CN117321082A

CN117321082A - anti-KLRG 1 antibodies

Info

Publication number: CN117321082A
Application number: CN202280031752.7A
Authority: CN
Inventors: 斯特凡诺·V·古拉; 肯尼思·埃文·汤普森
Original assignee: Apoco Ltd
Current assignee: Apoco Ltd
Priority date: 2021-03-26
Filing date: 2022-03-25
Publication date: 2023-12-29

Abstract

The present disclosure relates to antibodies or antigen-binding fragments thereof that specifically bind to the killer lectin-like receptor G1 (KLRG 1). Such antibodies, or antigen binding fragments thereof, may be used for a variety of therapeutic or diagnostic purposes, including treating cancer and increasing the effectiveness of the vaccine.

Description

anti-KLRG 1 antibodies

Technical Field

The field of technology relates to inhibition of lymphocyte co-inhibitor receptor killing cell lectin-like receptor subfamily G member 1.

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application Ser. No. 63/166,663, filed on day 26, 3, 2021, and U.S. provisional application Ser. No. 63/294,436, filed on day 29, 12, 2021, each of which is incorporated herein by reference in its entirety.

Reference to sequence Listing

The present application contains a sequence listing in computer readable form. The computer readable form is incorporated herein by reference. The ASCII copy was created at 2022, 3 months, 1 day, named 770808_000043_sl. Txt, of size 32,100 bytes.

Background

Lymphocyte co-suppression receptors modulate the effects of the adaptive immune system (see, e.g., WO 2020/060781). The action of co-inhibitory receptors is generally performed by binding a ligand to the extracellular domain of the co-inhibitory receptor, followed by recruitment of intracellular phosphatases via an immunoreceptor tyrosine-based inhibitory motif (ITIM) located in the intracellular domain of the co-inhibitory receptor. The role of co-inhibitory receptors is generally to attenuate the immune response of the T Cell Receptor (TCR) participation. In recent years, agents that block the activity of co-inhibitory receptors have proven useful in the effective treatment of cancer and infectious diseases.

Disclosure of Invention

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

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

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

The antigen binding fragment may be a F (ab) fragment. The antigen binding fragment may be F (ab) ₂ ) Fragments. The antibody or antigen binding fragment thereof may be bispecific.

In one aspect, the present disclosure relates to a method of treating cancer comprising administering an antibody or antigen binding fragment disclosed herein to a cancer patient. The method may further comprise administering a checkpoint inhibitor to the cancer patient. The checkpoint inhibitor may be administered concurrently with administration of the antibody or antigen-binding fragment thereof. The checkpoint inhibitor may be administered within one day, one week or one month of the antibody or antigen binding fragment thereof. The cancer can be breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, and cancer of the brain primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian cancer, and cervical cancer lung cancer, small cell lung cancer, wilms 'tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, gastric cancer, colon cancer, prostate cancer, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenal cortical cancer, and malignant pancreatic insulinoma, malignant carcinoid, choriocarcinoma, mycosis fungoides, hypercalcemia of malignancy, cervical hyperplasia, leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, kaposi's (Kaposi) sarcoma, polycythemia vera, idiopathic thrombocythemia, hodgkin's disease, non-Hodgkin's lymphoma, soft tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma.

The present disclosure also relates to adjuvant therapy comprising administering an antibody or antigen binding fragment thereof disclosed herein to a subject undergoing treatment with a checkpoint inhibitor, thereby performing the adjuvant therapy.

Drawings

The present disclosure will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings.

FIG. 1 is a graph showing the binding of PA015 to CHOK1 cells expressing human KLRG 1.

FIG. 2 is a graph showing that PA015 inhibits binding of E-cadherin to KLRG1 expressing CHOK1 cells in a relative binding assay.

FIG. 3 is a graph showing that PA015 inhibited E-cadherin binding to KLRG 1-expressing CHOK1 cells in comparison to HG1N01 and HG1N02 in a relative binding assay.

FIG. 4 shows a graph of PA015 binding to human CD8+ T cells from two donors compared to HG1N02 in a FACS binding assay.

Fig. 5 is a graph showing the serum concentration of PA015 over time in a cynomolgus monkey (cynomolgus monkey) pharmacokinetic assay described in example 9.

FIG. 6 is a graph showing the serum concentration of HG1N01 over time in the cynomolgus monkey pharmacokinetic assay described in example 9.

FIG. 7 shows a graph of deamidation at specific Light Chain (LC) and Heavy Chain (HC) residues of PA015 compared to HG1N 02.

Detailed Description

Certain exemplary embodiments are described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the antibodies, fragments thereof, compositions of matter, kits, and related methods and therapies disclosed herein.

The killer lectin-like receptor G1 (KLRG 1) is a type II transmembrane protein that can act as a co-inhibitory receptor by modulating the activity of T cells and NK cells. The extracellular portion of KLRG1 contains a C-lectin domain, a known ligand of cadherin. The intracellular portion of KLRG1 contains an immunoreceptor tyrosine based inhibitory motif (ITIM) domain responsible for co-suppression of T Cell Receptor (TCR) mediated signaling. KLRG1 ligands include E-cadherin, N-cadherin, R-cadherin, and combinations thereof.

In humans KLRG1 expression is generally limited to cells of the immune system and in particular to CD8 positive T cells, NK cells and to a lesser extent to CD4 positive T cells. KLRG1 expression is associated with a late differentiation phenotype. As antigen-specific T cells differentiate, they may acquire increased expression of cytotoxic molecules and thus may have increased cytotoxic potential. The biological function of KLRG1 is to inhibit the cytotoxicity and proliferation of these T cells in some cases. In murine cancer models, blockade of KLRG1 interaction with its ligand has proven beneficial in controlling tumor growth or metastasis.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only, and is not intended to be limiting of the disclosure and any invention described or otherwise provided herein.

Amino acids are referred to herein as single letter codes or three letter codes, both of which conform to 37c.f.r. ≡1.822 and established usage.

All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for all purposes to obtain teachings relating to the sentences and/or paragraphs in which the references are provided.

Definition of the definition

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

As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in an alternative manner ("or").

The terms "about" and "about" as used herein in reference to a measurable value such as the amount of a polypeptide, dose, time, temperature, enzymatic activity or other biological activity, and the like, are intended to encompass variations of the specified amounts of + -20%, + -10%, + -5%, + -1%, + -0.5%, or even + -0.1%.

The transitional phrase "consisting essentially of … …" means that the scope of the claims should be construed to cover the specified materials or steps recited in the claims, "as well as those that do not materially affect the basic and novel characteristics of the claimed invention. See Inre Herz,537F.2d 549, 551-52, 190USPQ 461, 463 (CCPA 1976) (highlighted by text); see also MPEP 2111.03.

The term "consisting essentially of … …" (and grammatical variants) as applied to polynucleotide or polypeptide sequences of the present disclosure means a polynucleotide or polypeptide consisting of the recited sequence (e.g., SEQ ID NO) and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids at the N-and/or C-terminus of the recited sequence such that the function of the polypeptide is not substantially altered. The total of 10 or less additional amino acids may include the total number of additional amino acids added at both ends.

An "effective amount" as used herein is an amount that provides the desired effect. The term "effective amount" refers to a dose or amount sufficient to reduce KLRG1 activity to result in an improvement in the symptoms of the patient or to achieve a desired biological result, such as a reduction in KLRG1 activity, modulation of lymphocyte co-suppression response, increased or decreased activation of cytotoxic T cells and NK cells, or increased or decreased ifnγ release by cytotoxic T cells or NK cells.

A "therapeutically effective" amount as used herein is an amount that provides some improvement or benefit to a subject. In other words, a "therapeutically effective" amount is an amount that will provide some alleviation, relief, or reduction of at least one clinical symptom in a subject. Those skilled in the art will appreciate that the therapeutic effect need not be complete or therapeutic so long as some benefit is provided to the subject.

The term "treatment" or "treatment of" means to reduce, or at least partially improve or ameliorate, the severity of a disorder in a subject, and to achieve some alleviation, alleviation or reduction of at least one clinical symptom.

The term "cancer" as used herein refers to any benign or malignant abnormal growth of cells. Examples include, but are not limited to, breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, wilms ' tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, prostate cancer, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal gland cancer, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid tumor, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, cytoblast, sarcomas, kaposi's sarcoma, polycythemia, hodgkin's sarcoma, lymphomatosis, and lymphomatosis. In some embodiments, the cancer is selected from the group of tumors that form the cancer. Those skilled in the art will recognize which cancers fall within the scope of this group.

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

The term "fragment" as applied to a polypeptide is understood to mean an amino acid sequence having a reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or nearly identical (e.g., about 90%, about 92%, about 95%, about 98%, about 99% identical) to the reference polypeptide or amino acid sequence. Such polypeptide fragments according to the present disclosure may, where appropriate, be comprised in a larger polypeptide of which they are a component. In some embodiments, such fragments may comprise, consist essentially of, and/or consist of a peptide of at least about 4, about 6, about 8, about 10, about 12, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 75, about 100, about 150, about 200 or more contiguous amino acids in length of a polypeptide or amino acid sequence according to the present disclosure.

As used herein, the terms "protein" and "polypeptide" are used interchangeably, and encompass both peptides and proteins, unless otherwise indicated.

The term "antibody (antibodies)" or "antibodies" as used herein refers to all types of immunoglobulins, including IgG, igM, igA, igD and IgE. Antibodies may be monoclonal or polyclonal and may belong to any source of species (including, for example, mouse, rat, rabbit, horse, goat, sheep, camel, or human), or may be chimeric antibodies. The antibody may be a recombinant monoclonal antibody produced according to the methods disclosed in, for example, U.S. patent No. 4,474,893 or U.S. patent No. 4,816,567. Antibodies can also be chemically constructed, for example, according to the methods disclosed in U.S. Pat. No. 4,676,980.

The terms "antigen binding domain", "antigen binding fragment" and "binding fragment" refer to the portion of an antibody molecule that contains amino acids responsible for specific binding between the antibody and antigen. In the case of larger antigens, the antigen binding domain may bind to only a portion of the antigen. The portion of the antigen molecule responsible for specific interactions with the antigen binding domain is referred to as an "epitope" or "antigenic determinant".

Although the antigen binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), it is not necessarily required to comprise both. For example, fd antibody fragments consist of VH domains only, but still retain some of the antigen-binding function of an intact antibody.

The anti-KLRG 1 antibody comprises an antibody constant region or part thereof. For example, a VL domain may be attached at its C-terminus to an antibody light chain constant domain comprising a human ck or cλ chain. Similarly, specific antigen binding domains based on VH domains may attach all or part of an immunoglobulin heavy chain derived from any antibody isotope, e.g., igG, igA, igE and IgM, and any isotopic subclass, including but not limited to IgG1 and IgG4. The DNA and amino acid sequences of the C-terminal fragment are well known in the art.

