WO2000028001A1

WO2000028001A1 - Modified exosomes and uses

Info

Publication number: WO2000028001A1
Application number: PCT/FR1999/002691
Authority: WO
Inventors: Philippe Benaroch; Hélène VINCENT-SCHNEIDER; Pamela Stumptner; Sebastian Amigorena; Christian Bonnerot; Graça RAPOSO
Original assignee: Institut National De La Sante Et De La Recherche Medicale; Institut Curie; Centre National De La Recherche Scientifique
Priority date: 1998-11-05
Filing date: 1999-11-04
Publication date: 2000-05-18
Also published as: AU1051400A; FR2785543A1; IL142634A0; FR2785543B1; AU772451B2; AU2004203482A1; EA200100509A1; TWI224138B; CA2349679A1; EA004428B1; EP1127110A1; CN1325441A; JP2002529074A

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

MODIFIED EXOSOMES AND USES

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 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. 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 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 MHC molecules. 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, to stimulate in vivo the production of cytotoxic T lymphocytes and to cause 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 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 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. 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 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.

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 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.

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) the exocytosis (ie, the release) of the 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 particularly advantageous property for the implementation of the present invention. 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, 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. 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 et 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 (Rat Basophicil Leukemia) cells, deposited at ATCC under number CRL1378 (Kulczycki et al., J. Exp. Med. 139 (1974) 600), line KU-812 (Butterfield et al, Leukemia Res. 12 (1988) 345), or cells a line of human immature mast cells, 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. (1 1 (1 981) 317). It is understood that any other cell exhibiting the properties set out above can be brought into use. artwork.

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. Human MHC molecules fall into two distinct classes, MHC class I 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 IL In this regard, the molecules of human MHC class II are composed of two chains, an α chain and a β chain, the β 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 express more particularly a recombinant α chain and a β chain of a molecule of the major histocompatibility complex of class IL

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. 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). Preferably, the exosomes according to the invention therefore express a MHC class II molecule comprising an α chain and a β 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 α and / or β chain of a CMH-II molecule of haplotype DR1. In another embodiment, 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 α chain, and β2-microglobulin, which is constant and soluble. In humans, three genetic loci code for the α chain, designated A, B and C. In conventional MHC-I molecules, each locus A, B and C of the α chain is subject to allelic variation. Thus, the 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).

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

In a particular embodiment, the exosomes according to the invention express an α chain of a non-conventional MHC-I molecule, that is to say non-polymorphic. In fact, in contrast to the so-called "classical" CMH-I, subjected to significant polymorphism, there are "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.

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 class I and / or IL molecules. Thus, 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. In this regard, in a more preferred embodiment 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 secretory 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 of classical infection, and therefore weakly immunogens under natural conditions or also 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), GplOO, 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. 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 anticoφs 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 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 anticoφs (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 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. 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.

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. 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, 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 5OOμg of exosomes, for example from 5 to 200 μg.

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, there may be mentioned by way of example the immediate early promoter of the cytomegalovirus (CMV), the promoter of SV40, the promoter of the thymidine kinase gene, in particular HSV-1 TK, the promoter of the LTR of a retro virus, in particular LTR-RSV, or a strong endogenous promoter of mast cell cells. A particularly preferred embodiment comprises the use of the SRα 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 having the sequence of a part of the following genes: Lampl, CD63, LIMPII, Cdlc, FcγR. 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. In this context, 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. Thus, 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. In this regard, the invention therefore relates to any exosome comprising a recombinant molecule of fusion between a polypeptide of interest and an addressing signal. It can be an exosome produced from a mast cell, dendritic cell, tumor cell or also a B lymphocyte, for example. 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.

In a particular embodiment of the invention, 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

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 α chain and a β chain of MHC-II). In this embodiment, each type of vector used advantageously comprises a different selection system, making it possible to easily select multiple transfectants. In another embodiment, a vector may comprise several expression cassettes as defined above, for example one encoding an α chain and the other, a β 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, 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. 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 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. These cells can be used extemporaneously for the production of exosomes of the invention, or else be stored for later 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 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. 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. 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.

Preferably, the recombinant nucleic acid codes for an α and / or β 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 mode of embodiment, the cell comprises several nucleic acids coding respectively for an α chain and a β 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. 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 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. 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.

