WO2001082959A1

WO2001082959A1 - Omp protein associated with autologous and/or heterologous tumour cell lysate

Info

Publication number: WO2001082959A1
Application number: PCT/FR2001/001348
Authority: WO
Inventors: Toufic Renno; Isabelle Invernizzi; Jean-Yves Bonnefoy
Original assignee: Pierre Fabre Medicament
Priority date: 2000-05-04
Filing date: 2001-05-03
Publication date: 2001-11-08
Also published as: EP1278539A1; AU2001258481A1; FR2808445A1

Abstract

The invention concerns a pharmaceutical composition comprising an Omp membrane protein, in particular an OmpA membrane protein of Klebsiella pneumoniae, associated with lysate of autologous and/or heterologous tumour cells and the use of said compositions for preventing and treating cancer. The invention also concerns a method for isolating tumour antigens using said Omp.

Description

OMP PROTEIN ASSOCIATED WITH A LYSATE OF AUTOLOGOUS AND / OR HETEROLOGOUS TUMOR CELLS

The invention relates to a pharmaceutical composition comprising an Omp membrane protein, in particular an OmpA membrane protein of Klebsiella pneumoniae, associated with a lysate of autologous and / or heterologous tumor cells as well as the use of these compositions for the prevention and treatment cancer.

Vaccination is an effective way to prevent or reduce viral or bacterial infections. The success of vaccination campaigns in these fields has made it possible to extend the concept of vaccine previously used in the field of infectious diseases to the fields of cancer and autoimmune diseases. Vaccine antigens administered alone in the host are often not immunogenic enough to induce an immune response, and must therefore be combined with an adjuvant or coupled to a carrier protein to induce (or increase) their immunogenicity. Under these conditions, only a humoral type immune response can be induced. However, in the context of antiviral therapy, the generation of cytotoxic T lymphocytes (CTL) capable of recognizing and destroying the virus is of great importance (Bachmann et al., 1994, Eur. J. Immunol., 24, 2228 -2236; Borrow P., 1997, J. Virol. Hepat, 4, 16-24), as attested by numerous studies showing, in vivo, the protective role of responses directed against viral epitopes (Arvin AM, 1992, J. Inf. Dis., 166, S35-S41; Koszinowski et al., 1987, Immunol. Lett., 16, 185-192).

The importance of CTL responses has also been strongly documented in anti-tumor responses, in particular those directed against melanoma cells (reviewed in Rivoltini et al., 1998, Crit. Rev. Immunol., 18, 55-63). The CTL epitope (s) (peptide sequences interacting with Let class molecules presented to CD8 + T lymphocytes) have been defined for several antigens. However, the difficulty lies in the generation of CTL in vivo, due to the low immunogenicity of these peptides (Melief, 1992, Adv. Cancer Res., 58, 143-175; Nandaz and Sercaz, 1995, Cell, 82, 13- 17). Thanks to their efficiency in presenting antigens and stimulating the immune system, dendritic cells, for example, have been used to generate CTL anticancer responses (Nestlé FO et al., 1998, Nat. Med., 4, 328-332). The approaches consisted of loading the dendritic cells ex vivo with the antigen of interest (peptides or cell lysate) and re-implanting these cells in the patient. Other approaches consist in transfecting the dendritic cells ex vivo with the gene coding for the antigen of interest and in reinjecting these transfected cells (Gilboa E. et al., 1998, Cancer Immunol. Immunother., 46, 82-87 ). These approaches have been successfully used in mice and recently in humans (Hsu FJ et al., 1996, Nat. Med., 2, 52-58) but nevertheless remain complex insofar as these cells must be treated ex vivo (transformation of cells or internalization of antigens) and transplanted into the host organism. Likewise, the use of viral type particles (Layton GT et al., 1993, J. Immunol, 151, 1097-1107) or of incomplete Freund's adjuvant (IF A) (Nalmori et al., Eur. J. Immunol., 1994, 24, 1458-1462) makes it possible to generate CTL responses. However, an anti-tumor vaccination carried out with peptides corresponding to CTL epitopes and in the presence of such an adjuvant can lead to a specific state of tolerance which can lead in certain cases to the desired opposite effect, that is to say to a decrease in the immune response (Toes et al., Proc. Νat. Acad. Sci., USA, 1996, 93, 7855-7860).

