EP1127110A1 - Modifizierte exosome und verwendungen - Google Patents

Modifizierte exosome und verwendungen

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Publication number
EP1127110A1
EP1127110A1 EP99954055A EP99954055A EP1127110A1 EP 1127110 A1 EP1127110 A1 EP 1127110A1 EP 99954055 A EP99954055 A EP 99954055A EP 99954055 A EP99954055 A EP 99954055A EP 1127110 A1 EP1127110 A1 EP 1127110A1
Authority
EP
European Patent Office
Prior art keywords
exosomes
cells
mhc
molecule
peptide
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.)
Withdrawn
Application number
EP99954055A
Other languages
English (en)
French (fr)
Inventor
Philippe Benaroch
Hélène VINCENT-SCHNEIDER
Pamela Stumptner
Sebastian Amigorena
Christian Bonnerot
Graça RAPOSO
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.)
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
Original Assignee
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Curie
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Filing date
Publication date
Application filed by Centre National de la Recherche Scientifique CNRS, Institut National de la Sante et de la Recherche Medicale INSERM, Institut Curie filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP1127110A1 publication Critical patent/EP1127110A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • 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
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • 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

Definitions

  • the present invention relates to the fields of biology and immunology. It relates to membrane vesicles comprising molecules, in particular antigenic, of predetermined structure, and to their uses. It relates more particularly to vesicles comprising recombinant molecules of the major histocompatibility complex, and their use as an immunogen or as a diagnostic or therapeutic tool.
  • the invention also relates to methods of producing these vesicles, genetic constructs, cells and compositions, which can be used for implementing the methods of the invention.
  • the specificity of antigenic recognition is a major characteristic of cells of the immune system. B cells recognize antigens in native form.
  • T lymphocytes recognize the complexes formed by the association of peptides resulting from the degradation of antigens with molecules of the Major Histocompatibility Complex (MHC).
  • MHC Major Histocompatibility Complex
  • Peptides derived from antigens synthesized by body cells (tumor or viral antigens), associate with MHC class I molecules which are recognized by cytotoxic T lymphocytes.
  • Peptides derived from exogenous antigens associate with MHC class II molecules which are recognized by T helper cells.
  • the identification of peptides presented by MHC molecules and recognized by cytotoxic (CD8) or helper (CD4) T lymphocytes was at the origin of new therapeutic and vaccine strategies. This evolution of immunotherapies requires the development of techniques for assessing the specific immune response of antigens.
  • Antigenic peptides associate with MHC molecules in intracellular compartments.
  • MHC molecules For class II molecules, these are composed of vesicles, contained in a larger granule belonging to the endocytic pathway (Peters et al., Nature 349 (1991) 669). Their fusion with the plasma membrane leads, on the one hand, to the expression of peptide-MHC complexes on the cell surface and, on the other hand, to the secretion of these vesicles called exosomes.
  • the present invention relates to new methods and compositions which can be used in the fields of biology and immunology. More particularly, the present invention describes new membrane vesicles whose composition has been modified in a determined manner. In particular, the present invention describes a new method allowing the production, on a custom basis, of vesicles expressing molecules of the MHC complex of known composition, possibly complexed with antigenic peptides of determined structure. The present invention therefore makes it possible to modify the composition of membrane vesicles in a controlled manner, and therefore to create products which are particularly advantageous from the therapeutic, diagnostic or even experimental standpoint.
  • the vesicles described so far comprise, in the best of cases, the endogenous MHC molecules, that is to say the MHC molecules expressed by the cell from which they originate.
  • these molecules are of varied structure, not always identified, and generally multiple, depending on the HLA type of the organism from which they originate.
  • the present invention makes it possible to produce membrane vesicles carrying MHC molecules of defined composition.
  • the vesicles of the invention have the advantage of being very rich in MHC molecules thus determined, and of offering a powerful immunogenic power.
  • the present invention relates in particular to membrane vesicles comprising molecules of predetermined structure, in particular MHC molecules of predetermined structure.
  • the present invention relates in particular to membrane vesicles comprising CMHpeptide complexes of predetermined structure.
  • the present invention also relates to a method of modifying the composition of a membrane vesicle comprising the introduction, into a producer cell of such a vesicle, of a nucleic acid comprising a hybrid region composed of a coding region fused to a addressing region, or of a nucleic acid coding for a protein or polypeptide which, alone or associated with one or more proteins, is naturally addressed in these membrane vesicles.
  • the present invention also relates to membrane vesicles comprising defined antigenic molecules, anchored in the membrane part.
  • the present invention also relates to membrane vesicles comprising molecules of predetermined structure, exposed on their surface, allowing their purification, in particular by affinity methods.
  • the current the invention also relates to membrane vesicles as defined above, further comprising a marker. Such a marker allows in particular the detection of vesicles in a sample, for example their monitoring in vivo.
  • the invention also relates to a process for the preparation of the vesicles defined above, as well as the use of these vesicles.
  • these vesicles can be used as an immunogen, for the preparation of antibodies.
  • these vesicles are used to produce antibodies restricted to MHC, that is to say, specific for a peptide-molecule complex of MHC.
  • a first object of the invention more particularly resides in a membrane vesicle, characterized in that it comprises a recombinant molecule of the major histocompatibility complex.
  • membrane vesicle designates within the meaning of the invention in particular any vesicle composed of a lipid bilayer containing a cytosolic fraction. These vesicles are generally produced by salting out, from cells, and are therefore also designated in the present application by the term "exosome".
  • the membrane vesicles (or exosomes) according to the invention generally have a diameter of approximately 60 to 80 nm.
  • these vesicles advantageously carry membrane proteins which are in the same orientation as in the plasma membrane of the cells from which they originate.
  • the present invention now shows that it is possible to modify the composition of exosomes, in a controlled and specific manner. More particularly, the present invention shows that it is possible to produce membrane vesicles expressing recombinant molecular complexes of (pre) determined composition.
  • such vesicles have particularly advantageous properties, both therapeutically, diagnostically and experimentally.
  • the present invention firstly arises from the selection of particular cell populations for the production of membrane vesicles.
  • the present invention also results from the demonstration that it is possible to introduce recombinant molecules into these cells, and that these molecules are then expressed in a functional and dense manner in the exosomes.
  • the cell used is a cell comprising internal secretion vesicles, cultivable, genetically modifiable, and, preferably, whose internal vesicles can be secreted under the effect of external stimulation.
  • They are essentially mammalian cells, in particular animal cells, but also cells of human origin.
  • it can be primary cultures or immortalized lines.
  • the starting cells are essentially devoid of MHC molecules, that is to say do not express little or no endogenous MHC molecules. This characteristic can prove to be very important in certain applications, as will be illustrated below.
  • the vesicles of the present invention are more preferably capable of being obtained from mast cells or derived from mast cells.
  • the membrane vesicles according to the invention are preferably prepared from cells of mast cells or derived from mast cells.
  • mast cells group together a set of cell types derived from spinal precursors, residing, after differentiation, in epithelia such as the skin, lung, intestine or spleen (Smith and Weis, Immunology Today 17 (1996) 60). These cells are essentially characterized by the fact that their cytoplasm is mainly made up of granules which contain histamine, as well as heparin or proteases, and in that they express on their surface receptors of high affinity for immunoglobulins E (IgE).
  • IgE immunoglobulins E
  • another advantage of the use of mast cells according to the invention lies in the possibility of triggering (in particular strongly stimulating) the exocytosis (ie, the release) of the exosomes by different treatments.
  • vesicles by treatment in the presence of a calcium ionophore or, more physiologically, by stimulation of receptors of high affinity for IgE.
  • These cells have particularly advantageous properties for the implementation of the present invention, namely the presence of internal secretory vesicles, the possibility of cultivating them and of inducing massive exocytosis.
  • these cells can also be genetically modified in a stable manner, which represents a particularly advantageous property for the implementation of the present invention.
  • the vesicles according to the invention have a diameter of approximately 60 to 80 nm, and are produced from cells of mast cells or derived from mast cells.
  • the membrane vesicles of the invention are essentially devoid of endogenous MHC molecules.
  • endogenous MHC molecules that is to say MHC molecules of the vesicle-producing cell
  • the vesicles of the invention are, in a particularly advantageous mode, capable of inducing, in animals, production of antibodies specific for the defined recombinant molecules expressed by them, without detection of directed antibodies against genetically unmodified cells.
  • the term "essentially” lacking designates the fact that certain MHC molecules can be present in very small quantities, difficult to detect by conventional methods, and without notable interference on the antigenic specificity of the vesicles of the invention.
  • recombinant molecules of defined structure for example recombinant peptide-MHC complexes, of defined composition.
  • Such vesicles of the invention are advantageously produced from cells derived from mast cells, which are essentially devoid of endogenous MHC molecules.
  • mast cells accumulate MHC class II molecules in their secretory granules.
