FR2785543A1 - MODIFIED EXOSOMES AND USES - Google Patents

Abstract

The invention concerns the fields of biology and immunology. It concerns membrane vesicles comprising molecules, in particular antigenic molecules, with predetermined structure, and their uses, More particularly, it concerns vesicles (exosomes) comprising recombinant molecules of the major histocompatibility complex, and their use as immunogen or for diagnostic and therapeutic purposes. The invention also concerns methods for producing said vesicles, genetic constructs, cells and compositions, useful for implementing said methods.

Description

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 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).

Peptides, derived from antigens synthesized by body cells (tumor or viral antigens), associate with MHC class # 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 cells 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. 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 peptideCMH complexes on the cell surface and on the other hand, to the secretion of these vesicles called exosomes.

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 The work of Raposo et al. (J. Exp. Med. 183 (1996) 1161) have shown that B lymphocytes are capable of secreting exosome vesicles, carrying class II molecules of the MHC. In addition, Zitvogel et al. (Nature Medicine 4 (1998) 594) have highlighted the production of particular membrane vesicles by dendritic cells (designated dexosomes), having advantageous properties. Thus, these vesicles express molecules of classes I and II of the MHC, and are capable, after sensitization to the corresponding antigens, of stimulating in vivo the production of cytotoxic T lymphocytes and of causing the total or partial regression of tumors.

 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.

 Thus, 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. As a result, these molecules are of varied structure, not always identified, and generally multiple, depending on the HLA type of the organism from which they originate. On the contrary, the present invention makes it possible to produce membrane vesicles carrying MHC molecules of defined composition. In addition, 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

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 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. Such molecules can be exposed outside the vesicles or, on the contrary, enclosed in the cytosolic fraction. 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 present 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. Thus, these vesicles can be used as an immunogen, for the preparation of antibodies. In a particularly advantageous manner, these vesicles are used to produce antibodies restricted to MHC, that is to say, specific for a peptidemolecule 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.

 The term 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

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 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. In addition, 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. As illustrated later in the present description, 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.

 One of the first elements of the invention therefore lies in the definition and identification of the cell population used for the production of membrane vesicles. Advantageously, 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.

In addition, it can be primary cultures or immortalized lines.

 In a particularly advantageous manner, 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.

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 Different types of exosome-producing cells have been described in the literature, such as, for example, dendritic cells or B lymphocytes.

However, these cells are generally difficult to transfect and rich in endogenous MHC molecules. Therefore, although they can be used for the implementation of the invention, the vesicles of the present invention are more preferably capable of being obtained from mast cells or derived from mast cells.

 In a particular embodiment, 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). In addition, another advantage of the use of mast cells according to the invention lies in the possibility of triggering (in particular strongly stimulating) exocytosis (i.e., the release) of exosomes by different treatments. Thus, it is possible to regulate the production of 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. In addition, it has now been shown, as described in the examples, that these cells can also be genetically modified in a stable manner, which represents a property which is particularly advantageous for the implementation of the present invention.

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 More specifically, 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.

 In a preferred embodiment, the membrane vesicles of the invention are essentially devoid of endogenous MHC molecules. The absence of endogenous MHC molecules (that is to say MHC molecules of the vesicle-producing cell) can be demonstrated by means of specific antibodies, by conventional techniques. It can also be demonstrated by the selectivity of the antibodies obtained by immunization with the vesicles. As indicated in the examples, 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.

 Specific membrane vesicles according to the invention are more specifically characterized by the following properties: - they are essentially devoid of endogenous MHC molecules, - they carry one or more recombinant molecules of defined structure, for example recombinant peptide-MHC complexes, defined composition.

 Such vesicles of the invention are advantageously produced from cells derived from mast cells, which are essentially devoid of endogenous MHC molecules. In this regard, it is known that mast cells accumulate MHC class II molecules in their secretory granules.

In particular, mast cells are capable of accumulating preferentially the MHC-II-peptide complexes in particular multivesicular intracellular compartments, the secretory granules (Raposo and

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 al., Mol. Biol. Cell 8 (1997) 2619). These cells, taken from a mammal, therefore contain endogenous MHC molecules. Particularly advantageously, in the context of the present invention, cell lines derived from mast cells are used, essentially devoid of endogenous MHC molecules. Different mast cell lines have been described in the literature. The present application now shows that some of these lines have low levels of MHC molecules, and are therefore particularly advantageous for the implementation of the invention. By way of illustration, mention may in particular be made of lines derived from RBL cells (Rat Basophicil Leukemia), deposited at the ATCC under the number CRL1378 (Kulczycki et al., J. Exp. Med. 139 (1974) 600), the line KU-812 (Butterfield et al., Leukemia Res. 12 (1988) 345), or cells of a human immature mast cell line, such as the HMC line (Nilsson et al., Scand. J.

Immunol. 39 (1994) 489). A particular example of a line is the RBL-2H3 line (Barsumian et al, Eur. J. Immunol. (11 (1981) 317). It is understood that any other cell exhibiting the properties stated above can be used.

 In the context of the present invention, the expression "defined composition" more particularly designates the fact that the vesicles of the invention have, for example, great antigenic and haplotype specificity. Thus, the vesicles described in the prior art generally express MHC molecules of various and unknown haplotypes. On the contrary, 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.

 As indicated above, the vesicles of the invention advantageously express defined molecules of the MHC.

