JP2002529074A - Modified exosomes and uses - Google Patents

Abstract

  (57) [Summary] The present invention relates to the fields of biology and immunology. The present invention relates to membrane vesicles containing molecules having a predetermined structure, especially antigenic molecules, and uses thereof. More specifically, the present invention relates to vesicles (exosomes) containing recombinant major histocompatibility complex molecules and their use as immunogens or for diagnostic or therapeutic purposes. The invention also relates to methods for making the vesicles, gene constructs, cells and compositions useful for performing the method.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the fields of biology and immunology. The invention relates to membrane vesicles containing molecules having a predetermined structure, in particular antigenic molecules, and their use. The invention more particularly relates to vesicles containing recombinant major histocompatibility complex molecules,
And their use as immunogenic agents or as diagnostic or therapeutic tools. The invention can be used to carry out the method of the invention,
It also relates to methods for making these vesicles, genetic constructs, cells and compositions.

[0002] The specificity of antigen recognition is a major feature of immune system cells. B lymphocytes recognize the natural form of the antigen. T lymphocytes recognize a complex formed by binding of a peptide resulting from the degradation of an antigen to a major histocompatibility complex (MHC) molecule. Peptides derived from antigens (tumor or viral antigens) synthesized by cells of an organism are associated with MHC class I molecules, which are recognized by cytotoxic T lymphocytes. Peptides derived from exogenous antigens are associated with MHC class II molecules, which are recognized by helper T lymphocytes. The identification of peptides presented by MHC molecules and recognized by cytotoxic (CD8) or helper (CD4) T lymphocytes was the starting point for new therapeutic and vaccine strategies. Advances in this immunotherapy require the development of techniques to evaluate antigen-specific immune responses.

[0003] Antigenic peptides associate with MHC molecules in intracellular compartments.
In the case of class II molecules, these are composed of vesicles contained in relatively large granules belonging to the endocytic pathway (Peters et al., Na
true 349 (1991) 669). Their fusion with the plasma membrane leads firstly to the expression of the peptide-MHC complex on the cell surface and secondly to the secretion of these vesicles called exosomes.

A study by Raposo et al. (J. Exp. Med. 183 (1996) 116)
1) showed that B lymphocytes can secrete exosome vesicles that carry MHC class II molecules. In addition, Zitvogel et al. (Nature
medicine 4 (1998) 594) demonstrated the production of specific membrane vesicles (called dexosomes) by dendritic cells with advantageous properties. Thus, these vesicles express class I and class II MHC molecules and, after sensitization to the corresponding antigens, stimulate in vivo (in vivo) production of cytotoxic T lymphocytes, Can cause partial tumor resorption.

[0005] The present invention relates to novel methods and compositions that can be used in the fields of biology and immunology. More specifically, the present invention describes novel membrane vesicles whose composition has been modified in a defined manner. More specifically, the present invention provides for the purposeful production of vesicles expressing MHC complex molecules of known composition, optionally complexed with an antigenic peptide having a defined structure. A new way of enabling is described.
Thus, with the present invention, it is possible to modify the composition of membrane vesicles in a controlled manner, thereby creating a product that is particularly advantageous at the therapeutic, diagnostic or even experimental level.

[0006] The vesicles described to date contain, at most, endogenous MHC molecules, ie, MHC molecules expressed by their source cells. Therefore, these molecules have various structures that are not necessarily identified, and generally vary depending on the HLA type of the organism that produced them. Conversely, by using the present invention, it is possible to produce a membrane vesicle holding MHC molecules having a predetermined composition. In addition, the vesicles of the present invention have the advantage of containing a large number of MHC molecules thus defined, and provide strong immunogenic properties.

[0007] The present invention particularly relates to membrane vesicles containing molecules having a predetermined structure, especially MHC molecules having a predetermined structure. The invention particularly relates to membrane vesicles containing an MHC-peptide complex having a predetermined structure. The present invention is directed to a nucleic acid containing a hybrid region composed of a coding region fused to an addressing region, or alone or in combination with one or more proteins, to be naturally addressed to these membrane vesicles It also relates to a method for altering the composition of a membrane vesicle, comprising inserting a nucleic acid encoding a protein or polypeptide into a cell that produces the vesicle.

[0008] The invention also relates to membrane vesicles containing defined antigenic molecules anchored to a membrane portion. The molecule may be exposed outside the vesicle or, conversely, may be encapsulated in the cytosolic fraction. The invention further relates to membrane vesicles containing molecules with a predetermined structure, which are exposed on the surface, allowing purification using, inter alia, affinity methods. The invention also relates to a membrane vesicle as defined above which also comprises a tracer. With the tracer it is particularly possible to detect vesicles in a sample, for example for in vivo tracking.

The invention also relates to a method for preparing vesicles as defined above, and to the use of these vesicles. For example, these vesicles can be used as immunogenic agents for the preparation of antibodies. Particularly advantageously, these vesicles are MHC-restricted antibodies,
That is, it is used to prepare an antibody specific to the peptide-MHC molecule complex.

A first object of the present invention is more specifically a membrane vesicle characterized by containing a recombinant major histocompatibility complex molecule.

The term membrane vesicle in the sense of the present invention particularly refers to any vesicle composed of a lipid bilayer encapsulating a cytosolic fraction. These vesicles generally result from delivery from the cell and are therefore also referred to herein as "exosomes". The membrane vesicles (or exosomes) of the present invention generally have a diameter of approximately 60-80 nm. Also, these vesicles advantageously retain membrane proteins that have the same orientation as the plasma membrane of their source cells.

The invention demonstrates below that it is possible to modify the composition of exosomes in a controlled and specific manner. More specifically, the present invention shows that it is possible to produce membrane vesicles expressing recombinant molecular complexes of a (pre) defined composition. As exemplified later herein, the vesicles have particularly advantageous properties both from a therapeutic point of view as well as from a diagnostic and experimental point of view.

The present invention stems primarily from the selection of a particular cell population for the production of membrane vesicles. The present invention relates to the ability to insert recombinant molecules into these cells by a genetic route, and the ability of these recombinant molecules to be subsequently functionally expressed at high density in exosomes. Born from discovery.

Thus, one of the first elements of the present invention lies in the definition and identification of the cell population used for the production of membrane vesicles. Advantageously, the cells used are cells containing internal secretory vesicles, cells which can be cultured and genetically modified, whose internal vesicles can be secreted, preferably by the effect of external stimuli It is. This is mainly due to mammalian cells,
It particularly relates to animal cells, but also to cells of human origin. Primary cultures or immortalized systems can also be used.

[0015] Particularly advantageously, the initial cells are essentially free of MHC cells, ie they express no or very few endogenous MHC molecules. This feature may be of great importance in some applications, as exemplified below.

[0016] Different types of exosome-producing cells have been described in the literature, such as, for example, dendritic cells or B lymphocytes. Nevertheless, it is generally difficult to transfect these cells to contain large numbers of endogenous MHC cells. Thus, although they can be used to practice the invention, the vesicles of the invention can more preferably be obtained from mast cells or cells derived from mast cells.

[0017] In one particular embodiment, the membrane vesicles of the invention are preferably prepared from mast cells or cells derived from mast cells.

Mast cells are a set of cell types that, after differentiation, are derived from bone marrow precursors located in the epithelium such as skin, lung, intestine or spleen (Smith and Weis,
Immunology Today 17 (1996) 60). These cells express on their surface receptors whose cytoplasm is largely composed of histamine and granules containing heparin or protease, and have a strong affinity for immunoglobulin E (IgE). Is essentially characterized. A further advantage of the use of mast cells according to the invention is also that different treatments can initiate (especially strongly stimulate) exosome exocytosis (ie release). Therefore, by treatment in the presence of calcium ionophore,
Or, more physiologically, it is possible to control the production of vesicles by stimulating receptors with strong affinity for IgE.

These cells exhibit properties that are particularly important for the practice of the invention, namely the presence of endocrine vesicles, the potential for culture, and the induction of large amounts of exocytosis. Also, as described in the Examples, these cells can be stably genetically modified and have been shown to provide particularly advantageous properties for the practice of the present invention. I have.

More specifically, the vesicles of the invention have a diameter of approximately 60-80 nm and are made from mast cells or cells derived from mast cells.

[0021] In one preferred embodiment, the membrane vesicles of the invention are essentially free of endogenous MHC molecules. The lack of endogenous MHC molecules (ie, MHC molecules from vesicle-producing cells) can be manifested by specific antibodies using traditional techniques. It can also be demonstrated by the selectivity of the antibodies obtained by immunization with vesicles. As shown in the Examples, the vesicles of the present invention, in one particularly advantageous embodiment, have the ability, in animals, to detect, without detecting antibodies against cells that have not been genetically modified, to express them in a predetermined manner. The production of specific antibodies against the recombinant molecule can be induced. The term "essentially free" refers to the fact that MHC molecules are present in extremely low amounts, which are difficult to detect by traditional methods, and are marked for the antigenic specificity of the vesicles of the present invention. Means no effect.

[0022] Certain membrane vesicles of the present invention more specifically include-essentially free of endogenous MHC molecules-one or more recombinant molecules having a predetermined structure, such as a predetermined composition. It is characterized by having the property of retaining the recombinant peptide-MHC complex.

The vesicles of the invention are advantageously made from cells derived from mast cells essentially free of endogenous MHC molecules. In this regard, mast cells are known to accumulate MHC class II molecules within secretory granules. In particular, mast cells can preferentially accumulate MHC-II-peptides in a special multivesicular intracellular compartment, secretory granules (Raposo et al., Mol. Biol).
. Cell 8 (1997) 2619). Thus, these cells taken from mammals contain endogenous MHC molecules. Particularly advantageously, the cell lines used in the present invention are derived from mast cells essentially free of endogenous MHC molecules. Different mast cell lines have been described in the literature. The present invention demonstrates below that some of these systems have low levels of MHC molecules and are therefore particularly advantageous for the practice of the present invention. For example, RBL cells (rat basophil leukemia) deposited with the ATCC under the number CRL1378 (Kulc
zykki et al. , J. et al. Exp. Med. 139 (1974) 600)
KU-812 (Butterfield et al., Le.
ukemia Res. 12 198) 345), or even an immature human mast cell line such as the MHC line (Nilsson et al., Scand.
Immunol. 39 (1994) 489). An example of a particular system is the RBL-2H3 system (Barsumian et al., Eur. J.I.
mmunol. (11 (1981) 317). Obviously, any other cells having the above-mentioned properties can be used.

In the context of the present invention, the expression “predetermined composition” refers in particular to the fact that the vesicles of the invention have, for example, considerable antigenic and haplotype specificity. Vesicles described in the prior art generally express MHC molecules of various unknown haplotypes. Conversely, preferred vesicles of the present invention express recombinant molecules whose haplotypes are exactly predetermined. The term "recombinant" indicates that the molecule results from expression in a vesicle-producing cell of a recombinant nucleic acid encoding the molecule.
Thus, the membrane vesicles of the present invention are more preferably made from cell lines that have been genetically modified to express components having a predetermined structure.

As already indicated, the vesicles of the invention advantageously express a given MHC molecule.

[0026] Human MHC molecules fall into two distinct classes, MHC class I molecules and MHC class II molecules.

In certain embodiments, vesicles of the invention express one or more recombinant major histocompatibility complex class II molecules. In this regard, human MHC class I
The I molecule is composed of two chains, an α-chain and a β-chain, the β-chain conferring allele specificity to the complex.

In a particular variant embodiment, the vesicles of the invention express the recombinant α-chain, especially of the major histocompatibility complex class II molecule. In another particular variant embodiment, the vesicles of the present invention are more specifically a major histocompatibility complex class II
Expresses the recombinant α and β chains of the molecule.