The terms "specific interaction" and "specific binding" refer to two molecules that form a complex that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to medium capacity (capacity), which is distinguished from non-specific binding, which generally has low affinity and medium to high capacity. Typically, when the affinity constant KA is greater than about 10 ⁶ M ^-1 Or more preferably above about 10 ⁸ M ^-1 Binding is considered specific when it is. If desired, non-specific binding may be reduced without substantially affecting specific binding, e.g., by altering binding conditions. Suitable binding conditions such as concentration of antibody, ionic strength of solution, temperature, time allowed for binding, concentration of blocking agent (e.g., serum albumin, milk casein), etc. can be optimized by one skilled in the art using conventional techniques.

In certain embodiments, the antibody can specifically bind to an epitope within the extracellular domain (ECD) of human or monkey KLRG1 with an affinity (expressed as KD) of at least about 2nM, about 1nM, about 100pM, about 10pM, or about 5 pM. The amino acid sequences of the ECDs of human and cynomolgus KLRG1 are set forth in SEQ ID NO. 1 and SEQ ID NO. 2, and the amino acid sequences of human E-cadherin are set forth in SEQ ID NO. 3 below.

Antibodies and compositions

The present disclosure describes antibodies and fragments thereof that specifically bind to the extracellular domain of KLGR1 of a cell by interfering with the binding of E-cadherin, R-cadherin, and/or N-cadherin to the extracellular domain of KLRG1. Such antibodies prolong cytotoxicity of T cells and NK cells bearing KLRG1 on their surface by interfering with binding of E-cadherin, R-cadherin, and/or N-cadherin ligands to KLRG1. The antibodies described herein can be used as effective therapeutics for treating cancer as monotherapy or in combination with other immunotherapeutic agents (such as checkpoint inhibitors, e.g., anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA 4 antibodies) or in combination with chemotherapeutic agents or cancer vaccines. Such antibodies or fragments thereof may be used as an adjunct therapy for cancer treatment, regardless of whether the cancer cells express KLRG1.

Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. Briefly, each light chain may consist of an N-terminal variable domain (VL) and a constant domain (CL). Each heavy chain may consist of an N-terminal variable domain (VH), three or four constant domains (CH), and a hinge region. The CH domain closest to VH is designated CH1. The VH and VL domains consist of or comprise four regions of relatively conserved sequences, termed framework regions (FR 1, FR2, FR3 and FR 4), which form a scaffold of three regions of hypervariable sequences termed Complementarity Determining Regions (CDRs). CDRs may contain most of the residues responsible for specific interactions with antigens. These three CDRs are referred to as CDR1, CDR2 and CDR3. The CDR components on the heavy chain are referred to as H1, H2 and H3, while the CDR components on the light chain are correspondingly referred to as L1, L2 and L3.CDR3 (particularly H3) is the largest source of molecular diversity within the antigen binding domain. For example, H3 may be as short as 2 amino acid residues or greater than 26 amino acid residues.

The Fab fragment (antigen binding fragment) or F (ab) consists of or comprises a VH-CH1 domain and a VL-CL domain covalently linked by disulfide bonds between constant regions. As known to those skilled in the art, fab (50,000 daltons) is a monovalent fragment produced from IgG and IgM, composed of or comprising VH, CH1 and VL, CL regions linked by intramolecular disulfide bonds. To overcome the propensity of non-covalently linked VH and VL domains in Fv to dissociate upon co-expression in a host cell, so-called single chain (sc) Fv fragments (scFv) can be constructed. In scFv, a flexible and sufficiently long polypeptide connects the C-terminus of VH to the N-terminus of VL, or connects the C-terminus of VL to the N-terminus of VH. Most commonly, the 15-residue (Gly 4 Ser) 3 peptide (SEQ ID NO: 42) may be used as the linker, but other linkers are also known in the art.

Antibody diversity is the result of the combined assembly of multiple germline genes encoding variable regions and multiple somatic events. Somatic events can include recombining variable gene segments with diversity (D) and joining (J) gene segments to produce complete VH regions, and recombining variable gene segments and joining gene segments to produce complete VL regions. The recombination process itself is imprecise, resulting in the deletion or addition of amino acids at the V (D) J junction. These diversity mechanisms occur in developing B cells prior to antigen exposure. After antigen stimulation, the antibody genes expressed in B cells may undergo somatic mutation.

Based on the estimated number of germline gene segments, random recombination and random VH-VL pairing of these segments can be generated up to about 1.6x10 according to Fundamental Immunology, 3 rd edition, paul edit, raven Press, new York, N.Y.,1993 ⁷ Different antibodies. When considering other processes that contribute to antibody diversity, such as somatic mutations, it is believed that potentially more than about 1 x 10 can be produced ¹⁰ Different antibodies were used, as supported by Immunoglobulin Genes, version 2, jonio et al, academic Press, san Diego, calif., 1995. Due to the many processes involved in antibody diversity, it is highly unlikely that an independently generated antibody will have identical amino acid sequences in the CDRs.

The present disclosure provides novel antibody variable heavy and light chain regions (VH and VL) and antibody heavy and light chains derived from a human immunoglobulin gene library that are effective in blocking KLRG1 signaling in cells expressing cell surface KLGR 1. These heavy and light chains and their variable regions provide a scaffold for carrying CDRs. The CDRs may generally be, but are not limited to, antibody heavy or light chains or portions thereof, wherein the CDRs are located at positions corresponding to CDRs of the naturally occurring VH and VL. The structure and position of the immunoglobulin variable domain can be determined, for example, as described in Kabat et al, sequences of Proteins of Immunological Interest, 91-3242, national Institutes of Health Publications, bethesda, md., 1991.

TABLE 1 amino acid sequences of human and cynomolgus KLRG1 ECD and human E-cadherin

Antibody binding specificity

It is contemplated that antibodies of the present disclosure may also bind to other proteins, including, for example, recombinant proteins comprising all or a portion of KLRG 1.

The KLRG1 antibody may also be a multispecific antibody, such as a bispecific or tetraspecific antibody, diabody, or similar molecule (see, e.g., PNAS USA 90 (14), description of diabodies in 6444-8 (1993)). Indeed, the bispecific antibodies, diabodies, and the like provided herein may bind to any suitable target other than a portion of KLRG 1. For example, in one embodiment, the bispecific antibody can bind KLRG1 and HER2, which will activate klrg1+ T cells and NK cells and redirect them to her2+ cancer cells. In another embodiment, the bispecific antibody binds to KLRG1 and PD-1, which will activate T cells and NK cells by blocking both checkpoint receptors (KLRG 1 and PD-1).

The present disclosure also provides multi-specific antibody fusion proteins comprising an antibody moiety, e.g., a Fab or Fab' fragment, having a first specificity for an antigen of interest, and further comprising at least one single domain antibody having a second specificity for the antigen of interest. A multispecific antibody fusion will be capable of selectively binding to two or more antigens of interest.

In one embodiment, the first antigen and the second antigen are the same antigen.

In one embodiment, the antigen of interest bound by the antibody or antigen binding fragment thereof may be a cell-associated protein, for example a cell surface protein on a cell (such as a bacterial cell, a yeast cell, a T cell, an endothelial cell or a tumor cell), or it may be a soluble protein. The antigen of interest may also be any medically relevant protein, such as those proteins that are up-regulated during a disease or infection, e.g. receptors and/or their corresponding ligands. Specific examples of cell surface proteins include adhesion molecules, e.g., integrins such as 131 integrins, e.g., VLA-4, E-selectin, P-selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CDW52, CD69, CD134 (0X 40), ICOS, BCMP7, CD137, CD27L, CDCP1, DPCR 2, FLJ20584, FLJ40787, HEK2, KIAA 064, KIAA0659, KIAA1246, KIAA1455, LTBP2, LTK, MAL2, MRP2, adhesion protein like2 (nectin-like 2), CCI, PTK7, RAIG1, TCAM1, SC6, BCMP101, BCMP84, BCMP11, DTP 11, DTD, human, and MHC antigens (FG) and, as appropriate, and antigens for human, and MHC class (FG and MHC) antigens. Soluble antigens include interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-16 or IL-17, viral antigens such as respiratory syncytial virus or cytomegalovirus antigens, immunoglobulins such as IgE, interferons such as interferon a, interferon p or interferon y, tumor necrosis factor-a, tumor necrosis factor-I3, colony stimulating factors such as G-CSF or GM-CSF, and platelet-derived growth factors such as PDGF-a and PDGF-I3, and their receptors, as appropriate. Other antigens include bacterial cell surface antigens, bacterial toxins, viruses (such as influenza, EBV, hepA, hepB and HepC), bioterrorism agents, radionuclides and heavy metals, as well as snake and spider venom and toxins. In particular embodiments, the antigen of interest includes IL-2, soluble IL-15 or membrane-bound IL-15Ra/IL-15 complexes and IL-12.

Those of skill in the art will recognize that the antibodies of the present disclosure may be used to detect, measure and inhibit proteins somewhat different from KLRG 1. Antibodies are expected to retain binding specificity as long as the target protein comprises a sequence that is at least about 60%, about 70%, about 80%, about 90%, about 95% or greater percent identical to any of at least about 130, about 100, about 80, about 60, about 40 or about 20 consecutive amino acids of the sequence set forth in SEQ ID NO. 1 or SEQ ID NO. 2. Percent identity is determined by standard alignment algorithms, such as the basic local alignment tool (BLAST) described by Altshul et al, (1990) J.mol.biol.,215:403-410, needleman et al, (1970) J.mol.biol.,48:444-453, or the algorithm of Meyers et al, (1988) Comput.appl.biosci., 4:11-17.

In addition to sequence homology analysis, epitope mapping (see, e.g., epitope Mapping Protocols, morris editions, humana Press, 1996) and secondary and tertiary structure analysis can be performed to identify the specific 3D structure presented by the disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) biochem. Exp. Biol., 11:7-13) and computer modeling of virtual representations of presently disclosed antibodies (Fletterick et al, (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, cold Spring Harbor Laboratory, cold Spring Harbor, N.Y.).

Antibody variants

The disclosure also provides methods for obtaining KLRG1 specific antibodies. The Complementarity Determining Regions (CDRs) in such antibodies are not limited to the specific sequences of VH and VL identified in the examples below, and may include variants of these sequences that retain the ability to specifically bind KLRG 1. Such variants can be derived from the sequences listed in the examples by a person skilled in the art using techniques well known in the art. For example, amino acid substitutions, deletions or additions may be made in the Framework Regions (FR) and/or CDRs. While changes in FR can generally be designed to improve the stability and immunogenicity of an antibody, changes in CDR can generally be designed to increase the affinity of an antibody for its target.

Changes made to FR include, but are not limited to: antibodies of non-human origin are humanized or certain framework residues important for antigen exposure or for stable binding sites are engineered, for example, to alter the class or subclass of constant regions, to alter specific amino acid residues that may alter effector functions such as Fc receptor binding (e.g., as described in U.S. Pat. Nos. 5,624,821 and 5,648,260 and Lund et al, (1991) J.Immun.147:2657-2662 and Morgan et al, (1995) Immunology 86:319-324), or to alter the species from which the constant regions are derived.

