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EP4602372A1 - Cd81 utilisé en tant que biomarqueur et cible biologique dans des malignités de lymphocytes t - Google Patents

Cd81 utilisé en tant que biomarqueur et cible biologique dans des malignités de lymphocytes t

Info

Publication number
EP4602372A1
EP4602372A1 EP23790555.9A EP23790555A EP4602372A1 EP 4602372 A1 EP4602372 A1 EP 4602372A1 EP 23790555 A EP23790555 A EP 23790555A EP 4602372 A1 EP4602372 A1 EP 4602372A1
Authority
EP
European Patent Office
Prior art keywords
cell
antibody
cells
cell lymphoma
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23790555.9A
Other languages
German (de)
English (en)
Inventor
Armand Bensussan
Jérôme GIUSTINIANI
Nicolas Ortonne
Adèle DE MASSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Est Creteil Val de Marne
Universite Paris Cite
Universite de Marne la Vallee
Original Assignee
Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Paris Est Creteil Val de Marne
Universite Paris Cite
Universite de Marne la Vallee
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Assistance Publique Hopitaux de Paris APHP, Institut National de la Sante et de la Recherche Medicale INSERM, Universite Paris Est Creteil Val de Marne, Universite Paris Cite, Universite de Marne la Vallee filed Critical Assistance Publique Hopitaux de Paris APHP
Publication of EP4602372A1 publication Critical patent/EP4602372A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • CD81 AS A BIOMARKER AND BIOTARGET IN T-CELL MALIGNANCIES
  • the present invention is in the field of medicine, in particular oncology.
  • T-cell malignancies are a broad, heterogenous group of diseases and include T-cell lymphomas and T-cell leukemias.
  • T-cell malignancies primary cutaneous T-cell lymphomas are a heterogeneous group of lymphomas primarily affecting the skin.
  • cutaneous epidermotropic T-cell lymphomas mycosis fungoides and Sezary syndrome
  • Sezary syndrome is defined as erythroderma (erythema of the entire skin covering), and circulating blood damage (1).
  • the circulating T lymphocyte tumor cell expresses CD4 and can lose the expression of CD7 and CD26, while presenting in the majority of cases an aberrant expression of CD 158k (KIR3DL2) (2).
  • the initial diagnosis of the disease is difficult and the follow-up of the blood involvement is complicated because the international criteria use the loss of the CD7 and CD26 markers (CD4+ CD26- and CD4+ CD7- cells) (3) which we know as non-specific for tumor cells (4).
  • CD158k a marker expressed aberrantly by Sezary cells
  • KIR3DL2 a marker expressed aberrantly by Sezary cells
  • CCR4 is expressed not only by Sezary cells but also by peripheral blood memory regulatory T cells and its use is associated with the occurrence of autoimmune adverse effects (7).
  • autoimmune adverse effects (7).
  • the present invention is defined by the claims.
  • the present invention relates to methods for the diagnosis and treatment of T cell-malignancies.
  • T cell has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity.
  • T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility.
  • TCR T cell receptor for the antigen
  • There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells.
  • CD8+ T cell has its general meaning in the art and refers to a subset of T cells which express CD8 on their surface. They are MHC class I-restricted, and function as cytotoxic T cells.
  • CD8+ T cells are also called cytotoxic T lymphocytes (CTL), T-killer cells, cytolytic T cells, or killer T cells.
  • CD8 antigens are members of the immunoglobulin supergene family and are associative recognition elements in major histocompatibility complex class I-restricted interactions.
  • tumor infiltrating CD8+ T cell refers to the pool of CD8+ T cells of the patient that have left the blood stream and have migrated into a tumor.
  • CD4+ T cells also called T helper cells or TH cells refers to T cells which express the CD4 glycoprotein on their surfaces and which assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • CD4+ T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete cytokines that regulate or assist in the active immune response.
  • APCs antigen-presenting cells
  • TH1, TH2, TH3, TH17, TH9, TFH or Treg which secrete different cytokines to facilitate different types of immune responses.
  • Signaling from the APC directs T cells into particular subtypes.
  • the TH cell surface biomarkers known in the art include CXCR3 (Thl), CCR4, Crth2 (Th2), CCR6 (Th 17), CXCR5 (Tfh) and as well as subtype-specific expression of cytokines and transcription factors including T-bet, GATA3, HOMES, RORyT, BCL6 and FoxP3
  • gamma delta T cell has its general meaning in the art.
