDRL2, and CDRL3 shown in SEQ ID NO: 61, 62, and 63, respectively; or SEQ ID NO: 69, 70, and 71, having at most two cumulative substitutions in the CDRs of the VL, wherein the substitutions in the VH and VL do not degrade the binding properties of the antibody.

[0126] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises VH and VL, wherein the VH comprises CDRH1, CDRH2, and CDRH3 regions; and the VL comprises CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise, respectively, the amino acid sequences shown in SEQ ID NO: 58, 59, 60, 61, 62, and 63; or 66, 67, 68, 69, 70, and 71. In some embodiments, this disclosure provides a humanized variant of an antibody that specifically binds to KLRG1 (e.g., human KLRG1), wherein the antibody comprises VH and VL, the VH comprising CDRH1, CDRH2, and CDRH3 regions; the VL comprising CDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regions comprise, respectively, the amino acid sequences shown in SEQ ID NO: 58, 59, 60, 61, 62, and 63; or 66, 67, 68, 69, 70, and 71, having at most four cumulative substitutions in the CDR of the VH and at most two cumulative substitutions in the CDR of the VL, wherein the substitutions in the VH and VL do not degrade the binding properties of the antibody.

[0127] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VH containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 56 or 64. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a humanized variant of VH, said VH comprising at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 56 or 64. In some embodiments, the amino acid sequence of said VH consists of the amino acid sequence shown in SEQ ID NO: 56 or 64.

[0128] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a VL containing at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 57 or 65. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a humanized variant of VL, said VL comprising at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) the amino acid sequence shown in SEQ ID NO: 57 or 65. In some embodiments, the amino acid sequence of said VL consists of the amino acid sequence shown in SEQ ID NO: 57 or 65.

[0129] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 57 or 65. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a humanized variant of VH and a humanized variant of VL, wherein the VH comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the amino acid sequence shown in SEQ ID NO: 57 or 65. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising VH and VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 56 or 64; and the VL comprises the amino acid sequence of SEQ ID NO: 57 or 65. In some embodiments, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 56 or 64; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO: 57 or 65.

[0130] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising humanized variants of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, having up to fifteen cumulative substitutions in each of said VH and VL outside the CDR region, wherein said substitutions in said VH and VL do not degrade the binding properties of said antibody. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65. In some embodiments, the amino acid sequences of VH and VL consist of the amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, respectively.

[0131] In some embodiments, this disclosure provides an antibody that binds to the same or overlapping epitopes of KLRG1 (e.g., epitopes of human KLRG1) as described herein, such as antibodies comprising the VH and VL amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33 or 42 and 43, respectively.

[0132] In some implementations, the epitopes of the antibody can be determined by, for example, NMR spectroscopy, surface plasmon resonance (BIAcore) methods. ®It can be determined by X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assay and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be performed using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Part 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; ChayenNE (1997) Structure 5: 1269-1274; McPherson A (1976) Journal of Biochemistry 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody: Antigen crystals can be studied using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, refined by Molecular Simulations, Inc.; see, for example, *Meth Enzymol* (1985), vols. 114 and 115, edited by Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) *Acta Crystallographica D-Biocrystalline* 49 (Part 1): 37-60; Bricogne G (1997) *Meth Enzymol* 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) *Acta Crystallographica D-Biocrystalline* 56 (Part 10): ). 1316-1323 (all references cited herein are incorporated herein by reference in their entirety). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) ibid. and Cunningham BC and Wells JA (1989) ibid. In one specific embodiment, alanine scanning mutagenesis studies are used to determine the epitopes of the antibody. Alternatively, conventional techniques such as immunoassays can be used, for example by demonstrating the ability of one antibody to block the binding of another antibody to the target antigen, i.e., competitive binding assays, to identify antibodies that recognize and bind to the same or overlapping epitopes of KLRG1 (e.g., human KLRG1).Competitive binding assays can also be used to determine whether two antibodies have similar binding specificity to epitopes. Competitive binding can be determined in an assay where the immunoglobulin-inhibiting reference antibody being tested specifically binds to a common antigen such as KLRG1 (e.g., human KLRG1). Various types of competitive binding assays are known, such as: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competitive assay (see Stahli C et al., (1983) Enzymatic Methods 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) Journal of Immunology 137:3614-9); solid-phase direct labeling assay, solid-phase direct labeling sandwich assay (see Harlow E and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct labeling RIA using I-125 labeling (see MorelGA et al., (1988) Molecular Immunology 25(1): 7-15); solid-phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176: 546-52); and directly labeled RIA (see Moldenhauer G et al., (1990) Scandinavian Journal of Immunology 32: 77-82), all of which are incorporated herein by reference in their entirety. Typically, such assays involve the use of a purified antigen (e.g., KLRG1, such as human KLRG1) or cells carrying either of these bound to a solid surface, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when a competitive antibody is present in excess, it will inhibit the specific binding of the reference antibody or antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a wide variety of different forms using labeled antigens or labeled antibodies. In a common version of this assay, the antigen is immobilized on a 96-well plate. The ability of an unlabeled antibody to block the binding of the labeled antibody to the antigen is then measured using radioactive or enzyme labeling.For further details, see, for example, Wagener C et al., (1983) *Journal of Immunology* 130: 2308-2315; Wagener C et al., (1984) *Journal of Immunol Methods* 68: 269-274; Kuroki M et al., (1990) *Cancer Research* 50: 4872-4879; Kuroki M et al., (1992) *Immunol Invest* 21: 523-538; Kuroki M et al., (1992) *Hybridoma* 11: 391-407; and *Antibodies: A Laboratory Manual*, edited by Ed Harlow E and Lane D, ibid., pp. 386-389. All of these references are incorporated herein by reference in their entirety.

[0133] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a heavy chain containing the amino acid sequence shown in SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59, or 60. In some embodiments, the amino acid sequence of the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59, or 60.

[0134] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a light chain containing the amino acid sequence shown in SEQ ID NO: 10, 15, 28, 30, 41, or 47. In some embodiments, the amino acid sequence of the light chain consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 15, 28, 30, 41, or 47.

[0135] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a heavy chain and a light chain, the heavy chain and the light chain comprising the amino acid sequences of SEQ ID NO: 9 and 10, 56 and 10, 14 and 15, 57 and 15, 27 and 28, 58 and 28, 27 and 30, 58 and 30, 40 and 41, 59 and 41, 46 and 47 or 60 and 47, respectively.

[0136] In some embodiments, the amino acid sequences of the heavy chain and the light chain are each composed of amino acid sequences selected from the group consisting of: SEQ ID NO: 9 and 10, 56 and 10, 14 and 15, 57 and 15, 27 and 28, 58 and 28, 27 and 30, 58 and 30, 40 and 41, 59 and 41, 46 and 47, or 60 and 47.

[0137] The anti-KLRG1 antigen-binding molecule disclosed herein can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof can be functionally linked to one or more other molecular entities, such as another antibody or antibody fragment (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise), to produce a bispecific or multispecific antibody with a second or additional binding specificity.

[0138] In some embodiments, the antibodies disclosed herein are conjugated to cytotoxic agents, cell inhibitors, toxins, radionuclides, or detectable markers. In some embodiments, the cytotoxic agent is capable of inducing cell death or destruction upon contact with it. In some embodiments, the cell inhibitor is capable of preventing or significantly reducing proliferation and / or inhibiting the activity or function of cells upon contact with it. In some embodiments, the cytotoxic agent or cell inhibitor is a chemotherapeutic agent. In some embodiments, the radionuclide is selected from the group consisting of isotopes. 3 H, 14 C 32 P, 35 S, 36 Cl、 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 90 Y、 99 Tc, 111 In、 117 Lu、 121 I, 124 I, 125 I, 131 I, 198 Au、 211 At、 213 Bi、 225 Ac and 186 Re. In some implementations, the detectable marker includes a fluorescent group or a click chemical connector.

[0139] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In some embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG). 2a and IgG 2b Immunoglobulin molecules.

[0140] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a heavy chain constant region, optionally selected from the group consisting of human IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0141] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), the antibody comprising a heavy chain constant region that is a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region binds to FcγR.

[0142] In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a heavy chain, said light chain comprising a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 48, 49, 50, or 51. In some embodiments, this disclosure provides an antibody that specifically binds to KLRG1 (e.g., human KLRG1), said antibody comprising a heavy chain constant region consisting of the amino acid sequence of SEQ ID NO: 52, 53, 54, or 55.

[0143] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1 according to the EU numbering system) and / or the hinge region (residues 216-230 according to the EU numbering system) of the antibody to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding and / or antigen-dependent cytotoxicity.

[0144] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the antibody described herein, such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered, for example, to facilitate the assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0145] In one specific embodiment, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the constant region of IgG or its FcRn binding fragment (preferably Fc or hinge Fc fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745, all of which are incorporated herein by reference in their entirety, for example, mutations that alter (e.g., reduce or increase) the half-life of the antibody in vivo. In some embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge Fc fragment) to reduce the antibody's half-life in vivo. In other embodiments, one, two, or more amino acid mutations (e.g., substitution, insertion, or deletion) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge Fc fragment) to increase the antibody's half-life in vivo. In one specific embodiment, numbered according to the EU numbering system, the antibody may have one or more amino acid mutations (e.g., substitution) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1). In one specific embodiment, numbered according to the EU numbering system, the constant region of the IgG1 of the antibody described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamate (E) substitution at position 256. See U.S. Patent No. 7,658,921, which is incorporated herein by reference in its entirety. Compared to the wild-type version of the same antibody, this type of IgG (referred to as the “YTE mutant”) has been shown to exhibit a fourfold increase in half-life (see Dall'Acqua WF et al., (2006) Journal of Biochemistry 281: 23514-24, which is incorporated herein by reference in its entirety). In some embodiments, the antibody is numbered according to the EU numbering system and comprises an IgG constant region containing one, two, three or more amino acid substitutions at positions 251-257, 285-290, 308-314, 385-389 and 428-436.

[0146] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1)) and / or the CH3 domain (residues 341-447 of human IgG1 according to the EU numbering system) and / or the hinge region (residues 216-230 according to the EU numbering system) of the antibody described herein to increase or decrease the antibody's affinity for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of antibodies that decrease or increase the antibody's affinity for Fc receptors, and techniques for introducing such mutations into the Fc receptor or fragments thereof, are known to those skilled in the art. Examples of mutations in the Fc receptor of antibodies that can alter the affinity of an antibody for the Fc receptor are described, for example, in the following literature: Smith P et al., (2012) Proceedings of the National Academy of Sciences 109: 6181-6186; U.S. Patent No. 6,737,056; and International Publications No. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0147] In some embodiments, the antibody comprises a heavy chain constant region that is a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγRIIB with a higher affinity than the wild-type heavy chain constant region. In some embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as the variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In some embodiments, according to the EU numbering system, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations: G236D, P238D, S239D, S267E, L328F, and L328E. In some embodiments, according to the EU numbering system, the constant region of the variant human IgG heavy chain contains a set of amino acid mutations selected from the following groups: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the following groups: E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F. In some embodiments, FcγRIIB is expressed on cells selected from the following groups: macrophages, monocytes, B cells, dendritic cells, endothelial cells, and activated T cells.

[0148] In another embodiment, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332, and 396, numbered according to the EU numbering system, can be substituted with different amino acid residues to give the antibody a modified affinity for the effector ligand, but retain the antigen-binding ability of the parent antibody. The effector ligand whose affinity is modified can be, for example, an Fc receptor or the C1 component of complement. This method is further described in detail in U.S. Patents 5,624,821 and 5,648,260, each of which is incorporated herein by reference in its entirety. In some embodiments, the deletion or inactivation of the constant region domain (through point mutations or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety, for describing mutations that result in the deletion or inactivation of the constant region and thereby increase tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) Journal of Biochemistry 276: 6591-604, which is incorporated herein by reference in its entirety). In various embodiments, numbered according to the EU numbering system, one or more of the following mutations can be made in the constant region of the antibody described herein: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; C236 deletion; P238A substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution.

[0149] In some embodiments, mutations selected from the group consisting of D265A, P329A, and combinations thereof can be made in the constant region of the antibody described herein, according to the EU numbering system. In some embodiments, mutations selected from the group consisting of L235A, L237A, and combinations thereof can be made in the constant region of the antibody described herein, according to the EU numbering system. In some embodiments, mutations selected from the group consisting of S267E, L328F, and combinations thereof can be made in the constant region of the antibody described herein, according to the EU numbering system. In some embodiments, mutations selected from the group consisting of S239D, I332E, optionally A330L, and combinations thereof can be made in the constant region of the antibody described herein, according to the EU numbering system. In some embodiments, mutations selected from the group consisting of L235V, F243L, R292P, Y300L, P396L, and combinations thereof can be made in the constant region of the antibody described herein, according to the EU numbering system. In some implementations, numbering is performed according to the EU numbering system, and mutations selected from groups consisting of S267E, L328F, and combinations thereof can be performed in the constant region of the antibody described herein.

[0150] In one specific embodiment, numbered according to the EU numbering system, the antibody described herein comprises a constant region of IgG1 having an amino acid substitution of N297Q or N297A. In some embodiments, numbered according to the EU numbering system, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of D265A, P329A, and combinations thereof. In another embodiment, numbered according to the EU numbering system, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of L234A, L235A, and combinations thereof. In yet another embodiment, numbered according to the EU numbering system, the antibody described herein comprises a constant region of IgG1 having a mutation selected from the group consisting of L234F, L235F, N297A, and combinations thereof. In some embodiments, numbered according to the EU numbering system, the amino acid residues in the constant region of the antibody described herein at positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are not L, L, and D, respectively. This method is described in detail in International Publication No. WO 14 / 108483, which is incorporated herein by reference in its entirety. In some embodiments, the amino acids corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain are F, E, and A, respectively; or A, A, and A.

[0151] In some embodiments, one or more amino acid residues selected from amino acid residues 329, 331, and 322 in the constant region of the antibody described herein, numbered according to the EU numbering system, may be substituted with different amino acid residues, such that the antibody exhibits altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is further described in detail in U.S. Patent No. 6,194,551 (Idusogie et al.), which is incorporated herein by reference in its entirety. In some embodiments, one or more amino acid residues at positions 231 to 238 in the N-terminal region of the CH2 domain of the antibody described herein, numbered according to the EU numbering system, are altered, thereby changing the antibody's ability to bind complement. This method is further described in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In some embodiments, the Fc region of the antibodies described herein is modified to increase the antibody-mediated antibody-dependent cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor by mutating one or more amino acids at the following positions (e.g., introducing amino acid substitutions): 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, according to the EU numbering system. 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This method is further described in International Publication No. WO 00 / 42072, which is incorporated herein by reference in its entirety.

