Anti-KLRG1 antibodies

Abstract

The present invention relates to an anti-KLRG1 antibody. The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to a 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, as well as for increasing the effectiveness of vaccines.

Description

[0001] This application is a divisional application of the patent application "Anti-KLRG1 Antibody" with an application date of September 6, 2019 and application number 201980060735.4 (International Application Number PCT / US2019 / 050110). Technical Field

[0002] The technical field relates to the inhibitory lymphocyte co-inhibitory receptor killer cell lectin-like receptor subfamily G member 1 (KLRG1). Background Art

[0003] In response to activation signals such as antigenic peptides in the case of major histocompatibility complex (MHC) and T cell receptor (TCR) binding, lymphocyte co-inhibitory receptors regulate the effect of adaptive immune system (such as T cells and NK cells). Co-inhibitory receptors include PD-1, LAG-3, TIM-3 and CTLA4. The effect of co-inhibitory receptors is usually by binding the ligand to the extracellular domain of co-inhibitory receptors, and then recruiting intracellular phosphatases by immunoreceptor tyrosine-based inhibitory motifs (ITIMs) located in the intracellular domain of co-inhibitory receptors. The effect of co-inhibitory receptors is usually to suppress the immune response of TCR engagement. In recent years, it has been shown that agents blocking the activity of co-inhibitory receptors can be used as effective treatments for cancer and infectious diseases.

[0004] Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane protein that acts 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 ligand is cadherin, and its intracellular portion contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) domain responsible for co-inhibition of T cell receptor (TCR)-mediated signaling. The KLRG1 ligand can be E-cadherin, N-cadherin, R-cadherin, or a combination thereof.

[0005] The receptor killer cell lectin-like receptor G1 (KLRG1) is expressed on T cells and NK cells and binds to ligands on epithelial and mesenchymal cells. Ligands for KLRG1 have been described as E-cadherin, N-cadherin, and R-cadherin.

[0006] In humans, KLRG1 expression is restricted to cells of the immune system, and in particular CD8-positive T cells, NK cells, and to a lesser extent CD4-positive T cells. KLRG1 expression is associated with a late differentiation phenotype. As antigen-specific T cells differentiate, they acquire increased expression of cytotoxic molecules and therefore have increased cytotoxic potential. The biological function of KLRG1 is to inhibit the cytotoxicity and proliferation of these T cells. In cancer and infectious diseases, it has been shown to be beneficial for restoring T cell activity.

[0007] In general, there is a need to provide safe and effective treatments for cancer or infectious diseases. Modulation of cytotoxic (or CD8+) T cell and NK cell activation involved in these disorders can be accomplished by manipulating the KLRG1 pathway. Summary of the invention

[0008] The present disclosure provides characterization of new antibodies or antigen-binding fragments thereof that bind to the extracellular domain (ECD) of KLRG1 and inhibit its interaction with the ligands E-cadherin, N-cadherin and R-cadherin. The antibodies described herein have been obtained by mouse hybridoma technology and can be humanized by transplanting their complementary determining regions (CDRs) into a human framework. The present disclosure also provides antibodies or binding fragments that regulate (e.g., activate) CD8+ cytotoxic T cells and NK cells and achieve this purpose by regulating (e.g., neutralizing) the interaction between KLRG1 and its ligand. The antibodies described herein can be used as a monotherapy or in combination with other immunotherapeutics (such as anti-PD-1 antibodies, anti-PD-L1 antibodies, or anti-CTLA4 antibodies) or in combination with chemotherapeutics or cancer vaccines as an effective therapeutic agent for treating cancer. The antibodies described herein can be used as an effective treatment for infectious diseases or to enhance the effectiveness of vaccines against infectious diseases.

[0009] Non-limiting illustrative embodiments of antibodies are referred to as 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 comprise a V fragment of an Fv fragment of ABC_HG1N01, ABC_HG1N02, ABC_HG1N07, ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07, or ABC_G1N08. H and / or V L Other embodiments include these V H and V L Other embodiments comprise V or V sequences 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 domain of the H3 fragment.

[0010] The present disclosure also provides compositions comprising KLRG1 antibodies, and uses thereof in methods of modulating immune responses, including methods of treating humans or animals. In specific embodiments, anti-KLRG1 antibodies are used to treat or prevent cancer by activating CD8+ cytotoxic T cells and NK cells. Conditions susceptible to treatment with the compositions of the present invention include, but are not limited to, cancer and infectious diseases.

[0011] In addition, anti-KLRG1 antibodies can be used to diagnose and detect KLRG1 or fragments thereof in biological samples. The amount of KLRG1 detected can be correlated with the expression level of KLRG1, which in turn is correlated with the activation state of lymphocytes (e.g., cytotoxic T cells or natural killer cells) in the subject.

[0012] The present disclosure also provides isolated nucleic acids comprising V sequences encoding 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 or V L Also provided is an isolated nucleic acid comprising a sequence encoding a V domain from the presently disclosed H and V L The disclosure also provides vectors and host cells comprising such nucleic acids.

[0013] The present disclosure also provides a method for generating a new V H and V L Methods for producing domains and / or functional antibodies comprising all or part of such domains, wherein the domains are derived from V and 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 Structural domain.

[0014] Additional aspects of the present disclosure will be listed in part in the following description, and in part will become apparent from the description, or can be learned by practicing the present invention. The present invention is listed and particularly pointed out in the appended claims, and the present disclosure should not be interpreted as limiting the scope of the claims in any way. The following detailed description includes exemplary representations of various embodiments of the present invention, which do not limit the present invention as claimed. The accompanying drawings constitute a part of this specification and, together with the specification, are only used to illustrate various embodiments without limiting the present invention. The citation of the references is not an admission that these references are prior art of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown are the results of an interferon-γ (IFNγ) secretion assay, demonstrating that anti-KLRG1 antibodies activate CD8+ human T cells.

[0016] Figure 2 The results of a CD8+ T cell proliferation assay are shown, demonstrating that anti-KLRG1 antibodies induce proliferation of CD8+ T cells.

[0017] Figure 3 Shown is the relationship between the results of the IFNγ secretion assay in CD8+ T cells treated with anti-KLRG1 antibodies and the blocking activity of the antibodies (shown as IC50). DETAILED DESCRIPTION

[0018] definition

[0019] The term "antibody" as used in the present disclosure refers to an immunoglobulin or a fragment or derivative thereof, and encompasses any polypeptide comprising an antigen binding site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single chain, chimeric, synthetic, recombinant, hybrid, mutant and transplanted antibodies. For the purposes of the present disclosure, unless the term "complete" is otherwise modified, as in "complete 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 contain an antigen binding domain.

