Treatment of T-cell lymphoma

Abstract

The present invention relates to methods for treating T-cell lymphomas using compounds that specifically bind to KIR3DL2 in combination with chemotherapeutic agents, particularly gemcitabine and / or oxaliplatin. The treatment is particularly useful in the treatment of peripheral T-cell lymphomas, such as PTCL-NOS.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 795,194, filed on January 22, 2019, which is incorporated herein by reference in its entirety (including any drawings).

[0003] References to sequence listings

[0004] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a 54KB file named "KIR-9PCT_ST25" created on January 14, 2020. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to use of a KIR3DL2 targeting agent for diagnosing and treating T-cell lymphoma. Background Art

[0006] KIR3DL2 / CD158k is a cell surface receptor expressed on a subset of healthy circulating NK and CD8+ T lymphocytes. However, KIR3DL2 has also been found on the surface of malignant T cells, particularly malignant CD4+ T cells, and has therefore also emerged as a target for the treatment of T-cell lymphomas (TCL).

[0007] TCL expressing KIR3DL2, including cutaneous T-cell lymphomas (CTCL), such as mycosis fungoides (MF) and Sézary Syndrome (SS) (see, e.g., PCT Publications WO2010 / 081890 (Innate Pharma) and WO02 / 50122 (National Institute of Health and Medical Research (INSERM))), and peripheral T-cell non-Hodgkin lymphomas (see, e.g., PCT Publication WO2014 / 128221, Innate Pharma). Although MF and SS are relatively rare, PTCL accounts for 15% to 20% of aggressive lymphomas and 7% to 10% of all non-Hodgkin lymphomas (NHL) in Western countries. It usually occurs in middle-aged to elderly patients, and the presentation characteristics are characterized by 68% of patients suffering from disseminated disease, wherein almost half (45%) of patients present with systemic symptoms, a quarter (25.8%) of patients involve bone marrow (BM), and a third (37%) of patients involve extranodal disease. Despite aggressive therapy, more than half of patients still die from the disease they suffer from. Although some unique disease entities (if treated) have an improved prognosis, the prognosis of many aggressive PTCLs remains relatively unchanged by using second-generation and third-generation chemotherapy regimens, and, for example, for PTCL-NOS, the 5-year overall survival (OS) remains between 25% and 47%.

[0008] Antibodies that specifically bind to KIR3DL2 are well known in the art. Innate Pharmaceuticals, for example, has described numerous anti-KIR3DL2 antibodies against a range of different epitopes on KIR3DL2, such as antibodies 15C11, 19H12, 22B2, 18B10, 12B11, 13H1, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, and 20E9 described in PCT Publication Nos. WO2014 / 044681 and WO2014 / 044686. Positive results of anti-KIR3DL2 antibodies in human patients with mycosis fungoides and Sezary syndrome have been demonstrated by clinical trials at Innate Pharmaceuticals. Specifically, data from refractory SS patients revealed strong clinical activity when treated with repeated administrations of a single agent, IPH4102 (ADCC-mediated anti-KIR3DL2 antibody), as demonstrated by an overall response rate (ORR) of 42.9%, a median duration of response (DoR) of 13.8 months, and a median progression-free survival (PFS) of 11.7 months. In addition, these encouraging results, with few side effects and the ability to deplete malignant KIR3DL2-expressing cells without causing depletion of healthy KIR3DL2-expressing NK and T cells, provide an important advance in the treatment of TCL. However, some patients exhibit progressive disease and / or an inadequate response to initial treatment.

[0009] Therefore, there is a need in the art to improve the benefits to patients suffering from TCL. Summary of the invention

[0010] The present invention arises, inter alia, from the discovery that gemcitabine and oxaliplatin can each cause increased KIR3DL2 expression in malignant T cells and that these agents, when combined with depleting anti-KIR3DL2 antibodies, result in a significant increase in the ability of immune cells (NK cells) to deplete malignant T cells.

[0011] Interestingly, not only did each of gemcitabine and oxaliplatin increase KIR3DL2 expression on TCL cells individually, but the combination of gemcitabine + oxaliplatin caused an even greater increase in KIR3DL2 expression on TCL cells. It was also observed that the triplet of gemcitabine + oxaliplatin + anti-KIR3DL2 antibody resulted in a strong ability of immune cells (NK cells) to deplete malignant T cells compared to the independent agents. Therefore, gemcitabine (or nucleoside analogs) and / or oxaliplatin (or other platinum compounds) can be used to enhance the activity of anti-KIR3DL2 antibodies, or specifically to enhance the ability of anti-KIR3DL2 antibodies to cause lymphocytes (e.g., T cells, NK cells) to eliminate TCL cells.

[0012] The combination of gemcitabine and / or a platinum-based compound with an anti-KIR3DL2 antibody may therefore provide a particularly advantageous treatment for patients with TCL (eg, KIR3DL2-expressing TCL, PTCL, CTCL, SS or MF).

[0013] Gemcitabine Approved as a nucleoside metabolic inhibitor and used for a variety of cancers, including:

[0014] - in combination with carboplatin, for the treatment of advanced ovarian cancer that has relapsed at least 6 months after completing platinum-based therapy;

[0015] -In combination with paclitaxel, for the first-line treatment of metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless anthracyclines are clinically contraindicated;

[0016] - in combination with cisplatin for the treatment of non-small cell lung cancer; and

[0017] -As a single agent for the treatment of pancreatic cancer.

[0018] Oxaliplatin is a platinum-based agent approved for:

[0019] -Adjuvant treatment of stage III colon cancer in patients who have undergone complete resection of the primary tumor; and

[0020] -Treatment of advanced colorectal cancer.

[0021] Thus, in one aspect, the invention provides improved methods of mediating anti-tumor immune responses against malignant T cells expressing KIR3DL2 by using antibodies that bind KIR3DL2 in combination with an agent or treatment (e.g., a chemotherapeutic agent) that induces or increases expression of KIR3DL2 at the surface of malignant T cells.

[0022] In one aspect, the present disclosure provides an improved method for enhancing anti-tumor immune responses by using an antibody capable of binding to KIR3DL2 in combination with a device for inducing or increasing the expression of KIR3DL2 at the surface of malignant T cells. In one embodiment, the device for inducing or increasing the expression of KIR3DL2 is a pharmaceutical composition comprising a chemotherapeutic agent (e.g., a nucleoside analog and / or a platinum agent) and a pharmaceutically acceptable carrier.

[0023] In one aspect, provided herein is an antibody that binds KIR3DL2 for use in treating TCL, wherein the antibody that binds KIR3DL2 is administered in combination with an agent that induces or increases the expression of KIR3DL2 at the surface of malignant T cells.

[0024] In one aspect, provided herein is an agent that induces or increases the expression of KIR3DL2 on the surface of malignant T cells, for use in treating TCL, wherein the agent that induces or increases the expression of KIR3DL2 on the surface of malignant T cells is administered in combination with an antibody that binds to KIR3DL2.

[0025] In one embodiment, a method for treating or preventing TCL cells in an individual is provided, the method comprising administering to the individual: (a) an agent that induces or increases expression of KIR3DL2 on the surface of malignant T cells; and (b) an antibody that binds KIR3DL2.

[0026] In one embodiment, a method of enhancing the anti-tumor effect of an antibody that binds KIR3DL2 is provided, the method comprising administering to an individual an agent that induces or increases the expression of KIR3DL2 at the surface of malignant T cells.

[0027] In one embodiment, the agent or device for inducing or increasing the expression of KIR3DL2 at the surface of malignant T cells is a platinum-based agent. In one embodiment, the agent or device for inducing or increasing the expression of KIR3DL2 at the surface of malignant T cells is a nucleoside metabolic inhibitor, optionally gemcitabine or its analogs or derivatives. In one embodiment, the agent or device for inducing or increasing the expression of KIR3DL2 at the surface of malignant T cells is a combination of a platinum-based agent and a nucleoside metabolic inhibitor (e.g., gemcitabine or its analogs or derivatives).

[0028] In one embodiment, the antibody that binds KIR3DL2 is an antibody that mediates or directs effector cell-mediated lysis of cells (e.g., malignant T cells) expressing KIR3DL2. In one embodiment, the antibody includes an Fc domain that can mediate ADCC and / or ADCP. In one embodiment, the antibody is expressed at the surface of an immune effector cell (e.g., as part of a chimeric antigen receptor on an immune effector cell). Optionally, the antibody is a monospecific or multispecific (e.g., bispecific) antibody that directs ADCC and / or ADCP to cells expressing KIR3DL2. In any aspect, the antibody that binds to a KIR3DL2 polypeptide and can deplete cells expressing KIR3DL2 can be characterized as a composition that can deplete cells expressing KIR3DL2, wherein the composition includes an antibody that binds to a KIR3DL2 polypeptide. In one embodiment, the antibody that binds to KIR3DL2 is lacutamab.

[0029] In one aspect of any of the embodiments herein, the individual can be designated as a human.

[0030] In one embodiment, the anti-KIR3DL2 antibody and the agent or device for inducing or increasing the expression of KIR3DL2 at the surface of malignant T cells are each administered in a therapeutically effective amount.

[0031] In one embodiment, an anti-KIR3DL2 antibody is administered to an individual having cancer in an amount and frequency sufficient to mediate ADCC towards tumor cells expressing KIR3DL2.

[0032] In one embodiment, an agent or device for inducing or increasing expression of KIR3DL2 at the surface of malignant T cells is administered to an individual with cancer in an amount and frequency sufficient to cause increased expression of KIR3DL2 at the surface of malignant T cells.

[0033] In any of the aspects herein, the individual treated in accordance with the present disclosure may be a non-responder or an individual who has experienced a partial or incomplete response following a previous treatment for TCL.

[0034] In other embodiments, a method for predicting or evaluating the efficacy or suitability of an anticancer agent for use in combination with an antibody that binds to a KIR3DL2 polypeptide to treat TCL is provided, the method comprising determining or evaluating (e.g., in vitro) whether the anticancer agent is capable of inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells, wherein determining that the anticancer agent is capable of inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells indicates that the agent can be used in combination with an anti-KIR3DL2 antibody to treat cancer. Determining or evaluating whether an anticancer agent is capable of inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells can include, for example, contacting a malignant T cell (e.g., a tumor cell) expressing KIR3DL2 in vitro with the agent and evaluating the expression of KIR3DL2. In one embodiment, the anticancer agent is a chemotherapeutic agent, optionally a nucleoside analog or a platinum agent.

[0035] These aspects will be more fully described, and additional aspects, features, and advantages will be apparent, in the description of the invention provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Cytotoxicity of anti-KIR3DL2 antibodies against the HUT78 TCL cell line (Sézary's syndrome) was shown, and the anti-tumor activity of anti-KIR3DL2 was enhanced by each of gemcitabine and oxaliplatin, and even by the combination of gemcitabine and oxaliplatin.

[0037] Figure 2 It was shown that gemcitabine and oxaliplatin increased cell surface KIR3DL2 expression in tumor cells. RAJI-KIR3DL2 (48 hours incubation) ( Figure 2, left panel) or Hut 78 (transfected B-NHL cell line; 24 h incubation) ( Figure 2 , right panel) were incubated with increasing doses of gemcitabine and oxaliplatin. The median fluorescence intensity of KIR3DL2 on the surface of tumor cell lines was analyzed by flow cytometry.

[0038] Figure 3 Shown is the survival rate of mice (n=9 per group) transplanted IV with Raji-KIR3DL2 and treated twice weekly with isotype control mAb, IPH4102 (0.3 μg per injection), gemcitabine (50 mg / kg) or oxaliplatin (5 mg / kg), both IPH4102+gemcitabine, or both IPH4102+oxaliplatin. DETAILED DESCRIPTION

[0039] As used herein, "a" or "an" may mean one or more. As used in the claims, when used in conjunction with the word "comprising," the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0040] When "comprising" is used, this may optionally be replaced by "consisting essentially of" or "consisting of.

[0041] Whenever reference is made to an anti-KIR3DL2 binding agent (e.g., antibody) throughout this specification as "treating TCL" or the like, this means: (a) a method of treating TCL comprising the steps of administering (for at least one treatment) an anti-KIR3DL2 binding agent (e.g., in a pharmaceutically acceptable carrier material) to a warm-blooded animal, especially a human, in need of such treatment, in a dosage that allows for the treatment of TCL (therapeutically effective amount), e.g., in a dosage (amount) as specified above and below; (b) use of an anti-KIR3DL2 binding agent for treating TCL, or an anti-KIR3DL2 binding agent for treating TCL. (c) use of an anti-KIR3DL2 binder for the treatment (especially in humans); (c) use of an anti-KIR3DL2 binder for the manufacture of a pharmaceutical preparation for the treatment of TCL, a method of using an anti-KIR3DL2 binder to manufacture a pharmaceutical preparation for the treatment of TCL, the method comprising mixing the anti-KIR3DL2 binder with a pharmaceutically acceptable carrier or a pharmaceutical preparation comprising an effective dose of the anti-KIR3DL2 binder suitable for the treatment of TCL; or (d) any combination of a), b) and c), according to the subject matter of a patent application allowed in the country where the present application is filed.

[0042] As used herein, the term "antibody" refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are designated as one of the following five major classes: IgA, IgD, IgE, IgG and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light chains and heavy chains, respectively. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called "α", "δ", "ε", "γ" and "μ", respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgG is an exemplary antibody class used herein because it is the most common antibody in physiological situations and because it is the easiest to prepare in a laboratory environment. In one embodiment, the antibody is a monoclonal antibody. Humanized, chimeric, human or other suitable antibodies are provided. "Antibody" includes full-length antibodies and any fragment or derivative of any antibody of the antibodies described herein.

[0043] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region generally includes amino acid residues from a "complementarity determining region" or "CDR" (e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991) and / or those residues from a "hypervariable loop" (e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987; 196:901-917), or an analogous system for defining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed by the methods described above by Kabat et al., supra. Phrases such as "Kabat position," "variable domain residue numbering as in Kabat," and "according to Kabat" refer herein to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids, corresponding to shortening or insertion of a FR or CDR of a variable domain. For example, a heavy chain variable domain may include a single amino acid insert after residue 52 of CDR H2 (residue 52a according to Kabat) and an insertion residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc., according to Kabat). The Kabat residue numbering of a given antibody may be determined by comparing the homologous region of an antibody sequence with a "standard" Kabat numbering sequence.

[0044] As used herein, "framework" or "FR" residues mean the region of the antibody variable domains except those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into continuous regions (FR1, FR2, FR3 and FR4) separated by CDRs. The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, for example, from about amino acid (aa) 230 to about aa 450 of a human γ (γ) heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ of human antibodies), or its naturally occurring allotype. Unless otherwise indicated, the generally accepted Kabat amino acid numbering of immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) "Sequences of Protein of Immunological Interest with Immunological Significance", 5th edition, United States Public Health Service, National Institutes of Health, Bethesda, Maryland).

[0045] The term "specifically binds to" means that the antibody can bind to a binding partner, e.g., KIR3DL2, in a competitive binding assay as assessed using a recombinant form of the protein, an epitope therein, or the native protein as present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are further described below and are well known in the art.

[0046] When an antibody is said to "compete" with a particular monoclonal antibody, this means that the antibody competes with the monoclonal antibody in a binding assay using recombinant KIR3DL2 molecules or surface expressed KIR3DL2 molecules. For example, if the test antibody reduces the binding of AZ158, 19H12, 2B12 or 12B11 to KIR3DL2 polypeptide or cells expressing KIR3DL2 in a binding assay, the antibody is said to "compete" with AZ158, 19H12, 2B12 or 12B11, respectively.

[0047] As used herein, the term "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is given by the dissociation constant, Kd, ​​defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K aDefined by 1 / Kd. Methods for determining the affinity of mAbs can be found in: Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); Coligan et al., eds., Current Protocols in Immunology, Green Publishing Associates and Wiley Interscience, New York, (1992, 1993) and Muller, Meth. Enzymol. 92: 589-601 (1983), which are incorporated herein by reference in their entirety. A standard method for determining the affinity of mAbs well known in the art is to use surface plasmon resonance (SPR) screening (e.g., by analysis with a BIAcoreTM SPR analysis device).

[0048] "Determinant" refers to a site of interaction or binding on a polypeptide.

[0049] The term "epitope" refers to an antigenic determinant, and is a region or area on an antigen to which an antibody binds. Protein epitopes may include amino acid residues that are directly involved in binding and amino acid residues that are effectively blocked by specific antigen-binding antibodies or peptides, i.e., amino acid residues within the "footprint" of an antibody. It is the simplest form or smallest structural region on a complex antigen molecule, which can be combined with, for example, an antibody or a receptor. An epitope may be linear or conformational / structural. The term "linear epitope" is defined as an epitope consisting of continuous amino acid residues on a linear sequence (primary structure) of amino acids. The term "conformational or structural epitope" is defined as an epitope consisting of incompletely continuous amino acid residues, and therefore represents a separate portion (secondary, tertiary and / or quaternary structure) of a linear sequence of amino acids that is close to each other by folding the molecule. Conformational epitopes depend on 3-dimensional structures. Therefore, the term "conformational" is usually used interchangeably with "structural".