[0152] In some embodiments, the antibodies described herein comprise a modified constant region of IgG1, wherein said modification enhances the antibody-mediated antibody-dependent cytotoxicity (ADCC). In some embodiments, 0.1 µg / mL, 1 µg / mL, or 10 µg / mL antibodies are capable of inducing cell death in at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of KLRG1-expressing cells within 1 hour, 2 hours, or 3 hours, as assessed by methods described herein and / or known to those skilled in the art. In some embodiments, the modified constant region of IgG1, numbered according to the EU numbering system, comprises S239D and I332E substitutions. In some embodiments, the modified constant region of IgG1, numbered according to the EU numbering system, comprises S239D, A330L, and I332E substitutions. In some embodiments, the modified constant region of IgG1, numbered according to the EU numbering system, comprises L235V, F243L, R292P, Y300L, and P396L substitutions. In some embodiments, the antibody is capable of inducing cell death in effector T cells and regulatory T cells (Tregs), wherein the percentage of regulatory T cells undergoing cell death is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times the percentage of effector T cells undergoing cell death.

[0153] In some embodiments, numbered according to the EU numbering system, the antibodies described herein contain the constant region of the IgG4 antibody, and the serine at amino acid residue 228 of the heavy chain is replaced by proline.

[0154] In some implementations, any constant region mutation or modification described herein may be introduced into one or both heavy chain constant regions of an antibody having two heavy chain constant regions as described herein.

[0155] Pharmaceutical Composition This document provides compositions comprising, for example, a physiologically acceptable carrier, excipient, or stabilizer at a desired concentration of the disclosed anti-KLRG1 antibody (see, for example, Remington's Pharmaceutical Sciences (1990), Mack Publishing Co., Easton, PA). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; such as serum... Proteins such as albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0156] In one embodiment, the pharmaceutical composition comprises the anti-KLRG1 antibody disclosed herein, and optionally one or more other prophylactic or therapeutic agents (in a pharmaceutically acceptable carrier form). In some embodiments, the antibody is the sole active ingredient contained in the pharmaceutical composition. The pharmaceutical compositions described herein can be used to reduce or block KLRG1 (e.g., human KLRG1) activity and to treat conditions such as cancer. In some embodiments, this disclosure relates to pharmaceutical compositions comprising the anti-KLRG1 antibody of this disclosure used as a medicament. In another embodiment, this disclosure relates to pharmaceutical compositions of this disclosure used in methods for treating cancer.

[0157] Pharmaceutically acceptable carriers used in parenteral preparations include aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, barrier agents or chelating agents, and other pharmaceutically acceptable substances. Examples of aqueous carriers include sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, dextran, and lactated Ringer's injection. Non-aqueous parenteral carriers include non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents at concentrations that inhibit bacteria or fungi can be added to parenteral preparations packaged in multi-dose containers, including phenol or cresol, mercury, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride, and benzyl chloride. Isotonic agents include sodium chloride and dextran. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspensions and dispersants include sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN). ® 80). Metal ion blocking or chelating agents include EDTA. Drug carriers also include ethanol, polyethylene glycol, and propylene glycol for water miscible media; and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

[0158] Pharmaceutical compositions can be formulated for any route of administration to the subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal, and parenteral administration. Parenteral administration characterized by subcutaneous, intramuscular, or intravenous injection is also considered herein. Injectable formulations can be prepared in conventional forms as liquid solutions or suspensions, solid forms suitable for dissolving or suspending in a liquid prior to injection, or as emulsions. Injectables, solutions, and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextran, glycerol, or ethanol. Additionally, if desired, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, pH buffers, stabilizers, solubilizers, and other such agents, such as sodium acetate, sorbitol monolaurate, triethanolamine oleate, and cyclodextrin.

[0159] Preparations of antibodies for parenteral administration include sterile solutions for injection, sterile dried soluble products such as lyophilized powders (including subcutaneous tablets) prepared for use only before combination with a solvent, sterile dried insoluble products prepared for injection, sterile dried emulsions prepared for use only before combination with a carrier, and sterile emulsions. Solutions may be aqueous or non-aqueous.

[0160] If administered intravenously, suitable carriers include physiological saline or phosphate-buffered saline (PBS), as well as solutions containing thickeners and solubilizers such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[0161] Prepare topical mixtures containing antibodies as described for both topical and systemic application. The resulting mixtures may be solutions, suspensions, emulsions, etc., and may be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, rinses, sprays, suppositories, bandages, skin patches, or any other formulation suitable for topical application.

[0162] The anti-KLRG1 antibodies disclosed herein can be formulated as aerosols for topical application (e.g., by inhalation) (see, for example, U.S. Patents 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids that can be used to treat inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be, alone or in combination with an inert carrier such as lactose, in aerosol or solution form for use in nebulizers or as a fine powder for inhalation. In such cases, the particle diameter of the formulation will be less than 50 micrometers in some embodiments and less than 10 micrometers in others.

[0163] The anti-KLRG1 antibody disclosed herein can be formulated for local or topical applications, such as topical application to the skin and mucous membranes (e.g., eyes) in the form of gels, creams, and lotions, as well as for ocular or intracranial or spinal applications. Transdermal delivery and topical application, including ocular or mucous membrane administration or inhalation therapy, are considered. Nasal solutions containing the antibody alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[0164] Transdermal patches, including iontophoresis therapy and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0165] In some embodiments, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder, which can be reconstituted for administration in the form of a solution, emulsion, and other mixture. The lyophilized powder can also be reconstituted and formulated into a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that can be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, and in some embodiments, the buffer is approximately neutral pH. The solution is then sterilely filtered and then lyophilized under standard conditions known to those skilled in the art to provide the desired formulation. In some embodiments, the resulting solution is dispensed into vials for lyophilization. Each vial will contain a single or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature. The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. To perform reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends on the compound selected. Such amounts can be determined empirically.

[0166] The anti-KLRG1 antibody disclosed herein and other compositions provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of a subject to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated herein for use in the compositions of the invention. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In one specific embodiment, the antibody described herein targets a tumor.

[0167] Compositions intended for in vivo administration can be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0168] Usage and Purpose On the other hand, this disclosure provides a method of treating a subject using the anti-KLRG1 antibody disclosed herein. The anti-KLRG1 antibody disclosed herein can be used to treat any disease or condition in a subject that benefits from reduced KLRG1 (e.g., human KLRG1) function. In some embodiments, the disease or condition is resistant to checkpoint-targeting agents (e.g., antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, or antagonistic anti-PD-1 antibodies). In some embodiments, the disease or condition relapses after treatment with a checkpoint-targeting agent (e.g., antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, or antagonistic anti-PD-1 antibodies).

[0169] The anti-KLRG1 antibody disclosed herein is particularly suitable for suppressing the immune system's tolerance to tumors and can therefore be used as an immunotherapy for patients with cancer. For example, in some embodiments, this disclosure provides a way to increase the number of T cells (e.g., CD8+) that respond to antigens in a subject. + Cytotoxic T cells, CD4 + Methods for activating helper T cells, NKT cells, effector T cells, or memory T cells, the methods comprising administering to the subject an effective amount of an anti-KLRG1 antibody or a pharmaceutical composition thereof as disclosed herein. In some embodiments, this disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody or pharmaceutical composition as disclosed herein.

[0170] Cancers that can be treated with the anti-KLRG1 antibodies or pharmaceutical compositions disclosed herein include, but are not limited to, solid tumors, hematologic malignancies (e.g., leukemia, lymphoma, myeloma, such as multiple myeloma), and metastatic lesions. In some embodiments, the cancer is a solid tumor. Examples of solid tumors include malignant tumors such as sarcomas and carcinomas, such as adenocarcinomas of various organ systems, such as adenocarcinomas affecting the lungs, breasts, ovaries, lymph nodes, gastrointestinal tract (e.g., colon), anus, genitals and genitourinary tract (e.g., kidneys, urothelial cells, bladder cells, prostate), pharynx, CNS (e.g., brain, nerves, or glial cells), head and neck, skin (e.g., melanoma), and pancreas, as well as adenocarcinomas including malignant tumors such as colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), small bowel cancer, and esophageal cancer. The cancer can be early, intermediate, advanced, or metastatic. In some embodiments, the cancer is resistant to checkpoint-targeting agents (e.g., antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, or antagonistic anti-PD-1 antibodies). In some embodiments, the cancer recurs after treatment with checkpoint-targeting agents (e.g., antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, or antagonistic anti-PD-1 antibodies).

[0171] In some implementations, the cancer is selected from lung cancer (e.g., lung adenocarcinoma or non-small cell lung cancer (NSCLC) (e.g., NSCLC with squamous and / or non-squamous histology, or NSCLC adenocarcinoma)), melanoma (e.g., advanced melanoma), kidney cancer (e.g., renal cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), myeloma (e.g., multiple myeloma), prostate cancer, breast cancer (e.g., breast cancer that does not express one, two, or all of estrogen receptors, progesterone receptors, or Her2 / neu, such as triple-negative breast cancer), ovarian cancer, colorectal cancer, pancreatic cancer, head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), anal cancer, gastroesophageal cancer (e.g., esophageal squamous cell carcinoma), mesothelioma, nasopharyngeal carcinoma, thyroid cancer, cervical cancer, epithelial cancer, peritoneal cancer, or lymphoproliferative disorders (e.g., post-transplant lymphoproliferative disorders).

[0172] In some implementations, the cancer is a blood cancer, such as leukemia, lymphoma, or myeloma. In some implementations, the cancer is leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), acute myeloblastic leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), or hairy cell leukemia. In some embodiments, the cancer is a lymphoma, such as B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), activated B-cell-like (ABC) diffuse large B-cell lymphoma, germinal center B-cell (GCB) diffuse large B-cell lymphoma, mantle cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, relapsed follicular non-Hodgkin lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, or extranodal marginal zone lymphoma. In some embodiments, the cancer is a myeloma, such as multiple myeloma.

[0173] In another implementation, the cancer is selected from carcinoma (e.g., advanced or metastatic cancer), melanoma, or lung cancer, such as non-small cell lung cancer.

[0174] In some implementations, the cancer is lung cancer, such as lung adenocarcinoma, non-small cell lung cancer, or small cell lung cancer.

[0175] In some embodiments, the cancer is melanoma, such as advanced melanoma. In some embodiments, the cancer is advanced or unresectable melanoma that does not respond to other therapies. In other embodiments, the cancer is melanoma with a BRAF mutation (e.g., BRAF V600 mutation). In still other embodiments, the anti-KLRG1 antibody or pharmaceutical composition disclosed herein is administered after treatment with an anti-CTLA-4 antibody (e.g., ipilimumab) with or without a BRAF inhibitor (e.g., vemurafenib or dabrafenib).

[0176] In another implementation, the cancer is liver cancer, such as advanced liver cancer, with or without a viral infection, such as chronic viral hepatitis.

[0177] In another implementation, the cancer is prostate cancer, such as advanced prostate cancer.

[0178] In yet another implementation, the cancer is myeloma, such as multiple myeloma.

[0179] In yet another implementation, the cancer is kidney cancer, such as renal cell carcinoma (RCC) (e.g., metastatic RCC, clear cell renal cell carcinoma (CCRCC), or papillary renal cell carcinoma).

[0180] In yet another implementation, the cancer is selected from lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, leukemia, or metastatic lesions of cancer.

[0181] In some embodiments, these methods further include administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiation therapy agent, or a checkpoint target. In some embodiments, the chemotherapeutic agent is a hypomethylating agent (e.g., azacitidine). In some embodiments, the chemotherapeutic agent is a DNA damage inducer (e.g., gemcitabine). In some embodiments, the checkpoint target is selected from the group consisting of: antagonist-type anti-CTLA-4 antibody, antagonist-type anti-PD-L1 antibody, antagonist-type anti-PD-L2 antibody, antagonist-type anti-PD-1 antibody, antagonist-type anti-TIM-3 antibody, antagonist-type anti-LAG-3 antibody, antagonist-type anti-VISTA antibody, antagonist-type anti-CD96 antibody, antagonist-type anti-CEACAM1 antibody, agonist-type anti-CD137 antibody, agonist-type anti-GITR antibody, and agonist-type anti-OX40 antibody. In some embodiments, the checkpoint target is selected from the group consisting of: antagonistic anti-CTLA-4 antibodies, antagonistic anti-PD-L1 antibodies, antagonistic anti-PD-L2 antibodies, and antagonistic anti-PD-1 antibodies, wherein the KLRG1 (e.g., human KLRG1) antibody or pharmaceutical composition disclosed herein synergizes with the checkpoint target.

[0182] In some embodiments, this disclosure relates to antibody and / or pharmaceutical compositions of this disclosure used in methods of this disclosure, wherein the methods further include administering an additional therapeutic agent to the subject. In some embodiments, this disclosure relates to (a) an antibody and / or pharmaceutical composition of this disclosure, and (b) an additional therapeutic agent used as a medicine. In some embodiments, this disclosure relates to (a) an antibody and / or pharmaceutical composition of this disclosure, and (b) an additional therapeutic agent used in methods for treating cancer. In other embodiments, this disclosure relates to pharmaceutical compositions, kits, or kit-of-parts comprising (a) an antibody and / or pharmaceutical composition of this disclosure and (b) an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapy agent, or a checkpoint targeting agent.

[0183] In some embodiments, the subject is administered a combination of the anti-KLRG1 antibody and the anti-PD-L1 antibody disclosed herein. In some embodiments, the subject is administered a combination of the anti-KLRG1 antibody and the anti-PD-1 antibody disclosed herein.

[0184] In some embodiments, the anti-PD-1 antibody is used in the methods disclosed herein. In some embodiments, the anti-PD-1 antibody is nivolumab, also known as BMS-936558 or MDX1106, developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-1 antibody is pembrolizumab, also known as lambolizumab or MK-3475, developed by Merck & Co. In some embodiments, the anti-PD-1 antibody is pidilizumab, also known as CT-011, developed by CureTech. In some embodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514, developed by Medimmune. In some embodiments, the anti-PD-1 antibody is PDR001, developed by Novartis Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is REGN2810, developed by Regeneron Pharmaceuticals. In some embodiments, the anti-PD-1 antibody is PF-06801591, developed by Pfizer. In some embodiments, the anti-PD-1 antibody is BGB-A317, developed by BeiGene. In some embodiments, the anti-PD-1 antibody is TSR-042, developed by AnaptysBio and Tesaro. In some embodiments, the anti-PD-1 antibody is SHR-1210, developed by Hengrui Medicine.

