FR2827872A1 - In vitro preparation of membrane vesicles, useful for treatment of cancer and to induce immunological tolerance, from sample of mammalian body fluid - Google Patents

Abstract

Process (M1) for preparing, in vitro, membrane vesicles (MV), isolated from their natural environment, from a mammalian, particularly human, sample, previously taken in vivo, where the sample is a biological fluid. Independent claims are also included for the following; (1) MV produced using (M1); in vitro preparation (M2) of antigen-presenting cells (APC) loaded with MV; (2) APC produced by method (M2) and isolated texosomes (MV from tumor cells) secreted by them; in vitro preparation (M3) of T lymphocytes, activated natural killer (NK) or NKT cells or anergic T lymphocytes; (3) T lymphocytes and NK(T) cells produced by method (M3); in vitro diagnosis of cancer and/or its progression; in vitro preparation of a library of murine antibodies (Ab) directed against human antigens (Ag) carried by MV; in vitro preparation of a library of texosomes of murine or human origin; and (4) method for preparing in vitro-modified texosomes.

Description

as an emulsion.

  The present invention relates to a method for preparing a suspension of membrane vesicles isolated from a sample of a biological fluid sample, such as an ascites liquid, in a mammal, in particular in humans and use of these isolated membrane vesicles to charge in vitro or in vivo cells presenting antigens, in particular dendritic cells, antigens carried by said membrane vesicles and / or for the stimulation in vitro or in viro of T lymphocytes specific NK or NKT cells of antigens carried by Uites 0 membrane vesicles. The invention also includes the use of such isolated membrane vesicles, antigen presenting cells loaded by these vesicles or T lymphocytes NK or NKT cells stimulated by said antigen presenting cells charged according to the invention for prevention or treatment cancers as well as to induce tolerance in a recipient of an allogeneic graft. The present invention further comprises a method of diagnosing

  tumor based on the presence and characterization of such membrane vesicles.

  Cross presentation of antigens allows the presentation of an epitope from a peripheral cell to a T lymphocyte (also called T cell) via antigen presenting cells (APC). CPA, in particular dendritic cells (CD), placed in the presence of antigen, can indeed in vitro or in viro acquire and present peptides derived from these antigens Immunotherapy based on the use of CPA, in particular CD, was able to show for example its effectiveness in

  the treatment of certain tumors by studies carried out in mice.

  Among the methods for sensitizing dendritic cells to antigens of interest which are currently used, mention may be made of those which use antigenic peptides corresponding to epitopes presented in association with the molecules of the major histocompatibility complex (MHC) of class I and identified, for example, in tumor cells using tumor-specific CTL clones. Mention may be made, for example, of the document Mayordomo et al. (Nature Medecine, Vol. 1, 12, 1297-1302, 1995), which describes the protection conferred on mice against lethal tumors after injection of CD derived from bone marrow previously "pulsed" with tumor peptides characterized as being recognized by cytotoxic T lymphocytes (CTL) restricted to MHC class I. Mention may also be made of the document Zitvogel et al. (Nature Medecine, Vol. 4, 5, 594-600, 1998) which describes the regression of tumors established in mice by injection of exosomes isolated from syngeneic CDs previously loaded with isolated poptides

tumor.

  However, these methods are probably not optimal because: either, they do not take into account the epitopes recognized in the context of class II molecules which are critical for the proliferation of T 0 helper cells necessary for obtaining optimal cytotoxic responses; - Either, epitopes presented by tumor cells and those presented by antigen presenting cells (such as for example dendritic cells), which are probably not the same; and - finally, in the majority of tumors, specific antigens have not been identified. Indeed, tumor specific antigens are only known in cases of tumors induced by viruses (cervical carcinoma), in cases of melanoma (self antigens, mutated antigens, differentiation antigens) or in a small percentage breast tumors (oncogenes, or mutated tumor suppressor gene products). Thus, isolated poptids derived from tumor antigens recognized by the CTLs are only available for a limited number of tumors and often for a small percentage of patients with class I molecules.

of appropriate haplotype.

  Among the methods for sensitizing dendritic cells to antigens of interest which are currently used, mention may also be made of those which use exosomes secreted by tumor cells derived from culture of cell lines. The exosomes secreted by tumor cells (hereinafter referred to as "Tex" or "Texosomes") are vesicles of 60 to 90 nm containing in particular tumor antigens, molecules of class I and / or II of MHC and proteins of thermal shock ("Heat Shock Protein" or HSP). Tex derived from tumor cell lines are thus sources of tumor antigens which have been used for the

  loading of antigen presenting cells in vitro or in viro.

  Among the documents of the prior art describing such sources of antigens and methods of loading antigen-presenting cells from these sources derived from tumor cell lines, there may be mentioned in particular: - the international patent application published under the WO 99/03499 which describes a process for the preparation of texosomes isolated from the culture of tumor cell lines and the use of these texosomes to load dendritic cells and to obtain T lymphocytes directed specifically against antigens contained in the texosomes; and - the document Wolfers et al. (Nature Medecine, vol. 7, 3, 297-303, 2001) which describes the regression of tumors established in mice by injection of syngeneic dendritic cells previously loaded with texosomes isolated from

  culture of tumor cell lines.

  However, these antigenic sources when they come from tumor cell lines, require the cultivation in serum of tumor cells generally collected from tumor ascites fluids, a culture which is difficult to carry out and which generally results in a heterogeneous cell population whose repertoire antigen is no longer representative of the original cell population repertoire

  tumors from the ascites fluid collected.

  Thus, one of the drawbacks of the use of cell lines lies in the use of serum, the random nature of cell expansion and above all the fact that the lines, by the occurrence of spontaneous mutations, do not necessarily represent the

  antigenic repertoire of the patient's tumor cells.

  On the other hand, the use of cell lines to produce antigenic material (apoptotic bodies or exosomes) does not exploit the potential contribution of a ligand which can promote the uptake and cross presentation of the antigen by the presenting cell. antigen, including the dendritic cell. Indeed, it has already been reported that proteins such as immunoglobulins allow the acquisition of

  antigenic material by the dendritic cell.

  Finally, it would be advantageous to be able to have antigenic material available.

  greater quantity than that obtained from cell line culture.

  There is therefore a real need to be able to have methods of raising awareness of CPA with new quantitatively larger sources of tumor antigens having the essential advantage of being both representative of the antigenic repertoire of the patient's tumor and of exploiting in addition the potential presence of a natural ligand allowing the capture of the antigen by the CPA, in particular the dendritic cells, this in order to increase the effectiveness of these approaches and to widen their applications, as well as to develop new means of vectorization of antigens or

other molecules.

  Applied to antitumor immunotherapy, the ideal sensitization method for APCs, in particular dendritic cells, would be that this method can be applied to any tumor with a minimal risk of immunoselection, since it should not be restricted to a small number of tumor antigens identified and in which the tumor antigens need not be known. Likewise, such a method should be able to use intact protein antigens rather than peptides, in order to allow the dendritic cell to prepare them and to present the appropriate combination of peptides in association with MHC class I and class II molecules. , and this for any individual.

  This is precisely the object of the present invention.

  The inventors have surprisingly demonstrated in the examples below

  after the purification of membrane vesicles carried out from samples taken from a mammal, in particular from humans, made it possible to obtain not only a larger quantity of these vesicles (approximately 20 times more at equal volume) but also a source of with the advantages described above compared to membrane vesicles purified from line cultures

human cells.

  In addition, the process for preparing membrane vesicles carried out using 2 samples taken from a mammal according to the invention described below is a rapid process which does not require prior cell cultures, often long and tedious, not always possible, and required for the preparation of vesicles

  membranes purified from cultures of human cell lines.

  The inventors have indeed demonstrated that the membrane vesicles isolated directly from the biological fluid of a mammal, in particular human, containing cells capable of secreting such membrane vesicles, such as tumor cells, are more numerous and have properties particularly advantageous iromunogens compared to texosomes from cell lines such as

  as described for example in document WO 99/03499.

  These membrane vesicles correspond to an internal vesicle contained in an endosome of a living dividing cell and secreted by said cell following the fusion of the external membrane of said endosome with the cytoplasmic membrane of the cell from which it originates. Because of this formation mechanism, their original cell and their original functional characteristics and properties, these vesicles are designated in the following by the general term "membrane vesicle" or lo "exosome" and in particular by the term "texosome" when the cell whose vesicle

  membrane is a tumor cell.

  The present invention therefore relates to a process for the preparation of membrane vesicles, or of a suspension of membrane vesicles, said membrane vesicles being isolated from their natural environment, characterized in that it comprises the following steps: a) direct sampling viro of a sample in a mammal, in particular in humans, said sample being a biological fluid, or a sample capable of releasing a biological fluid containing in suspension membrane vesicles and / or cells capable of producing such membrane vesicles by exocytosis; b) in vitro isolation of said membrane vesicles from said sample taken. The secretion of membrane vesicles by certain types of mammalian cells is a phenomenon described in the prior art (such as by type 2s reticulocyte cells, B lymphocytes CPA, such as macrophages or dendritic cells, or even tumor cells ). These membrane vesicles are generally designated by the generic term "exosome" which reflects their mechanism of production by exocytosis of internal vesicles. However, the physiological role of these vesicles has not really been established. In addition, the structural features, properties and

  Functions of these vesicles are variable depending on the cell type from which they originate.

  By the expression "i sol ée of its natural environment", one intends to indicate i ci that the membrane vesicle is at least separated from the cell from which it comes by a physical treatment of the sample of the blological fluid taken. Generally, said membrane vesicle prepared by the method according to the invention is at least partially isolated and / or purified so as to obtain a composition or suspension enriched in membrane vesicles, devoid of intact cells and cellular debris. According to an advantageous embodiment, the removal of the sample of biological fluid, or capable of releasing a biological fluid, containing in suspension membrane vesicles and / or cells capable of producing such membrane vesicles by exocytosis is an in viro sample fluid sample chosen o from the following biological fluids: an abdominal fluid, a peritoneal fluid, a pleural fluid, an encephalo-cerebrospinal fluid (CSF), whole blood, plasma, serum or urine; or an in viro sample of tissue or organ sample capable of releasing after ex vivo treatment of said sample taken (in the case of

  particular solid tumor sample).

  According to an advantageous embodiment, the sample of the biological fluid sample containing in suspension membrane vesicles and / or cells capable of producing such membrane vesicles by exocytosis is a sample of fluid sample, or sample capable of releasing a fluid, chosen from exudative or transsudative fluids, in particular exudative or transsudative fluid taken from a cavity and resulting from an inflammatory reaction, a phenomenon of

  cancerization or transplant.

  According to a particular embodiment, said transudative fluid taken from a cavity, in particular a peritoneal cavity, may result from an inflammation caused by a pretreatment of the cavity comprising bringing this cavity into contact with a 2s abrasive chemical agent (irritant) , or by pre-treatment by chemotherapy of said cavity, or by transfer of suicide genes, such as the gene coding for

  TK (Thymidine Kinase) using for example the Herpes Simplex virus (HSV).

  By “exudative” or “exudate” fluid, is meant here designating a fluid escaped from a tissue, such as a liquid of ascites, or from blood vessels containing cells and / or cellular debris, and which can be found in a tissue or on its surface, and which usually results from inflammation, a phenomenon of cancerization or a phenomenon of rejection following a transplant (fluid whose protein concentration is greater than 40 g / 1, and whose pressure equals osmotic or hyperosmotic pressure). By “transsudative” or “transsudate” fluid, is meant here a fluid of plasma origin having passed through a membrane or a fluid extruded from a tissue in which is accumulated such a fluid (for example during a edema) and which can also result from a vascular hyperpressure (fluid whose protein concentration is

  less than 30 g / 1, and whose pressure is hypo-osmotic protein content <30 g / 1).

  When the biological fluid is a fluid, or a sample capable of releasing a fluid, containing in suspension membrane vesicles secreted by tumor cells and! Or tumor cells capable of producing membrane vesicles, the sample may be taken : - a sample of tumor ascites fluid or, in general, of an effusion of the tumor; or - an efferent blood sample from the vein of the target tumor organ; or - the drainage product of a tumor-bearing organ excised surgically and treated ex viro by an isolated circuit perfused for the drainage of the tumor it carries, or - the supernatant of a tumor explant taken in vivo and mechanically dissociated

in vitro.

  When it comes to an efferent blood sample from the target tumor organ, this sample will for example be a sample of approximately 20 to 50 ml of blood from the main efferent vein of the tumor organ, taken before the ablation procedure

  surgical treatment of the tumor if this should be done.