[0050] With respect to cells expressing KIR3DL2, the terms "depleting," "deplete," or "depletion" mean a process, method, or compound that can kill, eliminate, lyse, or induce such killing, elimination, or lysis (e.g., by inducing ADCC or ADCP), thereby negatively affecting the number of KIR3DL2-expressing cells present in a sample or subject.

[0051] The terms "immunoconjugate," "antibody conjugate," "antibody drug conjugate," and "ADC" are used interchangeably and refer to an antibody conjugated to another moiety (e.g., any non-antibody moiety, therapeutic agent, cytotoxic moiety, or label).

[0052] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, biological macromolecules, or extracts made from biological materials. The term "therapeutic agent" refers to an agent that has biological activity.

[0053] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well known in the art and refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. Nonspecific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

[0054] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0055] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid.

[0056] The term "recombinant" when used with respect to, for example, a cell or a nucleic acid, protein or vector, means that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes not found within the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed or not expressed at all.

[0057] In the present context, the term "an antibody that binds" a polypeptide or epitope refers to an antibody that binds to said determinant with specificity and / or affinity.

[0058] The terms "identity" or "identical" when used in the context of a relationship between sequences of two or more polypeptides refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, with gap alignments (if any) resolved by a particular mathematical model or computer program (i.e., an "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1993. Press), New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0059] The method for determining identity is designed to give the maximum match between the test sequences. The method for determining identity is described in a publicly available computer program. The computer program method for determining the identity between two sequences includes the GCG program package, including GAP (Devereux et al., " Nucleic Acids Research (Nucl.Acid.Res.) " 12,387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., " Journal of Molecular Biology " 215,403-410 (1990)). The BLASTX program can be publicly obtained from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB / NLM / NIH Bethesda, Maryland (Bethesda, Md.) 20894; Altschul et al., the same). The well-known Smith Waterman algorithm (SmithWaterman algorithm) can also be used to determine identity.

[0060] Treatment of T-cell malignancies

[0061] Described herein are methods useful in treating and preventing TCL in an individual by administering an agent that induces or increases KIR3DL2 expression at the surface of malignant T cells to enhance the activity of antibodies that bind to KIR3DL2 polypeptides. The treatment regimens and methods described herein can be used for a variety of T cell lymphomas, particularly CD4+ T cell lymphomas. In any aspect, TCL can be designated as TCL characterized by malignant cells expressing KIR3DL2 at their surface.

[0062] As shown in this article, gemcitabine ( Eli Lilly and Company) can induce or increase KIR3DL2 expression at the surface of malignant T cells. Gemcitabine is a nucleoside analog approved as a chemotherapy in the treatment of solid tumors. Like fluorouracil and other analogs of pyrimidines, the drug replaces one of the building blocks of nucleic acids, in this case cytidine, during DNA replication. Therefore, gemcitabine and its analogs and derivatives can be used to enhance the activity of antibodies that bind to KIR3DL2 polypeptides.

[0063] As shown herein, oxaliplatin can induce or increase KIR3DL2 expression at the surface of malignant T cells. Oxaliplatin and other platinum agents are well known in the art and include, for example, cisplatin, carboplatin, nedaplatin, phenoplatin, picoplatin, satraplatin. Therefore, platinum agents can be used to enhance the activity of antibodies that bind to KIR3DL2 polypeptides.

[0064] The platinum agent and / or gemcitabine agent can be in any suitable configuration or formulation, including, for example, as a free compound or as part of a conjugate, a nanoparticle formulation, encapsulated (e.g., in a liposome), in each case optionally further combined with an additional pharmaceutically active agent.

[0065] Thus, in one aspect, a method for treating an individual with TCL is provided, the method comprising administering to the individual an effective amount of each of: (a) an antibody that binds to a KIR3DL2 polypeptide; and (b) a platinum agent and / or gemcitabine. In one embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide, a platinum agent (e.g., oxaliplatin), and gemcitabine. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and a platinum agent (e.g., oxaliplatin) without administering or using gemcitabine in combination. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and gemcitabine without administering or using oxaliplatin in combination. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and gemcitabine without administering or using oxaliplatin in combination. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and gemcitabine without administering or using a platinum agent in combination.

[0066] In one embodiment, the platinum agent (eg, oxaliplatin) and / or gemcitabine is administered in an amount effective to cause increased expression of the KIR3DL2 polypeptide on the surface of malignant cells.

[0067] In one aspect, a method for increasing expression of a KIR3DL2 polypeptide on the surface of a malignant cell, optionally a TCL cell (e.g., in vitro or in vivo in an individual with TCL) is provided, the method comprising contacting the malignant cell with an effective amount of a platinum agent and / or gemcitabine. In one embodiment, the method further comprises a step of depleting malignant cells expressing KIR3DL2 (e.g., having increased KIR3DL2 expression), wherein the step comprises contacting the malignant cell with an effective amount of an antibody that binds to the KIR3DL2 polypeptide.

[0068] The combination therapies provided herein for treating cancer can include administering: (a) a depleting anti-KIR3DL2 antibody (e.g., an antibody or antibody fragment that binds KIR3DL2, an effector cell that expresses at its surface a chimeric activating receptor (e.g., a T cell receptor) that includes an antibody fragment that binds KIR3DL2); and (b) a platinum agent, optionally oxaliplatin, to treat an individual with a T cell malignancy, optionally a CD4+ T cell malignancy.

[0069] In another embodiment, the combination therapy provided herein for treating cancer comprises administering: (a) a depleting anti-KIR3DL2 antibody (e.g., an antibody or antibody fragment that binds KIR3DL2, an effector cell that expresses at its surface a chimeric activating receptor (e.g., a T cell receptor) comprising an antibody fragment that binds KIR3DL2); and (b) a nucleoside analog, optionally gemcitabine, to treat an individual with a T cell malignancy, optionally a CD4+ T cell malignancy.

[0070] In another embodiment, the combination therapy provided herein for treating cancer comprises administering: (a) a depleting anti-KIR3DL2 antibody (e.g., an antibody or antibody fragment that binds KIR3DL2, an effector cell that expresses at its surface a chimeric activating receptor (e.g., a T cell receptor) comprising an antibody fragment that binds KIR3DL2); and (b) a platinum agent, optionally oxaliplatin; and (c) a nucleoside analog, optionally gemcitabine, to treat an individual with a T cell malignancy, optionally a CD4+ T cell malignancy.

[0071] Provided are methods of treating individuals with TCL, susceptible to TCL, or who have experienced TCL. In one embodiment, TCL is aggressive or advanced TCL (e.g., stage IV, or more generally beyond stage II). In one embodiment, the patient suffers from recurrent or refractory disease. In one embodiment, the patient has a poor prognosis for disease progression (e.g., a poor prognosis for survival), has a poor prognosis for response to therapy, or has progressive or recurrent disease after prior treatment with prior therapy.

[0072] In one embodiment, the TCL is an aggressive T-cell tumor. In one embodiment, the TCL is an aggressive non-cutaneous TCL. In another embodiment, the TCL is an aggressive cutaneous TCL, optionally a primary cutaneous CD4+ small / medium T-cell lymphoma or a primary CD8+ small / medium T-cell lymphoma. In one embodiment, the TCL is a cutaneous T-cell lymphoma (CTCL). In one embodiment, the TCL is a peripheral T-cell lymphoma (PTCL), optionally a non-cutaneous PTCL. PTCL and PTCL-NOS may optionally be designated as a disease other than cutaneous T-cell lymphoma.

[0073] Cutaneous T-cell lymphoma (CTCL) (see the following image) is a group of lymphoproliferative disorders, characterized in that neoplastic T lymphocytes are localized in the skin. In general, CTCL is classified as the type of non-Hodgkin's lymphoma (NHL). The World Health Organization-European Organization for Research and Treatment of Cancer (WHO-EORTC) CTCL classification was reported in Willemze et al. (2005) "Blood (Blood)" 105:3768-3785. WHO-EORTC divides CTCL into those with indolent clinical behavior and those with aggressive subtypes. The third class is a precursor blood tumor of non-T cell lymphoma (CD4+ / CD56+ blood skin tumor, mother cell natural killer (NK) cell lymphoma or B cell derived primary skin tumor). CTCLs that may have indolent clinical behavior include mycosis fungoides (MF) and its variants, primary cutaneous CD30+ lymphoproliferative disorders (e.g., primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis), subcutaneous panniculitis-like T-cell lymphoma (provisional), and primary cutaneous CD4+ small / medium-sized polymorphic T-cell lymphoma (provisional). CTCLs with aggressive clinical behavior include Sezary syndrome (SS), adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, primary cutaneous peripheral T-cell lymphoma, unspecified primary cutaneous invasive epidermotropic CD8+ T-cell lymphoma (provisional), and cutaneous gamma / delta-positive T-cell lymphoma (provisional). The methods disclosed herein can be used to treat each of these conditions.

[0074] The most common CTLCs are MF and SS. Their characteristics are reviewed, for example, in Willemze et al. (2005) Blood 105:3768-3785, the disclosure of which is incorporated herein by reference. In most cases of MF, the diagnosis is made due to its clinical features, disease history, and histomorphological and cytomorphological findings. An additional diagnostic criterion for distinguishing CTCL from inflammatory skin diseases is the demonstration of a dominant T-cell clone in a skin biopsy specimen by molecular assays (e.g., Southern blot, polymerase chain reaction (PCR)). Genetic testing may also be considered. Classic mycosis fungoides is divided into three stages: (1) patches (atrophic or nonatrophic): nonspecific dermatitis, patches on the lower trunk and buttocks; mild / no pruritus; (2) plaques: intensely pruritic plaques, lymphadenopathy, and (3) tumors: prone to ulceration. Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin, lymphadenopathy, palmar and / or plantar hyperkeratosis, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly. For the diagnosis of Sézary syndrome, criteria usually include an absolute Sézary cell count in peripheral blood, an abnormal immunophenotype, loss of T-cell antigens, and / or T-cell clonality as demonstrated by molecular or cytogenetic methods.

[0075] According to the TNM classification, CTCL stages include I, II, III and IV, and, when appropriate, peripheral blood involvement. Peripheral blood involvement with mycosis fungoides or Sézary syndrome (MF / SS) cells is associated with more advanced skin stages, lymph node and visceral involvement, and shortened survival. MF and SS have a formal staging system proposed by the International Society of Cutaneous Lymphoma (ISCL) and the European Organization for Research and Treatment of Cancer (EORTC). See, Olsen et al., (2007) Blood 110 (6): 1713-1722; and Agar et al. (2010) Journal of Clinical Oncology (J. Clin. Oncol.) 28 (31): 4730-4739, the disclosures of which are incorporated herein by reference.

[0076] In one embodiment, TCL is a peripheral T-cell lymphoma (PTCL), optionally a non-cutaneous PTCL. PTCL and PTCL-NOS can optionally be designated as diseases other than Sezary syndrome and mycosis fungoides, which are considered to be different pathologies of the skin T-cell lymphoma. In one embodiment, PTCL is a nodal (e.g., a major or significant nodal) PTCL. Significant nodal PTCLs particularly include PTCL-NOS (peripheral T-cell lymphoma, not otherwise specified), anaplastic large cell lymphoma (ALCL) and angioimmunoblastic T-cell lymphoma (AITL). For example, PTCL can be an aggressive, non-cutaneous, significant nodal PCTL (the disease can additionally have extranodal presentation).

[0077] In one embodiment, the PTCL is an extranodal (eg, primary extranodal) PTCL. For example, the PTCL can be an aggressive, non-cutaneous, extranodal PCTL.

[0078] In one embodiment, the PTCL is adult T-cell leukemia or lymphoma (ATL), such as HTLV+ATL.

[0079] In one embodiment, the PTCL is extranodal NK / T-cell lymphoma, nasal type. In one embodiment, the PTCL is enteropathy-associated T-cell lymphoma.

[0080] In one embodiment, the PTCL is hepatosplenic T-cell lymphoma, optionally hepatosplenic αβ T-cell lymphoma, optionally hepatosplenic γδ T-cell lymphoma.

[0081] In one embodiment, PTCL is anaplastic large cell lymphoma (ALCL), optionally ALK+ALCL, optionally ALK-ALCL. ALK+ALCL generally enjoys a favorable prognosis (93% 5-year survival) with conventional therapy, but ALK-ALCL has a poor prognosis (37%). ALCL is generally characterized by uniform CD30 surface expression. In one embodiment, PTCL is angioimmunoblastic T-cell lymphoma (AITL), optionally cutaneous AITL, optionally primary cutaneous CD4+ small / medium T-cell lymphoma or primary CD8+ small / medium T-cell lymphoma, optionally non-cutaneous AITL.

[0082] In one embodiment, the PTCL is intestinal lymphoma, such as intestinal ALCL.

[0083] In one embodiment, the PTCL is T-cell prolymphocytic leukemia.

[0084] In one embodiment, the PTCL is PTCL-NOS (Peripheral T-cell Lymphoma, Not Otherwise Specified). PTCL-NOS, also known as PCTL-U or PTCL not otherwise specified, is an aggressive lymphoma that is predominantly nodal, but extranodal involvement is common. Most nodal cases are CD4 + and CD8 - , and CD30 may be expressed in large cell variants. Most PTCL-NOS patients present with nodal involvement; however, many extranodal sites (e.g., liver, bone marrow, gastrointestinal tract, skin) may also be involved. Studies typically report a 5-year overall survival of approximately 30%-35% using standard chemotherapy. In the past, many clear entities corresponding to identifiable T-cell tumor subtypes have been described, such as Lennert lymphoma, T-zone lymphoma, polymorphic T-cell lymphoma, and T-immunoblastic lymphoma, but there is still a lack of evidence that these correspond to unique clinicopathological entities. For this reason, the World Health Organization (WHO)'s recent classification of hematopoietic and lymphoid tumors has grouped these under a single broad category of PTCL-NOS / U. Therefore, the designation of PTCL-NOS can exclude certain unique clinicopathologic entities, such as T-cell prolymphocytic leukemia, ATL / adult T-cell leukemia, extranodal NK / T-cell leukemia, nasal type, EATL / enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, ALCL / anaplastic large cell lymphoma, and / or AITL / angioimmunoblastic T-cell lymphoma.

[0085] PTCL diagnostic criteria can be those of standard medical guidelines, for example, according to the World Health Organization (WHO) classification system (see, for example, World Health Organization WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue, 4th edition, Lyon, France: IARC Press, 2008). See also, for example, Foss et al. (2011) Blood 117:6756-6767, the disclosure of which is incorporated herein by reference.

[0086] In one embodiment, TCL is characterized by tumors or tumor cells that do not express significant and / or detectable CD30 polypeptide at their surface (CD30 negative). In other embodiments, TCL is characterized by tumors or tumor cells that express CD30 at their surface (CD30 positive); in one embodiment, the tumor or tumor cells express low levels of CD30 at their surface; in another embodiment, the tumor or tumor cells express high levels of CD30 at their surface. Optionally, CD30 expression is determined by immunohistochemistry.

[0087] In one exemplary aspect, a method of reducing the progression of TCL in a mammalian host (e.g., a human patient) having detectable levels of cancer cells is provided, the method comprising administering an anti-KIR3DL2 antibody, an anti-KIR3DL2 antibody composition, or a related composition (e.g., a nucleic acid encoding an anti-KIR3DL2 antibody, a cellular composition expressing an anti-KIR3DL2 antibody fragment) in an amount sufficient to detectably reduce the progression of a hematological malignancy in the host.

[0088] In one exemplary aspect, a method of treating TCL in an individual who has a poor disease prognosis and / or has relapsed, is resistant or unresponsive to therapy with a first therapeutic agent is provided.

[0089] PTCL (e.g., PTCL-NOS) is generally based on peripheral blood or tissue biopsy of histological features supplemented by detailed immunohistochemistry, flow cytometry, cytogenetics and molecular genetics. Inspection may include, for example, complete blood count and difference, renal and liver function tests, lactate dehydrogenase (LDH), β2 microglobulin, albumin, serum calcium, uric acid, bone marrow biopsy, chest X-ray and computed tomography (CT) scans of chest, abdomen and pelvis. Progress is optionally determined by evaluating the selective clonal expansion of starting cells. The method for detecting cancer and cancer progression can be achieved by any suitable technology, and several examples of the technology are known in the art. The example of suitable technology includes PCR and RT-PCR (e.g., cancer cell-related genes or "markers"), biopsy, imaging technology, karyotyping and other chromosome analysis, immunoassay / immunocytochemical detection technology, histology and / or histopathological determination, cell dynamics research and cell cycle analysis, flow cytometry and physical examination technology (e.g., for physical symptoms).