  • Gamma delta T cells normally account for 1 to 5% of peripheral blood lymphocytes in a healthy individual (human, monkey). They are involved in mounting a protective immune response, and it has been shown that they recognize their antigenic ligands by a direct interaction with antigen, without any presentation by MHC molecules of antigen- presenting cells.
  • Gamma 9 delta 2 T cells (sometimes also called gamma 2 delta 2 T cells) are gamma delta T cells bearing TCR receptors with the variable domains Vy9 and V52. They form the majority of gamma delta T cells in human blood. When activated, gamma delta T cells exert potent, non-MHC restricted cytotoxic activity, especially efficient at killing various types of cells, particularly pathogenic cells.
  • These may be cells infected by a virus (Poccia et al., J. Leukocyte Biology, 1997, 62: 1-5) or by 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; Foumie and Bonneville, Res. Immunol., 66th Forum in Immunology, 147: 338-347).
  • subtypes include peripheral T-cell lymphomas, Hepatosplenic T-cell lymphoma (HSTCL), Angioimmunoblastic T-cell lymphoma (AITL), NK/T-cell lymphoma (NKTL), Mycosis fungoide (MF) and Sezary syndrome (SS).
  • the T-cell lymphoma is Sezary syndrome, Mycosis Fungoides, Hepatosplenic T-cell lymphoma or NK/T-cell lymphoma.
  • the T-cell malignancy is a cutaneous T-cell lymphoma.
  • CTCL cutaneous T-cell lymphoma
  • MF Mycosis fungoides
  • SS Sezary Syndrome
  • the cutaneous T-cell lymphoma is Sezary syndrome or Mycosis Fungoides.
  • the cutaneous T-cell lymphoma is Sezary syndrome.
  • SS has its general meaning in the art and refers to an aggressive form of cutaneous T-cell lymphoma characterized by a triad of erythroderma, lymphadenopathy and circulating atypical lymphocytes (Sezary cells). SS develops most frequently in men, is more frequent in the elderly, and progresses rapidly. SS correspond to stages IV A2 and IVB of T-cell cutaneous lymphoma (see this term). Patients present with a scaling erythroderma and infiltration often manifesting with leonine facies and severe pruritus. Alopecia, ectropium, mild palmoplantar keratoderma and nail onychodystrophy may be present. Lymphadenopathy and hepatosplenomegaly are observed. Patients often shiver and complain of chills and general fatigue.
  • the T-cell malignancy is a T-cell leukemia.
  • T- cell leukemia has its general meaning in the art and denotes a malignant hematological condition including several types of lymphoid leukemia which affect T-cells.
  • Leukemias usually develop from young blood cells within the bone marrow and spread through the bloodstream.
  • Leukemias are of different subtypes: acute leukemia (AL) and chronic leukemia (CL).
  • acute leukemias include Acute Lymphoblastic Leukemias (ALL).
  • the leukemia is T-cell Acute Lymphoblastic Leukemia (T-ALL).
  • CD81 refers to a protein belonging to the tetraspanin family (Entrez Gene: 975; Ensembl: ENSG00000110651). CD81 is expressed by hematopoietic cells, endothelial cells and epithelial cells and has been described as one of the major entry receptors of the hepatitis C virus (8). CD81 is composed of four transmembrane domains connected by two loops: the small extracellular loop (SEL) and the large extracellular loop (LEL).
  • SEL small extracellular loop
  • LEL large extracellular loop
  • CD81LEL is a five-helix bundle fold composed of a stalk subdomain (helices A and E) and a head subdomain (helices B, C and D) (Kitadokoro et al., 2001).
  • An exemplary amino acid sequence for CD81 is represented by SEQ ID NO:1.
  • the protein comprises 2 extracellular domains: the first one ranges from the amino acid residue at position 34 to the amino acid residue at position 63 in SEQ ID NO:1 and the second one ranges from the amino acid residue at position 113 to the amino acid residue at position 201 in SEQ ID NO:1.
  • TGF- has its general meaning in the art and refers to the Transforming growth factor-p.
  • the term encompasses any isoform of TGF-P, provided the isoform has immunosuppressive activity.
  • Transforming growth factor-P functions indeed as an immune suppressor by influencing immune cells' development, differentiation, tolerance induction and homeostasis (Sheng J, Chen W, Zhu HJ. The immune suppressive function of transforming growth factor-P (TGF-P) in human diseases. Growth Factors. 2015 Apr;33(2):92-101. doi: 10.3109/08977194.2015.1010645. Epub 2015 Feb 25).
  • Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments.
  • the term “bind” indicates that the antibody has affinity for the surface molecule.
  • affinity means the strength of the binding of an antibody to an epitope.
  • the affinity of an antibody is given by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen.
  • Kd dissociation constant
  • Ka is defined by 1/Kd.
  • chimeric antibody refers to an antibody which comprises a VH domain and a VL domain of a non-human antibody, and a CH domain and a CL domain of a human antibody.
  • a “chimeric antibody” is an antibody molecule in which (a) the constant region (i.e., the heavy and/or light chain), or a portion thereof, is altered, replaced 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 an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
  • Chimeric antibodies also include primatized and in particular humanized antibodies. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • humanized antibody refers to an antibody having variable region framework and constant regions from a human antibody but retains the CDRs of a previous non-human antibody.
  • a humanized antibody contains minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies and antibody fragments thereof may be human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary -determining region (CDR) of the recipient 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.
  • donor antibody such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such antibodies are designed to maintain the binding specificity of the non-human antibody from which the binding regions are derived, but to avoid an immune reaction against the non-human antibody. These modifications can further refine and optimize antibody or antibody fragment performance.
  • the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • 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 chain and also two different antigen-binding sites.
  • chimeric antigen receptor has its general meaning in the art and refers to an artificially constructed hybrid protein or polypeptide containing the antigen binding domains of an antibody (e.g., scFv) linked to T- cell signaling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains.
  • TCR endogenous T cell receptor alpha and beta chains.
  • the chimeric antigen receptor the present invention typically comprises an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.
  • CAR-T cell refers to a T lymphocyte that has been genetically engineered to express a CAR.
  • the definition of CAR T-cells encompasses all classes and subclasses of T-lymphocytes including CD4+, CD8+ T cells, gamma delta T cells as well as effector T cells, memory T cells, regulatory T cells, and the like.
  • the T lymphocytes that are genetically modified may be "derived” or “obtained” from the patient who will receive the treatment using the genetically modified T cells or they may "derived” or “obtained” from a different patient.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]).
  • the term "therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result.
  • a therapeutically effective amount of the active agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the active agent to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of drug are outweighed by the therapeutically beneficial effects.
  • the efficient dosages and dosage regimens for the active agent depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen.
  • Such an effective dose will generally depend upon the factors described above.
  • a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease.
  • the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors.
  • a therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a patient.
  • An exemplary, non-limiting range for a therapeutically effective amount of a drug of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 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 a drug of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg.
  • Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a 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.
  • the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
  • the efficacy may be monitored by visualization of the disease area, or by other diagnostic methods described further herein, e.g. by performing one or more PET-CT scans, for example using a labeled antibody of the present invention, fragment or mini-antibody derived from the antibody of the present invention.
  • an effective daily dose of a pharmaceutical composition may be administered as two, three, four, five, six or more subdoses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • treatment according to the present invention may be provided as a daily dosage of a drug of the present invention in an amount of about 0.1-100 mg/kg, such as 0.2, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of days 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, 36, 37, 38, 39, or 40, or alternatively, at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof
  • a first object of the present invention relates to a method of diagnosing a T-cell malignancy in a patient comprising detecting the expression level of CD81 in a sample obtained from the patient.
  • the T-cell malignancy is Sezary syndrome, Hepatosplenic T-cell lymphoma, NK/T-cell lymphoma or T-cell Acute Lymphoblastic Leukemia.
  • the T-cell lymphoma is Sezary syndrome, Mycosis Fungoides, Hepatosplenic T- cell lymphoma or NK/T-cell lymphoma.
  • sample refers to any biological sample obtained for the purpose of evaluation in vitro.
  • the sample is sample is a blood sample.
  • the sample is PBMC sample.
  • the sample is a sample of (i) purified blood leukocytes, (ii) peripheral blood mononuclear cells or PBMC, (iii) purified lymphocytes, (iv) purified T cells, (v) purified CD4+ T cells or (vi) purified CD3+ T cells.
  • the biological sample is a tissue sample.
  • tissue sample includes sections of tissues such as biopsy or autopsy samples and frozen sections taken for histological purposes. Thus in some embodiments, the tissue sample may result from a biopsy performed in the subject’s skin.
  • one or more labels can be attached to the antibody, thereby permitting detection of the target protein (i.e the marker).
