Anti-KLRG1 Antibody

Abstract

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

Description

[Technical field]

[0001] The technical field relates to inhibition of the lymphocyte co-inhibitory receptor killer cell lectin-like receptor subfamily G member 1 (KLRG1). [Background technology]

[0002] Lymphocyte co-inhibitory receptors regulate the action of the adaptive immune system, e.g., T cells and NK cells, in response to activation signals, such as antigenic peptides associated with the major histocompatibility complex (MHC) binding to the T cell receptor (TCR). Co-inhibitory receptors include PD-1, LAG-3, TIM-3, and CTLA4. The action of co-inhibitory receptors is generally carried out by binding of a ligand to the extracellular domain of the co-inhibitory receptor, followed by recruitment of intracellular phosphatases by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular domain of the co-inhibitory receptor. The action of co-inhibitory receptors is generally to dampen TCR-mediated immune responses. In recent years, it has been shown that drugs that block the activity of co-inhibitory receptors can be used as effective treatments for cancer and infectious diseases.

[0003] Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane protein that functions as a co-inhibitory receptor by regulating the activity of T cells and NK cells. Its extracellular portion contains a C-type lectin domain whose known ligands are cadherins, and its intracellular portion contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain that is involved in the co-inhibition of T cell receptor (TCR)-mediated signaling. The KLRG1 ligand can be E-cadherin, N-cadherin, R-cadherin, or a combination thereof.

[0004] The receptor killer cell lectin-like receptor G1 (KLRG1) is expressed on T cells and NK cells that binds to ligands on epithelial and mesenchymal cells. The ligands for KLRG1 have been described to be E-cadherin, N-cadherin, and R-cadherin.

[0005] In humans, KLRG1 expression is restricted to cells of the immune system, specifically CD8+ T cells, NK cells, and to a lesser extent CD4+ T cells. KLRG1 expression is associated with a late differentiation phenotype. As antigen-specific T cells differentiate, they acquire increased expression of cytotoxic molecules, thus increasing their cytotoxic potential. The biological function of KLRG1 is to inhibit the cytotoxicity and proliferation of these T cells. Restoring T cell activity has been shown to be beneficial in cancer and infectious diseases.

[0006] Generally, there is a need to provide safe and effective treatment methods for cancer or infectious diseases. Modulation of cytotoxic (or CD8+) T and NK cell activation involved in these disorders can be achieved by manipulating the KLRG1 pathway.

[0007] Summary of the Invention The present disclosure provides characterization of novel antibodies or antigen-binding fragments thereof that bind to the extracellular domain (ECD) of KLRG1 and inhibit its interaction with ligands E-cadherin, N-cadherin, and R-cadherin. The antibodies described herein are derived by mouse hybridoma technology and can be humanized by grafting their complementarity determining regions (CDRs) into a human framework. The present disclosure also provides antibodies or binding fragments that modulate (e.g., activate) CD8+ cytotoxic T cells and NK cells by modulating (e.g., neutralizing) the interaction of KLRG1 with its ligands. The antibodies described herein can be used as effective therapeutic agents for treating cancer, either as monotherapy or in combination with other immunotherapeutic agents (such as anti-PD-1, anti-PD-L1, or anti-CTLA4 antibodies), or in combination with chemotherapeutic agents, or cancer vaccines. The antibodies described herein can be used as effective treatments for infectious diseases or to enhance the effectiveness of vaccines against infectious diseases.

[0008] Non-limiting exemplary embodiments of the antibodies are designated ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, or ABC_G1N08. Other embodiments are designated ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, or ABC_G1N08 Fv fragments. H and / or V L Further embodiments include these V domains. H and V L Other embodiments include one or more CDRs of any of the domains ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H Contains the H3 fragment of the domain.

[0009] The present disclosure also provides compositions comprising KLRG1 antibodies and their use in methods of modulating immune responses, including methods of treating humans or animals. In certain embodiments, anti-KLRG1 antibodies are used to treat or prevent cancer by activating CD8+ cytotoxic T cells and NK cells. Disorders susceptible to treatment with the compositions of the present invention include, but are not limited to, cancer and infectious diseases.

[0010] In addition, anti-KLRG1 antibody can be used diagnostically to detect KLRG1 or its fragment in biological sample.The amount of KLRG1 detected can be correlated with the expression level of KLRG1, which is then correlated with the activation state of lymphocytes (e.g., cytotoxic T cells or natural killer cells) in subject.

[0011] The present disclosure also provides VFv fragments of ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H and V L Also provided is an isolated nucleic acid comprising a sequence encoding the V domain of the present disclosure. H and V L Also provided is an isolated nucleic acid comprising a sequence encoding one or more CDRs from any of the domains. The disclosure also provides vectors and host cells comprising such nucleic acids.

[0012] This disclosure relates to the V of ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H or V L A new V that is derived from a domain and contains all or part of such domain H and V L Further provided are methods for producing the domains and / or functional antibodies.

[0013] Additional aspects of the present disclosure will be set forth in part in the description which follows and in part will be apparent from the description or may be learned by the practice of the present invention. The present invention is set forth and particularly pointed out in the appended claims, and the present disclosure should not be construed as limiting the scope of the claims in any way. The following detailed description does not limit the invention as claimed, but includes illustrative representations of various embodiments of the invention. The accompanying drawings constitute a part of this specification and, together with the description, serve only to illustrate various embodiments and not to limit the invention. Citation of references is not an admission that those references are prior art to the present invention. [Brief description of the drawings]

[0014] [Figure 1] 1 shows the results of an interferon-γ (IFNγ) secretion assay demonstrating that anti-KLRG1 antibodies activate CD8+ human T cells. [Diagram 2] 1 shows the results of a CD8+ T cell proliferation assay demonstrating that anti-KLRG1 antibodies induce proliferation of CD8+ T cells. [Diagram 3] 1 shows the relationship between the results of an IFNγ secretion assay in CD8+ T cells treated with anti-KLRG1 antibodies and the blocking activity of the antibodies (expressed as IC50). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] definition The term "antibody" as used in this disclosure refers to an immunoglobulin or a fragment or derivative thereof, whether produced in vitro or in vivo, and encompasses any polypeptide that contains an antigen-binding site. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. For the purposes of this disclosure, unless otherwise modified by the term "intact," as in "intact antibody," the term "antibody" also includes antibody fragments, such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to specifically bind to KLRG1. Typically, such fragments will include the antigen-binding domain.

[0016] The terms "antigen-binding domain", "antigen-binding fragment" and "binding fragment" refer to the portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. In cases where the antigen is large, the antigen-binding domain may only bind to a portion of the antigen. The portion of the antigen molecule that is involved in the specific interaction with the antigen-binding domain is called the "epitope" or "antigenic determinant".