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 Human MHC molecules fall into two distinct classes, MHC class 1 molecules and MHC class II molecules.

 In a particular embodiment, the vesicles according to the invention express one or more recombinant molecules of the major histocompatibility complex of class II. In this regard, the human MHC class II molecules are composed of two chains, an a chain and a p chain, the p chain conferring allelic specificity on the complex.

 In a specific variant, the vesicles according to the invention more specifically express a recombinant α chain of a molecule of the major histocompatibility class II complex. In another specific variant, the vesicles according to the invention more specifically express a recombinant a chain and a p chain of a molecule of the major histocompatibility class II complex.

 Different types of human MHC II molecules have been identified, characterized and sequenced (see for example Immunogenetics 36 (1992) 135). Mention may preferably be made of molecules of the DR1 to DR13 type, in particular DR1, DR2, DR3, DR4, DR5, DR6 and DR7. The DNA encoding human DR, in particular DR1 to 13, can be easily isolated from cells, banks or plasmids by standard molecular biology techniques. These sequences have in particular been described in Bodmer et al. (Tissue antigens 44 (1994) 1). Preferably, the exosomes according to the invention therefore express a MHC class II molecule comprising an a chain and a p chain selected from haplotypes DR1 to DR13, even more preferably DR1 to DR7.

 In a specific example, the invention relates to any membrane vesicle comprising a recombinant a and / or p chain of a molecule of the MHC-II of haplotype DR1.

 In another embodiment, the vesicles according to the invention express one or more recombinant molecules of the major histocompatibility complex of class I. The molecules of the MHC class # are composed

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 also of two chains, the transmembrane and polymorphic chain, and ss2-microglobulin, which is constant and soluble. In humans, three genetic loci code for the a chain, designated A, B and C. In conventional MHC-I molecules, each locus A, B and C of the a chain is subject to allelic variation. Thus, the alleles A1, A2, A3, etc. are denoted. A10, B1, B7, B37, B54, etc., CW3, CW6, etc. (see for example Bodmer et al. supra and Immunogenetics 36, 1992, supra).

 Preferably, the exosomes of the invention express an a chain of a molecule of the conventional MHC-I, that is to say transmembrane and polymorphic. Even more preferably, it is a chain a of a MHC-I molecule of allele A1, A2 or A3.

 In a particular embodiment, the exosomes according to the invention express a chain a of a non-conventional, that is to say non-polymorphic, molecule of the MHC-I. Indeed, in contrast to the so-called "classical" MHC-I, subjected to a significant polymorphism, there are in humans "non-conventional" MHC-I molecules, 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 a non-conventional MHC-I according to the invention is represented by the molecule Cd 1.

 It is understood that any other molecule of human MHC-I can be expressed in the context of the present invention.

 In a specific example, the invention therefore relates to any membrane vesicle comprising a recombinant protein of a CMH-I molecule.

 In a particular variant, the vesicles of the invention comprise several MHC molecules of class # and / or II. Thus, an advantageous vesicle comprises, for example, 2 or more MHC-II molecules of different haplotypes.

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. In this regard, in

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 a more preferred mode of the invention, 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. In this regard, in a particular variant, 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. The term 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 secretion vesicles). The term "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. As preferred examples of 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

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 of classical infection, and therefore weakly immunogenic under natural conditions or also of proteins or fragments of membrane proteins. As preferred examples of 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), Gp100, ras proteins (wild or mutated p21), etc. It is understood that 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.

 In this regard, the recombinant antigenic molecules can be present either on the surface of the (exposed) vesicles, or inside the vesicles. In fact, particularly surprisingly, the inventors have shown that vesicles of the invention containing, in their cytosol, a recombinant antigen (in particular p53) are capable of inducing, in animals, a very high production of antibodies. directed against this antigen.

 Mention may generally be made, among ligand receptors, of any natural ligand receptor or one derived from genetic manipulation. In particular, it can be any hormone receptor, growth factor, lymphokine, trophic factor, antigen, etc. Mention may more particularly be made of the receptors of interleukins IL1 to IL15, the receptor for growth hormone, or the receptor for factors stimulating colonies of granulocytes and / or macrophages (G-CSF, GM-CSF, CSF, etc.) . A particular example of a ligand receptor is composed of a single-chain antibody (ScFv), which allows interaction with a specific ligand. Another particularly advantageous example within the meaning of the invention is represented by the T cell antigen receptor (TcR). 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.

 As pharmacological product, mention may be made of any active substance, of a chemical nature, such as for example pharmaceutical products prepared by conventional chemistry techniques. We can also cite any

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 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. On the other hand, such vesicles can also be used to transport the substances of interest (protein, peptide, acid nucleic acid, chemical substance, etc.) to cells.

Thus, 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.

 In a more preferred mode, the exosomes of the invention are used for the oriented transfer of substances of interest to selected cell populations. Thus, it is possible to prepare 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. This approach therefore allows a targeted and efficient transfer.

 In this regard, 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.

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 The vesicles of the invention may also comprise a peptide or a recombinant protein allowing purification of the vesicles. Thus, 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. In particular, it is possible to obtain an exosome exhibiting a peptide of particular structure, which can be easily detected and picked up by a receptor molecule. In a particular example, an exosome is produced comprising, in its structure, a peptide molecule comprising the His6 motif (i.e., 6 consecutive histidine residues). The presence of such a residue on the surface of the exosomes allows their easy purification on a support functionalized with nickel. Other recombinant peptides of this type can be used, such as for example the tag c-myc, VSV or HA.

