Treatment with anti-KIR3DL2 agents

Abstract

The present disclosure relates to the use of KIR3DL2-targeted agents for the treatment of CTCL. The present disclosure provides advantageous treatment regimens using anti-KIR3DL2 antibodies for the treatment of CTCL, particularly in first-line CTCL.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 410,880, filed October 21, 2016, the disclosures of which are incorporated by reference in their 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 as a file entitled "KIR-7_ST25", created on October 18, 2017, which is 53KB 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 treatment of CTCL. [Background technology]

[0004] Various T-cell and B-cell tumors can invade the skin either primarily or secondarily. Primary cutaneous lymphomas present in the skin are not associated with any evidence of extradermal disease at the time of diagnosis. Primary cutaneous lymphomas often have a completely different clinical behavior and prognosis than histologically similar systemic lymphomas, which may secondarily invade the skin and therefore require different types of treatment. Cutaneous T-cell lymphoma (CTCL) is a group of lymphoproliferative disorders characterized by localization of neoplastic T lymphocytes to the skin. Collectively, CTCL is classified as a type of non-Hodgkin's lymphoma (NHL). Treatment options for CTCL typically vary depending on the extent of skin involvement, the type of skin lesion, and whether the cancer has spread to lymph nodes or other internal organs. In the case of mycosis fungoides, treatment may be targeted to the skin or the whole body. Sézary syndrome is generally characterized by blood involvement and is not usually treated with topical skin therapy alone. Treatments can be prescribed alone or in combination to achieve optimal long-term benefits. Topical skin treatments for CTCL are useful in cases of plaque and limited plaque disease, and include, among others, topical treatments such as corticosteroids, retinoids, or imiquimod, topical chemotherapy, topical radiation therapy, methotrexate, cyclophototherapy, and ultraviolet light (phototherapy).

[0005] In recent years, several antibody therapeutics that target proteins expressed on the surface of malignant cells have shown promise as treatments for CTCL.

[0006] Alemtuzumab is a humanized IgG1κ monoclonal antibody specific for CD52, an antigen expressed by most T and B cells, which is used to treat CTCL and PTCL, and the usual administration protocol is 30 mg three times a week. However, although some retrospective and prospective studies have shown good efficacy in Sézary syndrome, treatment with alemtuzumab causes extensive elimination of NK and T cells, leading to cytopenia and immune elimination. Furthermore, Non-Patent Document 1 reported that alemtuzumab treatment nevertheless does not completely eliminate T cells in the skin. Although alemtuzumab eliminated all T cells in the blood, a distinct population of skin-resident T effector memory cells remained in the skin after therapy. T cell elimination by alemtuzumab requires the presence of neutrophils, a cell type commonly present in the blood but rare in normal skin, suggesting that central memory T cells were eliminated because they recirculate between the blood and skin, whereas skin-resident effector memory T cells remained because they are sessile and do not recirculate.

[0007] More recently, mogamulizumab (KW-0761) has emerged as a therapeutic agent for relapsed / refractory CTCL and PTCL. Mogamulizumab is a humanized anti-CCR4 monoclonal antibody that eliminates CCR4 and is approved for use in Japan for the treatment of CCR4+ATLL, PTCL or CTCL. However, mogamulizumab also eliminates normal CCR4-expressing cells, thus causing the elimination of normal regulatory T (TReg) cells. The elimination of normal TReg cells results in the pre-elimination of subsequent or concomitant hematopoietic stem cell transplantation, or other therapeutic agents that require a properly functioning immune system, particularly for safety, due to the risk of graft-versus-host disease.

[0008] Another promising immunotherapeutic agent for the treatment of CTCL is brentuximab vedotin, an antibody-drug conjugate that targets the CD30 antigen and eliminates CD30-expressing cells. Adcetris™ (Brentuximab vedotin) is an anti-CD30 monoclonal antibody (clone cAC10) linked to the microtubule inhibitor monomethyl auristatin E (MMAE) by a protease-cleavable linker. Once bound to CD30, brentuximab vedotin is internalized and MMAE is released by the action of lysosomal enzymes on the linker, causing cell death. Brentuximab vedotin has shown high efficacy with manageable toxicity, but the treatment may also target normal CD30-expressing immune cells, especially activated B and T cells. Some authors have also suggested that MMAE released within the tumor environment may contribute to the mechanism of action by eliminating regulatory T (TReg) cells.

[0009] Finally, KIR3DL2 has been proposed as a target for CTCL (see, for example, Non-Patent Document 2; and Patent Document 1). KIR3DL2 / CD158k is a cell surface receptor expressed on normal circulating NK and CD8+ T lymphocytes. KIR3DL2 has also been found on the surface of CTCL cell lines and freshly isolated CD4+ PBL from SS patients, as well as in circulating malignant tumor cells from CTCL patients (Non-Patent Document 3). Non-Patent Document 4 reports a strong positive correlation between the percentage of CD158k+ blood lymphocytes analyzed by flow cytometry and the percentage of atypical circulating cells (Sezary cells) determined by cell morphology in a large group of Sezary syndrome patients, and reports that circulating CD4+CD158k+ lymphocytes are consistent with the malignant clonal cell population. Therefore, KIR3DL2 has been proposed as a marker for the assessment of circulating tumor burden and follow-up of Sezary syndrome patients. Patent Document 2 reports anti-KIR3DL2 antibodies, in particular antibodies that are efficient in mediating ADCC against circulating KIR3DL2-expressing tumor cells or tumor cell lines. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 02 / 50122 Brochure [Patent Document 2] International Publication No. 2014 / 044686 Brochure [Non-patent literature]

[0011] [Non-Patent Document 1] Clark et al.,2012 Sci.Trans.Med.4(117):117ra7(DOI:10.1126 / scitranslmed.3003008) [Non-Patent Document 2] Ortonne et al.(2006)Blood 107(10):4030-4038 [Non-Patent Document 3] Nikolova et al.(2002)Leuk Lymphoma.43(4):741-746 [Non-Patent Document 4] Poszepczynska-Guigne J Invest Dermatol.(2004)122(3):820-3 Summary of the Invention [Problem to be solved by the invention]

[0012] Although various treatments for CTCL are available, many or most of them have side effects that limit their use, such as antibodies that target proteins expressed on the surface of tumor cells.In particular, CD52 and CC4, which are targeted by alemtuzumab and mogamulizumab, respectively, are also expressed on normal cells, leading to side effects related to the elimination of normal T cells and NK cells, and further limiting the scope of use or combination with other available anti-CTCL treatments.Therefore, there is a need for improved CTCL treatments. [Means for solving the problem]

[0013] The present disclosure provides the use of anti-KIR3DL2 agents for clearance as immunomodulators in an amount effective to induce an immune response at extravascular, particularly cutaneous, sites of T cell proliferative disease. This treatment can, inter alia, eliminate malignant KIR3DL2 expressing cells without causing the elimination of normal KIR3DL2 expressing NK cells and T cells. These agents can be used in particular for the treatment of cutaneous T cell lymphoma (CTCL), regardless of the presence of detectable malignant cells in the circulation. These agents can be advantageously used in patients with overt or advanced disease who do not yet have detectable malignant KIR3DL2 expressing cells in the circulation. These agents can be advantageously used for the treatment of patients with indolent or early stage T cell lymphoma (e.g., CTCL) characterized by low or no significant malignant cells in the circulation. In one embodiment, the agents can be advantageously used as a first line treatment for T cell lymphoma (e.g., CTCL). Optionally, the subject has not yet been treated with a chemotherapeutic agent. Optionally, the subject has not yet been treated with an immunotherapy (e.g., mogamulizumab, alemtuzumab, and / or brentuximab vedotin). Optionally, the subject has not yet been treated with a bone marrow transplant or hematopoietic stem cell transplant. Optionally, the subject has progressive disease. In one embodiment, the agent can be advantageously used to treat patients prior to bone marrow transplant or hematopoietic stem cell transplant.

[0014] In clinical trials of relapsed / refractory CTCL in human patients with ADCC-inducing anti-KIR3DL2 antibodies, the inventors surprisingly observed potent anti-tumor effects in skin lesions in patients who received extremely low amounts of anti-KIR3DL2 antibodies (enough to reach only a small number of malignant cells in the skin and, at certain dose levels, a small fraction of malignant cells in the blood, if present).

[0015] Furthermore, a strong antitumor effect was observed in patients with dermatological disease (cutaneous erythroderma, plaques or spots) in the absence of detectable malignant KIR3DL2-expressing cells in the circulation.

[0016] In addition, analyses from clinical trials have revealed that when treated with clearing anti-KIR3DL2 agents at doses administered to result in only partial / minimal NK lytic activity against circulating KIR3DL2+ cells, and at amounts far below those that achieve significant occupancy of KIR3DL2 receptors on cells in skin tumors (e.g., tumors of patients with high tumor burden), patients experienced significant improvement in their skin disease, including restoration of normal skin architecture and a significant reduction in KIR3DL2-expressing cells at diseased skin sites.

[0017] These results suggest that KIR3DL2-binding agents, when administered to produce activity in the circulation for a sufficiently long period (e.g., 10 weeks or more), may be effective in treating disease in tissues (e.g., skin) despite low doses of therapeutic agent that are expected to act in diseased parts of the tissue. Moreover, this treatment can advantageously avoid elimination of normal KIR3DL2-expressing NK cells and / or T cells in the circulation, unlike that observed with other treatments. Thus, KIR3DL2-binding agents can be used with particular advantage for first-line treatment of CTCL, including, but not limited to, individuals with early and / or slowly progressing disease. In one embodiment, KIR3DL2-binding agents are used in conjunction with (e.g., prior to) hematopoietic stem cell or bone marrow transplantation in both early and late stages of the disease. In one embodiment, KIR3DL2-binding agents are used for first-line treatment of CTCL. In one embodiment, KIR3DL2-binding agents are used to treat CTCL in subjects who are ineligible for hematopoietic stem cell or bone marrow transplantation (e.g., due to high blood and / or skin tumor burden). In one embodiment, the KIR3DL2-binding agents achieve a reduction in blood and / or skin tumor burden without the elimination of normal NK and / or T cells, rendering the subject eligible for hematopoietic stem cell or bone marrow transplantation.

[0018] In our study, we used a new paradigm to determine the dosing of anti-KIR3DL2 antibodies. Rather than maintaining sufficient KIR3DL2 occupancy on malignant cells in solid tumors of the skin, which would require particularly high blood concentrations in individuals with high tumor burdens associated with advanced disease, anti-KIR3DL2 antibodies achieved significant anti-tumor responses at lower doses and frequencies sufficient to maintain blood (e.g., serum) concentrations that result in, for example, at least 60%, 80%, 90% or 100% NK lysis capacity (%), while allowing a single dosing regimen for all patients. These treatment regimens can be used over a prolonged treatment period and / or several treatment cycles, optionally preceded by an induction or loading period using a higher dosing frequency (or higher dose). In certain embodiments, a single treatment regimen (e.g., the same dose and frequency) can be advantageously used in both individuals with low blood and / or skin disease burden and individuals with high blood and / or skin disease burden.

[0019] In one embodiment, a common treatment regimen (e.g., the same dosage and frequency) that does not cause normal NK cell and / or T cell elimination may be advantageously used for individuals regardless of initial tumor burden and / or disease stage, in which case the common treatment regimen is preceded by an induction regimen or loading period in which an anti-KIR3DL2 antibody is administered to individuals (e.g., individuals with high tumor burden) at a higher dosing frequency (optionally, the dose per administration of the antibody in the common treatment regimen and the induction regimen is the same).

[0020] In one aspect, KIR3DL2 receptor may originate from the skin manifestation of disease, rather than clonally growing from circulating malignant and non-malignant CD4(+) T cell populations.Therefore, KIR3DL2 may be expressed sufficiently in cutaneous T cell malignancies, including indolent or early CTCL, that it can be therapeutically targeted by anti-KIR3DL2 binding agents when patients have no blood infiltration (no detectable KIR3DL2-expressing malignant cells in circulation).In addition or alternatively, tumor cells from skin lesions may enter (or re-enter) circulation, whereby the lysis of a small number of tumor cells in circulation helps contribute to a broader anti-tumor response in skin.

[0021] In one aspect, an agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells is provided for use in treating CTCL. In one embodiment, the CTCL has tissue manifestations of the disease, e.g., pruritus, erythroderma, and / or skin tumors. In one embodiment, the agent is used as a first line treatment. In one embodiment, a method is provided that includes administering to an individual with a T cell malignancy (e.g., CTCL) an agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells. In one aspect, an agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells is provided for use in pretreatment of an individual with a T cell malignancy (e.g., CTCL) in preparation for subsequent bone marrow or hematopoietic stem cell transplantation. In one aspect, an anti-KIR3DL2 binding agent is provided for use in treating an individual having a T cell malignancy with histologic manifestations of disease (e.g., CTCL with pruritus, erythroderma and / or skin tumors) but without detectable KIR3DL2-expressing malignant cells in the circulation. In one aspect, an anti-KIR3DL2 binding agent is provided for use in treating an individual with indolent or early stage CTCL.

[0022] In one embodiment, in patients with CTCL accompanied by skin manifestations of the disease, e.g., pruritus, erythroderma and / or skin tumors, the NK lytic capacity in the circulation may be sufficient to induce a significant antitumor effect, with only a small amount of EC 10 The anti-KIR3DL2 binding agent can be administered in an amount effective to achieve a small EC20 NK lytic capacity in the circulation. 10 The dose maintained the same level of NK cell proliferation, reducing blood tumor burden and showing only a slight EC 60 Doses that maintain this level of activity restore normal skin architecture and reduce KIR3DL2 expressing cells at diseased skin sites. Thus, in one embodiment, an anti-KIR3DL2 binding agent is provided for use in treating individuals with cutaneous symptoms of CTCL but with no or low levels of detectable malignant cells in the circulation, where the anti-KIR3DL2 binding agent is administered at a level of EC50 such that NK lytic capacity in the circulation may be sufficient to induce a significant anti-tumor effect. 10 In another embodiment, an anti-KIR3DL2 binding agent is provided for use in treating an individual having cutaneous manifestations of CTCL and detectable (e.g., higher levels) malignant cells in the circulation, wherein the treatment results in a small EC 10 The method includes administering multiple doses of an anti-KIR3DL2 binding agent in an amount effective to maintain a degree of inflammatory bowel disease.

[0023] Targeting KIR3DL2 with anti-KIR3DL2 binding agents is therefore advantageous in some therapeutic situations and does not require prior testing of KIR3DL2 expression on malignant cells in the circulation and / or skin. Furthermore, the use of anti-KIR3DL2 binding agents does not require a dose that maintains sufficient receptor occupancy on tumor cells of skin diseases (e.g., erythroderma, skin lesions) for all patients in a population with a range of tumor burdens, yet can benefit from the ability of normal immune cells (e.g., NK cells, CD8 T cells, gamma-delta T cells) to contribute to a broader anti-tumor response through clearance of small numbers of KIR3DL2-expressing tumor cells in the circulation (e.g., below the limit of detection), e.g., tumor cells that enter the circulation from skin lesions, and / or through induction of antibody-dependent cellular phagocytosis (ADCP) in skin lesions.

[0024] In one aspect, therapeutic regimens are provided for the administration of anti-KIR3DL2 agents capable of inducing such anti-tumor responses.

[0025] In one aspect, the therapeutic regimen disclosed herein has the advantage of being adapted to treat individuals with T cell lymphoma (e.g., CTCL) that have detectable malignant cells (e.g., KIR3DL2-expressing malignant cells) in the circulation, as well as individuals with T cell lymphoma that do not have such detectable malignant cells in the circulation. In particular, a single dosage and / or administration regimen can be used to treat such patients, avoiding the need to administer different treatments depending on the level (or lack thereof) of malignant cells in the circulation. Advantageously, the therapeutic regimen can be used to treat patients with high tumor burden, optionally repeatedly and / or continuously over a period of time, to generate a more extensive response to skin symptoms.

[0026] In one embodiment, the beneficial treatment is characterized by at least an EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100Optionally, the amount and frequency of the anti-KIR3DL2 agent is greater than or equal to EC for receptor saturation in the skin (or within a skin lesion or tumor, e.g., advanced disease stage, high tumor burden, or erythroderma). 90 , or E.C. 100 Lower than those that maintain.

[0027] In one aspect of any of the embodiments described herein, the beneficial treatment is an increase in NK lysis capacity of at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 The therapy includes multiple administrations of an anti-KIR3DL2 agent at an amount and frequency that results in a blood (e.g., serum) concentration that is greater than or equal to EC 2 + / - 10%. Optionally, the therapy is administered for a period of at least 10 weeks, 12 weeks, 3 months, 4 months, or 6 months. Optionally, administrations are spaced from about 1 week to about 2 months apart. Optionally, the anti-KIR3DL2 agent is administered at least 4, 6, 8, 10, or 20 times. Optionally, the amount and frequency of the anti-KIR3DL2 agent is greater than or equal to EC 2 + / - 10% for receptor saturation in the skin (or in a skin lesion or tumor, e.g., advanced disease stage, high tumor burden, or erythroderma). 90 , or E.C. 100 Lower than what results.

[0028] In one embodiment, the advantageous treatment includes administering to the individual an amount of an anti-KIR3DL2 agent that maintains a blood (e.g., serum) concentration that results in an NK% solvent capacity of at least 10%, optionally at least 60%, optionally at least 80%, optionally at least 90%, or optionally at least 100%) between two consecutive administrations.

[0029] In one embodiment, a beneficial treatment is one that reduces NK lysis capacity by at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C.100 In one embodiment, the treatment comprises administering to the individual an amount of an anti-KIR3DL2 agent that maintains a blood (e.g., serum) concentration of at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 and maintaining a trough concentration in blood (eg, serum) of at least

[0030] Optionally, the treatment is administered for a period of at least 10 weeks, 12 weeks, 3 months, 4 months, or 6 months.

[0031] Optionally, administrations are spaced apart by about one week to about two months.

[0032] Optionally, the treatment comprises at least 4, 6, 8, 10 or 20 consecutive administrations of the anti-KIR3DL2 agent.

[0033] In one embodiment, the individual is eligible (remains eligible or is made eligible) for hematopoietic stem cell transplant or bone marrow transplant prior to treatment with the anti-KIR3DL2 agent.

[0034] In one embodiment, the individual is ineligible for hematopoietic stem cell or bone marrow transplantation prior to treatment with the anti-KIR3DL2 agent, and is made eligible for hematopoietic stem cell or bone marrow transplantation following treatment with the anti-KIR3DL2 agent.

[0035] Optionally, the treatment with the anti-KIR3DL2 agent is performed before hematopoietic stem cell transplantation or bone marrow transplantation. Optionally, the treatment is combined with hematopoietic stem cell transplantation or bone marrow transplantation. In any embodiment, the method of treatment further comprises performing hematopoietic stem cell transplantation or bone marrow transplantation on the individual after treatment with the anti-KIR3DL2 agent.

[0036] In one embodiment, an agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells is provided for use in treating T cell malignancies with tissue manifestations, wherein treatment is effective in individuals with and without blood involvement.

[0037] In one embodiment, a method is provided that includes administering to an individual having a T-cell malignancy (e.g., CTCL) an agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells for at least one administration cycle, wherein the agent is administered at least twice in an amount that maintains a % solvent capacity in the circulation of at least 10%, optionally at least 60%, 80%, or 90%, or optionally 100% between two successive administrations of the agent. For example, the agent has an NK lysis capacity of at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 In one embodiment, the agent is administered intravenously. In one embodiment, the agent is administered at least 4, 6, 8, or 10 times, optionally with one week to one month between successive administrations. In one embodiment, the agent that binds to a KIR3DL2 polypeptide is administered such that the circulating concentration that results in the % lytic capacity (or EC value) in the circulation is maintained for at least 10 weeks, optionally at least 3 months, and optionally at least 6 months. In one embodiment, the method is a method of treating a T-cell malignancy with tissue manifestations, which is effective in both individuals with and without blood involvement. In one embodiment, the method is a method of preconditioning an individual with a T-cell malignancy (e.g., CTCL) for subsequent bone marrow or hematopoietic stem cell transplantation.

[0038] Optionally, in any embodiment, the treatment regimen is preceded by an induction or loading period in which the anti-KIR3DL2 binding agent is administered in a higher amount and / or frequency. Optionally, in any embodiment, the treatment regimen is preceded by an induction or loading period in which the anti-KIR3DL2 binding agent is administered in the same amount, but more frequently (e.g., in multiple successive administrations).

[0039] Optionally, the amount of the anti-KIR3DL2 agent is determined to be greater than or equal to the EC50 value for receptor saturation in a tissue (e.g., extravascular tissue, diseased tissue, skin, advanced disease stage, skin lesions including high tumor burden or erythroderma, or within a tumor). 80 , E.C. 90 , or E.C. 100 less than the amount that results.

[0040] In one embodiment, the anti-KIR3DL2 binding agent is an agent that mediates effector cell-mediated lysis of KIR3DL2-expressing cells (e.g., tumor cells). Optionally, the agent is an antigen-binding polypeptide, optionally an antibody or a fragment thereof (e.g., a protein comprising a VH and / or VL domain), that binds to a KIR3DL2 polypeptide, or to immune effector cells (e.g., chimeric antigen receptor immune effector cells), antibodies or other compounds expressing such a polypeptide. Optionally, the antibody is a clearance polypeptide (antibody). Optionally, the antibody is a monospecific or multispecific (e.g., bispecific) antibody that directs ADCC and / or ADCP against KIR3DL2-expressing cells.