[0017] An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but does not necessarily comprise both. For example, a so-called Fd antibody fragment consists only of a VH domain but still retains some antigen-binding function of an intact antibody.

[0018] The term "repertoire" refers to a genetically diverse collection of nucleotides derived entirely or partially from sequences encoding expressed immunoglobulins. The sequences are generated by in vivo rearrangement of, for example, V, D, and J segments for H chains, and, for example, V and J segments for L chains. Alternatively, the sequences can be generated from cell lines in response to in vitro stimulation to cause rearrangement. Alternatively, some or all of the sequences can be obtained by nucleotide synthesis, random mutagenesis, and other methods, such as those disclosed in U.S. Pat. No. 5,565,332, for example, by combining unrearranged V segments with D and J segments.

[0019] 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 usually characterized by a high affinity and a low to moderate capacity, as distinguished from nonspecific binding, which usually has a low affinity with a moderate to high capacity. Typically, binding is characterized by an affinity constant, K A 10 6 M - 1 Higher than, or more preferably, 10 8 M - 1 A specific binding is considered to be specific when it is higher than 0.01. If necessary, non-specific binding can be reduced by varying the binding conditions without substantially affecting the specific binding. Suitable binding conditions, such as the concentration of the antibody, the ionic strength of the solution, the temperature, the time allowed for binding, the concentration of the blocking agent (e.g., serum albumin, milk casein), etc., can be optimized by those skilled in the art using routine techniques.

[0020] The phrase "substantially depicted" means that the relevant CDR, VH, or VL domain of the invention is identical to the particular region (e.g., CDR) whose sequence is depicted or has only insubstantial differences, including minor amino acid changes, such as substitutions of one or two of any five amino acids in the sequence of the particular region.

[0021] The term "KLRG1 activity" refers to one or more lymphocyte co-inhibitory activities associated with KLRG1. For example, KLRG1 activity can refer to the regulation of cytotoxic T and NK cell activation.

[0022] The term "modulate" and similar terms refer to a decrease or increase in the activity of KLRG1 associated with T cell and NK cell activation resulting from interaction with an anti-KLRG1 antibody, which decrease or increase is compared to the activity of KLRG1 in the absence of the same antibody. The decrease or increase in activity is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. When KLRG1 activity is decreased, the terms "modulatory" and "modulate" are interchangeable with the terms "inhibitory" and "inhibit". When KLRG1 activity is increased, the terms "modulatory" and "modulate" are interchangeable with the terms "activation" and "activate". The activity of KLRG1 can be quantitatively determined using T cell and NK cell activation assays such as those described in Example 6.

[0023] The terms "treatment" and "treatment method" refer to both therapeutic treatment and prophylactic / preventative measures. Those in need of treatment can include those already suffering from a particular medical disorder as well as those who may eventually develop the disorder (i.e., those in need of preventative measures).

[0024] The term "effective amount" refers to a dosage or amount sufficient to reduce the activity of KLRG1, resulting in improvement of a patient's symptoms or to achieve a desired biological outcome, such as reducing the activity of KLRG1, modulating a lymphocyte co-inhibitory response, increasing the activation of cytotoxic T cells and NK cells, or increasing the release of IFNγ by cytotoxic T cells or NK cells.

[0025] The term "isolated" refers to a molecule that is substantially free of its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to preparations in which the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or is at least 70-80% (w / w) pure, more preferably at least 80-90% (w / w) pure, even more preferably 90-95% pure, and most preferably at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure.

[0026] Anti-KLRG1 antibody The present disclosure provides anti-KLRG1 antibodies, including novel antigen-binding fragments.

[0027] In general, antibodies can be produced using, for example, conventional hybridoma techniques (Kohler and Milstein (1975) Nature, 256:495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display techniques implemented using antibody libraries (Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597). For other antibody production techniques, see Antibodies: A Laboratory Manual, ed. Harlow et al., Cold Spring Harbor Laboratory, 1988. The invention is not limited to any particular source, species of origin, or method of production.

[0028] Intact antibodies, also known as immunoglobulins, are usually tetrameric glycosylated proteins composed of two light (L) chains of about 25 kDa each and two heavy (H) chains of about 50 kDa each. There are two types of light chains in antibodies, called lambda and kappa chains. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0029] The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a general review of antibody structure, see Harlow et al., supra. Briefly, each light chain contains an N-terminal variable domain (V L ) and the constant domain (C L Each heavy chain is composed of an N-terminal variable domain (V H ), three or four constant domains (C H ), and the hinge region. H C closest to H The domain is C H It is called 1. V H and V L A domain consists of four regions of relatively conserved sequence called framework regions (FR1, FR2, FR3, and FR4), which scaffold three regions of hypervariable sequence called complementarity determining regions (CDRs). The CDRs contain most of the residues responsible for specific interactions with the antigen. The three CDRs are called CDR1, CDR2, and CDR3. The CDR components of the heavy chain are called H1, H2, and H3, and the CDR components of the light chain are called L1, L2, and L3. CDR3, especially H3, is the greatest source of molecular diversity within the antigen-binding domain. H3 can be as short as, for example, two amino acid residues or longer than 26.

[0030] Fab fragments (fragment antigen binding) consist of V covalently linked by disulfide bonds between the constant regions. H-C H 1 and V L -C L When coexpressed in a host cell, the non-covalently linked V H and V L To overcome the tendency of the domains to dissociate, so-called single-chain (sc) Fv fragments (scFv) can be constructed. In scFvs, a flexible and sufficiently long polypeptide is H The C-terminus of L N-terminus or V L The C-terminus of H or the N-terminus of either. Most commonly, a 15 residue (Gly4Ser)3 peptide is used as the linker, although other linkers are known in the art.

[0031] Antibody diversity is the result of the combinatorial assembly of multiple germline genes that code for the variable regions and various somatic events. H Reassembly of variable region gene segments with diverse (D) and joining (J) gene segments to create a complete V region. L The process involves the rearrangement of variable and joining gene segments to create regions. The rearrangement process itself is imprecise, resulting in the loss or addition of amino acids at the V(D)J junctions. These mechanisms of diversity occur in developing B cells prior to antigen exposure. After antigen stimulation, antibody genes expressed in B cells undergo somatic mutation.

[0032] The estimated number of germline gene segments, random rearrangements of these segments, and random V H -V L Based on pairing, up to 1.6 × 10 7 (Fundamental Immunology, 3rd ed., ed. Paul, Raven Press, New York, NY, 1993). Taking into account other processes that contribute to antibody diversity (such as somatic mutations), the number of different antibodies produced is estimated at 1 × 10 10It is believed that more than 100 different antibodies could potentially be generated (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, Calif., 1995). Because of the many processes involved in antibody diversity, it is highly unlikely that independently generated antibodies will have identical amino acid sequences in the CDRs.