  • mast cells are capable of accumulating preferentially the MHC-II-peptide complexes in particular multivesicular intracellular compartments, the secretory granules (Raposo et al., Mol. Biol. Cell 8 (1997) 2619).
  • These cells taken from a mammal, therefore contain endogenous MHC molecules.
  • cell lines derived from mast cells are used, essentially devoid of endogenous MHC molecules.
  • a particular example of a line is the RBL-2H3 line (Barsumian et al, Eur. J. Immunol. (1 1 (1 981) 317). It is understood that any other cell exhibiting the properties set out above can be brought into use. artwork.
  • the expression "defined composition” more particularly designates the fact that the vesicles of the invention have, for example, great antigenic and haplotype specificity.
  • the vesicles described in the prior art generally express MHC molecules of various and unknown haplotypes.
  • the preferred vesicles of the invention express recombinant molecules whose haplotype is predetermined in a precise manner.
  • the term "recombinant” indicates that the molecule results from the expression, in the vesicle-producing cell, of a recombinant nucleic acid encoding this molecule.
  • the membrane vesicles according to the present invention are therefore more preferably produced from cells, established in lines, which are genetically modified to express constituents of predetermined structure.
  • the vesicles of the invention advantageously express defined molecules of the MHC.
  • Human MHC molecules fall into two distinct classes, MHC class I molecules and MHC class II molecules.
  • the vesicles according to the invention express one or more recombinant molecules of the major histocompatibility complex of class IL
  • the molecules of human MHC class II are composed of two chains, an ⁇ chain and a ⁇ chain, the ⁇ chain conferring allelic specificity on the complex.
  • the vesicles according to the invention more specifically express a recombinant ⁇ chain of a molecule of the major histocompatibility class II complex.
  • the vesicles according to the invention express more particularly a recombinant ⁇ chain and a ⁇ chain of a molecule of the major histocompatibility complex of class IL
  • DR1 to DR13 molecules of the DR1 to DR13 type, in particular DR1, DR2, DR3, DR4, DR5, DR6 and DR7.
  • DNA encoding human DRs, particularly DR1-13 can be easily isolated from cells, banks or plasmids by conventional molecular biology techniques. These sequences have in particular been described in Bodmer et al. (Tissue antigens 44 (1994) 1).
  • the exosomes according to the invention therefore express a MHC class II molecule comprising an ⁇ chain and a ⁇ chain selected from haplotypes DR1 to DR13, even more preferably DR1 to DR7.
  • the invention relates to any membrane vesicle comprising a recombinant ⁇ and / or ⁇ chain of a CMH-II molecule of haplotype DR1.
  • the vesicles according to the invention express one or more recombinant molecules of the major histocompatibility class I complex.
  • the MHC class I molecules are also composed of two chains, the transmembrane and polymorphic ⁇ chain, and ⁇ 2-microglobulin, which is constant and soluble.
  • three genetic loci code for the ⁇ chain designated A, B and C.
  • each locus A, B and C of the ⁇ chain is subject to allelic variation.
  • alleles A1, A2, A3, etc. are denoted. A10, Bl, B7, B37, B54, etc., CW3, CW6, etc. (see for example Bodmer et al. supra and Immunogenetics 36, 1992, supra).
  • the exosomes of the invention express an ⁇ chain of a molecule of the conventional MHC-I, that is to say transmembrane and polymorphic. Even more preferably, it is an ⁇ chain of a molecule of the MHC-I of allele Al, A2 or A3.
  • the exosomes according to the invention express an ⁇ chain of a non-conventional MHC-I molecule, that is to say non-polymorphic.
  • a non-conventional MHC-I molecule that is to say non-polymorphic.
  • non-classical CMH-I molecules in humans, which are essentially non-polymorphic.
  • Such molecules have for example been described in Bendelac et al. (Ann. Rev. Immunol. (1997) 535).
  • a preferred example of non-conventional CMH-1 according to the invention is represented by the molecule Cd 1.
  • the invention therefore relates to any membrane vesicle comprising a recombinant protein of a CMH-I molecule.
  • the vesicles of the invention comprise several MHC class I and / or IL molecules.
  • an advantageous vesicle comprises, for example, 2 MHC-II molecules of different haplotypes, or more. Any other combination of MHC molecules is of course possible, such as for example MHC-I and MHC-II.
  • the vesicles of the invention expressing one or more defined MHC complexes are particularly advantageous since they make it possible to present a given antigenic peptide, in a defined MHC context.
  • the membrane vesicles comprise a complex between a defined peptide and the recombinant molecule of the major histocompatibility complex.
  • the vesicles of the invention may also contain one or more other molecules of interest, heterologous, in addition to or in place of the MHC molecules mentioned above.
  • the invention relates to membrane vesicles produced from cells of mast cells or derived from mast cells, characterized in that they contain one or more heterologous molecules of interest.
  • cell "derived” from mast cells designates transformed and / or immortalized lines and / or obtained from cells of mast cells or basophils and having properties of mast cell cells (accumulation of internal secretory vesicles).
  • heterologous indicates that the molecule of interest is not present, in this form, in the exosomes of the invention in the natural state.
  • the molecules of interest carried by or contained in the exosomes of the invention can be any protein, polypeptide, peptide, nucleic acid, lipid, as well as any substance of interest (chemical, biological or synthetic in nature). These molecules can be of a recombinant nature, and can be introduced into the producer cell or directly into / on the exosomes. More particularly preferred types of molecules of interest are in particular MHC molecules, antigens (whole or in the form of peptides), receptor ligands, (specific) ligand receptors, nucleic acids, pharmacological products, markers or even peptides or proteins allowing purification of the vesicles. As antigen, there may be mentioned more particularly any protein, in particular cytoplasmic, of viral or tumor origin.
  • proteins of viral origin mention may in particular be made of any cytoplasmic or membrane protein expressed by the viruses EBV, CMV, HIV, measles, hepatitis, etc. It is more preferably cytoplasmic proteins, that is to say essentially invisible to the immune system in the process of classical infection, and therefore weakly immunogens under natural conditions or also proteins or fragments of membrane proteins.
  • proteins of tumor origin there may be mentioned in particular the proteins p53 (wild or any mutated form present in a tumor), MAGE (in particular MAGE 1, MAGE 2, MAGE 3, MAGE 4, MAGE 5 and MAGE 6), MART (notably MART 1), GplOO, ras proteins (wild or mutated p21), etc.
  • any other protein of interest can be expressed in or on the surface of the exosomes of the invention, following the teaching of the present application.
  • the recombinant antigenic molecules can be present either on the surface of the (exposed) vesicles, or inside the vesicles. Indeed, in a particularly suanterenant way, the inventors have shown that vesicles of the invention containing, in their cytosol, a recombinant antigen (in particular p53) were capable of inducing, in animals, a very high production of antico ⁇ s directed against this antigen.
  • Mention may generally be made, among ligand receptors, of any natural ligand receptor or one derived from genetic manipulation.
  • it can be any hormone receptor, growth factor, lymphokine, trophic factor, antigen, etc.
  • Mention may more particularly be made of the IL1 to IL15 interleukin receptors, the growth hormone receptor, or the receptor for factors stimulating granulocyte and / or macrophage colonies (G-CSF, GM-CSF, CSF, etc. ).
  • a particular example of a ligand receptor is composed of a single-chain antico ⁇ s (ScFv), which allows interaction with a specific ligand.
  • TcR T cell antigen receptor
  • Exosomes of the invention expressing on their surface one or more defined TcR constitute particularly advantageous analysis and diagnostic tools, as will be detailed below.
  • pharmaceutical product mention may be made of any active substance, of a chemical nature, such as for example pharmaceutical products prepared by conventional chemical techniques. Mention may also be made of any protein, polypeptide or peptide having a biological activity, such as for example a toxin, a hormone, a cytokine, a growth factor, an enzyme, a tumor suppressor, etc.
  • the nucleic acid can be any DNA or RNA coding for a pharmacological protein, polypeptide or peptide as mentioned above, as well as any other nucleic acid having a particular property (antisense, antigen, promoter, repressor, binding site).
  • a transcription factor etc.
  • It can be an oligonucleotide, a coding phase, an artificial chromosome, etc.
  • the vesicles of the invention carrying a ligand receptor can be used for the detection of any interaction of receptor-ligand type, in particular of low affinity, in any biological sample, as will be explained in more detail below.
  • such vesicles can also be used to transport the substances of interest (protein, peptide, acid nucleic acid, chemical substance, etc.) to cells.
  • the exosomes of the invention can be used, in general, for the transport and transfer of any molecule in cells, in vitro, ex vivo or in vivo.
  • the invention therefore relates to any vesicle as described above comprising a heterologous molecule of interest, usable as a vector for transferring said molecule into a cell.
  • the exosomes of the invention are used for the oriented transfer of substances of interest to selected cell populations.