The international patent application published on April 5, 1990 under the number WO 90/03183 describes immunotherapeutic vaccines comprising antigens originating from melanoma cell lysates, an adjuvant, called “DETOX” consisting of a detoxified endotoxin of the monophosphoryl lipid A type. (MPL) derived from Sahnonella minnesota and a carrier derived from the cell envelope of Mycobacterium bovis, strain BCG. The possible side effects of DETOX and of BCG derivative used in immunotherapeutic vaccines are known to those skilled in the art. The object of the present invention consists of new and improved immunotherapeutic compositions which in particular have the advantage of:

- allow prophylactic and therapeutic treatment;

- allow treatment at the onset of tumors at an early stage before the onset of symptoms associated with advanced stages of the disease; - allow a non-toxic treatment with more or less side effects; - allow to inhibit the growth of tumors.

Surprisingly, it has been demonstrated that an Omp protein, such as the P40 protein of Klebsiella pneumoniae, associated with a lysate of autologous and / or heterologous tumor cells, preferably without recourse to the addition of an adjuvant or a carrier, had the advantages mentioned above.

These properties of Omp could be explained in particular by the fact that the Omp proteins, unlike the carrier proteins conventionally used, make it possible to specifically retain antigens and in particular to better retain a larger number of tumor antigens compared to conventional carriers. Thus, the present invention relates to a pharmaceutical composition comprising in an acceptable pharmaceutical medium at least one Omp protein, or one of its fragments, associated with a lysate of autologous and / or heterologous tumor cells.

Proteins derived from said Omp protein whose amino acid sequence exhibits homology, as defined below, of at least 80%, preferably 85%, 90%, 95% and 99%, after optimal alignment with the sequence of the reference Omp protein and which are capable of generating or increasing a CTL and / or anti-tumor response can also be used as Omp protein in the pharmaceutical compositions according to the present invention. In the present invention, the term “protein” will also be understood to denote the peptides or polypeptides and the term “Omp” (for “Outer Membrane Protein”), the proteins of the outer membrane. As examples of Omp, the OmpA, the porines (in particular the OmpC and F) and the OmpX of E. coli. In a preferred embodiment, the Omp is chosen from among. OMP of Enterobacteriaceae.

In a particularly preferred embodiment, the Omp is chosen from OmpA, namely the proteins of the external membrane of type A, and more particularly those of Klebsiella pneumoniae. Even more preferably, the one called P40 will be used as described in patents WO 95/27787 and WO 96/14415. The term “fragment of an Omp protein” is intended to denote in particular any fragment of the amino acid sequence included in the amino acid sequence of the Omp protein which is capable of generating or increasing a CTL and / or anti-tumor response, said fragment Omp protein comprising at least 5 amino acids, preferably at least 10 amino acids or more preferably at least 15, 20, 25, 30, 40, 50, 75 and 100 consecutive amino acids of the sequence of said Omp protein .

In a particular embodiment, the composition according to the invention comprises an Omp protein or one of its fragments according to the invention, characterized in that said Omp protein, or one of its fragments, is obtained by a process extraction from a culture of said enterobacterium.

The methods for extracting bacterial membrane proteins are known to those skilled in the art and will not be developed in the present description. Mention may be made, for example, but not limited to, of the extraction process described by Haeuw J.F. et al. (EUT. J. Biochem, 255, 446-454, 1998).

In another preferred embodiment, the composition according to the invention comprises an Omp protein or one of its fragments, characterized in that said Omp protein or one of its fragments, is obtained by recombinant route. Particularly preferred are the Omp proteins obtained by the recombinant route. In fact, these recombinant proteins are particularly well defined, and can thus be used and marketed for administration in humans more easily and quickly, in particular for reasons of marketing authorization.

