Cellular vesicles denoted as 'exosomes', their preparation and use in the stimulation of an immune response

The invention relates to a new cell sensitization process antigen presenting, new means for the implementation of the process, and novel membrane vesicles having immunogenicity.

Since the demonstration of the existence of cytotoxic CD8 + T cells specific for tumor antigens presented in the context of class I molecules (Rosenberg et al., 1996; Boon, 1992), several laboratories have shown that immunotherapy anti tumor is an effective therapeutic strategy in animal models (Pardoll, 1995). The principle of immunotherapy is to induce an effective immune response against tumor-specific antigens. At present, this could be accomplished in different ways. First, tumor cells expressing recombinant costimulatory molecules and / or immunomodulatory cytokines are capable of stimulating anti-tumor responses capable of eradicating solid tumors<i>in vivo</i> (Zitvogel et al., 1996 [a]). Similarly, derivatives of the peptides of tumor antigens (or exogenous antigens expressed in tumor cells) injected in different chemical forms including the use of liposomes or viruses (adenovirus or poxvirus, for example) as vectors, are capable of regressing tumors. Finally, professional antigen-presenting cells, such as dendritic cells sensitized with peptides derived from tumor antigen, reinjected<i>in vivo</i> induce potent anti-tumor responses and regression of solid tumors established in mice (Butler et al., 1995).

Amirogena et al (references, p.59) have described the accumulation, in the endocytic compartment B cells, vesicles expressing MHC class II.

Zitvogel et al (1998) divulgent the exosones derived dendritic cells.

Immunotherapy based on the use of dendritic cells has been shown effective in studies in mice. Therefore, this therapy has recently been implemented clinically. In the United States, trials are underway to demonstrate that dendritic cells loaded with tumor peptides significantly increase the frequency of specific cytotoxic T cells (CTL).

A first limitation of this approach is the sensitization of the dendritic cells with peptides derived from tumor antigens. Indeed, in the majority of tumor specific antigens have not been identified. Specific antigens of the tumor are known only in cases of tumors induced by virus (cervical carcinoma), in cases of melanoma (self antigens, mutated antigens, differentiation antigens) or in a small percentage of tumors breast (oncogenes or products of tumor suppressor genes that have undergone mutations). However, the direct involvement of these peptides or tumor antigens in the elimination of tumors in humans remains to be demonstrated. New methods of sensitizing antigen presenting cells such as dendritic cells are therefore necessary. These methods are designed to induce specific anti-tumor responses in the context of class I molecules and MHC class II.

Most sensitization methods of dendritic cells used now use peptides corresponding to epitopes presented in association with class I molecules and identified in tumor cells through specific CTL clones of the tumor. However, these methods are unlikely to be optimal because they do not account epitopes recognized in the context of class II molecules that are critical for the proliferation of T helper cells required to obtain optimal cytotoxic responses. In addition, epitopes presented by the tumor cells and those presented by antigen presenting cells (eg dendritic cells) are probably not the same. Finally, tumor peptides recognized by CTL are only available to a small percentage of patients with class I molecules suitable haplotype.

The method ideally awareness, so as to be applicable to any tumor with a minimal risk of immunoselection, should not be restricted to a small number of identified tumor antigens. Similarly, such a method should use protein antigens intact rather than peptides, to allow the dendritic cell to prepare and present the right combination of peptides in association with molecules of class I and class II, and this for any individual.

Recently, Gilboa and coworkers (Boczkowsky et al., 1996) were able to show that messenger RNA prepared from tumor biopsies loaded in the dendritic cells may have an anti-tumor effect <i>in vivo.</i> However RNAs are very unstable and potentially interesting RNA amount compared to total RNA is probably very low. . Zitvogel et al, (. Zitvogel et al, 1996 [b]) showed that tumor peptides prepared from a tumor acid eluate (acidic peptide eluate: EPA) can be used to load dendritic cells. These cells thus charged, once injected, have the ability to regress tumors. However, in the case of tumors not expressing class I molecules (which represent the majority of metastatic human tumors), or in the case of tumors which can not be dissociated into a cell suspension, the approach using eluates acids is not very effective and is not reproducible.

A second limitation to immunotherapy based on the use of dendritic cells is linked to phenotypic changes that can occur when these cells are maintained in culture, or subjected to different treatments. This may indeed lead to very homogeneous cell populations and inadequately characterized for therapeutic use.

There is therefore a real need to improve the awareness of methods of antigen presenting cells, to increase the effectiveness of these approaches and expand their applications, and to develop new means of vectorization of antigens or other molecules.

The present invention provides solutions to these issues. The present invention indeed aim to provide new outreach methods of antigen-presenting cells, including dendritic cells, and the identification, isolation and characterization of the novel membrane vesicles having remarkable immunogenic properties.

One aspect of the invention is particularly to provide a novel reproducible process cell sensitization antigen presenting with tumor antigens.

One of other aspects of the invention is to provide a novel reproducible process cell sensitization by antigen presenting tumor antigens, wherein it is not necessary that the tumor antigens are known.

Another aspect is to provide the means to establish a bank of tumor antigens.

Another aspect of the invention resides in the membrane vesicles, the lipid produced by the tumor cells, and endowed with immunogenic properties, as well as their use for the production of antigen banks, sensitization of antigen presenting cells or the vectorization of antigens, particularly in the context of immunotherapeutic approaches.

In this regard, a first object of the invention relates to a vesicle derived from tumor cell having the following characteristics:<ul><li>it is freed from its natural environment,</li><li>it comprises a lipid bilayer (designated by "surface") which surrounds a cytosolic fraction,</li></ul>and eventually,<ul><li>it has on its surface class I molecules of the major histocompatibility complex (MHC) and of class II major histocompatibility complex (MHC), optionally loaded with antigenic peptides and / or adhesion molecules and / or molecules lymphocytic costimulatory and / or,</li><li>it contains in its cytosolic fraction tumor antigenic molecules and / or immunomodulatory and / or chemo-attractors and / or hormones and / or nucleic acids.</li></ul>

Secretion vesicles by cells is a phenomenon described in the prior art (reticulocytes, B lymphocytes, macrophages). These vesicles are usually designated by the generic term "exosome" which reflects their mechanism of production by exocytosis of internal vesicles. However, the physiological role of these vesicles was not really established. Moreover, structural characteristics, properties and functions of these vesicles vary depending on the cell type from which they arise.

Unexpectedly, the inventors have now demonstrated that the tumor cells are capable of secreting vesicles have particularly advantageous immunogenic properties. These vesicles generally correspond to an internal vesicle contained in an endosome of a tumor cell and secreted by said tumor cell following the merger of the outer membrane of the endosome aforesaid with the cytoplasmic membrane of the said tumor cell. Because of this formation mechanism of their cell of origin and their characteristics and unique functional properties, these vesicles are designated in what follows by the term "texosome".

The expression "freed from its natural environment" means that the bladder is physically separated from the cell from which it originated, or it is partially isolated or purified. Generally, the bladder is produced by the cell by exocytosis, then partially isolated or purified so as to obtain a composition enriched. This phrase can also mean that not only the vesicle was secreted by the cell during the fusion of multivesicular endosomes with the plasma membrane, but it is no longer surrounded by the soluble components which are in the lumen of the endosome , or is free of intact cells. The term "derived from tumor cell" means that the bladder has structural elements of a tumor cell. This bladder is generally "derivative" of a tumor cell in the sense that it is produced, at least in part, then released by a tumor cell, at a given stage of its development.

According to an advantageous embodiment, the texosomes of the invention exhibit MHC molecules loaded with antigenic peptides and / or express adhesion molecules and / or molecules express lymphocytic costimulatory, but lack in their fraction cytosolic tumor antigenic molecules and immunomodulatory and nucleic acids.

According to another advantageous embodiment, the texosomes of the invention are such that MHC molecules are "empty", that is to say not loaded with antigenic peptides and texosomes comprise in their cytosolic fraction, molecules tumor antigens, immunomodulators and / or nucleic acids. Texosomes having empty MHC molecules may be obtained either from tumor cells having such a deficiency for the peptide transporter (TAP), or by washing texosomes or tumor cells, in order to elute the peptide molecules associated MHC.

According to an advantageous embodiment of the invention, the texosomes of the invention are such that MHC molecules are loaded with antigenic peptides and / or express adhesion molecules and / or lymphocytic costimulatory molecules and texosomes contain in their cytosolic fraction tumor antigenic molecules, immunomodulators and / or nucleic acids.

The term "tumor cell" generally includes any cell derived from a tumor, for example a solid or liquid tumor, and transformed or immortalized cells in vitro. It is preferably a solid tumor, ascitic or hematopoietic response.

These include, for example, cells of malignant melanoma type of cancer (from primary lines established "<i>ex vivo</i>"Or of dissociated cells from the specimen) which express on their surface peptides as MART-1 / Melan-A, in the MHC context - class 1 HLA-A 02-01, and containing the protein antigen MART -1

Also exemplary cells from kidney cancer (clear cell adenocarcinoma) or leukemias whose cells express specific translocation products.

Thus, antigenic peptides likely to load MHC molecules derive, for example the following antigens: those from melanomas such as MART-1, tyrosinase, MAGE-1/2/3, P53 (in different tumors) or Her2 / Neu , PSA, CEA or PSMA. Other tumor antigens are cited for example in the article by<nplcit id="ncit0001" npl-type="s"><text>Rosenberg (Immunology Today 18 (1997) 175</text></nplcit>).

More generally include fusion products / translocation of oncogenes or anti-oncogenes, or differentiation antigens or peptides of self or mutated peptides.

Molecules by lymphocyte co-stimulation molecules such describes that give T lymphocytes signals complementary to those given when interacting molecule complex class I and II - peptide with the T cell receptor

These include, for example: CD80, CD86, ICAM, LFA, CD40, certain members of the TNF family R and adhesion molecules or chemo attraction (allowing contact between the professional antigen-presenting cell and the effector lymphocytes, or intracellular transport / localization specific ( "trafficking / homing") of other cells in the vaccine or inflammatory site.

Tumor antigenic molecules contained in the cytosol or presented by the texosomes derived from proteins expressed selectively and / or abundant in the tumor cells.

Immunomodulators which may be present in the cytosol of texosomes are for example:<ul><li>TNF-α, or</li><li>Interleukin 1, or</li><li>Interleukin 15, or</li><li>C-CR (chemokines).</li></ul>

The nucleic acids may be present in the cytosol of texosomes come from the tumor cell itself. These nucleic acids are found in the cytosol of texosomes direct consequence of their formation mechanism. It may also include heterologous nucleic acid.

More specific features of the invention texosomes are:<ul><li>these are small membrane vesicles of 60 to about 100 nm, usually 60 to about 90 nm, especially 60 to 80 nm, secreted by the tumor cells,</li><li>they possess molecules usually present in the endosomes,</li><li>they contain tumor antigens, such as MART-1 in the case of melanoma cells,</li><li>they are devoid of dead cells and / or cell debris</li><li>they are free of contaminants such as membrane contaminants, endoplasmic reticulum, Golgi apparatus, mitochondria or nuclei of constituents,</li><li>they bear at their membrane molecules of class I / II functional loaded with tumor antigen peptides,</li><li>they can stimulate in vitro the proliferation of specific T cells,</li><li>they can sensitize <i>in vivo</i> and <i>in vitro</i> dendritic cells can then activate specific T cells of the tumor,</li><li>they have the ability when inoculated <i>in vivo,</i> in particular intradermally, to regress established solid tumors,</li><li>they bear lymphocytic costimulatory molecules such as CD40 and CD80, and / or,</li><li>they contain protein ( "heat-shock") HSP70,</li><li>they lack gp96 protein,</li><li>they contain interleukins or chemo-attractants or immunomodulators.</li></ul>

Another interesting feature is texosomes they contain phosphatidylserine in their external layer. Phosphatidylserine (PS) is one of major components of cell membranes, normally present overwhelmingly in the inner leaflet of the lipid bilayer. In certain circumstances, such as early stages of apoptosis, PS is re-distributed to the outer leaflet. The presence of PS in the outer leaflet of the cytoplasmic membrane of apoptotic cells is a signal recognition by macrophages. To determine whether the PS is exposed at the surface of texosomes exosome preparations purified from supernatants of FON human melanoma cells were analyzed by the method described by<nplcit id="ncit0002" npl-type="s"><text>Aupeix et al. .. (J. Clin Invest 99: 1546-1554, 1997</text></nplcit>). The content of phosphatidylserine in the outer leaflet of the FON samples (containing 390 microg / ml protein) is 460 nM PS. Exosomes thus contain considerable quantities of PS in their external layer.