[0041] In one aspect of any of the embodiments herein, the KIR3DL2-binding agent comprises an anti-KIR3DL2 antibody of the human IgG isotype capable of mediating ADCC, and is administered to an individual at least twice in an amount effective to achieve (and / or maintain for a specified period of time, or between two successive administrations) a blood (serum) concentration of the anti-KIR3DL2 antibody of at least 0.1 μg / ml (or optionally at least 0.4, 1, 2, 10 μg / mL), optionally less than 60 μg / mL or less than 100 μg / mL, optionally between 2-30 μg / mL, optionally between 2-60 μg / mL. In one embodiment, the antibody is administered intravenously once every week, once every two weeks, once every month (or every four weeks), optionally once every month to once every two months.

[0042] These aspects are described in more detail in the description of the invention provided herein, and further aspects, features, and advantages will become apparent from the description of the invention provided herein. [Brief description of the drawings]

[0043] [Figure 1A] Incubation at 4° C., which inhibits receptor uptake / circulation, was expected to result in at least comparable levels of cell surface KIR3DL2, but staining with antibody 2B12 (human IgG1) shows that it was higher at 37° C. than at 4° C. Furthermore, higher median fluorescence was observed with longer incubation times, with maximal KIR3DL2 expression observed after 24 hours of incubation. [Figure 1B] The effect of antibody 2B12 (thick line / square) and isotype control (light line / circle) on KIR3DL2 levels is shown. It can be seen that free receptor and 2B12-bound KIR3DL2 receptor readouts correlate and have similar EC50s. The rightmost panel shows that a 20 hour incubation with 2B12 increases total KIR3DL2 receptor levels at the cell surface as detected by non-competitive anti-KIR3DL2 (mAb2) bound to APC. [Figure 1C]Incubation with antibody 2B12 at 37° C. shows that it increases the surface expression of KIR3DL2 (as detected by non-competitive anti-KIR3DL2 (mAb2) or by 2B12 itself plus a secondary Ab) in a dose-dependent manner. This increase is already observed after 1 hour at 37° C. and appears to reach its maximum after 24 hours. Staining is optimal after 24 hours (in terms of total staining and Ab-bound receptors detected). [Diagram 2] PK simulation model of IPH4102, a two-compartment model with parallel primary and saturable elimination pathways. [Diagram 3] This demonstrates that IPH4102 did not cause NK cell depletion as shown by the % change from baseline (Week 1 Day 1) in patients' NK cells over up to 50 weeks. [Figure 4] We demonstrate that IPH4102 did not cause NK cell elimination as indicated by patient NK cell counts (NK cells per μl) for up to 50 weeks. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] As used herein, "a" or "an" may mean one or more. When used in the claims, when used with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein, "other" may mean at least a second or more.

[0045] When "comprising" is used, this can optionally be exchanged for "consisting essentially of" or "consisting of."

[0046] At any time, "treatment" refers to a disease and an anti-KIR3DL2 binding agent (e.g., an antibody) and includes (a) a method of treatment of a disease comprising the step of administering (for at least one treatment) an anti-KIR3DL2 binding agent (e.g., in a pharma- ceutically acceptable carrier material) to a warm-blooded animal, particularly a human, in need of such treatment, in a dose (therapeutically effective amount) that allows for treatment of the disease, e.g., in the doses (amounts) specified above and below; (b) the use of an anti-KIR3DL2 binding agent for the treatment of a disease or in said treatment (e.g., in a human). (c) use of an anti-KIR3DL2 binding agent for the manufacture of a pharmaceutical preparation for the treatment of a disease, a method of using an anti-KIR3DL2 binding agent in the manufacture of a pharmaceutical preparation for the treatment of a disease, 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 a disease; or (d) 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.

[0047] The term "biopsy" as used herein is defined as the removal of tissue for the purpose of examination, such as to establish a diagnosis. Examples of types of biopsy include: by applying suction, such as through a needle attached to a syringe; by removing a piece of tissue with an instrument; by removing with a suitable instrument; by surgical removal, such as the entire lesion; and the like.

[0048] 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. Some 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 ... 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 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 VLC is a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and L ) and variable heavy chain (V HThe terms "antibody" and "antibody-specific constant domains" 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" also includes any fragment or derivative of the antibodies described herein. A "fragment" includes a portion of an intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of an uninterrupted sequence of contiguous amino acid residues (herein referred to as "single-chain antibody fragments" or "single-chain polypeptides"), such as, but not limited to, (1) single-chain Fv molecules; (2) single-chain polypeptides comprising only one light chain variable domain, without the associated heavy chain portion, or fragments thereof comprising the three CDRs of the light chain variable domain, and (3) single-chain polypeptides comprising only one heavy chain variable domain, without the associated light chain portion, or fragments thereof comprising the three CDRs of the heavy chain variable domain; and multispecific (e.g., bispecific) antibodies formed from antibody fragments. Among these are nanobodies, domain antibodies, single domain antibodies or "dAbs".

[0049] 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.

[0050] When an antibody is said to "compete with" a particular monoclonal antibody, it means that the antibody competes with the monoclonal antibody in a binding assay using either recombinant KIR3DL2 molecules or surface-expressed KIR3DL2 molecules. For example, if a test antibody reduces the binding of 19H12, 12B11, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9 to a KIR3DL2 polypeptide or a KIR3DL2-expressing cell in a binding assay, the antibody is said to "compete with" 19H12, 12B11, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9, respectively.

[0051] 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 fully incorporated herein by reference. 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).

[0052] The term "epitope" refers to an antigenic determinant, which 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., amino acid residues 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 become close to each other due to folding of the molecule (secondary, tertiary, and / or quaternary structure). Conformational epitopes are dependent on the three-dimensional structure. The term "conformational" is therefore often used interchangeably with "structural."

[0053] The term "intracellular uptake", or "uptake", when referring to KIR3DL2 polypeptide and / or antibodies that bind thereto, refers to a biochemical cellular event of a molecule that involves the process of transporting a molecule from the extracellular surface of a cell to the intracellular surface of a cell. These processes responsible for the intracellular uptake of molecules are well known and may include, among others, the uptake of extracellular molecules (such as hormones, antibodies, and small organic molecules); membrane-bound molecules (such as cell surface receptors); and the uptake of complexes of membrane-bound molecules bound to extracellular molecules (such as ligands bound to transmembrane receptors or antibodies bound to membrane-bound molecules). Thus, "inducing and / or enhancing intracellular uptake" includes events that initiate intracellular uptake and / or increase the rate and / or extent of intracellular uptake.

[0054] The terms "eliminating," "eliminate," or "elimination" with respect to KIR3DL2-expressing cells refer to a process, method, or composition that can kill, remove, lyse, or induce such killing, removal, or lysis to negatively affect the number of KIR3DL2-expressing cells present in a sample or subject. Elimination of cells can occur, for example, via ADCC.

[0055] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, a cell, or an extract made from biological material. The term "therapeutic agent" refers to an agent that has biological activity.

[0056] A "humanized" or "human" antibody refers to an antibody in which one or more human immunoglobulin constant and variable framework regions are fused with a binding region, e.g., the CDRs of an animal immunoglobulin. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding region is derived, but to avoid immune responses against the non-human antibody. Such antibodies can be obtained from transgenic mice or other animals that have been "engineered" to produce specific human antibodies in response to antigen challenge (see, e.g., Green et al. (1994) Nature Genet 7:13; Lonberg et al. (1994) Nature 368:856; Taylor et al. (1994) Int Immun 6:579, the entire teachings of which are incorporated herein by reference). Fully human antibodies can also be constructed by genetic or chromosomal transfection methods and phage display technology, all known in the art (see, e.g., McCafferty et al. (1990) Nature 348:552-553). Human antibodies may also be generated by in vitro activated B cells (see, eg, US Pat. Nos. 5,567,610 and 5,229,275, which are incorporated by reference in their entireties).

[0057] A "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof has been modified, replaced, or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or modified class, effector function, and / or species or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, drug, etc., that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof has been modified, replaced, or exchanged with a variable region having a different or modified antigen specificity.

[0058] The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, such as from about amino acid (aa) 230 to about aa 450 of the human gamma (gamma) heavy chain or its corresponding sequence in other types of antibody heavy chains (e.g., alpha, delta, epsilon, and mu for human antibodies) or naturally occurring allotypes thereof. Unless otherwise specified, the commonly 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).

[0059] The term "NK% lytic capacity" refers to the capacity of NK cells from a healthy donor to lyse tumor cells (e.g., HUT78 cells) in an in vitro cytotoxicity assay, which is 51 In a Cr release assay, it is measured by the percentage of maximum tumor cell lysis obtained (=tumor cell lysis / maximum tumor cell lysis at saturation x 100). An example of a suitable assay using PBMC and HUT78 cells as effector and target cells is described in the Examples section of this specification. The NK lysis capacity is referred to as "EC 10 ” (or “EC 60 ", E.C. 80 ", E.C. 90 " or "EC100 ") represents 10% of its maximal response or effect with respect to such NK lytic capacity (or "EC 60 ", E.C. 80 ", E.C. 90 " or "EC 100 " refers to the effective concentration of an anti-KIR3DL2 agent that produces 60%, 80%, 90% or 100%, respectively.

[0060] 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 non-specific cytotoxic cells expressing Fc receptors (FcR) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. Non-specific cytotoxic cells that mediate ADCC include natural killer (NK) cells, macrophages, monocytes, neutrophils, and eosinophils.

[0061] 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.

[0062] 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 the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0063] 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.

[0064] The term "modification" when referring to a sequence of amino acids (e.g., "amino acid modification") refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. "Modification" or "amino acid modification" refers to an amino acid substitution, insertion, and / or deletion in a polypeptide sequence. As used herein, an "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a given position in a protein sequence with another amino acid. For example, the substitution P14S refers to a variant of a parent polypeptide in which the proline at position 14 is replaced with a serine. A "variant" of a polypeptide refers to a polypeptide having an amino acid sequence that is substantially identical to a reference polypeptide, typically a native or "parent" polypeptide. A polypeptide variant may have one or more amino acid substitutions, deletions, and / or insertions at specific positions within a native amino acid sequence.

[0065] 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.

[0066] The term "identity" or "identical," when used in the context of two or more polypeptide sequences, refers 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 smaller sequences of two or more sequences, with gap alignments (if any), 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).

[0067] Methods for determining identity are designed to produce the greatest 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 may also be used to determine identity.

[0068] Treatment of Disease The anti-KIR3DL2 agents and dosing regimens disclosed herein can be advantageously used to treat KIR3DL2-expressing T-cell lymphomas, particularly CTCL, optionally as a first-line treatment, optionally with Sézary syndrome (SS), optionally with mycosis fungoides (MF), optionally with transformed MF, optionally with advanced disease (e.g., stage IIB, III, IIIA, IIIB, IVA1, IVA2 or IVB), optionally with peripheral blood involvement, optionally with detectable or high levels of KIR3DL2-expressing malignant cells in the peripheral blood, optionally with indolent or early disease, optionally with stage IA, IB or IIA disease, optionally without peripheral blood involvement, optionally with no or low levels of detectable KIR3DL2-expressing malignant cells in the peripheral blood. In another aspect, a method of preventing lymphoma in an individual with CTCL is provided. In another embodiment, a method is provided for suppressing the risk of disease progression in an individual with CTCL, or for reducing the risk of lymphoma within an initiated cell population. In another embodiment, a method is provided for conditioning or qualifying a subject for hematopoietic stem cell or bone marrow transplantation.

[0069] Cutaneous T-cell lymphoma (CTCL) (see image below) is a group of lymphoproliferative disorders characterized by the localization of neoplastic T cells to the skin. Collectively, CTCL are 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 described in Willemze et al. (2005) Blood 105:3768-3785. The WHO-EORTC divides CTCL into those with an indolent clinical behavior and those with an aggressive subtype. The third category is that of precursor hematologic neoplasms that are not T-cell lymphomas (CD4+ / CD56+ hematologic-cutaneous neoplasms, blastic natural killer (NK) cell lymphomas, or primary cutaneous neoplasms of B-cell origin). CTCL with 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 cell lymphoma (provisional). CTCL with 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 aggressive epidermotropic CD8+ T-cell lymphoma (provisional), and cutaneous gamma / delta positive T-cell lymphoma (provisional).

[0070] The most common CTCLs are MF and SS. Their characteristics are reviewed, for example, in Willemze et al. (2005) Blood 105:3768-3785, the disclosure of which is incorporated herein by reference. In most cases of MF, the diagnosis is reached by its clinical features, medical history, and histomorphological and cytomorphological findings. Another diagnostic criterion for distinguishing CTCL from inflammatory skin diseases is the demonstration of a predominant T-cell clone in skin biopsy material 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) plaques (atrophic or nonatrophic): nonspecific dermatitis, plaques on the lower trunk and buttocks; slight / absent pruritus; (2) plaques: plaques with intense pruritus, lymphadenopathy, and (3) tumors: tendency to ulceration. Sézary syndrome is defined by erythroderma and leukemia. Signs and symptoms include edematous skin tags, lymphadenopathy, palmar and / or plantar keratodermia, alopecia, nail dystrophy, ectropion, and hepatosplenomegaly. For the diagnosis of Sézary syndrome, criteria typically include absolute Sézary cell counts in peripheral blood demonstrated by molecular or cytogenetic methods, immunophenotypic abnormalities, loss of T cell antigens and / or T cell clones.

[0071] CTCL stages include I, II, III, and IV according to the TNM classification, as well as peripheral blood involvement, where appropriate. Peripheral blood involvement containing mycosis fungoides or Sézary syndrome (MF / SS) cells correlates with more advanced skin stages, lymph node and visceral involvement, and shorter survival times. MF and SS have official staging systems proposed by the International Society for Cutaneous Lymphoma (ISCL) and the European Organization for Research and Treatment of Cancer (EORTC). See Olsen et al., (2007) Blood. 110(6): 1713-1722; and Agar et al. (2010) J. Clin. Oncol. 28(31): 4730-4739, the disclosures of which are incorporated herein by reference. SS and MF, stage IV (IVA1, IVA2, and IVB) may include B2 peripheral blood infiltration (high blood-tumor burden; ≧1,000 / μL positive clones containing Sézary cells). SS and MF stages include I (IA and IB), II (IIA and IIB), III (III, IIIA, and IIIB), and IV (IVA1, IVA2, and IVB).

[0072] Treatments designed to achieve effector cell-mediated lysis of malignant KIR3DL2+ cells in the circulation may be through lysis of only a small number of circulating cells (e.g., compared to the total malignant cells in the extracellular manifestations of the disease), through activation of a limited number of effector cells in the circulation, and / or through antibody-dependent cellular phagocytosis (ADCP) directed at a limited number of malignant cells in the skin lesion, generating an anti-tumor response leading to elimination of the malignant cells and inducing general disease improvement of the skin lesion. The response may be expressed as NK lysis capacity, EC 10This was achieved by repeated dosing of KIR3DL2 binding antibodies using various treatment regimens designed to maintain a specific amount of anti-KIR3DL2 binding agent in the circulation effective to produce a cutaneous response. The dosing regimens improved the skin lesions in patients with low or no blood involvement, and the repeated dosing regimens also improved the skin lesions in patients with high blood involvement.

[0073] Upon diagnosis of CTCL, subjects can be treated with anti-KIR3DL2 binding agents. Treatment can be used regardless of tumor burden to eliminate malignant cells while maintaining normal NK and T cells. Thus, this treatment is compatible with subsequent BMT or HSCT. In one embodiment, the present disclosure provides the use of anti-KIR3DL2 binding agents as a first-line treatment to treat subjects with CTCL. The term "first-line treatment" as used herein refers to the first type of systemic drug therapy administered for the treatment of CTCL. This can be a single agent, combination or maintenance therapy that is first provided after diagnosis.

[0074] In one aspect, a method of treating CTCL in an individual is provided, the method comprising administering to the individual an anti-KIR3DL2 binding agent without a prior step of testing for KIR3DL2 expression on malignant cells from a blood sample.

[0075] In one aspect, a method of treating CTCL in an individual is provided, the method comprising administering to the individual an anti-KIR3DL2 binding agent without the step of prior testing for KIR3DL2 expression on malignant cells from a skin biopsy.

[0076] In one aspect, a method is provided for treating CTCL in an individual who has no detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells), comprising administering an anti-KIR3DL2 binding agent to the individual. In one aspect, a method is provided for treating CTCL in an individual who has low levels of detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells), comprising administering an anti-KIR3DL2 binding agent to the individual.

[0077] In one aspect, a method of treating CTCL in an individual with peripheral blood involvement less than stage B2 is provided, comprising administering an anti-KIR3DL2 binding agent to the individual. Optionally, the individual has a Sézary cell tumor burden of less than 1,000 / μL and / or no positive clones.

[0078] In one aspect, a method of treating indolent CTCL is provided, the method comprising administering to an individual an anti-KIR3DL2 binding agent.

[0079] In one aspect, a method of treating CTCL is provided, the method comprising: (a) assessing the stage and / or disease prognosis of CTCL in an individual having CTCL; and (b) if the individual has stage II or III disease, optionally IIB, IIIA or IIIB, administering an anti-KIR3DL2 binding agent to the individual.

[0080] In one aspect, a method of treating stage I CTCL is provided, the method comprising administering an anti-KIR3DL2 binding agent to an individual. In one aspect, a method of treating stage II CTCL is provided, the method comprising administering an anti-KIR3DL2 binding agent to an individual. In one aspect, a method of treating stage III CTCL is provided, the method comprising administering an anti-KIR3DL2 binding agent to an individual.

[0081] In one aspect, a method of treating CTCL in an individual with peripheral blood involvement less than stage B2 is provided, comprising administering an anti-KIR3DL2 binding agent to the individual. Optionally, the individual has no or low blood tumor burden, and optionally the individual has B0 (absence of significant blood involvement, e.g., ≦5% of peripheral blood lymphocytes are atypical (Sézary) cells) or B1 (low blood tumor burden, e.g., >5% of peripheral blood lymphocytes are atypical (Sézary) cells, not meeting the criteria for B2) peripheral blood involvement.

[0082] In one aspect of any of the foregoing, the individual with CTCL has skin lesions, optionally significant or advanced skin disease, optionally T2 (macules, papules, or plaques covering ≧10% of the skin surface, optionally also T2a (macules only) or T2b (plaques±macules), T3 (at least one tumor (≧1 cm diameter) or T4 stage cutaneous infiltrative erythroderma covering ≧80% of the body surface area). In one embodiment, the individual has multiple and / or high skin tumor burden. In one embodiment, the individual has one or more skin tumors greater than 1 cm in diameter.

[0083] In one embodiment of any of the above, the individual with CTCL has macules, papules, or plaques covering >10% of the skin surface. In one embodiment of any of the above, the individual with CTCL has at least one tumor >1 cm in diameter. In one embodiment of any of the above, the individual with CTCL has erythroderma covering >80% of the body surface area.

[0084] In an embodiment, a method of treating CTCL is provided, the method comprising: (a) assessing the stage and / or disease prognosis of CTCL in an individual having CTCL; and (b) if the individual has stage IV disease, optionally IVA1 or IVA2 disease, optionally IVB disease, administering an anti-KIR3DL2 binding agent to the individual.

[0085] It is understood that the treatment method of the present disclosure may or may not include a step of characterizing CTCL before treatment. In an embodiment, a method of treating CTCL is provided, which includes: (a) determining whether an individual has a CTCL including a skin symptom of CTCL (e.g., erythroderma, skin lesions or tumors), optionally a skin symptom characterized by pathogenic KIR3DL2-expressing cells; and (b) administering an anti-KIR3DL2 binding agent to the individual if the individual has a skin symptom of CTCL, optionally a skin symptom characterized by pathogenic KIR3DL2-expressing cells. Optionally, the step of determining whether an individual has a CTCL including a skin symptom of CTCL includes a step of determining the extent of skin lesions; optionally, if the individual has plaques and / or ulcerative tumors, administering an anti-KIR3DL2 binding agent to the individual. Optionally, the step of determining whether an individual has a CTCL including a skin symptom of CTCL includes a step of determining the stage of the skin disease, for example, T2, T3, or T4 disease. Optionally, if the individual has an advanced cutaneous manifestation of CTCL, such as T2, T3, or T4 disease, the method includes administering an anti-KIR3DL2 binding agent to the individual.

[0086] In an embodiment, a method of treating CTCL is provided, the method comprising: (a) assessing the stage and / or disease prognosis of CTCL in an individual having CTCL; and (b) if the individual has indolent CTCL, administering an anti-KIR3DL2 binding agent to the individual.

[0087] It will be appreciated that the treatment methods of the present disclosure may or may not include characterizing tumor cells for KIR3DL2 expression prior to treatment. In an embodiment, a method of treating CTCL is provided, comprising: (a) determining whether a skin manifestation of CTCL in an individual comprises pathogenic KIR3DL2-expressing cells (e.g., KIR3DL2-expressing cells in erythroderma and / or skin lesions); and (b) administering an anti-KIR3DL2 binding agent to the individual if the individual has a skin manifestation of CTCL comprising pathogenic KIR3DL2-expressing cells.