[0033] The present disclosure provides novel CDRs derived from a human immunoglobulin gene library. The structures for carrying the CDRs are generally antibody heavy or light chains or portions thereof, in which the CDRs are naturally occurring V H and V L The structure and location of immunoglobulin variable domains can be determined, for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.

[0034] Humanized anti-KLRG1 antibody V H and V L The amino acid sequences of the domains are shown in the sequence listing and are listed as set forth in Table 1. [Table 1]

[0035] Specific non-limiting exemplary embodiments of the antibodies are designated ABC_HG1N01, ABC_HG1N02, and ABC_HG1N07. H and V L The amino acid sequences of the CDRs within the domains are shown in the sequence listing and listed as set forth in Table 2. [Table 2]

[0036] Mouse anti-KLRG1 antibody V H and V L The amino acid sequences of the domains are shown in the sequence listing and are listed as set forth in Table 3. [Table 3-1] [Table 3-2]

[0037] Specific non-limiting exemplary embodiments of the antibodies are designated ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H and V L The amino acid sequences of the CDRs within the domains are shown in the sequence listing and are listed as set forth in Table 4. [Table 4-1] [Table 4-2]

[0038] The anti-KLRG1 antibody may optionally include an antibody constant region or a portion thereof. L The domain may have attached to its C-terminus an antibody light chain constant domain, including a human Cκ or Cλ chain. HA domain-based specific antigen-binding domain may be attached to all or a portion of an immunoglobulin heavy chain from any antibody isotype, such as any of the isotype subclasses, including but not limited to IgG, IgA, IgE, and IgM, as well as IgG1 and IgG4. In exemplary embodiments, the ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08 antibodies comprise C-terminal fragments of human IgG1λ or IgG1κ heavy and light chains. The DNA and amino acid sequences of the C-terminal fragments are well known in the art (see, e.g., Kabatetal., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991).

[0039] Certain embodiments include the V of Fv fragments from ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H and / or V L Further embodiments include these V domains. H and V LThe present invention encompasses within its scope at least one of the CDR sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38. One embodiment includes, for example, an antibody V selected from at least one of ABC_HG1N01, ABC_HG1N02, ABC_HG1N07ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, and ABC_G1N08. H Contains the H3 fragment of the domain.

[0040] In certain embodiments, V H and / or V L The domains may be germlined, i.e., the framework regions (FR) of these domains are mutated using conventional molecular biology techniques to match those produced by germline cells, hi other embodiments, the framework sequences remain divergent from the consensus germline sequences.

[0041] In certain embodiments, the antibody has a K of at least about 2 nM, 1 nm, 100 pM, 10 pM, or 5 pM for an epitope within the ECD of human or mouse KLRG1. D The amino acid sequences of the ECDs of human and cynomolgus KLRG1 are shown in SEQ ID NO:89 and SEQ ID NO:90, as listed in Table 6.

[0042] It is contemplated that the antibodies of the present invention may also bind to other proteins, including, for example, recombinant proteins that contain all or a portion of KLRG1.

[0043] Those skilled in the art will recognize that the antibodies of the present invention can be used to detect, measure, and inhibit proteins that are somewhat different from KLRG1. The antibodies are expected to retain binding specificity as long as the target protein contains a sequence that is at least about 60%, 70%, 80%, 90%, 95% or more identical to any sequence of at least 130, 100, 80, 60, 40, or 20 consecutive amino acids in the sequence shown in SEQ ID NO:89 or SEQ ID NO:90. Percent identity is determined by standard alignment algorithms, such as, for example, the Basic Local Alignment Tool (BLAST) described in Altshul et al. (1990) J. Mol. Biol., 215:403-410, the algorithm of Needleman et al. (1970) J. Mol. Biol., 48:444-453, or the algorithm of Meyers et al. (1988) Comput. Appl. Biosci., 4:11-17.

[0044] In addition to sequence homology analysis, epitope mapping (see, e.g., Epitope Mapping Protocols, ed. Morris, Humana Press, 1996), as well as secondary and tertiary structure analysis, can be performed to identify the specific 3D structures assumed by the disclosed antibodies and their complexes with antigens. Such methods include, but are not limited to, X-ray crystallography (Engstom (1974) Biochem. Exp. Biol., 11:7-13) and computer modeling of virtual representations of the presently disclosed antibodies (Fletterick et al. (1986) Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0045] derivative The present disclosure also provides a method for obtaining an antibody specific to KLRG1. The CDRs in such an antibody are those identified in Tables 1 and 3. H and V LThe present invention is not limited to the specific sequences of the disclosed CDRs, but may include variants of these sequences that retain the ability to specifically bind to KLRG1. Such variants may be derived from the sequences listed in Tables 1 and 3 by those skilled in the art using techniques well known in the art. For example, amino acid substitutions, deletions, or additions may be made within the FRs and / or CDRs. FR modifications are usually designed to improve the stability and immunogenicity of the antibody, while CDR modifications are typically designed to increase the affinity of the antibody for its target. FR variants also include naturally occurring immunoglobulin allotypes. Such affinity-increasing changes may be empirically determined by routine techniques that include modifying the CDRs and testing the affinity antibody for its target. For example, conservative amino acid substitutions may be made within any one of the disclosed CDRs. Various modifications may be made according to the methods described in Antibody Engineering, 2nd ed., Oxford University Press, ed. Borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that are modified by substitution of different codons that code for functionally equivalent amino acid residues within the sequence, thus resulting in a "silent" change. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs (see Table 5). Additionally, any naturally occurring residues in the polypeptide can also be substituted with alanine (see, e.g., MacLennan et al. (1998) Acta Physiol. Scand. Suppl. 643:55-67; ​​Sasaki et al. (1998) Adv. Biophys. 35:1-24). [Table 5]

[0046] Derivatives and analogs of antibodies of the invention can be produced by a variety of techniques known in the art, including recombinant and synthetic methods (Maniatis (1990) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Bodansky et al. (1995) The Practice of Peptide Synthesis, 2nd ed., Spring Verlag, Berlin, Germany).

[0047] In one embodiment, the V H Domain amino acid sequence variant V H The method for producing the domain is the V H adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence of the domain, and H A domain can be one or more V L and optionally combining with a V domain for specific binding to KLRG1. H Domain or V H / V L and, optionally, testing the ability of such antigen-binding domains to modulate KLRG1 activity. L The domains can have amino acid sequences that are identical or substantially identical to the amino acid sequences shown in Tables 1 and 3.