  • exemplary labels include radioactive isotopes, fluorophores, ligands, chemiluminescent agents, enzymes, and combinations thereof.
  • the label is a quantum dot.
  • Non-limiting examples of labels that can be conjugated to 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 e.g. luciferin, luciferase
  • haptens e.g. biotin
  • Affinity ligands can also be labeled with enzymes (e g. horseradish peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g. 3H, 14C, 32P, 35S or 1251) and particles (e.g. gold).
  • enzymes e g. horseradish peroxidase, alkaline phosphatase, beta-lactamase
  • radioisotopes e.g. 3H, 14C, 32P, 35S or 1251
  • particles e.g. gold
  • the obtained images can then be used for quantitatively or semi-quantitatively determining the amount of the marker in the sample.
  • Various automated sample processing, scanning and analysis systems suitable for use with immunohistochemistry are available in the art. Such systems can include automated staining and microscopic scanning, computerized image analysis, serial section comparison (to control for variation in the orientation and size of a sample), digital report generation, and archiving and tracking of samples (such as slides on which tissue sections are placed).
  • Cellular imaging systems are commercially available that combine conventional light microscopes with digital image processing systems to perform quantitative analysis on cells and tissues, including immunostained samples. See, e.g., the CAS-200 system (Becton, Dickinson & Co.).
  • detection can be made manually or by image processing techniques involving computer processors and software.
  • the images can be configured, calibrated, standardized and/or validated based on factors including, for example, stain quality or stain intensity, using procedures known to one of skill in the art (see e.g., published U.S. Patent Publication No. US20100136549).
  • the image can be quantitatively or semi-quantitatively analyzed and scored based on staining intensity of the sample.
  • Quantitative or semi -quantitative histochemistry refers to method of scanning and scoring samples that have undergone histochemistry, to identify and quantitate the presence of the specified biomarker (i.e. the marker).
  • Quantitative or semi -quantitative methods can employ imaging software to detect staining densities or amount of staining or methods of detecting staining by the human eye, where a trained operator ranks results numerically.
  • images can be quantitatively analyzed using a pixel count algorithms (e.g., Aperio Spectrum Software, Automated QUantitatative Analysis platform (AQUA® platform), and other standard methods that measure or quantitate or semi -quantitate the degree of staining; see e.g., U.S. Pat. No. 8,023,714; U.S. Pat. No. 7,257,268; U.S. Pat. No. 7,219,016; U.S. Pat. No. 7,646,905; published U.S.
  • a ratio of strong positive stain (such as brown stain) to the sum of total stained area can be calculated and scored.
  • the amount of the detected biomarker i.e. the marker
  • the amount is quantified and given as a percentage of positive pixels and/or a score. For example, the amount can be quantified as a percentage of positive pixels. In some examples, the amount is quantified as the percentage of area stained, e.g., the percentage of positive pixels.
  • 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 as compared to the total staining area.
  • fluorescence activated cell sorting may be therefore used, involves using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores, such as a BD Biosciences FACSCantoTM flow cytometer, used substantially according to the manufacturer's instructions.
  • the cytometric systems may include a cytometric sample fluidic subsystem, as described below.
  • the cytometric systems include a cytometer fluidically coupled to the cytometric sample fluidic subsystem.
  • Systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and/or speakers, softwares (e.g.
  • the sample is contacted with a panel of antibodies specific for the specific market of the population of cells of the interest.
  • antibodies or antigen-binding fragments are available commercially from vendors such as R&D Systems, BD Biosciences, e- Biosciences, Biolegend, Proimmune and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art.
  • an agent that specifically bind to a cell-surface marker such as an antibody or antigen-binding fragment, is labelled with a tag to facilitate the isolation and detection of population of cells of the interest.
  • label refers to a composition capable of producing a detectable signal indicative of the presence of a target, such as, the presence of a specific 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.
  • a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means needed for the methods to isolate and detect the cancer cells.
  • Non-limiting examples of fluorescent labels or tags for labeling the agents such as antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor 710, PE- CF594, Peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B,
  • the aforementioned assays may involve the binding of the antibodies to a solid support.
  • the solid surface could be a microtitration plate coated with the antibodies.
  • the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic.
  • the beads are preferably fluorescently labelled. In a preferred embodiment, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California).
  • the method further comprises detecting the expression level of at least one further marker.
  • the marker is selected from the group consisting of CD3, CD4, KIR3DL2, PLS3, Twist and NKp46.
