Methods for the diagnosis and treatment of T cell malignancies

Abstract

T-cell malignancies are a broad and heterogeneous group of diseases, including T-cell lymphomas and T-cell leukemias. T-cell lymphomas are a heterogeneous group of malignancies involving T lymphocytes, and are generally characterized by poor prognosis. Among them, cutaneous T-cell lymphoma mainly involves the skin. Mycosis fungoides and Sézary syndrome are the most frequent cutaneous T-cell lymphomas. The inventors have shown that both malignant and non-malignant T cells circulating in patients with Sézary syndrome express CD51. Therefore, CD51 can be a useful diagnostic, prognostic, and follow-up marker, as well as a potential therapeutic target in T-cell lymphoma.

Description

[Technical field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly oncology.

[0002] Background of the invention: T-cell malignancies are a broad and heterogeneous group of diseases, including T-cell lymphomas and T-cell leukemias. T-cell lymphomas are a heterogeneous group of malignancies involving T lymphocytes and are generally characterized by a poor prognosis. Among them, cutaneous T-cell lymphoma mainly involves the skin. Mycosis fungoides and Sézary syndrome are the most frequent cutaneous T-cell lymphomas. Sézary syndrome is defined as erythroderma (redness over the entire skin surface) and circulating tumor blood cells (1). Circulating tumor T cells express CD4 and may lose expression of CD7 and CD26, while the majority of cases show abnormal expression of CD158k (KIR3DL2), which is a surface marker of tumor T cells in Sézary syndrome (2). Early diagnosis of the disease is difficult and monitoring of hematologic complications is complex. This is because the international standard uses the loss of CD7 and CD26 markers (CD4+CD26- and CD4+CD7- cells) (3), which are not specific for tumor cells (4). The discovery of CD158k (KIR3DL2) expression by Sézary cells by Martine Bagot and Armand Bensussan allowed the use of the KIR3DL2 marker for diagnosis, disease monitoring (2), and the development of a therapeutic monoclonal antibody (lactamab). Lactamab has been tested in phase I trials with published results (5) and is currently being tested in an international, multicenter, prospective phase II trial in cutaneous T-cell lymphoma and other peripheral T-cell lymphomas. However, long-term responses are rare and new treatments are needed. Recently, treatment with an anti-CCR4 monoclonal antibody (mogamulizumab) has improved progression-free survival in cutaneous T-cell lymphoma (6). However, CCR4 is expressed not only by Sézary cells, but also by peripheral blood memory regulatory T cells, and its use has been linked to the development of autoimmune adverse reactions (7). In addition to CCR4, Sézary cells express several markers common to regulatory T lymphocytes, such as PD1 (8), CD39 (9), and TIGIT (10). Thus, there is a need to identify new markers and targets for the treatment of T cell lymphomas.

[0003] Summary of the invention: The invention is defined by the claims. In particular, the invention relates to a method for the diagnosis and treatment of T-cell malignancies, especially T-cell lymphomas or T-cell leukemias.

[0004] Detailed description of the invention: Key definitions: As used herein, the term "T cells" has its general meaning in the art and refers to an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as conventional lymphocytes because they recognize antigens with their TCR (T cell receptor for antigen), with presentation or restriction by complex major histocompatibility molecules. There are several subsets of T cells, each with different functions, such as CD8+ T cells, CD4+ T cells, and gamma delta T cells. As used herein, the term "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also referred to as cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is the relevant recognition element in major histocompatibility complex class I restricted interactions. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to the pool of CD8+ T cells of a patient that have left the bloodstream and migrated into the tumor. As used herein, the term "CD4+ T cells" (also called T helper cells or TH cells) refers to T cells that express the CD4 glycoprotein on their surface and assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD4+ T cells are activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or support active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9, TFH, or Treg, which secrete different cytokines to promote different kinds of immune responses. Signaling from APCs directs T cells to specific subtypes.In addition to CD4, TH cell surface biomarkers known in the art include CXCR3 (Th1), CCR4, Crth2 (Th2), CCR6 (Th17), CXCR5 (Tfh), and subtype-specific expression of cytokines and transcription factors including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3. As used herein, the term "gamma delta T cells" has its general meaning in the art. Gamma delta T cells normally account for 1-5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They are involved in initiating protective immune responses and it has been shown that they recognize their antigen ligands by direct interaction with the antigen without any presentation by MHC molecules of antigen presenting cells. Gamma 9 delta 2 T cells (sometimes also called gamma 2 delta 2 T cells) are gamma delta T cells that have a TCR receptor with variable domains Vγ9 and Vδ2. They form the majority of gamma delta T cells in human blood. When activated, gamma delta T cells exert a strong non-MHC restricted cytotoxic activity and are particularly efficient in killing various types of cells, especially pathogenic cells. These may be cells infected by viruses (Poccia et al., J. Leukocyte Biology, 1997, 62: 1-5) or other intracellular parasites, such as mycobacteria (Constant et al., Infection and Immunity, December 1995, vol. 63, no. 12: 4628-4633) or protozoa (Behr et al., Infection and Immunity, 1996, vol. 64, no. 8: 2892-2896). They may also be cancer cells (Poccia et al., J. Immunol., 159: 6009-6015; Fournie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147: 338-347).The possibility to modulate the activity of such cells in vitro, ex vivo or in vivo would therefore provide novel and effective therapeutic approaches in the treatment of various pathologies such as infectious diseases (especially viral or parasitic), cancer, allergies and even autoimmune and / or inflammatory disorders.

[0005] As used herein, the term "T cell malignancy" has its general meaning in the art and refers to a disease resulting from neoplastic transformation of T cells, affecting mature or immature T cells, leading to T cell lymphoma or T cell leukemia. In some embodiments, the T cell malignancy is a T cell lymphoma or T cell leukemia.

[0006] In some embodiments, the T cell malignancy is a T cell lymphoma. As used herein, the term "T cell lymphoma" has its general meaning in the art and refers to a rare form of cancerous lymphoma that affects T cells. Lymphomas arise primarily from uncontrolled proliferation of T cells and can become cancerous. T cell lymphomas are classified under non-Hodgkin's lymphoma (NHL) and account for less than 15% of all non-Hodgkin's diseases in this category. T cell lymphomas are often classified as either aggressive (fast growing) or indolent (slow growing) based on their growth patterns. In particular, T cell lymphomas include peripheral T cell lymphoma, angioimmunoblastic T cell lymphoma (AITL), and cutaneous T cell lymphoma (CTCL). In some embodiments, the T cell lymphoma is cutaneous, nodal, extranodal, or leukemic lymphoma. In some embodiments, the T cell lymphoma is peripheral T cell lymphoma, hepatosplenic T cell lymphoma (HSTCL), angioimmunoblastic T cell lymphoma (AITL), NK / T cell lymphoma (NKTL), gamma / delta T cell lymphoma, mycosis fungoides (MF) or Sézary syndrome (SS). In some embodiments, the T cell lymphoma is Sézary syndrome, NK / T cell lymphoma or gamma / delta T cell lymphoma. In some embodiments, the T cell lymphoma is HTLV1+.

[0007] As used herein, the term "cutaneous T-cell lymphoma" or "CTCL" has its general meaning in the art and refers to a rare heterogeneous group of non-Hodgkin's lymphomas derived from skin-homing mature T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) represent the most common subtypes of primary CTCL, with an incidence of 4.1 / 1,000,000 person-years and a male predominance.

[0008] As used herein, the term "Sezary syndrome" or "SS" has its common meaning in the art and refers to an aggressive form of cutaneous T-cell lymphoma characterized by the triad of erythroderma, lymphadenopathy, and circulating atypical lymphocytes (Sezary cells). SS occurs most frequently in men, is more frequent in older people, and progresses rapidly. SS corresponds to stages IVA2 and IVB of T-cell cutaneous lymphoma (see this term). Patients present with scaly erythroderma and infiltrates, often with lion face and severe pruritus. Alopecia, ectropion, mild palmoplantar keratosis, and nail dystrophy may be present. Lymphadenopathy and hepatosplenomegaly are observed. Patients often complain of shivers, chills, and general malaise.

[0009] In some embodiments, the T-cell malignancy is T-cell leukemia. As used herein, the term "T-cell leukemia" has its general meaning in the art and refers to a malignant hematological condition that affects T cells, including several types of lymphocytic leukemia. Leukemia usually develops from immature blood cells in the bone marrow and spreads through the bloodstream. There are different subtypes of leukemia: acute leukemia (AL) and chronic leukemia (CL). By way of example, acute leukemia includes acute lymphoblastic leukemia (ALL). In some embodiments, the leukemia is T-cell acute lymphoblastic leukemia (T-ALL).

[0010] As used herein, the term "T-cell acute lymphoblastic leukemia" or "T-ALL" has its general meaning in the art and refers to an aggressive hematological malignancy characterized by abnormal proliferation of immature thymocytes.

[0011] As used herein, the term "agent capable of inducing cell death of CD51-expressing cancer cells" refers to any molecule capable of inducing cell death of CD51-expressing cancer cells under cellular and / or physiological conditions. In particular, the agent is capable of inducing apoptosis of CD51-expressing cancer cells. In some embodiments, the agent is capable of depleting CD51 cancer cells. As used herein, the term "depletion" with respect to cancer cells refers to a measurable reduction in the number of CD51-expressing cancer cells in a patient. The reduction can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the term refers to an undetectable reduction in the number of CD51 cancer cells in a patient.