[0020] The terms "antigen binding domain", "antigen binding fragment" and "binding fragment" refer to a portion of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. In the case of a large antigen, the antigen binding domain may bind to only a portion of the antigen. The portion of the antigen molecule that is responsible for the specific interaction with the antigen binding domain is called an "epitope" or "antigenic determinant."

[0021] The antigen binding domain usually comprises the variable region of the antibody light chain (V L ) and antibody heavy chain variable region (V H ), but not necessarily both. For example, the so-called Fd antibody fragment consists only of V H domains, but still retains some of the antigen-binding function of the intact antibody.

[0022] The term "repertoire" refers to a genetically diverse collection of nucleotides derived in whole or in part from sequences encoding expressed immunoglobulins. These sequences are produced by in vivo rearrangements of, for example, V, D and J segments of H chains and, for example, V and J segments of L chains. Alternatively, the sequence can be produced from a cell line in response to in vitro stimulation in which rearrangement occurs. Alternatively, part or all of the sequence can be obtained by combining, for example, unrearranged V segments with D and J segments, by nucleotide synthesis, random mutagenesis and other methods (such as disclosed in U.S. Patent No. 5,565,332).

[0023] 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 moderate capacity, in contrast to nonspecific binding, which typically has low affinity and moderate to high capacity. In general, when the affinity constant K A Above 10 6 M -1 or more preferably higher than 10 8 M -1 When the binding is not specific, the binding is considered to be specific. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting specific binding. Appropriate binding conditions, such as antibody concentration, solution ionic strength, temperature, time allowed for binding, concentration of blocking agents (e.g., serum albumin, milk casein), etc., can be optimized by a skilled person using conventional techniques.

[0024] The phrase "essentially as described" refers to the relevant CDRs, V H or V L The domain will be identical to or have only insubstantial differences in the specified region (e.g., CDR) of the sequence. Insubstantial differences include minor amino acid changes, such as substitutions of 1 or 2 of any 5 amino acids in the sequence of the specified region.

[0025] The term "KLRG1 activity" refers to one or more lymphocyte co-suppressive activities associated with KLRG1. For example, KLRG1 activity may mean the regulation of cytotoxic T cell and NK cell activation.

[0026] The term "modulation" and its cognates refer to a decrease or increase in KLRG1 activity associated with the activation of T cells and NK cells due to the interaction of KLRG1 with an anti-KLRG1 antibody, wherein the decrease or increase is relative 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 reduced, the terms "modulatory / modulate" are interchangeable with the terms "inhibitory / inhibit". When KLRG1 activity is increased, the terms "modulatory / modulate" are interchangeable with the terms "activating / activate". The activity of KLRG1 can be quantitatively determined using T cell and NK cell activation assays such as those described in Example 6.

[0027] The terms "treatment" and "therapeutic method" refer to both therapeutic methods and prophylactic / preventative measures. Those in need of treatment can include individuals already suffering from a particular medical condition as well as individuals who may eventually acquire the condition (ie, individuals in need of preventative measures).

[0028] The term "effective amount" refers to a dose or amount sufficient to reduce KLRG1 activity to result in improvement of patient symptoms or to achieve a desired biological result, such as reducing KLRG1 activity, regulating lymphocyte co-inhibitory responses, increasing activation of cytotoxic T cells and NK cells, or increasing IFNγ release by cytotoxic T cells or NK cells.

[0029] The term "isolated" refers to a molecule that is substantially free of its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to a preparation in which the isolated protein is sufficiently pure to be administered as a pharmaceutical composition, or 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.

[0030] Anti-KLRG1 Antibody

[0031] The present disclosure provides anti-KLRG1 antibodies comprising novel antigen-binding fragments.

[0032] Typically, antibodies can be prepared, for example, using traditional hybridoma technology (Kohler and Milstein (1975) Nature, 256: 495-499), recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage display 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 also Antibodies: A Laboratory Manual, Harlow et al., ed., Cold Spring Harbor Laboratory, 1988. The present invention is not limited to any particular source, species of origin, or method of production.

[0033] Intact antibodies (also called immunoglobulins) are typically tetrameric glycosylated proteins consisting of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chains are found in antibodies, designated as lambda chains and kappa chains. Based on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be divided into five major classes: A, D, E, G, and M, and several of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0034] The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Harlow et al., supra. Briefly, each light chain consists of an N-terminal variable domain (V L ) and constant domain (C L Each heavy chain consists of an N-terminal variable domain (V H ), three or four constant domains (C H ) and hinge region. Closest to V H C H Domain named C H 1. V H and V LThe domain consists of four regions of relatively conserved sequence, called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequence, called complementarity determining regions (CDRs). The CDRs contain most of the residues responsible for specific interaction with the antigen. The three CDRs are called CDR1, CDR2, and CDR3. Thus, the CDR components on the heavy chain are called H1, H2, and H3, while the CDR components on the light chain are called L1, L2, and L3. CDR3, and particularly H3, is the largest source of molecular diversity within the antigen binding domain. For example, H3 can be as short as two amino acid residues or greater than 26.

[0035] The Fab fragment (fragment antigen binding) consists of V and V regions covalently linked by disulfide bonds between the constant regions. H -C H 1 and V L -C L In order to overcome the non-covalent linkage of V in Fv when co-expressed in host cells H and V L The tendency of the domains to dissociate allows the construction of so-called single-chain (sc) Fv fragments (scFv). In scFv, a flexible and sufficiently long polypeptide binds the V H The C terminal is connected to V L N terminal or V L The C terminal is connected to V H Most commonly, a 15-residue (Gly4Ser)3 peptide is used as a linker, but other linkers are also known in the art.

[0036] Antibody diversity is the result of the combinatorial assembly of multiple germline genes encoding variable regions and various somatic events. Somatic events include the recombination of variable gene segments with diversity (D) and joining (J) gene segments to form a complete V H region, and the recombination of variable gene segments with joining gene segments to form a complete V L The recombination process itself is imprecise, resulting in the loss or gain of amino acids at the V(D)J junction. These diversity mechanisms exist in developing B cells prior to antigen exposure. After antigen stimulation, somatic mutations occur in the antibody genes expressed in B cells.

