GARP as a biomarker and biotarget in T-cell malignancies

Abstract

This study of the regulatory T cell phenotype of Sézary cells led to the discovery of GARP (LRRC32) expression by Sézary cells. GARP was also shown to be overexpressed in samples from patients with acute lymphoblastic leukemia. Therefore, GARP appears as a diagnostic marker for monitoring T cell malignancies and as a therapeutic target. Accordingly, the present invention relates to methods for the diagnosis and treatment of T cell malignancies.

Description

[Technical Field]

[0001] Field of the invention: The present invention relates to the field of medicine, in particular to the field of 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. While T-cell acute lymphoblastic leukemia / lymphoma (T-ALL) originates from T-cell precursors termed thymocytes, other entities, such as T-cell large granular lymphocytic (LGL) leukemia, human T-cell leukemia virus type 1 (HTLV-1)-positive adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and most peripheral T-cell lymphomas (PTCLs), including angioimmunoblastic T-cell lymphoma (AITL), are derived from mature T cells (LH Sehn et al., Blood 2017). These subtypes are recognized by histological examination and immunophenotypic evaluation.

[0003] 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 lymphoma (mycosis fungoides and Sézary syndrome) is the most common. Their prognosis is poor in advanced stages of the disease. Sézary syndrome is defined as erythroderma (redness covering the entire skin) and circulating blood damage (1). Circulating T-lymphocyte tumor cells express CD4 and may lack expression of CD7 and CD26, but most cases exhibit abnormal expression of CD158k (KIR3DL2) (2). Early diagnosis of the disease is difficult, and follow-up of blood involvement is complicated because international criteria use the lack of CD7 and CD26 markers (CD4+CD26-negative cells and CD4+CD7-negative cells), which we know are not specific for tumor cells (3). The discovery of CD158k (KIR3DL2), a marker aberrantly expressed by Sézary cells, has enabled its use for diagnosis, disease monitoring (2), and the development of a therapeutic monoclonal antibody (lactamab), the results of which have been published in a phase I trial (5), and whose efficacy in cutaneous T-cell lymphoma and other peripheral T-cell lymphomas is currently being tested in an international, multicenter, prospective phase II trial. However, long-term responses are rare, and novel 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 not only expressed by Sézary cells but also by peripheral blood memory regulatory T cells, and its use is associated with the development of autoimmune adverse effects (7). In addition to CCR4, Sézary cells express several markers in common with regulatory T cells, such as PD1 (8), CD39 (9), and TIGIT (10) and CCR8 (11). Thus, there is a need to identify novel markers and targets for the treatment of T cell malignancies.

[0004] 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.

[0005] Detailed description of the invention: This study of the regulatory T cell phenotype of Sézary cells uncovered the expression of GARP (LRRC32) by Sézary cells. GARP has also been shown to be overexpressed in samples from patients with acute lymphoblastic leukemia. GARP is an anchoring receptor for latent, inactive transforming growth factor-β (TGFβ / LAP complex), which associates with integrin αV (CD51) and β1 / 3 / 6 or β8 integrins, allowing the release of active TGFβ (12, 13, 14, 15). GARP is expressed by activated regulatory T cells, platelets, and endothelial cells (16). TGFβ has been shown to cause profound immunosuppression in patients and contribute to survival and tumor cell migration. Therefore, GARP emerges as a diagnostic marker for monitoring T cell malignancies and as a therapeutic target. Therefore, the use of monoclonal antibodies (or antibody-drug conjugates, or cell therapy tools targeting GARP on chimeric antigen receptor cell lines) would allow the monoclonal antibodies to deplete tumor cells, but also activate anti-tumor immunity.

[0006] 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 use their TCR (T cell receptor for antigen) to recognize antigens presented or restricted by major histocompatibility complex molecules. There are several subsets of T cells, such as CD8+ T cells, CD4+ T cells, and γδ T cells, each with distinct functions. As used herein, the term "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I-restricted and function as cytotoxic T cells. "CD8+ T cells" are also referred to as cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is a recognition sequence associated with major histocompatibility complex class I-restricted interactions. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to a patient's pool of CD8+ T cells that leave the bloodstream and migrate into tumors. As used herein, the term "CD4+ T cells" (also called helper T cells or TH cells) refers to T cells that express the CD4 glycoprotein on their surface and assist other leukocytes in immune processes, including the maturation of B cells to plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. CD4+ T cells become activated upon presentation of peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist in active immune responses. These cells can differentiate into one of several subtypes, including T helper type 1 cells (TH1), T helper type 2 cells (TH2), T helper type 3 cells (TH3), T helper type 17 cells (TH17), T helper type 9 cells (TH9), T follicular helper cells (TFH), or regulatory T cells (Treg), which secrete different cytokines and promote different types of immune responses.Signaling from antigen-presenting cells (APCs) directs T cells toward specific subtypes. In addition to CD4, helper T cell surface biomarkers known in the art include CXCR3 (Th1), CCR4, Crth2 (Th2), CCR6 (Th17), CXCR5 (Tfh), and expression of subtype-specific cytokines and transcription factors, including T-bet, GATA3, EOMES, RORγT, BCL6, and FoxP3. As used herein, the term "γδ T cells" has its general meaning in the art. γδ T cells typically account for 1-5% of peripheral blood lymphocytes in healthy individuals (humans, monkeys). They are involved in the initiation of protective immune responses, and they have been shown to recognize their antigenic ligands through direct interaction with antigens, without any involvement of antigen-presenting cells (APCs) presenting them with MHC molecules. γ9δ2 T cells (sometimes called γ2δ2 T cells) are γδ T cells with a T cell receptor containing the variable domains Vγ9 and Vδ2. They constitute the majority of γδ T cells in human blood. Upon activation, γδ T cells exert potent, non-MHC-restricted cytotoxic activity that is particularly effective against various cell types, especially pathogenic cells. These may be cells infected with viruses (Poccia et al., J. Leukocyte Biology, 1997, 62: 1-5), or other intracellular parasites, such as mycobacteria (Constant et al., Infection and Immunity, December 1995, vol. 63, no. 12: 4628-4633), or protozoa (Behr et al., Infection and Immunity, 1996, vol. 64, no. 8: 2892-2896). They may also be cancer cells (Poccia et al., J. Immunol., 159: 6009-6015; Fournie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147: 338-347).Therefore, being able to modulate the activity of these cells in vitro, ex vivo, or in vivo would provide novel and effective therapeutic approaches in the treatment of a variety of pathologies, such as infectious diseases (especially viral or parasitic), cancer, allergies, and even autoimmune and / or inflammatory disorders.

[0007] As used herein, the term "T cell malignancy" has its general meaning in the art and refers to a disease that arises from neoplastic transformation of T cells, affects mature or immature T cells, and leads 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.

[0008] In some embodiments, the T-cell malignancy is human T-cell lymphotropic virus type 1 positive (HTLV1+). In some embodiments, the T-cell malignancy is human T-cell lymphotropic virus type 1 positive T-cell lymphoma. In some embodiments, the T-cell malignancy is human T-cell lymphotropic virus type 1 positive T-cell leukemia. As used herein, the term "HTLV1" or "human T-cell lymphotropic virus type 1" refers to an oncogenic retrovirus that possesses the classical gag, pol, and env genes encoding structural and enzymatic proteins, as well as a unique region encoding the regulatory proteins Tax and Rex. In particular, Tax plays a fundamental role in leukemogenesis by regulating the expression of many viral and intracellular genes through pathways related to CREB / ATF, SRF, and NF-κB. Most screening tests use immunoassays for diagnosis that rely on the detection of anti-HTLV-1 antibodies in serum. If the T-cell malignancy, T-cell lymphoma or T-cell leukemia is induced by human T-cell lymphotropic virus type 1, the T-cell malignancy, T-cell lymphoma or T-cell leukemia is HTLV1 positive.

[0009] 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 primarily arise from uncontrolled T-cell proliferation 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 within the category. T-cell lymphomas are often classified as either intermediate-grade (fast-growing) or low-grade (slow-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 hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), or Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is HTLV1 positive.