[0012] As used herein, the term "CD51" or "αV-integrin" has its general meaning in the art and refers to a protein encoded in humans by the ITGAV gene. An exemplary human amino acid sequence of CD51 is represented by SEQ ID NO:1. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain. αV undergoes post-translational cleavage to yield disulfide-linked heavy and light chains that combine with multiple integrin beta chains to form different integrins. Of the known associated beta chains (beta chains 1, 3, 5, 6, and 8; "ITGB1", "ITGB3", "ITGB5", "ITGB6", and "ITGB8"), each can interact with extracellular matrix ligands. The extracellular domain of CD51 extends from the amino acid residue at position 31 to the amino acid residue at position 992 in SEQ ID NO:1. [ka]

[0013] As used herein, the term "CD51 inhibitor" refers to a molecule that partially or completely blocks, inhibits, or neutralizes the biological activity or expression of CD51. A CD51 inhibitor can be any type of molecule that interferes with the signal transduction associated with CD51 in cells, for example, by reducing the transcription or translation of the nucleic acid encoding CD51, or by inhibiting or blocking CD51 polypeptide activity, or both. In particular, the CD51 inhibitor of the present invention is particularly suitable for blocking CD51-induced active TGF-beta production by T cells, which contributes to the immune evasion of tumor cells. Examples of CD51 inhibitors include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, CD51-specific aptamers, anti-CD51 antibodies, CD51-binding fragments of anti-CD51 antibodies, CD51-binding small molecules, CD51-binding peptides, and other polypeptides that specifically bind to CD51 (including, but not limited to, CD51-binding fragments of one or more CD51 ligands, optionally fused to one or more additional domains), such that interaction between the CD51 inhibitor and CD51 results in a reduction or cessation of CD51 activity or expression.

[0014] As used herein, the term "TGF-β" has its general meaning in the art and refers to transforming growth factor β. In particular, the term encompasses any isoform of TGF-β, provided that the isoform has immunosuppressive activity. Transforming growth factor β (TGF-β) actually functions as an immunosuppressant by affecting immune cell development, differentiation, tolerance induction and homeostasis (Sheng J, Chen W, Zhu HJ. The immune suppressive function of transforming growth factor-β(TGF-β)in human diseases. Growth Factors. 2015 Apr;33(2):92-101. doi: 10.3109 / 08977194.2015.1010645. Epub 2015 Feb 25).

[0015] As used herein, the term "antibody" is thus used to refer to any antibody-like molecule having an antigen-binding region, and this term includes any antibody-like molecule having an antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DAB), TandAbs dimers, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabodies; kappa(lambda)bodies (scFv-CL fusions); BiTEs (bispecific T cell engagers, scFv-scFv tandems that attract T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIPs (small immune proteins, a type of minibody); SMIPs ("small modular immunopharmaceuticals" scFv-Fc These include antibody fragments, including dimers; DARTs (ds stabilized diabodies "dual affinity retargeting"); small antibody mimetics containing one or more CDRs; and the like. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies are further described in EP 404,097 and WO 93 / 11161; whereas linear antibodies are described in Zapata et al. al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating antibodies with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments can also be synthesized recombinantly or chemically. Techniques for producing antibody fragments are well known and described in the art.For example, Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 each further describe and enable the production of effective antibody fragments. In some embodiments, the antibodies of the invention are single chain antibodies. As used herein, the term "single domain antibody" has its general meaning in the art and refers to a single heavy chain variable domain of a type of antibody that is naturally devoid of light chains and can be found in mammals of the Camelidae family. Such single domain antibodies are also "Nanobodies®". For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341(6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220, WO 06 / 003388. In natural antibodies, two heavy chains are linked to each other by disulfide bridges, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains a different sequence domain. The light chain contains two domains, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CHI, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine antigen binding recognition and specificity. The constant region domains of the light chain (CL) and the heavy chain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental transfer, complement fixation, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain.The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues that are mainly from the hypervariable or complementarity determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or may affect the overall domain structure and therefore the binding site. Complementarity determining regions or CDRs refer to the amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. An antigen binding site thus typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences inserted between the CDRs. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, United States Department of Health and Human Services, NIH, USA (hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not always correspond directly to the linear numbering of the amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, corresponding to shortening of or insertion into structural components, whether framework or complementarity determining regions (CDRs) of the basic variable domain structure. The exact Kabat numbering of residues may be determined for a given antibody by alignment of the homologous residues in the antibody's sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2), and residues 95-102 (H-CDR3) according to the Kabat numbering system.The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2), and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0016] As used herein, the term "bind" indicates that an antibody has affinity for a surface molecule. The term "affinity" as used herein means the strength of binding of an antibody to an epitope. The affinity of an antibody is given by the dissociation constant Kd and is 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 Ka is defined by 1 / Kd. Preferred 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 references are incorporated herein by reference in their entireties. One preferred standard method well known in the art for determining the affinity of mAbs is the use of a Biacore instrument.

[0017] As used herein, the term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

[0018] As used herein, the term "chimeric antibody" refers to an antibody that comprises the VH and VL domains of a non-human antibody and the CH and CL domains of a human antibody. In some embodiments, a "chimeric antibody" is an antibody molecule in which (a) the constant region (i.e., heavy and / or light chain), or a portion thereof, has been modified, substituted, or exchanged such that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function, and / or species, or an entirely different molecule that confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, has been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized antibodies and particularly humanized antibodies. In addition, chimeric antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (See U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0019] As used herein, the term "humanized antibody" refers to an antibody having variable region framework and constant regions from a human antibody, but retaining the CDRs of the previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from a non-human immunoglobulin. For the most part, humanized antibodies and their antibody fragments can be human immunoglobulins (recipient antibody or antibody fragment) in which residues from the recipient complementarity determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences. 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. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Typically, a humanized antibody or antibody fragment thereof comprises substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or a significant portion of the FR regions being those of a human immunoglobulin sequence. A humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0020] As used herein, the term "bispecific antibody" has its general meaning in the art and refers to an artificial hybrid antibody having two different pairs of heavy and light chains and also two different antigen-binding sites.

[0021] As used herein, the term "chimeric antigen receptor" or "CAR" has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide that comprises an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. Characteristics of CARs include their ability to redirect the specificity and reactivity of T cells to selected targets in a non-MHC restricted manner, utilizing the antigen-binding properties of monoclonal antibodies. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. The chimeric antigen receptors of the present invention typically comprise an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0022] The term "CAR-T cells" as used herein refers to T lymphocytes that have been genetically engineered to express a CAR. The definition of CAR T cells encompasses all classes and subclasses of T lymphocytes, including CD4+, CD8+ T cells, gamma delta T cells, as well as effector T cells, memory T cells, regulatory T cells, and the like. The genetically modified T lymphocytes may be "derived" or "obtained" from the patient undergoing treatment with the genetically modified T cells, or they may be "derived" or "obtained" from a different patient.

[0023] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatments as well as curative or disease-modifying treatments, including treatment of patients at risk of or suspected of having a disease as well as patients who have been diagnosed as ill or suffering from a disease or medical condition, and includes suppression of clinical recurrence. Treatment may be administered to a patient who has a medical disorder or may eventually acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the patient's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, e.g., a pattern of medication used during treatment. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen", which may involve administering a larger dose of drug than the physician may use during a maintenance regimen, administering a drug more frequently than the physician may administer during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, e.g., to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a certain predetermined criterion (e.g., disease manifestation, etc.).

[0024] As used herein, the term "therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve a desired therapeutic result. The therapeutically effective amount of an active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the drug are outweighed by the therapeutically beneficial effects. Efficient dosages and administration regimens for the active agent depend on the disease or condition being treated and can be determined by one of ordinary skill in the art. A physician with ordinary skill in the art can easily determine and formulate the effective amount of the pharmaceutical composition required. For example, a physician may start the dosage of the active agent used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, the appropriate dosage of the composition of the present invention is the amount of the compound that is the lowest dosage effective to produce a therapeutic effect according to a particular dosing regimen. Such an effective dosage will generally depend on the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of a disease. Typically, the ability of a compound to inhibit cancer may be evaluated, for example, in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate symptoms in a patient. One of skill in the art would be able to determine such amounts based on factors such as the size of the patient, the severity of the patient's symptoms, and the particular composition or route of administration selected. Exemplary, non-limiting ranges for a therapeutically effective amount of an inhibitor of the present invention are about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg, such as about 0.5, such as about 0.3, about 1, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg. An exemplary, non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is 0.02-100 mg / kg, such as about 0.02-30 mg / kg, such as about 0.05-10 mg / kg or 0.1-3 mg / kg, for example, about 0.5-2 mg / kg.Administration may be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, e.g., proximal to the target site. The dosing regimen in the above methods and uses of treatment is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally lowered or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of the treatment is monitored during the treatment, e.g., at predefined time points. In some embodiments, the effectiveness may be monitored by visualization of the diseased area, or by other diagnostic methods further described herein, e.g., by performing one or more PET-CT scans using the labeled inhibitors of the present invention, fragments derived from the inhibitors of the present invention, or mini-antibodies. If desired, the effective daily dose of the pharmaceutical composition may be administered as 2, 3, 4, 5, 6 or more sub-doses administered separately at suitable intervals throughout the day, optionally in unit dosage forms. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, for example, more than 24 hours, to minimize any undesirable side effects. Effective doses of the inhibitors of the invention may also be administered using weekly, biweekly, or three-week dosing periods. The dosing period may be limited, for example, to 8 weeks, 12 weeks, or until clinical progression is established.As a non-limiting example, treatment according to the invention may be performed in an amount of about 0.1 to 100 mg / kg, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg / day of an inhibitor of the invention. The dose may be provided using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or a combination thereof, on at least one of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks after initiation of treatment, or any combination thereof.