[0185] Further non-limiting examples of anti-PD-1 antibodies that can be used for the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 6,808,710; U.S. Patent No. 7,332,582; U.S. Patent No. 7,488,802; U.S. Patent No. 8,008,449; U.S. Patent No. 8,114,845; U.S. Patent No. 8,168,757; U.S. Patent No. 8,354,509; U.S. Patent No. 8,686,119; U.S. Patent No. 8,735,553; U.S. Patent No. 8,747,847; U.S. Patent No. 8,779,105; U.S. Patent No. 8,927,697; U.S. Patent No. 8,993,731; U.S. Patent No. 9,102,727; U.S. Patent No. 9,205,148; U.S. Publication No. US US Patent Application No. 2013 / 0202623 A1; US ​​Publication No. 2013 / 0291136 A1; US ​​Publication No. 2014 / 0044738 A1; US ​​Publication No. 2014 / 0356363 A1; US ​​Publication No. 2016 / 0075783 A1; and PCT Publication No. 2013 / 033091 A1; PCT Publication No. 2015 / 036394 A1; PCT Publication No. 2014 / 179664 A2; PCT Publication No. 2014 / 209804 A1; PCT Publication No. 2014 / 206107 A1; PCT Publication No. 2015 / 058573 A1; PCT Publication No. 2015 / 085847 A1; PCT Publication No. WO 2015 / 200119 A1; PCT Publication No. WO 2016 / 015685 A1; and PCT Publication No. WO 2016 / 020856 A1.

[0186] In some embodiments, the anti-PD-L1 antibody is used in the methods disclosed herein. In some embodiments, the anti-PD-L1 antibody is atezolizumab, developed by Genentech. In some embodiments, the anti-PD-L1 antibody is durvalumab, developed by AstraZeneca, Celgene, and Medimmune. In some embodiments, the anti-PD-L1 antibody is avelumab, also known as MSB0010718C, developed by Merck Serono and Pfizer. In some embodiments, the anti-PD-L1 antibody is MDX-1105, developed by Bristol-Myers Squibb. In some embodiments, the anti-PD-L1 antibody is AMP-224, developed by Amplimmune and GSK.

[0187] Non-limiting examples of anti-PD-L1 antibodies that can be used in the treatments disclosed herein are disclosed in the following patents and patent applications, all of which are incorporated herein by reference in their entirety for all purposes: U.S. Patent No. 7,943,743; U.S. Patent No. 8,168,179; U.S. Patent No. 8,217,149; U.S. Patent No. 8,552,154; U.S. Patent No. 8,779,108; U.S. Patent No. 8,981,063; U.S. Patent No. 9,175,082; U.S. Publication No. US 2010 / 0203056 A1; U.S. Publication No. US 2003 / 0232323 A1; U.S. Publication No. US 2013 / 0323249 A1; U.S. Publication No. US 2014 / 0341917 A1; U.S. Publication No. US 2014 / 0044738 ... 2014 / 0044738 A1; U.S. 2014 / 00447 US Patent No. 2015 / 0203580 A1; US ​​Publication No. 2015 / 0225483 A1; US ​​Publication No. 2015 / 0346208 A1; US ​​Publication No. 2015 / 0355184 A1; PCT Publication No. 2014 / 100079A1; PCT Publication No. 2014 / 022758 A1; PCT Publication No. 2014 / 055897 A2; PCT Publication No. 2015 / 061668 A1; PCT Publication No. 2015 / 109124 A1; PCT Publication No. 2015 / 195163 A1; PCT Publication No. 2016 / 000619 A1; and PCT Publication No. 2016 / 030350 A1.

[0188] In some embodiments, an anti-CTLA-4 antibody is used in the methods disclosed herein. In some embodiments, the anti-CTLA-4 antibody is ipilimumab developed by Bristol-Myers Squibb.

[0189] In some embodiments, the subject is administered the anti-KLRG1 antibody disclosed herein in combination with a compound that targets immunomodulatory enzymes such as IDO (indoleamine-(2,3)-dioxygenase) and / or TDO (tryptophan 2,3-dioxygenase). Therefore, in some embodiments, the additional therapeutic agent is a compound that targets immunomodulatory enzymes such as an inhibitor of tryptophan 2,3-dioxygenase (IDO). In some embodiments, such compounds are selected from the group consisting of: epacadostat (Incyte Corp; see, for example, WO 2010 / 005958, which is incorporated herein by reference in its entirety), F001287 (Flexus Biosciences / Bristol-Myers Squibb), indoximod (NewLink Genetics), and NLG919 (NewLink Genetics). In some embodiments, the compound is epacadostat. In another embodiment, the compound is F001287. In another embodiment, the compound is indomod. In another embodiment, the compound is NLG919. In one specific embodiment, a combination of the anti-KLRG1 antibody disclosed herein and an IDO inhibitor is administered to a subject to treat cancer. The IDO inhibitors for treating cancer as described herein are available in solid dosage forms such as tablets, pills, or capsules, wherein the pharmaceutical composition comprises an IDO inhibitor and a pharmaceutically acceptable excipient. Therefore, the antibody as described herein and the IDO inhibitor as described herein can be administered alone, sequentially, or simultaneously as separate dosage forms. In some embodiments, the antibody is administered parenterally, and the IDO inhibitor is administered orally. In a particular embodiment, the inhibitor is selected from the group consisting of: icardostat (Inset), F001287 (Fox Biosciences / Bristol-Myers Squibb), indomod (Newlink Genetics), and NLG919 (Newlink Genetics). Ekadostat has been described in PCT Publication WO 2010 / 005958, which is incorporated herein by reference in its entirety for all purposes. In some embodiments, the inhibitor is icadocostat. In another embodiment, the inhibitor is F001287. In yet another embodiment, the inhibitor is indomod. In yet another embodiment, the inhibitor is NLG919.

[0190] In some embodiments, the subject is administered a combination of the anti-KLRG1 antibody disclosed herein and a vaccine. The vaccine may be, for example, a peptide vaccine, a DNA vaccine, or an RNA vaccine.

[0191] In some embodiments, the subject is administered a combination of the anti-KLRG1 antibody disclosed herein with an adjuvant. Various adjuvants may be used depending on the treatment context. Non-limiting examples of suitable adjuvants include, but are not limited to, complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), montanide ISA (incomplete Seppic adjuvant), Ribi adjuvant system (RAS), Titer Max, muramyl peptide, Syntex Adjuvant Formulation (SAF), alum (aluminum hydroxide and / or aluminum phosphate), aluminum salt adjuvants, Gerbu ® Adjuvants, nitrocellulose-adsorbed antigens, encapsulated or embedded antigens, 3D-O-acylated monophosphoryllipid A (3D-MPL), immunostimulatory oligonucleotides, Toll-like receptor (TLR) ligands, mannan-binding lectin (MBL) ligands, STING agonists, immunostimulatory complexes such as saponins, Quil A, QS-21, QS-7, ISCOMATRIX, etc. Other adjuvants include CpG oligonucleotides and double-stranded RNA molecules such as poly(A) and poly(U). Combinations of the above adjuvants may also be used. See, for example, U.S. Patent Nos. 6,645,495, 7,029,678, and 7,858,589, all of which are incorporated herein by reference in their entirety. In some embodiments, the adjuvant used herein is QS-21 STIMULON.

[0192] In some embodiments, a combination of the anti-KLRG1 antibody disclosed herein and an additional therapeutic agent comprising a TCR is administered to a subject. In some embodiments, the additional therapeutic agent is a soluble TCR. In some embodiments, the additional therapeutic agent is cells expressing a TCR. Therefore, in some embodiments, this disclosure relates to a combination of the antibody and / or pharmaceutical composition of this disclosure with an additional therapeutic agent comprising a TCR, used as a medicine and / or used in methods for treating cancer.

[0193] In some embodiments, the subject is administered a combination of the anti-KLRG1 antibody disclosed herein and cells expressing a chimeric antigen receptor (CAR). In some embodiments, the cells are T cells.

[0194] In some embodiments, a combination of the anti-KLRG1 antibody disclosed herein and a TCR mimic antibody is administered to the subject. In some embodiments, the TCR mimic antibody is an antibody that specifically binds to a peptide-MHC complex. For non-limiting examples of TCR mimic antibodies, see, for example, U.S. Patent No. 9,074,000 and U.S. Publications Nos. US 2009 / 0304679A1 and US 2014 / 0134191A1, all of which are incorporated herein by reference in their entirety.

[0195] In some embodiments, the subject is administered an anti-KLRG1 antibody disclosed herein in combination with a BiTE (e.g., as described in WO2005061547A2, which is incorporated herein by reference in its entirety) and / or a biaffinity retargeting antibody (DART) (e.g., as described in WO 2012162067A2, which is incorporated herein by reference in its entirety). In some embodiments, the BiTE and / or DART specifically bind to tumor-associated antigens (e.g., peptides overexpressed in tumors, peptides derived from tumor viruses, peptides containing tumor-specific post-translational modifications, peptides specifically mutated in tumors) and molecules on effector cells (e.g., CD3 or CD16). In some embodiments, the tumor-associated antigen is EGFR (e.g., human EGFR), optionally wherein the BiTE and / or DART comprises the VH and VL sequences of cetuximab. In some embodiments, the tumor-associated antigen is Her2 (e.g., human Her2), optionally wherein BiTE and / or DART comprises the VH and VL sequences of trastuzumab. In some embodiments, the tumor-associated antigen is CD20 (e.g., human CD20). In some embodiments, BiTE and / or DART specifically bind to CD3 and Her2. In some embodiments, the tumor-associated antigen is CD19 (e.g., human CD19). In some embodiments, BiTE and / or DART specifically bind to CD3 and CD19, optionally wherein BiTE and / or DART comprises the VH and VL sequences of bonnetumab.

[0196] Anti-KLRG1 antibodies and other therapeutic agents (e.g., chemotherapeutic agents, radiotherapy agents, checkpoint targets, IDO inhibitors, vaccines, adjuvants, soluble TCRs, TCR-expressing cells, chimeric antigen receptor-expressing cells, and / or TCR mimic antibodies) can be administered alone, sequentially, or simultaneously as separate dosage forms. In some embodiments, the anti-KLRG1 antibody is administered parenterally, and the IDO inhibitor is administered orally.

[0197] The antibody or pharmaceutical compositions described herein can be delivered to subjects via a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, intraarterial, and subcutaneous routes. Lung administration can also be employed, for example, by using an inhaler or nebulizer and a nebulizer formulation. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered subcutaneously or intravenously. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intraarterially. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered intratumorally. In some embodiments, the antibody or pharmaceutical compositions described herein are delivered to tumor-draining lymph nodes.

[0198] The amount of antibody or composition that will be effective in the treatment and / or prevention of the condition will depend on the nature of the disease and can be determined by standard clinical techniques.

[0199] The precise dosage used in the composition will also depend on the route of administration and the severity of the infection or disease it causes, and should be determined based on the practitioner's judgment and the individual subject's circumstances. For example, the effective dosage can also vary depending on the route of administration, target site, patient's physiological state (including age, weight, and health), whether the patient is human or animal, and whether other drugs or treatments administered are prophylactic or therapeutic. Typically, the patient is human, but treatment can also be given to non-human mammals, including transgenic mammals. The therapeutic dose can be titrated in an optimal manner to optimize safety and efficacy.

[0200] The anti-KLRG1 antibody described herein can also be used to determine the level of KLRG1 (e.g., human KLRG1) protein in biological samples using classical immunohistochemical methods known to those skilled in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; iodine (… 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99Radioactive isotopes such as Tc; luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, as well as biotin. Such labels can be used to label the antibodies described herein. Alternatively, a second antibody recognizing the anti-KLRG1 antibody described herein can be labeled, and the second antibody can be used in combination with the anti-KLRG1 antibody to detect KLRG1 (e.g., human KLRG1) protein levels. Therefore, in some embodiments, this disclosure relates to the use of the anti-KLRG1 antibody of this disclosure in the in vitro detection of KLRG1 (e.g., human KLRG1) protein in biological samples. In other embodiments, this disclosure relates to the use of the anti-KLRG1 antibody of this disclosure in the in vitro determination and / or detection of KLRG1 (e.g., human KLRG1) protein levels in biological samples, optionally wherein the anti-KLRG1 antibody is conjugated with a radionuclide or a detectable label, and / or carries a label described herein, and / or is used using immunohistochemical methods.

[0201] Determining the expression level of KLRG1 (e.g., human KLRG1) protein aims to include a qualitative or quantitative measurement or estimation of the level of KLRG1 (e.g., human KLRG1) protein in a first biological sample, either directly (e.g., by determining or estimating an absolute protein level) or relatively (e.g., by comparing it with disease-associated protein levels in a second biological sample). The expression level of the KLRG1 (e.g., human KLRG1) peptide in the first biological sample can be measured or estimated and compared with a standard KLRG1 (e.g., human KLRG1) protein level, which is derived from a second biological sample obtained from a disease-free individual or determined by the average level of a disease-free population. As will be understood in the art, once the "standard" KLRG1 (e.g., human KLRG1) peptide level is known, it can be repeatedly used as a comparison standard. Therefore, in another embodiment, this disclosure relates to an in vitro method for determining and / or detecting the level of KLRG1 protein, such as human KLRG1 protein, in a biological sample, the method comprising qualitatively or quantitatively measuring or estimating the level of KLRG1 protein, such as human KLRG1 protein, in the biological sample by immunohistochemical methods.

[0202] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cellular source that potentially expresses KLRG1 (e.g., human KLRG1). Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans or cynomolgus monkeys) are well known in the art. Biological samples include peripheral blood mononuclear cells (PBMCs).

[0203] The anti-KLRG1 antibody described herein can be used for prognostic, diagnostic, monitoring, and screening applications, including in vitro and in vivo applications well-known and standard to those skilled in the art and based on this specification. Prognostic, diagnostic, monitoring, and screening assays and kits for in vitro assessment and evaluation of immune system status and / or immune response can be used to predict, diagnose, and monitor patient samples, including those known to have or suspected of having immune system dysfunction or those concerning expected or desired immune system responses, antigen responses, or vaccine responses. Assessment and evaluation of immune system status and / or immune response can also be used to determine a patient's suitability for a drug clinical trial or for a specific chemotherapeutic agent, radiotherapy agent, or antibody (including combinations thereof) relative to the administration of different agents or antibodies. This type of prognostic and diagnostic monitoring and assessment has been utilized in practice with antibodies against the HER2 protein in breast cancer (HercepTest). TM Dako, where the assay is also used to evaluate the use of Herceptin ® Patients undergoing antibody therapy. In vivo application includes targeted cell therapy and immunomodulation, as well as radiographic imaging of immune responses. Therefore, in some embodiments, this disclosure relates to the anti-KLRG1 antibody and / or pharmaceutical composition of this disclosure as a diagnostic agent. In some embodiments, this disclosure relates to the anti-KLRG1 antibody and / or pharmaceutical composition of this disclosure used in methods for predicting, diagnosing, and / or monitoring subjects with or suspected of having immune system dysfunction and / or with respect to expected or desired immune system responses, antigen responses, or vaccine responses. In another embodiment, this disclosure relates to the use of the anti-KLRG1 antibody of this disclosure in predicting, diagnosing, and / or monitoring subjects with or suspected of having immune system dysfunction and / or with respect to expected or desired immune system responses, antigen responses, or vaccine responses by in vitro assaying and / or detecting human KLRG1 protein levels in a biological sample of a subject.

