EP1368457A1 - Use of gram-negative bacterial membrane fraction for inducing the maturation of dendritic cells - Google Patents
Use of gram-negative bacterial membrane fraction for inducing the maturation of dendritic cellsInfo
- Publication number
- EP1368457A1 EP1368457A1 EP02716890A EP02716890A EP1368457A1 EP 1368457 A1 EP1368457 A1 EP 1368457A1 EP 02716890 A EP02716890 A EP 02716890A EP 02716890 A EP02716890 A EP 02716890A EP 1368457 A1 EP1368457 A1 EP 1368457A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- dendritic cells
- membrane fraction
- use according
- maturation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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Definitions
- the present invention relates to the use of a membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae to induce the maturation of dendritic cells and thus promote the development of a specific immune response.
- Dendritic cells play a role in the development of an immune response and in the initiation of a specific T lymphocyte response (Steinman RM et al Immuno Rev (1997) 156, 25 & Cella M et al Curr Opin Immunol (1997) 9, 10). Immature dendritic cells are located in non-lymphoid tissue. At the skin level, in all epidemics except the intestine, they are then called Langerhans cells; dendritic cells, in smaller quantities, are also located in organs such as the lung, liver and intestine. These immature dendritic cells capture and digest antigens very efficiently.
- the dendritic cells After antigenic stimulation in vivo or stimulation by proinflammatory molecules, the dendritic cells which have captured the antigens migrate into the secondary lymphoid organs. During this migration, the dendritic cells undergo functional and phenotypic modifications which are grouped under the term of maturation.
- This maturation is characterized by an increase on the surface of dendritic cells of molecules involved in the activation of T lymphocytes (such as CD40, CD54, CD58, CD86 and MHC class I / O), the production of proinflarnmatory cytokines (IL-6 ) and chemokines (such as TNF ⁇ , E -8, MCP-1 and MlP-la) and lose their ability to process the antigen.
- T lymphocytes such as CD40, CD54, CD58, CD86 and MHC class I / O
- IL-6 proinflarnmatory cytokines
- chemokines such as TNF ⁇ , E -8, MCP-1 and MlP
- Chemokines recruit inflammatory cells and promote the initiation of an immune response.
- IL-8, MCP-1 and MlP-1 ⁇ attract not only inflammatory cells but also naive and memory T lymphocytes.
- dendritic cells by secreting chemokines increase their chances of coming into contact with T lymphocytes.
- the proinflammatory cytokines will also act by directly activating CD.
- These mature dendritic cells present the antigens to the T lymphocytes and initiate the specific T responses very efficiently, the costimulation molecules being expressed in large quantities on these cells. As cells mature, they lose their ability to capture and process the antigen. In the thymo-dependent areas of the lymphoid organs, the migrated dendritic cells have acquired powerful immunostimulatory properties and will therefore very effectively activate the circulating naive T lymphocytes.
- the membrane fraction of K. pneumoniae 1145 is used in the composition of a pharmaceutical preparation preventing the occurrence and recurrence of respiratory infections of bacterial origin and used in humans for 20 years. As such, there is a decline in non-toxicity of the product.
- the present invention thus relates to the use of at least one membrane fraction of gram negative bacteria, preferably of Klebsiella pneumoniae to induce the maturation of dendritic cells.
- the present invention also relates to the use of at least one membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae for the manufacture of a medicament intended to induce the maturation of dendritic cells.
- bacterial membrane fraction is intended to denote in the present invention any purified or partially purified membrane fraction or extract obtained from a culture of said bacteria and the preparation process of which comprises at least one step of lysis of the bacteria obtained after culture. and a step of separating the fraction containing the membranes of said bacteria from the total lysate obtained after the lysis step, in particular by centrifugation or filtration.
- a membrane fraction comprises at least membrane proteoglycans.
- a membrane fraction can also be defined as a fraction comprising at least membrane proteins associated with LPS (lipopolysaccharides), known to those skilled in the art.
- proteoglycan titer of the membrane fractions according to the invention is represented by the sum of the galactose and protein contents, is preferably understood:
- membrane fractions according to the invention are capable of being obtained by the methods described below or by any other equivalent method, in particular those described in patent applications FR9903154 and FR9903153.
- membrane fractions can be used at concentrations of 0.1 to 100, preferably from 1 to 50, and more preferably from 1 to 20 micrograms per milliliter of culture medium.
- the gram-negative bacteria are chosen from bacteria of the genus Klebsiella and preferably of pneumoniae species.