[0088] It is understood that the treatment method of the present disclosure may or may not include characterizing tumor cells for KIR3DL2 expression prior to treatment. In an embodiment, a method of treating CTCL is provided, comprising: (a) obtaining a blood sample or biopsy (e.g., skin biopsy) from an individual to determine whether the sample contains pathogenic KIR3DL2-expressing cells (KIR3DL2+ tumor cells); and (b) administering an anti-KIR3DL2 binding agent to the individual if the sample contains pathogenic KIR3DL2-expressing cells. In another embodiment, a method of treating CTCL is provided, comprising: (a) obtaining a blood sample from an individual to determine whether the sample contains pathogenic KIR3DL2-expressing cells (KIR3DL2+ tumor cells); and (b) administering an anti-KIR3DL2 binding agent to the individual if the sample does not contain detectable pathogenic KIR3DL2-expressing cells.

[0089] Optionally, the method further includes determining whether the diseased cells do not express other markers of abnormal lymphocytes on their surface, e.g., determining whether the cells are CD4, CD30, CD3, CD8 cells.

[0090] In some embodiments, an anti-KIR3DL2 binding agent can be administered to an individual who is in remission after treatment of CTCL or who has otherwise responded well to a first(s) anti-CTCL therapy (i.e., non-KIR3DL2), and optionally has a low blood tumor burden.

[0091] In some embodiments, anti-KIR3DL2 binding agents can be administered to individuals who have a poor disease prognosis and / or have relapsed and are resistant or unresponsive to a first therapeutic agent(s).

[0092] Provided herein are treatment regimens that can be used to treat both CTCL with low or no blood tumor burden (and / or no detectable KIR3DL2+ tumor cells), or CTCL with blood invasion or with high blood tumor burden (and / or with detectable KIR3DL2+ tumor cells). However, it will be understood that these regimens can be used separately for one or the other subgroup.

[0093] In one embodiment, optionally, the anti-KIR3DL2 binding agent is administered at a low dose, optionally at an amount designed to be below the amount that maintains sufficient receptor occupancy on tumor cells of the skin disease (e.g., erythroderma, skin lesion or tumor) in all patients, including those with high blood and skin tumor burden; such a dose may have the advantageous property of providing a broader anti-tumor response via elimination of low numbers of circulating KIR3DL2-expressing tumor cells (e.g., below the limit of detection), e.g., tumor cells entering the circulation from skin tumors, and / or via induction of antibody-dependent cellular phagocytosis (ADCP) in skin tumors. In one embodiment, the dose of the anti-KIR3DL2 binding agent is repeated, in particular, the treatment includes first, second, and optionally further administrations of the anti-KIR3DL2 binding agent. Optionally, the administration schedule (e.g., the interval between two successive administrations) and doses are at least EC for NK lysis capacity. 10 , E.C. 60 , E.C.80 , E.C. 90 , or E.C. 100 The anti-KIR3DL2 binding agent is selected to maintain a trough level that results in a blood (eg, serum) concentration corresponding to

[0094] In some embodiments, the anti-KIR3DL2 agent has an NK lysis potency of at least EC 10 , optionally, roughly or at least roughly EC 60 , E.C. 80 , E.C. 90 , or E.C. 100 at a dose and frequency to obtain and / or maintain a blood (eg, serum) concentration equivalent to

[0095] Optionally, in any of the embodiments described herein, the amount and frequency of the anti-KIR3DL2 agent is less than an amount that, when administered weekly, results in a blood (e.g., serum) concentration equivalent to that provided in increments of 25 mg / kg, 20 mg / kg, 15 mg / kg, 10 mg / kg, 7.5 mg / kg, or 6 mg / kg body weight. Optionally, in any of the embodiments described herein, the amount or dose of the anti-KIR3DL2 agent administered can be specified to be less than 25 mg / kg, 20 mg / kg, or 15 mg / kg body weight.

[0096] In another embodiment of any aspect described herein, the method of treatment is a method of inhibiting or preventing progression of, maintaining remission of, or preventing recurrence of CTCL, or preventing recurrence of CTCL lymphoma. In another embodiment of any aspect described herein, the method of treatment is a method of increasing the likelihood of survival over the relevant period. In another embodiment of any aspect described herein, the method of treatment is a method of improving the quality of life of an individual. In another embodiment of any aspect described herein, the method of treatment is a method of reducing the number of circulating lymphoma cells (e.g., Sézary cells) in an individual. In another embodiment of any aspect described herein, the method of treatment is a method of reducing blood tumor burden in an individual.

[0097] In another embodiment of any aspect described herein, the method of treatment is a method of preventing progression from early stage CTCL to a more advanced stage CTCL. In another embodiment of any aspect described herein, the method of treatment is a method of preventing progression from early stage I, II or III CTCL to stage IV CTCL. In another embodiment of any aspect described herein, the method of treatment is a method of preventing progression from CTCL with no hematological tumor or low hematological tumor burden to CTCL with hematological tumor burden or high hematological tumor burden. In another embodiment of any aspect described herein, the method of treatment is a method of preventing progression from CTCL with B0 or ​​B1 hematological tumor burden to CTCL with B2 hematological tumor burden.

[0098] Delivery of an anti-KIR3DL2 agent (e.g., an antibody or fragment thereof) to a subject (either by direct administration as an isolated proteinaceous binding agent, administration as a cell, such as a CAR effector cell, expressing the anti-KIR3DL2 binding protein on its surface, or expression of the proteinaceous binding agent from an internal nucleic acid, such as a poxvirus gene transfer vector comprising an anti-KIR3DL2 antibody-encoding nucleic acid sequence), as well as practice of other methods described herein, can be used to alleviate, treat, prevent, or otherwise ameliorate any suitable aspect of CTCL disclosed herein. Treatments can be administered parenterally, e.g., intravenously, and can be particularly useful in inhibiting and / or ameliorating the proliferation of abnormal lymphocytes in skin lesions, restoring normal skin structure, and significantly reducing pathogenic T cells.

[0099] In certain embodiments described herein, the KIR3DL2-binding agent is administered to the individual for at least one administration cycle, in which the agent exhibits a NK lysis capacity of at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100Optionally, between two successive drug administrations, the NK lytic capacity is at least equal to the EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 The agent is administered in an amount effective to achieve and / or maintain a concentration resulting in a blood (e.g., serum) concentration equivalent to . Optionally, the administration cycle includes at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administration of the agent. Optionally, the agent is administered intravenously. Optionally, the treatment has a duration of at least 10 weeks, 2 months, 3 months, 4 months, or 6 months.

[0100] In one aspect of any of the embodiments described herein, the NK lysis capacity is at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 The KIR3DL2-binding agent is administered to the individual in an amount that results in (eg, achieves and / or maintains) a blood (eg, serum) concentration that is:

[0101] In one aspect of any of the embodiments described herein, the NK lysis capacity is determined by measuring the EC 10 ~EC 70 , E.C. 10 ~EC 80 , E.C. 10 ~EC 90 , or E.C. 60 ~EC 100 The KIR3DL2-binding agent is administered to the individual in an amount that maintains a blood (eg, serum) concentration corresponding to for at least one week, at least two weeks, at least one month, or at least two months.

[0102] In one aspect of any of the embodiments described herein, the KIR3DL2-binding agent is administered to the individual in an amount that is less than an amount that maintains substantially sufficient KIR3DL2 occupancy on CTCL cells in the skin (e.g., skin lesion or tumor) between two successive administrations of the agent. In one aspect of any of the embodiments described herein, the NK lytic capacity is at least EC 50 , E.C. 70 , E.C. 80 , E.C. 90 , or E.C. 100 In one aspect of any of the embodiments described herein, an anti-KIR3DL2 antibody of the human IgG isotype, optionally for HuT78 tumor lysis by PBMCs from healthy volunteers, is administered to the individual in an amount below that which maintains a concentration in the skin (e.g., skin lesion or tumor) equivalent to 51 an EC of less than 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 1 and 50 ng / ml, optionally about 50 ng / ml in a Cr-release assay; 50 The antibody is administered to the individual in an amount (eg, weekly) of less than 15, 20, or 30 mg / kg body weight.

[0103] In one aspect of any of the embodiments described herein, the KIR3DL2-binding agent is an anti-KIR3DL2 antibody of the human IgG isotype, optionally associated with HuT78 tumor lysis by PBMCs from healthy volunteers. 51 an EC of less than 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 1 and 50 ng / ml, optionally about 50 ng / ml in a Cr-release assay; 50and is administered to an individual in an amount effective to achieve (and / or maintain for a specified period of time or between two successive administrations) a blood (serum) concentration of anti-KIR3DL2 antibody of at least 0.1 μg / ml (or, optionally, at least 0.4, 1.2 or 10 μg / ml). In one embodiment, the antibody is administered intravenously once every week, once every two weeks, once every three weeks, once every month, optionally once every month to once every two months. In one embodiment, the antibody is administered to the individual in an amount effective to maintain a blood (serum) concentration of the anti-KIR3DL2 antibody of at least 7 ng / ml (e.g., 10% lytic capacity), optionally at least 70 ng / ml (e.g., 60% lytic capacity), optionally at least 0.4 μg / ml (e.g., 80% lytic capacity), optionally at least 2 μg / ml (e.g., 90% lytic capacity), optionally at least 10 μg / ml (e.g., 100% lytic capacity), or optionally at least 20 μg / ml, 50 μg / ml or 80 μg / ml (between two successive administrations).

[0104] In one aspect of any of the embodiments described herein, the KIR3DL2 binding agent comprises an anti-KIR3DL2 antibody of the human IgG isotype and is administered to an individual in an amount effective to maintain a minimum (trough) blood (serum) concentration of the anti-KIR3DL2 antibody of 0.1-0.5 μg / ml, optionally 0.4-2 μg / ml, optionally 2-7 μg / ml, optionally 2-10 μg / ml, optionally 2-50 μg / ml, optionally 10-20 μg / ml, optionally 20-50 μg / ml, or optionally 50-100 μg / ml, over a specified period of time or between two successive administrations. In one embodiment, the antibody is administered intravenously once monthly, optionally once monthly to once every two months.

[0105] The amount of antibody required to achieve a particular blood concentration can be determined based on the characteristics of the particular antibody. In one aspect of any of the embodiments described herein, the KIR3DL2 binding agent is an anti-KIR3DL2 antibody of the human IgG isotype, optionally associated with HuT78 tumor lysis by PBMCs from healthy volunteers. 51EC20 values ​​comparable to those of the anti-KIR3DL2 antibodies disclosed herein in a Cr-release assay. 50 (e.g., an EC of an antibody disclosed herein (e.g., 2B12 antibody) 50 EC 50 , optionally less than 100ng / ml, optionally between 1 and 100ng / ml, optionally between 1 and 50ng / ml, optionally about 50ng / ml 50 In one aspect of any of the embodiments described herein, the KIR3DL2-binding agent is administered intravenously to an individual at a dose of 0.1-0.75 mg / kg, optionally 0.2-0.75 mg / kg, optionally 0.4-1 mg / kg, optionally 0.75-1.5 mg / kg, optionally about 0.01 mg / kg, optionally about 0.2 mg / kg, optionally about 0.75 mg / kg, or optionally about 1.5 mg / kg body weight. In one embodiment, the antibody is administered intravenously once per month, optionally once per month to once every two months, at a dose of 0.1-0.75 mg / kg, optionally 0.2-0.75 mg / kg, optionally 0.4-1 mg / kg, optionally 0.75-1.5 mg / kg, optionally about 0.01 mg / kg, optionally about 0.2 mg / kg, optionally about 0.75 mg / kg, optionally about 1 mg / kg, or optionally about 1.5 mg / kg body weight.

[0106] In one aspect of any of the embodiments described herein, the KIR3DL2-binding agent is administered intravenously to an individual at a dose of 0.75-10 mg / kg, optionally 0.75-1.5 mg / kg, optionally 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 6 mg / kg, or optionally about 10 mg / kg body weight. In one embodiment, the antibody is administered intravenously once weekly (optionally once every two weeks) or once weekly to once monthly (or once every four weeks) at a dose of 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 1.5-8 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 4 mg / kg, optionally about 6 mg / kg, optionally less than 10 mg / kg body weight, or optionally about 10 mg / kg body weight.

[0107] In one aspect of any of the embodiments described herein, the KIR3DL2-binding agent is administered intravenously to an individual at a dose of 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 6 mg / kg, or optionally about 10 mg / kg body weight. In one embodiment, the antibody is administered intravenously once every month to once every two months at a dose of 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 6 mg / kg, optionally less than 10 mg / kg body weight, or optionally about 10 mg / kg body weight.

[0108] In any embodiment, mg / kg can be expressed as any fixed dose equivalent of the dose, e.g., using a body weight of 65 kg or 75 kg, e.g., a fixed dose equivalent of 10 mg / kg can be defined as 750 mg.

[0109] In one embodiment, a method of treating CTCL in an individual (e.g., an individual having CTCL as described herein) is provided, the method comprising administering to the individual a KIR3DL2-binding agent for at least one administration cycle, wherein the agent has an NK lysis capacity of at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 Optionally, between two successive drug administrations, the NK lysis capacity is at least EC 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 The agent is administered in an amount effective to achieve and / or maintain a concentration resulting in a blood (e.g., serum) concentration of: Optionally, the administration cycle includes at least a first and a second (and optionally a third, fourth, fifth, sixth, seventh, and / or eighth or more) administrations of the agent. Optionally, the agent is administered intravenously.

[0110] Optionally, the treatment regimen may include induction cycles, e.g., anti-KIR3DL2 antibodies of human IgG isotype, optionally for HuT78 tumor lysis by PBMCs from healthy volunteers. 51 EC50 values ​​comparable to those of the anti-KIR3DL2 antibodies disclosed herein in a Cr-release assay 50 (e.g., that of the EC of the 2B12 antibody disclosed herein). 50 EC 50and optionally, for HuT78 tumor lysis by PBMCs from healthy volunteers. 51 EC50 of less than 100 ng / ml, optionally between 1 and 100 ng / ml, optionally between 1 and 50 ng / ml, optionally about 50 ng / ml in a Cr-release assay. 50 The regimen for the antibody comprising: (a) an induction treatment cycle comprising multiple antibody administrations, wherein the antibody is administered intravenously to the individual in an amount effective to maintain a minimum (trough) blood (serum) concentration of the anti-KIR3DL2 antibody of at least 50, 80, 90, 100, 200 or 300 μg / ml, optionally between 50-200 μg / ml, optionally between 50-100 μg / ml, over a specified period of time or between two successive administrations; followed by (b) a treatment cycle comprising multiple antibody administrations, wherein the antibody is administered intravenously to an individual in an amount effective to maintain a minimum (trough) blood (serum) concentration of the anti-KIR3DL2 antibody of less than 100 μg / ml, optionally less than 50 μg / ml, optionally at least 0.1-0.5 μg / ml, optionally 0.4-2 μg / ml, optionally 2-7 μg / ml, optionally 2-10 μg / ml, optionally 2-50 μg / ml, optionally 10-20 μg / ml, optionally 20-50 μg / ml (over a specified period of time or between two successive administrations); In one embodiment, the amount administered in treatment cycle (b) is the same as the amount administered in treatment cycle (a), but administered less frequently.

[0111] In another exemplary treatment regimen for anti-KIR3DL2 antibodies of the human IgG isotype, the treatment comprises the following: (a) an induction treatment cycle comprising multiple (e.g., at least 2, 4, 8, or 10) administrations of the antibody, wherein the antibody is administered intravenously to the individual at a dose of 1-20 mg / kg, optionally 1-10 mg / kg, optionally 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 6 mg / kg, or optionally about 10 mg / kg of body weight, about 2, 3, or 4 times per month, optionally once weekly, followed by (b) a treatment cycle (e.g., a maintenance cycle) comprising multiple (e.g., at least 2, 4, 8, or 10) administrations of the antibody, in which the antibody is administered intravenously to the individual at a dose of 1-20 mg / kg, optionally 1-10 mg / kg, optionally 1-3 mg / kg, optionally 1.5-3 mg / kg, optionally 3-6 mg / kg, optionally 6-10 mg / kg, optionally about 1 mg / kg, optionally about 1.5 mg / kg, optionally about 3 mg / kg, optionally about 6 mg / kg, or optionally about 10 mg / kg of body weight, about once every 1-3 months, optionally about once every month. In one embodiment, the dose administered in treatment cycle (b) (e.g., 1, 1.5, 3, 6 or 10 mg / kg) is the same as the dose administered in treatment cycle (a).

[0112] In one embodiment, a common treatment regimen (e.g., the same dose and same frequency of administration) that does not cause normal NK and / or T cell elimination may be advantageously used for individuals regardless of initial tumor burden and / or disease stage, in which case the common treatment regimen is preceded by an induction regimen or loading period in which an anti-KIR3DL2 antibody is administered to the individual (e.g., an individual with a high tumor burden) at a higher dosing frequency (optionally, the dose per administration of the antibody in the common treatment regimen and the induction regimen is the same).

[0113] In one embodiment, a method of treating an individual having cancer (e.g., a solid tumor) is provided, the method comprising administering to the individual an anti-KIR3DL2 antibody of the human IgG isotype for at least one administration cycle, the method comprising: a. an induction period (or cycle) during which the antibody is administered by multiple successive intravenous injections at a dose of 0.75-10 mg / kg body weight, with a frequency of 2-4 doses per month (e.g., one dose per week); and b. A maintenance period (or cycle) in which the antibody is administered by multiple consecutive intravenous injections at a dose of 0.75-10 mg / kg body weight, once every month or every two months (e.g., once every week). In one embodiment, the first administration in the maintenance period is administered within one month of the last administration in the loading period. In one embodiment, the dose for each administration during the induction cycle of (a) and each administration during the maintenance period of (b) is the same (e.g., 0.75 mg / kg, 1.5 mg / kg, 6 mg / kg, or 10 mg / kg is used in both the induction cycle and the maintenance period).

[0114] In one embodiment of any of the treatments comprising induction cycles or periods, the induction period comprises 4, 5, 6, 7, 8 or more administrations. In one embodiment, the subsequent (e.g., maintenance) period comprises at least 2, 3, 4, 5, 6, 7 or 8 administrations. In one embodiment, the antibody is administered at the same dose in both the loading period and the maintenance period. In one embodiment, each of the induction period and the maintenance period comprises administering the antibody at a dose of 0.75 mg / kg body weight. In one embodiment, each of the induction period and the maintenance period comprises administering the antibody at a dose of 1.5 mg / kg body weight. In one embodiment, each of the induction period and the maintenance period comprises administering the antibody at a dose of 3 mg / kg body weight. In one embodiment, each of the induction period and the maintenance period comprises administering the antibody at a dose of 6 mg / kg body weight. In one embodiment, each of the induction period and the maintenance period comprises administering the antibody at a dose of 10 mg / kg body weight. In one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25. In one embodiment, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26.

[0115] Optionally, the treatment of the present disclosure does not cause elimination of normal immune cells expressing KIR3DL2 (e.g., NK cells, CD8T cells, γδT cells). Optionally, the amount of the agent is effective to provide a broader anti-tumor response via elimination of circulating KIR3DL2-expressing tumor cells (e.g., where such cells are few or undetectable, e.g., in individuals with low / no blood tumor burden), e.g., via elimination of tumor cells that invade the circulation from skin lesions. Optionally, the amount of the agent is effective to provide a broader anti-tumor response via induction of antibody-dependent cellular phagocytosis (ADCP) in skin lesions.

[0116] In one embodiment, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same administration dose and frequency of the anti-KIR3DL2 agent) is used for individuals with SS and individuals with MF.

[0117] In one embodiment, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same administration dose and frequency of the anti-KIR3DL2 agent) is used for individuals with indolent disease and individuals with aggressive disease.

[0118] In one aspect, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same administration dose and frequency of the anti-KIR3DL2 agent) is used in individuals who do not have detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells) and in individuals who have detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells).

[0119] In one aspect, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same administration dose and frequency of anti-KIR3DL2 agent) is used for individuals with small numbers of detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells) and for individuals with large numbers of detectable KIR3DL2-expressing malignant cells in the circulation (e.g., KIR3DL2-expressing Sézary cells).

[0120] In one aspect, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same dose and frequency of administration of the anti-KIR3DL2 agent) is used for individuals with low or no blood tumor burden and individuals with blood tumor burden (or high blood tumor burden). In one embodiment, no or low blood tumor burden is B0 (absence of significant blood infiltration, e.g., ≦5% of peripheral blood lymphocytes are atypical (Sézary) cells) or B1 (low blood tumor burden, e.g., >5% of peripheral blood lymphocytes are atypical (Sézary) cells). In one embodiment, those with blood tumor burden or high blood tumor burden are B2 (high blood tumor burden: ≧1,000 / μL positive clone-containing Sézary cells).

[0121] In one aspect, any of the treatment regimens described herein are used to treat individuals with CTCL, where the treatment regimen (e.g., the same administration dose and frequency of the anti-KIR3DL2 agent) is used for individuals with early stage CTCL (e.g., stages I, II and / or III) as well as individuals with late stage CTCL (e.g., stage IV).