[0010] As used herein, "cutaneous T-cell lymphoma" or "CTCL" has its general meaning in the art and refers to a rare and heterogeneous group of non-Hodgkin's lymphomas derived from skin-homing mature T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) represent the most frequent subtypes of primary cutaneous T-cell lymphoma, with an incidence rate of 4.1 / 1,000,000 people / year, with a male predominance. In some embodiments, the cutaneous T-cell lymphoma is Sézary syndrome.

[0011] As used herein, the term "Sézary syndrome" or "SS" has its common meaning in the art and refers to an intermediate-grade form of cutaneous T-cell lymphoma characterized by the triad of erythroderma, lymphadenopathy, and circulating atypical lymphocytes (Sézary cells). Sézary syndrome occurs most frequently in men, is more common in older adults, and progresses rapidly. Sézary syndrome corresponds to stages IVA2 and IVA of T-cell cutaneous lymphoma (see this term). Patients present with scaly erythroderma and infiltrates, often manifesting as lion facies and severe pruritus. Alopecia, ectropion, mild palmoplantar keratosis, and nail dystrophy may be present. Lymphadenopathy and hepatosplenomegaly are observed. Patients often present with shivering, chills, and general fatigue.

[0012] As used herein, the term "T-cell leukemia" has its general meaning in the art and refers to a malignant hematological condition affecting T cells, including several types of lymphocytic leukemia. Leukemia usually arises from immature blood cells in the bone marrow and spreads through the bloodstream. There are different subtypes of leukemia: acute leukemia (AL) and chronic leukemia (CL). As an example, acute leukemia includes acute lymphoblastic leukemia (ALL). In some embodiments, the leukemia is T-cell acute lymphoblastic leukemia (T-ALL).

[0013] As used herein, the term "T-cell acute lymphoblastic leukemia" or "T-ALL" has its general meaning in the art and refers to an aggressive hematological malignancy characterized by the abnormal proliferation of immature thymocytes.

[0014] As used herein, "GARP" or "Glycoprotein-A repetitions Predominant" or "LRRC32" or "Leucine-Rich Repeat-Containing Protein 32" has its general meaning in the art and is an anchoring receptor for latent, inactive transforming growth factor-β (TGFβ / LAP complex), which associates with integrin αV (CD51) and β1 / 3 / 6 or β8 integrin, allowing the release of active transforming growth factor-β (12, 13, 14, 15) (Entrez Gene: 2615; Ensemble: ENSG00000137507). GARP is expressed by activated regulatory T cells, platelets, and endothelial cells (16). An exemplary amino acid sequence of GARP is shown by SEQ ID NO: 1.

[0015] [ka]

[0016] As used herein, the term "agent capable of inducing cell death of GARP-expressing cancer cells" refers to any molecule that can induce cell death of GARP-expressing cancer cells under intracellular and / or physiological conditions. In particular, the agent can induce apoptosis of GARP-expressing cancer cells. In some embodiments, the agent can deplete GARP cancer cells.

[0017] As used herein, the term "depletion" with respect to cancer cells refers to a measurable reduction in the number of GARP-expressing cancer cells in a patient. The reduction can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the term refers to a reduction in the number of GARP-expressing cancer cells in a patient to below detectable limits.

[0018] As used herein, the term "GARP inhibitor" refers to a molecule that partially or completely blocks, inhibits, or neutralizes the biological activity or expression of GARP. A GARP inhibitor can be any kind of molecule that interferes with GARP-related signal transduction in cells, for example, by reducing the transcription or translation of a nucleic acid encoding GARP, or by inhibiting or blocking GARP polypeptide activity, or both. In particular, the GARP inhibitor of the present invention is particularly suitable for blocking the active production of GARP-induced transforming growth factor-β by T cells, which is responsible for the immune escape of tumor cells. Examples of GARP inhibitors include, but are not limited to, antisense polynucleotides, interfering RNAs, catalytic RNAs, RNA-DNA chimeras, GARP-specific aptamers, anti-GARP antibodies, GARP-binding fragments of anti-GARP antibodies, GARP-binding small molecules, GARP-binding peptides, and other polypeptides that specifically bind to GARP (including, but not limited to, GARP-binding fragments of one or more GARP ligands, optionally fused to one or more additional domains), such that interaction between the GARP inhibitor and GARP results in a reduction or cessation of GARP activity or expression.

[0019] As used herein, the term "transforming growth factor-β (TGF-β)" has its common meaning in the art and refers to transforming growth factor-β. In particular, the term encompasses any isoform of TGF-β, provided that the isoform has immunosuppressive activity. Transforming growth factor-β (TGF-β) actually functions as an immune suppressor 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).

[0020] Thus, the term "antibody" as used herein is used to refer to any antibody-like molecule having an antigen-binding region, and this term also includes antibody fragments comprising the antigen-binding domain, such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimers, Fv, scFv (single-chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibodies, tribodies (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabodies; kappa (lambda) bodies (scFv-CL fusions); antibody fragments, including: BiTEs (bispecific T cell engagers, scFv-scFv tandems that attract T cells); DVD-Ig (dual variable domain antibodies, bispecific format); SIPs (small immune proteins, a type of minibody); SMIPs ("small modular immunopharmaceuticals" scFv-Fc dimers; DARTs (two-chain stabilized diabodies "dual affinity retargeting"); miniature antibody mimetics containing one or more CDRs, etc. Techniques for preparing and using various antibody-based constructs and fragments are well known in the art (Kabat et al., 2004). (See, e.g., E. et al., 1991, specifically incorporated herein by reference). Diabodies are further described, inter alia, in European Patent No. 404,097 and WO 93 / 11161; while linear antibodies are further described in Zapata et al. (1995). Antibodies may be fragmented using conventional techniques. For example, F(ab')2 fragments may be generated by treating antibodies with pepsin. The resulting F(ab')2 fragment may be treated to reduce disulfide bridges and generate Fab' fragments. Fab fragments may be formed by digestion with papain. Fab, Fab', and F(ab')2, scFv, Fv, dsFv, Fd, dAb, TandAb, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments, and other fragments may also be synthesized by recombinant techniques or chemically synthesized. Techniques for generating 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 generation 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 the type of antibody that can be found in camelids, which naturally lack light chains. Such single-domain antibodies are also "Nanobodies®." For a general description of (single) domain antibodies, see the prior art cited above, as well as EP 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06 / 030220 and WO 06 / 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 (λ) and kappa (κ). There are five main heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules. 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: one variable domain (VH) and three constant domains (CH1, 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, placental transfer, complement binding, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region.The specificity of an antibody resides in the structural complementarity between the antibody-binding site and an antigenic determinant. Antibody-binding sites are primarily composed of residues derived from hypervariable or complementarity-determining regions (CDRs). Occasionally, residues derived from non-hypervariable or framework regions (FRs) may participate in the antibody-binding site or influence the overall domain structure and thus the binding site. Complementarity-determining regions (CDRs) refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs, designated L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including the CDR sets derived from the heavy and light chain V regions, respectively. Framework regions (FRs) refer to the amino acid sequences interposed between the CDRs. Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is set forth in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, National Institutes of Health, 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 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, corresponding to shortening of, or insertion into, structural elements of the basic variable domain structure (whether framework regions or complementarity-determining regions (CDRs)). The correct Kabat numbering of residues for a given antibody can be determined by alignment of homologous residues in the antibody sequence with the "standard" Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (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.

[0021] As used herein, the term "bind" indicates that an antibody has affinity for a surface molecule. As used herein, the term "affinity" refers to the binding strength of an antibody to an epitope. The affinity of an antibody is indicated by the dissociation constant Kd, defined as [antibody (Ab)] × [antigen (Ag)] / [antibody-antigen (Ab-Ag)], where [antibody-antigen] is the molar concentration of the antibody-antigen complex, [antibody] is the molar concentration of unbound antibody, and [antigen] is the molar concentration of unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of monoclonal antibodies (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 monoclonal antibodies is the use of a Biacore instrument.

[0022] 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.