  • vesicles of the invention comprising a substance of interest (a toxin, a hormone, a cytokine, a recombinant nucleic acid, etc.) and expressing on its surface a ligand receptor or a ligand of receptor, and bringing said vesicles into contact with cells expressing the ligand or the corresponding receptor.
  • a substance of interest a toxin, a hormone, a cytokine, a recombinant nucleic acid, etc.
  • This approach therefore allows a targeted and efficient transfer.
  • a particular object of the invention resides in a vesicle as defined above, characterized in that it expresses a ligand receptor and in that it comprises a heterologous molecule of interest.
  • the vesicles of the invention may also comprise a peptide or a recombinant protein allowing purification of the vesicles.
  • the invention indeed describes the possibility of genetically modifying the composition of exosomes, and therefore of making them express a particular “label” molecule, allowing its purification.
  • an exosome is produced comprising, in its structure, a peptide molecule comprising the His6 motif (i.e., 6 consecutive histidine residues).
  • the vesicles according to the invention further comprise a marker.
  • the marker may be of a different nature (enzymatic, fluorescent, radioactive, etc.) and present in the vesicle or on its surface.
  • a preferred labeling is non-radioactive, such as for example a fluorescent labeling. More preferably, the labeling used is a fluorochrome or an enzyme with a chromogenic substrate. The labeling can be carried out directly on the producer cell, or else on the exosomes produced.
  • the invention also relates to any composition comprising one or more membrane vesicles as defined above.
  • the compositions of the invention can also comprise a plurality of membrane vesicles as defined above, carrying different recombinant molecules.
  • a composition according to the invention may comprise membrane vesicles as defined above, carrying recombinant MHC molecules of different haplotypes in association with the same antigenic peptide. They may also be compositions comprising membrane vesicles as defined above, carrying recombinant MHC molecules of the same haplotype, associated with different antigenic peptides, for example.
  • Other combinations of vesicles according to the invention are of course possible.
  • compositions of the invention generally comprise a vehicle such as a buffer, saline, physiological solution, etc., making it possible to preserve the structure of the vesicles. They may also include any stabilizing agent, surfactant, etc., preferably compatible with biological use (in vitro or in vivo). These compositions can be packaged in any suitable device, such as tube, bottle, vial, flask, pouch, etc., and stored at 4 ° C or at -20 ° C, for example. Typical compositions according to the invention comprise from 5 to 5OO ⁇ g of exosomes, for example from 5 to 200 ⁇ g.
  • the vesicles of the invention are obtained from genetically modified cells.
  • the present invention in fact results from the demonstration that it is possible to introduce recombinant molecules into certain cells, by genetic means, and that these molecules are then expressed in a functional and dense manner in the exosomes.
  • a first step therefore consists in introducing, into a vesicle-producing cell, as defined above, the genetic constructions allowing the expression of the (or selected recombinant molecule (s).
  • the genetic constructs used for the production of cells can generally comprise a coding region placed under the control of a promoter functional in the cell used (expression cassette).
  • the promoter used is therefore a promoter functional in mammalian cells. It can be a viral, cellular or bacterial promoter, for example. It can be a constitutive or regulated promoter, preferably allowing expression at high levels of protein in the cell.
  • CMV cytomegalovirus
  • HSV-1 TK the promoter of the thymidine kinase gene
  • LTR-RSV retro virus
  • a strong endogenous promoter of mast cell cells a particularly preferred embodiment comprises the use of the SR ⁇ promoter, as described in more detail in the examples.
  • the coding region used is generally composed of complementary, genomic or synthetic DNA (for example modified to contain certain introns or to have a preferential use of codons). More generally, it is a cDNA.
  • This nucleic acid can be obtained by all the techniques known in molecular biology, and in particular by bank screening, amplification, synthesis, cleavages and enzymatic ligations, etc.
  • a signaling sequence can be positioned upstream (5 '), downstream (3') or inside the coding region.
  • the addressing signal is positioned 3 'to the coding region, more particularly in its cytoplasmic region, and in reading phase with the coding region.
  • an addressing signal can be particularly useful for promoting the accumulation of the expression product in or on the surface of a given intracellular compartment, in particular in or on the surface of the secretory vesicles.
  • This embodiment is particularly suitable for the expression of a molecule such as a tag peptide, an antigen, a molecule of MHC-I or even a receptor ligand.
  • the present application shows that human MHC-II molecules can be expressed directly, without the addition of a particular signal, in vesicles which secrete mast cell cells, even xenogenic cells.
  • an addressing signal a nucleic acid fragment having the sequence of a part of the following genes: Lampl, CD63, LIMPII, Cdlc, Fc ⁇ R. These genes in fact comprise regions coding for signals for addressing the protein towards the compartments of the endosome of the cells (Sandoval and Bakke, Trends in Cell Biol. 4 (1994) 292).
  • An addressing signal usable in the present invention corresponds for example to the formula GYXXI, in which X represents any amino acid residue.
  • An addressing signal which is particularly suitable for the present invention is composed of the signal peptide of the protein LAMP1 of sequence SHAGYQTI.
  • Another type of signal allowing the addressing towards the membrane compartments includes all or part of a transmembrane region of protein.
  • the addressing of the expression product towards the cellular compartments allowing the presence of this product in the exosomes can also be achieved by fusing the coding region to all or part of a region coding a membrane or transmembrane protein, in particular a membrane protein or trans-membrane expressed in exosomes.
  • a particular embodiment of the invention comprises the introduction of a recombinant product into an exosome by expression of this product in the producer cell, in the form of a fusion with a membrane or trans-membrane protein.
  • a particular example of such a protein is, for example, the recombinant MHC protein introduced into the producer cell, in particular a beta chain, preferably a MHC beta chain of class IL.
  • the results presented in the examples show that such a fusion allows efficiently accumulate any polypeptide of interest in an exosome, without altering its properties or those of the MHC molecule.
  • This aspect of the present invention constitutes a new concept of vectorization of recombinant products in an exosome, and can be applied to any recombinant product, introduced into any type of exosome.
  • the invention therefore relates to any exosome comprising a recombinant molecule of fusion between a polypeptide of interest and an addressing signal.
  • the polypeptide of interest can be an antigen (or fragment of antigen) or any other biological product of interest.
  • the addressing signal can be any peptide, polypeptide or protein having the property of directing the fusion product towards a membrane compartment, in particular intracellular, as defined above. It is advantageously a chain of a molecule of the MHC.
  • these vesicles are produced by introduction into the producer cell of a chimeric nucleic acid, coding for a fusion protein comprising the recombinant product linked to the C-terminal end of a beta chain of a MHC molecule, preferably MHC class IL
  • the coding region is operably linked to the promoter, so as to allow its expression in cells.
  • the constructs of the invention may advantageously comprise a region, placed 3 ′ from the coding region, which specifies an end of transcription signal (polyA region for example).
  • the expression cassettes according to the invention advantageously form part of a vector, of plasmid, viral, episomal type, artificial chromosome, etc.
  • a vector advantageously comprises a system allowing the selection of the cells containing it.
  • the vectors advantageously comprise a gene coding for a product conferring resistance to an agent, for example to an antibiotic (ampicillin, hygromycin, geneticin, neomycin, zeocin, etc.).
  • each vector comprises a single expression cassette as described above.
  • the cells are therefore modified by the introduction of several vectors, when several molecules are to be expressed in the vesicles (for example an ⁇ chain and a ⁇ chain of MHC-II).
  • each type of vector used advantageously comprises a different selection system, making it possible to easily select multiple transfectants.
  • a vector may comprise several expression cassettes as defined above, for example one encoding an ⁇ chain and the other, a ⁇ chain of MHC-II.
  • the vectors used are preferably of the plasmid type, and comprise for example an origin of bacterial replication allowing their easy manipulation and production in vitro.
  • Such vectors can in particular be constructed from plasmids of the pBR322, pUC, pBS, pSR, etc. type.
  • genetically modified cells are therefore used, expressing the selected molecules.
  • These genetically modified cells are prepared by introducing, into cells chosen as defined above, the genetic constructs defined above.
  • the introduction of genetic constructs can be carried out in different ways, essentially depending on the type of cell used.
  • the transfer of nucleic acids can be carried out by any known technique such as electroporation, precipitation with calcium phosphate, chemical agent (cationic peptide, polymers, lipids, etc.), ballistics, etc.
  • transfer is generally obtained by simple infection of cells.
  • the amounts of vector used can moreover be adapted by a person skilled in the art according to the type of transfer and the cells used.
  • a particularly effective method for introducing nucleic acids into mast cells includes electroporation of the vectors.
  • constructs vectors
  • these can be transferred simultaneously or sequentially.
  • the cells that have effectively incorporated the nucleic acids are selected and cloned on the basis of their resistance to a compound (e.g., antibiotic) using the resistance gene present in the DNA that has been transferred.
  • a compound e.g., antibiotic
  • These cells can be used extemporaneously for the production of exosomes of the invention, or else be stored for later use.