[0090] Delivery of an anti-KIR3DL2 antibody to a subject (by direct administration as an isolated or purified antibody (e.g., an antibody in solution), as an antibody fragment expressed by a CAR cell, or by expression from a nucleic acid therein, such as from a poxvirus gene transfer vector comprising an anti-KIR3DL2 antibody-encoding nucleic acid sequence) and practice of the other methods herein can be used to reduce, treat, prevent, or otherwise improve any suitable aspect of cancer progression (notably TCL progression). The methods herein can be particularly useful for reducing and / or improving tumor growth (e.g., percentage (tumor cells compared to healthy T cells), number of circulating tumor cells) and any parameters or symptoms associated therewith (e.g., biomarkers). Methods that independently and collectively reduce, prevent, or otherwise improve these aspects of cancer progression are advantageous features.

[0091] In a further aspect, a method of promoting remission of TCL in a mammalian host (eg, a human patient) is provided, the method comprising administering to the host a composition comprising an anti-KIR3DL2 antibody so as to promote remission of TCL in the host.

[0092] In an even further aspect, a method for reducing the risk of developing TCL, reducing the time of onset of cancerous symptoms and / or reducing the severity of TCL diagnosed at an early stage is provided, the method comprising administering to a host a prophylactically effective amount of an anti-KIR3DL2 antibody or related composition so as to achieve a desired physiological effect.

[0093] In a further aspect, a method for increasing the likelihood of survival of a human patient diagnosed with TCL within a relevant time period is provided. In another aspect, a method for improving the quality of life of a TCL patient is provided, the method comprising administering a composition to the patient in an amount effective to improve the quality of life thereof. In a further aspect, the methods described herein can be applied to significantly reduce the number of TCL cells in a vertebrate host, such as to reduce the total number of TCL cells, for example. In a related sense, a method for killing TCL cells of a vertebrate, such as a human cancer patient (e.g., directly or indirectly causing their death) is provided.

[0094] As used herein, adjuvant or combined administration includes administering the compounds simultaneously in the same or different dosage forms or administering the compounds separately (e.g., sequentially). Thus, an antibody that binds KIR3DL2 can be used in combination with a platinum agent and / or gemcitabine. For example, an anti-KIR3DL2 antibody and a platinum agent and / or gemcitabine can be administered simultaneously in a single formulation. Alternatively, an anti-KIR3DL2 antibody and a platinum agent and / or gemcitabine can be formulated for separate administration and administered simultaneously or sequentially.

[0095] A suitable dose of gemcitabine is 800-1200 mg / m 2 The administration of gemcitabine can be repeated every 2 weeks. Alternatively, gemcitabine can be administered on the 1st and 8th day of a 3-week cycle (which can be repeated).

[0096] A suitable dose of oxaliplatin is 75-150 mg / m2 administered intravenously. 2 (e.g., 75 mg / m 2 , 100mg / m 2 , 130mg / m 2 , 75-130mg / m 2 ) dose. Administration of oxaliplatin may be repeated every 2 weeks, for example at 75-100 mg / m 2 (e.g., 75 mg / m 2 , 100mg / m 2 Alternatively, oxaliplatin can be administered once every three weeks (e.g., on days 1 and 8 of a 3-week cycle, where the cycle can be repeated). When oxaliplatin is administered once every three weeks, the dosage range can optionally be higher than twice weekly administration, e.g., for a 3-week regimen, a dosage of 100-150 mg / m 2 (For example, 100 mg / m 2 , 130mg / m 2 ).

[0097] Typically, treatment includes at least one administration cycle (e.g., a period of eight weeks or less), wherein for each of at least one cycle, two, three, or four doses of an anti-KIR3DL2 antibody are administered and at least two, three, or four doses of a platinum agent (e.g., oxaliplatin) and / or gemcitabine are administered.

[0098] Gemcitabine and oxaliplatin can be administered on the same day (eg, Day 1), or gemcitabine can be administered, for example, on the first day (Day 1) followed by oxaliplatin on the second day (Day 2).

[0099] Suitable treatment regimens for treating humans with CTLs (e.g., PTCL, CTCL, MF, or SS) comprise, e.g., administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and gemcitabine, wherein the treatment comprises at least one administration cycle in which (e.g., weekly, every two weeks, every four weeks) at least one dose, optionally at least two doses of the anti-KIR3DL2 antibody is administered and at least one dose, optionally at least two doses of gemcitabine is administered, optionally wherein gemcitabine is administered at about 800-1200 mg / m 2 , optionally about 800-1000 mg / m 2 Optionally, gemcitabine is administered once every 2 weeks, or on days 1 and 8 of repeated 3-week cycles. Optionally, the treatment regimen is characterized by not combining treatment with a platinum agent or oxaliplatin.

[0100] An exemplary treatment regimen for treating a human with MF or SS can comprise, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and gemcitabine, wherein the treatment comprises at least one administration cycle in which at least one dose, optionally at least two doses of the anti-KIR3DL2 antibody is administered (e.g., weekly, every two weeks, every four weeks) and at least one dose, optionally at least two doses of gemcitabine is administered, wherein gemcitabine is administered at about 800-1200 mg / m 2 on weeks 1, 2, and 3 (e.g., on about days 1, 8, and 15) of repeated 4-week cycles. 2 The 4-week cycle may be repeated, for example for a period of 3, 4, 5, 6 or more months. The regimen may be characterized by not using a platinum agent or oxaliplatin for combination therapy.

[0101] A further suitable treatment regimen for treating a human with CTL (e.g., PTCL) comprises, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and oxaliplatin, wherein the treatment comprises at least one dosing cycle in which at least one dose, optionally at least two doses of the anti-KIR3DL2 antibody is administered (e.g., weekly, every two weeks, every four weeks) and at least one dose, optionally at least two doses of oxaliplatin is administered, optionally wherein the oxaliplatin is administered at about 75-130 mg / m 2 Optionally, oxaliplatin is administered once every 2 weeks or once every 3 weeks.

[0102] Another suitable treatment regimen for treating a human with PCTL comprises, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody, gemcitabine, and oxaliplatin, wherein the treatment comprises at least one administration cycle comprising administering:

[0103] - at least one dose, optionally at least two doses of anti-KIR3DL2 antibody (e.g., every week, every two weeks, every four weeks);

[0104] - at least one dose, optionally at least two doses of gemcitabine, optionally wherein gemcitabine is administered at about 800-1000 mg / m 2 optionally wherein gemcitabine is administered once every 2 weeks or on days 1 and 8 of repeated 3 week cycles; and

[0105] - at least one dose, optionally at least two doses of oxaliplatin, optionally wherein oxaliplatin is administered at about 75-130 mg / m 2 In one embodiment, oxaliplatin and gemcitabine are administered once every two weeks, optionally further on the same day. In another embodiment, oxaliplatin and gemcitabine are administered once every three weeks, wherein oxaliplatin is administered on the first day of a three-week period, and gemcitabine is administered on the first and eighth days of a three-week period.

[0106] It should be understood that the administration cycle can be a suitable time period consistent with the administration frequency described herein. For example, the administration cycle can be a time period of 4 weeks, 8 weeks, more than 8 weeks, less than 8 weeks, etc.

[0107] In one embodiment, the anti-KIR3DL2 antibody is administered on the same day as gemcitabine and / or oxaliplatin. In one embodiment, the anti-KIR3DL2 antibody is administered at least one day (e.g., one day, two days, three days) after the administration of gemcitabine. In one embodiment, the anti-KIR3DL2 antibody is administered at least one day (e.g., one day, two days, three days) after the administration of oxaliplatin. In one embodiment, the anti-KIR3DL2 antibody is administered at least one day (e.g., one day, two days, three days) after the administration of both gemcitabine and oxaliplatin (e.g., at least one day after the administration of the compound later).

[0108] Anti-KIR3DL2 antibodies (e.g., humanized 2B12 antibodies referred to as IPH4102 or lacotumab) can advantageously be administered by intravenous infusion at a dose of 1-20 mg / kg body weight, optionally 1-10 mg / kg body weight, or at a fixed dose of 750 mg. Anti-KIR3DL2 antibodies can advantageously be administered 1-4 times per month, for example once a week, once every two weeks, or once every four weeks. In one embodiment, anti-KIR3DL2 antibodies are administered according to a regimen comprising a first phase, during which anti-KIRDL2 antibodies are administered weekly (e.g., four times per week), followed by a second phase, during which anti-KIRDL2 antibodies are administered less than once per week, for example 1-2 times per month, once every two weeks, or once per month. In one embodiment, the anti-KIR3DL2 antibody is administered according to a regimen comprising a first phase during which the anti-KIRDL2 antibody is administered weekly (e.g., four times per week), followed by a second phase during which the anti-KIRDL2 antibody is administered once every two weeks (e.g., at least 2 consecutive administrations every two weeks, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more administrations every two weeks), followed by a third phase during which the anti-KIRDL2 antibody is administered once a month (e.g., at least 2 consecutive administrations every month, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more administrations every month).

[0109] When combined with gemcitabine and oxaliplatin, the treatment method can, for example, be specified to include at least one administration cycle (e.g., a period of eight weeks or less, optionally eight weeks, optionally four weeks), wherein for each of at least one cycle, two, three, or four doses of anti-KIR3DL2 antibody (e.g., humanized 2B12 antibody is administered at a fixed dose of 750 mg at a dose of 1-20 or 1-10 mg / kg body weight) and at least two, three, or four doses of gemcitabine and oxaliplatin are administered. Gemcitabine and oxaliplatin can be specified to be administered on the same day or consecutive days (e.g., on gemcitabine at 800-1000 mg / m 2 Oxaliplatin was administered at a dose of 75-130 mg / m on the day after administration. 2 Administration). The KIR3DL2-binding antibody can be designated for administration on the same day as gemcitabine and / or oxaliplatin, on consecutive days before or after gemcitabine and / or oxaliplatin, or on several days before or after gemcitabine and / or oxaliplatin.

[0110] In the treatment method, the antibody binding to KIR3DL2 and platinum and / or gemcitabine agents can be administered alone, together or sequentially, or in the form of a cocktail. In some embodiments, the antibody binding to KIR3DL2 is administered before the administration of platinum and / or gemcitabine agents. For example, the antibody binding to KIR3DL2 can be administered about 0 to 30 days, optionally 1 to 7 days before the administration of gemcitabine or 0 to 30 days, optionally 1 to 7 days before the administration of platinum agents. In some embodiments, the antibody binding to KIR3DL2 is administered about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day or about 1 to 5 days before the administration of gemcitabine (or platinum agents). In some embodiments, the antibody binding to KIR3DL2 is administered simultaneously with the administration of platinum agents and / or gemcitabine. In some embodiments, an antibody that binds KIR3DL2 is administered after administration of a platinum and / or gemcitabine agent. For example, an antibody that binds KIR3DL2 can be administered about 0 to 30 days, optionally 1 to 7 days after administration of a platinum and / or gemcitabine agent. In some embodiments, an antibody that binds KIR3DL2 is administered about 30 minutes to about 2 weeks, about 30 minutes to about 1 week, about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 6 hours, about 6 hours to about 8 hours, about 8 hours to 1 day, or about 1 to 5 days after administration of a platinum and / or gemcitabine agent.

[0111] In one embodiment, the anti-KIR3DL2 antibody and platinum and / or gemcitabine agents are administered at the following doses:

[0112] (a) 750 mg of anti-KIR3DL2 antibody and (i) 75-130 mg / m 2 Platinum-based agents (e.g., oxaliplatin) or (ii) 800-1200 mg / m 2 of gemcitabine;

[0113] (b) 1-10 mg / kg or 750 mg of anti-KIR3DL2 antibody and 75-130 mg / m 2 Oxaliplatin;

[0114] (c) 1-10 mg / kg or 750 mg of anti-KIR3DL2 antibody and 800-1200 mg / m 2 Gemcitabine;

[0115] (d) 1-10 mg / kg or 750 mg of anti-KIR3DL2 antibody, 75-100 mg / m 2 Oxaliplatin and 800-1000 mg / m 2 Gemcitabine;

[0116] (e) 1-10 mg / kg or 750 mg of anti-KIR3DL2 antibody, 100-130 mg / m 2 Oxaliplatin and 800-1000 mg / m 2 Gemcitabine.

[0117] Also provided are kits, such as a kit comprising:

[0118] (i) a pharmaceutical composition comprising an anti-KIR3DL2 antibody and a pharmaceutical composition comprising gemcitabine;

[0119] (ii) a pharmaceutical composition comprising an anti-KIR3DL2 antibody and a pharmaceutical composition comprising a platinum agent (e.g., oxaliplatin);

[0120] (iii) a pharmaceutical composition containing an anti-KIR3DL2 antibody, a pharmaceutical composition containing a platinum agent (e.g., oxaliplatin), and a pharmaceutical composition containing gemcitabine;

[0121] (iv) a first pharmaceutical composition comprising an anti-KIR3DL2 antibody and instructions for administering the anti-KIR3DL2 antibody together with a platinum agent (e.g., oxaliplatin) and / or gemcitabine;

[0122] (v) a pharmaceutical composition comprising a platinum agent (e.g., oxaliplatin) and / or gemcitabine, and instructions for administering the composition together with an anti-KIR3DL2 antibody;

[0123] (vi) a pharmaceutical composition containing a platinum agent (e.g., oxaliplatin), and instructions for administering the composition with an anti-KIR3DL2 antibody (and optionally additional gemcitabine);

[0124] (vii) a pharmaceutical composition containing gemcitabine, and instructions for administering the composition together with an anti-KIR3DL2 antibody (and optionally an additional platinum agent (eg, oxaliplatin).

[0125] The pharmaceutical composition may optionally be specified to include a pharmaceutically acceptable carrier. Anti-KIR3DL2 antibodies, platinum agents and / or gemcitabine may optionally be specified to be present in a therapeutically effective amount suitable for any method herein, optionally present in an amount that increases the level of KIR3DL2 at the surface of malignant TCL cells. The kit may optionally also include instructions, such as including an administration regimen, to allow a practitioner (e.g., a physician, a nurse, or a patient) to administer the composition contained therein to a patient suffering from cancer (e.g., in a specific CTLC or PTCL disclosed herein). In any embodiment, the kit may also include a syringe.

[0126] Optionally, the kit comprises a plurality of packaged single-dose pharmaceutical compositions, each of which contains an effective amount of an anti-KIR3DL2 antibody and / or a platinum agent (e.g., oxaliplatin) and / or gemcitabine for single administration according to the methods provided above. The instruments or devices necessary for administering the pharmaceutical compositions may also be included in the kit. For example, the kit may provide one or more prefilled syringes or vials containing a certain amount of an anti-KIR3DL2 antibody, a platinum agent, or gemcitabine.

[0127] In one embodiment, the present invention provides a kit for treating TCL in a human patient, the kit comprising:

[0128] (a) a dose of an anti-KIR3DL2 antibody comprising H-CDR1, H-CDR2 and H-CDR3 domains of a heavy chain variable region and L-CDR1, L-CDR2 and L-CDR3 domains of a light chain variable region, wherein the heavy chain variable region has a sequence as shown in any one of SEQ ID NOs: 32 or 48-51, and the light chain variable region has a sequence as shown in SEQ ID NOs: 33 or 43-47; and / or

[0129] (b) a dose of a platinum agent (e.g., oxaliplatin) and / or a dose of gemcitabine; and

[0130] (c) Optionally, instructions for using the anti-KIR3DL2 antibody and / or the platinum agent (eg, oxaliplatin) and / or gemcitabine in any of the methods described herein.

[0131] In one embodiment, treatment comprises administration of an anti-KIR3DL2 antibody, a platinum agent (eg, oxaliplatin), and gemcitabine without administration of any additional anti-cancer agent.

[0132] It should be understood that the treatment methods of the present disclosure may or may not involve a step of characterizing tumor cells for KIR3DL2-expression prior to treatment. In any embodiment, KIR3DL2 expression may be determined by immunohistochemistry.

[0133] In one embodiment, the method comprises: (a) determining whether the individual has TCL, optionally PTCL; and (b) if the individual has TCL, optionally PTCL, determining whether the individual has TCL cells that express a KIR3DL2 polypeptide. Determining that the individual has TCL cells that express a KIR3DL2 polypeptide indicates that the individual can be treated with a treatment method of the present disclosure. Optionally, the method comprises the step of administering a treatment of the present disclosure to the individual.

[0134] In one embodiment, the KIR3DL2 polypeptide is expressed in a large number of tumor cells (e.g., TCL cells) taken from a given patient. For example, KIR3DL2 can be present on at least 30%, 40%, 50%, 60%, 70%, 80% or more of the TCL cells taken from the patient.