[0023] 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 altered class, effector function, and / or species, or to an entirely different molecule, such as an enzyme, toxin, hormone, growth factor, or drug, that confers new properties to the chimeric antibody; or (b) the variable region or portions thereof have been modified, substituted, or exchanged with a variable region having a different or altered antigen specificity. Chimeric antibodies also include primatized, particularly humanized, antibodies. Furthermore, chimeric antibodies may contain residues not found in the recipient antibody or 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)).

[0024] As used herein, the term "humanized antibody" refers to an antibody having variable region framework and constant regions derived from a human antibody but retaining the CDRs of a previous non-human antibody. In some embodiments, a humanized antibody contains minimal sequence derived from a non-human immunoglobulin. Humanized antibodies and antibody fragments thereof may be, for the most part, 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 cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments may 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) while avoiding 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 substantial portion of the FR regions are those of a human immunoglobulin sequence. A humanized antibody or antibody fragment may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0025] 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.

[0026] 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 containing an antibody antigen-binding domain (e.g., scFv) linked to a T cell signaling domain. Characteristics of CARs include their ability to redirect T cell specificity and reactivity toward selected targets in an MHC-unrestricted manner, leveraging the antigen-binding properties of monoclonal antibodies. Furthermore, when expressed in T cells, CARs advantageously do not dimerize with the α and β chains of the endogenous T cell receptor (TCR). Chimeric antigen receptors of the present invention typically comprise an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0027] 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+ T cells, CD8+ T cells, γδ T cells, as well as effector T cells, memory T cells, regulatory T cells, etc. Genetically modified T lymphocytes may be "derived" or "obtained" from a patient who will receive treatment with the genetically modified T cells, or they may be "derived" or "obtained" from a different patient.

[0028] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment, as well as curative or disease-modifying treatment (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatment can be administered to patients with a medical disorder or who are likely to eventually develop a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring 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 dosing pattern used during therapy. 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 in 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 higher 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 a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., symptoms of disease, etc.)).

[0029] As used herein, the term "therapeutically effective amount" refers to an amount effective, at a dosage and for a period of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an active agent may vary depending on 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 any toxic or adverse effects of the drug are outweighed by the therapeutically beneficial effects. The effective dose and dosage regimen for an active agent depends on the disease or condition to be 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 pharmaceutical composition required. For example, a physician can start the dose of the active agent used in the pharmaceutical composition at a level lower than required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Generally, an appropriate dose of the composition of the present invention will be the amount of the compound that is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend 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 cancer-inhibiting ability of a compound can be evaluated in an animal model system that is predictive of efficacy in, for example, human tumors. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise ameliorate symptoms in a patient. One of ordinary skill in the art would be able to determine such amounts based on such factors as the patient's size, the severity of the patient's symptoms, and the specific 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 0.1-100 mg / kg, e.g., about 0.1-50 mg / kg, e.g., about 0.1-20 mg / kg, e.g., about 0.1-10 mg / kg, e.g., 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, for example, about 0.02 to 30 mg / kg, for example, 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, and can be administered, for example, near the target site.Dosage regimens in the above-described methods of treatment and use are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may 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, e.g., at predetermined time points. In some embodiments, effectiveness may be monitored by visualization of diseased areas or by other diagnostic methods further described herein, e.g., by performing one or more PET-CT scans using, for example, a labeled antibody of the invention, a fragment derived from an antibody of the invention, or a miniantibody. If desired, the effective daily amount of the pharmaceutical composition may be administered as two, three, four, five, six, or more divided doses administered separately at appropriate intervals throughout the day, optionally in unit dosage form. In some embodiments, the human monoclonal antibodies of the invention are administered by slow continuous infusion over an extended period of time, such as more than 24 hours, to minimize undesirable side effects. Effective doses of the drugs of the invention can also be administered using weekly, biweekly, or triweekly dosing periods, which 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, e.g., 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg / day, at 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, 40, 45, 50, 60, 70, 80, 90, or 100 mg / kg / day, at least 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, 40, 45, 50, 60, 70, 80, 90, or , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or alternatively for at least one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 weeks, or any combination thereof, using a single or divided dose every 24, 12, 8, 6, 4, or 2 hours.

[0030] Diagnostic Methods: A first object of the present invention relates to a method for diagnosing a T-cell malignancy in a patient, comprising detecting GARP expression levels in a sample obtained from the patient. In some embodiments, the T-cell malignancy is Sézary syndrome, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia. In some embodiments, the T-cell malignancy is Sézary syndrome. In some embodiments, the T-cell malignancy is human T-cell lymphotropic virus type 1 positive.

[0031] In some embodiments, the present invention relates to a method for diagnosing T-cell lymphoma in a patient, comprising detecting GARP expression levels in a sample obtained from the patient. Accordingly, 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 hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), or Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is HTLV-1 positive. In some embodiments, the methods of the present invention are particularly suitable for diagnosing cutaneous T-cell lymphoma, and more particularly, for diagnosing Sézary syndrome.

[0032] In some embodiments, the present invention relates to a method for diagnosing T-cell leukemia in a patient, comprising detecting the expression level of GARP in a sample obtained from the patient. Thus, in some embodiments, the T-cell malignancy is T-cell leukemia. In some embodiments, the T-cell leukemia is T-cell acute lymphoblastic leukemia. In some embodiments, the T-cell leukemia is HTLV1 positive.

[0033] 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 peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample is a sample of (i) purified blood leukocytes, (ii) peripheral blood mononuclear cells or PBMCs, (iii) purified leukocytes, (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 tissue sections, such as biopsy or autopsy samples, and frozen sections taken for histological examination. Thus, in some embodiments, the tissue sample may be obtained from a biopsy performed on the subject's skin.

[0034] In some embodiments, the level of a marker is determined by immunohistochemistry (IHC). Immunohistochemistry typically includes 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 to the marker; v) rinsing the sections; vi) incubating the sections with a biotinylated secondary antibody; and vii) revealing the antigen-antibody complex using 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 an appropriate technique, depending on the type of label produced by the labeled antibody, such as a radioactive label, a fluorescent label, or an enzyme label. Multiple labels may be used simultaneously. Alternatively, the method of the present invention may use a secondary antibody and an enzyme molecule linked to an amplification system (to enhance the staining signal). Such conjugated secondary antibodies are commercially available, for example, from Dako's EnVision system. Counterstains, such as hematoxylin and eosin (H&E), DAPI, or Hoechst, may be used. Other staining methods may 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, allowing for 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 wide 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, β-lactamase), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S, or 125The affinity ligand may be labeled with amines and particles (e.g., gold). Different types of labels can be conjugated to the affinity ligand using various chemical reactions, such as amine or thiol reactions. However, other reactive groups besides amines and thiols, such as aldehydes, carboxylic acids, and glutamine, may also be used. Various enzymatic staining methods for labeling proteins of interest are known in the art. For example, enzymatic interactions can be visualized using various enzymes, such as peroxidase, alkaline phosphatase, or various chromophores, such as DAB (3,3'-diaminobenzidine), AEC (aminoethylcarbazole), or Fast Red. In another example, an antibody can be conjugated to a peptide or protein, which can be detected via a labeled binding partner or antibody. In indirect immunohistochemical (IHC) assays, a secondary antibody or secondary binding partner is required to detect binding of the first binding partner because the first binding partner is unlabeled. The resulting stained specimens are then imaged using a system for detecting detectable signals and acquiring images, such as digital staining images. Methods for image acquisition are well known to those skilled in the art. For example, once a sample has been stained, 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, can be used to detect the stain or biomarker label. In some instances, images can be acquired digitally. The resulting images can then 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 may include automated staining and microscopic scanning, computerized image analysis, comparison of serial sections (to control for variations in sample orientation and size), generation of digital reports, and recording and tracking of samples (e.g., slides on which tissue sections are placed).Cell imaging systems that combine conventional optical microscopes with digital image processing systems to perform quantitative analysis of cells and tissues, including immunostained samples, are commercially available. See, for example, the CAS-200 system (Becton Dickinson). 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 constructed, calibrated, standardized, and / or verified based on factors including staining quality or intensity, using procedures known to those skilled in the art (see, for example, published U.S. Patent Publication No. US20100136549). Images can be analyzed and scored quantitatively or semi-quantitatively based on the staining intensity of the sample. Quantitative or semi-quantitative histochemistry refers to methods in which a histochemically tested sample is scanned and scored to identify and quantify the presence of specific biomarkers (i.e., markers). Quantitative or semi-quantitative methods can use imaging software to detect staining density or amount, or by visual detection of staining, in which a trained operator ranks the results numerically. For example, images can be quantitatively analyzed using pixel counting algorithms (e.g., Aperiospectrum software), automated quantitative analysis platforms (AQUA® platform), and other standard methods to measure, quantify, or semi-quantify 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 strong positive staining (e.g., brown staining) to the sum of all stained areas can be calculated and scored. The amount of biomarker (ie, marker) detected is quantified and expressed as a percentage of positive pixels and / or 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., as 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, the sample is assigned a score, which is a numerical indication of the intensity or amount of histochemical staining of the sample, indicating the amount of target biomarker (e.g., marker) present in the sample. The numerical value of the optical density or area ratio may be given a converted score, e.g., on an integer scale. Thus, in some embodiments, the method of the present invention comprises the steps of: i) providing one or more immunostained thin sections of tissue sections obtained by an automated slide staining system using a binding partner capable of selectively interacting with the marker (e.g., an antibody as described above); ii) proceeding with the digitization of the slides of step a. by high-resolution scan capture; iii) detecting the thin sections of the tissue sections on the digital photograph; iv) preparing a size reference grid with uniformly distributed units having the same surface area, the grid adapted to the size of the tissue section to be analyzed; and v) detecting, quantifying, and measuring the intensity of stained cells within each unit, thereby assessing the number or density of stained cells in each unit.