  • the cells can be stored at 4 ° C. in a usual conditioning medium for a period sufficient to allow different batches of exosome production.
  • the cells can also be stored in frozen form (for example in nitrogen), for later use. In this regard, it is thus possible according to the present invention to constitute banks of cells producing exosomes having particular properties.
  • a particular object of the invention resides in an exosome-producing cell as defined above, in particular a mast cell, characterized in that it comprises a recombinant nucleic acid encoding a molecule of the complex major histocompatibility.
  • the invention also relates to any cell producing exosomes as defined above, in particular a mast cell, characterized in that it comprises a recombinant nucleic acid coding for an invariant chain li, in particular modified to include an antigenic peptide in place of the CLIP region, or for a peptide allowing the purification of the exosome.
  • it is a mammalian cell, in particular of animal origin, in particular a rodent. It can also be a cell of human origin. In a more particular mode, it is a cell line derived from a mast cell, such as in particular a mast cell line of basophilic leukemia. AT As a specific example, mention may be made of cells of the RBL line, in particular RBL-2H3, cells of the KU-812 or HMC-1 line.
  • the recombinant nucleic acid codes for an ⁇ and / or ⁇ chain of a molecule of the major histocompatibility complex of class II and / or for a molecule of the major histocompatibility complex of class I.
  • the cell comprises several nucleic acids coding respectively for an ⁇ chain and a ⁇ chain of a molecule of the major histocompatibility complex of class IL
  • the present invention makes it possible to produce, in a simple and reproducible manner, large quantities of exosomes of known composition.
  • the genetically modified cells described above are cultured in an appropriate medium, and the exosomes are recovered.
  • a particular object of the invention thus resides in a process for the production of an exosome comprising a defined recombinant molecule, comprising the following steps: a) the culture of a mast cell or mast cell derivative comprising a recombinant nucleic acid encoding said defined recombinant molecule, c) recovering the exosomes produced by said cells, these exosomes comprising said defined recombinant molecule.
  • the method according to the invention further comprises an intermediate step b) during which the cells are stimulated to induce and / or increase the secretion of exosomes.
  • the method of the invention allows the production of vesicles in which the defined recombinant molecule is exposed outside the exosome, or is included, in part or in whole, in the cytosolic fraction of the exosome.
  • the recombinant molecule can be, for example, a molecule of the major histocompatibility complex, an antigenic molecule, a receptor ligand, a ligand receptor or a purification peptide , or any other polypeptide of interest.
  • the nucleic acid used in the method further comprises a region coding for an address signal to the membrane compartments, in particular the internal secretory vesicles, of the mast cell.
  • Another particular object of the invention resides in a process for producing a membrane vesicle, comprising the culture of an exosome-producing cell, comprising a recombinant nucleic acid coding for a recombinant MHC molecule, in particular of class I or II, in particular human, and
  • the invention also relates to a process for the preparation of an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises:
  • the peptide or peptides used can be synthetic peptides, mixtures of peptides, cellular extracts, for example a mixture of peptides extracted from tumor cells.
  • the peptide (s) can be in isolated form, or purified or, as indicated above, in a mixture.
  • the exosomes can be isolated or purified according to conventional methods.
  • the invention relates to a process for the preparation of an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises:
  • an exosome-producing cell comprising one or more recombinant nucleic acids coding for a defined recombinant molecule of the MHC and a nucleic acid comprising a region coding for a defined recombinant peptide
  • the nucleic acid comprising a region coding for the recombinant peptide codes for a derivative of the invariant chain li, in which the CLIP region has been deleted and substituted by said peptide.
  • the nucleic acid comprises a region coding for the peptide and a region targeting to the intracellular compartments.
  • the nucleic acid can comprise several regions coding for the same or for different antigenic peptides.
  • the producer cells used in the process are mast cell or mast cell derived cells.
  • the stimulation of the cells to induce a release of the exosomes is preferably carried out by means of one or more calcium ionophores, or by IgE.
  • the producer cells used in the process are essentially devoid of endogenous MHC molecules.
  • Another subject of the invention relates to a process for modifying the composition of an exosome, comprising
  • exosomes of the invention can be used in numerous applications, such as for example as analysis, diagnostic, therapeutic or experimental tools. Thus, they can be used for analysis of the specific antigen T response; for the study of receptor / ligand interactions with low affinity where multimerization of the different partners is necessary in order to increase the avidity of these molecular complexes, thus exceeding the scope of immunological applications; on the diagnostic or therapeutic level, as well as for the production of specific antico ⁇ s, in particular antico ⁇ s restricted to MHC.
  • One of the first applications of the exosomes of the invention lies in the production of antico ⁇ s. Indeed, due to the defined composition of the exosomes of the invention, it is possible to produce antico ⁇ s of specific specificity. In addition, as the examples show, the exosomes of the invention have very strong immunogenic properties, in particular due to the high density of the complexes. MHC-peptide on their surface, their functionality, and their effective presentation to the immune system.
  • the antico ⁇ s thus produced can be polyclonal or monoclonal. They can be prepared by conventional immunology techniques, including the immunization of animals, and the collection of sera (polygonal antico )s) and / or the fusion of spleen lymphocytes with myeloma cells which do not produce immunoglobulins (for generate hybridomas producing monoclonals).
  • Another object of the invention therefore relates to antico ⁇ s or fragments of antico ⁇ s produced by immunization with exosomes as described above.
  • the fragments of antico ⁇ s can be for example Fab, (Fab ′) 2, ScFv, etc., and, more generally, any fragment retaining the specificity of the antico ⁇ s.
  • the invention relates to a process for the preparation of antibodies, comprising the immunization of an animal with an exosome as described above, carrying a defined peptide-MHC complex and the recovery of antibodies and / or cells producing antibodies or involved in the immune response.
  • the method of the invention allows the production of monoclonal antico ⁇ s, in particular restricted to MHC, that is to say specific to the MHC-peptide association.
  • exosomes essentially devoid of endogenous MHC molecules, which express recombinant MHC-peptide complexes, and which are produced from an autologous cell vis-à-vis the animal in which immunization is carried out.
  • this method makes it possible to obtain, without the need for an adjuvant, powerful antico ⁇ s directed against the peptide, in particular antico ⁇ s restricted to MHC, that is to say specific to the peptide in its associated conformation to the defined MHC molecule.
  • antico ⁇ s are particularly advantageous on the experimental, diagnostic and therapeutic level.
  • the antico ⁇ s according to the invention can be marked by any known technique (enzymatic, fluorescent, radioactive, etc.), according to methods known to those skilled in the art.
  • exosomes and antico ⁇ s of the invention have advantageous properties for diagnostic use.
  • the antico ⁇ s or fragments of antico ⁇ s obtained according to the invention can be used in any diagnostic application, for the detection, in a biological sample, of the presence of corresponding specific antigens, thanks to the use of different conventional techniques, such as flow cytometry, immunohistochemistry or immunofluorescence, for example.
  • antico ants restricted to MHC they advantageously allow the detection of the MHC-peptide complexes corresponding, and therefore the diagnosis of corresponding pathologies.
  • These antico ⁇ s can in particular be applied to the diagnosis of pathologies implying a defect of response or an inappropriate response of the immune system in order to determine the expression of an antigen, defined beforehand, in a form recognizable by T lymphocytes.
  • exosomes according to the invention can also be used for the detection of specific partners of a protein molecule in a biological sample.
  • the exosomes of the invention carrying MHC-peptide complexes can be used to detect T lymphocytes specific for these complexes in biological samples, for example in different pathological situations, in particular in the pathologies described above.
  • the exosomes can be labeled by any labeling system known to those skilled in the art (enzymatic, fluorecent, radioactive, etc.) to allow their detection in biological samples.
  • the invention therefore resides in the use of labeled exosomes, in particular fluorescent, as described above, for the detection of T lymphocytes specific for antigenic peptide -CMH complexes in a biological sample.
  • the biological sample can be any sample of blood, serum, tissue, tumor, biopsy, skin, urine, etc.
  • the biological sample can be pretreated for example to dissociate the cells, amplify the cells in culture, prepare membrane fractions, etc.
  • the biological sample comes from a human organism.
  • the invention also relates to a method for the detection of the presence of T lymphocytes specific for antigen-MHC complexes in a biological sample, comprising bringing said sample into contact with a labeled exosome as defined above, comprising said antigen-MHC complex, and highlighting the labeling T cells in said sample.
  • the detection of these T lymphocytes not only makes it possible to detect and therefore diagnose a pathophysiological condition, but also to monitor, for example, the effectiveness of immunization protocols and the state of the immune response at different stages of the disease and thus assess the effectiveness of the therapeutics undertaken.
  • the exosomes of the invention carrying a TcR receptor are used for the detection of peptide-MHC complexes specific for this receptor in a biological sample.
