CA2349679A1 - Modified exosomes and uses - Google Patents

Abstract

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

Description

MODIFIED EXOSOMES AND USES
The present invention relates to the fields of biology and immunology.
It relates to membrane vesicles comprising molecules, especially antigenic, of predetermined structure, and their uses. She concerns more particularly vesicles containing recombinant molecules of the complex major histocompatibility, and their use as immunogen or as tool diagnostic or therapeutic. The invention also relates to methods of production of these vesicles, genetic constructs, cells and compositions, usable for the implementation of the methods of (invention.
The specificity of antigenic recognition is a characteristic major of immune system cells. B cells recognize antigens under native form. T cells recognize the complexes formed by the association of peptides from the degradation of antigens with molecules of the Major Complex Histocompatibility (CMH). Peptides; derived from synthesized antigens by the body cells (tumor or viral antigens), associate with molecules of MHC class I which are recognized by cytotoxic T cells. The peptides derived from exogenous antigens associate with MHC class II molecules which are recognized by helper T cells. Identification of peptides presented by MHC molecules and recognized by cytotoxic T lymphocytes (CD8) or auxiliaries (CD4) was at the origin of new therapeutic strategies and vaccine.
This evolution of immunotherapies requires the development of techniques to assess 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 pathway endocytic (Peters et al., Nature 349 (1991) 669). Their fusion with the plasma membrane results, from part, to the expression of peptide-MHC complexes on the cell surface and on the other hand at the secretion of these vesicles called exosomes.
The work of Raposo et al. (J. Exp. Med. I 83 (1996) 1161) have shown that the B cells are capable of secreting exosome vesicles, carrying molecules class II of the MHC. In addition, Zitvogel et al. (Nature Medicine 4 (1998) 594) have put in evidence of the production of particular membrane vesicles by cells dendritics (designated dexosomes), having advantageous properties. So, these vesicles express MHC classes I and II molecules, and are capable, after

2 sensitization to the corresponding antigens, to stimulate in vivo the production of cytotoxic T cells and cause total or partial regression tumors.
The present invention relates to new methods and compositions usable in the fields of biology and immunology. More particularly, the present invention describes new membrane vesicles whose composition has been modified in a determined way. In particular, the present invention describes a new method allowing the production, on a contract basis, of vesicles expressing molecules of the complex MHC of known composition, possibly complexed with peptide antigenics of determined structure. The present invention therefore makes it possible to modify the composition of 1o membrane vesicles in a controlled manner, and therefore to create products particularly advantageous from a therapeutic, diagnostic or even experimental point of view.
Thus, the vesicles described so far include, in the best of case the endogenous MHC molecules, i.e. the MHC molecules expressed by the cell from which they come. Therefore, these molecules are structured varied, not always identified, and generally multiple, depending on the HLA type of the organization whose they came out. On the contrary, the present invention makes it possible to produce vesicles membranes carrying MHC molecules of defined composition. In addition, the vesicles of the invention have (advantage of being very rich in molecules of the CMH as well determined, and offer a powerful immunogenic power.
The present invention relates in particular to membrane vesicles comprising molecules of predetermined structure, in particular molecules of the MHC of structure predetermined. The present invention relates in particular to vesicles membranes comprising CMHpeptide complexes of predetermined structure. The current also relates to a method for modifying the composition of a membrane vesicle comprising the introduction into a producer cell of such vesicle, of a nucleic acid comprising a hybrid region composed of a region coding fused to an addressing region, or a coding nucleic acid for a protein or polypeptide which, alone or associated with one or more proteins, East naturally addressed in these membrane vesicles.
The present invention also relates to membrane vesicles comprising defined antigenic molecules, anchored in the membrane part. Of such molecules can be exposed on the outside of the vesicles or, conversely, enclosed in the cytosolic fraction. The present invention also relates to vesicles membranes comprising molecules of predetermined structure, exposed to their surface, allowing their purification, in particular by affinity methods.
The current

3 invention also relates to membrane vesicles as defined above including additionally a marker. Such a marker allows in particular the detection of vesicles in a sample, for example their in vivo monitoring.
The invention also relates to a process for the preparation of vesicles defined here above, as well as the use of these vesicles. So these vesicles can be used as an immunogen, for the preparation of antibodies. Particularly advantageous, these vesicles are used to produce antibodies restricted to CMH, that is to say, specific for a peptide-molecule complex of the MHC.
A first object of (invention more particularly resides in a vesicle 1o membrane, characterized in that it comprises a recombinant molecule of 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 fraction cytosolic. These vesicles are usually produced by salting out, from cells, and are therefore also designated in the present application by the term "exosome". The vesicles membranes (or exosomes) according to the invention generally have a diameter of 60 to 80 nm approx. In addition, these vesicles advantageously carry proteins membranes which are in the same orientation as in the plasma membrane of cells from which they come.
2o The present invention now shows that it is possible to modify the composition of the exosomes, in a controlled and specific manner. More particularly the present invention shows that it is possible to produce vesicles membranes expressing recombinant molecular complexes of composition (pre) determined.
As illustrated later in the present description, such vesicles present 2s particularly advantageous properties, both therapeutically that diagnostic and experimental.
The present invention stems first of all from the selection of populations particular cells for the production of membrane vesicles. The present invention also results from the demonstration that it is possible to introduce into 30 these cells, genetically, recombinant molecules, 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 resides in the definition and identification of the cell population used for the production of vesicles membranes. Advantageously, the cell used is a cell comprising of 3s internal secretion vesicles, cultivable, genetically modifiable, and,

4 preferentially, the internal vesicles of which can be secreted under (effect of external stimulation. They are essentially mammalian cells, in particular particular of animal cells, but also cells of human origin. Otherwise, he can be primary cultures or immortalized lines.
Particularly advantageously, the starting cells are essentially devoid of MHC molecules, i.e. do not express or little molecules of Endogenous MHC. This feature can be very important in some applications, as will be illustrated below.
Different types of exosome-producing cells have been described in the l0 literature, such as for example dendritic cells or B cells.
However, these cells are generally difficult to transfect and rich in molecules endogenous MHC. Therefore, although they can be used for the setting work of (invention, the vesicles of the present invention are more preferentially likely to be obtained from mast cells or derived from mast cells.
In a particular embodiment, the membrane vesicles according to (invention are preferably prepared from cells of mast cells or derivatives mast cells.
Mast cells are a group of cell types derived from precursors medullary, residing, after differentiation, in epithelia like skin, lung, intestine or spleen (Smith and Weis, Immunology Today 17 (1996) 60). These cells get essentially characterized by the fact that their cytoplasm is predominantly constituted granules which contain (histamine, as well as heparin or proteases, and in what they express on their surface receptors of high affinity for immunoglobulins E (IgE). In addition, another benefit of using mast cells according to the invention resides in the possibility of triggering (in particular of stimulate strongly) fexocytosis (ie, release) of exosomes by different treatments. So, it is possible to regulate the production of vesicles by treatment in presence of a calcium ionophore or, more physiologically, by stimulating receivers of high affinity for IgE.
These cells have particularly advantageous properties for setting work of the present invention, namely the presence of internal vesicles of secretion, the possibility of cultivating them and inducing massive exocytosis. In addition, he now been shown, as described in the examples, that these cells can also to be genetically modified in a stable way, which represents a property particularly advantageous for the implementation of the present invention.

More specifically, the vesicles according to (invention have a diameter of 60 to 80 nm approximately, and are produced from 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 molecules

5 endogenous MHC (i.e. MHC molecules of the producer cell of vesicles) can be demonstrated by specific antibodies, by the techniques classics. It can also be demonstrated by the selectivity of the antibody obtained by immunization with vesicles. As shown in the examples, the vesicles of the invention are, in a particularly advantageous mode, capable 1o to induce, in (animal), a production of antibodies specific for molecules defined recombinants expressed by them, without detection of directed antibodies against the genetically unmodified cells. The term "essentially" devoid means the fact that some MHC molecules can be present in very large amounts weak, hardly detectable by conventional methods, and without interference notable on the antigenic specificity of the vesicles of (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, by example recombinant peptide-MHC complexes, of defined composition.
Advantageously, such vesicles of the invention are produced from cells derived from mast cells, which are essentially devoid of molecules of the CMH
endogenous. In this regard, it is known that mast cells accumulate in their granules of secretion of MHC class II molecules. In particular, mast cells are capable preferentially accumulate the MHC-II-peptide complexes in specific multivesicular intracellular compartments, the granules of secretion (Raposo et al., Mol. Biol. Cell 8 (1997) 2619). These cells, taken from a mammal, therefore contain endogenous MHC molecules. So particularly advantageous, used in the context of this invention of lineages 3o cells derived from mast cells, essentially devoid of molecules CMH
endogenous. Different mast cell lines have been described in the literature. The present application now shows that some of these bloodlines have low levels of MHC molecules, and are therefore particularly advantageous for the implementation of the invention. By way of illustration, we can quote in particular lines derived from RBL (Rat Basophicil Leukemia) cells, deposited at

6 fATCC under number CRL1378 (Kulczycki et al., J. Exp. Med. 139 (1974) 600), the line KU-812 (Butterfield et al., Leukemia Res. 12 (1988) 345), or cells 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 lineage is the RBL line -2H3 (Barsumian et al, Eur. J. Immunol. (1 1 (1 981) 317). It is understood that any other cell having the properties set out above can be implemented.
In the context of the present invention, (expression "defined composition"
designates more particularly the fact that the vesicles of (invention have by example one great antigenic and haplotype specificity. So the vesicles described in fart 1o anterior generally express MHC molecules of various haplotypes and unknown. On the contrary, the preferred vesicles of (invention express molecules recombinants whose fhaplotype is precisely predetermined. The term "recombinant" indicates that the molecule results from expression, in the cell producer vesicles, of a recombinant nucleic acid encoding this molecule.
The membrane vesicles according to the present invention are therefore more preferentially produced from cells, established in lines, which are modified genetically for express constituents of predetermined structure.
As indicated above, the vesicles of the invention advantageously express defined molecules of the MHC.
Human MHC molecules are grouped 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 complex histocompatibility of class II. In this regard, class II human MHC molecules are composed of two chains, an a chain and a Vii chain, the chain (3 conferring specificity allelic to complex.
In a specific variant, the vesicles according to the invention express more particularly a recombinant a chain of a molecule of the major complex class II histocompatibility. In another specific variant, the vesicles according the invention more particularly express an a chain and a chain (3 recombinant of a molecule of the major class II histocompatibility complex.
Different types of human MHC II molecules have been identified, characterized and sequences (see for example Immunogenetics 36 (1992) 135). We can cite to title preferential molecules of type DR1 to DR13, in particular DR1, DR2, DR3, DR4, DRS, DR6 and DR7. DNA encoding human DR, in particular DR1 to 13, may be