[0035] In some embodiments, the level of the marker is determined by flow cytometry. As used herein, the term "flow cytometry" refers to a technique for counting cells of interest by suspending them in a liquid stream and passing them through an electronic detection device. Flow cytometry allows for simultaneous multiparameter analysis of physical and / or chemical parameters, such as fluorescence parameters, at up to thousands of events per second. Modern flow cytometry instruments typically have multiple lasers and fluorescence detectors. A common type of flow cytometry technique uses "fluorescence-activated cell sorting" to physically separate 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, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell, providing rapid, objective, and quantitative recording of fluorescent signals from individual cells and physical separation of cells of particular interest. Thus, FACS can be used in conjunction with the methods described herein to isolate and detect the cell populations of the present invention. For example, therefore, fluorescence-activated cell sorting (FACS) can be used, which involves the use of 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 can include a cytometry sample fluidic subsystem, as described below. Additionally, the cytometry system includes a cytometer fluidically coupled to the cytometry sample fluidic subsystem. Systems of the present disclosure can include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers; software (e.g., Flowjo, Laluza, etc.); data input devices, e.g., interface ports, mice, keyboards, etc.; fluid handling components; power supplies, etc. More particularly, the sample is contacted with a panel of antibodies specific for particular markers of the cell population of interest.Such antibodies or antigen-binding fragments are commercially available from vendors such as R&D Systems, BD Biosciences, eBiosciences, BioLegend, ProImmune, and Miltenyi, or can be generated against these cell surface markers by methods known to those of skill in the art. In some embodiments, substances that specifically bind to cell surface markers, such as antibodies or antigen-binding fragments, are labeled with a tag that facilitates isolation and detection of the cell population of interest. As used herein, the terms "label" or "tag" refer to a composition capable of producing a detectable signal that is indicative of the presence of a target, e.g., 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. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means required for methods for isolating and detecting cancer cells.Non-limiting examples of fluorescent labels or tags for labeling substances 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 (nitrobenzoxadiazole), NBD-X, R-phycoerythrin (PE), PE-Cy5 conjugates (cyclochrome, R670, Tricolor, Quantum Red), PE-Cy7 conjugates, Red 613, PE-Texas Red, PerCP (peridinin chlorophyll), PerCPeFluor710, 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, Examples of suitable antibodies include 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, BV785, BV711, BV421, BV605, BV510, and 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 or magnetically responsive beads. The beads may be made of a variety of materials, including, but not limited to, glass, plastic, polystyrene, and acrylic. Furthermore, the beads are preferably fluorescently labeled.In a preferred embodiment, the fluorescent beads are those contained in TruCount™ tubes available from Becton Dickinson Biosciences (San Jose, Calif.).

[0036] In some embodiments, the method further comprises detecting the expression level of at least one additional marker, typically selected from the group consisting of CD3, CD4, KIR3DL2, PLS3, Twist, and NKp46.

[0037] As used herein, the names of the various markers of interest refer to the internationally recognized names of the corresponding genes, as found in internationally recognized databases of gene and protein sequences, including, in particular, the database of the Human Genetic Organization (HUGO) Gene Nomenclature Committee, available at the following internet address: http: / / www.gene.ucl.ac.uk / nomenclature / index.html. As used herein, the names of the various markers of interest may also refer to the internationally recognized names of the corresponding genes, as found in the internationally recognized database of gene and protein sequences, Genbank. Through these internationally recognized sequence databases, nucleic acid and amino acid sequences corresponding to each marker of interest described herein can be found by those skilled in the art.

[0038] Multiplexed tissue analysis techniques are particularly useful for quantifying several markers in a tissue sample. Such techniques should enable the measurement of at least five, or at least ten or more, biomarkers from a single tissue sample. Furthermore, it would be beneficial for the technique to preserve the location of biomarkers and to be able to distinguish between their presence in cancerous and non-cancerous cells. Such methods include layered immunohistochemistry (L-IHC), layered expression scanning (LES), or multiplexed tissue immunoblot (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 that the generation of up to eight, up to nine, up to ten, up to eleven, or more tissue section images on layered and blotted membranes, papers, filters, and the like, may be used. Coated membranes useful for carrying out the L-IHC / MTI process are available from 20 / 20 Gene Systems, Inc. (Rockville, MD).

[0039] In some embodiments, the L-IHC method can be performed on any of a variety of tissue samples, whether fresh or preserved. Samples include core needle biopsies, which were routinely fixed in 10% normal buffered formalin and processed by the pathology department. Standard 5-μm-thick tissue sections were cut from the tissue blocks onto charged slides and used for L-IHC. L-IHC thus allows for the examination of multiple markers within a tissue section by obtaining molecular copies transferred from the tissue section onto multiple biocompatible coated membranes, essentially creating a copy of the tissue "image." In the case of paraffin sections, the tissue sections are deparaffinized as known in the art, for example, by exposing the sections to xylene or a xylene substitute, such as NEO-CLEAR®, and graded ethanol solutions. Sections can be treated with proteinases, such as papain, trypsin, proteinase K, and the like. A stack of membrane substrates, e.g., comprising multiple sheets of 10 μm-thick coated polymer scaffolds with 0.4 μm-diameter pores for tissue molecules such as proteins to pass through the stack, is then placed on the tissue section. The stack is positioned so that fluid and tissue molecule movement is substantially perpendicular to the membrane surface. The sandwich of the section, membrane, spacer paper, absorbent paper, weight, etc., can be exposed to heat to promote the transfer of molecules from the tissue into the membrane stack. A portion of the tissue's proteins is captured on the biocompatible coated membrane of each stack (available from 20 / 20 Gene Systems, Inc., Rockville, MD). Thus, each membrane contains a copy of the tissue and can be probed for different biomarkers using standard immunoblotting techniques, allowing for unlimited amplification of the marker profile, as performed on a single tissue section.Because membranes further away from the tissue in the stack may have lower amounts of protein, which may be due to, for example, differences in the amount of the molecule in the tissue sample, differences in the mobility of molecules released from the tissue sample, differences in the binding affinity of the molecule to the membrane, or the length of transcription, the procedure can include normalization of values, running controls, and assessment of transcription levels of tissue molecules, etc., to correct for variations that occur within, between, or between membranes, allowing direct comparison of information within, between, or between membranes. Thus, total protein per membrane can be determined by exposing the membrane to labeled avidin or streptavidin; protein stains known in the art, such as Blot fastStain, Ponceau Red, or brilliant blue stain, using any means for quantifying protein, e.g., molecules available for biotinylation, e.g., protein groups, using standard reagents and methods, followed by revealing the bound biotin.