[0037] Based on the estimated number of germline gene segments, random recombination of these segments, and random V H -V L Paired, it can generate up to 1.6×10 7different antibodies (Fundamental Immunology, 3rd edition, Paul ed., Raven Press, New York, NY, 1993). When other processes that contribute to antibody diversity (such as somatic mutation) are taken into account, it is believed that it is possible to generate more than 1×10 10 Different antibodies (Immunoglobulin Genes, 2nd edition, Jonio et al., ed., Academic Press, San Diego, Calif., 1995). Due to the many processes involved in antibody diversity, it is extremely unlikely that independently generated antibodies will have identical amino acid sequences in the CDRs.

[0038] The present disclosure provides novel CDRs derived from human immunoglobulin gene libraries. The structure used to carry the CDRs is generally an antibody heavy chain or light chain or a portion thereof, wherein the CDRs are located in a region corresponding to a naturally occurring V H and V L The structure and position of the immunoglobulin variable domain can be determined, for example, as described in Rabat et al., Sequences of Proteins of Immunological Interest, pp. 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991.

[0039] V of humanized anti-KLRG1 antibody H and V L The amino acid sequences of the domains are listed in the sequence listing and are enumerated as shown in Table 1.

[0040] Table 1: Amino acid sequences of humanized anti-KLRG1 antibody variable regions

[0041]

[0042] Specific non-limiting illustrative embodiments of antibodies are referred to as ABC_HG1N01, ABC_HG1N02, and ABC_HG1N07. H and V L The amino acid sequences of the CDRs within the domain are listed in the sequence listing and enumerated as shown in Table 2.

[0043] Table 2. CDR amino acid sequences of humanized anti-KLRG1 antibodies

[0044]

[0045]

[0046] Mouse anti-KLRG1 antibody V H and V L The amino acid sequences of the domains are listed in the sequence listing and enumerated as shown in Table 3.

[0047] Table 3: Variable region amino acid sequences of mouse anti-KLRG1 antibodies

[0048]

[0049]

[0050]

[0051] Specific non-limiting illustrative embodiments of antibodies are referred to as 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 domain are listed in the sequence listing and enumerated as shown in Table 4.

[0052] Table 4: CDR amino acid sequences of mouse anti-KLRG1 antibodies

[0053]

[0054]

[0055] The anti-KLRG1 antibody may optionally comprise an antibody constant region or a portion thereof. L The domain can be connected at its C-terminus to an antibody light chain constant domain, including a human CK or CK chain. HThe specific antigen binding domain of the domain may be connected to all or part of the immunoglobulin heavy chain derived from any antibody isotype (e.g., IgG, IgA, IgE, and IgM) and any isotype subclass (including but not limited to IgG1 and IgG4). In an exemplary embodiment, 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 a C-terminal fragment of the heavy and light chains of human IgG1k or IgG1x. The DNA and amino acid sequences of the C-terminal fragments are well known in the art (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 91-3242, National Institutes of Health Publications, Bethesda, Md., 1991).

[0056] Certain embodiments comprise a V or Fv fragment 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 Other embodiments include these V H and V L The invention also includes at least one CDR of any of the following CDR sequences: 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. For example, one embodiment includes a V domain of an antibody selected from at least one of the following: HH3 fragments of the domain: ABC_HG1N01, AB C_HG1N02, ABC_HG1N07ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05, ABC_G1N06, ABC_G1N07 and AB C_G1N08.

[0057] In certain embodiments, V H and / or V L The domains are germlined, ie, the framework regions (FRs) of these domains are mutated to match those produced by germline cells using conventional molecular biology techniques. In other embodiments, the framework sequences remain distinct from the consensus germline sequences.

[0058] In certain embodiments, the antibody specifically binds to an epitope within the ECD of human or mouse KLRG1 with an affinity expressed as a KD of at least about 2 nM, 1 nm, 100 pM, 10 pM, or 5 pM. The amino acid sequences of the ECD of human and cynomolgus KLRG1 are set forth in SEQ ID NO: 89 and SEQ ID NO: 90, as shown in Table 6.

[0059] It is contemplated that the antibodies of the invention may also bind to other proteins including, for example, recombinant proteins comprising all or part of KLRG1.

[0060] Those of ordinary skill in the art will recognize that the antibodies of the present invention can be used to detect, measure and inhibit proteins that are different from KLRG1. As long as the target protein comprises 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 set forth in SEQ ID NO: 89 or SEQ ID NO: 90, the antibody is expected to retain specific binding. The percent identity is determined by a standard alignment algorithm, 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.

[0061] In addition to sequence homology analysis, epitope mapping (see, e.g., Epitope Mapping Protocols, Morris, ed., Humana Press, 1996) and secondary and tertiary structural analysis can be performed to identify the specific 3D structure 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 currently 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).

[0062] derivative

[0063] The present disclosure also provides a method for obtaining an antibody specific for KLRG1. The CDRs in such antibodies are not limited to the V sequences identified in Tables 1 and 3. H and V LSpecific sequences of, and 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 a skilled person using techniques well known in the art. For example, amino acid substitutions, deletions or additions may be made in FR and / or CDR. Changes in FR are usually designed to improve the stability and immunogenicity of the antibody, while changes in CDR are usually designed to increase the affinity of the antibody for its target. Variants of FR also include naturally occurring immunoglobulin allotypes. Such changes in affinity increase may be empirically determined by conventional techniques involving changing CDRs and testing the affinity of antibodies for their targets. For example, conservative amino acid substitutions may be made in any of the disclosed CDRs. Various changes may be made according to the methods described in Antibody Engineering, 2nd edition, Oxford University Press, edited by Borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that are altered by replacing different codons for functionally equivalent amino acid residues within the coding sequence to produce "silent" changes. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutes for amino acids within the sequence can be selected from other members of the class to which the amino acids belong (see Table 5). In addition, any native residue 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).

[0064] Table 5: Exemplary conservative substitutions:

[0065] Residue Conservative substitution Ala(A) Ser(S) Arg(R) Lys(K) Asn(N) Gln(Q);His(H) Asp(D) Glu(E) Cys(C) Ser(S) Gln(Q) Asn(N) Glu(E) Asp(D) Gly(G) Pro(P) His(H) Asn(N), Gln(Q) Ile(I) Leu(L), Val(V) Leu(L) Ile(I), Val(V) Lys(K) Arg(R), Gln(Q) Met(M) Leu(L), Ile(I) Phe(F) Met(M), Leu(L), Tyr(Y) Ser(S) Thr(T); Gly(G) Thr(T) Ser(S), Val(V) Trp(W) Tyr(Y) Tyr(Y) Trp(W), Phe(F) Val(V) Ile(I), Leu(L) Pro(P) —

[0066] Derivatives and analogs of the antibodies of the invention can be produced by a variety of techniques well 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).

