CD81 as a biomarker and biotarget in T-cell malignancies

Abstract

This study of the regulatory T phenotype of Sézary cells led to the discovery of overexpression of CD81 by Sézary cells. CD81 has also been shown to be a relevant therapeutic target in the treatment of Sézary syndrome, NK / T lymphoma, hepatosplenic T-cell lymphoma, and acute T-cell leukemia. CD81 is therefore seen as a diagnostic marker and therapeutic target in T-cell malignancies. The present invention therefore implicates CD81 as a biomarker and biotarget in T-cell malignancies.

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. Among these T-cell malignancies, primary cutaneous T-cell lymphoma is a heterogeneous group of lymphomas that primarily affect the skin. Among them, cutaneous epidermotropic T-cell lymphomas (mycosis fungoides and Sézary syndrome) are the most frequent entities. Their prognosis is poor in advanced stages of the disease. Sézary syndrome is defined as erythroderma (redness covering the entire skin) and circulating blood disorders (1). Circulating T-lymphocyte tumor cells express CD4 and may lose expression of CD7 and CD26, while most cases display abnormal expression of CD158k (KIR3DL2) (2). Early diagnosis of this disease is difficult, and tracking of blood involvement is complex. This is because international standards use the loss of CD7 and CD26 markers (CD4+CD26- and CD4+CD7- cells) (3), which we know as nonspecific for tumor cells (4). The discovery of CD158k (KIR3DL2), a marker aberrantly expressed by Sézary cells, has enabled its use for disease diagnosis, monitoring (2), and the development of a therapeutic monoclonal antibody (lactamab). Phase I results have been published (5), and its efficacy 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 a depleting 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 is associated with the occurrence of autoimmune side effects. (7) Thus, there is a need to identify new markers and targets for the treatment of T cell malignancies.

[0003] Summary of the Invention: The invention is defined by the claims. In particular, the invention relates to methods for the diagnosis and treatment of T-cell malignancies.

[0004] Detailed description of the invention: Key definitions: As used herein, the term "T cell" 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 using their TCR (T cell receptor for antigen), with presentation or restriction by complex major histocompatibility molecules. There are several subsets of T cells, such as CD8+ T cells, CD4+ T cells, and gamma delta T cells, each with different functions. As used herein, the term "CD8+ T cell" 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 a pool of a patient's CD8+ T cells 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 immune processes, including B cell maturation 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 rapidly divide 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, TGF-β, or Treg, which secrete different cytokines and promote different types 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 typically account for 1-5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They are involved in initiating protective immune responses, recognizing their antigen ligands through direct interaction with antigen, without any presentation by MHC molecules on antigen-presenting cells. Gamma delta 2 T cells (sometimes referred to as gamma delta 2 T cells) are gamma delta T cells with TCR receptors 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 potent, non-MHC-restricted cytotoxic activity and are effective 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 Mycobacterium tuberculosis (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 provide new 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 that results from neoplastic transformation of T cells, affecting mature or immature T cells, and leading to T-cell lymphoma or T-cell leukemia. In some embodiments, the T-cell malignancy is a T-cell lymphoma. In some embodiments, the T-cell malignancy is a T-cell leukemia.

[0006] As used herein, the term "T-cell lymphoma" has its common meaning in the art and refers to a rare form of cancerous lymphoma affecting T cells. Lymphomas arise primarily from the uncontrolled proliferation of T cells and can become cancerous. T-cell lymphomas are classified as non-Hodgkin's lymphomas (NHLs) and account for less than 15% of all non-Hodgkin's diseases in this category. T-cell lymphomas are often classified as either high-grade (rapidly growing) or low-grade (slowly growing) based on their growth pattern. In particular, T-cell lymphomas include cutaneous lymphoma, nodal lymphoma, extranodal lymphoma, and leukemic lymphoma. In particular, subtypes include peripheral T-cell lymphoma, hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), mycosis fungoides (MF), and Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is Sezary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma.

[0007] In some embodiments, the T-cell malignancy is cutaneous T-cell lymphoma. 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. In some embodiments, the cutaneous T-cell lymphoma is Sézary syndrome or mycosis fungoides. In some embodiments, the cutaneous T-cell lymphoma is Sézary syndrome.

[0008] As used herein, the term "Sézary syndrome" or "SS" has its common meaning in the art and refers to a malignant form of cutaneous T-cell lymphoma characterized by the triad of erythroderma, lymphadenopathy, and circulating atypical lymphocytes (Sézary cells). SS occurs most frequently in men, is more common 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 a lion's-face and severe pruritus. Alopecia, ectropion, mild palmoplantar keratosis, and onychodystrophy of the nails may be present. Lymphadenopathy and hepatosplenomegaly are observed. Patients often complain of shivering, 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 blood condition, including several types of lymphocytic leukemia, that affects T cells. Leukemia usually develops from young blood cells in the bone marrow and spreads through the bloodstream. Leukemia comes in different subtypes: 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 a malignant hematological malignancy characterized by the abnormal proliferation of immature thymocytes.

[0011] As used herein, the term "CD81" or "TAPA-1" refers to a protein belonging to the tetraspanin family (Entrez Gene: 975; Ensembl: ENSG00000110651). CD81 is expressed by hematopoietic cells, endothelial cells, and epithelial cells and has been described as one of the major entry receptors for hepatitis C virus (8). CD81 is composed of four transmembrane domains connected by two loops: the small extracellular loop (SEL) and the large extracellular loop (LEL). CD81 LEL CD81 is a five-helix bundle fold composed of a stalk subdomain (helices A and E) and a head subdomain (helices B, C, and D) (Kitadokoro et al., 2001). An exemplary amino acid sequence for CD81 is represented by SEQ ID NO: 1. The protein contains two extracellular domains: the first ranging from amino acid residue 34 to amino acid residue 63 in SEQ ID NO: 1, and the second ranging from amino acid residue 113 to amino acid residue 201 in SEQ ID NO: 1. [ka]

[0012] As used herein, the term "agent capable of inducing cell death of CD81-expressing cancer cells" refers to any molecule capable of inducing cell death of CD81-expressing cancer cells under cellular and / or physiological conditions. In particular, the agent is capable of inducing apoptosis of CD81-expressing cancer cells. In some embodiments, the agent is capable of depleting CD81 cancer cells.

[0013] As used herein, the term "depletion," with respect to cancer cells, refers to a measurable reduction in the number of CD81-expressing cancer cells in a patient. This 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 a reduction below the detectable limit in the number of CD81-expressing cancer cells in a patient.