[0040] In some embodiments, the methods of the present invention utilize multiplex tissue imprinting (MTI) technology to measure biomarkers, wherein the methods preserve accurate biopsy tissue by allowing for multiple biomarkers, in some cases at least six biomarkers.

[0041] In some embodiments, there are alternative multiplexed tissue analysis systems 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, Inc., Rockville, MD). The technology is described in U.S. Patent No. 7,473,532.

[0042] In some embodiments, the methods of the invention utilize multiplex IHC technology developed by GE Global Research (Niskayuna, NY) and described in U.S. Publication Nos. 2008 / 0118916 and 2008 / 0118934, in which sequential analysis is performed on a biological sample containing multiple targets, including binding a fluorescent probe to the sample, followed by signal detection, then inactivating the probe, followed by binding, detecting, and inactivating the probe to another target, and continuing this process until all targets are detected.

[0043] In some embodiments, when using fluorescence (e.g., fluorophores or quantum dots), multiplexed tissue imaging can be performed, in which 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, the system can acquire a series of images at various wavelengths, which can be electronically and sequentially selected, and then used with an analysis program designed to handle such data. In this way, the system can simultaneously obtain quantitative information from multiple dyes, even when their spectra are highly overlapping, co-localized, or present at the same point in the sample (but with different spectral curves). 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 simply increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can separate or separate autofluorescence from tissue, thereby increasing the achievable signal-to-noise ratio. Briefly, quantification can be performed by the following steps: i) providing a tumor tissue microarray (TMA) obtained from a subject; ii) subsequently staining the TMA sample with an anti-antibody specific for the protein(s) of interest; iii) further staining the TMA slide with an epithelial cell marker to aid in the automatic differentiation of tumor and stroma; iv) subsequently scanning the TMA slide using a multispectral imaging system; v) processing the scanned image using automated image analysis software (e.g., PerkinElmer Technologies) that allows for the detection, quantification, and differentiation of specific tissues through powerful pattern recognition algorithms. Machine learning algorithms are typically pre-trained to differentiate tumor from stroma and identify labeled cells.

[0044] 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 amplification methods include ligase chain reaction (LCR), transcription-modified amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA).

[0045] In some embodiments, the methods of the invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has a T-cell malignancy.

[0046] In some embodiments, the T-cell malignancy is a T-cell lymphoma. Accordingly, in some embodiments, the methods of the present invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has a T-cell lymphoma.

[0047] In some embodiments, the T-cell malignancy is T-cell leukemia. Accordingly, in some embodiments, the methods of the invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein detecting a difference between the expression level of the marker and the predetermined reference value indicates whether the subject has T-cell leukemia.

[0048] In some embodiments, the predetermined reference value is a comparison with a number or numerical 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 disease. Such predetermined reference values can be derived from statistical analysis and / or population risk prediction data obtained from mathematical algorithms and computer-calculated indices. In some embodiments, retrospective measurements of marker levels in appropriately deposited historical subject samples can 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 function of the test and the benefit / risk balance (false-positive and false-negative clinical outcomes). Typically, optimal sensitivity and specificity (and thus the threshold) can be determined using a receiver operating characteristic (ROC) curve based on experimental data. For example, after determining the marker levels of a reference group, algorithmic analysis can be used for statistical processing of the measured marker levels in the samples to be tested, thereby obtaining a meaningful classification standard for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operating characteristic curve. It is primarily used for clinical biochemical 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). It uses image synthesis to reveal the relationship between sensitivity and specificity. A series of different cutoff values (threshold or critical value, which is the numerical boundary between normal and abnormal results of a diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. The curve is then drawn using sensitivity as the vertical axis coordinate and specificity as the horizontal axis 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 plot is the critical point, which has both high sensitivity and high specificity values.The AUC value of the ROC curve is 1.0 to 0.5. When AUC>0.5, the diagnostic result is better as AUC approaches 1. When AUC is 0.5 to 0.7, the accuracy is low. When AUC is 0.7 to 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is very high. This algorithm method is preferably implemented using a computer. Existing software or systems in the art, 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 VI 0.0 (Dynamic Microsystems, Silver Spring, Maryland, USA), can be used to draw the ROC curve.

[0049] Typically, as demonstrated in the Examples, the expression level of GARP is higher than the expression level determined in samples from healthy individuals. Thus, in some embodiments, the methods of the invention further comprise comparing the expression level of the marker with a predetermined reference value, wherein detection of an expression level of the marker higher than the predetermined reference value indicates that the subject has a T-cell malignancy.

[0050] In some embodiments, the methods of the present invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein an expression level of the marker higher than the predetermined reference value indicates that the subject has T-cell lymphoma. In some embodiments, the T-cell lymphoma is Sézary syndrome.

[0051] In some embodiments, the methods of the present invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein an expression level of the marker higher than the predetermined reference value indicates that the subject has T-cell leukemia.

[0052] In some embodiments, the methods of the present invention further comprise comparing the expression level of the marker to a predetermined reference value, wherein an expression level of the marker higher than the predetermined reference value indicates that the subject has T-cell lymphoma or T-cell leukemia.

[0053] In some embodiments, the GARP expression level is determined using fluorescence intensity. In some embodiments, the GARP expression level is determined using GARP mean fluorescence intensity. In some embodiments, the method comprises the further step of determining the GARP mean fluorescence intensity and concluding that the patient has a T-cell malignancy if the GARP mean fluorescence intensity is greater than 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, or 600. In some embodiments, the method further comprises the further step of determining a GARP mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the GARP mean fluorescence intensity is greater than 400. In some embodiments, the GARP expression level is determined using a GARP delta mean fluorescence intensity. In some embodiments, the GARP delta mean fluorescence intensity is calculated relative to an IgG2a control isotype expression level. In some embodiments, the GARP delta mean fluorescence intensity is calculated relative to an IgG2a control isotype mean fluorescence intensity. In some embodiments, the method comprises the further step of determining a GARP delta mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the GARP mean fluorescence intensity is greater than 400. In some embodiments, the GARP expression level is determined using a GARP delta mean fluorescence intensity. In some embodiments, the GARP delta mean fluorescence intensity is calculated relative to an IgG2a control isotype mean fluorescence intensity. In some embodiments, the method comprises the step of determining a GARP delta mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the GARP delta mean fluorescence intensity is greater than 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, 54 , 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 or 600, concluding that the patient is afflicted with a T-cell malignancy.In some embodiments, the method comprises the further step of determining the GARP delta mean fluorescence intensity and concluding that the patient is afflicted with a T-cell malignancy if the GARP delta mean fluorescence intensity is higher than a predetermined reference value. In some embodiments, the T-cell malignancy is Sézary syndrome.

[0054] Monitoring the effect of a drug (e.g., a drug compound) on the expression level of GARP can be used to monitor the status of a patient's T-cell malignancy over time.For example, the effectiveness of a drug that causes marker expression can be monitored during treatment of a subject undergoing anti-T-cell malignancy treatment.

[0055] Therefore, the present invention also provides the following: (vi) obtaining a pre-dose sample from the patient prior to administration of the agent; (ii) detecting the level of GARP expression in the pre-administration sample; (iii) obtaining one or more pre-dose samples from the patient; (iv) detecting the expression level of the same marker(s) in the post-administration sample; (v) comparing the GARP expression level in the pre-administration sample with the GARP expression level in the post-administration sample or samples; and (vi) varying the administration of the drug to the patient accordingly. Also provided is a method for monitoring the effectiveness of treatment of a patient suffering from a T-cell malignancy, comprising:

[0056] 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.

[0057] For example, a diagnosis of worsening, as determined by assessing GARP expression levels over the course of treatment, may indicate ineffective dosing and the desirability of increasing the dose. Conversely, a diagnosis of success, as determined by assessing GARP expression levels, may indicate effective treatment and no need for dose modification.