[0122] In one aspect, any of the treatment regimens described herein are used to treat an individual with CTCL with skin lesions, optionally significant or progressive skin disease, optionally with T2 (macula, papules, or plaques covering ≧10% of the skin surface, optionally also with T2a (macula only) or T2b (plaques±macula), T3 (at least one tumor (≧1 cm diameter) or T4 stage cutaneous infiltrative erythroderma covering ≧80% of the body surface area). In one embodiment, the individual has multiple and / or high skin tumor burden. In one embodiment, the individual has one or more skin tumors greater than 1 cm in diameter.

[0123] The anti-KIR3DL2 binding agent may be used in combination with one or more other treatments or treatments, such as treatments and agents typically used for the particular treatment purpose for which the agent is administered. The additional treatments or agents are typically administered in amounts and treatment regimens typically used for the treatments or agents in monotherapy for the particular disease or condition to be treated. In the treatment method, the KIR3DL2 binding compound and the second therapeutic agent or treatment may be administered sequentially. The KIR3DL2 binding compound may be administered prior to administration of the second therapeutic agent or treatment. For example, the KIR3DL2 binding compound may be administered about 0 to 30 days prior to administration of the second therapeutic agent or treatment. In some embodiments, the KIR3DL2 binding compound 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 prior to administration of the second therapeutic agent or treatment. In one embodiment, the treatment is bone marrow transplant or hematopoietic stem cell transplant.In some embodiments, the KIR3DL2 binding compound is administered simultaneously with the administration of the therapeutic agent.

[0124] In one embodiment, a subject is treated with an anti-KIR3DL2 agent prior to treatment with a bone marrow transplant or hematopoietic stem cell transplant. For example, the transplant can be administered within 1, 2, or 3 months after the end of treatment with the anti-KIR3DL2 agent.

[0125] In one embodiment, treatment with an anti-KIR3DL2 agent may be in combination with any of the following: corticosteroids, nitrogen mustard, carmustine, topical tacrolimus (Protopic®), imiquimod (Aldara®; 3M Inc.), topical retinoids, and rexinoids (bexarotene; Targretin®; Ligand Pharmaceuticals, San Diego, CA)), mogamulizumab, alemtuzumab, brentuximab vedotin, and ultraviolet light therapy (Psoralen+UVA (PUVA), narrowband UVB, and UVB), photodynamic therapy (PDT) and total body irradiation, histone deacetylase inhibitors, such as vorinostat (suberoylanilide hydroxamic acid, Zolinza®) and Romidepsin (depsipeptide, FK-228, Istodax®), histone deacetylase isotype 1, 2, Prior to treatment with an additional CTCL therapeutic agent or treatment selected from the group consisting of cyclic peptides selectively inhibiting 4 and 6, chemotherapy or combination therapy, gemcitabine, antifolate analogs such as Pralatrexate (Folotyn®), IMiDs (immunosuppressants), CC-5013 (lenalidomide; Revlimid®), CC-4047 (Actimid), and ENMD-0995, proteosome inhibitors, and bortezomib (Velcade®). Anti-KIR3DL2 agents can be advantageously used in individuals who have not received one or more (or any) of the aforementioned treatments.

[0126] In one embodiment, the anti-KIR3DL2 agent composition optionally does not include another therapeutic agent. In one embodiment, the anti-KIR3DL2 agent composition may be used as a monotherapy, e.g., without the concomitant administration of another therapeutic agent for the particular treatment purpose for which the anti-KIR3DL2 agent is administered, particularly for the treatment of CTCL.

[0127] KIR3DL2 binding agents An agent that binds to a KIR3DL2 polypeptide (terms used interchangeably: anti-KIR3DL2 agent, KIR3DL2 binding agent, anti-KIR3DL2 binding agent, etc.) may be any agent suitable for binding to KIR3DL2 and has functionality according to the present disclosure.

[0128] 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]

[0129] Also encompassed are any nucleic acids or proteins that are allelic variants of KIR3DL2 set forth in SEQ ID NO:1, eg, KIR3DL2 proteins having at least 95%, 97%, 98%, 99% or more amino acid identity.

[0130] 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.

[0131] KIR3DL2-binding agents can be readily obtained from any suitable source, for example, KIR3DL2-binding agents can be produced from a variety of immunoglobulin or non-immunoglobulin scaffolds, such as antibodies based on the Z domain of Staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins (designed ankyrin repeat domains, multimerized LDLR-A modules, avimers or cysteine-rich knottin peptides. See, for example, Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference. In certain embodiments, the KIR3DL2-binding agent comprises an antibody (or an antibody fragment).

[0132] KIR3DL2 binding agents (e.g., antibodies, antibody fragments) for use in treating CTCL may be, for example, in the form of isolated proteins or may be present on the surface of a cell (e.g., a CAR effector cell such as a T cell, NK cell, or NKT cell) or may be encoded by an internal nucleic acid, such as a poxvirus 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 comprises an extracellular single-chain antibody (scFv) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain, and is engineered to have the ability to redirect antigen recognition (i.e., KIR3DL2 recognition) based on the specificity of the monoclonal antibody when expressed in an effector cell such as a T cell, NKT cell, or NK cell. In one aspect, genetically modified immune cells are provided that express and carry on their cell surface membrane 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, e.g., one of the antibodies disclosed herein. Additionally provided are KIR3DL2-specific chimeric immune receptors, DNA constructs encoding the receptors, and plasmid expression vectors containing the constructs in the correct orientation for expression.

[0133] In one embodiment, the KIR3DL2 binding antibody is an antibody that directs ADCC, and optionally ADCP, to KIR3DL2 expressing cells.

[0134] In one embodiment, the antibody used in any of the embodiments described 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 ) is its characteristic.

[0135] Optionally, the antibody is a polypeptide that inhibits HuT78 tumor lysis by PBMCs from healthy volunteers. 51 an EC of less than 100ng / ml, optionally between 1 and 100ng / ml, optionally between 1 and 50ng / ml, optionally between 25 and 75ng / ml, optionally about 50ng / ml in a Cr-release assay 50 The antibody is optionally characterized as being related to HuT78 tumor lysis by PBMCs from healthy volunteers. 51 EC20 values ​​comparable to those of the anti-KIR3DL2 antibodies disclosed herein in a Cr-release assay. 50 (e.g., having a VH of SEQ ID NO: 31 and a VL of SEQ ID NO: 25 or 26, comprising an Fc domain of wild-type or modified human IgG1 isotype, and having an EC50 that is 1-log or 0.5-log less or within that of the EC50 of the 2B12 antibody disclosed herein for mediating ADCC).

[0136] Exemplary anti-KIR3DL2 antibodies have a potency of 1×10 for KIR3DL2, as determined, for example, by surface plasmon resonance (SPR) screening (such as analysis using a BIAcore™ SPR analyzer). -9 The average dissociation constant (K D Optionally, the anti-KIR3DL2 antibody may be about 1×10 for KIR3DL2. -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -11 Has a KD of M.

[0137] In one embodiment, an antibody that specifically binds to KIR3DL2 may be characterized by one or more of the following properties (including any combination thereof, unless such combination is inconsistent): (a) Binding to KIR3DL2 polypeptide, 10 -8 Less than M, preferably 10 -9 have a Kd of less than M, or preferably less than 10-10 M; (b) binds to at least one residue in a segment corresponding to residues 1-98 or residues 193-292 of a KIR3DL2 polypeptide; (c) competes with antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 and / or 20E9 for binding to a KIR3DL2 polypeptide; (d) competes with a natural ligand of KIR3DL2 (e.g., an HLA polypeptide, optionally HLA-B27) for binding to a KIR3DL2 polypeptide (e.g., in a polypeptide interaction assay); (e) does not substantially increase or induce uptake of a KIR3DL2 polypeptide into KIR3DL2-expressing cells and / or is not taken up into KIR3DL2-expressing cells; (f) inhibiting or not inhibiting KIR3DL2 signaling induced by a natural ligand of KIR3DL2 (e.g., an HLA polypeptide; HLA-B27); (g) does not substantially bind to KIR3DL1, KIR3DS1, KIR3DL3, KIR2DS1, KIR2DS2, KIR2DL3, KIR2DL1 and / or KIR2DS4 polypeptides; (h) binds to an epitope including any one or more of amino acid residues R13, P14, S15, H23, A25, Q27, H32, G33, I60, G62, R78, L82, W226, I231, and / or R246 of a KIR3DL2 polypeptide; and (i) has low binding to a KIR3DL2 polypeptide having a mutation in one or more of residues R13, P14, S15, H23, A25, Q27, H32, G33, I60, G62, R78, L82, W226, I231 and / or R246 of the KIR3DL2 polypeptide.

[0138] In any of the embodiments described herein, the antibody may be characterized by any one or more of features (a)-(i) above. In any of the embodiments described herein, the antibody may be further characterized by features (a), (b), (c) and (g), optionally in combination with feature (d) or (f), and optionally also with feature (e) above. Optionally, the antibody is further characterized by features (h) and / or (i).

[0139] In one embodiment, the antibody is suitable for humans. In one embodiment, the antibody is chimeric, e.g., comprises variable regions of non-human or mouse origin and constant regions of human or non-mouse origin. In one embodiment, the antibody is a human or humanized antibody.

[0140] In one embodiment, the antibody is an isotype that contains an Fc domain or is bound by an FcγR (eg, FcγRIIIA), such as an IgG1 or IgG3 isotype.

[0141] Examples of antibodies that bind to human KIR3DL2 include antibodies 19H12, 12B11, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9. These and other antibodies are described 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 but not to KIR3DL1 (or KIR3DS1). Antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 can be used, for example, as therapeutic agents administered to an individual for the elimination of KIR3DL2-expressing targets, for example by inducing ADCC towards pathogenic KIR3DL2-expressing cells, whereas antibodies 12B11 and 19H12 may be advantageous for use in detecting KIR3DL2 expression on the surface of cells (e.g., in vitro assays) since antibodies 12B11 and 19H12 are particularly efficient for detecting KIR3DL2-positive cells in detection assays, 12B11 being advantageous for immunohistochemical assays using frozen tissue sections and 19H12 being advantageous for flow cytometric detection.

[0142] The amino acid sequences of the heavy and light chain variable regions of antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9 are listed in Table C. In specific embodiments, the anti-KIR3DL2 antibody binds to approximately the same epitope or determinant as any of monoclonal antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9; optionally, the antibody comprises the antigen-binding region of antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9. In any of the embodiments described herein, the antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 may be characterized by its amino acid sequence and / or the nucleic acid sequence encoding it. In one embodiment, the monoclonal antibody may comprise the Fab or F(ab')2 portion of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. The monoclonal antibody comprises the heavy chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. According to one embodiment, the monoclonal antibody comprises three CDRs of the heavy chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. The monoclonal antibody may further comprise a variable light chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, or one, two or three CDRs of the light chain variable region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. Optionally, any one or more of the light or heavy chain CDRs may comprise one, two, three, four or five or more amino acid modifications (e.g., substitutions, insertions or deletions).Optionally, either the light and / or heavy chain variable regions comprising part or all of the antigen binding region of antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 are fused to a human IgG-type immunoglobulin constant region, optionally a human constant region, optionally a human IgG1 or IgG3 isotype.

[0143] In another embodiment, the antibody comprises the following: an HCDR1 region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9 comprising an amino acid sequence set forth in Table A, or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof (optionally, one or more of these amino acids may be substituted with a different amino acid; an HCDR1 region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9 comprising an amino acid sequence set forth in Table A, or a sequence of at least 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids thereof HCDR2 regions of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, optionally with one or more of these amino acids replaced with a different amino acid; HCDR3 regions of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, comprising an amino acid sequence set forth in Table A, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof, optionally with one or more of these amino acids replaced with a different amino acid. the LCDR1 region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an amino acid sequence as set forth in Table B, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with a different amino acid; the LCDR1 region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an amino acid sequence as set forth in Table B, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof the LCDR2 region of 3H1, 5H1, 1E2, 1C3 or 20E9 (optionally, one or more of these amino acids may be substituted with a different amino acid); the LCDR3 region of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 comprising an amino acid sequence set out in Table B, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (optionally, one or more of these amino acids may be deleted or substituted with a different amino acid).The HCDR1, 2, 3 and LCDR1, 2, 3 sequences may optionally be designated all together (or each independently) as according to the Kabat numbering system (as shown in Tables A and / or B for each CDR), the Chotia numbering system (as shown in Table A for each CDR), the IMGT numbering system (as shown in Table A for each CDR), or any other suitable numbering system.

[0144] [Table 1]

[0145] [Table 2]

[0146] [Table 3]

[0147] [Table 4]

[0148] [Table 5]

[0149] Examples of humanized VH and VL amino acid sequences of antibody 10G5 are shown in Table D as SEQ ID NOs: 13 to 17 and 8 to 12, respectively. In one aspect, an isolated humanized antibody that binds to a human KIR3DL2 polypeptide is provided, wherein the antibody comprises: an HCDR1 region comprising the amino acid sequence SYTMH as set forth in SEQ ID NO:2, or a sequence of at least 3 or 4 amino acids thereof; an HCDR2 region comprising the amino acid sequence YINPSSGYTENNRKF as set forth in SEQ ID NO:3, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof; an HCDR3 region comprising the amino acid sequence LGKGLLPPFDY as set forth in SEQ ID NO:4, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof; an LCDR1 region comprising the amino acid sequence RASENIYSNLA as set forth in SEQ ID NO:5, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof; an LCDR2 region comprising the amino acid sequence AATNLAD as set forth in SEQ ID NO:6, or a sequence of at least 3, 4 or 5 consecutive amino acids thereof; an LCDR3 region comprising the amino acid sequence QHFWGTPYT as set forth in SEQ ID NO:7, or a sequence of at least 4, 5, 6, 7, or 8 consecutive amino acids thereof.

[0150] In one embodiment, the humanized 10G5 antibody that binds to a human KIR3DL2 polypeptide has the following structure: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:6; (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and (g) Human framework sequences Includes.

[0151] In one embodiment, the humanized antibody comprises a heavy chain framework from human subgroup VH1 together with JH6, optionally the antibody comprises IGHV1-46*03 together with IGHJ6*01.In one embodiment, the humanized antibody comprises a light chain framework from human subgroup VK1, optionally IGKV1-NL1*01.

[0152] Optionally, the human framework comprises one or more mutations, e.g., back mutations that indicate a retained ability to bind KIR3DL2. Thus, embodiments of the invention include the following residues, using Abnum numbering: 10G5 VH:5, 11, 12, 13, 20, 38, 40, 48, 66, 67, 69, 71, 72a, 75 and m is an integer from 1 to 3. The backmutated 10G5 heavy chain mutants include those having backmutations in any one or more (or any combination) of:

[0153] The Abnum amino acid numbering nomenclature is described in Abhinandan and Martin, (2008) Molecular Immunology 45:3832-3839, the disclosure of which is incorporated herein by reference. Sequence numbering using the Abnum system can also be generated automatically at http: / / www.bioinfo.org.uk / abs / abnum. However, one of skill in the art will appreciate that alternative numbering systems can be used to find identifying positions that correspond to the Abnum numbering, for example, the Kabat numbering system (Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0154] Yet another embodiment of the invention is therefore a compound comprising the following residues: 10G5 VL:17, 18, 40, 45, 48, 70, 76, 100 and m is an integer from 1 to 3. The backmutated 10G5 light chain variants include those having backmutations in any one or more (or any combination) of:

[0155] The humanized antibody may further comprise one or more additional mutations (e.g., backmutations) in the human framework sequences to, for example, enhance the affinity, stability, or other properties of the humanized antibody.

[0156] In one embodiment, the humanized 10G5 antibody that binds to a human KIR3DL2 polypeptide has the following structure: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO:2; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:3; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO:6; (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 7; and (g) a human framework sequence in which a glutamine (Q) residue is present at position 39 of the VH domain and at position 38 of the VL domain Optionally, the human framework sequences include one or more back mutations.

[0157] The glutamine (Q) residue at position 39 may be naturally occurring in the human VH framework sequence or may be introduced by amino acid substitution or other modification of the sequence.

[0158] In another embodiment, the humanized antibody may comprise 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 10G5 of SEQ ID NOs: 13-17. In another particular embodiment, the humanized antibody may comprise: (a) a VH domain comprising non-human CDR residues incorporated into a human VH domain, wherein the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 10G5 VH of SEQ ID NOs: 13-17, and (b) a VL domain comprising non-human CDR residues incorporated into a human VL domain, wherein the VL domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to the humanized 10G5 VL of SEQ ID NOs: 8-12.

[0159] Examples of humanized VH and VL amino acid sequences of antibody 2B12 are shown in Table D as SEQ ID NOs: 24 to 28 and 30 to 33, respectively. In one embodiment, the humanized antibody comprises: an HCDR1 region comprising the amino acid sequence TAGMQ as set forth in SEQ ID NO: 18, or a sequence of at least 3 or 4 consecutive amino acids thereof; an HCDR2 region comprising the amino acid sequence WINSHSGVPKYAEDFK as set forth in SEQ ID NO: 19, or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof; an HCDR3 region comprising the amino acid sequence GGDEGVMDY as set forth in SEQ ID NO: 20, or a sequence of at least 5, 6, 7, or 8 consecutive amino acids thereof; an LCDR1 region comprising the amino acid sequence KASQDVSTAVA 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; an LCDR2 region comprising the amino acid sequence WTSTRHT as set forth in SEQ ID NO: 22, or a sequence of at least 3, 4 or 5 consecutive amino acids thereof; and / or an LCDR3 region comprising the amino acid sequence QQHYSTPWT as set forth in SEQ ID NO: 23, or a sequence of at least 4, 5, 6, 7, or 8 consecutive amino acids thereof.

[0160] In any embodiment 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 70%, 80%, 85%, 90%, or 95% sequence identity with a particular CDR or set of CDRs listed in the corresponding SEQ ID NO:

[0161] In one aspect, the humanized 2B12 antibody has the following structure: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and (g) Human framework sequences Includes.

[0162] In one embodiment, the humanized antibody comprises a heavy chain framework from human subgroups VH1 and / or VH7 together with JH6, optionally the antibody comprises IGHV7-4-1*02 and / or IGHV1-c*01 together with IGHJ6*01. In one embodiment, the humanized antibody comprises a light chain framework from human subgroups VK1 and / or VK4, optionally IGKV4-1*01 and / or IGKV1-39*01 together with JH4, optionally IGKJ4*01.

[0163] Optionally, the human framework comprises one or more mutations, e.g., back mutations. Optionally, the 2B12 heavy chain variant of the amino acid sequence below (SEQ ID NO:29) comprises the following residues, using Abnum numbering: 2B12 VH:2, 38, 39, 40, 43, 48, 68, 72c, 91, 108 The mutant may have back mutations in any one or more (or any combination) of: [ka]

[0164] Yet another embodiment of the invention is therefore a compound comprising the following residues: 2B12 VL:3, 8, 9, 21, 43, 71, 78, 104 and m is an integer from 1 to 3. The backmutated 2B12 light chain variants include those having backmutations in any one or more (or any combination) of:

[0165] The humanized antibody may further comprise one or more additional mutations (e.g., backmutations) in the human framework sequences to, for example, enhance the affinity, stability, or other properties of the humanized antibody.

[0166] In one aspect, the humanized 2B12 antibody has the following structure: (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19; (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; (d) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 21; (e) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 22; (f) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 23; and (g) a human framework sequence in which a glutamine (Q) residue is present at position 39 of the VH domain and at position 38 of the VL domain Optionally, the human framework sequence further comprises one or more back mutations.

[0167] In another embodiment, the humanized antibody comprises a VH domain having at least 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or more identity) to the VH domain of 2B12 or humanized 2B12 of SEQ ID NOs: 30-33. In another specific embodiment, the humanized antibody comprises: (a) a VH domain comprising non-human CDR residues incorporated into a human VH domain, wherein the VH domain is at least about 80% (e.g., at least 90%, 95%, 97%, 98%) identical to a humanized 2B12 VH of SEQ ID NOs: 30-33, and (b) a VL domain comprising non-human CDR residues incorporated into a human VH domain, wherein 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: 24-28.

[0168] The glutamine (Q) residue at position 39 may be naturally occurring in the human VH framework sequence or may be introduced by amino acid substitution or other modification of the sequence.

[0169] The 10G5 or 2B12 antibody may further comprise a native or modified human IgG constant domain. Optionally, the constant domain is an IgG1 domain, optionally further comprising a modification to increase Fc receptor binding.

[0170] [Table 6]

[0171] [Table 7]

[0172] In one embodiment, the humanized 2B12 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:25.

[0173] In one embodiment, the humanized 2B12 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0174] In one embodiment, the humanized 2B12 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:32; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0175] In one embodiment, the humanized 2B12 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:33; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:26.

[0176] In one embodiment, the humanized 10G5 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:13; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:8.

[0177] In one embodiment, the humanized 10G5 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:14; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0178] In one embodiment, the humanized 10G5 monoclonal antibody comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:15; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:9.

[0179] In one aspect, an anti-KIR3DL2 agent used in accordance with the methods of treatment described herein binds to an epitope on a KIR3DL2 polypeptide that at least partially overlaps with or includes at least one residue in a segment (or a subsequence thereof) corresponding to residues 1-192, residues 1-98, or residues 99-192 of the KIR3DL2 polypeptide of SEQ ID NO:1. In one embodiment, all of the key 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.