 Finally, in a particular variant, 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. In particular, 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.

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 The 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, flask, ampoule, flask, pouch, etc., and stored at 4 ° C. or at -20 ° C., for example. Typical compositions according to the invention comprise from 5 to 500 μg of exosomes, for example from 5 to 200 g.

 The vesicles of the invention are obtained from genetically modified cells. As indicated above, 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.

 For the production of the vesicles of the invention carrying recombinant molecules of determined composition, 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).

 Generally, 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. Among the promoters which can be used, mention may be made, by way of example, of the immediate early cytomegalovirus (CMV) promoter, the SV40 promoter, the thymidine kinase gene promoter, in particular HSV-1 TK, the LTR promoter of a retrovirus, in particular LTR-RSV, or a

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 strong endogenous promoter of mast cells. A particularly preferred embodiment includes the use of the SRa 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.

 Depending on the type of coding region used, certain modifications may also be made to the construction. Thus, it may be particularly advantageous in certain cases to introduce into the coding region a signaling sequence, making it possible to address the expression product in a particular compartment of the cell, in particular towards a membrane compartment (internal, plasma, etc.). This addressing signal can be positioned upstream (5 '), downstream (3') or inside the coding region.

Preferably, the addressing signal is positioned 3 'to the coding region, more particularly in its cytoplasmic region, and in reading phase with the coding region. The use of 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.

On the other hand, in a particularly advantageous manner, 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.

 For the implementation of the present invention, it is in particular possible to use as an addressing signal a nucleic acid fragment

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 having the sequence of part of the following genes: Lamp1, CD63, LIMPII, Cd1 c, FcyR. 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 G-Y-X-X-I, 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.

 In the constructs used, the coding region is operably linked to the promoter, so as to allow its expression in cells.

 Furthermore, 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. In this regard, such a vector advantageously comprises a system allowing the selection of the cells containing it. In particular, 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.). In a particular embodiment, each vector comprises a single expression cassette as described above.

In this embodiment, the cells are therefore modified by the introduction of several vectors, when several molecules are to be expressed in the vesicles (for example an a chain and an ss chain of MHC-II). In this embodiment, each type of vector used advantageously comprises a different selection system, making it possible to easily select the multiple transfectants.

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 In another embodiment, a vector can comprise several expression cassettes as defined above, for example one encoding an a chain and the other, a p 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.

 For the production of exosomes according to the invention, 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. Thus, 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. In the case of viral vectors, the transfer is generally obtained by simple infection of the 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. In this regard, a particularly effective method for introducing nucleic acids into mast cells includes electroporation of the vectors.

 On the other hand, when several constructs (vectors) have to be introduced into the cells, these can be transferred simultaneously or sequentially.

 After transfer, the cells which have effectively incorporated the nucleic acids are selected and cloned on the basis of their resistance to a compound (eg, antibiotic) using the resistance gene present in the DNA which has been transferred. These cells can be used extemporaneously for the production of exosomes of the invention, or else be stored for

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 subsequent use. In this regard, the cells can be stored at 4 ° C. in a usual conditioning medium for a period sufficient to allow different batches of production of exosomes. 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. In particular, it is possible according to the invention to constitute banks of cells expressing the main HLA types of MHC class II molecules. Therefore, it is then possible, according to the applications envisaged and according to the HLA type of the subject, to choose from the bank the cells producing the corresponding MHC molecules, without having to reconstruct these cells on a case-by-case basis.

 In this regard, 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.

 More particularly, 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. As a particular example, mention may be made of cells of the RBL line, in particular RBL-2H3, cells of the KU-812 or HMC-I line.

 Preferably, the recombinant nucleic acid codes for an a chain and / or a P 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. In another embodiment, the cell comprises several acids

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 nucleic acid coding respectively for an a chain and a p chain of a molecule of the major class II histocompatibility complex.

 The present invention makes it possible to produce, in a simple and reproducible manner, large quantities of exosomes of known composition.

For the production of exosomes, 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 producing an exosome comprising a defined recombinant molecule, comprising the following steps: a) culturing a mast cell or mast cell derivative comprising a recombinant nucleic acid coding for said defined recombinant molecule, c) recovering the exosomes produced by said cells, these exosomes comprising said defined recombinant molecule.

 Advantageously, 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.

 On the other hand, 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.

 As indicated above, in the process of the invention, 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. In addition, as explained above, in certain embodiments, 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.

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 Another particular object of the invention resides in a process for the production of 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 # or II, and - recovery of the exosomes produced, where appropriate after stimulation of the exocytosis.

 In this regard, 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 culture of an exosome-producing cell comprising one or more Recombinant nucleic acids encoding a defined recombinant MHC molecule, - stimulation of cells to induce release of exosomes, - recovery of exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant MHC molecule, and, - bringing the exosomes into contact with the peptide (s).

 For the implementation of the invention, 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. Furthermore, after contacting the exosomes with the peptides, the exosomes can be isolated or purified according to conventional methods.

 In another variant, 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: - the culture of an exosome-producing cell comprising one or more Recombinant nucleic acids encoding a molecule

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 defined recombinant MHC and a nucleic acid comprising a region coding for a defined recombinant peptide, - stimulation of cells to induce release of exosomes, - recovery of exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant molecule of the MHC associated with said recombinant peptide.