  • the fluorescent exosomes according to the invention carrying any type of protein of defined composition also represent fluorescent probes allowing the detection of potential receptors.
  • the new field of exosomes can thus be generalized to the demonstration, in vivo, of any protein / protein interaction of low affinity.
  • the invention therefore also relates to the use of exosomes, preferably labeled, in particular fluorescent, as described above,
  • the exosomes used therefore comprise, on their surface, said biological molecule of defined structure.
  • the exosomes used therefore comprise, on their surface, a specific receptor for said ligand.
  • Restricted antico ⁇ s and exosomes can therefore be used in all situations where one wishes to reduce or suppress an immune response mediated by T lymphocytes which proves to be deleterious for the organism as is the case for example in: - organ transplants or marrow transplants in which an attempt is made to neutralize the host's response against the graft, generally by means of large doses of immunosuppressive agents;
  • the exosomes of the invention expressing on their surface a defined peptide-MHC complex, the implication of which is known in the development of the pathological situation, can therefore be used to block the development of the immune response and therefore the development of the pathological response.
  • exosomes of the invention carrying MHC-peptide complexes can also be used for the amplification (expansion) of the population of cytotoxic T lymphocytes ex vivo. Used directly from blood samples, they can thus be the basis of cell therapies against different cellular targets. Thus exosomes can be used to sort specific T cells from various combinations of complexes expressed by cells which represent a therapeutic target, such as tumor cells or cells infected with a virus. Another object of the invention therefore lies in the use of the exosomes described above for the clonal amplification and / or the stimulation of cytotoxic and / or helper T lymphocytes.
  • the invention also relates to a method for the clonal amplification (or expansion) ex vivo of T lymphocytes, in particular cytotoxic cells, comprising bringing a biological sample comprising T lymphocytes into contact with exosomes as described above. , comprising a defined peptide-MHC complex, the recovery of specific T lymphocytes and their amplification.
  • This method is particularly advantageous for the clonal amplification of cytotoxic T lymphocytes specific for complexes between MHC molecules and peptides of tumor or viral antigens.
  • the vesicles according to the invention resides in the transfer of molecules to cells. Indeed, due to their composition, the vesicles of the invention are capable of playing the role of vector for the transfer of molecules to cells, in vitro, ex vivo and in vivo.
  • the invention relates to the use of exosomes as described above, comprising a substance of interest, for the preparation of a composition intended for the transfer of said substance into a cell.
  • it is an exosome comprising a ligand receptor or a receptor ligand on its surface, thus making it possible to orient the transfer to one or more selected cell populations.
  • the invention also relates to a method for the transfer of a substance into a cell, in vitro, ex vivo or in vivo, comprising bringing said cell into contact with a vesicle according to the invention comprising said substance. More preferably, the vesicle used also expresses a ligand receptor and the method of the invention allows an oriented transfer of the substance to cells expressing the corresponding ligand.
  • the vesicles of the invention are administered to a subject (preferably a mammal, in particular man), by any conventional route of administration (intravenous, intraarterial, intramuscular, subcutaneous injection, etc.).
  • the cells are contacted by incubation in an appropriate device (box, flask, pouch, vial, etc.), preferably under sterile conditions.
  • an appropriate device box, flask, pouch, vial, etc.
  • the parameters of the contacting step can be easily adjusted by a person skilled in the art according to the aims pursued and the teaching of the present application .
  • the exosomes of the invention can be used either as such or in immobilized form on a support.
  • the results presented in the examples indeed show that it is possible to fix the exosomes on supports, without altering their functional properties, in particular their antigenic specificity for example.
  • a particular object of the present invention resides in a composition comprising exosomes immobilized on a support.
  • the support is preferably a solid or semi-solid support, of the ball, filter or similar type. It is preferably a support of plastic material, of the polymer type, for example latex beads, or magnetic beads. It is understood that any other synthetic or biological material can be used, as long as it does not induce alteration substantial qualities of exosomes or cells.
  • beads with a diameter of 1 to 10 ⁇ m, for example from 2 to 5 ⁇ m, are used.
  • the immobilization of the exosomes on the supports is advantageously obtained by covalent bonding, for example by activation with an aldehyde, or any other chemical coupling reagent.
  • the immobilization of the exosomes is carried out by incubating the exosomes with the support in solution, under the conditions allowing the fixation, then the supports are recovered by centrifugation.
  • the functionalized supports thus obtained can be used to characterize exosomes or to detect or amplify T cells in vitro, as will be described in detail in the experimental section. Other aspects and advantages of the present invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.
  • FIG. 1 Production of functional MHC-DR1-HA peptide complexes in the RBL2H3 line.
  • the DRI molecules are detected by the antico ⁇ s L243 (black line) itself revealed by a goat anti-mouse IgG serum coupled to the FITC.
  • DRI invariant chain
  • DR11iHA and B-EBV Hom2 were diluted in culture plates with a T lymphocyte specific for the DRI HA complex.
  • the B-EBV Hom2 line was also incubated in the presence of a saturated concentration (10 mM) of the HA peptide.
  • the production of IL2 in culture supernatants makes it possible to evaluate the stimulation of THA lymphocytes (T lymphocytes specific for the HA peptide).
  • IL2 is measured via of a tritiated thymidine inco ⁇ oration test of the CTLL2 line, the proliferation of which is IL2-dependent.
  • Figure 2 Accumulation of DRI molecules in a secretion compartment of RBL2H3.
  • the RBL DRLHA cells were fixed with 0.5% glutaraldehyde and then permeabilized with 0.05% Saponin.
  • the DRI molecules and the invariant chain were detected respectively with the antico ⁇ s L243 and PLN1 and then an anti-mouse IgG donkey serum coupled to the FITC.
  • Serotonin was detected using a specific rabbit serum revealed with an anti rabbit IgG donkey serum coupled to Texas red.
  • the images were obtained by confocal microscopy (Leica). The thickness of the section planes was 0.5 microns.
  • the RBL DRI liHA cells were incubated in the presence of 5 mM of “Green Tracker” (fluorescent lipid accumulating in the lysosomal compartments of the cells) for 30 minutes at 37 ° C. then washed and reincubated for one hour at 37 ° C. in the absence of fluorescent marker. The cells were fixed (3% paraformaldehyde), then analyzed by confocal microscopy.
  • Green Tracker fluorescent lipid accumulating in the lysosomal compartments of the cells
  • DRI HA exosomes were purified from the cells described in A.
  • the fluorescence present in the samples was quantified using a fluorimeter and viewed directly by confocal microscopy.
  • the DRI HA fluorescent exosomes were incubated at 50 mg / ml with THA lymphocytes specific for the DRI HA complex or TH30 lymphocytes specific for another complex (D) for two hours at 37 "C in the presence of azide to block the internalization
  • the fluorescence of the cells was evaluated by flow cytometry.
  • FIG. 5 Production of exosomes carrying class II molecules of MHC A
  • the expression of class II molecules IAb is detected by the monoclonal antibody Y3P and analyzed by flow cytometry.
  • the transfectants obtained in the RBL2H3 cell express similar levels of class II molecules recognized by Y3P that a B lymphoma controls B414.
  • Latex beads were coated either with fetal calf serum (FCS) or with exosomes from RBL 2H3 cells (exos RBL) or with a transfectant of this cell with murine class II molecules lAbli (Exos lAbli) or human DRlliHA (ex DRlliHA).
  • FCS fetal calf serum
  • exosomes from RBL 2H3 cells exos RBL
  • a transfectant of this cell with murine class II molecules lAbli Exos lAbli
  • human DRlliHA ex DRlliHA
  • FIG. 6 Mo ⁇ hological characterization of the exosomes produced by RBL-2H3: A The RBL-2H3 cells transfected with HLA-DR1 were fixed with paraformaldehyde. Frozen ultra-thin sections were prepared and immunolabeled with polyclonal antibodies directed against the HLA-DR molecules. These antico ⁇ s are visualized with protein A coupled to 10 nm colloidal gold particles. Class II molecules are mainly detected in compartments filled with vesicular-looking membranes. Bar: 250 nm.
  • Figure 7 Manipulation of the internal composition of exosomes, introduction of a recombinant protein.
  • class II molecules DRI is detected by the monoclonal antibody L243 and analyzed by flow cytometry.
  • the transfection of molecules in the RBL2H3 cell induces the expression of similar levels of class II recognized by Y3P that a B lymphoma controls B414.
  • Latex beads were coated with either fetal calf serum (FCS) or exosomes from the RBL DRI GFP cells. DRI molecules are detected by antico ⁇ s L243 and secondary antico ⁇ s coupled to phycoerythrin while the presence of GFP is detected directly in the FL1 channel.
  • FCS fetal calf serum
  • FIG. 8 A Links of fluorescent exosomes by specific T cells.
  • Exosomes produced from RBL DRI liHA cells labeled with the green cell tracker were incubated in the presence of two types of T cells: HATs which have a TCR specific for the HLA-DR1 / HA complexes and wild Jurkat Ts lacking a like receiver.