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

- recovery of the exosomes produced, if necessary 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 coding for a defined recombinant molecule of the MHC,

- stimulation of cells to induce release of exosomes,

recovering the exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant molecule of the MHC, 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 coding for a defined recombinant molecule of the MHC and a nucleic acid comprising a region coding for a defined recombinant peptide,

- stimulation of cells to induce 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.

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.

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 exceeding the scope of immunological applications; on the diagnostic or therapeutic level, as well as for the production of specific anticoφs, in particular anticoφs restricted to MHC. These different applications and others are illustrated below.

a) Use for the production of anticoφs

One of the first applications of the exosomes of the invention lies in the production of anticoφs. Indeed, due to the defined composition of the exosomes of the invention, it is possible to produce anticoφs 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 anticoφs 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 anticoφs or fragments of anticoφs produced by immunization with exosomes as described above. The fragments of anticoφs can be for example Fab, (Fab ′) 2, ScFv, etc., and, more generally, any fragment retaining the specificity of the anticoφs. In particular, 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. Advantageously, the method of the invention allows the production of monoclonal anticoφs, 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 anticoφs directed against the peptide, in particular anticoφs restricted to MHC, that is to say specific to the peptide in its associated conformation to the defined MHC molecule. Such anticoφs are particularly advantageous on the experimental, diagnostic and therapeutic level. In addition, the anticoφs 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.

b) Diagnostic applications

The exosomes and anticoφs of the invention have advantageous properties for diagnostic use.

Thus, the anticoφs or fragments of anticoφs 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 antico ants restricted to MHC, they advantageously allow the detection of the MHC-peptide complexes corresponding, and therefore the diagnosis of corresponding pathologies. These anticoφs 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. For example and so non-exhaustive, we can consider the diagnosis: - of tumor pathologies where the detection on tumor samples of different peptides derived from proteins such as p53, Her2, MAGE, BAGE, Mart, GP100 associated with MHC class I molecules can allow phenotyping 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 can 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 -CMH 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 the cells, amplify the 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 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. 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 specific receptors 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.

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 anticoφs 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;

- 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 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.

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 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. 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, flask, pouch, vial, 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 anticoφs which make 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 a MHC-peptide complex determined by the use of fluorescent exosomes in their ability to detect T receptors for MHC-peptide complexes. In these different applications (diagnostic, therapeutic, experimental, production of T lymphocytes, etc.), the exosomes of the invention can be used either as such or in immobilized form on a support. Thus, 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. In this regard, 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. Advantageously, beads with a diameter of 1 to 10 μm, for example from 2 to 5 μm, 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. In general, 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.

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

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

C. Stimulation of T cells specific for the DRI -HA complex by the RBL line expressing this complex or B-EBVs of the same haplotype. RBL lines

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 incoφoration test of the CTLL2 line, the proliferation of which is IL2-dependent.

D. Analysis of the saturation in HA peptide of the RBL DRI liHA line. Hom2 and RBL DRI liHA 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 DRI molecules in a secretion compartment of RBL2H3. A. Analysis of the intracellular site of accumulation of DRI molecules in RBL

2H3. 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 anticoφs 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.

B. Purification of RBL DRlliHA 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 β chain of DRI was detected with the monoclonal anticoφs IB5 in the preparation of exosome and as a control in the lysates of RBLDRLHA and Hom2 cells (equivalent to 10 ⁵ cells per well) migrated under the same conditions.

Figure 3: Use of exosomes for the production of antiDRI 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 DRI HA molecules. The labeling thus obtained was analyzed by flow cytometry.

B. The sera of the rats (diluted to 1 / l00) immunized with the exosomes were incubated with the RBL cells expressing or not (left) the DRI HA molecules. On the right, the cells expressing DRI 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 X63A8 line under standard conditions for the manufacture of monoclonal antibodies. The supernatant of the various hybridomas was tested by immunofluorescence on the RBL2H3 cells expressing or not the DRI or DRI HA molecules. The clones a4O, b82 and al 5 are representative examples of the anticoφs obtained.

Figure 4 -. Use of exosomes for the detection of T lymphocytes specific for the DRI HA complex

A. 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.

B. In parallel, the 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.

CD. 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.

Figure 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.

B Western blot analysis of the expression of the IAb molecule in RBL. 10 mg of a cell lysate and of an exosome preparation originating from the RBL lAbli cell were analyzed by western blot with a rabbit serum specific for the cytoplasmic region of the a chain of the molecule IA.