 More particularly, in this method, 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. This embodiment ensures great specificity in the formation of the peptide-MHC complex.

 In another variant, the nucleic acid comprises a region coding for the peptide and a region targeting to the intracellular compartments.

In addition, the nucleic acid can comprise several regions coding for the same or for different antigenic peptides.

 Preferably, the producer cells used in the process are mast cell or mast cell derived cells. In this embodiment, 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.

 In a particularly preferred embodiment, 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 - the introduction into a producer cell of exosomes of a nucleic acid coding for a defined molecule, linked to an addressing signal in the membrane compartments, and - the production of exosomes from said cell.

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 This process advantageously makes it possible to produce exosomes expressing defined and varied recombinant molecules.

The 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 going beyond the fields of immunological applications; on the diagnostic or therapeutic level, as well as for the production of particular antibodies, in particular antibodies restricted to MHC. These different applications and others are illustrated below. a) Use for the production of antibodies
One of the first applications of the exosomes of the invention lies in the production of antibodies. Indeed, due to the defined composition of the exosomes of the invention, it is possible to produce antibodies 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 MHC-peptide complexes on their surface, their functionality, and their efficient presentation to the immune system. .

 The antibodies thus produced can be polyclonal or monoclonal. They can be prepared by conventional immunology techniques, including animal immunization, and the collection of sera (polyclonal antibodies) and / or the fusion of spleen lymphocytes with myeloma cells that do not produce immunoglobulins (for generate hybridomas producing monoclonals).

 Another subject of the invention therefore relates to antibodies or antibody fragments produced by immunization with exosomes as described above. The antibody fragments can be for example Fab, (Fab ') 2, ScFv, etc., and, more generally, any fragment retaining the specificity of

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the antibody. In particular, the invention relates to a method for preparing 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. Advantageously, the method of the invention allows the production of monoclonal antibodies, in particular restricted to MHC, that is to say specific to the MHC-peptide association. Preferably, in the process of the invention, use is made of 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. Thus, as shown in the examples, this method makes it possible to obtain, without the need for an adjuvant, powerful antibodies directed against the peptide, in particular antibodies restricted to MHC, that is to say specific to the peptide in its conformation associated with the defined molecule of the MHC. Such antibodies are particularly advantageous from an experimental, diagnostic and therapeutic point of view. In addition, the antibodies according to the invention can be labeled by any known technique (enzymatic, fluorescent, radioactive, etc.), according to the methods known to those skilled in the art. b) Diagnostic applications
The exosomes and antibodies of the invention have advantageous properties for diagnostic use.

 Thus, the antibodies or antibody fragments 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. In the particular case of antibodies restricted to MHC, they advantageously allow the detection of MHC-peptide complexes, and therefore the diagnosis of corresponding pathologies. These antibodies can

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 in particular be applied to the diagnosis of pathologies involving a defect of response or an inappropriate response of the immune system in order to determine the expression of an antigen, previously defined, in a form recognizable by T lymphocytes. For example and in a non-exhaustive manner, we can consider the diagnosis: - of tumor pathologies where the detection on tumor samples of different peptides from proteins such as p53, Her2, MAGE, BAGE, Mart, GP100 associated with molecules of class # MHC can allow phenotyping of the tumor and facilitate the choice of therapy; - viral diseases at a pre-infectious or latent stage, where the virion cannot be detected (hepatitis, HIV infection, CMV and other viruses); - autoimmune diseases such as multiple sclerosis, autoimmune diabetes, autoimmune thyroiditis, rheumatoid arthritis, systemic lupus erythematosus, where the detection of MHC molecules with peptides derived from autoantigens may be the warning sign of 'an progressive outbreak of the disease.

 The exosomes according to the invention can also be used for the detection of specific partners of a protein molecule in a biological sample. Thus, 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. In this regard, 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.

 In a particular embodiment, 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-MHC complexes in a biological sample. The biological sample can be any sample of blood, serum, tissue, tumor, biopsy, skin, urine, etc. In addition, the biological sample can be pretreated for example to dissociate cells, amplify

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 cells in culture, prepare membrane fractions, etc. Advantageously, the biological sample comes from a human organism. To this end, the invention also relates to a method for detecting 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 CMH antigen complex, and the demonstration of the labeling of T lymphocytes in said sample.

 In addition, 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.

 In a particular application, 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.

 Furthermore, 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, - for the detection of receptors specific for a protein molecule in a biological sample. In this embodiment, the exosomes used therefore comprise, on their surface, said biological molecule of defined structure.

 - for the detection of the presence of a ligand in a biological sample. In this embodiment, the exosomes used therefore comprise, on their surface, a specific receptor for said ligand.

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c) Therapeutic applications
Restricted antibodies or fragments thereof are potentially capable of inhibiting the interaction between the T cell receptor and the MHC-peptide complex for which it is specific. In parallel, exosomes carrying on their surface a single type of MHC-peptide complex can, by interacting with T lymphocytes specific for these complexes, compete with their natural ligands, T lymphocytes and cause their inactivation.

 Restricted antibodies and exosomes can therefore be used in all situations where one wishes to reduce or suppress an immune response mediated by T lymphocytes which is harmful for the organism as is the case for example in: - organ transplants or marrow transplants in which attempts are made to neutralize the host response against the graft, generally by means of large doses of immunosuppressive agents; - autoimmune diseases or viral pathologies during which the T CD8 or CD4 immune response chronically leads to tissue destruction; - allergies and asthma.