Variants of FR also include naturally occurring immunoglobulin allotypes. Such changes to increase affinity can be determined empirically by conventional techniques involving altering CDRs and testing targets of affinity antibodies. For example, conservative amino acid substitutions may be made in any one of the disclosed CDRs. Various modifications may be made according to the method described, for example, in Antibody Engineering, 2 nd edition, oxford University Press, borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that are altered by substitution of different codons within the sequence encoding functionally equivalent amino acid residues, thereby producing "silent" changes. For example, nonpolar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions (subunits) of amino acids within the sequence may be selected from other members of the class to which the amino acid belongs. In addition, any of the natural residues in the polypeptide may also be substituted with alanine (see, e.g., macLennan et al (1998) Acta Physiol. Scand. Suppl.643:55-67; sasaki et al (1998) adv. Biophys. 35:1-24).

The phrase "substantially as set forth" means that the relevant CDR, VH or VL domains of the present disclosure are identical or only have insubstantial differences from the stated sequences in a designated region (e.g., CDR). Insubstantial differences include minor amino acid changes, such as substitutions of one (1) or two (2) of any five (5) amino acids in the sequence of the designated region.

The term "KLRG1 activity" refers to one or more lymphocyte co-inhibitory activities associated with KLRG 1. For example, KLRG1 activity may mean modulation of cytotoxic T cell and NK cell activation.

The term "modulate" and its cognate terms refer to a decrease or increase in KLRG1 activity associated with activation of T cells and NK cells due to its interaction with an anti-KLRG 1 antibody, wherein the decrease or increase is relative to KLRG1 activity in the absence of the same antibody. The decrease or increase in activity is preferably at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more. The terms "modulator" and "modulator" are interchangeable with the terms "inhibit" and "inhibit" when KLRG1 activity is reduced. The terms "modulator" and "modulator" are interchangeable with the terms "activation" and "activation" when KLRG1 activity is increased.

The term "percent (%) sequence identity" as used herein refers to the percentage of nucleotides or amino acids in a candidate sequence that are identical to nucleotides or amino acids in a reference nucleic acid sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. The alignment for determining the percent sequence identity can be accomplished in a variety of ways known to those of skill in the art, for example, using publicly available computer software such as ClustalW, clustal Omega, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software.

Antibody fragments included within the scope of the present disclosure include, for example: fab, fab ', F (ab') 2, and Fv fragments; domain antibodies, diabodies; vaccine antibodies, linear antibodies; single chain antibody (ScFv) molecules; and multispecific (e.g., bispecific, tetraspecific) antibodies formed from antibody fragments. Such fragments may be generated by known techniques. For example, F (ab ') 2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reduction of disulfide bonds of F (ab') 2 fragments. Alternatively, fab expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (see Huse et al, science 12, 8, 1989; 246 (4935): 1275-1281).

Antibodies of the present disclosure are humanized versions of non-human (e.g., murine) antibodies, and may include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, fab, fab1, F (ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are present in neither the recipient antibody nor the imported CDR or framework sequences. Generally, a humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the Framework Region (FR) regions (i.e., the sequence between the CDR regions) are those of a human immunoglobulin consensus sequence. Preferably, the humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

In certain embodiments, VH and/or VL domains may be germlined (germlined), i.e., the Framework Regions (FR) of these domains are mutated to match those produced by germline cells using conventional molecular biology techniques. In other embodiments, the backbone sequence remains offset from the consensus germline sequence (dived).

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

Monoclonal antibodies useful in the practice of the present disclosure can be produced in hybridoma cell lines according to the technique of Kohler and Milstein, nature265:495 (1975). For example, a solution containing the appropriate antigen may be injected into mice, and after a sufficient time, the mice may be sacrificed and spleen cells obtained. Spleen cells are then immortalized to produce hybridoma cells by fusing the spleen cells, for example, with myeloma cells or lymphoma cells, typically in the presence of polyethylene glycol. The hybridoma cells can then be grown in a suitable medium and the supernatants screened for monoclonal antibodies of the desired specificity. Monoclonal Fab fragments can be produced in e.coli (e.coli) by recombinant techniques known to those skilled in the art. Antibodies specific for the target polypeptide can also be obtained by phage display techniques known in the art.

Various immunoassays can be used for screening to identify antibodies with the desired specificity for the extracellular domain of KLRG 1. Numerous protocols for competitive binding or immunoradiometric assays using monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve measuring complex formation between an antigen and its specific antibody (e.g., antigen/antibody complex formation). Dual site, monoclonal-based immunoassays, and competitive binding assays using monoclonal antibodies reactive with two non-interfering epitopes on a polypeptide or peptide of the present disclosure can be used.

In addition to the conjugates described above, the anti-KLRG 1 antibodies described herein can be conjugated to a solid support (e.g., a bead, plate, slide, or well formed from a material such as latex or polystyrene) according to known techniques. The anti-KLRG 1 antibodies described herein can also be conjugated to a detectable group, such as a radiolabel (e.g., ³⁵ S、 ¹²⁵ I、 ¹³¹ i or ^99m Tc, which may also be attached to an antibody using conventional chemical reactions), an enzyme label (e.g., horseradish peroxidase, alkaline phosphatase), and a fluorescent label (e.g., fluorescein). The detectable label further includes a chemical moiety (such as biotin) that is detectable via binding to a specific cognate detectable moiety (e.g., labeled avidin). Determination of antibody/antigen complex formation in the methods of the present disclosure may be performed by detecting, for example, precipitation, aggregation, flocculation, radioactivity, coloration or discoloration, fluorescence, luminescence, and the like, and such methods are well known in the art.

As described above, the anti-KLRG 1 antibodies described herein may be linked to another functional molecule, e.g., another peptide or protein (albumin, another antibody, etc.), a toxin, a radioisotope, a cytotoxic agent, or a cytostatic agent. Antibodies as described herein may be conjugated to toxins. Antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are three well-known antibody-mediated mechanisms for killing target cells.

For example, antibodies may be linked by chemical cross-linking or by recombinant methods. Antibodies may also be attached to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol or polyalkylene oxide, in the manner set forth in U.S. Pat. nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 and 4,179,337. Antibodies can be chemically modified by covalent conjugation with polymers, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching them are also shown in U.S. Pat. nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546.

PA015 composition

In some embodiments, an anti-KLRG 1 antibody comprises a combination of any six CDRs of any one of the antibodies described herein. In some embodiments, the anti-KLRG 1 antibody comprises (i) three heavy chain CDRs (i.e., CDR1, CDR2, and CDR 3) from table 2, and (ii) three light chain CDRs from table 2.

TABLE 2

There are a variety of antibody variable chain numbering schemes, including those set forth by: kabat (Wu and Kabat,1970, J Exp Med. (1970) 132:211-50.); kabat EA, te Wu T, foeller C, perry HM, gottesman KS. Sequences of Proteins of Immunological interest. Diode Publishing Company (1992)); chothia (1987,Chothia C,Lesk a M.Canonical structures for the hypervariable regions of immunoglobulins.J Mol Biol. (1987) 196:901-17); al-Lazikani et Al, (1997Standard conformations for the canonical structures of immunoglobulins.J Mol Biol (1997)) 273:927-48), lefranc (IMGT numbering; lefranc,1997Unique database numbering system for immunogenetic analysis.Immunol Today (1997) 18:509; lefranc et Al, 2005IMGT-ONTOLOGY for immunogenetics and immunoinformation ics.In Silico biol. (2004) 4:17-29, and Honyger (Honyger and Pluckthun, 2001,Yet another numbering scheme for immunoglobulin variabledomains:an automatic modeling and analys is tool.J Mol Biol. (2001) 309:657-70), which further complicates it.

Chothia and his colleagues define CDRs based on structure (i.e., ring position) (Chothia and Lesk,1987 supra; al-Lazikani et Al, 1997 supra).

The CDRs were initially defined by amino acid sequence variability analysis early in the 70 s of the 20 th century by Kabat and Wu (Wu and Kabat,1970, supra; kabat and Wu,1971, supra). Since then, detailed genetic and three-dimensional structural analyses have more clearly defined the structural basis of antigen binding sites in terms of HVL/CDR, resulting in the definition of contact residues (MacCallum et al, 1996, anti-body-antigen interactions: contact analysis and binding site topogram.J Mol biol. (1996) 262:732-745; martin and Allen,2007,Bioinformatics tools for antibody engineering; in Dubel S eds., handbook of Therapeutic antibodies.Weinheim: wiley-VCH Verlag GmbH (2008), pages 95-117).

AbM definitions of CDRs (which are a compromise between Kabat and Chothia definitions) were used in AbM modeling software (Martin and Allen,2007, supra). Martin and his colleagues also formed the definition of CDRs based on the actual paratopes (i.e., those residues that actually contact the antigen) (Martin and Allen,2007, supra), and developed and tested an automated application called "Abnum" for variable strand numbering and CDR determination based on Chothia method (abhinan and Martin,2008,Analysis and prediction of VH/VL packaging in anti bodies.

The Contact definition of a CDR is based on the observation that only 20-33% of the amino acids within the CDR are in direct Contact with the antigen (Padlan EA. Anatomy of the anti-body molecular. Mol immunol. (1994) 31:169-217). These residues, termed "Specificity Determining Residues (SDR)", were first described by Padlan et al (Padlan EA, abergel C, tipper JP. Identification of specificity-determining residues in antibodies.FASEB JOffPubl Fed Am Soc Exp biol. (1995) 9:133-9). Their results show that these SDRs are involved in interactions with antigens and in most cases in matching with the most variable positions present in the CDRs. Using this SDR concept, macCallum and colleagues propose a new method of defining CDRs and rename the SDR to "contact residues". They also suggested that the contact residues are more often located in the center of the paratope and that, as mentioned in Chothia, the non-contact residues play a role in shaping the conformation of the CDR loop, so that the contact residues can be optimally oriented for efficient and specific antigen binding.

The unique number of IMGT for V-REGION allows redefining the limitations of complementarity determining and framework REGIONs in IMGT designated CDR-IMGT and FR-IMGT, respectively. The unique numbers of IMGT for V-DOMA IN are the CD R3-IMGT and FR4-IMGT for rearranged V-J-REGION and V-D-J-REGION, providing standardized numbers.

The complementarity determining region (CDR-IMGT) is the loop region of V-DOMAIN (variable (V) DOMAIN) defined by the IMGT unique number of the V DOMAIN (Lefranc M. Et al, (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable DOMAINs and Ig superfamily V-like DOMAINs. Dev Comp Immunol 27:55-77). There are three CDR-IMGT in V-DOMAIN: CDR1-IMGT (loop BC), CDR2-IMGT (loop C') and CDR3-IMGT (loop FG).