  • the fluorescent exosomes were incubated for 3 hours at 37 ° C with the two T lymphocyte lines and the resulting labeling was analyzed with FACS.
  • Figure 9 Characterization of HMC-1 cells and exosomes Analysis of HMC-1 cells by flow cytofluorometry, solid black line: single cells; solid clear line: anti-mouse anti-FITC only; tight dotted lines: specific antico ⁇ s + anti-mouse-FITC.
  • the cells used for the production of exosomes in the experimental part are cells of murine or human mast cells. More particularly, a basophil cell line with the mucosal mast cell phenotype, designated RBL-2H3, was used (Barsumian et al, Eur. J. Immunol. (1 1 (1 981) 317), as well as a line of mast cells immature humans (HMC-1). Other mast cells, in particular established in line, can be used, such as lines derived from RBL (Rat Basophicil Leukemia) cells, deposited at ATCC under number CRLI378 (Kulczycki et al., J. Exp. Med. 139 (1974) 600).
  • RBL-2H3 Basophil cell line with the mucosal mast cell phenotype
  • T lymphocyte lines capable of recognizing a particular antigen in a human MHC II context (DRI) have also been used.
  • the Jurkatt line transfected with the cDNA encoding the T cell receptor ("T-HA") specific for the influenza virus Hemagglutinin peptide 306-318 in combination with HLA-DRI (Sidhu et al. ., J. Exp. Med. 176 (1 992) 875).
  • T-HA T cell receptor
  • the human B cell line transformed with the Epstein Barr virus (Hom-2 line) was used as a control for the restricted response to HLA-DRI.
  • the cells were cultured in DMEM medium (Gibco BRL), RPMI, or "CLICK": RPMI medium supplemented with 10% fetal calf serum (Sigma), 1 mg / ml of penicillin-streptomycin, 1 mg / ml of glutamine , 5 mM sodium pyruvate and 50 ⁇ M b-mercaptoethanol. Any other medium suitable for the culture of eukaryotic cells, in particular mammals, can of course be used.
  • the cells were mainly cultured in a 25 or 15 cm3 culture flask.
  • the RBL-2H3 cells being adherent cells, they are detached from the support thanks to the action of Trypsin-EDTA (Seromed). In order to produce these in large quantities, it is also possible to cultivate them in "spinner", at the density of 10 6 cells / ml.
  • CDNAs coding for the human HLA-DRI ⁇ chain (Larhammer et al., Ceil 30 (1982) 153), the human HLA-DRI ⁇ chain (Bell et al., PNAS 82 (1985) 3405) and the human invariant chain p33 li (Ciaesson et al., PNAS 80 (1983) 7395) were isolated.
  • the cDNA coding for the invariant chain p331i was then modified by PCR to replace the region coding for the CLIP peptide (residues 87-102) with a restriction site.
  • This cDNA thus makes it possible to insert, in place of the peptide CLIP, any fragment of cDNA of interest coding for an antigenic peptide (Stumptner et al., EMBO J. 16 (1997) 5807).
  • a DNA fragment coding for the peptide HA308-319 of the hemagglutinin of the influenza virus was inserted into this cDNA, coding for a chimeric polypeptide li (HA308-319).
  • the nucleic acids described above were then cloned, separately, in the plasmid pSR ⁇ under the control of the promoter SR ⁇ (Takebe et al., Mol. Cell Bio. 8 (1988) 466).
  • Each of the plasmids was then modified so as to incorporate a different resistance gene, allowing selection for each of the plasmids, and therefore for each of the chains; the ⁇ chain with the neomycin resistance gene; the ⁇ chain with the hygromycin resistance gene, and the invariant chain with the zeocin resistance gene.
  • the corresponding plasmid vectors were linearized by the restriction enzyme Seal. 50 ⁇ g of each plasmid were linearized then precipitated with ethanol, and the pellets were resuspended in the presence of RBL-2H3 cells at a concentration of 1.10 7 cells / ml. Stable transformants were obtained by electroporation of 5.10 6 cells using a "draw gene" (Bio-Rad, Richmond, CA) under the following conditions: 260V, 960 ⁇ F.
  • the transfectants are selected by culture in a selection medium comprising 250 ⁇ g: ml of G418 (Geneticin, Gibco) lmg / ml of hygromycin and 500 ⁇ g / ml of zeocine. After 8 days of culture in the selection medium, 60 to 90% of the cells present are transfected. The transfectants were then seeded on a petri dish in the selection medium at a concentration allowing the appearance of individualized adherent colonies. The clones thus obtained were removed and cultured separately. These clones can be stored in frozen form for later use.
  • Y3P (MHC II (IA)) is a mouse monoclonal antibody recognizing the IAb a ⁇ complex (Janeway et al., 1984).
  • Anti IA a is a rabbit serum directed against the cytoplasmic part of the IA chain.
  • the anti-GFP is a mixture of two monoclonal antibodies (clones 7.1 and 13.1) directed against the "green fluorescent protein" sold by Boehringer Mannheim.
  • the second antico ⁇ s used are F (ab ') 2 fragments coupled to (-Phycoerythrin, produced in donkeys and directed against mouse IgG (H + L) (Jackson Immunoresearch Laboratories) .
  • Latex beads Surfactant-free white aldehyde / latex sulfate, D: 3.9 ⁇ m, Interfacial Dynamics Co ⁇ ., Portland, Or. USA
  • exosomes are fixed with 2% paraformaldehyde in 0.2M phosphate buffer pH 7.4. (PB buffer) and deposited on electron microscopy grids covered with a film of carbonaceous formvar.
  • PB buffer phosphate buffer pH 7.4.
  • exosomes are either contrasted and coated in a solution of 4% uranyl acetate and methylcellulose or are immunolabelled with antico ⁇ s directed against the class II molecules before coating. Antico ⁇ s are visualized with protein A coupled to 10 nm colloidal gold particles. Results
  • RBL-2H3 DRI cells were then sensitized to a given peptide, of precise composition, in order to generate MHC-peptide complexes of defined composition.
  • the peptide can be incubated directly with the exosomes.
  • a nucleic acid coding for the peptide can be introduced into the cells, so as to also express this peptide.
  • the chosen antigen peptide was introduced into the cells in the form of a genetic fusion with the human invariant chain li.
  • the CLIP peptide of the invariant chain has been replaced by the sequence of the chosen peptide, derived from hemagglutinin (HA 308-319) of the influenza virus, known to bind with the molecule DRI.
  • This construct (liHA) was transfected into cells under the conditions described in the Materials and Methods.
  • the hybrid chain expressed in RBL-2H3 DRI cells made it possible to build cells which express DRI molecules recognized by the antico ⁇ s L243 at a similar level as a B-EBV (Hom2) control line of DRI haplotype (Figure 1B ).
  • the functional character of the peptide-MHC complexes expressed by the cells of the invention was confirmed in a test for stimulation of T lymphocytes specific for the DRI -HA combination carried by the cells.
  • the cells of the invention were incubated in the presence of THA lymphocytes, and the stimulation was determined by measuring the interleukin-2 released in the supernatant, by a growth test of an IL-2 cell line. -dependent.
  • a line of B lymphocytes transformed by EBV (Hom-2), of DRI haplotype, pulsed by a saturating concentration (10 mM) of the HA peptide was used.
  • FIG. 1C and 1D show that the mast cell cells of the invention express a DRI -HA complex, capable of stimulating a T lymphocyte specific for this combination. They also show that the stimulation obtained in the presence of the cells of the invention is more effective than that produced by the control cells (B-EBV of haplotype DRI) obtained by a saturating concentration (10 mM) of the peptide HA. Finally, the results obtained show that the DRI molecules seem to present only the HA peptide since the addition of a saturated concentration of peptide does not significantly increase the capacity of the RBL DRI liHA cells to stimulate a T HA lymphocyte ( Figure 1 D).
  • the cells were cultured in the presence of a calcium ionophore, and the production of membrane vesicles was monitored. More particularly, the cells were centrifuged at 300 g for 5 minutes at room temperature. On each cell pellet, a solution of calcium ionophore (1 mM lonomycin) was added (approximately 300 ⁇ l) and the incubation was continued for 30 minutes at 37 ° C. The exocytosis was stopped by rapid cooling in ice and addition of 300 ⁇ l of a cold 1 mM PBS-EGTA solution. The cells were then centrifuged at 300g at 4 ° C for 5 minutes.
  • a calcium ionophore 1 mM lonomycin
  • the supernatants were recovered and recentrifuged, first 5 minutes at 1200 g, then 5 minutes at 10 000 g, then finally 1 hour at 70 000 g. After this differential centrifugation, the pellets (including the exosomes) are taken up and dissolved in a buffer solution (30 ⁇ l of Laemmlî-DDT buffer (IX or 2X)). A fraction of the pellets is also dissolved in lysis buffer to determine the protein concentration.
