Treatment of T-cell lymphoma

Abstract

The present invention relates to methods of treating T-cell lymphoma 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

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 795,194, filed January 22, 2019, which is incorporated by reference in its entirety, including any drawings.

[0002] Reference to sequence listing This application is submitted with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled "KIR-9 PCT_ST25", created on January 14, 2020, which is 54KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present invention relates to the use of KIR3DL2 targeting agents for the diagnosis and treatment of T cell lymphoma. [Background technology]

[0004] KIR3DL2 / CD158k is a cell surface receptor expressed on healthy circulating NK lymphocytes and a subpopulation of CD8+ T lymphocytes. However, KIR3DL2 has also been found on the surface of malignant T cells, particularly malignant CD4+ T cells, and as a result has further emerged as a target for the treatment of T cell lymphoma (TCL).

[0005] KIR3DL2-expressing TCLs include cutaneous T-cell lymphomas (CTCLs) such as mycosis fungoides (MF) and Sézary syndrome (SS) (see, e.g., US Pat. No. 5,399,633 (Innate Pharma) and US Pat. No. 5,499,663 (INSERM)) and peripheral T-cell non-Hodgkin's lymphomas (see, e.g., US Pat. No. 5,499,663 (Innate Pharma)). While MF and SS are relatively rare, PTCLs account for 15%-20% of high-grade lymphomas and 7%-10% of all non-Hodgkin's lymphomas (NHLs) in Western countries. They usually occur in middle-aged to elderly patients and are primarily characterized by disseminated disease in 68% of patients, with systemic manifestations in almost half (45%), bone marrow (BM) involvement in a quarter (25.8%) and extranodal disease in a third (37%). Despite aggressive therapy, more than half of patients die from their disease. Although certain disease states have improved prognostic signs when treated, prognosis for many aggressive PTCLs remains relatively unchanged with second and third generation chemotherapy regimens, with 5-year overall survival (OS) remaining at 25%-47% for PTCL-NOS, for example.

[0006] Antibodies that specifically bind to KIR3DL2 are well known in the art. Innate Pharma, for example, has described many anti-KIR3DL2 antibodies directed against a series 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 US Pat. No. 5,999,211 and US Pat. No. 5,999,211. Clinical trials by Innate Pharma have demonstrated promising results of anti-KIR3DL2 antibodies in human mycosis fungoides and Sezary syndrome patients. In particular, data from refractory SS patients revealed strong clinical activity upon treatment with repeated doses of single-agent IPH4102 (an 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. These encouraging results, with few side effects, and the ability to eliminate malignant KIR3DL2-expressing cells without causing the elimination of healthy KIR3DL2-expressing NK and T cells, represent an important advance in the treatment of TCL. However, some patients showed disease progression and / or did not respond adequately to initial treatment. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2010 / 081890 Brochure [Patent Document 2] International Publication No. 02 / 50122 Brochure [Patent Document 3] International Publication No. 2014 / 128221 Brochure [Patent Document 4] International Publication No. 2014 / 044681 Brochure [Patent Document 5] International Publication No. 2014 / 044686 Brochure Summary of the Invention [Problem to be solved by the invention]

[0008] As a result, there is a need in the art for improved benefits for patients with TCL. [Means for solving the problem]

[0009] The present invention stems from the discovery, inter alia, that gemcitabine and oxaliplatin can each cause increased KIR3DL2 expression in malignant T cells, and that when these agents are combined with anti-KIR3DL2 depleting antibodies, this results in a dramatic increase in the ability of immune cells (NK cells) to eliminate malignant T cells.

[0010] Interestingly, not only did gemcitabine and oxaliplatin each increase KIR3DL2 expression on TCL cells separately, but the combination of gemcitabine + oxaliplatin caused a further increase in KIR3DL2 expression on TCL cells. It was also observed that the triple combination of gemcitabine + oxaliplatin + anti-KIR3DL2 antibody resulted in a stronger ability of immune cells (NK cells) to eliminate malignant T cells compared to the individual drugs. Gemcitabine (or nucleoside analogues) and / or oxaliplatin (or other platinum compounds) can therefore be used to enhance the activity of anti-KIR3DL2 antibodies, or to enhance the ability of anti-KIR3DL2 antibodies to specifically cause lymphocytes (e.g., T cells, NK cells) to eliminate TCL cells.

[0011] The combination of gemcitabine and / or platinum-based compounds with anti-KIR3DL2 antibodies may therefore provide a particularly advantageous treatment of patients with TCL (eg, KIR3DL2-expressing TCL, PTCL, CTCL, SS or MF).

[0012] Gemcitabine (GEMZAR®) is a nucleoside metabolism inhibitor approved for many cancers, including: - in combination with carboplatin, for the treatment of advanced ovarian cancer that has recurred at least 6 months after completion of platinum-based therapy; - in combination with paclitaxel, for the first-line treatment of metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless an anthracycline is clinically contraindicated; in combination with cisplatin for the treatment of non-small cell lung cancer, and -As a single agent for the treatment of pancreatic cancer.

[0013] Oxaliplatin (ELOXATIN®) is a platinum-based drug approved for: - Adjuvant treatment of stage III colon cancer in patients who have undergone complete resection of the primary tumor; and - Treatment of advanced colorectal cancer.

[0014] Thus, in one aspect, the present invention provides an improved method for mediating anti-tumor immune responses against KIR3DL2-expressing malignant T cells through the use of antibodies that bind to KIR3DL2 in combination with an agent or treatment (e.g., a chemotherapeutic agent) that induces or increases expression of KIR3DL2 on the surface of malignant T cells.

[0015] In one aspect, the present disclosure provides an improved method for enhancing anti-tumor immune responses through the use of an antibody capable of binding to KIR3DL2 in combination with a means for inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells. In one embodiment, the means 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.

[0016] In one aspect, provided herein is an antibody that binds to KIR3DL2 for use in the treatment of TCL, wherein the antibody that binds to KIR3DL2 is administered in combination with an agent that induces or increases expression of KIR3DL2 on the surface of malignant T cells.

[0017] In one aspect, provided herein is an agent that induces or increases expression of KIR3DL2 on the surface of malignant T cells for use in the treatment of TCL, wherein the agent that induces or increases expression of KIR3DL2 on the surface of malignant T cells is administered in combination with an antibody that binds to KIR3DL2.

[0018] 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 to KIR3DL2.

[0019] In one embodiment, a method is provided for enhancing the anti-tumor effect of an antibody that binds to KIR3DL2, the method comprising administering to an individual an agent that induces or increases expression of KIR3DL2 on the surface of malignant T cells.

[0020] In one embodiment, the agent or means for inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells is a platinum-based agent. In one embodiment, the agent or means for inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells is a nucleoside metabolic inhibitor, optionally gemcitabine or an analog or derivative thereof. In one embodiment, the agent or means for inducing or increasing the expression of KIR3DL2 on the surface of malignant T cells is a combination agent or a platinum-based agent and a nucleoside metabolic inhibitor (e.g., gemcitabine or an analog or derivative thereof).

[0021] In one embodiment, the antibody that binds to KIR3DL2 is an antibody that mediates or induces effector cell-mediated lysis of KIR3DL2-expressing cells (e.g., malignant T cells). In one embodiment, the antibody comprises an Fc domain that can mediate ADCC and / or ADCP. In one embodiment, the antibody is expressed on 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 induces ADCC and / or ADCP toward KIR3DL2-expressing cells. In any aspect, an antibody that binds to a KIR3DL2 polypeptide and is capable of ablation of KIR3DL2-expressing cells can be characterized as a composition that is capable of ablation of KIR3DL2-expressing cells, the composition comprising an antibody that binds to a KIR3DL2 polypeptide. In one embodiment, the antibody that binds to KIR3DL2 is a lacutamab.

[0022] In one aspect of any of the embodiments herein, an individual may be designated as a human.

[0023] In one embodiment, the anti-KIR3DL2 antibody and the agent or means for inducing or increasing expression of KIR3DL2 on the surface of malignant T-cells are each administered in a therapeutically effective amount.

[0024] In one embodiment, anti-KIR3DL2 antibodies are administered to an individual with cancer in an amount and frequency sufficient to mediate ADCC towards KIR3DL2-expressing tumor cells.

[0025] In one embodiment, an agent or means for inducing or increasing expression of KIR3DL2 on 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 on the surface of malignant T cells.

[0026] In any embodiment herein, the individual treated according to the present disclosure may be an individual who is a non-responder or has experienced a partial or incomplete response to prior treatment for TCL.

[0027] In another embodiment, a method is provided for predicting or evaluating the efficacy or suitability of an anti-cancer agent for use in combination with an antibody that binds to a KIR3DL2 polypeptide for the treatment of TCL, comprising determining or assessing (e.g., in vitro) whether the anti-cancer agent can induce or increase the expression of KIR3DL2 on the surface of malignant T cells, where the determination that the anti-cancer agent can induce or increase the expression of KIR3DL2 on the surface of malignant T cells indicates that the agent can be used to treat cancer in combination with an anti-KIR3DL2 antibody. Determining or assessing whether the anti-cancer agent can induce or increase the expression of KIR3DL2 on the surface of malignant T cells may, for example, include contacting malignant KIR3DL2-expressing T cells (e.g., tumor cells) with the agent in vitro and assessing the expression of KIR3DL2. In one embodiment, the anti-cancer agent is a chemotherapeutic agent, optionally a nucleoside analog or a platinum agent.

[0028] These aspects are described in more detail in the description of the invention provided herein, and other aspects, features and advantages will become apparent from the description of the invention provided herein. [Brief description of the drawings]

[0029] [Figure 1] We demonstrated the cytotoxicity of anti-KIR3DL2 antibodies against HUT78 TCL cell line (Sezary syndrome), and the antitumor activity of anti-KIR3DL2 was enhanced by gemcitabine and oxaliplatin, respectively, and even more enhanced by the combination of gemcitabine and oxaliplatin. [Diagram 2]Figure 2 shows that gemcitabine and oxaliplatin increase cell surface KIR3DL2 expression in tumor cells. RAJI-KIR3DL2 (48 h incubation) (Figure 2, left panel) or Hut78 (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 tumor cell line surface was analyzed by flow cytometry. [Diagram 3] Shown is survival time for mice (n=9 per group) implanted 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 OF THE PREFERRED EMBODIMENTS

[0030] As used herein, "a" or "an" may mean one or more. When used in the claims, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more.

[0031] When "comprising" is used, this can optionally be exchanged for "consisting essentially of" or "consisting of."

[0032] At any time throughout this specification, "treatment of TCL" or the like will be referred to in relation to an anti-KIR3DL2 binding agent (e.g. an antibody) and refers to (a) a method of treatment of TCL comprising (for at least one treatment) the step of administering an anti-KIR3DL2 binding agent (e.g. in a pharma- ceutically acceptable carrier material) to a warm-blooded animal, in particular a human, in need of such treatment, in a dose (therapeutically effective amount) that allows for treatment of TCL, for example in the doses (amounts) specified above and below; (b) an anti-KIR3DL2 binding agent for the treatment of TCL or in said treatment (in particular (c) use of an anti-KIR3DL2 binding agent for use in a human (in a human); (d) use of an anti-KIR3DL2 binding agent for the manufacture of a pharmaceutical preparation for the treatment of TCL, a method of using an anti-KIR3DL2 binding agent in the manufacture of a pharmaceutical preparation for the treatment of TCL, the method comprising mixing an anti-KIR3DL2 binding agent with a pharma- ceutical carrier, or a pharmaceutical preparation comprising an effective dose of an anti-KIR3DL2 binding agent suitable for the treatment of TCL; or (e) any combination of a), b) and c) in accordance with subject matter acceptable for a patent in the country in which this application is filed.

[0033] The term "antibody" as used herein refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are assigned to one of 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, and the like. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The variable light chain (VLC) is a tetramer that ... variable light chain (VLC) is a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. L ) and variable heavy chain (V HThe terms "antibody" and "antibody-specific" refer to these light and heavy chains, respectively. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated "alpha", "delta", "epsilon", "gamma" and "mu", respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgGs are the exemplary class of antibodies used herein because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting. In one embodiment, the antibody is a monoclonal antibody. Humanized, chimeric, human or otherwise human-suitable antibodies are provided. "Antibody" includes full-length antibodies and any fragment or derivative of the antibodies described herein.

[0034] 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 comprises 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 amino acid 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 a similar system which determines the essential amino acids responsible for antigen binding. Typically, the numbering of the amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "Kabat variable domain residue numbering" and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or the light chain variable domain. 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 the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) as well as inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the homologous regions of the sequence of the antibody with the "standard" Kabat numbering sequence.