[0067] In one embodiment, a method for preparing V H Variant of amino acid sequence of domain V H The method of structural domain includes V H The step of adding, deleting, substituting or inserting one or more amino acids into the structural domain, optionally converting the V H domain with one or more V L Domain combination, and test V H domain or one or more V H / V L Combining antigen binding domains with specific binding to KLRG1 and optionally testing the ability of such antigen binding domains to modulate KLRG1 activity. L The domain may have an amino acid sequence identical to or substantially as described in Tables 1 and 3.

[0068] A similar approach can be employed, wherein the V disclosed herein is L One or more sequence variants of a domain and one or more V H Domain combination.

[0069] Another aspect of the present disclosure provides a method for preparing an antigen-binding fragment that specifically binds to KLRG1. The method comprises:

[0070] (a) Provide code V H The starting library of nucleic acids of the domain, the V H The domain contains the CDR3 to be replaced or lacks the CDR3 coding region;

[0071] (b) Target the library and code to V H The donor nucleic acids are combined with the amino acid sequences of CDR3 (i.e., H3) substantially as described herein, such that the donor nucleic acids are inserted into the CDR3 region of the library to provide the V H A product library of nucleic acids of a structural domain;

[0072] (c) nucleic acids expressing a library of products;

[0073] (d) selecting a binding fragment that is specific for KLRG1; and

[0074] (e) recovering the specific binding fragment or the nucleic acid encoding it.

[0075] Again, a similar method can be used, wherein V of the present invention is L CDR3 (i.e., L3) and encoding V L The library of nucleic acids of the domain is composed of the V L The domain includes the CDR3 to be replaced or lacks the CDR3 coding region.The donor nucleic acid may be selected from nucleic acids encoding an amino acid sequence substantially as set forth in SEQ ID NOs: 7-24 and 41-88.

[0076] Sequences encoding the CDRs (e.g., CDR3) of the present invention can be introduced into a library of variable domains lacking the corresponding CDRs (e.g., CDR3) using recombinant DNA techniques, for example, using the method described by Marks et al. (Bio / Technology (1992) 10:779-783). In particular, common primers for the 5' end of the variable domain region or adjacent to the 5' end of the variable domain region can be used with human V H The consensus primers for the third framework region of the gene were used in combination to provide a V sequence lacking CDR3. H The library can be combined with the CDR3 of a particular antibody. Using similar techniques, CDR3-derived sequences can be combined with V sequences lacking CDR3. H or V L The library of structural domains is reorganized, and the reorganized complete V H or V L Domain structure and homologous V L or V H The domains are combined to prepare KLRG1 specific antibodies of the invention. The library can then be displayed in a suitable host system such as a phage display system (such as described in WO92 / 01047) so that suitable antigen binding fragments can be selected.

[0077] Similar shuffling or combinatorial techniques were also disclosed by Stemmer (Nature (1994) 370:389-391), who described the technique in relation to the beta-lactamase gene but observed that the method could be used to generate antibodies.

[0078] In other embodiments, one or more selected V H and / or V L Random mutagenesis of genes to generate new V sequences carrying one or more sequences derived from the sequences disclosed herein H or VL One such technique, error-prone PCR, is described by Gram et al. (Proc. Nat. Acad. Sci. USA (1992) 89:3576-3580).

[0079] Another approach that can be used is to direct mutagenesis to V H or V L Such techniques are disclosed by Barbas et al. (Proc. Nat. Acad. Sci. USA (1994) 91: 3809-3813) and Schier et al. (J. Mol. Biol. (1996) 263: 551-567).

[0080] Similarly, one or more or all three CDRs may be ported to V H or V L The library of structural domains was then screened for antigen-binding fragments that were specific for KLRG1.

[0081] A portion of an immunoglobulin variable domain will comprise at least one of the CDRs substantially as described herein, and optionally an intervening framework region from a scFv fragment as described herein. The portion may include at least about 50% of one or both of FR1 and FR4, the 50% being the C-terminal 50% of FR1 and the N-terminal 50% of FR4. Other residues at the N-terminal or C-terminal end of the substantial portion of the variable domain may be those residues that are not usually associated with naturally occurring variable domain regions. For example, constructing antibodies by recombinant DNA technology may result in the introduction of N-terminal or C-terminal residues encoded by a joint introduced to facilitate cloning or other manipulation steps. Other manipulation steps include introducing a joint to connect the variable domain to other protein sequences, including an immunoglobulin heavy chain constant region, other variable domains (e.g., in the production of double antibodies) or protein tags, as discussed in further detail below.

[0082] Although the embodiment shown in the examples comprises a pair of "matched" V H and V L domain, but the skilled person will recognize that alternative embodiments may include only V L or V HAny of the single chain specific binding domains can be used to screen for complementary domains that can form two domain specific antigen binding fragments that can bind, for example, to KLRG1. Screening can be accomplished by phage display screening methods using the so-called hierarchical dual combinatorial approach disclosed in WO92 / 01047, in which a single colony containing either the H chain or the L chain clone is used to infect the entire library of clones encoding the other chain (L or H), and the resulting two chain specific binding domains are selected according to phage display technology as described.

[0083] The anti-KLRG1 antibodies described herein can be linked to another functional molecule, for example, another peptide or protein (albumin, another antibody, etc.), a toxin, a radioisotope, a cytotoxic or cytostatic agent. For example, the antibody can be linked by chemical cross-linking or by a recombinant method. The antibody can also be linked to one of a variety of non-protein polymers in the manner listed in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene. The antibody can be chemically modified by covalent conjugation with a polymer, for example, to increase its circulation half-life. Exemplary polymers and methods of linking them are also shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546.

[0084] The disclosed antibodies may also be altered to have a glycosylation pattern that is different from the natural pattern. For example, one or more sugar moieties may be deleted and / or one or more glycosylation sites may be added to the original antibody. Adding glycosylation sites to the currently disclosed antibodies may be achieved by changing the amino acid sequence to contain a glycosylation site consensus sequence known in the art. Another way to increase the number of sugar moieties on an antibody is by chemical or enzymatic coupling of glycosides to antibody amino acid residues. Such methods are described in WO 87 / 05330 and Aplin et al. (1981) CRC Crit. Rev. Biochem., 22: 259-306. Removal of any sugar moiety from an antibody may be accomplished by chemical or enzymatic methods, for example, as described in Hakimuddin et al. (1987) Arch. Biochem. Biophys., 259: 52; and Edge et al. (1981) Anal. Biochem., 118: 131 and Thotakura et al. (1987) Meth. Enzymol., 138: 350. The antibodies may also be labeled with a detectable or functional label. Detectable labels include radioactive labels such as 131 I or 99Tc, it can also be attached to the antibody using conventional chemical methods. Detectable labels also include enzyme labels, such as horseradish peroxidase or alkaline phosphatase. Detectable labels also include chemical moieties, such as biotin, which can be detected by binding to a specific homologous detectable moiety (e.g., labeled avidin).