In V-DOMAIN (the V DOMAIN of Immunoglobulin (IG) or antibody and T cell receptor (TR), the amino acids of CDR-IMGT bind to the antigen (epitope) and confer specificity for IG and TR (paratope, IMGT-ONTOLOGY, type of specificity). The first two CDR-IMGT are part of a V-REGION (encoded by a variable (V) gene), whereas CDR3-IMGT corresponds to a junction and is produced by a rearrangement between the V gene and the junction (J) gene (V-J rearrangement) or a rearrangement between the V gene, the diversity (D) gene and the J gene (V-D-J rearrangement) (immunoglobulin synthesis).

PA015 sequence

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

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

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

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

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

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

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

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

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

One particular embodiment provides an antibody (preferably a monoclonal antibody) or antigen binding fragment thereof having (i) a light chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9 in a region other than the Complementarity Determining Region (CDR) region, such as including but not limited to the Framework Region (FR) region, and (ii) a heavy chain comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-H1, CDR-H2, CDR-H3 are SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively; SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, respectively; SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, respectively; SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, respectively; or SEQ ID NO 34, SEQ ID NO 35 and SEQ ID NO 36, respectively.

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

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

One embodiment provides an antibody (preferably a monoclonal antibody) or antigen binding fragment thereof having (i) a light chain variable region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:9 in a region other than the Complementarity Determining Region (CDR) region, such as including but not limited to the Framework Region (FR) region, and (ii) a heavy chain having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:8 in a region other than the Complementarity Determining Region (CDR) region, such as including but not limited to the Framework Region (FR) region.

One embodiment provides an antibody (preferably a monoclonal antibody) or antigen binding fragment thereof having (i) a light chain variable region comprising CDRs (CDR-L1, CDR-L2, CDR-L3) with amino acid sequences SEQ ID Nos 13, 14 and 15, respectively; (ii) Heavy chain variable regions comprising CDRs (CDR-H1, CDR-H2, CDR-H3) having the amino acid sequences SEQ ID Nos. 10, 11 and 12, respectively; (iii) A heavy chain constant region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 6, and (iv) a light chain constant region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 7.

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

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

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

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

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

One embodiment provides an antibody (preferably a monoclonal antibody) or antigen binding fragment thereof having (i) a light chain variable region comprising CDRs (CDR 1, CDR2, CDR 3) having the amino acid sequences SEQ ID nos. 13, 14 and 15, respectively; (ii) A heavy chain variable region comprising CDRs (CDR 1, CDR2, CDR 3) having the amino acid sequences SEQ ID Nos. 10, 11 and 12, respectively; (iii) A heavy chain constant region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 6, and (iv) a light chain constant region having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO. 7.

In certain aspects of all of the aforementioned embodiments, when a light or heavy chain, variable region, or constant region is described as having a certain percentage of sequence identity to a reference SEQ ID NO, such identity is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Immunotoxins

In some aspects, the disclosure provides antibodies and/or antigen binding fragments thereof conjugated to a toxic agent. Immunoconjugates comprising one or more cytotoxins are referred to as "immunotoxins". Antibodies conjugated to cytotoxic agents, drugs, etc. are also known as antibody-drug conjugates (ADCs). Immunoconjugates can beHas a half-life of sufficient period of time to enable the antibody-drug conjugate to be internalized, degraded and induce cell killing by the released toxin. The cytotoxin or cytotoxic agent may include any agent that is detrimental to (e.g., kills) the cell. Suitable cytotoxic agents for forming the immunoconjugates of the present disclosure include paclitaxel, tubulysin, dolastatin (duloxetine), cytochalasin B, poncirin D, ethidium bromide, ipecine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin (doxorubicin), daunorubicin, dihydroxyanthracenedione, maytansine or an analog or derivative thereof, mitoxantrone (mitoxantrone), mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin; calicheamicin or analogues or derivatives thereof, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dicarbazine (decacarbamine), hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as nitrogen mustard, thiapiprazole, chlorambucil, melphalan (melphalan), carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin, and analogues or derivatives of duocarmycin (duocarmycin) A, duocarmycin SA, CC-1065 (also known as rachelmin) or CC-1065), sea toxin, auristatin (auristatin), pyrrolo [2,1-C ] pyrrole ][1,4]Benzodiazepines(PDB), indole benzodiazepine->(IGN) or an analog thereof, antibiotics such as dactinomycin (formerly actinomycin), bleomycin (bleomycin), daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, and Amphotericin (AMC), antimitotics (e.g.,tubulin targeting agents) such as diphtheria toxins and related molecules (such as diphtheria a chain and active fragments and hybrid molecules thereof); ricin (such as ricin a or deglycosylated ricin a chain toxin), cholera toxin, shiga-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-birk type protease inhibitors, pseudomonas exotoxin, alorin, saporin, mo Disu (modeccin), gelan, abrin a chain, mo Disu a chain, α -sarcin (sarcosine), tung (Aleurites fordii proteins) protein, carnation toxin (dianin) protein, pokeweed (Phytolacca americana) protein (PAPI, PAPII and PAP-S), balsam pear (momordica charantia) inhibitors, curcin (crotin), saporin (sapaonaria officinalis) inhibitors, gelonin (gelonin), mitomycin (trigemin), restrictocin (mycotoxin), and enomycin (enomycin). Other suitable conjugated molecules include antimicrobial/lytic peptides such as CLIP, bombesin 2, melittin, antibacterial peptides and P18; ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed antiviral protein, diphtheria toxin and Pseudomonas endotoxin.

Supplementary therapeutic agent

The antibodies of the disclosure (including fragments thereof and conjugates thereof) may optionally be delivered to a patient in combination with other therapeutic agents. The additional therapeutic agent may be delivered before, simultaneously with, or after the antibodies of the present disclosure. As used herein, the term "parallel" means sufficiently close in time to be able to produce a combined effect (i.e., parallel may be simultaneous, or it may be two or more events occurring within a short period of time before or after each other).

In some embodiments, the antibodies of the present disclosure may be administered in combination with an anti-cancer agent, such as: 1) Vinca alkaloids (e.g., vinblastine, vincristine); 2) Epipodophyllotoxins (e.g., etoposide and teniposide); 3) Antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubdomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) Enzymes (e.g., L-asparaginase); 5) Biological response modifiers (e.g., interferon- α); 6) Platinum coordination complexes (e.g., cisplatin and carboplatin); 7) Anthracenediones (e.g., mitoxantrone); 8) Substituted ureas (e.g., hydroxyurea); 9) Methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); 10 Adrenocortical inhibitors (e.g., mitotane (o, p' -DDD) and aminoglutethimide); 11 An adrenocorticosteroid (e.g., prednisone); 12 Progestins (e.g., medroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13 Estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14 Antiestrogens (e.g., tamoxifen); 15 Androgens (e.g., testosterone propionate and fluoxytestosterone); 16 Anti-androgens (e.g., flutamide): and 17) gonadotropin releasing hormone analogues (e.g., leuprorelin). In some embodiments, the antibodies of the present disclosure may be administered in combination with an anti-angiogenic agent such as an antibody to VEGF (e.g., bevacizumab (AVASTIN), ranibizumab (LUCENTIS)) and other promoters of angiogenesis (e.g., bFGF, angiopoietin-1), an antibody to α -v/β -3 vascular integrin (e.g., VITAXIN), angiostatin, endostatin, dalteparin, ABT-510, CNGRC peptide (SEQ ID NO: 43), tnfα conjugates, cyclophosphamide, combretastatin (combretastatin) A4 phosphate, dimethylxanthenone acetic acid, docetaxel, lenalidomide, enzatoin, paclitaxel, nanoparticulate formulations stabilized with paclitaxel albumin (Abraxane), soy isoflavone (Genistein), citric acid tamoxifen, thalidomide, ADH-1 (herceptin), heat-01376, BMS 217d, 217a, guanfacitinib, asatin, tsuccinide, tsubide, tsubiquitos, tsubide, atacamide, and ataxin-582664.

In some embodiments, the antibodies of the present disclosure may be administered in combination with immunosuppressants including, for example, cyclosporin a, rapamycin, glucocorticoids, azathioprine, mizoribine, aspirin derivatives, hydroxychloroquine, methotrexate, cyclophosphamide, and FK506 (tacrolimus).

Nucleic acid, cloning and expression systems

The disclosure further provides isolated nucleic acids encoding the disclosed antibodies. The nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic or recombinant. Unless the context requires otherwise, reference to a nucleotide sequence as set forth herein encompasses DNA molecules having the specified sequence, and encompasses RNA molecules having the specified sequence, wherein U replaces T.

The nucleic acids provided herein comprise coding sequences for CDRs, VH domains, and/or VL domains as disclosed herein. The disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcripts or expression cassettes comprising at least one nucleic acid encoding a CDR, VH domain and/or VL domain disclosed herein. The present disclosure further provides host cells comprising one or more constructs as described above.

In some embodiments, the coding sequence of the heavy chain is the nucleotide sequence of SEQ ID NO. 40.

In some embodiments, the coding sequence of the light chain is the nucleotide sequence of SEQ ID NO. 41.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. For cells suitable for antibody production, see Gene Expression Systems, academic Press, fernandez et al, eds., 1999. Briefly, suitable host cells include bacterial, plant, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines useful in the art for expressing heterologous polypeptides include chinese hamster ovary cells, heLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many others. A common bacterial host is E.coli. Any protein expression system compatible with the present invention may be used to produce the disclosed antibodies. Suitable expression systems include the transgenic animals described in Gene Expression Systems, academic Press, fernandez et al, 1999.

Suitable vectors may be selected or constructed such that they contain suitable regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other suitable sequences. The vector may be a plasmid or a virus, e.g., a phage or phagemid, as the case may be. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual, 2 nd edition, cold Spring Harbor Laboratory Press,1989 for further details. Many known techniques and protocols for manipulating nucleic acids (e.g., preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and protein analysis) are described in detail in Current Protocols in Molecular Biology, 2 nd edition, ausubel et al, eds., john Wiley & Sons, 1992.

A further aspect of the disclosure provides a host cell comprising a nucleic acid as disclosed herein. Still further aspects provide methods comprising introducing such nucleic acids into host cells. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia virus or baculovirus (for insect cells). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection with phage. Following introduction of the nucleic acid into the cell, expression of the nucleic acid may then be caused or allowed, for example, by culturing the host cell under conditions of gene expression.

Therapeutic method

The disclosed anti-KLRG 1 antibodies are capable of modulating an immune response associated with KLRG 1. In particular embodiments, activation of cytotoxic T cells and NK cells is mediated by modulation of KLRG1 signaling. The disclosed antibodies may act as agonists or antagonists of KLRG1, depending on their method of use. The antibodies are useful for preventing, diagnosing or treating medical conditions in mammals, particularly humans. Antibodies of the disclosure may also be used to isolate KLRG1 or KLRG1 expressing cells. Furthermore, the antibodies may be used to treat a subject at risk of or susceptible to or suffering from a disorder associated with aberrant KLRG1 expression or function.

The antibodies of the present disclosure may be used in situations where modulation of cytotoxic T cell and NK cell activation may be desirable, for example, in certain types of cancers and infectious diseases.

