Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K40/00—Cellular immunotherapy
  • A61K40/10—Cellular immunotherapy characterised by the cell type used
  • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K40/00—Cellular immunotherapy
  • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
  • A61K40/22—Immunosuppressive or immunotolerising
• • A—HUMAN NECESSITIES
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
• • A—HUMAN NECESSITIES
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/418—Antigens related to induction of tolerance to non-self
• • A—HUMAN NECESSITIES
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  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
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  • A61K40/00—Cellular immunotherapy
  • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
  • A61K40/41—Vertebrate antigens
  • A61K40/42—Cancer antigens
  • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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• • A—HUMAN NECESSITIES
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  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/515—Animal cells

Definitions

• the present invention relates to the fields of biology, genetics and medicine.
• the invention describes methods and compositions for the identification, production and ex vivo and in vivo manipulation of suppressor T cells or lymphocytes (Ts, including their pTs precursors), also called regulatory T (or Treg), and use of said suppressor lymphocytes to control various pathological conditions, including diseases associated with abnormal activity of effector lymphocytes and / or regulatory / suppressor T cells.
• Ts suppressor T cells or lymphocytes
• Treg regulatory T
• the invention relates to the preparation of such compositions based on Ts and pTs lymphocytes, and their use in the context of cellular and / or gene therapies.
• compositions or cell populations based on Ts and pTs lymphocytes obtained in the context of the invention are in particular suitable for the treatment of genetic or acquired diseases, in particular tumors, autoimmune diseases, allergies, graft disease against the host, transplant against infection (GNI) or transplant against leukemia (GNL) effects, inflammatory diseases including, for example, atherosclerosis, diabetes, viral, bacterial or parasitic infections, immune reconstitution or induction of tolerance in the event of transplantation of stem cells, tissues or organ in a mammal.
• diseases including, for example, atherosclerosis, diabetes, viral, bacterial or parasitic infections, immune reconstitution or induction of tolerance in the event of transplantation of stem cells, tissues or organ in a mammal.
• Ts cells express a T receptor specific for the antigen, like other T lymphocytes, but their overall action is partly non-specific with the possibility of recruiting other additional suppressor T lymphocytes by a phenomenon called "infectious suppression".
• CD4 + / CD25 + regulatory cell population which represents less than 5% of CD4 + T cells, has also been described.
• Ts cells play a major role in the control of autoimmune diseases such as type I diabetes or graft versus host disease (GNH) induced by allogenic T cells.
• Addition of Ts cells to grafts containing allogeneic hematopoietic stem cells and effector T cells can control the onset or emergence of GNH.
• Injection of Ts cells can reduce the autoimmune response in autoimmune polymyositis (unpublished).
• Ts cells also play a major role in establishing or inducing tolerance during the transplantation of tissues or organs and / or in the presence of immunogenic molecules such as transgenes.
• Ts cells also play an important role in modulating the response to infectious agents, including intracellular bacteria and viruses.
• Ts cells play a role in several inflammatory diseases such as atherosclerosis. In this case, the absence or reduction of the number of Ts cells leads to an acceleration of the development of the disease and an increase in its severity (unpublished results).
• Ts cells prevent the development of effector anti-tumor responses, which can otherwise lead to the eradication of tumors.
• depletion of Ts cells leads in many cancer models to the eradication of tumors by an effector immune response.
• Ts are associated with reduced survival.
• the drug modulation of Ts improves treatments based on Tumor Infiltrating Lymphocytes.
• Ts cells are also important during vaccination since they can suppress the development of a specific immune response. Likewise, depletion or reduction in the number of Ts cells very significantly improves the effects of an anti-cancer vaccination.
• Ts cells The characterization of Ts cells is therefore of major importance. Although few elements are known concerning the homeostasis and the regulation of this population of Ts lymphocytes, it appears that the transcription factor Foxp3 plays an important role in the development and the functioning of suppressor T lymphocytes CD4 + / CD25 +. It is not established that Foxp3 is expressed on all Ts cells but the absence of expression of Foxp3 in mice is correlated with a dramatic loss of functions of Ts cells, while the forced expression of Foxp3 in lymphocytes T effectors transform these into Ts cells.
• CD4 and CD25 characterize a population of cells which contains the suppressor T lymphocytes, it appears in fact that the suppressive functions are not entirely carried by the CD4 + / CD25 + cells and especially that all the CD4 + / CD25 + cells are not suppressor.
• CD25 is a marker also expressed by activated effector T cells. The identification and purification of Ts cells on the basis of such a marker is a major problem since they present the risk of identifying and actually purifying activated effector T cells.
• activated T lymphocytes expressing CD4 and CD25 have a high probability of precisely containing the effector T against which a therapeutic intervention is desirable.
• the use of CD4 and CD25 in a diagnostic setting (identification) would not be reliable, and in a therapeutic setting (purification, injection) would run the risk of being ineffective or even increasing the pathology.
• the best marker currently known to be able to differentiate Ts cells from activated effector T lymphocytes is the expression of the transcription factor Foxp3.
• This intracellular transcription factor cannot be used in the context of simple immunophenotypic identification and purification methods.
• Other markers such as CD62L allow better characterization of Ts cells but they are far from allowing perfect identification.
• suppressive activities exist in the CD4 + / CD25- population as well as in certain CD8 + cells. It therefore appears that the diagnostic and therapeutic use of Ts cells is clearly dependent on their own identification and that current knowledge has so far described no specific marker for suppressor T lymphocytes.
• Ts lymphocytes In addition, if the differentiation of certain Ts lymphocytes seems to be carried out in the thymus (they are often called “natural” Ts), other Ts lymphocytes could be generated at the periphery and nothing is known about the ontogenic development of Ts at from the T progenitors
• the present invention offers for the first time the possibility of identifying, isolating, analyzing (transcriptome, proteome, etc.) and of manipulating (culture, activation, depletion, genetic modifications, etc.) of T cell populations. suppressive, in particular human, and in particular (i) populations of Ts precursors, and (ii) populations of pure Ts among CD4 + and CD8 + cells.
• CD90 also called THY-1
• THY-1 represents a characteristic marker of human Ts cells CD4 + and / or CD8 +, and of their precursors and can be used effectively to identify these cell populations.
• the THY-1 antigen (Seki et al., 1985; Planelles et al., 1995) corresponds to a well-characterized surface glycoprotein anchored to the membrane by a phosphatidyl-inositol bridge. This protein belongs to the immunoglobulin superfamily and contains around 140 amino acids (25-30 kDa). This antigen was first identified as a differentiation marker expressed in the thymus and the mouse brain. In humans, THY-1 is expressed on a small percentage of fetal thymocytes, on immature hematopoietic progenitors CD34 + and on less than 1% of CD3 + lymphocytes present in the peripheral circulation. THY-1 is also expressed on mesenchymal cells, endothelial cells as well as on several continuous cell lines. The function of THY-1 is not known.
• THY-1 is expressed at the level of the thymus on the progenitors and precursors T. It is also expressed on regulatory cells (Mukasa et al., Clin. Exp. Immunol. 96 (1994) 138; Torre-Amione and al., Cell. Immunol. 124 (1989) 50; Sakatsume et al., Int. Immunol. 3 (1991) 377) as well as on all circulating T lymphocytes. Therefore, it cannot constitute a discriminating marker of a particular cell type. Furthermore, two isoforms Thy-1.1 and Thy-1.2 have been described in mice.