[0035] As used herein, "framework" or "FR" residues refer to the regions of an antibody variable domain excluding the regions defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3 and FR4) separated by the CDRs. The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., from about amino acid (aa) 230 to about aa 450 of the human γ (gamma) heavy chain or its corresponding sequences in other types of antibody heavy chains (e.g., α, δ, ε and μ for human antibodies) or their naturally occurring allotypes. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0036] The term "specifically binds to" means that the antibody is capable of binding 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 present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are described further below and are well known in the art.

[0037] When an antibody is said to "compete with" a particular monoclonal antibody, it is meant that the antibody competes with the monoclonal antibody in a binding assay using recombinant KIR3DL2 molecules or surface-expressed KIR3DL2 molecules. For example, if a test antibody reduces binding of AZ158, 19H12, 2B12, or 12B11 to a KIR3DL2 polypeptide or a KIR3DL2-expressing cell in a binding assay, the antibody is said to "compete with" AZ158, 19H12, 2B12, or 12B11, respectively.

[0038] The term "affinity" as used herein refers to the strength of binding of an antibody 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 a is defined 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, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993) and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. One standard method well known in the art for determining the affinity of mAbs is the use of surface plasmon resonance (SPR) screening (such as by analysis with a BIAcore™ SPR analysis device).

[0039] "Determinant" refers to a site of interaction or binding on a polypeptide.

[0040] The term "epitope" refers to an antigenic determinant and is the area or region on an antigen to which an antibody binds. A protein epitope may include amino acid residues directly involved in binding and those effectively blocked by a specific antigen-binding antibody or peptide, i.e., within the "footprint" of an antibody. It is the simplest form or smallest structural area on a complex antigen molecule that can be associated with, for example, an antibody or a receptor. Epitopes can be linear or conformational / structural. The term "linear epitope" is defined as an epitope composed of amino acid residues that are contiguous on a linear sequence of amino acids (primary structure). The term "conformational or structural epitope" is defined as an epitope composed of amino acid residues that are not all contiguous and therefore correspond to separated portions of a linear sequence of amino acids that are brought into close proximity with each other by folding of the molecule (secondary, tertiary and / or quaternary structure). Conformational epitopes depend on the three-dimensional structure. The term "conformational" is therefore often used interchangeably with "structural".

[0041] The terms "removing," "remove," or "removal" with respect to KIR3DL2-expressing cells refer to a process, method, or compound that can kill, remove, lyse, or induce such killing, removal, or lysis (e.g., through induction of ADCC or ADCP) so as to negatively affect the number of KIR3DL2-expressing cells present in a sample or subject.

[0042] The terms "immunoconjugate," "antibody conjugate," "antibody drug conjugate," and "ADC" are used interchangeably and refer to an antibody that is conjugated to another moiety (e.g., any non-antibody moiety, therapeutic agent, cytotoxic moiety, or label).

[0043] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological material. The term "therapeutic agent" refers to an agent that has biological activity.

[0044] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a term well understood in the art and refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Nonspecific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils and eosinophils.

[0045] 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 isoelectric focusing or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0046] 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 in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0047] The term "recombinant," for example, when used with reference to a cell or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found within the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are otherwise abnormally expressed, poorly expressed, or not expressed at all.

[0048] Within the context of this specification, the term antibody "binds to" a polypeptide or epitope denotes an antibody that binds to said determinant with specificity and / or affinity.

[0049] The terms "identity" or "identical," when used in the context of two or more polypeptide sequences, refer to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between a series of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller sequence of two or more sequences, with gap alignments (if any), as processed 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, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988).

[0050] Methods for determining identity are designed to produce the largest match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0051] Treatment of T-cell malignancies Described herein is a method useful for treating and preventing TCL in individuals through administration of an agent that induces or increases KIR3DL2 expression on the surface of malignant T cells to enhance the activity of an antibody that binds to KIR3DL2 polypeptide.The therapeutic regimen and treatment method described herein can be used for various T cell lymphomas, particularly CD4+ T cell lymphomas.In any embodiment, TCL can be designated as TCL characterized by malignant cells that express KIR3DL2 on their surface.

[0052] As shown herein, gemcitabine (Gemzar®, Eli Lilly and Company) can induce or increase KIR3DL2 expression on the surface of malignant T cells. Gemcitabine is a nucleoside analogue approved as a chemotherapy in the treatment of solid tumors. Like fluorouracil and other analogues of pyrimidine, this drug replaces one of the building blocks of nucleic acid, in this case cytidine, during DNA replication. Thus, gemcitabine and its analogues and derivatives can be used to enhance the activity of antibodies that bind to KIR3DL2 polypeptides.

[0053] As shown herein, oxaliplatin can induce or increase KIR3DL2 expression on the surface of malignant T cells. Oxaliplatin and other platinum agents are well known in the art, including, for example, cisplatin, carboplatin, nedaplatin, phenanthriplatin, picoplatin, satraplatin. Thus, platinum agents can be used to enhance the activity of antibodies that bind to KIR3DL2 polypeptides.

[0054] The platinum formulation and / or gemcitabine formulation may be in any suitable shape or form, including, for example, as a free compound or as part of a complex, in a nanoparticle formulation, encapsulated (e.g., in a liposome), in each case optionally further combined with an additional pharma- ceutically active agent.

[0055] Thus, in one aspect, a method of treating an individual with TCL is provided, comprising administering to the individual an effective amount of each of the following: (a) an antibody that binds to a KIR3DL2 polypeptide, and (b) a platinum drug and / or gemcitabine. In one embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide, a platinum drug (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 drug (e.g., oxaliplatin) without combination with the administration or use of gemcitabine. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and gemcitabine without combination with the administration or use of oxaliplatin. In another embodiment, the method comprises administering to the individual an antibody that binds to a KIR3DL2 polypeptide and gemcitabine without combination with the administration or use of a platinum drug.

[0056] In one embodiment, the platinum agent (eg, oxaliplatin) and / or gemcitabine are administered in an amount effective to cause increased expression of the KIR3DL2 polypeptide on the surface of the malignant cells.

[0057] In one aspect, a method is provided 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), comprising contacting the malignant cell with an effective amount of a platinum agent and / or gemcitabine. In one embodiment, the method further comprises removing the KIR3DL2-expressing malignant cell (e.g., having increased expression of KIR3DL2), which comprises contacting the malignant cell with an effective amount of an antibody that binds to the KIR3DL2 polypeptide.

[0058] Combination therapies for the treatment of cancer provided herein can include administration of (a) an anti-KIR3DL2 ablating antibody (e.g., a KIR3DL2-binding antibody or antibody fragment, an effector cell expressing on its surface a chimeric activating receptor (e.g., a T cell receptor) comprising a KIR3DL2-binding antibody fragment), and (b) a platinum agent, optionally oxaliplatin, to treat an individual having a T cell malignancy, optionally a CD4+ T cell malignancy.

[0059] In another embodiment, a combination therapy for the treatment of cancer provided herein comprises administration of (a) an anti-KIR3DL2-depleting antibody (e.g., a KIR3DL2-binding antibody or antibody fragment, an effector cell expressing on its surface a chimeric activating receptor (e.g., a T cell receptor) comprising a KIR3DL2-binding antibody fragment), and (b) a nucleoside analog, optionally gemcitabine, to treat an individual having a T cell malignancy, optionally a CD4+ T cell malignancy.

[0060] In another embodiment, a combination therapy for the treatment of cancer provided herein comprises administration of (a) an anti-KIR3DL2 depleting antibody (e.g., a KIR3DL2 binding antibody or antibody fragment, an effector cell expressing on its surface a chimeric activating receptor (e.g., a T cell receptor) comprising a KIR3DL2 binding antibody fragment), and (b) a platinum agent, optionally oxaliplatin, and (c) a nucleoside analog, optionally gemcitabine, to treat an individual having a T cell malignancy, optionally a CD4+ T cell malignancy.

[0061] Therapeutic methods are provided for treating individuals who have, are susceptible to, or have experienced TCL. In one embodiment, the TCL is high-grade or advanced TCL (e.g., greater than stage IV or more commonly stage II). In one embodiment, the patient has relapsed or refractory disease. In one embodiment, the patient has a poor prognosis for disease progression (e.g., poor prognosis for survival), a poor prognosis for response to therapy, or disease progression or recurrence after prior treatment with a prior therapy.

[0062] In one embodiment, the TCL is a high-grade T-cell neoplasm. In one embodiment, the TCL is a high-grade non-cutaneous TCL. In another embodiment, the TCL is a high-grade 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 diseases other than cutaneous T-cell lymphoma.

[0063] Cutaneous T-cell lymphoma (CTCL) (see image below) is a group of lymphoproliferative disorders characterized by the localization of neoplastic T-lymphocytes to the skin. Overall, CTCL is classified as a type of non-Hodgkin's lymphoma (NHL). The World Health Organization-European Organization for Research and Treatment of Cancer (WHO-EORTC) classification of CTCL is reported in Willemze et al. (2005) Blood 105:3768-3785. The WHO-EORTC divides CTCL into those with indolent clinical behavior and those with aggressive subtypes. The third category is that of hematologic precursor tumors that are not T-cell lymphomas (CD4+ / CD56+ hematologic tumors, blastic natural killer (NK) cell lymphomas, or primary tumors of B-cell origin). CTCLs that may have an 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 pleomorphic T-cell lymphoma (provisional). CTCLs that have an aggressive clinical behavior include Sézary syndrome (SS), adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, nasal type, primary cutaneous peripheral T-cell lymphoma, not otherwise specified, primary cutaneous high-grade 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.

[0064] 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 clinical features, medical history, and histomorphological and cytomorphological findings lead to the diagnosis. An additional diagnostic criterion to distinguish inflammatory skin diseases from CTCL is the demonstration of a predominant T-cell clone in skin biopsy specimens by molecular assays (e.g., Southern blot, polymerase chain reaction (PCR)). Genetic testing may also be considered. Classical mycosis fungoides is divided into three stages: (1) erythema (atrophic or nonatrophic): nonspecific dermatitis, erythema on the lower trunk and buttocks; slight pruritus / absent; (2) plaque: itch-inducing plaque, lymphadenopathy, and (3) mass: prone to ulceration. Sézary syndrome is characterized by erythroderma and leukemia. Signs and symptoms include skin edema, lymphadenopathy, palmar and / or plantar hyperkeratosis, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly. For the diagnosis of Sézary syndrome, criteria typically include absolute Sézary cell counts, immunophenotypic abnormalities, loss of T cell antigens, and / or T cell clones in peripheral blood as demonstrated by molecular or cytogenetic methods.

[0065] CTCL stages include I, II, III, and IV according to the TNM classification, including peripheral blood involvement, where appropriate. Peripheral blood involvement with mycosis fungoides or Sezary syndrome (MF / SS) cells correlates with more advanced skin stage, lymph node and visceral involvement, and shorter survival. MF and SS have formal staging systems proposed by the International Society for Cutaneous Lymphomas (ISCL) and the European Organization of Research and Treatment of Cancer (EORTC). See Olsen et al., (2007) Blood. 110(6): 1713-1722 and Agar et al. (2010) J. Clin. Oncol. 28(31): 4730-4739, the disclosures of which are incorporated herein by reference.

[0066] In one embodiment, the TCL is peripheral T-cell lymphoma (PTCL), optionally a non-cutaneous PTCL. PTCL and PTCL-NOS may be optionally designated as diseases other than cutaneous T-cell lymphoma, Sézary syndrome, and mycosis fungoides, which are considered separate pathologies. In one embodiment, the PTCL is a nodal (e.g., primarily or predominantly nodal) PTCL. Predominantly nodal PTCL includes, among others, PTCL-NOS (peripheral T-cell lymphoma, not otherwise specified), anaplastic large cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL). For example, the PTCL may be a high-grade, non-cutaneous, predominantly nodal PCTL (the disease may have extranodal manifestations as well).

[0067] In one embodiment, the PTCL is an extranodal (e.g., primarily extranodal) PTCL. For example, the PTCL can be high-grade non-cutaneous extranodal PCTL.

[0068] In one embodiment, the PTCL is adult T-cell leukemia or lymphoma (ATL), for example HTLV+ATL.

[0069] In one embodiment, the PTCL is extranodal NK / T-cell lymphoma, nasal type. In one embodiment, the PTCL is enteropathy-associated T-cell lymphoma.

[0070] In one embodiment, the PTCL is hepatosplenic T-cell lymphoma, optionally hepatosplenic αβ T-cell lymphoma, optionally hepatosplenic γδ T-cell lymphoma.

[0071] In one embodiment, the PTCL is anaplastic large cell lymphoma (ALCL), optionally ALK+ALCL, optionally ALK-ALCL. ALK+ALCL generally enjoys a good prognosis with conventional therapy (5-year survival rate 93%), whereas ALK-ALCL has a poor prognosis (37%). ALCL is generally characterized by uniform CD30 surface expression. In one embodiment, the 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.