[0085] Antibodies whose CDR sequences differ only insubstantial ways from those listed 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 encompassed within the scope of the invention. Typically, amino acids are substituted with related amino acids having similar charge, hydrophobicity or stereochemical characteristics. Such substitutions will be within the ordinary skill of the artisan. Unlike in CDRs, more substantial changes can be made in FRs without adversely affecting the binding properties of the antibody. Alterations to the FRs include, but are not limited to, humanizing a non-human derived framework or engineering certain framework residues that are important for antigen contact or stabilization of the binding site, for example, changing the class or subclass of the constant region, changing specific amino acid residues that may alter effector functions such as Fc receptor binding, for example, 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.

[0086] Those skilled in the art will appreciate that the above modifications are not exhaustive and that many other modifications will be apparent to those skilled in the art based on the teachings of the present disclosure.

[0087] Nucleic Acids, Cloning and Expression Systems

[0088] The present disclosure also provides isolated nucleic acids encoding the disclosed antibodies. The nucleic acids may comprise DNA or RNA, and may be synthesized or recombinant in whole or in part. Unless the context otherwise requires, reference to nucleotide sequences as described herein encompasses DNA molecules with the specified sequence, and encompasses RNA molecules with the specified sequence in which U replaces T.

[0089] The nucleic acids provided herein comprise the CDRs, V H Domain and / or V L Domain coding sequence.

[0090] The present disclosure also provides constructs in the form of plasmids, vectors, phagemids, transcription or expression cassettes comprising at least one CDR, V H Domain and / or V L Domain nucleic acid.

[0091] The present disclosure also provides a host cell comprising one or more of the above constructs.

[0092] Also provided are sequences encoding any CDR (H1, H2, H3, L1, L2 or L3), V H or V L The invention relates to a nucleic acid encoding a domain, and a method for preparing the encoded product. The method comprises expressing the encoded product from the encoding nucleic acid. Expression can be achieved by culturing a recombinant host cell containing the nucleic acid under appropriate conditions. After production by expression, V can be isolated and / or purified using any suitable technique. H or V L The domain or specific binding member is then used appropriately.

[0093] Antigen binding fragment, V H and / or V L Domains as well as encoding nucleic acid molecules and vectors can 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 desired function.

[0094] Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. For cells suitable for producing antibodies, see Gene Expression Systems, Academic Press, Fernandez et al., ed., 1999. In short, suitable host cells include bacteria, plant cells, mammalian cells, and yeast and baculovirus systems. Mammalian cell lines available in the art for expressing heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse myeloma cells, and many other cells. Common bacterial hosts are Escherichia coli. Any protein expression system compatible with the present invention can be used to produce the disclosed antibodies. Suitable expression systems include transgenic animals described in Gene Expression Systems, Academic Press, Fernandez et al., ed., 1999.

[0095] Suitable vectors can be selected or constructed so that they contain appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other appropriate sequences. Where appropriate, the vector can be a plasmid or a virus, such as a phage or a phagemid. For more details, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989. Many known techniques and protocols are described in detail in Current Protocols in Molecular Biology, 2nd edition, Ausubel et al., John Wiley & Sons, 1992, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression and protein analysis to manipulate nucleic acids.

[0096] Another aspect of the present disclosure provides a host cell comprising a nucleic acid as disclosed herein. Another aspect provides a method comprising introducing such a nucleic acid into a host cell. The introduction may be carried out using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and transduction using retrovirus or other viruses (e.g., vaccinia or baculovirus for insect cells). For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. After the nucleic acid is introduced into the cell, expression from the nucleic acid may be caused or allowed, for example, by culturing the host cell under conditions that express the gene.

[0097] How to use

[0098] The disclosed anti-KLRG1 antibodies are capable of modulating KLRG1-related immune response regulation. In specific embodiments, the activation of cytotoxic T cells and NK cells is mediated by modulating KLRG1 signaling. The disclosed antibodies can act as agonists or antagonists of KLRG1, depending on their method of use. The antibodies can be used to prevent, diagnose or treat medical conditions in mammals, particularly humans. The antibodies of the present invention can also be used to isolate KLRG1 or KLRG1-expressing cells. In addition, the antibodies can be used to treat subjects who are at risk of or susceptible to a condition or who are at risk of or susceptible to a condition or who are suffering from a condition associated with abnormal KLRG1 expression or function or who are suffering from the condition.

[0099] The antibodies of the invention are useful in situations where modulation of cytotoxic T cell and NK cell activation may be desirable, for example, in certain types of cancer and infectious diseases.

[0100] When attenuated lymphocyte activation is desired, the anti-KLRG1 antibodies of the invention can be used as agonists of KLRG1 to enhance the attenuation of KLRG1-associated cytotoxic (or CD8+) T cell and NK cell activation.

[0101] In some cases, it may be desirable to elicit or enhance a patient's immune response in order to treat cancer or infectious diseases. Diseases treated or prevented by the disclosed methods include, but are not limited to, infections by microorganisms (e.g., bacteria), viruses (e.g., systemic viral infections such as influenza, viral skin diseases such as herpes or herpes zoster), or parasites; and cancers (e.g., melanoma and prostate cancer).

[0102] Anti-KLRG1 antibodies enhance T cell and NK cell responses to the activation of cytotoxic T cells and NK cells. In such cases, the antibody acts as an antagonist of KLRG1. Therefore, in some embodiments, the antibody can be used to inhibit or reduce the down-regulation activity associated with KLRG1, i.e., the activity associated with the down-regulation of cytotoxic T cells and NK cell activation. As demonstrated in the examples, blocking the interaction of KLRG1 / E-cadherin with antagonistic anti-KLRG1 antibodies leads to enhanced T cell proliferation response and IFNγ secretion of these cells, which is consistent with the down-regulation of KLRG1 pathways in cytotoxic T cells and NK cells. In various embodiments, the antibody inhibits the binding of E-cadherin to KLRG1 with an IC50 of less than about 50nM, and more preferably less than about 40nM, 30nM, 20nM, 10nM or 5nM. The inhibition of E-cadherin binding can be measured as described in Example 6 or using techniques known in the art.