The methods for preparing recombinant proteins are today well known to those skilled in the art and will not be developed in the present description. Among the cells which can be used for the production of these recombinant proteins, mention must of course be made of bacterial cells (Olins PO and Lee SC, 1993, Recent advances in heterologous gene expression in E. coli. Curr. Op. Biotechnology 4: 520-525 ), but also yeast cells (Buckholz RG, 1993, Yeast Systems for the Expression of Heterologous Gene Products, Curr. Op. Biotechnology, 4: 538-542), as well as animal cells, in particular cell cultures mammalian (Edwards CP and Aruffo A., 1993, Current applications of COS cell based transient expression Systems, Curr. Op. Biotechnology, 4: 558-563) but also insect cells in which methods using, for example, baculoviruses can be used (Luckow N? A., 1993, Baculovirus Systems for the expression of human gene products, Curr. Op. Biotechnology, 4: 564-572). Most preferably, the Omp protein used in the compositions according to the invention is the OmpA protein from Klebsiella pneumoniae.

In particular, the invention relates to the use in the compositions according to the invention, of the amino acid sequence of said OmpA protein of Klebsiella pneumoniae, or one of its fragments, which comprises: a) the sequence of amino acids of sequence SEQ ID Ν ° 1; b) the amino acid sequence of a sequence having a homology of at least 80%, preferably 85%, 90%, 95% and 99%, after optimal alignment with the sequence SEQ ID No. 1; or c) the amino acid sequence of a fragment of at least 5 amino acids, preferably 10, 15, 20, 25, 30, 40, 50, 75 and 100 amino acids, of a sequence as defined in a).

By nucleic acid or amino acid sequence having a homology of at least 80% after optimal alignment with a determined nucleic acid or amino acid sequence, is meant a sequence which after optimal alignment with said determined sequence includes a percentage identity of at least 80% with said determined sequence.

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. Optimal alignment of sequences for comparison can be carried out, in addition to manually, using the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math., 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol., 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci., USA, 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST N or BLAST P comparison software).

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.

For example, we could use the BLAST program, "BLAST 2 sequences", available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extension gap penaltie”: 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program. Among said sequences having a homology of at least 80% with the reference Omp sequence, or among the amino acid sequences of fragment of reference Omp sequence, it is preferred:

- the peptide sequences capable of inducing CTL activity (Cytotoxic T Lymphocytes), as it can be evaluated with a standard method for measuring CTL activity based for example on the measurement of percentage of specific lysis of co- incubated with effector lymphocytes from animals immunized with a peptide derived from the reference Omp sequence associated with a relevant peptide; and or

- the sequences of peptides capable of generating or increasing an antitumor activity, as measured in particular in the examples below.

Preferably, said homologous peptides or said Omp fragments will be capable of inducing a CTL activity and / or of generating or increasing an antitumor activity at least equal to 10%, preferably 20%, 30%, 50% and 75% of the activity measured under the same conditions for the reference Omp sequence.

The other essential element of the pharmaceutical compositions according to the invention relates to a lysate of autologous and / or heterologous tumor cells. For the purposes of the present invention, the term “autologous tumor cells” means tumor cells belonging to the subject who will receive the compositions according to the invention.

The term “heterologous tumor cells” should be understood to mean cells originating from tumors originating from an individual different from that for which the composition according to the invention is intended. The use of heterologous cells makes it possible to obtain pharmaceutical compositions which make it possible in particular to treat patients suffering from cancer from whom the removal of tumor cells is not possible. The use of heterologous cells also makes it possible to obtain standardized compositions according to the invention comprising antigens found in many types of cancer and thus usable in a majority of patients.

Tumor cells can be obtained from a sample of cancerous tissue, for example following a biopsy or surgical resection.

These cells can then be used as such or be cultured before being lysed. A cell lysate can be defined within the meaning of the present invention as a mixture of intracellular and / or membrane antigens, preferably intracellular and membrane.

Among the lysates of tumor cells forming part of the pharmaceutical composition according to the invention, preferred are the lysates of tumor cells expressing an associated tumor antigen, in particular a tumor antigen whose expression is induced by the tumor or potentially tumor character of the cell. Among said tumor cells expressing an associated tumor antigen, particular preference is given to tumor tumor cells originating from cancers with tumor cells expressing an associated tumor antigen, as mentioned below.

Said autologous and / or heterologous tumor cell lysate according to the invention can be obtained by mechanical, chemical or enzymatic lysis of tumor cells.