  • the name of each of the various markers of interest refers to the internationally recognised name of the corresponding gene, as found in internationally recognised gene sequences and protein sequences databases, including in the database from the HUGO Gene Nomenclature Committee that is available notably at the following Internet address: http://www.gene.ucl.ac.uk/nomenclature/index.html.
  • the name of each of the various markers of interest may also refer to the internationally recognised name of the corresponding gene, as found in the internationally recognised gene sequences and protein sequences database Genbank. Through these internationally recognised sequence databases, the nucleic acid and the amino acid sequences corresponding to each of the marker of interest described herein may be retrieved by the one skilled in the art.
  • Multiplex tissue analysis techniques are particularly useful for quantifying several markers in the tissue sample. Such techniques should permit at least five, or at least ten or more biomarkers to be measured from a single tissue sample. Furthermore, it is advantageous for the technique to preserve the localization of the biomarker and be capable of distinguishing the presence of biomarkers in cancerous and non-cancerous cells.
  • Such methods include layered immunohistochemistry (L-IHC), layered expression scanning (LES) or multiplex tissue immunoblotting (MTI) taught, for example, in U.S. Pat. Nos. 6,602,661, 6,969,615, 7,214,477 and 7,838,222; U.S. Publ. No.
  • the L-IHC method can be performed on any of a variety of tissue samples, whether fresh or preserved.
  • the samples included core needle biopsies that were routinely fixed in 10% normal buffered formalin and processed in the pathology department. Standard five pm thick tissue sections were cut from the tissue blocks onto charged slides that were used for L-IHC.
  • L-IHC enables testing of multiple markers in a tissue section by obtaining copies of molecules transferred from the tissue section to plural bioaffmity- coated membranes to essentially produce copies of tissue "images."
  • the tissue section is deparaffinized as known in the art, for example, exposing the section to xylene or a xylene substitute such as NEO-CLEAR®, and graded ethanol solutions.
  • the section can be treated with a proteinase, such as, papain, trypsin, proteinase K and the like. Then, a stack of a membrane substrate comprising, for example, plural sheets of a 10 pm thick coated polymer backbone with 0.4 pm diameter pores to channel tissue molecules, such as, proteins, through the stack, then is placed on the tissue section.
  • tissue molecules such as, proteins
  • the movement of fluid and tissue molecules is configured to be essentially perpendicular to the membrane surface.
  • the sandwich of the section, membranes, spacer papers, absorbent papers, weight and so on can be exposed to heat to facilitate movement of molecules from the tissue into the membrane stack.
  • each membrane comprises a copy of the tissue and can be probed for a different biomarker using standard immunoblotting techniques, which enables open-ended expansion of a marker profile as performed on a single tissue section.
  • the amount of protein can be lower on membranes more distal in the stack from the tissue, which can arise, for example, on different amounts of molecules in the tissue sample, different mobility of molecules released from the tissue sample, different binding affinity of the molecules to the membranes, length of transfer and so on, normalization of values, running controls, assessing transferred levels of tissue molecules and the like can be included in the procedure to correct for changes that occur within, between and among membranes and to enable a direct comparison of information within, between and among membranes.
  • total protein can be determined per membrane using, for example, any means for quantifying protein, such as, biotinylating available molecules, such as, proteins, using a standard reagent and method, and then revealing the bound biotin by exposing the membrane to a labeled avidin or streptavidin; a protein stain, such as, Blot fastStain, Ponceau Red, brilliant blue stains and so on, as known in the art.
  • biotinylating available molecules such as, proteins
  • the present methods utilize Multiplex Tissue Imprinting (MTI) technology for measuring biomarkers, wherein the method conserves precious biopsy tissue by allowing multiple biomarkers, in some cases at least six biomarkers.
  • MMI Multiplex Tissue Imprinting
  • alternative multiplex tissue analysis systems exist that may also be employed as part of the present invention.
  • One such technique is the mass spectrometry-based Selected Reaction Monitoring (SRM) assay system ("Liquid Tissue” available from OncoPlexDx (Rockville, MD). That technique is described in U.S. Pat. No. 7,473,532.
  • SRM Selected Reaction Monitoring
  • Multispectral imaging can unmix, or separate out, autofluorescence from tissue and, thereby, increase the achievable signal -to-noise ratio.
  • a series of different cut-off values are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis.
  • AUC area under the curve
  • the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values.
  • the AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate.