7 easily isolated from cells, banks or plasmids by techniques classics of molecular biology. These sequences have in particular been described in Bodmer and al.
(Tissue antigens 44 (1994) 1). Preferably, the exosomes according to the invention express therefore a MHC class II molecule comprising an a chain and a chain (3 selected from the DR1 to DR13 haplotypes, even more preferably DR1 to DR ?.
In a specific example, (invention relates to any membrane vesicle comprising a recombinant a and / or (3 chain of a MHC-II molecule haplotype DR1.
l0 In another embodiment, the vesicles according to (invention express one or several recombinant molecules of the major histocompatibility complex of class I.
MHC class I molecules are also composed of two chains, the chain a transmembrane and polymorphic, and (32-microglobulin, which is constant and soluble.
In (man, three genetic loci code for the a chain, designated A, B and C. In the classic MHC-I molecules, each locus A, B and C of the chain a is subject to an allelic variation. Thus, the alleles A1, A2, A3, etc. are denoted. A10, B1, B7, B37, B54, etc., CW3, CW6, etc. (see for example Bodmer et al. above and Immunogenetics 36, 1992, supra).
Preferably, the exosomes of (invention express an a chain of a classic CMH-I molecule, that is to say transmembrane and polymorphic.
Again more preferably, it is a chain of a molecule of the MHC-I
Al, A2 allele or A3.
In a particular embodiment, the exosomes according to (invention express a chain of a non-classical, i.e. non-conventional, MHC-I molecule polymorphic. In fact, as opposed to the so-called "classic" CMH-I, a important polymorphism, in humans there are molecules of MHC-I "non "which are essentially non-polymorphic. Such molecules have by example have 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 3o Cd 1.
It is understood that any other molecule of human MHC-I can be expressed in the part of the present invention.
In a specific example, the invention therefore relates to any vesicle membrane comprising a recombinant protein from a MHC-I molecule.

8 In a particular variant, the vesicles of the invention comprise many MHC class I and / or II molecules. Thus, an advantageous vesicle behaves by example 2 or more MHC-II molecules of different haplotypes. Any other combination of MHC molecules is of course possible, such as by example of CMH-I and CMH-II.
The vesicles of the invention expressing one or more MHC complexes defined are particularly advantageous since they make it possible to present a peptide given antigen, in a defined MHC context. In this regard, in a more prefer of (invention, the membrane vesicles comprise a complex between a peptide 1o defined and the recombinant molecule of the major histocompatibility complex.
The vesicles of (invention can also include one or more other molecules of interest, heterologous, in addition to or instead of MHC molecules mentioned above. In this regard, in a particular variant, the invention relates membrane vesicles produced from mast cell cells or derived from mast cells, characterized in that they contain one or more molecules heterologists of interest. The term cell "derived" from mast cells designates lines transformed and / or immortalized and / or obtained from mast cells or basophils and with properties of mast cells (accumulation of vesicles internal secretion). The term "heterologous" indicates that the molecule of interest is not present, in this form, in the exosomes of the invention in their 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). These molecules can be recombinant in nature, and can be introduced into the cell producer or directly into / on the exosomes. More particularly preferred types of molecules of interest include MHC molecules, antigens {whole or as peptides), receptor ligands, receptors (specific) of ligands, nucleic acids, pharmacological products, markers or peptides or proteins allowing purification of the vesicles.
3o As antigen, there may be mentioned more particularly any protein, especially cytoplasmic, of viral or tumor origin. As preferred examples of protein of viral origin, mention may in particular be made of any cytoplasmic protein or membrane expressed by the viruses EBV, CMV, HIV, measles, hepatitis, etc. It is more preferably cytoplasmic proteins, i.e. essentially invisible to immune system in the classic infection process, and therefore weakly

9 immunogenic under natural conditions or also protein or fragments of membrane proteins. As preferred examples of original proteins tumor, we may especially cite the p53 proteins (wild or any mutated form present in tumor), MAGE (in particular MAGE l, MAGE 2, MAGE 3, MAGE 4, MAGE 5 and MAGE 6), MART (notably MART 1), Gp 100, ras proteins (wild p21 or transferred), etc. It is understood that any other protein of interest can be expressed in or at the surface of the exosomes of (invention, following the teaching of the this request.
In this regard, recombinant antigenic molecules may be present either at the surface of the (exposed) vesicles, i.e. inside (vesicles.
effect so lo particularly surprising, the inventors have shown that vesicles of (invention containing, in their cytosol, a recombinant antigen (in particular p53) were capable to induce, in (animal), a very high production of antibodies directed against this antigen.
Among the ligand receptors, mention may be made, in general, of any receiver of natural ligand or from genetic manipulation. In particular, it can be all hormone receptor, growth factor, lymphokine, trophic factor, antigen, etc.
Mention may more particularly be made of the ILI interleukin receptors for IL15, the growth hormone receptor, or the factor receptor stimulation of granulocyte and / or macrophage colonies (G-CSF, GM-CSF, CSF, etc.). A
example particular ligand receptor is composed of a single-chain antibody (ScFv), which allows (interaction with a specific ligand. Another example particularly advantageous in the sense of (invention is represented by the receptor for (antigen) of T lymphocytes (TcR). Exosomes of the invention expressing on their surface a or several defined TcR constitute analysis and diagnostic tools particularly advantageous, as will be detailed below.
Mention may be made, as pharmaceutical product, of any active substance, of a nature chemical, such as for example pharmaceutical products prepared by techniques of conventional chemistry. We can also cite any protein, polypeptide or peptide having a biological activity, such as for example a toxin, a hormone, a cytokine, growth factor, enzyme, tumor suppressor, etc.
The nucleic acid can be any DNA or RNA coding for a protein, pharmacological polypeptide or peptide as mentioned above, as well as other nucleic acid with a particular property (antisense, antigen, promoter, repressor, transcriptional factor binding site, etc.). he can be a oligonucleotide, coding phase, artificial chromosome, etc.

t0 The vesicles of the invention carrying a ligand receptor can be used for the detection of any receptor-ligand interaction, in particular low affinity, in any biological sample, as will be explained more in details in the continuation of the text. On the other hand, such vesicles can also be used for convey the substances of interest (protein, peptide, nucleic acid, chemical substance, etc.) to cells. Thus, the exosomes of (invention can be used, so general, for the transport and transfer of any molecule into cells, in vitro, ex vivo or in vivo. The invention therefore relates to any vesicle as described above.
before comprising a heterologous molecule of interest, usable as a vector for transfer from said molecule in a cell.
In a more preferred mode, the exosomes of (invention are used for the oriented transfer of substances of interest to cell populations selected.
Thus, it is possible to prepare vesicles of the invention comprising a substance of interest (a toxin, a hormone, a cytokine, a nucleic acid recombinant, etc.) and expressing on its surface a ligand receptor or a receptor ligand, and bring into contacting said vesicles with cells expressing the ligand or the receiver corresponding. This approach therefore allows a targeted and efficient transfer.
In this regard, a particular object of the invention resides in a vesicle such as defined above, characterized in that it expresses a ligand receptor and in that she 2o comprises a heterologous molecule of interest.
The vesicles of the invention can also comprise a peptide or a protein recombinant allowing purification of the vesicles. So, (invention indeed describes the possibility of genetically modifying the composition of exosomes, and therefore of Make them express a particular "label" molecule, allowing its purification. In particular, it is possible to obtain an exosome exposing a structural peptide which can be easily detected and picked up by a receptor molecule. In an example particular, an exosome is produced comprising, in its structure, a peptide molecule comprising the His6 motif (ie, 6 consecutive histidine residues). The presence of such residue on the surface of exosomes allows their easy purification on a support functionalized with nickel. Other recombinant peptides of this type may be used, like for example the tag c-myc, VSV or HA.
Finally, in a particular variant, the vesicles according to the invention behave in in addition to a marker. The marker can be of a different nature (enzymatic, fluorescent, radioactive, etc.) and present in or on the vesicle. A marking preferred is non radioactive, such as, for example, 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 exosomes products.
The invention also relates to any composition comprising one or more membrane vesicles as defined above. The compositions of (invention may further comprise a plurality of membrane vesicles such as defined above, carrying different recombinant molecules. In particular, a composition according to (invention can comprise membrane vesicles such than defined above, carrying recombinant MHC molecules of haplotypes different 1o in association with the same antigenic peptide. It can also be of compositions comprising membrane vesicles as defined above above, carrying recombinant MHC molecules of the same haplotype, associated with different antigenic peptides, for example. Other combinations of vesicles according finventîon are of course possible.
The compositions of the invention generally comprise a vehicle such that buffer solution, saline, physiological, etc., to preserve the structure of vesicles. They may also include any stabilizing agent, tensio-active, etc., from preferably compatible with biological use (in vitro or in vivo). These compositions can be packaged in any suitable device, such as a tube, vial, ampoule, 2o flange, pocket, etc., and stored at 4 ° C or -20 ° C, by example. Compositions typical according to the invention include from 5 to SOOpg of exosomes, for example from 5 to 200 pg.
The vesicles of the invention are obtained from genetically engineered cells modified. As indicated above, the present invention indeed results from setting evidence that it is possible to introduce into certain cells, by way genetics, recombinant molecules, and that these molecules are then expressed from way functional and dense in exosomes.
For the production of the vesicles of the invention carrying molecules recombinant of determined composition, a first step therefore consists in introducing, into a cell vesicle producer, as defined above, the constructs genetic allowing the expression of the recombinant (or molecules) selected.
The genetic constructs used for cell production can generally understand a coding region under control of a promoter functional in the cell used (expression cassette).