[0072] In one embodiment, the PTCL is an intestinal lymphoma, such as intestinal ALCL.

[0073] In one embodiment, the PTCL is T-cell prolymphocytic leukemia.

[0074] In one embodiment, the PTCL is PTCL-NOS (peripheral T-cell lymphoma, not otherwise specified). PTCL-NOS, also called PCTL-U or PTCL-not otherwise specified, is an aggressive lymphoma that is predominantly nodal, although extranodal disease is common. The majority of nodal cases are CD4 + and CD8 -and CD30 may be expressed in the large cell variant. Most patients with PTCL-NOS present with nodal disease, but many extranodal sites may also be involved (e.g., liver, bone marrow, gastrointestinal, skin. Studies have generally reported that the 5-year overall survival is approximately 30%-35% with standard chemotherapy. Although many distinct entities have been described thus far that correspond to distinct subtypes of T-cell neoplasms, such as Lennert lymphoma, T-zone lymphoma, pleomorphic T-cell lymphoma, and T-immunoblastic lymphoma, evidence that these correspond to unique clinicopathological entities remains lacking. For these reasons, recent World Health Organization recommendations on hematopoietic and lymphoid neoplasms have not been fully addressed. In the classification by the World Health Organization (WHO), these have been combined under a single broad category, PTCL-NOS / U, whereby PTCL-NOS may be designated to exclude certain unique clinicopathologic entities such as T-cell prolymphocytic leukemia, ATL / adult T-cell leukemia, extranodal NK / T-cell leukemia nasal type, EATL / enteropathic 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.

[0075] The PTCL diagnostic criteria may be, for example, those of standard treatment guidelines according to the classification system of the World Health Organization (WHO) (e.g., World Health Organization. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4 th ed. Lyon, France: IARC Press, 2008.) See also, e.g., Foss et al. (2011) Blood 117:6756-6767, the disclosure of which is incorporated herein by reference.

[0076] In one embodiment, TCL is characterized by tumors or tumor cells that do not express significant and / or detectable CD30 polypeptide on their surface (CD30 negative). In another embodiment, TCL is characterized by tumors or tumor cells that express CD30 on their surface (CD30 positive); in one embodiment, the tumor or tumor cells express low levels of CD30 on their surface; in another embodiment, the tumor or tumor cells express high levels of CD30 on their surface. Optionally, CD30 expression is determined by immunohistochemistry.

[0077] In one exemplary aspect, a method is provided for reducing the progression of TCL in a mammalian host (e.g., a human patient) having detectable levels of cancer cells, 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.

[0078] In one exemplary embodiment, a method is provided for treating TCL in an individual who has a poor disease prognosis and / or has relapsed or is resistant or non-responsive to therapy with a first therapeutic agent.

[0079] Diagnosis of PTCL (e.g., PTCL-NOS) is typically based on peripheral blood examination or tissue biopsy for histological features, supplemented with detailed immunohistochemistry, flow cytometry, cytogenetic and molecular genetic testing. Examination may include, for example, complete blood count and differential, renal and liver function tests, lactate dehydrogenase (LDH), beta 2 microglobulin, albumin, serum calcium, uric acid, bone marrow biopsy, chest x-ray and computed tomography (CT) scans of the chest, abdomen and pelvis. Progression is optionally determined by evaluating selective clonal expansion of occult cells. Methods of detecting cancer and cancer progression can be achieved by any suitable technology, some examples of which are known in the art. Examples of suitable techniques include PCR and RT-PCR (e.g., of genes or "markers" associated with cancer cells), biopsies, imaging techniques, karyotyping and other chromosome analyses, immunoassays / immunocytochemical detection techniques, histological and / or histopathological assays, cell kinetic studies and cell cycle analysis, flow cytometry, and physical examination techniques (e.g., for physical symptoms).

[0080] Delivering an anti-KIR3DL2 antibody to a subject (either as an isolated or purified antibody (e.g., an antibody in solution), by direct administration as an antibody fragment expressed by a CAR cell, or by expression from a nucleic acid in the subject, such as from a poxvirus gene transfer vector comprising a nucleic acid sequence encoding an anti-KIR3DL2 antibody) and practicing other methods herein can be used to reduce, treat, prevent or otherwise ameliorate any suitable aspect of cancer progression (particularly TCL progression). The methods herein can be particularly useful in reducing and / or ameliorating tumor growth (e.g., percentage (tumor cells compared to healthy T cells), number of tumor cells in circulation) and any parameter or symptom (e.g., biomarker) associated with tumor growth. Methods that reduce, prevent or otherwise ameliorate such aspects of cancer progression individually and collectively are advantageous features.

[0081] In a further aspect, a method is provided for promoting remission of a TCL in a mammalian host, such as a human patient, comprising administering to the host a composition comprising an anti-KIR3DL2 antibody to promote TCL remission in the host.

[0082] In a further aspect, there is provided a method for reducing the risk of developing TCL, reducing the chance of developing a cancerous condition, and / or reducing the severity of TCL diagnosed at an early stage, comprising administering to a host a prophylactically effective amount of an anti-KIR3DL2 antibody or related composition to achieve a desired physiological effect.

[0083] In a further aspect, a method is provided for increasing the chances of survival over a reasonable period of time in a human patient diagnosed with TCL. In another aspect, a method is provided for improving the quality of life of a TCL patient, comprising administering to the patient an effective amount of a composition to improve the quality of life. In a further aspect, the method described herein can be applied to significantly reduce the number of TCL cells in a vertebrate host, for example, so that the total number of TCL cells is reduced. In a related sense, a method is provided for killing (e.g., directly or indirectly causing the death of) TCL cells in a vertebrate, such as a human cancer patient.

[0084] As used herein, adjunctive or combined administration includes simultaneous administration of the compounds in the same or different dosage forms or separate administration of the compounds (e.g., sequential administration). Thus, KIR3DL2 binding antibodies can be used in combination with platinum agents and / or gemcitabine. For example, anti-KIR3DL2 antibodies and platinum agents and / or gemcitabine can be administered simultaneously in a single dosage form. Alternatively, anti-KIR3DL2 antibodies and platinum agents and / or gemcitabine can be formulated for separate administration, administered in parallel or sequentially.

[0085] The optimal dose for gemcitabine administration is 800-1200 mg / m 2and is administered intravenously. Gemcitabine administration may be repeated once every two weeks. Alternatively, gemcitabine may be administered on days 1 and 8 of a three-week cycle, which may be repeated.

[0086] The appropriate dose for administration of oxaliplatin is 75-150 mg / m 2 (e.g., 75 mg / m 2 , 100 mg / m 2 ,130mg / m 2 , 75-130mg / m 2 ) and is administered intravenously. Oxaliplatin is administered at a dose of, for example, 75 to 100 mg / m 2 (e.g., 75 mg / m 2 , 100 mg / m 2 ) every 2 weeks. Alternatively, oxaliplatin can be administered once every 3 weeks (e.g., on days 1 and 8 of a 3-week cycle, if the cycle can be repeated). When oxaliplatin is administered once every 3 weeks, the dose range can optionally be higher than when administered every 2 weeks, e.g., 100-150 mg / m for an every 3 week regimen. 2 (e.g., 100 mg / m 2 , 130 mg / m 2 ) dosage.

[0087] Generally, the treatment includes at least one administration cycle (e.g., a duration of 8 weeks or less), in which, for each of the at least one cycle, 2, 3, or 4 doses of an anti-KIR3DL2 antibody are administered and at least 2, 3, or 4 doses of a platinum agent (e.g., oxaliplatin) and / or gemcitabine are administered.

[0088] The gemcitabine and oxaliplatin can be administered on the same day (e.g., day 1), or gemcitabine can be administered, for example, on the first day (day 1), followed by oxaliplatin the following day (day 2).

[0089] A suitable treatment protocol for treating a human with CTL (e.g., PTCL, CTCL, MF or SS) includes, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and gemcitabine, where the treatment comprises administering at least one dose, optionally at least two doses, of an anti-KIR3DL2 antibody (e.g., weekly, every two weeks, every four weeks) and at least one dose, optionally at least two doses, of gemcitabine, optionally at about 800-1200 mg / m 2 , optionally about 800-1000 mg / m 2 Optionally, gemcitabine is administered once every two weeks or on days 1 and 8 of a repeated three-week cycle. Optionally, the treatment protocol is characterized as not involving combination treatment with a platinum agent or oxaliplatin.

[0090] An exemplary treatment protocol for treating a human with MF or SS can include, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and gemcitabine, where the treatment comprises administering at least one dose, and optionally at least two doses, of an anti-KIR3DL2 antibody (e.g., every week, every two weeks, every four weeks) and at least one dose, and optionally at least two doses of gemcitabine, where the gemcitabine is administered at about 800-1200 mg / m on weeks 1, 2, and 3 (e.g., on days 1, 8, and 15) of a repeated four-week cycle. 2 The protocol includes at least one dosing cycle in which 4-week cycles are administered at a dose of 0.01 mg / kg / day. The 4-week cycles can be repeated, for example, for 3, 4, 5, 6 or more months. The protocol can be characterized as not involving combination treatment with platinum agents or oxaliplatin.

[0091] Further suitable treatment protocols for treating humans with CTL (e.g., PTCL) include, for example, administering to the patient an effective amount of each of an anti-KIR3DL2 antibody and oxaliplatin, where the treatment comprises administering at least one dose, optionally at least two doses, of an anti-KIR3DL2 antibody (e.g., every week, every two weeks, every four weeks) and at least one dose, optionally at least two doses of oxaliplatin, optionally at a dose of about 75-130 mg / m 2 Optionally, the oxaliplatin is administered once every two weeks or once every three weeks.

[0092] Another suitable treatment protocol 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, the treatment comprising at least one administration cycle comprising administration of: - at least one dose, optionally at least two doses (e.g., every week, every two weeks, every four weeks) of an anti-KIR3DL2 antibody; at least one dose of gemcitabine, optionally at least two doses, optionally wherein the gemcitabine is about 800-1000 mg / m 2 and optionally gemcitabine is administered once every two weeks or on days 1 and 8 of a repeated three-week cycle; and at least one dose, optionally at least two doses, of oxaliplatin, optionally wherein the oxaliplatin is about 75-130 mg / m 2 and optionally oxaliplatin is administered once every two weeks or once every three weeks (eg, ie, on day 1 of a repeated three-week cycle). In one embodiment, oxaliplatin and gemcitabine are each administered once every two weeks, optionally on the same day. In another embodiment, oxaliplatin and gemcitabine are each administered every three weeks, with oxaliplatin administered on day 1 of the three week period and gemcitabine administered on days 1 and 8 of the three week period.

[0093] It will be understood that the administration cycle can be of any suitable duration consistent with the administration frequency described herein. For example, the administration cycle can be 4 weeks, 8 weeks, more than 8 weeks, less than 8 weeks, etc.

[0094] 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., 1, 2, 3 days) after administration of gemcitabine. In one embodiment, the anti-KIR3DL2 antibody is administered at least one day (e.g., 1, 2, 3 days) after administration of oxaliplatin. In one embodiment, the anti-KIR3DL2 antibody is administered at least one day (e.g., 1, 2, 3, 4, 5, 6, or 7 days) after administration of both gemcitabine and oxaliplatin (e.g., at least one day after the latter is the compound administered).

[0095] Anti-KIR3DL2 antibodies (e.g., the humanized 2B12 antibody known as IPH4102 or lactamab) may be advantageously administered by intravenous infusion at a dose of 1-20 mg / kg body weight, optionally 1-10 mg / kg body weight, or at a flat dose of 750 mg. Anti-KIR3DL2 antibodies may be advantageously administered 1-4 times per month, for example, once per week, once every 2 weeks, or once every 4 weeks. In one embodiment, the anti-KIR3DL2 antibodies are administered according to a regimen comprising a first phase in which the anti-KIRDL2 antibody is administered on a weekly basis (e.g., administration every 4 weeks), followed by a second phase in which the anti-KIRDL2 antibody is administered less than once per week, for example, 1-2 times per month, once every 2 weeks, or once per month. In one embodiment, the anti-KIR3DL2 antibody is administered according to a regimen comprising a first phase in which the anti-KIRDL2 antibody is administered weekly (e.g., administration every 4 weeks), followed by a second phase in which the anti-KIRDL2 antibody is administered once every two weeks (e.g., at least two sequential administrations administered every two weeks, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more administrations administered once every two weeks), followed by a third phase in which the anti-KIRDL2 antibody is administered once per month (e.g., at least two sequential administrations administered once per month, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more administrations administered once per month).