[0204] In some embodiments, anti-KLRG1 antibodies can be used for immunohistochemistry of biopsy samples. In some embodiments, the method is an in vitro method. In another embodiment, anti-KLRG1 antibodies can be used to detect levels of KLRG1 (e.g., human KLRG1), or levels of cells containing KLRG1 (e.g., human KLRG1) on their membrane surface, levels that may be associated with certain disease symptoms. The anti-KLRG1 antibodies described herein may carry detectable or functional markers and / or may be conjugated to radionuclides or detectable markers. When fluorescent markers are used, the specific binding members can be identified and quantified using currently available microscopy and fluorescence activated cell sorting analysis (FACS) or a combination of both methods known in the art. The anti-KLRG1 antibodies described herein may carry fluorescent markers or may be conjugated thereto. Exemplary fluorescent markers include, for example, reactive and conjugated probes, such as aminocoumarins, fluorescein and Texas Red, Alexa fluorescent dyes, Cy dyes and DyLight dyes. Anti-KLRG1 antibodies may carry radiolabels or radionuclides or may be conjugated thereto, such as isotopes. 3 H, 14 C 32 P, 35 S, 36 Cl、 51 Cr 57 Co、 58 Co、 59 Fe、 67 Cu、 90 Y、 99 Tc, 111 In、 117 Lu、 121 I, 124 I, 125 I, 131 I, 198 Au、 211 At、 213 Bi、 225 Ac and 186Re. When using radiolabeling, the specific binding of anti-KLRG1 antibody to KLRG1 (e.g., human KLRG1) can be identified and quantified using currently available counting procedures known in the art. In the case of an enzyme-based label, detection can be performed using any of the currently employed colorimetric, spectrophotometric, fluorescence spectrophotometric, electrotitration, or gas quantification techniques known in the art. This can be achieved by contacting a sample or control sample with the anti-KLRG1 antibody under conditions that allow for the formation of a complex between the anti-KLRG1 antibody and KLRG1 (e.g., human KLRG1). Any complexes formed between the anti-KLRG1 antibody and KLRG1 (e.g., human KLRG1) are detected, and said complexes are compared between the sample and the control. Given the specific binding of the anti-KLRG1 antibody to KLRG1 (e.g., human KLRG1) described herein, the anti-KLRG1 antibody can be used for the specific detection of KLRG1 (e.g., human KLRG1). The anti-KLRG1 antibody described herein can also be used to purify KLRG1 (e.g., human KLRG1) via immunoaffinity purification. This document also includes assay systems that can be prepared in the form of assay kits, reagent kits, or multipart kits for the quantitative analysis of the presence, for example, of KLRG1 (e.g., human KLRG1) / KLRG1 (e.g., human KLRG1) ligand complexes. The systems, assay kits, reagent kits, or multipart kits may contain labeled components, such as labeled antibodies, and one or more additional immunochemical reagents.

[0205] Polynucleotides, vectors and methods for antibody production On the other hand, this document provides: polynucleotides comprising nucleotide sequences or fragments thereof encoding antibodies or portions thereof as described herein (e.g., VL and / or VH; and light and / or heavy chains), said antibodies or portions thereof specifically binding to KLRG1 (e.g., human KLRG1) antigens; and vectors, for example, comprising such polynucleotides for use in host cells (e.g., *Escherichia coli*). E. coli Vectors for recombinant expression in host cells, such as mammalian cells. This document provides polynucleotides and vectors comprising nucleotide sequences encoding the heavy and / or light chains of any antibody provided herein, and vectors comprising such polynucleotide sequences for efficient expression, for example, in host cells, such as mammalian cells.

[0206] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a molecule isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules (e.g., mice or humans). Furthermore, "isolated" nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material, or substantially free of culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the term "substantially free" includes formulations of polynucleotide or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (especially less than about 10%) of other materials, such as cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals. In one specific embodiment, one or more nucleic acid molecules encoding the antibodies described herein are isolated or purified.

[0207] In certain respects, this document provides polynucleotides comprising nucleotide sequences encoding antibodies that specifically bind to a KLRG1 (e.g., human KLRG1) peptide and comprise the amino acid sequences described herein, as well as antibodies that compete with such antibodies for binding to a KLRG1 (e.g., human KLRG1) peptide (e.g., in a dose-dependent manner), or antibodies that bind to an epitope identical to that of such antibodies.

[0208] In some aspects, this document provides polynucleotides comprising nucleotide sequences encoding a light chain or heavy chain of an antibody described herein. The polynucleotide may comprise a nucleotide sequence encoding a light chain (see, for example, Table 1) encoding a VL FR and CDR of an antibody described herein, or a nucleotide sequence encoding a heavy chain (see, for example, Table 1) encoding a VH FR and CDR of an antibody described herein. In some embodiments, the polynucleotide encodes the VH, VL, heavy chain, and / or light chain described herein. In another embodiment, the polynucleotide encodes a first VH and a first VL described herein. In another embodiment, the polynucleotide encodes a second VH and a second VL described herein. In another embodiment, the polynucleotide encodes a first heavy chain and a first light chain described herein. In another embodiment, the polynucleotide encodes a second heavy chain and a second light chain described herein. In yet another embodiment, the polynucleotide encodes the VH and / or VL or the heavy chain and / or light chain of an antibody described herein.

[0209] This document also provides polynucleotides encoding anti-KLRG1 antibodies, optimized, for example, through codon / RNA optimization, substitution with heterologous signal sequences, and elimination of unstable elements in the mRNA. Therefore, optimized nucleic acids for recombinant expression encoding anti-KLRG1 antibodies or fragments thereof (e.g., light chain, heavy chain, VH domain, or VL domain) can be generated by adapting optimization methods described in the following literature: e.g., U.S. Patent Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, all of which are incorporated herein by reference in their entirety. For example, potential splicing sites and unstable elements (e.g., A / T or A / U enriched elements) in the RNA can be mutated without altering the amino acids encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These alterations exploit the degeneracy of the genetic code, such as the use of alternative codons for the same amino acid. In some implementations, it may be desirable to change one or more codons to encode a conserved mutation, such as a similar amino acid having a similar chemical structure and properties and / or function to the original amino acid. Such methods can increase the expression of anti-KLRG1 antibodies or fragments thereof by at least 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times or more relative to the expression of anti-KLRG1 antibodies encoded by unoptimized polynucleotides.

[0210] In some embodiments, an optimized polynucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof (e.g., VL domain and / or VH domain) as described herein can hybridize with an antisense (e.g., complementary) polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof (e.g., VL domain and / or VH domain) as described herein. In specific embodiments, an optimized nucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof as described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof as described herein under high-tightness conditions. In specific embodiments, an optimized nucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof as described herein hybridizes with an antisense polynucleotide sequence encoding an unoptimized polynucleotide sequence encoding an anti-KLRG1 antibody or a fragment thereof as described herein under high-tightness, medium-tightness, or low-tightness hybridization conditions. Information regarding hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. 2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0211] Polynucleotides can be obtained and their nucleotide sequences determined by any method known in the art. Methods well-known in the art can be used to determine the nucleotide sequences encoding antibodies described herein (e.g., antibodies described in Table 1 and modified versions of these antibodies), i.e., assembling nucleotide codons known to encode specific amino acids in such a manner to generate nucleic acids encoding antibodies. Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, which is incorporated herein by reference in its entirety), briefly involving the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating those oligonucleotides, and then amplifying the ligated oligonucleotides by PCR.

[0212] Alternatively, polynucleotides encoding the antigen-binding region or antibody described herein can be generated from nucleic acids from a suitable source (e.g., hybridoma) using methods well-known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification can be performed using synthetic primers that hybridize to the 3' and 5' ends of a known sequence, using genomic DNA obtained from hybridoma cells that produce the antibody of interest. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding the light chain and / or heavy chain of an antibody. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions of an antibody. The amplified nucleic acids can be cloned into a vector for expression in a host cell and further cloned.

[0213] If a clone containing a nucleic acid encoding a specific antigen-binding region or antibody is unavailable, but the sequence of the antigen-binding region or antibody molecule is known, the nucleic acid encoding an immunoglobulin can be obtained by PCR amplification using synthetic primers capable of hybridizing to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to the specific gene sequence, or by obtaining a cDNA clone encoding an immunoglobulin from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from nucleic acids (preferably poly A+ RNA) isolated from any tissue or cell expressing the antibody (such as hybridoma cells selected for expressing the antibody described herein), to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a reproducible cloning vector using any method well known in the art.

[0214] DNA encoding the anti-KLRG1 (e.g., human KLRG1) antibody described herein can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of an anti-KLRG1 (e.g., human KLRG1) antibody. Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells (e.g., CHO cells from CHO GSSystem™ (Lonza),) or myeloma cells, to obtain the synthesis of anti-KLRG1 antibodies in recombinant host cells.

[0215] To generate a complete antibody or antigen-binding region, the VH or VL sequence in the scFv clone can be amplified using PCR primers containing the VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region (e.g., human γ1 or human γ4 constant region), and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region (e.g., human κ or λ constant region). In some embodiments, the vector for expressing the VH or VL domain includes an EF-1α promoter, a secretion signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. Alternatively, the VH and VL domains can be cloned into a single vector expressing the necessary constant region. The heavy chain transformation vector and the light chain transformation vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0216] DNA can also be modified in the following ways: for example, by replacing mouse sequences with coding sequences of human heavy and light chain constant regions, or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence.

[0217] Also provided are polynucleotides that hybridize with polynucleotides encoding the antibodies described herein under high-strictness, medium-strictness, or low-strictness hybridization conditions. In specific embodiments, the polynucleotides described herein hybridize with polynucleotides encoding the VH domain and / or VL domain provided herein under high-strictness, medium-strictness, or low-strictness hybridization conditions.

[0218] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization with membrane-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing once or more in 0.2x SSC / 0.1% SDS at about 50–65°C; hybridization under highly stringent conditions may involve hybridization with membrane-bound nucleic acids in 6x SSC at about 45°C, followed by washing once or more in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described, see, for example, Ausubel FM et al., eds., (1989), Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1–6.3.6 and 2.10.3, which are incorporated herein by reference in their entirety.

[0219] In some aspects, this document provides (e.g., recombinantly) cells (e.g., host cells) for expressing antibodies described herein that specifically bind to KLRG1 (e.g., human KLRG1), along with associated polynucleotides and expression vectors. This document provides vectors (e.g., expression vectors) containing polynucleotides encoding nucleotide sequences encoding anti-KLRG1 antibodies or fragments for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells). This document also provides host cells containing such vectors for recombinant expression of the anti-KLRG1 antibodies (e.g., human or humanized antibodies) described herein. In a particular aspect, this document provides methods for generating the antibodies described herein, the methods comprising expressing the antibodies from host cells.

[0220] Recombinant expression of antibodies (e.g., full-length antigen-binding regions or heavy and / or light chains of antibodies or antibodies described herein) that specifically bind to KLRG1 (e.g., human KLRG1) typically involves constructing expression vectors containing polynucleotides encoding antibodies. Once polynucleotides encoding antibody molecules, heavy and / or light chains of antibodies or fragments thereof (e.g., variable regions of heavy and / or light chains) as described herein are obtained, vectors for generating antibody molecules can be produced using recombinant DNA techniques well known in the art. Therefore, methods for preparing proteins by expressing polynucleotides containing antibodies or antibody fragments (e.g., light or heavy chains) encoding nucleotide sequences are described herein. Expression vectors containing sequences encoding antibodies or antibody fragments (e.g., light or heavy chains) and appropriate transcription and translation control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Also provided are reproducible vectors comprising a nucleotide sequence operatively linked to a promoter encoding a variable region or CDR of an antibody molecule, a heavy or light chain of an antibody, a fragment thereof, or a heavy or light chain thereof, as described herein. Such vectors may, for example, comprise a nucleotide sequence encoding a constant region of an antibody molecule (see, for example, International Publications WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464, which are incorporated herein by reference in their entirety), and the variable region of an antibody may be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0221] In some embodiments, the vector comprises polynucleotides encoding the VH, VL, heavy chain, and / or light chain of the antibody described herein. In another embodiment, the vector comprises polynucleotides encoding the VH and VL of the antibody described herein. In yet another embodiment, the vector comprises polynucleotides encoding the heavy chain and light chain of the antibody described herein.

[0222] Expression vectors can be transferred to cells (e.g., host cells) using conventional techniques, and the resulting cells can then be cultured using conventional techniques to produce cells containing antibodies or fragments thereof as described herein. Therefore, this document provides host cells containing polynucleotides encoding antibodies or fragments thereof, or their heavy or light chains or fragments thereof, or single-chain antibodies as described herein, operably linked to a promoter for expression in the host cell.

[0223] In some embodiments, the host cell contains polynucleotides encoding VH and VL of the antibody described herein. In another embodiment, the host cell contains a vector containing polynucleotides encoding VH and VL of the antibody described herein. In yet another embodiment, the host cell contains a first polynucleotide encoding VH of the antibody described herein and a second polynucleotide encoding VL of the antibody described herein. In yet another embodiment, the host cell contains a first vector and a second vector, the first vector containing a first polynucleotide encoding VH of the antibody described herein; and the second vector containing a second polynucleotide encoding VL of the antibody described herein.

[0224] In specific embodiments, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / light chain variable region of the second cell to form the anti-KLRG1 (e.g., human KLRG1) antibody described herein. In some embodiments, a host cell population comprising such a first host cell and such a second host cell is provided herein.

[0225] In some embodiments, a vector community is provided herein comprising: a first vector comprising a polynucleotide encoding a light chain / light chain variable region of an anti-KLRG1 (e.g., human KLRG1) antibody described herein; and a second vector comprising a heavy chain / heavy chain variable region of a polynucleotide encoding a heavy chain / heavy chain variable region of an anti-KLRG1 (e.g., human KLRG1) antibody described herein.