- the membrane fractions according to the invention can serve to promote the maturation of dendritic cells in vitro, the mature cells then being able to be reinjected in vivo.
- this medicament can be used to promote the generation and maturation of dendritic cells in vitro, after contact with a biological agent.
- the invention relates to the use of a membrane fraction according to the invention and in addition to at least one biological agent for the manufacture of a medicament. The latter may for example increase the immunological response vis-à-vis this biological agent.
- This biological agent can be chosen from nucleic acids, proteins, lipids, lipopeptides or polysaccharides.
- It can more particularly be chosen from vaccinating antigens and / or antigens from bacteria, viruses, yeasts, parasites, fungi.
- the biological agent is a tumor antigen.
- a tumor antigen is defined as a tumor protein or peptide, and in particular as an epitope, in particular a CTL epitope (peptide sequences interacting with class I molecules and presented to CD8 + T lymphocytes) or as the nucleic acid sequence encoding such proteins, peptides or epitopes.
- tumor antigens may be mentioned without limitation: MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens ON- TL3 and MON 18, TUA ⁇ , alpha-feto protein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associated antigen G250, EGP-40 (or EpCAM), S100 (malignant melanoma -associated antigen), p53, prostate tumor-associated antigens (eg, PSA and PSMA), and p21ras.
- the biological agent can also be chosen from a lysate of autologous and / or heterologous tumor cells.
- autologous tumor cells means tumor cells belonging to the subject who will receive the compositions according to the invention.
- 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 compositions according to the invention standardized 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 they are or can be cultured before being lysed.
- a cell lysate can be defined within the meaning of the present invention as a mixture of intra-cellular and / or membrane antigens, preferably intra and membrane.
- Said autologous and / or heterologous tumor cell lysate according to the invention can be obtained by mechanical, chemical or enzymatic lysis of tumor cells. 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 and thawing.
- freeze / thaw 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 dendritic cells are thus modified using these antigens or cell lysates so that they express tumor antigens.
- said drug can be injected at the same time as the dendritic cells or, preferably, said drug can be used in vitro, in order to promote the maturation of the dendritic cells before reinjecting them into patients.
- dendritic cells in the presence of a membrane fraction of gram-negative bacteria can be used to generate CTL anticancer responses (Nestlé FO et al., 1998, Nat. Med., 4, 328-332).
- This approach called “ex vivo” therefore consists in loading the dendritic cells ex vivo with the antigen of interest (peptides or cell lysate) in the presence of a membrane fraction of bacteria with gram negative and to re-implant these cells in the patient.
- a step of washing the dendritic cells so as to remove the membrane fraction of gram-negative bacteria from the medium containing the dendritic cells can be carried out before re-implanting these cells in the patient.
- Another approach according to the invention consists in transfecting ex vivo the dendritic cells with the gene coding for the antigen of interest and in particular with a gene coding for a bacteria, virus, yeast, parasite, fungus antigens, or for a tumor antigen, such as those described above, and / or with a gene encoding a cytokine or a growth factor, such as those described below, and to put the dendritic cells, before and / or during and / or after transfection, in the presence of a membrane fraction of gram-negative bacteria and to reinject these transfected cells (Gilboa E.
- a step of washing the dendritic cells so as to remove the membrane fraction of gram-negative bacteria from the medium containing the dendritic cells can be carried out before re-implanting these cells in the patient.
- Such approaches have been successfully used in mice and in humans (Hsu FJ et al., 1996, Nat. Med., 2, 52-58).
- the medicament according to the invention may also comprise a cytokine or a growth factor, in particular interferon alpha or gamma, TNF ⁇ , GM-CSF, 1TL- 2, 1 "IL-12, ⁇ IL-4, ⁇ IL- 6 and IL-IS, an HSP (Heat Shock Protein) such as for example hsp70, hsp90, hsp96, which makes it possible to potentiate the immune response; and / or fibroblasts genetically modified so as to release a cytokine or a growth factor.
- a cytokine or a growth factor in particular interferon alpha or gamma, TNF ⁇ , GM-CSF, 1TL- 2, 1 "IL-12, ⁇ IL-4, ⁇ IL- 6 and IL-IS, an HSP (Heat Shock Protein) such as for example hsp70, hsp90, hsp96, which makes it possible to potentiate the immune response; and / or fibroblast
- the medicament according to the invention may also comprise an adjuvant making it possible to increase the immune response, in particular chosen from the group of adjuvant comprising MPL-A, Quil-A, ISCOM, CpG, Leif, CT (cholera toxin) or LT (Heat labile enterotoxin), just like the detoxified versions of CT or LT, or membrane proteins bacteria such as OMPC of Neisseria meningitidis (Nella et al., Infect. Immun. 60, 1992, 4977-4983), TraT of Escherichia coli (Croft et al, J. Immunol.