Different types of human MHC II molecules have been identified, characterized, and sequenced (see, eg, Immunogenetics 36 (1992) 135). Preferentially, DR1 to DR13, in particular, DR1, DR2, DR3, DR4,
Mention may be made of molecules of the type DR5, DR6 and DR7. Human DR, especially D
DNA encoding R1-13 can be readily isolated from cells, banks or plasmids using traditional molecular biology techniques. These sequences are, in particular, Bod
(Tissue antigens 44 (1994) 1). Thus, preferably, the exosomes of the invention express MHC class II molecules comprising α and β chains selected from haplotypes DR1 to DR13, or more preferably DR1 to DR7.

In one particular example, the invention relates to any membrane vesicle comprising the recombinant α and / or β chains of the MHC-II molecule of the DR1 haplotype.

[0031] In another embodiment, the vesicles of the invention express one or more recombinant major histocompatibility complex class I molecules. MHC class I molecules are also composed of two chains: a transmembrane and polymorphic α-chain and β2-microglobulin which is poorly polymorphic and soluble. In humans, three loci, designated A, B, and C, encode α-chains. In traditional MHC-I molecules, each locus A, B, and C of the α-chain has an allelic variation (allelic v
aria). Therefore, alleles are A1, A2, A3, etc., A1
0, B1, B7, B37, B54, etc., CW3, CW6, etc. (for example,
See Bodmer et al. , (Supra) and Immunogenetics 3
6, 1992 (supra)).

Preferably, the exosomes of the invention express the α-chain of a traditional MHC-I molecule that is transmembrane and polymorphic. More preferably, it comprises allele A1,
It is the α chain of the MHC-I molecule having A2 or A3.

In one particular embodiment, the exosomes of the invention express the non-traditional, ie, non-polymorphic, alpha chain of the MHC-I molecule. Unlike so-called “traditional” MHC-I molecules, which are susceptible to substantial polymorphism, there are “non-traditional” MHC-I molecules in humans that have essentially no polymorphism. Such molecules are, for example,
Bendelac et al. (Ann. Rev. Immunol. (1997) 535).
It is described in. One preferred example of a non-traditional MHC-I according to the present invention is
The molecule is Cd1.

[0034] Obviously, any other human MHC-I molecule can be expressed within the scope of the present invention.

Thus, in one particular example, the invention relates to any membrane vesicle containing a recombinant protein of the MHC-I molecule.

[0036] In one particular variant embodiment, the vesicles of the present invention comprise several class I and / or
Or class II MHC molecules. One advantageous vesicle comprises, for example, two or more MHC-II molecules of different haplotypes. For example, MHC-I and MHC-
Clearly, any other combination of MHC molecules, such as II, is possible.

The vesicle of the present invention that expresses one or more predetermined MHC complexes comprises a predetermined MHC
It is particularly advantageous because it allows the presentation of certain antigenic peptides in the environment.
In this regard, in one more preferred embodiment of the present invention, the membrane vesicle contains a complex of a given peptide and a recombinant major histocompatibility complex molecule.

The vesicles of the present invention may also include one or more heterologous molecules of interest in addition to or instead of the aforementioned MHC molecules. In this regard, in one particular variant embodiment, the invention relates to membrane vesicles made from mast cells or cells derived from mast cells, characterized in that they comprise one or more heterologous molecules of interest. The term "derived" mast cell refers to a system that has been transformed and / or immortalized and / or obtained from a mast cell or basophil and has the properties of a mast cell (accumulation of endocrine vesicles). Means The term "heterologous" indicates that the molecule of interest is not present in its native form in the exosome of the invention in this form.

The target molecules carried or contained by the exosomes of the present invention include any protein, polypeptide, peptide, nucleic acid, lipid, and any target substance (having chemical, biological or synthetic properties) It is. These molecules may have recombinant properties and may be inserted into the producing cell or directly into / on the exosome. More specifically, preferred types of molecules of interest include, among others, MHC molecules, antigens (in full or peptide form), receptor ligands, (specific) receptors for ligands, nucleic acids, pharmacological products , Tracers or even peptides or proteins that allow the purification of vesicles.

Antigens can include in particular any proteins, especially cytoplasmic proteins, or proteins of viral or tumor origin. Preferred examples of proteins of viral origin include cytoplasmic proteins or membrane proteins produced by EBV, CMV, HIV virus, cysts, hepatitis and the like. These are more preferably cytoplasmic proteins, ie proteins which are essentially not recognized by the immune system in traditional infection processes and therefore show little immunogenicity under natural conditions, or they are membrane proteins or It may be a protein fragment. Preferred examples of proteins of tumor origin include p53 protein (wild type or any mutant type present in the tumor), MAGE (especially MAGE1, MAGE
2, MAGE3, MAGE4, MAGE5 and MAGE6), MART (in particular,
MART1), Gp100, ras protein (wild-type or mutant p21), and the like. Obviously, any other protein of interest can be expressed inside or on the exosome of the invention using the teachings of the invention.

In this regard, the recombinant antigenic molecule can be either on the surface of the vesicle (exposed) or inside the vesicle. Indeed, particularly surprisingly, we have found that vesicles of the present invention containing a recombinant antigen (particularly p53) in the cytosol induce very high production of antibodies against this antigen in animals. I found that I can do it.

Among the ligand receptors, mention may generally be made of any ligand receptor, natural or derived from genetic engineering. In particular, they can be any hormones, growth factors, lymphokines, trophic factors, antigen receptors and the like. In particular, interleukin IL1-IL15 receptor, growth hormone receptor, or receptor for granulocyte and / or macrophage colony stimulating factor (G-CSF, GM-CSF, CSF, etc.) can be mentioned. One particular example of a ligand receptor consists of a single-chain antibody (ScFv) that allows for interaction with a specific ligand.
Another particularly advantageous example in the sense of the present invention is the T lymphocyte antigen receptor (Tc
R). Exosomes of the present invention that express one or more predetermined TcRs on a surface form particularly advantageous analytical and diagnostic tools, as described in more detail below.

Pharmaceutical products can include any active substance with chemical properties, such as, for example, pharmaceutical products prepared using traditional chemical techniques. For example, such as toxins, hormones, cytokines, growth factors, enzymes, tumor suppressors, etc.
Mention may also be made of any protein, polypeptide or peptide having biological activity.

A nucleic acid is any DNA or RNA that encodes a protein, polypeptide or pharmacological peptide, as described above, and any other nucleic acid (eg,
Antisense, antigen, promoter, repressor, binding site of transcription factor, etc.). It can be an oligonucleotide, coding phase, artificial chromosome and the like.

[0045] The vesicles of the invention that retain a ligand receptor can be any of the ligand-receptor type, in any biological sample, as described in more detail elsewhere herein. It can be used to detect interactions, especially low affinity interactions. In addition, such vesicles can be used to transport a target substance (protein, peptide, nucleic acid, chemical substance, etc.) to cells. Accordingly, the exosomes of the invention are generally prepared in vitro, ex vivo or in vivo.
Can be used for the transport and introduction of any molecule into cells. Accordingly, the present invention relates to any vesicle as described above containing a heterologous target molecule, which can be used as a vector for introducing a heterologous target molecule into cells.

In a more preferred embodiment, the exosomes of the invention are used for the directed introduction of a substance of interest into a selected cell population. Therefore, the target substance (toxin,
Preparing vesicles of the present invention containing a hormone, cytokine, recombinant nucleic acid, etc., and expressing a ligand receptor or receptor ligand on the surface, and cells expressing the ligand or receptor corresponding to the vesicle It is possible to contact with. Therefore, by using this technique, it is possible to achieve efficient targeted introduction.

In this regard, one particular object of the invention resides in a vesicle as defined above, characterized in that it expresses a ligand receptor and comprises a heterologous molecule of interest.

[0048] The vesicles of the present invention may also contain peptides or recombinant proteins that allow for purification of the vesicles. Thus, the present invention makes it possible, in effect, to genetically modify the composition of exosomes and, therefore, to express them in vesicles with special "tag" molecules which allow the purification of the vesicles. It is described. In particular, it is possible to obtain exosomes that expose peptides with a particular structure that can be easily detected and captured by receptor molecules. In one particular example, the His6 pattern (ie,
An exosome is created that contains in its structure a peptide molecule containing six consecutive histidine residues). The presence of such residues on the surface of exosomes facilitates their purification in a nickel-functionalized support medium. Other recombinant peptides of this type may also be used, such as, for example, a cmyc tag, VSV or HA.

Finally, in one particular variant embodiment, the vesicles of the invention may also contain a tracer. The tracer may have different properties (such as enzymatic, fluorescent, radioactive, etc.) and may be present in the vesicle or on its surface. One preferred label is
Non-radioactive, such as fluorescent labels. More preferably, the tracer used is an enzyme with a fluorescent dye or a chromogenic substrate. Labeling can be done directly on the producer cells or on the produced exosomes.

The present invention also relates to any composition comprising one or more membrane vesicles as defined above. The composition of the invention may also comprise a plurality of membrane vesicles as defined above, carrying different recombinant molecules. In particular, the composition of the invention may comprise a plurality of membrane vesicles as defined above, carrying different haplotypes of recombinant MHC molecules bound to the same antigenic peptide. The composition may be, for example, a composition comprising membrane vesicles as defined above, carrying a recombinant MHC molecule of the same haplotype bound to a different antigenic peptide. Other combinations of the vesicles of the invention are clearly possible.

The compositions of the present invention generally include a medium, such as a buffer, saline or physiological solution, that allows for preservation of the vesicle structure. They are preferably (in
Any stabilizer, surfactant, etc., compatible with in vitro or in vivo biological uses may also be included. These compositions may be stored in any suitable equipment, such as tubes, bottles, ampules, flasks, pouches, etc.
C. can be stored. A typical composition according to the present invention comprises 5-500 μg, for example 5
Contains ２００200 μg of exosomes.

The vesicles of the present invention are obtained from genetically modified cells. As already indicated, the invention is based on the fact that it is possible, by means of a genetic pathway, to insert recombinant molecules into some cells, and that these molecules then have a high functional density. It was born from the discovery that it is expressed in exosomes.

To produce vesicles of the invention that carry a recombinant molecule having a defined composition,
The first step consists in inserting into a vesicle-producing cell as defined above a genetic construct allowing the expression of the selected recombinant molecule (s).

[0054] The genetic construct used to create the cell may generally include a coding region (expression cassette) placed under the control of a functional promoter in the cell used.

Thus, in general, the promoter used is a promoter that is functional in mammalian cells. It can be, for example, a viral, cellular or bacterial promoter. It can be a constitutive or regulatable promoter, preferably allowing high levels of protein expression in the cell. Among the promoters that can be used, for example, the immediate early promoter of cytomegalovirus (CMV),
Mention may be made of the promoter of the SV40, the promoter of the thymidine kinase gene, in particular of the HSV-1 TK, the promoter of the retroviral LTR, in particular of the LTR-RSV, or even the strong endogenous promoter of mast cells. One particularly preferred embodiment involves the use of the SRα promoter, as described in further detail in the Examples.

The coding region used is generally complementary DNA (eg, modified to include certain introns or to acquire preferential codon usage),
It is composed of genomic DNA or synthetic DNA. More generally, it is c
DNA. The nucleic acid may be obtained by any known molecular biology technique, in particular by screening, amplification, synthesis, enzymatic cleavage and ligation of banks.

Depending on the type of coding region used, certain modifications can be made to the construct. For example, in some cases, it may be particularly desirable to insert a signaling sequence into the coding region that allows the expression product to be addressed to a particular compartment of the cell, especially a membrane compartment (internal, cytoplasmic, etc.). It may be advantageous. This addressing signal is upstream of the coding region (5
'), Downstream (3') or internal. Preferably, the addressing signal is located 3 'of the coding region, more specifically in its cytoplasmic region, in a reading phase consistent with the coding region. The use of addressing signals may be particularly useful for promoting the accumulation of expression products inside or on certain intracellular compartments, especially inside or on secretory vesicles. This embodiment is particularly suited for the expression of molecules such as labeled peptides, antigens, MHC-I molecules or even receptor ligands. On the other hand, the present invention demonstrates that the human MHC-II molecule can be directly expressed in vesicle-secreting mast cells, particularly advantageously, without adding specific signals, even if heterologous.