[0226] Various host expression vector systems can be used to express the antibody molecules described herein (see, for example, U.S. Patent No. 5,807,715, which is incorporated herein by reference in its entirety). Such host expression systems represent media by which the coding sequence of interest can be generated and subsequently purified, and also represent cells that can express the antibody molecules described herein in situ upon transformation or transfection with appropriate nucleotide coding sequences. These systems include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors containing antibody coding sequences (e.g., *Escherichia coli* and *Bacillus subtilis*). B. subtilis Yeast transformed with a recombinant yeast expression vector containing antibody-encoding sequences (e.g., *Saccharomyces* genus). Saccharomyces ) and Pichia pastoris ( PichiaInsect cell systems infected with recombinant viral expression vectors containing antibody-encoding sequences (e.g., baculovirus); plant cell systems infected with, for example, recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV); tobacco mosaic virus (TMV)) or transformed with, for example, recombinant plasmid expression vectors containing antibody-encoding sequences (e.g., Ti plasmids) (e.g., Chlamydomonas reinhardtii). Chlamydomonas reinhardtii(e.g., green algae); or mammalian cell systems having, for example, recombinant expression constructs (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells), said recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In one specific embodiment, the cells used to express the antibodies described herein are Chinese hamster ovary (CHO) cells, such as CHO cells from CHO GS System™ (Lonza). In some embodiments, the heavy and / or light chains of antibodies produced by CHO cells may have N-terminal glutamine or glutamate residues replaced by pyroglutamic acid. In some embodiments, the cells used to express the antibodies described herein are human cells, such as human cell lines. In specific embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, particularly for the expression of the whole recombinant antibody molecule, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) are used to express the recombinant antibody molecule. For example, the binding of mammalian cells (such as CHO cells) to vectors (such as major immediate early gene promoter elements from human cytomegalovirus) is an efficient antibody expression system (Foecking MK and Hofstetter H (1986 Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8(7): 662-7, each of which is incorporated herein by reference in its entirety). In some embodiments, the antibodies described herein are produced by CHO cells or NSO cells. In one specific embodiment, the expression of the nucleotide sequence encoding the antibody described herein that specifically binds to KLRG1 (e.g., human KLRG1) is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0227] In bacterial systems, a number of expression vectors can be advantageously selected depending on the intended use of the expressed antibody molecule. For example, when producing large quantities of such antibodies, a vector that directs the high-level expression of an easily purified fusion protein product may be required to generate a pharmaceutical composition of the antibody molecule. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruether U and Mueller-Hill B (1983) *European Organization for Molecular Biology Journal (EMBO J)* J 2:1791-1794), in which the coding sequence can be individually linked to a vector within a framework containing the lacZ coding region to produce a fusion protein; the pIN vector (Inouye S and Inouye M (1985) *Nucleic Acid Research* 13: 3101-3109; VanHeeke G and Schuster SM (1989) *Journal of Biochemistry* 24: 5503-5509); and so on, all of which are incorporated herein by reference in their entirety. For example, the pGEX vector can also be used to express foreign peptides as fusion proteins containing glutathione 5-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to and binding to a matrix of glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to contain thrombin or factor Xa protease cleavage sites, allowing the cloned target gene product to be released from the GST portion.

[0228] In insect systems, for example, the alfalfa silver-striped noctuid moth ( Autographa californica Nucleopolyhedrovirus (AcNPV) is used as a vector to express foreign genes. The virus is present in the fall armyworm (AcNPV). Spodoptera frugiperda It grows in cells. The coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedral protein gene) and placed under the control of an AcNPV promoter (e.g., the polyhedral protein promoter).

[0229] In mammalian host cells, numerous virus-based expression systems can be utilized. When adenovirus is used as an expression vector, the coding sequence of interest can be linked to an adenoviral transcription / translation control complex (e.g., a late promoter and a triple leader sequence). This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., E1 or E3 regions) will produce a viable recombinant virus capable of expressing the molecule in an infected host (see, for example, Logan J and Shenk T (1984), Proceedings of the National Academy of Sciences (PNAS) 81(12): 3655-9, which is incorporated herein by reference in its entirety). For efficient translation of the inserted coding sequence, specific start signals may also be required. These signals contain the ATG start codon and adjacent sequences. Furthermore, the start codon must correspond to the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can have a variety of sources, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcription terminators, etc. (see, for example, Bitter G et al.) , (1987) Enzymatic Methods 153: 516-544, the reference of which is incorporated herein by reference in its entirety.

[0230] Additionally, host cell strains can be selected to regulate the expression of the inserted sequence or to modify and process gene products in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for protein function. Different host cells possess characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of expressed foreign proteins. For this purpose, eukaryotic host cells with cellular mechanisms for appropriate processing of primary transcripts, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In some embodiments, the anti-KLRG1 (e.g., human KLRG1) antibody described herein is generated in mammalian cells such as CHO cells.

[0231] In one specific embodiment, the antibody described herein has a reduced fucose content or no fucose content. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibody can be expressed in cells lacking or devoid of fucosylation capacity. In a specific example, an antibody with reduced fucose content can be produced using a cell line with two alleles knocked out of α1,6-fucosyltransferase. ® The system (Lonza) is an example of such a system that can be used to produce antibodies with reduced fucose content.

[0232] To achieve long-term, high-yield production of recombinant proteins, stable expression cells can be generated. For example, cell lines stably expressing the anti-KLRG1 (e.g., human KLRG1) antibody described herein can be engineered. In specific embodiments, the cells provided herein stably express light chain / light chain variable regions and heavy chain / heavy chain variable regions, which associate to form the antigen-binding region or antibody described herein.

[0233] In some respects, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and optional markers, without using expression vectors containing viral origins of replication. After the introduction of foreign DNA / polynucleotides, engineered cells can be allowed to grow in enriched media for 1–2 days and then switched to selective media. Optional markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form lesions, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing anti-KLRG1 (e.g., human KLRG1) or fragments thereof as described herein. Such engineered cell lines are particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

[0234] A variety of selection systems can be used, including but not limited to those for herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1): 223-32), hypoxanthine-guanine phosphoribosyltransferase (Szybalska EH and Szybalski W (1962) Proceedings of the National Academy of Sciences 48(12): 2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3): 817-23) genes, all of which are incorporated herein by reference in their entirety. Furthermore, antimetabolite resistance can be used as a basis for selection of the following genes: dhfr This confers resistance to methotrexate (Wigler M et al. (1980), Proceedings of the National Academy of Sciences 77(6): 3567-70; O'Hare K et al. (1981), Proceedings of the National Academy of Sciences 78: 1527-31). gpt Mulligan RC and Berg P (1981), Proceedings of the National Academy of Sciences (PNAS) 78(4): 2072-6; neo, which conferred resistance to aminoglycoside G-418 (Wu GY and Wu CH (1991), Biotherapy 3:87-95; Tolstoshev P (1993), Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993), Science 260: 926-932; Morgan RA and Anderson WF (1993), Ann Rev Biochem 62: 191-217; and Nabel GJ and Felgner PL (1993). Trends in Biotechnology 11(5): 211-5); and hygroThis confers resistance to hygromycin (Santerre RF et al., (1984) Genes 30(1-3): 147-56), and all references in the cited literature are incorporated herein by reference in their entirety. The desired recombinant clone can be selected using methods commonly known in the field of recombinant DNA technology, and such methods are described in the following literature: e.g., Ausubel FM et al., (ed.), *Current Protocols in Human Genetics*, John Wiley & Sons, NY (1993); Kriegler M, *Gene Transfer and Expression, A Laboratory Manual*, Stockton Press, NY (1990); and in Chapters 12 and 13, Dracopoli NC et al., (ed.), *Current Protocols in Human Genetics*, John Wiley & Sons, NY (1994); Colbère-Garapin F et al., (1981) *Journal of Molecular Biology* 150: 1-14, all of which are incorporated herein by reference in their entirety.

[0235] Vector amplification can increase the expression level of antibody molecules (see Bebbington CR and Henschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the marker in the vector system is amplifiable, the increased level of inhibitors present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the gene of interest, protein production will also increase (CrouseGF et al., (1983) Molecular Cell Biology 3: 257-66, which is incorporated herein by reference in its entirety).

[0236] Host cells can be co-transfected with two or more expression vectors described herein, wherein a first vector encodes a heavy chain-derived polypeptide and a second vector encodes a light chain-derived polypeptide. Both vectors may contain the same optional marker that enables equal expression of the heavy chain and light chain polypeptides. Host cells can be co-transfected with varying amounts of the two or more expression vectors. For example, host cells can be co-transfected with the first and second expression vectors at any of the following ratios: approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0237] Alternatively, a single vector encoding both the heavy and light chain polypeptides and capable of expressing both can be used. In such cases, the light chain should be placed before the heavy chain to avoid excessive non-toxic heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Köhler G (1980) Proceedings of the National Academy of Sciences 77: 2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequences for the heavy and light chains can comprise cDNA or genomic DNA. The expression vector can be monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gene / nucleotide sequences, or in the range of 2-5, 5-10 or 10-20 gene / nucleotide sequences. For example, a bicistronic nucleic acid construct may contain a promoter, a first gene (e.g., the heavy chain of the antibody described herein), and a second gene (e.g., the light chain of the antibody described herein) in the following order. In such expression vectors, transcription of both genes can be driven by the promoter, while translation of mRNA from the first gene can be driven by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can be driven by a cap-independent mechanism (e.g., via IRES).

[0238] Once the antibody molecule described herein has been generated through recombinant expression, it can be purified using any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity (especially affinity for a specific antigen after Protein A purification), and fractionation column chromatography), centrifugation, differential dissolution, or any other standard technique for protein purification. Furthermore, the antibody described herein can be fused with a heterologous polypeptide sequence described herein or known in the art to facilitate purification.

[0239] In specific embodiments, the antibodies described herein are isolated or purified. In some embodiments, the isolated antibody is an antibody substantially free of other antibodies having different antigen specificity compared to the isolated antibody. For example, in some embodiments, the formulations of the antibodies described herein are substantially free of cellular material and / or chemical precursors. The language “substantially free of cellular material” includes formulations of antibodies in which the antibody is separated from cellular components of cells from which the antibody was isolated or recombined to produce the antibody. Thus, antibodies substantially free of cellular material comprise antibody formulations and / or antibody variants having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as “contaminating protein”), such as different post-translational modifications of the antibody or other different versions of the antibody (e.g., antibody fragments). When the antibody is recombined to produce the antibody, the TCR is also generally substantially free of culture medium, i.e., the culture medium constitutes less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein formulation. When antibodies are produced through chemical synthesis, they are typically substantially free of chemical precursors or other chemicals; that is, the antibodies are isolated from the chemical precursors or other chemicals involved in protein synthesis. Therefore, such antibody formulations, apart from the antibody of interest, contain less than about 30%, 20%, 10%, or 5% (on a dry weight basis) of chemical precursors or compounds. In one specific embodiment, the antibody described herein is isolated or purified.

[0240] Anti-KLRG1 antibodies (e.g., human KLRG1) or fragments thereof can be produced by any method known in the art for the synthesis of proteins or antibodies, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ conventional techniques from molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the scope of this art. These techniques are described, for example, in the references cited herein and are fully explained therein. See, for example, Maniatis T et al., (1982) *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989) *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., *Experimental Guide to Contemporary Molecular Biology*, John Willie & Son Publishing (1987 and annual updates); *Current Protocols in Immunology*, John Willie & Son Publishing (1987 and annual updates); Gait (ed.) (1984) *Oligonucleotide Synthesis: A Practical Approach*, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Publishing; Birren B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Publishing. All references cited herein are incorporated herein by reference in their entirety.

[0241] In one embodiment, the antibodies described herein are prepared, expressed, generated, or isolated by any means involving generation (e.g., synthesis of DNA sequences, genetic engineering). In some embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the animal or mammalian (e.g., human) phylogenetic lineage.

[0242] On one hand, this document provides a method for preparing an anti-KLRG1 (e.g., human KLRG1) antibody, the method comprising culturing cells or host cells as described herein. In some embodiments, the method is performed in vitro. On another aspect, this document provides a method for preparing an anti-KLRG1 (e.g., human KLRG1) antibody, the method comprising expressing (e.g., recombinantly expressing) the antibody using cells or host cells as described herein (e.g., cells or host cells containing polynucleotides encoding the antibody described herein). In some embodiments, the cells are isolated cells. In some embodiments, exogenous polynucleotides have been introduced into the cells. In some embodiments, the method further includes the step of purifying the antibody obtained from the cells or host cells.

[0243] In some embodiments, antibodies are generated by expressing polynucleotides encoding VH and VL of the antibody described herein in cells under suitable conditions, thereby expressing polynucleotides and generating antibodies. In another embodiment, antibodies are generated by expressing polynucleotides encoding the heavy chain and light chain of the antibody described herein in cells under suitable conditions, thereby expressing polynucleotides and generating antibodies. In some embodiments, antibodies are generated by expressing a first polynucleotide encoding VH of the antibody described herein and a second polynucleotide encoding VL of the antibody described herein in cells under suitable conditions, thereby expressing polynucleotides and generating antibodies. In some embodiments, antibodies are generated by expressing a first polynucleotide encoding the heavy chain of the antibody described herein and a second polynucleotide encoding the light chain of the antibody described herein in cells under suitable conditions, thereby expressing polynucleotides and generating antibodies.

[0244] Methods for generating polyclonal antibodies are known in the art (see, for example, Chapter 11 of the following literature: Short Protocols in Molecular Biology, (2002) 5th edition, edited by Ausubel FM et al., John Willie Fathers and Sons, New York, which is incorporated herein by reference in its entirety).

[0245] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma, recombinant, and phage display techniques, or combinations thereof. For example, hybridoma techniques can be used to generate monoclonal antibodies, including those known in the art and taught in the following literature: e.g., Harlow E and Lane D, *Antibodies: A Laboratory Handbook*, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling GJ et al., *Monoclonal Antibodies and T-Cell Hybridomas*, 563 681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma techniques. For example, monoclonal antibodies can be recombinantly generated from host cells expressing exogenous antibodies or fragments thereof (e.g., light and / or heavy chains of such antibodies).

[0246] In specific embodiments, as used herein, a “monoclonal antibody” is an antibody produced by a single cell (e.g., a hybridoma or host cell that produces recombinant antibodies) that specifically binds to KLRG1 (e.g., human KLRG1), as determined by, for example, an ELISA or other antigen-binding or competitive binding assay known in the art or in the examples provided herein. In some embodiments, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In some embodiments, the monoclonal antibody is a monovalent or multivalent (e.g., bivalent) antibody. In some embodiments, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method described in, such as Kohler G and Milstein C (1975), Nature 256: 495, which is incorporated herein by reference in its entirety, or can be isolated from a phage library, for example, using techniques described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed therefrom are well known in the art (see, for example, Chapter 11 of the following literature: The Concise Guide to Molecular Biology, 5th Edition, Ausubel FM et al., ibid.).

[0247] As used herein, when an antibody contains at least two (e.g., two or more) monovalent binding regions, the antibody binds to an antigen in a multivalent (e.g., bivalent) manner, each monovalent binding region capable of binding to an epitope on the antigen. Each monovalent binding region may bind to the same or different epitopes on the antigen.

[0248] Methods for generating and screening specific antibodies using hybridoma technology are routine and well-known in the art. For example, in hybridoma methods, mice or other suitable host animals (such as sheep, goats, rabbits, rats, hamsters, or macaques) are immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., KLRG1 (e.g., human KLRG1)). Alternatively, lymphocytes can be immunized in vitro. Then, lymphocytes are fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding JW (ed.), *Monoclonal Antibodies: Principles and Practice*, pp. 59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety. Alternatively, RIMMS (Repeated Immunization Multiple Sites) technology can be used to immunize animals (Kilpatrick KE et al., *Hybridoma*, 16:381-9, 1997), which is incorporated herein by reference in its entirety.