 In this type of pathology, 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.

 The 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 or infected cells.

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 by 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.

 Another particularly interesting application of 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.

In this regard, 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. Advantageously, it is an exosome comprising a ligand receptor or a receptor ligand on its surface, thus making it possible to direct 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. For an in vivo implementation, 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.). For in vitro or ex vivo use, the cells are contacted by incubation in an appropriate device (box,

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flask, pocket, bulb, etc. ), preferably under sterile conditions. The parameters of the contacting step (quantity of vesicle, contact time, temperature, medium, etc.) can be easily adjusted by a person skilled in the art according to the aims pursued and the teaching of the present application . d) Applications in the field of research
They obviously relate to all the applications mentioned above in the analysis of the molecular mechanisms of antigenic presentation by the use of antibodies making it possible to detect and analyze the different stages of the formation of MHC-peptide complexes in different normal situations. or pathological.

 Furthermore, they also relate to the analysis and molecular characterization of T lymphocyte populations capable of recognizing an MHC-peptide complex determined by the use of fluorescent exosomes in their capacity to detect T receptors for MHCpeptide complexes.

 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. In addition, all publications cited in the application are incorporated herein by reference.

Legends of figures
Figure 1: Production of functional MHC-DR1-HA peptide complexes in the RBL2H3 line.

 A. Analysis of the surface expression by flow cytometry of the human MHC II molecules DR1 before (left) and after (right) transfection of the cDNAs coding for the a and b chains of DR1 in the RBL 2H3 line. The DR1 molecules are detected by the antibody L243 (black line) itself revealed by a goat anti-mouse IgG serum coupled to the FITC.

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 B. Surface expression of DR1 in the line expressing an invariant chain (IiHA) where the peptide CLIP has been replaced by the peptide 308-319 derived from the hemagglutinin of the influenza virus. DR1 molecules are detected on the RBL DR1liHA line and a B lymphocyte transformed by EBV (Hom2) with the same haplotype by the antibody L243 (black line).

 C. Stimulation of T lymphocytes specific for the DR1-HA complex by the RBL line expressing this complex or B-EBVs of the same haplotype. The RBL DR1liHA and B-EBV Hom2 lines were diluted in culture plates with a T lymphocyte specific for the DR1 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 by means of a test for incorporation of tritiated thymidine from the CTLL2 line, the proliferation of which is IL2-dependent.

 D. Analysis of the saturation in HA peptide of the RBL DR1 liHA line.

Hom2 and RBL DRUiHA cells were incubated (100 cells per well) in the presence of increasing concentrations of the HA peptide and THA lymphocytes. The stimulation of the lymphocytes was evaluated as previously.

 Figure 2: Accumulation of DR1 molecules in a secretion compartment of RBL2H3.

 A. Analysis of the intracellular site of accumulation of DR1 molecules in RBL 2H3. The RBL DR1 HA cells were fixed with 0.5% glutaraldehyde and then permeabilized with 0.05% Saponin. The DR1 molecules and the invariant chain were detected respectively with the L243 and PIN1 antibodies and then a mouse anti-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.

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 B. Purification of RBL DR1IiHA exosomes. After washing in DMEM, the cells were incubated for 30 minutes in the presence of 1 mM ionomycin at 37 C. The exosomes were purified by differential ultracentrifugation from the cell supernatant. The exosome pellet, resuspended in PBS, was separated (5 mg) by SDS-PAGE and then transferred to a nylon membrane. The DR1 b chain was detected with the monoclonal antibody IB5 in the preparation of exosome and in control in the lysates of RBLDR1HA and Hom2 cells (equivalent to 105 cells per well) migrated under the same conditions.

Figure 3: Use of exosomes for the production of antiDR1 HA antibodies
A. Increasing dilutions of the sera of mice immunized with the exosomes were incubated with the RBL cells expressing (right) or not (left) the DR1 HA molecules. The labeling thus obtained was analyzed by flow cytometry.

 B. The sera of the rats (diluted 1/100) immunized with the exosomes were incubated with the RBL cells expressing or not (left) the DR1 HA molecules. On the right, the cells expressing DR1 were incubated beforehand or not for two hours at 37 ° C. with 10 mM of the HA peptide and then with the same dilution of the serum of the immune rats.

 C. The spleen of the immune rat was fused with the line X63A8 under standard conditions for the production of monoclonal antibodies. The supernatant of the various hybridomas was tested by immunofluorescence on the RBL2H3 cells expressing or not the DR1 or DR1 HA molecules. Clones a40, b82 and a15 are representative examples of the antibodies obtained.

Figure 4: Use of exosomes for the detection of T lymphocytes specific for the DR1 HA complex
A. The DR1liHA RBL cells were incubated in the presence of 5 mM of Green Tracker (fluorescent lipid accumulating in the compartments

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 lysosomal cells) for 30 minutes at 37 ° C. then washed and reincubated for one hour at 37 ° C. in the absence of fluorescent label. The cells were fixed (3% paraformaldehyde), then analyzed by confocal microscopy.

 B. In parallel, the DR1 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.

 CD The DR1 HA fluorescent exosomes were incubated at 50 mg / ml with THA lymphocytes specific for the DR1 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.