  The drainage product of an organ carrying tumors surgically excised 2s and treated ex viro by isolated-perfused circuit can be obtained in the following way in the case of organ having an afferent artery and an efferent vein: - we characterize the artery by a plastic tube connected to a proclive pocket containing physiological saline with possibly other agents; then - the organ is drained and the biological fluid comes out through another tubing catheterizing the vein, in declivity (for example in the case of kidney cancer, or of a

cerebral glioblastoma).

  The supernatant of a tumor explant dissociated in vitro can be obtained mechanical dissociation of the tumor generally leading to a single-cell suspension containing tumor cells, cells of the tumor stroma and cells of the

immune system.

  s In a particular embodiment, the biological fluid directly taken in vivo, or obtained from the sample taken in vivo can be frozen and then thawed before step b) of isolation of the membrane vesicles of the process according to the invention . Among the biological fluids, or samples capable of releasing a biological o fluid, containing in suspension membrane vesicles secreted by tumor cells and / or tumor cells capable of producing membrane vesicles, there may be mentioned in particular: - samples of solid tumors or effusion of these tumors such as those observed in cancer of the kidney, breast, colon, lung, stomach, liver, melanomas, sarcomas, etc .; - biological fluid samples from hematopoietic tumors such as blood lemmas, Hodgkin's and non-Hodgkin's malignant lymphomas; - samples of tumor ascites fluid, pleural fluid, CSF or urine. The term "tumor cells" generally encompasses any cell originating from a tumor, for example a solid or liquid tumor. It's about

  preferably a solid, ascites or hematopoietic tumor.

  According to a particularly advantageous embodiment, the removal of

  the biological fluid sample is a sample of ascites fluid sample.

  2s By fluid or liquid "ascites" is meant here a fluid which can be found for example in the abdominal, peritoneal or pleural cavity of a mammal and which usually results from an effusion resulting from an inflammation , from a tumor

or venous hypertension.

  According to an even more particularly advantageous embodiment, the taking of the biological fluid sample is an in viro taking

  of tumor ascites fluid sample from cancer patient.

  In a particular mode of implementation of the process of the invention, the biological fluid sample taken in vivo, or obtained from the in viro sample, can be treated if necessary before step b) d in vitro isolation of said membrane vesicles by one or more agents stimulating the production of membrane vesicles, in particular texosomes if they are membrane vesicles derived from tumor cells. This treatment can include, for example, when it comes to tumor fluids, the addition of steroid agents (for example dexamethasone which aims to increase cellular stress and the exocystosis of texosomes outside the tumor cell) , pharmacological agents (for example cytotoxic agents such as taxanes, cisplatin, etc.), agents capable of increasing the amount of endosomes

  multivesicular and / or sample irradiation.

  With regard to irradiation, it must be sufficient to cause the cytostatic action of the tumor cells contained in the sample taken. The irradiation of the tumor cells must be done when the tumor cells are alive, that is to say: - either on the biological fluid containing the tumor cells in suspension, - or on the excised organ carrying the tumor before the perfusion, - either on the cell suspension obtained after mechanical dissociation of the tumor; but, in all cases, before tumor cell suffering due to

  hypoxia and / or vascular nocrosis and / or dehydration.

  Regarding treatment with steroids, it can cause

  cellular activation leading to the exocytosis of the texosomes.

  Regarding treatment with pharmacological agents, it allows: 2s - to modify the cytoskeleton and rearrange the intracellular compartments to disrupt the phenomena of internalization and exocytosis, or

- depolymerize the microtubules.

  An advantageous process for the preparation of texosomes according to the invention from the biological fluid sampled in vivo containing a suspension of tumor cells will comprise, after step a) of sampling the sample in viro, the following step: - irradiation, an intensity sufficient to cause the cytostatic action of the tumor cells and not exceeding 15,000 rads, advantageously at approximately 10,000 rads, from the suspension of tumor cells; or - a treatment of the suspension of tumor cells, using steroids, for example dexamethasone, or cytotoxic agents, for example 5-fluorouracil (5 Fu) or cisplatin, docetaxel, anthracycline, spindle poison , antipyrimide, or interleukin for example IL-10, IL-2, IL-15, GM-CSF; or - treatment with an agent capable of increasing the quantity of multivesicular endosomes, for example nocodazole (Gruenberg et al., Journal of Cell Biology,

  o 108: 1301-1316, 1989) and therefore increase the production of texosomes.

  An equally advantageous process for the preparation of texosomes according to the invention: either from a sample of physiological serum draining an organ excised surgically and treated ex viro by an isolated-perfused circuit for the drainage of the tumor which it carries, or - either from the supernatant of a tumor explant dissociated in vitro will comprise, after step a) of in viro sampling of the sample, the following step: - treatment using steroids, for example dexamethasone, or cytotoxic agents, for example 5-fluorouracil (5-Fu) or cis-platinum, taxanes, or

  Interlenkin for example IL-10, IL-2, GM-CSF.

  The invention also relates to the use of a texosome as defined above, or of an antigen presenting cell such as defined above, or of a dexosome as defined above, for the preparation of a medicine for

  treatment of tumors, especially solid, ascites and hematopoetic.

  2s According to a preferred embodiment, step b) of isolation of the membrane vesicles is carried out by centrifugation, electrophoresis, chromatography and / or nanofiltration, or else by capture using beads coupled to directed antibodies

  specifically against proteins expressed on the outer surface of these vesicles.

  The isolation of the membrane vesicles in step b) of the process according to the present invention can be carried out according to different techniques for the separation of biological materials. These techniques are generally based on the differences in size, mass, charge, density or on the immunological properties (i.e. specific recognition of antibodies) of the membrane vesicles compared to the other elements.

contained in the biological fluid.

  Among these techniques, the technique of isolation by centrifugation of the biological fluid taken in vivo, or obtained from the sample taken in vivo, is preferred. It can be, for example, a differential centrifugation and / or a density gradient centrifugation, followed by recovery of the fraction (s) containing said membrane vesicles. This type of methodology is based on the separation, by successive centrifugations, of membrane vesicles on the one hand and cells, cellular debris, internal vesicles, etc. on the other. In this particular mode of implementation, the fraction comprising the membrane vesicles is generally that obtained after ultracentrifugation at 100,000 g. This method is illustrated in particular

in the examples below.

  The step of isolating the membrane vesicles can also be carried out by chrom atography, electrophoresis and / or nanofiltration, or even by capture on b i ll es coated with specific antibodies. It can advantageously be an electrophoresis in the fluid phase and / or in density gradient, which can, if necessary, be carried out on the supernatant obtained.

  after a first differential ultracentrifugation of the biological fluid.

  Fluid phase electrophoresis, which allows the separation of biological materials according to their charge, is quite advantageous. For this technique, one can advantageously refer to example 11, page 58, of document WO 99/03499 showing that this technique can be used for the isolation of membrane vesicles with good yields. This technique is also

  particularly advantageous on the industrial level.

  It can also be a purification by chromatography. Mention may in particular be made of ion exchange chromatography, gel permeation (or exclusion) chromatography or hydrophobic chromatography. Given the lipid nature of the membrane vesicles, ion exchange chromatography is particularly interesting. Nanofiltration can be carried out according to known techniques, from the biologic fluid, preferably from a supernatant obtained after a first

  differential ultracentrifugation.

  The use of these chromatography and / or electrophoresis and / or nanofiltration techniques allows, in particular with respect to current centrifugation technologies, production of improved quality, in quantities suitable for a

  industrial use (especially pharmacological).

  In this regard, the invention also relates to a process for the preparation of membrane vesicles comprising at least in step b) of isolation of the process according to the invention a step of separation by electrophoresis, chromatography or nanofiltration. Such a process will therefore be more particularly suitable for the preparation of membrane vesicles meeting the qualitative standards required for a

o therapeutic administration.

  According to an embodiment which is also advantageous, the membrane vesicles obtained in step b) of the method according to the invention may be stored frozen, preferably at a temperature less than or equal to −80 ° C., then thawed before use. In another aspect, the present invention relates to membrane vesicles isolated from their natural environment capable of being obtained by the process according to the invention as described above, in particular in the form

frozen.

  In a preDerated manner, said membrane vesicles according to the present invention are characterized in that they mainly contain membrane vesicles with a diameter between 30 and 150 nm, preferably between 50 and 100 nm and between 60 and nm. Also preferably, said membrane vesicles according to the present invention are characterized in that the flotation of these vesicles is obtained for a density of between 1.14 and 1.18 g / ml in sucrose gradient, in particular

  for membrane vesicles from tumor cells.

  More preferably, said membrane vesicles according to the present invention are characterized in that they carry molecules involved in the presentation of antigens, in particular MHC class I and / or II molecules, and / or chaperone molecules of tvpe HSP and / or antigens, especially tumor antigens, and / or adhesion molecules capable of targeting CPA and / or molecules of the type

  tetraspane associated with class I or II with MHC molecules of class I or II.

  Among the HSP type molecules, there may be mentioned in particular HSP 70, HSP 80

or the HSP 90.

  Among the adhesion or targeting molecules, mention may be made in particular of isoforms of lactadherin and human immunoglobulins (in particular of the IgG type). Among the tetraspane-type molecules associated with the MHC, mention may be made of

  CD63 molecule, molecule involved in lymphocyte co-stimulation.

  More preferably, said membrane vesicles according to the present

  The invention are characterized in that they do not carry the gp96 glycoprotein.

  In general, the subject of the invention relates to a membrane vesicle isolated from an in viro sample from a mammal of a sample of biological fluid, in particular of ascites, of tumor prerence, or of a sample capable of releasing such a fluid, having the following characteristics: - it is rid of its natural environment, - it comprises on the surface a lipid bilayer which surrounds a cytosolic fraction, - it presents on its surface MHC class I molecules and / or class II, optionally charged with antigenic peptides, in particular tumor, and / or adhesion molecules and / or lymphocytic co-stimulation molecules, and / or, - it contains in its cytosolic fraction antigenic molecules, in particular, tumor , and / or immunomodulators and / or chemo-attractors and / or

  hormones and / or nucleic acids.

  Preferably, when it is a membrane vesicle originating from tumor cells, the membrane vesicle (texosome) of the invention consists of a texosome as isolated by a process of the invention defined above and having on its surface MHC class I and / or II molecules, possibly loaded with antigenic peptides and containing in its cytosolic fraction antigenic tumor molecules. Also preferably, when it is a membrane vesicle originating from tumor cells, the membrane vesicles (texosomes) of the invention have MHC class I and / or II molecules loaded with antigenic peptides and / or express adhesion molecules and / or express lymphocytic co-stimulation molecules, but are devoid, in their cytosolic fraction, of molecules

  tumor antigen and immunomodulators and nucleic acids.

  In a manner also claimed, the texosomes of the invention are such that the MHC molecules of class I and / or II are "empty", that is to say not charged with antigenic peptides and the texosomes comprise, in their fraction cytosolic,

  tumor antigen molecules, immunomodulators and / or nucleic acids.

  Texosomes having empty MHC molecules can be obtained by elution with acid at pH 2 to 4 from the texosomes in order to elute the poptides associated with MHC molecules. In a manner also claimed, the texosomes of the invention are such that the MHC molecules of class I and / or II are loaded with antigenic peptides and / or express mo lules of adhesi on and / or target, and / or in lymphocytic co stimulation stimulation molecules and the texosomes contain in their cytosolic fraction tumor antigenic molecules, immunomodulators and / or nucleic acids. Among the adhesion and / or cell targeting molecules, there may be mentioned in particular the CD11b molecules, the tetaspanes, the various isoforms of lactadherin or also immunoglobulins, in particular of the IgG type, which associate with the Fc gamma receptors present on target cells, or Fc receptors

  gamma (Cassard et al., Immunol. Lett., 75 (1), 1-8, 2000).

  Of course, these tumor antigen molecules capable of being contained in the cytosolic fraction or antigenic peptides capable of loading the MHC molecules may be specific for the cancerous nature and the type of tissue of the cell from which said membrane vesicle is derived. The tumor antigen 2s molecules presented on their surface by the texosomes or contained in their cytosol will generally come from proteins expressed selectively and / or

  abundant by tumor cells.

  Mention may be made, by way of example as tumor antigen molecules which may be contained in the cytosolic fraction or as antigenic peptides capable of loading MHC class I and / or II molecules, those originating from melanomas such as: MART- 1, tyrosinase, MAGE-1-12, PS3 (expressed in different tumors) or Her2 / Neu, PSA, CEA or even PSMA. Other tumor antigens are cited for example in the article by Rosenberg et al. (Immunology Today,

  18, 175, 1997) incorporated herein by reference.