[0014] As used herein, the term "CD81 inhibitor" refers to a molecule that partially or completely blocks, inhibits, or neutralizes the biological activity or expression of the CD81 gene. A CD81 inhibitor can be any type of molecule that interferes with CD81-associated signal transduction in cells, for example, by reducing the transcription or translation of a nucleic acid encoding CD81, or by inhibiting or blocking CD81 polypeptide activity, or both. In particular, the CD81 inhibitors of the present invention are particularly suitable for blocking CD81-induced active TGF-beta production by T cells, which contributes to immune evasion of tumor cells. Examples of CD81 inhibitors include, but are not limited to, antisense polynucleotides, interfering RNA, catalytic RNA, RNA-DNA chimeras, CD81-specific aptamers, anti-CD81 antibodies, CD81-binding fragments of anti-CD81 antibodies, CD81-binding small molecules, CD81-binding peptides, and other polypeptides that specifically bind to CD81 (including, but not limited to, CD81-binding fragments of one or more CD81 ligands, optionally fused to one or more additional domains), wherein interaction of the CD81 inhibitor with CD81 results in a reduction or cessation of CD81 activity or expression.

[0015] As used herein, the term "TGF-β" has its common meaning in the art and refers to transforming growth factor β. In particular, this 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).

[0016] 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 antibody fragments comprising the antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DAB), TandAb 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-dibodies. Examples of antibody-based fragments include diabodies; kappa (lambda) bodies (scFv-CL fusions); BiTEs (bispecific T cell engagers; scFv-scFv tandems that attract T cells); DVD-Igs (dual variable domain antibodies, bispecific formats); SIPs (small immune proteins, a type of minibody); SMIPs ("small modular immunopharmaceuticals"); scFv-Fc dimers; DARTs (ds-stabilized diabodies "dual affinity retargeting"), miniature 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, inter alia, in EP 404,097 and WO 93 / 11161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating an antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bonds to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, 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 found in mammals of the Camelidae family, which naturally lacks light chains. Such single-domain antibodies are also "Nanobodies®." For a general description of (single) domain antibodies, reference is made to the prior art cited above, as well as to EP0368684, Ward et al. (Nature 1989 Oct 12;341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO06 / 030220, WO06 / 003388. In natural antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (I) and kappa (K). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains a distinct sequence domain. The light chain contains two domains, a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains, a 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 transport, complement fixation, and Fc receptor (FcR) binding. 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. Antibody specificity resides in the structural complementarity between the antibody combining site and antigenic determinants.Antibody combining sites are primarily composed of residues from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody combining site or influence the overall domain structure and therefore the combining site. Complementarity-determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin combining site. Each immunoglobulin light and heavy chain has three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. An antigen-binding site therefore typically contains six CDRs, including the CDR sets from each of the heavy and light chain V regions. Framework regions (FRs) refer to the amino acid sequences interposed between the CDRs. Residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set out in Kabat et al. (1987, Sequences of Proteins of Immunological Interest, U.S. 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 NO: sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, which corresponds to shortening of, or insertion into, structural components of the basic variable domain structure, whether framework or complementarity determining region (CDR). The exact Kabat numbering of residues The numbering can be determined for a given antibody by aligning the homologous residues in the antibody 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.

[0017] As used herein, the term "binding" indicates that an antibody has affinity for a surface molecule. As used herein, the term "affinity" refers to the strength of antibody binding to an epitope. The affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant 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 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.

[0018] 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 a human form of the antibody or immunoglobulin.

[0019] As used herein, the term "chimeric antibody" refers to an antibody comprising 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 regions (i.e., heavy and / or light chains), or portions thereof, have been modified, substituted, or exchanged so that the antigen-binding site (variable region) is linked to a constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or portions thereof, have been modified, substituted, or exchanged with a variable region having a different or modified antigen specificity. Chimeric antibodies also include primatized antibodies, particularly humanized antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient antibody or in the donor antibody. These modifications are made to further refine 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)).

[0020] 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 original non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies and their antibody fragments are human immunoglobulins (recipient antibody or antibody fragment) in which residues from the recipient's 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 found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to retain the binding specificity of the non-human antibody from which the binding region is derived but to avoid an immune response against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment will also typically comprise at least a portion of an immunoglobulin constant region (Fc) 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.

[0021] 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.

[0022] 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 comprising the antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. Characteristics of CARs include their ability to utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells toward a selected target in an MHC-unrestricted manner. 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.

[0023] As used herein, the term "CAR-T cells" 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. 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.

[0024] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients diagnosed as ill or suffering from a disease or medical condition, including the suppression of clinical recurrence. Treatment may be administered to patients who have a medical disorder or who may ultimately acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or alleviate 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 disease treatment, e.g., a pattern of medication used during treatment. A therapeutic regimen can include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. An induction regimen may use (in part or in whole) a "loading regimen," which may involve administering a larger dose of drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would administer a drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or part of a treatment regimen) used to maintain a patient between disease treatments, for example, to keep a patient in remission for an extended period (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., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., disease onset, etc.)).

[0025] 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. A therapeutically effective amount of an active agent may vary according to factors such as the individual's disease state, age, sex, and weight, as well as the ability of the active agent to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which the toxic or adverse effects of the drug outweigh the therapeutically beneficial effects. The effective dosage and administration regimen for an active agent depends 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 prescribe the effective amount of the required pharmaceutical composition. For example, a physician can start the dosage of the active agent used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Generally, the appropriate dose of the composition of the present invention is the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular administration regimen. Such an effective dose generally depends on the factors described above. For example, a therapeutically effective amount for therapeutic use can be measured by its ability to stabilize the progression of a disease. Typically, the ability of a compound to inhibit cancer can be assessed, for example, in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a patient. One of skill in the art can 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. An exemplary, non-limiting range for a therapeutically effective amount of a drug of the present invention is about 0.1 to 100 mg / kg, such as about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, such as about 0.1 to 10 mg / kg, for example, about 0.5, e.g., 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 the drug of the present invention is 0.02 to 100 mg / kg, such as about 0.02 to 30 mg / kg, such as about 0.05 to 10 mg / kg or 0.1 to 3 mg / kg, for example, about 0.5 to 2 mg / kg.Administration can be, for example, intravenous, intramuscular, intraperitoneal, or subcutaneous, but can also be administered proximal to the target site. The dosage regimen in the above methods and uses of treatment is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the effectiveness of treatment is monitored during treatment, for example, at predetermined time points. In some embodiments, effectiveness can be monitored by visualization of diseased areas or by other diagnostic methods further described herein, for example, by performing one or more PET-CT scans using a labeled antibody of the present invention, a fragment derived from an antibody of the present invention, or a miniantibody. If desired, the effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day, optionally in a unit dosage form. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period, such as more than 24 hours, to minimize any unwanted side effects. An effective dose of the drug of the invention may also be administered using a weekly, biweekly, or triweekly dosing period. 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 present invention may be carried out in an amount of about 0.1 to 100 mg / kg / day, such as 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, at 1 ... , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, for at least one week of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, using a single dose or divided doses every 24 hours, 12 hours, 8 hours, 6 hours, 4 hours, or 2 hours, or any combination thereof.