[0180] In one aspect, an anti-KIR3DL2 agent used in accordance with the treatment methods described herein binds to an epitope comprising one, two, three, four, five or more residues selected from the group consisting of R13, P14, S15, H23, A25, Q27, I60 and G62 (with respect to SEQ ID NO:1), and / or has low binding to a KIR3DL2 polypeptide comprising a mutation at a residue selected from the group consisting of R13, P14, S15, H23, A25, Q27, I60 and G62 (with respect to SEQ ID NO:1).

[0181] The abbreviation used for the mutations described herein is as follows: wild type residue:position in polypeptide:mutated residue, using the numbering of the residues shown in SEQ ID NO:1.

[0182] In one embodiment, the anti-KIR3DL2 agent binds to an epitope that includes residues R13, A25, and / or Q27 of a KIR3DL2 polypeptide, and / or has low binding to a KIR3DL2 polypeptide having mutations (with respect to SEQ ID NO:1) at residues R13, A25, and / or Q27. For example, the antibody has low binding to a KIR3DL2 polypeptide having mutations R13W, A25T, and / or Q27R. Optionally, the epitope additionally includes one or more of residues I60 and / or G62 (with respect to SEQ ID NO:1), and / or the antibody has low binding to a KIR3DL2 polypeptide having mutations (with respect to SEQ ID NO:1, e.g., I60N, G62S) at residues I60 and / or G62. Optionally, the epitope additionally or alternatively includes one or more of residues P14, S15 and / or H23 (with respect to SEQ ID NO:1), and / or the antibody has reduced binding to KIR3DL2 polypeptides having mutations at residues P14, S15 and / or H23 (e.g., P14S, S15A, H23S with respect to SEQ ID NO:1). Optionally, the epitope does not include residues R32 and / or G33 (with respect to SEQ ID NO:1), and / or the antibody does not have reduced binding to KIR3DL2 polypeptides having mutations at residues R32 and / or G33 (e.g., R32H and / or G33R with respect to SEQ ID NO:1). Optionally, the epitope does not include residues F50 and / or R53 (with respect to SEQ ID NO:1), and / or the antibody does not have reduced binding to a KIR3DL2 polypeptide having mutations at residues F50 and / or R53 (e.g., F50A, R53S with respect to SEQ ID NO:1).The antibody may bind to residues Q56 and / or E57, and / or residues F9 and / or S11 (e.g., KIR3DL2-HLA B27 and HLA A3 interaction), or may not bind (e.g., a non-internalizing antibody); thus, in one embodiment, optionally the epitope does not include residues F9, S11, Q56 and / or E57 (with respect to SEQ ID NO:1), and / or the antibody has low binding to KIR3DL2 polypeptides having mutations at residues F9, S11, Q56 and / or E57 (e.g., F9S and S11A, Q56S and E57A with respect to SEQ ID NO:1); in another embodiment, optionally the epitope includes residues F9, S11, Q56 and / or E57 (with respect to SEQ ID NO:1), and / or the antibody has low binding to KIR3DL2 polypeptides having mutations at residues F9, S11, Q56 and / or E57 (e.g., F9S and S11A, Q56S and E57A with respect to SEQ ID NO:1). Optionally, the epitope does not include residues H29 and / or F34 (with respect to SEQ ID NO:1), and / or the antibody does not have reduced binding to KIR3DL2 polypeptides having mutations at residues H29 and / or F34 (e.g., H29S, F34A with respect to SEQ ID NO:1). Optionally, the epitope does not include one or more of residues F9 and / or S11 (with respect to SEQ ID NO:1), and / or the antibody does not have reduced binding to KIR3DL2 polypeptides having mutations at residues F9 and / or S11 (e.g., F9S, S11A with respect to SEQ ID NO:1).

[0183] In one aspect, the anti-KIR3DL2 agent binds to an epitope that includes residues I60 and / or G62 of the KIR3DL2 polypeptide of SEQ ID NO: 1, and / or has low binding to KIR3DL2 polypeptides having mutations (with respect to SEQ ID NO: 1) at residues I60 and / or G62. For example, the antibody may have low binding to KIR3DL2 polypeptides having mutations I60N and / or G62S. Optionally, the epitope additionally or alternatively includes one or more of residues P14, S15 and / or H23 (with respect to SEQ ID NO: 1), and / or the antibody has low binding to KIR3DL2 polypeptides having mutations (with respect to SEQ ID NO: 1, e.g., P14S, S15A, H23S) at residues P14, S15 and / or H23. Optionally, the antibody does not bind to residues R13, A25 and / or Q27 of a KIR3DL2 polypeptide and / or does not have low binding to a KIR3DL2 polypeptide having mutations at residues R13, A25 and / or Q27 (e.g., a KIR3DL2 polypeptide having mutations at R13W, A25T and / or Q27R).

[0184] In one aspect, the anti-KIR3DL2 agent binds to an epitope including residues P14, S15 and / or H23 of the KIR3DL2 polypeptide of SEQ ID NO: 1, and / or has low binding to a KIR3DL2 polypeptide having mutations at residues P14, S15 and / or H23 (e.g., P14S, S15A, H23S with respect to SEQ ID NO: 1).

[0185] In one aspect, the anti-KIR3DL2 agent exhibits low binding to (1) a KIR3DL2 polypeptide having a mutation at residues I60 and / or G62 (e.g., I60N, G62S with respect to SEQ ID NO:1), and (2) a KIR3DL2 polypeptide having a mutation at residues P14, S15 and / or H23 (e.g., P14S, S15A, H23S with respect to SEQ ID NO:1).

[0186] In one embodiment, the anti-KIR3DL2 agent binds to an epitope of a KIR3DL2 polypeptide that includes (a) one, two or three of residues R13, A25 and / or G27, and (b) one or both of residues I60 and / or G62. In one embodiment, the antibody has reduced binding to a KIR3DL2 polypeptide having (a) a mutation at one, two or three of residues R13, A25 and / or G27, and (b) a mutation at one or both of residues I60 and / or G62.

[0187] In one embodiment, an anti-KIR3DL2 agent binds to an epitope that includes residues R78 and / or L82 of the KIR3DL2 polypeptide of SEQ ID NO: 1, and / or has reduced binding to a KIR3DL2 polypeptide having mutations (with respect to SEQ ID NO: 1) at residues R78 and / or L82. For example, an antibody may have reduced binding to a KIR3DL2 polypeptide having mutations R78H and L82P. Optionally, the epitope additionally includes or excludes one or more of residues K7, Y30, R31, P79, H80, S81, T83, G84, W85, S86 and / or A87 (with respect to SEQ ID NO:1), and / or the antibody has low or no binding to a KIR3DL2 polypeptide having mutations at residues K7, Y30, R31, P79, H80, S81, T83, G84, W85, S86 and / or A87 (with respect to SEQ ID NO:1). In one embodiment, the antibody binds to an epitope including one, two, three, four, five, six, seven or more residues in the segment corresponding to residues 1-98 of the KIR3DL2 polypeptide (with respect to SEQ ID NO:1), and optionally the epitope further includes one or more (e.g., one, two, three, four, five) of residues K7, Y30, R31, R78, P79, H80, S81, L82, T83, G84, W85, S86 and / or A87.

[0188] In one aspect, the anti-KIR3DL2 agent binds to an epitope that includes residue W226 of the KIR3DL2 polypeptide of SEQ ID NO: 1, and / or has reduced binding to KIR3DL2 polypeptides having a mutation (with respect to SEQ ID NO: 1) at residue W226. Optionally, the epitope additionally includes one or more of residues I231 and / or R246 (with respect to SEQ ID NO: 1), and / or the antibody has reduced binding to KIR3DL2 polypeptides having a mutation (with respect to SEQ ID NO: 1, e.g., I231M, R246P) at residues I231 and / or R246. Optionally, the epitope additionally includes residue E239 (with respect to SEQ ID NO: 1), and / or the antibody has reduced binding to KIR3DL2 polypeptides having a mutation (with respect to SEQ ID NO: 1, e.g., E239G) at residue E239.

[0189] In one aspect, the anti-KIR3DL2 agent binds to an epitope including residues I231 and / or R246 of the KIR3DL2 polypeptide of SEQ ID NO:1, and / or has low binding to a KIR3DL2 polypeptide having a mutation at residues I231 and / or R246 (with respect to SEQ ID NO:1).

[0190] In one embodiment, the anti-KIR3DL2 agent binds to an epitope that includes residue W226 and one or more of residues I231 and / or R246 of a KIR3DL2 polypeptide.

[0191] In one embodiment, an anti-KIR3DL2 agent has reduced binding to a KIR3DL2 polypeptide having a mutation at residue W226 and a mutation at one or both of residues I231 and / or R246.

[0192] The binding of the anti-KIR3DL2 antibody to cells transfected with the KIR3DL2 mutant is measured, and then the ability of the anti-KIR3DL2 antibody to bind to the wild-type KIR3DL2 polypeptide (SEQ ID NO: 1) is compared (see WO 2014 / 044686, the disclosure of which is incorporated herein by reference). As used herein, reduced binding between the anti-KIR3DL2 antibody and the 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 reduction in binding indicates that the mutated residue is directly involved in binding to the anti-KIR3DL2 antibody or is in close proximity to the binding protein when the anti-KIR3DL2 antibody binds to KIR3DL2. An antibody epitope may thus include such residues and may also include further residues which are spatially adjacent to such residues.

[0193] 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.

[0194] In some embodiments, the anti-KIR3DL2 antibodies exhibit significantly reduced binding to mutant KIR3DL2 polypeptides in which residues of a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO:1) have been substituted, e.g., mutants as described in Example 1. In the shorthand notation used herein, the format is as follows: wild-type residue:position in polypeptide:mutated residue, using the numbering of the residues shown in SEQ ID NO:1.

[0195] Optionally, the antibody has reduced binding to a KIR3DL2 polypeptide having substitutions at residues N99, H100, E130, H131, F132, V178, P179, H180, S181, P182, Y183, and / or Q184 of SEQ ID NO:1.

[0196] 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., 1, 2, 3, or 4) 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.

[0197] In some embodiments, an anti-KIR3DL2 antibody exhibits significantly reduced binding to a mutant KIR3DL2 polypeptide in which residues in the segment corresponding to residues 1-98, residues 99-292, or residues 99-192 in a wild-type KIR3DL2 polypeptide (e.g., SEQ ID NO:1) (or a subsequence thereof) are replaced with different amino acids.

[0198] In one aspect, the antibody is capable of competing with monoclonal antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 and recognizes, binds to or has immunospecificity for substantially the same or nearly the same epitope or "epitope site" on the KIR3DL2 molecule as monoclonal antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9. In other embodiments, the monoclonal antibody consists of antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, or is a derivative or fragment thereof.

[0199] Although the antibody may bind to the same epitope as antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, it will be understood that suitable antibodies may 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, may be used as an immunogen to raise antibodies, and the antibodies may recognize epitopes anywhere within the KIR3DL2 polypeptide, so long as they behave similarly 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 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9. Additionally, antibodies recognizing distinct epitopes within KIR3DL2 can be used in combination, e.g., to bind KIR3DL2 polypeptide with maximal efficacy and targeting range among different individuals.

[0200] 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 the 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 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 antibody. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence or at least about 10 consecutive amino acids thereof. The fragments typically are essentially derived 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. In another embodiment, the polypeptide is a recombinant KIR3DL2 polypeptide.

[0201] The step of immunizing a non-human mammal with an antigen may 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). In the case of an exemplary monoclonal antibody, the next step is to isolate splenocytes from the immunized non-human mammal, and then fuse these splenocytes with immortalized cells to form antibody-producing hybridomas. Once isolated and present in a single cell suspension, the lymphocytes can be fused with an immortalized cell line.

[0202] Antibodies may 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.

[0203] Identification of one or more antibodies that bind to KIR3DL2 can be easily determined using any of a variety of immunological screening assays that can evaluate antibody competition.Many such assays are routinely performed and are well known in the art (see, for example, U.S. Patent No. 5,660,827, issued August 26, 1997, which is specifically incorporated herein by reference).It will be understood that the actual determination of the epitope that the antibody described herein binds is in no way necessary to identify an antibody that binds to the same or substantially the same epitope as the monoclonal antibody described herein.

[0204] For example, if the test antibodies to be examined are obtained from different animal origins or are of different Ig isotypes, a simple competition assay can be used in which a control (e.g., 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9) and the test antibody are mixed (or serum adsorbed) before being applied to a sample containing a KIR3DL2 polypeptide.Protocols based on the use of Western blotting and BIACORE analysis are suitable for use in such competition studies.

[0205] In certain embodiments, a control antibody (e.g., 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9) is premixed with varying amounts of test antibody (e.g., about 1:10 or about 1:100) for a predetermined period of time prior to application 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. So long as it is possible to distinguish bound antibody from free antibody (e.g., using separation or washing techniques to remove unbound antibody) and 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 from the test antibody (e.g., by using a species- or isotype-specific secondary antibody or by specifically labeling 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 with a detectable label), it can be determined whether the test antibody reduces binding of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to the antigen. Binding of a (labeled) control antibody in the absence of a completely unrelated antibody can serve as a high control value. A control low value can be obtained by incubating a labeled (10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9) antibody with an unlabeled antibody of the exact same type (10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9), where competition occurs and reduces binding of the labeled antibody. A significant reduction in labeled antibody reactivity in the presence of the test antibody in the test assay indicates the test antibody is capable of recognizing substantially the same epitope.The test antibody may reduce binding of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to the KIR3DL2 antigen by at least about 50%, e.g., at least about 60%, or more preferably at least about 80% or 90% (e.g., about 65-100%), at any ratio of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9:test antibody ranging from about 1:10 to about 1:100. For example, such a test antibody may reduce binding of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 to the KIR3DL2 antigen by at least about 90% (eg, about 95%).

[0206] Competition can also be assessed, for example, by flow cytometry assays, in which cells bearing a given KIR3DL2 polypeptide can be first incubated with, for example, 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, and then with a test antibody labeled with a fluorescent dye or biotin. An antibody is said to compete with 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 if the binding obtained upon preincubation with saturating amounts of 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 is about 80%, about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained by the antibody without preincubation with 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 (as measured using fluorescence). Alternatively, an antibody is said to compete with 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 if the binding (by fluorescent dye or biotin) achieved with labeled 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9 antibody on cells preincubated with a saturating amount of the test antibody is about 80%, about 50%, about 40% or less (e.g., about 30%, 20% or 10%) of the binding obtained without preincubation with the test antibody.

[0207] Also, a simple competitive assay may be used in which a test antibody is serum-adsorbed and then applied at saturating concentration onto a surface on which KIR3DL2 antigen is immobilized. The surface in a simple competitive assay is, for example, a BIACORE chip (or other medium suitable for surface plasmon resonance analysis). A control antibody (e.g., 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9) is then contacted with the surface at KIR3DL2 saturating concentration, and the binding of KIR3DL2 to the control antibody is measured. The binding of this control antibody is compared to the binding of the control antibody to the KIR3DL2-containing surface in the absence of the test antibody. In the test assay, a significant reduction in the binding of the control antibody to the KIR3DL2-containing surface in the presence of the test antibody indicates that the test antibody can compete and recognize substantially the same epitope as the control antibody. Any test antibody can be selected that reduces the binding of a control antibody (such as 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9) to the KIR3DL2 antigen by at least about 30% or more, or about 40%. For example, such a test antibody will reduce the binding of a control antibody (such as 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3, or 20E9) to the KIR3DL2 antigen by at least about 50% (e.g., at least about 60%, at least about 70%, or more). It will be appreciated that the order of the control and test antibodies can be reversed; i.e., in a competitive assay, the control antibody can be bound to the surface first, and then the test antibody can be contacted with the surface. For example, an antibody with a higher affinity for the KIR3DL2 antigen is bound to the surface first, since the reduction in binding observed with the secondary antibody (assuming the antibodies are cross-reacting) is expected to be of a greater magnitude. Another example of such an assay is described in Saunal (1995) J. Immunol. Methods 183:33-41, the disclosure of which is incorporated herein by reference.

[0208] 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 may be determined by epitope "footprinting" using chemical modification of exposed amines / carboxyls in the KIR3DL2 protein. 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 that participate 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 interaction with the antigen-binding peptide shift in position in the spectrum of the complex compared to the spectrum of the free antigen, and the amino acids involved in binding can thus be identified.See, e.g., Ernst Schering Res Found Workshop. 2004;(44):149-67; Huang et Journal of Molecular Biology, Vol. 281(1) pp. 61-67 (1998); and Saito and Patterson, Methods. 1996 Jun;9(3):516-24.

[0209] Epitope mapping / characterization can also be performed using mass spectrometry methods. See, for example, Downward, J Mass Spectrom. 2000 Apr;35(4):493-503 and Kiselar and Downard, Anal Chem. 1999 May 1;71(9):1792-801. 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 (thereby revealing a footprint for the binder) by comparison of the samples subjected to trypsin digestion and the antibody, for example, followed by digestion with trypsin. Other enzymes such as chymotrypsin, pepsin, etc. can also or instead be used in similar epitope characterization methods. Furthermore, enzymatic digestion can provide a rapid method to analyze whether potential antigenic determinant sequences are within regions of the KIR3DL2 polypeptide that are not surface exposed and therefore perhaps not relevant in terms of immunogenicity / antigenicity. For a discussion of similar techniques, see, e.g., Manca, Ann Ist Super Sanita. 1991;27:15-9.

[0210] 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 results on binding affinity are 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.

[0211] 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.

[0212] 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. Chromatogr. 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.

[0213] 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.

[0214] Once antibodies capable of binding to KIR3DL2 and / or having other desired properties are identified, they will also typically be evaluated for their ability to bind to other polypeptides, including unrelated polypeptides, using standard methods, including those described herein.Ideally, an antibody will only bind to KIR3DL2, such as human KIR3DL2, with substantial affinity and will not bind to unrelated polypeptides at significant levels.However, it will be understood that an antibody is suitable for use in the methods of the present invention as 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.

[0215] In one aspect of any of the embodiments, the antibodies prepared according to the methods of the invention are monoclonal antibodies. In another aspect, the non-human animal used to produce the antibodies is a mammal, such as a rodent, cow, pig, poultry, horse, rabbit, goat, or sheep.

[0216] 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.

[0217] DNA encoding a monoclonal antibody, such as antibody 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 5H1, 1E2, 1C3 or 20E9, can be easily 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 mouse 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.

[0218] 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)).

[0219] In one embodiment, the antibody can mediate elimination of pathogenic KIR3DL2-expressing cells (e.g., tumor cells) via ADCC (and optionally also via ADCP). Once an antigen-binding compound is obtained, it may be evaluated for its ability to induce ADCC toward, inhibit the activity and / or proliferation of, and / or cause the elimination of, KIR3DL2-expressing target cells. Evaluation of the ability of the antigen-binding compound to induce ADCC or generally lead to the elimination or inhibition of the activity of KIR3DL2-expressing target cells can be performed at any suitable stage of the method. This evaluation may 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 may be assessed in the context of a screening method to identify candidate antigen-binding compounds or in a method in which an antigen-binding compound is selected and made suitable for human (e.g., in the case of an antibody, 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. The steps may involve testing multiple (e.g., a very large number or a small number using high throughput screening methods) antigen-binding compounds.

[0220] Testing for ADCC can be performed and determined by various assays, including those known in the art and described in the experimental examples herein. Testing for ADCC typically involves assessing cell-mediated cytotoxicity, in which anti-KIR3DL2 antibody-bound KIR3DL2-expressing target cells (e.g., celiac disease 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 increased cytokine production, expression of a cytotoxicity marker, or at least 20%, 50%, 80%, 100%, 200%, or 500% target cell lysis in the presence of target cells compared to 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.

[0221] In one embodiment, the anti-KIR3DL2 antibody does not substantially increase or induce cellular uptake of KIR3DL2 expressed on the surface of a cell. As used herein, a "non-internalizing" or "non-internalizing" anti-KIR3DL2 antibody is one that, upon binding to KIR3DL2 on a mammalian cell (i.e., cell surface KIR3DL2), is not substantially internalized by the cell (i.e., does not enter the cell).

[0222] In one embodiment, an anti-KIR3DL2 antibody can cause an increase in cell surface KIR3DL2 polypeptide available for binding by an anti-KIR3DL2 antibody, particularly on malignant cells. An antibody can, in one embodiment, increase the expression level of KIR3DL2 polypeptide on the cell surface (e.g., of a malignant cell). An antibody can, in one embodiment, increase the amount or number of KIR3DL2 polypeptide on the cell surface available for binding by an anti-KIR3DL2 antibody. An antibody can, in one embodiment, stabilize and / or cause accumulation of KIR3DL2 polypeptide present on the cell surface, e.g., an antibody can reduce receptor circulation or uptake of KIR3DL2 polypeptide. For example, an antibody that increases cell surface KIR3DL2 on pathogenic CD4+ T cells has high potency because a greater number of antibodies can bind to KIR3DL2 expressing cells (e.g., target cells, malignant cells). In one embodiment, an isolated monoclonal antibody is provided that binds to a KIR3DL2 polypeptide on the surface of a KIR3DL2-expressing cell, where the antibody causes an increase in the amount or number of KIR3DL polypeptide detectable on the cell surface after contacting the cell (in vivo or in vitro) for at least 1 hour, 3 hours, 6 hours, 12 hours, or 24 hours, which increase may be in comparison to a control antibody, e.g., an isotype control, or another antibody that binds to KIR3DL2 (e.g., an antibody having a different heavy and / or light chain variable region amino acid sequence).