[0135] The method may optionally further comprise a step of assessing the level of progression of TCL (staged disease), which allows for assessing the proportion (e.g., percentage) of malignant TCL cells present in a certain body compartment of the patient. According to this method, cells from a biological sample collected from the body compartment are contacted with an anti-KIR3DL2 antibody, and tumor cells (e.g., proportion of cells) expressing a KIR3DL2 polypeptide at their surface are measured. The cells may be, for example, CD4+ cells or CD4-CD8+ cells. The finding that the tumor cells express or predominantly express KIR3DL2 indicates that the TCL is an aggressive or advanced TCL (e.g., stage IV, or more generally beyond stage II). Such TCL can advantageously be treated using the methods of treatment disclosed herein.

[0136] In any embodiment, the method may include a step of TCL diagnosis, comprising contacting cells from a biological sample of an individual with an anti-KIR3DL2 antibody, and measuring the proportion (e.g., percentage) of T cells expressing KIR3DL2 polypeptides at their surfaces, and comparing this proportion with the average proportion (e.g., percentage) of T cells expressing KIR3DL2 polypeptides at their surfaces observed in non-TCL humans (e.g., in healthy humans), wherein when the measured proportion is significantly higher than the average proportion, a positive diagnosis of TCL is made.

[0137] In another embodiment, the method includes the step of determining the expression level of a KIR3DL2 nucleic acid or polypeptide in a biological sample from an individual who has received administration of a platinum agent or gemcitabine, such as on tumor cells found in the biological sample.

[0138] In another embodiment, the method includes the step of determining whether an anti-cancer agent (eg, a platinum agent or gemcitabine) increases the expression level of a KIR3DL2 nucleic acid or polypeptide in a biological sample from an individual, such as on tumor cells found in the biological sample.

[0139] Determining the expression level of a KIR3DL2 nucleic acid or polypeptide in a biological sample can include comparing the level to a reference level (e.g., a value, weak cell surface staining, etc.) corresponding to a healthy individual or to an individual prior to treatment with an anti-cancer agent (e.g., a platinum agent or gemcitabine). Determining that the biological sample expresses a KIR3DL2 nucleic acid or polypeptide at an increased level compared to the reference level indicates that the individual has a TCL that may benefit from treatment with an anti-KIR3DL2 antibody, or optionally the individual has a TCL that may benefit from treatment with an anti-KIR3DL2 antibody in combination with an anti-cancer agent (e.g., a platinum agent and / or gemcitabine, respectively). Optionally, detecting a KIR3DL2 polypeptide in a biological sample comprises detecting a KIR3DL2 polypeptide expressed on the surface of a malignant lymphocyte.

[0140] Antibody production

[0141] KIR3DL2 (CD158k) is a disulfide-linked homodimer of a three-Ig domain molecule of approximately 140 kD, described in Pende et al. (1996) J. Exp. Med. 184:505-518, the disclosure of which is incorporated herein by reference. Several allelic variants have been reported for the KIR3DL2 polypeptide, each of which is encompassed by the term KIR3DL2. The amino acid sequence of mature human KIR3DL2 (allele *002) is shown below in SEQ ID NO: 1, corresponding to Genbank accession number AAB52520, with the 21 amino acid residue leader sequence omitted.

[0142]

[0143] The cDNA of KIR3DL2 (allele *002) is shown in Genbank accession number U30272. The amino acid sequence of human KIR3DL2 allele *003 is shown below, corresponding to Genbank accession number AAB36593:

[0144]

[0145]

[0146] Also encompassed are any nucleic acid or protein sequences that share one or more biological properties or functions with wild-type, full-length KIR3DL2 and share at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more nucleotide or amino acid identity, respectively.

[0147] The closely related KIR3DL1 (CD158e1) is a monomeric molecule of approximately 70 kD, which is described in Colonna and Samaridis (1995) Science 268 (5209), 405-408. The cDNA encoding the KIR3DL1 (CD158e2) polypeptide (allele *00101) is shown in Genbank Accession No. L41269; the encoded amino acid sequence is shown in Genbank Accession No. AAA69870. In one embodiment, the KIR3DL1 polypeptide referred to herein is allele *00101.

[0148] Examples of antibodies that bind to human KIR3DL2 include antibodies 19H12, 12B11, 10F6, 2B12, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9. These and additional antibodies are provided in PCT / EP2013 / 069302 and PCT / EP2013 / 069293, both filed on September 17, 2013, the disclosures of which are incorporated herein by reference. These antibodies selectively bind to KIR3DL2 and do not bind to KIR3DL1 (or KIR3DS1). While antibodies 10F6, 2B12, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 can be used as therapeutic agents, e.g., administered to an individual, for depletion of targets expressing KIR3DL2, e.g., by inducing ADCC towards pathogenic KIR3DL2-expressing cells, antibodies 12B11 and 19H12 will be advantageously used for detecting (e.g., in vitro assays) KIR3DL2 expression on the surface of cells because 12B11 and 19H12 are particularly effective for detecting KIR3DL2-positive cells in detection assays, 12B11 being advantageous for immunohistochemical assays using frozen tissue sections, and 19H12 being advantageous for flow cytometry assays.

[0149] Each of 2B12, 10G5, 19H12 and 12B11 is also suitable for use as a therapeutic agent administered to an individual for eliminating target cells expressing KIR3DL2. 19H12 and 12B11, as well as other antibodies disclosed in PCT / EP2013 / 069293, are capable of internalization into cells by KIR3DL2 and can be advantageously used as depleting antibodies configured as antibody-drug conjugates, for example when the antibody is conjugated to a toxic moiety. 2B12 and other antibodies disclosed in PCT / EP2013 / 069302 do not induce any KIR3DL2 internalization into tumor cells, thereby providing advantageous uses when effector cell-mediated activities are sought, such as for depletion of antibodies that induce ADCC.

[0150] The amino acid sequences of the heavy chain variable region and the light chain variable region of antibody 10F6, 9E10, 10G5, 13H1, 1E2, 1C3 or 20E9 are listed in Table A.

[0151] Table A

[0152]

[0153] In a specific embodiment, an antibody is provided that binds to substantially the same epitope or determinant as any of the monoclonal antibodies AZ158, 19B12, 10G5, 12B11, 10G5 or 2B12; optionally, the antibody comprises an antigen binding region of the antibody AZ158, 10G5, 19B12, 12B11 or 2B12. In any embodiment herein, the antibody AZ158, 19B12, 12B11, 10G5 or 2B12 can be characterized by its amino acid sequence and / or the nucleic acid sequence encoding it. In one embodiment, the monoclonal antibody comprises a Fab or F(ab') of AZ158, 19B12, 12B11, 10G5 or 2B12. 2Part. A monoclonal antibody is also provided, comprising a heavy chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. According to one embodiment, the monoclonal antibody comprises three CDRs of the heavy chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. A monoclonal antibody is also provided, wherein the monoclonal antibody further comprises one, two or three of the CDRs of the variable light chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12 or the light chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. Optionally, any one or more of the light chain or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (e.g., substitutions, insertions or deletions). Optionally, an antibody is provided in which any light chain and / or heavy chain variable region comprising part or all of the antigen binding region of antibody AZ158, 19B12, 12B11, 10G5 or 2B12 is fused to an immunoglobulin constant region of the human IgG type, optionally a human constant region, optionally of the human IgG1 or IgG3 isotype.

[0154] The antibody (or antibody fragment) for treating TCL binding KIR3DL2 can be, for example, in the form of an isolated and / or purified protein composition, or the antibody can be present on the surface of a cell (e.g., CAR effector cell, such as T cell, NK cell or NKT cell) or in combination with it, or still further in the form of a nucleic acid encoding an antibody, such as from a poxvirus gene transfer vector or other viral gene transfer vectors including anti-KIR3DL2 antibody-encoding nucleic acid sequences. Cells expressing chimeric antigen receptors (CARs) can be constructed. Examples of CARs are engineered to include an extracellular single-chain antibody (scFv) fused to the intracellular signaling domain of the ζ chain of the T cell antigen receptor complex, and when expressed in effector cells, such as T cells, NKT cells or NK cells, have the ability to specifically redirect antigen recognition (i.e., KIR3DL2 recognition) based on monoclonal antibodies. In one aspect, a genetically engineered immune cell is provided, which expresses and carries a KIR3DL2-specific chimeric immune receptor on the cell surface membrane, the chimeric immune receptor comprising an intracellular signaling domain, a transmembrane domain (TM), and a KIR3DL2-specific extracellular domain (e.g., a domain derived from a variable heavy chain region and a light chain region of a monoclonal antibody that specifically binds to KIR3DL2, e.g., one of the antibodies disclosed herein). Also provided are a KIR3DL2-specific chimeric immune receptor, a DNA construct encoding the receptor, and a plasmid expression vector containing the construct in an appropriate orientation for expression.

[0155] In one embodiment, antibodies that bind KIR3DL2 include antibodies or antibody fragments that direct ADCC, and optionally additionally ADCP, toward cells expressing KIR3DL2.

[0156] In one embodiment, the antibody used in any of the embodiments herein binds to a KIR3DL2 polypeptide, optionally wherein the antibody does not substantially bind to a KIR3DL1 polypeptide, characterized by a binding affinity (K affinity) for a human KIR3DL2 polypeptide. D ) is less than (better than) 100 ng / ml, optionally between 1 and 100 ng / ml.

[0157] The antibody is optionally characterized by lysis of HuT78 tumors by PBMC from healthy volunteers. 51 EC in Cr-release assay 50 Less than 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 1 and 50 ng / ml, optionally between 25 and 75 ng / ml, optionally about 50 ng / ml. The antibody is optionally characterized by: lysis of HuT78 tumors by PBMCs from healthy volunteers 51 EC in Cr-release assay 50 EC with the anti-KIR3DL2 antibodies disclosed herein 50 Equivalent (e.g., its EC 50 Lower than the EC of the 2B12 antibody disclosed herein 50 The 2B12 antibody has a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 44 or 45, includes a wild-type or modified Fc domain of human IgG1 isotype, and mediates ADCC.

[0158] Antibody AZ158

[0159] AZ158 binds to human KIR3DL2 and human KIR3DL1 polypeptides (see PCT Patent Publication No. WO2010 / 081890). The VH of AZ158 is shown below, with CDR1, 2 and 3 underlined respectively:

[0160] QVQLKESGPG LVAPSQSLSI TCTVS GFSLTSFGVH WVRQP PGKGLEWLG V IWAGGSTNYN SALMS RLSIS KDNSKSQVFL KMNSLQNDDT AMYYCAR GNS NHYVSSFYYF DY WGQGTTLT VSS

[0161] (SEQ ID NO:3).

[0162] The VL of AZ158 is shown below, with CDR1, 2, and 3 underlined:

[0163] DIQMTQSPSS LSASLGGKVT ITC KASQDINKYIA WYQHKP GKGPRLLIH Y TSTLQP GIPSRFSGSGSGRD YSFSISNLEP EDITTYYC LQ FGGG TKLEIK

[0164] (SEQ ID NO:4).

[0165] The anti-KIR3DL2 antibody may comprise an antibody having a variable region or CDR sequence from such an AZ158 antibody (e.g., a heavy chain and / or light chain variable region fused to a human constant region; a heavy chain variable region fused to a human IgG1 heavy chain constant region); alternatively, the anti-KIR3DL2 antibody may be an antibody other than an antibody having a variable region or CDR sequence from an AZ158 antibody.

[0166] Antibody 19H12

[0167] The amino acid sequence of the heavy chain variable region of antibody 19H12 is listed below:

[0168] QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDMATYFCARNGNFGYYFDYWGQGTTLTVSS

[0169] (SEQ ID NO:5).

[0170] The amino acid sequence of the light chain variable region of antibody 19H12 is listed below:

[0171] DVLMTQTPLSLPVSLGDQASSFSCRSSQNIVHSNGNTYLEWYLQKPGQSPSLLIYKVSNRFSGVPDRFSGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVPFTFGSGTKLEIK

[0172] (SEQ ID NO:6).

[0173] In one aspect, a purified polypeptide encoding an antibody is provided, wherein the antibody comprises: a HCDR1 region comprising the amino acid sequence GYTFTNFGMN as shown in SEQ ID NO:9 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., NFGMN (SEQ ID NO:7), GYTFTN (SEQ ID NO:8)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR2 region comprising the amino acid sequence WINTYTGEPTYADDF as shown in SEQ ID NO:10 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINTYTGE (SEQ ID NO:11)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR3 region comprising the amino acid sequence WINTYTGEPTYADDF as shown in SEQ ID NO:10 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINTYTGE (SEQ ID NO:11)), wherein one or more of these amino acids can be substituted with a different amino acid; The amino acid sequence NGNFGYYFDY as shown in SEQ ID NO:12 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by a different amino acid; a LCDR1 region comprising the amino acid sequence RSSQNIVHSNGNTYLE as shown in SEQ ID NO:13 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by a different amino acid; a LCDR2 region comprising the amino acid sequence KVSNRFS as shown in SEQ ID NO:14 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by a different amino acid; and / or a LCDR3 region comprising the amino acid sequence RSSQNIVHSNGNTYLE as shown in SEQ ID NO:15 The amino acid sequence FQGSHVPFT shown in NO:15 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids may be deleted or substituted by different amino acids, or wherein the sequence may include an insertion of one or more amino acids.

[0174] In another aspect, an antibody that binds to human KIR3DL2 is provided, the antibody comprising:

[0175] (a) the heavy chain variable region of SEQ ID NO: 5, wherein one, two, three or more amino acid residues may be substituted by different amino acids; and / or

[0176] (b) a light chain variable region of SEQ ID NO: 6, wherein one, two, three or more amino acid residues may be substituted by different amino acids; and / or

[0177] (c) a heavy chain variable region of SEQ ID NO:5, in which one, two, three or more amino acid residues may be substituted with different amino acids; and a light chain variable region of SEQ ID NO:6, in which one or more of these amino acids may be substituted with different amino acids; and / or

[0178] (d) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NOs: 7-9, 10-11 and 12, respectively, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0179] (e) the light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NO: 13, 14 or 15, respectively, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0180] (f) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NO:7, 8 or 9, 10 or 11 and 12, respectively, wherein one, two, three or more amino acid residues of any CDR can be substituted by different amino acids; and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NO:13, 14 or 15, wherein one, two, three or more amino acid residues can be substituted by different amino acids; and / or

[0181] (g) a heavy chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the variable region having the amino acid sequence of SEQ ID NO:5, wherein one, two, three or more amino acid residues may be substituted by a different amino acid; and / or

[0182] (h) a light chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the variable region having the amino acid sequence of SEQ ID NO:6, wherein one, two, three or more amino acid residues may be substituted by a different amino acid.

[0183] Antibody 12B11

[0184] The amino acid sequence of the heavy chain variable region of antibody 12B11 is listed below:

[0185] QLVQSGPELKNPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCAHGPWLAYWGQGTLVTVS

[0186] (SEQ ID NO: 16).

[0187] The amino acid sequence of the light chain variable region of antibody 12B11 is listed below:

[0188] DIKMTQSPSSMYASLGERVTITCKASQDINVYLSWFQQKPGKSPKTLIYRAIRLVDGVPSRFSGSGSGQDYSLTISSLDYEDMGIYYCLQYDELPYTFGGGTKLEIE

[0189] (SEQ ID NO: 17).

[0190] In one aspect, a purified polypeptide encoding an antibody is provided, wherein the antibody comprises: a HCDR1 region comprising the amino acid sequence GYTFTNYGMN as shown in SEQ ID NO: 20 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., NYGMN (SEQ ID NO: 18), GYTFTN (SEQ ID NO: 19)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR2 region comprising the amino acid sequence WINTYTGEPTYADDFKG as shown in SEQ ID NO: 21 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINTYTGEPT (SEQ ID NO: 22)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR3 region comprising the amino acid sequence WINTYTGEPTYADDFKG as shown in SEQ ID NO: 23 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINTYTGEPT (SEQ ID NO: 24)), wherein one or more of these amino acids can be substituted with a different amino acid; The amino acid sequence GPWLAY as shown in SEQ ID NO:23 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by different amino acids; the LCDR1 region, which includes the amino acid sequence KASQDINVYLS as shown in SEQ ID NO:24 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by different amino acids; the LCDR2 region, which includes the amino acid sequence RAIRLVD as shown in SEQ ID NO:25 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted by different amino acids; the LCDR3 region, which includes the amino acid sequence LQYDELPYT as shown in SEQ ID NO:26 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be deleted or substituted by different amino acids.