Tests to verify that the texosomes of the invention possess molecules usually present in the endosomes consist of electron microscopy and immunoblotting (Western Blot). These tests show that the texosomes of the invention express the transferrin receptor, the LAMP molecules ( "lysozome associated membrane protein": membrane-associated protein lysozome) class of molecules I / II, tumor antigens.

A test to check that the texosomes of the invention lack contaminants is electron microscopy and immunoblotting with anti-calnexin antibody which is present in the endoplasmic reticulum.

A test to check that the texosomes bear at their membrane class I molecules / functional II loaded with tumor antigenic peptides consists of antigenic presentation to T lymphocytes specific for antigens of the relevant tumor (proliferation assays clones specific T d antigen and MHC class I-restricted).

One can also use a test of secretion of cytokines (IFN, GM-CSF, TNFβ) by the above-mentioned T clones.

A test to verify that there is awareness <i>in vivo</i> and <i>in vitro</i> dendritic cells capable of activating T cells specific to the tumor is given by <figref idrefs="f0011">Figure 7</figref> (Proliferation assay and / or cytokine secretion by the T clones specific antigens by cross-sensitization method ( "cross-priming"): texosomes of a MART-1 positive tumors, HLA-A2- loaded onto a dendritic cell MART-1, HLA-A2 +).

A test to check that the texosomes have the ability, when inoculated, in particular intradermally, to regress established solid tumors is given in <figref idrefs="f0010">6</figref>.

For example, an injection used from 10 to 40 micrograms of tumor texosomes intradermal ipsilateral side to the established tumor from 3 to 10 days; observed the carrier of the animal tumor and the progressive disappearance of the established tumor within 7 to 10 days (mice type rodents).

An advantageous texosome of the invention is constituted by a texosome as defined above and having on its surface molecules of class I and / or MHC class II, optionally loaded with antigenic peptides and containing in its cytosolic fraction of the molecules tumor antigenic. More particularly, the preferred texosome also comprises one or more molecules of lymphocytic costimulatory and / or HSP70 protein. In a particular embodiment, the texosome lacks the protein gp96.

According to an advantageous embodiment, the invention relates to a texosome as defined above,<ul><li>expressing on its surface molecules of class I and class II major histocompatibility complex (MHC), and / or tumor antigens characteristic and / or molecules of lymphocytic costimulatory / adhesion and / or immunomodulators, and / or chemo-attractants, exogenous with respect to the tumor cell, the exosome is derived, or</li><li>containing tumor antigens and / or immunomodulatory and / or nucleic acids or cytotoxic agents or exogenous hormones relative to the tumor cell from which the exosome is derived.</li></ul>

The invention also relates to a texosome preparation process as defined above. This method advantageously comprises a step of providing a biological sample and an isolation step of texosomes from said sample.

The biological sample is advantageously constituted of membrane fractions, culture supernatants or lysates of tumor cells, or fresh tumor suspensions.

The biological sample may be derived from tumor operative parts after surgical excision (1 case) or of tumor-bearing organs (surgically excised organ) (2nd case), which is treated by mechanical disruption (1 case) or by prolonged infusion (2nd case).

The final cell suspension was treated in the same manner as the culture supernatants.

It can also be cells treated by freeze / thaw in several successive cycles.

According to an advantageous embodiment, the biological sample used in the method of the invention is:<ul><li>an efferent blood sample from the vein of the isolated tumor-bearing organ, or</li><li>a plasma or serum sample of the circulating blood of a patient, or</li><li>the drainage of product (physiological saline optionally containing dexamethasone or cytotoxic agent stimulating exocytosis texosomes) a surgically excised organ and treated <i>ex vivo</i> by isolated-perfused circuit for the drainage of the tumor it bears, or</li><li>the supernatant of a tumor explant dissociated <i>in vitro.</i></li></ul>

The efferent blood sample of the isolated tumor-bearing organ corresponds to 20 to 50 ml of blood from the efferent vein of the tumor main organ, taken prior to surgical ablation procedure.

Drainage product of an organ excised surgically and treated <i>ex vivo</i> by isolated-perfused circuit place as follows.

In the case member having an artery afferent and efferent vein, artery is characterized by a plastic tubing connected to a Trendelenburg bag containing physiological serum with optionally other agents. It drains the liquid member and the spring by another manifold cathétérisant the vein, in a dependent (eg in the case of renal cancer, or a brain glioblastoma).

Dexamethasone, possibly contained in the drainage product, is to increase the cellular stress and exocystose texosomes out of the tumor cell.

The supernatant of a tumor explant dissociated <i>in vitro</i> is obtained as follows:<ul><li>one proceeds to the mechanical dissociation of the tumor leading to a unicellular suspension containing tumor cells and cells of the tumor stroma and immune system cells; this suspension may be irradiated and recovered for differential centrifugations.</li></ul>

As indicated above, in a particular mode of implementation of the inventive method, the biological sample may be treated with one or more agents stimulating the production of texosomes. This treatment may include the addition of steroid agents (eg dexamethasone), pharmacological agents (eg, cytotoxic agents such as taxanes, cisplatin, etc.), agents that increase the amount of endosomes multivesicular and / or irradiation of the sample.

Regarding radiation, it should be sufficient to induce the cytostatic action of the tumor cells. Irradiation of tumor cells can be made before the cell culturing or during or after the cultivation of tumor cells. Furthermore, it should be irradiated when the tumor cells are alive, that is to say:<ul><li>either on the tumor-bearing excised organ prior to infusion,</li><li>or on cells in culture, </li><li>or on the cell suspension dissociated mechanically; but in any event before tumor cell stress due to hypoxia / vascular necrosis / dehydration.</li></ul>

As regards the treatment of steroid using, it can cause cell activation leading to exocytosis texosomes.

As regards the treatment with pharmacological agents, it allows:<ul><li>modify the cytoskeleton and rearrange the intracellular compartments to disrupt the phenomena of internalization and exocytosis,</li><li>depolymerize microtubules.</li></ul>

As regards the treatment with an agent capable of increasing the quantity of multivesicular endosomes, it occurs during cell culturing, as agent include nocodazole (drug to depolymerize microtubules) bafilomycin ( drugs inhibiting vacuolar ATPases) ( "<nplcit id="ncit0003" npl-type="s"><text>Bafilomycins: A class of inhibitors of membrane ATPase from microorganisms, animal cells, and plant cells "(1988) Proc Natl Acad Sci USA 85:.... 7972-7976</text></nplcit>)

An advantageous process for the preparation of texosomes according to the invention is carried out:<ul><li>a) from cultures of tumor cells, and comprises:<ul><li>irradiation, an intensity sufficient to induce the cytostatic action of the tumor cells and not exceeding 15,000 rads advantageously about 10,000 rads, the tumor cells before, during or after culturing them, or</li><li>a treatment during culture of the tumor cells, using steroids, for example dexamethasone or cytotoxic agents, such as 5-fluorouracil (5-Fu) or cisplatin, docetaxel, anthracycline, spindle poison, antipyrimidique, or interleukin for example IL10, IL2, IL15, GM-CSF,</li></ul>or,<ul><li>treatment with an agent capable of increasing the quantity of multivesicular endosomes, for example nocodazole (<nplcit id="ncit0004" npl-type="s"><text>Gruenberg J. et al, (1989) "Characterization of the Early Endosome and Putative endocytic Carrier Vesicles In vivo and with an Assay of Vesicle Fusion in vitro" The Journal of Cell Biology 108:. 1301-1316</text></nplcit>) And therefore increase production texosomes,</li></ul></li><li>b) from a saline sample draining a surgically excised organ and treated <i>ex vivo</i> by isolated-perfused circuit for the drainage of the tumor it bears or</li><li>c) from the supernatant of a tumor explant dissociated <i>in vitro</i> and comprising:<ul><li>a treatment with steroids, for example dexamethasone or cytotoxic agents, for example 5-fluorouracil (5-Fu); cisplatin, taxanes, or interleukin for example IL-10, IL-2, GM-CSF.</li></ul></li></ul>

The isolation step texosomes can be carried out using various techniques such as centrifugation, chromatography, electrophoresis, nanofiltration etc. This is for example of differential centrifugation of membrane fractions of culture supernatants or lysates of tumor cells or fresh tumor suspensions and recovery or fraction (s) containing said exosomes (<nplcit id="ncit0005" npl-type="s"><text>Raposo et al., J. Exp. Med. 1996, 183: 1161-1172</text></nplcit>). In this particular implementation, the membrane fraction supernatants is that obtained after ultracentrifugation at 100,000 g. It may advantageously be a liquid phase electrophoresis, which allows the separation of biological materials according to their charge. The following examples show that this technique can be advantageously used for the isolation of texosomes with good yields. This technique is also particularly advantageous industrially.

The invention also relates to a texosome preparation process as defined above, further comprising:<ul><li>or the genetic modification of tumor cells with exogenous genes coding for molecules of class I and / or class II major histocompatibility complex (MHC), and / or genes encoding tumor antigens characteristic and / or genes coding for molecules of costimulatory / adhesion or attractant chemokines, the products of these exogenous genes can be expressed on the surface texosomes and / or be sequestered within texosomes,</li><li>either modification <i>in vitro</i> products texosomes by tumor cells, such as the introduction (by electroporation, by fusion with a synthetic liposome, recombinant virus or chemical method), proteins or nucleic acids or pharmaceutically defined medicines in and / or with texosomes.</li></ul>

The invention also relates to the texosomes obtainable by the process described above.

The texosomes transfected tumor cells as indicated above is collected and used as tumor vaccines.

The modified texosomes <i>in vitro</i> as indicated above are designed to deliver the exogenous material to a target cell <i>in vitro</i> or <i>in vivo.</i>

Regarding the fusion with a synthetic liposome, this process is carried out for example as described in Nabel et al. (1996) or in<nplcit id="ncit0006" npl-type="s"><text>Walker et al. (1997, Nature 387, pages 61 and following</text></nplcit>).

The invention also relates to antigen-presenting cells, including B cells, macrophages, monocytes or dendritic cells, loaded with texosomes as defined above. It is advantageously dendritic cells.

Dendritic cells include the following attributes:<ul><li>in tumor models which do not express class I molecules and therefore do not have the ability to stimulate CD8 + T cells, dendritic cells loaded with tumor cell texosomes may present these tumor peptides to cytotoxic T cells in the context of class I MHC molecules, (characteristic # 1)</li><li>loaded dendritic cells of texosome tumor cells injected intravenously or subcutaneously are also very effective (characteristic # 2).</li></ul>

A test to highlight the characteristic # 1 is as follows.

In the human system, a texosome class I negative, incubated in the presence of a dendritic cell class I positive, can allow the stimulation of specific CD8 + T cell clones of antigen in the texosome (see <figref idrefs="f0011">7</figref>).

A test to highlight the characteristic # 2 is as follows.

In a murine system, where the tumor is classified negative I and where the texosomes are also lacking in class I molecules, these texosomes may, when they are incubated and loaded on dendritic cells, mediate anti-tumor immune response while only by intradermal injection, they do not.