[0026] How it's diagnosed: A first object of the present invention relates to a method of diagnosing a T-cell malignancy in a patient, comprising detecting the expression level of CD81 in a sample obtained from the patient. In some embodiments, the T-cell malignancy is Sézary syndrome, hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia. In some embodiments, the T-cell lymphoma is Sézary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma.

[0027] In some embodiments, the present invention relates to a method for diagnosing T-cell lymphoma in a patient, comprising detecting the expression level of CD81 in a sample obtained from the patient. Thus, in some embodiments, the T-cell malignancy is T-cell lymphoma. In particular, T-cell lymphomas include cutaneous lymphoma, nodal lymphoma, extranodal lymphoma, and leukemic lymphoma. T-cell lymphomas also include peripheral T-cell lymphoma, hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), mycosis fungoides (MF), and Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is Sézary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma. In some embodiments, the methods of the present invention are particularly suitable for diagnosing cutaneous T-cell lymphoma. In some embodiments, the cutaneous T-cell lymphoma is Sézary syndrome or mycosis fungoides. More specifically, the method of the present invention is particularly suitable for diagnosing Sézary syndrome.

[0028] In some embodiments, the T-cell malignancy is T-cell leukemia. In some embodiments, the present invention relates to a method for diagnosing T-cell leukemia in a patient, comprising detecting the expression level of CD81 in a sample obtained from the patient. In some embodiments, the T-cell leukemia is T-cell acute lymphoblastic leukemia.

[0029] 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 result from a biopsy performed on the skin of a subject.

[0030] In some embodiments, the level of a marker is determined by immunohistochemistry (IHC). Immunohistochemistry typically involves the following steps: i) fixing the tissue sample with 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 coupled to an enzyme molecule. Such coupled secondary antibodies are commercially available, for example, from Dako, EnVision systems. Counterstains, e.g., H&E, DAPI, Hoechst, can be used. Other staining methods can be achieved 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 can be attached to the antibody, thereby enabling 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), chromophoric 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 also include enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S, or 125IHC can be labeled using amines, thiols, and particles (e.g., gold). Different types of labels can be conjugated to affinity ligands using various chemistries, such as amine or thiol reactions. However, reactive groups other than amines and thiols, such as aldehydes, carboxylic acids, and glutamine, can also be used. Various enzyme staining methods are known in the art for detecting proteins of interest. For example, enzymatic 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 secondary antibody or second binding partner is required to detect binding of the first binding partner because it is unlabeled. The resulting stained specimens are each imaged using a system for viewing detectable signals and acquiring images, such as digital images of the staining. Methods for image acquisition are well known to those skilled in the art. For example, once a sample has been stained, the stain or biomarker label can be detected using any optical or non-optical imaging device, such as an upright or inverted optical microscope, a scanning confocal microscope, a camera, a scanning or tunneling electron microscope, a scanning probe microscope, and an imaging infrared detector. In some cases, the image can be captured digitally. The resulting image can be used to quantitatively or semi-quantitatively determine the amount of 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 include automated staining and microscope scanning, computerized image analysis, serial section comparison (to control for variations in sample orientation and size), digital report generation, and archiving and tracking of samples (e.g., slides on which tissue sections are placed).Cell 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 performed manually or by image processing techniques involving a computer processor and software. Using such software, for example, images can be configured, calibrated, standardized, and / or verified based on factors including staining quality or staining intensity using procedures known to those skilled in the art (see, e.g., 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 refers to methods of scanning and scoring histochemically tested samples to identify and quantify the presence of specific biomarkers (i.e., markers). Quantitative or semi-quantitative methods can use image software to detect staining density or amount, or can use methods of detecting staining by human eye, where a trained operator numerically ranks the results. 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 for measuring or quantifying or semi-quantifying the degree of staining; see, for example, 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 strongly positive staining (e.g., brown staining) to the sum of the total stained area can be calculated and scored.The amount of detected biomarker (i.e., 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 the stained area, e.g., a percentage of positive pixels. For example, a sample can 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 assigned to the sample, which is a numerical representation of the intensity or amount of histochemical staining of the sample, representing the amount of target biomarker (e.g., marker) present in the sample. The optical density or area percentage values ​​can be given a score scaled, for example, on an integer scale. Thus, in some embodiments, the method of the present invention includes: i) providing one or more immunostained thin sections of a tissue section obtained by an automated slide staining system by using a binding partner (e.g., an antibody described above) capable of selectively interacting with the marker; ii) proceeding to digitize the slide in step a by high-resolution scan capture; iii) detecting slices of the tissue section on the digital photograph; iv) providing a size reference grid with uniformly distributed units having the same surface, where the grid is 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.

[0031] 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 use "fluorescence-activated cell sorting" to physically sort particles based on their properties to purify or detect a population of interest. 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 at a time, based on the specific light scattering and fluorescence properties of each cell, providing rapid, objective, and quantitative recording of fluorescent signals from individual cells and physical separation of specifically targeted cells. Thus, FACS can be used in conjunction with the methods described herein to isolate and detect populations of cells of the present invention. Thus, for example, fluorescence-activated cell sorting (FACS) may be used, 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 may also include a cytometer fluidically coupled to the cytometry sample fluidics subsystem. 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); data input devices, e.g., interface ports, mice, keyboards, etc.; fluid handling components; power supplies; and the like.More specifically, the sample is contacted with a panel of antibodies specific for a 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 skilled in the art. In some embodiments, an agent that specifically binds to a cell surface marker, such as an antibody or antigen-binding fragment, is labeled with a tag 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 as required for methods to isolate 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, Tricolor, 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 involve binding of 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, etc. The beads may be made of different materials, including, but not limited to, glass, plastic, polystyrene, and 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.).

[0032] 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 CD3, CD4, KIR3DL2, PLS3, Twist, and NKp46.

[0033] As used herein, the names of each of the various markers of interest refer to the internationally recognized names of the corresponding genes found in internationally recognized gene and protein sequence databases, including in the databases from the HUGO Gene Nomenclature Committee, available at the following notable internet address: http: / / www.gene.ucl.ac.uk / nomenclature / index.html. As used herein, the names of each of the various markers of interest may also refer to the internationally recognized names of the corresponding genes found in the internationally recognized gene and protein sequence database, Genbank. Through these internationally recognized sequence databases, nucleic acid and amino acid sequences corresponding to each of the markers of interest described herein can be retrieved by those skilled in the art.

[0034] Multiplexed tissue analysis techniques are particularly useful for quantifying multiple markers in a tissue sample. Such techniques should enable the measurement of at least five, or even at least ten or more, biomarkers from a single tissue sample. Advantageously, the techniques also preserve the localization of the biomarkers and allow for the differentiation of their presence 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 teaches creating up to eight, nine, ten, eleven, or more images of tissue sections on layered and blotted membranes, paper, filters, and the like. Coated membranes useful for performing the L-IHC / MTI process are available from 20 / 20 GeneSystems, Inc. (Rockville, MD).