[0096] When combined with gemcitabine and oxaliplatin, the method of treatment can be specified, for example, as including at least one dosing cycle (e.g., of 8 weeks or less, optionally 8 weeks, optionally 4 weeks duration), in which for each of the at least one cycle, 2, 3, or 4 doses of anti-KIR3DL2 antibody are administered (e.g., a flat dose of 750 mg of humanized 2B12 antibody at a dose of 1-20 or 1-10 mg / kg body weight) and at least 2, 3, or 4 doses of gemcitabine and oxaliplatin are administered. The gemcitabine and oxaliplatin can be specified as being administered on the same day or on consecutive days (e.g., 75-130 mg / m 2 The oxaliplatin is 800-1000mg / m 2(The KIR3DL2 binding antibody may be specified as being administered on the same day as gemcitabine and / or oxaliplatin, on consecutive days before or after gemcitabine and / or oxaliplatin, or several days before or after gemcitabine and / or oxaliplatin.

[0097] In the treatment method, the KIR3DL2 binding antibody and the platinum agent and / or gemcitabine agent can be administered separately, together, or sequentially or in a cocktail. In some embodiments, the KIR3DL2 binding antibody is administered prior to administration of the platinum agent and / or gemcitabine agent. For example, the KIR3DL2 binding antibody can be administered approximately 0-30 days, optionally 1-7 days, prior to administration of gemcitabine or 0-30 days, optionally 1-7 days, prior to administration of the platinum agent. In some embodiments, the KIR3DL2 binding antibody 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 1-5 days prior to administration of gemcitabine (or the platinum agent). In some embodiments, the KIR3DL2 binding antibody is administered in parallel with administration of a platinum agent and / or gemcitabine. In some embodiments, the KIR3DL2 binding antibody is administered after administration of a platinum agent and / or gemcitabine. For example, the KIR3DL2 binding antibody can be administered approximately 0 to 30 days, optionally 1 to 7 days, after administration of a platinum agent and / or gemcitabine. In some embodiments, the KIR3DL2 binding antibody 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 agent and / or gemcitabine.

[0098] In one embodiment, the anti-KIR3DL2 antibody and the platinum and / or gemcitabine agent are administered in the following doses: (a) 750 mg of anti-KIR3DL2 antibody and (i) 75 to 130 mg / m 2or (ii) 800 to 1200 mg / m 2 of gemcitabine; (b) 1 to 10 mg / kg or 750 mg of an anti-KIR3DL2 antibody and 75 to 130 mg / m 2 of oxaliplatin; (c) 1 to 10 mg / kg or 750 mg of an anti-KIR3DL2 antibody and 800 to 1200 mg / m 2 of gemcitabine; (d) 1 to 10 mg / kg or 750 mg of an anti-KIR3DL2 antibody, 75 to 100 mg / m 2 of oxaliplatin and 800-1000mg / m 2 of gemcitabine; (e) 1 to 10 mg / kg or 750 mg of an anti-KIR3DL2 antibody, 100 to 130 mg / m 2 of oxaliplatin and 800-1000mg / m 2 of gemcitabine.

[0099] Kits are also provided, for example kits that include: (i) a pharmaceutical composition containing an anti-KIR3DL2 antibody and a pharmaceutical composition containing gemcitabine; (ii) a pharmaceutical composition containing an anti-KIR3DL2 antibody and a pharmaceutical composition containing a platinum agent (e.g., oxaliplatin); (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; (iv) a first pharmaceutical composition containing an anti-KIR3DL2 antibody and instructions for administering the anti-KIR3DL2 antibody together with a platinum agent (e.g., oxaliplatin) and / or gemcitabine; (v) a pharmaceutical composition containing a platinum agent (e.g., oxaliplatin) and / or gemcitabine and instructions for administering said composition together with an anti-KIR3DL2 antibody; (vi) a pharmaceutical composition containing a platinum agent (e.g., oxaliplatin) and instructions for administering said composition together with an anti-KIR3DL2 antibody (and optionally further with gemcitabine); (vii) A pharmaceutical composition containing gemcitabine and instructions for administering said composition together with an anti-KIR3DL2 antibody (and optionally further with a platinum agent (e.g., oxaliplatin)).

[0100] The pharmaceutical composition may optionally be specified as including a pharma- ceutically acceptable carrier. The anti-KIR3DL2 antibody, platinum agent and / or gemcitabine may optionally be specified as being present in a therapeutically effective amount suitable for use in any method herein, optionally in an amount that increases KIR3DL2 levels on the surface of malignant TCL cells. The kit may also optionally include instructions, including an administration schedule, for example, to enable a practitioner (e.g., a doctor, a nurse, or a patient) to administer the composition contained therein to a patient with cancer (e.g., CTLC or PTCL, particularly as disclosed herein). In any embodiment, the kit may also include a syringe.

[0101] Optionally, the kit includes multiple packages of single-dose pharmaceutical compositions, each containing an effective amount of an anti-KIR3DL2 antibody and / or a platinum agent (e.g., oxaliplatin) and / or gemcitabine for a single administration according to the methods provided above. The kit may also include an instrument or device required for administering the pharmaceutical composition. For example, the kit may provide one or more pre-filled syringes or vials containing an amount of an anti-KIR3DL2 antibody, a platinum agent, or gemcitabine.

[0102] In one embodiment, the invention provides a kit for treating TCL in a human patient, the kit comprising: (a) a dose of an anti-KIR3DL2 antibody comprising the H-CDR1, H-CDR2 and H-CDR3 domains of a heavy chain variable region having a sequence as set forth in any one of SEQ ID NOs: 32 or 48 to 51, and the L-CDR1, L-CDR2 and L-CDR3 domains of a light chain variable region having a sequence as set forth in any one of SEQ ID NOs: 33 or 43 to 47; and / or (b) a dose of a platinum agent (e.g., oxaliplatin) and / or a dose of gemcitabine; and (c) optionally, instructions for using said anti-KIR3DL2 antibody and / or said platinum agent (e.g., oxaliplatin) and / or gemcitabine in any of the methods described herein.

[0103] In one embodiment, the treatment comprises administration of an anti-KIR3DL2 antibody, a platinum agent (eg, oxaliplatin) and gemcitabine, without any additional anti-cancer agents being administered.

[0104] It will be appreciated that the treatment methods of the present disclosure may or may not involve characterizing the tumor cells for KIR3DL2 expression prior to treatment. In any embodiment, KIR3DL2 expression may be determined by immunohistochemistry.

[0105] In one embodiment, the method includes: (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 expressing KIR3DL2 polypeptide. The determination that the individual has TCL cells expressing KIR3DL2 polypeptide indicates that the individual can be treated by the treatment method of the present disclosure. Optionally, the method includes administering the treatment of the present disclosure to the individual.

[0106] In one embodiment, KIR3DL2 polypeptides are expressed in a significant number of tumor cells (e.g., TCL cells) taken from a given patient. For example, KIR3DL2 may be present in at least 30%, 40%, 50%, 60%, 70%, 80% or more of TCL cells taken from a patient.

[0107] The method can optionally further comprise a step of assessing the level of TCL development, allowing assessment of the proportion (e.g., percentage) of malignant TCL cells present in a body compartment of the patient (disease staging). According to this method, cells from a biological sample collected from said body compartment are contacted with an anti-KIR3DL2 antibody and the tumor cells (e.g., the proportion of cells) expressing KIR3DL2 polypeptide on their surface are measured. The cells can be, for example, CD4+ cells or CD4-CD8+ cells. The finding that tumor cells express or predominantly express KIR3DL2 indicates that the TCL is an aggressive or advanced TCL (e.g., above stage IV or more generally stage II). Such TCL can be advantageously treated using the treatment method of the present disclosure.

[0108] In any embodiment, the method may include a step of diagnosing TCL comprising contacting cells from a biological sample from the individual with an anti-KIR3DL2 antibody and measuring the proportion (e.g., percentage) of T cells that express a KIR3DL2 polypeptide on their surface and comparing such proportion with the average proportion (e.g., percentage) of T cells that express a KIR3DL2 polypeptide on their surface observed in non-TCL humans (e.g., in healthy humans), where a diagnosis of TCL positivity is made if the measured proportion is significantly higher than the average proportion.

[0109] In another embodiment, the method includes determining the expression level of a KIR3DL2 nucleic acid or polypeptide in a biological sample from an individual who has been administered a platinum drug or gemcitabine, for example, for tumor cells found in the biological sample.

[0110] In another embodiment, the method includes a step of determining, for example, for tumor cells found in the biological sample, whether an anti-cancer drug (e.g., a platinum agent or gemcitabine) increases the expression level of a KIR3DL2 nucleic acid or polypeptide in a biological sample from the individual.

[0111] Determining the expression level of a KIR3DL2 nucleic acid or polypeptide in the biological sample may include comparing the level to a reference level (e.g., value, weak cell surface staining, etc.) corresponding to a healthy individual or to the individual prior to treatment with an anti-cancer agent (e.g., platinum or gemcitabine). A determination that the biological sample expresses a KIR3DL2 nucleic acid or polypeptide at a level that is increased compared to the reference level indicates that the individual has a TCL that may benefit from treatment with an anti-KIR3DL2 antibody, or optionally, that the individual has a TCL that may benefit from treatment with an anti-KIR3DL2 antibody in combination with an anti-cancer agent (e.g., platinum and / or gemcitabine, respectively). Optionally, detecting a KIR3DL2 polypeptide in the biological sample includes detecting a KIR3DL2 polypeptide expressed on the surface of a malignant lymphocyte.

[0112] Antibody production KIR3DL2 (CD158k) is a disulfide-linked homodimer of three Ig domain molecules 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 and corresponds to Genbank accession number AAB52520, with the 21 amino acid residue leader sequence omitted. [ka]

[0113] The cDNA for KIR3DL2 (allele *002) is shown in Genbank accession number U30272. The amino acid sequence of human KIR3DL2 allele *003 is shown below and corresponds to Genbank accession number AAB36593. [ka]

[0114] 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 with the wild-type, full-length KIR3DL2, respectively.

[0115] The closely related KIR3DL1 (CD158e1) is a monomeric molecule of about 70 kD 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 number L41269, and the encoded amino acid sequence is shown in Genbank accession number AAA69870. In one embodiment, the KIR3DL1 polypeptide referred to herein is allele *00101.

[0116] Examples of antibodies that bind to human KIR3DL2 include 19H12, 12B11, 10F6, 2B12, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9. These and further 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). For example, antibodies 10F6, 2B12, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 can be used as therapeutic agents administered to an individual to eliminate KIR3DL2 expressing targets, for example by inducing ADCC against pathogenic KIR3DL2 expressing cells, whereas antibodies 12B11 and 19H12 will be advantageous for detecting KIR3DL2 expression on the surface of cells (e.g. in vitro assays) since 12B11 is particularly efficient in detecting KIR3DL2 positive cells in detection assays, with 12B11 being advantageous for immunohistochemistry assays using frozen tissue sections, while 19H12 is advantageous for flow cytometry detection.

[0117] Each of 2B12, 10G5, 19H12 and 12B11 is suitable as a therapeutic agent administered to an individual for the elimination of KIR3DL2-expressing target cells. 19H12 and 12B11, as well as other antibodies disclosed in PCT / EP2013 / 069293, can be internalized into cells via 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 use when effector cell-mediated activity is required for the depleting antibody to induce, for example, ADCC.

[0118] The amino acid sequences of the heavy and light chain variable regions of antibodies 10F6, 9E10, 10G5, 13H1, 1E2, 1C3, and 20E9 are listed in Table A.

[0119] [Table 1]

[0120] In certain embodiments, an antibody is provided that essentially binds to the same epitope or determinant as any of the monoclonal antibodies AZ158, 19B12, 10G5, 12B11, 10G5 or 2B12, and optionally the antibody comprises the antigen-binding region of the antibody AZ158, 19B12, 12B11, 10G5 or 2B12. In any of the embodiments 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 the Fab or F(ab')2 portion of AZ158, 19B12, 12B11, 10G5 or 2B12. Also provided are monoclonal antibodies that comprise the heavy chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. According to one embodiment, the monoclonal antibody comprises the three CDRs of the heavy chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. Also provided are monoclonal antibodies further comprising the variable light chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12 or one, two or three CDRs of the light chain variable region of AZ158, 19B12, 12B11, 10G5 or 2B12. Optionally, any one or more of said light or heavy chain CDRs may contain one, two, three, four or five or more amino acid modifications (e.g., substitutions, insertions or deletions). Optionally, antibodies are provided in which either the light 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 a human IgG type immunoglobulin constant region, optionally a human constant region, optionally a human IgG1 or IgG3 isotype.