In order to lyse the cells mechanically, mention may in particular be made of the techniques known to those skilled in the art, namely in particular sonication, ultrasonication or freezing / thawing. Particularly preferred is freeze / thaw, and most particularly the use of multiple freeze / thaw cycles.

The cells can also be lysed using chemical compounds or enzymes, such as for example digitonin lysis buffer, triton X-100 or Nonidet P40. Any method of breaking the cell membrane of tumor cells can be used to obtain a lysate.

The pharmaceutical composition according to the invention can also comprise a cytokine or a growth factor, in particular alpha or gamma interferon, TNF, GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), IL-2, IL-4, IL-6 and IL-18, an HSP (Heat Shock Protein) such as for example hsρ70, hsρ90, hsρ96, which makes it possible to potentiate the immune response, and / or fibroblasts genetically engineered to release a cytokine or growth factor. Mention may be made of fibroblasts expressing GM-CSF sold by the company Immune Response Corporation. In one embodiment of the invention, the compositions according to the invention can also contain antigen presenting cells. The latter can be chosen in particular from macrophages, B lymphocytes or dendritic cells and preferably from macrophages.

Antigen presenting cells can be obtained by in vitro production from stem cells from human blood or spinal cord (see

Inaba et al., 1992, J. Exp. Med., 176: 1693-1702). We can use macrophages humans obtained from blood mononuclear cells preferably isolated from the patient to be treated. The number of macrophages can be increased using M-CSF (Macrophage Colony Stimulating Factor).

It may be noted that the use of antigen presenting cells is optional in the present invention. Thus, the invention also relates to compositions according to the invention without antigen presenting cells allowing a therapeutic treatment which does not require ex vivo manipulation of antigen presenting cells and whose implementation is thus easier and more simplified and not having the possible disadvantages of those using antigen presenting cells.

The compositions according to the invention can also comprise an adjuvant making it possible to increase the immune response, in particular chosen from the group of adjuvant comprising Qs21 (mixture of saponins marketed by the company

Aquila), ISCOM (Immunostimularoty complex marketed by the company CSL Ltd.), CpG (DNA sequences of bacteria marketed by the company Coley

Pharmaceutical Group), Leif (Leishmania major Initiation Factor marketed by the company Corixa), CT (cholera toxin) or LT (LT for “Heat labile enterotoxin”, just like the detoxified versions of CT or the LT.

In another aspect, the composition according to the invention is characterized in that it contains no other adjuvant making it possible to increase the immune response such as those mentioned above, apart from said Omp protein, in particular said enterobacterium OmpA protein , or one of its fragments.

Within the meaning of the present invention, the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium injectable into humans. It can for example consist of water, an aqueous saline solution or an aqueous solution based on dextrose and or glycerol.

In a particular embodiment, the composition according to the invention also contains a detergent.

The compositions according to the invention can also contain a detergent, and in particular any type of pharmaceutically acceptable surfactant, such as for example anionic, cationic, nonionic or amphoteric surfactants. We preferably uses Zwittergent 3-12 detergents and octylglucopyrannoside and even more preferably Zwittergent 3-14.

The invention also relates to the compositions according to the invention, characterized in that they are conveyed in a form which makes it possible to ensure and / or improve their stability, and thus also comprise, for example, vectors making it possible to ensure and / or to improve the stability of the Omp protein association or one of its fragments and of the lysate of autologous and / or heterologous tumor cells, in particular the vectors chosen from liposomes, virosomes, nanospheres, microspheres or microcapsules. The invention also includes the use of an Omp protein or a fragment thereof associated with a lysate of autologous and / or heterologous tumor cells for the preparation of a pharmaceutical composition intended to generate or enhance an immune response against a tumor cell.

In a preferred embodiment according to the invention, the Omp protein or one of its fragments is used in combination with a lysate of autologous tumor cells for the preparation of a pharmaceutical composition intended to generate or increase an immune response against said cell autologous tumor.

More generally, the use according to the invention relates to the preparation of a pharmaceutical composition intended for preventing or treating cancers, preferably cancers associated with a tumor antigen.