[0103] The antibody or antibody composition of the present invention is administered in a therapeutically effective amount. Generally, the therapeutically effective amount can vary with the age, condition and sex of the subject and the severity of the subject's medical condition. The therapeutically effective amount of the antibody is from about 0.001 mg / kg body weight to about 30 mg / kg body weight, preferably from about 0.01 mg / kg body weight to about 25 mg / kg body weight, from about 0.1 mg / kg body weight to about 20 mg / kg body weight or from about 1 mg / kg body weight to about 10 mg / kg body weight. The dosage can be adjusted as needed to adapt to the observed therapeutic effect. The treating physician selects a suitable dosage based on clinical indications.

[0104] The antibody can be administered as a bolus dose to maximize circulating levels of the antibody for the maximum length of time after administration. A bolus dose followed by a continuous infusion can also be used.

[0105] Immune cells (e.g., T cells or NK cells) can also be isolated from the patient and incubated with the antibodies of the present invention in vitro. In some embodiments, the activation of T cells and NK cells can be regulated by removing immune cells from the subject and contacting the immune cells with the anti-KLRG1 antibodies of the present invention in vitro. In such embodiments, the anti-KLRG1 antibodies can be used in a multivalent form so that the KLRG1 molecules on the surface of the immune cells become "cross-linked" after binding to such antibodies. For example, the anti-KLRG1 antibodies can be bound to a solid support such as beads, or cross-linked by a second antibody. The immune cells can then be isolated using methods known in the art and re-implanted into the patient.

[0106] In another aspect, the antibodies of the invention can be used as targeting agents for delivering another therapeutic agent or cytotoxic agent (e.g., toxin) to cells expressing KLRG1. The method comprises administering an anti-KLRG1 antibody coupled to a therapeutic agent or cytotoxic agent, or administering under conditions that allow the antibody to bind to KLRG1.

[0107] The antibodies of the present invention can also be used to detect the presence of KLRG1 in a biological sample. The amount of KLRG1 detected can be correlated with the expression level of KLRG1, which in turn is correlated with the activation state of immune cells (eg, activated T cells or NK cells) in a subject.

[0108] Detection methods using antibodies are well known in the art and include, for example, ELISA, radioimmunoassay, immunoblot, western blot, immunofluorescence, immunoprecipitation. The antibodies can be provided in a diagnostic kit that combines one or more of these techniques to detect KLRG1. Such a kit may contain other components, packaging, instructions or other materials to aid in the detection of the protein.

[0109] When antibodies are used for diagnostic purposes, it may be desirable to modify them, for example, with ligand groups (such as biotin) or detectable labeling groups (such as fluorescent groups, radioisotopes or enzymes). If desired, antibodies of the present invention 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 tetramethylbenzidine (TMB) into a blue pigment, which can be quantitatively measured with a spectrophotometer. For detection, suitable binding partners include, but are not limited to, biotin and avidin or streptavidin, IgG and protein A, and numerous receptor-ligand pairs known in the art. For those of ordinary skill in the art, other arrangements and possibilities will be apparent and are considered equivalents within the scope of the present invention.

[0110] The antibodies of the present invention can be used in screening methods to identify KLRG1 pathway inhibitors that are effective as therapeutic agents. In such a screening assay, a first binding mixture is formed by combining KLRG1 and the antibodies of the present invention; and the amount of binding (M0) in the first binding mixture 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 (M1) in the second binding mixture is measured. As shown in the examples, the compound to be tested may be another anti-KLRG1 antibody. 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 compared to the first binding mixture, it is considered that the compound or agent is able to modulate the downregulation of the immune response associated with KLRG1. The preparation and optimization of the binding mixture is within the technical level of 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 greater than about 30%, can be identified and then, if desired, screened for the ability to improve the condition in other assays or animal models as described below. The strength of binding between KLRG1 and the antibody can be measured using, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), surface plasmon resonance-based technology (e.g., Biacore), all of which are well known in the art.

[0111] The compound can then be tested in vitro or in an animal model as described in the examples. For example, the preliminary dose is determined according to an animal experiment, and the dose scaling for human administration is performed according to the practice recognized in the art. Toxicity and therapeutic efficacy can be determined by standard drug procedures in cell culture or experimental animals. The data obtained by cell culture assays or animal studies can be used in the dosage range for preparation for humans. The therapeutically effective dose obtained in an animal model can be converted to another animal, including humans, using conversion factors known in the art (see, for example, Freireich et al. (1966) Cancer Chemother. Reports, 50 (4): 219-244).

[0112] Pharmaceutical compositions and methods of administration

[0113] The present disclosure provides compositions comprising anti-KLRG1 antibodies. Such compositions may be suitable for pharmaceutical use and administration to patients. The compositions generally comprise one or more antibodies of the present invention 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, etc. that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. The pharmaceutical composition may also be contained in a container, package or dispenser together with instructions for administration.

[0114] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Methods for accomplishing administration are known to those of ordinary skill in the art. Administration may be, for example, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous or transdermal administration. Compositions that may be topically or orally administered or that may be capable of transmucosal transmission may also be obtained.

[0115] Solutions or suspensions for intradermal or subcutaneous application typically contain one or more of the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Such preparations can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0116] Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be a fluid that is easy to inject. The composition should be stable under manufacturing and storage conditions and must prevent the contamination of microorganisms such as bacteria and fungi during storage. Various antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.) can be used to prevent the action of microorganisms. In many cases, it is preferred to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol and sodium chloride in the composition. The carrier can be a solvent or a dispersion medium, and the solvent or dispersion medium contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) 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 dispersion and / or by the use of surfactants. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0117] Oral compositions typically include an inert diluent or edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For oral administration, the antibody can be combined with an excipient and used in the form of tablets, lozenges or capsules. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar properties; binders such as microcrystalline cellulose, 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 flavorings such as mint, methyl salicylate or orange flavoring.

[0118] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a permeant suitable for the barrier to be penetrated is used in the formulation. Such permeants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be completed, for example, by using lozenges, nasal sprays, inhalants or suppositories; for example, in the case of antibodies comprising Fc parts, the composition may be able to cross the mucosal transmission in the intestine, mouth or lungs (for example, by FcRn receptor-mediated pathways as described in U.S. Patent No. 6,030,613). For transdermal administration, the active compound can be formulated into an ointment, salves, gels or emulsions, as generally known in the art. For inhalation administration, the antibody can 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.