• the inventors have now discovered that, surprisingly, the expression of the THY-1 molecule in humans is very closely correlated with Ts activity, and that the THY-1 molecule constitutes a specific marker for suppressor T lymphocytes, allowing in particular the identification, selection, expansion or depletion in vitro or in vivo of precursors of Ts and / or pure Ts populations among CD4 + or CD8 + lymphocytes.
• a first aspect of the present invention therefore relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors), comprising a step of selection, separation, and / or isolation of T lymphocytes expressing the THY-1 molecule. This step can be carried out from any biological samples comprising lymphocytes.
• a more particular subject of the present invention relates to a method for obtaining, preparing or producing suppressor T lymphocytes (and / or their precursors) comprising:
• the T lymphocytes expressing the THY-1 antigen are preferably selected, separated, isolated, recovered or eliminated by means of a specific ligand for THY-1.
• the ligand is chosen from an antibody or an antibody fragment.
• the ligand can for example be immobilized on a support or placed in solution. Such a ligand is more fully defined in the following description of the invention.
• step (b) can be preceded and / or followed by a step of amplification of the T lymphocytes and / or of a step of purification of subpopulation (s) of lymphocytes, such as for example the CD4 + lymphocytes. or CD8 +, or even lymphocytes specific for a given antigen.
• Another subject of the invention relates to a method of identifying and / or quantifying suppressor T lymphocytes (and / or their precursors) in a cell population, comprising the exposure of said cell population to a specific ligand for THY-1 and determining and / or quantifying the formation of a complex between the ligand and the cells, the formation of such complexes indicating the presence and / or the amount of suppressor T lymphocytes (and / or their precursors) within the cell population.
• Ligand-binding cells can be separated from non-ligand-binding cells.
• Another subject of the invention relates to the use of a specific ligand for the THY-1 antigen for enriching or eliminating ex vivo the suppressor T lymphocytes (and / or their precursors) of a cell population.
• the THY-1 antigen can itself be used as a marker for the selection of Ts or pTs lymphocytes within a cell population.
• Another object of the invention resides in a method of diagnosing a patient, comprising determining the presence, number or state of activity of Ts cells in this patient using a specific ligand for THY-1 .
• Such a diagnosis can be carried out in vitro, ex vivo or in vivo, and allow the detection of a pathological condition linked to the activity of the immune system, or the monitoring of the effectiveness of a treatment, or the selection of a patient for inclusion in a particular therapeutic program.
• Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for selecting, identifying, sorting or preparing (in vitro or ex vivo) Ts or pTs lymphocytes.
• the invention further relates to suppressor T lymphocytes (and / or their precursors) expressing the THY-1 antigen capable of being obtained by a method according to the invention.
• Another object of the invention resides in the use of a specific ligand for the THY-1 antigen for preparing a diagnostic composition intended for selecting, identifying or quantifying in vivo suppressor T lymphocytes (including their precursors).
• Another subject of the invention relates to the use of a specific ligand for the THN -1 antigen for preparing a therapeutic composition intended to modify, stimulate or suppress suppressor T lymphocytes in vivo.
• a particular object of the invention relates to the use of a specific THY-1 ligand for enriching or eliminating ex vivo or in vivo suppressor T lymphocytes (including their precursors) from a cell population.
• THY-1 antigen as a selection marker for enriching or eliminating, in vivo, in vitro or ex vivo, Ts or pTs lymphocytes within a cell population.
• the invention further relates to suppressor T lymphocytes (including their precursors) expressing the THY-1 antigen capable of being obtained by a method as defined above, as well as a population of cells enriched in Ts or pTs cells, in which at least 30%, preferably at least 50%, even more preferably at least 65% of the T cells express the THY-1 antigen.
• Particularly preferred compositions or cell populations according to the present invention comprise at least 75%, preferably at least 80% of Ts or pTs cells expressing THY-1, more preferably at least 85, 90 or 95%.
• the invention also relates to an isolated human T lymphocyte, characterized in that it exhibits suppressive activity and in that it expresses the markers CD8 or CD4 and THY-1, as well as a population of cells comprising suppressive T cells CD8 + THY-1 + or CD4 + THY-1 +, preferably a population comprising at least 50, 60, 70, 80, 85, 90 or 95% of CD8 + THY-1 + T cells.
• Such cells can, in part, also express the CD25 antigen.
• the T lymphocytes present in the mammalian cell population or the Ts or pTs lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express biological products of interest , allowing in particular to improve their efficiency and / or their safety of use.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising cells or cell populations as defined above, typically in combination with a pharmaceutically acceptable vehicle or excipient.
• Another particular subject of the invention relates to a pharmaceutical composition
• a pharmaceutical composition comprising suppressor T cells (and / or their precursors) amplified ex vivo and an adjuvant or a pharmaceutically acceptable medium, said amplified cells being enriched in cells expressing the THY-1 antigen and, possibly, in specific cells of a particular antigen, such as allergens, auto-antigens, allo-antigens or antigens of infectious agents.
• the antigen is involved in or specific to a pathological condition chosen from an immune disease, in particular autoimmune diseases, inflammatory diseases, graft versus host disease, allergy and rejection of 'transplant.
• the invention also relates to the preparation of a composition consisting of at least one such suppressor T lymphocyte, of a population enriched in Ts cells and / or pTs as defined above or on the contrary of a population depleted in cells.
• Ts and / or pTs and an adjuvant or a pharmaceutically acceptable medium as well as the composition itself intended for the implementation of a therapeutic method.
• a particular subject of the invention thus also relates to a method of producing a pharmaceutical composition, comprising:
• Another particular object of the invention thus also relates to a method for producing a pharmaceutical composition, comprising: (a) obtaining a biological sample comprising T lymphocytes, (b) depletion of T lymphocytes expressing the THY-1 antigen within this biological sample, and (c) the conditioning of said T lymphocytes obtained which do not express the THY-1 antigen in an adjuvant or a pharmaceutically acceptable medium.
• the invention further relates to a kit for isolating or characterizing Ts cells comprising a specific ligand for THY-1, optionally placed on a support or placed in solution as well as, possibly, reagents for detecting the ligand.
• the ligand is typically placed in a container, such as a plate, syringe, tube, pipette, vial, etc.
• a kit can also be used to diagnose the presence of such Ts cells and pTs from a biological sample taken from the individual to be tested or directly in vivo.
• the invention further relates to a kit or a composition for eliminating Ts cells and pTs in vivo, in vitro or ex vivo, comprising a specific ligand for THY-1, optionally placed in solution or on a support, and coupled to a product. toxic (radioactive, toxins ).
• the invention also relates to the use of a Thy-1 ligand to specifically target a viral or non-viral vector in Ts and pTs in order to express genes.
• the invention further relates to a kit or a composition for activating Ts and pTs cells in vivo, ex vivo or in vitro, comprising a specific ligand for THY-1, optionally placed in solution or on a support, coupled to a product capable activate T cells (eg cytokme, such as IL2, IL7, IL 10, IL15).
• a product capable activate T cells eg cytokme, such as IL2, IL7, IL 10, IL15.
• the invention also relates to the use of a THY-1 ligand to specifically target a viral or non-viral vector in Ts in order to express activator genes or all therapeutic genes.
• Ts cells the compositions made up of isolated or amplified Ts and pTs cells and the compositions enriched in Ts and pTs cells obtained within the framework of the present invention can be advantageously used within the framework of experimental or therapeutic applications.
• the cells used in the context of the present invention are mammalian cells, typically human.
• the invention can also be used in particular in primates, and therefore also relates to primate suppressor T cells, in particular monkey.