Among the cancers whose tumors express an associated tumor antigen which can be prevented or treated by the uses according to the present invention, there may be mentioned in particular, but not limited to:

• breast, lung, colon cancer and gastric carcinoma (Kawashima et al., 1999, Cancer Res., 59: 431-5);

• mesothelioma, osteosarcoma, brain cancer (Xie et al., 1999, J. Natl. Cancer. Inst, 91: 169-75);

• melanoma (Zheuten et al., 1998, Bratilsl. Lek. Listy, 99: 426-34);

• pancreatic adenocarcinoma (Hammel et al., 1998, Eur. J. Gastroenterol. Hepatol., 10: 345-8);

• colorectal cancer (Ogura et al., 1998, Anticancer Res., 18: 3669-75); • renal cell carcinoma (Jantzer et al., 1998, Cancer Res., 58: 3078-86); and

• ovarian and cervical cancer (Sonoda et al., 1996, Cancer., 77: 1501-9).

Another subject of the invention is the use of an Omp protein or of a fragment thereof associated with a lysate of autologous and / or heterologous tumor cells for the preparation of a pharmaceutical composition intended for inhibiting the growth of tumors .

These different uses use the compositions described above.

The therapeutic method of implementing the compositions according to the invention consists in particular in:

- assess in a patient the type of cancer he has;

- to choose according to the type of cancer a treatment using autologous and / or heterologous tumor cells;

- if necessary to collect tumor cells; - optionally and not preferably to carry out primary or secondary cultures of said tumor cells;

- To prepare the compositions according to the invention with adequate concentrations of protein Omp or one of its fragments and lysate of tumor cells;

- injecting the patient with said composition, preferably subcutaneously or intradermally;

- if necessary to repeat these injections several times, preferably 3 injections one month apart and thereafter a booster every 3 to 6 months.

The appropriate concentrations of Omp protein or of a fragment thereof and of tumor cell lysate may in particular be chosen so as to obtain compositions comprising for example from 0.01 to 50 mg, and preferably from 0.1 to 25 mg of cell lysate, and from 0.1 to 50 mg, and preferably from 1 to 25 mg of Omp. The Lysat / Omp ratio can be between 1/2 and 1/10, and preferably between 1/5 and 1/8.

When antigen presenting cells are used, these are incubated in vitro with the Omp protein, or a fragment thereof, associated with a lysate of autologous and / or heterologous tumor cells. They could for example be incubated at 37 ° C for a period of between 5 minutes and 24 hours. The amounts and concentrations of antigen presenting cells to be used are those described in the prior art. We can thus use for example 10 ⁷ antigen presenting cells per ml. The antigen presenting cells brought into contact with the Omp protein, or a fragment thereof, associated with a lysate of autologous and / or heterologous tumor cells are then injected into the patient, preferably the patient from which they originated. Depending on the patient's physical condition, for example, 10 ⁶ to 10 ¹² antigen presenting cells may be injected.

The subject of the invention is also a device suitable for implementing the above-mentioned therapeutic method, such as for example a kit, comprising at least the Omp and the means necessary to prepare the compositions according to the invention, such as for example a protocol, a pharmaceutically acceptable medium or a lysis buffer. When antigen presenting cells are used, this device may also contain these cells, preferably purified. Finally, the subject of the invention is a method for detecting tumor antigens, characterized in that it consists in:

- associate an Omp protein or one of its fragments with a lysate of autologous and / or heterologous tumor cells so as to form complexes between the Omp proteins or their fragments and the tumor antigens of the lysate of autologous and / or heterologous tumor cells ;

- isolating the complexes thus formed;

- to separate from these complexes the tumor antigens; and

- to identify said tumor antigens.

The first step of this process consists in mixing the tumor extracts with proteins or fragments of Omp, in particular in the quantities and proportions mentioned above. Prior to this step, it is possible to modify the Omp or their fragment so as to make them suitable for the step of isolating the Omp-antigen complexes, it is for example possible to biotinylate the Omp.

To isolate the complexes thus formed, an affinity chromatography column can be used, then by carrying out successive elutions, the tumor antigens will be separated and isolated. Conventional techniques are then used to identify said antigens, such as, for example, the sequencing of said antigens by techniques known to those skilled in the art using mass spectrometry, a Maldi-Tof and bioinformatics. The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope.