[0048] The V disclosed herein L One or more sequence variants of the domain have one or more V H A similar method can be used in combination with domains.

[0049] A further aspect of the present disclosure provides a method for preparing an antigen-binding fragment that specifically binds to KLRG1, the method comprising: (a) a V that contains a replaced CDR3 or lacks a CDR3 coding region; H providing a starting repertoire of nucleic acids encoding domains; (b) Repertoire, V H A donor nucleic acid encoding an amino acid sequence substantially as described herein for CDR3 (i.e., H3), and a donor nucleic acid inserted within the CDR3 region of the repertoire to form a V H combining to provide a product repertoire of nucleic acids encoding the domains; (c) expressing the nucleic acids of the product repertoire; (d) selecting a binding fragment specific to KLRG1; (e) recovering the specific binding fragment or a nucleic acid encoding same.

[0050] In this case, the V L The CDR3 (i.e., L3) is either a V that contains a substituted CDR3 or that lacks the CDR3 coding region. L A similar method can be used in which a repertoire of nucleic acids encoding domains is combined. The donor nucleic acids can be selected from nucleic acids encoding the amino acid sequences substantially as set forth in SEQ ID NOs: 7-24 and 41-88.

[0051] Sequences encoding the CDRs (e.g., CDR3) of the invention can be introduced into a repertoire of variable domains lacking the respective CDRs (e.g., CDR3) using recombinant DNA techniques, for example, using the methodology described by Marks et al. (Bio / Technology (1992) 10:779-783). In particular, consensus primers directed to or adjacent to the 5' end of the variable domain region are inserted into the repertoire of human V H Combined with a consensus primer to the third framework region of the gene, a V lacking CDR3 was generated. HA repertoire of variable domains can be provided. The repertoire can be combined with the CDR3 of a particular antibody. Using similar techniques, sequences derived from the CDR3 can be combined with Vs lacking a CDR3. H or V L The domain repertoire can be shuffled and the shuffled complete V H or V L The domain is homogenous V L or V H The repertoire can then be displayed in a suitable host system, such as a phage display system as described in WO92 / 01047, to allow selection of suitable antigen-binding fragments.

[0052] A similar shuffling or combinatorial technique has also been disclosed by Stemmer (Nature (1994) 370:389-391), who describes the technique in relation to the β-lactamase gene, but observes that this approach can be used for the generation of antibodies.

[0053] In a further embodiment, one or more selected V H and / or V L Random mutagenesis of the gene can be used to generate novel V sequences carrying one or more sequences derived from the sequences disclosed herein. H or V L One such technique, error-prone PCR, is described by Gram et al. (Proc. Nat. Acad. Sci. USA (1992) 89:3576-3580).

[0054] Another method that can be used is H or V L The first approach is to direct mutagenesis to the CDRs of a gene. Such techniques are described by Barbas et al. (Proc. Nat. Acad. Sci. USA (1994) 91:3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263:551-567).

[0055] Similarly, one or more or all three CDRs may be V H or V L The domains can be grafted into a repertoire of domains which are then screened for antigen-binding fragments specific for KLRG1.

[0056] A portion of an immunoglobulin variable domain comprises at least one of the CDRs substantially as described herein, and optionally an intervening framework region from a scFv fragment as described herein. This portion may comprise at least about 50% of either or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Additional residues at the N- or C-terminus of a substantial portion of a variable domain may be residues that are not normally associated with naturally occurring variable domain regions. For example, the construction of antibodies by recombinant DNA techniques may result in the introduction of N- or C-terminal residues that are encoded by linkers introduced to facilitate cloning or other engineering steps. Other engineering steps include the introduction of linkers to join the variable domain to additional protein sequences, including immunoglobulin heavy chain constant regions, other variable domains (e.g., in the production of diabodies), or protein tags as discussed in more detail below.

[0057] Although the embodiment illustrated in the examples includes a "matching" pair of VH and VL domains, one skilled in the art will recognize that alternative embodiments may include antigen-binding fragments containing only a single CDR from either the VL or VH domain. Any one of the single-chain specific binding domains can be used to screen for complementary domains that can form two-domain specific antigen-binding fragments capable of binding, for example, to KLRG1. Screening can be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach, as disclosed in WO92 / 01047, in which individual colonies containing either H or L chain clones are used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain specific binding domains are selected according to the phage display techniques described.

[0058] The anti-KLRG1 antibodies described herein can be linked to another functional molecule, such as another peptide or protein (albumin, another antibody, etc.), a toxin, a radioisotope, a cytotoxic agent, or a cytostatic agent. For example, the antibodies can be linked by chemical cross-linking or recombinant methods. The antibodies can also be linked to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in the manner described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. The antibodies can be chemically modified by covalent conjugation to a polymer, for example, to increase their circulating half-life. Exemplary polymers and methods for attaching them are also set forth in U.S. Patent Nos. 4,766,106, 4,179,337, 4,495,285, and 4,609,546.

[0059] The disclosed antibodies may also be modified to have a glycosylation pattern that differs from the native pattern. For example, one or more carbohydrate moieties can be deleted and / or one or more glycosylation sites can be added to the original antibody. Addition of glycosylation sites to the antibodies of the present disclosure can be accomplished by modifying the amino acid sequence to contain a glycosylation site consensus sequence known in the art. Another means of increasing the number of carbohydrate moieties on an antibody is by chemically or enzymatically coupling glycosides to amino acid residues of the antibody. Such methods are described in WO87 / 05330 and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22:259-306. Removal of any carbohydrate moieties from the antibody can be accomplished chemically or enzymatically, for example, as described by Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259:52, and Edge et al. (1981) Anal. Biochem., 118:131 and by Thotakura et al. (1987) Meth. Enzymol., 138:350. The antibody may also be tagged with a detectable or functional label. The detectable label may be attached to the antibody using conventional chemistry. 131 I or 99 Detectable labels include radiolabels such as Tc. Detectable labels also include enzyme labels such as horseradish peroxidase or alkaline phosphatase. Detectable labels further include chemical moieties such as biotin that can be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin.

[0060] Antibodies whose CDR sequences differ only insubstantially from those shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40 are included within the scope of the present invention. Typically, amino acids are replaced by related amino acids with similar charge, hydrophobic, or stereochemical properties. Such substitutions would be within the ordinary skill of the craftsman. Unlike CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of the antibody. Modifications to the FR include, but are not limited to, humanization of non-human origin, or manipulation of certain framework residues important for stabilizing antigen contact or binding sites, e.g., changing the class or subclass of the constant region, changing certain amino acid residues that may alter effector functions such as Fc receptor binding, as described in U.S. Pat. Nos. 5,624,821 and 5,648,260, and Lund et al. (1991) J. Immun. 147:2657-2662, and Morgan et al. (1995) Immunology 86:319-324, or changing the species from which the constant region is derived.