- an adjuvant comprising MPL-A, Quil-A, ISCOM, CpG, Leif, CT (cholera toxin) or LT (Heat labile enterotoxin), just like the detoxified versions of CT or LT, or membrane proteins bacteria such as OMPC of Neisseria meningitidis (Nella et al., Infect.
- PorB of Neisseria meningitidis (Fusco et al, J. Infect. Dis. 175, 1997, 364-372), and preferably an OmpA of a bacterium of the genus Klebsiella, a major protein of the outer membrane called P40, exhibiting an adjuvant activity for peptide subunit antigens (WO 95/27787 and WO 96/14415; Haeuw et al, Eur. J. Biochem. 255, 1998, 446-454; Plornicky-Gilquin et al, J. Virol. 73, 1999, 5637- 5645).
- the medicament according to the invention can also comprise a pharmaceutically acceptable medium.
- the pharmaceutically acceptable medium is the medium in which the compounds of the invention are administered, preferably a medium injectable into humans. It can consist of water, an aqueous saline solution or an aqueous solution based on dextrose and / or glycerol.
- the medicament according to the invention can also be conveyed in a form making it possible to improve its stability and / or its immunogenicity; thus, it can be conveyed in the form of liposomes, virosomes, nanospheres, microspheres or microcapsules.
- the medicament or the association of a membrane fraction of gram-negative bacteria-biological agent may also be in an easily administered form such as an ointment, a lotion, a solution or even in the form of an adhesive composition: plaster , "Patch".
- the medicament according to the invention may be used in particular for the treatment:
- virus for example the human immunodeficiency virus (HIV), hepatic viruses, in particular hepatic viruses A, B , C and D and parainfluenza virus, bacteria, parasites and yeasts
- HAV human immunodeficiency virus
- hepatic viruses in particular hepatic viruses A, B , C and D and parainfluenza virus, bacteria, parasites and yeasts
- the invention particularly relates to cellular immunotherapy using anti-cancer and anti-infectious vaccines in particular, by generating autologous dendritic cells in vitro, by introducing a tumor antigen there and by reinjecting them after a step of optional washing. Exposing dendritic cells in vitro to the membrane fraction of gram-negative bacteria will increase the maturation of dendritic cells and therefore increase the effectiveness of the vaccine.
- the present invention relates particularly to the use of a membrane fraction of gram-negative bacteria, and more particularly of the membrane fraction of Klebsiella pneumoniae for the preparation of a medicament for increasing the immunological response vis-à-vis a biological agent as defined above.
- This medicine can be a vaccine for the treatment or prevention of infectious diseases of viral origin - like in particular the NIH, the hepatic viruses and the parainfluenza virus -, bacterial, fungal, or caused by a yeast or a parasite.
- the present invention also relates to the use of a membrane fraction of gram-negative bacteria with at least one biological agent for the manufacture of a medicament for the treatment or prevention of cancers and particularly cancers among myelomas, lymphomas, leukemias , carcinomas of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, and for the manufacture of a medicament for the treatment or prevention of skin cancers chosen from keratinomas and carcinomas.
- the cancer cells are in direct contact with the Langerhans cells, located in the epidemic, the use according to the present invention by skin application makes it possible to act directly, locally on Langerhans cells.
- the medicament according to the invention may be applied to the skin, in particular by the cutaneous, subcutaneous, transdermal, intra-epidermal route or to the mucous membranes, it then acts systemically by the maturation of dendritic cells and locally by the maturation of Langerhans cells.
- the treatment of these cancers can also be envisaged by injecting autologous dendritic cells, and in particular dendritic cells.
- autologous which have been modified to express tumor antigens.
- the maturation of dendritic cells will be ensured by the membrane fraction of gram negative bacteria.
- the invention also relates to a device for the maturation of dendritic cells, such as for example a kit, comprising at least the membrane fraction of gram negative bacteria.
- This kit can be adapted for the maturation of dendritic cells in vitro and can be used for example in research laboratories.