Cell Materials and Methods
The cells used for the production of exosomes in the experimental part are murine mast cell cells. More particularly, a basophil cell line with a mucosal mast cell phenotype, designated RBL-2H3, was used (Barsumian et al, Eur. J. Immunol. (11 (1981) 317).) Other mast cell cells, in particular established in line, can be used, such as lines derived from RBL cells (Rat Basophicil Leukemia), deposited at ATCC under the number CRL1378 (Kulczycki et al., J.

Exp. Med. 139 (1974) 600).

 T lymphocyte lines capable of recognizing a particular antigen in a human MHC II context (DR1) have also been used.

In particular, 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-DR1 (Sidhu et al. ., J. Exp. Med. 176

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 (1992) 875). 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-DR1.

 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 µM 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 the latter in large quantities, it is also possible to cultivate them in a "spinner", at a density of 106 cells / ml.

Plasmids
To genetically modify the cells of mast cells, the following genetic constructions were carried out.

 CDNAs encoding the human HLA-DR1a chain (Larhammer et al., Cell 30 (1982) 153), the human HLA-DR1ss chain (Bell et al., PNAS 82 (1985) 3405) and the human invariant chain p33 li (Claesson et al., PNAS 80 (1983) 7395) have been 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). In a specific example, a DNA fragment coding for the peptide HA308-319 of

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 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, into the plasmid pSRa under the control of the promoter SRa (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 a chain with the neomycin resistance gene; the p chain with the hygromycin resistance gene, and the invariant chain with the zeocin resistance gene.

Transfections
To introduce the different nucleic acids into mast cell cells, the corresponding plasmid vectors were linearized by the restriction enzyme Scal. 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 1,107 cells / ml. Stable transformants were obtained by electroporation of 5,106 cells using a "gene pulser" (Bio-Rad, Richmond, CA) under the following conditions: 260V, 960 F. 72 hours after electroporation, the transfectants are selected by culture in a selection medium comprising 250 G: ml of G418 (Geneticin, Gibco) 1 mg / 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.

 Results

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1-Production of DR1HA exosomes
1.1. Construction and characterization of genetically modified producer cells
In order to produce, in a controlled manner, exosomes carrying MHC-peptide complexes of defined composition, the a and b chains of molecules of class Idu MHC, DR1, were expressed in the mast cell line RBL2H3, resulting from a basophilic leukemia of the Rat. For this, two vectors carrying respectively a nucleic acid coding for each chain, under the control of the SRa promoter were transfected simultaneously in the cells (see materials and methods). The results obtained by flow cytometry show that the transfected cells express the DR1 molecules well (Figure 1 A).

 These RBL-2H3 DR1 cells were then sensitized to a given peptide, of precise composition, in order to generate MHC-peptide complexes of defined composition. For this, different techniques can be envisaged. In a simple embodiment, the peptide can be incubated directly with the exosomes. In another variant, a nucleic acid coding for the peptide can be introduced into the cells, so as to also express this peptide. In this particular implementation example, to manufacture a presenting cell comprising a single antigen specificity, the chosen antigen peptide was introduced into the cells in the form of a genetic fusion with the human invariant chain li. More particularly, 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 DR1 molecule. This construct (liHA) was transfected into cells under the conditions described in the Materials and Methods. The hybrid chain expressed in RBL-2H3 DR1 cells, allowed to construct

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 cells which express DR1 molecules recognized by the L243 antibody at a level similar to that of a B-EBV (Hom2) control line with the DR1 haplotype (FIG. 1B). These results therefore show that the mast cell cells of the invention do indeed express a functional human peptide-MHC complex, of predetermined and controlled composition.

 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 DR1-HA combination carried by the cells. For this, 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.

As control, a line of B lymphocytes transformed by the EBV (Hom-2), of haplotype DR1, pulsed by a saturating concentration (10mM) of the peptide HA was used.

 The results obtained are presented in FIG. 1 C and 1 D. They show that the mast cell cells of the invention express a DR1-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 DR1) pulsed by a saturating concentration (10 mM) of the HA peptide. Finally, the results obtained show that the DR1 molecules seem to only present the HA peptide since the addition of a saturated concentration of peptide does not significantly increase the capacity of the RBL DR1 liHA cells to stimulate a T HA lymphocyte (FIG. 1 D).

 All of these results therefore demonstrate the functional nature of the peptide-MHC complexes produced. They also illustrate the specific character of the cells obtained, and therefore the specific character of the process of the invention,

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 which makes it possible to obtain cells (and exosomes) carrying molecules of defined and controlled composition.

1. 2. Production of functionalized exosomes
An immunofluorescence study has shown that the recombinant MHC-peptide complexes (DR1 HA) accumulate in the secretory granules of the RBL-2H3 cell line. Figure 2A indeed demonstrates the co-localization of DR1 molecules with serotonin in intracellular vesicular structures.

 The possibility that functional exosomes may be released by these cells has therefore been studied. For this, 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 NI) 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 1 mM PBS-EGTA cold solution. The cells were then centrifuged at 300g at 4 ° C for 5 minutes. 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 Laemmli-DDT buffer (1X 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 20mA and then transferred to Immobilon. The analysis of the exosomes is then carried out by western blotting with antibodies specific for the different chains of MHC class II molecules.