In some cases, it may be desirable to elicit or enhance an immune response in a patient to treat cancer or an infectious disease. Disorders treated or prevented by the disclosed methods include, but are not limited to, microbial (e.g., bacterial), viral (e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or shingles) or parasitic infections; and cancers (e.g., melanoma and prostate cancer). Cytotoxic T cell and NK cell activation with anti-KLRG 1 antibodies disclosed herein enhances T cell and NK cell responses. In such cases, the antibody acts as an antagonist of KLRG 1. Thus, in some embodiments, the antibodies can be used to inhibit or reduce down-regulating activity associated with KLRG1, i.e., activity associated with down-regulation of cytotoxic T cell and NK cell activation.

As demonstrated in the examples, blocking the interaction of KLRG 1/E-cadherin with antagonistic anti-KLRG 1 antibodies resulted in enhanced T cell proliferation response and IFNy secretion of these cells, consistent with down-regulation of the KLRG1 pathway in cytotoxic T cell and NK cell activation. In various embodiments, the antibody inhibits binding of E-cadherin to KLRG1 with an IC50 of less than about 50nM, more preferably less than about 40nM, 30nM, 20nM, 10nM, or 5 nM. Inhibition of E-cadherin binding can be measured using techniques known in the art.

The antibodies or antibody compositions of the present disclosure are administered in a therapeutically effective amount. In general, a therapeutically effective amount can vary with the age, condition, and sex of the subject, and the severity of the medical condition of the subject. The therapeutically effective amount of the antibody ranges from about 0.001 to about 30mg/kg body weight, preferably from about 0.01 to about 25mg/kg body weight, from about 0.1 to about 20mg/kg body weight, or from about 1 to about 10mg/kg body weight. The dosage may be adjusted, if desired, to accommodate the observed therapeutic effect.

The appropriate dose is selected by the treating physician based on the clinical indication. The antibody may be administered in bolus doses to maximize circulating levels of the antibody for the longest period of time following administration. Continuous infusion may also be used after bolus doses.

Immune cells (e.g., T cells or NK cells) can also be isolated from a patient and incubated ex vivo with the antibodies of the present disclosure. In some embodiments, T cell and NK cell activation can be modulated by removing immune cells from a subject, contacting immune cells in vitro with an anti-KLRG 1 antibody of the present disclosure. In such embodiments, the anti-KLRG 1 antibodies may be used in multivalent form such that KLRG1 molecules on the surface of immune cells become "crosslinked" upon binding to such antibodies. For example, the anti-KLRG 1 antibody may be bound to a solid support such as a bead, or crosslinked via a second antibody. The immune cells can then be isolated and re-implanted into a patient using methods known in the art.

The antibodies of the present disclosure can also be used to detect the presence of KLRG1 in a biological sample. The amount of KLRG1 detected can be related to the level of expression of KLRG1, which in turn is related to the activation status of immune cells (e.g., activated T cells or NK cells) in the subject.

Detection methods employing antibodies are well known in the art and include, for example, ELISA, radioimmunoassay, immunoblot, western blot, immunofluorescence, immunoprecipitation. Antibodies may be provided in a diagnostic kit incorporating one or more of these techniques for detecting KLRG 1. Such kits may contain additional components, packaging, instructions or other materials that aid in the detection of proteins.

When an antibody is intended for diagnostic purposes, it may be desirable to modify the antibody, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, radioisotope, or enzyme). If desired, the antibodies of the present disclosure may be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron dense reagents, enzymes, and ligands with specific binding partners. Enzymes are usually detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethyl benzidine (TMB) to a blue pigment that can be quantified by a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, igG and protein a, and many receptor-ligand pairs (receptor-ligand pairs) known in the art. Other arrangements and possibilities will be apparent to those of ordinary skill in the art and are considered equivalent within the scope of the present disclosure.

The antibodies of the present disclosure can be used in screening methods to identify inhibitors of the KLRG1 pathway that are effective as therapeutic agents. In such screening assays, a first binding mixture is formed by combining KLRG1 and the antibodies of the present disclosure; and measuring the amount of binding in the first binding Mixture (MO). A second binding mixture is also formed by combining KLRG1, an antibody and the compound or agent to be screened, and the amount of binding in the second binding mixture (M1) is measured. The compound to be tested may be another anti-KLRG 1 antibody as illustrated in the examples. The amount of binding in the first binding mixture and the second binding mixture is then compared, for example by calculating the M1/M0 ratio. If a decrease in binding in the second binding mixture is observed as compared to the first binding mixture, the compound or agent is believed to be capable of modulating the down-regulation of the immune response associated with KLRG 1.

Formulation and optimization of binding mixtures it is within the skill in the art that such binding mixtures may also contain buffers and salts necessary to enhance or optimize binding, and additional control assays may be included in the screening assays of the present disclosure. Thus, compounds that were found to reduce KLRG 1-antibody binding by at least about 10% (i.e., M1/MO < 0.9), preferably greater than about 30%, can be identified and then, if desired, subjected to a secondary screening for the ability to ameliorate the condition in other assays or animal models as described below. The binding strength between KLRG1 and antibody can be measured using, for example, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance based techniques (e.g., biacore), all of which are well known in the art.

The compounds can then be tested in vitro or in animal models as described in the examples. Dose scaling as determined, for example, from animal trials, for human administration is performed according to accepted practices in the art. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The data obtained from cell culture assays or animal studies can be used in formulating a range of dosage for use in a human.

A therapeutically effective dose achieved in one animal model can be transformed using transformation factors known in the art for use in another animal, including humans (see, e.g., freireich et al, (1966) Cancer reports,50 (4): 219-244).

Pharmaceutical composition

The present disclosure provides pharmaceutical compositions comprising anti-KLRG 1 antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions generally comprise one or more antibodies of the present disclosure and a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds that provide supplemental, additional or enhanced therapeutic functions. The pharmaceutical composition may also be contained in a container, package, or dispenser together with instructions for administration.

The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Methods of accomplishing administration are known to those of ordinary skill in the art. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. Compositions that can be administered topically or orally, or that can be delivered across mucous membranes, can also be obtained.

Solutions or suspensions for intradermal or subcutaneous application (application) typically contain one or more of the following components: sterile diluents such as water for injection, saline solutions, non-volatile oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for modulating tonicity, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Such preparations may be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyoxyethylated castor oil (Cremophor EL) (BASF, parippanyy, n.j.), or Phosphate Buffered Saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy injectability (easy syringeability) exists. It should be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars in the compositions; polyols such as mannitol, sorbitol; and sodium chloride. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and/or by the use of surfactants. Prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption (e.g., aluminum monostearate and gelatin).

Administration and dosage

Methods of accomplishing administration are known to those of ordinary skill in the art. The administration may be, for example, parenteral (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous).

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of, for example, lozenges, nasal sprays, inhalants, or suppositories; for example, in the case of an antibody comprising an Fc portion, the composition may be capable of transport across a mucosal membrane in the intestine, mouth, or lung (e.g., via an FcRn receptor-mediated pathway as described in U.S. patent No. 6,030,613). For transdermal administration, the active compounds may be formulated as ointments, salves, gels or creams as generally known in the art. For administration by inhalation, the antibody may be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant (e.g., a gas such as carbon dioxide), or a nebulizer.

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

For ease of administration and uniformity of dosage, it may be advantageous to formulate parenteral compositions in dosage unit form. The term "dosage unit form" as used herein refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound calculated to be sufficient to produce the desired therapeutic effect when combined with the desired pharmaceutical carrier.

Toxicity and therapeutic efficacy of the compositions of the present disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., standard pharmaceutical procedures for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and it can be expressed as the LD50/ED50 ratio.

Compositions exhibiting a large therapeutic index are preferred. For any composition used in the present disclosure, a therapeutically effective dose may be initially estimated from cell culture assays. Examples of suitable bioassays include DNA replication assays, cytokine release assays, transcription-based assays, KLRG 1/cadherin binding assays, immunological assays, other assays (as described in the examples, for example). The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosages may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of antibody that achieves half-maximal inhibition of symptoms). Circulating levels in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay. The dosage is preferably in the circulating concentration range with little or no toxicity. The dosage may vary depending upon the dosage form employed and the route of administration employed.

The following examples are not intended to limit the scope of the present disclosure in any way. Those of ordinary skill in the art will recognize that many modifications and changes may be made without changing the spirit or scope of the present disclosure. Such modifications and variations are intended to be included within the scope of the present disclosure. All references, patents and published patent applications cited throughout this application are hereby incorporated by reference in their entirety.

Recombinant proteins were produced by standard molecular cloning and expression protocols, including human KLRG1ECD (SEQ ID NO: 1), cynomolgus monkey KLRG1ECD (SEQ ID NO: 2) and human E-cadherin (SEQ ID NO: 3), the amino acid sequences of which are known in the art and disclosed in PCT publication WO2020060781, the entire disclosure of which is incorporated herein by reference. Recombinant proteins were produced as FC fusions or HIS-tagged versions by cloning the corresponding cDNA into pCDNA4 vector (Invitrogen) and transiently transfection in mammalian HEK 293. Purification of the expressed protein was performed by chromatography using protein a affinity resin for the FC fusion version and nickel-NTA resin for the HIS-tagged protein. All purified proteins were characterized by SDS-PAGE electrophoresis to verify purity and molecular weight.

Exemplary sequence

PA015 heavy chain variable region sequence

＞PA015_VH(SEQ ID NO:4)

QVTLRESGPALVKPTQTLTLTCTVSGFSLSTFGMGVGWIRQPPGKALEWLAHIWWDDDKWYELALKSRLTISKDTSKNQVVLTITNMDPVDTATYYCARVIYYGSRSAYYSMDYWGQGTTVTVSS

PA015 light chain variable region sequence

＞PA015_VL(SEQ ID NO:5)

DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSNAHTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPVTFGQGTKLEIKRTV

PA015 heavy chain constant region sequence

> PA 015-heavy chain constant region (SEQ ID NO: 6)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

PA015 light chain constant region sequence

> PA 015-light chain constant region (SEQ ID NO: 7)

AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

PA015 heavy chain sequence

＞PA015_HC(SEQ ID NO:8)

QVTLRESGPALVKPTQTLTLTCTVSGFSLSTFGMGVGWIRQPPGKALEWLAHIWWDDDKWYELALKSRLTISKDTSKNQVVLTITNMDPVDTATYYCARVIYYGSRSAYYSMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

PA015 light chain sequence

＞PA015_LC(SEQ ID NO:9)

DVVMTQTPLSLSVTPGQPASISCKSSQSIVHSNAHTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPVTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

> PA015_VH_CDR1 (SEQ ID NO: 10) -Chothia format

GFSLSTFGM

> PA015_VH_CDR2 (SEQ ID NO: 11) -Chothia format

WWDDD

> PA015_VH_CDR3 (SEQ ID NO: 12) -Chothia format

VIYYGSRSAYYSMDY

> PA015_VL_CDR1 (SEQ ID NO: 13) -Chothia format

KSSQSIVHSNAHTYLE

> PA015_VL_CDR2 (SEQ ID NO: 14) -Chothia format

KVSNRFS

> PA015_VL_CDR3 (SEQ ID NO: 15) -Chothia format

FQGSHVPVT

＞PA015_HC(SEQ ID NO:40)

ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCGGCGCTCATTCTCAAGTGACACTGAGAGAGTCTGGCCCCGCTCTGGTCAAGCCTACACAGACCCTGACACTGACCTGCACCGTGTCTGGCTTCTCCCTGTCTACCTTTGGCATGGGCGTCGGCTGGATTAGACAGCCTCCTGGAAAAGCCCTGGAATGGCTGGCCCACATTTGGTGGGACGACGACAAGTGGTACGAGCTGGCTCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAATCAGGTGGTGCTGACAATCACCAACATGGACCCTGTGGACACCGCCACCTACTACTGCGCCAGAGTGATCTACTACGGCTCCAGATCCGCCTACTACTCCATGGATTATTGGGGCCAGGGCACCACCGTGACCGTGTCCTCTGCTTCTACAAAGGGCCCCTCTGTGTTCCCTCTGGCTCCTTCCTCTAAATCCACCTCTGGCGGAACCGCTGCTCTGGGCTGTCTCGTGAAGGATTACTTCCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGTGCTCTGACATCCGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCTCTGGCCTGTACTCTCTGTCCTCTGTCGTGACAGTGCCTTCCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGTCCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAAGCTGCTGGCGCTCCCTCCGTGTTTCTGTTCCCTCCAAAGCCTAAGGACACCCTGATGATCTCTCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCTCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACTCCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAAAAGACCATCAGCAAGGCTAAGGGCCAGCCTCGGGAACCTCAGGTTTACACCCTGCCTCCATCTCGGGAAGAGATGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGACATTGCCGTGGAATGGGAGTCCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACTCCGACGGCTCATTCTTCCTGTACTCCAAGCTGACAGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTCTCCTGTTCTGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCTCTGTCCCCTGGCAAGTGATGA

＞PA015_LC(SEQ ID NO:41)

ATGGGCTGGTCCTGCATCATTCTGTTTCTGGTGGCTACCGCCACCGGCGCTCATTCTGATGTGGTCATGACACAGACCCCTCTGAGCCTGTCTGTGACACCTGGACAGCCTGCCTCCATCTCCTGCAAGTCCTCTCAGTCCATCGTGCACAGCAACGCCCACACCTACCTGGAATGGTATCTGCAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACAAGGTGTCCAACAGATTCTCTGGCGTGCCCGACAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGAAGATCTCTAGAGTGGAAGCCGAGGACGTGGGCGTGTACTACTGTTTCCAAGGCTCTCACGTGCCCGTGACCTTTGGCCAGGGAACAAAGCTGGAAATCAAGCGGACCGTGGCCGCTCCTTCCGTGTTCATCTTTCCACCTTCCGACGAGCAGCTGAAGTCCGGCACAGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAAGTGGATAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACTCTCTGTCCTCCACACTGACCCTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGCTGATGA

Examples

Example 1: production of anti-KLRG 1 antibodies

The PA015 antibody is a humanized IgGl antibody against the extracellular domain of KLRG 1. Mouse Monoclonal Antibodies (MAB) against human KLRG1 were generated by standard immunization of female B ALB/c mice and SJL mice with human and cynomolgus KLRG1 followed by hybridoma screening. Several immunization strategies have been employed to generate different numbers of antibody hits. Briefly, SJL and Balb/c mice were repeatedly immunized with cDNA, recombinant antigen, or CHO cells expressing the antigen of interest. Antigen-specific antibody titers were monitored periodically by ELISA, and animals were sacrificed when appropriate titers were reached, typically between 1:1000 and 1:10000 dilution factors. Spleen cells from the sacrificed mice were fused with mouse myeloma cells to generate hybridoma cells, which were then cultured and subcloned into single cells. Stable clones were scaled up, conditioned medium was collected and tested for expression of anti-KLRG 1 antibodies by ELISA and FACS.

Example 2: PA015 binds to CHOK1 cells expressing human KLRG 1.

Binding of anti-KLRG 1 monoclonal antibodies to cells expressing human and cynomolgus KLRG1 was performed by FACS. Chinese Hamster Ovary (CHO) cells were stably transfected to express full length human KLRG1 (CHOK 1-hKLRG 1)). Cells expressing CHOK1-hKLRG1 were cultured and incubated with PA015 or control human IgG1 at different concentrations. The cells were then centrifuged, the supernatant discarded, and washed with incubation buffer (hbss+2% fbs+10mm ca2+). Cells were resuspended and incubated with anti-human IgG-Alexa 488, washed, resuspended, and FACS studied. The Mean Fluorescence Intensity (MFI) of cells incubated with PA015 and with control human IgG1 is shown in figure 1. The EC50 of PA015 was 1.33nM.

Example 3: ligation of PA015 to E-cadherin with KLRG 1-expressing CHOK1 cells in a relative binding assay Inhibition of the synthesis.

The ability of the monoclonal antibodies to inhibit E-cadherin/KLRG 1 binding was measured by FACS.

Cells expressing CHOK1-hKLRG1 were cultured and incubated with PA015 or control human IgG1 at different concentrations. Then 0.3. Mu.M recombinant human E-cadherin-ECD-FC-biotin was added and incubated. The cells were then centrifuged, the supernatant discarded, and washed with incubation buffer (hbss+2% fbs+10mm ca2+). Cells were resuspended and incubated with streptavidin-Alexa 488, washed, resuspended, and FACS studied. The Mean Fluorescence Intensity (MFI) of cells incubated with PA015 and with control human IgG1 is shown in figure 2. IC50 values were determined by monitoring the loss of E-cadherin binding as the concentration of monoclonal antibody was changed from 0.1nM to 100 nM. The IC50 of PA015 for inhibiting E-cadherin binding was 8.05nM.

Example 4: PA015 has better purity than HG1N02

Examples 4-6 relate to antibody PA015 and antibody designated HG1N02 as described herein. HG1N02 is an anti-KLRG 1 antibody described in WO 2020/060781.

PA015 and HG1N02 were produced on a 1L scale using an ExpiCho expression system (Thermo Fisher). Reduced capillary electrophoresis (rCE) and non-reduced capillary electrophoresis (nrCE) were performed and the percentage of the main peak was measured in 3 independent experiments. The results showed better purity during CHO cell production of PA015 compared to HG1N02 (table 3).

TABLE 3 purity comparison of PA015 and HG1N02

Example 5: PA015 has better thermal Properties (melting and aggregation temperatures) than HG1N02

PA015 and HG1N02 were produced on a 1L scale using an ExpiCho expression system (Thermo Fisher). Melting temperature (Tm) was measured by total spectral fluorescence, and in 3 independent experiments, small molecule aggregate formation (Tagg 266) and large molecule aggregate formation (Tagg 473) were measured by Static Light Scattering (SLS) using une (Unchained Labs, inc.) at 4 hours, 7 days and 28 days time points. The results show that PA015 has better thermal properties (higher melting point and aggregation temperature) than HG1N02 (table 4).

Table 4. Thermal properties of pa015 compared to HG1N02. Temperature is shown in degrees celsius

Example 6: PA015 has better thermal properties (melting and aggregation temperatures) after stress testing compared to HG1N02

PA015 and HG1N02 were subjected to stress testing with thermal stress, low pH and high pH stress for 4 hours, 7 days and 28 days. Melting temperature (Tm) was measured by full spectrum fluorescence, and small molecule aggregate formation (Tagg 266) and large molecule aggregate formation (Tagg 473) were measured by Static Light Scattering (SLS) using une (Unchained Labs, inc.) at 4 hours, 7 days and 28 days time points. The results show that PA015 has better thermal stability (higher melting point and aggregation temperature) after stress testing compared to HG1N02 (tables 5-7).

Table 5. Thermal properties of pa015 and HG1N02 after stress at 40 ℃.

Temperature is shown in degrees celsius

Table 6. Thermal property temperatures of pa015 and HG1N02 after low pH (3.5) stress are shown in degrees celsius.

Table 7. Thermal property temperatures of pa015 and HG1N02 after high pH (8.5) stress are shown in degrees celsius.

Example 7: PA015 vs HG1N01 and HG1N02 on E-cadherin and expression in relative binding assays CHOK1 cells of KLRG1 had better blocking.

Cells expressing CHOK1-hKLRG1 were cultured and incubated with different concentrations of PA015, HG1N01, HG1N02 or control human IgG 1. Then 0.3. Mu.M recombinant human E-cadherin-ECD-FC-biotin was added and incubated. The cells were centrifuged, the supernatant discarded, and washed with incubation buffer (hbss+2% fbs+10mm ca2+). Cells were resuspended and incubated with streptavidin-Alexa 488, washed, resuspended, and FACS studied. The Mean Fluorescence Intensity (MFI) of cells incubated with the antibodies is shown in fig. 3. The IC50 for PA015 was 5.3nM, for HG1N01 was 8.3nM, and for HG1N02 was 7.9nM.

Example 8: PA015 has better binding to human CD8+ T cells in FACS binding assays than HG1N02 And (5) combining.

Human cd8+ T cells were isolated from 3 healthy donors by magnetic separation and incubated with different concentrations of PA015, HG1N02 or control human IgG1 and detected with a fluorescent-labeled anti-human IgG secondary antibody. The percentage of positive cells and EC50 values are shown in figure 4.

Example 9: PA015 has better pharmacokinetic properties in non-human primates compared to HG1N 01.

PA015 and HG1N01 (3 mg/kg) were each administered intravenously to two cynomolgus monkeys in a single dose. Serum concentrations of each antibody were measured over 42 days (fig. 5 and 6). PA015 showed better clearance (6.99 mL/day/kg vs 38.6 mL/day/kg), area under the curve (AUC; 429 days. Ug/mL vs 77.6 days. Ug/mL) and β half-life (6.23 days vs 4.7 days) compared to HG1N01 (tables 8 and 9).

Table 8 pharmacokinetic parameters of pa015

Table 9 pharmacokinetic parameters of hg1n01

Example 10: PA015 has improved deamidation properties compared to HG1N 02.

HG1N02 had a theoretical deamidation risk (NG at light chain residue N33). However, in the study of 131 clinically approved therapeutic mAbs (Lu Y et al, MAbs.2019Jan;11 (1): 45-57), the 48% (13/27) CDR NG sites were virtually unamidated under stress conditions, and thus the sequence was virtually unpredictable for the development of deamidation. Tryptic peptide mapping of unstressed PA015 and HG1N02 showed that PA015 had less deamidation at the critical N33 light chain CDR residues than HG1N02 (fig. 7). Incorporated by reference

The disclosures of each of the U.S. and foreign patents and pending patent applications and publications mentioned herein are expressly incorporated by reference in their entirety, as are the contents of the sequence listing and drawings. The teachings of PCT/US2019/050110 (published as WO 2020060781) are hereby incorporated by reference in their entirety.