[0119] In certain embodiments, the antibodies disclosed herein are prepared together with a carrier that prevents the rapid elimination of the compound from the body, such as a controlled release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The preparation methods of such preparations are obvious to those skilled in the art. Liposomal suspensions containing the antibodies disclosed herein can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0120] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, the term "dosage unit form" refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0121] The toxicity and therapeutic efficacy of the compositions of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, for determining LD50 (50% population lethal dose) and ED50 (50% population effective therapeutic dose). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the LD50 / ED50 ratio. Compositions that exhibit high therapeutic indices are preferred.

[0122] For any composition used in the present invention, the therapeutically effective dose can be initially estimated by 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 embodiments. Data obtained from cell culture assays and animal studies can be used in the formulation of dosage ranges for humans. Doses can be formulated in animal models to achieve a circulating plasma concentration range including IC50 (i.e., antibody concentrations that achieve half-maximal inhibition of symptoms). Circulating levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dose can be monitored by suitable bioassays. The dose is preferably within the range of circulating concentrations with minimal or no toxicity. The dose may vary depending on the dosage form employed and the route of administration utilized.

[0123] The following examples do not limit the scope of the present invention in any way. Those of ordinary skill in the art will recognize that various modifications and variations 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 disclosed patent applications cited in this application are incorporated herein by reference.

[0124] Example

[0125] Example 1: Production of recombinant protein

[0126] Recombinant proteins were produced by standard molecular cloning, and expression protocols included human KLRG1 ECD (SEQ ID NO: 89), cynomolgus KLRG1 ECD (SEQ ID NO: 90), and human E-cadherin (SEQ ID NO: 91), whose amino acid sequences are shown in Table 6 below. Recombinant proteins were produced as FC fusion proteins or HIS spiked forms by cloning the corresponding cDNA into pCDNA4 vector (Invitrogen) and transiently transfected in mammalian HEK293. Purification of the expressed proteins was performed by chromatography using protein A affinity resin for FC fusion protein forms and nickel-NTA resin for HIS spiked proteins. All purified proteins were characterized by SDS-PAGE electrophoresis to verify purity and molecular weight.

[0127] Table 6: Amino acid sequences of human and cynomolgus monkey KLRG1 ECD and human E-cadherin

[0128]

[0129]

[0130] Stable cell lines were developed as immunizing antigens to test the binding of antibodies to the full-length antigen and as target cells in functional T cell assays. The cell lines developed included CHO expressing full-length human KLRG1 and CHO expressing full-length cynomolgus monkey KLRG1. Stable cell lines were obtained by transfecting CHO cells with pCDNA4 plasmids encoding the protein of interest. After transfection, the cells were exposed to 500ug / ml of G418 to select for stably integrated plasmids. The cell lines were further characterized for expression by FACS and sorted to select for homogeneous and stable expression.

[0131] Stable CHO cells double transfected with CD3 agonist and E-cadherin were used in a functional assay to demonstrate the blocking effect of KLRG1 in human T cells. Co-expression of CD3 agonist and E-cadherin was verified by two-color FACS using appropriate antibodies.

[0132] Example 2: Generation of anti-KLRG1 antibodies

[0133] 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 produced by standard immunization of female BALB / c mice and SJL mice with human and cynomolgus monkey KLRG1, followed by hybridoma screening. Several immunization strategies have been used to produce various antibody hits. In short, SJL and Balb / c mice were repeatedly immunized with cDNA, recombinant antigens or CHO cells expressing the antigen of interest. The titer of antigen-specific antibodies was regularly monitored by ELISA, and animals were killed when the appropriate titer (usually between 1:1000 and 1:10000 dilution factors) was reached. Splenocytes from mice killed were fused with mouse myeloma cells to produce hybridoma cells, which were then cultured and subcloned into single cells. Stable clones were scaled up and conditioned medium was harvested, and the expression of anti-KLRG1 antibodies was tested by ELISA and FACS.

[0134] Example 3: Selection of anti-KLRG1 blocking antibodies

[0135] The antibodies produced by hybridomas were screened for binding to human KLRG1 ECD and cynomolgus KRG1 ECD. Antibodies with cross-reactivity between the two antigens were selected to enter the next screening stage to determine their ability to neutralize the interaction between KLRG1 and E-cadherin. Antibodies were ranked according to their EC50 binding to human and cynomolgus (cyno) KLRG1 and further sorted according to their IC50 in the E-cadherin competition assay. According to these criteria, a total of 48 antibodies were selected. Functional characterization of eight antibodies was preferred in cell-based assays. Table 3 summarizes the variable region amino acid sequences of the selected 8 mouse antibodies, and Table 4 shows the CDR regions of the selected 8 mouse antibodies. Antibodies were further ranked according to the presence of the sequence liability motifs outlined in Table 7. Deamidation sites (NG) were found in the CDR-L1 of ABC_G1N01 and ABC_G1N02, which may affect the stability, manufacturability and activity of the drug material. In order to remove this potential liability, a series of mutants were constructed and tested. Specifically, the NG motif can be replaced by NA, QG and KG sequences without losing binding. Mouse antibodies can be humanized by transplanting the CDR region 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). In addition, conservative mutations can be made to the CDR region to improve affinity, efficacy or biophysical characteristics. Table 5 summarizes a list of conservative mutations that can be made to the CDR region. According to the ability of monoclonal antibodies to compete with each other, they are epitope binned. It was found that antibodies can be divided into 3 different bins; BIN1 includes: ABC_G1N01, ABC_G1N02, ABC_G1N03, ABC_G1N04, ABC_G1N05. BIN2 includes: MAB25, MAB031. BIN3 includes: ABC_G1N07, ABC_G1N08. Chimeric forms of the antibodies were generated 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, as described herein.

[0136] Table 7: Sequence-responsible motifs used to screen antibodies for potential manufacturability issues

[0137]

[0138] Example 4: Characterization of Antibody Binding to KLRG1 (EC50) and Inhibition of KLRG1 / E-cadherin Interaction (IC50) by FACS

[0139] Binding of anti-KLRG1 monoclonal antibodies to cell-expressed 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 in the concentration range of 1-100 nM and measuring the fluorescence of the labeled cells using an anti-mouse detection antibody directly conjugated to a fluorescent probe.

[0140] The ability of monoclonal antibodies to inhibit E-cadherin / KLRG1 binding was measured by FACS. The binding of E-cadherin to CHO-hKLRG1 cells was first determined by incubating KLRG1 expressing cells with recombinant E-cadherin spiked with different concentrations of HIS and detecting it by FACS using an anti-HIS detection antibody. The EC50 for the 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 the concentration of the monoclonal antibody was changed from 0.1 nM to 100 nM. EC50 and IC50 values ​​were calculated using SigmaPlot software and are summarized in Table 8.