[0025] How it's diagnosed: A first object of the present invention relates to a method for diagnosing T-cell malignancies comprising detecting the expression level of CD51 in a sample obtained from a patient.

[0026] In some embodiments, the present invention relates to a method for diagnosing T-cell lymphoma in a patient, comprising detecting the expression level of CD51 in a sample obtained from the patient. In some embodiments, the method of the present invention is particularly suitable for diagnosing Sézary syndrome, NK / T-cell lymphoma, or gamma / delta T-cell lymphoma. In some embodiments, the method of the present invention is particularly suitable for diagnosing cutaneous T-cell lymphoma. More specifically, the method of the present invention is particularly suitable for diagnosing Sézary syndrome.

[0027] In some embodiments, the present invention relates to a method for diagnosing T-cell leukemia in a patient, comprising detecting the expression level of CD51 in a sample obtained from the patient. In some embodiments, the T-cell leukemia is T-cell acute lymphoblastic leukemia.

[0028] As used herein, the term "sample" refers to any biological sample obtained for the purpose of in vitro evaluation. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a PBMC sample. In some embodiments, the sample is a sample of (i) purified blood leukocytes, (ii) peripheral blood mononuclear cells or PBMCs, (iii) purified lymphocytes, (iv) purified T cells, (v) purified CD4+ T cells, or (vi) purified CD3+ T cells. In some embodiments, the biological sample is a tissue sample. The term "tissue sample" includes sections of tissue, such as biopsy or autopsy samples, and frozen sections taken for histological purposes. Thus, in some embodiments, the tissue sample may come from a biopsy performed on the skin of a subject.

[0029] In some embodiments, the level of the marker is determined by immunohistochemistry (IHC). Immunohistochemistry typically includes the following steps: i) fixing the tissue sample in formalin, ii) embedding the tissue sample in paraffin, iii) cutting the tissue sample into sections for staining, iv) incubating the sections with a binding partner specific for the marker, v) rinsing the sections, vi) incubating the sections with a biotinylated secondary antibody, and vii) revealing the antigen-antibody complex with an avidin-biotin-peroxidase complex. Thus, the tissue sample is first incubated with the binding partner. After washing, the labeled antibody bound to the marker of interest is revealed by a suitable technique, depending on the type of label carried by the labeled antibody, e.g., radioactive, fluorescent, or enzymatic label. Multiple labels can be performed simultaneously. Alternatively, the method of the present invention may use an amplification system (to enhance the staining signal) and a secondary antibody bound to an enzymatic molecule. Such bound secondary antibodies are commercially available, e.g., from Dako, EnVision system. A counterstain may be used (e.g., H&E, DAPI, Hoechst). Other staining methods may be accomplished using any suitable method or system, including automated, semi-automated, or manual systems, as would be apparent to one of skill in the art. For example, one or more labels may be attached to the antibody, thereby allowing detection of the target protein (i.e., marker). Exemplary labels include radioisotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof. In some embodiments, the label is a quantum dot. Non-limiting examples of labels that can be conjugated to the primary and / or secondary affinity ligands include fluorescent dyes or metals (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromogenic dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole), and bioluminescent proteins (e.g., luciferin, luciferase), haptens (e.g., biotin).A variety of other useful fluorescers and chromophores are described in Stryer L (1968) Science 162:526-533 and Brand L and Gohlke JR (1972) Annu. Rev. Biochem. 41:843-868. Affinity ligands can also be labeled with enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3H, 14C, 32P, 35S, 125I) and particles (e.g., gold). Different types of labels can be conjugated to affinity ligands using various chemical reactions, e.g., amine reactions or thiol reactions. However, other reactive groups besides amines and thiols can be used, e.g., aldehydes, carboxylic acids, and glutamines. A variety of enzymatic staining methods are known in the art for detecting proteins of interest. For example, enzyme interactions can be visualized using different enzymes, such as peroxidase, alkaline phosphatase, or different chromogens, such as DAB, AEC, or Fast Red. In other examples, antibodies can be conjugated to peptides or proteins that can be detected via labeled binding partners or antibodies. In indirect IHC assays, a second antibody or a second binding partner is required to detect the binding of the first binding partner, since it is unlabeled. The resulting stained specimens are imaged using a system that displays a detectable signal and obtains an image, such as a digital image of the staining, each of which displays a detectable signal. Methods for image acquisition are well known to those skilled in the art. For example, once a sample is stained, any optical or non-optical imaging device can be used to detect the stain or biomarker label, such as an upright or inverted optical microscope, a scanning confocal microscope, a camera, a scanning or tunneling electron microscope, a canning probe microscope, and an imaging infrared detector. In some examples, the image can be captured digitally. The resulting image can then be used to quantitatively or semi-quantitatively determine the amount of the marker in the sample.A variety of automated sample processing, scanning, and analysis systems suitable for use in immunohistochemistry are available in the art. Such systems can include automated staining and microscope scanning, computer image analysis, serial section comparison (to control for variations in sample orientation and size), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed). Cellular imaging systems are commercially available that combine conventional optical microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, for example, the CAS-200 system (Becton, Dickinson & Co.). In particular, detection can be made manually or by image processing techniques involving computer processors and software. Using such software, for example, images can be configured, calibrated, standardized, and / or verified based on factors including, for example, staining quality or staining intensity, using procedures known to those skilled in the art (see, for example, published U.S. Patent Publication No. US20100136549). Images can be quantitatively or semi-quantitatively analyzed and scored based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry is a method in which a histochemically tested sample is scanned and scored to identify and quantify the presence of specific biomarkers (i.e., markers). Quantitative or semi-quantitative methods can use imaging software to detect the density or amount of staining, or can use methods to detect staining by the human eye, where a trained operator ranks the results numerically.For example, images can be quantitatively analyzed using pixel counting algorithms (e.g., Aperio Spectrum Software, Automated Quantitative Analysis platform (AQUA® platform), and other standard methods to measure or quantify or semi-quantify the degree of staining; see, e.g., U.S. Patent No. 8,023,714; U.S. Patent No. 7,257,268; U.S. Patent No. 7,219,016; U.S. Patent No. 7,646,905; Published U.S. Patent Publication Nos. US20100136549 and 20110111435; Camp et al. (2002) Nature Medicine, 8:1323-1327; Bacus et al. (1997) Analyt Quant Cytol Histol, 19:316-328). The ratio of strong positive staining (e.g., brown staining) to the sum of the total stained area can be calculated and scored. The amount of detected biomarker (marker) is quantified and given as a percentage of positive pixels and / or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as a percentage of stained area, e.g., a percentage of positive pixels. For example, a sample may have at least or about at least or about 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more positive pixels compared to the total stained area. In some embodiments, a score is given to the sample, which is a numerical representation of the intensity or amount of histochemical staining of the sample, and represents the amount of target biomarker (e.g., marker) present in the sample. The optical density or area percentage values ​​can be given a scaled score, for example on an integer scale.Thus, in some embodiments, the method of the invention comprises the steps of: i) providing one or more immunostained slices of a tissue section obtained by using a binding partner (e.g. an antibody as described above) capable of selectively interacting with a marker by an automated slide staining system; ii) proceeding to digitize the slide of step a by high resolution scan capture; iii) detecting the slices of the tissue section on the digital image; iv) providing a size reference grid with uniformly distributed units having the same surface, said grid being adapted to the size of the tissue section to be analyzed; and v) detecting, quantifying and measuring the intensity of stained cells in each unit, thereby assessing the number or density of stained cells in each unit.