In order to carry out the present invention, the following genes: Lamp1, CD63, LIMPII
, Cdlc, and a nucleic acid fragment having a partial sequence of FcγR can be particularly used as an addressing signal. These genes contain regions encoding protein addressing signals to the endosomal compartment of the cell (Sandoval and Bakke, Trends in Cell).
Biol. 4 (1994) 292). The addressing signal that can be used in the present invention satisfies, for example, the formula GYXXI (where X represents any amino acid residue). One addressing signal that is particularly suited for the present invention is the sequence S
Consists of the peptide signal of the LAMPI protein with HAGYQTI. Another type of signal that allows addressing to the membrane compartment involves all or part of the protein transmembrane region.

The addressing of the expression product into the cell compartment, which allows the expression product to be present in the exosome, comprises a membrane protein or a transmembrane protein, in particular a region encoding a membrane protein or a transmembrane protein expressed in an exosome. May be implemented by fusing all or a part of the coating with the coating region. In this regard, one particular embodiment of the present invention involves insertion of the recombinant product into an exosome by expression of the recombinant product in a producer cell in the form of a fusion with a membrane protein or transmembrane protein. I do. One particular example of such a protein is, for example, a recombinant MHC protein inserted into a producer cell, in particular the beta chain of MHC, preferably the beta chain of MHC class II. Thus, the results given in the Examples show that such fusions allow for efficient accumulation of any target peptide in the exosome without affecting the properties of the target peptide or the MHC molecule. It indicates that you want to. This aspect of the invention provides a novel concept for vectoring recombinant products in exosomes and can be applied to any recombinant product inserted into any type of exosome. Thus, in this regard, the present invention relates to any exosome comprising a recombinant fusion molecule of a polypeptide of interest and an addressing signal. It may be an exosome made from mast cells, dendritic cells or tumor cells, or it may be an exosome made, for example, from B lymphocytes.
The polypeptide of interest can be an antigen (or a fragment of an antigen) or any other biological product of interest. The addressing signal can be any peptide, polypeptide or protein having the property of directing the fusion product to a membrane compartment as defined above, in particular an intracellular compartment. Advantageously, it is a chain of an MHC molecule.

In one particular embodiment of the present invention, these vesicles comprise a chimeric nucleic acid encoding a fusion protein comprising a recombinant product linked to the MCH molecule, preferably the C-terminus of the beta chain of MCH class II. Produced by insertion into producer cells.

In the construct used, the coding region is operably linked to a promoter to allow its expression in a cell.

The constructs of the invention also advantageously include a region located 3 ′ of the coding region, which indicates a transcription termination signal (eg, a polyA region).

The expression cassette of the present invention is advantageously a part of a vector such as a plasmid type, virus type, episomal type, artificial chromosome type and the like. In this regard, the vector advantageously comprises a system that allows the selection of the cells containing it. In particular, the vector advantageously contains a gene that encodes a product that confers resistance to drugs, eg, antibiotics (ampicillin, hygromycin, geneticin, neomycin, zeocin, etc.). In one particular embodiment, each vector contains a single expression cassette as described above. Thus, in this embodiment, if some molecules (eg, the α and β chains of MHC-II) are to be expressed in the vesicle, the cell is modified by the insertion of several vectors. In this embodiment, each vector type used advantageously comprises a different selection system that allows for easy selection of multiple transfectants.

In another embodiment, the vector comprises an expression cassette as defined above, eg, an expression cassette encoding the α chain of MHC-II and a β cassette of MHC-II.
And an expression cassette encoding the chain.

The vector used is preferably a plasmid-type vector and contains, for example, a bacterial origin of replication which allows easy manipulation and in vitro production. The vector can be constructed from plasmids of the type, in particular pBR322, pUC, pBS, pSR and the like.

Thus, for the production of exosomes according to the present invention, genetically modified cells that express the selected molecule are used. These genetically modified cells are prepared by inserting a genetic construct, also as described above, into a selected cell as defined above.

The insertion of the genetic construct can be made in different ways, depending mainly on the type of cell used. Therefore, introduction of nucleic acid is performed by electroporation, calcium phosphate precipitation, chemical drugs (cationic peptides, polymers, lipids, etc.), marking agents (marking).
agent and the like can be done using any known technique. For viral vectors, transduction is generally obtained by simple cell infection. The amount of vector used can be adapted by one skilled in the art, depending on the type of transfer and the cell used. In this regard, one particularly efficient method for introducing nucleic acids into mast cells involves vector electroporation.

If several constructs (vectors) need to be inserted into a cell, they can be introduced simultaneously or sequentially.

After transfection, cells that have efficiently taken up the nucleic acid are selected based on resistance to the compound (eg, an antibiotic) by a resistance gene present in the introduced DNA,
Cloned. These cells can be used immediately for production of the exosomes of the invention, or can be stored for future use. In this regard, the cells are maintained for a period sufficient to achieve several exosome production batches,
It can be stored at 4 ° C. in normal storage media. Cells can also be stored in frozen form (eg, under nitrogen) for later use. Thus, in this regard, it is possible in accordance with the present invention to form a bank of exosome-producing cells having particular properties. In particular, according to the present invention, it is possible to form a bank of cells that primarily express the HLA type of MHC class II molecules. Then, depending on the intended application and HLA type, cells producing the corresponding MHC molecule can be selected from the bank without having to reconstitute these cells on a case-by-case basis.

In this regard, one particular object of the present invention is to provide an exosome-producing cell as defined above, in particular a mast cell, characterized in that it comprises a recombinant nucleic acid encoding a major histocompatibility complex molecule. It is in. The present invention specifically includes recombinant nucleic acids encoding a li invariant chain that has been modified to include an antigenic peptide in place of the CLIP region, or a peptide that enables purification of exosomes. Characterized by any exosome-producing cell as defined above,
In particular, it relates to mast cells.

More specifically, it is a mammalian cell, especially of animal, especially rodent origin.
It may be a cell of human origin. In one particular embodiment, it is a cell line derived from a mast cell, such as a mast cell line, especially for basophil leukemia. Particular examples include cells of the RBL line, especially RBL-2H3, KU-812 line or HMC-1.

Preferably, the recombinant nucleic acid encodes the α-chain and / or β-chain of a major histocompatibility complex class II molecule and / or a major histocompatibility complex class I molecule. In another embodiment, the cell contains several nucleic acids, each encoding the α and β chains of a major histocompatibility complex class II molecule.

With the present invention, exosomes having a known composition can be produced in a considerable amount in a simple and reproducible manner. For the production of exosomes, the genetically modified cells are cultured in a suitable medium and the exosomes are collected.

Accordingly, one particular object of the present invention is a method of making exosomes containing a given recombinant molecule, comprising: a) a mast cell containing a recombinant nucleic acid encoding the given recombinant molecule Or a step of culturing mast cell-derived cells; and c) a step of recovering exosomes containing said predetermined recombinant molecule produced by said cells.

Advantageously, the method of the invention also comprises an intermediate step b) of stimulating the cells to induce and / or increase secretion of exosomes.

Further, according to the method of the present invention, vesicles in which a predetermined recombinant molecule is exposed outside the exosome, or which is completely or partially contained in the cytosol fraction of the exosome, can be used. It can be made.

As already indicated, in the method according to the invention, the recombinant molecule is, for example, a major histocompatibility complex molecule, an antigenic molecule, a receptor ligand, a ligand receptor or a purified peptide or any other purpose. It can be a polypeptide. Also, as described above, in some embodiments, the nucleic acids used in the present invention include:
It further comprises a region coding for an addressing signal to the membrane compartment of mast cells, especially to endocrine vesicles.

A further specific object of the present invention is a method for producing membrane vesicles, comprising a recombinant nucleic acid encoding a recombinant MHC molecule, in particular of class I or II, in particular of humans. A method comprising culturing exosome-producing cells and, optionally, recovering the produced exosomes after stimulation of exocytosis.

In this regard, the present invention provides a method for preparing an exosome containing a peptide-MHC complex having a predetermined composition, comprising: one or more sets encoding a predetermined recombinant MHC molecule. Culturing exosome-producing cells containing the recombinant nucleic acid,-stimulating the cells to induce the release of exosomes,-collecting exosomes produced by the cells and expressing on the surface a given recombinant MHC molecule, And-contacting the exosome with the peptide (s).

For the practice of the invention, the peptide (s) used may be a synthetic peptide, a mixture of peptides, a cell extract, for example a mixture of peptides extracted from tumor cells. The peptide (s) may be in isolated form, or purified, or mixed as indicated above. Also, after contacting the exosome with the peptide, the exosome can be isolated or purified using traditional methods.

In another variant embodiment, the present invention provides a peptide having a particular composition
A method for preparing an exosome containing an MHC complex, comprising:-one or more recombinant nucleic acids encoding a given recombinant MHC molecule; and a nucleic acid comprising a region encoding a given recombinant peptide. Culturing exosome-producing cells containing:-stimulating cells to induce the release of exosomes-predetermined recombinant MHC produced by the cells and bound to the recombinant peptide
Recovering exosomes expressing the molecule on the surface.

More specifically, in this method, the nucleic acid containing the region encoding the recombinant peptide encodes an invariant li chain derivative in which the CLIP region has been deleted and replaced with the peptide. This embodiment ensures considerable specificity in the formation of the peptide-MHC complex.

[0083] In another variation, the nucleic acid comprises a region encoding the peptide and a region addressing the intracellular compartment. Also, nucleic acids are identical,
Alternatively, it may contain several regions encoding different antigenic peptides.

[0084] Preferably, the producer cells used in this method are mast cells or cells derived from mast cells. In this embodiment, the stimulation of the cells to induce the release of exosomes is preferably with one or more ionophores, or IgE.

[0085] In a particularly preferred embodiment, the producer cells used in the method are substantially free of endogenous MHC molecules.

A further object of the present invention is a method for altering the composition of an exosome, comprising: inserting a nucleic acid encoding a molecule of interest bound to an addressing signal to a membrane compartment into an exosome-producing cell; And- including the production of exosomes from said cells.

Using this method, it is advantageous to be able to produce exosomes that express various predetermined recombinant molecules.

The exosomes of the present invention can be used in a number of applications, for example, as a tool for analysis, diagnosis, therapy or experiment. For example, they may require low affinity receptor / ligand interactions that require multimerization of various partners to increase the avidity of these molecular complexes, for analysis of specific T antigen responses. For studying effects (and therefore beyond immunological application areas)
, For diagnosis and therapy, and for the production of specific antibodies, especially MHC-restricted antibodies. These and other different applications are illustrated below.

A) Uses for the Production of Antibodies One of the first applications of the exosomes of the invention is in the production of antibodies. Given a given composition of the exosome of the present invention, it is possible to produce an antibody having a defined specificity. Also, as shown in the examples, especially the MH on the surface
Due to the high density of the C-peptide complex, its functionality, and its efficient presentation to the immune system, the exosomes of the present invention have extremely strong immunogenic properties.

The antibodies thus produced may be polyclonal or monoclonal. They include traditional methods, including immunization of animals, collection of serum (polyclonal antibodies), and / or fusion of spleen lymphocytes with myeloma cells that do not produce immunoglobulins (to produce monoclonal-producing hybridomas). It can be prepared using immunological techniques.

Accordingly, a further object of the present invention relates to antibodies or antibody fragments produced by immunization with exosomes as described above. Antibody fragments include, for example, Fab fragments,
It may be (Fab ') 2 fragment, ScFv fragment, or the like, and more generally, any fragment that maintains the specificity of the antibody. In particular, the present invention relates to certain peptide-MHC
The present invention relates to a method for preparing an antibody, including immunization of an animal with an exosome as described above carrying the complex, and recovery of the antibody and / or cells producing the antibody or participating in an immune response. Advantageously, using the method of the present invention, it is possible to produce MHC-restricted monoclonal antibodies, in particular, monoclonal antibodies specific for MHC-peptide binding. Preferably, exosomes expressing recombinant MHC-peptide complexes and made essentially from cells of the same species as the immunized animal and essentially free of endogenous MHC molecules are used in the method of the invention. You. Thus, as shown in the Examples, using this method, without the need for additives, it is possible to identify a strong antibody against the peptide, especially an antibody restricted to MHC, that is, a peptide in a conformation bound to a given MHC molecule. It is possible to obtain a specific antibody. Such antibodies are particularly advantageous at the experimental, diagnostic and therapeutic levels. Also, the antibodies of the present invention can be labeled by any known technique (enzyme, fluorescence, radioactivity, etc.) using methods known to those skilled in the art.