Legends of the figures:

Figure 1: Measurement of tumor volume following immunization with a B16.F10 lysate with or without P40.

Figures 2A and 2B: Measurement of the percentage of eyes with pathology (Figure 2A) and the degree of pathology (Figure 2B) in TRP / Tag mice following immunization with an eye lysate with or without P40. Degree 0: no visible pathology; Degree 1: moderate pathology; Degree 2: severe pathology.

Figures 3A and 3B: SDS-PAGE gel showing the fractions of the mixtures of the lysate of B16.F10 with P40-biotin (Figure 3 A) or TT-biotin (Figure 3B) eluted with detergent from a streptavidin-sepharose column (' TT 'for Tetanus Toxoid).

EXAMPLE I Measurement of the Antitumor Activity in C57BL / 6 Mice

2,500 melanoma B16.F10 cells were implanted subcutaneously on day 0 in C57BL / 6 mice, from which the B16.F10 cells were originally derived. Also on day 0, the C57BL / 6 mice were immunized subcutaneously at the base of the tail with P40 (350 μg) mixed with a lysate of 10 B16.F10 cells (ground with a Dounce homogenizer, sonicated and passed over nylon screen) or with lysate alone. A second immunization 10 days apart from the first was also done using the same injection method. From day 18 post-implantation, the volume of the subcutaneous tumor was measured.

As shown in FIG. 1, the immunization of C57BL / 6 mice which received melanoma B 16F 10 cells on the same day with P40 mixed with the lysate of B 16F 10, but not with the lysate alone, significantly slows the development of subcutaneous melanoma.

EXAMPLE II Measurement of the Antitumor Activity in the TRP / Tag Mouse

TRP / Tag mice develop tumors in the eyes, which can therefore be easily seen.

A mixture of P40 (350 μg) and tumor eye lysate (ground with a Dounce homogenizer, sonicated and passed through a nylon sieve) or lysate alone were injected subcutaneously at the base of the tail in TRP / Tag mice at 4 weeks of age. The frequency and severity of eye tumors were measured daily.

The frequency and severity of eye tumors in TRP / Tag mice are significantly reduced after immunization with the mixture of P40 and the lysate (see Figures 2A and 2B). However, no effect was observed in mice treated with the lysate alone. This result is all the more surprising since the malignant transformation process of the pigmented retinal epithelium in TRP / Tag mice begins from before birth, therefore at least 3 weeks before the first immunization. Thus, even when the tumor is already at an advanced stage, the present invention makes it possible to obtain a significant reduction in this tumor.

Example III Comparison of the Ability to Retain P40 Antigens with That of TT

The biotinylation of the P40 or TT proteins is carried out in a mixture containing 340 μl of P40 or TT at 6 milligrams (mg) / ml, 100 μl of NaHCO3, 0.5 Mole / liter (M), pH 8.5, 100 μl of S-NHS biotin (2 mg / ml) and 460 μl of ultra pure water.

The mixture is then placed for 2 hours (h) at 4 ° C in order to stop the biotinylation reaction and is then dialyzed overnight against PB S in dialysis cassettes at room temperature with magnetic stirring in order to remove the biotin in excess.

The P40 biotin (2 mg / ml) + lysate (0.9 x 10 ⁶ cells per ml) or TT biotin (2 mg / ml) + lysate (0.9 x 10 ⁶ cells per ml) mixture is dialyzed in a cassette dialysis against ultra pure water overnight, at room temperature, with magnetic stirring. This step makes it possible to promote the hydrophobic interactions between P40 and the lysate. TT serves as a negative control. The support used for this chromatography low pressure affinity is an analytical column with a stationary phase of high performance sepharose-streptavidin previously saturated with BSA in order to eliminate all artefactual interactions. The samples used are either P40 biotin + lysate or TT biotin + lysate obtained after the dialysis step and are incubated with the resin for half an hour at room temperature so that the biotin streptavidin interactions take place correctly.

The washing step is carried out with five column volumes of PBS IX and the elution of the proteins and peptides retained by P40 is carried out with five column volumes of PBS IX + 0.1% of chaps. The resin is then washed with 2 column volumes of PBS IX. The samples are lyophilized and taken up in Tris Glycine / SDS buffer, heated at 99 ° C for 5 min and centrifuged for 1 min at 13,000 rpm ("rpm" for revolutions per minute). The proteins are analyzed by denaturing electrophoresis on polyacrylamide gel.