[0249] In some implementations, mice (or other animals such as rats, monkeys, donkeys, pigs, sheep, hamsters, or dogs) can be immunized with an antigen (e.g., KLRG1 (e.g., human KLRG1)), and once an immune response is detected, such as the detection of antigen-specific antibodies in mouse serum, the mouse spleen is harvested and spleen cells are isolated. The spleen cells are then combined with any suitable myeloma cells (e.g., from the American Type Culture Collection, ATCC) using well-known techniques. ® Cells from the SP20 cell line (Manassas, Virginia, VA) are fused to form hybridomas. Hybridomas are selected and cloned using limiting dilutions. In some embodiments, lymph nodes from immunized mice are harvested and fused with NSO myeloma cells.

[0250] The hybridoma cells thus prepared are inoculated and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (HAT medium) to prevent the growth of HGPRT-deficient cells.

[0251] The specific implementation method employs myeloma cells that are effectively fused, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. These myeloma cell lines include mouse myeloma lines, such as the NSO cell line, or cell lines derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, San Diego, CA, USA), as well as SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heterologous myeloma cell lines for producing human monoclonal antibodies have also been described (Kozbor D (1984) *Journal of Immunology* 133: 3001-5; Brodeur et al., *Monoclonal Antibody Production Techniques and Applications*, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), each of which is incorporated herein by reference in its entirety).

[0252] The production of monoclonal antibodies against KLRG1 (e.g., human KLRG1) in the culture medium for growing hybridoma cells is measured. The binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by methods known in the art, such as immunoprecipitation or by in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0253] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned using a limited dilution procedure and grown using standard methods (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, ibid.). Suitable media for this purpose include, for example, D-MEM or RPMI 1640 media. Additionally, in animals, hybridoma cells can be grown in vivo as ascites tumors.

[0254] Monoclonal antibodies secreted by subclones can be appropriately separated from culture medium, ascites, or serum using conventional immunoglobulin purification procedures, such as protein A-agarose gel chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0255] The antibodies described herein comprise, for example, antibody fragments that recognize KLRG1 (e.g., human KLRG1) and can be generated using any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of an immunoglobulin molecule using enzymes such as papain (which produces the Fab fragment) or pepsin (which produces the F(ab')2 fragment). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of the antibody molecule linked by disulfide bonds in a hinge region.

[0256] Furthermore, antibodies described herein can be generated using various phage display methods known in the art. In phage display, functional antibody domains are displayed on the surface of a phage particle carrying a multinucleotide sequence encoding the phage particle. Specifically, DNA sequences encoding the VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of an affected tissue). The DNA encoding the VH and VL domains is recombined with an scFv adapter by PCR and cloned into a phage particle vector. The vector is electroporated into *E. coli*, and the *E. coli* is infected with a helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and the VH and VL domains are typically recombinantly fused with phage gene III or gene VIII. Phages expressing antigen-binding domains that bind to a specific antigen can be selected or identified using an antigen (e.g., using a labeled antigen or an antigen that binds to or captures a solid surface or bead). Examples of phage display methods that can be used to prepare the antibodies described herein include those described in the following literature: Brinkman U et al., (1995) *Journal of Immunology Methods* 182: 41-50; Ames RS et al., (1995) *Journal of Immunology Methods* 184: 177-186; Kettleborough CA et al., (1994) *Eur J Immunol* 24: 952-958; Persic L et al., (1997) *Genes* 187: 9-18; Burton DR and Barbas CF (1994) *Advans in Immunology* 57: 191-280; PCT application No. PCT / GB91 / 001134; International Publication No. WO 90 / 02809, No. WO No. 91 / 10737, No. 92 / 01047, No. 92 / 18619, No. 93 / 11236, No. 95 / 15982, No. 95 / 20401 and No. 91 / 10737 U.S. Patent Nos. 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, all of which are incorporated herein by reference in their entirety.

[0257] As described in the references above, after phage selection, antibody-coding regions from the phage can be isolated and used to generate complete antibodies (containing human antibodies or any other desired antigen-binding fragments) and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria), for example, as described below. Antibody fragments (such as Fab, Fab', and F(ab')2 fragments) can also be generated using methods known in the art, such as those disclosed in the following documents: PCT Publication WO 92 / 22324; Mullinax RL et al., (1992) Biotechnology 12(6): 864-9; Sawai H et al., (1995) American Journal of Reproductive Immunology 34: 26-34; and Better M et al., (1988) Science 240: 1041-1043, all of which are incorporated herein by reference in their entirety.

[0258] In some embodiments, to generate complete antibodies, PCR primers containing the VH or VL nucleotide sequence, a restriction site, and flanking sequences protecting the restriction site can be used to amplify the VH or VL sequence from a template (e.g., an scFv clone). Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing the VH constant region, and the PCR-amplified VL domain can be cloned into a vector expressing the VL constant region (e.g., the human κ or λ constant region). Alternatively, the VH and VL domains can be cloned into a single vector expressing the necessary constant region. The heavy chain transformation vector and the light chain transformation vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0259] Chimeric antibodies are molecules in which different portions of an antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a mouse or rat monoclonal antibody fused to a constant region of a human antibody. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison SL (1985) Science 229: 1202-7; Oi VT and Morrison SL (1986) Biotechnology 4: 214-221; Gillies SD et al., (1989) Journal of Immunological Methods 125: 191-202; and U.S. Patents 5,807,715, 4,816,567, 4,816,397, and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0260] Humanized antibodies are capable of binding to a predetermined antigen, wherein the predetermined antigen comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a core region (CDR) having substantially the amino acid sequence of a non-human immunoglobulin (e.g., mouse immunoglobulin). In a particular embodiment, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. The antibody may also comprise CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotypes, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be generated using a variety of techniques known in the art, including but not limited to CDR transplantation (European Patent EP 239400; International Publication WO 91 / 09967; and US Patents 5,225,539, 5,530,101 and 5,585,089), veneer or surface resurfacing (European Patents EP 592106 and EP 519596; Padlan EA (1991) *Molecular Immunology* 28(4 / 5): 489-498; Studnicka GM et al., (1994) *Protein Engineering* 7(6): 805-814; and Roguska MA et al., (1994) *Proceedings of the National Academy of Sciences* 91: 969-973), chain truncation (US Patent No. 5,565,332), and techniques disclosed in the following documents: for example, US Patent No. 6,407,213; US Patent No. 5,766,886; International Publication No. WO 93 / 17105; Tan P et al., (2002) Journal of Immunology 169: 1119-25; Caldas C et al., (2000) Protein Engineering 13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M et al., (1997) Journal of Molecular Biology 272(16): 10678-84; Roguska MA et al., (1996) Protein Engineering 9(10): 895 904; Couto JR et al., (1995) Cancer Research 55 (Supplement 23): 5973s-5977s; Couto JR et al., (1995) Cancer Research 55(8): 1717-22; Sandhu JS (1994) Genes 150(2): 409-10; and Pedersen JT et al., (1994) Journal of Molecular Biology 235(3): 959-73, all of which are incorporated herein by reference in their entirety. See also U.S. Application Publication No. US 2005 / 0042664 A1 (February 24, 2005), which is incorporated herein by reference in its entirety.

[0261] Methods for preparing multispecific antibodies (e.g., bispecific antibodies) have been described, see, for example, U.S. Patent Nos. 7,951,917; 7,183,076; 8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992; and 8,586,713, all of which are incorporated herein by reference in their entirety.

[0262] Bispecific bivalent antibodies and methods for their preparation are described, for example, in the following documents: U.S. Patent Nos. 5,731,168, 5,807,706, and 5,821,333; and U.S. Application Publications Nos. 2003 / 020734 and 2002 / 0155537; each of these documents is incorporated herein by reference in its entirety. Bispecific tetravalent antibodies and methods for their preparation are described, for example, in the following documents: International Patent Publications Nos. WO 02 / 096948 and WO 00 / 44788; the disclosures of both of these documents are incorporated herein by reference in their entirety. See also International Patent Publications Nos. 93 / 17715, 92 / 08802, 91 / 00360 and 92 / 05793; Tutt et al., Journal of Immunology 147:60-69 (1991); U.S. Patents Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920 and 5,601,819; and Kostelny et al., Journal of Immunology 148:1547-1553 (1992); each of these references is incorporated herein by reference in its entirety.

[0263] The bispecific antibodies described herein can be generated using the DuoBody technology platform (Genmab A / S), as described, for example, in the following publications: International Publications Nos. WO 2011 / 131746, WO 2011 / 147986, WO 2008 / 119353 and WO 2013 / 060867; and Labrijn AF et al., (2013) Proceedings of the National Academy of Sciences 110(13): 5145-5150. DuoBody can be used to bind half of a first monospecific antibody or a first antigen-binding region containing two heavy chains and two light chains to half of a second monospecific antibody or a second antigen-binding region containing two heavy chains and two light chains. The resulting heterodimer contains one heavy chain and one light chain from the first antibody or the first antigen-binding region paired with one heavy chain and one light chain from the second antibody or the second antigen-binding region. When two monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0264] DuoBody technology requires that each of the monospecific antibody or antigen-binding regions contains a heavy-chain constant region with a single-point mutation in the CH3 domain. The point mutation makes the interactions between the CH3 domains in the resulting bispecific antibody stronger than the interactions between the CH3 domains in the monospecific antibody or antigen-binding region. Numbered according to the EU numbering system, the single-point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407, or 409 in the CH3 domain of the heavy-chain constant region, as described, for example, in International Publication No. WO 2011 / 131746. Furthermore, the single-point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to another. For example, according to the EU numbering system, one monospecific antibody or antigen-binding region may contain the mutant F405L (i.e., a mutation from phenylalanine to leucine at residue 405), while another monospecific antibody or antigen-binding region may contain the mutant K409R (i.e., a mutation from lysine to arginine at residue 409). The heavy chain constant region or antigen-binding region of a monospecific antibody may be an IgG1, IgG2, IgG3, or IgG4 isoform (e.g., human IgG1 isoform), and bispecific antibodies produced by DuoBody technology can retain Fc-mediated effector function.

[0265] Another method for generating bispecific antibodies is known as the "mortar and pestle" strategy (see, for example, International Publication WO2006 / 028936). In this technique, mismatches in the Ig heavy chains are reduced by mutating selected amino acids at the interface that forms the CH3 domain in IgG. At the site where the two heavy chains directly interact within the CH3 domain, an amino acid with a small side chain (mortar) is introduced into the sequence of one heavy chain, and an amino acid with a large side chain (mortar) is introduced into the corresponding interacting residue site on the other heavy chain. In some embodiments, the compositions of this disclosure have immunoglobulin chains in which the CH3 domain has been modified by mutating selected amino acids that interact at the interface between the two polypeptides, thereby preferentially forming bispecific antibodies. Bispecific antibodies may consist of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3 or IgG3 and IgG4).

[0266] In some cases, bispecific antibodies may contain heterodimers of IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chains. Such heterodimeric heavy chain antibodies can often be engineered, for example, by modifying selected amino acids at the interface that forms the CH3 domain in human IgG4 and IgG1 or IgG3, thereby facilitating the formation of heterodimeric heavy chains.

[0267] In some embodiments, the antibodies described herein that bind to the same epitope of KLRG1 (e.g., human KLRG1) as the anti-KLRG1 (e.g., human KLRG1) antibody described herein are human antibodies. In some embodiments, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) the binding of any of the antibodies described herein to KLRG1 (e.g., human KLRG1) are human antibodies. Human antibodies can be generated using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. Specifically, human heavy chain and light chain immunoglobulin gene complexes can be randomly introduced or introduced via homologous recombination into mouse embryonic stem cells. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. When introducing mouse heavy chain and light chain immunoglobulin genes into human immunoglobulin gene loci via homologous recombination, the mouse heavy chain and light chain immunoglobulin genes can be rendered nonfunctional, individually or simultaneously. Specifically, J HThe homozygous absence of the region prevents the production of endogenous antibodies. Modified embryonic stem cells are expanded and microinjected into blastocysts to generate chimeric mice. The chimeric mice are then fed to produce homozygous offspring expressing human antibodies. The transgenic mice are immunized in the normal manner with a selected antigen (e.g., all or part of an antigen such as KLRG1 (e.g., human KLRG1)). Monoclonal antibodies against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgenes carried by the transgenic mice undergo rearrangement during B cell differentiation and are subsequently subjected to class switching and somatic mutations. Therefore, using such techniques, therapeutically useful IgG, IgA, IgM, and IgE antibodies can be generated. For an overview of this technique for generating human antibodies, see Lonberg N and Huszar D (1995), *Int Rev Immunol* 13:65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of the techniques and protocols for generating human antibodies and human monoclonal antibodies, see, for example, International Publications WO 98 / 24893, WO 96 / 34096, and WO 96 / 33735; and U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of generating human antibodies include XenoMouse. TM (Amgenix, Inc.; U.S. Patents Nos. 6,075,181 and 6,150,184), HuaAb-Mouse TM (Medarex, Inc. / GenPharm; U.S. Patent Nos. 5,545,806 and 5,569,825), Trans Chromo Mouse TM (Kirin Corporation) and KM Mouse TM (Medarex / Kirin), all references cited herein are incorporated herein by reference in their entirety.

[0268] Human antibodies that specifically bind to KLRG1 (e.g., human KLRG1) can be prepared using a variety of methods known in the art, including the phage display method described above using an antibody library derived from a human immunoglobulin sequence. See also U.S. Patents 4,444,887, 4,716,111, and 5,885,793; and International Publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741, all of which are incorporated herein by reference in their entirety.

[0269] In some embodiments, human antibodies can be generated using mouse-human hybridomas. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBV) can be fused with mouse myeloma cells to generate mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to identify hybridomas that secrete human monoclonal antibodies that specifically bind to a target antigen (e.g., KLRG1 (e.g., human KLRG1)). Such methods are known and described in the art, see, for example, Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is incorporated herein by reference in its entirety.

[0270] Reagent test kit Kits are also provided that contain one or more antibodies or pharmaceutical compositions or conjugates described herein. In one specific embodiment, a pharmaceutical package or kit is provided that comprises one or more containers filled with one or more components of the pharmaceutical compositions described herein (such as one or more antibodies provided herein). In some embodiments, the kit contains pharmaceutical compositions described herein, such as those described herein, and any prophylactic or therapeutic agent. In some embodiments, the kit may contain T-cell mitogens, such as phytohemagglutinin (PHA) and / or phorbol myristate acetate (PMA), or TCR complexes that stimulate antibodies such as anti-CD3 antibodies and anti-CD28 antibodies. Optionally associated with such containers may be a notice in the form prescribed by a government agency regulating the manufacture, use, or sale of a drug or biological product, reflecting the agency's permission for its manufacture, use, or sale for human use.