  The term “lymphocytic co-stimulation molecules” denotes, for example, molecules which give T lymphocytes signals complementary to those given during the interaction of MHC class I and II peptide molecules with the T cell receptor. , for example: CD63, CD80, CD86, ICAM, LFA, CD40, certain members of the TNF R family and adhesion or chemo attraction molecules (allowing contact between the professional cell 0 presenting antigen and effector lymphocytes, or intracellular transport / specific localization ("trafficking / homing") of other cells to the site

vaccine or inflammatory.

  Immunomodulators that may be present in the cytosol of texosomes

  are for example: TNF-a, IL-1, IL-15 or a C-CR (chemokine).

  The nucleic acids that may be present in the cytosol of the texosomes come from the tumor cell itself. These nucleic acids are found in the

  cytosol of texosomes as a direct result of their formation mechanism.

  In a particular embodiment, nucleic acids or heterologous proteins may be introduced into said texosomes, in particular by electroporation, by fusion with a synthetic liposome, by recombinant virus or by

chemical method.

  More specific characteristics of the texosomes of the invention are the following: - these are small membrane vesicles with a diameter between 30 and 150 2s nm, with a pre-eminence between 50 and 100 nm and between 60 and 90 nm secreted by the cells - they contain tumor antigens, such as MART-1 in the case of melanoma cells, - they are devoid of dead cells, and / or cellular debris, - they are devoid of contaminants such as membrane contaminants, endoplasmic reticulum , Golgi apparatus, mitochondria or constituents of nuclei, - they carry class I and / or II functional molecules loaded with tumor antigen peptides on their membranes, - they can stimulate the proliferation of specific T lymphocytes in vitro , - they can sensitize in viro and in vitro autologous dendritic cells capable then of activating autologous T cells specific for the tumor, - they p adhere adhesion molecules of the lactadherin type, - they carry molecules of the tetaspane CD63 type associated with the MHC class II, - they contain the protein HSP70 and / or HSP80, and

  - they lack the gp96 protein.

  0 The analysis techniques making it possible to verify that said membrane vesicles according to the invention express or do not express the expected molecular markers, such as the presence of HSP, CD63, MHC class I and / or II molecules, tumor-specific antigens, or the non-presence of gp96, are well known to those skilled in the art and will not be developed here in an exhaustive manner. We can for example refer to the technique using electron microscopy and immunoblotting (Western Blotting technique) using antibodies specific for these different markers as described in the examples below or

  in examples 2 and 3 and in FIG. 2 of document WO 99/03499.

  A test which makes it possible to verify that the texosomes according to the invention carry at their 20 m membrane of functional molecules I and / or II loaded with antigenic tumor peptides consists of an antigenic presentation to lymphocytes T specific to the antigens of the tumor concerned (such as, for example, pro-proliferation tests of specific T clones of anti genes and MHC cl as se I restricted as well as

  described in the examples below or in example 4 of document WO 99/03499).

  2s It is also possible to use a cytokine secretion test (IFN, GM, CSF, TNFa) by the abovementioned T clones described in the examples below or in FIG.

WO 99/03499).

  A test making it possible to verify that there is sensitization in viro or in vitro of dendritic cells capable of activating T cells specific for the tumor is given in the examples below (test of proliferation and / or secretion of cytokines by the antigen-specific T clones, by the cross priming method: texosomes of a MART-1 +, HLA-A2-loaded tumor on a dendritic cell MART-1-, HLA-A2 +), as described in the examples below or at the

FIG. 5 of document WO 99/03499).

  A test making it possible to verify that the texosomes according to the invention exhibit the capacity, when they are inoculated, in particular intradermally, of causing established solid tumors to regress is given in Example 6 and in FIGS. 6A, 6B, and 7 of

document WO 99/03499.

  By way of example, an injection of 10 to 40 g of isolated texosomes of ascitic tumor fluid is performed intradermally on the side homolateral to the tumor established for 3 to 10 days; the animal carrying the tumor is observed and the progressive disappearance of the

  o tumor established in 7 to 10 days (in rodents of the mouse type).

  In another aspect, the present invention comprises the use of membrane vesicles according to the invention for the loading and / or activation (stimulation) in vitro or in viro of antigen presenting cells, in particular of dendritic cells. By "antigen presenting cells" or CPA, we will mean very particularly macrophages, monocytes or dendritic cells. It will be

  advantageously in the present invention of dendritic cells.

  Preferably, said membrane vesicles and said cells

  Antigen presenters are autologous or syngeneic.

  By "autologous" is meant to mean that the two compositions are from A ...

same mdvdu.

  Also preferably, said membrane vesicles and said

  Antigen presenting cells are allogenic.

  By "allogeneic" is meant to denote that the two compositions are derived 2s from individuals having a genetic variation (in general, within the same species). The invention also relates to the use of membrane vesicles according to the invention for stimulation in vitro or in viro, and, where appropriate, amplification (here equivalent to proliferation or expansion) or anergization in vitro or in viro T cells or for in vitro or in vivo stimulation, and, where appropriate, amplification of NK or NKT cells specific for antigens contained in said cells

membrane vesicles.

  Preferably, said membrane vesicles and said T lymphocytes

  NK or NKT cells are autologous or syngeneic.

  Also preferably, said membrane vesicles and said

  T lymphocytes NK or NKT cells are allogenic.

  In another aspect, the invention relates to the use of a membrane vesicle according to the invention, for the transfer of biological material into a cell

in vitro or in viro.

  In yet another aspect, the present invention relates to a process for the in vitro preparation of antigen presenting cells, in particular of o dendritic cells, loaded with membrane vesicles, characterized in that it comprises the following steps: a) incubation in vitro of antigen presenting cells in the presence of membrane vesicles according to the invention (step of sensitization of CPA); and b) recovering said cells presenting antigens loaded with membrane vesicles thus obtained. Step a) of the process for the in vitro preparation of antigen-presenting cells, loaded with membrane vesicles, generally requires a step

  prerequisite for the preparation of CPA, in particular of dendritic cells.

  This preliminary step comprises, in the case of dendritic cells, the provision of a culture (s) of dendritic cells. They can be cell cultures enriched in dendritic cells, or even cell cultures essentially comprising dendritic cells. Advantageously, these are human dendritic cells. By dendritic cells, we will also mean to include here the

  dendritic cells of the plasmacytode type (having the CDl lc- / CD4 + phenotype).

  The preparation of dendritic cells has been well documented in the literature.

  Thus, it is known that these cells can be obtained from stem cells of the immune system or from monocyte precursors, or even isolated

  directly in differentiated form (Review by Hart, Blood, 90, 3245, 1997).

  Obtaining dendritic cells from stem cells is illustrated for example by Inaba et al. (J. Exp. Med., 176, 1693, 1992), Caux et al. (Nature 360, 258,

  1992) or Bernhard et al. (Cancer Res., 55, 1099, 1995).

  Obtaining dendritic cells from monocyte precursors is

  illustrated for example by Romani et al. (J. Exp. Med. 180, 83, 1994), Sallusto et al. (J.

  Exp. Med. 179, 1109, 1994), Inaba et al. (J. Exp. Med. 175, 1157, 1992) or Jansen et al. (J. Exp. Med. 170, 577, 1989). These methodologies are essentially based on the removal of mononuclear cells from the blood (PBL) and the cultivation in the presence of different combinations of cytokines. One particular method consists in treating precursors of blood monocytes in the presence of combinations of cytokines such as IL-4 with GM-CSF, or IL-13 with GM-CSF for example. This

  technique is also illustrated by Mayordomo et al., 1995.

  o Another approach for obtaining dendritic cells consists in isolating, from biological samples, already differentiated plasmacytode dendritic cells called ppDC (for "plasmacytoid precursor dendritic cells"). This approach has been described for example by Hsu et al. (Nature Medecine 2, 52, 1996). The methodology described by this team essentially consists of collecting peripheral blood samples and processing them with different gradients and

  centrifugations so as to extract the dendritic cells therefrom.

  The preferred methodology in the context of the present invention is based on the production of dendritic cells from monocyte precursors or bone marrow. These methodologies are illustrated in the examples below or in document WO 99/03499. More particularly, it is preferred to use, within the framework of the present invention, dendritic cells obtained by treatment of monocyte precursors (contained in peripheral blood or marrow) in the presence of a

  combination GM-CSF + IL-4 or GM-CSF + IL-13.

  Furthermore, for the implementation of the method of the present invention, it is very particularly advantageous to use a population of dendritic cells comprising immature dendritic cells. Advantageously, a population of dendritic cells is used which is composed mainly (ie, at least 60%, preferably 70%) of immature dendritic cells. The immature state of dendritic cells corresponds to an early stage of their development, at which they exhibit strong endocytic activity and express low levels of molecules

  of class I and II of the MHC and of lymphocytic co-stimulation molecules on their surface.

  Thus, the prior step of the process of the invention can therefore advantageously comprise the preparation of a population of dendritic cells comprising immature dendritic cells, in particular from monocyte precursors, more particularly by treatment with a combination of cytokines such as GM

s CSF + IL-4 or GM-CSF + IL-13.

  Furthermore, it is also possible to use, in the context of the present invention, immortalized dendritic cell populations. They can be immortalized dendritic cell lines. They can also be dendritic cells prepared and then immortalized in vitro. The interest of dendritic or immortalized cells lies in the constitution of banks of cells sensitized to groups of given antigens, usable industrially and likely to be

  administered (or their dexosomes) to entire families of patients.

  When the dendritic cells are prepared, they can be maintained in culture, further purified, stored or used directly in the following process steps. Thus, according to a preferred embodiment, the antigen presenting cells in step a) of the process for the in vitro preparation of antigen presenting cells, loaded with membrane vesicles, according to the invention, are dendritic cells, in particular immature, dendritic cells obtained from monocyte or bone marrow precursors, or dendritic cells are known

  immortalized dendritic cells.

  According to an also preferred embodiment, said membrane vesicles and the antigen presenting cells in step a) of the method are autologous or syngeneic. 2s According to an also preferred embodiment, said membrane vesicles

  and the antigen presenting cells in step a) of the method are allogenic.

  Step a) of the process for the in vitro preparation of antigen presenting cells, loaded with membrane vesicles, and consisting in incubating the CPA in vitro in the presence of membrane vesicles according to the invention corresponds to the step of

  awareness (or even stimulation) of CPA.

  The incubation of CPA, in particular of dendritic cells, with the membrane vesicles according to the present invention and containing antigens or antigenic peptides (in particular the texosomes of tumor cells according to the present invention) is particularly advantageous insofar as it does not require the knowledge of particular antigens and where the charged antigenic peptides are in

a native conformation.

  s For the conditions for implementing this incubation step, reference may be made to the examples below or to the examples described in document WO 99/03499 for the incubation step of dendritic cells in the presence of texosomes obtained from cultures of tumor cell lines. In general, contacting is

  performed for a period of 30 min to 5 h, preferably 1 h 30 min to 2 h 30 min.

  o After step a) of sensitization, the CPA thus sensitized, in particular the dendritic cells, are generally washed in a serum-free medium (NaC10.9%) by centrifugation. In yet another aspect, the present invention relates to a process for the in vitro preparation of membrane vesicles secreted by antigen presenting cells (here called “dexosomes”) loaded with membrane vesicles, characterized in that it comprises the following steps: a) the in vitro incubation of antigen presenting cells in the presence of membrane vesicles according to the invention; b) a step of differential centrifugation of membrane fractions of culture supernatants of the abovementioned cells presenting antigens loaded with membrane vesicles according to the invention; and

  c) recovering the fraction containing said isolated dexosomes.

  It has in fact already been demonstrated (cf. document WO 99/03499) that the CPA, in particular the dendritic cells, produce membrane vesicles and that these 2 vesicles constitute powerful immunogenic vesicles for tumor immunization. These vesicles are particularly advantageous since they make it possible to avoid the step of in vivo injection of the whole dendritic cells from which they come. In vitro preparation of membrane vesicles secreted by antigen presenting cells (here called "dexosomes") loaded with vesicles

membranes according to the invention.

  When the populations of dendritic cells are sensitized, said dexosomes can be isolated according to the methods described for example in document WO 99/03499 for the preparation of dexosomes isolated from cells

  dendritics previously sensitized.

  s Briefly, this preparation involves an optional first step of

  treatment of CPA cells, followed by a second step of isolation of dexosomes.

  The first step in processing CPA cells results from the fact that the production of dexosomes by CPA, in particular dendritic cells, is a regulated phenomenon. Thus, in the absence of treatment, the quantities of lo dexosomes produced are relatively low, in particular, when a population of mature dendritic cells which are not previously stimulated is used. This stimulating treatment can be carried out either by cell culture in the presence of certain cytokines, or by cell irradiation, or by lowering the pH of the culture, or by combining these

different types of treatment.