[0030] In some embodiments, the level of the marker is determined by a flow cytometry method. As used herein, the term "flow cytometry method" refers to a technique for counting cells of interest by suspending them in a fluid stream and passing them through an electronic detection device. Flow cytometry methods allow simultaneous multiparameter analysis of physical and / or chemical parameters, such as fluorescence parameters, of up to thousands of events per second. Modern flow cytometry instruments typically have multiple lasers and fluorescence detectors. A common variation of flow cytometry techniques is to physically sort particles based on their properties to purify or detect populations of interest using "fluorescence-activated cell sorting." As used herein, "fluorescence-activated cell sorting" (FACS) refers to a flow cytometry method for sorting a heterogeneous mixture of cells from a biological sample into two or more containers one cell at a time based on the specific light scattering and fluorescence properties of each cell, providing fast, objective, and quantitative recording of the fluorescent signals from individual cells, as well as physical separation of the cells of particular interest. Thus, FACS can be used with the methods described herein to isolate and detect populations of cells of the invention. For example, fluorescence activated cell sorting (FACS) can be used accordingly, including using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores, such as a BD Biosciences FACSCanto™ flow cytometer, used substantially in accordance with the manufacturer's instructions. The cytometry system may include a cytometry sample fluidics subsystem, as described below. The cytometry system also includes a cytometer fluidically coupled to the cytometry sample fluidics subsystem. The systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, software (e.g., Flowjo, Laluza, etc.), data input devices, e.g., interface ports, mice, keyboards, etc., fluid handling components, power sources, etc.More specifically, the sample is contacted with a panel of antibodies specific for the particular target of the cell population of interest. Such antibodies or antigen-binding fragments are commercially available from vendors such as R&D Systems, BD Biosciences, e-Biosciences, Biolegend, Proimmune, and Miltenyi, or can be produced against these cell surface markers by methods known to those of skill in the art. In some embodiments, agents that specifically bind to cell surface markers, such as antibodies or antigen-binding fragments, are labeled with tags to facilitate isolation and detection of the cell population of interest. As used herein, the term "label" or "tag" refers to a composition capable of producing a detectable signal indicative of the presence of a target, such as the presence of a particular cell surface marker in a biological sample. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means required for the method to separate and detect cancer cells.Non-limiting examples of fluorescent labels or tags for labeling agents, such as antibodies for use in the methods of the invention include hydroxycoumarin, succinimidyl ester, aminocoumarin, succinimidyl ester, methoxycoumarin, succinimidyl ester, cascade blue, hydrazide, Pacific blue, maleimide, Pacific orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), PE-Cy5 conjugates (Cychrome, R670, Tri-Color, Quantum Red), PE-Cy7 conjugates, Red 613, PE-Texas Red, PerCP, PerCPeFluor. 710, PE-CF594, peridinin chlorophyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, fluorescein isothiocyanate (FITC), BODIPY-FL, TRITC, X-rhodamine (XRITC), lissamine rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV 785, BV711, BV421, BV605, BV510, or BV650. The assay may include binding the antibody to a solid support. The solid surface may be a microtiter plate coated with the antibody. Alternatively, the solid surface may be beads, such as activated beads, magnetically responsive beads, and the like. The beads may be made of different materials, including, but not limited to, glass, plastic, polystyrene, acrylic.The beads are also preferably fluorescently labeled. In a preferred embodiment, the fluorescent beads are those contained in TruCount™ tubes available from Becton Dickinson Biosciences (San Jose, Calif.).

[0031] In some embodiments, the method further comprises detecting the expression level of at least one additional marker. Typically, the marker is selected from the group consisting of KIR3DL2, PLS3, Twist, and NKp46.

[0032] Herein, the name of each of the various markers of interest refers to the internationally recognized name of the corresponding gene found in internationally recognized gene and protein sequence databases, in particular those contained in the database from the HUGO Gene Nomenclature Committee available at the following Internet address: http: / / www.gene.ucl.ac.uk / nomenclature / index.html. Herein, the name of each of the various markers of interest may also refer to the internationally recognized name of the corresponding gene found in the internationally recognized gene and protein sequence database Genbank. Through these internationally recognized sequence databases, the nucleic acid and amino acid sequences corresponding to each of the markers of interest described herein can be searched by those skilled in the art.

[0033] Multiple tissue analysis techniques are particularly useful for quantifying several markers in tissue samples. Such techniques should allow the measurement of at least five, or even at least ten or more, biomarkers from a single tissue sample. Moreover, it is advantageous that the technique preserves the localization of the biomarkers and is able to distinguish between the presence of the biomarkers in cancerous and non-cancerous cells. Such methods include layered immunohistochemistry (L-IHC), layered expression scanning (LES), or multiple tissue immunoblotting (MTI), as taught, for example, in U.S. Pat. Nos. 6,602,661, 6,969,615, 7,214,477, and 7,838,222; U.S. Patent Publication No. 2011 / 0306514 (incorporated herein by reference); and Chung & Hewitt, Meth Mol Biol, Prot Blotting Detect, Kurlen & Scofield, eds. 536: 139-148, 2009, each of which is taught to produce up to eight, nine, ten, eleven or more images of tissue sections on layered and blotted membranes, papers, filters, and the like, and can be used. Coated membranes useful for carrying out the L-IHC / MTI process are available from 20 / 20 GeneSystems, Inc. (Rockville, Md.).

[0034] In some embodiments, the L-IHC method can be performed on any of a variety of tissue samples, either fresh or preserved. Samples included core needle biopsies routinely fixed in 10% normal buffered formalin and processed in the pathology department. Standard 5 μm thick tissue sections were cut from tissue blocks onto charged slides used for L-IHC. Thus, L-IHC allows for testing multiple markers in tissue sections by obtaining copies of multiple bioaffinity-coated membrane-transferred molecules from the tissue section, essentially producing a copy of the tissue "image". In the case of paraffin sections, the tissue sections are deparaffinized as known in the art, for example by exposing the sections to xylene or a xylene substitute, such as NEO-CLEAR®, and graded ethanol solutions. Sections can be treated with proteinases, such as papain, trypsin, proteinase K, and the like. A stack of membrane substrates, including, for example, multiple sheets of 10 μm thick coated polymer scaffolds with 0.4 μm diameter pores for passing tissue molecules, such as proteins, through the stack, is then placed on the tissue section. The movement of fluids and tissue molecules is configured to be essentially perpendicular to the membrane surface. The sandwich of section, membrane, spacer paper, absorbent paper, weight, etc. can be exposed to heat to promote the transfer of molecules from the tissue into the membrane stack. A portion of the tissue's proteins is captured on each of the bioaffinity coated membranes (available from 20 / 20 GeneSystems, Inc. Rockville, MD) of the stack. Thus, each membrane contains a copy of the tissue and can be probed for different biomarkers using standard immunoblotting techniques, allowing unlimited expansion of marker profiles as would be performed on a single tissue section.Since the amount of protein may be lower on membranes more distal in the stack from the tissue, which may result from, for example, different amounts of molecules in the tissue sample, different mobility of molecules released from the tissue sample, different binding affinity of molecules to the membrane, length of migration, etc., standardization of values, running controls, assessing the migration level of tissue molecules, and the like, may be included in the procedure to correct for changes occurring within, between, and between membranes and allow direct comparison of information within, between, and between membranes. Thus, total protein may be determined per membrane using, for example, any means for quantifying protein, for example, biotinylating available molecules, such as proteins, using standard reagents and methods, and then revealing bound biotin by exposing the membrane to labeled avidin or streptavidin; protein stains, such as Blot fastStain, Ponceau Red, Brilliant Blue stain, etc., as known in the art.

[0035] In some embodiments, the methods utilize multiple tissue imprinting (MTI) technology to measure biomarkers, which conserves valuable biopsy tissue by allowing for multiple biomarkers, in some cases at least six biomarkers.

[0036] In some embodiments, there are alternative multiple tissue analysis systems that may also be used as part of the present invention. One such technology is the mass spectrometry-based Selected Reaction Monitoring (SRM) assay system ("Liquid Tissue", available from OncoPlexDx, Rockville, MD). This technology is described in U.S. Patent No. 7,473,532.

[0037] In some embodiments, the methods of the invention utilized multiplex IHC technology developed by GE Global Research (Niskayuna, NY), which is described in U.S. Publication Nos. 2008 / 0118916 and 2008 / 0118934, in which a sequential analysis is performed on a biological sample containing multiple targets, including the steps of binding a fluorescent probe to the sample, followed by signal detection, then inactivation of the probe, followed by binding, detection and inactivation of the probe to another target, and continuing this process until all targets are detected.

[0038] In some embodiments, multi-tissue imaging can be performed when using fluorescence (e.g., fluorophores or quantum dots), where the signal can be measured with a multispectral imaging system. Multispectral imaging is a technique that collects spectral information at each pixel of an image and analyzes the resulting data with spectral image processing software. For example, the system can obtain a series of images at different wavelengths that are electronically and sequentially selectable, and then utilize an analysis program designed to handle such data. This system thus allows quantitative information to be obtained simultaneously from multiple dyes, even when the dye spectra are highly overlapping, or even when they are co-localized or occur at the same point in the sample, provided that the spectral curves are different. Many biological materials autofluoresce or emit low-energy light when excited by high-energy light. This signal can result in lower contrast images and data. A high-sensitivity camera without multispectral imaging capabilities increases only the autofluorescence signal along with the fluorescence signal. Multispectral imaging allows the autofluorescence to be separated or separated from the tissue, thereby increasing the achievable signal-to-noise ratio. Briefly, quantification can be performed by the following steps: i) providing a tumor tissue microarray (TMA) obtained from a subject, ii) the TMA sample is then stained with an anti-antibody with specificity for the protein of interest, iii) the TMA slide is further stained with an epithelial cell marker to aid in the automated segmentation of tumor and stroma, iv) the TMA slide is then scanned using a multispectral imaging system, v) the scanned image is processed using automated image analysis software (e.g., Perkin Elmer Technology), thereby allowing detection, quantification, and segmentation of specific tissues through powerful pattern recognition algorithms. The machine learning algorithm is typically pre-trained to separate tumor from stroma and identify labeled cells.