Generally, the promoter used is therefore a functional promoter in the mammalian cells. It can be a viral, cellular or bacterial, by example. It can be a constitutive or regulated promoter, preferably allowing a expression at high levels of protein in the cell. From promoters usable, we can cite by way of example the immediate early promoter of cytomegalovirus (CMV), the SV40 promoter, the gene promoter thymidine kinase, in particular HSV-1 TK, the promoter of the LTR of a retrovirus, in particular LTR
RSV, or a strong endogenous promoter of mast cell cells. A way of particularly preferred embodiment includes the use of the SRa promoter, such as i0 describes in more detail in the examples.
The coding region used is generally composed of complementary DNA, genomic or synthetic (eg modified to include certain introns or for have a preferential use of codons). More generally, it is a CDNA. This acid nucleic acid can be obtained by all techniques known in biology molecular, and in particular by bank screening, amplification, synthesis, cuts and ligatures enzymatic, etc.
Depending on the type of coding region used, certain modifications may elsewhere be made to the construction. So it can be particularly advantageous in some cases of introducing into the coding region a sequence of signaling, allowing to address the expression product in a particular compartment of the cell, in particularly towards a membrane compartment (internal, plasma, etc.). This signal can be positioned upstream (5 '), downstream (3') or inside of the region coding. Preferably, the addressing signal is positioned 3 'from the region coding, more particularly in its cytoplasmic region, and in the phase of reading with the coding region. The use of an addressing signal can be particularly useful for promote the accumulation of the expression product in or on the surface of a compartment intracellular given, especially in or on the surface of the vesicles of secretion. This mode of implementation is particularly suitable for the expression of a molecule such that a label peptide, an antigen, a MHC-I molecule or a ligand of receiver. On the other hand, in a particularly advantageous manner, the present request shows that human MHC-II molecules can be expressed directly, without addition of particular signal, in vesicles secreting cells of mast cells, even xenogenic.
For the implementation of the present invention, it is possible by particular to use as an addressing signal a nucleic acid fragment having the sequence of a part of the following genes: Lampl, CD63, LIMPII, Cdlc, FcyR. These genes behave in effect of regions coding for protein addressing signals to cell fendosome compartments (Sandoval and Bakke, Trends in Cell Biol. 4 (1994) 292). An address signal usable in the present invention responds with example with the formula GYXXI, in which X represents any acid residue amine.
An addressing signal particularly suited to the present invention is composed of signal peptide of the protein LAMP 1 of sequence SHAGYQTI. Another type of signed allowing addressing to the membrane compartments, includes all or a part of a transmembrane region of protein.
Addressing the expression product towards the cell compartments allowing the presence of this product in exosomes can also be achieved by merging the coding region to all or part of a region coding for a membrane protein or trans-membrane, in particular a expressed membrane or trans-membrane protein in exosomes. In this context, a particular embodiment of the invention includes the introduction of a recombinant product into an exosome by expression of this produced in the producer cell, as a fusion with a protein membrane or trans-membrane. A particular example of such a protein is through example the recombinant MHC protein introduced into the cell producer, in particular a beta chain, preferably a beta chain of the MHC class II.
So, the results presented in the examples show that such a fusion allows efficiently accumulate any polypeptide of interest in an exosome, without alter its properties neither those of the MHC molecule. This aspect of this 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 could be of an exosome produced from a mast cell, dendritic, tumor or also from a B cell, for example. The polypeptide of interest can be an antigen (or fragment antigen) or any other biological product of interest. The addressing signal maybe everything 3o peptide, polypeptide or protein having the property of directing the product to a comparatively membrane, in particular intracellular, as defined above.
It's about preferably 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 a fusion protein comprising the recombinant product linked to the C- terminus terminal of a beta chain of a MHC molecule, preferably MHC class II.
In the constructions used, the coding region is linked in a way functional to the promoter, so as to allow its expression in cells.
Furthermore, the constructions of (invention can advantageously understand a region, placed 3 'from the coding region, which specifies an end signal transcription (polyA region for example).
The expression cassettes according to (invention are advantageously part of a vector, plasmid, viral, episomal, artificial chromosome, etc. AT
this regard a l0 such a vector advantageously comprises a system allowing the selection of cells the container. In particular, the vectors advantageously comprise a gene coding for a product conferring resistance to an agent, for example to a antibiotic (ampicillin, hygromycin, geneticin, neomycin, zeocin, etc.). In a mode particular implementation, each vector has a single cassette expression as described above. In this embodiment, the cells are therefore modified by the introduction of several vectors, when several molecules are at express in vesicles (for example an a chain and a chain (3 of CMH-II).
fashion of realization each type of vector used advantageously comprises a system of different selection, allowing easy selection of multiple transfectants.
In another embodiment, a vector can comprise several cassettes of expression as defined above, for example one encoding a string a and the other, a chain (3 of CMH-II.
The vectors used are preferably of the plasmid type, and behave by example an origin of bacterial replication allowing their manipulation and their easy in vitro production. Such vectors can in particular be constructed from pBR322-type plasmids, pUC, pBS, pSR, etc.
For the production of exosomes according to the invention, modified cells genetically are therefore implemented, expressing the molecules selected. These genetically modified cells are prepared by introduction, in cells chosen as defined above, genetic constructs defined above before.
The introduction of genetic constructs can be carried out in different ways manners, 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, rush to 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 by elsewhere be adapted by (skilled person depending on the type of transfer and cells used.
In this regard, a particularly effective method for the introduction of acids nucleic acid in mast cells includes electroporation of vectors.
5 On the other hand, when several constructions (vectors) must be introduced in cells, these can be transferred simultaneously or sequential.
After transfer, the cells having effectively incorporated the acids nucleic acids are selected and cloned based on their resistance to a compound (eg, antibiotic) thanks to the resistance gene present in the DNA that has been transferred. These cells can lo be used extemporaneously for the production of exosomes of (invention, or be stored for later use. In this regard, the cells can be stored at 4 ° C in a usual conditioning medium for one sufficient period to allow different batches of exosome production. Cells can also be kept in frozen form (for example in nitrogen), for of uses 15 subsequent. In this regard, it is thus possible according to the present invention to constitute banks of exosome-producing cells with properties particular. In in particular, it is possible according to (invention to constitute banks of cells expressing the main HLA types of MHC class II molecules. From this done it is then possible, depending on the applications envisaged and according to the HLA type of the subject, to choose 2o in the bank the cells producing MHC molecules correspondent, without having to rebuild these cells on a case-by-case basis.
In this regard, a particular object of the invention resides in a cell producer of exosomes as defined above, in particular a cell of mast cell, characterized in that it comprises a recombinant nucleic acid coding for a molecule of the major histocompatibility complex. The invention also relates to all exosome-producing cell as defined above, in particular a cell mast cell, characterized in that it comprises a recombinant nucleic acid coding for a li invariant chain, in particular modified to include a peptide antigenic in place of the CLIP region, or for a peptide allowing the purification of fexosome.
More particularly, it is a mammalian cell, in particular of origin animal, especially rodent. It can also be a cell original human. In a more particular mode, it is a cell line derived from mast cell, such as in particular a mast cell line of leukemia with basophile. AT

As a specific example, mention may be made of cells of the RBL line, including RBL-2H3, cells of the KU-812 or HMC-1 line.
Preferably, (recombinant nucleic acid codes for an a chain and / or ~ i of a molecule of the major histocompatibility complex of class II and / or for a molecule of the major histocompatibility class I complex. In another fashion of embodiment, the cell comprises several nucleic acids encoding respectively for an a chain and a ~ i chain of a molecule of the major complex histocompatibility of class II.
The present invention makes it possible to produce, in a simple and reproducible manner, of large quantities of exosomes of known composition. For the production of exosomes, the genetically modified cells described above are grown in a appropriate medium, and the exosomes are recovered.
A particular object of the invention thus lies in a production process of a exosome comprising a defined recombinant molecule, comprising the steps Following 1s:
a) the culture of a mast cell or mast cell derived 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 a step intermediate b) during which cells are stimulated to induce and or increase secretion of exosomes.
On the other hand, the method of (invention allows the production of vesicles in which the defined recombinant molecule is exposed to outside of fexosome, or is included, in part or in whole, in the cytosolic fraction of fexosome.
As indicated above, in the process of the invention, the molecule recombinant may be, for example, a molecule of the major histocompatibility complex, a antigenic molecule, a receptor ligand, a ligand receptor or a peptide purification, or any other polypeptide of interest. In addition, as explained above before, 3o in certain embodiments, the nucleic acid used in the process includes in addition to a region coding for an address signal to the compartments membranes, in particular the internal secretion vesicles, of the mast cell.
Another particular object of the invention resides in a production process of a membrane vesicle, comprising - the culture of an exosome-producing cell, comprising an acid nucleic recombinant encoding 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 fexocytosis.
In this regard, (the invention also relates to a process for preparing a exosome comprising a peptide-MHC complex of defined composition, characterized in what he understands:
- the cultivation of an exosome-producing cell comprising one or more acids lo recombinant nucleic acid encoding a defined recombinant molecule CMH, - stimulation of cells to induce release of exosomes, - the recovery of exosomes produced by said cells, these exosomes expressing on their surface said defined MHC recornbinating molecule, and, - bringing the exosomes into contact with the peptide (s).
For the implementation of the invention, the peptide or peptides used can to be synthetic peptides, mixtures of peptides, cell extracts, by example one mixture of peptides extracted from tumor cells. The peptide (s) can to be under isolated, or purified, or, as noted above, mixture. Through elsewhere, after bringing the exosomes into contact with the peptides, the exosomes can be isolated or purified by conventional methods.
In another variant, the invention relates to a process for the preparation of a exosome comprising a peptide-MHC complex of defined composition, characterized in what he understands:
- the culture of an exosome-producing cell comprising one or more acids recombinant nucleic acid encoding a defined recombinant MHC molecule and one nucleic acid comprising a region coding for a recombinant peptide defined, - stimulation of cells to induce release of exosomes, - recovery of the exosomes produced by said cells, these exosomes expressing on their surface said defined recombinant molecule of the associated MHC
audit 3o recombinant peptide.
More particularly, in this method, the nucleic acid comprising a region coding for the recombinant peptide coding for a derivative of the invariant chain li, in which the CLIP region has been deleted and substituted by said peptide. This mode of achievement ensures great specificity in the formation of the complex peptide-MHC.

In another variant, the nucleic acid comprises a region coding for the peptide and a region for addressing to the intracellular compartments. In outraged, (nucleic acid can include several regions coding for the same or for of different antigenic peptides.
Preferably, the producer cells used in the process are of mast cell or mast cell derivative. In this embodiment, the stimulation of cells to induce release of exosomes is preferentially performed with one or more calcium ionophores, or with IgE.
In a particularly preferred embodiment, the cells producers lo used in the process are essentially devoid of molecules of the CMH
endogenous.
Another object of the invention relates to a method for modifying the composition of an exosome, including - introduction into a producer cell of exosomes of a nucleic acid coding for a defined molecule, linked to an addressing signal in the compartments membranes, 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 (invention can be used in many applications, such as for example as analysis, diagnostic, therapeutic tools or experimental. Thus, they can be used for the analysis of the T response antigen specific; for the study of low affinity receptor / ligand interactions or a multimerization of different partners is necessary in order to increase the greed of these molecular complexes, thus going beyond the fields of applications immunological;
on the diagnostic or therapeutic level, as well as for the production antibody individuals, including antibodies restricted to MHC. These different applications and others are illustrated below.
a) Use for the production of antibodies One of the first applications of the exosomes of the invention lies in the antibody production. Indeed, due to the defined composition of exosomes of (invention, it is possible to produce specificity antibodies determined. In addition, as the examples show, the exosomes of the invention have properties very strong immunogens, especially due to the high density of complex MHC-peptide on their surface, their functionality, and their presentation effective at immune system.
The antibodies thus produced can be polyclonal or monoclonal.
They can be prepared by standard immunology techniques, including (immunization of animals, and collection of sera (polygonal antibodies) and / or the fusion of spleen lymphocytes with non myeloma cells producers immunoglobulins (to generate hybridomas producing monoclonals).
Another subject of the invention therefore relates to antibodies or fragments antibody produced by immunization with exosomes as described above. The fragments of antibodies can be for example Fab, (Fab ') 2, ScFv, etc., and, more usually, any fragment retaining the specificity of the antibody. In particular, (invention relates to a method for preparing antibodies, comprising immunizing a animal with an exosome as described above, carrying a peptide-MHC complex defined and the recovery of antibodies and / or cells producing antibodies or involved in the immune response. Advantageously, the method of (invention allows the production of monoclonal antibodies, in particular restricted to MHC, that is to say specific for (MHC-peptide association. Preferably, in the method of the invention, we use exosomes essentially devoid of molecules CMH
endogenous, 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. So, as shown in the examples, this method allows to get, without the need for an adjuvant, strong antibodies directed against the peptide, including MHC-restricted antibodies, i.e. specific for the peptide in its conformation associated with the defined MHC molecule. Such antibodies are particularly advantageous from an experimental, diagnostic and therapeutic point of view. In addition, the antibodies according to the invention can be labeled by any known technique (enzymatic, fluorescent, radioactive, etc.), according to known methods of (man of Job.
b) Diagnostic applications The exosomes and antibodies of the invention have advantageous properties for diagnostic use.
Thus, the antibodies or antibody fragments obtained according to the invention can be used in any diagnostic application, for detection, in a sample biological, the presence of corresponding specific antigens, thanks to the employment of wo aonsoo ~ Pc'r ~ 99ioi69 ~
different classical techniques, such as flow cytometry, immunohistochemistry or immunofluorescence, for example. In the particular case of antibodies restricted at MHC, they advantageously allow the detection of MHC-peptide complexes corresponding, and therefore the diagnosis of corresponding pathologies. These antibody 5 can in particular be applied to the diagnosis of pathologies involving a failure immune response or inappropriate response to determine (expression of an antigen, previously defined, in a recognizable form by T lymphocytes For example and in a non-exhaustive manner, one can consider the diagnostic:
lo - of tumor pathologies where the detection on tumor samples of different peptides from proteins such as p53, Her2, MAGE, BAGE, Mart, GP