- This kit may also contain the membrane fraction of gram-negative bacteria from immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means for purifying blood mononuclear cells. It may thus include, for example, different culture media, washing solutions, culture plates, reagents, controls such as, for example, antibodies, and an explanatory notice for the implementation of the method according to the invention. maturation of dendritic cells.
- a kit according to the invention can also be adapted to the implementation of the therapeutic method mentioned above.
- This kit can also contain the membrane fraction of gram-negative bacteria from immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means for purifying blood mononuclear cells. It may thus include, for example, different culture media, washing solutions, culture plates, reagents, controls, and an explanatory note for the implementation of the therapeutic method mentioned above. If necessary, it may also contain heterologous antigens or means for obtaining a lysate of autologous cells.
- the legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope.
- FIG. 1 illustrates the fact that:
- the membrane fraction of Klebsiella Pneumonia induces the expression of the CD83 molecule, increases CD86 expression as well as the production of IL-8.
- results are also expressed as a percentage of positive cells.
- results of the IL-8 assay are expressed in ng / ml and are expressed as an average ⁇ n.d. of 4 experiments.
- Immature DCs were treated or not for 24 hours with membrane fraction of Klebsiella Pneumonia or LPS. Then they were irradiated and cultured with allogeneic T lymphocytes from 2 different donors. The proliferation of T lymphocytes was measured after 5 days. The results are expressed in counts per minute (cpm) and represent the average of 5 values and are representative of one of 3 experiments.
- Example 1 Obtaining a membrane fraction of K. pneumomae
- a constant volume dialysis is then carried out on PUF 100 up to 800 ⁇ cm, followed by a concentration of the membrane suspension (SM) thus obtained, at 11 1 / kg of cells. dry.
- the SM is then autoclaved at + 121 ° C for 35 min which can be stored at + 4 ° C for 6 weeks.
- proteoglycan titer is equal to the sum of the galactose and protein contents.
- Example 2 The membrane fraction of Klebsiella Pneumonia increases the expression of CD 83 A.
- Human dendritic cells are generated from monocytes isolated from peripheral blood.
- the blood is taken by leukopheresis in the presence of an anticoagulant such as, for example, lithium heparinate.
- the monocytes are purified by positive selection using a magnetic cell separator (MACS TM; Miltenyi Biotex, Bergisch Gladbach, Germany) in accordance with the manufacturer's instructions.
- CMNs are incubated for 20 minutes at 4 ° C with magnetic beads on which are fixed an anti-human CD 14 monoclonal antibody. After washing, the cell suspension plus beads is placed on a column and subjected to a magnetic field. After three washes, the column is no longer subjected to the magnetic field and the monocytes are collected by gravitation.
- the purity of the monocytes is evaluated by cytofluorometry (FACScan cytofluorometer; Becton Dickinson, Erembodegem, Belgium) on the basis of the size-granulosity parameters of the cells. The purity is greater than 98%.
- the monocytes are then cultured at a concentration of 5 ⁇ 10 6 cells / ml in the following medium (hereinafter referred to as complete culture medium): RPMI 1640 medium supplemented with 10% serum fetal calf (heating at 56 ° C for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates (Nunc, Roskilde, Denmark ) at a rate of 5 ml of medium per well.
- complete culture medium hereinafter referred to as complete culture medium: RPMI 1640 medium supplemented with 10% serum fetal calf (heating at 56 ° C for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates (Nunc, Roskilde, Denmark ) at a rate of 5 ml
- the cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom). After 5 to 7 days of culture (37 ° C, 5% CO 2 in a humid atmosphere), the phenotype of the cells is defined by cytofluorometry. Briefly, an aliquot of the cell suspension is taken.
- the cells are washed in FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) and then distributed in wells of a 96-well culture plate at the bottom conical (Nunc) at the rate of 2 ⁇ 10 5 cells in a volume of 50 ml of FACS buffer.
- FACS buffer 10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide
- CDla versus CD83 The analysis of the expression of CDla versus CD83 is evaluated by FACS. Only immature dendritic cells characterized by expression of the CDla molecule (mean fluorescence intensity (IMF)> 100) and the absence of expression of the CD83 molecule were used.
- IMF mean fluorescence intensity
- Immature dendritic cells were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 ⁇ l in 96-well flat-bottomed culture plates (Costar, Cabridge, USA).
- the cells are activated with the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms, 20 micrograms and 50 micrograms per milliliter of final medium.