B) Diagnostic Applications Exosomes and antibodies of the present invention have advantageous properties for use in diagnostics.

For example, the antibodies or antibody fragments obtained according to the invention may be used for any diagnostic application, for example by using different traditional techniques such as flow cytometry, immunohistochemistry or immunofluorescence. Can be used for the detection of the presence of the corresponding specific antigen in the target sample. In the particular example of MHC-restricted antibodies, they advantageously allow the detection of the corresponding MHC-peptide complex,
Enables diagnosis of the corresponding pathology. These antibodies determine the expression of previously defined antigens in a form that can be recognized by T lymphocytes, and thus may be particularly applied in the diagnosis of pathologies involving defective or inappropriate responses of the immune system. For example, but not exhaustively, the following: p53, Her2, MAGE, BAGE, M bound to MHC class I molecules
Detection of different peptides derived from proteins such as ART, GP100 in tumor samples allows for phenotyping of tumors and facilitates the choice of treatment, tumor pathology-pre-infection or latency in which virions cannot be detected. Viral diseases (hepatitis, HI
V, CMV or other viral infections)-Multiple sclerosis, autoimmune diabetes, autoimmune thyroid function, where detection of MHC molecules representing peptides derived from self-antigens can be a precursor to further progression of the disease Diagnosis of autoimmune diseases such as insufficiency, rheumatoid arthritis, and lupus erythematosus can be considered.

The exosomes of the present invention can also be used to detect specific partners of a protein molecule in a biological sample. Thus, exosomes of the invention that carry MHC-peptide complexes detect T lymphocytes specific for these complexes in biological samples, for example, in different pathological situations, especially in the aforementioned pathologies. Can be used for In this regard, the exosomes can be labeled by any labeling system (enzyme, fluorescence, radioactive, etc.) known to those skilled in the art that allows their detection in biological samples.

Thus, in one particular embodiment, the present invention provides a label, as described above, in particular a label, as described above, for the detection of T lymphocytes specific for an antigenic peptide-MHC complex in a biological sample. It relates to the use of fluorescently labeled exosomes. Biological samples include blood,
It can be any sample such as serum, tissue, tumor, biopsy specimen, skin, urine and the like. In addition, the biological sample, for example, for dispersion of cells, amplification of cells in culture, preparation of membrane fraction, etc.
It can be pre-processed. Advantageously, the biological sample is from a human organism.
To this end, the invention involves contacting a biological sample with a labeled exosome as defined above containing an antigen-MHC complex, and detecting the labeling of T lymphocytes in the sample. And a method for detecting the presence of T lymphocytes specific for an antigen-MHC complex in a biological sample.

Furthermore, the detection of these T lymphocytes not only allows detection, and thus diagnosis of pathophysiological conditions, but also eg the effectiveness of immunization protocols and the status of the immune response at different stages Also allows tracking of the efficacy of a given treatment.

In one particular embodiment, an exosome of the invention that carries a TcR receptor is used for the detection of a specific peptide-MHC complex of this receptor in a biological sample.

[0098] The fluorescent exosomes of the present invention, which carry any type of protein having a given composition, also form fluorescent probes that allow detection of potential receptors. Therefore,
The novel regions developed by exosomes can generally be extended to in vivo detection of any protein / protein interaction with low affinity. Accordingly, a further object of the invention is to detect a specific receptor for a protein molecule in a biological sample (therefore, in this embodiment, the exosomes used are Detection of the presence of the ligand in the biological sample (therefore, in this embodiment,
The exosomes used comprise, on the surface, a specific receptor for the ligand), the use of exosomes as described above, preferably labeled by fluorescence, in particular by fluorescence.

C) Therapeutic applications Restricted antibodies or fragments thereof can potentially inhibit the interaction of T lymphocyte receptors with their specific MHC-peptide complexes. at the same time,
Exosomes, which retain a single type of MHC-peptide complex on the surface, interact with T lymphocytes specific for these complexes to form the natural ligand, T
They can compete with lymphocytes and cause their inactivation.

Thus, the constrained antibodies and exosomes can be used, for example, in organ transplantation or bone marrow transplantation, T cells, which are likely to neutralize the host's response to the graft, typically with large amounts of immunosuppressive drugs. CD8 or CD4 immune response causes chronic tissue destruction, is mediated by T lymphocytes, which is known to be harmful to organisms, such as in autoimmune diseases or viral pathologies, allergies and asthma Can be used in any situation where it is needed to reduce or suppress an immune response.

Thus, in these types of pathologies, exosomes of the present invention that express on the surface certain peptide-MHC complexes known to be involved in the development of pathological conditions, Can be used to block the development of pathological reactions and thus the development of pathological reactions.

The exosomes of the present invention that carry the MHC-peptide complex can also be used to amplify (expand) a population of cytotoxic T lymphocytes ex vivo. Thus, when used directly from blood samples, they form the basis for cell therapy against different cellular targets. Thus, exosomes are specific for complexes expressed by cells targeted for therapy, such as tumor cells or virus-infected cells.
It can be used to sort various combinations of T cells. Accordingly, a further object of the present invention is the use of said exosomes for clonal amplification and / or stimulation of cytotoxic and / or helper T lymphocytes. The present invention involves contacting a biological sample containing T lymphocytes with such exosomes containing a predetermined peptide-MHC complex, collecting specific T lymphocytes, and amplifying them. E of T lymphocytes, especially cytotoxic T lymphocytes, including
It also relates to a method for clonal amplification (or expansion) in x vivo. This method is particularly advantageous for the clonal amplification of cytotoxic T lymphocytes specific for complexes of MHC molecules with peptides of tumor or viral antigens.

A particularly important further application of the vesicles of the invention is the introduction of molecules into cells. Due to their composition, the vesicles of the invention can be used in vitro, ex vivo and in vivo.
It can serve as a vector in the introduction of a molecule of the vivo into a cell. In this regard, the present invention relates to the use of such exosomes containing the substance of interest for the preparation of a composition aimed at introducing said substance into cells.
Advantageously, it is an exosome containing a ligand receptor or receptor ligand on its surface, which is capable of directing the introduction of one or more selected cell populations. The invention also relates to a method for the introduction of an in vitro, ex vivo and in vivo substance into a cell, including contacting the cell with a vesicle of the invention containing the substance. More preferably, the vesicles used also express the ligand receptor, and the method of the invention allows the directed introduction of a substance into cells expressing the corresponding ligand. For in vivo practice, the vesicles of the present invention may be administered by any of the traditional routes (intravenous, intraarterial, intramuscular,
(Preferably to mammals, especially humans). in
For use in vitro or ex vivo, the cells are contacted, preferably under sterile conditions, by incubation in a suitable device (dish, flask, pouch, ampoule, etc.). The parameters of the contacting step (volume of vesicles, contact time, temperature, medium, etc.) can be easily adjusted by those skilled in the art in relation to the purpose set and the teachings of the present application.

D) Applications in the research area These are clearly due to the use of antibodies capable of detecting and analyzing different stages of MHC-peptide complex formation in different normal or pathological situations.
It concerns all the aforementioned applications for the analysis of the molecular mechanisms in antigen presentation.

They are defined by the use of fluorescent exosomes and their ability to detect
It also relates to the analysis and molecular characterization of T lymphocyte populations that can recognize the HC-peptide complex.

In these different applications (diagnosis, therapy, experiments, production of T lymphocytes, etc.), the exosomes of the invention can be carried out as such or in a form immobilized on a supporting medium. Thus, the results given in the examples show that it is possible to immobilize exosomes on a supporting medium without compromising functional properties, in particular, for example, antigenic specificity. In this regard, one particular object of the invention resides in a composition containing exosomes immobilized on a support medium. The support medium is preferably a solid or semi-solid support, such as a bead, a filter or the like. It is preferably a support in a plastic material of polymer type, for example latex beads or magnetic beads. Obviously, any other synthetic or biological material that does not substantially impair exosome or cell quality can be used. Advantageously, beads having a diameter of 1 to 10 μm, for example 2 to 5 μm, can be used. Immobilization of the exosomes on the support medium is advantageously obtained by covalent bonding, by activation with an aldehyde or any other chemical binding reagent.
Generally, immobilization of the exosomes is performed by incubating the exosomes with the support in a solution under conditions that allow immobilization, and then collecting the support by centrifugation. The functionalized support thus obtained can be used to characterize exosomes, as described in the experimental section, or in vivo.
It can be used to detect or amplify T lymphocytes in vitro.

[0107] Other aspects and advantages of the invention will become apparent by reference to the following examples. The examples should be considered as illustrative and not limiting of the invention. All publications cited in this application are also incorporated herein by reference.

Materials and Methods In the cell examples section, the cells used to make the exosomes are mouse or human mast cells. More specifically, a basophil tumor line of the mucosal mast cell phenotype called RBL-2H3 (Barsumian et al., Eur. J.
. Immunol. (11 (1981) 317) and an immature human mast cell line (MHC-1) were used. RBL cells (rat basophil leukemia) deposited with the ATCC under the number CRL1378 (Kulczicki et al.,
J. Exp. Med. 139 (1974) 600), other mast cells may also be used, particularly in the system.

The T that can recognize certain antigens in the human MHC-II environment (DR1)
The lymphoid system was also used. In particular, the Jurkat strain (Sidhu) transfected with a cDNA encoding a receptor for a T ("T-HA") cell specific for the flu virus hemagglutinin peptide 306-318 bound to HLA-DR1.
et al. , J. et al. Exp. Med. 176 (1992) 875). The human B cell line transformed by Epstein Barr virus (Hom2 line)
It was used as a control for a restricted response to A-DR1.

The cells were cultured in DMEM medium (GibcoBRL), RPMI or “CLICK”. The RPMI medium contained 10% fetal bovine serum (Sigma), 1 mg
/ Ml penicillin-streptomycin, 1 mg / ml glutamine, 5 mM sodium pyruvate and 50 mM β-mercaptoethanol. Obviously any other medium that is compatible with the culture of eukaryotes, especially mammalian cells, can be used.

Cells were cultured mainly in 25 or 15 cm ³ culture flasks. RBL-2H3
The cells are detached from the support using trypsin-EDTA (Seromed) because they are adherent cells. To make the latter in large quantities, it is also possible to culture them in a "spinner" to a density of 10 ⁶ cells / ml.

To genetically modify plasmid mast cells, the following gene constructs were made.

The human HLA-DR1α chain (Larhammer et al., Cell 3
0 (1982) 153), human HLA-DR1 β chain (Bell et al.,
PNAS 82 (1985) 3405) and the human invariant chain p33li.
(Ciases et al., PNAS 80 (1983) 7395) was isolated. The cDNA encoding the invariant chain p33li was then modified by PCR to replace the region encoding the CLIP peptide (residues 87-102) with a restriction site. Using this cDNA, CL
Instead of the position of the IP peptide, any cD of interest encoding the antigenic peptide
It is possible to insert NA fragments (Stuntner et al., EM
BOJ. 16 (1997) 5807). In one precise example, the DNA fragment encoding the HA virus 308-319 of the flu virus hemagglutinin is
Inserted into this cDNA encoding the i polypeptide (HA308-319).

The nucleic acids were then cloned separately into the pSRα plasmid under the control of the SRα promoter (Takebe et al., Mol. Cell).
Bio. 8 (1988) 466). Then, each plasmid can be selected,
Thus, each plasmid was modified to incorporate a different resistance gene that allowed for the selection of each strand. A neomycin resistance gene was incorporated into the chain α plasmid, a hygromycin resistance gene was introduced into the chain β plasmid, and a zeocin resistance gene was incorporated into the invariant chain plasmid.