associated with MHC class I molecules may allow phenotyping of the tumor and facilitate the choice of therapy;
- viral diseases at a pre-infectious or latent stage, where the virion cannot to be 15 detected (hepatitis, HIV infection, CMV and other viruses) - autoimmune diseases such as Multiple Sclerosis, Autoimmune Diabetes, the Autoimmune thyroiditis, Rheumatoid arthritis, Lupus erythematosus disseminated, where the detection of MHC molecules with peptides derived from auto-antigens may be the harbinger of an active flare-up.
The exosomes according to the invention can also be used for the detection of specific partners of a protein molecule in a sample organic. So, the exosomes of the invention carrying MHC-peptide complexes are usable for detect T cells specific for these complexes in samples biological, for example in different pathological situations, in particular in the pathologies described above. In this regard, exosomes can be labeled all over labeling system known to (skilled in the art (enzymatic, fluorecent, radioactive, etc.) to allow their detection in biological samples.
In a particular mode, (invention therefore resides in (use of exosomes marked, in particular fluorescent, as described above, for detection of T lymphocytes specific for -CMH antigenic peptide complexes in a sample organic. The biological sample can be any blood sample, serum, tissue, tumor, biopsy, skin, urine, etc. In addition, the biological sample can to be pretreated for example to dissociate cells, amplify cells in culture, prepare membrane fractions, etc. Advantageously, (biological sample comes from of 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- complexes CMH
in a biological sample, including contacting said sample with a labeled exosome as defined above, comprising said antigen complex-CMH, and the demonstration of the labeling of T lymphocytes in said sample.
In addition, the detection of these T lymphocytes not only allows the detection and therefore the diagnosis of a pathophysiological condition, but also to follow by example the effectiveness of immunization protocols and the state of the immune response to different stages of the disease and thus assess the effectiveness of therapeutics businesses.
In a particular application, the exosomes of (invention bearing a receiver 1o TcR 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 what type protein of defined composition also represent probes fluorescent allowing the detection of potential receptors. The new field of exosomes is thus generalizable to the demonstration, in vivo, of any interaction protein / protein of low affinity. The subject of the invention is therefore also (use of exosomes, of preferably marked, in particular fluorescent, as described above, - for the detection of specific receptors of a protein molecule in a biological sample. In this embodiment, the exosomes used 2o therefore comprise, on their surface, said biological molecule of structure defined.
- for the detection of the presence of a ligand in a biological sample.
In this mode of implementation, the exosomes used therefore comprise, at their surface a specific receptor for said ligand.
c) Therapeutic applications Restricted antibodies or fragments thereof are potentially capable of inhibiting (interaction between the T cell receptor and the complex MHC-peptide for which it is specific. In parallel, the exosomes bearing to their surface only one type of MHC-peptide complex can, by interacting with the T cells specific for these complexes, to compete with their ligands natural T cells and cause their inactivation.
Restricted antibodies and exosomes can therefore be used in all the situations where you want to reduce or suppress an immune response mediated by T cells which is harmful to (organism as it is the case by example in:

- organ transplants or bone marrow transplants in which looking for to neutralize the response of (host against the graft, usually by means of strong doses of immunosuppressive agents;
- autoimmune diseases or viral pathologies during which the reply immune system CD8 or CD4 chronically leads to the destruction of fabrics;
- allergies and (asthma.
In this type of pathology, the exosornes of (invention expressing at their area 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 lo 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 population of lymphocytes T cytotoxic ex vivo. Used directly from blood samples, they can thus be the basis of cell therapies against different targets cellular.
So exosomes can be used to sort specific T cells from combinations variety of complexes expressed by cells that represent a target therapeutic, like tumor cells or virus infected cells. Another object of (invention therefore resides in (use of the exosomes described above for clonal amplification and / or stimulation of cytotoxic and / or helper T cells.
The invention 2o also relates to a method for clonal amplification (or (expansion)) ex vivo T cells, in particular cytotoxic cells, comprising bringing a biological sample comprising T lymphocytes with exosomes such as described above, comprising a defined peptide-MHC complex, the recovery of specific T cells and their amplification. This method is everything particularly advantageous for the clonal amplification of T lymphocytes specific cytotoxics of complexes between MHC molecules and peptides tumor or viral antigens.
Another particularly interesting application of vesicles according to the invention lies in the transfer of molecules to cells. Indeed, from share their 3o composition, the vesicles of (invention are capable of playing the role of vector of transfer of molecules to cells, in vitro, ex vivo and in vivo. In this respect, (invention relates to the use of exosomes as described above, with a substance of interest, for the preparation of a composition intended for transfer of said substance in a cell. Advantageously, it is an exosome comprising a ligand receptor or a receptor ligand on its surface, thus allowing to orient the transfer to one or more selected cell populations. The invention concerned also a method for transferring a substance into a cell, in vitro, ex vivo or in vivo, comprising contacting said cell with a vesicle according (invention comprising said substance. More preferably, the vesicle used further expresses a ligand receptor and the method of (invention allows a transfer directed from the substance to cells expressing the corresponding ligand.
For a implemented in vivo, the vesicles of (invention are administered to a subject of preferably a mammal, especially humans), by any route of administration classic (intravenous, intraarterial, intramuscular, sub-skin, etc.).
1o For in vitro or ex vivo use, the cells are contacted by incubation in an appropriate device (box, flask, pocket, bulb, etc.), preferably in sterile conditions. The parameters of the contacting step (quantity of vesicle, contact time, temperature, medium, etc.) can be easily adjusted by (man of profession according to the aims pursued and (teaching of this request.
d) Applications in the field of research They obviously concern all the applications mentioned above in (analysis of the molecular mechanisms of antigen presentation by (use antibodies to detect and analyze the different stages of formation of 2o MHC-peptide complexes in different normal situations or pathological.
In addition, they also relate to analysis and characterization molecular of T cell populations capable of recognizing an MHC-peptide complex determined by (use of fluorescent exosomes in their ability to detect from T-receptor for MHC-peptide complexes.
In these different applications (diagnostic, therapeutic, experimental, production of T lymphocytes, etc.), the exosomes of the invention can be put in work either as is or in immobilized form on a support. So 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 specificity 3o antigen for example. In this regard, a particular object of this invention resides in a composition comprising exosomes immobilized on a support. The support is preferably a solid or semi-solid support, of the ball type, filter or the like.
It is preferably a plastic support, of the type polymer, by example of latex beads, or magnetic beads. It is understood that everything other synthetic or biological material can be used, as long as it does not no alteration substantial qualities of exosomes or cells. Advantageously, we uses beads with a diameter of 1 to 10 μm, for example from 2 to 5 μm. Immobilization of exosomes on the supports is advantageously obtained by covalent bond, through example by activation with an aldehyde, or any other coupling reagent chemical.
Generally, the immobilization of exosomes is carried out by incubation of exosomes with the support in solution, under conditions allowing fixing and then the supports are recovered by centrifugation. The supports thus functionalized obtained can be used to characterize exosomes or to detect or amplify in vitro T cells, as will be described in detail in the section experimental.
lo Other aspects and advantages of the present invention will become apparent from reading examples which follow, which should be considered as illustrative and not limiting.
In addition, all publications cited in the application are incorporated into the present by reference.
Legends of figures Figure.l. Production of functional MHC-DR1-HA peptide complexes in the RBL2H3 line.
A. Analysis of surface expression by flow cytometry of the molecules of the CMH
It human DR1 before (left) and after (right) transfection of cDNA encoding for the chains a and (3 of DR1 in the RBL 2H3 line. The DRI molecules are detected by the L243 antibody (black line) itself revealed by an anti-IgG goat serum mouse coupled with FITC.
B. Surface expression of DR1 in the line expressing an invariant chain (IiHA) where the CLIP peptide has been replaced by the 308-319 peptide from hemagglutinin from the influenza virus. DR1 molecules are detected on the line RBL DR1 IiHA and a B lymphocyte transformed by fEBV (Hom2) of the same haplotype by (L243 antibody (black line).
C. Stimulation of T-cells specific for the DR1-HA complex by the line 3o RBL expressing this complex or B-EBV of the same haplotype. RBL lines DRIIiHA and B-EBV Hom2 were diluted in culture plates with a DR1 HA complex specific T cell. 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 the culture supernatants makes it possible to evaluate the stimulation of lymphocytes HAT (HA peptide specific T cells). IL2 is measured by the intermediary of a test for incorporation of tritiated thymidine from the CTLL2 line, the proliferation is IL2-dependent.
D. Analysis of the HA peptide saturation of the RBL DR1 IiHA line. The Hom2 and RBL DR1 IiHA cells were incubated (100 cells per well) in presence 5 of increasing concentrations of HA peptide and THA lymphocytes. The stimulation lymphocytes was evaluated as before.
Figure 2: Accumulation of DRI molecules in a secretion compartment of RBL2H3.