[0061] Those of ordinary skill in the art will recognize that the modifications described above are not exhaustive and that many other modifications will be apparent to those of ordinary skill in the art in light of the teachings of this disclosure.

[0062] Nucleic acids, cloning and expression systems The present disclosure further provides the isolated nucleic acid that encodes the disclosed antibody.Nucleic acid may comprise DNA or RNA, and may be wholly or partially synthetic or recombinant.Reference to the nucleotide sequence shown herein includes the DNA molecule with the specified sequence, and includes the RNA molecule with the specified sequence with U substituted for T, unless otherwise required by the context.

[0063] The nucleic acids provided herein comprise coding sequences for the CDRs, VH domains, and / or VL domains disclosed herein.

[0064] The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes comprising at least one nucleic acid encoding the CDRs, VH domains, and / or VL domains disclosed herein.

[0065] The present disclosure further provides a host cell comprising one or more of the constructs described above.

[0066] Nucleic acids encoding any of the CDRs (H1, H2, H3, L1, L2 or L3), VH or VL domains, as well as methods of making the encoded products are also provided. The methods include expressing the encoded product from the encoding nucleic acid. Expression may be achieved by culturing recombinant host cells containing the nucleic acid under appropriate conditions. Following production by expression, the VH or VL domain, or specific binding member, may be isolated and / or purified using any suitable technique and then used as required.

[0067] Antigen-binding fragments, VH and / or VL domains, and the encoding nucleic acid molecules and vectors, may be isolated and / or purified from their natural environment in substantially pure or homogeneous form, or, in the case of nucleic acids, free or substantially free of nucleic acids or genes of origin other than sequences encoding a polypeptide having the required function.

[0068] Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. For suitable cells for producing antibodies, see Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999. Briefly, suitable host cells include bacteria, plant cells, mammalian cells, as well as yeast and baculovirus systems. Mammalian cell lines available in the art for the expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse myeloma cells, and many others. A common bacterial host is E. coli. Any protein expression system compatible with the present invention may be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals as described in Gene Expression Systems, Academic Press, eds. Fernandez et al., 1999.

[0069] Suitable vectors can be selected or constructed to contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Vectors can be plasmids or viruses, such as phages or phagemids, as needed. For further details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989. For example, many known techniques and protocols for the manipulation of nucleic acids in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins are described in detail in Current Protocols in Molecular Biology, 2nd Edition, eds. Ausubel et al., John Wiley & Sons, 1992.

[0070] A further aspect of the disclosure provides a host cell comprising a nucleic acid disclosed herein. Yet a further aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction can use any available technique. For eukaryotic cells, suitable techniques can include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as vaccinia, or for insect cells, baculovirus. For bacterial cells, suitable techniques can include calcium chloride transformation, electroporation, and transfection using bacterial phages. Following introduction of the nucleic acid into the cell, expression from the nucleic acid can be caused or allowed, for example, by culturing the host cell under conditions for expression of the gene.

[0071] How to use The disclosed anti-KLRG1 antibodies can modulate KLRG1-related regulation of immune responses. In certain embodiments, the activation of cytotoxic T cells and NK cells is mediated by the modulation of KLRG1 signaling. The disclosed antibodies can act as either agonists or antagonists of KLRG1 depending on their method of use. The antibodies can be used to prevent, diagnose, or treat medical disorders in mammals, particularly humans. The antibodies of the present invention can also be used to isolate KLRG1 or KLRG1-expressing cells. Furthermore, the antibodies can be used to treat subjects at risk or susceptible to disorders or having disorders associated with abnormal KLRG1 expression or function.

[0072] The antibodies of the invention can be used in situations in which modulation of cytotoxic T and NK cell activation is desirable, for example, in certain cancers and infectious diseases.

[0073] When decreased lymphocyte activation is desired, the anti-KLRG1 antibodies of the invention can be used as agonists for KLRG1 to enhance KLRG1-associated attenuation of cytotoxic (or CD8+) T and NK cell activation.

[0074] In certain circumstances, it may be desirable to induce or enhance a patient's immune response to treat cancer or infectious diseases. Disorders that may be treated or prevented by the disclosed methods include, but are not limited to, infections caused by microorganisms (e.g., bacteria), viruses (e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or shingles), or parasites, as well as cancers (e.g., melanoma and prostate cancer).

[0075] Cytotoxic T and NK cell activation by anti-KLRG1 antibodies enhances T and NK cell responses. In such cases, the antibodies act as antagonists of KLRG1. Thus, in some embodiments, the antibodies can be used to inhibit or reduce downregulatory activities associated with KLRG1, i.e., activities associated with downregulation of cytotoxic T and NK cell activation. As shown in the Examples, blocking KLRG1 / E-cadherin interactions with antagonistic anti-KLRG1 antibodies results in enhanced T cell proliferative responses and IFNγ secretion by these cells, consistent with a downregulatory role of the KLRG1 pathway in cytotoxic T and NK cell activation. In various embodiments, the antibodies inhibit E-cadherin binding to KLRG1 with an IC50 of less than about 50 nM, and more preferably less than about 40, 30, 20, 10, or 5 nM. Inhibition of E-cadherin binding can be measured as described in Example 6 or using techniques known in the art.

[0076] The antibody or antibody composition of the present invention is administered in a therapeutically effective amount. In general, the therapeutically effective amount may vary depending on the age, condition, and sex of the subject, as well as the severity of the subject's symptoms. The therapeutically effective amount of the antibody ranges from about 0.001 to about 30 mg / kg body weight, preferably about 0.01 to about 25 mg / kg body weight, about 0.1 to about 20 mg / kg body weight, or about 1 to about 10 mg / kg. The dosage may be adjusted as necessary to suit the observed effects of the treatment. The appropriate dose is selected by the treating physician based on clinical indications.

[0077] The antibody may be given as a bolus dose to maximize circulating levels of antibody for the longest period of time following administration. Continuous infusion may also be used following the bolus dose.

[0078] 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 invention. In some embodiments, T cell and NK cell activation can be modulated by removing immune cells from a subject and contacting the immune cells in vitro with an anti-KLRG1 antibody of the invention). In such embodiments, anti-KLRG1 antibodies can be used in a multivalent form, such that KLRG1 molecules on the surface of immune cells are "cross-linked" upon binding to such antibodies. For example, anti-KLRG1 antibodies can be bound to a solid support, e.g., beads, or cross-linked via a secondary antibody. Immune cells can then be isolated using methods known in the art and re-implanted into the patient.