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 The results obtained show that exosomes can be released from the RBL-2H3 line, significantly, after stimulation with an appropriate agent. These exosomes can be isolated and purified for example by differential ultracentrifugation for the preparation of composition of exosomes.

Finally, the results presented in Figure 2B demonstrate that these exosomes are functional. Indeed, western blotting analyzes show that the exosomes obtained express the different recombinant chains of MHC class II molecules. In addition, these results also show the high density of peptide-MHC complexes on the surface of the exosomes of the invention.

 The examples which follow illustrate in particular the use of exosomes DR1 HA for the production of antibodies specific for this combination and the capacity of exosomes DR1 HA to bind T lymphocytes specific for this same combination.

2 - Generation of antibodies specific for the DR1 HA complex
This example illustrates the use of the exosomes of the invention for the production of antibodies, in particular so-called "restricted" antibodies, 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 antibodies in the absence of any adjuvant.

 The exosomes purified from the supernatant of RBL DR1 HA cells (Example 1) were resuspended in PBS. These 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 at three weeks interval, then 30ug per channel

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 intraperitoneally and finally 30 g intravenously 3 days before the collection of the sera.

 - The rats were injected with exosomes intraperitoneally (10 μg), twice twice three weeks apart, then intravenously (50 μg) three days before the collection of the sera.

 As shown in FIG. 3A, the sera collected from the immunized mice exhibited a very high reactivity against the RBL line expressing or not the DR1 HA complex but which was detectable up to a dilution to thirty thousandths of the sera only in the case of DR1 HA cell.

 As shown in FIG. 3B, the sera of the rats thus immunized, showed, surprisingly, a reactivity against the RBL line expressing the DR1 HA complex while the same sera reacted with a lower intensity with the initial line RBL-2H3. In addition, the addition of the HA peptide to cells expressing DR1 (RBL-2H3 DR1) significantly increases the reactivity of the antisera thus produced (FIG. 3B).

 These results therefore show that the exosomes of the RBL line are capable of inducing an antibody response which, unexpectedly, is mainly directed in the rat against the DR1 HA complexes.

 The rats of the immune rats were fused with cells of the X63A8 line. 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 antibodies were thus obtained, for some directed against proteins of the RBL line, for others, against monomorphic determinants of molecules of human class II of haplotype DR1 and finally against the complex constituted by molecules DR1 associated with peptide derived from the HA protein of the influenza virus (Figure 3C). These latter antibodies

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 monoclonals constitute restricted antibodies, and therefore have particularly advantageous properties for diagnostic or therapeutic applications.

3 - Detection of T lymphocytes specific for the DR1HA complex
This example illustrates the use of the exosomes of the invention for the detection of specific T lymphocytes in a biological sample. This example also shows 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 antibodies described in Example 2, as well as to the detection of any receptor specific for a ligand.

 For the implementation of this application, labeled exosomes were produced. For this, before purification of the exosomes of the DR1 HA line, the latter was incubated with 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 cells. Analysis by confocal microscopy of the cells, after fixation, shows the presence of the fluorescent labeling in the secretion granules of the cells (FIG. 4A). The fluorescent exosomes were then produced and purified from these cells, under the conditions described in Example 1. Made thus fluorescent, these exosomes (Figure 4B) were used to detect, in a biological sample, the presence of lymphocytes T specific to the DR1 HA combination (HAT lymphocytes). It is understood that any other labeling can be used in the context of the invention, applied either to the producer cells, or to the exosomes produced.

 For this, the exosomes were incubated in the presence of a sample of HAT lymphocytes and TH30 lymphocytes, specific for another complex. The

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 results obtained by flow cytofluorometry show that the exosomes expressing the DR1 HA bind, in a specific way, to the HAT lymphocytes (FIG. 4C) whereas these same exosomes are unable to recognize lymphocytes having another specificity (FIG. 4D). These results demonstrate the unique and unexpected ability of the exosomes produced by the mast cell line according to the invention to detect T lymphocytes specific for this same complex. This application can be implemented from any type of biological sample.

 In addition, 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 class I and class II molecules of the MHC, of peptides derived from antigens expressed by the 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, with a view to their therapeutic use. Indeed, different approaches to immunotherapies for cancers or viral infections, for example, have been developed, based on the collection, from a subject, of lymphocytes and on the ex vivo expansion of particular clones of T lymphocytes specific for an antigen involved in the pathology (tumor or viral antigen, for example). These amplified clones are then re-administered to the subject, as a therapeutic agent. The present invention makes it possible to greatly facilitate the selection and amplification of specific T cell clones, and therefore the potential and the implementation of these therapeutic approaches.

Claims (44)