[0039] In some embodiments, the level of the marker is determined at the nucleic acid level. Typically, the level of a gene may be determined by determining the amount of mRNA. Methods for determining the amount of mRNA are well known in the art. For example, nucleic acids contained in a sample (e.g., cells or tissues prepared from a subject) are first extracted according to standard methods, e.g., using lytic enzymes or chemical solutions, or extracted with a nucleic acid binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis, in situ hybridization) and / or amplification (e.g., RT-PCR). Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0040] In some embodiments, the method of the present invention further comprises comparing the expression level of the marker with a predetermined reference value, and detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has a T-cell malignancy. In some embodiments, the method of the present invention further comprises comparing the expression level of the marker with a predetermined reference value, and detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has a T-cell lymphoma. In some embodiments, the method of the present invention further comprises comparing the expression level of the marker with a predetermined reference value, and detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has a T-cell leukemia.

[0041] In some embodiments, the predefined reference value is a relative value to a number or value derived from a population test, including, but not limited to, subjects of the same or similar age range, subjects of the same or similar ethnic group, and subjects with the same severity of the pathology. Such predefined reference value can be derived from statistical analysis of the population and / or risk prediction data obtained from mathematical algorithms and calculated indices. In some embodiments, retrospective measurements of the levels of the markers in appropriately stored past subject samples may be used in establishing these predefined reference values. Thus, in some embodiments, the predefined reference value is a threshold or cut-off value. The threshold value must be determined to obtain optimal sensitivity and specificity according to the function of the test and the benefit / risk balance (clinical outcomes of false positive and false negative). Typically, the optimal sensitivity and specificity (and thus the threshold value) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the levels of the markers in the reference group, an algorithmic analysis can be used for statistical processing of the measured levels of the markers in the samples to be tested, thus obtaining classification criteria with significance for sample classification. The full name of ROC curve is Receiver Operating Characteristic Curve, and it is also known as Receiver Operating Characteristic Curve. It is mainly used for clinical biochemistry diagnostic tests. ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). It uses image synthesis method to reveal the relationship between sensitivity and specificity. A series of different cut-off values ​​(threshold or critical value, boundary value between normal and abnormal results of diagnostic test) are set as continuous variables, and a series of sensitivity and specificity values ​​are calculated. Then, the curve is drawn using sensitivity as the vertical coordinate and specificity as the horizontal coordinate. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the top left of the coordinate diagram is the decisive point with both high sensitivity and specificity values. The AUC value of ROC curve is between 1.0 and 0.5. If AUC>0.5, the diagnostic result is better as AUC approaches 1. If the AUC is between 0.5 and 0.7, the accuracy is low. If the AUC is between 0.7 and 0.9, the accuracy is moderate.If AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably performed on a computer. Existing software or systems in the art may be used to plot ROC curves, such as: MedCalc 9.2.0.1 medical statistics software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (Dynamic Microsystems, Inc. Silver Spring, Maryland, USA), etc.

[0042] Typically, as demonstrated in the examples, the expression level of CD51 is higher than the expression level measured in a sample from a healthy individual.Thus, in some embodiments, the method comprises the further step of concluding that the subject suffers from a T-cell malignancy when the expression level of CD51 is higher than a predetermined reference value.In some embodiments, the method comprises the further step of concluding that the subject suffers from a T-cell lymphoma when the expression level of CD51 is higher than a predetermined reference value.In some embodiments, the method comprises the further step of concluding that the subject suffers from a T-cell leukemia when the expression level of CD51 is higher than a predetermined reference value.In some embodiments, the predetermined reference value is measured in a sample from a healthy individual.

[0043] Monitoring the effect of an agent (e.g., a drug compound) on the level of expression of CD51 can be applied to monitor the status of a T cell malignancy, particularly a T cell lymphoma or T cell leukemia, in a patient over time. For example, the effectiveness of an agent that affects marker expression can be monitored during treatment of a subject undergoing anti-T cell malignancy, such as anti-T cell lymphoma or anti-T cell leukemia treatment.

[0044] Thus, the present invention also provides a method for monitoring the effectiveness of the treatment of a patient suffering from a T-cell malignancy, in particular a T-cell lymphoma or a T-cell leukemia, comprising the steps of: (i) obtaining a pre-dose sample from the patient prior to administration of the drug; (ii) detecting the level of expression of CD51 in the pre-administration sample; (iii) obtaining one or more post-dose samples from the patient; (iv) detecting the level of expression of the same markers in post-administration samples; (v) comparing the level of CD51 expression in the pre-treatment sample with the level of CD51 expression in the post-treatment sample; and (vi) Modifying the administration of medication to the patient accordingly.

[0045] For example, a worse diagnosis as determined by assessing CD51 expression levels over the course of treatment may indicate ineffective dosing and the desirability of increasing the dose, whereas a better diagnosis as determined by assessing CD51 expression levels may indicate effective treatment and no need to change the dose.

[0046] The present invention therefore also relates to a method for adapting therapy in a patient suffering from a T-cell malignancy, in particular a T-cell lymphoma or a T-cell leukemia, comprising the following steps: a) performing an in vitro diagnostic method as disclosed herein on at least one sample collected from the patient; and b) administering it to the patient, thereby adapting the treatment of the patient;

[0047] The present invention also relates to a kit for carrying out the diagnostic method described above. The kit comprises a number of reagents, in particular at least one agent capable of specifically binding to the CD51 marker. Suitable reagents for binding to marker proteins include antibodies, antibody derivatives, antibody fragments, and the like. Suitable reagents for binding to marker nucleic acids (e.g., genomic DNA, mRNA, spliced ​​mRNA, cDNA, or the like) include complementary nucleic acids. For example, nucleic acid reagents may include oligonucleotides (labeled or unlabeled) immobilized on a substrate, labeled oligonucleotides not bound to a substrate, pairs of PCR primers, molecular beacon probes, and the like. The kit of the present invention may optionally include additional components useful for carrying out the method of the present invention. As an example, the kit may include a suitable fluid (e.g., SSC buffer) for annealing complementary nucleic acids or binding an antibody to a protein to which it specifically binds, one or more sample compartments, instructions describing the performance of the in vitro diagnostic method of the present invention, and the like.

[0048] Method of treatment: A further object of the present invention is a method for treating a T-cell malignancy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD51 inhibitor. In some embodiments, the present invention is a method for treating a T-cell lymphoma in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD51 inhibitor. In some embodiments, the present invention is a method for treating a T-cell leukemia in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD51 inhibitor.

[0049] In particular, the CD51 inhibitors of the present invention are suitable for blocking CD51-induced active TGF-beta production by T cells, which contributes to immune evasion of tumor cells.

[0050] A further object of the present invention relates to a method of treating a T-cell malignancy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD51-expressing cancer cells.

[0051] Another object of the present invention is to provide a method for treating T-cell lymphoma in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a drug capable of inducing cell death of CD51-expressing cancer cells. In some embodiments, the T-cell lymphoma is cutaneous T-cell lymphoma. More specifically, the T-cell lymphoma is Sezary syndrome.

[0052] Another object of the present invention is a method for treating T-cell leukemia in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD51-expressing cancer cells. In some embodiments, the T-cell leukemia is acute lymphoblastic leukemia.

[0053] In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult. In some embodiments, the patient is an elderly human. In some embodiments, the patient is an immature human infant.

[0054] CD51 inhibitors: A significant number of CD51 inhibitors (Goodman et al, Trends in Pharmacological Sciences, 2012, 33, 405; Tucker, Gordon C. "Alpha v integrin inhibitors and cancer therapy." Current opinion in investigational drugs(London, England: 2000)4.6(2003): 722-731.; Hatley, Richard JD, et al. "An αv‐RGD Integrin Inhibitor Toolbox: Drug Discovery Insight, Challenges and Opportunities." Angewandte Chemie International Edition 57.13(2018): 3298-3321.) have been disclosed in the literature, including inhibitory antibodies, peptides, and small molecules.

[0055] In some embodiments, the CD51 inhibitor is cilengitide. Cilengitide is a cyclic peptide antagonist that inhibits both ανβ1, ανβ3, and ανβ5. The IUPAC name for cilengitide is 2-[(2S,5R,8S,11S)-5-benzyl-11-{3-[(diaminomethylidene)amino]propyl}-7-methyl-3,6,9,12,15-pentaoxo-8-(propan-2-yl)-1,4,7,10,13-pentaazacyclopentadecan-2-yl]acetic acid. In some embodiments, the CD51 inhibitor is not cilengitide.

[0056] In some embodiments, each of the CD51 inhibitors is an inhibitor of CD51 expression. In particular, the inhibitor of gene expression is an siRNA, an antisense oligonucleotide, or a ribozyme. For example, antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, will act to directly block the translation of CD51 mRNA by binding to CD51 mRNA and thus preventing protein translation or increasing mRNA degradation, thus reducing the level and thus activity of CD51 in cells. For example, antisense oligonucleotides complementary to unique regions of the mRNA transcript sequence encoding CD51 of at least about 15 bases can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, e.g., U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. CD51 gene expression can be reduced by contacting a patient or cells with small double-stranded RNA (dsRNA), or a vector or construct that causes the production of small double-stranded RNA, such that CD51 gene expression is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs, and ribozymes of the present invention may be delivered in vivo alone or in association with a vector.

[0057] CD51 antibody: In some embodiments, the inhibitor or agent is an antibody having binding affinity to CD51, hi some embodiments, the agent is an antibody directed against at least one extracellular domain of CD51.

[0058] In some embodiments, the antibodies lead to inhibition of TGF-beta production by T cells, which contributes to immune evasion of tumor cells.

[0059] In some embodiments, the antibody leads to depletion of CD51-expressing cancer cells.

[0060] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0061] In some embodiments, the antibody is a fully human antibody. Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference.