[0058] Therefore, the present invention also relates to a method for adapting a treatment in a patient suffering from a T-cell malignancy, wherein said method comprises: a) performing an in vitro diagnostic method disclosed herein on at least one sample collected from the patient; and b) administering to said patient thereby adapting the treatment of said patient Includes:

[0059] 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.

[0060] The present invention also relates to a kit for carrying out the diagnostic method described above. The kit includes multiple reagents, particularly at least one substance capable of specifically binding to a GARP marker. Reagents suitable for binding to a marker protein include antibodies, antibody derivatives, antibody fragments, etc. Reagents suitable for binding to a marker nucleic acid (e.g., genomic DNA, mRNA, spliced mRNA, cDNA, etc.) include complementary nucleic acids. For example, nucleic acid reagents include oligonucleotides (labeled or unlabeled) immobilized on a substrate, labeled oligonucleotides not bound to a substrate, PCR primer pairs, molecular beacon probes, etc. The kit may optionally contain additional components useful for carrying out the method of the present invention. For example, the kit may include a liquid (e.g., sodium citrate saline buffer) suitable for annealing to a complementary nucleic acid or suitable for binding of an antibody to a specifically binding protein, one or more sample compartments, instructional materials explaining how to carry out the in vitro diagnostic method of the present invention, etc.

[0061] Treatment: A further object of the present invention is a method for treating a T-cell malignancy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of a GARP-expressing cancer cell. In some embodiments, the T-cell malignancy is Sézary syndrome, hepatosplenic T-cell lymphoma, autoimmunoblastic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia. In some embodiments, the T-cell malignancy is human T-cell lymphotropic virus type 1 positive.

[0062] In some embodiments, the present invention relates to a method of treating T-cell lymphoma in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of GARP-expressing cancer cells. In some embodiments, the T-cell lymphoma is cutaneous T-cell lymphoma. More particularly, the T-cell lymphoma is Sézary syndrome. 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 hepatosplenic T-cell lymphoma (HSTCL), angioimmunoblastic T-cell lymphoma (AITL), NK / T-cell lymphoma (NKTL), or Sézary syndrome (SS). In some embodiments, the T-cell lymphoma is HTLV-1 positive.

[0063] In some embodiments, the present invention relates to a method of treating T-cell leukemia in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of GARP-expressing cancer cells. In some embodiments, the T-cell leukemia is T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the T-cell leukemia is HTLV1-positive.

[0064] 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.

[0065] GARP inhibitors: In another aspect, the invention relates to a method of treating a T-cell malignancy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a GARP inhibitor.

[0066] In some embodiments, the present invention relates to a method of treating T-cell lymphoma in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a GARP inhibitor.

[0067] In some embodiments, the present invention relates to a method of treating T-cell leukemia in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a GARP inhibitor.

[0068] In some embodiments, the inhibitor is an antibody that has binding affinity for GARP.

[0069] In some embodiments, the GARP inhibitor is a GARP expression inhibitor. Particularly, the gene expression inhibitor is siRNA, antisense oligonucleotide, or ribozyme. For example, antisense oligonucleotide (including antisense RNA molecule and antisense DNA molecule) will directly block the translation of GARP mRNA by binding to it, thereby preventing protein translation or increasing mRNA degradation, thereby reducing the level of GARP in cells and thus reducing its activity. For example, antisense oligonucleotides that are at least about 15 bases long and complementary to the unique region of the mRNA transcript sequence encoding GARP can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically suppress 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 expression inhibitors for use in the present invention. GARP 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, thereby specifically suppressing GARP gene expression (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, small hairpin RNAs (shRNAs), and ribozymes of the present invention can be delivered in vivo alone or in association with a vector.

[0070] GARP antibody: In some embodiments, the agent is an antibody having binding affinity for GARP. In some embodiments, the agent is an antibody directed against at least one extracellular domain of GARP. In some embodiments, the antibody is an anti-GARP neutralizing antibody. In some embodiments, the antibody results in depletion of GARP-expressing cancer cells. In some embodiments, the antibody results in the suppression of transforming growth factor beta production by T cells, which is responsible for immune escape of tumor cells. In some embodiments, the antibody results in depletion of GARP-expressing cancer cells. In some embodiments, the antibody is directed against at least one extracellular domain of GARP. In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0071] 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, 6,150,584, and the references cited therein, the contents of which are incorporated herein by reference.

[0072] Anti-GARP antibodies are well known in the art. For example, antibodies targeting GARP are described in WO2018 / 206790, WO2017 / 051888, WO2017 / 173091, WO2016 / 125017 or WO2015 / 015003.

[0073] In some embodiments, the antibody is the DS-1055a antibody as disclosed in Satoh K, Kobayashi Y, Fujimaki K, et al. The novel anti-GARP antibody DS-1055a enhances antitumor immunity by depleting highly suppressive GARP-positive regulatory T cells. Int Immunol. 2021;33(8):435-446.

[0074] In some embodiments, the antibody is an ARGX-115 (or ABBV-115 or livmoniplimab) antibody, as described in Cuende, Julia et al. "Monoclonal antibodies against GARP / TGF-β1 complexes inhibit the immunosuppressive activity of human regulatory T cells in vivo." Science translational medicine vol. 7,284 (2015): 284ra56.

[0075] In particular, the heavy chain of ribmoniplimab is set forth as SEQ ID NO:2 and the light chain of ribmoniplimab is set forth as SEQ ID NO:3. [ka]

[0076] Antibodies that deplete GARP In some embodiments, antibodies suitable for depleting GARP cancer cells mediate antibody-dependent cell-mediated cytotoxicity.

[0077] In some embodiments, the antibody comprises the VH and VL domains of DS-1055a.

[0078] In some embodiments, the antibody comprises the VH and VL domains of ribmoniplimab.

[0079] 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 antibodies on target cells and subsequently cause lysis of the target cells. Without wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs).

[0080] As used herein, the term "Fc region" includes the polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, plus the flexible hinge at the N-terminus of these domains. In IgA and IgM, Fc may include the J chain. In 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 might vary, the human IgG heavy chain Fc region is usually defined to include from residue C226 or P230 to the carboxy terminus, where numbering is according to the EU index as in Kabat et al. (1991, National Institutes of Health Publication 91-3242, National Scientific and Technical Information Service, Springfield, VA). The "EU index as set forth in Kabat" refers to the residue numbering of the human IgG1 EU antibody as 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 containing variant Fc regions that are variants of non-naturally occurring Fc regions. The amino acid sequence of a non-naturally occurring Fc region (also referred to herein as a "variant Fc region") contains at least one amino acid substitution, insertion, and / or deletion compared to the wild-type amino acid sequence. Any new amino acid residues that appear in the variant Fc region sequence as a result of insertion or substitution may be referred to as non-naturally occurring amino acid residues. Note: Polymorphisms have been observed in the numbering of Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, and thus slight differences may exist between the presented sequence and prior art sequences.

[0081] As used herein, the term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. Natural killer cells, the primary cells for mediating ADCC, express FcγIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. Fc receptor expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To assess the ADCC activity of a molecule, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, can 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).

[0082] As used herein, the term "effector cell" refers to a leukocyte that expresses one or more Fc receptors and exerts effector function. Such cells express at least FcγRI, FcγRII, FcγRIII, and / or FcγRIV and perform 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.

[0083] 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.

[0084] In some embodiments, antibodies suitable for depletion of cancer cells comprise a variant Fc region having 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 increases 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, where amino acid residues are numbered according to the EU index. In some embodiments, the antibodies of the present invention comprise 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 1332E, where amino acid residues are numbered according to the EU index.