[0035] In some embodiments, the L-IHC method can be performed on any of a variety of tissue samples, whether fresh or preserved. Samples routinely include core needle biopsies 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. In this way, L-IHC allows for the testing of multiple markers in a tissue section by obtaining copies of molecules transferred from the tissue section to multiple bioaffinity-coated membranes, 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. Next, 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 structure is such that the movement of liquid and tissue molecules is essentially perpendicular to the membrane surface. The sandwich of section, membrane, spacer paper, absorbent paper, and weight 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 in the stack (available from 20 / 20 GeneSystems, Inc., Rockville, MD). Each membrane thus contains a copy of the tissue and can be probed for different biomarkers using standard immunoblotting techniques, thereby enabling open-ended expansion of the marker profile as performed on a single tissue section.Because protein amounts may be lower on membranes more distal from the tissue in the stack, which may result from, for example, different amounts of molecules in the tissue sample, different mobilities of molecules released from the tissue sample, different binding affinities of molecules to the membrane, migration lengths, etc., standardization of values, running controls, assessing the migration levels of tissue molecules, etc., may be included in the procedure to correct for variations that occur within membranes, between membranes, and between multiple membranes, allowing direct comparison of information within membranes, between membranes, and between multiple membranes. Thus, total protein can be determined per membrane using any means for quantifying protein, such as by biotinylating available molecules, such as proteins, using standard reagents and methods, followed by 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.

[0036] In some embodiments, the methods of the present invention utilize multiplex tissue imprinting (MTI) technology to measure biomarkers, which allows for multiple biomarkers, in some cases at least six biomarkers, thereby preserving valuable biopsy tissue.

[0037] In some embodiments, alternative multiplex tissue analysis systems exist that can 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.

[0038] In some embodiments, the methods of the invention utilize 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 sequential analyses are performed on biological samples 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.

[0039] In some embodiments, multitissue imaging can be performed using fluorescence (e.g., fluorophores or quantum dots), where signals can be measured using a multispectral imaging system. Multispectral imaging is a technique that collects spectral information at each pixel of an image and analyzes the resulting data using spectral image processing software. For example, this system can acquire a series of images at different, electronically and sequentially selectable wavelengths and utilize analysis programs designed to handle such data. This system thus allows for simultaneous quantitative information from multiple dyes, even when their spectra are highly overlapping, colocalized, or occurring at the same point in the sample, provided that their spectral curves are different. Many biological materials autofluoresce or emit low-energy light when excited by high-energy light. This signal can result in images and data with lower contrast. High-sensitivity cameras without multispectral imaging capabilities only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can unmix or separate autofluorescence from tissues, thereby increasing the achievable signal-to-noise ratio. Briefly, quantification can be performed in 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 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 enabling detection, quantification, and segmentation of specific tissues through powerful pattern recognition algorithms.Machine learning algorithms are typically pre-trained to segment tumor from stroma and identify labeled cells.

[0040] In some embodiments, the level of the marker is determined at the nucleic acid level. Typically, the level of a gene can 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, for example, 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).

[0041] In some embodiments, the methods of the present invention further comprise comparing the expression level of CD81 with a predetermined reference value, and detecting a difference between the expression level of CD81 and the predetermined reference value indicates whether or not the subject has a T-cell malignancy.

[0042] In some embodiments, the T cell malignancy is a T cell lymphoma. Thus, in some embodiments, the method of the present invention further comprises comparing the expression level of CD81 with a predetermined reference value, and detecting a difference between the expression level of CD81 and the predetermined reference value indicates whether the subject has a T cell lymphoma.

[0043] In some embodiments, the T-cell malignancy is T-cell leukemia. Thus, in some embodiments, the method of the present invention further comprises comparing the expression level of CD81 with a predetermined reference value, and detecting a difference between the expression level of CD81 and the predetermined reference value indicates whether the subject has T-cell leukemia.

[0044] In some embodiments, the predetermined reference value is relative to a number or value derived from a population study, 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 lesion. Such predetermined reference values ​​can be derived from statistical analysis of a population and / or risk prediction data obtained from mathematical algorithms and calculated indices. In some embodiments, retrospective measurements of marker levels in correctly deposited past subject samples may be used to establish these predetermined reference values. Thus, in some embodiments, the predetermined reference value is a threshold or cutoff value. The threshold must be determined to obtain optimal sensitivity and specificity according to the test's function and benefit / risk balance (clinical consequences of false positives and false negatives). Typically, optimal sensitivity and specificity (and therefore threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the levels of a marker in a reference group, algorithmic analysis can be used to statistically analyze the measured levels of the marker in the samples to be tested, and thus a classification criterion with significance for sample classification can be obtained. The official name of the ROC curve is the receiver operating characteristic curve, also known as the receiver operating characteristic curve. It is mainly used for clinical biochemistry diagnostic tests. The ROC curve is a comprehensive index that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1-specificity). The relationship between sensitivity and specificity is revealed using the image synthesis method. A series of different cutoff values ​​(thresholds or critical values, the boundary value between normal and abnormal results of a diagnostic test) are set as continuous variables, and a series of sensitivity and specificity values ​​are calculated. The curve is then drawn using sensitivity as the vertical coordinate and specificity as the horizontal coordinate. The higher the area under the curve (AUC), the higher the diagnostic accuracy. On the ROC curve, the point closest to the top left of the coordinate diagram is the decisive point, with both high sensitivity and high specificity. The AUC value of the ROC curve is between 1.0 and 0.5. If AUC > 0.5, the diagnostic result is better as the 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 the AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably performed using a computer. Existing software or systems may be used to plot the ROC curve, 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.

[0045] Typically, as demonstrated in the Examples, the expression level of CD81 is higher than that determined in samples from healthy individuals. Thus, in some embodiments, the methods of the present invention further comprise comparing the expression level of CD81 with a predetermined reference value, and detecting an expression level of CD81 higher than the predetermined reference value indicates that the subject has a T-cell malignancy.

[0046] In some embodiments, the methods of the present invention further comprise comparing the expression level of CD81 with a predetermined reference value, and detecting an expression level of CD81 higher than the predetermined reference value indicates that the subject has a T-cell lymphoma. In some embodiments, the T-cell lymphoma is Sézary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma. In some embodiments, the T-cell lymphoma is Sézary syndrome.

[0047] In some embodiments, the methods of the present invention further comprise comparing the expression level of the marker with a predetermined reference value, and detecting an expression level of the marker higher than the predetermined reference value indicates that the subject has T-cell leukemia. In some embodiments, the T-cell leukemia is T-cell acute leukemia.

[0048] In some embodiments, the methods of the present invention further comprise comparing the expression level of CD81 with a predetermined reference value, and detecting an expression level of CD81 higher than the predetermined reference value indicates that the subject has T-cell lymphoma or T-cell leukemia.