Transfection To insert different nucleic acids into mast cells, the corresponding plasmid vector was
Linearization was performed with the a1 restriction enzyme. 50 μg of each plasmid was linearized, followed by ethanol precipitation, and the residue was resuspended in the presence of RBL-2H3 cells at a concentration of 1 × 10 ⁷ cells / ml. "Gene pulser" under the condition of 260V, 960μF
Stable transformants were obtained by electroporation of 5 × 10 ⁶ cells using (Bio-Rad, Richmond, Calif.). 72 hours after electroporation, transfectants were treated with 250 μg / ml G418 (Geneticin, Gibco), 1 mg / ml.
Was selected by culturing in a selective medium containing 500 g / ml of zeocin. After 8 days of culture in selective medium, 60 to 90% of the cells present were transfected. The transfectants were then plated in Petri dishes containing a selective medium at a concentration that allowed the initiation of individualized adherent colonies. The clones thus obtained were collected and placed separately in culture. These clones can be stored in frozen form for future use.

Antibody Y3P (MHC-I (IA) is a mouse monoclonal antibody that recognizes the IAbαβ complex (Janeway et al., 1984). Anti-IAa is a rabbit serum against the cytoplasmic portion of the a chain of IA. The anti-GFP is commercially available from Boehringer Mannheim, a “green fluorescent protein”.
in)) is a mixture of two monoclonal antibodies (clone 7.1 and 13.1). The secondary antibody used in the flow cytometry experiments was mouse IgG (H + L) (Jackson Immunoresearch Lab).
F. (ab ') 2 fragment produced by donkeys against phycoerythrin.

Bead latex Bead: white aldehyde / sulfate latex containing no surfactant, D: 3.9 μm, manufactured by Interfacial Dynamics (Interf
Aial Dynamics Corp. , Portland, Or. USA
).

Electron Microscope HLA-DR1 transfected RBL-2H3 cells were fixed with 0.2 M phosphate buffer (pH 7.4) containing 2% paraformaldehyde (PB buffer). After fixation, cells were washed with PB buffer-50 mM glycine and then coated with 10% gelatin. After coagulation, blocks were prepared, poured into 2.3M sucrose and frozen in liquid nitrogen. Ultrathin frozen sections were prepared and immunolabeled with a polyclonal antibody against the HLA-DR molecule. These antibodies are visualized with A protein bound to 10 nm colloidal gold particles.

The exosomes were prepared in 0.2 M phosphate buffer containing 2% paraformaldehyde (p
H7.4) is mounted on an electron microscope plate fixed with (PB buffer) and covered with carbonated formvar film.

Exosomes are contrasted and coated in a 4% solution of uranyl acetate and methylcellulose, or immunolabeled with antibodies to class II molecules before coating. The antibody was A coupled to 10 nm colloidal gold particles.
Visualized with protein.

Results I-DR1 HA Exosome Generation 1.1 Construction and Characterization of Genetically Modified Producer Cells An exosome carrying an MHC-peptide complex with a given composition is generated in a controlled manner. To do this, MHC class II molecules, chains a and b of DR1, were expressed in the RBL2H3 lomast cell line from rat basophil leukemia. To this end, cells were co-transfected with two vectors, each carrying a nucleic acid encoding each chain, under the control of the SRa promoter (see Materials and Methods). The results obtained by flow cytometry indicate that the transfected cells efficiently express the DR1 molecule (FIG. 1A).

These RBL2H3 DR1 cells were then made sensitive to specific peptides with the correct composition in order to generate MHC-peptide complexes with the required composition. Different techniques are conceivable for this purpose. In a simple embodiment, the peptide can be incubated directly with the exosome. In another alternative embodiment, a nucleic acid encoding the peptide can be inserted into a cell such that the peptide is also expressed. In this particular example, a selected antigenic peptide is inserted into a cell in the form of a gene fusion with a human invariant li chain to create a presentation cell containing a single antigenic specificity. did. More specifically, the CLIP peptide of the invariant chain was replaced with the sequence of a selected peptide derived from the flu virus hemagglutinin known to bind to the DR1 molecule (HA308-319). This construct (liHA) was transfected into cells under the conditions described in Materials and Methods. RBL-2H
The hybrid chains expressed in the 3DR + cells show a level of D3 that is recognized by the L243 antibody at levels similar to the control B-EBV (Hom2) system of the DR1 haplotype.
This enabled the construction of cells expressing the R1 molecule (FIG. 1B). Thus, these results indicate that the mast cells of the present invention efficiently express a human functional peptide-MHC complex with a predetermined controlled composition.

The functional characteristics of the peptide-MHC complex expressed by the cells of the present invention were confirmed by a T lymphocyte stimulation test specific for the DR1-HA combination retained by the cells. To this end, the cells of the invention are incubated in the presence of THA lymphocytes and stimulated by measuring interleukin-2 released into the supernatant by a proliferation test of an IL-2-dependent cell line. Were determined. The control used was the EBV-transformed B lymphocyte lineage of the DR1 haplotype (Hom2) pulsed with a saturating concentration (10 mM) of the HA peptide.

The results obtained are given in FIGS. 1C and 1D. They show that the mast cells of the invention express a DR1-HA complex that can stimulate T lymphocytes specific for this combination. They show that the stimuli obtained in the presence of the cells according to the invention are control cells (DR) pulsed with a saturating concentration (10 mM) of the HA peptide.
It also shows that it is more efficient than stimulation caused by one haplotype (B-EBV). Finally, the addition of saturating peptide does not significantly increase the ability of RBL DR1 liHA cells to stimulate THA lymphocytes, so only DR1 molecules are HA
The results obtained indicate that it is believed to present the peptide (FIG. 1D).
.

Thus, all of these results demonstrate the functional characteristics of the generated peptide-MHC complex. They also exemplify the specific characteristics of the obtained cells, and thus the specificity of the method of the invention, which makes it possible to obtain cells (and exosomes) carrying molecules having a defined and controlled composition. Features are also illustrated.

1.2 Preparation of Functionalized Exosomes Immunofluorescence studies showed that recombinant MHC-peptide conjugate (DR1 HA)
It has been shown to accumulate in secretory granules of the 2H3 cell line. FIG. 2A provides evidence of co-localization of DR1 molecule and serotonin in vesicle intracellular structures.

Thus, the possibility that functional exosomes were released from these cells was investigated. For this purpose, cells were cultured in the presence of calcium ionophore and the production of membrane vesicles was followed. More specifically, cells were centrifuged at 300 g for 5 minutes at room temperature.
A solution of calcium ionophore (1 mM ionomycin) was added to each cell residue (
(Approximately 300 μl), incubation continued for 30 minutes at 37 ° C. By rapid cooling on ice and addition of 300 μl of cold 1 mM PBS-EGTA solution,
Exocytosis was stopped. The cells were then centrifuged at 300g for 5 minutes at 4 ° C. The supernatant was collected and the first was 1200 g for 5 minutes, then 1000 g for 5 minutes.
At 0 g, finally re-centrifuged at 70000 g for 1 hour. After this stepwise centrifugation, the residue (including exosomes) is collected and buffered (30 μl of Laemmli-DDT)
Solubilized in buffer (1 × or 2 ×). A portion of the residue was solubilized in lysis buffer and the protein concentration was determined. The exosome solution was separated by gel migration at 20 mA (12% polyacrylamide minigel) and then immobilon (Immobilon).
mobilon). The analysis of exosomes was then performed using MHC class I
This was performed by Western blotting using specific antibodies of different chains of the I molecule.

The results obtained show that after stimulation with the appropriate agent, the exosomes
2H3 can be released from the system. These exosomes can be isolated and purified, for example, by stepwise ultracentrifugation for the preparation of an exosome composition. Finally, the results given in FIG. 2B indicate that these exosomes are functional. Western blotting analysis demonstrates that the resulting exosomes express different recombinant chains of MHC class II molecules. Furthermore, these results also indicate that the peptide-MHC complex on the surface of the exosome of the present invention is dense.

The following examples specifically illustrate the use of DR1 HA exosomes for producing antibodies specific for this combination, and the ability of DR1 HA exosomes to bind to T lymphocytes specific for this combination. I do.

[0130] 2. Generation of Antibodies Specific for the DR1 HA Complex This example demonstrates the use of the present invention for the production of antibodies, particularly so-called "restricted" antibodies, ie, antibodies specific for antigenic peptides bound to MHC molecules. The use of exosomes is exemplified. This example particularly illustrates that the exosomes of the present invention allow for the production of antibodies without additives, so that the immunogenic properties of the exosomes of the present invention are extremely potent.

Exosomes purified from the supernatant of RBL DR1 HA cells (Example 1) were resuspended in PBS. Then Bal without additives according to the following protocol
These exosomes were used to immunize b / c mice or LOU rats.

For two injections at three week intervals, 10 μg of exosomes are injected into the mice by the subcutaneous route, then 30 μg by the intraperitoneal route and finally 30 μg by the intravenous route, on the third day Later the serum was collected.

Mice were injected with exosomes by the intraperitoneal route (10 μg) in two injections at three week intervals followed by the intravenous route (50 μg), and three days later the serum was collected.

As shown in FIG. 3A, serum collected from immunized mice was DR1
It showed extremely strong reactivity against both the RBL system expressing the HA complex and the non-expressing RBL system.
It became detectable only with HA.

As shown in FIG. 3B, the sera of immunized rats surprisingly
It showed reactivity to the RBL line expressing the R1 HA complex, and the serum reacted with relatively low intensity to the original RBL-2H3 line. In addition, DR1-expressing cells (
Addition of the HA peptide to RBL-2H3 DR1) resulted in a significant increase in the reactivity of the antiserum produced (FIG. 3B).

Thus, these results indicate that RBL-based exosomes can unexpectedly induce an antibody response in rats primarily against the DR1 HA complex.

The spleen of the immunized rat was fused with cells of the X63A8 line. The resulting hybridomas were selected by clonal dilution using traditional immunological techniques and then selected by immunofluorescence for the specificity of the monoclonal antibodies produced. Thus, an antibody against an RBL-based protein, an antibody against a monomorphic determinant of a human class II molecule of the DR1 haplotype, and an antibody against a complex consisting of a DR1 molecule bound to a peptide derived from the HA protein of flu virus are included. A different monoclonal antibody was obtained (FIG. 3C). Monoclonal antibodies against the last conjugate form bound antibodies and therefore have particularly advantageous properties for diagnostic or therapeutic applications.

3- Detection of T Lymphocytes Specific for the DR1 HA Complex This example illustrates the use of exosomes of the invention for the detection of specific T lymphocytes in a biological sample. This example also shows how exosomes can be used to select and amplify specific T lymphocyte populations, particularly for reinjection (cell therapy) into an individual. This approach can obviously be extended to the use of constrained antibodies as described in Example 2, and to the detection of any ligand-specific receptor.

For the performance of this application, labeled exosomes were made. For this purpose, the DR1 HA system was incubated with a fluorescent tracer that strongly accumulates in the exosomes contained in secretory granules before purification of the exosomes of the DR1 HA system. The tracer "Green Tracker" used is a fluorescent lipid that accumulates in cellular lysosomes. Analysis of the cells under a confocal microscope after fixation indicates the presence of a fluorescent label within the secretory granules of the cells (FIG. 4A). Then, fluorescent exosomes were prepared and purified from these cells under the conditions described in Example 1. These exosomes, thus rendered fluorescent (FIG. 4B), were converted to DR1 in a biological sample.
It was used to detect the presence of T lymphocytes specific for the HA combination (THA lymphocytes). It is clear that any other label can be used within the scope of the invention and applied to the producer cells or the exosomes produced.

For this purpose, the exosomes are converted to THA lymphocytes and other complexes specific T cells.
Incubated in the presence of a sample of H30 lymphocytes. The results obtained by flow cytometry show that exosomes expressing DRLHA specifically express THA.
Binding to lymphocytes (FIG. 4C), indicating that these same exosomes cannot recognize lymphocytes with another specificity (FIG. 4D). These results demonstrate the unique and unexpected ability of exosomes produced by the mast cell lines of the present invention to detect T lymphocytes specific for this complex. This application can be performed using any type of biological sample.