Equivalent scheme

Those skilled in the art will recognize that the disclosure described herein and illustrated in the accompanying drawings is a non-limiting example embodiment and that the scope of the disclosure is limited only by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of embodiments disclosed in any of the various dependent claims or examples is contemplated as being within the scope of the present disclosure.

Other embodiments

From the foregoing description, it will be apparent that the invention described herein may be altered and modified to suit various uses and conditions, including the use of different signal sequences and nucleic acid sequences to express antibodies of the invention having the same or similar protein sequences are considered within the scope of the invention. Other embodiments according to the invention are within the appended claims.

The recitation of a list of elements in any definition of a variable herein includes the definition of that variable as any single element or combination (or sub-combination) of the listed elements. The recitation of embodiments herein includes the embodiments as any single embodiment or in combination with any other embodiment or portion thereof.

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

Sequence listing

<110> Apiaceae, inc. (ABCURO, INC.)

<120> anti-KLRG 1 antibodies

<130> 770808.000043

<140>

<141>

<150> 63/294,436

<151> 2021-12-29

<150> 63/166,663

<151> 2021-03-26

<160> 43

<170> PatentIn version 3.5

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Leu Cys Gln Gly Ser Asn Tyr Ser Thr Cys Ala Ser Cys Pro Ser Cys

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Pro Asp Arg Trp Met Lys Tyr Gly Asn His Cys Tyr Tyr Phe Ser Val

20 25 30

Glu Glu Lys Asp Trp Asn Ser Ser Leu Glu Phe Cys Leu Ala Arg Asp

35 40 45

Ser His Leu Leu Val Ile Thr Asp Asn Gln Glu Met Ser Leu Leu Gln

50 55 60

Val Phe Leu Ser Glu Ala Phe Cys Trp Ile Gly Leu Arg Asn Asn Ser

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Gly Trp Arg Trp Glu Asp Gly Ser Pro Leu Asn Phe Ser Arg Ile Ser

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Ser Asn Ser Phe Val Gln Thr Cys Gly Ala Ile Asn Lys Asn Gly Leu

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Gln Ala Ser Ser Cys Glu Val Pro Leu His Trp Val Cys Lys Lys Val

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Arg Leu

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Leu Cys Gln Gly Ser Lys Tyr Ser Thr Cys Ala Ser Cys Pro Ser Cys

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Pro Asp His Trp Met Lys Tyr Gly Asn His Cys Tyr Tyr Phe Ser Val

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Glu Lys Lys Asp Trp Ile Ser Ser Leu Glu Phe Cys Leu Ala Arg Asp

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Ser His Leu Leu Met Ile Thr Asp Lys Gln Glu Met Ser Leu Leu Gln

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Asp Phe Leu Ser Glu Ala Phe His Trp Val Gly Leu Arg Asn Asn Ser

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Gly Trp Arg Trp Glu Asp Gly Ser Pro Leu Asn Phe Ser Arg Ile Tyr

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Ser Asn Ser Leu Val Gln Thr Cys Gly Ala Ile Asn Lys Asn Ser Leu

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Gln Ala Ser Ser Cys Glu Val Ser Leu Gln Trp Val Cys Lys Lys Val

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Ser Pro

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Asp Trp Val Ile Pro Pro Ile Ser Cys Pro Glu Asn Glu Lys Gly Pro

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Phe Pro Lys Asn Leu Val Gln Ile Lys Ser Asn Lys Asp Lys Glu Gly

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Lys Val Phe Tyr Ser Ile Thr Gly Gln Gly Ala Asp Thr Pro Pro Val

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Gly Val Phe Ile Ile Glu Arg Glu Thr Gly Trp Leu Lys Val Thr Glu

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Pro Leu Asp Arg Glu Arg Ile Ala Thr Tyr Thr Leu Phe Ser His Ala

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Val Ser Ser Asn Gly Asn Ala Val Glu Asp Pro Met Glu Ile Leu Ile

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Thr Val Thr Asp Gln Asn Asp Asn Lys Pro Glu Phe Thr Gln Glu Val

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Phe Lys Gly Ser Val Met Glu Gly Ala Leu Pro Gly Thr Ser Val Met

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Glu Val Thr Ala Thr Asp Ala Asp Asp Asp Val Asn Thr Tyr Asn Ala

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Ala Ile Ala Tyr Thr Ile Leu Ser Gln Asp Pro Glu Leu Pro Asp Lys

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Asn Met Phe Thr Ile Asn Arg Asn Thr Gly Val Ile Ser Val Val Thr

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Thr Gly Leu Asp Arg Glu Ser Phe Pro Thr Tyr Thr Leu Val Val Gln

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Ala Ala Asp Leu Gln Gly Glu Gly Leu Ser Thr Thr Ala Thr Ala Val

195 200 205

Ile Thr Val Thr Asp Thr Asn Asp Asn Pro Pro Ile Phe Asn Pro Thr

210 215 220

Thr Tyr Lys Gly Gln Val Pro Glu Asn Glu Ala Asn Val Val Ile Thr

225 230 235 240

Thr Leu Lys Val Thr Asp Ala Asp Ala Pro Asn Thr Pro Ala Trp Glu

245 250 255

Ala Val Tyr Thr Ile Leu Asn Asp Asp Gly Gly Gln Phe Val Val Thr

260 265 270

Thr Asn Pro Val Asn Asn Asp Gly Ile Leu Lys Thr Ala Lys Gly Leu

275 280 285

Asp Phe Glu Ala Lys Gln Gln Tyr Ile Leu His Val Ala Val Thr Asn

290 295 300

Val Val Pro Phe Glu Val Ser Leu Thr Thr Ser Thr Ala Thr Val Thr

305 310 315 320

Val Asp Val Leu Asp Val Asn Glu Ala Pro Ile Phe Val Pro Pro Glu

325 330 335

Lys Arg Val Glu Val Ser Glu Asp Phe Gly Val Gly Gln Glu Ile Thr

340 345 350

Ser Tyr Thr Ala Gln Glu Pro Asp Thr Phe Met Glu Gln Lys Ile Thr

355 360 365

Tyr Arg Ile Trp Arg Asp Thr Ala Asn Trp Leu Glu Ile Asn Pro Asp

370 375 380

Thr Gly Ala Ile Ser Thr Arg Ala Glu Leu Asp Arg Glu Asp Phe Glu

385 390 395 400

His Val Lys Asn Ser Thr Tyr Thr Ala Leu Ile Ile Ala Thr Asp Asn

405 410 415

Gly Ser Pro Val Ala Thr Gly Thr Gly Thr Leu Leu Leu Ile Leu Ser

420 425 430

Asp Val Asn Asp Asn Ala Pro Ile Pro Glu Pro Arg Thr Ile Phe Phe

435 440 445

Cys Glu Arg Asn Pro Lys Pro Gln Val Ile Asn Ile Ile Asp Ala Asp

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Leu Pro Pro Asn Thr Ser Pro Phe Thr Ala Glu Leu Thr His Gly Ala

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Ser Ala Asn Trp Thr Ile Gln Tyr Asn Asp Pro Thr Gln Glu Ser Ile

485 490 495

Ile Leu Lys Pro Lys Met Ala Leu Glu Val Gly Asp Tyr Lys Ile Asn

500 505 510

Leu Lys Leu Met Asp Asn Gln Asn Lys Asp Gln Val Thr Thr Leu Glu

515 520 525

Val Ser Val Cys Asp Cys Glu Gly Ala Ala Gly Val Cys Arg Lys Ala

530 535 540

Gln Pro Val Glu Ala Gly Leu Gln Ile Pro Ala Ile Leu Gly Ile Leu

545 550 555 560

Gly Gly Ile Leu Ala Leu Leu Ile Leu Ile Leu Leu Leu Leu Leu Phe

565 570 575

Leu Arg Arg Arg Ala Val Val Lys Glu Pro Leu Leu Pro Pro Glu Asp

580 585 590

Asp Thr Arg Asp Asn Val Tyr Tyr Tyr Asp Glu Glu Gly Gly Gly Glu

595 600 605

Glu Asp Gln Asp Phe Asp Leu Ser Gln Leu His Arg Gly Leu Asp Ala

610 615 620

Arg Pro Glu Val Thr Arg Asn Asp Val Ala Pro Thr Leu Met Ser Val

625 630 635 640

Pro Arg Tyr Leu Pro Arg Pro Ala Asn Pro Asp Glu Ile Gly Asn Phe

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Ile Asp Glu Asn Leu Lys Ala Ala Asp Thr Asp Pro Thr Ala Pro Pro

660 665 670

Tyr Asp Ser Leu Leu Val Phe Asp Tyr Glu Gly Ser Gly Ser Glu Ala

675 680 685

Ala Ser Leu Ser Ser Leu Asn Ser Ser Glu Ser Asp Lys Asp Gln Asp

690 695 700

Tyr Asp Tyr Leu Asn Glu Trp Gly Asn Arg Phe Lys Lys Leu Ala Asp

705 710 715 720

Met Tyr Gly Gly Gly Glu Asp Asp

725

<210> 4

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic polypeptides

<400> 4

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Trp Tyr Glu Leu Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 5

<211> 115

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

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<400> 5

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Ala His Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val

115

<210> 6

<211> 330

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic polypeptides

<400> 6

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 7

<211> 104

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic polypeptides

<400> 7

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

1 5 10 15

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

20 25 30

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

35 40 45

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

50 55 60

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

65 70 75 80

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

85 90 95

Lys Ser Phe Asn Arg Gly Glu Cys

100

<210> 8

<211> 455

<212> PRT

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<220>

<223> description of artificial sequence:

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<400> 8

Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Ser Thr Phe

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Trp Tyr Glu Leu Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val

65 70 75 80

Val Leu Thr Ile Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Arg Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

130 135 140

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

195 200 205

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

210 215 220

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

225 230 235 240

Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

245 250 255

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

260 265 270

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

275 280 285

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

290 295 300

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

305 310 315 320

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

325 330 335

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

340 345 350

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

435 440 445

Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 9

<211> 219

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<220>

<223> description of artificial sequence:

synthetic polypeptides

<400> 9

Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Ala His Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Val Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 10