The invention also relates to a presenting cells process for preparation of antigens as defined above, comprising the steps of incubation of antigen presenting cells in the presence of texosomes as defined above and recovery of the above cells antigen-presenting the aforesaid loaded texosome.

The invention also relates to presenting cells loaded with texosomes and capable of being obtained by the method described above.

The invention also relates to the use of texosomes as defined above for the sensitization of antigen presenting cells, including B cells, macrophages, monocytes or dendritic cells or for the stimulation of specific T lymphocytes.

The specification divulge a membrane vesicle freed from its natural environment, secreted by antigen-presenting cells loaded with texosomes as defined above.

To obtain these membrane vesicles defined above, may be used in a method comprising:<ul><li>a step of preparing a texosome as defined above, </li><li>a step of incubating a texosome with antigen presenting cells,</li><li>differential centrifugation of membrane fractions of step culture supernatants or lysates of the above antigen-presenting cells loaded with texosomes and</li><li>a step of recovering the fraction containing the aforesaid membrane vesicles.</li></ul>

The specification also divulge membrane vesicles as defined above and obtainable by the process described above.

Dexosomes advantageously comprise molecules lymphocyte co-stimulation, and in particular CD63 molecules and / or CD82 and / or CD86, preferably at least CD86. The studies presented in the examples show that the dexosomes are strongly marked by antibodies directed specifically against these costimulatory molecules.

Furthermore, analysis by electron microscopy shows that the dexosomes are homogeneous and have a diameter between 60 and about 100 nm, most often between 60 and about 90 nm.

Dexosomes further comprise one or more antigenic peptides and / or are obtained from immature dendritic cells.

The dexosomes may lack H2-M markers, li chain and calnexin (a specific marker of the endoplasmic reticulum).

On the other hand, the dexosomes may also include phosphatidylserine (PS) in their external layer. Thus exosome preparations purified from supernatants derived dendritic cells from bone marrow were analyzed by the method described by<nplcit id="ncit0007" npl-type="s"><text>Aupeix et al. .. (J. Clin Invest 99: 1546-1554, 1997</text></nplcit>). The content of phosphatidylserine to the outer leaflet of BMDC samples (containing 35 microg / ml of proteins), it is 80 nM PS. The dexosomes thus contain considerable quantities of PS in their external layer.

Dexosomes may be prepared using methodology comprising a first step of obtaining dendritic cells or a cell culture comprising dendritic cells, a second optional step, during which the cells can be sensitized to antigens of interest, and a third step comprising the production of dexosomes from these cell cultures. These different steps can be performed advantageously according to the methodologies described hereinafter.

Preparation of Dendritic Cells

The first step of the process comprises the provision of (a) culturing (s) dendritic cells. It may be cell cultures enriched in dendritic cells or cell cultures consisting essentially of dendritic cells. Advantageously, it is of course of human dendritic cells.

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 isolated directly in a differentiated form (Review by<nplcit id="ncit0008" npl-type="s"><text>Hart, Blood 90 (1997) 3245</text></nplcit>).

The production of dendritic cells from stem cells is illustrated for example by <nplcit id="ncit0009" npl-type="s"><text>Inaba et al. (J. Exp. Med. 176 (1992) 1693</text></nplcit>) <nplcit id="ncit0010" npl-type="s"><text>Caux et al. (Nature 360 ​​(1992) 258</text></nplcit>) or <nplcit id="ncit0011" npl-type="s"><text>Bernhard et al. (Cancer Res. 55 (1995) 1099</text></nplcit>). These work particularly show that dendritic cells can be produced by culture of bone marrow in the presence of colony stimulating factor Granocytes-Macrophages (GM-CSF) or, more precisely, from hematopoietic stem cells (CD34 +) by culturing in presence of a combination of cytokines (GM-CSF + TNF alpha).

The production of dendritic cells from monocyte precursors is illustrated for example by <nplcit id="ncit0012" npl-type="s"><text>Romani et al. (J. Exp. Med. 180 (1994) 83</text></nplcit>) <nplcit id="ncit0013" npl-type="s"><text>Sallusto et al. (J. Exp. Med. 179 (1994) 1109</text></nplcit>) <nplcit id="ncit0014" npl-type="s"><text>Inaba et al. (J. Exp. Med. 175 (1992) 1157</text></nplcit>) or <nplcit id="ncit0015" npl-type="s"><text>Jansen et al. (J. Exp. Med. 170 (1989) 577</text></nplcit>). These methods are essentially based on the collection of mononuclear cells in the blood and cultured in the presence of various combinations of cytokines. One particular method involves treating the precursor monocytes of the blood in the presence of combinations of cytokines such as Interleukin-4 + GM-CSF or interleukin-13 + GM-CSF, for example. This technique is also illustrated by Butler et al., 1995. Furthermore, it is also possible to treat the monocyte precursors with pharmacological agents of cell differentiation, such as activators of calcium channels.

Another approach for obtaining dendritic cell comprises isolating, from biological samples, dendritic cells already differentiated. This approach has been described for example by<nplcit id="ncit0016" npl-type="s"><text>Hsu et al. (Nature Medicine 2 (1996) 52</text></nplcit>). The methodology described by this team essentially consists of harvesting peripheral blood samples and process by different gradients and centrifugations to extract dendritic cells.

The methodology is based on the production of dendritic cells from monocyte precursors or bone marrow. These methods are illustrated in the examples. More particularly, it is preferred in the context of the present invention dendritic cells obtained by treating monocyte precursors (contained in blood or bone marrow) in the presence of GM-CSF + IL-4 combination or GM-CSF + IL -13.

Moreover, for the implementation of the present invention, it is particularly advantageous to use a population of dendritic cells comprising immature dendritic cells. Advantageously, a dendritic cell population is used consisted primarily (ie, at least 60%, preferably 70%) of immature dendritic cells. The immature dendritic cells corresponds to an early stage of their development, in which they exhibit a high endocytic activity and express low levels of class I and II molecules of the MHC and lymphocytic costimulatory molecules on their surface. Surprisingly, the inventors have found that only immature dendritic cells were capable of producing membrane vesicles in significant quantities. This discovery is all the more surprising that dendritic cells immature stage are known for their low capacity to stimulate T cells, and thus their low biological activity (<nplcit id="ncit0017" npl-type="s"><text>Cella, Nature London, 388 (1997) 782</text></nplcit>).

The first step of the process of the invention can advantageously include the preparation of a dendritic cell population comprising immature dendritic cells, especially from monocyte precursors, especially by treatment with a combination of cytokines such as GM-CSF + IL-4 or GM-CSF + IL-13.

Furthermore, it is also possible to use in the context of this invention the populations of immortalized dendritic cells. It may be immortalized dendritic cell lines (line D1 used in the examples, or any other line produced for example by introduction of the oncogene myc in dendritic cells). It can also be dendritic cells prepared in vitro and then immortalized. The interest of immortalized dendritic cells resides in the constitution of cell banks sensitized to particular groups of antigens, used industrially to prepare dexosomes may be administered to whole families of patients.

When dendritic cells are prepared, they can be maintained in culture, purified advantage, stored or used directly in the following process steps.

Awareness of dendritic cells

Dexosomes may be prepared from non-loaded dendritic cell antigens, that is to say containing no determined antigens in their membranes or their cytosol. Such dexosomes are then designated "naive" or "virgin".

In a preferred implementation, the dexosomes of the invention are however prepared from dendritic cells sensitized to an antigen or group of antigens. In this embodiment, the dexosomes are indeed themselves carrying said one or more antigens and are thus able to induce a response against the latter.

Various techniques can be used to educate dendritic cells to antigens. These techniques have been discussed above and include:<ul><li>contacting dendritic cells with antigenic peptides ( "peptide pulsing"). This approach involves incubating dendritic cells during a variable time (generally 30 minutes to 5 hours) with one or more antigenic peptides, that is to say with a peptide derived from an antigen, such as could result from the treatment of said antigen with an antigen-presenting cell. This approach has been described for example for antigenic peptides of the HIV virus, the influenza or HPV or for peptides derived from Mut1 antigens Mart, or Her2 Neu example (<nplcit id="ncit0018" npl-type="s"><text>Macatonia et al., J. Exp. Med. 169 (1989) 1255</text></nplcit>; <nplcit id="ncit0019" npl-type="s"><text>Takahashi et al., Int. Immunol. 5 (1993) 849</text></nplcit>; <nplcit id="ncit0020" npl-type="s"><text>Porgador and Gilboa, J. Exp. Med. 182 (1995) 255</text></nplcit>; <nplcit id="ncit0021" npl-type="s"><text>Ossevoort et al., J. Immunother. 18 (1995) 86</text></nplcit>; Butler et al, supra.<nplcit id="ncit0022" npl-type="s"><text>Mehta-Damani et al., J. Immunol. (1994) 996</text></nplcit>). It is also possible to incubate the dendritic cells with an acidic peptide eluate of tumor cell using the methodology described by Zitvogel et al. (1996, supra).</li><li>contacting dendritic cells with one or more antigens ( "antigen pulsing"). This approach involves incubating dendritic cells not with one or several antigenic peptides, but with the</li></ul> or intact antigens. The advantage of this technique lies in the fact that the antigen will be transformed into antigenic peptides by the natural mechanisms of the dendritic cell, so that the resulting antigenic peptides presented by the dendritic cell should provide better immunogenicity. This approach has been illustrated for example by<nplcit id="ncit0023" npl-type="s"><text>Inaba et al. (J. Exp. Med. 172 (1990) 631</text></nplcit>) or by <nplcit id="ncit0024" npl-type="s"><text>Hsu et al., (Nature Medicine 2 (1996) 52</text></nplcit>).<ul><li>contacting dendritic cells with one or more antigenic protein complexes. This approach is similar to the above but can improve the transformation efficiency and / or antigen presentation. In particular, the antigen may be used in soluble form or complexed with the targeting elements, permitting in particular target membrane receptors such as the mannose receptor or the immunoglobulin receptors (Rfc). It is also possible to make the antigen particulate so as to improve its penetration or its phagocytosis by cells.</li><li>contacting dendritic cells with cells or cell membranes expressing antigens or antigenic peptides. This technique relies on the direct transfer of antigens or antigenic peptides by fusion of cells or cell membranes. This approach has been illustrated for example by fusion of dendritic cells and tumor cell membranes (<nplcit id="ncit0025" npl-type="s"><text>Zou et al., Cancer Immunol. Immunother. 15 (1992) 1</text></nplcit>).</li><li>contacting dendritic cells with membrane vesicles containing antigens or antigenic peptides (including exosomes from tumor cells as described above). This awareness approach dendritic cells using exosomes, as demonstrated in the present invention is particularly advantageous insofar as it does not require knowledge of the particular antigens and wherein the antigenic peptides are loaded in a native conformation. This technology is illustrated in the examples.</li><li>contacting dendritic cells with liposomes containing antigens or antigenic peptides (<nplcit id="ncit0026" npl-type="s"><text>Nair et al., J. Exp. Med. 175 (1992) 609</text></nplcit>).</li><li>contacting dendritic cells with RNAs coding for antigens or antigenic peptides (see Boczkowsky et al., 1996, supra).</li><li>contacting dendritic cells with DNAs coding for antigens or antigenic peptides (possibly incorporated in the plasmid type vector, viral or chemical). Thus, a method of sensitizing dendritic cells consists for example of infecting dendritic cells with a virus against which protection is sought. This has been described for example for the Influenza virus (<nplcit id="ncit0027" npl-type="s"><text>Bhardwaj et al., J. Clin. Invest. 94 (1994) 797</text></nplcit>; Macatonia et al., Supra). Another approach is to deliver, by means of a virus or other transfer vectors of nucleic acids, DNA encoding the antigen or antigenic peptides of interest. Such an approach has been illustrated for example by<nplcit id="ncit0028" npl-type="s"><text>Arthur et al. (Cancer Gene Therapy, 1995</text></nplcit>) or by <nplcit id="ncit0029" npl-type="s"><text>Alijagie et al. (Eur. J. Immunol. 25 (1995) 3100</text></nplcit>). Some viruses such as adenoviruses, AAVs or retroviruses appear to be used for this purpose, to deliver a nucleic acid into a dendritic cell.</li></ul>

Preferred techniques in the context of the present invention are the sensitization methods using membrane vesicles (of exosome type), antigenic peptides, vectors, RNAs or peptide eluates tumor acid (EPA). The use of membrane vesicles and that the "peptide pulsing" and the EPA method are illustrated in the examples and are particularly preferred.