• a particular subject of the invention thus also relates to methods of analysis and of obtaining genetic sequences specifically expressed in suppressor T lymphocytes (or their precursors), a method comprising the isolation of RNA from a population of T lymphocytes expressing the Thy-1 antigen, comparison of said RNA to RNA extracted from a population of non-suppressor T cells and recovery of RNA specific for suppressor T cells.
• the invention also relates to a method as described above, further comprising the manufacture of a probe from the RNA specific for suppressor T lymphocytes (or their precursors) and the screening of a population of nucleic acids intended to be hybrid to said probe.
• One method also corresponds to the analysis of the transcriptome by RNA hybridization on biochips in order to establish expression profiles.
• a particular object of the invention thus relates to a method for obtaining proteins specifically expressed in suppressor T lymphocytes (or their precursors).
• One method includes isolating proteins from a population of T cells expressing the Thy-1 antigen, comparing these proteins to those extracted from a population of non-suppressor T cells.
• a particular object of the invention thus relates to a method of identifying new molecules specifically expressed in suppressor T lymphocytes (or their precursors) by immunization using Ts lymphocytes (or pTs) expressing the Thy- antigen. 1, or cell or protein fractions of these same cells.
• Ts cells or pTs
• compositions made up of isolated or amplified Ts cells or pTs
• compositions enriched in Ts cells obtained in the context of the present invention, for example for treating numerous subjects, for example human patients suffering from or at risk of developing an immune disease, in particular a disease caused by a abnormal T response.
• Ts cells are thus suitable for the treatment of various pathologies or conditions caused by a disorder affecting T lymphocytes and in particular a tumor, an autoimmune disease, an allergy, graft versus host disease, a inflammatory disease, type I diabetes, viral or bacterial infection, etc.
• Treatment can be preventive or curative. It can also be combined with other treatments.
• suppressor T lymphocytes designates a population of T cells which are characterized by their capacity to suppress or reduce the immune reactions mediated by effector T cells, such as CD4 + T cells or CD8 +.
• This term includes conventional Ts cells, which strongly express the CD25 marker, as well as their precursors, designated pTs, which are endowed with suppressive activity and can give rise, in culture, to conventional Ts cells.
• the invention indeed demonstrates the existence of a population of suppressive T cells, designated pTs, expressing the markers THY-1 and CD25, endowed with suppressive property, and capable of giving rise in culture to conventional Ts cells.
• suppressor T lymphocytes also includes Ts lymphocytes originating from total lymphocyte populations (or CD25-), of the CD4 + or CD8 + type, expressing THY-1.
• CD4 and CD25 characterize suppressor T lymphocytes (or their precursors) on an immunophenotypic level, it appears in fact that the suppressive functions are not entirely carried by CD4 + cells / CD25 + and that all CD4 + / CD25 + cells are not suppressive.
• CD25 is indeed a marker also expressed by activated effector T cells.
• THY-1 represents a characteristic marker human Ts and pTs cells and can be used effectively to identify this cell population.
• the present invention thus relates, as indicated above, to a method for obtaining, preparing, selecting or producing human suppressor T lymphocytes (including their precursors) comprising:
• the cell population can be obtained, in the context of step (a), from biological samples comprising lymphocytes, in particular from samples of a tissue chosen from bone marrow, spleen, liver, thymus , blood that may or may not have been previously enriched for T lymphocytes, umbilical cord blood, peripheral fetal blood, newborns or adults, plasma, a lymph node, a tumor, a inflammatory site, a transplanted organ, or a cell culture established with either of these tissues. Lymphocytes are typically isolated or collected from peripheral blood.
• the T lymphocytes expressing THY-1 can be recovered, selected, isolated, removed or sorted, in particular during step (b), using any specific ligands for THY-1, that is to say typically of all molecules capable of selectively binding Thy-1 to the surface of a cell.
• the ligand is preferably chosen from an antibody, preferably an anti-THY-1 antibody, an analog or a fragment of such an antibody.
• THY-1 is a molecule devoid of an intra-cytoplasmic domain which interacts with the cell membrane via a glycophosphatidylinositol (GPI) which attaches to the membrane by its C-terminal end.
• GPI glycophosphatidylinositol
• the sequence of Thy-1 has been determined and is accessible in the literature, such as for example the nucleotide sequence (no NM 006288 (gi: 199 233 61)) and the amino acid sequence (no P 006279 (gi: 199 233 62)) of human protein.
• a specific ligand according to the invention is preferably a molecule capable of selectively binding a polypeptide comprising all or part of the sequence of the human Thy-1 protein, preferably a molecule comprising an epitope of the human Thy-1 protein.
• Such ligands are naturally chosen from recognized molecules and / or capable of interacting with the extracellular part of THY-1.
• a preferred ligand for THY-1 which can be used in the context of the present invention, is an anti-THY-1 antibody (that is to say an antibody specific for THY-1).
• the antibody can be polyclonal or monoclonal. It can also be fragments and derivatives of a fragment or derivative of antibodies having substantially the same antigenic specificity, in particular fragments of antibodies (eg, Fab, Fab'2, CDRs), of humanized antibodies, human antibodies, polyfunctional, single-stranded (ScFv), or multimeric antibodies (C4bp coupling for example), etc.
• the antibodies, and therefore the recognition sites of the THY-1 molecule which can be used to generate a specific ligand can be produced using conventional methods, comprising the immunization of a non-human animal with a THY-1 polypeptide or a fragment thereof comprising an epitope, and the recovery of its serum (polyclonal) or of spleen cells (so as to produce hybridomas by fusion with appropriate cell lines).
• a non-human animal with a THY-1 polypeptide or a fragment thereof comprising an epitope
• serum polyclonal
• spleen cells so as to produce hybridomas by fusion with appropriate cell lines.
• Various methods of producing polyclonal antibodies from various species have been described in the prior art.
• the antigen is combined with an adjuvant (eg Freund's adjuvant) and administered to an animal, for example by subcutaneous injection. Repeated injections can be given. Blood samples are collected and immunoglobulin or serum are separated.
• an adjuvant e
• monoclonal antibodies include immunizing a non-human animal with an antigen, followed by the recovery of spleen cells which are then fused with immortalized cells, such as myeloma cells.
• the resulting hybridomas produce monoclonal antibodies and can be selected by limiting dilutions so as to isolate the individual clones.
• the Fab or F (ab ') 2 fragments can be produced by digestion using a protease according to conventional techniques.
• the preferred antibodies are antibodies specific for the THY-1 protein, that is to say having a higher affinity for this protein than for other antigens, even if a non-specific or less affine bond cannot be excluded.
• the term “specific” or “selective” indicates in particular that the binding of the ligand to the THY-1 protein can be discriminated from the possible binding of the ligand to other molecules.
• Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with specific ligands, such as those defined above.
• specific ligands such as those defined above.
• specific ligands according to the invention are in particular the monoclonal antibodies produced by the hybridomas Kl 7 (ATCC n ° HB- 8553), the clones 5E10, F15-42-1, Thy-1/310, FIB1 (clone AS02 ), as well as any fragments or derivatives of these antibodies.
• ligands according to the invention are for example artificial ligands, having a particular affinity for THY-1.
• Such ligands can be of varied nature, such as nucleic acids (for example aptamers) or synthetic chemical molecules.
• Such molecules can be generated for example on the basis of the sequences of the recognition sites of the THY-1 molecule by the specific antibodies defined above.
• Ts and pTs cells can thus be isolated, in the context of step (b), by bringing the cell population into contact with one or more specific ligands, such as those defined above.