10 A. Analysis of the intracellular site of accumulation of DR1 molecules in RBL
2H3. The RBL DRLHA cells were fixed with 0.5% glutaraldehyde and then permeabilized with 0.05% Saponin. DR1 molecules and the invariant chain have been detected with L243 and PIN 1 antibodies respectively, followed by donkey serum anti-IgG
mouse coupled to FITC. Serotonin was detected using a serum from rabbit 15 specific revealed with anti rabbit IgG donkey serum coupled to Texas red. The images were obtained by confocal microscopy (Leica). The thickness of plans of cut were 0.5 micron.
B. Purification of RBL DRIIiHA exosomes. After washing in DMEM, the cells were incubated for 30 minutes in the presence of 1 mM ionomycin at 20 37 ° C. Exosomes were purified by ultracentrifugation differential from supernatant of cells. The pellet of exosomes, resuspended in PBS, has been separated (5 mg) by SDS-PAGE and then transferred to a nylon membrane. The chain ~ i of DR1 was detected with the monoclonal antibody IBS in the preparation of exosome and in control in the lysates of RBLDRLHA and Hom2 cells (equivalent of Os cells 25 per well) migrated under the same conditions.
Figure 3: Use of exosomes for the production of antiDRI HA antibodies A. Increasing dilutions of sera from mice immunized with exosomes were incubated with RBL cells expressing (right) or not (left) the molecules DRl HA. The labeling thus obtained was analyzed by flow cytometry.
B. The sera of rats (diluted 1/100) immunized with the exosomes were incubated with RBL cells expressing or not (left) the DR1 HA molecules.
AT
right, DR1 expressing cells have been incubated or not for two hours at 37 ° C. with 10 mM of the HA peptide then with the same dilution of the rat serum immune.

C. The spleen of the immune rat was fused with the X63A8 line in conditions conventional monoclonal antibody manufacturing. The supernatant of different hybridomas was tested by immunofluvrescence on RBL2H3 cells expressing or not the DR1 or DR1 HA molecules. Clones a40, b82 and a15 are examples representative of the antibodies obtained.
Figure 4 - Use of exosomes for the detection of T lymphocytes specific to the DR1 HA complex A. The RBL DR1 IiHA cells were incubated in the presence of “Green” SmM
1o Tracker ”(fluorescent lipid accumulating in the compartments lysosomal cells) for 30 minutes at 37 ° C then washed and reincubated for one hour to 37 ° C in the absence of fluorescent marker. The cells were fixed (3%
paraformaldehyde), then analyzed by confocal microscopy.
B. In parallel, the DR1 HA exosomes were purified from the cells described at A. The fluorescence present in the samples was quantified at (help of a fluorometer and viewed directly by confocal microscopy.
CD The DR1 HA fluorescent exosomes were incubated at SOmg / ml with THA lymphocytes specific for the DR1 HA complex or TH30 lymphocytes specific for another complex (D) for two hours at 37 "C in the presence azide 2o to block (internalization. The fluorescence of the cells was evaluated by cytometry flow.
Figure 5: Production of exosomes carrying MHC class II molecules A The expression of class II molecules IAb is detected by (antibody monoclonal Y3P and analyzed by flow cytometry. Transfectants obtained in the RBL2H3 cell express similar levels of class II molecules recognized by Y3P that B lymphoma controls B414.
B Western blot analysis of (expression of the IAb molecule in RBL. 10 mg a cell lysate and an exosome preparation from the cell RBL IAbIi were analyzed by western blot with a rabbit serum specific for the region cytoplasmic a chain of the IA molecule.
C Analysis by flow cytometry of the composition of exosomes. Marbles latex were coated with either fetal calf serum (FCS) or exosomes derived from RBL 2H3 cells (exos RBL) or a transfectant of this cell with the molecules Class II marine IAbIi (Exos IAbIi) or human DRIIiHA (exos DRIIiHA). The rat CD63 molecule is detected with the AD1 antibody, IAb molecules with (Y3P antibody while DR1 molecules are detected by (L243 antibody.
These different antibodies are revealed by secondary antibodies coupled to the phycoeryrhrin.
Figure 6: Morphological characterization of the exosomes produced by RBL-2H3:
A The RBL-2H3 cells transfected with HLA-DR1 were fixed with paraformaldehyde. Ultra-thin frozen sections have been prepared and immunostained with polyclonal antibodies to the HLA-DR molecules. These antibodies are visualized with protein A coupled to particles of colloidal gold 10 nm. The class II molecules are mainly detected in compartments filled with membranes of vesicular appearance. Bar: 250 nm.
B C. Morphological characterization of the exosomes secreted by RBL cells 2H3 Exosomes are fixed with 2% paraformaldehyde in buffer phosphate 0.2M pH 7.4. (PB buffer) and deposited on microscopy grids electronic covered with a carbon formvar film. The exosomes are either contrasted and coated in a solution of 4% uranyl acetate and methylcellulose (b) either immunolabelled with antibodies against class II molecules before coating (vs). As in figure (a), the antibodies are visualized with protein A
coupled with 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 DR1 is detected by (antibody monoclonal L243 and analyzed by flow cytometry. The transfection of molecules in the RBL2H3 cell induces expression of similar levels of recognized class II
by Y3P
that B lymphoma controls B414.
B Western blot analysis of (expression of the DR1 GFP molecule in RBL. IO
pg of a cell lysate and 20 ~, g of an exosome preparation from the cell RBL DR1 GFP were analyzed by western blot a couple of antibodies monoclonals specific to PFM.
C Analysis by flow cytometry of the composition of exosomes. Marbles latex were coated with either fetal calf serum (FCS) or exosomes derived from the RBL DR1GFP cells. DR1 molecules are detected by (L243 antibody and secondary antibodies coupled to phycoerythrin while the presence the GFP is detected directly in channel FL1.
Figure 8 A Links of fluorescent exosomes by specific T cells. Of exosomes produced from RBL DRl IiHA cells labeled with the green cell tracker have summer incubated in the presence of two types of T cells: HATs which have a TCR
specific for HLA-DR1 / HA complexes and wild Jurkat T without Phone receiver. The fluorescent exosomes were incubated for 3 hours at 37 ° C with 1o two T lymphocyte lines then the resulting labeling was analyzed with FACS.
B Acquisition of an exosome marker by T cells specifically marked. The same markings as in A were carried out but the connection of exosomes (not marked with green cell tracker) to T cells was detected with a antibody monoclonal mouse AD 1 anti CD63 rat, then revealed by antibodies donkey anti Phycoerythrin-labeled mouse IgG (Jackson Immuno Research, West Grove, PA).
The same labeling was carried out in parallel on the T cells which had not been exposed to exosomes.
C Stimulation of Lymphocytes by exosomes. Exosomes purified from DR1GFP cells were crosslinked onto latex beads prepared from the same 2o as for flow cytofluorometry but washed in complete medium.
Each base of beads was taken up in 100 ~, L, SO ~, L deposited in the first well of a plate 96 well and SOp.L diluted two by two. T cells (Jurkat T and THA1.7) have been adjusted at 106 cells / mL and SOpL were deposited per well in the presence or absence peptide HA307-319 at S ~ tM. The culture plate was placed in (incubator (37 ° C, S% C02, H20) 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 HMC-1 cell analysis by flow cytofluorometry, dark solid line : cells alone; solid solid line: anti-mouse antibody-FITC alone;
tight dotted lines specific antibody + anti-mouse-FITC.
B analysis of HMC-1 exosomes bonded to latex beads by cytofluorometry flow, dark solid line: latex beads-HMC-I exosomes only; Full line clear: antibody anti-mouse-FITC alone; tight dotted lines: latex-SVF + control beads specific antibody + anti-mouse-FITC; loose dotted lines: latex-exosome beads + antibody specific + anti mouse-FITC
C Western blot analysis of a lysate of HMC-1 cells compared to their exosomes; 10 or 3 g of protein per put; HC10: 1 / 10e supernatant; 1B5:
supernatant 1/10; CD63: 5 ~ .g / mL; Lampl: 2 ~, g / mL; H68.4: 1 / 10th supernatant. The exosomes appear to be enriched in CD63, Lampl, TfR. The absence of MHC class II both in the lysate that in the exosomes confirms the analysis by cytofluorometry. The proportion of MHC class I seems identical for the cell lysate and the exosomes.
1o Materials and Methods Cells Cells used for the production of exosomes in the part experimental are cells of marine or human mast cells. More specifically, a line tumor of basophils with a mucosal mast cell phenotype, designated RBL-2H3, a summer used (Barsumian et al, Eur. J. lmmunol. (1 1 (1 981) 317}), as well as a lineage of human immature mast cells (HMC-1). Other mast cells, in particular established in line, can be used, such as derived lines RBL cells (Rat Basophicil Leukemia), deposited at fATCC under number CRLI378 (Kulczycki and 2o al., J. Exp. Med. 139 (1974) 600).
T lymphocyte lines capable of recognizing a particular antigen in a human MHC II context (DR1) were also used. In particular, line Jurkatt transfected with cDNA encoding the T cell receptor ("T-HA") specific of the influenza virus fHemagglutinin peptide 306-318 in combination with HLA-DRI (Sidhu et al., J. Exp. Med. 176 (1 992) 875). The B cell line humans transformed by the Epstein Barn virus (Hom-2 line) has been used as control for the restricted response to HLA-DRl.
The cells were cultured in DMEM (Gibco BRL), RPMI, or "CLICK" medium.
RPMI medium supplemented with 10% fetal calf serum (Sigma), 1 mg / ml of 3o penicillin-streptomycin, 1 mg / ml glutamine, 5 mM sodium pyruvate and 50 ~ tM of b-mercaptoethanol. Any other medium suitable for cell culture eukaryotes, in particular mammals, can of course be used.
The cells were mainly cultured in culture flasks of 25 or 15 cm3.
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 grow them in a "spinner", the density of 106 cells / ml.
Plasmids 5 To genetically modify mast cells, the constructs The following genetics have been performed.
CDNAs encoding the human HLA-DRIa chain (Larhammer et al., Ceil 30 (1982) 153), the HLA-DRI chain (3 human (Bell et al., PNAS 82 (1985) 3405) and the human invariant chain p33 li (Ciaesson et al., PNAS 80 (1983) 7395) have been isolated.
10 The cDNA encoding the invariant chain p331i was then modified by PCR
for replace the region coding for the CLIP peptide (residues 87-102) with a site of restriction. This cDNA thus makes it possible to insert, instead of the peptide CLIP, everything cDNA fragment of interest coding for an antigenic peptide (Stumptner and al., EMBO
J. 16 (1997) 5807). In a specific example, a DNA fragment coding for the peptide 15 HA308-319 flu virus hemagglutinin has been inserted into this CDNA, coding for a chimeric polypeptide li (HA308-319).
The nucleic acids described above were then cloned, separately, in the plasmid pSRa under the control of the SRa promoter (Takebe et al., Mol. Cell Bio.