  • the method comprises a further step consisting determining CD81 delta mean fluorescence intensity and concluding that the patient suffers from a T-cell malignancy when CD81 delta mean fluorescence intensity is higher 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.
  • a worse diagnosis that is determined by assessing the expression level of CD81 during the course of treatment may indicate ineffective dosage and the desirability of increasing the dosage.
  • a better diagnosis that is determined by assessing the expression level of CD81 may indicate efficacious treatment and no need to change dosage.
  • the present invention also relates to a method for adapting a therapy in a patient suffering from a T-cell malignancy, wherein said method comprises the steps of: a) performing, on at least one sample collected from said patient, the in vitro diagnosis method that is disclosed herein; and b) adapting the therapy of said patient by administering to said patient.
  • the T-cell malignancy is a T-cell lymphoma
  • the T-cell lymphoma is Sezary Syndrome.
  • the T-cell malignancy is a T-cell leukemia.
  • the invention also relates to a kit for performing the diagnosis methods as described above.
  • the kit comprises a plurality of reagents, in particular at least one agent that is capable of binding specifically to the CD81 marker.
  • Suitable reagents for binding with a marker protein include antibodies, antibody derivatives, antibody fragments, and the like.
  • Suitable reagents for binding with a marker nucleic acid e.g.
  • a genomic DNA, an mRNA, a spliced mRNA, a cDNA, or the like include complementary nucleic acids.
  • the nucleic acid reagents may include oligonucleotides (labeled or non-labeled) fixed to a substrate, labeled oligonucleotides not bound with a substrate, pairs of PCR primers, molecular beacon probes, and the like.
  • the kit of the invention may optionally comprise additional components useful for performing the methods of the invention.
  • the kit may comprise fluids (e.g. SSC buffer) suitable for annealing complementary nucleic acids or for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of the in vitro diagnosis method of the invention, and the like.
  • a further object of the present invention relates to a method of treating a T-cell malignancy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent capable of inducing cell death of CD81 expressing cancer cells.
  • the T-cell lymphoma is Sezary syndrome, Mycosis Fungoides, Hepatosplenic T- cell lymphoma or NK/T-cell lymphoma.
  • the T-cell malignancy is Sezary syndrome, Hepatosplenic T-cell lymphoma, NK/T-cell lymphoma or T-cell Acute Lymphoblastic Leukemia.
  • the present invention relates to a method of treating a 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 CD81 expressing cancer cells.
  • T-cell lymphomas include cutaneous, nodal, extranodal and leukemic lymphomas.
  • subtypes include peripheral T-cell lymphomas, Hepatosplenic T-cell lymphoma (HSTCL), Angioimmunoblastic T-cell lymphoma (AITL), NK/T-cell lymphoma (NKTL), Mycosis fungoide (MF) and Sezary syndrome (SS).
  • the T-cell lymphoma is Sezary syndrome, Mycosis Fungoides, Hepatosplenic T-cell lymphoma or NK/T- cell lymphoma. In some embodiments, the T-cell lymphoma is a cutaneous T-cell lymphoma. In some embodiments, the T-cell lymphoma is Sezary syndrome. In some embodiments, the present invention relates to a method of treating a 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 CD81 expressing cancer cells. In some embodiments, the T-cell leukemia is T-cell Acute Lymphoblastic Leukemia (T-ALL).
  • T-ALL T-cell Acute Lymphoblastic Leukemia
  • 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.
  • the present invention relates to a method of treating a T-cell malignancy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.
  • the present invention relates to a method of treating a T-cell lymphoma in patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.
  • the present invention relates to a method of treating a T-cell leukemia in patient in need thereof comprising administering to the patient a therapeutically effective amount of a CD81 inhibitor.
  • the CD81 inhibitor is an inhibitor of CD81 gene expression.
  • said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme.
  • anti-sense oligonucleotides including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of CD81 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of CD81, and thus activity, in a cell
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding CD81 can be synthesized, e.g., by conventional phosphodiester techniques.
  • RNAs small double stranded RNA
  • dsRNA small double stranded RNA
  • RNAi RNA interference or RNAi
  • Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector.