• the invention uses a combination of a specific ligand for THY-1 and a specific ligand for CD25.
• the second ligand can be specific for any other marker of T cells, in particular of suppressor T cells, for example markers identified by the genomics and proteomics techniques described in the present application.
• the ligand (s) can (can) be immobilized on a support, for example a column or a ball (in particular a magnetic ball), or placed (s) in solution.
• the ligand can optionally be labeled.
• the labeling can be carried out using a fluorescent, radioactive, luminescent, phosphorescent, chemical or enzymatic detection marker.
• the detection marker is preferably chosen from fluorescein, Texas red, rhodamine, phycoerythrin, dlophycocyanin, biotin and streptavidin, Cyanine.
• the complexes formed by the ligand and the labeled cells can then be used to visualize, detect, quantify, sort, isolate and / or deploy the cells, according to various techniques which are known per se to those skilled in the art.
• the cells can be recovered, selected, sorted, separated, isolated, depleted for example by a method chosen from flow cytometry, affinity chromatography, FACS (fluorescent activated cell sorting "Fluorescent Activated Cell Sorting"), MACS (magneticbead cell sorting "Magnetic bead Cell Sorting"), D / MACS (double magnetic bead cell sorting), affinity chromatography "Double Magnetic bead Cell Sorting”), a selection method on solid surface (“panning ”), An ELIS A test, an RIA test, etc.
• the MACS procedure is described in detail by Miltenyi et al., "High Gradient Magnetic Cell Separation ith MACS,” Cytometry 11: 231-238 (1990).
• the cells labeled with magnetic beads pass through a paramagnetic separation column.
• the separation column is placed near a magnet, thereby creating a magnetic field inside the column.
• the magnetically marked cells are trapped in the column, the others cross it.
• the cells trapped inside the column are then eluted.
• a sample of cells is labeled using magnetic beads comprising antibodies, and the cells are harvested or sorted by application of a magnetic field.
• the cells for example peripheral blood
• the cells are incubated sequentially with saturating amounts of functionalized anti-THY-1 antibody (eg, labeled with biotin) and with a solid support (for example microbeads) functionalized (eg, coated with streptavidin).
• the cells are then purified by recovery of the support, eg, by magnetic separation of the cells.
• the cells of the positive fraction can be subsequently separated on another column.
• the purification is generally carried out in a phosphate salt buffer, although other suitable media can be used.
• the cells can be cultured or maintained in any suitable buffer or medium, such as a saline solution, a buffer, a culture medium, in particular DMEM, RPMI etc. They can be frozen or kept in a cold situation. They can be formulated in any suitable device or apparatus, such as a tube, a flask, a vial, a dish, a syringe, a bag, etc., preferably under sterile conditions suitable for pharmaceutical use.
• step (b) of the method described above can advantageously be preceded and / or followed by a step of purifying a subpopulation of T lymphocytes (CD4 + and or CD8 + for example) and / or a lymphocyte amplification step (which can be carried out ex vivo or in vitro.).
• a step of purifying a subpopulation of T lymphocytes CD4 + and or CD8 + for example
• a lymphocyte amplification step which can be carried out ex vivo or in vitro.
• Amplification can be obtained by activation of lymphocytes.
• This activation can be non-specific (obtained for example by anti-CD3 and / or anti-CD28 antibodies, with or in the presence of an interleukin, for example IL2) or specific (obtained by antigens or allo-antigens presented adequately to Ts or pTs cells, for example by antigen presenting cells (dendritic cells, B lymphocytes, macrophage monocytes, genetically modified cells capable of presenting the antigen and activating lymphocytes), exosomes, dexosomes, artificial structures, etc.).
• antigen presenting cells dendritic cells, B lymphocytes, macrophage monocytes, genetically modified cells capable of presenting the antigen and activating lymphocytes, exosomes, dexosomes, artificial structures, etc.
• the amplification step makes it possible to increase the number of T lymphocytes present in the starting T lymphocyte population (which includes effector T lymphocytes and suppressor T lymphocytes) before proceed to the selection of Ts and pTs lymphocytes, and / or to increase the number of Ts and pTs lymphocytes after having selected the T lymphocytes expressing the THY-1 antigen. It is also possible to carry out two amplification stages, one concerning the general T lymphocyte population present in the population of mammalian cells, the other concerning the population of Ts and pTs lymphocytes.
• the amplification step is carried out under conditions which favor the Ts (or pTs), thus allowing their enrichment.
• the present invention shows that culture in the absence of N-acetyl cysteine promotes the proliferation of Ts ( Figure 1).
• the cells are amplified by culture in a medium devoid of N-acetyl cysteine.
• the use of certain populations of natural or modified (in particular genetically modified) antigen presenting cells can promote the proliferation of Ts (or pTs).
• the examples show that the dendritic cells derived from CD34 + hematopoietic progenitors and having an interstitial DC type phenotype promote the proliferation of Ts (or pTs) (FIG. 2).
• the cells are amplified by culture in the presence of dendritic cells, in particular interstitial dendritic cells.
• the population obtained at the end of step (a) can also be enriched in T cells belonging to the general population of T cells, ie, comprising effector T lymphocytes and suppressor T lymphocytes or their precursors.
• the population of step (a) can thus be enriched in T cells, possibly in one or more specific subpopulations of lymphocytes (for example CD4 + and / or CD8 +). It can also be rid of certain subpopulations of lymphocytes, if necessary.
• the population obtained at the end of step (a) then, optionally amplified and / or sorted, thus preferably comprises at least 30%, preferably at least 50%, even more preferably at least 65% of cells. T.
• compositions enriched in T cells capable of being used in the context of step (b) comprise at least 75%, preferably at least 80% of T cells.
• the population of T lymphocytes expressing the THY-1 antigen can also be amplified. It is also possible, as indicated above, to carry out two amplification steps, one concerning the general T lymphocyte population, the other concerning the Ts or pTs lymphocyte population.
• a particular object of the present invention thus relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising. : (a) obtaining a population of mammalian cells comprising T lymphocytes, (a ') amplifying T lymphocytes within said population of cells, and (b) recovering T lymphocytes expressing the THY-1 antigen.
• Another particular object of the present invention relates to a method for obtaining suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) recovery of T lymphocytes expressing the THY-1 antigen, and (b ') amplification of said T lymphocytes expressing the THY-1 antigen.
• Cellular amplification of lymphocytes belonging to the general population of T lymphocytes is preferably carried out by culturing the cells in the presence of a cytokine and optionally a stimulating agent.
• a cytokine and optionally a stimulating agent.
• the culture is maintained for a period sufficient to obtain the amplification of said cell population within CD4 + and / or CD8 + T lymphocyte populations.
• Activation usually involves culture in the presence of a cytokine, such as, for example, interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-10 (IL-10 ) or Interleukin-15 (IL-15), preferably of human origin.
• the stimulating agent can be an antigen presenting cell (“APC”), Le., Any antigen presenting cell or any cell promoting activation of T cells, in particular Ts cells.
• APCs are preferably irradiated before their use in order to avoid their amplification.
• CPA can be cells isolated from a donor or from the patient himself. They can be chosen to produce Ts and pTs cells with a chosen activity profile.
• CPA peripheral blood mononuclear cells, dendritic cells, splenocytes, umbilical cord blood cells, tissue or organ samples, etc.
• Ts and pTs cell stimulants include MHC polymers, lectins (such as PHA), antibodies (such as anti-CD3 and / or anti-CD28 antibodies) or fragments thereof, autoantigens (including tissues, cells, cell fragments or debris, purified polypeptides or peptides, etc., preferably in combination with CPA), etc.