(1988) 466). Each of the plasmids was then modified so as to incorporate a 20 different resistance genes, allowing selection for each of the plasmids, and therefore for each of the chains; chain a with the resistance gene neomycin;
the ~ i chain with the fhygromycin resistance gene, and the chain invariant with the zeocin resistance gene.
25 Transfections To introduce the different nucleic acids into the cells of mast cells, corresponding plasmid vectors have been linearized by the enzyme Scal restriction.
50 μg of each plasmid were linearized and then precipitated with fethanol, and the caps have were resuspended in the presence of RBL-2H3 cells at a concentration of 1.10 ' 3o cells / ml. Stable transformants were obtained by electroporation from 5.106 cells using a "gene pulser" (Bio-Rad, Richmond, CA) in conditions following: 260V, 960 pF. 72 hours after electroporation, the transfectants are selected by culture in a selection medium comprising 250 μg: ml of 6418 (Geneticin, Gibco) lmg / ml of hygromycin and 500 ~ .g / ml of zeocine. After 8 days of culture in a selection medium, 60 to 90% of the cells present are transfected. The transfectants were then sown on a petri dish in the middle of one selection concentration allowing (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.
Antibody Y3P (MHC II (IA)) is a mouse monoclonal antibody recognizing the IAb af3 complex (Janeway et al., 1984). Anti IA a is rabbit serum directed against the cytoplasmic part of the α chain of IA. Anti-GFP is a mixture of two antibody 1o monoclonals (clones 7.1 and 13.1) directed against the "fluorescent green protein "sold by Boehringer Mannheim. In flow cytofluorometry experiments, the seconds antibodies used are F (ab ') 2 fragments coupled to (-Phycoerythrin, products at (donkey and directed against mouse IgG (H + L} (Jackson Immunoresearch Laboratories).
Balls Latex beads: Surfactant-free white aldehyde / latex sulfate, D: 3.9p, m, Interfacial Dynamics Corp., Portland, Or. USA
Electron microscopy RBL-2H3 cells transfected with HLA-DR1 were fixed with 2% parafonmaldehyde in 0.2M phosphate buffer pH 7.4. (PB buffer) After fixation the cells were washed with PB-glycine SOmM buffer and then coated in 10% gelatin. After solidification of the blocks were prepared, infused in the 2.3M sucrose and frozen in liquid nitrogen. Ultra-thin frozen cuts have been prepared and immunolabelled with polyclonal antibodies against HLA-DR molecules. These antibodies are visualized with protein A coupled Has 10 nm colloidal gold particles.
Exosomes are fixed with 2% paraforrnaldehyde in buffer 0.2M phosphate pH 7.4. (PB buffer) and deposited on microscopy grids electronics covered with a carbonaceous formvar film.
The exosomes are either contrasted and coated in an acetate solution uranyl 4% and methylcellulose be immunolabeled with antibodies against the class II molecules before coating. Antibodies are visualized with protein A coupled to 10 nm colloidal gold particles.

Results 1 -Production of DR1 HA exosomes 1.1. Construction and characterization of genetically producing cells modified In order to produce exosomes carrying MHC- complexes in a controlled manner peptide of defined composition, the a and b chains of class II molecules CMH, DR1, were expressed in the mast cell line RBL2H3, derived from a leukemia at basophile du Rat. For this, two vectors carrying respectively an acid nucleic coding for each chain, under the control of the SRa promoter were transfected simultaneously in cells (see materials and methods). The results obtained by flow cytometry show that the transfected cells express the molecules DR1 (Figure lA).
These RBL-2H3 DRI cells were then sensitized to a given peptide, precise composition, in order to generate MHC-peptide complexes of composition defined. For this, different techniques can be envisaged. In one fashion of simple implementation, the peptide can be incubated directly with the exosomes.
In 2o another variant, a nucleic acid encoding the peptide can be introduced in cells, so as to also express this peptide. In this example particular of implementation, to manufacture a presenting cell comprising a single specificity antigenic, the chosen antigenic peptide was introduced into the cells 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 peptide sequence selected, derived from hemagglutinin (HA 308-319) from the influenza virus, known to bind with the DR1 molecule. This construct (IiHA) has been transfected into cells in the conditions described in the Materials and Methods. The hybrid chain expressed in the RBL-2H3 DR1 cells, allowed to build cells that express molecules DR1 recognized by (L243 antibody at a level similar to that of a B-EBV line (Hom2) DR1 haplotype control (Figure 1B). These results therefore show that the cells of mast cells of the invention express a human peptide-MHC complex well functional, of predetermined and controlled composition.
The functional character of the peptide-MHC complexes expressed by the cells of (invention was confirmed in a T cell stimulation test specific to the DR1-HA combination carried by the cells. For this, the cells of the invention have were incubated in the presence of HAT lymphocytes, and stimulation was determined by measurement of finterleukin-2 released in the supernatant, by a test of growth of a IL-2-dependent cell line. As a control, a line of B lymphocytes transformed by fEBV (Hom-2), of haplotype DR1, pulsed by a concentration saturated (IOmM) of the HA peptide was used.
The results obtained are presented in Figure 1 C and 1 D. They show that mast cell of the invention express a DR1-HA complex, capable of stimulate a T cell specific for this combination. They show also that l0 the stimulation obtained in the presence of the cells of the invention is more effective than that produced by control cells (B-EBV of haplotype DR1) pulsed by a saturation concentration (IOmM) of the HA peptide. Finally, the results obtained show that the DR1 molecules seem to only present the HA peptide since the addition of a saturated concentration of peptide does not significantly increase the capacity RBL DR1 IiHA cells to stimulate a T HA lymphocyte (Figure 1 D).
All of these results therefore demonstrate the functional nature of the complex peptide-MHC products. They also illustrate the specific nature of cells obtained, and therefore the specific nature of the process of (invention which allows to get cells (and exosomes) carrying molecules of defined composition and controlled.
1.2. Production of functionalized exosomes An immunofluorescence study has shown that the complexes MHC-peptide recombinants (DRl HA) accumulate in secretory granules of the RBL-2H3 cell line. Figure 2A indeed demonstrates co-location of DR1 molecules with serotonin in intracellular structures vesicular.
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 ionophore calcium, and the production of membrane vesicles was monitored. More particularly, the cells were centrifuged at 300 g for 5 minutes at temperature ambient. Sure each cell pellet, a solution of calcium ionophore (1 mM lonomycin) has been added (approximately 300 ~ 1) and the incubation was continued for 30 minutes at 37 ° C.
The exocytosis was stopped by rapid cooling in ice and addition of 300 ~ 1 of a cold 1 mM PBS-EGTA solution. The cells were then centrifuged at 300g at 4 ° C for 5 minutes. Supernatants have been recovered is recentrifugees, all 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 solubilized in a buffer solution (30p.1 of Laemmlî-DDT buffer (1X or 2X)). A fraction of the pellets is also dissolved in a lysis buffer for determine the protein concentration. Exosome solutions can be separated by migration on gel (mirai-gel 12% polyacrylamide) at 20mA then transferred to linmobilon. Exosome analysis is then carried out by western blotting with some antibodies specific for the different chains of class IIdu molecules CMH.
1o The results obtained show that exosomes can be released from line RBL-2H3, significantly, after stimulation with an appropriate agent. These exosomes can be isolated and purified for example by ultracentrifugation differential for the preparation of exosome composition. Finally, the results shown in Figure 2B demonstrate that these exosomes are functional. In indeed the Western blotting analyzes show that the exosomes obtained express the different recombinant chains of MHC class II molecules. In addition, these results also show the high density of peptide-MHC complexes at the area exosomes of the invention.
The following examples illustrate in particular the use of the DR1 exosomes HA
for the production of antibodies specific for this combination and the ability of DRl HA exosomes to bind specific T cells from that same combination.
2 - Generation of antibodies specific for the DR1 HA complex This example illustrates (use of the exosomes of (invention for the production of antibodies, in particular of so-called "restricted" antibodies, that is to say peptide specific antigenic in association with the MHC molecule. This example illustrates especially the very important immunogenic power of the exosomes of the invention, since they allow the production of antibodies in the absence of any adjuvant.
Exosomes purified from the supernatant of RBL DRI HA cells (example 1) were resuspended in PBS. These exosomes were then used for immunize Balb / c mice or LOU rats in the absence of any adjuvant, the following protocols - The mice were injected subcutaneously with the Opg of exosomes, twice three weeks apart, then 30p.g intraperitoneally and finally 30 pg per channel intravenously 3 days before collection of the sera.
- The rats were injected with exosomes intraperitoneally (lOp.g), by 5 twice at three-week intervals, then intravenously (SOp, g) three days before serum collection.
As shown in Figure 3A, the sera collected from immunized mice showed a very strong reactivity against the RBL line expressing or not the complex DRl HA but which was detectable to a dilution of thirty thousandths will be 1o only in the case of the DR1 HA cell.
As shown in FIG. 3B, the sera of the rats thus immunized, showed, of surprisingly, reactivity against the RBL line expressing the complex HA while the same sera reacted with lower intensity with line initial RBL-2H3. In addition, the addition of the HA peptide to cells expressing 15 (RBL-2H3 DRl) significantly increases the reactivity of antisera as well products (Figure 3B).
These results therefore show that the exosomes of the RBL line are capable to induce an antibody response which, unexpectedly, is mainly directed in rats against DR1 HA complexes.
2o The spleens of the immune rats were fused with cells of the line X63A8.
The hybridomas thus obtained were sorted by clonal dilution, according to the techniques immunology classics, then selected by immunofluorescence for the specificity monoclonal antibodies produced. Different monoclonal antibodies have summer obtained, for some directed against proteins of the RBL line, for others, 25 against monomorphic determinants of human class II molecules DRl haplotype and finally against the complex formed by the DR1 molecules associated to the peptide derived from the HA protein of the influenza virus (FIG. 3C). These money monoclonal antibodies are restricted antibodies, and therefore have of particularly advantageous properties for diagnostic applications or 3o therapeutic.
3 - Detection of specific T lymphocytes of the DR1 HA complex This example illustrates the use of the exosomes of the invention for the detection of 35 specific T cells in a biological sample. This example watch WO 00/28001 PCT / FR99l02691 also how exosomes can be used to select and amplify a population of particular T lymphocytes, in particular with a view to their reinjection to a subject (cell therapy). This approach can of course be extended to use restricted antibodies described in (Example 2, as well as the detection of any receiver specific for a ligand.
For the implementation of this application, labeled exosomes have been products.
For this, before purification of the exosomes of the DRl HA line, this was incubated with a fluorescent tracer accumulating very strongly in the exosomes contained in secretory granules. The marker used, "Green Tracker ', is a lipid fluorescent which accumulates in the lysosomes of cells. Analysis in microscopy confocal of cells, after fixation, shows the presence of fluorescent labeling in the granules of cell secretion (Figure 4A). The fluorescent exosomes were then products and purified from these cells, under the conditions described in the example 1. Renderings thus fluorescent, these exosomes (Figure 4B) were used to detect, in one biological sample, the presence of T cells specific for the combination DRI HA (HAT lymphocytes). It is understood that any other marking may be used in the context of (invention, applied either to producer cells or on 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 flow cytofluorometry results show that exosomes expressing the DRLHA bind specifically to HAT lymphocytes (Figure 4C) then than these same exosomes are unable to recognize lymphocytes with a other specificity (Figure 4D). These results demonstrate the unique ability and unexpected exosomes produced by the mast cell line according to (invention to detect of T cells specific for this same complex. This app can be setting works from any type of biological sample.
In addition, this technology can be applied in the same way to the manufacturing and to (use of exosomes expressing CMHI-peptide complexes. The present invention thus makes it possible to detect, on the surface of presenting cells, even to cells presentation, by class I and class II molecules of the CMH, from peptides from antigens expressed by tumors. The present invention allows also detect or even purify T lymphocytes capable of recognize these same complexes. They can thus amplify a population of T cells specific for a particular peptide-MHC complex, for example a wo oor ~ soai pcr, ~~ ioz69 ~