[0223] Whether an anti-KIR3DL2 antibody is internalized upon binding to KIR3DL2 on a mammalian cell, or whether a KIR3DL2 polypeptide undergoes cellular uptake (e.g., upon being bound by an antibody) can be determined by various assays, such as those described in the experimental examples of PCT / EP2013 / 069302 and PCT / EP2013 / 069293, both filed on September 17, 2013. For example, cells can be incubated in tissue culture dishes in the presence or absence of relevant antibodies added to the medium and processed for microscopic analysis at desired time points. The presence of labeled antibodies that have been internalized into cells can be directly visualized by microscopy or autoradiography when using radiolabeled antibodies. Optionally, co-localization with known polypeptides or other cellular components can be assessed by microscopy; for example, the endosomal / lysosomal marker LAMP-1 (CD107a) can provide information on the subcellular localization of the internalized antibodies.

[0224] Testing whether an antibody can increase the number of KIR3DL2 polypeptides on the cell surface can be performed by incubating the test antibody with KIR3DL2-expressing cells (e.g., T-cell lymphoma) and detecting KIR3DL2 polypeptides on the cell surface after an incubation period. KIR3DL2 polypeptides can be detected using a suitable affinity reagent, e.g., one or more antibodies. Exemplary assays are shown in PCT / EP2013 / 069302 and PCT / EP2013 / 069293. For example, an antibody can induce at least a 20%, 50%, 75% or 100% increase in the number of KIR3DL2 polypeptides detectable on the cell surface after incubation (e.g., for at least 1, 3, 6, 12, 24 or 48 hours) in the presence of the test antibody compared to a control antibody (e.g., an antibody that does not bind to KIR3DL2, a different anti-KIR3DL2 antibody). Optionally, the number of KIR3DL2 polypeptides detectable on the cell surface after incubation is the number detectable using the test antibody. Optionally, the number of KIR3DL2 polypeptides detectable on the cell surface after incubation is the number detectable using a second anti-KIR3DL2 antibody that does not compete with the test antibody for binding to KIR3DL2.

[0225] In one embodiment, an anti-KIR3DL2 antibody can be tested for its ability to detectably reduce (or eliminate) binding between KIR3DL2 and its HLA natural ligand. Exemplary assays are set forth in PCT / EP2013 / 069302 and PCT / EP2013 / 069293. In one embodiment, an antibody that binds to a KIR3DL2 polypeptide is provided, wherein the antibody detectably reduces (or eliminates) binding between KIR3DL2 and a first HLA natural ligand of KIR3DL2, but does not detectably reduce (or eliminate) binding between KIR3DL2 and a second HLA natural ligand of KIR3DL2.

[0226] In one embodiment, the antibody optionally detectably reduces binding between KIR3DL2 and its HLA class I-ligand (e.g., HLA-B27). In one embodiment, the antibody optionally detectably reduces binding between KIR3DL2 and HLA-B27, but does not detectably reduce binding between KIR3DL2 and HLA-A3.

[0227] If an agent that binds to a KIR3DL2 polypeptide is identified, it can be tested to 51 By testing the ability of NK cells to lyse tumor cells (e.g., HUT78 cells) in an in vitro cytotoxicity assay, as measured by a Cr release assay, the concentration that achieves the designated "NK% lytic capacity" can be determined by the percentage of maximum tumor cells obtained (=tumor cell lysis / maximum tumor cell lysis at saturation x 100). Examples of suitable assays using PBMC and HUT78 cells as effector and target cells are described in the Examples herein. The EC of such maximum response or effect for NK lytic capacity can be determined by the percentage of maximum tumor cells obtained (=tumor cell lysis / maximum tumor cell lysis at saturation x 100). 10 , E.C. 60 , E.C. 80 , E.C. 90 , or E.C. 100 Anti-KIR3DL2 agents can be tested to determine the activity of NK cells. Typically, the NK cells are from healthy human donors, e.g., NK cells in PBMCs. A suitable number of experiments can be performed using samples from different donors, e.g., 10, 20 or more donor samples.

[0228] 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 for 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.

[0229] Thus, according to other embodiments, the antibody is a humanized antibody. "Humanized" forms of antibodies are specific chimeric immunoglobulins, immunoglobulin chains thereof, or fragments (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.

[0230] In some cases, Fv framework residues of the human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies can contain residues that 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, 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 those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally 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.

[0231] The selection of the human variable domains, both light and heavy, that will be used to make 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)).

[0232] It is further important that the antibody is 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 parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that show and display likely three-dimensional structures of candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the likely role of 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 target antigen, are achieved. In general, CDR residues are directly and most substantially involved in influencing antigen binding.

[0233] Another method of making "humanized" monoclonal antibodies is to use XenoMouse (Abgenix, Fremont, CA) as the mouse used for immunization. XenoMouse is a mouse host whose immunoglobulin genes have been replaced with functional human immunoglobulin genes. Thus, the antibodies produced by this mouse or in hybridomas made from B cells of this mouse are already humanized. XenoMouse is described in U.S. Patent No. 6,162,963, the entirety of which is incorporated herein by reference.

[0234] Human antibodies may also be produced according to a variety of other techniques, such as by using for immunization other transgenic animals engineered to express a human antibody repertoire (Jakobovitz et al., Nature 362 (1993) 255) or by selection of an antibody repertoire 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 the present application.

[0235] Considering the ability of anti-KIR3DL2 antibodies to induce ADCC, the antibodies can be modified to increase their ability to bind to Fc receptors, which can affect effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation, and phagocytosis, as well as immunoregulatory signals such as the regulation of lymphocyte proliferation and antibody secretion. Exemplary modifications include modified human IgG1 constant regions that contain at least one amino acid modification (e.g., substitution, deletion, insertion) and / or modified types of glycosylation, such as hypofucosylation. Such modifications can affect interactions with 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. The modification may, for example, increase binding of the Fc domain to FcγRIIIa on effector (eg, NK) cells.

[0236] The anti-KIR3DL2 antibody may comprise an Fc domain (or a portion thereof) of a human IgG1 or IgG3 isotype, optionally modified. Residues 230-341 (Kabat EU) are the Fc CH2 region. Residues 342-447 (Kabat EU) are the Fc CH3 region. The anti-KIR3DL2 antibody may comprise a mutant Fc region having one or more amino acid modifications (e.g., substitutions, deletions, insertions) in one or more portions that increase the affinity and avidity of the mutant Fc region for FcγR (including activating and inhibitory FcγR). In some embodiments, the one or more amino acid modifications increase the affinity of the mutant Fc region for FcγRIIIA and / or FcγRIIA. In other embodiments, the mutant Fc region further specifically binds FcγRIIB with a lower affinity than the Fc region of a comparable parent antibody (i.e., an antibody having the same amino acid sequence as the antibody except for one or more amino acid modifications in the Fc region). For example, one or both of the histidine residues at amino acid positions 310 and 435 may be substituted, e.g., with lysine, alanine, glycine, valine, leucine, isoleucine, proline, methionine, tryptophan, phenylalanine, serine, or threonine (see, e.g., PCT Publication WO 2007 / 080277), such substituted constant regions resulting in reduced binding to inhibitory FcγRIIB without reduced binding to activating FcγRIIIA. In some embodiments, such modifications increase the affinity of the mutant Fc region for FcγRIIIA and / or FcγRIIA and also enhance the affinity of the mutant Fc region for FcγyRIIB, as compared to the parent antibody. In other embodiments, the one or more amino acid modifications increase the affinity of the mutant Fc region for FcγRIIIA and / or FcγRIIA, but do not alter the affinity of the mutant Fc region for FcγRIIB, as compared to the Fc region of the parent antibody. In other embodiments, the one or more amino acid modifications enhance the affinity of the variant Fc region for FcγRIIIA and FcγRIIA but decrease affinity for FcγRIIB relative to the parent antibody.The increase in affinity and / or avidity results in detectable FcγR-binding or FcγR-associated activity in cells expressing low levels of FcγR, where the binding activity of the parent molecule (without the modified Fc region) cannot be detected in the cells.

[0237] The affinity and binding characteristics of a molecule for FcγR can be determined using in vitro assays (biochemical or immunological based assays) known in the art to determine antibody-antigen or Fc-FcγR interactions, i.e., specific binding of an antigen to an antibody or specific binding of an Fc region to an FcγR, respectively, including but not limited to ELISA assays, surface plasmon resonance assays, immunoprecipitation assays.

[0238] Also shown below are specific mutations (in the IgG1 Fc domain) that affect (enhance) FcγRIIIa or FcRn binding.

[0239] [Table 8]

[0240] In some embodiments, the molecule comprising the mutated Fc region comprises at least one amino acid modification in the CH3 domain of the Fc region (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications). In other embodiments, the molecule comprising the mutated Fc region comprises at least one amino acid modification in the CH2 domain of the Fc region, defined as spanning amino acids 231-341 (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications). In some embodiments, the molecule comprises at least two amino acid modifications (e.g., having 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), with at least one such modification in the CH3 region and at least one such modification in the CH2 region. The amino acid modification may be made, for example, in the hinge region. In certain embodiments, the invention encompasses amino acid modifications in the CH1 domain of the Fc region, defined as spanning amino acids 216-230.

[0241] Any combination of Fc modifications, e.g., U.S. Pat. Nos. 7,632,497; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7,183,387; 7,122,637; 6,821,505, and 6,737,056; WO 2011 / 109400; WO 2008 / 105886; WO 20 WO 08 / 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 Presta, LGet Any combination of the various modifications disclosed in (Shields, RLet al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields, RLet al. (2002) J. Biol. Chem. 26;277(30):26733-26740, and Shields, RLet al. (2001) J. Biol. Chem. 276(9):6591-6604) can be made.

[0242] The anti-KIR3DL2 antibody may comprise a variant Fc region, where the variant Fc region comprises at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications) compared to a wild-type Fc region, such that the molecule has enhanced effector function compared to a molecule comprising a wild-type Fc region, and optionally the variant Fc region comprises at least one amino acid modification (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), , 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 308, 309, 310, 311, 312, 316, 320, 322, 326, 329, 330, 332, 331, 332, 333, 334, 335, 337, 338, 339, 340, 359, 360, 370, 373, 376, 378, 392, 396, 399, 402, 404, 416, 419, 421, 430, 434, 435, 437, 438, and / or 439.

[0243] The anti-KIR3DL2 antibody may comprise a mutant Fc region, where the mutant Fc region comprises at least one amino acid modification compared to a wild-type Fc region (e.g., having 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acid modifications), and the molecule has enhanced effector function compared to a molecule comprising a wild-type Fc region, and optionally the mutant Fc region comprises a substitution at any one or more of positions 329, 298, 330, 332, 333, and / or 334 (e.g., an S239D, S298A, A330L, I332E, E333A, and / or K334A substitution).

[0244] In one embodiment, an antibody having a mutant or wild-type Fc region may have an altered glycosylation pattern that increases the antibody's Fc receptor binding ability. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express recombinant antibodies, thereby producing antibodies with altered glycosylation. See, for example, Shields, R Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, and EP 1,176,195; WO 06 / 133148; WO 03 / 035835; WO 99 / 54342, each of which is incorporated herein by reference in its entirety.

[0245] Generally, such antibodies with modified glycosylation are "glycooptimized", that is, the antibodies have specific N-glycan structures that confer certain desirable properties, including, but not limited to, enhanced ADCC and effector cell receptor binding activity, when compared to unmodified antibodies or antibodies with naturally occurring constant regions, produced by mouse myeloma NSO and Chinese Hamster Ovary (CHO) cells (Chu and Robinson, Current Opinion Biotechnol. 2001, 12:180-7), HEK293T expressing antibodies produced in the Examples section herein, or other mammalian host cell systems commonly used to produce recombinant therapeutic antibodies.

[0246] Monoclonal antibodies produced in mammalian host cells contain an N-linked glycosylation site at Asn297 of each heavy chain. The glycans on antibodies are typically complex biantennary structures with very low or no bisecting N-acetylglucosamine (bisecting GlcNAc) and high levels of core fucosylation. The glycan termini contain very low or no terminal sialic acid and variable amounts of galactose. For a review of the effects of glycosylation on antibody function, see Wright & Morrison, Trend Biotechnol. 15:26-31 (1997). Considerable research indicates that changes to the sugar composition of antibody glycan structures can alter Fc effector function. The key carbohydrate structure contributing to antibody activity is thought to be the fucose residue attached via an alpha-1,6 linkage to the innermost N-acetylglucosamine (GlacNAc) residue of the Fc region N-linked oligosaccharide (Shields et al., 2002).

[0247] FcγR binding requires the presence of an oligosaccharide covalently attached to the conserved Asn297 in the Fc region of human IgG1, IgG2, or IgG3 types. Nonfucosylated oligosaccharide structures have recently been associated with a dramatic increase in in vitro ADCC activity. "Asn297" refers to the amino acid asparagine located at about position 297 in the Fc region; based on minor sequence variations in the antibody, Asn297 can also be located a few amino acids upstream or downstream (usually +3 amino acids or less).

[0248] Historically, antibodies produced in CHO cells contain approximately 2-6% nonfucosylated populations. YB2 / 0 (rat myeloma) and Lecl3 cell lines (CHO-based lectin mutants) with defective GDP-mannose 4,6-dehydratase leading to a lack of GDP-fucose or GDP sugar intermediates, which are substrates for alpha 6-fucosyltransferase, have been reported to produce antibodies with 78-98% nonfucosylated species. In other examples, RNA interference (RNAi) or knockout techniques can be used to engineer cells to reduce FUT8 mRNA transcript levels or completely knock out gene expression, and such antibodies have been reported to contain up to 70% nonfucosylated glycans.

[0249] An antibody that binds to KIR3DL2 may be glycosylated with a carbohydrate chain at Asn 297. In one embodiment, the antibody will comprise a constant region comprising at least one amino acid modification in the Fc region that improves antibody binding to FcyRIIIa and / or ADCC.

[0250] In one aspect, the antibodies are hypofucosylated in their constant regions. Such antibodies may or may not contain amino acid modifications, but may be produced or treated under conditions to result in such hypofucosylation. In one aspect, an antibody composition comprises a chimeric, human, or humanized antibody as described herein, wherein at least 20, 30, 40, 50, 60, 75, 85, 90, 95%, or substantially all of the antibody species in the composition have a constant region that comprises a core carbohydrate structure that lacks fucose (e.g., complex, hybrid, and high mannose structures). In one embodiment, an antibody composition is provided in which no antibody comprises a core carbohydrate structure with fucose. The core carbohydrate will preferably be the glycan at Asn297.

[0251] In one embodiment, an antibody composition, e.g., a composition comprising an antibody that binds to KIR3DL2, is glycosylated with a glycan at Asn297, and the antibody is partially fucosylated. A partially fucosylated antibody is characterized by the percentage of anti-KIR3DL2 antibodies in the composition that lack fucose in the glycan at Asn297 being between 20% and 90%, between 20% and 80%, between 20% and 50%, between 55%, between 60%, between 70%, between 75%, between 35% and 50%, between 55%, between 60%, between 70%, between 75%, between 75%, between 80%, between 90%, between 100%, between 15 ...

[0252] The glycan can further exhibit any characteristic (e.g., the presence and proportion of complex, hybrid, and high mannose structures), including the characteristic of the N-linked glycan attached to Asn297 of an antibody derived from a human cell or of an antibody recombinantly expressed in a rodent cell, a murine cell (e.g., a CHO cell), or an avian cell.

[0253] In one embodiment, the antibody is expressed in cells that lack fucosyltransferase enzyme, so that the cell line produces proteins that lack fucose on their core carbohydrate. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6) fucosyltransferase), so that the antibodies expressed in Ms704, Ms705, and Ms709 cell lines lack fucose on their core carbohydrate. These cell lines were created by targeted disruption of the FUT8 gene in CHO / DG44 cells using two exchange vectors (see U.S. Patent Application Publication No. 20040110704 by Yamane et al.; and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22, the disclosures of which are incorporated herein by reference). Other examples include the use of antisense suppression, double-stranded RNA (dsRNA) interference, hairpin RNA (hpRNA) interference, or intron-containing hairpin RNA (ihpRNA) interference to functionally disrupt the FUT8 gene. In one embodiment, the antibody is expressed in a cell line having a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that the antibody expressed in such a cell line exhibits hypofucosylation by reducing or eliminating the alpha 1,6 bond-related enzyme.

[0254] In one embodiment, the antibody is expressed in a cell line engineered to express a glycoprotem-modifying glycosyltransferase (e.g., beta(1,4)-N-acetylglucosaminyl-transferase III (GnTHI)), such that the antibody expressed in the engineered cell line exhibits an increase in bisecting GlcNac structures resulting in increased ADCC activity of the antibody (Umana et al., WO 99 / 54342; and Umana et al. (1999) Nat. Biotech. 17:176-180, the disclosures of which are incorporated herein by reference).

[0255] In other embodiments, the antibody is expressed and the fucosyl residues are cleaved using a fucosidase enzyme. For example, the fucosidase alpha-L-fucosidase removes fucosyl residues from antibodies (Tarentino, et al. (1975) Biochem. 14:5516-5523). In other examples, cell lines producing the antibody can be treated with glycosylation inhibitors; Zhou et al. Biotech. and Bioengin. 99:652-665 (2008) described treatment of CHO cells with the alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with non-fucosylated oligomannose-type N-glucans.

[0256] In one embodiment, the antibody is expressed in a cell line that naturally has low or no enzymatic activity for adding fucosyl to the N-acetylglucosamine attached to the Fc region of the antibody, such as the rat myeloma cell line YB2 / 0 (ATCC CRL1662). Other examples of cell lines include Led3 cells, a mutant CHO cell line that has a reduced ability to add fucosyl to the Asn(297)-linked carbohydrate, which also results in hypofucosylation of antibodies expressed in the host cell (WO 03 / 035835 (Presta et al); and Shields, RX. et al. (2002) J. Biol. Chem. 277:26733-26740, the disclosures of which are incorporated herein by reference). In other embodiments, the antibody is expressed in avian cells that naturally result in antibodies with low fucose content, such as EBx® cells (Vivalis, France) (e.g. WO 2008 / 142124). Low fucosylated glycans can also be produced in cell lines of plant origin (e.g., WO 07 / 084926 A2 (Biolex Inc.) and WO 08 / 006554 (Greenovation Biotech GMBH), the disclosures of which are incorporated herein by reference).

[0257] Antibody preparations The pharma- ceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphoric acid, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, salts or electrolytes such as zinc salts, colloidal silicic acid, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.The method includes contacting the composition with the patient.Such a method will be useful for both preventive and therapeutic purposes.

[0258] For use in administration to a patient, the composition is formulated for administration to a patient. The composition may be administered parenterally, particularly by intravenous injection or infusion techniques.

[0259] Sterile injectable forms of the composition may be aqueous or oily suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and physiological saline solution. Moreover, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans, and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms may also be used for formulation purposes.

[0260] Some monoclonal antibodies have been shown to be effective in clinical settings, such as Rituxan™ (rituximab), Herceptin™ (trastuzumab), or Xolair™ (omalizumab), and similar dosing regimens (i.e., formulations and / or doses and / or administration protocols) may be used with the antibodies. For example, the antibodies present in the pharmaceutical composition may be supplied at a concentration of 10 mg / mL in 100 mg (10 mL) or 500 mg (50 mL) single-use vials.

[0261] Further aspects and advantages are disclosed in the following experimental section, which should be considered as illustrative and not limiting the scope of this application. EXAMPLES

[0262] Example 1 - Generation of KIR3DL2-selective antibodies Immunization and Screening Antibodies that bind to KIR3DL2 but not to the closely related KIR3DL1 were generated by immunizing mice with recombinant KIR3DL2-Fc fusion protein, as described in US Patent Publication No. 2015-0232556-A1. Supernatants (SN) of growing hybridomas were tested by flow cytometry against Sézary syndrome cell lines (HUT78, COU-L) and HEK-293T / KIR3DL2 domain 0-eGFP. Potentially interesting hybridomas selected from the first screening were cloned by limiting dilution technique in 96-well plates. The second screening involved the selection of hybridomas of interest by testing the supernatants of subclones by flow cytometry against HUT78, COU-L, HEK-293T / KIR3DL1 domain 0-eGFP, and HEK-293T / KIR3DL2 domain 0-eGFP. Positive subclones were injected into mice to produce ascites fluid and antibodies of interest were purified before being tested in Biacore assays using rec KIR3DL2 chips followed by various assay formats based on binding to human KIR3DL2-expressing cells.

[0263] The sequences of the variable domains of the antibody heavy (VH) and light (VL) chains were amplified by PCR from the cDNA of each antibody. The amplified sequences were run on an agarose gel and then purified using a Qiagen Gel Extraction kit. The VH and VL sequences were then subcloned into Lonza expression vectors (Double-Gene Vectors) using the InFusion system (Clontech) according to the manufacturer's instructions. After sequencing, the vectors containing the VH and VL sequences were prepared as Maxipreps using the Promega PureYield™ Plasmid Maxiprep System. The vectors were then used for HEK-293T cell transfection using Invitrogen's Lipofectamine 2000 according to the manufacturer's instructions. The antibodies generated included 10G5, 2B12, 19H12 and 12B11, among others.