• Ts and pTs cells can be amplified in different ways, whether or not they are specific antigens.
• large quantities of the complete repertoire of T cells are preferably used (e.g., injected).
• This technique is, in particular, suitable for patients who have an overall deficit (quantitative or functional) in Ts and pTs cells.
• the Ts and pTs cells are preferably amplified, for example, using autologous CPA and PHA cells or anti-CD3 and / or anti-CD25 antibodies (or any other activator of T cells or Ts) in the presence of cytokines of identical or different nature.
• Ts and pTs lymphocytes in humans and in mice, can be cultured and amplified in vitro in the presence of a culture medium containing interleukin 2, anti-CD3 and anti-CD28 antibodies.
• Specific Ts and pTs lymphocytes can also be isolated, generated for example by stimulation in the presence of cells presenting allogenic antigens, followed by a culture by interleukin 2.
• a more specific amplification can be envisaged, in particular when suppression of specific effector T cells is desired, such as in the context of autoimmune diseases, allergies, transplant rejection, GNHD, etc.
• the cells are preferably amplified in the presence of CPA presenting particular antigens, for example allogenic or of infectious origin, in order to favor the amplification of Ts cells preferentially active against pathogenic effector T cells.
• the antigens are presented in peptide form or after transfer of AR ⁇ or DNA.
• Ts and pTs cells preferably come from the patient and are stimulated using autologous CPA and autoantigens from the target tissue, in the presence of cytokines.
• Autoantigens can be from tissues, cells, cell fragments, purified proteins, peptides, nucleic acids, etc.
• Ts cells preferably come from the patient and are stimulated by APCs or tissues from the donor, in the presence of cytokines. Ts cells from the patient can also be stimulated by autologous APCs in the presence of tissues, cells, cell fragments, purified proteins, or peptides from the donor and cytokines.
• Ts cells typically come from the patient and are stimulated by CPA and allergens in the presence of cytokines.
• the cytokines preferentially used are 1TL-2, IL-10 and / or IL-15.
• the Ts and pTs cells used to treat various pathologies are preferably autologous, Le. , they come from the subject to be treated. Syngene cells can also be used. In other situations, for example in the treatment of GNHD or other pathologies, Ts and pTs cells are typically allogenic, ie, they come from a different human being. In these cases, it is preferable to use Ts and pTs cells originating from a donor subject (eg, the donor subject of effector cells). Genetic modification of T cells and in particular of Ts and pTs cells
• the T lymphocytes (general population of T lymphocytes) present in the population of mammalian cells or the suppressor T lymphocytes (carrying the THY-1 marker) can be genetically modified so as to express organic products of interest.
• the expression "genetically modified” indicates that the cells comprise a nucleic acid molecule which is not naturally present in unmodified T cells, or which is present in these cells when they are not in their natural state ( eg, when amplified).
• the nucleic acid molecule may have been introduced into said cells or into a parent or progenitor cell.
• a particular subject of the invention thus relates to a method for obtaining or producing suppressor T lymphocytes (and / or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, ( b) recovery of the T lymphocytes expressing the THY-1 antigen, and (c) the genetic modification of said T lymphocytes expressing the THY-1 antigen by bringing said lymphocytes into contact with a recombinant nucleic acid molecule.
• Another particular object of the invention relates to a method for obtaining or producing suppressor T lymphocytes (or their precursors) comprising: (a) obtaining a population of mammalian cells comprising T lymphocytes, (b) the genetic modification of said T lymphocytes by bringing said population of cells into contact with a recombinant nucleic acid molecule, and (c) the recovery of T lymphocytes expressing the THY-1 antigen.
• Several approaches can be used to genetically modify the T cells belonging to the mammalian cell population [comparable to the general population of T lymphocytes (effector T cells and Ts cells)] or Ts and pTs lymphocytes, such as, for example, gene delivery via virus, naked DNA, physical treatments, etc.
• the nucleic acid is generally incorporated into a vector, such as a recombinant virus, a plasmid, a phage, an episome, an artificial chromosome, etc.
• the T cells as defined in the preceding paragraph are genetically modified using a viral vector (or a recombinant virus).
• the heterologous nucleic acid is, for example, introduced into a recombinant virus which is then used to infect T lymphocytes.
• Different types of recombinant viruses can be used, in particular recombinant refroviruses or ANAs.
• the T lymphocytes are preferably modified using a recombinant refrovirus. The use of refrovirus is particularly appreciated insofar as refroviral infection allows stable integration of the nucleic acid into the genome of the cells.
• refractive viruses susceptible of being used come from the family of oncoviruses, lentiviruses or spumaviruses.
• Particular examples of the oncovirus family are MoMLN, ALN, BLN or MMTN but also RSN, etc.
• Examples of the lentivirus family are HIN, SIN, FIN, ELAN or CAEN, etc.
• the recombinant refrovirus comprises the envelope of the GALN virus (refrovirus pseudotyped with GALN).
• T cells can be infected using recombinant viruses and various protocols, such as incubation with a virus supernatant, with purified viruses, by coculture of T cells with viral packaging cells, Transwell techniques, etc.
• a particularly effective method comprising a centrifugation step has been described by Movassagh et al. (Movassagh M, Desmyter C, Baillou C, Chapel-Fernandes S, Guigon M, Klatzmann D, Lemoine FM. Hum Gène Ther. 1998; 9: 225-234).
• Non-viral techniques include the use of cationic lipids, polymers, peptides, synthetic agents, etc.
• Alternative methods use the “gun gene” technique, electric fields, bombardment, precipitation, etc.
• Different selection techniques can be used, including the use of antibodies recognizing specific markers present on the surface of the modified cells, the use of resistance genes (such as the neomycin resistance gene and the G418 molecule), or the use of compounds that are toxic to cells that do not express fransgene (ie, thymidine kinase).
• the selection is preferably carried out using a marker gene expressing a membrane protein. The presence of this protein allows selection using techniques separation techniques such as separation using magnetic beads, the use of columns or flow cytometry.
• the nucleic acid used to genetically modify T cells can be a therapeutic fransgene and can code for various active biological products, including polypeptides (e.g., proteins, peptides, etc.), RNAs, etc.
• the nucleic acid encodes a polypeptide having immunosuppressive activity.
• the nucleic acid encodes a polypeptide that is toxic or has conditional toxicity to cells.
• Preferred examples include thymidine kinase (which confers toxicity in the presence of nucleoside analogs), such as HSN-1 TK, cytosine deaminase, gprt, etc.
• nucleic acids can also be a non-toxic polypeptide but which can allow the elimination of the injected cells if necessary (such as for example a molecule expressed at the cell membrane and a monoclonal antibody fixing the complement).
• nucleic acids are those for targeting. They may be nucleic acids encoding a T or B cell receptor or a subunit or a functional equivalent thereof. For example, the expression within Ts cells of a specific recombinant TCR of an autoantigen produces Ts cells and pTs which can act more specifically on effector T cells which destroy tissue in a subject.
• T-bodies ie, hybrid receptors between T cell receptors and an immunoglobulin. Such "T-bodies” allow targeting of antigenic complexes, for example.
• the suppressor T lymphocytes are genetically modified and comprise a recombinant nucleic acid coding for a product having a conditional toxicity for these cells, such as thymidine kinase.
• the genetically modified Ts and pTs cells comprise a recombinant nucleic acid molecule encoding a T cell receptor or a subunit or a functional equivalent thereof.