[0049] In some embodiments, the CD81 expression level is determined using fluorescence intensity. In some embodiments, the CD81 expression level is determined using CD81 mean fluorescence intensity. In some embodiments, the method comprises the further step of determining CD81 mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the CD81 mean fluorescence intensity is greater than 6500, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, or 8000. In some embodiments, the method comprises the further step of determining CD81 mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the CD81 mean fluorescence intensity is greater than a predetermined reference value. In some embodiments, the method comprises the further step of determining CD81 mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the CD81 mean fluorescence intensity is greater than 7500. In some embodiments, the CD81 expression level is determined using CD81 delta mean fluorescence intensity. In some embodiments, the CD81 delta mean fluorescence intensity is calculated relative to an IgG2a control isotype expression level. In some embodiments, the CD81 delta mean fluorescence intensity is calculated relative to an IgG2a control isotype mean fluorescence intensity. In some embodiments, the method determines the CD81 delta mean fluorescence intensity, and determines whether the CD81 delta mean fluorescence intensity is 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600, and concluding that the patient is afflicted with a T-cell malignancy if the CD81 delta mean fluorescence intensity is greater than 0, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, or 600. In some embodiments, the method comprises the further step of determining a CD81 delta mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the CD81 delta mean fluorescence intensity is greater than a predetermined reference value.In some embodiments, the T-cell malignancy is Sezary syndrome.

[0050] Monitoring the effect of an agent (e.g., a drug compound) on CD81 expression levels can be applied to monitor the status of a T-cell malignancy 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 treatment.

[0051] Thus, the present invention also provides a method for monitoring the effectiveness of treatment of a patient suffering from a T-cell malignancy, said method comprising the steps of: (i) obtaining a pre-dose sample from the patient prior to administration of the drug; (ii) detecting the level of CD81 expression in pre-administration samples; (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 CD81 expression in the pre-administration sample with the level of CD81 expression in the post-administration sample or samples; and (vi) modifying the patient's medication accordingly;

[0052] In some embodiments, the T cell malignancy is a T cell lymphoma. In some embodiments, the T cell lymphoma is Sezary syndrome. In some embodiments, the T cell malignancy is a T cell leukemia.

[0053] For example, a worse diagnosis, as determined by assessing CD81 expression levels over the course of treatment, may indicate an ineffective dose and the desirability of increasing the dose. Conversely, a better diagnosis, as determined by assessing CD81 expression levels, may indicate an effective treatment and no need to change the dose.

[0054] Therefore, the present invention also relates to a method for adapting therapy in a patient suffering from a T-cell malignancy, said method comprising the steps of: a) performing an in vitro diagnostic method as disclosed herein on at least one sample taken from the patient; and b) administering it to the patient to treat the patient;

[0055] In some embodiments, the T cell malignancy is a T cell lymphoma. In some embodiments, the T cell lymphoma is Sezary syndrome. In some embodiments, the T cell malignancy is a T cell leukemia.

[0056] The present invention also relates to a kit for carrying out the diagnostic method described above. The kit includes multiple reagents, in particular at least one agent capable of specifically binding to the CD81 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 include substrate-immobilized oligonucleotides (labeled or unlabeled), labeled oligonucleotides not bound to substrates, PCR primer pairs, 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. For example, the kit may include a fluid (e.g., SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody to its specific binding protein, one or more sample compartments, instructions describing how to carry out the in vitro diagnostic method of the present invention, and the like.

[0057] Treatment method: A further object of the present invention relates to a method of treating a T-cell malignancy in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD81-expressing cancer cells. In some embodiments, the T-cell lymphoma is Sézary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma. In some embodiments, the T-cell malignancy is Sézary syndrome, hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia.

[0058] In some embodiments, the present invention relates to a method of treating T-cell lymphoma in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD81-expressing cancer cells. In particular, T-cell lymphomas include cutaneous lymphoma, nodal lymphoma, extranodal lymphoma, and leukemic lymphoma. In particular, subtypes include peripheral T-cell lymphoma, hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), mycosis fungoides (MF), and Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is Sézary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, or NK / T-cell lymphoma. In some embodiments, the T-cell lymphoma is cutaneous T-cell lymphoma. In some embodiments, the T-cell lymphoma is Sézary syndrome.

[0059] In some embodiments, the present invention relates to a method of treating T-cell leukemia in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD81-expressing cancer cells. In some embodiments, the T-cell leukemia is T-cell acute lymphoblastic leukemia (T-ALL).

[0060] 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 a premature human infant.

[0061] CD81 inhibitors: In another aspect, the present invention relates to a method of treating a T-cell malignancy in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.

[0062] In some embodiments, the present invention relates to a method of treating T-cell lymphoma in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.

[0063] In some embodiments, the present invention relates to a method of treating T-cell leukemia in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.

[0064] In some embodiments, the CD81 inhibitor is an inhibitor of CD81 gene expression. In particular, the gene expression inhibitor is an siRNA, an antisense oligonucleotide, or a ribozyme. For example, antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, can act to directly block CD81 mRNA translation by binding to CD81 mRNA and thus preventing protein translation, or by increasing mRNA degradation, thereby reducing the level and therefore activity of CD81 in the cell. For example, antisense oligonucleotides of at least about 15 bases complementary to unique regions of the mRNA transcript sequence encoding CD81 can be synthesized, for example, by conventional phosphodiester techniques. 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. CD81 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 CD81 gene expression is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, shRNAs, and ribozymes of the present invention can be delivered in vivo alone or in combination with a vector.

[0065] CD81 antibody: In some embodiments, the agent is an antibody having binding affinity for CD81. In some embodiments, the agent is an antibody directed against at least one extracellular domain of CD81. In some embodiments, the agent is an antibody directed against the C and / or D helix of the CD81 large extracellular loop. In some embodiments, the agent is an antibody directed against SEQ ID NO:2 (LTALTTSVLKNNLCPSGSNIISNLFKE). In some embodiments, the agent is an antibody directed against SEQ ID NO:3 (LTALTTSVLKN). In some embodiments, the agent is an antibody directed against SEQ ID NO:4 (NIISNLFKE). In some embodiments, the anti-CD81 antibody binds to the same epitope as the 5A6 antibody. In some embodiments, the anti-CD81 binds to at least residues Ser179, Asn180, and Phe186 of SEQ ID NO:1. In some embodiments, the anti-CD81 binds to at least residues His151, Ala164, Ser168, and Asn172 of SEQ ID NO:1. In some embodiments, the anti-CD81 antibody is the 5A6 antibody. In some embodiments, the anti-CD81 antibody comprises a VH domain consisting of the sequence set forth in SEQ ID NO:5 and a VL domain consisting of the sequence set forth in SEQ ID NO:6. In some embodiments, the anti-CD81 antibody comprises a VH domain consisting of the sequence set forth in SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:9 and a VL domain consisting of the sequence set forth in SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:10. In some embodiments, the anti-CD81 antibody comprises a VH-CDR1 defined by SEQ ID NO:11 (DDSIH), a VH-CDR2 defined by SEQ ID NO:12 (WINTETGEPTYADDFKG), a VH-CDR3 defined by SEQ ID NO:13 (LSPVVVIFIY), a VL-CDR1 defined by SEQ ID NO:14 (KSSQSLLHSRTRKNYLA), a VL-CDR2 defined by SEQ ID NO:15 (WASTRES), and a VL-CDR3 defined by SEQ ID NO:16 (KQSYNLYA). [ka]