C Analysis by flow cytometry of the composition of exosomes. 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). The CD63 molecule of the rat is detected with the anticoφs AD1, IAb molecules with the anticoφs Y3P while the DRI molecules are detected by the anticoφs L243. These different anticoφs are revealed by secondary anticoφs coupled to phycoerythrin.

Figure 6: Moφhological 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 anticoφs 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.

B C. Moφhological characterization of the exosomes secreted by RBL-2H3 cells The 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. The exosomes are either contrasted and coated in a solution of 4% uranyl acetate and methylcellulose (b) or immunolabeled with anticoφs directed against the class II molecules before coating (c). As in Figure (a), the anticoφs are visualized with protein A coupled to 10 nm colloidal gold particles. Bars: 250 nm.

Figure 7: Manipulation of the internal composition of exosomes, introduction of a recombinant protein.

A The expression of 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.

B Western blot analysis of the expression of the DRI GFP molecule in RBL. 10 μg of a cell lysate and 20 μg of an exosome preparation originating from the RBL DRI GFP cell were analyzed by western blot a pair of monoclonal anticoφs specific for GFP.

C Analysis by flow cytometry of the composition of exosomes. Latex beads were coated with either fetal calf serum (FCS) or exosomes from the RBL DRI GFP cells. DRI molecules are detected by anticoφs L243 and secondary anticoφs coupled to phycoerythrin while the presence of GFP is detected directly in the FL1 channel.

Figure 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.

B Acquisition of an exosome marker by specifically labeled T cells. The same markings as in A were carried out but the binding of exosomes (not marked with the green cell tracker) to the T cells was detected with a monoclonal antibody of mouse AD1 anti CD63 of rat, then revealed by anticoφs of donkey 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.

C Stimulation of Lymphocytes by exosomes. The exosomes purified from DRI GFP cells were crosslinked on latex beads prepared in the same way as for flow cytofluorometry but washed in complete medium. Each pellet of beads was taken up in 100 μL, 50 μL deposited in the first well of a 96-well plate and 50 μL diluted two by two. T cells (Jurkat T and THAI 7) were adjusted to 106 cells / mL and 50 μL were deposited per well in the presence or absence of the HA307-319 peptide at 5 μM. 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.

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 anticoφs + anti-mouse-FITC.

B analysis of HMC-1 exosomes bonded to latex beads by flow cytofluorometry, dark solid line: latex beads-HMC-1 exosomes alone; solid clear line: anti-mouse anti-FITC only; tight dotted lines: latex control beads - S VF + specific anticoφs + anti-mouse-FITC; loose dotted lines: latex-exosome beads + specific anticoφs + anti-mouse-FITC

C Western blot analysis of a lysate of HMC-1 cells compared to their exosomes; 10 or 3μg of proteins per put; HC10: supernatant at 1/10; 1B5: 1 / 10th supernatant; CD63: 5μg / mL; Lampl: 2μg / mL; H68.4: supernatant to 1/10. Exosomes appear to be enriched in CD63, Lampl, TfR. The absence of MHC class II in both the lysate and the exosomes confirms the analysis by cytofluorometry. The proportion of MHC class I seems identical for the cell lysate and the exosomes.

Materials and methods

Cells

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).

T lymphocyte lines capable of recognizing a particular antigen in a human MHC II context (DRI) 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-DRI (Sidhu et al. ., J. Exp. Med. 176 (1 992) 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-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 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 these in large quantities, it is also possible to cultivate them in "spinner", at the density of 10 ⁶ cells / ml.

Plasmids To genetically modify mast cell cells, the following genetic constructs have been made.

CDNAs coding for the human HLA-DRI α chain (Larhammer et al., Ceil 30 (1982) 153), the human HLA-DRI β 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). In a specific example, 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 pSRα under the control of the promoter SRα (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 α chain with the neomycin resistance gene; the β 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 Seal. 50 μg 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 μF. 72 hours after the electroporation, the transfectants are selected by culture in a selection medium comprising 250 μg: ml of G418 (Geneticin, Gibco) lmg / ml of hygromycin and 500 μg / 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.