• the nucleic acid which is introduced into the T cells according to the invention typically comprises, in addition to the coding region, regulatory sequences, such as a promoter and a polyadenylation sequence.
• a particular object of this invention is a composition comprising at least one suppressor T lymphocyte according to the invention, eg isolated, genetically modified and / or amplified ex vivo, or a population enriched in suppressor T cells as defined above or, on the contrary, a population depleted in suppressor T cells, as well as a pharmaceutically acceptable adjuvant or medium.
• Another particular object of the invention is a composition comprising suppressor T lymphocytes (including their precursors) transduced using a first suicide gene and effector T cells which are transplanted using a second suicide gene, different from the first.
• compositions may include other cell types, without significantly affecting the therapeutic benefit of said compositions.
• the cells are packaged in a composition comprising between approximately 10 5 and 10 10 suppressor T cells depending on the pathology to be treated, more generally between 10 5 and approximately 10 9 suppressor T cells.
• a particular composition within the meaning of the invention comprises a population of THY-1 positive human lymphocytes, endowed with suppressive properties with regard to effector T cells.
• the medium or adjuvant can be any culture medium, defined medium, aqueous solution or suspension, buffered, optionally supplemented with preservatives.
• the compositions according to the invention can be administered by any suitable route, such as intravenous, intraarterial, subcutaneous, fransdermal, etc. Repeated administrations of such compositions can be implemented.
• compositions according to the invention comprise a specific Thy-1 ligand coupled or conjugated to an effector molecule, for example a molecule exhibiting a toxicity (conditional or not, for example "a TK, castor toxin, etc.) or a stimulating activity for T lymphocytes (for example a cytokine, in particular IL-2, IL-7, IL-15, etc.).
• an effector molecule for example a molecule exhibiting a toxicity (conditional or not, for example "a TK, castor toxin, etc.) or a stimulating activity for T lymphocytes (for example a cytokine, in particular IL-2, IL-7, IL-15, etc.).
• a cytokine for example a cytokine, in particular IL-2, IL-7, IL-15, etc.
• the administration of a conjugate comprising a toxic molecule can make it possible to inactivate or reduce the suppressor T cell activity in a subject, and therefore to increase the activity
• the administration of a conjugate comprising an activating molecule can make it possible to stimulate the activity of suppressor T cells in a subject, and therefore to reduce the activity of effector cells. be covalent or not.
• compositions of the invention include a transfection agent coupled to a specific Thy-1 ligand.
• the transfection agent can be a viral particle (for example recombinant, defective, attenuated, synthetic, etc.) or a non-viral transfection agent, such as a liposome, a canonical lipid, a polymer, etc.
• the coupling can be covalent or not.
• Such compositions make it possible to modify in a targeted manner the suppressor T cells of a subject, for example to confer on them new properties. uses
• the present invention makes it possible to obtain cell populations which can be used for the treatment of various pathologies associated with the activity of T cells, as indicated above. Treatment can be preventive or curative.
• suppressor T cells including pTs
• cell populations enriched in suppressor T cells including pTs
• the compositions according to the invention can be used in combination with other agents or active ingredients. , such as other cell populations, immunosuppressive molecules or conditions, radiation, gene therapy products, etc.
• treatment means the reduction of symptoms or causes of a disease, the regression of a disease, the delay of a disease, the improvement of the condition of patients, the reduction of their suffering, the increase in their lifespan, etc.
• Suppressor T cells including pTs cells
• cell populations enriched in suppressor T cells including pTs cells
• the compositions according to the invention are particularly suitable for delaying or preventing graft versus host disease (GNHD) in subjects who have undergone an allogeneic organ transplant, in particular bone marrow (or hematopoietic stem cells or non-hematopoietic stem cells).
• GNHD graft versus host disease
• GNHD and the frequent complications caused by transplantation of hematopoietic stem cells are due to the presence of mature donor T cells in the fransplant.
• the elimination of these cells before the transplant leads to a failure of the latter, prolongs immunosuppression and the recurrence of leukemia.
• Ts cells according to the invention at the time of the graft delays or even prevents GNHD.
• This depletion of THY-1 cells may or may not be associated with a depletion using other antibodies such as those specific for CD25.
• Suppressor T cells including pTs
• cell populations enriched in suppressor T cells including pTs
• compositions according to the invention are also suitable for the treatment of autoimmune diseases (including chronic inflammatory diseases ), such as systemic lupus erythematosus, rheumatoid arthritis, polymyositis, multiple sclerosis, diabetes, atherosclerosis etc.
• autoimmune diseases including chronic inflammatory diseases
• Autoimmune diseases have an immunological component as numerous biological and histological investigations have been able to demonstrate. For these diseases, the central element is an inadequate immune response.
• the present invention can be used to prevent, treat, reduce or alleviate such pathologies by administering to the subject an effective amount of suppressor T cells (including pTs cells) to suppress or reduce the activity of these deleterious T cells. Repeated administrations can be carried out if necessary.
• suppressor T cells including pTs cells
• Suppressor T cells including pTs cells
• cell populations enriched in suppressor T cells including pTs cells
• the compositions according to the invention can also be used in the context of the treatment of infectious diseases and in particular pathologies immune systems induced by viruses.
• the immune response against infectious agents can have immunopathological consequences which can lead to death.
• One example is the response to certain viruses responsible for hepatitis. These viruses replicate in hepatocytes and the destruction of these infected hepatocytes by the immune system causes hepatitis, which is sometimes fatal.
• the progression of this chronic hepatitis is accompanied by biological signs and an abnormal immune response (for example, the presence of anti-DNA antibodies or cryoglobulinemia).
• the suppressor T cells (including the pTs), the cell populations enriched in suppressor T cells (including the pTs) and the compositions according to the invention make it possible to eliminate, delete or reduce the active T lymphocytes responsible for the pathology and thus reduce the consequences of immune pathologies induced by viruses.
• Suppressor T cells (including pTs), cell populations enriched in suppressor T cells (including pTs) and compositions according to the invention can also be used for the treatment or prevention of rejection of transplanted organs such as heart, liver, kidneys, lungs, pancreas, etc.
• the usual treatment for a certain number of disorders affecting the organs consists, when it becomes necessary, in replacing the organ by a healthy organ coming from a deceased donor (or from a living donor in certain cases, or even from a donor of another species). This is also the case for the treatment of insulin-dependent diabetes, through the transplantation of cells or of an insulin-producing organ, such as the pancreas or the pancreatic islets. Even if great care is taken in the selection of organ donors with maximum compatibility with histocompatibility antigens, the transplanted organ always leads, with the exception of transplants made between homozygous twins, to development of an immune response directed against the antigens specifically expressed by this organ.
• transplanted organ This is the main cause of failure of allogenic transplants.
• rejection of the transplanted organ is, in the majority of cases, essentially mediated by effector T lymphocytes.
• the present invention now makes it possible to envisage treatment (eg reduction or delay) of organ rejection using suppressor T cells (including pTs cells).
• suppressor T cells including pTs cells.
• Such cells can be prepared from patient cells, stimulated with donor antigens and re-administered to the patient, before or during organ transplantation. Repeated administrations can be carried out if necessary.
• Ts cells are amplified and activated by culture in the presence of autoantigens from the donor tissue. These cells can be produced for example by culture in the presence of dendritic cells autologous to the graft. These amplified and activated suppressor T cells (including pTs cells) can be injected into the patient before, during and / or after organ transplantation, thereby reducing the destructive activity of effector T cells.