[0121] A KIR3DL2-binding antibody (or antibody fragment) for use in treating TCL can be in the form of, for example, an isolated and / or purified protein composition, or it can be present on or bound to the surface of a cell (e.g., a CAR effector cell such as a T cell, NK cell, or NKT cell), or it can further take the form of a nucleic acid encoding the antibody, such as from a pox or other viral gene transfer vector that contains an anti-KIR3DL2 antibody-encoding nucleic acid sequence. Cells expressing chimeric antigen receptors (CARs) can be constructed. An example of a CAR is engineered to contain an extracellular single-chain antibody (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and has the ability to redirect antigen recognition (i.e., KIR3DL2 recognition) based on the specificity of a monoclonal antibody when expressed in an effector cell such as a T cell, NKT cell, or NK cell. In one aspect, genetically engineered immune cells are provided that express a KIR3DL2-specific chimeric immune receptor that includes an intracellular signaling domain, a transmembrane domain (TM), and a KIR3DL2-specific extracellular domain (e.g., a domain derived from the variable heavy and light chain regions of a monoclonal antibody that specifically binds to KIR3DL2, such as one of the antibodies disclosed herein), carried on the cell surface membrane. Also provided are KIR3DL2-specific chimeric immune receptors, DNA constructs encoding the receptors, and plasmid expression vectors containing the constructs in the proper orientation for expression.

[0122] In one embodiment, a KIR3DL2 binding antibody comprises an antibody or antibody fragment that directs ADCC, and optionally further ADCP, against KIR3DL2-expressing cells.

[0123] In one embodiment, the antibody used in any of the embodiments herein binds to a KIR3DL2 polypeptide, and optionally the antibody does not substantially bind to a KIR3DL1 polypeptide, and has a binding affinity (K D)

[0124] The antibody optionally has a potency of 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, for HuT78 oncolysis by PBMCs from healthy volunteers. 51 EC in Cr release measurement 50 The antibody is optionally characterized by an EC of the anti-KIR3DL2 antibodies disclosed herein. 50 for HuT78 tumor lysis by PBMCs from healthy volunteers, comparable to that of 51 EC in Cr release measurement 50 (e.g., the EC of the 2B12 antibody disclosed herein having a VH of SEQ ID NO: 49 and a VL of SEQ ID NO: 44 or 45, comprising an Fc domain of wild-type or modified human IgG1 isotype, and mediating ADCC. 50 EC lower than or within 1-log or 0.5-log of 50 has.

[0125] Antibody AZ158 AZ158 binds to human KIR3DL2 and KIR3DL1 polypeptides (see WO 2010 / 081890). The VH of AZ158 is shown below with CDR1, 2 and 3 underlined, respectively. [ka]

[0126] The VL of AZ158 is shown below with CDR1, 2 and 3 underlined respectively. [ka]

[0127] An anti-KIR3DL2 antibody can include an antibody having variable region or CDR sequences from such an AZ158 antibody (e.g., a heavy 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, an anti-KIR3DL2 antibody can be an antibody other than an antibody having variable region or CDR sequences from an AZ158 antibody.

[0128] Antibody 19H12 The amino acid sequence of the heavy chain variable region of antibody 19H12 is listed below. [ka]

[0129] The amino acid sequence of the light chain variable region of antibody 19H12 is listed below. [ka]

[0130] In one aspect, a purified polypeptide encoding an antibody is provided, the antibody comprising an HCDR1 region comprising the amino acid sequence GYTFTNFGMN as set forth 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 may be replaced by a different amino acid; an HCDR2 region comprising the amino acid sequence WINTYTGEPTYADDF as set forth 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 may be replaced by a different amino acid; an HCDR3 region comprising the amino acid sequence NGNFGYYFDY as set forth in SEQ ID NO:12, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (e.g. an LCDR1 region comprising the amino acid sequence RSSQNIVHSNGNTYLE as set forth 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 may be replaced by a different amino acid); an LCDR2 region comprising the amino acid sequence KVSNRFS as set forth 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 may be replaced by a different amino acid); and / or an LCDR3 region comprising the amino acid sequence FQGSHVPFT as set forth in SEQ ID 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 replaced by a different amino acid, or the sequence may include an insertion of one or more amino acids).

[0131] In another aspect, (a) a heavy chain variable region of SEQ ID NO:5, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (b) a light chain variable region of SEQ ID NO: 6, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (c) a heavy chain variable region of SEQ ID NO: 5, in which one, two, three or more amino acid residues may be replaced by 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 replaced by different amino acids; and / or (d) the 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, in which one, two, three or more amino acid residues of any CDR may be replaced by different amino acids; and / or (e) the light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as set forth in SEQ ID NOs: 13, 14 or 15, respectively, in which one, two, three or more amino acid residues of any CDR may be replaced by different amino acids; and / or (f) the heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NOs: 7, 8 or 9, 10 or 11 and 12, respectively, in which one, two, three or more amino acid residues of any CDR may be replaced by different amino acids; and the light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 13, 14 or 15, in which one, two, three or more amino acid residues of any CDR may be replaced by different amino acids; and / or (g) a heavy chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having the amino acid sequence of SEQ ID NO:5, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (h) An antibody that binds to human KIR3DL2 is provided, comprising a light chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having the amino acid sequence of SEQ ID NO:6, wherein one, two, three or more amino acid residues may be replaced by different amino acids.

[0132] Antibody 12B11 The amino acid sequence of the heavy chain variable region of antibody 12B11 is listed below. [ka]

[0133] The amino acid sequence of the light chain variable region of antibody 12B11 is listed below. [ka]

[0134] In one aspect, a purified polypeptide encoding an antibody is provided, the antibody comprising: an HCDR1 region comprising the amino acid sequence GYTFTNYGMN as set forth 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)), where one or more of these amino acids may be replaced by a different amino acid; an HCDR2 region comprising the amino acid sequence WINTYTGEPTYADDFKG as set forth 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)), where one or more of these amino acids may be replaced by a different amino acid; an HCDR3 region comprising the amino acid sequence GPWLAY as set forth in SEQ ID NO: 23, or a sequence of at least 4, 5, 6, 7, 8 or 10 consecutive amino acids thereof (e.g., an LCDR1 region comprising the amino acid sequence KASQDINVYLS shown in SEQ ID NO:24 or a sequence of at least 4, 5, 6, 7, 8 or 10 consecutive amino acids thereof (wherein one or more of these amino acids may be replaced by a different amino acid); an LCDR2 region comprising the amino acid sequence RAIRLVD shown in SEQ ID NO:25 or a sequence of at least 4, 5, 6, 7, 8 or 10 consecutive amino acids thereof (wherein one or more of these amino acids may be replaced by a different amino acid); an LCDR3 region comprising the amino acid sequence LQYDELPYT shown in SEQ ID NO:26 or a sequence of at least 4, 5, 6, 7, 8 or 10 consecutive amino acids thereof (wherein one or more of these amino acids may be deleted or replaced by a different amino acid).

[0135] In another aspect, (a) a heavy chain variable region of SEQ ID NO: 16, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (b) a light chain variable region of SEQ ID NO: 17, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (c) a heavy chain variable region of SEQ ID NO: 16, in which one or more amino acid residues may be replaced by a different amino acid; and a light chain variable region of SEQ ID NO: 17, in which one, two, three or more of these amino acids may be replaced by a different amino acid; and / or (d) the heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as set forth in SEQ ID NOs: 18, 19 or 20, 21 or 22 and 23, respectively, in which one, two, three or more amino acid residues of any CDR may be replaced by different amino acids; and / or (e) the light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as set forth in SEQ ID NOs: 24, 25 and 26, in which one, two, three or more amino acid residues of any CDR may be replaced by a different amino acid; and / or (f) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NOs: 18, 19 or 20, 21 or 22 and 23, respectively, in which one or more amino acid residues of any CDR may be replaced by a different amino acid; and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 24, 25 and 26, in which one, two, three or more amino acid residues of any CDR may be replaced by a different amino acid; and / or (g) a heavy chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having the amino acid sequence of SEQ ID NO: 16, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (h) a light chain variable region that is at least 60%, 70%, 80%, 85%, 90% or 95% identical to a variable region having the amino acid sequence of SEQ ID NO: 17, wherein one, two, three or more amino acid residues may be replaced by different amino acids. The present invention provides an antibody that binds to human KIR3DL2, comprising:

[0136] Antibody 2B12 The amino acid sequence of the heavy chain variable region of antibody 2B12 is listed below (Kabat-defined CDRs are underlined): [ka]

[0137] The amino acid sequence of the light chain variable region of antibody 2B12 is listed below (Kabat CDRs are underlined): [ka]

[0138] In one aspect, a purified polypeptide encoding an antibody is provided, the antibody comprising an HCDR1 region comprising the amino acid sequence GYTFTTAGMQ as set forth in SEQ ID NO: 36, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof (e.g., GYTFTT (SEQ ID NO: 34) or TAGMQ (SEQ ID NO: 35)), wherein one or more of these amino acids may be replaced by a different amino acid; an HCDR2 region comprising the amino acid sequence WINSHSGVPKYAEDFKG as set forth in SEQ ID NO: 37, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof (e.g., WINSHSGVP (SEQ ID NO: 38)), wherein one or more of these amino acids may be replaced by a different amino acid; an HCDR3 region comprising the amino acid sequence GGDEGVMDY as set forth in SEQ ID NO: 39, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids thereof (e.g., , 9 or 10 consecutive amino acids thereof (wherein one or more of these amino acids may be replaced by a different amino acid); an LCDR1 region comprising the amino acid sequence KASQDVSTAVA 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 may be replaced by a different amino acid); an LCDR2 region comprising the amino acid sequence WTSTRHT 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 may be replaced by a different amino acid); an LCDR3 region comprising the amino acid sequence QQHYSTPWT 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 may be deleted or replaced by a different amino acid).

[0139] In another aspect, (a) the heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NOs: 34, 35 or 36 (HCDR1), 37 or 38 (HCDR2) and 39 (HCDR3), respectively, in which one, two, three or more amino acid residues of any CDR may be replaced by a different amino acid; and / or (b) the light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 40, 41 and 42, in which one, two, three or more amino acid residues of any CDR may be replaced by a different amino acid; and / or (c) heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) amino acid sequences as shown in SEQ ID NOs: 34, 35 or 36 (HCDR1), 37 or 38 (HCDR2) and 39 (HCDR3), respectively, in which one or more amino acid residues of any CDR may be replaced by a different amino acid; and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) amino acid sequences as shown in SEQ ID NOs: 40, 41 and 42, in which one, two, three or more amino acid residues of any CDR may be replaced by a different amino acid; and / or (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, in which one, two, three or more amino acid residues may be replaced by different amino acids; and / or (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, in which one, two, three or more amino acid residues may be replaced by different amino acids. An antibody that binds human KIR3DL2 is provided, comprising:

[0140] In other aspects of any of the embodiments herein, any of the heavy and light chain CDR1, 2 and 3 may be characterized by its sequence of at least 4, 5, 6, 7, 8, 9 or 10 contiguous amino acids and / or as having an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 85%, 90% or 95% sequence identity with a particular CDR or set of CDRs listed in the corresponding SEQ ID NO:

[0141] In another embodiment, for any of the above antibodies, there is provided an antibody that competes with the monoclonal antibodies of (a) to (h) for KIR3DL2 binding.

[0142] Humanized antibodies In another embodiment, the anti-KIR3DL2 antibody used in the methods of treatment of the invention is a humanized antibody, such as a humanized 2B12 or 10G5 antibody having a VH and VL selected from the 2B12 and 10G5 variable regions shown in Table B below.

[0143] For example, the humanized 2B12 antibody can include: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, 35, or 36; (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37 or 38; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 40; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 41; (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; and (g) Human framework sequences.

[0144] In one embodiment, the humanized antibody comprises a heavy chain framework from human subgroups VH1 and / or VH7 together with JH6, and optionally the antibody comprises IGHJ6 * 01 together with IGHV7-4-1 * 02 and / or IGHV1-c *In one embodiment, the humanized antibody comprises a light chain framework from human subgroups VK1 and / or VK4, optionally with JH4, comprising IGKV4-1 * 01 and / or IGKV1-39 * 01. Optionally select IGKJ4 * Including 01.

[0145] Optionally, the human framework includes, for example, one or more mutations, such as back mutations.

[0146] In another embodiment, 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 more identity) to the VH domain of humanized 2B12 of SEQ ID NOs: 48-51. In another particular embodiment, the humanized antibody comprises (a) a VH domain comprising non-human CDR residues incorporated within a human VH domain, where the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to a humanized 2B12 VH of SEQ ID NOs: 48-51, and (b) a VL domain comprising non-human CDR residues incorporated within a human VL domain, where the VL domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to a humanized 2B12 VL of SEQ ID NOs: 43-47.

[0147] In another embodiment, 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 more identity) to the VH domain of humanized 10G5 of SEQ ID NOs: 57-61. In another particular embodiment, the humanized antibody comprises (a) a VH domain comprising non-human CDR residues incorporated within a human VH domain, where the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98% etc.) identical to the humanized 10G5 VH of SEQ ID NOs: 57-61, and (b) a VL domain comprising non-human CDR residues incorporated within a human VL domain, where the VL domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98% etc.) identical to the humanized 10G5 VL of SEQ ID NOs: 52-56.