Anticoφs

Y3P (MHC II (IA)) is a mouse monoclonal antibody recognizing the IAb aβ 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. In flow cytofluorometry experiments, the second anticoφs used are F (ab ') 2 fragments coupled to (-Phycoerythrin, produced in donkeys and directed against mouse IgG (H + L) (Jackson Immunoresearch Laboratories) .

Balls

Latex beads: Surfactant-free white aldehyde / latex sulfate, D: 3.9μm, Interfacial Dynamics Coφ., Portland, Or. USA

Electron microscopy The RBL-2H3 cells transfected with HLA-DRI were fixed with 2% paraformaldehyde in 0.2M phosphate buffer pH 7.4. (PB buffer) After fixing, the cells were washed with PB-glycine buffer 50 mM and then coated in 10% gelatin. After solidification, the blocks were prepared, infused in 2.3M sucrose and frozen in liquid nitrogen. Frozen ultra-thin sections were prepared and immunolabeled with polyclonal antibodies directed against the HLA-DR molecules. These anticoφs are visualized with protein A coupled to 10 nm colloidal gold particles.

The 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.

The exosomes are either contrasted and coated in a solution of 4% uranyl acetate and methylcellulose or are immunolabelled with anticoφs directed against the class II molecules before coating. Anticoφs are visualized with protein A coupled to 10 nm colloidal gold particles. Results

1 - Production of DRI HA 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 MHC class II molecules, DRI, were expressed in the lo mast cell line RBL2H3, originating from a leukemia basophile du 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 DRI molecules well (Figure IA).

These RBL-2H3 DRI 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 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 anticoφs L243 at a similar level as a B-EBV (Hom2) control line of DRI haplotype (Figure 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 DRI -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 a control, 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.

The results obtained are presented in FIG. 1C and 1D. They 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).

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 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 (DRI HA) accumulate in the secretory granules of the RBL-2H3 cell line. Figure 2A indeed demonstrates the colocation of DRI molecules with serotonin in vesicular intracellular 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 μl) and the incubation was continued for 30 minutes at 37 ° C. The exocytosis was stopped by rapid cooling in ice and addition of 300 μl of a cold 1 mM PBS-EGTA 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 μl 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 anticoφs of the various chains of the class II molecules of the MHC. The results obtained show that exosomes can be released from the line

RBL-2H3, 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, analyzes by westem blotting show that the exosomes obtained express the different recombinant chains of class II molecules of the MHC. 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 the exosomes DRI HA for the production of specific antico cettes of this combination and the capacity of the exosomes DRI HA to bind T lymphocytes specific of this same combination.

2 - Generation of specific anticoφs of the DRI HA complex

This example illustrates the use of the exosomes of the invention for the production of anticoφs, in particular so-called “restricted” anticoφs, 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 anticoφs in the absence of any adjuvant. The exosomes purified from the RBL DRI HA cell supernatant (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 μg of exosomes, twice three weeks apart, then 30 μg intraperitoneally and finally 30 μg intravenously 3 days before the collection of the sera.

- The rats were injected with exosomes by the intraperitoneal route (10 μg), twice twice at three weeks apart, then by the intravenous route (50 μg) three days before the collection of the samples.

As shown in FIG. 3A, 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.

As shown in FIG. 3B, 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. In addition, the addition of the HA peptide to cells expressing DRI (RBL-2H3 DRI) significantly increases the reactivity of the antisemms thus produced (FIG. 3B).

These results therefore show that the exosomes of the RBL line are capable of inducing an anticoφs response which, unexpectedly, is mainly directed in the rat against the DRI 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 anticoφs have thus been obtained, for some directed against proteins of the RBL line, for others, against monomoφhical 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 anticoφs constitute restricted anticoφs, and therefore have particularly advantageous properties for diagnostic or therapeutic applications.

3 - Detection of T lymphocytes specific to the DRI HA 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 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 anticoφs 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 DRI HA line, it 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 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. Made thus fluorescent, these exosomes (Figure 4B) were used to detect, in a biological sample, the presence of T lymphocytes specific for the DRI HA combination (THA 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 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). 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 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. Indeed, different approaches to immunotherapies for cancers or viral infections, for example, have been developed, based on the removal, 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 clones of T lymphocytes, and therefore the potential and the implementation of these therapeutic approaches.

4- Production of exosomes carrying murine MHC class II molecules (Figures 5A, 5B).