[0079] In another embodiment, the antibodies of the present invention can be used as targeting agents for the delivery of another therapeutic or cytotoxic agent (e.g., a toxin) to cells expressing KLRG1. The method includes administering an anti-KLRG1 antibody coupled to a therapeutic or cytotoxic agent or under conditions that allow binding of the antibody to KLRG1.

[0080] The antibody of the present invention can also be used to detect the presence of KLRG1 in biological samples.The amount of KLRG1 detected can be correlated with the expression level of KLRG1, which then correlates with the activation state of immune cells (e.g., activated T cells or NK cells) in subjects.

[0081] Detection methods using antibodies are well known in the art, and include, for example, ELISA, radioimmunoassay, immunoblot, Western blot, immunofluorescence, immunoprecipitation.Antibody can be provided in a diagnostic kit that incorporates one or more of these techniques to detect KLRG1.Such kits can contain other components, packaging, instructions, or other materials to assist in the detection of protein.

[0082] If the antibodies are for diagnostic purposes, it may be desirable to modify them, for example, with a ligand group (such as biotin) or a detectable marker group (such as a fluorescent group, a radioisotope, or an enzyme). If necessary, the antibodies of the present invention can be labeled using conventional techniques. Suitable detectable labels include, for example, fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands with specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase can be detected by its ability to convert tetramethylbenzidine (TMB) to a blue pigment quantifiable by a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, as well as the numerous receptor-ligand pairs known in the art. Other permutations and possibilities will be readily apparent to those skilled in the art and are considered equivalents within the scope of the present invention.

[0083] The antibodies of the present invention can be used in screening methods to identify inhibitors of the KLRG1 pathway that are effective as therapeutic agents. In such screening assays, a first binding mixture is formed by combining KLRG1 with an antibody of the present invention, and the amount of binding in the first binding mixture (M0) is measured. A second binding mixture is also formed by combining KLRG1, the antibody, and the compound or agent to be screened, and the amount of binding in the second binding mixture (M1) is measured. The compound to be tested may be another anti-KLRG1 antibody, as shown in the examples. The amount of binding in the first and second binding mixtures is then compared, for example, by calculating the M1 / M0 ratio. A compound or agent is considered to be capable of modulating KLRG1-related downregulation of immune responses if a decrease in binding is observed in the second binding mixture compared to the first binding mixture. Formulation and optimization of binding mixtures is within the level of one of ordinary skill in the art, and 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 invention. Thus, compounds found to reduce KLRG1 antibody binding by at least about 10% (i.e., M1 / M0<0.9), preferably by more than about 30%, may be identified and then, if necessary, secondarily screened for their ability to ameliorate the disorder in other assays or animal models described below. The strength of binding between KLRG1 and the antibody can be measured, for example, using enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance-based technology (e.g., Biacore), all of which are techniques well known in the art.

[0084] Compounds can then be tested in vitro or in animal models as described in the examples. For example, preliminary doses determined according to animal tests, and dosage scaling for human administration are performed according to accepted practices in the art. Toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. Data obtained from cell culture assays or animal studies can be used to formulate dosage ranges for use in humans. The therapeutically effective dosage achieved in one animal model can be converted for use in another animal, including humans, using conversion factors known in the art (e.g., Freireich et al. (1966) Cancer Chemother. Reports, 50(4):219-244).

[0085] Pharmaceutical Compositions and Methods of Administration The present disclosure provides compositions comprising anti-KLRG1 antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions typically comprise one or more antibodies of the present invention and a pharma- ceutically acceptable excipient. The phrase "pharma- ceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharma- ceutical active substances is well known in the art. The compositions may also contain other active compounds that provide supplementary, additional, or enhanced therapeutic functions. The pharmaceutical compositions may be contained in a container, pack, or dispenser together with instructions for administration.

[0086] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration.Methods for achieving administration are known to those skilled in the art.Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.It may also be possible to obtain a composition that may be topically or orally administered, or that may be capable of being transmitted across mucous membranes.

[0087] Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetate, citrate or phosphate; and agents for adjusting osmolarity such as sodium chloride or dextrose. pH can be adjusted using acids or bases such as hydrochloric acid or sodium hydroxide. Such formulations may be enclosed in ampoules, disposable syringes, or multiple 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 saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonicity agents, for example, sugars, polyalcohols, for example, mannitol, sorbitol, and sodium chloride, in the composition. The carrier can be a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining 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 an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0088] Oral compositions generally include an inert diluent or an edible carrier. These can be enclosed in gelatin capsules or compressed into tablets. For oral administration, the antibody can be combined with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose; disintegrants such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterotes; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0089] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved, for example, by using lozenges, nasal sprays, inhalers, or suppositories. For example, in the case of antibodies that contain an Fc portion, the composition may be capable of being delivered across the mucous membranes of the intestine, mouth, or lungs (e.g., via the FcRn receptor-mediated pathway described in U.S. Pat. No. 6,030,613). For transdermal administration, the active compound can be formulated into ointments, salves, gels, or creams, as generally known in the art. For administration by inhalation, the antibody can be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, a gas such as carbon dioxide, or a nebulizer.

[0090] In certain embodiments, the antibody of the present disclosure is prepared with a carrier that protects the compound from rapid elimination from the body, for example, a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be clear to those skilled in the art. Liposomal suspensions containing the antibody disclosed herein can also be used as pharma-ceutically 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.

[0091] It may be advantageous to formulate oral or parenteral compositions into dosage unit form for ease of administration and uniformity of dosage.The term "dosage unit form" as used herein refers to physically discrete units suitable for use as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0092] Toxicity and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit large therapeutic indices are preferred.

[0093] For any composition used in the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. Examples of suitable bioassays include DNA replication assays, cytokine release assays, transcription-based assays, KLRG1 / cadherin binding assays, immunological assays and other assays, such as those described in the Examples. Data obtained from cell culture assays and animal studies can be used to formulate a dosage range for use in humans. Doses can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of antibody that achieves half-maximal inhibition of symptoms). Circulating levels in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be monitored by suitable bioassays. Dosages are preferably within a range of circulating concentrations with little or no toxicity. Dosages can vary depending on the dosage form used and the route of administration used.