  For example, in the first embodiment, the dendritic cells are incubated in the presence of a cytokine chosen to be preferable from interferon gamma (IFN), interlenkin-10 (IL-10) and interlenkin-12 (IL-12), preferably gamma interferon and IL-10. In this embodiment, the cytokines are used at doses adaptable by a person skilled in the art according to (i) the cytokine, (ii) the cell population and (iii) the possible implementation of other treatments. . It is understood that the cytokines are preferentially used at sub-toxic doses. Interleukin doses are generally between 1 and 100 ng / ml, preferably between 1 and 50 ng / ml. Interferon can be used at doses between 1 and

  1,000 IU / ml, preferably between 100 and 1,000 IU / ml.

  2s In the second embodiment, the dendritic cells are subjected to irradiation. The irradiation is generally carried out between 1,000 and 5,000 rads, of

  Preference between 2,000 and 4,000 rads, advantageously around 3,000 rads.

  The second step involves the isolation of dexosomes. The purpose of this step is to separate the dexosomes from the dendritic cells and / or from the culture medium. This step makes it possible in particular to obtain a composition enriched in dexosomes and essentially devoid of intact cells. The isolation of the dexosomes at this second stage can be carried out by following the methods applied to the isolation of the membrane vesicles as described above (difierential centrifugation

  in gradient, fluid electrophoresis, nanofiltration and / or immunocapture).

  The dexosomes indeed have remarkable immunogenic properties since they are capable of stimulating the proliferation and the activity of cytotoxic T lymphocytes, both in vitro and in viro, and that they make it possible to suppress the growth of tumors in viro. established, in a T cell dependent manner and restricted to the type of MHC (cú document WO 99/03499). The dexosomes therefore constitute active principles which are particularly suitable for non-cellular approaches to immunotherapy. o Preferably, the process for the preparation of dexosomes according to the present invention results in a composition comprising at least 70% of dexosomes,

preferably 85% at least.

  The present invention also includes the antigen presenting cells loaded with membrane vesicles capable of being obtained by the method of in vitro preparation of antigen presenting cells according to the invention. The present invention further relates to the isolated dexosomes obtained by the process of in vitro preparation of membrane vesicles secreted by cells.

  presenting antigens loaded with membrane vesicles according to the invention.

  In yet another aspect, the present invention comprises a process for the in vitro preparation of activated (stimulated) NK or NKT T-cell lymphocytes, characterized in that it comprises the following steps: a) the in vitro incubation of T-cells. NK or NKT cells in the presence of cells presenting antigens loaded with membrane vesicles according to the invention or isolated dexosomes, secreted by cells presenting antigens loaded with membrane vesicles according to the invention, and

  b) the recovery of said T lymphocytes NK or NKT cells thus activated.

  In a particular embodiment, the present invention comprises a method for the in vitro preparation of anergic T lymphocytes, characterized in that it comprises the following steps: a) the in vitro incubation of T lymphocytes in the presence of cells presenting with antigens loaded in membrane vesicles according to the invention or isolated dexosomes, secreted by cells presenting antigens loaded in membrane vesicles according to the invention; and

  b) the recovery of said T cells thus anergized.

  The term “anergy” is intended to denote the detail of the T response which can also be observed when the T cells are placed in the presence of the antigen presented by the CPA. According to a claimed embodiment of the process for the in vitro preparation of T lymphocytes NK cells or NKT according to the invention described above, the cells

  Antigen presenting and T lymphocytes are autologous or syngeneic.

  o The present invention furthermore comprises the T lymphocytes NK or NKT cells capable of being obtained by the method of in vitro preparation of lymphocytes

  T. NK or NKT cells according to the invention described above.

  In another aspect, the present invention comprises the use of membrane vesicles, in particular texosomes, according to the invention, antigen presenting cells or dexosomes secreted by antigen presenting cells according to the invention, or lymphocytes T according to the invention, for the preparation of a pharmaceutical composition intended to be administered to a patient and to induce or increase an immune response of the CTL type specific for antigens contained in said membrane vesicles according to the invention, and in which use, said membrane vesicles, antigen presenting cells and T lymphocytes are

  autologous or syngeneic of the patient to be treated.

  In another aspect, the present invention comprises the use of membrane vesicles, in particular texosomes, according to the invention, antigen presenting cells or dexosomes secreted by antigen presenting cells according to the invention, or NK or NKT cells according to the invention, for the preparation of a pharmaceutical composition intended to be administered to a patient and to induce or increase an innate response of Npe or NKt Npe or NKT specific for antigens contained in said membrane vesicles according to invention, and in which use, said membrane vesicles, antigen presenting cells and NK cells or

  NKT are autologous or syngeneic to the patient to be treated.

  For preference, said pharmaceutical composition is intended for prevention

  or treating cancer patients.

  In another aspect, the present invention includes the use of antigen presenting cells, in particular dendritic cells, taken from a first patient (the recipient) or from a patient whose antigen presenting cells are syngeneic (having haplotypes common HLA) to the antigen presenting cells of the first patient, for the preparation of a pharmaceutical composition intended to be administered to this first patient, characterized in that the said antigen presenting cells were previously loaded in vitro with membrane vesicles according to the invention isolated from a biological fluid, in particular ascites, of tumor-like appearance, taken from a second

  o patient (the donor), said recipient and donor being allogenic.

  In another aspect, the present invention comprises the use of T lymphocytes taken from a first patient (the recipient) or from a patient whose T lymphocytes are syngeneic to the T lymphocytes of the first patient for the preparation of a pharmaceutical composition intended for be administered to this first patient, characterized in that said T lymphocytes were previously activated in viro with antigen presenting cells previously loaded with membrane vesicles according to the invention, said membrane vesicles having been isolated from a fluid biological sample taken from a second patient (the donor),

  said recipient and donor being allogenic.

  The present invention further relates to the use of antigen presenting cells, or their dexosomes, or T lymphocytes according to the invention, for the preparation of a pharmaceutical composition intended to induce tolerance by said

  recipient of an allogeneic graft taken from said donor.

  The present invention further relates to the use of antigen presenting cells, or their dexosomes, or T lymphocytes according to the invention, for the preparation of a pharmaceutical composition intended to induce tolerance by said recipient of an allogenic graft. taken from said donor by the method called "adoptive transfer of dendritic cells from the recipient previously loaded in vitro with membrane vesicles from the donor" or by the method called "adoptive transfer of T lymphocytes from the recipient previously activated in viro by CD of the recipient previously charged (pulsed) by membrane vesicles of the

donor ".

  In yet another aspect, the present invention comprises the use of membrane vesicles, antigen presenting cells or dexosomes according to the invention, for the ex viro selection of a repertoire of T lymphocytes and / or NK cells or NKT, capable of recognizing specific antigens, glycolipids or

  s phospholipids contained in said membrane vesicles according to the invention.

  The invention also comprises a medicament comprising, with active substance, one or more membrane vesicles, cells presenting antigens or dexosomes, or also T lymphocytes of NK or NKT cells according to the present invention, in combination with a pharmaceutically acceptable vehicle. , especially

0 for the treatment of cancers.

  As an appropriate dosage form, the membrane vesicles, cells presenting antigens or dexosomes, or even T lymphocytes according to the present invention can be contained in physiological saline, in an ampoule or any other appropriate means (syringe, pocket, etc.). . They can be prepared immediately or stored, for example in frozen form at -80 C. The solutions used can be composed of saline solutions, optionally supplemented with stabilizing agents and / or adjuvants. The stabilizing agents can be in particular proteins or molecules of high molecular weight. Mention may more particularly be made of proteins such as human serum albumin, or molecules

  such as dextran or poloxamer for example.

  An appropriate mode of administration of the medicaments or pharmaceutical compositions according to the invention consists of injections, and in particular intradermal or subcutaneous injections. This mode of administration is particularly suitable when the active substance of the drug consists of 2s of CPA, in particular dendritic cells, loaded with membrane vesicles

  according to the invention (in particular in texosomes) or by dexosomes according to the invention.

  The appropriate dosages are from 0.01 to 10 μg / kg, and in particular 0.10 to g / kg, even more particularly from 0.15 to 2 μg / kg of body weight, and from 10 μg

  for vaccinations and 100 times less for intradermal reaction tests (HSR).

* Preferably, said medicament according to the present invention will comprise

  in addition a synthetic or natural immunostimulating adjuvant.

  The compositions of the invention can also comprise or be used in combination with one or more adjuvants. The adjuvant can more particularly be any immunostimulating pharmacological agent, such as for example a cytokine (in particular interlenkin-12). Such agents are conventionally used in clinical protocols or in vaccine compositions. Furthermore, the adjuvant according to the invention can also be an agent capable of stimulating the production of cells.

  dendritics in viro. By way of example, mention may be made of the compound Flt3L + 4-IBBL.

  The combined use of this type of agent makes it possible to increase the number of dendritic cells, and therefore potentially to improve the effectiveness of the compositions of the invention. Another subject of the invention also relates to a combination of membrane vesicles and / or cells presenting antigens (or their dexosomes) and a

  adjuvant, for simultaneous, separate or time-separated use.

  The invention also relates to the creation of texosome banks according to the invention derived from tumor cells of common or different histological type. These may be composed of mixtures of texosomes made from in viro samples from patients with a given type of cancer. These texosome banks can make it possible to sensitize antigen-presenting cells,

  especially dendritic cells, against all tumors of this type.

  The invention also relates to mixtures of texosomes or dexosomes according to

  the present invention, in particular as a medicament.

  Mention may be made, for example, of mixtures of texosomes according to the invention for genetically linked tumors (cancer of the breast and of the ovary) or having

  known pS3, pl6 mutations (breast cancer, melanomas, glioblasms, sarcomas).

  2s One can also mention mixtures of texosomes according to the invention with membrane vesicles originating from immortalized cells and transfected to express costimulation molecules, adhesion molecules,

  attractive chemokines (different from those expressed on the texosomes).

  In another aspect, the present invention relates to the use of texosomes, or of CPA, in particular of dendritic cells according to the invention, for the manufacture of a delayed hypersensitivity test for cancer, or as a diagnostic tool for researching frequency of specific precursors CTL, NK or NKT (immunomonitoring). It is a question here of using the CPA, in particular the dendritic, autologous or allogeneic cells preincubated with the texosomes of the invention, as targets of peripheral lymphocytes of subjects carrying the tumor, before, during and after

  anti-tumor treatment (conventional treatment or specific active immunization).

  In another aspect, the present invention relates to a method of in vitro diagnosis of cancer and / or its evolution in a patient, characterized in that the presence of specific markers for cancer and / or its cancer is determined. evolution on the surface and / or in the cytosol of membrane vesicles obtained from fluid

  biological sample taken in viro from said patient.

  Said biological fluid is ascites or effusion, in particular emanating from a cavity, such as a peritoneal, abdominal cavity.

or pleural.

  Preferably, the determination of the presence of markers of a cancer and / or of its evolution is carried out by an immunoeuzymatic technique of the immunocapture type, by Western Blot or ELISA using antibodies specific for these markers. The membrane vesicles according to the present invention, in particular those derived from ascites fluid, can thus not only be used as a source of natural tumor antigens leading to new therapeutic pathways (as mentioned above and also as a source of antigens for active prophylactic immunization after the second "look" in Figo IIIc or mesothelial ovarian cancer, passive transfer into the peritoneal cavity of award-winning 2s T cells activated in situ as an adjuvant therapy against residual disease (see examples and following )) but also as a new entity in human pathophysiology that can be used to control the status of the tumor (diagnosis and prognosis, chemoresistance and detoxification). All of these uses are good

  understood included in the present invention.

  In another aspect, the present invention relates to a process for the in viro preparation of a bank of texosomes derived from peritoneal ascites fluid, characterized in that it comprises the following step: a) implantation intraperitoneally d 'a tumor, tissue or human tumor cells in a SCID mouse, in particular SCID / NOD; In another aspect, the present invention relates to a process for the in vitro preparation of a bank of antibodies of murine origin directed against human antigens, in particular tumor antigens, characterized in that it comprises the following steps: a) implantation by intraperitoneal route of a tumor, tissue or human tumor cells in a SCID mouse, in particular SCID / NOD; b) the in viro removal from the SCID mouse of a sample of peritoneal ascites fluid resulting from the implantation and containing said bank of antibodies; and

  C) where appropriate, the purification of the antibodies contained in said sample.

  In a particular embodiment, the lymphocytes secreting such antibodies contained in the sample of peritoneal ascites fluid taken in viro from the SCID mouse in step b) may be used to obtain cell lines or hybridomas by fusion with for example a tumor cell according to the well-known technique of Kohler and Milstein for the production of monoclonal antibodies. In another aspect, the present invention relates to a process for the in vitro preparation of a library of isolated texosomes of murine and human origin, characterized in that it comprises the following steps: a) implantation by intraperitoneal route of a human tumor, tissue or tumor cells in a SCID mouse; b) in viro sampling in SCID mice of a sample of peritoneal ascites fluid resulting from implantation; and c) in vitro isolation of said texosomes from said sample taken by centrifugation, electrophoresis, chromatography, nanofiltration and / or by

2s immunocapture.