[0148] The 10G5 or 2B12 antibody may further comprise a native or engineered human IgG constant domain. Optionally, the constant domain is an IgG1 domain, optionally further comprising a modification to increase Fc receptor binding.

[0149] [Table 2]

[0150] [Table 3]

[0151] In one embodiment, the humanized 2B12 monoclonal antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 Includes.

[0152] In one embodiment, the humanized 2B12 monoclonal antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 45 Includes.

[0153] In one embodiment, the humanized 10G5 monoclonal antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:58, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53 Includes.

[0154] In one embodiment, the humanized 10G5 monoclonal antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:59, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 53 Includes.

[0155] In one embodiment, the humanized 2B12 antibody is or comprises the heavy and light chain amino acid sequences of the antibody IPH4102 (Innate Pharma, France), also known as lactamab (see WHO Drug Information, Vol. 32, No. 4, 2018). Lactamab is a humanized 2B12 antibody (having the Kabat heavy and light chain CDRs 1, 2 and 3 of 2B12) and comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44.

[0156] It will be appreciated that any suitable antibody may be used, but in one aspect, the antibody used binds to the same area or region as a known anti-KIR3DL2 antibody, including any of the anti-KIR3DL2 antibodies described herein. The anti-KIR3DL2 antibody may bind to an epitope that at least partially overlaps or includes at least one residue in the segment corresponding to residues 1-192, residues 1-98, or residues 99-192 of the KIR3DL2 polypeptide of SEQ ID NO:1 (or a subsequence thereof). In one embodiment, all of the significant residues of the epitope are in the segment corresponding to residues 1-192, residues 1-98, or residues 99-192 of the 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 the segment corresponding to residues 1-192, 1-98, or 99-192 of the KIR3DL2 polypeptide of SEQ ID NO:1. Preferably, the residues to which the antibody binds are present on the surface of the KIR3DL2 polypeptide.

[0157] Optionally, the antibody binds to an epitope that includes residue P179 and / or residue S181 of SEQ ID NO: 1. Optionally, the antibody binds to an epitope that includes 1, 2, 3, 4, 5, 6, 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.

[0158] PCT / EP2013 / 069302 and PCT / EP2013 / 069293 describe the testing of a series of 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 polypeptide (SEQ ID NO: 1). As used herein, reduced binding between an anti-KIR3DL2 antibody and a mutant KIR3DL2 polypeptide means that there is a reduced binding affinity (e.g., as measured by known methods such as FACS testing of cells expressing a particular mutant or by Biacore testing for binding to the mutant polypeptide) and / or a reduced overall binding ability of the anti-KIR3DL2 antibody (e.g., as evidenced by a reduced Bmax in a plot of anti-KIR3DL2 antibody concentration versus polypeptide concentration). A significant decrease in binding indicates that the mutated residue is either 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 thus include such a residue and may include additional residues that are spatially adjacent to such a residue.

[0159] In some embodiments, a significant decrease in binding means that the binding affinity and / or ability between the anti-KIR3DL2 antibody and the mutant KIR3DL2 polypeptide is reduced by more than 40%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% compared to the binding between the antibody and a wild-type KIR3DL2 polypeptide (e.g., the polypeptide set forth in SEQ ID NO:1). In certain embodiments, the binding is reduced to below detectable limits. In some embodiments, a significant decrease in binding is evidenced 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 a wild-type KIR3DL2 polypeptide (e.g., the extracellular domain set forth in SEQ ID NO:1). Such binding measurements can be generated using a variety of binding assays known in the art. A specific example of one such assay is described in the Examples section.

[0160] In some embodiments, anti-KIR3DL2 antibodies exhibit significantly lower binding to mutant KIR3DL2 polypeptides in which residues in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO: 1) have been substituted. In the shorthand notation used herein, the format is wild-type residue:position in polypeptide:mutant residue, with the numbering of the residues as shown in SEQ ID NO: 1.

[0161] Optionally, the antibody has reduced binding to a KIR3DL2 polypeptide having a substitution at residues N99, H100, E130, H131, F132, V178, P179, H180, S181, P182, Y183 and / or Q184 of SEQ ID NO:1.

[0162] In some embodiments, an 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., one, two, three, or four) of the following mutations: P179T and / or S181T (with respect to SEQ ID NO: 1). In one embodiment, binding to the mutant KIR3DL2 is significantly reduced compared to binding to wild-type KIR3DL2.

[0163] In some embodiments, an anti-KIR3DL2 antibody exhibits significantly reduced binding to a mutant KIR3DL2 polypeptide in which residues in the segment (or a subsequence thereof) corresponding to residues 1-98, residues 99-292, or residues 99-192 in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO:1) are replaced with different amino acids.

[0164] In one aspect, the antibody is capable of competing with monoclonal antibody AZ158, 10G5, 19H12, 2B12 or 12B11 and recognizes, binds to or has immunospecificity for substantially or essentially the same or the same epitope or "epitopic site" on the KIR3DL2 molecule as monoclonal antibody AZ158, 10G5, 19H12, 2B12 or 12B11. In other embodiments, the monoclonal antibody consists of or is a derivative or fragment of antibody AZ158, 10G5, 19H12, 2B12 or 12B11.

[0165] Although the antibody may bind to the same epitope as antibody AZ158, 10G5, 19H12, 2B12 or 12B11, it will be understood that suitable antibodies can recognize and be raised against any portion of the KIR3DL2 polypeptide, so long as the antibody binds to KIR3DL2 and has the desired functionality. For example, any fragment of KIR3DL2, e.g., human KIR3DL2, or any combination of KIR3DL2 fragments, can be used as an immunogen to generate antibodies, which can recognize epitopes anywhere within the KIR3DL2 polypeptide, so long as they can be recognized on KIR3DL2-expressing NK cells as described herein. In one embodiment, the recognized epitopes are present on the cell surface, i.e., they are accessible to antibodies present on the outside of the cell. Optionally, the epitope is an epitope specifically recognized by antibody AZ158, 10G5, 19H12, 2B12 or 12B11. Moreover, antibodies that recognize distinct epitopes within KIR3DL2 can be used in combination, for example, to bind KIR3DL2 polypeptide with maximal potency and breadth across different individuals.

[0166] Antibodies may be produced by various techniques known in the art. Typically, they are produced by immunization of 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 an immunogenic fragment, i.e., a portion of the polypeptide that comprises an epitope exposed on the surface of a cell expressing the KIR3DL2 polypeptide, optionally comprising an epitope recognized by the AZ158, 10G5, 19H12, 2B12 or 12B11 antibody. Such fragments typically contain at least about 7 contiguous amino acids of the mature polypeptide sequence or at least about 10 contiguous amino acids thereof. The fragments are typically derived essentially from the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human KIR3DL2 polypeptide, typically in a lipid membrane on the surface of a cell. In one embodiment, the immunogen comprises an intact cell, particularly an intact human cell, optionally treated or lysed, hi another embodiment, the polypeptide is a recombinant KIR3DL2 polypeptide.

[0167] The step of immunizing a non-human mammal with an antigen can be carried out by any method well known in the art to stimulate the production of antibodies in mice (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, type of buffer and amount of adjuvant are well known to those skilled in the art and are in no way limiting. These parameters may vary for different immunogens, but are easily ascertained.

[0168] Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies are also well known in the art. In a typical immunization protocol, non-human animals are injected intraperitoneally with antigen on day 1 and again about one week later. This is followed by a recall injection of antigen on about day 20, optionally with an adjuvant such as incomplete Freund's adjuvant. The recall injection is performed intravenously and can be repeated on consecutive days. This is followed by an intravenous or intraperitoneal booster injection on day 40, typically without an adjuvant. This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols can also be used, so long as they result in the production of B cells expressing antibodies directed to the antigen used in immunization.

[0169] For polyclonal antibody preparation, serum is obtained from the immunized non-human animal and the antibodies present therein are isolated by well known techniques. The serum can be affinity purified using any of the immunogens described above linked to a solid support to obtain antibodies reactive with the KIR3DL2 polypeptide.

[0170] In an alternative embodiment, lymphocytes from a non-immunized non-human mammal are isolated and grown in vitro and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.

[0171] For an exemplary monoclonal antibody, the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of those splenocytes with immortalized cells to form antibody-producing hybridomas. Isolation of splenocytes from a non-human mammal is well known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces, and forcing the splenocytes from the splenic membrane through the nylon mesh of a cell strainer in an appropriate buffer to produce a single-cell suspension. The cells are washed, centrifuged, and resuspended in a buffer that lyses any red blood cells. The solution is centrifuged again, and the remaining lymphocytes in the pellet are finally resuspended in fresh buffer.

[0172] Once isolated and in single cell suspension, lymphocytes can be fused to immortal cell lines. This is typically a mouse myeloma cell line, although many other immortal cell lines useful for making hybridomas are known in the art. Mouse myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, USA, X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland USA. Fusion is accomplished using polyethylene glycol or the like. The resulting hybridomas are then grown in a selection medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin and thymidine (HAT medium), which substances prevent growth of HGPRT-deficient cells.

[0173] Hybridomas are typically grown on a feeder layer of macrophages. The macrophages may be from a litter of the non-human mammal used to isolate the splenocytes, and are typically primed with incomplete Freund's adjuvant or similar several days before plating the 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.

[0174] The cells are allowed to grow in the selection medium for a time sufficient for colony formation and antibody production, which is usually from about 7 to about 14 days.

[0175] Hybridoma colonies are then assayed for the production of antibodies that specifically bind to the KIR3DL2 polypeptide gene product, optionally the epitope specifically recognized by the antibodies AZ158, 19H12, 2B12 or 12B11. While any assay that can be adapted to the wells in which the hybridomas are grown can be used, the assay is typically a colorimetric ELISA type assay. Other assays include radioimmunoassay or fluorescent activated cell sorting. Wells positive for the desired antibody production are examined to determine whether one or more distinct colonies are present. If two or more colonies are present, the cells may be recloned and grown to ensure that only a single cell has resulted in a colony that produces the desired antibody. Typically, the antibodies will also be tested for their ability to bind to the KIR3DL2 polypeptide, e.g., KIR3DL2-expressing cells.

[0176] Hybridomas confirmed to produce monoclonal antibodies can be grown in large quantities in an appropriate medium, such as DMEM or RPMI-1640. Alternatively, hybridoma cells can be grown in vivo as ascites tumors in animals. After sufficient growth to produce the desired monoclonal antibodies, the growth medium (or ascites fluid) containing the monoclonal antibodies is separated from the cells and the monoclonal antibodies present therein are purified. Purification is typically achieved by gel electrophoresis, dialysis, chromatography using Protein A or Protein G-Sepharose or anti-mouse Ig linked to a solid support such as agarose or Sepharose beads (all as described, for example, in Antibody Purification Handbook, Biosciences, publication No. 18-1037-46, Edition AC, the disclosures of which are incorporated herein by reference). Bound antibodies are typically eluted from Protein A / Protein G columns by using a low pH buffer (glycine or acetate buffers at or below pH 3.0) and the antibody-containing fractions are immediately neutralized. These fractions are optionally pooled, dialyzed and concentrated.

[0177] Positive wells, which have a single distinct colony, are typically recloned and reassayed to ensure that only one monoclonal antibody is being detected and produced.

[0178] Antibodies can also be produced by selection of 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.

[0179] Identification of one or more antibodies that bind to the antigen of interest, i.e., KIR3DL2, specifically or essentially the same region, determinant or epitope as monoclonal antibody AZ158, 19H12, 2B12 or 12B11, can be readily determined using any one of a variety of immunological screening assays that can assess antibody competition. Many such assays are routinely performed and are well known in the art (see, e.g., U.S. Patent No. 5,660,827, issued August 26, 1997, specifically incorporated herein by reference).

[0180] For example, if the test antibodies to be examined are obtained from different sources of animals or even different Ig isotypes, 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 added to a sample containing a KIR3DL2 polypeptide. Western blot-based protocols and the use of BIACORE analysis are suitable for use in such competition studies.

[0181] In some embodiments, a control antibody (e.g., AZ158, 19H12, 2B12, or 12B11) is premixed with varying amounts of test antibody (e.g., about 1:10 or about 1:100) for a period of time prior to addition to the KIR3DL2 antigen sample. In other embodiments, the control and varying amounts of test antibody can simply be mixed during exposure to the KIR3DL2 antigen sample. As long as it is possible to distinguish bound antibody from free antibody (e.g., by using a separation or washing technique to remove unbound antibody) and 2B12 from test antibody (e.g., by using a species- 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. Binding of the (labeled) control antibody in the absence of any unrelated antibody can serve as a control high value. A control low value can be obtained by incubating the labeled (AZ158, 19H12, 2B12 or 12B11) antibody with an unlabeled antibody of the exact same type (AZ158, 19H12, 2B12 or 12B11), which will result in competition and reduced binding of the labeled antibody. In the test assay, a significant reduction in labeled antibody reactivity in the presence of the test antibody indicates that the test antibody "cross-reacts" or competes with the labeled (AZ158, 19H12, 2B12 or 12B11) antibody. Any test antibody can be selected that reduces 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%) at any ratio of AZ158, 19H12, 2B12 or 12B11:test antibody from about 1:10 to about 1:100. In one embodiment, such a test antibody will reduce binding of AZ158, 19H12, 2B12 or 12B11 to the KIR3DL2 antigen by at least about 90% (e.g., about 95%).