Production dexosomes

When the populations of dendritic cells are obtained and optionally sensitized to one or more antigens, may be prepared dexosomes.

This preparation included a first step, optional cell treatment, followed by a second step of isolating dexosomes.

The first cell processing step results from the demonstration by the inventors that the production of dexosomes by dendritic cells is a regulated phenomenon. Thus, in the absence of treatment, the quantities of dexosomes produced are relatively small. In particular, when using a population not previously stimulated mature dendritic cells, the production of dexosomes is virtually undetectable. The inventors have therefore shown that the production of dexosomes was essentially dependent on the type of dendritic cells and the implementation of a treatment of these cells. These are the prerequisites that achieve dexosomes having advantageous properties, in significant quantities for industrial use. Treatment of dendritic cells is advantageously carried out so as to stimulate the production of dexosomes by these cells. This stimulating treatment can be done either by culturing the cells in the presence of certain cytokines or by irradiating the cells, or by decreasing the pH of the culture, or by combining these different types of treatment.

In the first implementation mode, the dendritic cells are incubated in the presence of a cytokine selected preferably from gamma interferon (IFN), interleukin-10 (IL-10) and interleukin-12 (IL -12), preferably interferon gamma and IL-10. As illustrated in the examples, these cytokines appear to exert a stimulatory effect rather pronounced on producing dexosomes (factor 3 to 5). Moreover, surprisingly, no stimulant effects were observed in the presence of the following cytokines: IL-1β, IL-2, IL-4, IL-6 and IL-15, and an inhibitory effect was even observed presence of lipopolysaccharide (LPS) or TNFa, which are nevertheless described as stimulating the maturation of dendritic cells. These results show (i) the controlled character of the production of dexosomes and (ii) the specific effect of certain cytokines on this production. These results illustrate the surprising more interest to use of immature dendritic cells, and the use in the stimulation step, cytokines inducing an immature state of the cells, such as IL-10 in particular. In this implementation mode, cytokines are used to suitable doses by the skilled person based on (i) the cytokine, (ii) the cell population and (iii) conducting any other treatment . It is understood that cytokines are preferably used at sub-toxic doses. Doses of interleukin are generally between 1 and 100 ng / ml, preferably between 1 and 50 ng / ml. Interferon can be carried out at doses of between 1 and 500 IU / ml, preferably between 5 and 200 IU / ml.

In the second mode of implementation, the dendritic cells are subjected to irradiation. The results presented in the examples indeed show that irradiation of the cells also increases the dexosomes production levels. Irradiation is generally carried out between 1000 and 5000 rads, preferably between 2000 and 4000 rads, preferably about 3,000 rads.

The second step comprises isolating dexosomes. This step aims to separate dexosomes dendritic cells and / or culture medium. This step in particular allows to obtain a composition enriched dexosome and essentially free of intact cells. Preferably, this step leads to a composition comprising at least 70% of dexosomes, preferably at least 85%.

Isolation dexosomes can be performed according to various techniques of separation of biological materials. As described above for texosomes tumor cells, these techniques may be based on differences in size, mass, charge or density dexosomes.

Thus, the dexosomes can be isolated by centrifuging the culture medium or from the culture supernatant or membrane fractions of dendritic cells or lysates. It may be for example a differential centrifugation and / or centrifugation in density gradient, followed by (s) a fetch or fraction (s) containing said dexosomes. This type of methodology is based on the separation by successive centrifugation, membrane vesicles one hand and cells, cell debris, internal vesicles, etc., on the other. In this particular implementation, the fraction comprising the dexosomes is usually that obtained after ultracentrifugation at 100,000 g. This method is illustrated in particular in Examples 1 and 8.

The isolating step of the dexosomes can also be achieved by chromatography, electrophoresis and / or nanofiltration.

It may advantageously be a liquid phase electrophoresis and or density gradient. The liquid phase electrophoresis, which enables the separation of biological materials according to their charge, is quite advantageous. Example 11 below shows that this technique can be advantageously used for the isolation of exosomes in good yields. This technique is also particularly advantageous industrially.

It may also be a purification by chromatography. Mention may be made especially ion exchange chromatography, gel permeation (or exclusion) or hydrophobic chromatography. Given the lipid nature of dexosomes, ion-exchange chromatography is particularly interesting. Nanofiltration may be performed according to known techniques, from a cell supernatant.

The use of techniques of chromatography and / or electrophoresis and / or nanofiltration is another important aspect of the present invention, since it allows, compared to current technologies, improved production quality, in quantities adapted to industrial use (including pharmacological).

In this regard, the invention also relates to a membrane vesicle preparation process comprising at least one step of separation by electrophoresis, chromatography or nanofiltration. This process is particularly suitable for the preparation of membrane vesicles of the exosome type, such as texosomes or dexosomes. In this process, electrophoresis or chromatographic separation step can be carried out directly on culture supernatant, cell lysate, or a pre-purified preparation. Electrophoresis is most preferably a liquid phase electrophoresis.

The dexosomes have remarkable properties which are illustrated in the examples. Thus, the dexosomes stimulate proliferation of cytotoxic T lymphocytes in vitro. Furthermore, in vivo, the dexosomes are able to block tumor growth. These vesicles are able to present very effectively, in association with MHC class I and class II antigens of interest. The dexosomes therefore have many applications in the field of cancer, infectious and parasitic diseases, for example. In addition, at high doses (which may induce tolerance), the dexosomes may also be used in the treatment of conditions such as allergy, asthma or autoimmune diseases. In addition, the "naive" dexosomes can also be used as adjuvant to stimulate and / or modulate an immune response.

The invention also relates to the use:<ul><li>texosome as defined above, for stimulation and optionally amplification <i>in vitro</i> of T cells specific for antigens contained in the above texosomes - or B cells, and in particular for the stimulation and amplification <i>in vitro</i> T cell</li></ul>

The invention also relates to the use of texosomes as defined above, for the selection <i>ex vivo</i> a T cell repertoire, capable of recognizing specific antigens contained in above-mentioned texosomes.

The invention further relates to a medicament comprising as active ingredient at least one texosome as defined above, in association with a pharmaceutically acceptable carrier.

Advantageously, the invention relates to a medicament as defined above for use in the treatment of cancers, infectious and parasitic diseases.

More preferably, the medicament comprises texosomes as defined above.

According to another mode of implementation, the invention relates to a medicament as defined above for use in treating type of pathology allergy, asthma and autoimmune disease.

As appropriate galenic form, the texosomes may be contained in physiological saline in an ampoule or any other suitable means (syringe, pouch, etc.). They can be prepared extanporanément or stored, for example in frozen form at -80 ° C. Solutions used may consist of salt solutions, optionally supplemented with stabilizing agents and / or adjuvants. The stabilizers may include proteins or high molecular weight molecules. Mention may more particularly proteins such as human serum albumin, or molecules such as dextran or poloxamer for example.

The compositions of the invention may also include or be used in combination with one or more adjuvants. The adjuvant may be more particularly any immunostimulating pharmacological agent, such as for example a cytokine (especially interleukin-12). Such agents are conventionally used in clinical protocols or in vaccine compositions. Furthermore, the adjuvant of the invention may also be an agent capable of stimulating the production of dendritic cells in vivo. Include for example the Flt3 compound. The combined use of this type of agent used to increase the number of dendritic cells, and thus potentially improving the efficiency of compositions of the invention.

Another object of the invention therefore relates to a texosomes association and an adjuvant, for simultaneous, separate or spread over time.

A suitable administration method of the invention drugs is by injection, and particularly intradermal or subcutaneous. This mode of administration is particularly suitable when the active drug substance is formed by dendritic cells loaded with texosomes or dexosomes.

Suitable dosages are 0.01 to 10 and particularly from 0.10 to 5, still more preferably 0.15 2 ug / kg body weight, and 10 .mu.g for the intradermal reaction test.

The medicaments of the invention may also be used to 100 mcg for the treatment prophylactic vaccination.

The objectives of the use of drugs of the invention are:<ul><li>delayed hypersensitivity (tests in cancer patients), or</li><li>prophylactic therapy, or</li><li>use in the context of detecting the frequency of specific cytotoxic lymphocyte precursors or secreting interferon by the limiting dilution technique.</li></ul>

It involves using dendritic cells allogeneic or autologous pre-incubated with the texosomes of the invention as targets of peripheral lymphocytes of subjects with the tumor before, during and after anti-tumor treatment (standard treatment or specific active immunization ).

The invention also relates to the use of a texosome as defined above, for the preparation of a medicament for the treatment of tumors, especially solid, ascitic and hematopoietic.

As solid tumors include: cancer of the kidney, breast, colon, lung, stomach, liver, melanoma, sarcomas, etc ...

As hematopoietic tumors include: leukemias, malignant lymphomas Hodgkin or non-Hodgkin.

The invention also relates to the use of a texosome as defined above, in the context of a delayed hypersensitivity test cancer or as a diagnostic tool frequency tracking of specific cytotoxic CTL precursors.

The invention also relates to the use of a texosome, or a fraction or constituent component of a texosome as defined above, for the transfer of biological material into a cell <i>in vitro</i>, Or for the manufacture of a medicament for transfer of biological material in a cell in vivo.

The invention also concerns the creation of texosomes banks derived from tumor cells of common or different histological type.

They are composed of mixtures of texosomes made from tumor cell lines for a given type of cancer. These texosomes banks can afford to raise awareness of antigen presenting cells, including dendritic cells, against all tumors of this type.

The invention also relates to mixtures of texosomes.

Include for example mixtures of texosomes for genetically related tumors (breast cancer and ovarian cancer) or with p53 mutations, known p16 (breast cancer, sarcoma).

tumor texosome mixtures can also be mentioned with vesicles derived from immortalized cells transfected to express costimulatory molecules, adhesion molecules, attractant chemokines (different from those expressed on texosomes).

The present invention will be described in more detail using the following examples, which should be considered as illustrative and not restrictive.

LEGENDS OF FIGURES

<b>Table 1.</b> Cell tumor lines were incubated for 24 h at a density of one million cells per milliliter. The texosomes were then prepared (see Example) from culture media by differential ultracentrifugation. Texosomal the protein concentration is measured by the Bradford assay (BioRad Protein Assay [BioRad].

MZ-2 is described in Traversari et al. (1992)

The * means that the different primary lines were established and characterized in the clinical laboratory of the Gustave Roussy Institute and are available on request.