  In another aspect, the present invention relates to a method for preparing texosomes modified in vitro, characterized in that it comprises the following steps: a) the in vitro isolation of membrane vesicles (texosomes) from a sample of tumor ascites fluid by a process for the in vitro preparation of membrane vesicles isolated from their natural environment according to the invention; b) the elution, in particular acidic, of said natural texosomes obtained in step a); c) bringing said eluted natural texosomes into contact with peptides, in particular s, synthetic, glycolipids or phospholipids of interest, preferably exogenous which it is desired to load into the texosomes; and

  d) recovering said texosomes thus modified.

  In a last aspect, the present invention relates to the use of texosomes: - isolated from their natural environment from a sample of tumor ascites fluid by a process for the in vitro preparation of membrane vesicles according to the invention; or lo - obtained by a process for the preparation of texosomes isolated in vitro derived from peritoneal ascites fluid after implantation of a tumor, tissue or human tumor cells in a SCID mouse according to the invention; or also obtained by a process for preparing texosomes modified in vitro according to the invention, for the preparation of antibodies or banks of antibodies directed against antigens

  tumors contained in or carried by said texosomes.

  The present invention will be described in more detail with the aid of the examples which

  follow, which should be considered as illustrative and not limiting.

LEGENDS OF FIGURES

  Fiaure 1: Peritoneal carcinosis secretes vesicles from 50 to 100 nm in ascites. FIG. 1 represents vesicles purified from ascites of a carrier patient

  of melanoma with peritoneal extension seen by electron microscopy.

  Fiures 2A to 2D: The ascites vesicles contain class I molecules,

  80 heat shock proteins and tumor antigens.

  FIGS. 2A to 2C show immunoblots produced on samples of ascites vesicles (5 to 20 l / well). Figure 2A shows the presence of molecules of the major histocompatibility class I complex and heat shock proteins (HSP80) in the vesicles of patients with ovarian cancer or melanoma. Figure 2B shows the absence of gp96 protein in ascites vesicles, unlike the cell lysate. Figure 2C shows the presence of the Martl protein

  in melanoma ascites vesicles.

  Figure 2D shows the presence of the two dimerized lactadherin isoforms

in the ascites vesicles.

  s Fiures 3A and 3B: The ascites vesicles carry a cross presentation of antigen

Martl tumor.

  Figure 3A shows the experimental mode. Ascites vesicles (10 to 20 µl) from an HLA-A2 patient with melanoma with peritoneal extension were loaded onto 104 A2 + dendritic cells. These dendritic cells were cocultivated for 24 h with 104 cells of a CD8 + T clone specific for Martl and restricted for the haplotype A2 (LT11). The activation of the clone was evaluated on the secretion of interferon. FIG. 3B shows the levels of interferon secreted by the LT11 clones after 24 h of coculture with the uncharged dendritic cells, loaded with vesicles from patients carrying ovarian carcinoma. or melanoma. The positive control was represented by dendritic cells loaded with exosomes from a Martl + melanoma cell line, HLA-A2- (Mel 888). Rates are the average

of triplicate.

  Figure 4: Experimental mode of in vitro stimulation of lymphocytes.

  The experimental mode of stimulation [n vitro is shown in Figure 4. Monocytes and lymphocytes were taken from peripheral blood, vesicles and tumor cells were isolated from ascites. Dendritic cells were derived from GM-CSF and IL-4 monocytes. The circulating lymphocytes were stimulated 3 times (OJ, J7, J14) with dendritic cells loaded or not with 10 or 2s 20 μl of ascites vesicles. The cocultures were carried out in 96-well plates with a round bottom. The specificity of the lymphocytes was assessed on their capacity to lyse the tumor.

  autologous and secrete interferon-y in the presence of tumor cells.

  Figures SA and 5B: Lymphocytes induced by ascites vesicles specifically recognize tumor cells via an interaction mediated by molecules of the

CMH I.

  FIG. 5A represents the lysis of tumor cells and K562 cells by the lymphocytes of patient I after 3 stimulations by dendritic cells loaded with ascites vesicles. Lysis against autologous tumor cells was assessed in the presence of cold K562 cells. Values represent the mean of

three wells.

  FIG. 5B represents the inhibition of the lysis of tumor cells by an anti-class I antibody (w632). Lymphocytes stimulated three times with dendritic cells loaded with 10/20 μl of vesicles were cocultivated in the presence of chromium 51-labeled tumor cells, in the presence of cold K562. The tumor cells were preincubated with 100 μl of w632 antibody for 1 h. Fieure 6: The injection of ascites vesicles increases the survival of DBA2 mice

lo carrying leukemia L1210.

  The L1210 vesicles were purified from ascites from mice carrying the Ll210 serum with peritoneal extension. These vesicles were injected subcutaneously into mice carrying L1210 leukemia. Survival was assessed on D12 and D24. (gray) Tex injection of non-tumor peritoneal fluid

(pristanized mice).

  (in black) Injection of purified exosomes from ascites from

L1210.

  Figures 7A to 7C: Panel A: Phenotype of lymphocytes after 3 stimulations Figure 7A: lymphocytes stimulated in the presence of CD loaded by exosomes

ascites (DC / ExAs).

  Figure 7B: Lymphocytes stimulated in the presence of CD loaded by cells

autologous irradiating tumors.

  Figure 7C: Functional test of lymphocytes stimulated 3 times.

  2s Figures 8A to 8C: The stimulated lymphocytes recognize the tumor cells via

  an interaction mediated by MCH I molecules: Example 2.

  FIGS. 8A and B show the phenotypes of the lymphocytes after 3 stimulations with CD loaded (panel A) or not (panel B) with ascites vesicles. FIG. 8C represents a test for the secretion of interferon-y by lymphocytes in the presence of autologous tumor cells. Two lymphocyte conditions were tested. The first condition was patient 3's lymphocytes stimulated three times with uncharged dendritic cells. The second condition was 1yTnphocytes from patient 3 stimulated by dendritic cells loaded with ascites vesicles. 104 Iyrnphocytes were cocultivated with 104 tumor cells preincubated or not 1 h with

  w632 antibody (100 µl / well). Values represent the average of three wells.

  Standard deviations are shown as bars.

EXAMPLES

  Examples 1 to 4 relate to the detection of membrane vesicles in ascites liquids from patients with peritoneal carcinosis and the use

  of these vesicles for the cross presentation of antigens.

  0 Example 1: Material and methods A) Cell cultures The dendritic cells are obtained from monocytes differentiated into GM-CSF (1000 IU / ml) and IL-4 (1000 IU / ml, Shering Plow). They are used on D7

  for in viro stimulation and cross presentation tests.

  The tumor cells used for the cytotoxicity tests are obtained by centrifugation of ascites and cultured in RPMI1640 medium, 10% fetal calf serum, Ullml of penicillin, 50 g / ml of streptomycin, 2 mM L-glutamine, I mM Na pyruvate. B) Obtaining ascites and purifying the vesicles of as cites Ascites is taken from patients with peritoneal carcinosis diagnosed on the presence of tumor cells in the centrifugation pellet and

  a protein content greater than 30 g / 1.

  The first steps consist in recovering the supernatant from the centrifugations

  do 1,200 rpm (10 min), 3,000 rpm (15 ') twice, 10,000 rpm (30').

  - The vesicles are recovered after ultracentrifugation at 29,000 rpm for 1 h. The vesicles are isolated on a D20 + sucrose cushion at a density of 1.077 during an ultracentrifugation of 23,500 rpm (duration: 1 h 15 min). The vesicles are then collected in the cushion and concentrated using ultracentrifugation at 49,000 rpm for 1 h. The whole of this process makes it possible to obtain what will be called subsequently the

  ascites vesicles (or texosomes according to the invention).

  C) Electron microscopy The vesicles are fixed in 4% paraformaldehyde. The vesicles are spread out

  on microscopy grids and visualized (see Figure 1) (see also Example 5C).

D) Western blot

  The vesicles are reduced to 5% DMSO and changed at 96 ° C. for 10 min.

  A migration is made on Novex gel. The migrated proteins are then transferred to a nitrocellulose membrane. The proteins are then hybridized with an anti-MHC class I antibody (HC10, Soldano Ferrone, NY, USA), anti HSC-80 (SPA 815, Stressgen), anti-gp96 (SPA850, Stressgen, anti Martl (clone A103, Novocastra ) or anti lactadherin (see example 2D). The revelation is made using an antibody coupled to the

  o peroxidase (see Figures 2A to 2D).

  E) Cross presentation tests in Yitro The vesicles are purified from ascites from an HLA-A2 patient with peritoneal melanoma carcinosis. The vesicles are incubated with 10,000 HLA-A2 + dendritic cells (30 µl) for 2 h. After 2 h, 10,000 LTl 1 (clone restricted by HLA-A2 and specific for Martl) are cocultivated with the dendritic cells for 24 h. The supernatant is removed and an ELISA assay of interferon-y is then carried out in order to

  to evaluate the activation of the clone (cf. FIGS. 3A and 3B)).

  F) In Vitro Stimulation Tests The vesicles (20 μl of a 2,000-fold concentrated ascites liquid) are loaded onto the autologous dendritic cells (30,000 / well) in 30 μl of medium (RPMI1640, 10% human serum AB ', 50 U / ml of penicillin, 50 1lg / ml of streptomycin, 2 mM L-glutamine, 1 mM Na pyruvate). After 2 h of incubation, the dendritic cells are cocultivated with autologous circulating lymphocytes in 96-well round bottom culture plates. Lymphocyles are cultivated at 100,000 2s per well. The cultures are made in the presence of interleukin 2 (prolenkin, Chiron) at 50 IU / ml and of stimulation factors T. The lymphocytes are stimulated by the dendritic cells three times at OJ, D7, D15 and are tested for their ability to lyse them

  autologous tumor cells (see Table 1, also for Figure 5A).

  Negative stimulation controls include tumor cells

  irradiated (30,000 cells / well) or uncharged autologous dendritic cells.

  For cytotoxicity, autologous tumor cells or K562 cells are chromated for 1 h with s'Cr. The cells are washed three times and 2,000 cells are placed in conical bottom wells. The tumor cells are incubated with 50,000 or 100,000 unchromed K562 cells. The lysis is the result of the following equation: (number of strokes-min) / (MAX-min) where min is the number of strokes released in the supernatants of those tumor cells and MAX is the number of counts of tumor cells incubated with cetrimide (see Table 1, also for Figure SA) Example 2: Demonstration of membrane vesicles containing tumor antigens in the ascites of patients with peritoneal carcinosis L ' electron microscopy study shows that the differential centrifugation of ascites allows membrane vesicles to be obtained (see Figure 1). Microscopy studies also show that these vesicles are between 150 nm and have a tendency to aggregate. Biochemical analysis of the vesicles highlights the presence of MHC I molecules (see Figure 2A)., HSP 70, HSP 80 (see Figure 2A). and lactadherin (see Figure 2D). In the patient with melanoma, the presence of a Martl tumor antigen has also been demonstrated (cf. FIG. 2C). On the other hand, ascites vesicles do not contain gp96 (cf. figure 2B) this

  which means that there is no contamination by cellular detris.

  EXAMPLE 3 Membrane Vesicles of the Texosome Type Induce Cross Presentation of Antigen by Dendritic Cells FIG. 3B shows that the vesicles purified from ascites from an HLA A2 patient carrying MARTI + melanoma loaded onto HLA-A2 + dendritic cells clone LT11 (restricted HLA-A2 and specific to Martl). This means that the ascites vesicles are effectively picked up by dendritic cells and induce cross presentation of antigen. On the contrary, CD-A2 + pulses by texosomes

  2s (Tex) of the ascites of ovarian eancer Martl-, do not induce stimulation of LT11. Example 4: The aseite vesicles loaded on dendritic cells

  induce cytotoxic T lymphocytes recognizing the epitopes presented by autologous tumor cells In order to evaluate their immunogenicity, the ascites vesicles of a patient carrying an ovarian adenocarcinoma were loaded on autologous dendritic cells, and a co culture with circulating lymphocytes were performed (see Figure 4). Table 1 of results (see below) highlights on the one hand: - the significant proliferation of lymphocytes obtained in the presence of autologous dendritic cells loaded by the ascites vesicles of a patient carrying a comparative ovarian adenocarcinoma to the coculture obtained in the presence of the autologous tumor; and on the other hand s - the lysis of the autologous tumor by the lymphocytes stimulated by the cells

  dendritics loaded with ascites vesicles.