[0066] In some embodiments, the anti-CD81 antibody comprises a heavy chain having at least 70% identity with the heavy chain of the 5A6 antibody and / or a light chain having at least 70% identity with the light chain of the 5A6 antibody. In some embodiments, the anti-CD81 antibody is produced by antibody-producing cells (e.g., lymphocytes, plasma cells, immortalized B cells, or hybridomas) contacted with an immunogenic agent. In some embodiments, the immunogenic agent is an OCI-LY8 cell.

[0067] In some embodiments, the antibody is an anti-CD81 neutralizing antibody. In some embodiments, the antibody leads to the depletion of CD81-expressing cancer cells. In some embodiments, the antibody leads to the inhibition of TGF-beta production by T cells, which contributes to immune evasion of tumor cells. In some embodiments, the antibody leads to the depletion of CD81-expressing cancer cells. In some embodiments, the antibody is directed against at least one extracellular domain of CD81. In some embodiments, the antibody is directed against the C-helix and / or D-helix of the CD81 large extracellular loop. In some embodiments, the antibody is a humanized antibody or a chimeric antibody. Anti-CD81 antibodies are well known in the art. By way of example, antibodies targeting CD81 are described in WO2017 / 218691, US8440797B2, or WO2012 / 077649.

[0068] Other examples of anti-CD81 antibodies include those described in Fofana I, Xiao F, Thumann C, Turek M, Zona L, Tawar RG, Grunert F, Thompson J, Zeisel MB, Baumert TF. A novel monoclonal anti-CD81 antibody produced by genetic immunization efficiently inhibits Hepatitis C virus cell-cell transmission. PLoS One. 2013 May 21;8(5):e64221. doi: 10.1371 / journal.pone.0064221. PMID: 23704981; PMCID: PMC3660333; Nelson, Bryce, et al. "Structure-guided combinatorial engineering facilitates affinity and specificity optimization of anti-CD81 antibodies." Journal of molecular biology 430.14 (2018): 2139-2152.

[0069] In some embodiments, the antibody is fully human. 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, and 6,150,584, and the references cited therein, the contents of which are incorporated herein by reference.

[0070] CD81 depletion antibody In some embodiments, antibodies suitable for depletion of CD81 cancer cells mediate antibody-dependent cell-mediated cytotoxicity.

[0071] In some embodiments, the antibody comprises the VH and VL domains of the 5A6 antibody.

[0072] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cells and subsequently cause lysis of the target cells. While not wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).

[0073] 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, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes 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, numbering according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). The "EU index as shown in Kabat" refers to the residue numbering of the human IgG1 EU antibody described in Kabat et al. (supra). Fc can refer to this region alone or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein can be an antibody, an Fc fusion, or any protein or protein domain comprising an Fc region. Particularly preferred are proteins comprising variant Fc regions that are non-naturally occurring variants of Fc regions. The amino acid sequence of a non-native 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 a variant Fc region as a result of an insertion or substitution may be referred to as a non-naturally occurring amino acid residue. Note: Polymorphisms have been observed at numerous Fc positions, including, but not limited to, Kabat positions 270, 272, 312, 315, 356, and 358; thus, slight differences between the presented sequence and sequences in the prior art may exist.

[0074] 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 for 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 ADCC activity of a molecule, an in vitro ADCC assay, such as that 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).

[0075] As used herein, the term "effector cell" refers to a leukocyte that expresses one or more FcRs 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 (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.

[0076] In some embodiments, the antibody suitable for depleting 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.

[0077] In some embodiments, antibodies suitable for cancer cell depletion comprise a variant Fc region with increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In some embodiments, antibodies of the invention comprise a variant Fc region comprising at least one amino acid substitution, insertion, or deletion, wherein the substitution, insertion, or deletion of at least one amino acid residue results in increased affinity for FcγRIA, FcγRIIA, FcγRIIB, FcγRIIIA, FcγRIIIB, and FcγRIV. In some embodiments, antibodies of the invention comprise 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, wherein the at least one amino acid substitution is selected from the group consisting of S239D, A330L, A330Y, and I332E, wherein amino acid residues are numbered according to the EU index.

[0078] In some embodiments, the glycosylation of antibodies suitable for cancer cell depletion is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such sugar modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made that result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for an antigen. Such approaches are described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co et al. Additionally or alternatively, antibodies with altered glycosylation can be generated, such as hypofucosylated or nonfucosylated antibodies with reduced or no fucosyl residues, or antibodies with increased bisecting GlcNAc structures. Such altered 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 an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells for expressing recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, EP 1176195 by Hang et al. describes cell lines in which the FUT8 gene, which encodes fucosyltransferase, has been functionally disrupted, and antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Thus, in some embodiments, human monoclonal antibodies of the invention can be produced by recombinant expression in cell lines exhibiting hypofucosylated or non-fucosylated patterns, for example, mammalian cell lines with defective expression of the FUT8 gene, which encodes fucosyltransferase.PCT Publication WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to add fucose to Asn(297)-linked sugars, 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 cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180). Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with an altered mammalian glycosylation pattern lacking 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 engineered for a mammalian-like glycosylation pattern and capable of producing antibodies lacking fucose as a glycosylation pattern (see, e.g., EP1297172B1).

[0079] In some embodiments, antibodies suitable for depletion of cancer cells mediate complement dependent cytotoxicity.

[0080] 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).

[0081] In some embodiments, antibodies suitable for depletion of cancer cells mediate antibody-dependent phagocytosis.

[0082] 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, followed by phagocytosis of the target cell.

[0083] CD81 multispecific antibody: In some embodiments, suitable antibodies for depletion of CD81 cancer cells are multispecific antibodies comprising a first antigen-binding site directed against CD81 and at least one second antigen-binding site directed against effector cells, as described above.

[0084] In some embodiments, the first antigen binding domain comprises the VH domain and the VL domain of the 5A6 antibody.