<figref idrefs="f0001"><b>1A</b></figref><b>and</b><figref idrefs="f0002"><b>1B</b></figref><b>.</b> Morphology of the multi-vesicular endosomes and texosomes derived from TS / A cells.<ol><li>A. Sections ultrafine TS / A cells analyzed by electron microscopy. Details of the cytoplasm showing an endosomal compartment containing vesicles 60-80 nm in diameter.</li><li>B cell texosome Preparation TS / A analyzed by electron microscopy by the technique intact vesicle (Raposo et al. (1996)). texosome preparations contain a major population of vesicles of 60-80 nm diameter size and morphology similar to the internal vesicles of multivesicular endosomes shown in A.</li></ol>

<figref idrefs="f0003 f0004 f0005 f0006"><b>Figure 2</b></figref><b>.</b> Presence of different markers in the texosomes tumor cells. <ul><li>A. Two micrograms of texosomal proteins (Exos) or 2x10⁵ tumor cells were analyzed by Western blot using specific monoclonal antibodies: class I MHC molecules (<nplcit id="ncit0030" npl-type="s"><text>Machold Robert P. et al. (1995) "Peptide Influences the Folding and Intracellular Transport of Major Histocompatibility Complex Class Free I Heavy Chains" J. Exp. Med. 181: 1111-1122</text></nplcit>), Transferrin receptor (TfR) (corresponding antibodies are described in H68.4 <nplcit id="ncit0031" npl-type="s"><text>Biochimica et Biophysica Acta (1992) 1136 (1): 28-34</text></nplcit>), Lamp 1 and 2 (anti-mouse rat monoclonal antibodies, Pharmingen) and calnexin (Hebert <nplcit id="ncit0032" npl-type="s"><text>Daniel N. et al. (1995) "and Glucose Trimming Reglucosylation determined glycoprotein Association with Calnexin in the Endoplasmic Reticulum" Cell 81: 425-433</text></nplcit>).</li><li>B. Ten micrograms of texosomal proteins of a melanoma cell line (FON) or 10 micrograms of total proteins of the same cells were analyzed by Western blotting using an anti-MART-1 antibody (<nplcit id="ncit0033" npl-type="s"><text>Marincola F. et al. (1996) "Analysis of Expression of the melanoma associated antigens gp100 and MART-1 in metastatic melanoma cell lines and in" in situ "lesions" Journal of Immunotherapy 19: 192-205</text></nplcit>).</li><li>C. texosomal proteins of a melanoma cell line (FON) or total protein from the same cells were analyzed by Western blotting using an anti-HSP70 antibody.</li><li>D. texosomal proteins of a melanoma cell line (FON) or MZ-2 line were analyzed by Western blotting using an anti-gp96 antibody.</li></ul>

<figref idrefs="f0007"><b>Figure 3</b></figref><b>.</b> The texosomes derived from a tumor line MART-1 positive (FON) stimulate a T clone specific for MART-1.

Twenty thousand cells of clone LT8 T (or LT12, results not shown) were incubated with texosomes derived from FON (MART-1 cell lines and HLA-A2 positive), or GIAM (nephroma cells of a line, negative MART-1) as a negative control, for 48 h. TNFβ production by cells of the T clone was measured by bioassay with WEHI cells (Espavik et al.). Texosomes induce the production of IFN.gamma by clone T, thus revealing the presence of HLA-A2 / peptide derived from MART-1 on the surface of the texosomes.<ul><li>"LT8 TexGIAM +" is LT8 T clones incubated in the presence of tumor cells derived texosomes GIAM;</li><li>"LT8 TexFON +" is LT8 T clones incubated in the presence of texosomes derived from FON tumor cells;</li><li>"LT8 TumFON +" is LT8 T clones incubated in the presence of texosomes derived from FON tumor cells;</li></ul>

Abscissa, the conditioned cells are shown and ordered the produced amount of TNFβ (pg / ml).

<figref idrefs="f0008"><b>Figure 4</b></figref><b>.</b> The texosomes of P815 cells expressing βGal stimulate splenocytes of mice immunized with recombinant βGal adenovirus.

Splenocytes (10⁵) Of BALB / c mice immunized with 10 2 months before⁶ pfu recombinant adenovirus βGal rejected a tumor expressing βGal were incubated with texosomes derived P815 cells (open squares 0) or P815-βGal cells (solid squares ■). Splenocytes not incubated in texosomes give the background symbolized by filled dots (●). After 5 days of culture, 1 uCi of tritiated thymidine was added per culture well. Tritium incorporation into cellular DNA was measured 18 h later. The significantly different results from the Fisher exact test are marked *.

The abscissa shows the amount texosomes derived P815 tumor cells (ug / ml) and the ordinate the counts per minute (CPM).

<figref idrefs="f0009"><b>Figure 5</b></figref><b>.</b> The tumor antigen MART-1 contained in texosomes may be presented to T lymphocytes by dendritic cells.

increasing doses texosomes derived from the tumor cell lines FON (MART-1 +, HLA-A2 + A1) and MZ2 (MART-1 +, HLA-A2-, A1 +) were incubated with the clones (20 000 cells 96 per well of microplates) LT12 T (specific for HLA-A2 / MART-1 peptide) in the presence of dendritic cells HLA-A2 + derived from circulating macrophages (DCA2) (<nplcit id="ncit0034" npl-type="s"><text>Sallusto, F. A. And Lanzavecchia, 1994 Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by GMCSF and IL-4 and down regulated by TNF. J. Exp.Med. 179: 1109-1118</text></nplcit>). Secretion IFN.gamma represented on the ordinate (pg / ml) was measured in the culture supernatants after 2 days. Texosomes derived from FON, as well as those derived from MZ2 induced secretion by IFN.gamma LT8 and LT12 (results not shown). FON cells also induced a higher secretion of IFN.gamma by the T clones while MZ2 cells, which do not express the haplotype adequately HLA molecule (HLA-A2) did not induce the production of IFN.gamma.<ul><li>"LT12 DCA2 +" corresponds to LT12 T clones incubated in the presence of dendritic cells HLA-A2 +;</li><li>"LT12 + + DCA2 TexFON" corresponds to LT12 T clones incubated in the presence of dendritic cells loaded with texosomes derived from FON tumor cells;</li><li>"LT12 + + DCA2 TexMZ2" corresponds to TL12 clones incubated in the presence of dendritic cells loaded with texosomes derived from MZ2 tumor cells.</li></ul>

<figref idrefs="f0010"><b>6A and 6B</b></figref><b>.</b> Anti-tumor effects texosomes <i>in vivo</i>.

Hundred thousand tumor TS / A cells were injected BALB / c mice (A) or Nude (B). Three days later, each mouse has received two successive injections, in a 24 h interval (shown by D3 and D4 in the figure), 20 mcg to 30 mcg of texosomes intradermally. Tumor size was then measured twice per week. Statistical analyzes were made by the Fisher exact test (the 95% significance is indicated by an *).<ol><li>A. Two groups of 5 mice received texosomes derived from TS / A (filled triangles) or MCA38 (open triangles Δ) (a colon adenocarcinoma cell line derived from a C57BU6 mouse) as negative control. Only texosomes derived from TS / A have an anti-tumor effect<i>in vivo.</i></li><li>B. Two nude mice groups received the same doses in parallel the same texosome preparations (■: exosomes TS / A: □: exosomes MC38).</li></ol>

No anti-tumor effects were observed on the Nude Mice. T cells are required for anti-tumor effects texosomes<i>in vivo.</i>

Abscissae, it was reported the day and ordered the mean tumor size (mm²).

<figref idrefs="f0011"><b>Figure 7</b></figref><b>.</b> Of bone marrow-derived dendritic cells sensitized by the texosomes derived from tumor cells induce the total eradication in vivo of established solid tumors.

Five hundred thousand P815 tumor cells were injected into the right flank of DBA / 2 mice 10 days before treatment. The treatment consisted of a single injection of texosomes (10 mcg / mouse) in the same flank but at a distance from the tumor. Another group was injected intravenously with dendritic cells (derived from bone marrow by treatment for 5 days in GM-CSF + IL-4 (Butler et al., 1995), incubated in advance for 3 hours with texosomes P815. The tumors were measured and the results analyzed as described in the<figref idrefs="f0010">6</figref>. P815 of the texosomes sensitized dendritic cells to induce the rejection of established tumors. Microsomes had no significant effect on tumor growth. The insert shows the percentage of mice without tumor (ordinate), the only solid bar corresponding to the groups of filled squares type animals, the abscissa is days. These mice did not develop tumors after reinjection twice the minimum tumor dose, showing that they developed an anti-tumor immunity. The symbols used in the figure are:<ul><li>○: dendritic cells incubated with texosomes witnesses </li><li>■: dendritic cells incubated with P815 texosomes, filled triangles: P815 texosomes intradermally</li><li>X: untreated animals.</li></ul>

Abscissa, days and ordered the mean tumor volume was noted.

<figref idrefs="f0012"><b>8A, 8B and 8C</b></figref><b>.</b> Chemotherapy / cytotoxic agents and irridiation can stimulate exocytosis tumor texosomes.

The L1210 murine leukemia and GIAM primitive human renal cancer cell line were used (<figref idrefs="f0012">8C</figref>). Two million tumor cells were incubated in the presence of increasing amounts of 5-Fu (for L1210) or cis-platinum (CDDP) (for GIAM) per ml for 16 hours.

The supernatant was recovered and then was subject to differential centrifugations as described in Table I.

GIAM was also incubated in IL-2 1,000 IU / ml, or dexamethasone (10^-6 M), or irradiated with 10,000 rads.

High-dose chemotherapy and irridiation are good examples of upregulation exocytosis texosomes in these tumor models. The results were found in other tumors as well (<figref idrefs="f0012">8A and 8B</figref>); in particular, irridiation seems to be the most potent exocytosis stimulus.

The <figref idrefs="f0012">8A</figref> corresponds to the FON melanoma described above-and <figref idrefs="f0012">8B</figref> corresponds to a murine lymphoma designated EL4 (<nplcit id="ncit0035" npl-type="s"><text>J. Nat. Cancer Ins. (1972) 48: 265-271</text></nplcit>).

On the <figref idrefs="f0012">8A</figref>The FON cells incubated under different conditions is shown:<ol><li>1) CM: Basic culture medium (RPMI containing 10% fetal calf serum),</li><li>2) DXM = in presence of dexamethasone,</li><li>3) Irradiation: irradiated at 10,000 rads,</li><li>4) without serum, </li><li>5) IL-10 = in the presence of IL-10 at 10 ng / ml.</li></ol>

The radiation above are also valid for the EL4 cells, L1210 and GIAM.

<b>9A,</b><figref idrefs="f0013 f0014"><b>9B and 9C</b></figref>. Membrane vesicles (dexosomes) produced by dendritic cells sensitized to tumor antigens of melanomas are effective in stimulating T cells specific for these melanomas, and their production is regulated by cytokines.<ul><li>LT8 (CM) corresponds to LT8 T clones incubated in basal culture medium as defined in <figref idrefs="f0012">8A</figref>,</li><li>DexTexNUN matches dexosome derived dendritic cells loaded with texosome from NUN tumor cells,</li><li>DexTexFON matches dexosome derived dendritic cells loaded with texosome from FON tumor cells,</li><li>TumFON corresponds to the FON tumor cells.</li></ul>

A. Proliferation Assays: the texosomes Fon (used <figref idrefs="f0007">3</figref>) Were incubated with HLA-A2 dendritic cells for 3 hours, then washed with 9% saline pH6.3 then incubated in acid medium for 18 hours. Membrane vesicles of dendritic cells (dexosomes) are thus recovered from the culture supernatant of the aforesaid HLA-2 dendritic cells.

Membrane vesicles derived from dendritic cells loaded with texosomes (DexTex) are then incubated with the LT8 clones specific for MART-1 presented in the HLA-A2 context (FON texosomes contain antigen MART-1). As negative control, the texosomes Nun (cancer line of the HLA-A2 negative kidney, MART-1 negative) were used. As a positive control, we used the irradiated tumor line FON (TumFON) or anti-CD3 antibody, préasorbé on plastic (anti-CD3Ab). Incubation of DexTex with LT8 clones lasts 48 hours and then 1 .mu.Ci of tritiated thymidine was added per well of 200 .mu.l. The lymphocyte proliferation LT8 measured 18 hours later.

The ordinate shows the blows per minute.

B. Same manipulations, but IFN.gamma is measured in the culture supernatant at 48 hours by ELISA. The ordinate shows the content IFN.gamma (pg / ml).

C. dexosomes were isolated from dendritic cell supernatants after 48 hours of incubation in the presence or absence of LPS (20 ug / ml), IFN-γ (100 U / ml) or IL-10 (10 ug / mL).