[0141] This technique can be similarly applied to the production and use of exosomes that express MHCI-peptide complexes. Thus, using the present invention, the MH of peptides derived from antigens expressed by tumors on the surface of presenting cells and even tumor cells
It is possible to detect presentation by C class I and class II molecules. Accordingly, the present invention provides detection of T lymphocytes capable of recognizing these same complexes,
It also allows for purification. Thus, they can be used to amplify a population of T lymphocytes specific for a particular peptide-MHC complex, such as a population of CTL lymphocytes for use in therapy. For example, different immunotherapies against cancer or viral infections may involve collecting lymphocyte samples from the individual and in particular T lymphocyte clones specific for antigens involved in pathology (eg, tumor or viral antigens)
It is being developed based on in vivo amplification. These amplified clones are then re-administered to the individual as a therapeutic. The present invention makes it much easier to select and amplify specific T lymphocyte clones, and thus to make the efficacy and implementation of these therapies much easier.

4-Generation of Exosomes Retaining Mouse MHC Class II Molecules (FIGS. 5A, 5B ) Complementary DNAs encoding the chains α and β of the mouse class II molecule of the IAb haplotype, and the mouse invariant chain, Transcription was inserted into an NT eukaryotic expression vector under the control of the alpha SR promoter. Each plasmid is
Hygromycin (αIAb chain), neomycin (βIAb chain) or zeocin (
(Variant chain). RBL by electroporation
After transfection of 2H3 cells (materials and methods), cells were selected based on resistance to the three antibiotics, then resistant cells were cloned by limiting dilution and the IAb molecules were analyzed by flow cytometry using specific Y3P antibodies. Characterized for expression.

FIG. 5 shows a part of the obtained results. Flow cytometry analysis was performed using RBL
IAbIi cells, as well as B lymphocyte lineage B414 (specific for IAb molecules)
Recognized by the Y3P antibody, indicating that no label is detected in the early RBL system (FIG. 5A). We established that mouse class II IAb molecules, like the human DR1 molecule, accumulate in the secretory granules of cells (not shown), so we have determined their exosomes in exosomes. It was decided to search for a localized location. Exocytosis of the cells was initiated by the addition of 1 mM ionomycin, and the resulting exosomes were purified by stepwise ultracentrifugation (see Example 1.2). Western blotting of these exosome preparations
They are the same mouse MHC class II as the molecules detected in control cell lysates
It shows that it contains a molecule (FIG. 5B).

These results demonstrate that human class II molecules (DR1) and mouse molecules (IAb) can be expressed and accumulated in the corresponding exosomes of the RBL 2H3 system.

5- Morphological Characterization of Exosomes Produced by RBL 2H3 (FIG. 6 ) Observation of immunolabeled cryosections of RBL cells under a confocal microscope revealed that many membrane-filled cells The existence of the compartment was revealed. The majority of these membranes corresponded to vesicles filling the lumen of the compartment (FIG. 6A). When transfected into these cells, class II molecules accumulate in compartments with internal vesicles and are specifically visualized in association with the membranes of these vesicles (FIG. 6A).

When stimulating RBL cells to induce degranulation (IgE antigen or ionomycin), the intracellular compartment fuses with the plasma membrane. The inner vesicles to which the class II molecules are bound are released into the extracellular medium. Thereafter, these vesicles are called exosomes.

A selection method in electron microscopy to investigate the morphology of exosomes and their protein content is the “whole mount” method. Using this technique, it is possible to visualize complete exosomes without any other cellular content. Using this method, it is also possible to very efficiently detect molecules bound to the exosome membrane. By using this technique, we have found that exosomes secreted by RBL cells can
It was observed to have a non-uniform size of 20 nm and various electron densities (FIG. 6B). Class II molecules were 80-100 nm in size and were abundant in populations of vesicles with average electron density. Most of these vesicles enriched for class II molecules were dish-shaped (FIG. 6C).

6— Immobilization of exosomes on a support medium (FIG. 5C) This example describes the immobilization of exosomes on a solid support, the exosomes thus immobilized on their functional properties It is shown that can be maintained. These new products (exosome supports) can be used to characterize and analyze exosomes; or as diagnostic or reagent products, for example for detecting and / or stimulating T lymphocytes in vitro. Can be done.

Incubation of different preparations of exosomes produced by RBL cells expressing or not expressing human or mouse Class II molecules with 4 micron latex beads activated by aldehyde sulfate did. More specifically, the exosomes purified from the degranulated supernatant of RBL 2H3 were washed with PBS (centrifugation at 50,000 rpm in TLA100.4 for 30 minutes). 30 μg of exosomes were mixed with 10 μl of aseptically collected latex beads, homogenized and then incubated at room temperature for 10-15 minutes. The bead volume was then made up to 1 ml with 1 × PBS and then incubated at room temperature for 2 hours. The exosome-crosslinked beads were then saturated-by adding a final 100 mM glycine (30 min room temperature),
- centrifuged for 2 minutes at 4 ° C. at 2200 g, then collecting the beads residue in 1 × PBS 3% SVF 0.01% NaN 3 in 1 ml.

[0150] For use of beads to cells fluorometry: - beads residue was washed 2 times with 3 times 1 × PBS 3% SVF 0.01% NaN 3, - 1 × PBS 0.01% NaN of 1mL Collected in _3- Secondary antibody was traditionally incubated in the first well, using between 5 and 20 μL per point. Reading is performed by Facscalelibur (Becto)
n Dickinson).

Using this technique, it was possible to cover the surface of these latex beads with exosomes while preserving the structure.

If exosomes are present on the beads, their handling will be easier.
They can be centrifuged at low speed, for example, and detected by traditional flow cytometry techniques. FIG. 5 shows an example of detection by flow cytometry of different proteins incorporated in the composition of the exosome. Latex beads activated with aldehyde sulfate were added to untransfected RBL 2
Exosomes produced by H3 cells, or human class II molecules DR1 and I
Incubation with exosomes of RBL 2H3 cells expressing the iHA construct or mouse class II molecule IAb, or fetal calf serum (FCS) as a control. The latex beads thus prepared were then combined with different monoclonal antibodies; AD1 recognizing the rat CD63 molecule present in RBL 2H3 secretory granules, Y3P antibody specific for molecule IAb, and DR1 molecule specific. L2
Incubated with 43 antibodies. These different antibodies are exposed by a secondary antibody conjugated to phycoerythrin and then FACScalibur (Beck
The resulting label was analyzed by flow cytometry using M. man).

Thus, CD63 molecules are clearly present in all exosomes from RBL cells expressing or not expressing class II molecules, and latex beads coated with IAb exosomes It was observed that the DRIiHA exosome was specifically recognized by Y3P and not by L243, whereas the DRIiHA exosome was recognized by L243 and not by Y3P. None of these antibodies recognize latex beads covered with fetal calf serum (FIG. 5C).

These results indicate that this technique allows sensitive and specific detection of the expression of different proteins incorporated into the composition of the exosome. Example 8
Also show that such products (exosome supports) can also detect or stimulate the proliferation of specific T lymphocytes.

Engineering of 7- Exosomal Composition (FIG. 7) Exosomes are vesicles bound by a lipid bilayer into which multiple molecules such as the aforementioned MHC class II molecules or CD63 have been inserted. Inside these vesicles, not only the cytoplasmic region of the aforementioned transmembrane molecule, but also soluble proteins derived from the cell cytosol are found. To demonstrate that the contents of these vesicles can be modified at will, we used green fluorescent protein (GFP) as a tracer.

The cDNA encoding GFP was fused to the COOH terminus of the beta chain of the human DR1 molecule. This construct was inserted into the NT expression vector carrying the hygromycin resistance gene and co-transfected into cells RBL 2H3 with the vector carrying the alpha chain of DR1 and the neomycin resistance gene. Cells resistant to these two antibiotics were selected and then screened positive for GFP expression.

More specifically, the present inventors have created a construct that connects the cytoplasmic portion (C-terminus) of the DRβ chain to the N-terminus of GFP. DRβ cDNA has Pst at position 565
Has an I site. An approximately 200 base pair fragment on the 3 'side of this cDNA was obtained from the vector pcDNA3 / RSV / Dra by using a 5' PstI site and a 3 'NcoI
P using two oligonucleotides containing a site and excluding the stop codon
Amplified by CR. The obtained PCR fragment was digested with PstI and NcoI, and cloned into the same site of the vector pEGFP N1 (Clontech). P
The resulting plasmid, digested with stI and XbaI, allowed the release of a fragment corresponding to the last 30 amino acids of DRa followed by GFP. At the same time, the plasmid pcDNA3 / RSV is digested with EcoRV / PstI,
The fragment corresponding to the rest of the Dra chain (from the beginning to the PstI site) was released. The two fragments were then assembled and cloned between EcoRV and XbaI of pcDNA3 / CMV.

Analysis of these cells by flow cytometry (DR1 GFP)
Cells recognized by the L243 antibody specific for one molecule also emit green fluorescence detected in tube FL1, while cells transfected with the alpha and beta chains of GFP-free DR1 FL1 does not emit fluorescence (FIG. 7A).

To demonstrate that GFP is truly contained in the exosomes of cell RBL 2H3, exosomes from cell RBL DR1 GFP were prepared by stepwise ultracentrifugation and then analyzed by Western blot (FIG. 7B). After cross-linking with latex beads, the samples were analyzed by flow cytometry (FIG. 7C). In Western blotting, GFP specific antibodies are
1 A 65 kDa protein corresponding to the molecular weight of the beta chain of DR1 fused to GFP was detected in GFP and its exosomes (FIG. 7B), and cells RBL 2H3 expressing only DR1 molecule or cells RBL not expressing DR1 molecule were detected. No signal was detected in the cell lysate of 2H3. Comparison of latex beads cross-linked with exosomes derived from fetal calf serum or DR1 GFP cells indicates that only beads coated with exosomes induce fluorescence in tube FL1 (FITC) and are detected by a secondary antibody conjugated to phycoerythrin L1 specific to DR1
43 shows that the antibody is recognized by the 43 antibody.

Thus, these results demonstrate that it is possible to insert exogenous proteins inside exosomes produced by RBL 2H cells. These results further show that expression in producer cells, in the form of a fusion with a transmembrane molecule, such as an MHC molecule, can direct the protein to exosomes.

8-Example Functional Characterization of Exosomes (FIG. 8) This example demonstrates that exosomes produced by RBL 2H3 cells carrying MHC class II molecules bind to an antigenic peptide and bind to the peptide-MHC class II Figure 4 shows that T lymphocytes expressing the specific receptor of the complex can be stimulated.

The exosomes produced from RBL DR1 IiHA cells labeled with Green Cell Tracker were converted to HLA-D
Incubation was performed in the presence of two types of T cells, THA with a TCR specific for the R1 / HA complex, and wild-type T Jurkat without the receptor.
Using a 96-well multidish with round bottom wells, 50 μl per well
Binding experiments were performed at 37 ° C. for 3 hours at 37 ° C. with RPMI 1640 supplemented with 10% fetal bovine serum containing 10 mM Hepes as a buffer in a final volume of 10 l, 10 ⁵ T cells per well and various amounts of fluorescent exosomes. did. Next, after washing twice with the same medium, cells collected by FACS in 400 μl of PBS were analyzed.

The dose-effect range was plotted and showed that the labeling intensity of THA was proportional to the amount of exosomes used. 10 ⁸ RBL DR1 IiHA cells are 700
μl of exosomes were given. Exosomal doses ranging from 32 to 2 μl per well were tested. The label obtained at 16 μl per point was retained, which corresponded to exosome production by 2.3 million cells (results not shown).

The results obtained indicate that the THA population is labeled with fluorescent exosomes, produces fluorescence detectable with FL1 by cytofluorometry, and does not alter wild-type Jurkat (FIG. 8A).