[0191] In another aspect, an antibody that binds to human KIR3DL2 is provided, the antibody comprising:

[0192] (a) the heavy chain variable region of SEQ ID NO: 16, wherein one, two, three or more amino acid residues may be substituted by different amino acids; and / or

[0193] (b) the light chain variable region of SEQ ID NO: 17, wherein one, two, three or more amino acid residues may be substituted by different amino acids; and / or

[0194] (c) the heavy chain variable region of SEQ ID NO: 16, wherein one or more amino acid residues may be substituted by a different amino acid; and the light chain variable region of SEQ ID NO: 17, wherein one, two, three or more of the amino acids may be substituted by a different amino acid; and / or

[0195] (d) the heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NO: 18, 19 or 20, 21 or 22 and 23, respectively, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0196] (e) light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 24, 25 and 26, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0197] (f) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NO: 18, 19 or 20, 21 or 22 and 23, respectively, wherein one or more amino acid residues of any CDR can be substituted by a different amino acid; and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NO: 24, 25 and 26, wherein one, two, three or more amino acid residues of any CDR can be substituted by a different amino acid; and / or

[0198] (g) a heavy chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the variable region having the amino acid sequence of SEQ ID NO: 16, wherein one, two, three or more amino acid residues may be substituted by a different amino acid; and / or

[0199] (h) a light chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to the variable region having the amino acid sequence of SEQ ID NO: 17, wherein one, two, three or more amino acid residues may be substituted by a different amino acid.

[0200] Antibody 2B12

[0201] The amino acid sequence of the heavy chain variable region of antibody 2B12 is listed below (Kabat-defined CDRs are underlined):

[0202] QIQLVQSGPELKKPGETVRISCKASGYTFT TAGMQ WVQKTPGKGLKWIG WINSHSGVPKYAEDFKG RFAFSLETSASTAYLQISTLKNEDTATYFCAR GGDEGVMDY WGQGTSVTVS

[0203] (SEQ ID NO:32).

[0204] The amino acid sequence of the light chain variable region of antibody 2B12 is listed below (Kabat CDRs are underlined):

[0205] DIVMTQSHKFMSTSLGDRVSFTC KASQDVSTAVA WYQQKPGQSPKLLIY WTSTRHT GVPDRFTGSGSGTDYTLTISSVQAEDLALYYC QQQHYSTPWT FGGGTKLEIK

[0206] (SEQ ID NO:33).

[0207] In one aspect, a purified polypeptide encoding an antibody is provided, wherein the antibody comprises: a HCDR1 region comprising the amino acid sequence GYTFTTAGMQ as shown in SEQ ID NO: 36 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., GYTFTT (SEQ ID NO: 34) or TAGMQ (SEQ ID NO: 35)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR2 region comprising the amino acid sequence WINSHSGVPKYAEDFKG as shown in SEQ ID NO: 37 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINSHSGVP (SEQ ID NO: 38)), wherein one or more of these amino acids can be substituted with a different amino acid; a HCDR3 region comprising the amino acid sequence WINSHSGVPKYAEDFKG as shown in SEQ ID NO: 39 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g., WINSHSGVP (SEQ ID NO: 34)), wherein one or more of these amino acids can be substituted with a different amino acid; The amino acid sequence GGDEGVMDY as shown in SEQ ID NO:39 (optionally further comprising a tryptophan residue) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted with different amino acids; a LCDR1 region comprising the amino acid sequence KASQDVSTAVA as shown in SEQ ID NO:40 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted with different amino acids; a LCDR2 region comprising the amino acid sequence WTSTRHT as shown in SEQ ID NO:41 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted with different amino acids; a LCDR3 region comprising the amino acid sequence WTSTRHT as shown in SEQ ID NO:42 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids can be substituted with different amino acids; The amino acid sequence QQHYSTPWT shown in NO:42 or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, wherein one or more of these amino acids may be deleted or substituted by a different amino acid.

[0208] In another aspect, an antibody that binds to human KIR3DL2 is provided, the antibody comprising:

[0209] (a) the heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NO:34, 35 or 36 (HCDR1), 37 or 38 (HCDR2) and 39 (HCDR3), respectively, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0210] (b) light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 40, 41 and 42, wherein one, two, three or more amino acid residues in any CDR may be substituted by a different amino acid; and / or

[0211] (c) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NO:34, 35 or 36 (HCDR1), 37 or 38 (HCDR2) and 39 (HCDR3), respectively, wherein one or more amino acid residues of any CDR may be substituted by a different amino acid; and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NO:40, 41 and 42, wherein one, two, three or more amino acid residues of any CDR may be substituted by a different amino acid; and / or

[0212] (d) a heavy chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having an amino acid sequence of SEQ ID NO: 32 or 48-51, wherein one, two, three or more amino acid residues may be substituted by a different amino acid; and / or

[0213] (e) a light chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having an amino acid sequence of SEQ ID NO:33 or 43-47, wherein one, two, three or more amino acid residues may be substituted by a different amino acid.

[0214] In another aspect of any of the embodiments herein, any CDR1, 2 and 3 of the heavy and light chains can be characterized by a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids, and / or an amino acid sequence having at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with the specific CDR or the corresponding CDR group listed in the SEQ ID NO.

[0215] In another aspect, an antibody is provided that competes with the monoclonal antibodies of (a) to (h) for KIR3DL2 binding against any of the above antibodies.

[0216] Humanized Antibodies

[0217] In another aspect, the anti-KIR3DL2 antibody used in the therapeutic methods of the invention is a humanized antibody, such as a humanized 2B12 or 10G5 antibody having VH and VL variable regions selected from the 2B12 and 10G5 variable regions shown in Table B below.

[0218] For example, a humanized 2B12 antibody may include:

[0219] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, 35 or 36;

[0220] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37 or 38;

[0221] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39;

[0222] (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:40;

[0223] (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:41;

[0224] (f) CDR-L3 comprising the amino acid sequence of SEQ ID NO:42; and

[0225] (g) Human framework sequences.

[0226] In one embodiment, the humanized antibody comprises a heavy chain framework from human subgroup VH1 and / or VH7 together with JH6, optionally the antibody comprises IGHV7-4-1*02 and / or IGHV1-c*01 together with IGHJ6*01. In one embodiment, the humanized antibody comprises a light chain framework from human subgroup VK1 and / or VK4 (optionally IGKV4-1*01 and / or IGKV1-39*01) together with JH4 (optionally IGKJ4*01).

[0227] Optionally, for example, the human framework includes one or more mutations, such as back mutations.

[0228] On the other hand, the humanized antibody comprises a VH domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or higher identity) with the VH domain of humanized 2B12 of SEQ ID NOs: 48-51. In another specific aspect, the humanized antibody comprises: (a) a VH domain comprising non-human CDR residues incorporated into a human VH domain, wherein the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 2B12 VH of SEQ ID NOs: 48-51; and (b) a VL domain comprising non-human CDR residues incorporated into a human VL domain, wherein the VL domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 2B12 VL of SEQ ID NOs: 43-47.

[0229] On the other hand, the humanized antibody includes a VH domain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or higher identity) with the VH domain of humanized 10G5 of SEQ ID NOs: 57-61. In another specific aspect, the humanized antibody includes: (a) a VH domain including non-human CDR residues incorporated into a human VH domain, wherein the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 10G5 VH of SEQ ID NOs: 57-61; and (b) a VL domain including non-human CDR residues incorporated into a human VL domain, wherein the VL domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 10G5 VL of SEQ ID NOs: 52-56.

[0230] The 10G5 or 2B12 antibody may further include a native or engineered human IgG constant domain. Optionally, the constant domain is an IgG1 domain, optionally further including modifications to increase Fc receptor binding.

[0231] Table B

[0232]

[0233]

[0234] In one embodiment, the humanized 2B12 monoclonal antibody comprises:

[0235] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49; and

[0236] (b) a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 44.

[0237] In one embodiment, the humanized 2B12 monoclonal antibody comprises:

[0238] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49; and

[0239] (b) a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 45.

[0240] In one embodiment, the humanized 10G5 monoclonal antibody comprises:

[0241] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 58; and

[0242] (b) a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 53.

[0243] In one embodiment, the humanized 10G5 monoclonal antibody comprises:

[0244] (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 59; and

[0245] (b) a light chain variable region, which comprises the amino acid sequence of SEQ ID NO: 53.

[0246] In one embodiment, the humanized 2B12 antibody is or includes the heavy chain and light chain amino acid sequences of antibody IPH4102 (Innet Pharmaceuticals, France) (also known as lactumab) (see WHO Drug Information, Vol. 32, No. 4, 2018). Lactumab is a humanized 2B12 antibody (having Kabat heavy and light chain CDR1, 2 and 3 of 2B12) and includes a heavy chain variable region and a light chain variable region, the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 49, and the light chain variable region includes the amino acid sequence of SEQ ID NO: 44.

[0247] While it will be appreciated that any suitable antibody may be used, in one aspect, the antibody used binds to the same region or region as a known anti-KIR3DL2 antibody (including any anti-KIR3DL2 antibody described herein). The anti-KIR3DL2 antibody may bind to an epitope that at least partially overlaps with or includes at least one residue in a segment corresponding to residues 1-192, residues 1-98, or residues 99-192 of a KIR3DL2 polypeptide of SEQ ID NO: 1 (or a subsequence thereof). In one embodiment, all of the key residues of the epitope are located in a segment corresponding to residues 1-192, residues 1-98, or residues 99-192 of a KIR3DL2 polypeptide of SEQ ID NO: 1. In one embodiment, the antibody binds to an epitope that includes 1, 2, 3, 4, 5, 6, 7 or more residues in a segment corresponding to residues 1-192, 1-98, or 99-192 of a KIR3DL2 polypeptide of SEQ ID NO: 1. Preferably, the residues bound by the antibody are present on the surface of the KIR3DL2 polypeptide.

[0248] Optionally, the antibody binds to an epitope comprising residue P179 and / or residue S181 of SEQ ID NO: 1. Optionally, the antibody binds to an epitope comprising 1, 2, 3, 4, 5, 6 or 7 or more residues selected from the group consisting of N99, H100, E130, H131, F132, V178, P179, H180, S181, P182, Y183 and / or residue Q184 of SEQ ID NO: 1.

[0249] PCT / EP2013 / 069302 and PCT / EP2013 / 069293 describe a series of tests for mutant human KIR3DL2 polypeptides. The binding of anti-KIR3DL2 antibodies to cells transfected with KIR3DL2 mutants was measured and compared to the ability of the anti-KIR3DL2 antibodies to bind to wild-type KIR3DL2 polypeptides (SEQ ID NO: 1). As used herein, reduced binding between an anti-KIR3DL2 antibody and a mutant KIR3DL2 polypeptide means a decrease in binding affinity (e.g., as measured by known methods, such as FACS testing of cells expressing a specific mutant, or by Biacore testing of binding to a mutant polypeptide) and / or a decrease in the total binding capacity of the anti-KIR3DL2 antibody (e.g., as demonstrated by a decrease in Bmax in a graph of anti-KIR3DL2 antibody concentration versus polypeptide concentration). A significant reduction in binding indicates that the mutated residue is directly involved in binding to the anti-KIR3DL2 antibody or is in close proximity to the binding protein when the anti-KIR3DL2 antibody binds to KIR3DL2. The antibody epitope may therefore comprise such a residue and may comprise additional residues that are spatially adjacent to such a residue.

[0250] In some embodiments, a significant reduction in binding means that the binding affinity and / or capacity between the anti-KIR3DL2 antibody and the mutant KIR3DL2 polypeptide is reduced by greater than 40%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95% relative to the binding between the antibody and the wild-type KIR3DL2 polypeptide (e.g., the polypeptide shown in SEQ ID NO: 1). In certain embodiments, the binding is reduced to below the detectable limit. In some embodiments, a significant reduction in binding is demonstrated when the binding of the anti-KIR3DL2 antibody to the mutant KIR3DL2 polypeptide is less than 50% (e.g., less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10%) of the binding observed between the anti-KIR3DL2 antibody and the wild-type KIR3DL2 polypeptide (e.g., the extracellular domain shown in SEQ ID NO: 1). Such binding measurements can be performed using various binding assays known in the art. A specific example of such an assay is described in the Examples section.

[0251] In some embodiments, the anti-KIR3DL2 antibody exhibits significantly lower binding to a mutant KIR3DL2 polypeptide in which a residue in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO: 1) is substituted. In the simplified notation used herein, the format is: wild-type residue: position in polypeptide: mutant residue, where the residues are numbered as shown in SEQ ID NO: 1.

[0252] Optionally, the antibody has reduced binding to a KIR3DL2 polypeptide having a substitution at residue N99, H100, E130, H131, F132, V178, P179, H180, S181, P182, Y183 and / or residue Q184 of SEQ ID NO:1.

[0253] In some embodiments, the anti-KIR3DL2 antibody binds to a wild-type KIR3DL2 polypeptide having the sequence of SEQ ID NO: 1, but has reduced binding to a mutant KIR3DL2 polypeptide having any one or more (e.g., 1, 2, 3, or 4) of the following mutations: P179T and / or S181T (with reference to SEQ ID NO: 1). In one embodiment, binding to a mutant KIR3DL2 is significantly reduced compared to binding to wild-type KIR3DL2.

[0254] In some embodiments, the anti-KIR3DL2 antibody exhibits significantly lower binding to a mutant KIR3DL2 polypeptide in which residues in a segment corresponding to residues 1-98, residues 99-292, or residues 99-192 (or a subsequence thereof) in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO: 1) are substituted with different amino acids.

[0255] In one aspect, the antibody can compete with monoclonal antibody AZ158, 10G5, 19H12, 2B12 or 12B11 and recognize binding to or have immunospecificity for an epitope or "epitope site" on the KIR3DL2 molecule that is substantially the same or essentially the same or identical to monoclonal antibody AZ158, 10G5, 19H12, 2B12 or 12B11. In other embodiments, the monoclonal antibody consists of antibody AZ158, 10G5, 19H12, 2B12 or 12B11 or is a derivative or fragment thereof.

[0256] It should be understood that although the antibody can bind to the same epitope as antibody AZ158, 10G5, 19H12, 2B12 or 12B11, a suitable antibody can recognize and be produced against any portion of the KIR3DL2 polypeptide, as long as the antibody binds to KIR3DL2 and has the desired function. For example, any fragment of KIR3DL2 (e.g., human KIR3DL2) or any combination of KIR3DL2 fragments can be used as an immunogen to produce an antibody, and the antibody can recognize an epitope at any position within the KIR3DL2 polypeptide, as long as it can do so on NK cells expressing KIR3DL2 as described herein. In one embodiment, the recognized epitope is present on the cell surface, i.e., the epitope is accessible to antibodies present outside the cell. Optionally, the epitope is an epitope specifically recognized by antibody AZ158, 10G5, 19H12, 2B12 or 12B11. Further, antibodies that recognize different epitopes within KIR3DL2 can be used in combination, for example, to bind to a KIR3DL2 polypeptide with maximum efficacy and breadth across different individuals.

[0257] Antibodies can be produced by various techniques known in the art. Typically, the antibodies are produced by immunizing a non-human animal, optionally a mouse, with an immunogen comprising a KIR3DL2 polypeptide, optionally a human KIR3DL2 polypeptide. The KIR3DL2 polypeptide may comprise the full-length sequence of a human KIR3DL2 polypeptide or a fragment or derivative thereof, typically the full-length sequence of an immunogenic fragment, i.e., a portion of a polypeptide comprising an epitope exposed on the surface of a cell expressing the KIR3DL2 polypeptide, optionally the epitope being recognized by AZ158, 10G5, 19H12, 2B12 or 12B11 antibody. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, or at least about 10 consecutive amino acids thereof. The fragments are typically substantially derived from the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human KIR3DL2 polypeptide in a lipid membrane, typically on a cell surface. In one embodiment, the immunogen comprises intact cells, particularly intact human cells, that are optionally treated or lysed. In another embodiment, the polypeptide is a recombinant KIR3DL2 polypeptide.

[0258] The step of immunizing a non-human mammal with an antigen can be performed in any manner well known in the art for stimulating the production of antibodies in mice (see, e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988), the entire disclosure of which is incorporated herein by reference). The immunogen is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete or incomplete Freund's adjuvant. Methods for determining the amount of immunogen, the type of buffer, and the amount of adjuvant are well known to those skilled in the art and are not limiting in any way. These parameters may vary for different immunogens, but are readily elucidated.

[0259] Similarly, the immunization position and frequency sufficient to stimulate antibody production are also well known in the art. In a typical immunization protocol, non-human animals are injected intraperitoneally with antigens on the 1st day and again about a week later. The antigen injection is then recalled on the 20th day or so, optionally with an adjuvant such as incomplete Freund's adjuvant. The recall injection is performed intravenously and can be repeated for several consecutive days. Subsequently, on the 40th day, a booster injection is usually given intravenously or intraperitoneally in the absence of an adjuvant. This protocol results in the production of B cells that produce antigen-specific antibodies after about 40 days. Other protocols may also be used, as long as they result in the production of B cells that express antibodies to the antigens used in immunization.

[0260] For polyclonal antibody preparations, serum is obtained from an immunized non-human animal and the antibodies present therein are isolated by well-known techniques. The serum may be affinity purified using any of the above-described immunogens attached to a solid support to obtain antibodies reactive with the KIR3DL2 polypeptide.