population of CTL lymphocytes, for their therapeutic use. In effect, different approaches to cancer immunotherapy or viral infections, for example, have been developed based on the removal of lymphocytes from a subject and on ex vivo expansion of particular T cell clones specific for a antigen involved in the pathology (tumor or viral antigen, for example). These clones amplified are then re-administered to the subject as a therapeutic agent. The present invention greatly facilitates the selection and amplification of clones specific for T cells, and therefore the potential and implementation of these approaches therapeutic.
lo 4- Production of exosomes carrying class II molecules from the marine MHC
(Figures 5A, 5B).
The complementary DNAs encoding the chains a and (3 of class II molecules IAb haplotype marine as well as the invariant marine chain were introduced in NT eukaryotic expression vectors in which transcription of cDNA
is under the control of an SR alpha promoter. Each of the plasmids, moreover, carries a gene resistance to fhygromycin (for the IAb chain), to neomycin (for IAb ~ i chain), or zeocin (for the invariant chain). After transfection of cells RBL2H3 by 2o electroporation (materials and methods), cells have been selected based on their resistance to the three antibiotics then the resistant cells were cloned by limiting dilution and characterized for the expression of IAb molecules by flow cytometry using the specific antibody Y3P.
Figure 5 shows some of the results obtained. Analysis by flow cytometry shows that the RBL IAbIi cell is recognized by the Y3P antibody (specific for IAb molecules) in an equivalent way to the line B B4 lymphocyte 14, while no marking is detectable on the original RBL line (Figure 5A).
Morphological analysis by microscopy having established that the molecules of class II IAb like the human molecules DR1, accumulated in the granules of secretion of the cell (not shown), this led us to look for their location in exosomes. The exocytosis of the cells being triggered by the addition of 1 mM
of ionomycin, the exosomes thus obtained were purified by ultracentrifugation differential (see example 1.2). Western blot analysis of these exosome preparations shows that they contain MHC class II marine molecules identical to those detected in a control cell lysate (Figure SB).

These results establish that human class II molecules (DR1) but also marine (IAb) can be expressed and accumulate in the exosomes of RBL lines 2H3 corresponding.
S-Morphological characterization of the exosomes produced by RBL 2H3 (Figure 6) Electron microscopy observations on immuno-labeled frozen section RBL cells reveal the presence of many compartments intracellular filled with membranes. Most of these membranes correspond to 10 vesicles that fill the lumen of the compartments (Figure 6A). The molecules of class II once transfected in these cells accumulate in compartments having internal vesicles and are visualized in particular, in association with the membrane of these vesicles (Figure 6A).
When RBL cells are stimulated to induce their degranulation (IgE
Antigen or ionomycin), the intracellular compartments merge with the plasma membrane. The internal vesicles with which the molecules of class II are released into the extracellular medium. These vesicles are then called exosomes.
The method of choice in electron microscopy to study morphology of 2o exosomes and their protein content is the "whole mount" method ". This technique allows to visualize entire exosomes devoid of any other content cellular. This method also makes it possible to detect, with a large efficiency, molecules associated with the exosome membrane. Using this technique we we observed that the exosomes secreted by RBL cells have a size heterogeneous from 30 to 120 nm and a variable electron density (Figure 6B). The molecules of class II are abundant in the population of vesicles having a size from 80 to 100 nm having an average electron density. The vast majority of these vesicles enriched in class II molecules have a plate shape (Figure 6C).
6- Immobilization of exosomes on supports (Figure SC).
This example describes the fixation of exosomes on solid supports, and shows than the exosomes thus fixed retain their functional properties. These new products (supports-exosomes) can be used to characterize and analyze exosomes; or as diagnostic or reagent products to detect edou stimulate in in vitro T cells, for example.
Different preparations of exosomes produced by RBL cells expressing or not human or marine class II molecules have been incubated with marbles 4 micron latex activated by (aldehyde sulfate. More specifically, exosomes, purified from degranulation supernatants of RBL 2H3 were washed in PBS
(centrifugation at SOOOOrpm on TLA 100.4 for 30 minutes). 30g of exosomes are mixed with 10 μL of Latex beads taken sterile, homogenized then incubated for 10 min to 1 S min at room temperature. The ball volume is then completed lo at 1 mL with PBS lx then incubated on a wheel at room temperature for 2h. Then the "crosslinked" beads with exosomes are:
-Saturated by adding 100 mM final glycine (30 min at temperature ambient), -Centrifuged at 2200xg for 2 min at 4 ° C then the pellet of beads is included in 1 mL of PBSIx 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 1 x 0.01% NaN3.
-Use between SpL and 20pL per point and incubate conventionally in the first then the second antibody. The reading is done on Facscalibur (Becton Dickinson).
This technique made it possible to cover the surface of these latex beads with of exosomes, while preserving their structure.
When the exosomes are on the beads, their handling is easier. In effect, they can be centrifuged at low speed and detected by techniques of cytometry classic flow. Figure 5C shows some examples of detection, by cytometry of flux, of different proteins used in the composition of exosornes. Of marbles latex activated with aldehyde sulfate were thus incubated either with exosomes produced by RBL 2H3 cells not transfected, either with cell exosomes expressing human DR1 class II molecules and the IiHA construct or ies class II marine molecules IAb, either with fetal calf serum (FCS) in control.
The latex beads thus prepared were then incubated with different antibody monoclonals; AD1 recognizing the rat CD63 molecule present in the granules of secretion of RBL 2H3, Y3P antibody specific for IAb molecules, and L243 antibody specific for DR1 molecules. These different antibodies were revealed by antibody secondary coupled to phycoerythrin then the markings obtained were analyzed by flow cytometry thanks to a FACScalibur (Beckman).
It is thus observed that the molecule CD63 is of course present on all exosomes originating from the RBL cell expressing or not class II molecules, whereas marbles 5 of latex coated with IAb exosomes are specifically recognized by Y3P and no by L243 while (opposite, the DRIiHA exosomes are recognized by L243 and not by Y3P.
None of these antibodies recognize latex beads coated with Serum of calf fetal (Figure SC).
These results show that this technique makes it possible to detect in a sensitive 10 and specific (expression of different proteins entering the composition of exosomes. Example 8 also shows that such products (exosomes-support) also make it possible to detect or stimulate the proliferation of T cells specific.
15 7-Manipulation of the composition of exosomes (Fi ~ ure7) Exosomes are vesicles delimited by a lipid bilayer in which are inserted a large number of molecules like the molecules of class II of CMH or CD63 cited above. A (inside these vesicles, we find the regions 20 cytoplasmic of the preceding transmembrane molecules but also of protein soluble from the cytosol of cells. To demonstrate that it is possible to change to will the content of these vesicles we used as a tracer the Green Fluorescent Protein (GFP).
CDNA encoding GFP has been fused to the COOH terminus of the chain 25 beta of the human DR1 molecule. This construction, inserted in the vectors of expression NT carrying the gene for resistance to fhygromycin, was cotransfected in the RBL 2H3 cell with a vector carrying the alpha chain of DR1 and the gene for resistance neomycin. Cells resistant to these two antibiotics have been selected then sorted positively for GFP expression.
30 More specifically, we carried out a construction linking the part cytoplasmic of the DR chain (3 (in C-ter) at the N-ter end of the GFP.
CDNA from DR (3 has a PstI site at position 565. A fragment of 200 base pairs about from side 3 ′ of this cDNA was amplified by PCR from the vector pcDNA3 / RSV / DRa at means of 2 oligonucleotides including the PstI site for the 5 'and the NcoI site for the 3 'which 35 plus eliminated the stop codon. The PCR fragment obtained was digested with PstI and NcoI and WO 00! 28001 PCT1FR99 / 02691 cloned into the same sites of the pEGFP N 1 vector (Clontech). The plasmid resulting, digested with PstI and XbaI, released a fragment corresponding to last 30 amino acids of DRa followed by GFP. In parallel, the plasmid pcDNA3 / RSV has summer digested with EcoRVlPstI, thus releasing a fragment corresponding to the rest of the chain DRa (from start to PstI site). The two fragments were then assembled then cloned into pcDNA3 / CMV between EcoRV and XbaI.
Analysis of these cells (DR1 GFP) by flow cytometry shows that the cells recognized by (L243 antibody, specific for DR1 molecules, also emit a green fluorescence detected in the FL1 channel while cells transfected with DR1 alpha and beta chains without GFP do not emit any fluorescence in channel FL1 (Figure 7A).
In order to show that GFP is actually contained in the exosomes of the RBL 2H3 cell, exosomes from RBL DR1 GFP cells have been prepared through differential ultracentrifugation then analyzed by western blot (Figure 7B) and cytometry flow after "crosslinking" on latex beads (Figure 7C). A
specific antibody of GFP detects, by western blot, in the DR1 GFP cell and in its exosomes a 65 kDa protein corresponding to the molecular weight of the DR1 beta chain merged with GFP (Figure 7 B) while no signal is detected in lysates cells of the RBL2H3 cell expressing or not expressing the DR1 molecule alone. Through comparison between "crosslinked" latex beads with calf serum fetal or exosomes from the DR1 GFP cell, we observe that only the beads covered of exosomes induce fluorescence in the FL 1 channel (FITC) and are recognized by (L243 antibody, specific for DR1, detected by a coupled secondary antibody to the phycoerythrine.
These results therefore demonstrate that it is possible to introduce a protein exogenous to 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 cell producer, in the form of fusion with a transmembrane molecule, such that MHC molecule.
8-Functional characterization of exosomes (Figure 8) This example shows that the exosomes produced by the RBL2H3 cell, carrying of MHC class II molecules, are capable of binding an antigenic peptide and of stimulate a T lymphocyte expressing a specific receptor for this complex peptide-CMH class II.
Exosomes produced from RBL DR1 IiHA cells labeled with green cells tracker were incubated in the presence of two types of T cells: HAT which own a specific TCR for HLA-DR1 / HA complexes and wild Jurkat T without of such a receiver. The bonding experiments are carried out on plate 96 bottom holes round, in RPMI 1640 medium supplemented with 10% fetal calf serum, stamped by IOmM Hépès at the rate of final SOpI per well, 105 T cells per well and quantities fluorescent exosome variables for 3 hours at 37 ° C. Then, two washes are l0 carried out in the same medium before analyzing the cells taken up in 400 ~, I of PBS
at FACS.
A dose effect range was produced showing that the intensity of the labeling of THA
was proportional to the amount of exosomes used. 10g RBL DR1 cells IiHA have gave 700 wl of exosomes. Doses ranging from 32 to 2 p, l of exosomes per well have been tested. The marking obtained with 16 pl per point was retained, which corresponds to the 2.3 million cells produce exosomes (results not exposed).
The results obtained show that the HAT population is marked by fluorescent exosomes which gives a detectable fluorescence in FL1 by cytofluorimetry, while wild Jurkats are not modified (Figure 8A).
The same type of labeling was carried out, but after the binding of the exosomes, the T cells were incubated with anti AD 1 mouse monoclonal antibody Rat CD63, then revealed by donkey-labeled anti mouse IgG donkey antibodies phycoerythrin (Jackson Immuno Research, West Grove, PA). The same marking was carried out in parallel on T cells that have not been exposed to exosomes. We observe (figure 8B) that only HAT cells which have linked exosomes such as (attests to 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 for visualize by flow cytometry specific T cell populations of HLA-DR / antigenic peptide complexes carried by exosomes.
To assess the ability of exosomes to stimulate a T cell in a way dependent on the presence of a peptide, the exosomes obtained from the DRl cell GFPs were crosslinked onto latex beads and then incubated in the presence of cell of the Jurkat T lymphocyte line whether or not expressing a T receptor specific for complex DR1-peptide HA 307-319.