- 24 hours after stimulation the expression of the CD83 and CD86 molecules is evaluated by cytofluorometry using specific monoclonal antibodies labeled with fluorescein (Becton Dickinson).
- the control isotypic antibodies used come from Becton Dickinson.
- the cells are washed in FACS buffer and then distributed in wells of a 96-well culture plate with a conical bottom at the rate of 2 ⁇ 10 5 cells in a volume of 50 ⁇ l of FACS buffer. An antibody is added to each well. After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 ⁇ l of FACS buffer and then are resuspended in 200 ⁇ l of this same buffer. The analysis of the expression of the surface markers is evaluated by FACS.
- FIG. 1 show that the membrane fraction of Klebsiella pneumoniae increases the expression by immature dendritic cells of surface molecules involved in the activation of T lymphocytes:
- the membrane fraction of Klebsiella pneumoniae also increases the expression of other molecules involved in the activation of T lymphocytes, such as CD86.
- Example LU. The membrane fraction of Klebsiella Pneumonia induces the expression of IL-8 by the CD A.
- Methodology Immature human dendritic cells were generated as described in Example 1. After 5 to 7 days of culture, the cells are re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 ml in 96-well flat-bottomed culture plates. The cells are activated with the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms and 50 micrograms per milliliter of culture medium and after 24 hours of culture, the culture supernatants are centrifuged at 10,000 rpm for 15 minutes at 4 ° C.
- Immature human dendritic cells are generated as described in Example 1. After 5 to 7 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 ⁇ 10 5 cells / well in a 6-well culture plate (5 ml / well). The cells are not stimulated or are stimulated in the presence of the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms and 50 micrograms or 10 ng / ml LPS. After 24 hours of culture, the cells are collected and washed three times in RPMI 1640 medium. The cells are then irradiated at 3000 rad. Human T cells freshly isolated from peripheral blood were prepared by the rosette technique with sheep erythrocytes.
- the mononuclear cells are isolated on a Ficoll-Hypaque gradient, as described in Example 1.
- the mononuclear cells are resuspended in complete medium at the concentration of 200 ⁇ 10 6 cells / ml and mixed with 1 ml of a 50% suspension of sheep erythrocytes (BioMérieux, Marcy l'Etoile, France). The cell suspension is incubated at 4 ° C overnight. After gentle resuspension, the T cells are isolated by centrifugation on a Ficoll-Hypaque gradient (1500 rpm for 30 minutes at room temperature). The T cell / erythrocyte complexes are collected at the bottom of the tube.
- the red blood cells are lysed by two successive hypotonic shocks.
- the purity of the cells thus isolated is evaluated by cytofluorometry using a human anti-CD3 antibody labeled with fluorescein (Becton Dickinson). Purity is> 95%.
- the cells are resuspended in the complete culture medium at a concentration of 2.5 ⁇ 10 5 cells / ml.
- the activated and irradiated dendritic cells are cultured at the concentration of 10 4 cells / 200 microliters in a 96-well culture plate in the presence or not of 5 ⁇ 10 4 allogenic lymphocytes. After 5 days of culture, the proliferation of T cells is evaluated by measuring the incorporation of tritiated thymidine ( 3 H-Thy) (Amersham, Amersham, United Kingdom). Briefly, 0.25 mCi of 3 H-Thy are added to each culture well. The incorporation of 3 H-Thy is measured by a liquid scintillation counter (Packard Instruments, Australia). The results are presented in counts per minute (cpm). Mixed lymphocyte reactions are performed with T lymphocytes from two different healthy donors. B. Results
- results presented in FIG. 1 show that the dendritic cells stimulated by the membrane fraction of Klebsiella pneumoniae have an increased capacity to supply costimulation signals to the T lymphocytes and induce a greater proliferation of T lymphocytes than the unstimulated CD.
- Example V KpOmpA acts in synergy with LPS to induce the maturation of dendritic cells
- KpOmpA membrane proteins contained in the FMKp (membrane fraction)
- the ability of which to induce the maturation of human dendritic cells has already been shown (Jeannin et al., 2000, Nat hnmunol, 1 (6), 502-9) .
- FMKp containing a protein level between 1.2 g / 1 and 3.4 g / 1 and galactose between 7.5 and 14.9 g / 1, is much more effective in inducing the maturation of dendritic cells as protein alone, as shown in Table ⁇ , where the maturation of human dendritic cells is objectified by the production of dTL-12.
- FMKp induces the production of 134 pg / ml dTL-12 at 0.1 ⁇ g of proteins / ml whereas among these total proteins KpOmpA represents only a small percentage (approximately 10%).