<figref idrefs="f0015"><b>Figure 10</b></figref><b>.</b> Images in immuno-electron microscopy dexosomes. Dexosomes provide a uniform diameter of between 50 and 90 nm and are intensely labeled by anti-CD63 antibodies (<figref idrefs="f0015">10A</figref>). Most of these dexosomes is also labeled with anti-MHC-I antibodies (page 15,<figref idrefs="f0015">FIG 10B</figref>) And anti-MCH-II (Page 15, <figref idrefs="f0015">10C</figref>). Bar: 250 nm.

<figref idrefs="f0016"><b>Figure 11</b></figref><b>.</b> Measurements of gamma secreted interferon levels by T cells incubated in the presence of dexosomes loaded with peptides or control dexosomes. Dendritic cells (2X10⁶ / Ml) were incubated for 3 to 12 hours, either in the presence of 10 ug / ml of antigen peptide MART-1 / MelanA_(27-35)Or in the presence of 10 ug / ml of gp100 peptide_(280-288) (Control) were suspended in citric acid pH 3.7, then the dexosomes were isolated. Cells LT12 clone (CTL clone restricted HLA-A2, MART-1 (27-35) specific) were then incubated (100 000 CTL per well) with increasing doses of dexosomes or gp100 peptides (control) in 96-well plates for 5 days. The secretion of interferon gamma production by cells was then measured by ELISA (Genzyme).

<figref idrefs="f0017"><b>Figure 12</b></figref><b>.</b> Western Blot analysis of markers present on the dexosomes (1.4 to 10 mcg) produced by dendritic cells derived from bone marrow: H-2K (MHC-I), I-Aα (MHC-II), CD86, CD63, TfR (transferrin receptor), Clx (calnexin), p31 Ii (invariant chain).

<figref idrefs="f0018"><b>Figure 13</b></figref><b>.</b> antitumor effect in vivo dexosomes on a mast cell tumor model (P815). Dex-H2d-APE-P815: Dexosomes derived from bone marrow dendritic cells loaded with peptide eluate acid of the P815 tumor. Dex-H2d-APE-Spleens: Dexosomes derived from bone marrow dendritic cells loaded with peptide eluate acid spleen.

<figref idrefs="f0019"><b>Figure 14</b></figref><b>.</b> anti-tumor effect in vivo dexosomes derived from dendritic bone marrow cells loaded with acidic tumor peptide eluate on a model of breast tumor (TS / A). Caption: see<figref idrefs="f0018">Figure 13</figref>. (A) Experiment performed on immunocompetent mice. (B) Experiment performed on nude mice.

<figref idrefs="f0020"><b>Figure 15</b></figref><b>.</b> radioactive chromium release assay (51 Cr). The test is to show that dexosomes of the invention trigger a specific CTL response in vivo. target cells: P815 leukemic cell line L1210, YAC line insensitive to NK cells.

<figref idrefs="f0021"><b>Figure 16</b></figref><b>.</b> Comparative effectiveness dexosomes and dendritic cells. This figure shows that the dexosomes are more powerful than immature dendritic cells from which they are derived to eradicate established tumors in vivo. 5 million dendritic cells loaded with peptide eluate spleen acid (open squares) or the P815 tumor (open triangles) were administered intravenously or intradermally to mice with established tumors P815 on day 8 to 10. In parallel the supernatant of these cells was harvested after 18h incubation with the peptides of the spleen (open squares) or the P815 tumor (solid triangles), ultracentrifuged and characterized for its content in dexosomes. 5 million dendritic cells have resulted in 5 to 10 ug of dexosomes, which enabled the immunization of 5 mice by intradermal administration in the ipsilateral side. A single administration of dexosome was performed on day 8-10. Tumor size was measured twice per week and is shown in FIG. the (*) represent significant results to 95% depending on the Fisher exact test, as compared with injections of saline (open squares) or pulsed dendritic cells. In the insert are shown the percentages of mice immunized against P815 showing a complete absence (disappearance) of tumor during (day 21) and end (day 60) of the experiment, in the various groups of 5 mice.

<figref idrefs="f0022"><b>Figure 17</b></figref><b>.</b> Dexosomes purification by liquid phase electrophoresis.

EXAMPLES

1. Production of texosomes by lines of human and murine tumor cells.

This example illustrates the ability of tumor cells to produce lipid vesicles.

Murine or human tumor cells from leukemia or solid tumors (melanoma kidney or colon cancer) (see Table 1) were incubated for 24 h at a density of one million cells per milliliter. The culture medium (RPMI containing 10% fetal calf serum) was then cleared from cells by centrifugation at 300 g for 10 minutes. Cell debris were then removed by two successive centrifugations of 15 min. each 800 g (and an optional centrifugation for 30 minutes at 10 000 g). Texosomes were finally collected by centrifugation for 60 minutes at 100,000 g, then washed once with PBS under the same conditions. The protein concentration in the texosome preparations was measured by the Bradford method (BioRad Protein Assay [BioRad]).

All tumor lines tested, human and murine (solid or hematopoietic, primary or established in culture or originating from dissociated fresh tumors) produce texosomes (Table 1). However, production efficiencies are variable between different lines. The lines of murine tumor cells produce between 100 and 200 micrograms of texosome protein per 50 million cells in 24 hours. Human melanoma lines and nephroma produce between 10 and 100 micrograms of texosome protein per 20 million cells in 24 hours

2. The vesicles produced by the tumor cells are of endocytic origin.

To determine whether the vesicles purified from the supernatants of tumor cell lines are of endocytic origin, we performed a morphological study using electron microscopy of one of these tumor lines, TS / A (mouse mammary carcinoma line ) (<nplcit id="ncit0036" npl-type="s"><text>Nanni P. et al. (1983) "TS / A: a new metastasizing cell line originate from a BALB / c mammary adenocarcinoma sponteneous" Clin. Exp. Metastasis 1: 373-380</text></nplcit>). Tumor cells were fixed and primed for electron microscopy as described above. The texosomes were deposited directly on grids and analyzed.

The <figref idrefs="f0001">1A</figref> shows examples of intracellular compartments of multivesicular appearance observed in tumor cells. These endocytic compartments have a diameter of 200-300 nm (the bottom bar of the panels A and B is 200 nm) and consist of an outer membrane surrounding multiple internal vesicles of 68-80 nm in diameter. texosome preparations contain a major population of vesicles 60-80 nm in diameter (<figref idrefs="f0002">1B</figref>), Sometimes aggregate, and morphologically similar to the internal vesicles of multivesicular endosomes observed inside the cells (<figref idrefs="f0001">1A</figref>). These results suggest that the texosomes are secreted into the extracellular medium after fusion of the outer membrane of endosomes with the cytoplasmic membrane. Indeed, such exocytosis profiles are observed in these cells (data not shown).

To determine whether the texosomes are actually of endocytic origin, we then conducted a Western blot analysis of these markers defined below in texosomes derived from tumor lines, TS / A and P815 (mastocytoma data by T. Boon, Ludwig Institute, Brussels, Belgium) (murine mast cell tumor). To do this, two micrograms of texosome proteins or the cell lysate 200 000 TS / A cells were separated by polyacrylamide gel, then transferred onto a nylon membrane (Amersham). The possible presence of different markers was then revealed using specific antibodies. Texosomes TS / A and P815 contain class I MHC molecules and different markers of the endocytic pathway (the transferrin receptor, the lysosomal glycoproteins Lamp 1 and 2) (<figref idrefs="f0003">2A</figref>). By cons, a marker characteristic of the Endoplasmic Reticulum (ER), calnexin, is not present in preparations of texosomes showing that ER membranes do not contaminate texosomes.

3. texosomes produced by melanoma cells contain a cytosolic tumor antigen.

These results show that the texosomes secreted by tumor cells correspond to internal membranes of multivesicular endosomes. Now these intraendosomal vesicles are formed by invagination and budding of the outer membrane of endosomes inwardly of the endosome. These vesicles intraendosomal and consequently the texosomes should contain a cytosol fraction. This is particularly important in the context of anti-tumor immunotherapy, since a number of tumor antigens including MART-1 (one of the most studied) are cytosolic proteins. So we tested the presence of MART-1 in the texosomes.

To do this, ten micrograms of texosome or cell lysate protein of 200 000 cells of a human melanoma line (M10) (T. Boon, Ludwig Institute, Brussels, Belgium) were analyzed by Western blotting, as previously. The possible presence of MART-1 was then revealed using anti-MART-1 specific antibody (S. Rosenberg, NCI, Bethesda, USA). The texosomes secreted by the tumor line FON (FON melanoma patient from F. Faure, Pasteur Institute, Paris, France) contain the MART-1 tumor antigen. Protection experiments with proteinase K (Sigma) showed that the epitope of MART-1 recognized by this monoclonal antibody is within texosomes (results not shown).

This first part of the work shows that:<ul><li>tumor cells produce and secrete vesicles,</li><li>that these vesicles are original vesicles having an outer endosomal membrane where different membrane molecules are located (MHC class I, different labels endosomes),</li><li>These vesicles contain a cytosol fraction, including cytosolic tumor antigens, such as MART-1.</li></ul>

These results led to verify the following biological activities of the vesicles, designated texosomes.

4. texosomes can stimulate CD8 <i>in vitro</i>.

Since texosomes bear class I molecules on their surface, it was tested whether they are able to stimulate CD8 T lymphocytes. We used two clones T LT8 and LT12 given by F. Faure, Pasteur Institute, Paris, France, recognizing a peptide derived from MART-1 in association with HLA-A2 (Dufour et al., 1997). To this end, since the FON tumor cells are HLA-A2 incubated cells LT8 T clones or LT12 with texosomes prepared from supernatants of FON, or, as a positive control, intact FON cells. Activation of T cells was measured by the secretion of TNFβ. FON's texosomes induced secretion by TNFβ LT8 and LT12 in a dose-dependent manner (<figref idrefs="f0007">Figure 3</figref>). FON cells also induced secretion TNFβ, while texosomes derived from tumor cells not MART-1 do not background expressing (<figref idrefs="f0007">3</figref>).

Thus, HLA-A2 complex / MART-1 peptide are present on the surface of the texosomes.

Similar results were obtained in mice with a mouse spleen T cells immunized with the β-galactosidase (β-gal).

Texosomes were produced from P815 mastocytoma cell supernatants or P815 cells expressing β-gal (lines A. Albina, Gustave Roussy Institute, Villejuif, France), or cells of another tumor (L1210) murine leukemia H2α) not expressing β-gal, L210. Increasing concentrations (0.3 ug / ml to 20 * g / ml) of these different texosome preparations were then incubated for 4 days with spleen cells of mice immunized with the β-gal expressed in a recombinant adenovirus. Only texosomes P815 cells (the highest concentration of 20 ug / ml) expressing β-gal induced significant proliferation (see<figref idrefs="f0008">4</figref>) (Measured by tritiated thymidine incorporation), although not strong, spleen cells. These results show that the texosomes produced by the P815 cells expressing β-gal bear at their complex area H2^α/ Peptides derived from β-gal and are able to activate murine T lymphocytes.

5. The texosomes may issue cytosolic antigens they contain antigen-presenting cells for presentation to T cells

For this purpose, we used the T clones LT8 and LT12 specifically recognizing a peptide derived from MART-1 (MART-1_{27-25 =} AAGIGILTV, <nplcit id="ncit0037" npl-type="s"><text>And E. Dufour al., Diversity of the melanoma-specific cytotoxic immune response. J. Immunol. 1997 158: 3787-3795</text></nplcit>) In combination with HLA-A2. It has been shown that the texosomes produced by the human melanoma line FON (who is HLA-A2) contain the MART-1 tumor antigen (see<figref idrefs="f0003 f0004 f0005 f0006">2</figref>) And are able to directly activate LT8 clones and LT12. In order to have texosomes also containing MART-1, but unable to directly stimulate the LT12 and LT8 clones was used the MZ2 melanoma line (T. Boon, Ludwig Institute, Brussels, Belgium), MART-1 positive but expressing another restriction element that HLA-A2 (HLA-A1 in this case). Indeed, MZ2 cells, as well as texosomes derived from these cells do not activate LT8 and LT12 clones (results not shown), unlike the FON cells and texosomes derived from these cells (<figref idrefs="f0007">Figure 3</figref>). Against by, when these same texosomes derived from MZ2 are incubated in the presence of dendritic cells expressing HLA-A2, stimulation LT12 T clones and LT8 is observed, as in the case texosomes derived FON (<figref idrefs="f0009">5</figref>).