[0062] Antibodies with specificity for CD51 are known in the art and typically include intetumumab and abituzumab.

[0063] In particular, the heavy chain of intetumumab is set forth as SEQ ID NO:2 and the light chain of intetumumab is set forth as SEQ ID NO:3. [ka]

[0064] In particular, the heavy chain of avituzumab is set forth as SEQ ID NO:4 and the light chain of intetumumab is set forth as SEQ ID NO:5. [ka]

[0065] CD51 depletion antibody In some embodiments, antibodies suitable for depletion of CD51 cancer cells mediate antibody-dependent cell-mediated cytotoxicity.

[0066] In some embodiments, the antibody comprises the VH and VL domains of intetumumab.

[0067] In some embodiments, the antibody comprises the VH and VL domains of avituzumab.

[0068] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcR).

[0069] As used herein, the term "Fc region" includes the polypeptides comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinges at the N-terminus of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 toward its carboxyl terminus, with numbering according to the EU index, as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "EU index as shown in Kabat" refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al. (supra). Fc may refer to this region alone or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein is an antibody, an Fc fusion, or any protein or protein domain comprising an Fc region. Particularly preferred are proteins comprising variant Fc regions, which are non-natural variants of Fc regions. The amino acid sequence of a non-natural Fc region (also referred to herein as a "variant Fc region") comprises a substitution, insertion, and / or deletion of at least one amino acid residue compared to the wild-type amino acid sequence. Any new amino acid residue that appears in the sequence of the variant Fc region as a result of the insertion or substitution may be referred to as a non-natural amino acid residue. Note: polymorphisms have been observed at many Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358; thus, slight differences between the presented sequence and those in the prior art may exist.

[0070] As used herein, the term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. NK cells, the primary cells mediating ADCC, express FcγRIII, whereas monocytes express FcγRI, FcγRII, FcγRIII and / or FcγRIV. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess the ADCC activity of a molecule, an in vitro ADCC assay, such as those described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

[0071] As used herein, the term "effector cell" is a leukocyte that expresses one or more FcR and performs effector function. The cell expresses at least FcγRI, FcγRII, FcγRIII and / or FcγRIV and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

[0072] In some embodiments, the antibody suitable for depletion of cancer cells is a full-length antibody. In some embodiments, the full-length antibody is an IgG1 antibody. In some embodiments, the full-length antibody is an IgG3 antibody.

[0073] In some embodiments, an antibody suitable for depletion of cancer cells comprises a variant Fc region having increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In some embodiments, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, wherein the substitution, insertion, or deletion of the at least one amino acid residue results in increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In some embodiments, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, wherein the at least one amino acid residue is selected from the group consisting of residues 239, 330, and 332, wherein the amino acid residues are numbered according to the EU index. In some embodiments, an antibody of the invention comprises a variant Fc region comprising at least one amino acid substitution selected from the group consisting of S239D, A330L, A330Y, and I332E, where the amino acid residues are numbered according to the EU index.

[0074] In some embodiments, the glycosylation of an antibody suitable for depletion of cancer cells is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be modified, for example, to increase the affinity of the antibody for an antigen. Such sugar modifications can be achieved, for example, by modifying one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such approaches are described in more detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, antibodies with altered types of glycosylation can be made, such as hypofucosylated or nonfucosylated antibodies with reduced amounts or no fucosyl residues, or antibodies with increased bisecting GlcNAc structures. Such modified glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such sugar modifications can be achieved, for example, by expressing the antibody in a host cell with modified glycosylation machinery. Cells with modified glycosylation machinery have been described in the art and can be used as host cells for expressing the recombinant antibodies of the invention, thereby producing antibodies with modified glycosylation. For example, EP1176195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene encoding a fucosyltransferase, and antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Thus, in some embodiments, the human monoclonal antibodies of the invention may be produced by recombinant expression in a cell line exhibiting hypofucosylation or non-fucosylation patterns, for example, a mammalian cell line with defective expression of the FUT8 gene encoding a fucosyltransferase.PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lecl3 cells, with a reduced ability to attach fucose to Asn(297)-linked sugars and resulting in hypofucosylation of antibodies expressed in the host cells (see also Shields, RL et al, 2002 J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes engineered cell lines that express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) and antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNAc structures, which results in increased ADCC activity of the antibodies (see also Umana et al, 1999 Nat. Biotech. 17: 176-180). Eureka Therapeutics further describes engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation patterns that lack fucosyl residues (http: / / www.eurekainc.com / a&boutus / companyoverview.html). Alternatively, the human monoclonal antibodies of the invention can be produced in yeast or filamentous fungi that have been engineered for a mammalian-like glycosylation pattern and are capable of producing antibodies that lack fucose as a glycosylation pattern (see, e.g., EP1297172B1).

[0075] In some embodiments, antibodies suitable for depletion of cancer cells mediate complement dependent cytotoxicity.

[0076] As used herein, the term "complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santaru et al., J. Immunol. Methods, 202:163 (1996).

[0077] In some embodiments, antibodies suitable for depletion of cancer cells mediate antibody-dependent phagocytosis.

[0078] As used herein, the term "antibody-dependent phagocytosis" or "opsonization" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently phagocytosis of the target cell.

[0079] CD51 multispecific antibody: In some embodiments, antibodies suitable for depletion of CD51 cancer cells are multispecific antibodies comprising a first antigen-binding site for CD51 and at least one second antigen-binding site for an effector cell, as described above.

[0080] In some embodiments, the first antigen binding comprises the VH domain and the VL domain of intetumumab.

[0081] In some embodiments, the first antigen binding comprises the VH domain and the VL domain of avituzumab.

[0082] In particular, the second antigen binding site is used to recruit killing mechanisms, for example by binding to antigens on human effector cells. In some embodiments, the effector cells are capable of inducing ADCC, such as natural killer cells. For example, monocytes, macrophages, which express FcR, are involved in the specific killing of target cells and in presenting antigens to other components of the immune system. In some embodiments, the effector cells may phagocytose target antigens or target cells. The expression of certain FcRs on effector cells may be regulated by humoral factors, such as cytokines. Effector cells may phagocytose target antigens or phagocytose or lyse target cells. Suitable cytotoxic agents and second therapeutic agents are exemplified below and include toxins (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs. In some embodiments, the second binding site binds to an Fc receptor as defined above. In some embodiments, the second binding site may bind to a surface molecule on NK cells and activate the cells. In some embodiments, the second binding site binds to NKp46.Exemplary formats of the multispecific antibody molecules of the invention include, but are not limited to, (i) two antibodies (one with specificity for a particular surface molecule of ILCs and the other with specificity for a second antigen) cross-linked by a chemical heterolinkage; (ii) a single antibody comprising two different antigen-binding regions; (iii) a single chain antibody comprising two different antigen-binding regions (e.g., two scFvs linked in tandem by an extra peptide linker); (iv) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain comprises two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin, Germany). Heidelberg (2010); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandab, which is the fusion of two single chain diabodies resulting in a tetravalent bispecific antibody with two binding sites for each of the target antigens; (vii) Flexibodies, which is a combination of scFv and diabody resulting in a multivalent molecule; (viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" in protein kinase A, which when applied to Fab results in a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (ix) so-called Scorpion molecules, which for example comprise two scFvs fused to both ends of a human Fab arm; and (x) diabodies. Another exemplary format for bispecific antibodies is an IgG-like molecule with complementary CH3 domains to force heterodimerization.Such molecules can be prepared using known techniques such as, for example, Triomab / Quadroma (Trion Pharma / Fresenius Biotech), Knob-into-Hole (Genentech), CrossMAb (Roche) and electrostatically-matched (Amgen), LUZ-Y (Genentech), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono), Biclonic (Merus), and DuoBody (Genmab A / S) technologies.

[0083] In some embodiments, a multispecific antibody is thus a bispecific antibody.

[0084] In some embodiments, the bispecific antibody is a BiTE. As used herein, the term "bispecific T cell engager" or "BiTE" refers to a bispecific antibody that is a recombinant protein structure composed of two flexibly connected single chain antibodies (scFv). One of the scFv antibodies specifically binds to a selected target cell expressed tumor antigen (i.e., CD51) and the second specifically binds to another molecule, such as CD3, a subunit of the T cell receptor complex on T cells. In some embodiments, the BiTE antibody is capable of transiently binding the T cell to the target cell and simultaneously activating the cytolytic activity of the T cell. BiTE-mediated activation of the T cell does not require a specific T cell receptor on the T cell, or an MHC I molecule, peptide antigen, or costimulatory molecule on the target cell.

[0085] CD51 antibody-drug conjugates: In some embodiments, antibodies suitable for depletion of cancer cells are conjugated to a therapeutic moiety, ie, a drug.

[0086] In some embodiments, the antibody-drug conjugate comprises the VH and VL domains of intetumumab.

[0087] In some embodiments, the antibody-drug conjugate comprises the VH and VL domains of avituzumab.