  • the exosome solutions can be separated by migration on gel (12% polyacrylamide mini-gel) at 20 mA and then transferred to lmmobilon. The analysis of the exosomes is then carried out by westem blotting with specific antico ⁇ s of the various chains of the class II molecules of the MHC. The results obtained show that exosomes can be released from the line
  • This example illustrates the use of the exosomes of the invention for the production of antico ⁇ s, in particular so-called “restricted” antico ⁇ s, that is to say specific for the antigenic peptide in association with the MHC molecule.
  • This example illustrates in particular the very important immunogenic power of the exosomes of the invention, since they allow the production of antico ⁇ s in the absence of any adjuvant.
  • the exosomes purified from the RBL DRI HA cell supernatant (Example 1) were resuspended in PBS.
  • exosomes were then used to immunize Balb / c mice or LOU rats in the absence of any adjuvant, according to the following protocols: - The mice were injected subcutaneously with 10 ⁇ g of exosomes, twice three weeks apart, then 30 ⁇ g intraperitoneally and finally 30 ⁇ g intravenously 3 days before the collection of the sera.
  • the rats were injected with exosomes by the intraperitoneal route (10 ⁇ g), twice twice at three weeks apart, then by the intravenous route (50 ⁇ g) three days before the collection of the samples.
  • the semms taken from the immunized mice exhibited a very high reactivity against the RBL line expressing or not the DRI HA complex but which was detectable up to a dilution to thirty thousandths of the semms only in the case of the DRI HA cell.
  • the semms of the rats thus immunized showed, surprisingly, a reactivity against the RBL line expressing the DRI HA complex whereas the same semms reacted with a lower intensity with the initial line RBL-2H3.
  • the addition of the HA peptide to cells expressing DRI significantly increases the reactivity of the antisemms thus produced (FIG. 3B).
  • the hybridomas thus obtained were sorted by clonal dilution, according to conventional immunology techniques, then selected by immunofluorescence for the specificity of the monoclonal antibodies produced.
  • Different monoclonal antico ⁇ s have thus been obtained, for some directed against proteins of the RBL line, for others, against monomo ⁇ hical determinants of human class II molecules of DRI haplotype and finally against the complex constituted by DRI molecules associated with peptide derived from the HA protein of the flu virus ( Figure 3C).
  • These latter monoclonal antico ⁇ s constitute restricted antico ⁇ s, and therefore have particularly advantageous properties for diagnostic or therapeutic applications.
  • This example illustrates the use of the exosomes of the invention for the detection of specific T lymphocytes in a biological sample.
  • This example shows also how exosomes can be used to select and amplify a population of particular T lymphocytes, in particular with a view to their reinjection into a subject (cell therapy).
  • This approach can of course be extended to the use of the restricted antico ⁇ s described in Example 2, as well as to the detection of any receptor specific for a ligand.
  • labeled exosomes were produced.
  • a fluorescent tracer which accumulates very strongly in the exosomes contained in the secretory granules.
  • the marker used "Green Tracker” is a fluorescent lipid which accumulates in the lysosomes of the cells. Analysis by confocal microscopy of the cells, after fixation, shows the presence of the fluorescent marking in the secretory granules of the cells ( Figure The fluorescent exosomes were then produced and purified from these cells, under the conditions described in Example 1.
  • the exosomes were incubated in the presence of a sample of HAT lymphocytes and TH30 lymphocytes, specific for another complex.
  • the results obtained by flow cytofluorometry show that the exosomes expressing DRLHA bind, in a specific way, to HAT lymphocytes (FIG. 4C) whereas these same exosomes are unable to recognize lymphocytes having another specificity (FIG. 4D).
  • this technology can be applied in the same way to the manufacture and use of exosomes expressing CMHI-peptide complexes.
  • the present invention thus makes it possible to detect, on the surface of presenting cells, or even tumor cells, the presentation, by MHC class I and class II molecules, of peptides derived from antigens expressed by tumors.
  • the present invention also makes it possible to detect or even to purify the T lymphocytes capable of recognizing these same complexes. They can thus make it possible to amplify a population of T lymphocytes specific for a particular peptide-MHC complex, for example a population of CTL lymphocytes, for their therapeutic use.
  • the complementary DNAs encoding the ⁇ and ⁇ chains of the murine class II molecules of haplotype IAb as well as the murine invariant chain were introduced into the eukaryotic expression vectors NT in which the transcription of the cDNA is under the control of an SR alpha promoter.
  • Each of the plasmids moreover, carries a gene for resistance to hygromycin (for the ⁇ chain IAb), to neomycin (for IAb ⁇ chain), or to zeocine (for the invariant chain).
  • the cells were selected on the basis of their resistance to the three antibiotics and then, the resistant cells were cloned by limiting dilution and characterized for the expression of the IAb molecules by cytometry of flow using the specific Y3P antico ⁇ s.
  • Figure 5 shows some of the results obtained.
  • Analysis by flow cytometry shows that the RBL lAbli cell is recognized by the antico ⁇ s Y3P (specific for IAb molecules) in an equivalent manner to the B B4 lymphocyte line 14, while no labeling is detectable on the RBL line of origin ( Figure 5 A).
  • Mo ⁇ hological analysis by microscopy having established that murine class II IAb molecules are, like human DRI molecules, accumulated in the secretory granules of the cell (not shown), this led us to seek their location in exosomes.
  • the exocytosis of the cells being triggered by the addition of ImM of ionomycin, the exosomes thus obtained were purified by differential ultracentrifugation (see example 1.2).
  • the intracellular compartments fuse with the plasma membrane.
  • the internal vesicles with which the class II molecules are associated are released into the extracellular medium. These vesicles are then called exosomes.
  • the method of choice in electron microscopy to study the mo ⁇ hology of exosomes as well as their protein content is the “whole mount” method.
  • This technique makes it possible to visualize entire exosomes devoid of any other cellular content.
  • This method also makes it possible to detect, with great efficiency, molecules associated with the exosome membrane.
  • the exosomes secreted by RBL cells have a heterogeneous size of 30 to 120 nm and a variable electron density (Figure 6B).
  • Class II molecules are abundant in the population of vesicles with a size of 80 to 100 nm having an average electron density. The vast majority of these vesicles enriched with class II molecules have a plate shape (Figure 6C).
  • This example describes the fixation of exosomes on solid supports, and shows that the exosomes thus fixed retain their functional properties.
  • These new products can be used to characterize and analyze the exosomes; or as diagnostic or reagent products for detecting and / or stimulating T cells in vitro, for example.
  • exosomes produced by RBL cells expressing or not expressing human or murine class II molecules were incubated with 4 micron latex beads activated by aldehyde sulfate. More particularly, the exosomes, purified from degranulation supernatants of RBL 2H3, were washed in PBS (centrifugation at 50,000 ⁇ m on TLA 100.4 for 30 minutes). 30 ⁇ g of exosomes are mixed with 10 ⁇ L of Latex beads taken sterile, homogenized and then incubated for 10 min to 15 min at room temperature. The bead volume is then made up to 1 ml with 1x PBS and then incubated on a wheel at room temperature for 2 h. Then the "crosslinked" beads with exosomes are:
  • FIG. 5C shows some examples of detection, by flow cytometry, of different proteins used in the composition of exosomes.
  • Latex beads activated with aldehyde sulfate were thus incubated either with exosomes produced by untransfected RBL 2H3 cells, or with exosomes of RBL 2H3 cells expressing human class II molecules DRI and the constellation LiHA or molecules murine class II IAb, or with fetal calf semm (FCS) in control.
  • the latex beads thus prepared were then incubated with different monoclonal antibodies; AD1 recognizing the rat CD63 molecule present in the secretion granules of RBL 2H3, Y3P antico ⁇ s specific for IAb molecules, and L243 antico ⁇ s specific for DRI molecules. These different antico ⁇ s were revealed by antico ⁇ s secondary coupled to phycoerythrin then the markings obtained were analyzed by flow cytometry using a FACScalibur (Beckman).
  • CD63 molecule is of course present on all the exosomes originating from the RBL cell expressing or not class II molecules, while latex beads coated with IAb exosomes are specifically recognized by Y3P and not by L243 while in contrast, the DRIiHA exosomes are recognized by L243 and not by Y3P. None of these antico ⁇ s recognizes latex beads covered with fetal calf semm (Figure 5C).
  • Example 8 shows that such products (support exosomes) also make it possible to detect or stimulate the proliferation of specific T lymphocytes.
  • Exosomes are vesicles delimited by a lipid bilayer into which are inserted a large number of molecules such as the class II molecules of the MHC or CD63 mentioned above. Inside these vesicles, we find the cytoplasmic regions of the preceding transmembrane molecules but also of the soluble proteins obtained from the cytosol of the cells. To demonstrate that it is possible to modify the content of these vesicles at will, we used the Green Fluorescent Protein (GFP) as a tracer.