[0094] The following examples are not intended to limit the scope of the present invention in any way. Those skilled in the art will recognize numerous modifications and variations that can be made without changing the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the present invention. The entire contents of all references, patents, and published patent applications cited throughout this application are incorporated herein by reference. EXAMPLES

[0095] Example 1: Production of recombinant proteins Recombinant proteins were produced by standard molecular cloning and expression protocols and included human KLRG1 ECD (SEQ ID NO: 89), cynomolgus KLRG1 ECD (SEQ ID NO: 90), and human E-cadherin (SEQ ID NO: 91), the amino acid sequences of which are shown in Table 6 below. Recombinant proteins were produced as FC fusion or HIS-tagged versions by cloning the respective cDNA into pCDNA4 vector (Invitrogen) and transiently transfecting in mammalian HEK293. Purification of the expressed proteins was performed by chromatography using Protein A affinity resin for the FC fusion versions and Nickel-NTA resin for the HIS-tagged proteins. All purified proteins were characterized by SDS-PAGE electrophoresis to confirm purity and molecular weight. [Table 6]

[0096] To test the binding of antibodies to full-length antigens, stable cell lines were developed to be used as immunization antigens and as target cells in functional T cell assays. The developed cell lines include CHO expressing full-length human KLRG1 and CHO expressing full-length cynomolgus KLRG1. The stable cell lines were obtained by transfecting CHO cells with pCDNA4 plasmids encoding the proteins of interest. After transfection, cells were exposed to 500ug / ml G418 to select stable integrated plasmids. The cell lines were further characterized by FACS for expression and sorted to select homogeneous and stable expression.

[0097] Stable CHO cells double-transfected with CD3 agonist and E-cadherin were used in functional assays to demonstrate the effect of blocking KLRG1 in human T cells. Co-expression of CD3 agonist and E-cadherin was confirmed by two-color FACS using appropriate antibodies.

[0098] Example 2: Generation of anti-KLRG1 antibodies Antibodies ABC_HG1N01, ABC_HG1N02 and ABC_HG1N07 are humanized IgG1 antibodies against the extracellular domain of KLRG1. Mouse monoclonal antibodies (MABs) against human KLRG1 were generated by standard immunization of female BALB / c and SJL mice with human and cynomolgus KLRG1 and subsequent screening of hybridomas. Several immunization strategies have been employed to generate a diverse number of antibody hits. Briefly, SJL and Balb / c mice were repeatedly immunized with either cDNA, recombinant antigen, or CHO cells expressing the antigen of interest. Antigen-specific antibody titers were monitored periodically by ELISA, and animals were sacrificed when appropriate titers were reached, usually a dilution factor of 1:1000 to 1:10000. Spleen cells from sacrificed mice were fused to mouse myeloma cells to produce hybridoma cells, which were then cultured and subcloned into single cells. Stable clones were scaled up and conditioned media was collected and tested for expression of anti-KLRG1 antibodies by ELISA and FACS.

[0099] Example 3: Selection of anti-KLRG1 blocking antibodies Hybridoma-produced antibodies were screened for binding to human KLRG1 ECD and cynomolgus KRG1 ECD. Antibodies with cross-reactivity between both antigens were selected to proceed to the next stage of screening to determine their ability to neutralize the interaction between KLRG1 and E-cadherin. Antibodies were ranked according to their binding EC50 to human and cynomolgus KLRG1 and further prioritized according to their IC50 in E-cadherin competition assay. A total of 48 antibodies were selected according to these criteria. Eight antibodies were prioritized for functional characterization in cell-based assays. Table 3 summarizes the variable region amino acid sequences of the eight selected mouse antibodies, and Table 4 shows the CDR regions of the eight selected mouse antibodies. Antibodies were further ranked according to the presence of sequence liability motifs summarized in Table 7. A deamidation site (NG) was found in the CDR-L1 of ABC_G1N01 and ABC_G1N02, which may affect the stability manufacturability and activity of the drug substance. To remove this potential liability, a series of mutants were constructed and tested. Specifically, the NG motif can be replaced with NA, QG, and KG sequences without loose binding. Mouse antibodies can be humanized by grafting the CDR regions into a human framework. Table 1 shows examples of variable region sequences of humanized constructs of ABC_G1N01 (ABC_HG1N01), ABC_G1N02 (ABC_HG1N02), and ABC_G1N07 (ABC_HG1N07). Furthermore, conservative mutations can be made in the CDR regions to improve affinity, potency, or biophysical characteristics. Table 5 summarizes a list of conservative mutations that can be made in the CDR regions. The monoclonal antibodies were epitope binned according to their ability to compete with each other. It was found that the antibodies could be grouped into three different bins: BIN1 includes ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05; BIN2 includes MAB25, MAB031; BIN3 includes ABC_G1N07, ABC_G1N08.The antibodies were produced as chimeras by cloning the variable mouse regions onto a human IgG constant framework and tested in functional assays to determine their functional activity on human T cells and are described herein. [Table 7]

[0100] Example 4: Characterization of antibody binding to KLRG1 (EC50) and inhibition and KLRG1 / E-cadherin interactions (IC50) by FACS Binding of anti-KLRG1 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 (CHO-human-KLRG1) and cynomolgus-KLRG1 (CHO-cynomolgus-KLRG1). EC50 values ​​were determined by incubating various concentrations of anti-KLRG1 monoclonal antibodies at concentrations ranging from 1 to 100 nM and measuring the fluorescence of labeled cells using an anti-mouse detection antibody directly conjugated with a fluorescent probe.

[0101] The ability of the monoclonal antibodies to inhibit E-cadherin / KLRG1 binding was measured by FACS. Binding of E-cadherin to CHO-hKLRG1 cells was first determined by incubating KLRG1 expressing cells with various concentrations of HIS-tagged recombinant E-cadherin and detected by FACS using an anti-HIS detection antibody. The EC50 of E-cadherin / KLRG1 interaction was determined to be 1 μM by this method. The IC50 value of each monoclonal antibody was determined by monitoring the loss of E-cadherin binding as a function of varying monoclonal antibody concentrations from 0.1 to 100 nM. EC50 and IC50 values ​​were calculated using SigmaPlot software and are summarized in Table 8. [Table 8]

[0102] Example 5: Measurement of binding kinetics The binding kinetics of humanized antibodies to human and cynomolgus KLRG1 were determined by OCTET® measurements. OCTET® Systems uses Bio-Layer Interferometry (BLI) technology to directly monitor the binding of proteins and other biomolecules to their partners in real time and provide analysis of kinetic binding constants. See https: / / www.fortebio.com / bli-technology.html, the contents of which, including all links and sublinks associated therewith, are incorporated herein by reference in their entirety. The experimental setup consisted of immobilizing biotinylated recombinant antigen (human-KLRG-ECD or cynomolgus-KLRG1-ECD) on a streptavidin OCTET® sensor to produce an antigen loaded sensor. The loaded sensors were exposed to various concentrations of each humanized antibody, ranging from 100 to 0.1 nM, in an OCTET® instrument to measure the association rate constant (K on Data is collected for 600 seconds to measure the dissociation rate constant (K). In the next step, the sensor is exposed to a solution of 1× phosphate buffered saline (PBS) buffer without antibody for 600 seconds to measure the dissociation rate constant (K off The resulting data is then fitted to a 1:1 binding kinetics model using ForteBio® analysis software to obtain the K D The kinetic binding parameters derived by this method are summarized in Table 9 for the three humanized antibodies. [Table 9]