[0271] Kits that can be used in the methods described above are also provided. In some embodiments, the kit contains the antibody described herein, preferably a purified antibody, in one or more containers. In one specific embodiment, the kit described herein contains a substantially isolated KLRG1 (e.g., human KLRG1) antigen as a control. In another specific embodiment, the kit described herein further contains a control antibody that does not react with the KLRG1 (e.g., human KLRG1) antigen. In yet another specific embodiment, the kit described herein contains one or more elements for detecting the binding of the antibody to the KLRG1 (e.g., human KLRG1) antigen (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, enzyme substrate, radioactive compound, or luminescent compound, or a second antibody recognizing the first antibody may be conjugated to a detectable substrate). In specific embodiments, the kits provided herein may contain recombinant or chemically synthesized KLRG1 (e.g., human KLRG1) antigen. The KLRG1 (e.g., human KLRG1) antigen provided in the kit may also be attached to a solid-phase carrier. In a more specific embodiment, the detection device of the kit described above comprises a solid-phase carrier to which the KLRG1 (e.g., human KLRG1) antigen is attached. Such kits may also comprise an anti-human antibody or an anti-mouse / rat antibody without an attached reporter gene label. In this embodiment, the binding of the antibody to the KLRG1 (e.g., human KLRG1) antigen can be detected by the binding of the reporter gene-labeled antibody. In some embodiments, this disclosure relates to the use of the kits of this disclosure in the in vitro assay and / or detection of the KLRG1 (e.g., human KLRG1) antigen in biological samples.

[0272] Example The following embodiments are provided by way of illustration rather than by way of limitation.

[0273] Example 1: Generation of KLRG1 antibody KLRG1 antibodies were generated using mice from Lake Pharma and Abveris. Mice were immunized with either the recombinant human KLRG1 extracellular domain-hFc fusion protein or a mouse 3A9 cell line stably expressing full-length human KLRG1. Serum antibody titers following immunization were assessed using an ELISA of hKLRG1.hFc and a FACS of the KLRG1 Jurkat cell line. Mice showing the highest titers against human KLRG1 were sacrificed for hybridoma generation. Hybridomas were further screened using FACS, ELISA, and biolayer interferometry (BLI) to measure antibody affinity and specificity for human-KLRG1. Subcloning generated hybridomas with high affinity for human KLRG1 antibodies, and the antibodies were sequenced. The amino acid sequences of the selected antibody clones are shown in Table 1.

[0274] Example 2: Functional characterization of KLRG1 antibody Determination of antibody affinity by Octet® and FACS The interaction between KLRG1 and the anti-KLRG1 antibody was determined using the Octet® Red96 system via BLI. BLI technology is used to monitor protein binding and analyze the kinetics of molecular interactions in real time by detecting changes in the interference pattern of light reflected from the sensor surface (biological layer). See https: / / www.sartorius.com / en / pr / octet for details, including all links and sublinks associated with it, which is incorporated herein by reference in its entirety. The antibody in hIgG4 format was captured at 10 ug / mL on the AHC sensor. The hKLRG1.hFc ligand was diluted from 0 nM to 100 nM in Octet® kinetic buffer (1XPBS, 0.1% BSA, and 0.02% PS20). The hFc fragment was used as a sensor blocking agent prior to the association step. Octet® data were analyzed at a 1:1 binding ratio using Octet® software to determine the dissociation constant (K). D ) and K on and K off The results are summarized in Table 2 below.

[0275] On-cell binding affinity (EC50) was assessed using KLRG1-expressing cell lines by flow cytometry (Cytek Aurora, 5-laser). Chinese hamster ovary (CHO) cells were stably transfected to express full-length human or cynomolgus monkey (cynomolgus monkey) KLRG1. In the experiments, KLRG1-expressing ExpiCHO cell lines (0.1 M / 200 µL) were incubated together with antibodies at concentrations ranging from 0 nM to 133 nM in FACS buffer (dPBS + 2 mM EDTA + 2% FBS). The mixture was incubated at 4°C for 1 h and then washed three times with cold PBS. Fluorescently labeled anti-hFc was used as the detection antibody. The parental CHO cell line was used as a reference. EC50 was determined by a 4-parameter nonlinear fitting. Figure 1 The results are summarized in Table 2 below.

[0276] Antibodies binding to donor PBMCs The binding of anti-KLRG1 antibody to donor PBMCs was assessed by flow cytometry and compared with commercially available baseline antibodies. Following the manufacturer's protocol, the candidate antibody, hIgG4 isotype control, and positive control commercial anti-KLRG1 antibody clone SA231A2 were conjugated with Alexa Fluor™ 647 using the APEX™ antibody labeling kit. Antibody concentrations were determined using absorbance readings at 280 nm and 650 nm using a NanoDrop™ One UV-Vis spectrophotometer, and the following equation was used: (A280 - 0.03*A650) / 1.4. PBMCs from healthy donors were thawed and resuspended in 10 mL FACS buffer (1xPBS + 2% fetal bovine serum + 2 mM EDTA), then blocked on ice for 20 min with a 1:20 human TruStain FcX™ and 10% human serum. Eleven concentration points starting at 66.67 nM, serially diluted 1:3 with conjugated antibody or commercially available conjugated SA231A2, were added to 100 μL of PBMC characterization kit (CD3-BV421, CD8-BV510, CD14-BV605, CD19-FITC, CD56-PE-CF594, CD4-PE-Cy7) and 1:1000 Zombie NIR™ amine-active fluorescent dye, and incubated on ice for 30 min. Cells were washed three times with FACS buffer, resuspended in 150 μL of FACS buffer, and analyzed on a Cytek® Aurora flow cytometer. Data were analyzed using FlowJo™ and MFI were calculated. Plots and EC50 values ​​were determined by 4-parameter nonlinear fitting and plotted using GraphPad Prism. Figure 2 Table 2 shows the results from the three donors.

[0277] Human KLRG1 tetramer blocking assay (IC50) Biotinylated human KLRG1 was mixed with streptavidin-phycoerythrin (PE) to generate KLRG1 tetramer-PE. A cell line overexpressing human E-cadherin (K562 E-cadherin cells) was generated. KLRG1 tetramer-PE was added to K562 E-cadherin cells in calcium-containing cell culture medium. Different concentrations of antibody were added to cell suspensions on ice. After two hours, cells were washed with cell culture medium, and the ability of the antibody to block the interaction between human KLRG1 tetramer and E-cadherin-expressing cells was assessed by flow cytometry. Figure 3 The IC50 value for each antibody was determined by monitoring the loss of E-cadherin binding as a function of different antibody concentrations, and is summarized in Table 2 below. All KLRG1 antibodies tested showed effective ligand blocking activity.

[0278] Table 2. Characteristics of KLRG1 antibody KLRG1 -N-cadherin interaction The ability of the anti-KLRG1 antibody to block KLRG1-N-cadherin interaction on A375 cells was tested. A375 cells were harvested and resuspended in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% FBS and penicillin-streptomycin to prepare 1 x 10^6 cells / ml, and plated in 96-well round-bottom plates. 100 µl of human KLRG1-tetramer-PE solution (final concentration 6 nM) and 50 µl of anti-KLRG1 antibody (final concentration 20 µg / ml) were added to the cells. After incubating on ice for 2 hours, the cells were washed with 3 x 200 µl of IMDM. The cells were resuspended in 200 µl of IMDM and analyzed using Cytek® Aurora flow cytometry. Data were analyzed and plotted using FlowJo™. Figure 4D and 4E As shown, the anti-KLRG1 antibody blocks the KLRG1-N cadherin interaction on A375 cells, while the human IgG4 isotype control antibody does not block the interaction. Figure 4C ).

[0279] Anti-KLRG1 antibodies reverse E-cadherin inhibition in CD8+ T cells Human E-cadherin was overexpressed in artificial antigen-presenting cells (aAPCs), which also expressed T-cell receptor (TCR) activating protein. CD8+ T cells were isolated from healthy PBMCs, and CCR7-negative CD8+ T cells were separated using a cell sorter to enrich KLRG1+ CD8+ T cells. CCR7-negative CD8+ T cells were added to aAPCs and aAPCs overexpressing cadherin. Antibodies were then added to the cell cultures. After three days, the cell supernatant was harvested, and interferon-γ (IFN-γ) levels were determined by enzyme-linked immunosorbent assay (ELISA). Figure 5 As shown, compared with the control, the anti-KLRG1 antibody increased IFN-γ expression in E-cadherin-exposed cells.

[0280] Parental and CHOK1 cells overexpressing E-cadherin were treated with 10 μg / mL mitomycin C for 1 hour to limit potential interference with T cell activation signaling. CHOK1 cells were seeded at 200,000 cells / well and incubated overnight at 37°C. CCR7-memory and effector memory T cells of CD8+ T cells were sorted using a Sony® SH800 cell sorter, as CCR7-CD8+ cells are predominantly KLRG1+. Sorted CCR7-CD8+ cells were added at 50,000 cells / well, along with 10 serially diluted candidate antibodies at 1:3 concentrations, starting from 133.33 nM or the corresponding hIgG4 isotype control, and incubated at 37°C for 3 days. The supernatant was collected, and the activation of cytokines IFN-γ and TNF-α was measured using an ELISA kit. Data were collected using a plate reader at absorbance at 450 nm and analyzed using GraphPad Prism, with a standard curve of cytokine concentrations inserted. Figure 6 As shown, compared with the control, the anti-KLRG1 antibody increased the expression of IFN-γ and TNF-α in E-cadherin-exposed cells. Figure 5 and Figure 6 All of these studies demonstrate the ability of anti-KLRG1 antibodies to reverse the inhibition of human CD8+ T cell activation by E-cadherin.

[0281] Anti-KLRG1 antibodies enhance activation of KRLG1 -enriched CD8+ T cells in patient-derived PBMCs CHOK1 cells expressing T cell activation ligand and E-cadherin were harvested, treated with mitomycin C, and seeded in 96-well plates at a concentration of 2 x 10^5 cells / well and incubated overnight. PBMCs from cancer cells were obtained from Precision Medicine. CD8+ T cells were isolated from PBMCs using the Miltenyi CD8 T cell isolation kit to enrich T cells. The isolated CD8+ T cells were stained with anti-CD8, anti-CD16, anti-CD56, and anti-CCR7 antibodies. KLRG1-enriched cells were isolated by sorting CD8+ CD16- CD56- CCR7- cells. Cells were incubated with anti-KLRG1 antibody and cells expressing T cell activation ligand and E-cadherin. IFN-γ levels in the cell supernatant were detected by ELISA. Figure 7 As observed, the anti-KLRG1 antibody enhanced CD8+ T cell activation in PBMCs derived from cancer patients.

[0282] Anti-KLRG1 antibodies induce activation of KRLG1 -enriched CD8+ T cells in human renal tumor samples Dissociated tumor cells were incubated with cells expressing T cell activation ligands and E-cadherin, as well as anti-KLRG1 and / or anti-PD-1 antibodies. CD8+ T cell activation was analyzed using IFN-γ intracellular staining. Human kidney tumors obtained from the Collaborative Human Tissue Network (CHTN) were mechanically dissociated in RPMI medium without the use of enzymes using a Miltenyigentle MACS dissociator. The dissociated tumors were filtered through a cell filter, and the resulting single-cell suspension was frozen in Bambanker™ freezing medium before use. The frozen dissociated tumor suspension was thawed and plated in 24-well culture plates, each containing 1 ml of RPMI+ medium (RPMI supplemented with 10% FBS and penicillin-streptomycin). The dissociated tumor suspension was treated with any of the following: 1D21v1 (final concentration 10 μg / ml), anti-PD1 (final concentration 5 μg / ml), 1D21v1 (final concentration 10 μg / ml) and anti-PD1 (final concentration 5 μg / ml) or no antibody (negative control). After 2 hours, the dissociated tumor suspension in the treatment medium was transferred to the wells of a 24-well plate pre-coated with a monolayer of TCR-CHO-K1-E-cadherin cells. GolgiPlug™ was added at a rate of 1 μl / 1 mL of treatment medium to prevent IFNG release. After 22 hours of culture, the wells were harvested, rinsed, and stained on ice for 30 minutes with anti-CD45, anti-CD2, anti-CD4, anti-CD8, anti-KLRG1, and anti-PD-1 antibodies, as well as Zombie NIR™. The cells were then washed, fixed, and permeabilized using the BD Cytofix / Cytoperm™ kit and stained on ice for 30 minutes with anti-IFNG antibody. Stained cells were washed three times with BD permeabilization buffer, then resuspended in PBS containing 2% FBS and analyzed using Cytek® Aurora flow cytometry. Data were analyzed using FlowJo™. Anti-KLRG1 antibody induces T cell activation, as demonstrated by increased INF-γ expression as a monotherapy and in combination with an anti-PD-1 antibody. Figure 8 ).

[0283] Example 3: Developability The developability of KLRG1 antibodies was assessed through stress studies and characterization. Stress studies included 5X freezing / thawing, high pH 8.5 (50 mM Tris, 150 mM NaCl) and low pH 3.5 (100 mM citrate) for up to 12 hours at room temperature, thermal stress at 40°C for up to 2 weeks, and mechanical stress by shaking at 400 RPM for 24 hours at room temperature. Stressed samples were analyzed by appearance assays, A280 concentration assays, SDS-PAGE, SE-HPLC, WCEX-HPLC, Octet®, and mass spectrometry. Characterization of these anti-KLRG1 antibodies included DSC, DLS, icIEF, free thiol assays, nonspecific binding assays (including serum, RBC, and platelet binding assays), and solubility assays. Additionally, all antibodies purified using a single ProA column with the same procedure were evaluated for HCP, endotoxin, and recovery. Nivolumab was used as a reference.

[0284] In the assays described above, all tested antibodies performed similarly to or better than nivolumab. Notably, BLI analysis of 16F5 and 17D2, as well as cell-borne binding of 1D21v1 and 1D21v3, showed affinity K under all tested stress conditions. D Minor variations were observed in the measurements. Acidic stress conditions led to an increase in LMW (low molecular weight) species in 5P13v14, 17D2, and nivolumab, but no increase in other antibodies tested. In charge heterogeneity assessment using WCEX-HPLC, only 16F5, 1D21v1, and 1D21v3 exhibited a single dominant peak. As measured by icIEF, the isoelectric point of all antibodies was >7.4, with 1D21v1 showing an ideal pI of approximately 8.2. In DSC assays, 1D21v1 and 1D21v3 showed the highest Tm (approximately 78°C) across all tested Fabs, with nivolumab showing a Tm of 68°C in this assay.

[0285] Example 4: Combination Therapy Anti-KLRG1 antibodies enhance BiTE-mediated cytotoxicity of E-cadherin-expressing target cells Cells overexpressing HEK293CD19 and HEK293CD19 E-cadherin were generated via lentiviral transduction and used as target cells. Frozen CD8+ T cells from healthy donors were obtained from StemCell Technology, with 75% of the CD8+ T cells being KLRG1 positive. KLRG1+ cells were used as effector cells and resuspended in culture medium. Target cells were labeled with CellTrace™ violet and resuspended in culture medium. Anti-hCD19-CD3 (InvivoGen, various concentrations) and anti-KLRG1 antibody (10 μg / ml final concentration) were diluted with culture medium. Effector cells and target cells (E:T ratio 1:2) were incubated with the antibody and CD3 x CD19 BiTE for 48 hours. Target cells were harvested and viable cell percentage was analyzed by Cytek® Aurora flow cytometry. FlowJo™ was used for data analysis. Figure 9 As observed, CD3 x CD19 BiTE as a monotherapy reduced the cell viability of CD19 HEK cells, but did not reduce the cell viability of CD19 E-cadherin HEK cells; while the combination of adding anti-KLRG antibody with CD3 x CD19 BiTE showed a decrease in the viability of CD19 E-cadherin HEK cells.