• Suppressor T cells including pTs
• cell populations enriched in suppressor T cells including pTs
• compositions according to the invention are also suitable for treating allergies, which are mediated by directed immune responses against specific antigens called allergens.
• suppressor T cells including pTs
• Another subject of the invention relates to a method of decreasing the activity (and or the amount of effector T lymphocytes) in a host mammal, said method comprising the administration to the mammal of suppressor T lymphocytes (or their precursors) according to the invention compatible with said host mammal, preferably autologous.
• suppressor T lymphocytes may also be sought (for example in the context of the treatment of cancer).
• treatment methods can be used.
• a first approach consists in preparing ex vivo cells with activity (for example anti-cancer), depleted in suppressor T cells (including pTs cells). Such depletion can be carried out ex vivo in accordance with a method according to the invention as described above. Depletion can be carried out ex vivo without a prior culture phase and / or after a culture phase in a medium containing N-acetyl cysteine which decreases the proliferation of Ts lymphocytes (including pTs cells) (see FIG. 1).
• the treatment then consists in the re-administration to the patient of a population of T lymphocytes or of a composition comprising such a population lacking T suppressor cells (including the pTs cells), and having or not having been activated ex vivo.
• This treatment may be accompanied by one or more vaccinations (for example anti-tumor), combined or not with chemotherapy and / or radiotherapy, in a patient who has possibly received conditioning.
• This conditioning can in particular comprise lympho-ablative treatments, myeloablative or not, intended to eliminate T lymphocytes, in particular dividing T lymphocytes, and which include suppressor T cells (including pTs cells) responsible for the absence of an effective immune response (like for example the Ts which prevent the development of an effective anti-tumor response).
• Another modality consists in depleting the T suppressors in vivo by the use of ligand and of any adequate toxic activity or molecule (such as for example radioactivity or toxin).
• the treatment of all these pathologies can also be carried out by in vivo modulation (suppression or activation) of suppressor T cells (including ⁇ Ts cells) using any molecule having these activities, and in particular anti-THYl antibodies, or all molecules modulating the activity of suppressor T (including pTs cells) whose discovery results from knowledge of the franscriptome and proteome of suppressor T (including pTs cells).
• Suppressor T lymphocytes can also be activated in vivo, for example by thy-1 ligands coupled to lymphocyte activation molecules (IL2, IL10 for example).
• the treatment can then be administered by general route (intravenous for example) or on the site where the action is sought (in the synovial fluid during the treatment of rheumatoid arthritis for example).
• a particular object of the present application corresponds to the use, in the context of vaccination, of a suppressor T cell (including pTs cells), of a cell population enriched in suppressor T cells (including pTs cells) ) or of a composition according to the invention.
• Various routes of administration and protocols can be implemented in the context of the present invention. They can be adapted by a person skilled in the art depending on the pathology to be treated. Generally, systemic or local administrations can be envisaged and take the intravenous, infra-arterial, infra-peritoneal, infra-muscular or subcutaneous route, etc.
• Cells can be injected during the surgical operation or by any appropriate means, for example using a syringe.
• the cell composition can be administered before, during or after bone marrow transplantation bone (or organ).
• additional administrations may be performed after transplantation, in order to prevent or delay the pathology.
• CD3 + purified T lymphocytes were cultured in RPMI medium containing 10% human serum, interleukin 2, anti-CD3 antibodies and presence or absence of N-acetyl Cysteine (NAC). The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
• Figure 2 Effect of dendritic cells on the preferential expansion or not of human CD4 + CD90 + T cells.
• Dendritic cells derived from CD34 + cells and enriched on the CD la marker (Langerhansian DC) or on the CD 14 marker (interstitial DC) were cultured with allogenic T lymphocytes in a 1/5 ratio for 5 days. The percentage of CD3 + T cells expressing the CD90 marker was studied over time by flow cytometry.
• FIG. 3 Expression of CD25 and CD90 antigens by human CD4 + (A) and CD8 + (B) T cells.
• T cells were labeled with antibodies recognizing the CD4, CD8, CD25 and CD90 antigens. The expression of these different markers was carried out by flow cytometry.
• CD4 + / CD90 + and CD8 + / CD90 + T cells have a suppressive function
• the purified populations CD4CD90 +, CD4CD25 ++ and CD8CD90 + were irradiated with 15 grays, cultured at equal ratio for 4 days with autologous T lymphocytes depressed in CD25 (CD25-) cells stimulated (A) by a mixture of OKT3 / CD28 antibodies. , (B) by allogenic B lymphocytes transformed by EBN, (C) by allogenic dendritic cells (DC). CD25 cells stimulated alone under the same conditions represent the positive control. The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
• CD4, IL-10, CTLA-4 and FoxP3 genes was carried out by RT-PCR on the different CD4 + CD25-, CD4 + CD25 ++, CD4 + CD90 +, CD8 + CD90 + cell populations.
• CD90 identifies the precursors of suppressor lymphocytes CD4CD25 ++
• the cell populations CD4CD90 +, CD4CD25 +, CD4CD25 ++ were highly purified by cell sorting, cultured in RPMI medium containing human serum AB, 1TL-2 and a mixture of antibodies OKT3 / CD28 for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the populations were enriched by cell sorting, irradiated with 15 grays and tested for their suppressive capacity by co-cultivating them in equal ratio with allogenic T lymphocytes doubly depressed in CD25 and CD90 (CD25-CD90-) cells stimulated. by mixing OKT3 / CD28 antibodies.
• CD90 identifies suppressor CD4 + / CD90 + and CD8 + / CD90 + lymphocytes after 6 days of culture
• CD3 T cells were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 6 days. Analysis of the markers CD25 and CD90 on the populations CD4 and CD8 makes it possible to determine the percentages CD25 + and CD90 + (A).
• the CD4CD25 ++, CD4CD90 + and CD8CD90 + cells were then sorted, irradiated and tested for their suppressive capacity by co-culturing them in equal ratio with allogenic T lymphocytes depleted in CD25 (CD25-) cells stimulated by mixing of OKT3 / CD28 antibodies ( B). The proliferation is evaluated after 4 days by incorporation of tritiated thymidine.
• CD90 allows identification from CD25-CD90 T cell culture - the appearance of precursor cells and suppressor lymphocytes
• T lymphocytes doubly depleted in CD25 and CD90 cells were cultured in the presence of IL-2 and a mixture of OKT3 / CD28 antibodies for 7 days. Analysis of the CD90 and CD25 markers was carried out at different culture times. On day 7, the CD25 + CD90 + population was enriched by cell sorting, irradiated with 15 grays and tested for its suppressive capacity by co-cultivating it in equal ratio with allogenic T lymphocytes doubly depleted in CD25 and CD90 cells (CD25-CD90 -) stimulated by mixing OKT3 / CD28 antibodies. The figures indicate the percentage of inhibition of proliferation compared to the control (CD25-CD90- cells cultured and stimulated alone).
• Figure 9 Use of the CD90 marker in human pathology: example of multiple sclerosis.
• Figure 10 Use of the CD90 marker in human pathology: example of a patient suffering from LPEX syndrome.
• Mononuclear cells obtained after Ficoll from samples from healthy donors, from patients with LPEX syndrome confirmed by sequencing of the FoXP3 gene were labeled with monoclonal antibodies CD4, CD25, CD90.
• the percentage of CD4 + T cells expressing the marker CD25 and CD90 was studied by flow cytometry. The results of an LPEX patient are shown.