[0261] In an alternative embodiment, lymphocytes from a non-immune non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then collected and subjected to the fusion step described below.

[0262] For exemplary monoclonal antibodies, the next step is to separate splenocytes from the non-human mammals of immunity, and then these splenocytes are fused with immortalized cells to form hybridomas that produce antibodies. It is well known in the art to separate splenocytes from non-human mammals, and is generally related to removing the spleen from the non-human mammals of anesthesia, cutting it into small pieces, and passing through the nylon mesh of a cell strainer, the splenocytes from the spleen are squeezed into a suitable buffer to produce a single cell suspension. The cells are washed, centrifuged and resuspended in the buffer of any red blood cell of lysis. The solution is centrifuged again, and the lymphocytes remaining in the precipitation are finally resuspended in a fresh buffer.

[0263] Once separated and present in a single cell suspension, lymphocytes can be fused with immortalized cell lines. Although many other immortalized cell lines known in the art can be used to produce hybridomas, this is generally a mouse myeloma cell line. Murine myeloma cell lines include but are not limited to MOPC-21 and MPC-11 mouse tumors obtained from the Salk Institute Cell Distribution Center (SalkInstitute Cell Distribution Center, San Diego, USA) in San Diego, USA, and those of X63 Ag8653 and SP-2 cells obtained from the American Type Culture Collection (American Type Culture Collection, Rockville, Maryland USA) in Rockville, Maryland, USA. Use polyethylene glycol etc. to fuse. Then, the resulting hybridoma is grown in a selective culture medium containing one or more substances that suppress the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0264] Hybridomas are usually grown on a feeder layer of macrophages. Macrophages can be from the same littermates of non-human mammals used to isolate splenocytes and are usually primed with incomplete Freund's adjuvant or the like a few days before plating hybridomas. Fusion methods are described in Goding, "Monoclonal Antibodies: Principles and Practice", pp. 59-103 (Academic Press, 1986), the disclosure of which is incorporated herein by reference.

[0265] The cells are grown in the selection medium for a sufficient time for colony formation and antibody production. This is usually between about 7 days to about 14 days.

[0266] The hybridoma colonies are then assayed for the production of antibodies that specifically bind to the KIR3DL2 polypeptide gene product (optionally an epitope specifically recognized by antibodies AZ158, 19H12, 2B12 or 12B11). The assay is typically a colorimetric ELISA type assay, although any assay that can accommodate the wells in which the hybridomas are grown may be employed. Other assays include radioimmunoassays or fluorescence activated cell sorting. Wells that are positive for the production of the desired antibody are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be recloned and grown to ensure that only a single cell has produced a colony that produces the desired antibody. Typically, the antibody will also be tested for its ability to bind to a KIR3DL2 polypeptide, such as a cell expressing KIR3DL2.

[0267] Hybridomas that have been confirmed to produce monoclonal antibodies can be grown in a large number in appropriate culture medium, such as DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in animals. After fully growing to produce the required monoclonal antibody, the growth medium containing the monoclonal antibody (or ascites) is separated from the cells, and the monoclonal antibody present therein is purified. Purification is usually achieved by gel electrophoresis, dialysis, using protein A or protein G agarose or connecting solid support, such as the anti-mouse Ig chromatography of agarose or agarose beads (all described in, for example, "Antibody Purification Handbook", Biological Sciences, Publication No. 18-1037-46, AC Edition, the disclosure of the document is incorporated herein by reference). Usually by using low pH buffer (pH equal to or less than 3.0 glycine or acetate buffer) to elute the bound antibody from the protein A / protein G column, immediately neutralize the fraction containing the antibody. These fractions are merged, dialyzed and concentrated as required.

[0268] Positive wells with a single apparent colony are usually recloned and replated to ensure that only one monoclonal antibody is detected and produced.

[0269] Antibodies can also be produced by selecting combinatorial libraries of immunoglobulins, for example as disclosed in (Ward et al., Nature, 341 (1989), p. 544, the entire disclosure of which is incorporated herein by reference).

[0270] Identification of one or more antibodies that bind to an antigen of interest, i.e., KIR3DL2, particularly or substantially to the same region, determinant or epitope as monoclonal antibody AZ158, 19H12, 2B12 or 12B11, can be readily determined using any of a variety of immunological screening assays in which antibody competition can be assessed. Many such assays are routinely practiced and are well known in the art (see, e.g., U.S. Pat. No. 5,660,827, issued Aug. 26, 1997, expressly incorporated herein by reference).

[0271] For example, where the test antibody to be tested is obtained from a different source animal, or even a different Ig isotype, a simple competition assay can be used in which a control (e.g., AZ158, 19H12, 2B12, or 12B11) and test antibody are mixed (or pre-adsorbed) and applied to a sample containing a KIR3DL2 polypeptide. Western blot-based protocols and the use of BIACORE analysis are suitable for such competition studies.

[0272] In certain embodiments, a control antibody (e.g., AZ158, 19H12, 2B12, or 12B11) is premixed with varying amounts of a test antibody (e.g., about 1:10 or about 1:100) for a period of time prior to application to a KIR3DL2 antigen sample. In other embodiments, the control and varying amounts of the test antibody may be simply mixed during exposure to the KIR3DL2 antigen sample. As long as bound antibody can be distinguished from free antibody (e.g., by using a separation or washing technique to eliminate unbound antibody) and 2B12 can be distinguished from the test antibody (e.g., by using a species-specific or isotype-specific secondary antibody or by specifically labeling AZ158, 19H12, 2B12, or 12B11 with a detectable label), it can be determined whether the test antibody reduces the binding of AZ158, 19H12, 2B12, or 12B11 to the antigen. In the absence of a completely unrelated antibody, the binding of the (labeled) control antibody can serve as a control high value. A control low value is obtained by incubating a labeled (AZ158, 19H12, 2B12 or 12B11) antibody with an unlabeled antibody (AZ158, 19H12, 2B12 or 12B11) of exactly the same type, where competition will occur and reduce binding of the labeled antibody. A significant reduction in the reactivity of the labeled antibody in the presence of the test antibody in the test assay indicates a test antibody that "cross-reacts" or competes with the labeled (AZ158, 19H12, 2B12 or 12B11) antibody. Any test antibody can be selected at any ratio of AZ158, 19H12, 2B12 or 12B11:test antibody between about 1:10 and about 1:100 that reduces the binding of AZ158, 19H12, 2B12 or 12B11 to the KIR3DL2 antigen by at least about 50%, such as at least about 60% or more preferably at least about 80% or 90% (e.g., about 65-100%). In one embodiment, such test antibodies reduce the binding of AZ158, 19H12, 2B12 or 12B11 to the KIR3DL2 antigen by at least about 90% (e.g., about 95%).

[0273] Competition can also be assessed by, for example, flow cytometry tests. In such tests, cells carrying a given KIR3DL2 polypeptide can be first incubated with 2B12, for example, and then incubated with a test antibody labeled with a fluorescent dye or biotin. If the binding obtained by preincubation with a saturating amount of 2B12 is about 80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained by the antibody not preincubated with 2B12 (as measured by the average value of fluorescence), then the antibody is said to compete with 2B12. Alternatively, if the binding obtained with a labeled 2B12 antibody (by a fluorescent dye or biotin) on cells preincubated with a saturating amount of the test antibody is about 80%, preferably about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained without preincubation with the test antibody, then the antibody is said to compete with 2B12.

[0274] Determining whether an antibody binds within an epitope region can be performed in a manner known to those skilled in the art. As an example of such mapping / characterization methods, the epitope region of an anti-KIR3DL2 antibody can be determined by "foot-printing" of the epitope using chemical modification of exposed amine / carboxyl groups in the corresponding KIR3DL2 protein. A specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), in which hydrogen / deuterium exchange, binding and reverse exchange of receptor and ligand protein amide protons occur, wherein the main chain amide groups involved in protein binding are protected from reverse exchange and will therefore remain deuterated. At this point, the relevant region can be identified by digestive proteolysis, rapid micropore high performance liquid chromatography separation and / or electrospray ionization mass spectrometry. See, for example, Ehring H, Analytical Biochemistry, Vol. 267 (2), pp. 252-259, (1999); Engen, JR and Smith, DL (2001) Anal. Chem. 73, 256A-265A. Another example of a suitable epitope identification technique is nuclear magnetic resonance epitope mapping (NMR), in which the positions of signals in a two-dimensional NMR spectrum of free antigen and an antigen in complex with an antigen-binding peptide, such as an antibody, are generally compared. Antigens are typically selectively isotopically labeled with 15N so that only signals corresponding to the antigen are seen in the NMR spectrum, and signals from the antigen-binding peptide are not seen. Antigen signals derived from amino acids that participate in the interaction with the antigen-binding peptide will typically be shifted in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in the binding can be identified in the described manner. See, e.g., Ernst Schering Res Found Workshop. 2004;(44):149-67; Huang et al., Journal of Molecular Biology, Vol. 281(1), pp. 61-67 (1998); and Saito and Patterson, Methods, Jun. 1996;9(3):516-24.

[0275] Epitope mapping / characterization can also be performed using mass spectrometry. See, e.g., Downard, J Mass Spectrom. April 2000; 35(4):493-503; and Kiselar and Downard, Analytical Chemistry, May 1, 1999; 71(9):1792-1801. Protease digestion techniques can also be used for epitope mapping and identification. Antigenic determinant-associated regions / sequences can be determined by protease digestion, e.g., by digestion o / n with trypsin at a ratio of about 1:50 or at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by anti-KIR3DL2 binders (thereby revealing the footprint of the binder) can then be identified by incubating the sample subjected to trypsin digestion with the antibody and then comparing the sample with, e.g., trypsin digestion. Other enzymes such as chymotrypsin, pepsin, etc. can also or alternatively be used in similar epitope characterization methods. Furthermore, enzymatic digestion may provide a rapid method for analyzing whether potential antigenic determinant sequences are within regions of the KIR3DL2 polypeptide that are not surface exposed and, therefore, are most likely not relevant in terms of immunogenicity / antigenicity.

[0276] Site-directed mutagenesis is another technique that can be used to elucidate binding epitopes. For example, in "alanine scanning", each residue within a protein segment is replaced with an alanine residue, and the results of binding affinity are measured. If the mutation results in a significant decrease in binding affinity, it is likely to be involved in binding. Monoclonal antibodies specific for structural epitopes (i.e., antibodies that do not bind to unfolded proteins) can be used to verify that alanine substitutions do not affect the overall folding of the protein. See, for example, Clackson and Wells, Science 1995; 267: 383-386; and Wells, Proc Natl Acad Sci USA 1996; 93: 1–6.

[0277] Electron microscopy can also be used for epitope "footprinting". For example, Wang et al., Nature 1992; 355: 275-278 used a coordinated application of cryo-electron microscopy, three-dimensional image reconstruction, and X-ray crystallography to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0278] Other forms of "label-free" assays for epitope assessment include surface plasmon resonance (SPR, BIACORE) and reflectance interferometry spectroscopy (RifS). See, e.g., et al., Journal of Molecular Recognition 1990; 3:208-14; Nice et al., J. Chromatography 1993; 646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998; 37:3308-3311; et al., Biosensors and Bioelectronics 2002;17:937-944.

[0279] It should also be noted that antibodies that bind to the same or substantially the same epitope as an antibody can be identified in one or more of the exemplary competition assays described herein.

[0280] Once an antibody is identified that is capable of binding to KIR3DL2 and / or has other desired properties (e.g., the ability to mediate ADCC and / or antibody-dependent cellular phagocytosis (ADCP)), the antibody will also typically be evaluated for its ability to bind to other polypeptides (including unrelated polypeptides) using standard methods (including those described herein). Ideally, the antibody will only bind to KIR3DL2 (e.g., human KIR3DL2) with significant affinity, and will not bind to unrelated polypeptides at significant levels. However, it will be understood that as long as the affinity for KIR3DL2 is substantially greater (e.g., 5x, 10x, 50x, 100x, 500x, 1000x, 10,000x, or more) than the affinity for other unrelated polypeptides, then the antibody is suitable for use in the methods of the invention.

[0281] Binding of antibodies to cells expressing KIR3DL2 can also be assessed in non-human primates (e.g., cynomolgus monkeys or other mammals, such as mice).An antibody and fragments and derivatives thereof are provided, wherein the antibody, fragment or derivative specifically binds KIR3DL2 and further binds KIR3DL2 from a non-human primate (e.g., cynomolgus monkey).

[0282] After immunization and production of antibodies in vertebrates or cells, specific selection steps may be performed to isolate antibodies as claimed. In this regard, in specific embodiments, the disclosure also relates to methods of producing such antibodies, the methods comprising: (a) immunizing a non-human mammal with an immunogen comprising a KIR3DL2 polypeptide; and (b) preparing antibodies from the immunized animal; and (c) selecting antibodies from step (b) that are capable of binding to KIR3DL2.

[0283] In one aspect of any embodiment, the antibody prepared according to the method of the present invention is a monoclonal antibody. In another aspect, the non-human animal used to produce the antibody is a mammal, such as a rodent, cattle, pig, poultry, horse, rabbit, goat or sheep.

[0284] According to an alternative embodiment, DNA encoding an antibody that binds to an epitope present on a KIR3DL2 polypeptide is isolated from a hybridoma and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used to recombinantly produce the antibody or a variant thereof, such as a humanized form of the monoclonal antibody, an active fragment of the antibody, a chimeric antibody comprising an antigen recognition portion of the antibody, or a form comprising a detectable portion.

[0285] DNA encoding the monoclonal antibody can be easily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). After separation, the DNA can be placed in an expression vector and then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins, to obtain the synthesis of the monoclonal antibody in a recombinant host cell. As described elsewhere in this specification, such DNA sequences can be modified for any number of purposes, such as for humanized antibodies, for the production of fragments or derivatives, or for modifying the sequence of an antibody, such as in an antigen binding site, to optimize the binding specificity of the antibody.

[0286] Recombinant expression in bacteria of DNA encoding antibodies is well known in the art (see, e.g., Skerra et al., Curr. Opinion in Immunol., 5, 256 (1993); and Pluckthun, Immunol. 130, 151 (1992).

[0287] Once an antigen binding compound is obtained, its ability to induce ADCC, ADCP and / or CDC towards and / or deplete target cells expressing KIR3DL2 can be assessed. The ability of an antigen binding compound to induce ADCC, ADCP and / or CDC (complement dependent cytotoxicity) or generally result in the elimination or inhibition of the activity of a target cell expressing KIR3DL2 can be performed at any suitable stage of the method. Such an assessment can be used for one or more of the various steps involved in the identification, production and / or development of an antibody (or other compound) intended for therapeutic use. For example, the activity can be assessed in the context of a screening method for identifying a candidate antigen binding compound or in a method for selecting an antigen binding compound and making it suitable for humans (e.g., chimerizing or humanizing it in the case of an antibody), in the case where a cell expressing an antigen binding compound (e.g., a host cell expressing a recombinant antigen binding compound) has been obtained and its ability to produce a functional antibody (or other compound) has been assessed and / or in the case where a certain amount of an antigen binding compound has been produced and its activity will be assessed (e.g., to test a batch or lot number of a product). Typically, it is known that the antigen binding compound will specifically bind to a KIR3DL2 polypeptide. The steps may involve testing a large number (eg, a very large number or a smaller number using high throughput screening methods) of antigen binding compounds.

[0288] The tests ADCP, CDC and ADCC that can be performed can be determined by various assays, including those known in the art and those described in the experimental examples herein. Testing ADCC generally involves assessing cell-mediated cytotoxicity, in which target cells expressing KIR3DL2 (e.g., TCL cells or other cells expressing KIR3DL2) with bound anti-KIR3DL2 antibodies are recognized by effector cells carrying Fc receptors without involving complement. Cells that do not express KIR3DL2 antigens can optionally be used as controls. The activation of NK cell cytotoxicity is assessed by measuring the increase in cytokine production (e.g., IFN-γ production) or cytotoxicity markers (e.g., CD107 mobilization). In one embodiment, compared to a control antibody (e.g., an antibody not bound to KIR3DL2, a KIR3DL2 antibody with a murine constant region), the antibody will induce cytokine production, cytotoxicity marker expression or target cell lysis in the presence of target cells by at least 20%, 50%, 80%, 100%, 200% or 500%. In another example, lysis of target cells is detected, for example, in a chromium release assay, for example, the antibody will induce lysis of at least 10%, 20%, 30%, 40% or 50% of the target cells.