  • CD81 antibodies CD81 antibodies:
  • the agent is an antibody having binding affinity for CD81. In some embodiments, the agent is an antibody directed against at least one extracellular domain of CD81. In some embodiments, the agent is an antibody directed against the C and/or D helices of CD81 large extracellular loop. In some embodiments, the agent is an antibody directed against SEQ ID NO:2 (LTALTTSVLKNNLCPSGSNIISNLFKE). In some embodiments, the agent is an antibody directed against SEQ ID NO:3 (LTALTTSVLKN). In some embodiments, the agent is an antibody directed against SEQ ID NO:4 (NIISNLFKE). In some embodiments, the anti-CD81 antibody binds to the same epitope as 5A6 antibody.
  • the anti-CD81 binds at least Ser 179 , Asn 180 and Phe 186 residues of SEQ ID NO:1. In some embodiments, the anti-CD81 binds at least His 151 , Ala 164 , Ser 168 and Asn 172 residues of SEQ ID NO:1. In some embodiments, the anti-CD81 antibody is 5A6 antibody. In some embodiments, the anti-CD81 antibody comprises the VH domain that consists of the sequence as set forth in SEQ ID NO:5 and the VL domain that consists of the sequence as set forth in SEQ ID NO:6.
  • the anti-CD81 antibody comprises the VH domain that consists of the sequence as set forth in SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9 and the VL domain that consists of the sequence as set forth in SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10.
  • SEQ ID NO : 8 > VL domain of an anti-CD81 antibody
  • the antibody is an anti-CD81 neutralizing antibody. In some embodiments, the antibody leads to the depletion of CD81 -expressing cancer cells. In some embodiments, the antibody leads to the inhibition of the TGF-beta production by T cells that contribute to immune escape of the tumor cells. In some embodiments, the antibody leads to the depletion of CD81 expression cancer cells. In some embodiments, the antibody is directed against at least one extracellular domain of CD81. In some embodiments, the antibody is directed against the C and/or D helices of CD81 large extracellular loop. In some embodiments, the antibody is a humanized antibody or a chimeric antibody. Anti-CD81 antibodies are well- known in the art. As example, antibodies targeting CD81 are described in WO2017/218691, US8440797B2 or WO2012/077649.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • non-specific cytotoxic cells e.g., Natural Killer (NK) cells, neutrophils, and macrophages
  • NK Natural Killer
  • macrophages e.g., neutrophils, and macrophages
  • FcRs Fc receptors
  • Fc region includes the polypeptides comprising the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains
  • IgA and IgM Fc may include the J chain.
  • Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2).
  • PBMC peripheral blood mononuclear cells
  • NK Natural Killer
  • ADCC activity of the molecules 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).
  • effector cells are leukocytes which express one or more FcRs and perform effector functions.
  • the cells express at least FcyRI, FCyRII, FcyRIII and/or FcyRIV and carry out ADCC effector function.
  • human leukocytes which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.
  • the antibody suitable for depletion of cancer cells is a full-length antibody.
  • the full-length antibody is an IgGl antibody.
  • the full-length antibody is an IgG3 antibody.
  • the antibody suitable for depletion of cancer cells comprises a variant Fc region that has an increased affinity for FcyRIA, FcyRIIA, FcyRIIB, FcyRIIIA, FcyRIIIB, and FcyRIV.
  • the antibody of the present invention comprises a variant Fc region comprising at least one amino acid substitution wherein said at least one amino acid substitution is selected from the group consisting of: S239D, A330L, A330Y, and 1332E, wherein amino acid residues are numbered following the EU index.
  • the glycosylation of the antibody suitable for depletion of cancer cells is modified.
  • an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated or non-fucosylated antibody having reduced amounts of or no fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, 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 in which to express recombinant antibodies of the present invention to thereby produce an antibody with altered glycosylation.
  • EPl 176195 by Hang et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation or are devoid of fucosyl residues. Therefore, in some embodiments, the human monoclonal antibodies of the present invention may be produced by recombinant expression in a cell line which exhibit hypofucosylation or non-fucosylation pattern, for example, a mammalian cell line with deficient expression of the FUT8 gene encoding fucosyltransferase.
  • PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277:26733-26740).
  • PCT Publication WO 99/54342 by Umana et al.
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • Eureka Therapeutics further describes genetically engineered CHO mammalian cells capable of producing antibodies with altered mammalian glycosylation pattern devoid of fucosyl residues (http://www.eurekainc.com/a&boutus/companyoverview.html).
  • the human monoclonal antibodies of the present invention can be produced in yeasts or filamentous fungi engineered for mammalian- like glycosylation pattern and capable of producing antibodies lacking fucose as glycosylation pattern (see for example EP1297172B1).
  • the antibody suitable for depletion of cancer cells mediated complement dependant cytotoxicity.