After separation, the proteins are visualized by staining with silver nitrate. As shown in FIGS. 3 A and 3B, several proteins are eluted with the detergent in the case of the P40-lysate mixture (FIG. 3 A), but not in the case of the TT-lysate mixture (FIG. 3B), demonstrating the existence of specific hydrophobic interactions of Omp. Thus, Omp proteins, unlike the carrier proteins conventionally used such as TT, make it possible to specifically retain antigens and thereby isolate and analyze them. OMPs therefore make it possible to isolate a larger number of tumor antigens compared to conventional carriers.

Claims

1) Pharmaceutical composition characterized in that it comprises in a pharmaceutically acceptable medium at least one Omp protein, or one of its fragments, associated with a lysate of autologous and / or heterologous tumor cells.

2) Composition according to claim 1, characterized in that said Omp protein, or one of its fragments, is obtained by an extraction process from an enterobacterium culture.

3) Composition according to claim 1, characterized in that said Omp protein, or one of its fragments, is obtained by recombinant route.

4) Composition according to one of claims 1 to 3, characterized in that said Omp protein is the OmpA protein of Klebsiella pneumoniae.

5) Composition according to claim 4, characterized in that the amino acid sequence of said OmpA protein, or one of its fragments, comprises: a) the amino acid sequence of sequence SEQ ID No. 1; b) the amino acid sequence of a sequence having at least 80% homology with the sequence SEQ ID No. 1; or c) the amino acid sequence of a fragment of at least 5 amino acids of a sequence as defined in a). 6) Composition according to one of claims 1 to 5, characterized in that said lysate of autologous and / or heterologous tumor cells is a mixture of intracellular eJoxx membrane antigens, preferably intra and membrane.

7) Composition according to one of claims 1 to 6, characterized in that it further comprises a cytokine and / or a growth factor or fibroblasts releasing a cytokine and / or a growth factor.

8) Composition according to one of claims 1 to 7, characterized in that it further comprises cells presenting antigens and preferably macrophages. 9) Composition according to one of claims 1 to 8, characterized in that it further comprises an adjuvant.

10) Composition according to one of claims 1 to 8, without other adjuvant for inducing an immune response. 11) Composition according to one of claims 1 to 10, characterized in that said pharmaceutically acceptable medium consists of water, an aqueous saline solution or an aqueous solution based on dextrose and / or glycerol.

12) Composition according to one of claims 1 to 11, characterized in that said composition also contains a detergent. 13) Composition according to one of claims 1 to 12, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability.

14) Use of an Omp protein or of a fragment thereof associated with a lysate of autologous and / or heterologous tumor cells for the preparation of a pharmaceutical composition intended to generate or increase an immune response against a tumor cell.

15) Use of an Omp protein or of a fragment thereof associated with a lysate of autologous tumor cells for the preparation of a pharmaceutical composition intended to generate or increase an immune response against said autologous tumor cell.

16) Use of an Omp protein or of a fragment thereof associated with a lysate of autologous and / or heterologous tumor cells for the preparation of a pharmaceutical composition intended for preventing or treating cancers, preferably cancers associated with a tumor antigen. 17) Use of an Omp protein or of a fragment thereof associated with a lysate of autologous and / or heterologous tumor cells for the preparation of a pharmaceutical composition intended for inhibiting the growth of tumors.

18) Use according to any one of claims 14 to 17, characterized in that said pharmaceutical composition is as defined in any one of claims 1 to 13.

19) Device characterized in that it comprises at least one Omp and one means necessary for the preparation of the compositions according to the invention as defined in any one of claims 1 to 13.

20) Method for detecting tumor antigens characterized in that it consists in: - associating an Omp protein or one of its fragments with a lysate of autologous and / or heterologous tumor cells so as to form complexes between the proteins Omp or fragments thereof and tumor antigens of the lysate of autologous and / or heterologous tumor cells;

- isolate the complexes thus formed; - separate tumor antigens from these complexes; and

- identify said tumor antigens.