[0264] Epitope mapping The antibodies were further tested for binding to a series of KIR3DL2 mutants. Antibodies 19H12 and 12B11 did not show any loss of binding to non-mutated wild-type KIR3DL2 (WTaKIR3DL2), but lost binding to mutant 11 with P179T and S181T substitutions and mutant 11A1 with V178A and H180S substitutions. Thus, the primary epitopes of these antibodies 19H12, 18B10, and 12B11 include residues P179, S181, V178, and / or H180. These residues at positions 179 and 181 in mutant 11 correspond to residues present in KIR3DL1 (KIR3DL1 has T179 and T181). Residues P179 and S181 are specifically located within the D1 domain of KIR3DL2 and on opposite faces of the KIR3DL2 protein in the HLA binding region (i.e., HLA binding pocket). Antibodies 15C11, 19H12, 18B10, and 12B11 each have reduced binding to mutant M11A4 with substitutions E130S, H131S, and R145S (complete loss of binding for 15C11 and 19H12). These residues at positions 179 and 181 in mutant 11 correspond to residues present in KIR3DL1 (KIR3DL1 has T179 and T181). Residues P179 and S181 are specifically located within the D1 domain of KIR3DL2 and on opposite faces of the KIR3DL2 protein in the HLA binding region (i.e., HLA binding pocket). Surface exposed residues adjacent to these mutated residues may also contribute to the antibody epitope, including, for example, residues N99, H100, E130, H131, F132, V178, H180, P182, Y183, and Q184 (with respect to SEQ ID NO: 1), which are located on the surface of KIR3DL2 in the region of the P179 / S181 epitope but outside the region of KIR3DL2 mutations (e.g., mutant 5 (residue P66) and mutant 8 (residue V127)) that did not result in loss of antibody binding. Antibody 2B12 lost binding to mutants with I60N and G62S substitutions and had reduced binding to mutants with P14S, S15A, and H23S substitutions, but did not lose binding to any other mutants.Thus, the primary epitopes of these antibodies include residues I60 and / or G62 (and, optionally, the epitopes further include one or more of P14, S15, and H23). Residues 60 and 62 are within the DO domain of KIR3DL2. Residues 14, 15, 23, 60, and 61 are within the DO domain of KIR3DL2.

[0265] The antibody increases the number of available cell surface KIR3DL2 receptors Part 1: Effect of staining conditions on 2B12 labeling of KIR3DL2-expressing cells This study aimed to evaluate the effect of staining conditions on 2B12 labeling of KIR3DL2 expressing cells, gated on total cells, at 4°C or 37°C, and incubation times of 2h, 4h or 24h. Briefly, 100,000 HUT78 cells per well were incubated with a dose range of 2B12 antibody (serial dilutions 1 / 3 in complete medium) starting from 0.0005μg / ml up to 30μg / ml. The protocol used was as follows: 2h incubation at 4°C and 37°C; 4h and overnight; staining in RPMI 10% with or without PFA fixation; 2 washes with SB (150μl / w); addition of anti-human Fc PE for 30min at 4°C; 2 washes with SB (100μl / w); and detection using FACS CANTO II.

[0266] The results are shown in Figure 1A. Although incubation at 4°C, which inhibits receptor uptake / circulation, was expected to result in at least comparable levels of available cell surface KIR3DL2, staining with antibody 2B12 (human IgG1) was higher at 37°C than at 4°C. Furthermore, higher median fluorescence was observed with longer incubation times, with maximal KIR3DL2 expression observed after 24 hours of incubation.

[0267] Part 2: Total, free and 2B12-bound KIR3DL2 detection on HUT78 tumor cells after overnight incubation This study aimed to assess the effect of 20 hours incubation with antibody 2B12 on cell surface KIR3DL2 levels by observing the amount of bound 2B12 (human IgG1), free (non-antibody bound) cell surface KIR3DL2 polypeptide, and total cell surface KIR3DL2 polypeptide. Briefly, HUT78 (100,000 cells / well) were incubated for 20 hours at 37° C. with a dose range of 2B12 antibody starting at 8.88 μg / ml (decreasing), 1 / 3 serial dilutions, and 11 concentrations. Dose ranges were made in duplicate to perform the following two staining conditions: -Total KIR3DL2+ bound KIR3DL2 (GaH IgG Fc-PE+mAb2-APC (non-competitive anti-KIR3LD2 mAb) (10 μg / ml) -Free KIR3DL2:2B12-PE (10μg / ml)

[0268] Staining was performed in staining buffer for 1 hour at 4°C and then analyzed on a FACS Canto II HTS. The results are shown in Figure 1B. The dark line / square represents antibody 2B12 and the light line / circle represents the isotype control. It is shown that free KIR3DL2 receptor can be detected by incubating cells with 10 μg / ml 2B12-PE to detect free receptor together with a non-competitive anti-KIR3DL2 antibody. It is shown that 2B12-bound KIR3DL2 receptor can be detected by incubating cells with a goat anti-human IgG Fc-PE secondary Ab. Both readouts correlated and had similar EC 50 The rightmost panel shows that 20 hours of incubation with 2B12 increases cell surface KIR3DL2 receptor levels as detected by the non-competitive anti-KIR3DL2 antibody mAb2-APC. Antibody 2B12 may cause conformational changes upon binding, receptor stabilization / accumulation at the cell surface and / or inhibition of uptake / recycling.

[0269] Part 3: Detection of total, free and 2B12-bound KIR3DL2 on HUT78 tumor cells after 1, 24 or 48 hours This study aimed to evaluate the kinetics of KIR3DL2 receptor expression by observing the amount of total, free and 2B12-bound KIR3DL2 after different incubation periods with antibody 2B12. Briefly, HUT78 cells (50,000 cells / well) were incubated with 2B12 (human IgG1), a (decreasing) dose range starting from 10 μg / ml, 1 / 3 serial dilutions, 11 concentrations, or with an isotype control (IC), a (decreasing) dose range starting from 10 μg / ml, 1 / 3 serial dilutions, 11 concentrations, for 1, 24 or 48 hours at 37° C. in complete medium. The dose range was realised in triplicate to carry out the following three staining conditions: -Conjugated KIR3DL2 (30 min at 4°C): GaH IgG Fc-PE, - Free KIR3DL2 + Total KIR3DL2 (1 h at 4°C): 2B12-PE (10 μg / ml) + mAb2-APC (non-competitive anti-KIR3DL2) (10 μg / ml), - Total KIR3DL2 (1 h + 30 min at 4°C): 2B12 (10 μg / ml) + GaH IgG Fc-PE.

[0270] Staining was performed in staining buffer at 4°C and analysis was performed on an HTFC Intellicyt.

[0271] The results are shown in Figure 1C. Under these culture conditions (96-well plates, 50,000 HUT78 / well at T0), KIR3DL2 detection at the cell surface is reduced in the absence of either Ab (mAb2-APC, 2B12-PE or 2B12+GaH-PE, as detected by the dots on the Y-axis).

[0272] Incubation with 2B12 at 37°C increases the surface expression of KIR3DL2 in a dose-dependent manner (as detected by non-competitive mAb2 or by 2B12 itself plus a secondary Ab). The isotype control did not cause any change in KIR3DL2. This increase is already observed after 1 hour at 37°C and appears to reach its maximum after 24 hours. Staining is optimal after 24 hours (in terms of total staining and Ab-bound receptors detected).

[0273] Antibodies do not internalize in Sézary syndrome cell lines The internalization of antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 13H1, 4B5, 5H1, 1E2, 1C3 and 20E9, as well as antibody AZ158 (anti-domain 0 mAb as a control) and other anti-D1 antibodies disclosed in PCT applications WO 2014 / 044686 and WO 2014 / 044681, was assessed by fluorescence microscopy using the HUT78 SS cell line.

[0274] material and method Hut-78 cells were incubated with 10 μg / ml of different antibodies for 1 h at 4°C. After this incubation, cells were either fixed (t=0 h) or incubated for 2 h at 37°C. The cells incubated for 2 h were then fixed and stained. Antibodies were stained using goat anti-mouse antibodies conjugated to Alexa594 (Invitrogen, A11032). LAMP-1 compartments were stained using rabbit anti-LAMP-1 antibodies (Abcam, ab24170) and revealed by goat anti-rabbit polyclonal antibodies conjugated to FITC (Abcam, ab6717). Images were acquired using an Aptome instrument (Zeiss) and analyzed using Axiovision software.

[0275] result: Anti-KIR3DL2 mAb appeared red, whereas LAMP-1 compartment appeared green. At the time of antibody addition, red KIR3DL2 staining was visible on the cell surface, whereas green LAMP-1 appeared green intracellularly. However, 2 hours after antibody addition, antibodies AZ158, 13H1 and 4B5, as well as anti-D1 antibody, each caused a colocalization of red staining with green staining, along with a decrease in red staining on the cell surface, indicating that AZ158, 13H1 and 4B5, and anti-D1 antibody were rapidly internalized. However, antibodies 10F6, 2B12, 18C6, 9E10, 10G5, 5H1, 1E2, 1C3, and 20E9 were not internalized, and 2 hours after antibody addition, red staining remained throughout the cell surface.

[0276] Antibodies can kill KIR3DL2-expressing targets via antibody-dependent cellular cytotoxicity (ADCC) Cell lysis via the ADCC mechanism is based on radioactivity 51 The level of radioactivity released from preloaded target cells was monitored in Cr release experiments (the level of radioactivity released from preloaded target cells is proportional to their death). One million target cells were incubated at 37° C. for 1 h. 51 The cells were loaded with Cr and washed three times. 3,000 cells were seeded per well (U-bottom 96-well plate) and test mAbs were added at 10 or 20 μg / ml final concentration (or increasing concentrations if dose-response relationships are studied). Effector cells were added at a defined effector:target ratio (typically 10:1) and the mixture was incubated for 4 hours at 37°C. Supernatants were analyzed on a Lumaplate instrument.

[0277] The anti-KIR3DL2 mAbs selected in Example 1 were tested at the same final concentration (10 μg / ml) to kill KIR3DL2-transfected B221 target cells. The mAbs were effective in mediating ADCC against KIR3DL2-expressing B221 targets.

[0278] Example 2 - Activity in mouse xenograft models of KIR3DL2-expressing human tumors Tumor cell lines B221 and RAJI were generated to express human KIR3DL2. The immune-compromised mice used for the B221-KIR3DL2 and RAJI-KIR3DL2 models were NOD-SCID purchased from Charles River Laboratories. In the following models, 5 million human B221-KIR3DL2 or RAJI-KIR3DL2 tumor cells (in 100 μl PBS as vehicle) were IV implanted on day 0 (D0), i.e., one day before the start of treatment (D1). From D1, mice were IV treated with various doses of anti-KIR3DL2 mAb (doses adapted to mouse weight) diluted in PBS, 2 injections per week for the duration of the entire experiment.

[0279] Control groups included depending on the experiment: - PBS / placebo treated mice as normal / tumor growth unaffected controls; - Mice injected with the same dose of an isotype control matched mAb directed against an irrelevant antigen.

[0280] Mice were weighed and observed for clinical symptoms every 2-5 days depending on the model. The percentage of body weight change was calculated relative to the body weight at D0 before tumor implantation or the highest body weight reached during the experiment. Deaths or significant weight loss of mice were recorded and survival Kaplan-Meier curves were plotted and used to calculate improvements in survival compared to control groups of mice.

[0281] The efficacy of IgG2b isotype murine anti-KIR3DL2 19H12 antibody (given at 300 μg / mouse twice weekly) was tested separately against SC B221-KIR3DL2 or RAJI-KIR3DL2 xenografts (n=6 NOD-SCID mice per group).Animals treated with anti-KIR3DL2 antibody showed increased survival compared to mice treated with isotype control corresponding mAb.

[0282] Example 3 - Improved detection methods reveal KIR3DL2 positive tumors Tumor biopsies from the RAJI-KIR3DL2 model and the RAJI-KIR3DL2 cell line were obtained and staining was performed on frozen samples using the AZ158 antibody (see WO 2010 / 081890) or the antibody 12B11 (see Example 1). KIR3DL2 was stained with anti-KIR3DL2 antibody by DAB chromogenic detection according to a standard protocol adapted for immunostaining with BenchMark XT Ventana Roche. DAB was performed for all staining control isotypes (mIgG1) and controls. Surprisingly, AZ158 was negative, but the tumor was positive when the same concentration (5 μg / ml) of the antibody 12B11 was used (see FIG. 1). Increasing the concentration of the antibody AZ158 (up to 50 μg / ml) generated extensive background staining that did not allow to distinguish the tumor samples from healthy tissue.

[0283] Next, tumor biopsies from cancer patients that had previously been stained with AZ158 were retested using antibody 12B11: biopsies that were KIR3DL2 negative with AZ158 stained with 12B11 (i.e., became KIR3DL2 positive).

[0284] Example 4: NK lysis ability assay Cell lysis via the ADCC mechanism is based on radioactivity 51 The level of radioactivity released from preloaded target cells HUT78 (ATCC reference TIB-161™ available from LGC Standards Corp.) is proportional to their cell death, as monitored by Cr release experiments. Briefly, human peripheral blood mononuclear cells (PBMCs) from healthy donors were transfected with the HUT78 target cell line (KIR3DL2 + ) in the presence of a dose range of IPH4102 mAb (humanized 2B12 mAb). HUT78 cell lysis by PBMC was monitored in a 4-hour chromium release assay using an E:T ratio of 100.

[0285] Effector cell preparation Human blood was collected in CPT tubes (n=6-8 tubes / donor, containing 7-8 ml of blood). Within 30 min of collection, the CPT tubes were centrifuged at 1500 g for 30 min at room temperature (RT) with low acceleration and low brake. After centrifugation, the mononuclear cells in the supernatant above the separation gel were transferred to a 50 ml conical tube (pooling the contents of 2-3 CPT tubes into one 50 ml tube) and supplemented to 50 ml with RPMI-1640, then centrifuged at 600 g for 10 min at RT. All cell pellets were transferred to one 50 ml conical tube and washed with 50 ml of RPMI-1640 (RT, centrifugation at 130 g for 10 min). Lysis of remaining red blood cells (RBCs) could be performed at this step by adding 1 ml of cold NH4Cl to the cell pellet and incubating at RT for 5-10 min. If RBC lysis was required, an additional washing step was performed by filling the tube with 50 ml of RPMI-1640 (10 min centrifugation at 130 g at RT). After resuspending the cell pellet in 20 ml of CCM, PBMCs were counted using a Cellometer cell counter and by excluding dead cells with trypan blue dye.

[0286] PBMC concentrations were determined using a target cell lysis assay ( 51 Cr release, 50μl / w=3×10 5 For cells, 6 × 10 6 cells / ml and in NK cell activation assay (CD137 expression, 50 μl / w = 1.25 × 10 5 In the case of cells, 2.5 × 10 6 Adjusted to cells / ml.

[0287] Target cell preparation HUT78 target cells were counted using a Cellometer cell counter and trypan blue dye to exclude dead cells. 6 50 μCi per cell 51 2.10 by adding Cr to the cell pellet in a round-bottom 14-ml polypropylene tube and incubating at 37 °C for 1 h.6 Cells 51 After chromium labeling, cells were washed three times with 10 ml of CCM (centrifugation 5 min, 500 g, RT). Cells were counted using a Kovaslide and by excluding dead cells with trypan blue dye. The cell concentration was 3 × 10 4 cells / ml (100μl / w=3×10 3 cells).

[0288] mAb solution preparation A 4x solution (50 μl / w in 200 μl / w final) of anti-KIR3DL2 antibody (1.6 ml), negative isotype control, 1.6 ml) and alemtuzumab (anti-CD52, positive control, 1.2 ml) was prepared in CCM and centrifuged at maximum speed (16100 g) for 10 min at 4°C in a tabletop centrifuge (to eliminate potential aggregates).

[0289] The highest tested concentrations were 10 μg / ml for isotype control and alemtuzumab (i.e., 40 μg / ml as a 4× solution) and 8.88 μg / ml for anti-KIR3DL2 antibody (i.e., 35.5 μg / ml as a 4× solution). For isotype control and anti-KIR3DL2 antibody, 1 / 4 serial dilutions were performed in 96 deep-well plates by transferring 400 μl of mAb solution into 1.2 ml of CCM. Eleven concentrations were tested for both Abs, but alemtuzumab was only tested at 10 μg / ml.

[0290] Assay procedure mAb solutions (50 μl / w) were transferred from 96-deep well plates to U-bottom plates in triplicate. Effector cells (PBMCs, 50 μl / w) and 51Cr-loaded target cells (HUT78, 100 μl / w) were added to the wells. The final E / T ratio was 100 / 1. Spontaneous and maximum chromium release from target cells was measured in dedicated wells (n=8 per plate) containing target cells in medium and target cells in medium + 2% Triton X-100, respectively. Plates were centrifuged at 300 g for 1 min and then incubated at 37° C. for 4 h. After 4 h of incubation, plates were centrifuged at 300 g for 3 min and 50 μl of supernatant was transferred to a Lumaplate containing scintillator. The supernatant was dried at 56° C. and the chromium released in the culture supernatant was quantified using a TopCount NXT™ microplate scintillation counter (Perkin Elmer).

[0291] Specific lysis of target cells is determined according to the following formula:

number

[0292] Example 5 - Creation of a model based on NK cell % lysis capacity to determine dose setting of anti-KIR3DL2 antibodies The pharmacokinetics of therapeutic mAbs are usually modeled using a two-compartment model (Dirks and Meibohm, 2010; Lobo et al., 2004; Morell et al., 1970; Roskos et al., 2004). The anti-KIR3DL2 antibody 2B12 obtained according to Example 1 was humanized (VH and VL amino acid sequences shown in Table D; see also WO 2015 / 136052, the disclosure of which is incorporated herein by reference); the antibody (designated IPH4102) was generated as a full-length human IgG1 isotype antibody and evaluated in cynomolgus monkeys and mice. According to the preclinical PK results in both cynomolgus monkeys and mice, IPH4102 is expected to exhibit similar PK properties to other therapeutic mAbs in humans, except for compound-specific target-mediated effects. In the case of SS and MF patients, IPH4102 inhibits KIR3DL2 +KIR3DL2 in blood and tissues in addition to normal lymphocytes + It is anticipated that target-mediated pharmacokinetics (TMDD) may affect the PK of IPH4102 in humans. Therefore, a parameter to represent TMDD was included in the PK model.

[0293] The final PK simulation model was a two-compartment model with parallel primary and saturable elimination pathways, as illustrated in Figure 2. This TMDD model can be used to describe the expected human PK for IPH4102, which includes the following: - Two-compartment distribution (blood to periphery), characterized by the intercompartmental clearance (Q) and the volume of distribution for the central and peripheral compartments (Vc and Vp, respectively). -Primary elimination from the central compartment, characterized by a single clearance parameter, CL. - Central and peripheral maximum target binding capacities (TB, respectively), which represent the amount of IPH4102 that can be bound by the available KIR3DL2 antigen at full saturation in the central and peripheral compartments, respectively maxc and T.B. maxp ). The systemic kinetics is determined by the association rate constant, K on , dissociation rate constant, K off , and the turnover rate of KIR3DL2-positive cells, K cell In fact, the association rate constant, K on is K on =K off / K D is determined as, where K D is the affinity of IPH4102 for binding to KIR3DL2.

[0294] The PK model was extended to include association of predicted serum concentrations of IPH4102 in humans with human NK cell lytic capacity, as well as KIR3DL2 saturation predictions.

[0295] The percentage of maximum tumor cell lysis obtained (= tumor cell lysis / maximum tumor cell lysis at saturation × 100) allows, e.g. 51 To describe the association between IPH4102 concentration (Conc) and NK lytic capacity measured by the Cr release assay, a single potency parameter, EC 50 and using a standard Emax type relationship: %NK dissolving ability=100×Conc / (Conc+EC 50 )

[0296] Maximum intracompartmental target binding (TB) for therapeutic mAbs max ) is as follows:TB max =Rec×C cell ×V×A N ×MW mAb ×10 9 It can be calculated as follows: Where: Rec is receptor density = number of target receptors / cell C cell is the concentration of target-positive cells in the compartment (number / mL), V is the volume of the compartment, A N is Avogadro's number for converting number of entities to moles = 6.023 x 10 23 / mol, MW mAb is the molecular weight of IPH4102 = 150,000 g / mol, 10 9 converts g to ng.

[0297] The structure of the PK / PD model is shown in Figure 1. The parameters for each compartment of the model were obtained based on in vitro data and literature information, as further described below.

[0298] The values ​​of the parameters (CL, Vc, Q, Vp) were determined based on preclinical PK results in both cynomolgus monkeys and mice, which indicate that IPH4102 is expected to exhibit PK properties similar to other therapeutic mAbs in humans, which defines the standard two-compartment model for IgG in humans.