[0289] Fragments and derivatives of antibodies (which are covered by the terms "antibody" or "antibodies" as used in this application, unless otherwise specified or clearly contradictory in context) can be produced by techniques known in the art. "Fragments" include a portion of an intact antibody, typically the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; bifunctional antibodies; any antibody fragment is a polypeptide having a primary structure consisting of an uninterrupted sequence of continuous amino acid residues (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"), including but not limited to (1) single-chain Fv molecules (2) single-chain polypeptides containing only one light chain variable domain, or containing three CDRs of the light chain variable domain, without its associated heavy chain portion and (3) single-chain polypeptides containing only one heavy chain variable region, or containing three CDRs of the heavy chain variable region, without its associated light chain portion; and multispecific antibodies formed by antibody fragments. In particular, nanobodies, domain antibodies, single domain antibodies or "dAbs" are included.

[0290] In certain embodiments, the DNA of the hybridoma producing the antibody can be modified prior to insertion into an expression vector, for example, by replacing the homologous non-human sequences with coding sequences for human heavy and light chain constant domains (e.g., Morrison et al., Proceedings of the National Academy of Sciences (PNAS) 6851 (1984)), or by covalently linking the coding sequences of all or part of non-immunoglobulin polypeptides to the immunoglobulin coding sequences. In such a manner, a "chimeric" or "hybrid" antibody having the binding specificity of the original antibody is prepared. Typically, such non-immunoglobulin polypeptides replace the constant domains of the antibody.

[0291] Therefore, according to another embodiment, the antibody is humanized. The "humanized" form of the antibody is correspondingly a specific chimeric immunoglobulin, an immunoglobulin chain or fragment thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a murine immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (acceptor antibody) in which the residues from the complementary determining region (CDR) of the acceptor are replaced by residues from the CDR of the original antibody (donor antibody), while maintaining the specificity, affinity and ability required for the original antibody.

[0292] In some cases, the Fv framework residues of human immunoglobulin can be replaced by corresponding non-human residues. In addition, humanized antibodies can include residues not found in receptor antibodies or CDR or framework sequences imported. These modifications are carried out to further improve and optimize antibody performance. Usually, humanized antibodies will include substantially all at least one and usually two variable domains, wherein all or substantially all CDR regions correspond to those of the original antibody, and all or substantially all FR regions are those in the human immunoglobulin consensus sequence. Humanized antibodies will also optimally include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For additional details, see Jones et al., Nature, 321, p. 522 (1986); Reichmann et al., Nature, 332, p. 323 (1988); Presta, Curr. Op. Struct. Biol., 2, p. 593 (1992); Verhoeyen et al., Science, 239, p. 1534; and U.S. Pat. No. 4,816,567, the entire disclosures of which are incorporated herein by reference). Methods for humanizing antibodies are well known in the art.

[0293] For preparing the human variable domains of humanized antibodies, the selection of both light chain and heavy chain is very important for reducing antigenicity. According to the so-called "best fit" method, for the entire library of known human variable domain sequences, the sequence of the variable domains of the antibody is screened. Then the human sequence closest to the mouse is accepted as the human framework (FR) of humanized antibodies (Sims et al., "Journal of Immunology (J.Immunol.)" 151, p. 2296 (1993); Chothia and Lesk, "Journal of Molecular Biology (J.Mol.)" 196, 1987, p. 901). Another method uses a specific framework of the consensus sequence of all human antibodies from a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., "Proceedings of the National Academy of Sciences of the United States of America" ​​89, p. 4285, (1992); Presta et al., "Journal of Immunology", 151, p. 2623 (1993)).

[0294] More importantly, the antibody is humanized, retaining high affinity for the KIR3DL2 receptor and other favorable biological properties. To achieve this goal, according to a method, humanized antibodies are prepared by analyzing the process of parental sequences and various conceptual humanized products using three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that display and show the possible three-dimensional structures of selected candidate immunoglobulin sequences. Examining these displays allows analysis of the possible role of residues in the function of candidate immunoglobulin sequences, that is, analysis of residues that affect the ability of candidate immunoglobulins to bind to their antigens. In this way, FR residues can be selected and combined from consensus sequences and input sequences to achieve desired antibody properties, such as increased affinity for target antigens. Typically, CDR residues are directly and most substantially involved in affecting antigen binding.

[0295] Another method for preparing human or humanized monoclonal antibodies is to use genetically modified mice for immunization. Several murine hosts in which known immunoglobulin genes are replaced by functional human immunoglobulin genes are known. Therefore, the antibodies produced by the mice or the antibodies produced in the hybridomas made by the B cells of the mice are already human or humanized. Exemplary animals are described in U.S. Patent No. 6,162,963 and Jakobovitz et al., Nature 362 (1993) 255, which are incorporated herein by reference. Human antibodies can also be produced according to various other techniques, such as by selecting antibody repertoires using phage display methods. Such techniques are known to those skilled in the art and can be implemented starting from the monoclonal antibodies disclosed in the present application.

[0296] The KIR3DL2 binding compound (eg, an anti-KIR3DL2 antibody) can be further conjugated to a second moiety, wherein the antibody is capable of delivering the second moiety to cells expressing KIR3DL2. Optionally, the second moiety is a therapeutic agent, a toxic agent, and / or a detectable agent.

[0297] While antibodies in an underivatized or unmodified form (particularly of the IgG1 or IgG3 type) are expected to inhibit proliferation of hyperproliferative cells or be cytotoxic to hyperproliferative cells (such as those from PTCL patients), for example, by directing ADCC, ADCP and / or CDC toward PTCL cells expressing KIR3DL2, it is also possible to prepare cytotoxic derivatized antibody immunoconjugates. In one embodiment, once a KIR3DL2-specific antibody is isolated and optionally modified (e.g., humanized) in other ways, it will be derivatized to render it toxic to cells. In this way, administration of the antibody to a PTCL patient will result in relatively specific binding of the antibody to hyperproliferative cells, thereby directly killing or inhibiting the cells that are the cause of the condition.

[0298] Given the ability of anti-KIR3DL2 antibodies to induce ADCC and CDC, the antibodies can also be modified to increase their ability to bind to Fc receptors, which can affect effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation and phagocytosis, and immunomodulatory signals such as regulation of lymphocyte proliferation and antibody secretion. Typical modifications include a modified human IgG1 constant region that includes at least one amino acid modification (e.g., substitution, deletion, insertion) and / or an altered glycosylation type, such as hypofucosylation.

[0299] The anti-KIR3DL2 antibody may include an Fc domain (or portion thereof) of the human IgG1 or IgG3 isotype, optionally modified.

[0300] In certain embodiments, the CH2 and / or CH3 domains (or Fc domains comprising them) may include one or more amino acid modifications (e.g., amino acid substitutions) that increase binding to human CD16 and optionally another receptor, such as FcRn. Optionally, the modification will not significantly reduce or eliminate the ability of the Fc-derived polypeptide to bind to a neonatal Fc receptor (FcRn), such as human FcRn. Typical modifications include a modified human IgG1-derived constant region that includes at least one amino acid modification (e.g., substitution, deletion, insertion) and / or an altered glycosylation type, such as low fucosylation. Such modifications may affect interaction with Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A), and FcγRIII (CD16) are activating (i.e., immune system enhancement) receptors, while FcγRIIB (CD32B) is an inhibitory (i.e., immune system suppression) receptor. The modification can, for example, increase the binding of the Fc domain to FcγRIIIa on effector (e.g., NK) cells and / or reduce the binding to FcγRIIB. Examples of modifications are provided in PCT Publication No. WO2014 / 044686, the disclosure of which is incorporated herein by reference. Specific mutations (in IgG1 Fc domains) that affect (enhance) FcγRIIIa or FcRn binding are also described below.

[0301]

[0302] Variant Fc region can include at least one amino acid modification (e.g., with 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications) in the CH2 and / or CH3 domains of the Fc region, wherein the modification enhances binding to human CD16 polypeptides. In other embodiments, the Fc region includes at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications) in the CH2 domain of the Fc region from amino acids 237-341 or in the lower hinge-CH2 region including residues 231-341. In some embodiments, the Fc region includes at least two amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), wherein at least one modification of such modifications is located in the CH3 region, and at least one such modification is located in the CH2 region. Amino acid modifications in the hinge region are also contemplated. In one embodiment, amino acid modifications in the CH1 domain, optionally in the upper hinge region including residues 216-230 (Kabat EU numbering) are contemplated.Any suitable functional combination of Fc modifications may be made, such as any combination of the different Fc modifications disclosed in: U.S. Patent Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; any of 6,821,505 and 6,737,056; and / or PCT Publication Nos. WO 2011 / 109400; WO 2008 / 105886; WO 2008 / 002933; WO 2007 / 021841; WO 2007 / 106707;WO06 / 088494;WO 05 / 115452;WO 05 / 110474;WO 04 / 1032269;WO 00 / 42072;WO 06 / 088494;WO07 / 024249;WO 05 / 047327;WO 04 / 099249 and WO 04 / 063351; and / or Lazar et al. (2006), Proc. Nat. Acad. Sci. USA 103(11):405-410; Presta, LG et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, RL et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields, RL et al. (2001) J. Biol. Chem. 276(9):6591-6604).

[0303] Examples

[0304] Example 1— In vitro combination studies using chemotherapeutic agents

[0305] It is known that chemotherapeutic agents can negatively affect the host immune system and thus inhibit the efficacy of therapeutic antibodies that mediate ADCC. Therefore, it was attempted to evaluate whether chemotherapeutic agents affect the NK cell-mediated ADCC activity of the humanized 2B12 antibody lactuzumab, which has a human IgG1 isotype and VH and VL amino acid sequences shown in SEQ ID NOs: 49 and 44, respectively. Chemotherapeutic agents were added alone or in combination to an in vitro cytotoxicity (ADCC) assay using allogeneic NK cells and a Sezary syndrome cell line (Hut 78) expressing KIR3DL2, in the presence of increasing concentrations of anti-KIR3DL2 antibodies.

[0306] Figure 1The results are shown in . In this experimental setting, neither gemcitabine nor oxaliplatin interfered with anti-KIR3DL2 antibody-induced ADCC against the HUT78 CTCL cell line. In fact, anti-KIR3DL2 antitumor activity was surprisingly enhanced by each of gemcitabine and oxaliplatin, and even more by the combination of gemcitabine and oxaliplatin.

[0307] Example 2—Gemcitabine and oxaliplatin each enhance KIR3DL2 expression

[0308] To investigate the possible reasons for the enhanced ADCC induced by anti-KIR3DL2 antibodies against the HUT78 CTCL cell line, we evaluated whether KIR3DL2 expression could be modulated in vitro by the same chemotherapeutic agents.

[0309] Briefly, Hut78 cells (Sezary syndrome cell line) or RAJI-KIR3DL2 cells (RAJI B-NHL cell line transfected with human KIR3DL2) were incubated with increasing doses of gemcitabine and oxaliplatin. The median fluorescence intensity of KIR3DL2 on the surface of tumor cell lines was analyzed by flow cytometry.

[0310] Figure 2 Results are shown in Figure A. RAJI-KIR3DL2 cells are shown in Figure A (left hand side), and Hut78 cells are shown in Figure B (right hand side). Both oxaliplatin and gemcitabine were found to enhance KIR3DL2 surface expression in a dose-dependent manner on both cell lines, with up to a 2- to 3-fold plateau compared to baseline ( Figure 2 ). These figures represent two independent experiments. It is noteworthy that other chemotherapeutic agents (such as cyclophosphamide) did not increase KIR3DL2 expression under the same experimental conditions.

[0311] In conclusion, these results support exploration of the combination of anti-KIR3DL2 antibodies with gemcitabine and oxaliplatin, particularly in patients with relapsed PTCL who may be in particular need of improved treatments.

[0312] Example 3—In vivo efficacy

[0313] Xenograft model in CB17-SCID mice, an immunocompromised mouse strain that retains fully functional NK cells and macrophages. In addition, mouse Fc receptors efficiently bind human Fc, allowing murine effector cells to be recruited by humanized mAbs and perform ADCC or ADCP in vivo. The B-NHL cell line RAJI was transfected to stably express KIR3DL2 (RAJI-KIR3DL2), and the transfectants were inoculated IV (5×10 6Cells) were introduced into SCID mice to provide a disseminated tumor model. Initially, individual doses of gemcitabine and oxaliplatin were established in this xenograft model. However, although gemcitabine and oxaliplatin can be used together in humans, it is impossible to find a dose of gemcitabine and oxaliplatin that can be combined to provide activity but is tolerated in these mice. Therefore, an anti-KIR3DL2 antibody (lactuzumab) was combined with gemcitabine or oxaliplatin, respectively.

[0314] In this disseminated tumor model (n=9 mice per group), groups of mice were therefore treated twice a week with:

[0315] - Isotype control mAb;

[0316] - Anti-KIR3DL2 antibody (lactuzumab);

[0317] - Gemcitabine (50 mg / kg);

[0318] - Oxaliplatin (5 mg / kg);

[0319] - Anti-KIR3DL2 antibody (lactuzumab) + gemcitabine (50 mg / kg), or

[0320] - Anti-KIR3DL2 antibody (lactuzumab) + oxaliplatin (5 mg / kg).

[0321] Both anti-KIR3DL2 antibody and isotype control were administered at 0.3 μg per injection.

[0322] Figure 3 The results are shown in . The combination of anti-KIR3DL2 antibody and oxaliplatin is at least as active as the anti-KIR3DL2 antibody alone (oxaliplatin itself has very limited (if any) anti-tumor effect in the model). Interestingly, both anti-KIR3DL2 antibody and gemcitabine showed synergistic anti-tumor activity in vivo. Mice treated with anti-KIR3DL2 antibody and gemcitabine had significantly improved survival compared to mice treated with each agent separately. The effects shown above were reproduced in 2 independent experiments, with 9 mice per group each time.

[0323] These results, together with the in vitro combination data and the potential induction of KIR3DL2 by gemcitabine and oxaliplatin, support the therapeutic combination of anti-KIR3DL2 antibodies with gemcitabine and oxaliplatin in TCL patients. Although the combination with humanized 2B12 is particularly effective as an anticancer treatment, the ability of gemcitabine and / or oxaliplatin to increase KIR3DL2 expression may also be useful for the purpose of enhancing the anti-tumor effects of antibodies that are less potent than 2B12 alone. The combination may be particularly effective for patients with recurrent PTCL.

[0324] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of whether incorporation of a specific document referenced elsewhere herein is provided separately.

[0325] Unless otherwise indicated, all exact values ​​provided herein represent corresponding approximate values ​​(eg, all exact exemplary values ​​provided for a particular factor or measurement may be considered to also provide the corresponding approximate measurement, modified by "about," where appropriate).

[0326] The description of any aspect or embodiment herein using terms such as “comprising,” “having,” “having,” or “containing” is intended to provide support for similar aspects or embodiments of the invention “consisting of,” “consisting essentially of,” or “consisting essentially of,” unless otherwise stated or clearly contradicted by context (e.g., a composition described herein comprising a particular element should be understood to also describe a composition consisting of said element, unless otherwise stated or clearly contradicted by context).