The complementary DNAs encoding the α and β 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 α chain IAb), to neomycin (for IAb β chain), or to zeocine (for the invariant chain). After transfection of the RBL2H3 cells by electroporation (materials and methods), 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 anticoφs.

Figure 5 shows some of the results obtained. Analysis by flow cytometry shows that the RBL lAbli cell is recognized by the anticoφs 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). Moφhological 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). Westem blot analysis of these exosome preparations shows that they contain murine class II MHC molecules identical to those detected in a control cell lysate (Figure 5B). These results establish that human class II molecules (DRI) but also murine (IAb) can be expressed and accumulate in the exosomes of the corresponding RBL 2H3 lines.

5-Moφhological characterization of the exosomes produced by RBL 2H3 (Figure 6)

Electron microscopy observations on an immunolabelled frozen section of RBL cells reveal the presence of numerous intracellular compartments filled with membranes. The major part of these membranes correspond to vesicles which fill the lumen of the compartments (Figure 6A). Class II molecules once transfected into these cells accumulate in the compartments having internal vesicles and are visualized in particular, in association with the membrane of these vesicles (Figure 6A).

When the RBL cells are stimulated so as to induce their degranulation (IgE-Antigen or ionomycin), 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 moφhology 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. Using this technique we observed that 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).

6- Immobilization of exosomes on supports (Figure 5C).

This example describes the fixation of exosomes on solid supports, and shows that the exosomes thus fixed retain their functional properties. These new products (supports-exosomes) 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.

Different preparations of 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 μm on TLA 100.4 for 30 minutes). 30μg of exosomes are mixed with 10 μL 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:

-Saturated by adding 100 mM final glycine (30 min at room temperature), -Centrifuged at 2200xg for 2 min at 4 ° C then the pellet of beads is taken up in 1 ml of PBSlx SVF3% NaN3 0.01% To use the beads in cytofluorometry,

- Wash two to three times the pellet of beads in PBSlx SVF3% NaN3 0.01%. - Resume in 1 mL of PBS lx 0.01% NaN3.

-Use between 5μL and 20μL per point and incubate conventionally in the first then the second anticoφs. The reading is done on Facscalibur (Becton Dickinson).

This technique made it possible to cover the surface of these latex beads with exosomes, while preserving their structure.

When the exosomes are on the beads, their handling is easier. Indeed, they can be centrifuged at low speed and detected by conventional flow cytometry techniques. 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 anticoφs specific for IAb molecules, and L243 anticoφs specific for DRI molecules. These different anticoφs were revealed by anticoφs secondary coupled to phycoerythrin then the markings obtained were analyzed by flow cytometry using a FACScalibur (Beckman).

It is thus observed that the 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 anticoφs recognizes latex beads covered with fetal calf semm (Figure 5C).

These results show that this technique makes it possible to detect in a sensitive and specific way the expression of different proteins included in the composition of exosomes. Example 8 also shows that such products (support exosomes) also make it possible to detect or stimulate the proliferation of specific T lymphocytes.

7-Manipulation of the composition of exosomes (Figure 7)

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.

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 DRβ chain (in C-ter) to the N-ter end of the GFP. The DRβ 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 resulting plasmid, digested with PstI and Xbal, made it possible to release a fragment corresponding to the last 30 amino acids of DRa followed by GFP. In parallel, 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.

The analysis of these cells (DRI GFP) by flow cytometry shows that the cells recognized by the anticoφs L243, specific for DRI molecules, also emit a green fluorescence detected in the FL1 channel whereas cells transfected with the alpha and beta of DRI without GFP do not emit any fluorescence in the FL1 channel (Figure 7 A).

In order to show that GFP is actually contained in the exosomes of the RBL 2H3 cell, 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. By comparison between “crosslinked” latex beads with fetal calf semm or exosomes from the DRI GFP cell, it is observed that only the beads coated with exosomes induce fluorescence in the FL1 channel (FITC) and are recognized by antico24s L243, specific for DRI, detected by a secondary anticoφs coupled to phycoerythrin. These results therefore demonstrate that it is possible to introduce an exogenous protein inside the exosomes produced by the RBL 2H3 cell. These results also show that it is possible to direct a protein into an exosome by expression, in the producer cell, in the form of a fusion with a transmembrane molecule, such as a MHC molecule.