The same type of labeling was performed, but after exosome binding, the T cells were incubated with the AD1 mouse monoclonal antibody anti-rat CD63 followed by donkey antibody anti-mouse IgG labeled with phycoerythrin (Jackson Immunon).
o Research, Wst Grove, PA). at the same time,
The same labeling was performed on T cells that were not exposed to exosomes. Only exosome-bound THA cells showed FL2 as evidenced by fluorescence at FL1.
Are also shown to be labeled (Figure 8), indicating the new presence of rat CD63 on their surface.

Thus, our results indicate that fluorescent exosomes were used to visualize, by flow cytometry, T lymphocyte populations specific for the HLA-DR / antigenic peptide complex carried by the exosomes. It indicates that

To assess the ability of exosomes to stimulate T lymphocytes depending on the presence of the peptide, exosomes obtained from DR1 GFP cells were cross-linked to latex beads and then conjugated to the DR1-HA peptide 307- The cells were incubated in the presence of cells of T Jurkat lymphocytes expressing or not expressing the 319 specific T receptor.

Latex beads were prepared as for flow cytometry, complete medium (RPMI, 10% fetal bovine serum, 1% penicillin-streptomycin-glutamine, 0.1% β-mercaptoethanol, 4% pyruvate). Sodium). For stimulation of T lymphocytes, each bead residue was collected in 100 μL and
μL was placed in the first well of a 96-well multi-dish and 50 μl was diluted in half.
Controls were similarly performed using beads incubated in fetal calf serum. 10 ⁶ T cells (T Jurkat Pasteur and THA 1.7)
Adjusted to cells / mL and placed 50 μl in each well. Peptides diluted to 15 μM in complete medium were also added at 50 μL per well. For the peptide-free series, 50 μL of complete medium was added to each well. The culture dish was left in an incubator (37 ° C., 5% CO ₂ , O ₂ ) for 20 hours, then the supernatant was collected, and the CTL. The concentration of IL2 was evaluated by the L2 test.

Addition of the HA peptide (5 mM) resulted in T-expressing the receptor for the DR1-HA complex.
It specifically induces stimulation of Jurkat cells (THA), whereas control T lymphocytes (T
Jurkat) was observed to be ineffective. Also, some exosomes cannot stimulate THA in the absence of HA peptide (FIG. 8C).

Exosomes of 9 Human Mast Cell Lines (FIG. 9) This example shows that exosomes modified according to the invention can be produced from other cells, especially human cells. In particular, the results we have obtained show that human MHC
A mast cell line of origin I, under the impact of increased intracellular calcium,
It demonstrates that exosomes can be produced under the same conditions that induce exosome secretion by H3.

[0171] Characterization of the MHCI system by flow cytometry indicates that the surface of these cells expresses MHC class I molecules (W6.32) but class II molecules (L24
3) indicates that it is not expressed. They also include the molecules CD9, CD6
3 and CD81 were positive, but the molecules Lamp1 and Lamp2 were negative (FIG. 9A).

Creating exosomes from the MHCI system by addition of ionomycin (1 mM)
Purified from the supernatant by stepwise ultracentrifugation. Their composition was analyzed by flow cytometry and Western blot after cross-linking to latex beads.

The analysis of exosomes by the method using latex beads indicates that they
It shows that it retains amp1, CD9, CD63, CD81 and MHC class I molecules, but not class II or Lamp2 molecules (FIG. 9B). In addition, the morphology of these exosomes observed under an electron microscope indicates that R
Identical to exosomes produced by the BL2H3 system. Finally, the protein composition observed by Western blot confirmed these results. Because, using this technology, we have identified the molecules CD63, Lamp1, MHC
Class I was found, but Lamp2, class II molecules remained negative (FIG. 9C).

In conclusion, the system MHCI is able to produce exosomes with the same structural and molecular characteristics after increasing intracellular calcium, thus
It is considered to be a human homolog of the L-2H3 rat strain. Thus, using these cells, it is possible to produce recombinant human exosomes that express MHC class II molecules or any other molecule specifically addressed to them.

[Brief description of the drawings]

FIG. 1 shows the production of a functional MHC-DR1-HA peptide complex in the RBL2H3 system. A. Before (left) and after (right) transfection of cDNA encoding the α and β chains of DR1 in the RBL 2H3 system, human MHC-
Analysis of surface expression of DR1 molecule of II by flow cytometry. The DR1 molecule is detected by the L243 antibody developed by goat serum anti-mouse-IgG conjugated to FITC (black line). B. 308-3 wherein the CLIP peptide is derived from the flu virus hemagglutinin
Surface expression of DR1 in a system expressing an invariant chain (liHA) substituted with 19 peptides. The DR1 molecule was expressed in RBL by the L243 antibody (black line).
It is detected on B lymphocytes (Hom2) transformed by the DR1 liHA system and EBV of the same haplotype. C. R-BL or haplotype B-EBV expressing DR1-HA complex
Stimulation of T lymphocytes specific for the DR1-HA complex. RBL Dr1
iHA and B-EBV Hom2 were diluted in culture dishes containing T lymphocytes specific for the DR1 HA complex. B-EBV Home2 has a saturation concentration (10 mM
) Was also incubated in the presence of the HA peptide. The production of IL2 in the culture supernatant is used to evaluate the stimulation of THA lymphocytes (T lymphocytes specific for the HA peptide). IL2 is measured by a tritiated thymidine incorporation test in the CTLL2 system, where proliferation is IL2-dependent. D. Analysis of HA peptide saturation of the RBL DR1 liHA system. The cells Hom2 and RBL DR1 liHA were treated with increasing concentrations of HA peptide and THA.
Incubated in the presence of lymphocytes (100 cells per dish). Lymphocyte stimulation was evaluated as described above.

FIG. 2. Accumulation of DR1 molecule in the secretory compartment of RBL2H3. A. Analysis of intracellular accumulation site of DR1 molecule in RBL 2H3. RBL D
RLHA cells were fixed with 0.5% glutaraldehyde and then permeabilized with 0.05% saponin. The DR1 molecule and the invariant chain were isolated from the antibody L2, respectively.
Donkey serum anti-mouse IgG detected at 43 and PIN1, then conjugated to FITC
Was detected. Serotonin was obtained from donkey serum anti-rabbit IgG conjugated with Texas Red.
Detected using a specific rabbit serum developed at The image was taken with a confocal microscope (L
eica). Section thickness was 0.5 microns. B. Purification of RBL Dr1liHA exosomes. After washing with DMEM,
Cells were incubated for 30 minutes at 37 ° C. in the presence of 1 mM ionomycin.
Exosomes were purified from the cell supernatant by stepwise ultracentrifugation. Exosomal residues resuspended in PBS were separated by SDS-PAGE (5 mg) and then transferred to a nylon membrane. The β-chain of DR1 was detected with monoclonal antibody IB5 on exosome preparations migrated under the same conditions and controls in lysates of RBLDRLHA cells and Hom2 cells (equivalent to 10 ⁵ cells per dish).

FIG. 3 shows the use of exosomes for the production of anti-DR1 HA antibodies. A. Serial dilutions of serum from mice immunized with exosomes were added to DR1
RBL cells expressing HA molecule (right) or non-expressing RBL cells (left)
Incubated with The resulting label was analyzed by flow cytometry. B. Serum from rats immunized with exosomes (1/100 dilution) was added to D
RBL cells expressing or not expressing R1 HA molecule (left)
Incubated with On the right, cells expressing DR1 were identified as 1
Preincubated for 2 hours at 37 ° C. with 0 mM HA peptide or without preincubation and then incubated with the same dilution of serum from immunized rats. C. Under traditional monoclonal antibody production conditions, the immunized rat spleen was transformed with X6
It was fused with the 3A8 line. The supernatants of the different hybridomas were used as DR1 molecules or DR1
Tested by immunofluorescence on RBL2H3 cells expressing or not expressing HA molecules. Clones a40, b82 and a15 are representative examples of the antibodies obtained.

FIG. 4 shows the use of exosomes for the detection of T lymphocytes specific for the DR1 HA complex. A. Cell RBL DR1 liHA was incubated at 37 ° C. for 30 minutes in the presence of 5 mM “green tracker” (a fluorescent lipid that accumulates in the lysosomal compartment of the cell), then washed and incubated at 37 ° C. in the absence of a fluorescent tracer. Reincubated for 1 hour. Cells were fixed (3% paraformaldehyde) and then analyzed under a confocal microscope. B. At the same time, exosome DR1 HA was purified from the cells described in A. The fluorescence present in the sample was quantified with a fluorimeter and visualized directly under a confocal microscope. C. D. 50 mg / ml of the fluorescent exosome DR1 HA was converted to a TH1 lymphocyte specific for the DR1 Ha complex or to a TH30 lymphocyte specific for another complex (D).
And incubated for 2 hours at 37 ° C. in the presence of azide to block uptake. Cell fluorescence was assessed by flow cytometry.

FIG. 5 shows the generation of exosomes that carry MHC class II molecules. A. Expression of the class II molecule IAb is detected by the monoclonal antibody Y3P and analyzed by flow cytometry. The transfectants obtained in cells RBL2H3 showed similar levels of Y3 cells as B414 control B lymphomas.
It expresses a class II molecule recognized by P. B. Western blot analysis of expression of molecular IAb in RBL. Preparations of exosomes from 10 mg of cell lysate and cell RBL IAbIi were analyzed by Western blot using rabbit serum specific for the cytoplasmic region of chain a of molecule IA. C. Analysis of exosome composition by flow cytometry. Fetal bovine serum (
FCS) or exosomes derived from cell RBL 2H3 (exosome RBS)
Or the mouse class II molecule IAbIi (exosome IAbIi) or human D
Latex beads were coated with a transfectant of this cell using R1IiHA (exosome IiHA). Rat molecule CD63 is detected with antibody AD1, molecule IAb is detected with antibody Y3P, and molecule DR1 is detected with antibody L243. These different antibodies are developed by secondary antibodies conjugated to phycoerythrin.

FIG. 6A shows morphological characterization of exosomes produced by RBL-2H3. A. Cells RBL-2H3 transfected with HLA-DR1 were fixed with paraformaldehyde. Ultrathin frozen sections were prepared and immunolabeled with a polyclonal antibody against the molecule HLA-DR. These antibodies are visualized with Protein A bound to 10 nm colloidal gold particles. Class II molecules are mainly detected in compartments filled with vesicle-like membranes. Bar: 250 nm.

FIG. 6B. C. Morphological characterization of exosomes secreted by RBL-2H3 cells. Exosomes are 0.2 M phosphate buffer containing 2% paraformaldehyde (
pH 7.4) (PB buffer) fixed and carbonated foam bar (for
mvar) placed on an electron microscope plate covered with film. Exosomes are contrasted and coated in a 4% solution of uranyl acetate and methylcellulose, or (b) immunolabeled with antibodies to class II molecules prior to coating. As in Figure (a), the antibody is visualized with protein A bound to 10 nm colloidal gold particles. Bar: 250 nm.

FIG. 6C. C. Morphological characterization of exosomes secreted by RBL-2H3 cells. Exosomes are 0.2 M phosphate buffer containing 2% paraformaldehyde (
pH 7.4) (PB buffer) fixed and carbonated foam bar (for
mvar) placed on an electron microscope plate covered with film. Exosomes are contrasted and coated in a 4% solution of uranyl acetate and methylcellulose, or (b) immunolabeled with antibodies to class II molecules prior to coating. As in Figure (a), the antibody is visualized with protein A bound to 10 nm colloidal gold particles. Bar: 250 nm.