[0327] Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention. Any language in this specification should not be construed as indicating any unclaimed element as necessary to practice the present invention. Sequence Listing <110> Innate Pharma <120> Treatment of T-cell lymphoma <130> KIR-9 <150> US 62 / 795,194 <151> 2019-01-22 <160> 70 <170> PatentIn Version 3.5 <210> 1 <211> 434 <212> PRT <213> Homo sapiens <400> 1 Leu Met Gly Gly Gln Asp Lys Pro Phe Leu Ser Ala Arg Pro Ser Thr 1 5 10 15 Val Val Pro Arg Gly Gly His Val Ala Leu Gln Cys His Tyr Arg Arg 20 25 30 Gly Phe Asn Asn Phe Met Leu Tyr Lys Glu Asp Arg Ser His Val Pro 35 40 45 Ile Phe His Gly Arg Ile Phe Gln Glu Ser Phe Ile Met Gly Pro Val 50 55 60 Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Arg Gly Ser Arg Pro His 65 70 75 80 Ser Leu Thr Gly Trp Ser Ala Pro Ser Asn Pro Leu Val Ile Met Val 85 90 95 Thr Gly Asn His Arg Lys Pro Ser Leu Leu Ala His Pro Gly Pro Leu 100 105 110 Leu Lys Ser Gly Glu Thr Val Ile Leu Gln Cys Trp Ser Asp Val Met 115 120 125 Phe Glu His Phe Phe Leu His Arg Asp Gly Ile Ser Glu Asp Pro Ser 130 135 140 Arg Leu Val Gly Gln Ile His Asp Gly Val Ser Lys Ala Asn Phe Ser 145 150 155 160 Ile Gly Pro Leu Met Pro Val Leu Ala 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Gln Leu Asp His Cys Val 370 375 380 Phe Ile Gln Arg Lys Ile Ser Arg Pro Ser Gln Arg Pro Lys Thr Pro 385 390 395 400 Leu Thr Asp Thr Ser Val Tyr Thr Glu Leu Pro Asn Ala Glu Pro Arg 405 410 415 Ser Lys Val Val Ser Cys Pro Arg Ala Pro Gln Ser Gly Leu Glu Gly 420 425 430 Val Phe <210> 2 <211> 455 <212> PRT <213> Homo sapiens <400> 2 Met Ser Leu Thr Val Val Ser Met Ala Cys Val Gly Phe Phe Leu Leu 1 5 10 15 Gln Gly Ala Trp Pro Leu Met Gly Gly Gln Asp Lys Pro Phe Leu Ser 20 25 30 Ala Arg Pro Ser Thr Val Val Pro Arg Gly Gly His Val Ala Leu Gln 35 40 45 Cys His Tyr Arg Arg Gly Phe Asn Asn Phe Met Leu Tyr Lys Glu Asp 50 55 60 Arg Ser His Val Pro Ile Phe His Gly Arg Ile Phe Gln Glu Ser Phe 65 70 75 80 Ile Met Gly Pro Val Thr Pro Ala His Ala Gly Thr Tyr Arg Cys Arg 85 90 95 Gly Ser Arg Pro His Ser Leu Thr Gly Trp Ser Ala Pro Ser Asn Pro 100 105 110 Val Val Ile Met Val Thr Gly Asn His Arg Lys Pro Ser Leu Leu Ala 115 120 125 His Pro Gly Pro Leu Leu Lys Ser Gly Glu Thr Val Ile Leu Gln Cys 130 135 140 Trp Ser Asp Val Met Phe Glu His Phe Phe Leu His Arg Glu Gly Ile 145 150 155 160 Ser Glu Asp Pro Ser Arg Leu Val Gly Gln Ile His Asp Gly Val Ser 165 170 175 Lys Ala Asn Phe Ser Ile Gly Pro Leu Met Pro Val Leu Ala Gly Thr 180 185 190 Tyr Arg Cys Tyr Gly Ser Val Pro His Ser Pro Tyr Gln Leu Ser Ala 195 200 205 Pro Ser Asp Pro Leu Asp Ile Val Ile Thr Gly Leu Tyr Glu Lys Pro 210 215 220 Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Gln Ala Gly Glu Asn Val 225 230 235 240 Thr Leu Ser Cys Ser Ser Trp Ser Ser Tyr Asp Ile Tyr His Leu Ser 245 250 255 Arg Glu Gly Glu Ala His Glu Arg Arg Leu Arg Ala Val Pro Lys Val 260 265 270 Asn Arg Thr Phe Gln Ala Asp Phe Pro Leu Gly Pro Ala Thr His Gly 275 280 285 Gly Thr Tyr Arg Cys Phe Gly Ser Phe Arg Ala Leu Pro Cys Val Trp 290 295 300 Ser Asn Ser Ser Asp Pro Leu Leu Val Ser Val Thr Gly Asn Pro Ser 305 310 315 320 Ser Ser Trp Pro Ser Pro Thr Glu Pro Ser Ser Lys Ser Gly Ile Cys 325 330 335 Arg His Leu His Val Leu Ile Gly Thr Ser Val Val Ile Phe Leu Phe 340 345 350 Ile Leu Leu Leu Phe Phe Leu Leu Tyr Arg Trp Cys Ser Asn Lys Lys 355 360 365 Asn Ala Ala Val Met Asp Gln Glu Pro Ala Gly Asp Arg Thr Val Asn 370 375 380 Arg Gln Asp Ser Asp Glu Gln Asp Pro Gln Glu Val Thr Tyr Ala Gln 385 390 395 400 Leu Asp His Cys Val Phe Ile Gln Arg Lys Ile Ser Arg Pro Ser Gln 405 410 415 Arg Pro Lys Thr Pro Leu Thr Asp Thr Ser Val Tyr Thr Glu Leu Pro 420 425 430 Asn Ala Glu Pro Arg Ser Lys Val Val Ser Cys Pro Arg Ala Pro Gln 435 440 445 Ser Gly Leu Glu Gly Val Phe 450 455 <210> 3 <211> 123 <212> PRT <213> Mus musculus <400> 3 Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Phe 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ala Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Asn Asp Asp Thr Ala Met Tyr Tyr Cys Ala 85 90 95 Arg Gly Asn Ser Asn His Tyr Val Ser Ser Phe Tyr Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 4 <211> 106 <212> PRT <213> Mus musculus <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Thr Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 119 <212> PRT <213> Mus musculus <400> 5 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Met Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Asn Gly Asn Phe Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 6 <211> 112 <212> PRT <213> Mus musculus <400> 6 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Phe Ser Cys Arg Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Ser Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Thr Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 7 <211> 5 <212> PRT <213> Mus musculus <400> 7 Asn Phe Gly Met Asn 1 5 <210> 8 <211> 6 <212> PRT <213> Mus musculus <400> 8 Gly Tyr Thr Phe Thr Asn 1 5 <210> 9 <211> 10 <212> PRT <213> Mus musculus <400> 9 Gly Tyr Thr Phe Thr Asn Phe Gly Met Asn 1 5 10 <210> 10 <211> 15 <212> PRT <213> Mus musculus <400> 10 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 1 5 10 15 <210> 11 <211> 8 <212> PRT <213> House mouse (Mus musculus) <400> 11 Trp Ile Asn Thr Tyr Thr Gly Glu 1 5 <210> 12 <211> 10 <212> PRT <213> House mouse (Mus musculus) <400> 12 Asn Gly Asn Phe Gly Tyr Tyr Tyr Phe Asp Tyr 1 5 10 <210> 13 <211> 16 <212> PRT <213> House mouse (Mus musculus) <400> 13 Arg Ser Ser Gln Asn Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 <210> 14 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 14 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 15 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 15 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 16 <211> 112 <212> PRT <213> House mouse (Mus musculus) <400> 16 Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Asn Pro Gly Glu Thr Val 1 5 10 15 Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 20 25 30 Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp 35 40 45 Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys Gly 50 55 60 Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln 65 70 75 80 Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala His 85 90 95 Gly Pro Trp Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 <210> 17 <211> 107 <212> PRT <213> Mus musculus <400> 17 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Val Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Ile Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu 100 105 <210> 18 <211> 5 <212> PRT <213> Mus musculus <400> 18 Asn Tyr Gly Met Asn 1 5 <210> 19 <211> 6 <212> PRT <213> Mus musculus <400> 19 Gly Tyr Thr Phe Thr Asn 1 5 <210> 20 <211> 10 <212> PRT <213> Mus musculus <400> 20 Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 <210> twenty one <211> 17 <212> PRT <213> House mouse (Mus musculus) <400> twenty one Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe Lys 1 5 10 15 Gly <210> twenty two <211> 10 <212> PRT <213> House mouse (Mus musculus) <400> twenty two Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> twenty three <211> 6 <212> PRT <213> House mouse (Mus musculus) <400> twenty three Gly Pro Trp Leu Ala Tyr 1 5 <210> twenty four <211> 11 <212> PRT <213> House mouse (Mus musculus) <400> twenty four Lys Ala Ser Gln Asp Ile Asn Val Tyr Leu Ser 1 5 10 <210> 25 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 25 Arg Ala Ile Arg Leu Val Asp 1 5 <210> 26 <211> 9 <212> PRT <213> Mus musculus <400> 26 Leu Gln Tyr Asp Glu Leu Pro Tyr Thr 1 5 <210> 27 <211> 117 <212> PRT <213> Mus musculus <400> 27 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ile Ala 20 25 30 Gly Met Gln Trp Val Gln Lys Met Pro Gly Lys Gly Leu Lys Trp Ile 35 40 45 Gly Trp Ile Asn Thr His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Ile Ala Tyr 65 70 75 80 Leu Gln Ile Ser Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser 115 <210> 28 <211> 107 <212> PRT <213> Mus musculus <400> 28 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr His Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ala Leu Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Asn Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 29 <211> 120 <212> PRT <213> Mus musculus <400> 29 Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Thr Thr Leu Thr Ala Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Ser Thr Leu Thr Val Ser Ser 115 120 <210> 30 <211> 113 <212> PRT <213> Mus musculus <400> 30 Glu Ile Val Leu Thr Gln Ser Ile Pro Ser Leu Thr Val Ser Ala Gly 1 5 10 15 Glu Arg Val Thr Ile Ser Cys Lys Ser Asn Gln Asn Leu Leu Trp Ser 20 25 30 Gly Asn Gln Arg Tyr Cys Leu Val Trp His Gln Trp Lys Pro Gly Gln 35 40 45 Thr Pro Thr Pro Leu Ile Thr Trp Thr Ser Asp Arg Tyr Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Val Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln 85 90 95 His Leu His Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 31 <211> 119 <212> PRT <213> Mus musculus <400> 31 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser His Tyr Ser Phe Ile Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Arg His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Ile Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asn Trp Gly Tyr Pro Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser 115 <210> 32 <211> 117 <212> PRT <213> Mus musculus <400> 32 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Arg Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Gln Lys Thr Pro Gly Lys Gly Leu Lys Trp Ile 35 40 45 Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Thr Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser 115 <210> 33 <211> 107 <212> PRT <213> Mus musculus <400> 33 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Leu Gly 1 5 10 15 Asp Arg Val Ser Phe Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Leu Ala Leu Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 34 <211> 6 <212> PRT <213> House mouse (Mus musculus) <400> 34 Gly Tyr Thr Phe Thr Thr 1 5 <210> 35 <211> 5 <212> PRT <213> House mouse (Mus musculus) <400> 35 Thr Ala Gly Met Gln 1 5 <210> 36 <211> 10 <212> PRT <213> House mouse (Mus musculus) <400> 36 Gly Tyr Thr Phe Thr Thr Ala Gly Met Gln 1 5 10 <210> 37 <211> 17 <212> PRT <213> House mouse (Mus musculus) <400> 37 Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe Lys 1 5 10 15 Gly <210> 38 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 38 Trp Ile Asn Ser His Ser Gly Val Pro 1 5 <210> 39 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 39 Gly Gly Asp Glu Gly Val Met Asp Tyr 1 5 <210> 40 <211> 11 <212> PRT <213> House mouse (Mus musculus) <400> 40 Lys Ala Ser Gln Asp Val Ser Thr Ala Val Ala 1 5 10 <210> 41 <211> 7 <212> PRT <213> House mouse (Mus musculus) <400> 41 Trp Thr Ser Thr Arg His Thr 1 5 <210> 42 <211> 9 <212> PRT <213> House mouse (Mus musculus) <400> 42 Gln Gln His Tyr Ser Thr Pro Trp Thr 1 5 <210> 43 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 44 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 45 Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 46 Asp Ile Val Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 47 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 47 Asp Ile Val Met Thr Gln Ser His Lys Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Phe Thr Cys Lys Ala Ser Gln Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Val Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln His Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 48 <211> 118 <212> PRT <213> Artificial <220> <213> Synthetic <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Gln Lys Ser Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 49 <211> 118 <212> PRT <213> Artificial <220> <213> Synthetic <400> 49 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 50 <211> 118 <212> PRT <213> Artificial <220> <213> Synthetic <400> 50 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Gln Lys Ser Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 51 <211> 118 <212> PRT <213> Artificial <220> <213> Synthetic <400> 51 Gln Ile Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Ala 20 25 30 Gly Met Gln Trp Val Gln Lys Thr Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Trp Ile Asn Ser His Ser Gly Val Pro Lys Tyr Ala Glu Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Gly Asp Glu Gly Val Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val Thr Val Ser Ser 115 <210> 52 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 53 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 54 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 54 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 55 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 56 <211> 107 <212> PRT <213> Artificial <220> <213> Synthetic <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ala Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 57 <211> 120 <212> PRT <213> Artificial <220> <213> Synthetic <400> 57 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 58 <211> 120 <212> PRT <213> Artificial <220> <213> Synthetic <400> 58 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Lys Thr Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 59 <211> 120 <212> PRT <213> Artificial <220> <213> Synthetic <400> 59 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Lys Thr Thr Leu Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 60 <211> 120 <212> PRT <213> Artificial <220> <213> Synthetic <400> 60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 61 <211> 120 <212> PRT <213> Artificial <220> <213> Synthetic <400> 61 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 62 <211> 111 <212> PRT <213> Mus musculus <400> 62 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Phe 20 25 30 Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Met Glu Glu Asp Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys 85 90 95 Glu Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 63 <211> 117 <212> PRT <213> Mus musculus <400> 63 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ala Met Asn Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile 35 40 45 Gly Val Ile Ser Thr Tyr Tyr Gly Asp Ala Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys 85 90 95 Ala Leu Ile Tyr Tyr Asp Tyr Asp Gly Ser Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser 115 <210> 64 <211> 113 <212> PRT <213> House mouse (Mus musculus) <400> 64 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 65 <211> 118 <212> PRT <213> House mouse (Mus musculus) <400> 65 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Tyr Asp Gly Tyr Tyr His Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser 115 <210> 66 <211> 115 <212> PRT <213> Mus musculus <400> 66 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Trp Ser 20 25 30 Val Asn Gln Lys Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Gln Arg Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Trp Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val His Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu 100 105 110 Glu Ile Lys 115 <210> 67 <211> 120 <212> PRT <213> Mus musculus <400> 67 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ser Ser Gly Phe Thr Phe Thr Thr Tyr 20 25 30 Trp Met Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asp Gly Asp Thr Arg Tyr Thr Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ile Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Asp Tyr Gly Asn Tyr Gly Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 68 <211> 112 <212> PRT <213> Mus musculus <400> 68 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn His Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 69 <211> 139 <212> PRT <213> Mus musculus <400> 69 Gln Val Gln Leu Gln Gln Ser Ala Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Glu Asn Asn Arg Lys Phe 50 55 60 Lys Asp Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Lys Gly Leu Leu Pro Pro Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val 115 120 125 Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr 130 135 <210> 70 <211> 107 <212> PRT <213> Mus musculus <400> 70 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Ala Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105

Claims

1. Use of an antibody that binds to a KIR3DL2 polypeptide and is capable of depleting cells expressing KIR3DL2, oxaliplatin and gemcitabine in the preparation of a pharmaceutical combination for preventing or treating T-cell lymphoma, wherein the antibody, oxaliplatin and gemcitabine are formulated for separate administration.

2. The use according to claim 1, wherein the prevention or treatment comprises 800-1000 mg / m 2 Gemcitabine is administered every two weeks at a dose of .

3. The use according to claim 1, wherein the prevention or treatment comprises administering the antibody from once a week to once every four weeks.

4. The use according to any one of claims 1 to 3, wherein the prevention or treatment comprises administration of the antibody once a week in a first phase, followed by a second phase in which the antibody is administered once every two weeks or once a month.

5. The use according to any one of claims 1 to 3, wherein the T-cell lymphoma is peripheral T-cell lymphoma.

6. The use according to claim 5, wherein the peripheral T-cell lymphoma is adult T-cell leukemia, enteropathy-associated T-cell lymphoma, PTCL-NOS or anaplastic large cell lymphoma.

7. The use according to any one of claims 1-3, wherein the prevention or treatment comprises administering the antibody at a dose of 1-20 mg / kg.

8. The use according to any one of claims 1 to 3, wherein the prevention or treatment comprises administering the antibody in a regimen comprising at least one administration cycle, wherein for each cycle in the at least one administration cycle, two, three or four doses of the antibody are administered and at least two, three or four doses of gemcitabine are administered.

9. The use according to any one of claims 1 to 3, wherein the treatment of a T-cell lymphoma in an individual comprises: a) determining the KIR3DL2 polypeptide status of malignant cells in said individual suffering from T-cell lymphoma; as well as b) after determining that the individual has a KIR3DL2 polypeptide expressed on the surface of a malignant cell, administering to the individual the combination of the antibody that binds to the KIR3DL2 polypeptide and the oxaliplatin and / or gemcitabine.

10. The use according to any one of claims 1-3, wherein the antibody comprises a human IgG1 or IgG3 heavy chain Fc domain.

11. The use according to any one of claims 1 to 3, wherein the antibody that binds to the KIR3DL2 polypeptide comprises: (a) a heavy chain variable region, the heavy chain variable region being as shown in SEQ ID NO: 49; And (b) a light chain variable region, wherein the light chain variable region is shown in SEQ ID NO:44 or 45.

12. The use according to any one of claims 1 to 3, wherein the KIR3DL2 antibody and the oxaliplatin and gemcitabine are administered simultaneously, separately or sequentially.

13. A kit comprising an anti-KIR3DL2 antibody that binds to a KIR3DL2 polypeptide and is capable of depleting cells expressing KIR3DL2, oxaliplatin, and gemcitabine.

14. Use of a substance for determining or evaluating the expression of KIR3DL2 in the preparation of a reagent or kit for predicting or evaluating the efficacy or suitability of oxaliplatin and gemcitabine for use in combination with an antibody that binds to a KIR3DL2 polypeptide and is capable of depleting cells expressing KIR3DL2 to treat TCL, wherein the prediction or evaluation comprises determining or evaluating whether the oxaliplatin and gemcitabine are capable of inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells, wherein determining that the oxaliplatin and gemcitabine are capable of inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells indicates that the oxaliplatin and gemcitabine can be used in combination with the anti-KIR3DL2 antibody to treat cancer.

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