  • GFP Green Fluorescent Protein
  • the cDNA encoding GFP has been fused to the COOH terminus of the beta chain of the human DRI molecule.
  • This constuction inserted into the NT expression vectors carrying the hygromycin resistance gene, was cotransfected in the RBL 2H3 cell with a vector carrying the DRI alpha chain and the neomycin resistance gene. Cells resistant to these two antibiotics were selected and then sorted positively for the expression of GFP. More particularly, we have made a constuction linking the cytoplasmic part of the DR ⁇ chain (in C-ter) to the N-ter end of the GFP.
  • the DR ⁇ cDNA has a PstI site at position 565.
  • a fragment of approximately 200 base pairs on the 3 ′ side of this cDNA was amplified by PCR from the vector pcDNA3 / RSV / DRa using 2 oligonucleotides including the site PstI for 5 'and the Ncol site for 3' which also eliminated the stop codon.
  • the PCR fragment obtained was digested with PstI and Ncol and cloned into the same sites of the pEGFP NI vector (Clontech).
  • the plasmid pcDNA3 / RSV was digested with EcoRV / PstI, thus releasing a fragment corresponding to the rest of the DRa chain (from the start to the PstI site). The two fragments were then assembled and then cloned into pcDNA3 / CMV between EcoRV and Xbal.
  • exosomes originating from the RBL DRI GFP cells were prepared by differential ultracentrifugation and then analyzed by westem blot (FIG. 7B) and flow cytometry after "crosslinking" on latex beads (Figure 7C).
  • a specific antico ants of GFP detects, by westem blot, in the DRI GFP cell and in its exosomes a protein of 65 kDa corresponding to the molecular weight of the beta chain of DRI fused with GFP (FIG. 7 B) while no signal is detected in the cell lysates of the RBL2H3 cell expressing or not expressing the DRI molecule alone.
  • exosomes produced by the RBL2H3 cell, carrying class II molecules of the MHC are capable of binding an antigenic peptide and of stimulate a T lymphocyte expressing a receptor specific for this peptide-MHC class IL complex
  • Exosomes produced from RBL DRI liHA cells labeled with the green cell tracker were incubated in the presence of two types of T cells: HATs which have a TCR specific for the HLA-DRI / HA complexes and wild Jurkat T cells lacking a like receiver.
  • the binding experiments are carried out in a 96-hole plate with a round bottom, in RPMI 1640 medium supplemented with 10% of fetal calf semm, buffered with 10 mM Hepes at a rate of 50 ⁇ l final per well, 10 5 T cells per well and variable quantities fluorescent exosomes for 3 hours at 37 ° C. Then, two washes are carried out in the same medium before analyzing the cells taken up in 400 ⁇ l of PBS with FACS.
  • a dose effect range was produced showing that the intensity of the THA labeling was proportional to the quantity of exosomes used. 10 8 RBL DRI liHA cells gave 700 ⁇ l of exosomes. Doses ranging from 32 to 2 ⁇ l of exosomes per well were tested. The labeling obtained with 16 ⁇ l per point was retained, which corresponds to the production of exosomes carried out by 2.3 million cells (results not exposed).
  • the results obtained show that the THA population is marked by fluorescent exosomes which gives a detectable fluorescence in FL1 by cytofluorimetry, while the wild Jurkats are not modified (FIG. 8A).
  • the same type of labeling was carried out, but after the binding of the exosomes, the T cells were incubated with an anti-mouse monoclonal antibody AD1 anti CD63 rat, then revealed by anti-mouse asses anti mouse IgG labeled with phycoerythrin (Jackson Immuno Research, West Grove, PA). The same labeling was carried out in parallel on T cells which had not been exposed to exosomes. It is observed (FIG. 8B) that only the THA cells which have linked exosomes, as attested by their fluorescence in FL1, are also marked in FL2 which indicates the new presence of rat CD63 on their surface.
  • the exosomes obtained from the DRI GFP cell were crosslinked on latex beads and then incubated in the presence of cells of the lymphocyte line.
  • T Jurkat whether or not expressing a T receptor specific for the DRI-peptide HA 307-319 complex.
  • the latex beads were prepared in the same way as for flow cytofluorometry but washed in a complete medium (RPMI, 10% semen of fetal calf, Penicillin-Streptomycin-Glutamine 1%, ⁇ mercaptoethanol 0.1%, Sodium Pymvate 4% ).
  • each pellet of beads was taken up in 100 ⁇ L, 50 ⁇ L deposited in the first well of a 96-well plate and 50 ⁇ L diluted two by two. A check was made in the same way with beads incubated in semen of fetal calf.
  • the T cells (Jurkat Pasteur T and THAI 7) were adjusted to 10 6 cells / mL and 50 ⁇ L were deposited per well.
  • the peptide diluted to 15 ⁇ M in complete medium was also added at the rate of 50 ⁇ L per well. In the peptide-free series, 50 ⁇ L of complete medium were added per well.
  • the culture plate was placed in the incubator (37 ° C, 5% CO2, H2O) for 20 hours then the supernatant was removed and the concentration of IL2 was evaluated by a CTL.L2 test.
  • modified exosomes according to the invention can be produced from other cells, in particular human.
  • the results we have obtained demonstrate that the mast cell line of human origin HMC1 is capable of producing exosomes under the impulse of an increase in intracellular calcium and under conditions identical to those which induce the secretion of exosomes by the RBL 2H3 rat line.
  • HMC1 line The characterization of the HMC1 line by flow cytometry indicates that the surface of these cells express MHC class I molecules (W6.32) but not class II molecules (L243). Furthermore, they are positive for the molecules CD9, CD63 and CD81 but negative for the molecules Lampl and Lamp 2 ( Figure 9 A). Exosomes were produced from the HMC1 line by the addition of
  • Ionomycin then purified from the supernatants by differential ultracentrifugation. Their composition was analyzed by flow cytometry after "crosslinking" on latex beads and by westem blot.
  • the HMC1 line seems to be the human counterpart of the RBL-2H3 rat line because of its ability, after an increase in intracellular calcium, to produce exosomes which have the same structural and molecular characteristics. These cells therefore make it possible to produce recombinant human exosomes expressing class II molecules of the MHC or any other molecule which would be specifically addressed thereto.
  • Membrane vesicle characterized in that it comprises a recombinant molecule of the major human histocompatibility complex.
  • Vesicle according to claim 1 characterized in that the recombinant molecule of the major histocompatibility complex is a molecule of class IL
  • Vesicle according to claim 2 characterized in that the recombinant molecule of the major histocompatibility complex of class II is an ⁇ chain.
  • Vesicle according to claim 2 characterized in that the recombinant molecule of the major class II histocompatibility complex comprises an ⁇ chain and a ⁇ chain.
  • Vesicle according to one of claims 2 to 4 characterized in that the recombinant molecule of the major histocompatibility complex of class II is chosen from the serotypes DRI to DRI 3, preferably DRI to DR7. 6. Vesicle according to claim 1 characterized in that the recombinant molecule of the major histocompatibility complex is a class I molecule.
  • Vesicle according to one of claims 1 to 6 characterized in that it comprises a complex between a defined peptide and the recombinant molecule of the major histocompatibility complex.
  • Vesicle according to one of the preceding claims characterized in that it further comprises one or more heterologous molecules of interest.
  • Vesicle according to one of the preceding claims characterized in that it further comprises a recombinant peptide or protein allowing its purification.
  • Vesicle according to the preceding claims characterized in that it comprises a marker.
  • Membrane vesicle characterized in that it is obtained from a mast cell or derived from mast cell, and in that it comprises one or more heterologous molecules of interest.
  • Vesicle according to claim 12 characterized in that the molecule of interest is a protein, a polypeptide, a peptide, a nucleic acid, a lipid or a substance of chemical, biological or synthetic nature.
  • Membrane vesicle according to claim 13 characterized in that the heterologous molecule is a molecule of the major histocompatibility complex, a

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JP4939926B2 (ja) 2003-02-14 2012-05-30 アノシス・インコーポレーテッド 抗体を生成し抗体レパートリーをスクリーニングするための方法とコンパウンド
WO2005094571A1 (ja) * 2004-03-31 2005-10-13 Japan Science And Technology Agency 遺伝子改変ヘテロ動物及び該動物を用いたエキソサイトシスの計測方法
WO2006089572A1 (en) * 2005-02-28 2006-08-31 Charité - Universitätsmedizin Berlin Polyelectrolyte capsules for detecting antigen-antibody-reactions
CN1322115C (zh) * 2005-07-06 2007-06-20 清华大学 载有外源配体分子的胞外体及其制备方法与应用
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JP2010516786A (ja) 2007-01-26 2010-05-20 ユニバーシティー オブ ルイヴィル リサーチ ファウンデーション，インコーポレーテッド ワクチンとしての使用のためのエキソソーム成分の改変
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