FIG. 7 illustrates manipulation of the internal composition of exosomes and insertion of recombinant proteins. A. The expression of the class II DR1 molecule is detected by monoclonal L243 and analyzed by flow cytometry. Transfection of the molecule into cells RBL2H3 induces expression of class II molecules recognized by Y3P at levels similar to B414 control B lymphoma. B. Western blot analysis of the expression of the molecule DR1 GFP in RBL. 10 μg of cell lysate and 20 μg of exosome preparation from cell RBL DR1 GFP were Western blotted using a set of GFP specific monoclonal antibodies. C. Analysis of exosome composition by flow cytometry. Latex beads were coated with fetal calf serum (FCS) or exosomes from cell RBL DR1GFP. The DR1 molecule is detected by the L243 antibody and a secondary antibody bound to the L243 antibody and phycoerythrin, but the presence of GFP is detected by the channel FL.
1 directly detected.

FIG. Binding of fluorescent exosomes by specific T cells. Exosomes produced from RBL DR1 liHA cells labeled with Green Cell Tracker were used to convert two types of T cells (THA with a TCR specific for the HLA-DR1 / HA complex,
And wild-type T-Jurkat which does not contain this receptor. Fluorescent exosomes were incubated with two T lymphocyte lines for 3 hours at 37 ° C., and the resulting label was analyzed by FACS. B. Obtaining an exosome tracer with specifically labeled T cells. Labeling was performed in the same manner as in A, except that the binding of exosomes (not labeled with Green Cell Tracker) to T cells was determined by the mouse monoclonal antibody AD1 anti-rat CD63.
And phycoerythrin-labeled donkey antibody anti-mouse IgG (J
ackson Immuno Research, West Grove, PA
). At the same time, similar labeling was performed on T cells not exposed to exosomes. C. Stimulation of lymphocytes by exosomes. Exosomes purified from DR1GFP cells were cross-linked to latex beads prepared as in flow cytometry and washed with complete medium. Each bead residue was collected in 100 μL, 50 μL was placed in the first well of a 96-well multi-dish, and 50 μL was diluted 2-fold. T cells (T-Jurkat and THA1.7) were adjusted to 10 ⁶ cells / mL and 50 μM in the presence or absence of 5 μM HA307-319 peptide.
l was placed in each well. The culture dish was left in an incubator (37 ° C., 5% CO ₂ , H ₂ O) for 20 hours, then the supernatant was collected, and the CTL. The IL2 concentration was evaluated by the L2 test.

FIG. 9. Characterization of MHC-1 cells and exosomes. A. Analysis of MHC-I cells by flow cytometry (continuous thick line: cells alone; continuous thin line: anti-mouse antibody-FITC alone; dark dotted line: specific antibody + anti-mouse-FITC). B. Analysis of MHC-I exosomes adhered to latex beads by flow cytometry (continuous thick line: latex beads-MHC-I exosome alone; continuous thin line: anti-mouse antibody-FITC alone; dark dotted line: reference latex beads-SVF + Specific antibody + anti-mouse-FITC; white dotted line: exosome-latex beads + specific antibody + anti-mouse-FITC). C. Western blot analysis of lysates of MHC-1 cells compared to exosomes (10 or 3 μg of protein per well; HC 10: 1/10 supernatant;
1B5: 1/10 supernatant; CD63: 5 μg / l, Lamp 1: 2 μg / mL; H68
. 4: 1/10 supernatant). The exosomes appeared to be rich in CD63, Lamp1, TfR. The absence of MHC class II in both lysates and exosomes confirms the cytofluorimetric analysis. The percentage of MHC class I appeared to be identical in cell lysates and exosomes.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] January 8, 2001 (2001.1.8)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/12 A61P 37/04 4C086 35/00 37/06 4H045 37/04 37/08 37/06 C07K 14 / 705 37/08 C12Q 1/04 C07K 14/705 G01N 33/53 Y C12N 5/10 33/577 B C12Q 1/04 C12N 15/00 ZNAA G01N 33/53 5/00 B 33/577 A61K 37/02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, U , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH , CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE , SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Center National de la Rucherche Shanti Fig CENTRE NATIONAL DE LA RECHER CHE SCIENT IFIQUE France, 75794 Paris-Sédéx 16, Lu Michel Ange 3 (72) Inventor Benaro シ ュ s, Philippe F-75014, France Paris, Avigne Rene Coti 32 Bis (72) Inventor Vincent-Schneider 94-800 Villejuif, Are Berlioz, France, Hélène 13 (72) Inventor Stumnet, Pamela, France, F-92160 Antoni, Rie Pasteur 22 (72) Inventor Amigorna, Sebastian, France, F- 75013 Boulevard-A-Blancie, Paris 124 (72) Inventor Bonero, Christian F-94130 Nogent-sur-Marne, Ampas Marchand 6 (72) Inventor Laposo, Grasa F-75004 Paris, France , Ryu Saint-Martin 8F term (reference) 4B024 AA01 B A31 BA44 CA01 GA11 GA18 4B063 QA01 QA18 QQ02 QQ79 QQ96 QR48 QR72 QR82 QS15 QS32. ZB082 ZB092 ZB132 ZB332 4C086 AA02 EA16 MA01 MA05 MA21 MA56 MA66 NA14 ZA59 ZB05 ZB08 ZB09 ZB13 ZB33 4H045 AA10 CA40 DA50 EA28 EA50 FA74

Claims (46)

[Claims] 1. A membrane vesicle comprising a recombinant human major histocompatibility complex molecule. 2. The vesicle according to claim 1, wherein the recombinant major histocompatibility complex molecule is a class II molecule. 3. The vesicle according to claim 2, wherein the recombinant major histocompatibility complex class II molecule is an α-chain. 4. The vesicle according to claim 2, wherein the recombinant major histocompatibility complex class II molecule comprises an α-chain and a β-chain. 5. The method according to claim 5, wherein the class II molecule of the recombinant major histocompatibility complex is serotype D.
Vesicle according to any one of claims 2 to 4, characterized in that it is selected from R1 to DR13, preferably DR1 to DR7. 6. The vesicle according to claim 1, wherein the recombinant major histocompatibility complex molecule is a class I molecule. 7. The vesicle according to claim 1, which comprises a complex of a predetermined peptide and a recombinant major histocompatibility complex molecule. 8. The vesicle according to claim 1, which further comprises one or more heterologous target molecules. 9. The vesicle according to claim 1, which further comprises a peptide or a recombinant protein that allows its purification. 10. The vesicle according to claim 1, comprising a tracer. 11. The method according to claim 11, wherein the substance is substantially free of endogenous MHC molecules.
Vesicle according to any one of claims 1 to 10. 12. A membrane vesicle characterized by being obtained from a mast cell or a cell derived from a mast cell, and also containing one or more heterologous molecules of interest. 13. The target molecule may be a protein, polypeptide, peptide, nucleic acid,
13. The vesicle according to claim 12, characterized in that it is a lipid or a substance having chemical, biological or synthetic properties. 14. The heterologous molecule is a major histocompatibility complex molecule, an antigen, a receptor ligand, a ligand receptor, a nucleic acid, a pharmaceutical product, a tracer and / or a purified peptide. Membrane vesicles. 15. The vesicle according to claim 14, characterized in that it expresses a ligand receptor and contains another heterologous molecule of interest. 16. A membrane vesicle comprising a recombinant fusion molecule of a target polypeptide and an addressing signal. 17. An exosome-producing cell, comprising one or more recombinant nucleic acids encoding a major histocompatibility complex molecule. 18. The cell according to claim 17, which is a mast cell. 19. The cell according to claim 18, characterized in that it is a basophil leukemia mast cell line, in particular an RBL line, preferably RBL-2H3. 20. The method according to claim 10, comprising a major histocompatibility complex class II molecule α chain and / or β chain and / or a recombinant histocompatibility complex class I molecule encoding nucleic acid. Item 20. The cell according to any one of items 17 to 19. 21. A method for producing an exosome containing a predetermined recombinant molecule, comprising the steps of: a) a mast cell containing a recombinant nucleic acid encoding the predetermined recombinant molecule or a cell derived from a mast cell. Culturing the cells, c) recovering the exosomes produced by the cells and containing the predetermined recombinant molecule. 22. The method according to claim 21, comprising an intermediate step b) of stimulating cells and inducing and / or increasing secretion of exosomes. 23. The method according to claim 21, wherein the predetermined recombinant molecule is exposed to the outside of the exosome, or is completely or partially contained in the cytosol fraction of the exosome. The described method. 24. The recombinant molecule according to claim 21, wherein the recombinant molecule is a major histocompatibility complex molecule, an antigenic molecule, a receptor ligand, a ligand receptor, a purified peptide or any other polypeptide of interest. 24. The method according to any one of claims 23 to 23. 25. The method according to any one of claims 21 to 24, wherein the nucleic acid also comprises a region encoding an addressing signal for a mast cell membrane compartment. 26. A method for preparing an exosome containing a peptide-MHC complex having a predetermined composition, comprising:-one or more recombinant nucleic acids encoding a predetermined recombinant MHC molecule. Culturing of exosome-producing cells,-stimulating the cells and inducing the release of exosomes,-collecting the exosomes produced by the cells and expressing the desired recombinant MHC molecule on its surface, and-exosomes Contacting with one or more peptides. 27. A method for preparing an exosome containing a peptide-MHC complex having a predetermined composition, comprising:-one or more recombinant nucleic acids encoding a predetermined recombinant MHC molecule; Culturing exosome-producing cells containing a nucleic acid containing a region encoding the recombinant peptide of-stimulating cells to induce release of exosomes;-binding to the recombinant peptide produced by the cells. Predetermined recombinant MHC
A method comprising recovering an exosome expressing a molecule on its surface. 28. The method of claim 27, wherein the nucleic acid encoding the recombinant peptide encodes a derivative of the li invariant chain in which the CLIP region has been deleted and replaced with the peptide. 29. The method according to any one of claims 26 to 28, wherein the producer cell is a mast cell or a cell derived from a mast cell. 30. The method according to any one of claims 26 to 29, wherein the producer cells are substantially free of endogenous MHC molecules. 31. A method for altering the composition of an exosome, comprising: inserting a nucleic acid encoding a molecule of interest bound to an addressing signal in a membrane compartment into an exosome-producing cell; and A method comprising the production of exosomes. 32. A composition comprising one or more membrane vesicles according to any one of claims 1 to 16. 33. Use of the vesicle according to any one of claims 1 to 16 for producing a polyclonal antibody and / or a monoclonal antibody. 34. A method for producing an antibody, comprising immunizing an animal with the vesicle according to claim 7, and collecting the antibody and / or cells producing the antibody or participating in an immune response. 35. The method according to claim 34, particularly for the production of monoclonal antibodies specific for MHC-peptide binding. 36. Use of an antibody or a fragment of said antibody obtained according to claim 34 or 35 for detecting the presence of a corresponding specific antigen in a biological sample. 37. A method according to claim 34 or 35 for the preparation of a therapeutic composition aimed at inhibiting the interaction between the receptor of T lymphocytes and its specific MHC-peptide complex. Use of the obtained antibody, a fragment of the antibody, or the vesicle according to claim 1. 38. Use of a membrane vesicle according to any one of claims 1 to 16 for the detection of a partner specific for a protein molecule in a biological sample. 39. Use according to claim 38, of an exosome carrying an MHC-peptide complex for the detection of T lymphocytes specific for the MHC-peptide complex in a biological sample. 40. A peptide-MH specific for a TcR receptor in a biological sample
39. An exosome carrying a TcR receptor for detection of the C complex.
Use of the description. 41. Use according to claim 38, of an exosome retaining a ligand receptor for detecting the presence of a ligand in a biological sample. 42. A method for detecting the presence of a T lymphocyte specific for an antigen-MHC complex in a biological sample, wherein the sample contains the antigen-MHC complex. 7. A method comprising contacting with an exosome labeled according to 7, and demonstrating labeling of T lymphocytes in said sample. 43. Use of a vesicle according to claim 7 for clonal amplification and / or ex vivo stimulation of cytotoxic and / or helper T lymphocytes. 44. Use of a vesicle according to any one of claims 11 to 16 for the preparation of a composition aimed at delivering said molecule to cells. 45. A composition comprising one or more exosomes immobilized on a support. 46. Use of a membrane vesicle according to any one of claims 1 to 16 for the purification of cells, in particular in the form immobilized on a support.