In the case texosomes derived from MZ2, the activation of T clones can not be due to HLA-A2 / peptide complexes derived from pre-existing MART-1, since they do not express the appropriate restriction element (HLA A2). Therefore, it may be that the antigen contained in the texosomes which was taken up by antigen presenting cells, degraded into peptides which are then associated with HLA-A2 molecules presenting cell. Texosomes therefore permit the transfer of an antigen from a tumor cell and an antigen presenting cell. The texosomes thus have a function similar to that of "natural liposomes".

6. texosomes induce regression of established solid tumors

<u>in</u>

<i>vivo</i>.

Finally, since the texosomes are capable of stimulating T cells <i>in vitro</i> and sensitize dendritic cells to activate T cells specific for tumors, were tested for antitumor activity texosomes <u>in vivo</u>.

To analyze such an anti-tumor activity, mice were injected twice with (10⁵) The minimum tumorigenic dose of tumor cells from a breast tumor (TS / A cells H2 haplotype^d, Syngeneic BALB / c cells) into the flank. After 3 or 4 days, the animals with established tumors were injected twice (day 3 and 4) with texosomes prepared from cell supernatants TS / A or as negative control cells MC38 texosomes (S. Rosenberg NCI, Bethesda, USA) (tumor cell H2 haplotype^b) Or a similar volume of PBS. The average size of tumors in the mice inoculated group with TS / A texosome preparations is very diminished in comparison with control groups of mice. This anti-tumor effect is T cell-dependent, because Nude mice (mutant mice lacking T-cells) and tumor-bearing inoculated similarly with texosome preparations, show no decrease in tumor mass (<figref idrefs="f0010">6B</figref>).

In this series of experiments, there was an anti-tumor effect texosomes prepared from cells of mastocytoma P815 H2 haplotype^d which suggests that these tumors express common antigens. Similar results were obtained with another very immunogenic tumor model, the mastocytoma P815 (mouse syngeneic DBA / 2 and H2 haplotype^d). In this model, it was shown that texosomes injected intradermally in the flank of mice bearing tumors (P815) established the 10th day (tumor measuring 80-100 mm²) Have capacity to complete eradication of the tumor in 60% of cases. In addition, these mice show an anti-tumor immunity in the long term (results not shown). In this series of experiments, we observed anti-tumor effects of texosomes prepared from L1210 lymphocytes isolated from a murine leukemia H2 haplotype^d and TS / A cells, suggesting that common epitopes also exist between these three tumors (between P815 and TS / A, mutated p53 is common to both tumors).

Two mechanisms could be the basis for the anti-tumor effect texosomes.

First, once injected, the texosomes could directly activate the T cells of the host, the tumor-specific. In this way, a clonal expansion of specific T cells of the tumor or "priming" of T cells may occur. A second hypothesis involves direct interaction texosomes injected with host dendritic cells. This could then stimulate an anti-tumor response. To test this second hypothesis, monitoring tumor growth in mice injected intravenously with dendritic cells derived from bone marrow loaded with tumor cell texosomes. Of DBA / 2 mice (IFFA CREDO, Orleans, France) were thus injected with twice (50 x 10⁵) The minimum tumorigenic dose of P815 mastocytoma cells. Ten days later, each animal was injected with 5 x 10⁵ loaded dendritic cells, and thereby sensitized with 9 mcg texosomes. The average size of tumors in these conditions significantly reduces as compared to groups of mice injected with PBS or with dendritic cells loaded cells texosome tumor MC38 control. Indeed, over 60% of the treated animals no longer a tumor at the end of the experiment. Moreover, recurrences were not observed in long-term (in 80% to 100% of cases). It is interesting that such a sub-optimal dose of texosomes injected intradermally has no effect, suggesting that dendritic cells can prepare dexosomes containing tumor antigens much more efficient that dendritic cells dermal or the Langerhans cells. We obtained similar results with the cell model of breast tumor TS / A.

The results obtained in the framework of the invention show that texosomes sensitize dendritic cells effectively. These cells, sensitized, have the ability to induce potent anti-tumor responses<i>in vivo,</i> and that in the case of different tumors. These results suggest that anti-tumor effects observed after direct inoculation of texosomes<i>in vivo</i> are due to sensitization of the dendritic cells of the host. Remarkably, the effect is observed after a single injection of sensitized dendritic cells. The majority of treated mice showed complete tumor regression or prolonged survival (60 days against 20 days in the control) due to tumor regression.

Awareness method presenting cells of the invention has various advantages over methods of the prior art:<ul><li>i) it does not require prior knowledge of tumor antigens: this is particularly important since tumor antigens are not known to the vast majority of tumors;</li><li>ii) the process of the invention can be applied to any tumor occurring texosomes; more than 15 tumor cell lines tested to date, only one does not occur texosome (one of six melanoma lines tested).</li></ul><ul><li>iii) this method does not depend on the MHC haplotype of the patient and the tumor cells, since the tumor antigens present in the texosomes are re-prepared and presented to T cells by MHC molecules of the patient's antigen presenting cells ;</li><li>iv) the method of the invention may in principle be effective between tumors of different origins, since there are common tumor antigens not only in a particular type of tumor (MART-A for example in melanoma), as well as antigens common to completely different tumors (such as molecules involved in tumorigenesis, p53, for example);</li><li>v) the use of texosomes may also be effective in the treatment of tumors expressing low levels of class I MHC molecules, or does not express at all (these tumors represent 40-50% of human metastatic cancers) . Indeed, the texosomes permit the transfer of intact antigens between tumor cells and dendritic cells, these antigens are then presented to T cells by MHC molecules of the dendritic cell. Preliminary results show that the texosomes possible to induce the rejection of murine tumors expressing low levels of class I MHC (as MCA101, S. Rosenberg, NCI, Bethesda, USA). This is probably due to the fact that the expression levels of MHC molecules required for induction of an effective immune response are much higher than those required in the effector phase (cellular cytotoxicity);</li><li>vi) the only texosomes constitute in themselves, by their immunogenicity prophylactic vaccination or therapeutic modality new and effective.</li></ul>

7. Dendritic cells produce immunogenic membrane vesicles (Dexosomes)

This example demonstrates that the dendritic cells produce membrane vesicles and that these vesicles are powerful immunogens vesicles for antitumor immunization. These vesicles are particularly advantageous since they can prevent the injection stage<i>in vivo</i> whole dendritic cells from which they originate.

The dendritic cell therapy does not provide the certainty stable phenotype of the injected cell, or the homogeneity of cell compositions used. By administering a stable product secreted by these cells, that is to say, the above-described membrane vesicles, are offered to the tumor-bearing host a guarantee of efficacy and immunizing power.

In this example, dendritic cells derived from bone marrow by treatment for 5 days in GM-CSF + IL-4 (Mayordomo et al., 1995) were incubated for 3 hours with exosomes from tumor cells. And the sensitized cells were then grown in acid for 18 hours (to stimulate the production of vesicles) and vesicles were observed and harvested using the methodology described in Example 1. To determine their immunogenicity, these vesicles were then incubated with in vitro cytotoxic T lymphocytes specific for the antigen MART1. The results shown in<figref idrefs="f0013 f0014">9</figref> show that 0.6 .mu.g of these membrane vesicles, called DexTexFON (that is to say dexosome from dendritic cells incubated with texosomes from FON tumor cells), allow to directly stimulate the proliferation and secretion of IFN.gamma LT8 clones specific to MART-1 and this is what can not be 0.6 mcg dexosomes from dendritic cells incubated with tumor cells texosomes NUN: DexTexNUN (NUN being a kidney tumor, HLA-A2 negative, MART 1 negative). These results show (i) that the dendritic cells produce membrane vesicles and that these membrane vesicles constitute a potent immunogen.

In addition, the results presented in <figref idrefs="f0014">9C</figref> show that, unexpectedly, the production of dexosomes to dendritic cells is a controlled phenomenon, which can be stimulated in the presence of certain cytokines such as IFN-γ and IL-10. Thus, the results presented show that IFN-γ or IL-10 significantly increase (about 5-fold), production of dexosomes. Similar results were observed with IL-12.

8. Characterization of membrane vesicles produced by dendritic cells.

The ability of dendritic cells to produce membrane vesicles was first confirmed on dendritic cells produced from human monocyte precursors and on the murine D1 dendritic cells.

D1 cell line and ripening conditions of this line have been described by <nplcit id="ncit0038" npl-type="s"><text>Winzler et al. (J. Exp. Med. 185 (1997) 317</text></nplcit>).

Dendritic cells derived from human monocyte precursors were obtained from the adherent fraction of mononuclear cells, taken from healthy subjects, incubated 7-8 days in AIMV medium containing L-Glu, antibiotics, 1000 IU / ml rhGM-CSF and rhIL-4 (Schering Plough, Kenilworth, NJ, USA). After 8 days of culture, loosely adherent cells and the cells in suspension have a typical morphology of dendritic cells, express high levels of MHC I and II molecules, as well as CD40 and CD86. Most of these cells is positive for CD1a and CD11b and negative for CD2, CD3, CD14, CD19 and CD83.

Microscopic analyzes of these cells revealed the presence of membrane vesicles rich in MHC I and II. These vesicles were isolated by centrifugation and analyzed by immuno-electron microscopy. More particularly, the culture supernatants of the dendritic cells were harvested, centrifuged at 300 g for 20 minutes and then at 10 000 g for 30 minutes at 4 ° C to remove cells and cell debris. Dexosomes were then isolated by centrifugation at 100 000 g for 1 h at 4 ° C, followed by washing with PBS under the same conditions (centrifugation at 100 000 g for 1 h at 4 ° C). The protein concentration in the preparations of dexosomes was measured by the Bradford method (BioRad Protein Assay [BioRad]).

The results obtained show (<figref idrefs="f0015">Figure 10</figref>) A homogeneous population of vesicles having a diameter of between 60 and about 90 nm. Furthermore, over 95% of dexosomes are labeled with anti-CD63, anti-CD82, anti-MHC-I and anti-MHC-II.

These results confirm that the dendritic cells produce membrane vesicles exhibiting antigen-presenting molecules as well as lymphocytic costimulatory molecules.

9. dexosomes present antigens in a restricted MHC-I context.

One of the advantageous features of s dexosomes lies in the presence of MHC class I. The molecules are indeed necessary for an effective cellular response generation, and in particular the activation and cell expansion CTL. The ability of dexosomes to stimulate CD8 + lymphocytes and the specific character of lymphocytes obtained have thus been tested.

For this, dendritic cells obtained from precursor human monocytes (HLA-A2 subjects) were initially sensitized by "peptide pulsing" to a particular antigen. For this purpose, the cells (2X10⁶ / Ml) were incubated for 3 to 12 hours, either in the presence of 10 ug / ml of antigen peptide MART-1 / MelanA_(27-35) Or in the presence of 10 ug / ml of gp100 peptide_(280-288) (Control) were suspended in citric acid to pH 3.7. After this sensitization step, the dexosomes were isolated as described in Example 1. Cells of the LT12 clone (CTL clone restricted HLA-A2, MART-1 (27-35) specific) were then incubated (100 000 CTL per well) with increasing doses of dexosomes or gp100 peptides (control) in 96-well plates for 5 days. The secretion of interferon gamma production by cells was then measured by ELISA (Genzyme).