[0141] Table 8: Binding EC50 and E-cadherin inhibition IC50 of MABs measured by FACS.

[0142]

[0143] Example 5: Measurement of binding kinetics

[0144] The binding kinetics of humanized antibodies to human and cynomolgus monkey KLRG1 were determined by Measure to determine. The system uses Bio-Layer Interferometry (BLI) technology to directly monitor the binding of proteins and other biomolecules to their partners in real time, providing analysis of kinetic binding constants. 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 involved immobilizing the biotinylated recombinant antigen (human-KLRG-ECD or cynomolgus monkey-KLRG1-ECD) on streptavidin The loaded sensor is then The instrument was exposed to different concentrations of each humanized antibody ranging from 100 nM to 0.1 nM, and data were collected for 600 seconds to measure the association kinetics (K) of the antibody / antigen complex. onIn the next step, the sensor was exposed to 1X phosphate-buffered saline (PBS) buffer solution without antibody for 600 seconds to observe the dissociation kinetics (K off ). Then use The analysis software fits the obtained data to a 1:1 binding kinetic model to calculate K D Table 9 summarizes the kinetic binding parameters of three humanized antibodies obtained by this method.

[0145] Table 9: Pass Measuring the binding affinity of the selected MAB:

[0146]

[0147] Example 6: Characterization of the functional activity of KLRG1 blocking antibodies

[0148] Blocking the interaction of KLRG1 with its ligand has an activating effect on human CD8+ T cells by measuring the effect of antibody-mediated blockade of KLRG1 signaling on IFNγ production and on T cell proliferation (proliferation index). To demonstrate the effect of KLRG1 blockade on immune system cells, CD8+ T cells were isolated from healthy donors and tested in co-culture with a CHO cell line (eAPC) co-expressing a CD3 agonist and E-cadherin. The assay is run by providing 2 competing signals to T cells, in which CD3 stimulation is offset by the inhibitory effect of E-cadherin. When KLRG1 signaling is blocked by anti-KLRG1 antibodies, the inhibitory signal is disrupted and the T cells are activated based on their interaction with the CD3 agonist expressed on the cell surface. IFNγ secretion was measured by ELISA and the results are summarized in Tables 10 and Figure 1 middle.

[0149] Table 10: Restoration of IFNγ release in human CD8+ T cells by KLRG1 blocking antibodies

[0150]

[0151] This data suggests that E-cadherin has an inhibitory effect on human T cells that can be reversed by blocking KLRG1 signaling with a blocking antibody.

[0152] Table 11 and Figure 2The data presented in show that blocking the KLRG1 / E-cadherin interaction leads to the proliferation of human CD8+T cells by measuring the proliferation index in response to eAPC. In this assay, eAPCs were generated by stably transducing CHO cells with hE-cadherin and CD3 agonists. The eAPCs thus generated were then co-incubated with freshly isolated CD8+T cells from healthy volunteers in the presence of 10 micrograms / ml of test antibody or isotype control for 3 days. Positive control samples were prepared by incubating CD8+T cells with CHO cells that stably expressed anti-CD3 but lacked expression of the inhibitory ligand E-cadherin, so that anti-CD3 expressed on CHO cells stimulated CD8+T cells uninhibitedly. The results showed that CD8+T cells proliferated in response to CD3 stimulation as expected, and co-expression of the inhibitory ligand E-cadherin on eAPC inhibited the proliferation of CD8+T cells. In addition, the proliferation of CD8+T cells can be restored by blocking KLRG1 with neutralizing antibodies.

[0153] Table 11: Effect of KLRG1 blockade on CD8+ T cell proliferation

[0154]

[0155] Table 12 and Figure 3 The data presented in show the correlation between T cell activity of anti-KLRG1 neutralizer antibodies in IFNγ secretion assays and the blocking activity of the antibodies. The IC50 values ​​of each antibody are from E-cadherin binding inhibition studies and plotted against the IFNγ production levels measured in CD8+T cell IFNγ release assays. The correlation shows that antibodies with lower IC50 values ​​(e.g., more effective KLRG1 / E-cadherin binding blockers) lead to higher levels of IFNγ release from CD8+T cells. This data suggests that antibody blocking KLRG1 leads to T cell activity recovery in an E-cadherin-dependent manner.

[0156] Table 12: Correlation between E-cadherin IC50 competition values ​​and IFNγ secretion

[0157] mAbs IFNγ (pg / ml) IC50(nM) ABC_G1N07 1512.76 4.21 ABC_G1N02 1484.66 9.6 ABC_G1N01 1365.7 5 MAB034 1201.44 11.2 MAB024 1129.8 15.4 ABC_G1N05 1082.62 12.3 ABC_G1N08 1047.75 23 MAB036 1038.52 10 MAB031 975.34 18 Isotype 799.27 390

Claims

1. An antibody or an antigen-binding fragment thereof, comprising an amino acid sequence as set forth in SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:21, SEQ ID NO:43, SEQ ID NO:49, SEQ ID NO:55, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:73, SEQ ID NO:79 or SEQ ID NO:

85.

2. The antibody of claim 1, 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.

3. The antibody of claim 1, comprising an amino acid sequence substantially as 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, SEQ ID NO: 38, SEQ ID NO: 39, or SEQ ID NO:

40.

4. The antibody of claim 1, comprising an amino acid sequence selected from the group consisting of 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, and SEQ ID NO:

40.

5. The antibody of claim 1, wherein the antibody specifically binds to an amino acid sequence having at least 95% identity to any sequence of at least 100 contiguous amino acids of at least one sequence selected from the group consisting of SEQ ID NO: 89 and SEQ ID NO:

90.

6. The antibody of claim 5, wherein the antibody specifically binds to the extracellular domain of KLRG1 with an affinity expressed as K D Expressed as at least about 2 nM, about 1 nM, about 100 pM, about 10 pM, or about 5 pM.

7. The antibody of claim 5, wherein the antibody inhibits binding of E-cadherin to KLGR1 with an IC50 of less than about 50 nM, 40 nM, 30 nM, 20 nM or 10 nM.

8. The antibody of claim 1, wherein the antibody is humanized.

9. The antibody of claim 1, wherein the antibody is IgG1 or IgG4.

10. The antibody of claim 9, wherein the antibody is IgG 1λ or IgG 1κ .

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