[0085] In some embodiments, the glycosylation of antibodies suitable for cancer cell depletion is modified. For example, aglycosylated antibodies can be generated (i.e., the antibodies lack glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more glycosylation sites in one or more variable framework regions can be deleted, thereby making one or more amino acid substitutions that eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for an antigen. Such approaches are described in further detail by Co et al. in U.S. Pat. Nos. 5,714,350 and 6,350,861. Additionally or alternatively, antibodies can be generated with altered types of glycosylation, 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 carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the invention and thereby produce antibodies with altered glycosylation. For example, European Patent No. 1176195 by Hang et al. describes cell lines with a functionally disrupted FUT8 gene encoding a fucosyltransferase, such that antibodies expressed in such cell lines exhibit hypofucosylation or lack fucosyl residues. Thus, in some embodiments, the human monoclonal antibodies of the invention can be produced by recombinant expression in cell lines exhibiting hypofucosylated or non-fucosylated patterns, e.g., mammalian cell lines deficient in expression of the FUT8 gene encoding a fucosyltransferase.PCT Publication No. WO 03 / 035835 by Presta describes a mutant CHO cell line, Lecl3 cells, that has a reduced ability to attach fucose to glycans linked to Asn(297), also 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 No. WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., β(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, Inc. further describes genetically engineered CHO mammalian cells capable of producing antibodies with a modified 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 that have been engineered for mammalian-like glycosylation patterns and are capable of producing antibodies lacking fucose as a glycosylation pattern (see, e.g., EP 1297172 B1).

[0086] In some embodiments, antibodies suitable for depletion of cancer cells mediated complement dependent cytotoxicity.

[0087] 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 can be performed, for example, as described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0088] In some embodiments, antibodies suitable for depleting cancer cells mediate antibody-dependent phagocytosis.

[0089] As used herein, the term "antibody-dependent phagocytosis" or "opsonization" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fcγ receptors recognize antibody bound on target cells and subsequently cause phagocytosis of the target cells.

[0090] GARP multispecific antibody: In some embodiments, antibodies suitable for depletion of GARP cancer cells are multispecific antibodies comprising a first antigen-binding site directed against GARP and at least one second antigen-binding site directed against an effector cell as described above.

[0091] In some embodiments, the first antigen binding domain comprises the VH domain and VL domain of DS-1055a.

[0092] In some embodiments, the first antigen binding domain comprises the VH domain and VL domain of ribmoniplimab.

[0093] 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 can induce ADCC, such as a natural killer cell. For example, monocytes and macrophages, which express Fc receptors, are involved in the specific killing of target cells and presenting 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 specific Fc receptors on effector cells 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 (such as radiolabeled peptides), chemotherapeutic agents, and prodrugs. In some embodiments, the second binding site binds to an Fc receptor as defined above. In some embodiments, the second binding site can bind to a surface molecule on a natural killer cell, thereby activating the cell. In some embodiments, the second binding site binds to NKp46. Exemplary formats of the multispecific antibody molecules of the present invention include: (i) two antibodies crosslinked by chemical heteroconjugation, one with specificity for a particular surface molecule of ILCs and the other 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 contains two variable domains in tandem connected through a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)). TM) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010); (v) chemically linked bispecific (Fab')2 fragments; (vi) Tandabs (which are the fusion of two single-chain diabodies, resulting in tetravalent bispecific antibodies with two binding sites for each target antigen); (vii) Flexibodies (which are the combination of a single-chain Fv and a diabody, resulting in a multivalent molecule); (viii) so-called "dock and lock" molecules based on the "dimerization and docking domain" of protein kinase A (which, when applied to Fabs, can result in tetravalent bispecific binding proteins consisting of two identical Fab fragments linked to different Fab fragments); (ix) so-called scorpion molecules, which contain, for example, two scFvs fused to either end of a human Fab arm; and (x) diabodies. Another exemplary format of bispecific antibodies is an IgG-like molecule with complementary CH3 domains that force heterodimerization. Such molecules can be prepared using known techniques, 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 (SEED body) (EMD Serono), Biclonic (Mers), and DuoBody (Genmab) technologies.

[0094] In some embodiments, therefore, the multispecific antibody is a bispecific antibody.

[0095] 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 construct composed of two flexibly linked single-chain antibodies (scFv). One of the scFv antibodies specifically binds to a tumor antigen (i.e., GARP) expressed on a selected target cell, and the second specifically binds to another molecule, such as CD3, a subunit of the T cell receptor complex on a T cell. In some embodiments, the BiTE antibody can transiently bind a T cell to a target cell and simultaneously activate the cytolytic activity of the T cell. BiTE-mediated T cell activation does not require a specific T cell receptor on the T cell, nor an MHC1 molecule, peptide antigen, or costimulatory molecule on the target cell.

[0096] GARP antibody-drug conjugates: In some embodiments, antibodies suitable for depleting cancer cells are conjugated to a therapeutic moiety, ie, a drug.

[0097] In some embodiments, the antibody-drug conjugate comprises the VH and VL domains of DS-1055a.

[0098] In some embodiments, the antibody-drug conjugate comprises the VH and VL domains of ribmoniplimab.

[0099] In some embodiments, the therapeutic moiety can be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0100] In some embodiments, antibodies suitable for depleting 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; dihydroxyanthracenedione; tubulin inhibitors, such as maytansine or an analogue or derivative thereof; mitotic inhibitors, 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, flucloxin, fluoxetine ... Darabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents such as mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or analogs or derivatives thereof; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-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 shiga-like toxin type 1 (SLT I), shiga-like toxin type 2 (SLT II), shiga-like toxin type IIV (SLT IIV), LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxin, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins, such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, soapwort (sapaonaria officinalis) inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); deoxyribonuclease (DNase I), Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtheria toxin; and Pseudomonas endotoxin.

[0101] 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 cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer (U.S. Pat. No. 5,663,149) and antifungal (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965) activity. For example, auristatin E can react with para-acetylbenzoic acid or benzoylvaleric acid to generate auristatin B (AEB) and auristatin VB (AEVB), respectively. Other exemplary auristatin derivatives include auristatin FP (AFP), MMAF (monomethylauristatin F), and MMAE (monomethylauristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as linkers suitable for conjugating auristatins to antibodies (Abs), are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, and 6,214,345, and International Patent Application Publication Nos. WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968, and WO205082023.

[0102] In some embodiments, antibodies suitable for depleting cancer cells are conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or an analog, derivative, or prodrug thereof. Suitable PDBs and PDB derivatives, and 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.

[0103] In some embodiments, an antibody suitable for depleting cancer cells is conjugated to a cytotoxic moiety selected from the group consisting of anthracycline, maytansine, calicheamicin, duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0104] In some embodiments, the antibody suitable for depleting cancer cells is conjugated to an anthracycline or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to 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.

[0105] In some embodiments, antibodies suitable for depleting cancer cells are conjugated to a nucleic acid or nucleic acid-binding 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.

[0106] 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 Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58). See, e.g., PCT Publication No. 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 methods incorporating unnatural amino acids have been reported to improve conjugate homogeneity (Axup, JY, Bajjuri, KM, Ritland, M., Hutchins, BM, Kim, CH, Kazane, SA, Halder, R., Forsyth, JS, Santidrian, AF, Stafin, K., et al. (2012). Synthesis of site-specific antibody-drug conjugates using unnatural amino acids. Proc. Natl. Acad. Sci. USA 109, 16101-16106.; Junutula, JR, Flagella, KM, Graham, RA, Parsons, KL, Ha, E., Raab, H., Bhakta, S., Nguyen, T., Dugger, DL, Li, G., et al. (2010). Engineered Thio-trastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin. Cancer Res. 16, 4769-4778). Junutula et al. (2008) developed a cysteine-based site-specific conjugation, termed "THIOMAB" (TDC), which is claimed to exhibit an improved therapeutic index compared to traditional conjugation methods. Conjugation to unnatural amino acids incorporated into antibodies has also been explored for antibody-drug conjugates (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 an Fc-containing polypeptide engineered with an acyl donor glutamine-containing tag (e.g., a Gin-containing peptide tag or Q-tag) or an endogenous glutamine that has been made reactive by engineering the polypeptide (e.g., via deletion, insertion, substitution, or mutation of an amino acid on the polypeptide). A transglutaminase can then covalently crosslink an amine donor substance (e.g., a small molecule containing or attached to a reactive amine) to form a stable and homogenous population of conjugates of the amine donor substance and the engineered Fc-containing polypeptide that are site-specifically conjugated to the Fc-containing polypeptide through the acyl donor glutamine-containing tag or the accessible / exposed / reactive endogenous glutamine (WO2012059882).