  In contrast, lymphocytes stimulated by autologous tumor cells are not able to lyse tumor cells. Lymphocytes induced by vesicles lyse the autologous tumor cell, but not significantly K562 (cf. FIG. 5A). In addition, lysis can be blocked by an anti-MHC class I antibody (cf. FIG. 5B). This means that the dendritic cells loaded by ascites vesicles have induced cytotoxic T lymphocytes recognizing epitopes presented by autologous tumor cells. The ascites vesicles can therefore be used for therapeutic purposes as an autologous or allogenic vaccine, but also as a

  is a method of loading dendritic cells for therapeutic purposes.

  Table 1: Proliferation (or amplification) of CD3 / CD8 + cells and percentage of lysis against the autologous tumor obtained with dendritic cells

  loaded with ascites vesicles, or with autologous tumor cells.

  Number of cells% lysis against CD3 / CD8 autologous tumor * PBL JO 0, 6x105 0% PBL J21 7xlO 30% CD + Texosome PBL J21 0.8xlO '0% Tumor Effector / Target Ratio (E: T): 50/1 ; PBL D21: lymphocytes after three in stimulation

  non-specific lysis inhibited by non-chromated K562 cells (cold K562).

  FIG. 6 highlights the prolonged survival of DBA2 mice vaccinated with ascites texosomes according to the invention against autologous lencemia compared to a placebo control vaccine b (injection of "Tex" of peritoneal fluid from pristanized mice ). Examples 5 and following relate to the detection of membrane vesicles in pleural or peritoneal ascites liquids from carrier patients

  of adenocarcinomas (see Table 2), their characterization and their uses.

  Example 5: Patients and Methods A) Patients Patients with peritoneal carcinomatosis associated with ascitis lo were included in the present study. The presence of tumor cells in non-hemorrhagic ascites was a criterion for the selection of patients for this study. The clinical characteristics of the patients are summarized in Table 2 below. Most were women with stage IIIc Figo ovarian cancer. The ascites are recovered either during the first intervention ("debulking") and i5 observation by the surgeons or in the case of the appearance of symptoms associated with ascites. Patients were excluded from the study if a) they received chemotherapy within four weeks before the recovery of the above or if b) the protein level in exudates is less than 30 g / 1 or if c) hemorrhage is associated with carcinomatosis. The patients gave their informed consent following the approval by the ethics council of the Gustave-Roussy Institute of this protocol (Advisory Committee for the Protection of Persons in Biomedical Research (CCPPRB)). A patient (patient 5) with lung cancer associated with malignant pleural effusion has

also been included.

  3. $. = O% S o o Z Z o Z Z V À; . U) z 2 o. V) V)

S ó ó ó ó ó ó ó

  > (uS% O. = I. = ô -.g 5> è: - Àe vvvv lo vvm 1 ô id hi hi B) Purification of exosomes (membrane vesicles or here texosomes! The ascites are centrifuged at 1200 rpm for remove the cells in suspension, the supernatants are collected and centrifuged successively at 3,000 rpm for 30 min, 10,000 rpm for 30 min and 29,000 rpm for 1 hour Following the last centrifugation step, the pellet is recovered, resuspended in 30% in a gradient of 30% sucrose / D20 and ulkacentrifuged at 23,500 rpm for 1 h 15 min to allow unwanted cellular debris to be pelletized and then eliminated. The exosomes are contained in the cushion of 30% sucrose / D20 and this is verified by analysis by electron microscopy then washed in PBS and concentrated using o a final step of 1 h of ultracentrifugation at 40,000 rpm. The pellet of exosomes is resuspended in PBS and stored stably at - 80. A proc dice is similar

  performed to purify the exosomes of the supernatant from the autologous cell line.

  C) Electron microscopy The exosomes obtained after differential centrifugations are fixed in 4% paraformaldehyde and stored at 4 C. The exosomes are loaded onto "formvar-carbon" electron microscopy grids and labeled with immunogold. The contrast is obtained as described in Raposo et al. (1996) (see also Zitvogel et al., 1998 and

Wolfers et al., 2001).

  D) Analysis by Western Blot The exosomal and cell lysate proteins are extracted as previously described and then analyzed by Western blotting using a monoclonal antibody directed against human HLA class I (HC10, generously offered by Soldano Ferrone, Roswell Park Cancer Institute , NY, USA), an anti-HSP 70 antibody (SPA 815, Stressgen, Canada), an anti-gp96 antibody (SPA 850, Stressgen, Canada), and 2s an anti-MHC class II antibody or an anti- Mart-1 / MelanA (clone A103, Novocastra, United Kingdom) at the dilutions recommended by the manufacturer followed by a revelation using secondary antibodies coupled to horseradish peroxidase and by a revelation with chemoluminescence (Boehringer Mannheim, Penzberg, Germany). Anti-lactadherin antibodies are produced by rabbits immunized with

human lactadherin.

  E) Cell cultures a) Autologous primary tumor lines and tumor cells The tumor cells are pelleted by centrifugation at 1200 rpm for ascites and then cultured in RPMI 1640 supplemented with 50 U / ml of penicillin, 50 g / ml of streptomycin , 2 mM L-glutamine, 1 mM Na pyruvate (Gibco-BRL, France) and 10% decomplemented fetal calf serum (Seromed, France). These cells are characterized before freezing with May-Grunwald-Giemsa and staining with an anti-cytokeratin antibody by pathologists on cytospins. Most of the 5Cr release tests are carried out using tumor cells derived from autologous ascites which have undergone three to five passages. After six passages, the tumor lines are frozen for later use. The Mel888 line is an established MART1 + and HLA-A2 negative melanoma cell line which has been

  generously donated by Dr. Markus Maeurer, University of Meinz (Germany).

  b) Dendritic cells and lymphocytes (MD-DC) CD-MD (dendritic cells derived from monocytes) are obtained from the adherent fraction of peripheral blood mononuclear cells (PBMC) effusion of tumor carried by the patients described in Table 1 after their informed consent. Briefly, the PBMCs are isolated by centrifugation on a density gradient using Ficoll-Hypaque and then seeded at a concentration of 3 to 5 × 106 cells / ml in AIMV medium (Gibco-BRL, France) supplemented with U / ml of penicillin, 50, ug / ml streptomycin, 2 mM L-glutamine, 1 mM Na pyruvate (Gibco-BRL, France), 10% decomplemented fetal calf serum (Seromed, France). All of the lymphocytes are depleted by a 3 h adhesion step at 37 ° C., followed by three steps of washing in a saline buffer. The adherent cells are propagated in a complete AIMV medium containing 1000 IU / ml of recombinant human GM-C SF and of IL-4 (Novarti s (S chering Plow NJ, Kenil sworth, 2s USA). is frozen on day O and on day 5 respectively. CD-MDs are used on day 6, one day after

  thawing, for in vitro stimulation.

  F) In vitro cross-presentation tests LTl 1 (V, B9) is a CD-specific CD8 + T cell clone Mart-1 (27-35) specific, HLA-A2 + propagated as previously described (Dutour et al., J . Of Immunology, 158, 8, 3787-95, 1997) On day 6, CD-MD HLA-AA2 + are seeded at a concentration of 2 104 cells / well for 2 h with exosomes purified from patient ascites HLA-A2 melanoma or from Mel888 HLA-A2 negative melanoma cell line supernatants. 2,104 LTl 1 are added to the 2,104 CD-MDs in a final volume of 200 μl in 96-well plates having rounded wells. The 24 h supernatant is tested in an ELISA IFN test (Immunotech,

s Marseille, France).

  G) Stimulations of PBLs in viko Mixed in vitro micro cultures are carried out in 96-well microplates having rounded wells in a complete RPMI medium supplemented with 10% of pooled human serum AB, 100 IU / ml of IL2 ( Prolenkine, Chiron), T cell growth factors (Dutour et al., 1997). 3x104 autologous CD cells are pulsed with autologous exosomes derived from ascites or exosomes derived from autologous tumor cells for 2 h in 50 p1. The quantity of pulsed exosomes in the wells containing the DCs is equivalent to 30 ml of pleural / peritoneal effusion or of culture supernatant. The autologous PBLs (peripheral blood lymphocytes) are then added at a ratio of three lymphocytes for a CD in a final volume of 200 Ill / well. Three weekly in vitro stimulations of PBL are carried out. The alternative culture conditions and negative controls include stimulation with irradiated autologous tumor cells (104 / well) for patient 1, stimulation with unpulseed CD cells in the other six patients. On day 21, stimulated lymphocytes are recovered, counted with trypan blue and phenotyped by a Facscan analysis using anti-CD4, anti-CD8, anti-CD56 monoclonal antibodies (Coulter, Fullerton, USA) and tested for release. at 5'Cr and for tests for secretion of IFN against autologous tumor cells with or without a

  W6.32 monoclonal antibody or unrelated tumor cells.

  H) Relay test for IFNy x104 lymphocytes stimulated in vitro in a weekly manner after three stages of incubation with autologous CD cells pulsed or not with exosomes are incubated with or without 104 autologous tumor cells. To verify the restriction of MHC class I on the recognition of tumors, the antibody W6.32 is incubated for 1 h with the tumor cells before coculture with lymphocytes. 24 h supernatants are tested by an ELISA IFN test (Immunotech, Marseille, France). For the IFN1 / ELISPOT tests, the cells are cocultivated with an anti-IFN monoclonal antibody present on the surface of the 96-well microplates. After 24 h the plates are washed and a secondary antibody is added. The spots are counted using a reader

  ELISPOT automatic (Bioreader 2000, Biosys).

  I) Cytotoxicity Test Lymphocytes stimulated in vitro on a weekly basis after three stages of incuhation with an autologous CD cell pulsed or not with exosomes are used as effectors. The autologous tumor cells after three to five passages are labeled with 100 IlCi of 5iCr for 60 min and then washed three times before their use. To inhibit non-specific lysis, K562 cold cells are added at a ratio of 50 cold cells to a hot target cell. To check the restriction by MHC class I during tumor recognition, the W6.32 antibodies are incubated for 1 h with the tumor cells labeled with 5iCr before coculture with the 1mphocytes. Effector cells are added to the corresponding targets at different E: T cell ratios. After 4 h, 50 μl of each supernatant are recovered and the release of the sCr is determined (gamma counter; Packard Top-Count, Zurich, Switzerland). The mean of the triplicate samples is calculated and the percentage of specific sCr release is determined as follows: percentage of specific lysis = (experimental release of 5Cr - release of 5iCr control) / (maximum release of 5Cr - release of sCr contr81e) x 100. The experimental release of Cr represents the shots for the target cells mixed with the effector cells, the release of the 5iCr contr81e represents the shots of the target cells incubated with a medium alone (spontaneous release), and the release of the 5Cr maximum represents the

  shots of target cells exposed to 5% Triton X-100.

  Example 6: Exosomes Derived from Tumor Cells Are Naturally Secreted in Pleural or Peritoneal Effusions These results also show that the revised standard method described in the material and methods of application WO 99/03499 could be successfully applied to peritoneal effusions for isolate the exosomes. As indicated by the immunoelectronmicroscopy studies, the pellets obtained after ultracentrifugation have a flotation at a density between 1.14 and 1.18 glml in a sucrose / D20 gradient and contain a large number of membrane vesicles resembling those described for the texosomes from cell line cultures (see Figure 1). These vesicles are, for the most part, aggregated, of heterogeneous size (from 30 to 150 nm, predominantly comprised between 50 and 100 nm in diameter, and can be labeled with an anti-tetaspaspine CD63 antibody and with a class MHC anti-MHC monoclonal antibody. I (cú figure 2A). No significant contamination with similar debris was observed in these preparations (cf figure 2B). The images obtained by western blotting reveal the typical characteristics of the texosomes lO obtained from cultures of cell lines, in particular, a significant level of MHC class I molecules and of Hsp70 and 80 molecules compared with the lysates of autologous tumor cells collected from ascites fluids or from autologous tumor cells of cell line (cf. FIG. 2A). previously described in application WO 99/03499 for texosomes derived from cultures of melanoma cell lines in vitro, Texosomes purified from two melanoma ascites contain the complete native tumor antigen MARTl / MelanA (cf. Figure 2C). The presence of the MART1 tumor antigen can be attached to exosomes and not to cellular debris since no gp96 glycoprotein can be detected in exosome preparations from ascites. As previously described for exosomes secreted from murine dendritic cells propagated ex vivo (Srivastava, PK, Methods, 12 (2): 165-71), the two lactadherin isoforms are overexpressed in exosomes derived from ascites compared to lysates tumor cells (see Figure 2 D). For two of the five patients tested, exosomes as opposed to tumor cell lysates extracted from primary autologous 2s tumor cells, have levels of MHC class II molecules detectable by Western blots. This suggests the presence of exosomes derived from CPA

  professional (B. macrophage cells, CD) in the ascites of these two patients.