The latex beads were prepared in the same way as for the cytofluorometry flow but washed in a complete medium (RPMI, 10% fetal calf serum, Penicillin-Streptomycin-Glutamine 1%, l3mercaptoethanol 0.1%, Sodium Pyruvate 4%).
For the stimulation of T lymphocytes, each pellet of beads was taken up in 100 pL, SOpL
deposited in the first well of a 96-well plate and SOp.L diluted two-fold of them. A
control was done in the same way with incubated beads in serum of fetal calf. T cells (Jurkat Pasteur and THA1.7) were adjusted to cells / mL and SOp.L were deposited per well. The peptide diluted to lSp.M in middle complete was also added at the rate of SOpL per well. In the series devoid of the peptide, SOwL of complete medium were added per well. The plate culture was placed in the incubator (37 ° C, 5% C02, H2O) for 20 hours then the supernatant a was taken and the IL2 concentration was evaluated by a CTL.L2 test.
It is thus observed that the addition of the HA peptide (SmM) specifically induces the stimulation of the Jurkat T cell expressing the DR1-HA complex receptor (THA) when it has no effect on the control T lymphocyte (T Jurkat). Through elsewhere, exosomes are unable to stimulate THA in the absence of HA peptide (Figure 8C).
9-Exosomes of the human mast cell moon HMC 1 (Figure 9) This example shows that modified exosomes according to the invention can be produced from other cells, especially human cells. In particular, results that we obtained demonstrate that the mast cell line of human origin HMC
1 is able to produce exosomes under (stimulus of increased calcium intracellular and under conditions identical to those which induce secretion of exosomes by the RBL 2H3 rat line.
The characterization of the HMC 1 line by flow cytometry indicates that the area of these cells express MHC class I molecules (W6.32) but no class II molecules (L243). They are also positive for CD9 molecules, CD63 and CD81 but negative for the Lampl and Lamp 2 molecules (Figure 9 A).
3o Exosomes were produced from the HMC 1 line by the addition of Ionomycin (1mM) then purified from the supernatants by ultracentrifugation differential. Their composition was analyzed by flow cytometry after "
crosslinking "on latex beads and western blot.
Analysis of the exosomes by the method using latex beads shows that they carry the Lampl, CD9, CD63, CD81 molecules and the class I molecules of the CMH

but no class II molecules nor the Lamp2 molecule (Figure 9B). Through elsewhere, Ia morphology observed by electron microscopy of these exosomes is identical to those produced by the RBL 2H3 line. Finally the protein composition observed by western blot confirms these results since we found by this technique the molecules CD63, Lampl, CMH class I while Lamp2, CMH class II remain negative (Figure 9C).
In conclusion, the HMC 1 line seems (human homolog of the rat line RBL-2H3 due to its capacity, after an increase in calcium intracellular, to produce exosomes that have the same structural characteristics and molecular.
These cells therefore make it possible to produce recombinant human exosomes expressing MHC class II molecules or any other molecule therein specifically addressed.

Claims (47)

1. Membrane vesicle produced by a genetically modified cell, said vesicle comprising a recombinant molecule of the major complex of human histocompatibility. 2. Vesicle according to claim 1 characterized in that the molecule recombinant major histocompatibility complex is a molecule of class II. 3. Vesicle according to claim 2 characterized in that the molecule recombinant of the class II major histocompatibility complex is an .alpha. chain. 4. Vesicle according to claim 2 characterized in that the molecule recombinant of the class II major histocompatibility complex comprises a chain .alpha. and an .beta chain..~ 5. Vesicle according to one of claims 2 to 4 characterized in that the molecule recombinant class II major histocompatibility complex is selected from serotypes DR1 to DR13, preferably DR1 to DR7. 6. Vesicle according to claim 1 characterized in that the molecule recombinant major histocompatibility complex is a molecule of class I. 7. Vesicle according to one of claims 1 to 6 characterized in that it includes a complex between a defined peptide and the recombinant molecule of the complex major of histocompatibility. 8. Vesicle according to one of the preceding claims, characterized in that what further comprises one or more heterologous molecules of interest. 9. Vesicle according to one of the preceding claims, characterized in that what further comprises a recombinant peptide or protein allowing its purification. 10. Vesicle according to the preceding claims characterized in that it has a marker. 11. Vesicle according to one of the preceding claims, characterized in that what is essentially devoid of endogenous MHC molecules. 12. Membrane vesicle characterized in that it is obtained from a cell mast cell or genetically modified mast cell derivative, and in that it includes one or more heterologous molecules of interest. 13. Vesicle according to claim 12, characterized in that the molecule of interest is a protein, polypeptide, peptide, nucleic acid, lipid or a substance of a 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 antigen, receptor ligand, ligand receptor, nucleic acid, a product pharmacological agent, 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 according to any one of the claims above, characterized in that it comprises a recombinant molecule of merger between a polypeptide of interest and an addressing signal. 17. Cell producing exosomes, characterized in that it comprises one or several recombinant nucleic acids encoding a molecule of the complex major of histocompatibility. 18. Cell according to claim 17, characterized in that it is a cell of mast cell. 19. Cell according to claim 18, characterized in that it is a line mast cell of basophilic leukemia, in particular of the RBL line, of preference RBL-2H3. 20. Cell according to one of claims 17 to 19, characterized in that it comprises a recombinant nucleic acid encoding an .alpha. and or a .beta channel.
of a molecule of the class II major histocompatibility complex and/or for a class I major histocompatibility complex molecule. 21. A method of producing a membrane vesicle according to any of claims 1 to 16, comprising a defined recombinant molecule, including the following steps:
a) the culture of a mast cell or mast cell-derived cell comprising a recombinant nucleic acid coding for said defined recombinant molecule, c) recovering the vesicles produced by said cells, these vesicles comprising said defined recombinant molecule. 22. Method according to claim 21, characterized in that it comprises a stage intermediate b) during which the cells are stimulated to induce and or increase the secretion of exosomes. 23. Process according to claim 21 or 22, characterized in that the molecule defined recombinant 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 molecule recombinant is a major histocompatibility complex molecule, a molecule antigen, a receptor ligand, a ligand receptor, a peptide of cleansing or any other polypeptide of interest. 25. Method according to one of claims 21 to 24 characterized in that acid nucleic acid further comprises a region encoding an addressing signal towards them mast cell membrane compartments. 26. 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 acids recombinant nuclei coding for a defined recombinant MHC molecule, - the stimulation of cells to induce the release of exosomes, - the recovery of the exosomes produced by said cells, these exosomes expressing at their surface said defined recombinant MHC molecule, and, - 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:
- the culture of an exosome-producing cell comprising one or more acids recombinant nuclei encoding a defined recombinant MHC molecule and one nucleic acid comprising a region encoding a recombinant peptide defined, - the stimulation of cells to induce the release of exosomes, - the recovery of the exosomes produced by said cells, these exosomes expressing at their surface said defined recombinant MHC molecule associated audit recombinant peptide. 28. Method according to 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 cell producer is a mast cell or mast cell-derived cell. 30. Method according to one of claims 26 to 29 characterized in that the cell producer is essentially devoid of endogenous MHC molecule. 31. A method of altering the composition of an exosome, comprising - the introduction into an exosome-producing cell of a nucleic acid coding for a defined molecule, linked to an addressing signal in the compartments membranes, and, - the production of exosomes from said cell. 32. Composition comprising one or more membrane vesicles according to one of claims 1 to 16. 33. Use of a vesicle according to one of claims 1 to 16 for the production of antibodies, polyclonal and/or monoclonal. 34. A method of producing antibodies, comprising immunizing an animal with a vesicle according to claim 7 and recovering the antibodies and/or cells producing antibodies or involved in the immune response. 35. A method according to claim 34, for the production of antibodies monoclonal, in particular specific to the MHC-peptide association. 36. Use of an antibody obtained according to claim 34 or 35, or of a fragment of such an antibody, for detection, in a sample biological, presence of corresponding specific antigens. 37. Use of an antibody produced according to claim 34 or 35, of a fragment of such an antibody, or of a vesicle according to claim 1 for the preparation of a therapeutic composition intended to inhibit the interaction between the receptor of one T lymphocytes and the MHC-peptide complex for which it is specific. 38. Use of a membrane vesicle according to one of claims 1 to for the in vitro or ex vivo detection of specific partners of a protein molecule in a biological sample. 39. Use according to claim 38 of an exosome bearing an MHC-peptide 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 receptor TcR
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 receptor of ligand for detecting the presence of said ligand in a sample organic. 42. Method for the in vitro or ex vivo detection of the presence of T cells specific for antigen-MHC complexes in a biological sample, including the bringing said sample into contact with an exosome labeled according to the claim 7, comprising said antigen-MHC complex, and the demonstration of the labeling of T lymphocytes in said sample. 43. Use of a vesicle according to claim 7 for the amplification clonal and/or the ex vivo stimulation of cytotoxic or helper T lymphocytes. 44. Use of a vesicle according to one of claims 12 to 16 for the preparation of a composition intended to convey said molecule to a cell. 45. Composition comprising one or more membrane vesicles 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. 47. Composition according to claim 45, comprising one or more membrane vesicles immobilized on a bead, in particular a bead in latex or a magnetic ball.