[0182] Competition can also be evaluated, for example, by flow cytometry tests. In such tests, cells bearing a given KIR3DL2 polypeptide can be, for example, incubated first with 2B12 and then with a test antibody labeled with a fluorescent dye or biotin. An antibody is said to compete with 2B12 if the binding obtained upon 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 without preincubation with 2B12 (measured by fluorescence emission). Alternatively, an antibody is said to compete with 2B12 if the binding obtained by 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.

[0183] Determining whether an antibody binds within an epitope region can be performed by methods known to those skilled in the art. As one example of such a mapping / characterization method, the epitope region for an anti-KIR3DL2 antibody can be determined by epitope "footprinting" using chemical modification of exposed amines / carboxyls in the respective KIR3DL2 proteins. One specific example of such a footprinting technique is the use of HXMS (hydrogen-deuterium exchange detected by mass spectrometry), where hydrogen / deuterium exchange, binding and back-exchange of receptor and ligand protein amide protons occurs, and the backbone amide groups participating in protein binding are protected from back-exchange and therefore will remain deuterated. The relevant regions can be identified at this point by digestive proteolysis, fast microbore 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), which typically compares the positions of signals in two-dimensional NMR spectra of free antigen and antigen complexed with an antigen-binding peptide such as an antibody. The antigen is typically selectively isotopically labeled with 15N, and only signals corresponding to the antigen are seen in the NMR spectrum, and no signals from the antigen-binding peptide are seen. Antigen signals originating from amino acids typically involved in interactions with the antigen-binding peptide are shifted in position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in binding can be identified as such.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. 1996 Jun;9(3):516-24.

[0184] Epitope mapping / characterization can also be performed using mass spectrometry methods. See, for example, Downard, J Mass Spectrom. 2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May 1;71(9):1792-1801. Protease digestion techniques can also be useful in the context of epitope mapping and identification. Regions / sequences associated with antigenic determinants can be determined by protease digestion, for example using trypsin at a ratio of about 1:50 to KIR3DL2 or o / n digestion at pH 7-8, followed by mass spectrometry (MS) analysis for peptide identification. Peptides protected from trypsin cleavage by anti-KIR3DL2 binders can then be identified by comparison of samples that have been subjected to trypsin digestion and antibodies, and then subjected to digestion with, for example, trypsin (thereby revealing a footprint for the binder). Other enzymes such as chymotrypsin, pepsin, etc. can also or alternatively be used in similar epitope characterization methods. Furthermore, enzymatic digestion can provide a rapid method to analyze whether potential antigenic determinant sequences lie within regions of the KIR3DL2 polypeptide that are not surface exposed and therefore likely not relevant in terms of immunogenicity / antigenicity.

[0185] Site-directed mutagenesis is another technique useful for elucidating binding epitopes. For example, in "alanine scanning", each residue in a protein segment is replaced with an alanine residue and the result on binding affinity is measured. If the mutation leads to a significant decrease in binding affinity, it is likely 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 the alanine substitutions do not affect the overall fold 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.

[0186] Electron microscopy can also be used for epitope "footprinting." For example, Wang et al., Nature 1992;355:275-278 used cryo-electron microscopy, 3D image restoration, and X-ray crystallography applied in tandem to determine the physical footprint of Fab fragments on the capsid surface of native cowpea mosaic virus.

[0187] Other forms of "label-free" assays for epitope evaluation include surface plasmon resonance (SPR, BIACORE) and reflectance interferometry (RifS). See, e.g., Faegerstam et al., Journal Of Molecular Recognition 1990;3:208-14; Nice et al., J. Chroma-togr. 1993;646:159-168; Leipert et al., Angew. Chem. Int. Ed. 1998;37:3308-3311; Kroeger et al., Biosensors and Bioelectronics 2002;17:937-944.

[0188] 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.

[0189] Once antibodies capable of binding to KIR3DL2 and / or having other desired properties (e.g., the ability to mediate ADCC and / or antibody-dependent cellular phagocytosis (ADCP)) have been identified, they will typically also be evaluated for their 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 substantial affinity and will not bind to unrelated polypeptides at significant levels. However, it will be understood that the antibody is suitable for use in the methods of the invention so long as its affinity for KIR3DL2 is substantially greater (e.g., 5x, 10x, 50x, 100x, 500x, 1000x, 10,000x or more) than it is for other unrelated polypeptides.

[0190] Binding of antibodies to KIR3DL2-expressing cells can also be assessed in other mammals, such as non-human primates, e.g., cynomolgus monkeys or mice. Antibodies, as well as fragments and derivatives thereof, are provided, which specifically bind to KIR3DL2, which further bind to KIR3DL2 from a non-human primate, e.g., cynomolgus monkey.

[0191] In the immunization and production of antibodies in vertebrates or cells, certain selection steps may be performed to isolate the claimed antibodies. In this regard, in certain embodiments, the present disclosure relates to a method of producing such antibodies, 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.

[0192] In one aspect of any of the embodiments, the antibodies prepared according to the methods of the invention are monoclonal antibodies, hi other aspects, the non-human animals used to produce the antibodies are mammals such as rodents, cows, pigs, poultry, horses, rabbits, goats or sheep.

[0193] According to an alternative embodiment, DNA encoding an antibody that binds an epitope present on a KIR3DL2 polypeptide is isolated from the hybridoma and placed into a suitable expression vector for transfection into a suitable host. The host is then used for the recombinant production of the antibody or variants thereof, such as humanized versions of the monoclonal antibody, active fragments of the antibody, chimeric antibodies containing the antigen recognition portion of the antibody, or versions containing a detectable moiety.

[0194] DNA encoding a monoclonal antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of a murine antibody). Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, which do not otherwise produce immunoglobulin proteins, to achieve synthesis of the monoclonal antibody in the recombinant host cell. As described elsewhere herein, such DNA sequences can be modified for any of a number of purposes, such as to humanize the antibody, to produce fragments or derivatives, or to optimize the binding specificity of the antibody, e.g., to modify the sequence of the antibody in the antigen-binding site.

[0195] Recombinant expression in bacteria of DNA encoding the antibody is well known in the art (see, e.g., Skerra et al., Curr. Opinion in Immunol., 5, pp. 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992)).

[0196] Once an antigen-binding compound is obtained, it can be evaluated for its ability to induce ADCC, ADCP and / or CDC towards and / or remove KIR3DL2-expressing target cells. Evaluation of the ability of an antigen-binding compound to induce ADCC, ADCP and / or CDC (complement dependent cytotoxicity) or generally lead to removal or inhibition of activity of KIR3DL2-expressing target cells can be carried out at any suitable stage of the method. This evaluation can be useful at one or more of the various steps involved in the identification, production and / or development of antibodies (or other compounds) designated for therapeutic use. For example, activity can be evaluated in the context of a screening method to identify a candidate antigen-binding compound or in a method in which an antigen-binding compound is selected and made human-suitable (e.g., in the case of antibodies, chimeric or humanized), a cell expressing the antigen-binding compound (e.g., a host cell expressing a recombinant antigen-binding compound) is obtained and evaluated for its ability to produce a functional antibody (or other compound), and / or a quantity of antigen-binding compound is produced and evaluated for activity (e.g., testing a batch or lot of product). Generally, the antigen-binding compound will be known to specifically bind to a KIR3DL2 polypeptide. A step may involve testing a plurality (e.g., a very large number or a small number using high-throughput screening methods) of antigen-binding compounds.

[0197] Tests for ADCP, CDC and ADCC can be performed and determined by various assays, including those known in the art and described in the experimental examples herein. Tests for ADCC typically involve assessing cell-mediated cytotoxicity, in which anti-KIR3DL2 antibody-bound KIR3DL2-expressing target cells (e.g., TCL cells or other KIR3DL2-expressing cells) are recognized by effector cells bearing Fc receptors without complement involvement. Cells that do not express the KIR3DL2 antigen can optionally be used as a control. NK cytotoxicity activation is assessed by measuring an increase in cytokine production (e.g., IFN-γ production) or cytotoxicity markers (e.g., CD107 recruitment). In one embodiment, the antibody will induce at least 20%, 50%, 80%, 100%, 200% or 500% target cell lysis in the presence of target cells compared to increased cytokine production, expression of cytotoxic markers, or a control antibody (e.g., an antibody that does not bind KIR3DL2, a KIR3DL2 antibody with a mouse constant region). In other examples, target cell lysis is detected, for example, in a chromium release assay, e.g., the antibody will induce at least 10%, 20%, 30%, 40% or 50% lysis of target cells.

[0198] Antibody fragments and derivatives (included by the term "antibody" as used in this application unless otherwise specified or clearly contradicted by the context) can be produced by techniques known in the art. A "fragment" includes a portion of an intact antibody, generally the antigen-binding site or the variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; bispecific antibodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as "single-chain antibody fragments" or "single-chain polypeptides"), including, without limitation, (1) a single-chain Fv molecule, (2) a single-chain polypeptide containing only one light chain variable domain or a fragment thereof containing the three CDRs of the light chain variable domain, without associated heavy chain components, and (3) a single-chain polypeptide containing only one heavy chain variable region or a fragment thereof containing the three CDRs of the heavy chain variable region, without associated light chain components; and multispecific antibodies formed from antibody fragments. These include, inter alia, nanobodies, domain antibodies, single domain antibodies or "dAbs".

[0199] In certain embodiments, the DNA of the hybridoma producing the antibody can be modified prior to insertion into an expression vector, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous non-human sequences (e.g., Morrison et al., PNAS pp. 6851 (1984)) or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In that way, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the original antibody. Typically, such a non-immunoglobulin polypeptide is substituted for the constant domains of the antibody.

[0200] Thus, according to another embodiment, the antibody is a humanized antibody. "Humanized" forms of antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from mouse immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are exchanged for residues from the CDRs of the original antibody (donor antibody) while retaining the desired specificity, affinity and capacity of the original antibody.

[0201] In some cases, Fv framework residues of the human immunoglobulin may be replaced by corresponding non-human residues. Furthermore, humanized antibodies can include residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of the original antibody and all or substantially all of the FR regions being of human immunoglobulin consensus sequences. A humanized antibody optimally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321, pp. 522 (1986); Reichmann et al, Nature, 332, pp. 323 (1988); Presta, Curr. Op. Struct. Biol., 2, pp. 593 (1992); Verhoeyen et Science, 239, pp. 1534; and U.S. Patent No. 4,816,567, the entire disclosures of which are incorporated herein by reference.) Methods for humanizing antibodies are well known in the art.

[0202] The choice of human variable domains, both light and heavy chains, to be used to generate a humanized antibody is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of an antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to that of the mouse is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J.Immunol. 151, pp.2296 (1993); Chothia and Lesk, J.Mol. 196, 1987, pp.901). Other methods use a specific framework derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al., PNAS 89, pp.4285 (1992); Presta et al., J.Immunol., 151, p.2623 (1993)).

[0203] It is further important that antibodies are humanized while retaining high affinity for the KIR3DL2 receptor and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the 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 depict and display likely three-dimensional structures of candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from consensus and import sequences so that desired antibody characteristics, such as increased affinity for the target antigen, are achieved. In general, CDR residues are involved in directly and most substantially influencing antigen binding.

[0204] Another method for producing human or humanized monoclonal antibodies is to use genetically modified mice for immunization. Several mouse hosts are known in which the immunoglobulin genes have been replaced by functional human immunoglobulin genes. Thus, the antibodies produced by this mouse or in hybridomas produced from the B cells of such mice are already human or humanized antibodies. Exemplary animals are described in U.S. Pat. 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 selection of antibody repertoires using phage display methods. Such techniques are known to those skilled in the art and can be carried out starting from the monoclonal antibodies disclosed in this application.

[0205] The KIR3DL2 binding compound, e.g., an anti-KIR3DL2 antibody, can be further conjugated to a second moiety, and the antibody can deliver the second moiety to a KIR3DL2-expressing cell. Optionally, the second moiety is a therapeutic agent, a toxic agent, and / or a detectable agent.