[0085] In particular, the second antigen-binding site is used to recruit killing mechanisms, for example, by binding to an antigen on a human effector cell. In some embodiments, the effector cell is capable of inducing ADCC, e.g., a natural killer cell. For example, monocytes and macrophages express FcRs and are involved in the specific killing of target cells and the presentation of antigens to other components of the immune system. In some embodiments, the effector cell can phagocytose a target antigen or target cell. The expression of a particular FcR on an effector cell can be regulated by humoral factors, such as cytokines. The effector cell can phagocytose a target antigen or phagocytose or lyse a target cell. Suitable cytotoxic agents and second therapeutic agents are exemplified below and include toxins (e.g., 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 binds to a surface molecule on an NK cell, enabling the cell to be activated. 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 crosslinked by chemical heteroconjugation, one with specificity for a particular surface molecule of ILCs and another with specificity for a second antigen; (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; and (iv) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain comprises two variable domains in tandem via a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010); (v) Tandab, which is a chemically linked bispecific (Fab')2 fragment; (vi) the fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vii) Flexibody, which is a combination of an scFv and a 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 Fabs, can result 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, contain 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 that force heterodimerization. Such molecules can be prepared using known technologies, such as those known as 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.

[0086] In some embodiments, the multispecific antibody is thus a bispecific antibody.

[0087] 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., CD81), 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 T cells to target cells and simultaneously activating the cytolytic activity of the T cell. BiTE-mediated activation of T cells does not require a specific T cell receptor on the T cell, nor an MHC I molecule, peptide antigen, or costimulatory molecule on the target cell.

[0088] CD81 antibody-drug conjugates: In some embodiments, antibodies suitable for cancer cell depletion are conjugated to a therapeutic moiety, i.e., a drug.

[0089] In some embodiments, the antibody-drug conjugate comprises the VH and VL domains of the 5A6 antibody.

[0090] In some embodiments, the therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory agent, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0091] In some embodiments, antibodies suitable for depletion of cancer cells are conjugated to a cytotoxic moiety. Cytotoxic moieties include, 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 analogue or derivative thereof; antimitotic agents such as monomethyl auristatin E or F or an analogue or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analogue or derivative thereof; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, etc.; platinum derivatives such as cinnamoyl benzoate, 5-fluorouracil, decanoyl benzoate, hydroxyurea, asparaginase, gemcitabine, or cladribine; ... Suplatin or carboplatin, etc.; Duocarmycin A, Duocarmycin SA, Rakelmycin (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 its active fragments and hybrid molecules, ricin toxin, such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, Shiga-like toxins, such as SLT I, SLT II, ​​SLT IIV, and LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alphasarcin, Aleurites foedii protein, dianthin protein, Cornus kousa protein, such as PAPI, PAPII, and PAP-S, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, and enomycin toxins; ribonucleases (RNases); DNases I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas aeruginosa endotoxin.

[0092] In some embodiments, antibodies suitable for depleting cancer cells are conjugated to auristatin or its peptide analogs, derivatives, or prodrugs. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cytoplasmic division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer (US Pat. No. 5,663,149) 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 (monomethyl auristatin F), and MMAE (monomethyl auristatin 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, and in International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0093] In some embodiments, suitable antibodies for depletion of cancer cells are 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.

[0094] In some embodiments, suitable antibodies for cancer cell depletion are 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 analogs, derivatives, or prodrugs thereof.

[0095] In some embodiments, an antibody suitable for cancer cell depletion is conjugated to an anthracycline or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to maytansine or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, an antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to 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.

[0096] In some embodiments, antibodies suitable for cancer cell depletion are conjugated to a nucleic acid or 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 ribozyme.

[0097] 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 Antibodies In Cancer Therapy," in Monoclonal Antibodies For See 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 ​​residue on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.Conjugation using engineered cysteines or the incorporation of unnatural amino acids has been reported to improve conjugate uniformity (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 cysteine-based site-specific conjugations called "THIOMABs" (TDCs), which are claimed to exhibit improved therapeutic indices compared with traditional conjugation methods. Conjugation to 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 engineered Fc-containing polypeptides with an acyl donor glutamine-containing tag (e.g., a Gln-containing peptide tag or Q-tag) or endogenous glutamine made reactive by polypeptide engineering (e.g., via amino acid deletion, insertion, substitution, or mutation on the polypeptide). Transglutaminase can then covalently crosslink an amine donor agent (e.g., a small molecule containing or appended with a reactive amine) to form a stable and homogenous population of engineered Fc-containing polypeptide conjugates with the amine donor agent site-specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine-containing tag or accessible / exposed / reactive endogenous glutamine (WO 2012 / 059882).

[0098] CD81 CAR-T cells In some embodiments, the agent is a CAR-T cell, and the CAR comprises an extracellular antigen-binding domain specific for at least CD81. In some embodiments, the CAR comprises an extracellular antigen-binding domain specific for the C and / or D helices of CD81.

[0099] In some embodiments, the extracellular antigen-binding domain specific for CD81 comprises the VH and VL domains of the 5A6 antibody.

[0100] 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") that 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 one another. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to one another, 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 chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0101] In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain specific for CD81, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain specific for CD81, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain specific for CD81, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain specific for CD81, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.

[0102] 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 cellular processing and localization of the CAR to the cell membrane.

[0103] In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for CD81, fused to the transmembrane and endodomain of CD3-zeta. 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 eliminate T cells upon addition of a prodrug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0104] In some embodiments, a chimeric antigen receptor of the invention comprises at least one VH and / or VL sequence of an antibody specific for CD81. In some embodiments, the portion of a CAR of the invention comprising an antibody or antibody fragment thereof specific for CD81 can exist in various forms, with the antigen binding domain being expressed as part of a contiguous 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 specific for CD81. In a further aspect, the CAR comprises an antibody fragment comprising an scFv that is specific for CD81.

[0105] 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 may stably express the CAR. In some embodiments, cells (e.g., T cells) are transfected with a nucleic acid, e.g., mRNA, cDNA, or DNA, encoding a CAR. In some embodiments, the antigen-binding domain (e.g., scFv) of a CAR of the present invention is encoded by a nucleic acid molecule whose sequence has been 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 has been 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 encode 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. Various codon optimization methods are known in the art, including, for example, the methods disclosed in at least US Pat. Nos. 5,786,464 and 6,114,148.

[0106] 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.

[0107] In some embodiments, CAR activity can be controlled, if desired, to optimize the safety and efficacy of CAR therapy. There are many ways in which CAR activity can be regulated. For example, inducible apoptosis using a caspase fused to a dimerization domain (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.

[0108] Pharmaceutical Composition: Typically, the agent of the present invention is administered to a patient in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as 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, the term "injection" includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, 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. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. Sterile fixed oils are also conventionally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in formulating pharmaceutically 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 pharmaceutically 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 use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavorings, or colorings 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, thereby dissolving in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, particularly when the target of treatment includes areas or organs 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 composition 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 composition may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically 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. Patches may also be used. The compositions of the present 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 using 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 present 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 It will be understood, however, 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) can be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular) 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 a drug of the invention.