  Quantitative analyzes using the immunocapture method with an anti-MHC class I antibody reveal that more than 10 109 MHC class I exosomal molecules can be recovered in 1 ml of tumor ascites. All of these results thus demonstrate the feasibility and reproducibility of isolating a large quantity of authentic tumor exosomes from ascites from cancer patients. Exosomes derived from ascites carry tumor antigens for loading into CDs and their cross presentation to TCD8 + I1 lyrnphocytes has been previously shown that exosomes derived from melanoma cell lines contain the native MART1 antigen and HSP 70-80 which could be kansfered to CDs to stimulate specific HLA-A2 restricted MART1 CTLs. Therefore, it was tested whether the exosomes purified from ascites of a HLA-A2- melanoma patient, when loaded onto CD-MDs allowed such 0 CDs to trigger HLA- restricted CTL clones A2 specific MART1. As shown in FIG. 2A, unlike exosomes derived from ovarian ascites, the exosomes purified from melanoma ascites contain significant amounts of MART1 protein for the two patients tested. Interestingly, CD-MDs loaded with exosomes of ascites extracted from patients with HLA-A2- melanoma, but not from patients with ovarian cancer, induce the activation of CTL for example by the production IFN-gamma (see Figure 3B). These results suggest that exosomes derived from ascites transfer their antigen to CDs for presentation

  crossed to specific CD8 + T cells.

  Example 7: CD-MDs loaded with exosomes derived from ascites induce the activation of CTL against the autolocal tumor In order to test the capacity of exosomes derived from ascites to induce tumor-specific CTL responses in vitro, the experiment The following was carried out: PBLs of tumor ascites carried by cancer patients were stimulated each week with either irradiated autologous tumor cells, or with CD-MD loaded with either exosomes derived from autologous ascites, or with or exosomes derived from autologous tumor cells. After three rounds of in vitro stimulation, the PBLs were phenotyped by FACScan analysis and counted by the trypan blue exclusion technique before being tested against tumor cells using the s'Cr release technique and the secretion of IFN-gamma. These results are presented and

  summarized in Table 3 (see below).

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Z C P P P

  Key to Table 3: Table 3 summarizes the functional results obtained with blood lymphocytes stimulated 2 or 3 times with CD loaded or not with ascites vesicles. For patients 5, 6, 7, an arm was added testing the stimulation of lymphocytes by CD loaded with exosomes from cell lines

  autologous from a liquid volume identical to that of ascites vesicles.

  Cytotoxicity is achieved with stimulated Lymphocytes. The results presented are

  obtained at effector / target ratios equivalent to 50: 1, in the presence of cold K562.

  Standard deviations are written in parentheses. Interferon secretion tests are obtained by co-cultivating stimulated lymphocytes with or without autologous tumor cells for 24 hours. The rates are expressed in pg / ml. The tumor-specific interferon-y levels are obtained by subtracting the interferon-secreted spontaneously by lymphocytes from the interferon-y secreted by lymphocytes in the presence of tumor cells. "Tum Auto" for autologous tumor; "ExAs" for exosome derived from ascites; "ExLi" for exosome derived from cell lines. In all patients, a five to twelve fold increase in the absolute number of CD3 / CD8 + cells was observed after three stimulations carried out with CDs loaded with exosomes derived from ascites (ExAs). For six of the seven patients, lymphocytes stimulated with CD loaded with ExAs, an increase in the frequency and "priming" of the CTL precursor was demonstrated. For five of the patients, it was observed that the lymphocytes stimulated with CD loaded with ExAs are able to lyse autologous tumor cells and secrete IFN-gamma after 24 h of mixed lymphocyte / tumor coculture significantly , these results not having 2s - been observed for the lymphocytes stimulated with uncharged CD. These results were confirmed by ELISPOT analysis of IFN-gamma enumerating from 0.3% to 0.15% of tumor-specific lymphocytes after stimulation with CD loaded with ExAs in patients 5, 6 and 7. Recognition autologous tumor cells was tumor specific and restricted to MHC class I since lysis was performed with saturating amounts of cold K562 and completely canceled by an anti-MHC class I antibody (W6.32, see Figure 5B ). Direct lysis of K562 was attempted for one of the patients, but no significant lysis was obtained (cf. FIG. 5A). Similarly, the secretion of INF-gamma by in vitro stimulated lymphocytes (IVS) directed against homologous tumor cells could be successfully blocked by W6.32 (cf. figure 5B). Similar results have been obtained in proliferation tests against autologous tumor cells for some patients (not shown). The recognition of tumor cells is specific for the tumor since, for patients 1 and 2, CTL are not able to lyse irrelevant and allogenic tumor cell lines nor to induce the release of IFNgamma after coculture in the presence of these irrelevant cells (results not shown). On day 0, the lymphocytes before IVS showed no tumor-specific recognition characteristics. In addition, lymphocytes stimulated with exosomes alone in the absence of CD did not proliferate (results not shown). Interestingly, exosomes derived from ascites appear to be more immunogenic than exosomes derived from autologous tumor lines to induce CTL responses in vitro. Indeed, for two of the three patients tested, the specific levels of cytotoxicity and IFN-gamma secretion obtained using the exosomes derived from ExAs are significantly

  higher than those obtained using exosomes derived from tumor cells.

  Culture and ascites supernatants containing 106 cells / ml were ultracentrifuged

  to collect the exosomes used in IVS.

  By these results, it was highlighted new endogenous antigenic entities found in abundance in ascites and tumor effusions of cancer patients, these entities resemble the exosomal membrane vesicles described for the culture supernatants propagated ex viro shown as being antigenic and immunogenic in tumor-bearing mice (Raposo, G. et 2s - al., J. Exp. Med., 183 (3): 1161-72, March 1, 1996). In vivo, the exosomes could be relevant from an immunological point of view for given pathophysiological conditions since it has been demonstrated that these naturally secreted exosomes are bioactive, transferring tumor antigens to CD-DM to effectively activate n vitro

  CTL of cancer patients.

  The exosomes have been described as being membrane vesicles approximately 90 nm in diameter, originating from multivesicular endosomes spontaneously released into the extracellular microenvironment after fusion of the multivesicular body with the plasma membrane. On the morphological level, the membrane vesicles purified from tumor ascites typically resemble exosomes derived from tumor cell lines ex viro for example by their heterogeneous size, in the form of red blood cells on a grid, and sometimes aggregated, marked strongly by anti-CD63 antibodies and anti-MHC class I antibodies by immuno-electron microscopy. The biophysical properties such as for example their flotation at a density of between 1.14 and 1.18 glml in sucrose gradient is a characteristic property of these

  exosomes derived from ascites, characteristics used for their purification process.

  Biochemical studies reveal classic molecular markers such as those previously reported for exosomes purified from CD-MD and tumor cell cultures. Indeed, the HSP 70 found in the exosomes derived from ascites was previously described by the Srivastava team as being strongly immunogenic in viro and as being constitutive chaperone molecules overexpressed in late endosomes and exosomes derived from murine CD (Thery, C. et al., J. Cell Biol., 147: 599-610, 1999) and exosomes derived from tumor cell lines (Raposo, G. et al.). The exosomes derived from ascites also present a significant quantity of the two isoforms of lactadine, a membrane-associated protein with an affinity for CPA ligands (aV, B3 and aV, B5), isoforms which could target the exosomes towards their effector cells, the cells

dendritics (Thery, C. et al.).

  The systematic detection of the MART1 tumor antigen, already described as being contained in secreted exosomes of melanoma cell lines and the detection, if necessary, of MHC class II molecules support the fact that most of the exosomes derived from ascites are secreted naturally by tumor cells

rather than by CPA.

  The biological functions of exosomes and their relevance remain little detailed.

  In reticulocytes, the secretion of exosomes eliminates transterin and acetylcholinesterase receptors which are necessary for the differentiation function of red blood cells. Exosomes derived from B cells carry in abundance tetraspannin and MHC class II molecules, and selectively bind to follicular CD which do not neosynthesize MHC class II molecules in viro, suggesting a possible function of these lymphocyte exosomes B in the responses

  humoral immune systems (Denzer, K. et al., J. Immunol., 165 (3): 1259-65, 1 August 2000).

  Recently, it has been demonstrated that both exosomes derived from CD and from tumor cells transport and transit antigens (MHC class I and / or II functional complex - peptide and / or HSP) to the CD to effectively activate T cells in vitro or in vivo (André, F., article submitted for publication). In the event of a danger signal delivered to the periphery (tumor, pathogens), DCs have all the biological characteristics to be chemically attracted to acquire and process antigens at the periphery and to migrate to areas rich in T-cell lymph nodes

  in order to activate naval T cells (Banchereau, J. et al., Nature, 392: 245-52, 1998).

  o However, the molecular mechanisms or cellular pathways applied in the transfer of tumor antigens to CD remain unknown. It has been clearly shown in vitro that apoptotic bodies (Albert, ML et al., Nature, 392: 86-9, 1998), immune complexes (Regnault, A. et al., J. Exp. Med. , 189: 371-80, 1999) mRNAs (Nair, SK et al., Nat. Med., 6 (9): 1011-7, September 2000), HSP (Singh Jasuja, H. et al., J. Exp. Med., 191 (11): 1965-74, June 5, 2000) and tumor-derived exosomes (Raposo, G. et al.) Participate in the transfer of antigens from peripheral cells to CD. However, the antigen transfer allowing the restricted cross presentation MHC class I has never been described from biological materials purified directly from samples taken in viro. Here these results show from a MART1 tumor antigen model that exosomes derived from ascites carrying a non-relevant HLA haplotype can induce the activation of CTL clone once loaded on the CDs carrying the appropriate HLA-A2 allele. . In addition, in an autologous model system for which tumor antigens are not characterized (ovarian, lung or kidney cancer), CDs loaded with exosomes derived from ascites induce CTL activation for six of the seven patients tested. Therefore, it is conceivable that antigenic transfer may be mediated by such exosomes derived from local tumor, supporting the hypothesis that exosomes could be a pathway.

  potential to transfer antigens from the tumor compartment to the CPA network.

  Regardless of their in viro interest, exosomes derived from ascites may be a new strategy for the eX vivo loading of CD allowing the cross presentation of MHC class I and II restricted antigens, particularly in tumor models for which candidate tumor rejection antigens are scarce. Indeed, despite promising results obtained by the first clinical trials of cancer treatment (Nestle, FO et al., Nat. Med., 4: 328-32, 1998; Murphy, GP et al., Prostate, 39: 54-9, 1999; Hsu, FJ et al., Nat. Med., 2 (1): 52-8, January 1996; Kugler, A. et al., Nat. Med., 6: 332-6, 2000 ), an optimal strategy for CD awareness is required for broad spectrum cancer vaccines. So far, in ovarian cancers, few tumor antigens and CTL clones have been obtained, and few individuals have been able to benefit from immunotherapies based on adoptive transfer of TIL or LAK cells stimulated by IL -2 or activated macrophages (Steis, RG et al., J. Clin. Oncol. 8: 1618-29, 1990; Aoki, Y. et al., Cancer Res., 0 51: 1934-9, 1991; Freedman , RS et al., J. Immunother. Emphais Tumo. Immunol., 16: 198-210, 1994; Soda, H. et al., Oncol., 72: 211-17, 1999). New concepts such as the fusion of CDs with suspensions of tumor cells (Gong, J. et al., J. Immunol., 165: 1705-11, 2000) or of CDs loaded with tumor lysates (Santin, AD, and al., Am. J. Obstet. Gynecol. 183: 601-9, 2000) or with peptides eluted with acid (Santin, AD et al., Obstet. Gynecol., 96: 422-30, 2000) are awaiting the outcome of clinical trials. Here, it has been shown that the use of exosomes derived from ascites to sensitize CD is a new and effective applicable method since capable of inducing the activation of CTL directed against autologous tumor cells for six of the seven patients tested. , this using a process compatible with good laboratory practices. Results obtained (not presented) reveal on the one hand that this strategy can be implemented to clone new CTLs and thus to isolate new tumor antigens attached to rare tumor models, and on the other hand reveal the efficacy of peritoneal exosomes as an intradermal vaccine

  prophylactic against the establishment of syngeneic L1210 seremia.