[0103] Example 6: Characterization of the functional activity of KLRG1 blocking antibodies Blockade of KLRG1 interaction with its ligands has an activating effect in human CD8+ T cells by measuring the effect of antibody-mediated blockade of KLRG1 signaling on the production of IFNγ and the proliferation of T cells (proliferation index). To demonstrate the effect of KLRG1 blockade on cells of the immune system, CD8+ T cells were isolated from healthy donors and tested in co-culture with a CHO cell line co-expressing CD3 agonist and E-cadherin (eAPC). This assay works by providing two competing signals to T cells, where CD3 stimulation is countered by the inhibitory effect of E-cadherin. When KLRG1 signaling is blocked by anti-KLRG1 antibodies, the inhibitory signal is disrupted and T cells are activated following their interaction with the CD3 agonist expressed on the cell surface. IFNγ secretion was measured by ELISA and the results are summarized in Table 10 and Figure 1. [Table 10]

[0104] This data demonstrates that E-cadherin has an inhibitory effect on human T cells that can be reversed by blockade of KLRG1 signaling with a blocking antibody.

[0105] The data presented in Table 11 and FIG. 2 demonstrate that blocking KLRG1 / E-cadherin interaction results in the proliferation of human CD8+ T cells by measuring the proliferation index in response to eAPCs. In this assay, eAPCs were generated by stably transducing CHO cells with h-ecadherin and a CD3 agonist agent. The eAPCs thus generated were then co-incubated for 3 days with freshly isolated CD8+ T cells from healthy volunteers in the presence of 10 micrograms / ml of test antibody or isotype control. A positive control sample was prepared by incubating CD8+ T cells with CHO cells stably expressing anti-CD3 but lacking expression of the inhibitory ligand E-cadherin, thus allowing non-inhibitory stimulation of CD8+ T cells by anti-CD3 expressed on CHO cells. The results show that CD8+ T cells proliferate in response to CD3 stimulation as expected, and that co-expression of the inhibitory ligand E-cadherin on eAPCs inhibits the proliferation of CD8+ T cells. Moreover, CD8+ T cell proliferation can be restored by blockade of KLRG1 with neutralizing antibodies. [Table 11]

[0106] The data presented in Table 12 and FIG. 3 show a correlation between T cell activity in an IFNγ secretion assay and blocking activity of anti-KLRG1 neutralizing antibodies. The IC50 value of each antibody was derived from the E-cadherin binding inhibition study and plotted against IFNγ production levels measured in a CD8+ T cell IFNγ release assay. The correlation shows that antibodies with lower IC50 values ​​(e.g., more potent blockers of KLRG1 / E-cadherin binding) result in higher levels of IFNγ release by CD8+ T cells. This data shows that antibody blockade of KLRG1 results in restoration of T cell activity in an E-cadherin-dependent manner. [Table 12]

Claims

1. (a) a heavy chain variable region (VH) comprising CDR-H1, CDR-H2 and CDR-H3 having the amino acid sequences of SEQ ID NOs: 47, 48 and 49, respectively; (b) an antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) comprising CDR-L2 and CDR-L3 having the amino acid sequences of SEQ ID NOs: 51 and 52, respectively, and CDR-L1 having the amino acid sequence of SEQ ID NO: 50 or comprising an NG motif substituted by NA, QG or KG; The antibody has (i) a heavy chain variable region (VH) sequence of SEQ ID NO: 3 and a light chain variable region (VL) sequence of SEQ ID NO: 4; or (ii) having the sequence of the heavy chain variable region (VH) of SEQ ID NO: 27 and the sequence of the light chain variable region (VL) of SEQ ID NO: 28; and The antibody or antigen-binding fragment thereof, wherein the antibody specifically binds to the extracellular domain of human killer cell lectin-like receptor subfamily G member 1 (KLRG1) and the extracellular domain of cynomolgus monkey KLRG1.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein CDR-L1 has the amino acid sequence of SEQ ID NO:50, said amino acid sequence comprising an NG motif substituted by NA.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody specifically binds to an amino acid sequence that is at least 95% identical to any sequence of at least 100 consecutive amino acids of at least one sequence selected from the group consisting of SEQ ID NO:89 and SEQ ID NO:

90.

4. The antibody has a K of at least 2 nM, 1 nM, 100 pM, 10 pM, or 5 pM. Ｄ 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which specifically binds to the extracellular domain of KLRG1 with an affinity as represented by the following formula:

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody inhibits binding of E-cadherin to KLRG1 with an IC50 of less than 50 nM, 40 nM, 30 nM, 20 nM, or 10 nM.

6. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody is humanized.

7. The antibody is IgG ｉ or IgG ４ 7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6,

8. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8 for the treatment or prevention of cancer or an infectious disease.

10. 8. An isolated nucleic acid encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

11. An expression vector comprising the nucleic acid of claim 10.

12. A host cell comprising the vector of claim 11.

13. 13. The host cell of claim 12, wherein the host cell is selected from E. Coli bacteria, Chinese hamster ovary cells, HeLa cells, and NSO cells.

14. The nucleic acid of claim 10, wherein the nucleic acid encodes the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:27 or SEQ ID NO:

28.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, which is an anti-KLRG1 antibody or fragment thereof for modulating CD8+ T and NK cell activation by contacting lymphocytes, such as T cells or NK cells.

16. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

17. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, a pharmaceutical composition described in claim 8, an isolated nucleic acid described in any one of claims 10 and 14, an expression vector described in claim 11, a host cell described in claim 12 or 13 or a composition described in claim 16 in the manufacture of a medicine or kit for the treatment or prevention of cancer or an infectious disease.

18. Use of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, a pharmaceutical composition described in claim 8, an isolated nucleic acid described in any one of claims 10 and 14, an expression vector described in claim 11, a host cell described in claim 12 or 13 or a composition described in claim 16 in the manufacture of a pharmaceutical or kit for modulating CD8+ T and NK cell activation by contacting with lymphocytes.

19. The anti-KLRG1 antibody or a fragment thereof according to claim 15, which further inhibits binding of E-cadherin to human and cynomolgus monkey KLRG1.

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