As shown in <figref idrefs="f0016">Figure 11</figref>, The dexosomes with the MART-1 peptide is capable of stimulating the production of interferon gamma by the LT12 clone in a dose-dependent manner. Instead, dexosomes produced from dendritic cells pulsed with the peptide gp100 control exercise no stimulating effect on this clone.

These results confirm that the MHC-I molecules expressed by dexosomes of the invention are functional.

10. The dexosomes block tumor growth in vivo.

This example demonstrates the ability of dexosomes of the invention to induce an immune response in vivo and more particularly to induce proliferation of T cells specific for a tumor.

Dendritic cells obtained from bone marrow were loaded with a tumor acid eluate having different tumor antigen peptides. The preparation technology and awareness of dendritic cells has been described by Zitvogel et al (1996). The<figref idrefs="f0017">Figure 12</figref> shows the markers expressed by the dexosomes produced by dendritic cells. As indicated above, this figure shows the presence of abundant MHC class I and class II and CD86 markers and the transferrin receptor. On the contrary, although they appear in the cell lysates, the H2-M markers, Ii and calnexin are undetectable in exosomal preparations.

Two experimental tumor models were chosen to test the anti-tumor properties in vivo dexosomes of the invention. The first model, the P815, is an aggressive mastocytoma syngeneic DBA / 2 (H2^d) For which very little effective immunotherapies have been reported on established tumors on day 10. The TS-A model is a weakly immunogenic spontaneous mammary carcimome expressing lower levels of MHC Class I, syngeneic BALB / c (H2^d). Tumor peptides P815 tumors or TS / A eluted by acid treatment were loaded into syngeneic dendritic cells derived from bone marrow, as described above. Dexosomes were then prepared from the supernatants of these dendritic cells and used for in vivo immunization.

tumor cell lines and mouse

.

Female DBA / 2J (H2^d) And BALB / c (H2^d) Aged six to eight weeks were purchased from the Iffa Credo Laboratory, Lyon, France and maintained in pathogen-free conditions. Nude mice were maintained in a protected microenvironment. P815 cells were provided by T. Boon (Ludwig Institute, Belgium). Model TS / A was provided by Guido Fomi (Immunogenetic and Histocompatibility Center, Turin, Italy). All tumor lines were maintained in RPMI 1640 supplemented with 10% fetal calf serum endotoxin-free medium (Gibco BRL), 2 mM L-glutamine, 100 Units / ml penicillin, 100 mg / ml streptomycin essential amino acids and pyruvate. This medium is also referred to in the following: CM medium.

Protocol and results.

Twice the minimal tumorigenic dose of tumor cells (5x10⁵ P815, 10⁵ TS / A) were inoculated intradermally into the upper area of ​​the right flank of DBA / 2 and BALB / c respectively. Animals with tumors TS / A set of three to four days or established P815 tumors of six to ten days were then immunized by a single intradermal injection of 3 to 5 micrograms of dexosomes in the lower part of the ipsilateral blank. These procedures were performed similarly in both immunocompetent animals and in nude mice. One therapeutic injection was conducted for each mouse. Tumor size was monitored twice a week and the mice were sacrificed when tumors were ulcerated or too large. Each series of experiment was performed two to three times using five groups of mice for each individual treatment. The results obtained are shown in<figref idrefs="f0018">13</figref>. As shown in<figref idrefs="f0018">13B</figref>, Treatment of established P815 tumors on day 10 (having a size of 50 to 90 mm²) Was made possible by a single intradermal administration of 3 to 5 micrograms of dexosomes per mouse. Within a week, tumor growth stopped in the groups receiving dexosomes derived dendritic cells loaded with autologous tumor peptide, and in 40 to 60% of the mice, the tumors disappeared completely in 60 days.

These animals also have a lasting immune response and rejected an additional injection of P815 normally lethal to untreated mice. However, these mice are not protected against an injection of leukemic clone syngeneic L1210, which shows the immunospecific character of the effect. Finally, the groups of mice immunized with control dexosomes (loaded with peptides from the mouse spleen) show no anti-tumor effect as well as the untreated mice. These results therefore show that the dexosomes loaded with tumor peptide of the invention are capable of inducing tumor regression in vivo.

Similar antitumour effects have been obtained on the tumor model TS / A comprising established tumors at days 3/4. In this series of experiments, all mice showed a statistically significant tumor growth delay which prolonged their survival (<figref idrefs="f0019">Figure 14</figref>). This antitumor effect was not observed in athymic Nu / Nu as shown in<figref idrefs="f0019">14B</figref>, Which shows that the presence of T cells is required for expression of antitumor effect dexosomes of the invention.

In addition, the following experiment shows that the dexosomes directly stimulate a specific CTL response in animals with P815 tumor. The mouse splenocytes that rejected the P815 tumors after immunization with dexosomes were harvested on day 90 and cultured for five days in the presence of irradiated P815 cells expressing B7.1 to increase the frequency of specific precursors. These effector cells were tested in a chromium release assay against (i) autologous tumor cells P815 (H2^d), (Ii) unrelated L1210 cells and (ii) the YAC cells. A significant specific cytolytic activity was observed against the P815 cells in the splenocytes of mice immunized with the dexosomes (<figref idrefs="f0020">Figure 15</figref>). Interestingly, none of the spleens of mice spontaneously rejecting the tumor P815 or presenting P815 tumors exhibit this cytolytic activity under the same conditions. These results show that a single injection of dexosomes according to the invention derived from dendritic cells sensitized with an antigen or corresponding antigenic peptides, is capable of eliciting effectively anti-tumor specific CTL response<i>in vivo.</i>

To determine whether the immune response and anti-tumor induced dexosomes is restricted to MHC and not simply due to a direct effect of tumor peptides, dendritic cells derived from mouse H2^d (DBA / 2) or H2^b (C57BL / 6) were loaded in parallel with the eluted tumor peptides of the P815 tumor. The dexosomes produced by these mouse dendritic cells were then isolated and used separately for direct intradermal injections in the DBA / 2 mice bearing P815 tumors established in 6/10 day.

As shown in <figref idrefs="f0018">13B</figref>, Only dexosomes bearing the syngeneic tumor peptides are effective anti-tumor vaccines (inducing in mice a 60% disappearance of the tumor) while the dexosomes allogeneic dendritic cells induce substantially no anti-tumor effect. These results indicate that the dexosomes according to the invention induce an anti-tumor response<i>in vivo</i> MHC restricted.

Similar experiments to those described above were conducted by carrying out not intradermal but intravenous injections. The results obtained are shown in<figref idrefs="f0021">Figure 16</figref>. They initially showed tumor regression subsequent to intravenous injection of dexosomes. Furthermore, these results show that the dexosomes are more powerful than dendritic cells which they are derived to eradicate tumors in vivo. These results illustrate the remarkable and unexpected properties dexosomes.

The results presented above therefore show that immature human dendritic cells or murine secrete dexosomes these dexosomes present molecules not only MHC class II, but also class I MHC and co-stimulatory molecules, and finally dexosomes these are immunogenic and induce tumor regression <i>in vivo</i>.

Dexosomes can be obtained in relatively high amounts (1 .mu.g per million dendritic cells by eighteen hours, depending on the Bradford assay) from the middle of dendritic cell cultures (dendritic cells derived from bone marrow in the presence of GM-CSF + IL4, dendritic cell lines D1 or derived dendritic cell precursors human monocytes isolated from peripheral blood mononuclear cells). The dexosomes were characterized morphologically and biochemically. Membrane vesicles analyzed by immunoelectron microscopy represent a homogeneous population of vesicles having a diameter of about 60 to 90 nannomètres. The dexosomales preparations are apparently devoid of retrovirus, plasma membranes, constituting microsomal or apoptotic bodies. Dexosomes abundantly overexpress MHC class II, I, CD63 and CD86 compared to plasma membranes. No compartment endoplasmic reticulum derivative was detected in the dexosomes by Western blotting using anti-calnexin antibodies. Programmed cell death could not be detected in these cultures using different conditions. Interestingly, the production of these vesicles appears as a regulated phenomenon. The amount of vesicles appear to be reduced by inducing maturation of dendritic cells, as determined by the Bradford assay, Western blotting, and immuno-electron microscopy. In addition, the level of secretion of these vesicles can be significantly improved by lowering the pH of the culture medium, or by incubating the cells in the presence of certain cytokines, or by treating the dendritic cells by irradiation. This is particularly surprising insofar as immature dendritic cells are generally considered to have low antigen presenting power and therefore a low immunological activity. The results presented show that dendritic cells are immature at this stage that possess the property of producing efficiently dexosomes. The results presented show that finally these dexosomes are capable of eliciting a response Efficient T cells both<i>in vitro</i> and <i>in vivo</i> and they are also able to induce tumor regression <i>in vivo.</i> These vesicles are so many particularly attractive candidates for immunotherapy approaches in non-cellular system.

11.

Purification of exosomes by liquid phase electrophoresis

This example describes the use of a novel method of purification of exosomes based on liquid phase electrophoresis.

The liquid phase electrophoresis is a preparative method for separating organic compounds according to their charge. This method was used to separate proteins by isoelectric focusing. This method may have the following advantages:<ul><li>it is a preparative method for the continuous injection of material, and therefore the purification of large quantities of vesicles.</li><li>This method allows purification of dexosomes in one or two steps, potentially eliminating the centrifugation steps.</li></ul>

To determine whether this method is applicable to the purification of exosomes, we conducted the following experiment:

A preparation of dexosomes isolated from a supernatant of murine dendritic cells by differential ultracentrifugation was injected into the liquid phase electrophoresis in the usual conditions described by <nplcit id="ncit0039" npl-type="s"><text>Amigorena et al. (Nature, 369 (1994), 113</text></nplcit>). 40 fractions were collected and the protein concentration in each of these fractions was determined by the Bradford test (BioRad), to detect the presence of dexosomes. As shown in<figref idrefs="f0022">Figure 17</figref>, 90% of dexosomes were found concentrated in four fractions of EPF. Migration dexosomes in a narrow peak with this technology demonstrates the feasibility of electrophoresis as isolated dexosomes method.<ul><li><nplcit id="ncit0040" npl-type="s"><text>Amigorena, S. Drake, JR, Webster, P., and Mellman, I. (1994). Transient accumulation of new class It molecules in a novel endocytic compartment in B lymphocytes [see comments]. 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Nature Medicine, Vol. 4 May, 1998, 594-600</text></nplcit>.</li></ul><tables id="tabl0001" num="0001"><table frame="all"><title><b>Table 1. Production of texosomes by lines of murine and human tumor cells.</b></title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="74mm" /><colspec colnum="2" colname="col2" colwidth="74mm" /><thead><row><entry valign="top"><b>LINES TUMOR CELLS</b></entry><entry valign="top"><b>texosomes (</b>μ<b>G / 2x10⁷ CELL / 18 H)</b></entry></row></thead><tbody><row><entry>murine</entry></row><row><entry>MCA101</entry><entry>172</entry></row><row><entry>P815</entry><entry>163</entry></row><row><entry>MC38</entry><entry>120</entry></row><row><entry>L1210</entry><entry>150</entry></row><row><entry>TS / A</entry><entry>160</entry></row><row><entry>Human (melanoma)</entry></row><row><entry>VIO *</entry><entry>80</entry></row><row><entry>FON</entry><entry>90</entry></row><row><entry>MZ2-2</entry><entry>18</entry></row><row><entry>Human (néphromes)</entry></row><row><entry>RCC NUN *</entry><entry>120</entry></row><row><entry>RCC JOUA *</entry><entry>18</entry></row><row><entry>RCC MEG *</entry><entry>10</entry></row><row><entry>RCC GIAM *</entry><entry>100</entry></row></tbody></tgroup></table></tables>