[0107] GARP CAR-T cells In some embodiments, the agent is a CAR-T cell, wherein the CAR comprises at least one extracellular antigen-binding domain specific for GARP.

[0108] In some embodiments, the extracellular antigen-binding domain specific for GARP comprises the VH and VL domains of DS-1055a.

[0109] In some embodiments, the extracellular antigen-binding domain specific for GARP comprises the VH and VL domains of ribmoniplimab.

[0110] In some embodiments, the CAR comprises at least one 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., 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 a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0111] In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain specific for GARP, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain specific for GARP, 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, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain specific for GARP, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and one functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain specific for GARP, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and one functional signaling domain derived from a stimulatory molecule.

[0112] 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, where the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0113] In certain aspects, the CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for GARP fused to CD3-zeta, a transmembrane domain, and an endodomain. In some embodiments, the CAR comprises additional domains for costimulatory signaling, such as CD3-zeta, an Fc receptor, CD27, CD28, CD137, DAP10, and / or OX40. In some embodiments, molecules may 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.

[0114] In some embodiments, a chimeric antigen receptor of the invention comprises at least one VH and / or VL sequence of an antibody specific for GARP. In some embodiments, the portion of a CAR of the invention comprising an antibody or antibody fragment thereof specific for GARP can exist in a variety of forms, where the antigen binding domain is 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 GARP. In a further aspect, the CAR comprises an antibody fragment comprising an scFv that is specific for GARP.

[0115] Methods for preparing CAR-T cells are well known in the art. In some embodiments, cells (e.g., T cells) are transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the cells can stably express the CAR. In some embodiments, the cells (e.g., T cells) are transfected with a nucleic acid, such as mRNA, cDNA, or DNA, encoding a CAR. In some embodiments, the antigen-binding domain (e.g., scFv) of the CAR of the present invention is encoded by a nucleic acid molecule whose sequence is codon-optimized for expression in mammalian cells. In some embodiments, the complete CAR construct of the present invention is encoded by a nucleic acid molecule whose complete sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons encoding the same amino acid) within coding DNA is biased in different species. Such codon degeneracy allows the same polypeptide to be encoded by a wide variety of nucleotide sequences. A wide variety of 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.

[0116] In some embodiments, the chimeric antigen receptors of the present invention can 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.

[0117] In some embodiments, CAR activity may 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, such as using caspases fused to dimerization domains (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention.

[0118] Pharmaceutical Composition: Typically, the agents of the present invention are administered to patients in the form of pharmaceutical compositions 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 patients, the compositions will be formulated for administration to patients. The compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, or via an implanted reservoir. The term "use" herein 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 solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile, fixed oils are conveniently used as solvents or suspending media. For this purpose, any non-irritating, fixed oil may be used, including synthetic monoglycerides 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 oily 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. For tablets for oral use, commonly used carriers include lactose and cornstarch. Lubricants, such as magnesium stearate, are also typically added. Diluents useful for oral administration in capsule form include, for example, lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, flavors, or coloring agents may also be added. Alternatively, the compositions of the present invention may be administered in the form of rectal suppositories. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore will melt in the rectum to release the drug. Such excipients include cocoa butter, beeswax, and polyethylene glycol. The compositions of the present invention may also be administered topically, especially when the target of treatment includes areas or organs easily accessible by topical application, including diseases of the eyes, skin, or lower gastrointestinal tract. Suitable topical formulations for each of these areas or organs are easily prepared. The compositions for topical application may be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the compositions may be formulated in a suitable lotion or cream containing the active compounds 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 to 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 convenient solubilizing or dispersing agents. For example, the antibody present in the pharmaceutical composition of the present invention may be supplied at a concentration of 10 mg / mL in either 100 mg (10 mL) or 50 mg (50 mL) single-use vials. The product is formulated for intravenous 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 dose range for the antibody in the pharmaceutical composition of the invention is about 1 mg / mL. 2 to 500 mg / 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 present invention for injection (e.g., intramuscular, intravenous) can be prepared to contain sterile buffered water (e.g., 1 ml for intramuscular administration) and about 1 ng to about 100 mg, for example, about 50 ng to about 30 mg or more, preferably about 5 mg to about 25 mg, of the drug of the present invention.

[0119] The present invention will be further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0120] [Figure 1] Expression of GARP in fresh peripheral blood tumor cells from patients with Sézary syndrome. After obtaining and signing informed consent, GARP (clone 7B11) expression was examined by flow cytometry on peripheral blood mononuclear cells from patients with Sézary syndrome using anti-CD3, anti-CD4, and anti-CD158k antibodies (=KIR3DK2, a surface marker of Sézary cells), and GARP or control isotypes. [Figure 2] Median mean fluorescence intensity (MFI) of GARP obtained with anti-GARP-APC antibody (clone 7B11) for eight patients with Sézary syndrome. [Figure 3] Absence of GARP expression in the Sézary cell line. Cells were incubated with a control isotype or anti-GARP antibody (clone 7B11) for 15 minutes at 4°C, then washed with PBS and analyzed on a LSRX20 flow cytometer. [Figure 4] Expression of GARP in T-ALL cell lines and T-ALL patient samples. Cells were incubated with a control isotype or anti-GARP antibody (clone 7B11) for 15 minutes at 4°C, then washed with PBS and analyzed on a LSRX20 flow cytometer.

[0121] Working Example: Materials and Methods GARP expression was examined in peripheral blood mononuclear cells of patients with Sézary syndrome or T-ALL by flow cytometry using anti-CD3, anti-CD4, anti-CD158k, and anti-GARP antibodies (clone 7B11) after obtaining and signing informed consent.

[0122] result GARP expression was examined in peripheral blood mononuclear cells from patients with Sézary syndrome or T-cell acute lymphoblastic leukemia (T-ALL). The results are shown in Figures 1, 2, and 4 and demonstrate that GARP is overexpressed in T-cell malignancies. Surprisingly, we demonstrate herein that GARP is overexpressed in samples from patients with Sézary syndrome. These results were not apparent in light of the negative results obtained with cellular tools representative of this condition (Figure 3). See also, for example, WO2018 / 208888, where Sézary cell lines and other lymphoid lineages do not overexpress GARP, in contrast to our demonstration in patient samples.

[0123] 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 the present disclosure.

[0124] [Table 1] TIFF2025526336000004.tif245169

Claims

1. A method for providing an indicator for diagnosing T-cell malignancies in patients, comprising detecting the expression level of GARP in a sample obtained from the patient.

2. The method according to claim 1, wherein the T-cell malignant tumor is T-cell lymphoma or T-cell leukemia.

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

4. The method according to claim 1, wherein the indicator is for diagnosing cutaneous T-cell lymphoma.

5. The method according to claim 1, wherein the indicator is for diagnosing Sézary syndrome.

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

7. A pharmaceutical composition for treating a T-cell malignancy in a patient in need thereof, comprising a therapeutically effective amount of GARP inhibitor.

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

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

10. The pharmaceutical composition according to claim 7 or 8 for the treatment of Sézary syndrome, hepatosplenic T-cell lymphoma, angioimmunoblastic T-cell lymphoma, NK / T-cell lymphoma, or T-cell acute lymphoblastic leukemia.

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

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

13. The pharmaceutical composition according to claim 7 or 8, wherein the inhibitor or drug is an antibody having binding affinity to GARP.

14. The pharmaceutical composition according to claim 13, wherein the antibody is directed to at least one extracellular domain of GARP.

15. The pharmaceutical composition according to claim 13, wherein the antibody causes depletion of GARP-expressing cancer cells.

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

17. The pharmaceutical composition according to claim 13, wherein the antibody is a multispecific antibody comprising a first antigen-binding site directed toward GARP and at least one second antigen-binding site directed toward effector cells.

18. The pharmaceutical composition according to claim 13, wherein the antibody is conjugated to the cytotoxic portion.

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

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