[0109] The present invention is further illustrated by the following figures and examples, which, however, should not be construed in any way as limiting the scope of the present invention. [Brief explanation of the drawings]

[0110] [Figure 1] CD81 expression in normal or Sézary cells. (A) Gating strategy for Sézary cell phenotyping. Cells were stained with anti-CD3, anti-CD4, and anti-CD158k to determine the tumor cell population. CD81 expression was detected using the 5A6 anti-CD81 clone. (B) CD81 expression on healthy CD3+CD4+ T lymphocytes (n=2) or neoplastic Sézary cells (CD3+CD4+CD158k+, n=15) was tracked by flow cytometry. Mean fluorescence intensity (MFI) is reported here. Tumor cells have higher expression of CD81 compared to normal CD4+ T cells in 70% of cases. [Figure 2] The anti-CD81 antibody 5A6 induces cell death in Sézary and mycosis fungoides cells, but not in healthy peripheral blood cells. HUT78, Seax, or Myla cells were incubated with a mouse IgG isotype control (mIgc) or anti-CD81 antibody (5A6 clone) at 2 × 10 cells / ml in complete RPMI 1640 medium at 37°C and 5% CO for 48 hours. Cell viability was measured using the VD staining marker (viability dye e780). PBMCs from HD were treated similarly, with IL-2 added to the medium at 10 ng / ml. [Figure 3] The anti-CD81 antibody 5A6 induces cell death in tumor cells from patients. Purified PBMCs from Sézary patients were incubated with mouse IgG isotype control (mIgc) or anti-CD81 antibody (5A6 clone) at 1 μg / ml, with or without IL-2 at 10 ng / ml, at 2 x 10 cells / ml in complete RPMI 1640 medium at 37°C and 5% CO for 72 hours. Cell viability was measured using VD staining marker (viability dye e780) on CD4+CD158k+ tumor cells (SS cells). [Figure 4]The anti-CD81 antibody 5A6 induces cell death in NK / T lymphoma (SNK6), acute T-cell leukemia (MOLT-4), and HSTL (DERL2) cells. Cells were incubated with mouse IgG isotype control (mIgc) or anti-CD81 antibody (5A6 clone) at 2 x 10 cells / ml in complete RPMI 1640 medium at 37°C and 5% CO for 48 hours. Cell viability was measured using the VD staining marker (viability dye e780). IL-2 was added at 10 ng / ml for SNK-6 and DERL-2 cell line cultures. [Figure 5] Anti-CD81 5A6 mAb induces cell death through caspase 3 cleavage. Cells were incubated with 5A6 or IgG mab (2 μg / ml) for 2 hours before washing and fixation / permeabilization for caspase 3 staining. Cell death was monitored by viability dye staining (VD efluor780).

[0111] Working example: Materials and Methods CD81 expression Testing of CD81 expression was performed by flow cytometry in peripheral blood mononuclear cells (PBMCs) of patients with Sézary syndrome using anti-CD3, CD4, CD158k, and CD81 antibodies (clone 5A6) after information and signed informed consent.

[0112] Induction of apoptosis Induction of apoptosis was performed in cells of the mycosis fungoides (Myla) Sézary cell line (HUT78, Seax) for cutaneous T-cell lymphoma, and in cells of the NK / T (SNK-6), HSTL (DERL-2), and MOLT-4 cell lines for other types of lymphoma / leukemia.

[0113] result The following is demonstrated herein: - CD81 is overexpressed in neoplastic Sézary cells (Figure 1); - Anti-CD81 antibodies induce cell death in neoplastic Sézary cells and mycosis fungoides cells, but not in healthy peripheral blood cells (Figure 2); - Anti-CD81 antibodies induce cell death of tumor cells from patients (Figure 3); - Anti-CD81 antibodies induce cell death in NK / T cell lymphoma (SNK6), T cell acute leukemia (MOLT-4), and HSTL (DERL2) cells (Figure 4); - Anti-CD81 5A6 antibody induces cell death through caspase-3 cleavage (Figure 5).

[0114] conclusion It has previously been shown that CD81 is overexpressed in B lymphomas, and that the use of therapeutic antibodies (e.g., the 5A6 antibody) targeting the C and D helices of the CD81 LEL (large extracellular loop) induces apoptosis of tumor B cells while sparing healthy cells (8, 9). This study, involving CD81 in Sézary cells and other T-cell malignancies, demonstrates that CD81 is also overexpressed by tumor T cells, and that targeting this portion of the CD81 large extracellular loop leads to apoptosis of tumor cells, but not healthy PBMCs. CD81 is therefore viewed as a potential therapeutic target.

[0115] 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 of diagnosing a T-cell malignancy in a patient, comprising detecting the expression level of CD81 in a sample obtained from the patient.

2. 10. The method of claim 1, wherein the T-cell malignancy is a T-cell lymphoma or a T-cell leukemia.

3. 2. The method of claim 1, wherein the T-cell malignancy is Sezary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia.

4. The method of claim 1 for diagnosing cutaneous T-cell lymphoma.

5. The method of claim 1 for diagnosing Sézary syndrome.

6. 10. The method of claim 1, further comprising detecting the expression level of at least one additional marker selected from the group consisting of CD3, CD4, KIR3DL2, PLS3, Twist, and NKp46.

7. 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 CD81-expressing cancer cells.

8. 8. The method of claim 7, wherein the T-cell malignancy is a T-cell lymphoma or a T-cell leukemia.

9. 8. The method of claim 7 for the treatment of Sezary syndrome, mycosis fungoides, hepatosplenic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia.

10. 8. The method of claim 7, wherein the T-cell lymphoma is cutaneous T-cell lymphoma.

11. 11. The method of claim 10, wherein the T-cell lymphoma is Sezary syndrome.

12. The method of claim 7, wherein the inhibitor or the agent is an antibody having binding affinity for CD81.

13. The method of claim 12, wherein the antibody is directed against at least one extracellular domain of CD81.

14. The method of claim 12, wherein the antibody is directed against the C helix and / or D helix of the CD81 large extracellular loop.

15. The method of claim 12, wherein the antibody leads to depletion of CD81-expressing cancer cells.

16. 16. The method of claim 15, wherein the antibody suitable for depletion of CD81 cancer cells mediates antibody-dependent cell-mediated cytotoxicity.

17. The method of claim 12, wherein the antibody is a multispecific antibody comprising a first antigen-binding site directed against CD81 and at least one second antigen-binding site directed against an effector cell.

18. The method of claim 12, wherein the antibody is conjugated to a cytotoxic moiety.

19. 8. The method of claim 7, wherein the agent is a CAR-T cell, and the CAR comprises an extracellular antigen-binding domain specific for at least CD81.

20. 20. The method of claim 19, wherein the CAR comprises an extracellular antigen-binding domain specific for the C helix and / or D helix of CD81.

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