8-Functional characterization of exosomes (Figure 8)

This example shows that the 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 μl 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 μl 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 μl of exosomes. Doses ranging from 32 to 2 μl of exosomes per well were tested. The labeling obtained with 16 μl 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.

Our results therefore show that fluorescent exosomes can be used to visualize by flow cytometry populations of T lymphocytes specific for HLA-DR / antigenic peptide complexes carried by the exosomes.

To assess the ability of exosomes to stimulate a T cell dependent on the presence of a peptide, 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%, βmercaptoethanol 0.1%, Sodium Pymvate 4% ). For the stimulation of T lymphocytes, each pellet of beads was taken up in 100 μL, 50 μL deposited in the first well of a 96-well plate and 50 μL 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 μL were deposited per well. The peptide diluted to 15 μM in complete medium was also added at the rate of 50 μL per well. In the peptide-free series, 50 μL 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.

It is thus observed that the addition of the HA peptide (5mM) specifically induces the stimulation of the Jurkat T cell expressing the receptor for the DRI -HA complex (THA) while it has no effect on the control T lymphocyte (Jurkat T ). Furthermore, exosomes are unable to stimulate THA in the absence of the HA peptide (Figure 8C).

9-Exosomes of the human mast cell line HMC1 (Figure 9)

This example shows that modified exosomes according to the invention can be produced from other cells, in particular human. In particular, 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.

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 (ImM) then purified from the supernatants by differential ultracentrifugation. Their composition was analyzed by flow cytometry after "crosslinking" on latex beads and by westem blot.

Analysis of the exosomes by the method using latex beads shows that they carry the Lampl, CD9, CD63, CD81 molecules and the MHC class I molecules. but no class II molecules nor the Lamp2 molecule (Figure 9B). Furthermore, the moφhology observed by electron microscopy of these exosomes is identical to those produced by the RBL 2H3 line. Finally, the protein composition observed by westem blot confirms these results since we have found by this technique the molecules CD63, Lampl, MHC class I while Lamp2, MHC class II remain negative (Figure 9C).

In conclusion, 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.

I. Membrane vesicle, characterized in that it comprises a recombinant molecule of the major human histocompatibility complex. 2. Vesicle according to claim 1 characterized in that the recombinant molecule of the major histocompatibility complex is a molecule of class IL

3. Vesicle according to claim 2 characterized in that the recombinant molecule of the major histocompatibility complex of class II is an α chain.

4. Vesicle according to claim 2 characterized in that the recombinant molecule of the major class II histocompatibility complex comprises an α chain and a β 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 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.

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.

I I. Vesicle according to one of the preceding claims, characterized in that it is essentially devoid of endogenous MHC molecules.

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. Membrane vesicle according to claim 13, characterized in that the heterologous molecule is a molecule of the major histocompatibility complex, a

Claims

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 a recombinant molecule of fusion between a polypeptide of interest and an addressing signal.

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 α chain and / or a β 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: a) culturing a mast cell or mast cell derivative comprising a recombinant nucleic acid encoding said defined recombinant molecule, c) recovering 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. Process for the preparation of an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises:

- stimulation of cells to induce release of exosomes,

- bringing the exosomes into contact with the peptide (s).

27. Process for the preparation of an exosome comprising a peptide-MHC complex of defined composition, characterized in that it comprises:

- stimulation of cells to induce release of exosomes,

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 anticoφs, polyclonal and / or monoclonal.

34. A method of producing anticoφs, comprising immunizing an animal with a vesicle according to claim 7 and recovering the anticoφs and / or cells producing anticoφs or involved in the immune response.

35. The method of claim 34, for the production of monoclonal anticoφs, in particular specific for the MHC-peptide association.

36. Use of an anticoφs obtained according to claim 34 or 35, or a fragment of such an anticoφs, for the detection, in a biological sample, of the presence of corresponding specific antigens.

37. Use of an anticoφs product according to claim 34 or 35, a fragment of such an anticoφs, or a vesicle according to claim 1 for the preparation of a therapeutic composition intended to inhibit the interaction between the 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.

44. Use of a vesicle according to one of claims 11 to 16 for the preparation of a composition intended to transport said molecule to a cell. 45. Composition comprising one or more exosomes immobilized on a support.

46. Use of a membrane vesicle according to one of claims 1 to 16, in particular in the form immobilized on a support, for the purification of cells.