  In conclusion, these results show that the isolated membrane vesicles (exosomes) derived from biological fluids taken in viro, in particular derived from ascites fluids, according to the present invention, have many advantages compared to the texosomes isolated from cell lines described in particular. in document WO 99/03499, as in particular: - they are secreted naturally and in greater quantity in vivo and can be purified easily in vitro; - The proliferation (or amplification, or expansion) of T cells induced by CD charged by the membrane vesicles according to the present invention, is at least equal to or if not greater than that induced by CD charged by texosomes derived from cell lines; s - the percentage of specific lysis which is obtained between the tumor cells and the self by the T cells stimulated (pulsed) by CDs loaded by the membrane vesicles according to the present invention, is generally much higher (2 to 5 times more) than that obtained for effector cells pulsed by CDs loaded with texosomes derived from cell lines (see Table 3); o - the MHC molecules carried by the membrane vesicles according to the present invention are in much greater number than those carried by texosomes derived from cell lines. The greater immunogenic power of the membrane vesicles according to the present invention is particularly demonstrated for patient 6 (cf.

table 3);

  - the membrane vesicles according to the present invention secrete a greater number of targeting molecules, in particular the isoforms of lactadherin or IgG, than the texosomes derived from cell lines; and - the membrane vesicles according to the present invention have an antigenic repertoire more representative of the repertoire of the initial population of cells of the fluid from which they originate than the texosomes originating from cell lines. It has thus already been demonstrated that the tumor-specific cytotoxic type immune response obtained using tumor cell lines did not fully reflect the anti-tumor activity exerted in vivo by the tumor (Dutour et al., J. of Immunol ., 158

(8), 3787-95, 1997).

  The membrane vesicles according to the present invention, in particular those isolated from ascites fluid, can therefore and advantageously be used for therapeutic or diagnostic purposes, in particular as an autologous or allogenic vaccine, but also as a loading method. dendritic cells to

therapeutic aim.

j 2827872

Claims (41)

  1. Process for the in vitro preparation of membrane vesicles isolated from their natural environment from a sample previously taken in vivo from a sample in a mammal? in particular in humans, characterized in that it comprises the following stage: a) the ir vitro isolation of said membrane vesicles from said sampled sample, said sample being a sample of a biological fluid capable of containing in   suspension of membrane vesicles.   2. Method according to claim 1, characterized in that the sample is a sample chosen from the following biological fluids: an aDdominal fluid, a peritoneal fluid, a pleural fluid, a cerebrospinal fluid, whole blood, urine   or any fluid, effusion or effusion emanating from a cavity of said mammal.   3. Method according to claim 1 or 2, characterized in that the sample is a sample of exudative or transudative fluid.   4. Method according to one of claims 1 to 3, characterized in that   the sample is a sample of exudative or transudative fluid taken from a cavity and resulting from cancer, an inflammatory reaction, a transplant, or   a gene transfer or a chemical.   5. Method according to one of claims I to 4, characterized in that   the bioIogic sample is a sample of ascites fluid.   6. Method according to claim 5, characterized in that the sample   biological is a sample of tumor ascites fluid from cancer patients.   7. Method according to one of claims 1 to 6, characterized in that   the isolation of the membrane vesicles is carried out by centrifugation, electrophoresis,   chromatography, nanofiltration and / or immunocapture.   8. Membrane vesicles isolated from their natural environment   capable of being obtained by a process according to one of claims I to 7.   9. Membrane vesicles according to claim 8, characterized in that they mainly contain membrane vesicles with a diameter between   and 150 nm, preferably between 50 and 100 nm and between 60 and 90 nm.   10. Membrane vesicles according to claim 8 or 9, characterized in that said membrane vesicles carry molecules involved in the presentation of antigens, in particular MHC class I and / or II molecules, and / or HSPs and / or antigens, especially tumor antigens, and / or molecules involved s in antigen targeting.   1 1. Membrane vesicles according to one of claims 8 to 10,   characterized in that they do not contain gp96 glycoprotein.   12. Use of membrane vesicles according to one of claims 8 to   I 1 for the loading and / or activation in vitro of antigen presenting cells,   0 including dendritic cells.   13. Use of membrane vesicles according to claim 12, characterized in that said membrane vesicles and said presenting cells   antigens are autologous or syngeneic.   14. Use of membrane vesicles according to claim 12, characterized in that said membrane vesicles and said presenting cells antigens are allogenic.   15. Use of membrane vesicles according to one of claims 8 to   11 for in vitro stimulation and, if necessary, in vitro amplification or anergisation of T lymphocytes or also for in vitro stimulation and, if necessary, in vitro amplification of NK or NKT cells specific for antigens contained in said membrane vesicles.   16. Use of membrane vesicles according to claim 15, characterized in that said membrane vesicles and said T lymphocytes or   NK or NKT cells are autologous or syngeneic.   17. Use of a suspension of membrane vesicles according to claim 15, characterized in that said membrane vesicles and said   T lymphocytes or NK or NKT cells are allogenic.   18. Process for the in vitro preparation of antigen presenting cells loaded with membrane vesicles, characterized in that it comprises the following steps: a) in vitro incubation of antigen presenting cells in the presence of vesicles   membranes according to one of claims 8 to 11; and   b) the recovery of said cells presenting antigens loaded with vesicles membranes thus obtained.   19. Method for preparing antigen presenting cells according to claim 18, characterized in that the antigen presenting cells in step a)   s are dendritic cells, in particular immature cells.   ! 20. A method of preparing antigen presenting cells according to claim 18, characterized in that the antigen presenting cells in step a) are dendritic cells obtained from monocyte or bone marrow precursors. o 21. A method of preparing antigen presenting cells according to claim 18, characterized in that the antigen presenting cells in step a)   are dendritic cells from immortalized dendritic cells.   22. Method for preparing antigen presenting cells according to one   claims 18 to 21, characterized in that said membrane vesicles and   is said antigen presenting cells are autologous or syngeneic.   23. Method for preparing antigen presenting cells according to one   claims 18 to 21, characterized in that said membrane vesicles and   said antigen presenting cells are allogenic.   24. Antigen presenting cell loaded with membrane vesicles   capable of being obtained by a process according to one of claims 18 to 23.   25. Isolated dexosomes secreted by antigen-presenting cells according to claim 24.   26. Process for the in vitro preparation of activated NK or NKT T cell lymphocytes or anergic T lymphocytes, characterized in that it comprises the following steps 2s: a) the in vitro incubation of NK or NKT cell T lymphocytes in presence of antigen presenting cells loaded with membrane vesicles according to claim 24 or dexosomes according to claim 25; and b) recovering said T lymphocytes NK or NKT cells thus activated or from said anergic T cells.   27. A method of in vitro preparation of NK or NKT T cell lymphocytes according to claim 26, characterized in that the antigen presenting cells, f 2827872 or their dexosomes, and said T lymphocytes or NK or NKT cells, are autologous or syngemques.   28. T lymphocytes NK or NKT cells capable of being obtained by the   method according to claim 26 or 27.   29. Use of membrane vesicles according to one of claims 8 to   11, antigen presenting cells according to claim 24, dexosomes according to claim 25, or T lymphocytes according to claim 28, for the preparation of a pharmaceutical composition intended to be administered in a patient and to induce or increase a CTL-type immune response specific for antigens   o contained in said membrane vesicles according to one of claims 8 to 11 and   in which use, said membrane vesicles, presenting cells   of antigens and T-lymphocytes are autologous or syngeneic to the patient to be treated.   30. Use of membrane vesicles according to one of claims 8 to   11, antigen presenting cells according to claim 24, dexosomes according to claim 25, or NK or NKT cells according to claim 28, for the preparation of a pharmaceutical composition intended to be administered to a patient and to induce or increase an innate NK or NKT response specific for antigens contained in said membrane vesicles according to one of the   claims 8 to 11 and in which use, said membrane vesicles,   antigen presenting cells and NK or NKT cells are autologous or syngenic of the patient to be treated.   31. Use according to claim 29 or 30, for the preparation of a pharmaceutical composition intended for the prevention or treatment of affected patients of a cancer.   32. Use of antigen presenting cells, in particular dendritic cells, taken from a first patient (the recipient) or from a patient whose antigen presenting cells are syngeneic to the antigen presenting cells of the first patient, for the preparation of a pharmaceutical composition intended to be administered in this first patient, characterized in that said antigen-presenting cells have been previously loaded in vitro with   membrane vesicles according to one of claims 8 to 11 isolated from a fluid   biological sample taken from a second patient (the donor), said recipient and donor being allogenic.   33. Use of T lymphocytes taken from a first patient (the recipient) or from a patient whose T lymphocytes are syngeneic to the T lymphocytes of the first patient for the preparation of a pharmaceutical composition intended to be administered in this first patient, characterized in that said T lymphocytes were previously activated in viro avoe of antigen presenting cells   previously loaded with membrane vesicles according to one of claims   8 to 11, said membrane vesicles having been isolated from a biological fluid o taken from a second patient (the donor), said recipient and donor being allogenic. 34. Use of antigen presenting cells according to claim 32 or T lymphocytes according to claim 33, for the preparation of a pharmaceutical composition intended to induce tolerance by said recipient of an allogenic graft taken from said donor. 35. Use of antigen-presenting cells according to claim 34 for the preparation of a pharmaceutical composition intended to induce tolerance by said recipient of an allogenic graft taken from said donor by the method known as "adoptive transfer of dendritic cells from the catcher previously loaded in   in vitro by donor membrane vesicles ".   36. Use of T lymphocytes according to claim 34 for the preparation of a pharmaceutical composition intended to induce tolerance by said recipient of an allogeneic graft taken from said donor by the method called "adoptive transfer of T lymphocytes from the recipient previously activated in vivo by dendritic cells of the recipient previously charged (pulsed) by vesicles donor membranes ".   37. Use of membrane vesicles according to one of claims 8 to   11, antigen presenting cells according to claim 24 or dexosomes according to claim 25, for the ex vivo selection of a repertoire of T lymphocytes capable of recognizing specific antigens contained in said vesicles   membranes according to one of claims 8 to 11. 1 2827872   38. Medicinal product containing one or more as active substance   membrane vesicles according to one of claims 8 to 11, presenting cells   antigens according to claim 24, dexosomes according to claim 25 or T lymphocytes NK or NKT cells according to claim 28, in combination with a   s pharmaceutically acceptable vehicle.   39. Medicament according to claim 38 for the treatment of cancers.   40. Medicament according to claim 38 or 39, characterized in that it   further includes a synthetic or natural immunostimulating adjuvant.   41. A method of in vitro diagnosis of cancer and / or its development in a patient, characterized in that the presence of markers or specific precursors of cancer and / or its development on the surface is determined and / or in the cytosol of   membrane vesicles according to one of claims 8 to 11.   42. In vitro diagnostic method according to claim 41, characterized in that the membrane vesicles originating from a biological fluid taken from the patient, are originating from an ascites or effusion fluid, in particular emanating from a cavity, such   than a peritoneal, abdominal or pleural cavity.   43. A method of in vitro diagnosis according to claim 41 or 42, characterized in that the determination of the presence of markers or precursors specific for cancer and / or of its evolution is carried out by an immunoenzymatic technique of ELISA type, Western Blot , immunocapture or immunoelispot using antibodies specific for these markers.   44. Use of membrane vesicles (texosomes) derived from samples   of ascites tumor fluid according to one of claims 8 to 11, of CPA, in particular of   dendritic cells, according to claim 24, for the manufacture of a test   2s of delayed hypersensitivity of cancer.   45. Use of membrane vesicles (texosomes) derived from samples   tumor ascites fluid according to one of claims 8 to 11, CPA, in particular   dendritic cells according to claim 24 as a diagnostic research tool   frequency of specific CTL precursors.   46. Process for the in vitro preparation of a bank of antibodies of murine origin directed against human antigens carried by membrane vesicles   according to one of claims 8 to 11, in particular tumor, from a sample 1 2827872   previously taken in viro from a SCID mouse, in particular SCID / NOD, of a sample of peritoneal ascites fluid resulting from the intraperitoneal implantation of a tumor, of a tissue or of human tumor cells, said sample containing said antibody bank, characterized in that it comprises the following step:   a) the purification of the antibodies contained in said sample.   47. Process for the in vitro preparation of a bank of isolated texosomes of murine and human origin from a sample previously taken in vivo from a SCID mouse of a sample of peritoneal ascites fluid resulting from implantation 0 intraperitoneally of a tumor, tissue or human tumor cells, characterized in that it comprises the following stage: a) in vitro isolation of said texosomes from said sample taken by centrifugation, electrophoresis, chromatography , nanofiltration and / or by immunocapture. 48. A process for preparing texosomes modified in vitro, characterized in that it comprises the following steps: a) in vitro isolation of mernbran vesicles (texosomes) from a sample   tumor ascites fluid by a method according to one of claims 1 to 7;   b) the elution, in particular acidic, of said natural texosomes obtained in step a); c) bringing said eluted natural texosomes into contact with poptides, glycolipids or phospholipids of interest which it is desired to load into the texosomes; and