<211> 9

<212> PRT

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synthetic peptides

<400> 10

Gly Phe Ser Leu Ser Thr Phe Gly Met

1 5

<210> 11

<211> 5

<212> PRT

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<400> 11

Trp Trp Asp Asp Asp

1 5

<210> 12

<211> 15

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<400> 12

Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met Asp Tyr

1 5 10 15

<210> 13

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 13

Lys Ser Ser Gln Ser Ile Val His Ser Asn Ala His Thr Tyr Leu Glu

1 5 10 15

<210> 14

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 14

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 15

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 15

Phe Gln Gly Ser His Val Pro Val Thr

1 5

<210> 16

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 16

Gly Phe Ser Leu Ser Thr Phe Gly Met Gly Val Gly

1 5 10

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 17

His Ile Trp Trp Asp Asp Asp Lys Trp

1 5

<210> 18

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 18

Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met Asp Tyr

1 5 10 15

<210> 19

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 19

Lys Ser Ser Gln Ser Ile Val His Ser Asn Ala His Thr Tyr Leu Glu

1 5 10 15

<210> 20

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

Synthetic peptides

<400> 20

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 21

Phe Gln Gly Ser His Val Pro Val Thr

1 5

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 22

Thr Phe Gly Met Gly Val Gly

1 5

<210> 23

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 23

His Ile Trp Trp Asp Asp Asp Lys Trp Tyr Glu Leu Ala Leu Lys Ser

1 5 10 15

<210> 24

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 24

Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met Asp Tyr

1 5 10 15

<210> 25

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 25

Lys Ser Ser Gln Ser Ile Val His Ser Asn Ala His Thr Tyr Leu Glu

1 5 10 15

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 26

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 27

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 27

Phe Gln Gly Ser His Val Pro Val Thr

1 5

<210> 28

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 28

Ser Thr Phe Gly Met Gly Val Gly

1 5

<210> 29

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 29

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Trp

1 5 10

<210> 30

<211> 16

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 30

Ala Arg Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met Asp

1 5 10 15

<210> 31

<211> 12

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 31

Val His Ser Asn Ala His Thr Tyr Leu Glu Trp Tyr

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

Synthetic peptides

<400> 32

Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe

1 5 10

<210> 33

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 33

Phe Gln Gly Ser His Val Pro Val

1 5

<210> 34

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 34

Gly Phe Ser Leu Ser Thr Phe Gly Met Gly

1 5 10

<210> 35

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 35

Ile Trp Trp Asp Asp Asp Lys

1 5

<210> 36

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 36

Ala Arg Val Ile Tyr Tyr Gly Ser Arg Ser Ala Tyr Tyr Ser Met Asp

1 5 10 15

Tyr

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 37

Gln Ser Ile Val His Ser Asn Ala His Thr Tyr

1 5 10

<210> 38

<211> 2

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 38

Lys Val

1

<210> 39

<211> 9

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 39

Phe Gln Gly Ser His Val Pro Val Thr

1 5

<210> 40

<211> 1428

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthesis of polynucleotides

<400> 40

atgggctggt cctgcatcat tctgtttctg gtggctaccg ccaccggcgc tcattctcaa 60

gtgacactga gagagtctgg ccccgctctg gtcaagccta cacagaccct gacactgacc 120

tgcaccgtgt ctggcttctc cctgtctacc tttggcatgg gcgtcggctg gattagacag 180

cctcctggaa aagccctgga atggctggcc cacatttggt gggacgacga caagtggtac 240

gagctggctc tgaagtcccg gctgaccatc tccaaggaca cctccaagaa tcaggtggtg 300

ctgacaatca ccaacatgga ccctgtggac accgccacct actactgcgc cagagtgatc 360

tactacggct ccagatccgc ctactactcc atggattatt ggggccaggg caccaccgtg 420

accgtgtcct ctgcttctac aaagggcccc tctgtgttcc ctctggctcc ttcctctaaa 480

tccacctctg gcggaaccgc tgctctgggc tgtctcgtga aggattactt ccctgagcct 540

gtgacagtgt cctggaatag cggtgctctg acatccggcg tgcacacctt tccagctgtg 600

ctgcagtcct ctggcctgta ctctctgtcc tctgtcgtga cagtgccttc cagctctctg 660

ggcacccaga cctacatctg caacgtgaac cacaagcctt ccaacaccaa ggtggacaag 720

aaggtggaac ccaagtcctg cgacaagacc cacacctgtc ctccatgtcc tgctccagaa 780

gctgctggcg ctccctccgt gtttctgttc cctccaaagc ctaaggacac cctgatgatc 840

tctcggaccc ctgaagtgac ctgcgtggtg gtggatgtgt ctcacgagga tcccgaagtg 900

aagttcaatt ggtacgtgga cggcgtggaa gtgcacaacg ccaagaccaa gcctagagag 960

gaacagtaca actccaccta cagagtggtg tccgtgctga ccgtgctgca ccaggattgg 1020

ctgaacggca aagagtacaa gtgcaaggtg tccaacaagg ccctgcctgc tcctatcgaa 1080

aagaccatca gcaaggctaa gggccagcct cgggaacctc aggtttacac cctgcctcca 1140

tctcgggaag agatgacaaa gaaccaggtg tccctgacct gcctggtcaa gggcttctac 1200

ccttccgaca ttgccgtgga atgggagtcc aatggccagc ctgagaacaa ctacaagaca 1260

acccctcctg tgctggactc cgacggctca ttcttcctgt actccaagct gacagtggac 1320

aagtctcggt ggcagcaggg caacgtgttc tcctgttctg tgatgcacga ggccctgcac 1380

aaccactaca cccagaagtc cctgtctctg tcccctggca agtgatga 1428

<210> 41

<211> 720

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthesis of polynucleotides

<400> 41

atgggctggt cctgcatcat tctgtttctg gtggctaccg ccaccggcgc tcattctgat 60

gtggtcatga cacagacccc tctgagcctg tctgtgacac ctggacagcc tgcctccatc 120

tcctgcaagt cctctcagtc catcgtgcac agcaacgccc acacctacct ggaatggtat 180

ctgcagaagc ccggccagtc tcctcagctg ctgatctaca aggtgtccaa cagattctct 240

ggcgtgcccg acagattcag cggctctggc tctggcaccg acttcaccct gaagatctct 300

agagtggaag ccgaggacgt gggcgtgtac tactgtttcc aaggctctca cgtgcccgtg 360

acctttggcc agggaacaaa gctggaaatc aagcggaccg tggccgctcc ttccgtgttc 420

atctttccac cttccgacga gcagctgaag tccggcacag cttctgtcgt gtgcctgctg 480

aacaacttct accctcggga agccaaggtg cagtggaaag tggataatgc cctgcagtcc 540

ggcaactccc aagagtctgt gaccgagcag gactccaagg actctaccta ctctctgtcc 600

tccacactga ccctgtccaa ggccgactac gagaagcaca aggtgtacgc ctgtgaagtg 660

acccaccagg gactgtctag ccccgtgacc aagtctttca accggggcga gtgctgatga 720

<210> 42

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 42

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 43

<211> 5

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> description of artificial sequence:

synthetic peptides

<400> 43

Cys Asn Gly Arg Cys

1 5

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1, said antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID No. 4.

2. The antibody or antigen-binding fragment thereof of claim 1, further comprising a light chain comprising CDR-L1 selected from the group consisting of SEQ ID No. 13, SEQ ID No. 19, SEQ ID No. 25, SEQ ID No. 31, and SEQ ID No. 37; CDR-L2 selected from the group consisting of SEQ ID NO. 14, SEQ ID NO. 20, SEQ ID NO. 26, SEQ ID NO. 32 and SEQ ID NO. 38; and CDR-L3 selected from the group consisting of SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33 and SEQ ID NO:39.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the CDR-L1, the CDR-L2, and the CDR-L3 are SEQ ID No. 13, SEQ ID No. 14, and SEQ ID No. 15, respectively; SEQ ID NO 19, SEQ ID NO 20 and SEQ ID NO 21, respectively; SEQ ID NO. 25, SEQ ID NO. 26 and SEQ ID NO. 27, respectively; SEQ ID NO. 31, SEQ ID NO. 32 and SEQ ID NO. 33, respectively; or SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39, respectively.

4. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1, said antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID No. 5.

5. The antibody or antigen-binding fragment thereof of claim 4, further comprising a heavy chain comprising CDR-L1 selected from the group consisting of SEQ ID No. 10, SEQ ID No. 16, SEQ ID No. 22, SEQ ID No. 28, SEQ ID No. 34; CDR-L2 selected from the group consisting of SEQ ID NO. 11, SEQ ID NO. 17, SEQ ID NO. 23, SEQ ID NO. 29, SEQ ID NO. 35; and CDR-L3 selected from the group consisting of SEQ ID NO:12, SEQ ID NO:18, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO: 36.

6. The antibody or antigen binding fragment thereof of claim 5, wherein the CDR-H1, the CDR-H2, and the CDR-H3 are SEQ ID No. 10, SEQ ID No. 11, and SEQ ID No. 12, respectively; SEQ ID NO. 16, SEQ ID NO. 17 and SEQ ID NO. 18, respectively; SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24, respectively; SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, respectively; or SEQ ID NO 34, SEQ ID NO 35 and SEQ ID NO 36, respectively.

7. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1, said antibody or antigen-binding fragment thereof comprising:

(a) A heavy chain variable region comprising SEQ ID NO. 4, and

(b) Comprising the light chain variable region of SEQ ID NO. 5.

8. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1 and comprises a heavy chain comprising SEQ ID No. 8.

9. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1 and comprises a light chain comprising SEQ ID No. 9.

10. An antibody or antigen-binding fragment thereof that specifically binds to the extracellular domain of KLRG1 and comprises:

(a) A heavy chain comprising SEQ ID NO. 8, and

(b) Light chain comprising SEQ ID NO. 9.

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

12. The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein the antigen-binding fragment is F (ab) ₂ ) Fragments.

13. The antibody or antigen-binding fragment of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is bispecific.

14. A method of treating cancer comprising administering the antibody or antigen-binding fragment thereof of any one of the preceding claims to a cancer patient.

15. The method of claim 14, further comprising administering a checkpoint inhibitor to the cancer patient.

16. The method of claim 15, wherein the administration of the checkpoint inhibitor is concurrent with administration of the antibody or antigen-binding fragment thereof.

17. The method of claim 15, wherein the checkpoint inhibitor is administered within one day, one week, or one month of the antibody or antigen binding fragment thereof.

18. The method according to claim 14 to 17, wherein the cancer is breast cancer, prostate cancer, lymphoma, skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain cancer, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck cancer, breast cancer, ovarian cancer, lung cancer, small cell lung cancer, wilms 'tumor, cervical cancer, testicular cancer, bladder cancer, pancreatic cancer, stomach cancer, colon cancer, prostate cancer, genitourinary tract cancer, thyroid cancer, esophageal cancer, myeloma, multiple myeloma, adrenal cancer, renal cell carcinoma, endometrial cancer, adrenocortical carcinoma, malignant pancreatic insulinoma, malignant carcinoid tumor, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, cytoblast, sarcomas, kaposi's sarcoma, polycythemia, hodgkin's sarcoma, lymphomatosis, and lymphomas in a patient, one's disease, or a giant cell sarcoma.

19. An adjuvant therapy comprising administering the antibody or antigen-binding fragment thereof of any one of claims 1 to 13 to a subject undergoing treatment with a checkpoint inhibitor, thereby performing the adjuvant therapy.

20. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 13.

21. The isolated nucleic acid of claim 20, comprising SEQ ID NO. 40.

22. The isolated nucleic acid of claim 20, comprising SEQ ID NO. 41.

23. An expression vector comprising the nucleic acid of any one of claims 20 to 22.

24. A host cell comprising the expression vector of claim 23.