1. Membrane vesicle, characterized in that it comprises a recombinant molecule of the major histocompatibility complex.  2. Vesicle according to claim 1 characterized in that the recombinant molecule of the major histocompatibility complex is a molecule of class II.  3. Vesicle according to claim 2 characterized in that the recombinant molecule of the major histocompatibility complex of class II is an a chain.  4. Vesicle according to claim 2 characterized in that the recombinant molecule of the major histocompatibility complex of class II comprises an a chain and a p chain.  5. 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 DR1 to DR13, preferably DR1 to DR7.  6. Vesicle according to claim 1 characterized in that the recombinant molecule of the major histocompatibility complex is a class I molecule.  7. 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.  8. Vesicle according to one of the preceding claims, characterized in that it further comprises one or more heterologous molecules of interest.  9. Vesicle according to one of the preceding claims, characterized in that it further comprises a recombinant peptide or protein allowing its purification.  10. Vesicle according to the preceding claims, characterized in that it comprises a marker.  11. Vesicle according to one of the preceding claims, characterized in that it is essentially devoid of endogenous MHC molecules. <Desc / Clms Page number 42>  12. 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.  13. 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.  14. A membrane vesicle according to claim 13, characterized in that the heterologous molecule is a molecule of the major histocompatibility complex, an antigen, a receptor ligand, a ligand receptor, a nucleic acid, a pharmacological product, a marker and / or a purification peptide.  15. Vesicle according to claim 14, characterized in that it expresses a ligand receptor and in that it comprises another heterologous molecule of interest.  16. Membrane vesicle, characterized in that it comprises two MHC class II molecules of different haplotypes.  17. Exosome-producing cell, characterized in that it comprises one or more recombinant nucleic acids encoding a molecule of the major histocompatibility complex.  18. Cell according to claim 17, characterized in that it is a mast cell.  19. Cell according to claim 18, characterized in that it is a mast cell line of a basophilic leukemia, in particular of the RBL line, preferably RBL-2H3.  20. Cell according to one of claims 17 to 19, characterized in that it comprises a recombinant nucleic acid encoding an a chain and / or an ss chain of a molecule of the major histocompatibility complex of class II and / or for a molecule of the major histocompatibility class I complex.  21. A method of producing an exosome comprising a defined recombinant molecule, comprising the following steps: <Desc / Clms Page number 43>  a) culturing 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.  22. Method according to claim 21, characterized in that it comprises an intermediate step b) during which the cells are stimulated to induce and / or increase the secretion of exosomes.  23. The method of claim 21 or 22 characterized in that the defined recombinant molecule is exposed outside the exosome, or is included, in part or in whole, in the cytosolic fraction of the exosome.  24. Method according to one of claims 21 to 23, characterized in that the recombinant molecule is a molecule of the major histocompatibility complex, an antigenic molecule, a receptor ligand, a ligand receptor, a purification peptide or any other polypeptide of interest.  25. Method according to one of claims 21 to 24 characterized in that the nucleic acid further comprises a region coding for an addressing signal to the membrane compartments of the mast cell.  26. Method for preparing an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises: - the culture of an exosome-producing cell comprising one or more recombinant nucleic acids coding for a recombinant molecule defined MHC, - stimulation of cells to induce release of exosomes, - recovery of exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant MHC molecule, and, - bringing the exosomes into contact with the or peptides.  27. Process for the preparation of an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises: <Desc / Clms Page number 44>  - the culture of an exosome-producing cell comprising one or more recombinant nucleic acids coding for a defined recombinant molecule of MHC and a nucleic acid comprising a region coding for a defined recombinant peptide, - stimulation of cells to induce a release of exosomes, - the recovery of exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant molecule of the MHC associated with said recombinant peptide.  28. The method of claim 27, characterized in that the nucleic acid 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.  29. Method according to one of claims 26 to 28 characterized in that the producer cell is a mast cell or mast cell derived.  30. Method according to one of claims 26 to 29 characterized in that the producer cell is essentially devoid of endogenous MHC molecule.  31. A method of modifying the composition of an exosome, comprising - the introduction into a producer cell of exosomes of a nucleic acid coding for a defined molecule, linked to an addressing signal in the membrane compartments, and, - the production of exosomes from said cell.  32. Composition comprising one or more membrane vesicles according to one of claims 1 to 16.  33. Use of a vesicle according to one of claims 1 to 16 for the production of antibodies, polyclonal and / or monoclonal.  34. A method of producing antibodies, comprising immunizing an animal with a vesicle according to claim 7 and recovering the antibodies and / or cells producing antibodies or involved in the immune response. <Desc / Clms Page number 45>  35. The method of claim 34, for the production of monoclonal antibodies, in particular specific for the MHC-peptide association.  36. Use of an antibody obtained according to claim 34 or 35, or a fragment of such an antibody, for the detection, in a biological sample, of the presence of corresponding specific antigens.  37. Use of an antibody produced according to claim 34 or 35, a fragment of such an antibody, or a vesicle according to claim 1 for the preparation of a therapeutic composition intended to inhibit the interaction between T cell receptor and the MHC-peptide complex for which it is specific.  38. Use of a membrane vesicle according to one of claims 1 to 16 for the detection of specific partners of a protein molecule in a biological sample.  39. Use according to claim 38 of an exosome carrying a MHC-peptide complex for the detection of T lymphocytes specific for this complex in a biological sample.  40. Use according to claim 38 of an exosome carrying a TcR receptor for the detection of peptide-MHC complexes specific for this receptor in a biological sample.  41. Use according to claim 38 of an exosome carrying a ligand receptor for detecting the presence of said ligand in a biological sample.  42. Method for detecting the presence of T lymphocytes specific for antigen-MHC complexes in a biological sample, comprising bringing said sample into contact with a labeled exosome according to claim 7, comprising said antigen-MHC complex, and evidence of labeling of T lymphocytes in said sample.  43. Use of a vesicle according to claim 7 for clonal amplification and / or ex vivo stimulation of cytotoxic or helper T lymphocytes. <Desc / Clms Page number 46>  44. Use of a vesicle according to one of claims 11 to 16 for the preparation of a composition intended for the transfer of said molecule into a cell.