[0088] In some embodiments, the therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic drug, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0089] In some embodiments, antibodies suitable for depletion of cancer cells are conjugated to a cytotoxic moiety, such as, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracin dione; tubulin inhibitors, such as maytansine or an analog or derivative thereof; Antimitotic agents, such as monomethylauristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analog thereof; irinotecan or an analog thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; antimetabolites, such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabine, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents, such as mechlorethamine, thioepamine, chloramphenicol ... lorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, etc.; platinum derivatives such as cisplatin, carboplatin, etc.; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or analogs or derivatives thereof; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC), etc.; pyrrolo[2,lc][l,4]-benzodiazepines (PDB);Diphtheria toxin and related molecules, such as diphtheria A chain and active fragments and hybrid molecules thereof, ricin toxins, such as ricin A or deglycosylated ricin A chain toxins, cholera toxins, Shiga-like toxins, such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, alpha-sarcin, Abrasion fornia proteins, dianthin proteins, pokeweed proteins such as PAPI, PAPII, PAP-S, momordica charantia inhibitor, curtin, crotin, saponaria inhibitor, gelonin, mitgelin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase), DNase I, Staphylococcal enterotoxin A, pokeweed antiviral protein, diphtherin toxin, Pseudomonas endotoxin;

[0090] In some embodiments, the antibody suitable for depletion of cancer cells is conjugated to an auristatin or its peptide analog, derivative, or prodrug. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). For example, auristatin E can react with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethylauristatin F), and MMAE (monomethylauristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugating auristatins to Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, and 6,214,345, as well as in International Patent Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0091] In some embodiments, the antibody suitable for depletion of cancer cells is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or its analogs, derivatives or prodrugs. Suitable PDBs and PDB derivatives, as well as related techniques, are described, for example, in Hartley JA et al., Cancer Res 2010; 70(17): 6849-6858; Antonow D. et al., Cancer J 2008; 14(3): 154-169; Howard PW et al., Bioorg Med Chem Lett 2009; 19: 6463-6466 and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18): 2083-2086.

[0092] In some embodiments, an antibody suitable for depletion of cancer cells is conjugated to a cytotoxic moiety selected from the group consisting of anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or an analog, derivative, or prodrug of any of them.

[0093] In some embodiments, the antibody suitable for depletion of cancer cells is conjugated to an anthracycline or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin F or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative, or prodrug thereof.

[0094] In some embodiments, the antibody suitable for depletion of cancer cells is conjugated to a nucleic acid or a nucleic acid-associated molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease (RNase) or deoxyribonuclease (e.g., DNase I), an antisense nucleic acid, an inhibitory RNA molecule (e.g., an siRNA molecule), or an immunostimulatory nucleic acid (e.g., an immunostimulatory CpG motif-containing DNA molecule). In some embodiments, the antibody is conjugated to an aptamer or a ribozyme.

[0095] Techniques for conjugating molecules to antibodies are well known in the art (e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc., 2nd ed. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO 89 / 12624.) Typically, the nucleic acid molecule is covalently attached to a lysine or cysteine ​​on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.Methods of conjugation using engineered cysteines or incorporation of unnatural amino acids have been reported to improve conjugate homogeneity (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al.(2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.;Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G., et al.(2010). Engineered thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res.16, 4769-4778.). Junutula et al. (2008) developed a cysteine-based site-specific conjugation called "THIOMAB" (TDC) that is claimed to exhibit an improved therapeutic index compared to conventional conjugation methods. Conjugation with unnatural amino acids incorporated into antibodies has also been explored for ADCs; however, the generality of this approach has not yet been established (Axup et al., 2012). In particular, one skilled in the art can also envision Fc-containing polypeptides engineered with acyl-donating glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or endogenous glutamines that have been made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide).The transglutaminase can then covalently cross-link an amine-donating agent (e.g., a small molecule that contains or has attached a reactive amine) to form a stable, homogenous population of engineered Fc-containing polypeptide complexes with the amine-donating agent site-specifically conjugated to the Fc-containing polypeptide through an acyl-donating glutamine-containing tag or an accessible / exposed / reactive endogenous glutamine (WO 2012059882).

[0096] CD51 CAR-T cells In some embodiments, the agent is a CAR-T cell, and the CAR comprises at least an extracellular antigen-binding domain specific for CD51.

[0097] In some embodiments, the extracellular antigen binding domain specific for CD51 comprises the VH and VL domains of intetumumab.

[0098] In some embodiments, the extracellular antigen-binding domain specific for CD51 comprises the VH and VL domains of avituzumab.

[0099] In some embodiments, the CAR comprises at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain"), and comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides comprises a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to each other, e.g., can couple the antigen binding domain to the intracellular signaling domain. In some embodiments, the stimulatory molecule is a zeta chain associated with the T cell receptor complex. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some embodiments, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0100] In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain specific for CD51, and comprises a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain specific for CD51, and comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain specific for CD51, and comprises two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain specific for CD51, and comprises at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0101] In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, which is optionally cleaved from the antigen-binding domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0102] In certain aspects, the CAR comprises a fusion of a single chain variable fragment (scFv) derived from a monoclonal antibody specific for CD51, fused to a CD3-zeta transmembrane domain and endodomain. In some embodiments, the CAR comprises additional domains for costimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules can be co-expressed with the CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally remove T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0103] In some embodiments, the chimeric antigen receptor of the present invention comprises at least one VH and / or VL sequence of an antibody specific for CD51. In some embodiments, the chimeric antigen receptor comprises the VH and VL domains of intetumumab. In some embodiments, the chimeric antigen receptor comprises the VH and VL domains of avituzumab. In some embodiments, the portion of the CAR of the invention that comprises an antibody or antibody fragment thereof that is specific for CD51 can exist in a variety of forms in which the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, NY; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment that is specific for CD51. In a further aspect, the CAR comprises an antibody fragment that comprises an scFv that is specific for CD51.

[0104] Methods for preparing CAR-T cells are well known in the art. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR. In some embodiments, cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some embodiments, the antigen-binding domain of the CAR of the present invention (e.g., scFv) is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the entire CAR construct of the present invention is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods are known in the art and include, for example, the methods disclosed in at least US Pat. Nos. 5,786,464 and 6,114,148.

[0105] In some embodiments, the chimeric antigen receptors of the present invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., via disulfide bridges, or converted into acid addition salts, and / or optionally dimerized or polymerized.

[0106] In some embodiments, CAR activity can be controlled if desired to optimize the safety and efficacy of CAR therapy.There are many ways that CAR activity can be regulated.For example, inducible apoptosis using caspases fused to dimerization domains (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3; 365(18):1673-1683) can be used as a safety switch in the CAR therapy of the present invention.

[0107] Pharmaceutical Compositions: Typically, the agent of the present invention is administered to a patient in the form of a pharmaceutical composition containing a pharma- ceutically acceptable carrier.The pharma-ceutically acceptable carrier that can be used in these compositions includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool fat.For use in administering to a patient, the composition is formulated for administration to a patient.The composition of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or via an implanted reservoir. As used herein, it includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of the present invention 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. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable solvents and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Sterile fixed oils are also conventionally used as a solvent or suspending medium. For this purpose, any sterile 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 pharma- ceutical-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 preparation of pharma- ceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span, 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. The compositions of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. In the case of tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include, for example, lactose. When an aqueous suspension is required for oral administration, the active ingredient is combined with an emulsifier and suspending agent. If desired, certain sweeteners, flavors, or colorants may also be added. Alternatively, the compositions of the present invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature, but liquid at rectal temperature, and therefore melts in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs that are easily accessible by topical application, including diseases of the eye, skin, or lower intestinal tract. Suitable topical formulations are easily prepared for each of these areas or organs. For topical application, the compositions may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma- ceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. A patch may also be used. The compositions of the invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline with benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the invention can be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials. The product is formulated for IV administration in 9.0 mg / mL sodium chloride, 7.35 mg / mL sodium citrate dihydrate, 0.7 mg / mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. An exemplary suitable dosage range for the antibody in the pharmaceutical composition of the invention is about 1 mg / m. 2 ~500mg / m 2 The dosage may be between about 100 mg and about 500 mg. However, it will be understood that these schedules are exemplary, and that optimal schedules and regimens can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition, which must be determined in clinical trials. Pharmaceutical compositions of the invention for injection (e.g., intramuscular, intravenous) may be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular use) and between about 1 ng and about 100 mg, e.g., about 50 ng to about 30 mg, or more preferably about 5 mg to about 25 mg, of the inhibitor of the invention.