[0299] Target binding capacity to normal immune cells in blood was determined. Briefly, results from a non-interventional, single-center, descriptive, and prospective open-label study in healthy volunteers were used to determine the total number of KIR3DL2-expressing lymphocytes. A total of 40 volunteers were enrolled and divided into two cohorts, cohort 1 consisting of 20 volunteers under 60 years of age and cohort 2 consisting of 20 volunteers over 61 years of age. Flow cytometry data were normalized using the number of white blood cells (WBCs) given by the blood composition of each donor. Blood cell subsets were defined by processing fresh white blood cell samples and then analyzing them with a panel of fluorochrome-conjugated mAbs (8-color combination). The flow cytometry gating strategy aimed to express each cell subset as a percentage of WBCs. Using these percentages and the WBC counts from the blood composition, the various blood cell subsets were defined as cells per μl of blood. Using PE-labeled anti-KIR3DL2 mAb (average value of 1 to 10 tubes), KIR3DL2 in lymphocytes was detected. + Absolute numbers of immune cell populations (mean values ​​of 1–10 tubes) and MESFs at saturation were used to calculate the total number of KIR3DL2 receptors on human lymphocytes.

[0300] KIR3DL2 + Specific information on the tissue:blood ratio of lymphocytes was not initially available. Therefore, the target binding capacity of normal immune cells in tissues was determined by the number of blood lymphocytes expressing KIR3DL2, as well as the number of KIR3DL2-expressing lymphocytes. + The estimates were based on the assumption that the cells have a distribution similar to that of other lymphocytes in general, where the number of cells in tissues is about 50 times higher than in blood, and KIR3DL receptor density was assumed to be comparable between blood and tissues.

[0301] The target binding ability to leukemic tumor cells in the blood of SS patients was evaluated. To evaluate IPH4102 target binding to blood tumor cells of SS patients, blood composition count and CD3 + CD4 + KIR3DL2 + Based on the percentage of cells expressing KIR3DL2 in 9 SS patients + The total number of tumor cells was determined. The number of KIR3DL2 molecules expressed on the cell surface of primary Sézary tumor cells was determined for each patient. + Tumor cells (CD4 + CD3 + KIR3DL2 + The mean of all measurements of absolute numbers of KIR3DL2 was calculated. The cell surface KIR3DL2 density of SS tumor cells was assessed.

[0302] Specific information on the total number of tumor T cells in the skin with respect to their target binding ability to tumor cells in the tissue was not initially available for CTCL patients. The total skin-resident T cells were estimated to be 20 billion cells, so the total tumor KIR3DL2 + Assuming that T cells do not grow much beyond this level, the median density of KIR3DL2 on tumor cells was estimated to be similar to that of circulating tumor cells in SS patients. The results showed that the TBmax obtained for tumor cells in tissues was found to be in the same range as the target binding capacity of IPH4102 to KIR3DL2 on circulating tumor cells observed in SS patients.

[0303] The mechanism of target-mediated kinetics was assumed to be limited to normal turnover of NK and tumor cells, which were independent of IPH4102 concentration.

[0304] In vitro affinity (K D ) and dissociation rate (K off). IPH4102 in vitro binding affinity to KIR3DL2 was assessed using PE-labeled IPH4102 in concentration-response flow cytometry experiments performed on KIR3DL2-transfected cell lines, KIR3DL2-expressing Sézary syndrome (SS) tumor cell lines, and SS primary tumors collected from patient blood samples. Concentration-response of IPH4102 binding to KIR3DL2 was confirmed in flow cytometry experiments on whole blood from healthy volunteers gated on NK cells, as well as in surface plasmon resonance (SPR) experiments using recombinant human KIR3DL2 protein (mean bivalent binding affinity on Biacore).

[0305] KIR3DL2 + In vitro concentration-response binding experiments with IPH4102 against Sézary cell lines (such as HuT78 or COU-L) and primary Sézary tumor cells demonstrated that the EC 50was found to be similar (0.06 μg / mL for HuT78, 0.087 μg / mL for COU-L, and 0.07 μg / mL for primary tumor cells from patients). In conclusion, the binding affinity of IPH4102 to blood tumor and immune cells was set at 70 ng / mL in the PK / PD model. In overnight staining conditions, PE labeling had only a small effect on IPH4102 affinity for KIR3DL2. Importantly, similar affinities were found for SPR (IPH4102 average bivalent binding affinity for recombinant KIR3DL2 in Biacore, 0.146 nM, equivalent to 21.9 ng / ml, shown in the table below). The dissociation rate of IPH4102 from recombinant KIR3DL2 was obtained from SPR experiments. KIR3DL2 antigen binding activity was determined using a two-step experimental setup. First, IPH4102 samples were injected at a constant concentration against the Protein A chip (antibody capture step). Second, KIR3DL2-His antigen samples were injected at a constant concentration against the captured antibody (antigen binding step), followed by dissociation and then regeneration buffer for baseline correction (blank subtraction). For batch-to-batch comparison, the average (n=3) reflectance unit (RU) ratio of bound antigen to captured antigen was used as a comparison index (0.4).

[0306] The average of three measurements of the dissociation rate for the bivalent bond was used (1.4 × 10 -4 s -1 , 0.504h -1 (equivalent to).

[0307] [Table 9]

[0308] To assess the biological and potential toxic activity of IPH4102 in a physiological context, an in vitro concentration-response assay of IPH4102 was measured in vitro on 15 human normal donor PBMCs co-incubated with HuT78 cells and increasing doses of IPH4102 mAb. Three readouts were examined in parallel: activation of NK cells by CD137 expression (flow cytometry), lysis of target cells by PBMCs (classical 51 Cr release assay) and secretion of five cytokines and chemokines: IFN-γ, TNF-α, IL-6, IL-8, MCP-1. Briefly, PBMCs from healthy donors were transfected with the HuT78 target cell line (KIR3DL2 + ) after 20 hours of incubation. Activation of NK cells in PBMCs after 20 hours of incubation was monitored using the activation marker CD137, using an E:T (effector:target) ratio of 2.5:1. Cytokines produced by PBMCs in culture supernatants during the 20 hours of incubation (CD137 assay) were quantified using AlphaLISA technology (Perkin Elmer). In parallel, NK cells were incubated with 100:1 E:T ratio after 4 hours of incubation, as described in Example 4. 51 HuT78 cell lysis by PBMC was monitored by Cr release assay.

[0309] 51 HuT78 tumor cell lysis by healthy donor PBMCs in a Cr release assay (NK cell lytic capacity) was selected as the parameter most relevant to IPH4102 safety and pharmacological activity for dose-finding determination. 51 Central EC in Cr release assay 10 and E.C. 50 (±SD) were 2 (±2.8) ng / ml and 45 (±40) ng / ml, respectively.

[0310] Therefore, the IPH4102 concentration (Conc) and % of NK lysis capacity: NK dissolution capacity%=100×Conc / (Conc+EC 50 ) A single efficacy parameter, 51 EC of IPH4102 in Cr release assay 50 A standard Emax type relationship with , i.e. = 45 ng / ml was used.

[0311] The final parameters are summarized in the table below.

[0312] [Table 10]

[0313] PD / PK simulations were then performed using the software Phoenix WinNonLin version 6.4 and the results plotted using GraphPad Prism 5 version 5.04. A model was implemented in WinNonLin and used to simulate the PK over time after 1 hour iv infusion of IPH4102 in humans for various dose levels. Based on this, doses for first-in-human (FIH) studies were determined. The selected pharmacological parameters for MABEL calculations were: 51 Hut78 tumor cell lysis by healthy donor PBMCs in a Cr release assay was a conservative assessment of the biological IPH4102-mediated response in SS patients. We determined a dose that produced a low, but discernible effect in an in vitro assay of HuT78 tumor lysis (see Example 4). A 10% response in this assay was taken as the low MABEL response (E 10 = 2ng / ml). Therefore, C max 10% of the predefined 51 The dose resulting in Cr release was of particular interest. Based on PK simulations, max , C maxThe % of NK lytic capacity at t = 3-6 h and maximum KIR3DL2 occupancy achieved at t = 3-6 h were predicted for various doses in healthy donors, MF (no circulating tumor cells) and SS (circulating tumor cells) patients, helping to determine the FHD as 0.1 μg / kg.

[0314] Simulated AUC after 1st and 4th doses of a multiple dose Phase 1 clinical trial 0~7日 , C max and accumulation index are shown in the table below for MF and SS patients.

[0315] [Table 11]

[0316] In MF and SS patients, at a dose of 0.1 μg / kg, KIR3DL2 occupancy will remain below 3%, and therefore the % of NK lysis capacity mediated by IPH4102 simulated NK cells will remain below 6%. The table below shows the C-C ... max and C トラフ Simulations of the predicted % NK lytic capacity in the circulation are summarized below.

[0317] [Table 12]

[0318] [Table 13]

[0319] Example 6 - Human Phase I Clinical Trial for Relapsed / Refractory CTCL IPH4102 (humanized IgG1 anti-KIR3DL2 antibody 2B12) is currently being tested in a first-in-human, dose-ranging Phase 1 study (NCT02593045) evaluating repeated dosing of single-agent IPH4102 in patients with relapsed / refractory CTCL.

[0320] The primary objective is to evaluate the safety and tolerability of increasing doses of IPH4102 by characterizing dose-limiting toxicities and adverse events. Secondary objectives include PK, immunogenicity and signals of antitumor clinical activity. Exploratory biomarkers aim to characterize KIR3DL2 expressing and non-expressing cells in infiltrated tissues / compartments and monitor their changes with IPH4102 treatment. Measurements of molecular residual disease will be performed in skin, blood and / or lymph nodes. Ex vivo NK cell-mediated ADCC assessment against autologous tumor cells will also be performed prior to administration to SS patients.

[0321] The study will have two sequential parts: a dose escalation followed by a cohort expansion part. The dose escalation part will have a 3+3 design with accelerated escalation and will aim to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). The doses tested included: 0.0001mg / kg, 0.001mg / kg, 0.01mg / kg, 0.05mg / kg, 0.2mg / kg, 0.75mg / kg, 1.5mg / kg, 3mg / kg, 6mg / kg and 10mg / kg body weight. In the expansion part, two CTCL subtype-specific cohorts will be tested, with each cohort including 10 additional patients to further explore the MTD or RP2D. Eligible CTCL patients must have received at least two prior antitumor systemic therapies. Center-assessed KIR3DL2 expression on malignant cells in the skin or blood is required for participation.

[0322] Patients received weekly IPH4102 doses until progression or unacceptable toxicity. Intrapatient dose escalation was not permitted until after the first full clinical evaluation at week 5 and provided that the next higher dose level was declared safe by the safety committee.

[0323] Of the 14 patients who received (or are still receiving) treatment, 11 have SS, 2 have MF, and 1 has CD4+CTCL, Not Otherwise Specified (NOS). Clinical evaluation was performed according to the published recommended standardized scoring system for assessment of tumor burden and definition of response in skin, lymph nodes, blood, and viscera, using the composite overall response score and consensus definition of clinical endpoints described in Olsen et al. (2011) American Society of Clinical Oncology 29, 2598-2607. In this system, an overall complete response (CR) is defined as the complete disappearance of all clinical evidence of disease and can only be achieved when CR is documented in all involved organs, i.e., all TNMB categories. In contrast, any progressive disease (PD) in any TNMB category is considered as an overall PD. In the intermediate situation, a composite score of partial response (PR) or stable disease (SD) is achieved according to the TNMB categories (described in Olsen et al. (2011) supra. Clinical evaluations performed included the following: - Total TNMB scoring (imaging may be required) performed prior to dosing, then at week 5 (W5), W14, W26, and then every 4 weeks until cessation of treatment; - Skin-specific mSWAT measurements performed pre-dose, at week 5, then every 2 weeks until W26, then every 4 weeks; - Assessment of blood involvement (by Sezary cell count or immunophenotyping or cytomorphology) will also be performed pre-dose, at W5, then every 2 weeks until W26, then every 4 weeks thereafter.

[0324] The clinical trial is still ongoing. Clinical evaluations of patients remaining in the trial are detailed in the table below:

[0325] [Table 14]

[0326] The table above also indicates the dose level at which each patient enrolled in the study, the number of doses of IPH4102 the patient received, the CTCL subtype and TNMB stage at the time of study enrollment. Three patients experienced an overall PR that lasted for 28, 74 and 70 days, respectively, and is still ongoing. The timing of the onset of these responses and the dose levels administered at the time of onset are also listed in the same columns.

[0327] Particular attention was paid to hematologic clinical responses, especially for patients with Sézary syndrome. As shown in the table below, of the five patients enrolled in the study, two achieved a hematologic PR and one achieved a hematologic CR.

[0328] [Table 15]

[0329] Overall, only grade 1 or 2 related adverse events (AEs) were reported. No patients in the study experienced DLTs or any grade 3-5 related AEs. No IPH4102-related skin rashes or infections were observed up to the highest dose tested.

[0330] Ex vivo functional assay results confirmed that NK cells from SS patients were functional and capable of killing autologous tumor cells via ADCC.

[0331] IPH4102 did not cause NK cell elimination. Figure 3 shows the % change from baseline (week 1 day 1) in patients' NK cells over a period of up to 50 weeks. Figure 4 shows the number of NK cells (NK cells per μl) in patients over a period of up to 50 weeks.

[0332] Primary IHC results were obtained from skin biopsies taken before and after repeated doses of IPH4102. + Signals of IPH4102 pharmacological activity in skin lesions were observed in both SS and MF patients, with evidence of significant cell depletion. Representative examples include:

[0333] Patient 3 had MF and started the study at the 0.01 mg / kg dose level. He had two biopsies (B1 and B2) taken at screening, which showed KIR3DL2 proficiency of 54% and 26%, respectively. + At week 5, a decrease in KIR3DL2 staining was observed in B1 (0.5%) but not in B2 (32%), and at W14, both lesions contained KIR3DL2 + The patients showed cytopenia (1% and 16%, respectively). The patient was in overall PR from week 10.

[0334] Patient 4 had Sézary syndrome and began the study at the 0.05 mg / kg dose level, with 52% KIR3DL2 cytotoxicity in a skin biopsy taken at screening. + At week 5, a significant decrease in KIR3DL2 staining was observed, with only 4.4% of cells expressing KIR3DL2. + The patient achieved an overall PR and blood CR by week 10 of the study.

[0335] Patient 6 had SS and started on 0.05 mg / kg. Screening biopsy revealed 17.5% KIR3DL2 + The patient showed cytosis, which decreased to 3% at week 5. The course of the lesions also improved from plaques at screening to patches at week 5. However, the patient remains in total SD (with SD in the skin and SD in the blood).

[0336] Patient 7 had SS and started on 0.2 mg / kg. Screening biopsy showed 76% KIR3DL2 + The patient showed no signs of cytosis and remained stable (62%) at week 5. The course of the lesions also improved from plaques to patches, although the patient remains in total SD (SD of the skin and SD in the blood).

[0337] In conclusion, interim analysis of primary signs of clinical activity shows that IPH4102 can confer significant clinical benefit to patients with advanced CTCL at repeated doses; responding patients received doses as low as 0.0001 mg / kg (response to blood involvement) or 0.01 mg / kg (response to skin disease). Clinical responses in blood (SS patients) were also observed in patients with very high blood involvement (e.g., patient 2, who had more than 19,000 blood Sézary cells / μL of blood at study entry). Interestingly, during the treatment period, antitumor effects were observed at levels much lower than complete NK lysis activity. Moreover, at a dose level of 0.01 mg / kg, IPH4102 is expected to reach at most a very small number of malignant cells in the skin.

[0338] Interestingly, an antitumor effect (in the skin) was also observed in a patient without blood involvement (patient 3, 0.01 mg / kg), suggesting that IPH4102 may be useful in treating individuals with indolent or early stage CTCL without significant blood involvement.

[0339] Furthermore, it would be advantageous if IPH4102 could be administered intravenously, even at low doses, and without the elimination of NK cells (a significant portion of NK cells expressing KIR3DL2), regardless of low and high doses, to treat skin diseases, since a single dosing regimen could be used for patients with or without blood involvement and / or with different tumor burdens. Despite the very wide range of blood and skin tumor burdens in CTCL patients, IPH4102 is promising for use even in the case of high tumor burdens, at doses below the amount required to occupy KIR3DL2 on tumor cells in these high-burden patients, suggesting that high-dose treatments are not necessary for these patients to maintain saturation of KIR3DL2 on malignant cells (e.g., in the skin), and furthermore, a single non-NK elimination treatment regimen could be used regardless of the level of blood or skin tumor burden (achieving sufficient receptor occupancy in tissues is generally believed to require at least 10 times the blood concentration of antibody required to achieve sufficient occupancy in the circulation). Final results from the study confirmed the excellent safety profile and promising activity of IPH4102 in this elderly and heavily pretreated patient population (n=25). Saturation of blood tumor cells was achieved in all patients at dose levels of 1.5 mg / kg and above (1.5, 3, 6 and 10 mg / kg) and regardless of the injection schedule, regardless of blood tumor burden. The objective response rate in 20 patients with Sézary syndrome, respectively, was 50%; ORR4 (response rate lasting at least 4 months) was 40%, disease control rate (DCR) was 90%, median duration of response (DOR) was 9.9 months, and median progression-free survival (PFS) was 10.8 months. The data showed substantial improvement in pruritus not only in patients with overall clinical response but also in patients with stable disease.

[0340] 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.

[0341] Use of the terms "a," "an," and "the" and similar referents in the context of describing the invention should be construed to encompass both the singular and the plural, unless otherwise specified or clearly contradicted by context.

[0342] 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).

[0343] 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 be understood to also describe a composition consisting of that element, unless otherwise specified or clearly contradicted by context).

[0344] 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 agent capable of binding to a KIR3DL2 polypeptide and causing effector cell-mediated lysis of KIR3DL2-expressing cells for use in the treatment of T-cell malignancies with tissue manifestations in both individuals with and without blood involvement, said treatment comprising administering to said individual a therapeutically effective amount of said agent that has an NK lysis capacity of at least EC50 / C10 between two successive administrations. ６０ at least one administration cycle, in which the compound is administered at least twice in an amount that maintains a blood concentration of the agent is an antibody that specifically binds to a KIR3DL2 polypeptide and is administered at a fixed dose of 10 mg / kg body weight or 750 mg, and the treatment regimen is one of the following: a lead-in period during which said amount of said antibody is administered in multiple consecutive intravenous doses at a frequency of once weekly; and a treatment period in which said amount of said antibody is administered in multiple consecutive intravenous doses at a frequency of one or two doses per month; Including, the agent comprises heavy chain CDR1, 2 and 3 (HCDR1, HCDR2, HCDR3) comprising the sequences of SEQ ID NO: 18 (HCDR1), SEQ ID NO: 19 (HCDR2) and SEQ ID NO: 20 (HCDR3), respectively, and light chain CDR1, 2 and 3 (LCDR1, LCDR2, LCDR3) comprising the sequences of SEQ ID NO: 21 (LCDR1), 22 (LCDR2) and 23 (LCDR3), respectively; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the treatment is effective in both individuals with high and individuals with low blood tumor burden.

3. The pharmaceutical composition of claim 1 or 2, wherein the treatment is used as a first line treatment.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the individual has not undergone a bone marrow transplant or a hematopoietic stem cell transplant.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the medicament is administered intravenously.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the T-cell malignancy with tissue symptoms is CTCL.

7. The pharmaceutical composition according to claim 6, wherein the CTCL is indolent CTCL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein said treatment is used for the treatment or prevention of a T-cell proliferative disorder in an individual substantially free of detectable KIR3DL2-expressing malignant cells in the circulation.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein said treatment is used in the treatment or prevention of T-cell proliferative disorders in individuals with high blood tumor burden, optionally with B2 peripheral blood infiltration.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the same dosing regimen is used for the treatment or prevention of a T-cell proliferative disorder in an individual with stage 2 or 3 mycosis fungoides.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the agent is an antibody that specifically binds to a KIR3DL2 polypeptide and comprises an Fc domain of the human IgG isotype that binds to a human CD16 polypeptide.

12. The agent specifically binds to a KIR3DL2 polypeptide and comprises an Fc region derived from a human IgG1 isotype, and is effective in inhibiting HuT78 tumor lysis by PBMCs from healthy volunteers. ５１ (a) an EC50 of an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 25 or 26, and an Fc region of a human IgG1 isotype, ５０ and / or (b) less than 100 ng / ml, optionally between 1 and 100 ng / ml. ５０ The pharmaceutical composition according to any one of claims 1 to 11, which is an antibody characterized by:

13. The treatment regimen comprises: an induction period (or cycle) during which said amount of said antibody is administered in multiple successive intravenous doses at a frequency of once weekly; and a treatment period in which said amount of said antibody is administered in multiple consecutive intravenous doses at a frequency of two doses per month; The pharmaceutical composition according to any one of claims 1 to 12, comprising:

14. The treatment regimen comprises: an induction period (or cycle) during which said amount of said antibody is administered in multiple successive intravenous doses at a frequency of once weekly; and a treatment period in which said amount of said antibody is administered in multiple consecutive intravenous doses at a frequency of once every month; The pharmaceutical composition according to any one of claims 1 to 12, comprising:

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the disease is Sezary syndrome, mycosis fungoides or NK / T cell lymphoma.

16. The drug is selected from the group consisting of: (a) an antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:25; and (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 31; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

26. The pharmaceutical composition according to any one of claims 1 to 15, wherein the antibody is selected from the group consisting of:

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