• the CD90 marker is expressed by human CD4 + / CD25 + T cells and by human CD8 + / CD25 + lymphocytes
• CD90 marker compared to CD25 on the CD4 + and CD8 + T lymphocyte populations
• mononuclear cells from adult peripheral blood were obtained by Ficoll gradient and then labeled with the following antibodies directly to fluorochromes: anti-CD4 , anti-CD8, anti-CD25 and anti-CD90.
• the cells were analyzed by flow cytometry (FACscalibur), the events being re-analyzed on the Cellquest and FlowJo software.
• FIG. 3A shows the expression of the markers CD25 and CD90 on the CD4 + T lymphocytes as well as the co-expression of CD25 and CD90 within the CD4 + cells.
• 6% and 1.2% of the CD4 + T lymphocytes express, respectively, the markers CD25 and CD90.
• the majority (> 80%) of CD4 + / CD90 + cells express CD25 + at an intermediate level while approximately 5% and 15% of CD4 + / CD90 + cells are CD25 ++ and CD25- respectively.
• FIG. 3B shows the expression of the markers CD25 and CD90 on the CD8 + T lymphocytes as well as the coexpression of CD25 and CD90 within the CD8 +. As can be seen, 7% and 0.2% of CD8 + T cells express, respectively, the markers
• CD25 and CD90 are CD25 and CD90. It should be noted that no CD8 + cell strongly expresses CD25 unlike CD4 + cells. The majority of CD8 + / CD90 + cells (75%) are CD25- while about 25% of CD8 + / CD90- cells are CD25 +.
• the CD90 marker identifies human suppressor T lymphocytes in the CD4 + and CD8 + populations.
• CD4 + / CD90 + cells have a suppressive function
• autologous CD4 + T lymphocytes which have been screened in CD4 + / CD25 + cells (CD25-) were stimulated by a mixture of OKT3 / CD28 antibodies previously immobilized on the bottom of the culture wells.
• CD25- cells were cultured alone or in the presence of an equal number of CD4 + / CD90 + or CD4 + / CD25 + cells (positive control) for 4 days then the proliferation was measured by incorporation test for tritiated thymidine, the reading being made on ⁇ counter.
• Figure 4A shows the inhibition exerted populations CD4 + / CD90 + and CD4 + / CD25 ++ on the proliferation of T cells autologous CD25 ".
• CD8 + / CD90 + cells have a suppressive function
• these were brought into contact with an equal number of CD25- cells stimulated by mixing with a mixture of OKT3 / CD28 antibodies, the cell proliferation being evaluated four days later late by tritiated thymidine incorporation test.
• the results presented FIG. 4A show that the CD8 + / CD90 + population exerts a suppressive function on the proliferation of CD25- cells.
• CD4 + / 90 + and CD8 + CD90 + lymphocytes express Foxp3, CTLA4 and TGF ⁇
• CD90 makes it possible to identify a population of lymphocytes that are precursors of CD4 + / CD25-I- lymphocytes.
• the CD4 + / CD90 + cells were highly purified by flow cytometry (purity greater than 98%) and then cultured in a liquid medium in the presence of a mixture of antibodies OKT3 / CD28 and interleukin 2. cells were analyzed by flow cytometry sequentially between 1 day in culture and the 7th day of culture at markers CD4 / CD90 and CD4 / CD25.
• the sorted populations CD4 + / CD25 ++ and CD4 + / CD25 + / CD90- which represent respectively the conventional suppressor T cells and the activated T cells were cultivated in parallel under the same conditions. The results presented in FIG.
• CD4 + / CD90 + cells gradually lose the CD90 marker and become strongly positive for the CD25 marker.
• the CD4 + / CD25 + / CD90- population strongly acquires the CD25 marker without acquiring the CD90 marker.
• the cells were sorted, irradiated with 15 grays and placed in the presence of CD25-allogenic cells.
• CD4 + / CD90 + cells are capable of giving rise to CD4 + / CD25 ++ cells with suppressive activity; 2) CD4 + / CD25 ++ cells retain their suppressive activity; 3) CD4 + / CD25 + / CD90- give rise to CD25 ++ cells without suppressive activity.
• CD4 + / CD90 + cells are capable of giving rise to suppressive CD4 + CD25 ++ cells and can be considered as precursor cells (pTs) of suppressor lymphocytes (Ts).
• CD90 makes it possible to identify the CD4 + / CD90 + and CD8 + / CD90 + suppressor lymphocytes after culture.
• CD90 marker makes it possible, after activation and culture of T lymphocytes, to identify suppressor T lymphocytes unlike the CD25 marker also expressed by activated T lymphocytes.
• FIG. 7A shows the expression of the markers CD25 and CD90 on the T lymphocytes CD4 + and CD8 + after 6 days of culture. As can be seen, 6.9% and 10% of the CD4 + and CD8 + T lymphocytes express the CD90 marker respectively, while 84% and 94.3% of the CD4 + and CD8 + T lymphocytes express the CD25 marker.
• FIG. 7B shows the inhibition exerted by the CD4 + / CD90 + and CD8 + / CD90 + populations on the proliferation of autologous CD25 T lymphocytes, while the CD4 + / CD25 + lymphocytes no longer have any suppressing effect on CD25 cells after culture.
• CD90 + cells become CD90 + CD25 ++ while CD25 + CD90- cells become CD25 ++ CD90-.
• the sorting and the functional study of CD90 + CD25 ++ cells show that these cells inhibit the proliferation of autologous CD25-CD90 T lymphocytes stimulated by OKT3 / CD28 antibodies. These results show that it is possible to generate identifiable suppressor T lymphocytes using the CD90 marker from CD25- CD90- T lymphocytes.
• Ts tissue or organ of interest
• a biological fluid of interest blood, CSF, synovial fluid for example
• tissue or organ of interest tissue or organ of interest
• FIG. 9 shows an increase in CD4 + CD25 + T lymphocytes (activated T lymphocytes) and on the contrary a decrease in CD4 + CD90 + cells during MS in relapses compared to MS in chronic phase or in the control group.
• FIG. 10 shows the virtual absence in the blood of CD4 + / CD90 + cells in an LPEX patient compared to a healthy donor, while the use of the CD25 marker which also recognizes activated T lymphocytes does not make such a difference.
• Ts play an important role in the control of GNH and that it is possible to prepare specific Ts by allo-activation (Cohen et al. JEM 2003, Trénado et al., JCI 2003).
• the Ts can be obtained from blood, cord blood, bone marrow of all tissues containing T lymphocytes. In these applications, they may or may not be genetically modified.
• the Ts are obtained from the patient or from a compatible donor, preferably genome-identical. They are purified by immunomagnetic beads, flow cytometry, by adhesion to a solid support covered with specific antibodies (panning) and possibly frozen.
• the patient is the subject of biological monitoring to assess his number of Ts cells. The appearance of clinical signs indicating the beginning of a surge or fall in the number of Ts leads to the injection of Ts which are either prepared for this occasion, or those prepared previously.
• Ts prevent the development of effective anti-tumor immune reactions.
• the depletion of these cells allows these responses to develop.
• preparation of anti-tumor T cells ex vivo encounters the problem of their contamination by Ts.
• the principle of the treatment is therefore to eliminate the Ts in vivo, in particular using a CD90 ligand coupled to a toxic. It may also involve depleting all of the T lymphocytes by conventional treatments (anti-lymphocyte serum, anti-CD3, Campath antibody, irradiation, etc., for example). This treatment can be supplemented by the administration of lymphocytes activated ex vivo against tumor antigens after having been depleted in Ts.

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