[0108] The present invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the present invention. [Brief description of the drawings]

[0109] figure: [Figure 1] Immunolabeling was performed on blood from healthy donors (HD; n=13) and Sézary syndrome patients (SS; n=10) to analyze the expression of tumor markers KIR3DL2 and CD51, gated on CD3+CD4+ and CD3+CD8+ T cell populations. For SS patients, anti-TCR-Vβ mAb was added to distinguish malignant (Vβ+) from non-malignant (Vβ-) CD4+ T cells within the total CD4+ T cell population. Scatter plots from the resulting data show the % of KIR3DL2 and CD51 positive cells within (A) the total CD4+ T cell population of HD vs. SS patients, (B) the total CD4+ T cell population of malignant and non-malignant CD4+ T cells of HD vs. SS patients, and (C) the total CD8+ T cell population of HD vs. SS patients. Statistical analysis was performed using the Mann-Whitney t-test. **** p<0.0001, ** p<0.005, * p<0.05. [Diagram 2] Expression of CD51, CD29 and CD61 was examined by flow cytometry using anti-CD51 (clone NKI-M9), anti-CD29 (clone TS2 / 16) and anti-CD61 (clone VI-PL2) on MyLA (top) and Seax (bottom) cell lines after 15 min incubation at 4° C. Black histograms represent the fluorescence intensity of the control isotype. [Diagram 3] PBMCs from a Sézary patient were isolated by Ficoll gradient centrifugation, incubated with anti-CD4 (clone RPA-T4), anti-CD158k / e (clone REA970), anti-CD51, CD61, and CD29 antibodies for 15 min at 4° C., and analyzed by flow cytometry. The black histogram represents the fluorescence intensity of the control isotype. [Figure 4]The expression of CD51, CD29, CD61, and beta integrin 5 / 6 / 8 in DERL2, SNK-6, Molt-4, and Jurkat cell lines was examined by flow cytometry. For this purpose, cells were incubated with either anti-CD51 (NKI-M9), anti-CD29 (TS2 / 16), anti-CD61 (VPI-PL2), anti-beta 5 integrin (P5H9), anti-beta 6 integrin (437211), or anti-beta 8 integrin (416922) for 15 min at 4°C. [Diagram 5] Analysis of CD51 expression by flow cytometry on CD3+ CD4+ lymphocytes from healthy donors (HD) or Sézary patients (SS). Freshly isolated PBMCs from patients were stained with anti-CD3 (OKT3), anti-CD4 (clone RPA-T4), anti-CD158k / e (clone REA970), and anti-CD51 (NKI-M9) for 15 min at 4° C. and analyzed by flow cytometry. For HD, PBMCs were stained with anti-CD3, anti-CD4, and anti-CD51 antibodies only. [Figure 6] Absence of CD51 in activated or non-activated healthy CD4 LT. Freshly isolated PBMCs from HD were stained with anti-CD4, anti-CD25, and anti-CD51 antibodies before (A) or after (B) cell activation with CD3 / CD28 beads for 24 h. CD51 was not detected on resting CD4+ CD25- (A) or activated CD4+ CD25- or CD4 CD25+ (B) cell populations, except for resting CD4+ CD25+ cells with weak expression of CD51 (Tregs). [Figure 7] Co-immunoprecipitation of integrin β1 in Seax cells. Co-immunoprecipitation was performed on Seax cell lysates using anti-CD51 antibody (clone NKI-M9) to immunoprecipitate CD51. After running on an 8% SDS-Page gel, co-immunoprecipitated CD29 was detected by Western blot using anti-CD29 antibody (clone D6S1w). To control for CD51 immunoprecipitation, we detected CD51 protein by Western blot using anti-CD51 polyclonal purified antibody. [Figure 8] Expression of CD29 and CD61 on SS cells. CD29 and CD61 expression was tested on CD3+ CD4+ CD7- CD51+ tumor cells from blood samples of 15 SS patients. VPI-PL2 anti-CD61 antibody clone and anti-CD29 TS2 / 16 antibody clone were used. Mean fluorescence intensity (MFI) is reported in this table. [Figure 9] TGF-beta activation assay. Myla or Seax cells were incubated with 10 ng of recombinant latent TGF-beta alone or with IL-2 (10 ng / ml) or CD3 / CD28 activation (1 / 200) beads in complete RPMI 1640 medium for 24 hours. Supernatants were collected and active TGF-beta was measured by multi-analyte flow assay.

[0110] Example 1: T-cell lymphomas are a heterogeneous group of malignant tumors involving T lymphocytes and are generally characterized by a poor prognosis. Among them, cutaneous T-cell lymphoma mainly involves the skin. Mycosis fungoides and Sézary syndrome are the most frequent cutaneous T-cell lymphomas. Sézary syndrome is defined as erythroderma (redness of the entire skin surface) and circulating tumor blood cells (1). Circulating tumor T cells express CD4 and lack expression of CD7 and CD26 (2). In the majority of cases, there is abnormal expression of CD158k (KIR3DL2 / CD158k) (3). The anti-CD158k monoclonal antibody lactamab has been tested in phase I trials and is currently being tested in cutaneous T-cell lymphoma and other peripheral T-cell lymphomas in a phase II international multicenter prospective clinical trial. However, long-term responses are rare and new treatments are needed (4). Recently, treatment with a depleting anti-CCR4 monoclonal antibody (mogamulizumab) has improved progression-free survival in cutaneous T-cell lymphoma. However, CCR4 is expressed not only by Sézary cells, but also by memory regulatory T cells in the peripheral blood, and its use is associated with the occurrence of autoimmune adverse reactions. In addition to CCR4, Sézary cells express several other markers common to regulatory T lymphocytes, such as CD39, PD1, and TIGIT (5-7). This study led to the identification of CD51 (alpha v) by Sézary T lymphocytes in the blood of patients as well as non-Sézary T lymphocytes. Expression of this integrin is not found on lymphocytes of healthy donors, whether activated or not, but only when they migrate into tissues (8). It has also been shown that CD51 is involved in the release of the active form of TGF-β, a cytokine that leads to severe immunosuppression in patients (9). CD51 therefore appears as a potential marker for the diagnosis and follow-up of Sézary syndrome (Figure 1A, B, C), which may be associated with the production of active TGF-beta, independent of tumor burden. The results show that CD51 expression is a hallmark of cutaneous T-cell lymphoma, since it is absent in healthy donors.Blocking CD51 with antibodies that prevent the release of this cytokine may improve the patient's immune response against the tumor.

[0111] As demonstrated in Figures 2 and 3, CD51 expression correlates with CD29 expression in cutaneous T lymphoma lines or tumor CD4 T cells from patients with Sezary syndrome and forms heterodimers. CD51 expression does not correlate with CD61 expression in these cutaneous T lymphoma lines or tumor CD4 T cells from patients with Sezary syndrome.

[0112] Example 2: Expression of CD51, CD29, and CD61 was examined by flow cytometry on DERL2, SNK-6, Molt-4, and Jurkat cell lines (Figure 4). Analysis of CD51 expression by flow cytometry on CD3+ CD4+ lymphocytes from healthy donors (HD) or Sézary syndrome patients (SS) was also performed, demonstrating that CD51 is overexpressed in patients with Sézary syndrome compared to healthy donors (Figure 5). As demonstrated in Figures 6A-6B, CD51 is not present on activated or non-activated healthy CD4 LTs. CD51 co-immunoprecipitates with integrin β1 in Seax cells (Figure 7), and expression of CD29 and CD61 in SS cells was also examined (Figure 8). Figure 9 demonstrates that Myla and Seax cells can activate the TGFB pathway.

[0113] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into this disclosure. [Table 1]

Claims

1. A method for obtaining an index used in the diagnosis of a patient's T-cell malignancy, comprising detecting the expression level of CD51 in a sample obtained from the patient.

2. The method according to claim 1, further comprising the step of comparing the detected expression level with a criterion that indicates the subject has a T-cell malignancy when the CD51 expression level is higher than a predetermined reference value.

3. The method according to claim 1, wherein the T-cell malignancy is Sézary syndrome, NK / T-cell lymphoma, gamma / delta T-cell lymphoma, or acute lymphoblastic leukemia.

4. The method according to claim 1, wherein the T-cell malignant tumor is cutaneous T-cell lymphoma.

5. The method according to claim 1, wherein the T-cell malignant tumor is Sézary syndrome.

6. The method according to claim 1, further comprising detecting the expression level of at least one further marker selected from the group consisting of KIR3DL2, PLS3, Twist, and NKp46.

7. A pharmaceutical composition for treating T-cell malignancies in patients requiring such treatment, comprising a therapeutically effective amount of a CD51 inhibitor.

8. A pharmaceutical composition for treating T-cell malignancies in patients who require it, comprising a therapeutically effective amount of a drug capable of inducing cell death in CD51-expressing cancer cells.

9. The pharmaceutical composition according to claim 7 or 8, wherein the T-cell malignant tumor is Sézary syndrome, NK / T-cell lymphoma, gamma / delta T-cell lymphoma, or acute lymphoblastic leukemia.

10. The pharmaceutical composition according to claim 7 or 8, wherein the T-cell malignant tumor is cutaneous T-cell lymphoma.

11. The pharmaceutical composition according to claim 10, wherein the cutaneous T-cell lymphoma is Sézary syndrome.

12. The pharmaceutical composition according to claim 7 or 8, wherein the inhibitor or the agent is an antibody having binding affinity to CD51.

13. The pharmaceutical composition according to claim 12, wherein the antibody is an antibody against at least one extracellular domain of CD51.

14. The pharmaceutical composition according to claim 12, wherein the antibody leads to inhibition of TGF-beta production by T cells, which contributes to immune evasion of the tumor cells.

15. The pharmaceutical composition according to claim 12, wherein the antibody leads to the depletion of CD51-expressing cancer cells.

16. The pharmaceutical composition according to claim 15, wherein the antibody suitable for depleting CD51 cancer cells mediates antibody-dependent cell-mediated cytotoxicity.

17. The pharmaceutical composition according to claim 15, wherein the antibody is a multispecific antibody comprising a first antigen-binding site for CD51 and at least one second antigen-binding site for effector cells.

18. The pharmaceutical composition according to claim 15, wherein the antibody is complexed with the cytotoxic portion.

19. The pharmaceutical composition according to claim 8, wherein the drug is a CAR-T cell, and the CAR comprises at least a CD51-specific extracellular antigen-binding domain.

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