[0286] KLRG1-expressing CD8+ T cells were sorted from a large number of CD8+ T cells obtained from three healthy donors. The sorted KLRG1+ CD8+ T cells were stained with CellTrace™ Violet solution according to the manufacturer's instructions. Approximately 0.1 x 10⁻⁶ cells were used. 6 KLRG1+ CD8+ T cells compared to 0.05 x 10⁸ cells pre-coated the previous day. 6 HCC2935 lung adenocarcinoma cells were co-cultured. Titrated concentrations of recombinant anti-HER2 x anti-CD3 bispecific T-cell connector and 10 µg / mL anti-KLRG1 antibody (1D21v1) or human IgG4 isotype control antibody were added to the co-culture. After 48 hours of incubation, the co-culture was harvested and stained with SYTOX™ Deep Red Viability Stain according to the manufacturer's instructions. The percentage of viable CellTrace™ Violet-negative HCC2935 cells was determined by flow cytometry. Among all three donors, the addition of anti-KLRG1 antibody enhanced the cytotoxicity of CD3xHer2 BiTE treatment against target cells expressing E-cadherin. Figure 10 ).

[0287] *** This invention is not limited to the specific embodiments described herein. In fact, various modifications to the invention, in addition to those described, will become apparent to those skilled in the art from the preceding description and drawings. Such modifications are intended to fall within the scope of the appended claims.

[0288] All references cited herein (e.g., publications, patents, or patent applications) are incorporated herein by reference in their entirety and for all purposes, to the extent that each individual reference (e.g., publications, patents, or patent applications) is specifically or individually indicated by reference in its entirety for all purposes.

[0289] Other embodiments are described in the following claims.

Claims

1. An antibody that specifically binds to human KLRG1, the antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, 19, or 48; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, 31, or 49.

2. The antibody according to claim 1, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31 or 48 and 49, respectively.

3. The antibody according to claim 1 or 2, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 3, 4 and 5; 21, 22 and 23; or 34, 35 and 50, respectively.

4. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 6, 18 and 8; 24, 25 and 26; or 37, 51 and 39, respectively.

5. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 3, 4 and 5; 21, 22 and 23; 34, 35 and 36; or 34, 35 and 44, respectively.

6. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 6, 7 and 8; 6, 13 and 8; 24, 25 and 26; 37, 38 and 39; or 37, 45 and 39.

7. The antibody according to any one of the preceding claims, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 3, 4, 5, 6, 18 and 8; 21, 22, 23, 24, 25 and 26; or 34, 35, 50, 37, 51 and 39.

8. The antibody according to any one of the preceding claims, wherein the antibody comprises, respectively, the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8; 3, 4, 5, 6, 13 and 8; 21, 22, 23, 24, 25 and 26; 34, 35, 36, 37, 38 and 39; or 34, 35, 44, 37, 45 and 39.

9. The antibody according to any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO: 16, 19 or 48.

10. The antibody according to any one of the preceding claims, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO: 1, 11, 19, 32 or 42.

11. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region, optionally selected from the group consisting of: human IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

12. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain constant region, the heavy chain constant region being a variant of the wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to the FcγR with a lower affinity than the wild-type heavy chain constant region binding to FcγR.

13. The antibody according to any one of the preceding claims, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO: 52, 53, 54 or 55.

14. The antibody according to any one of the preceding claims, wherein the antibody comprises a heavy chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59 or 60.

15. The antibody according to any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO: 17, 31 or 49.

16. The antibody according to any one of the preceding claims, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33 or 43.

17. The antibody according to any one of the preceding claims, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 10, 15, 28, 30, 41 or 47.

18. The antibody according to any one of the preceding claims, wherein the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 16 and 17, 19 and 31 or 48 and 49.

19. The antibody according to any one of the preceding claims, wherein the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 1 and 2, 11 and 12, 19 and 20, 19 and 29, 32 and 33 or 42 and 43.

20. An antibody that specifically binds to human KLRG1, said antibody comprising a heavy chain and a light chain, said heavy chain and said light chain comprising the amino acid sequences shown in SEQ ID NO: 9 and 10, 56 and 10, 14 and 15, 57 and 15, 27 and 28, 58 and 28, 27 and 30, 58 and 30, 40 and 41, 59 and 41, 46 and 47 or 60 and 47, respectively.

21. A polypeptide comprising VH, said VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 16, 19 or 48.

22. The polypeptide of claim 19, wherein the VH comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 3, 4 and 5; 21, 22 and 23; or 34, 35 and 50, respectively.

23. The polypeptide of claim 19, wherein the VH comprises the amino acid sequences of SEQ ID NO: 3, 4 and 5; 21, 22 and 23; 34, 35 and 36; or the CDRH1, CDRH2 and CDRH3 sequences shown in 34, 35 and 44.

24. The polypeptide of claim 19, wherein the VH comprises the amino acid sequence of SEQ ID NO: 16, 19 or 48.

25. The polypeptide of claim 19, wherein the VH comprises the amino acid sequence of SEQ ID NO: 1, 11, 19, 32 or 42.

26. The polypeptide of claim 19, wherein the polypeptide comprises a heavy chain comprising an amino acid sequence of SEQ ID NO: 9, 14, 27, 40, 46, 56, 57, 58, 59 or 60.

27. A polypeptide comprising a VL, said VL comprising the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 17, 31, or 49.

28. The polypeptide of claim 25, wherein the VL comprises the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 6, 18 and 8; 24, 25 and 26; or 37, 51 and 39, respectively.

29. The polypeptide of claim 25, wherein the VL comprises the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 6, 7 and 8; 6, 13 and 8; 24, 25 and 26; 37, 38 and 39; or 37, 45 and 39.

30. The polypeptide of claim 25, wherein the VL comprises the amino acid sequence of SEQ ID NO: 17, 31 or 49.

31. The polypeptide of claim 25, wherein the VL comprises the amino acid sequence of SEQ ID NO: 2, 12, 20, 29, 33 or 43.

32. The polypeptide of claim 25, wherein the polypeptide comprises a light chain comprising an amino acid sequence of SEQ ID NO: 10, 15, 28, 30, 41 or 47.

33. The antibody or polypeptide according to any one of the preceding claims, wherein the antibody or polypeptide is conjugated with a cytotoxic agent, a cell inhibitor, a toxin, a radionuclide, or a detectable label.

34. A polynucleotide encoding: the VH, VL, heavy chain and / or light chain of the antibody according to any one of claims 1 to 18; or a polypeptide according to any one of claims 19 to 30.

35. A vector comprising the polynucleotide according to claim 32.

36. A recombinant host cell comprising: (a) The polynucleotide according to claim 32; (b) The carrier according to claim 33; (c) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes the heavy chain variable region of the antibody according to any one of claims 1 to 18 or the heavy chain, and the second polynucleotide encodes the light chain variable region of the antibody according to any one of claims 1 to 18 or the light chain; or (d) A first carrier and a second carrier, the first carrier comprising a first polynucleotide encoding the heavy chain variable region or the heavy chain of the antibody according to any one of claims 1 to 18, and the second carrier comprising a second polynucleotide encoding the light chain variable region or the light chain of the antibody according to any one of claims 1 to 18.

37. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 18 or 31, a polypeptide according to any one of claims 19 to 31, a polynucleotide according to claim 32, a carrier according to claim 33 or a host cell according to claim 34, and a pharmaceutically acceptable carrier or excipient.

38. A method for producing an antibody, the method comprising culturing a host cell according to claim 34 under suitable conditions to express the polynucleotide and produce the antibody.

39. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an antibody according to any one of claims 1 to 18 or 31, a polypeptide according to any one of claims 19 to 31, a polynucleotide according to claim 32, a carrier according to claim 33, a host cell according to claim 34, or a pharmaceutical composition according to claim 35.

40. Use of the antibody according to any one of claims 1 to 18 or 31, the polypeptide according to any one of claims 19 to 31, the polynucleotide according to claim 32, the carrier according to claim 33, the host cell according to claim 34, or the pharmaceutical composition according to claim 35 in the preparation of a medicament for treating cancer in a subject of need.

41. An antibody according to any one of claims 1 to 18 or 31, a polypeptide according to any one of claims 19 to 31, a polynucleotide according to claim 32, a carrier according to claim 33, a host cell according to claim 34, or a pharmaceutical composition according to claim 35, for use in medicine.

42. An antibody according to any one of claims 1 to 18 or 31, a polypeptide according to any one of claims 19 to 31, a polynucleotide according to claim 32, a carrier according to claim 33, a host cell according to claim 34, or a pharmaceutical composition according to claim 35, for the treatment of cancer in a subject in need.

43. A method for treating cancer, the method comprising administering a therapeutically effective amount to a subject suffering from cancer: (a) an antibody according to any one of claims 1 to 18 or 31, or a polypeptide according to any one of claims 19 to 31; and (b) Bispecific T cell connector.

44. The method of claim 41, wherein the bispecific T-cell connective specifically binds to the following: (a) Tumor-associated antigens; and (b) Molecules on effector cells.

45. The method of claim 42, wherein the tumor-associated antigen is Her2.

46. ​​The method of claim 42, wherein the tumor-associated antigen is CD19.

47. The method of claim 42, wherein the molecule on the effector cell is CD3.

48. The method of claim 42, wherein the tumor-associated antigen is Her2 and the molecule on the effector cell is CD3.

49. The method of claim 42, wherein the tumor-associated antigen is CD19 and the molecule on the effector cell is CD3.

50. The method of claim 41, wherein the bispecific T-cell connector comprises trastuzumab and a CD3 binder.

51. The method of claim 41, wherein the bispecific T-cell connective is bonnetumab.

52. A method, wherein the method comprises administering a therapeutically effective amount to a subject: (a) an antibody according to any one of claims 1 to 18 or 31, or a polypeptide according to any one of claims 19 to 31; and (b) PD-1 and / or PD-L1 agents.

53. The method of claim 52, wherein the PD-1 or PD-L1 agent is or comprises an antibody.

54. The method of claim 53, wherein the antibody is an antagonist antibody.

55. An antibody that specifically binds to human KLRG1, said antibody comprising VH and VL, wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 57 or 65, wherein: The VH contains up to four cumulative amino acid substitutions from CDRH1, CDRH2, and CDRH3, and the VL contains up to two cumulative amino acid substitutions from CDRL1, CDRL2, and CDRL3.

56. The antibody of claim 55, wherein the antibody comprises VH and VL, wherein the VH comprises the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence shown in SEQ ID NO: 56 or 64; and the VL comprises the CDRL1, CDRL2 and CDRL3 amino acid sequences of the VL amino acid sequence shown in SEQ ID NO: 57 or 65.

57. The antibody according to claim 55 or 56, wherein the antibody comprises, respectively, the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, wherein: The VH contains up to four cumulative amino acid substitutions from CDRH1, CDRH2, and CDRH3, and the VL contains up to two cumulative amino acid substitutions from CDRL1, CDRL2, and CDRL3.

58. The antibody according to any one of claims 55 to 57, wherein the antibody comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 amino acid sequences of the VH and VL amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, respectively.

59. The antibody according to any one of claims 55 to 58, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 58, 59 and 60 respectively; or the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in 66, 67 and 68, wherein the VH comprises at most four cumulative amino acid substitutions of CDRH1, CDRH2 and CDRH3.

60. The antibody according to any one of claims 55 to 59, wherein the antibody comprises the amino acid sequences of CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO: 58, 59 and 60, respectively; or 66, 67 and 68.

61. The antibody according to any one of claims 55 to 60, wherein the antibody comprises the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NO: 61, 62 and 63 respectively; or CDRL1, CDRL2 and CDRL3 shown in 69, 70 and 71, and wherein the VL comprises at most two cumulative amino acid substitutions of CDRL1, CDRL2 and CDRL3.

62. The antibody according to any one of claims 55 to 61, wherein the antibody comprises SEQ ID NO: 61, 62 and 63 respectively; or the CDRL1, CDRL2 and CDRL3 amino acid sequences shown in 69, 70 and 71.

63. The antibody according to any one of claims 55 to 62, wherein the antibody comprises, respectively, SEQ ID NO: 58, 59, 60, 61, 62 and 63; or the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in 66, 67, 68, 69, 70 and 71, wherein: The CDRH1, CDRH2, and CDRH3 contain up to four cumulative amino acid substitutions, and the CDRL1, CDRL2, and CDRL3 contain up to two cumulative amino acid substitutions.

64. The antibody according to any one of claims 55 to 63, wherein the antibody comprises SEQ ID NO: 58, 59, 60, 61, 62 and 63; or the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in 66, 67, 68, 69, 70 and 71.

65. The antibody according to any one of claims 55 to 64, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO: 56 or 64, wherein the VH comprises up to fifteen amino acid substitutions outside the CDR region.

66. The antibody according to any one of claims 55 to 65, wherein the antibody comprises the VH amino acid sequence of SEQ ID NO: 56 or 64.

67. The antibody according to any one of claims 55 to 66, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO: 57 or 65, wherein the VL comprises up to fifteen amino acid substitutions outside the CDR region.

68. The antibody according to any one of claims 55 to 67, wherein the antibody comprises the VL amino acid sequence of SEQ ID NO: 57 or 65.

69. The antibody according to any one of claims 55 to 68, wherein the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65, wherein the VH and the VL each comprise up to fifteen amino acid substitutions outside their respective CDR regions.

70. The antibody according to any one of claims 55 to 69, wherein the VH and the VL respectively comprise the amino acid sequences shown in SEQ ID NO: 56 and 57 or 64 and 65.

71. A polynucleotide encoding the VH or VL according to any one of claims 55 to 70.

72. A vector comprising the polynucleotide according to claim 71.

73. A recombinant host cell comprising: (a) The polynucleotide according to claim 71; (b) The carrier according to claim 72; (c) A first polynucleotide and a second polynucleotide, wherein the first polynucleotide encodes the heavy chain variable region or heavy chain of the antibody according to any one of claims 55 to 70, and the second polynucleotide encodes the light chain variable region or light chain of the antibody according to any one of claims 55 to 70; or (d) A first carrier and a second carrier, the first carrier comprising a first polynucleotide encoding the heavy chain variable region or the heavy chain of the antibody according to any one of claims 55 to 70, and the second carrier comprising a second polynucleotide encoding the light chain variable region or the light chain of the antibody according to any one of claims 55 to 70.

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