  • the chimeric antigen receptor of the present invention comprises at least one VH and/or VL sequence of an antibody that is specific for CD81.
  • the portion of the CAR of the invention comprising an antibody or antibody fragment thereof that is specific for CD81 may 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 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, N.Y.; Houston et al., 1988, Proc.
  • the agent of the present invention is administered to the patient in the form of a pharmaceutical composition which comprises a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.
  • the used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • an antibody present in a pharmaceutical composition of this invention can be supplied at a concentration of 10 mg/mL in either 100 mg (10 mL) or 500 mg (50 mL) single-use vials.
  • the product is formulated for IV administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and Sterile Water for Injection.
  • the pH is adjusted to 6.5.
  • An exemplary suitable dosage range for an antibody in a pharmaceutical composition of this invention may between about 1 mg/m 2 and 500 mg/m 2 .
  • these schedules are exemplary and that an optimal schedule and regimen can be adapted taking into account the affinity and tolerability of the particular antibody in the pharmaceutical composition that must be determined in clinical trials.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 CD81 expression on normal or Sezary cells.
  • A Gating strategy for Sezary cells phenotyping. Cells were stained with an anti-CD3, anti-CD4 and anti-CD158k to determine tumor cell population. CD81 expression was detected with the 5A6 anti-CD81 clone.
  • MFI mean fluorescence Intensity
  • Anti-CD81 antibody 5A6 induces cell death of Sezary and Mycosis Fungoi'de cells but not healthy peripheral blood cells. HUT78, Seax or Myla cells were incubated 48h at 37°C, 5% CO2, in complete RPMI1640 medium at 2x 10 5 cells/ml with mouse IgG isotype control (mlgc) or anti-CD81 antibody (5A6 clone). Cell viability was measured with VD staining marker (viability dye e780). PBMC from HD were treated similarly and IL-2 added to the medium at lOng/ml. Figure 3. Anti-CD81 antibody 5A6 induces cell death of tumor cells from patient.
  • Purified PBMC from Sezary patient were incubated 72h at 37°C, 5% CO2, in complete RPMI1640 medium at 2x 10 5 cells/ml with mouse IgG isotype control (mlgc) or anti-CD81 antibody (5A6 clone) at lug/ml, with or without IL-2 at lOng/ml.
  • Cell viability was measured with VD staining marker (viability dye e780) on CD4+ CD158k+ tumor cells (SS cells).
  • Anti-CD81 antibody 5A6 induces cell death of NK/T lymphoma (SNK6), acute T cell leukemia (MOLT-4) and HSTL (DERL2) cells.
  • SNK6 NK/T lymphoma
  • MOLT-4 acute T cell leukemia
  • DEL2 HSTL
  • Cells were incubated 48h at 37°C, 5% CO2, in complete RPMI1640 medium at 2x 10 5 cells/ml with mouse IgG isotype control (mlgc) or anti-CD81 antibody (5A6 clone). Cell viability was measured with VD staining marker (viability dye e780).
  • IL-2 was added at lOng/ml for SNK-6 and DERL-2 cell lines culture.
  • the anti-CD81 5A6 mAb induces cell death through caspase 3 cleavage.
  • Cells were incubated 2 hours with the 5A6 or IgG mab (2ug/ml) before washing and fixation/permeabilization for caspase 3 staining.
  • Cell death was monitored by Viability dye staining (VD efluor780).
  • CD81 is overexpressed on tumoral Sezary cells ( Figure 1);
  • the anti-CD81 A6 antibodies induce cell death through caspase 3 cleavage ( Figure 5).

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Abstract

La présente étude du phénotype T régulateur de cellules de Sézary a conduit à la découverte de la surexpression de CD81 par des cellules de Sézary. CD81 s'est également révélé être une cible thérapeutique pertinente dans le traitement du syndrome de Sézary, du lymphome NK/T, du lymphome T-cell hépatosplénique et de la leucémie aiguë des cellules T. Le CD81 apparaît donc en tant que marqueur diagnostique et en tant que cible thérapeutique dans des malignités des lymphocytes T. Par conséquent, la présente invention se rapporte à CD81 utilisé en tant que biomarqueur et cible biologique dans des malignités de lymphocytes T.
EP23790555.9A 2022-10-12 2023-10-11 Cd81 utilisé en tant que biomarqueur et cible biologique dans des malignités de lymphocytes t Pending EP4602372A1 (fr)

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