[0206] Although it is expected that the antibodies in their underivatized or unmodified form, particularly IgG1 or IgG3 type, will inhibit the proliferation of hyperproliferating cells or be cytotoxic to hyperproliferating cells, such as those from PTCL patients, for example by directing ADCC, ADCP and / or CDC against KIR3DL2-expressing PTCL cells, it is also possible to prepare derivatized antibody immunoconjugates that are cytotoxic. In one embodiment, once KIR3DL2-specific antibodies have been isolated and optionally modified to other states (e.g., humanized), they will be derivatized to make them toxic to cells. Thus, administration of the antibodies to PTCL patients will result in relatively specific binding of the antibodies to hyperproliferating cells, thereby directly killing or inhibiting the cells underlying the disorder.

[0207] In view of the ability of anti-KIR3DL2 antibodies to induce ADCC and CDC, the antibodies can be modified to increase their ability to bind to Fc receptors, which can also affect effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation and phagocytosis, and immunoregulatory signals such as modulation of lymphocyte proliferation and antibody secretion. Exemplary modifications include modified human IgG1 constant regions, including at least one amino acid modification (e.g., substitution, deletion, insertion) and / or altered types of glycosylation, e.g., hypofucosylation.

[0208] The anti-KIR3DL2 antibody may comprise an Fc domain (or a portion thereof) of the human IgG1 or IgG3 isotype, optionally modified.

[0209] In certain embodiments, the CH2 and / or CH3 domains (or the Fc domains comprising them) may comprise 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 modifications will not substantially reduce or eliminate the ability of the Fc-derived polypeptide to bind to neonatal Fc receptors (FcRn), e.g., human FcRn. Exemplary modifications include modified human IgG1-derived constant regions that include at least one amino acid modification (e.g., substitution, deletion, insertion) and / or an altered type of glycosylation, e.g., hypofucosylation. Such modifications can affect interactions with the Fc receptors: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). FcγRI (CD64), FcγRIIA (CD32A) and FcγRIII (CD16) are activating (i.e. immune system enhancing) receptors, whereas FcγRIIB (CD32B) is an inhibitory (i.e. immune system suppressing) receptor. Modifications may, for example, increase binding of the Fc domain to FcγRIIIa on effector (e.g. NK) cells and / or decrease binding to FcγRIIB. Examples of modifications are provided in WO 2014 / 044686, the disclosure of which is incorporated herein by reference. Specific mutations (in the IgG1 Fc domain) that affect (enhance) FcγRIIIa or FcRn binding are further described below.

[0210] [Table 4]

[0211] The mutant Fc region can comprise at least one amino acid modification (e.g., having 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, which modification enhances binding to a human CD16 polypeptide. In other embodiments, the Fc region comprises 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 at amino acids 237-341 or in the lower hinge-CH2 region comprising residues 231-341. In some embodiments, the Fc region comprises at least two amino acid modifications (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), at least one such modification being in the CH3 region and at least one such modification being in the CH2 region. Amino acid modifications in the hinge region are also encompassed. In one embodiment, an amino acid modification in the CH1 domain, optionally in the upper hinge region comprising residues 216-230 (Kabat EU numbering), is included.Any suitable functional combination of Fc modifications can be made, for example any combination of different Fc modifications, such as those described in U.S. Pat. No. 7,632,497; U.S. Pat. No. 7,521,542; U.S. Pat. No. 7,425,619; U.S. Pat. No. 7,416,727; U.S. Pat. No. 7,371,826; U.S. Pat. No. 7,355,008; U.S. Pat. No. 7,335,742; U.S. Pat. No. 7,332,581; U.S. Pat. No. 7,183,387; U.S. Pat. No. 7,122,637; U.S. Pat. No. 6,821,505 and U.S. Pat. No. 6,737,056; and / or WO 2011 / 109400; ... WO 08 / 105886;WO 2008 / 002933;WO 2007 / 021841;WO 2007 / 106707;WO 06 / 088494;WO 05 / 115452;WO 05 / 110474;WO 04 / 1032269;WO 00 / 42072;WO 06 / 088494;WO 07 / 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, L Get al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, R Let al. (2002) J. Biol. Chem. 26;277(30):26733-26740 and Shields, R Let al. (2001) J. Biol. Chem. 276(9):6591-6604). EXAMPLES

[0212] Example 1 – In vitro combination studies with chemotherapeutic agents It is known that chemotherapeutic agents can negatively affect the host immune system and, as a result, inhibit the efficacy of therapeutic antibodies in mediating ADCC. As a result, we sought to evaluate whether chemotherapeutic agents affect the NK cell-mediated ADCC activity of the humanized 2B12 antibody lactamab, which has a human IgG1 isotype and the 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 in the presence of increasing concentrations of anti-KIR3DL2 antibodies, together with allogeneic NK cells and a KIR3DL2-expressing Sézary syndrome cell line (Hut78).

[0213] The results are shown in Figure 1. In this experimental setting, neither gemcitabine nor oxaliplatin prevented anti-KIR3DL2 antibody-induced ADCC against the HUT78 CTCL cell line. Indeed, the anti-KIR3DL2 antitumor activity was surprisingly enhanced by gemcitabine and oxaliplatin, respectively, and further enhanced by the combination of gemcitabine and oxaliplatin.

[0214] Example 2 – Gemcitabine and oxaliplatin each enhance KIR3DL2 expression To investigate possible reasons for the enhanced anti-KIR3DL2 antibody-induced ADCC against the HUT78 CTCL cell line, we assessed whether KIR3DL2 expression could be modulated by the same chemotherapeutic agents in vitro.

[0215] Briefly, Hut78 cells (Sézary 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 the tumor cell lines was analyzed by flow cytometry.

[0216] The results are shown in Figure 2. RAJI-KIR3DL2 cells are shown in panel A (left side) and Hut78 cells are shown in panel B (right side). Both oxaliplatin and gemcitabine were found to enhance KIR3DL2 surface expression on both cell lines in a dose-dependent manner until it reached a plateau of 2-3 fold compared to baseline (Figure 2). These figures are representative of two independent experiments. Interestingly, other chemotherapeutic agents such as cyclophosphamide did not increase KIR3DL2 expression in the same experimental conditions.

[0217] Overall, these results support the utilization of anti-KIR3DL2 antibody combinations with gemcitabine and oxaliplatin, especially in patients with relapsed PTCL who may be particularly in need of improved treatment.

[0218] Example 3 – In vivo efficacy Xenograft models in CB17-SCID mice, an immunodeficient mouse strain that retains fully functional NK cells and macrophages. In addition, mouse Fc receptors efficiently bind human Fc, allowing mouse 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 transfectants were inoculated by IV into SCID mice (5 × 10 6 Cells), which resulted in 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 was not possible to find doses of gemcitabine and oxaliplatin that could be combined in these mice in an active but tolerable manner. As a result, anti-KIR3DL2 antibody (lactamab) was combined separately with gemcitabine or oxaliplatin.

[0219] In this disseminated tumor model (n=9 mice per group), groups of mice were therefore treated twice per week with: - isotype control mAb, - Anti-KIR3DL2 antibody (lactamab), - Gemcitabine (50mg / kg) - oxaliplatin (5mg / kg), - Anti-KIR3DL2 antibody (lactamab) + gemcitabine (50mg / kg), or - Anti-KIR3DL2 antibody (lactamab) + oxaliplatin (5mg / kg).

[0220] Both the anti-KIR3DL2 antibody and the isotype control were administered at 0.3 μg per injection.

[0221] The results are shown in Figure 3. The combination of anti-KIR3DL2 antibody and oxaliplatin was at least as active as anti-KIR3DL2 antibody alone (oxaliplatin alone had very limited, if any, anti-tumor effect in this model). Interestingly, 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 drug separately. The effects shown above were reproduced in two independent experiments, each time with 9 mice per group.

[0222] These results, together with the in vitro combination data and the possible induction of KIR3DL2 by gemcitabine and oxaliplatin, support the combination of anti-KIR3DL2 antibody treatment with gemcitabine and oxaliplatin in TCL patients. The combination with humanized 2B12 is particularly potent as an anti-cancer treatment, but the ability of gemcitabine and / or oxaliplatin to increase KIR3DL2 expression may also be useful to enhance the anti-tumor effect of antibodies that are less potent than 2B12 alone. The combination may be particularly effective in patients with relapsed PTCL.

[0223] All references, including publications, patent applications, and patents, cited in this specification are incorporated herein by reference in their entirety and to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein (to the maximum extent permitted by law), notwithstanding that the incorporation of particular documents elsewhere in this specification is provided separately.

[0224] Unless otherwise specified, all exact values ​​provided herein are representative of the corresponding approximations (e.g., all exact exemplary values ​​provided for a particular factor or measurement can also be considered to provide the corresponding approximation of the measurement, modified by "about" where appropriate).

[0225] Description herein of any aspect or embodiment of the invention using terms such as "comprising," "having," "including," or "containing" in reference to an element is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" that particular element, unless otherwise specified or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should also be understood to describe a composition consisting of that element, unless otherwise specified or clearly contradicted by context).

[0226] The use of any examples or exemplary language provided herein (e.g., "etc.") is intended merely to better clarify the invention and does not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any element not claimed as essential to the practice of the invention.

Claims

1. A pharmaceutical composition comprising an antibody capable of binding to a KIR3DL2 polypeptide and ablating KIR3DL2-expressing cells for use in the treatment of TCL, wherein the pharmaceutical composition is administered in combination with gemcitabine and oxaliplatin, and wherein the anti-KIR3DL2 antibody, oxaliplatin and gemcitabine are formulated for separate administration and are administered in parallel or sequentially.

2. Gemcitabine, 800-1000 mg / m2, once every 2 weeks ２ and oxaliplatin is administered at a dose of 75-100 mg / m once every 2 weeks. ２ The pharmaceutical composition for use according to claim 1 , wherein the pharmaceutical composition is administered in a dose of

3. Gemcitabine, 800-1000 mg / m2, once every 2 weeks ２ and oxaliplatin is administered at a dose of 75-130 mg / m once every 3 weeks. ２ The pharmaceutical composition for use according to claim 1 , wherein the pharmaceutical composition is administered in a dose of

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the antibody is administered once per week to once every four weeks.

5. 4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the antibody is administered once per week, optionally wherein the antibody is administered once per week in a first phase, followed by once every two weeks or once per month in a second phase.

6. 2. The pharmaceutical composition for use according to claim 1, wherein the TCL is peripheral T-cell lymphoma (PTCL).

7. The pharmaceutical composition for use according to claim 6, wherein the PTCL is adult T-cell leukemia (ATL), enteropathy-associated T-cell lymphoma (EATL), PTCL-NOS or anaplastic large cell lymphoma (ALCL).

8. The pharmaceutical composition for use according to claim 1, wherein the antibody is administered at a dose of 1 to 20 mg / kg, optionally at a dose of 750 mg.

9. The antibody is administered in a regimen comprising at least one administration cycle, wherein for each of said at least one cycle, two, three or four doses of the antibody that binds to said KIR3DL2 polypeptide are administered, and at least two, three or four doses of gemcitabine are administered, and optionally oxaliplatin is administered, wherein gemcitabine is administered at a dose of 800-1000 mg / m once every two weeks. ２ and oxaliplatin is administered at a dose of 75-130 mg / m once every 2 weeks. ２ The pharmaceutical composition for use according to claim 1 , wherein the pharmaceutical composition is administered in a dose of

10. said treating TCL in an individual comprising: a) determining the KIR3DL2 polypeptide status of malignant cells within said individual with TCL; b) once said individual is determined to have a KIR3DL2 polypeptide expressed on the surface of a malignant cell, administering to said individual said antibody that binds to a KIR3DL2 polypeptide in combination with oxaliplatin and gemcitabine; 2. The pharmaceutical composition for use according to claim 1, comprising:

11. 2. The pharmaceutical composition for use according to claim 1, wherein the antibody that binds to a KIR3DL2 polypeptide comprises (a) a VH domain comprising an amino acid sequence at least 90% identical to a 2B12 VH of any of SEQ ID NOs: 48-51, and (b) a VL domain comprising an amino acid sequence at least 90% identical to a 2B12 VL of any of SEQ ID NOs: 43-47.

12. The pharmaceutical composition for use according to claim 11 , wherein the antibody that binds to a KIR3DL2 polypeptide comprises (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 49, and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 44 or 45.

13. A pharmaceutical composition comprising gemcitabine and oxaliplatin, in combination with an antibody capable of binding to a KIR3DL2 polypeptide and ablating KIR3DL2-expressing cells, for use in the treatment of TCL, wherein the anti-KIR3DL2 antibody, oxaliplatin and gemcitabine are formulated for separate administration and are administered in parallel or sequentially.

14. A kit for use in the treatment of TCL, comprising a pharmaceutical composition containing an anti-KIR3DL2 antibody capable of removing KIR3DL2-expressing cells, a pharmaceutical composition containing oxaliplatin, and a pharmaceutical composition containing gemcitabine.

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