- Dendritic cells at 10 x 10 6 / ml prepared as described above were stimulated with increasing doses of FMKp or KpOmpA. After 24 hours, IL-12, an indicator of maturation of the dendritic cells, was assayed in the supernatants by ELISA (R&D Systems).
- Table HL Maturation of human dendritic cells with LPS. in the presence or absence of KpOmpA.
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Abstract
The invention relates to the use of a gram-negative bacterial membrane fraction, especially Klebsiella pneumoniae, for inducing the maturation of dendritic cells.
Description
UTILISATION D'UNE FRACTION MEMBRANAIRE DE BACTERD2S A GRAM NEGATIF POUR INDUIRE LA MATURATION DES CELLULES USE OF A MEMBRANE FRACTION OF BACTERD2S WITH NEGATIVE GRAM TO INDUCE CELL MATURATION
DENDRITIQUESDENDRITIC
La présente invention concerne l'utilisation d'une fraction membranaire de bactéries à gram négatif, notamment de Klebsiella pneumoniae pour induire la maturation des cellules dendritiques et ainsi favoriser le développement d'une réponse immune spécifique.The present invention relates to the use of a membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae to induce the maturation of dendritic cells and thus promote the development of a specific immune response.
Les cellules dendritiques jouent un rôle dans le développement d'une réponse immune et dans l'initiation d'une réponse lymphocytaire T spécifique (Steinman RM et al Immuno Rev (1997) 156, 25 & Cella M et al Curr Opin Immunol (1997) 9, 10). Les cellules dendritiques immatures sont localisées dans les tissus non lymphoïdes. Au niveau de la peau, dans tous les épidémies hormis l'intestin, elles sont alors appelées cellules de Langerhans ; des cellules dendritiques, en plus faible quantité, sont aussi localisées dans les organes tels que le poumon, le foie et l'intestin. Ces cellules dendritiques immatures captent et digèrent les antigènes de façon très efficace. Après une stimulation antigénique in vivo ou stimulation par des molécules proinflammatoires, les cellules dendritiques qui ont capté les antigènes migrent dans les organes lymphoïdes secondaires. Durant cette migration, les cellules dendritiques subissent des modifications fonctionnelles et phénotypiques qui sont regroupées sous le terme de maturation. Cette maturation se caractérise par une augmentation à la surface des cellules dendritiques de molécules impliquées dans l'activation des lymphocytes T (telles que CD40, CD54, CD58, CD86 et MHC classe I/O), la production de cytokines proinflarnmatoires (IL-6) et de chemokines (telles que TNFα, E -8, MCP-1 et MlP-la) et perdent leur capacité à processer l'antigène. Les chemokines recrutent des cellules inflammatoires et favorisent l'initiation d'une réponse immune. En particulier, l'IL-8, le MCP-1 et le MlP-lα attirent non seulement des cellules -inflammatoires mais également des lymphocytes T naïfs et mémoires. Ainsi, les cellules dendritiques en sécrétant des chemokines augmentent leurs chances d'entrer en contact avec les lymphocytes T. Les cytokines proinflammatoires vont également agir en activant directement les CD.
Ces cellules dendritiques matures présentent les antigènes aux lymphocytes T et initient les réponses T spécifiques de façon très efficace, les molécules de costimulation étant exprimées en quantité importante sur ces cellules. Les cellules en devenant matures perdent leur capacité à capter et processer l'antigène. Dans les zones thymo-dépendantes des organes lymphoïdes, les cellules dendritiques qui ont migré ont acquis de puissantes propriétées immunostimulatrices et vont donc activer de façon très efficace les lymphocytes T naïfs circulants.Dendritic cells play a role in the development of an immune response and in the initiation of a specific T lymphocyte response (Steinman RM et al Immuno Rev (1997) 156, 25 & Cella M et al Curr Opin Immunol (1997) 9, 10). Immature dendritic cells are located in non-lymphoid tissue. At the skin level, in all epidemics except the intestine, they are then called Langerhans cells; dendritic cells, in smaller quantities, are also located in organs such as the lung, liver and intestine. These immature dendritic cells capture and digest antigens very efficiently. After antigenic stimulation in vivo or stimulation by proinflammatory molecules, the dendritic cells which have captured the antigens migrate into the secondary lymphoid organs. During this migration, the dendritic cells undergo functional and phenotypic modifications which are grouped under the term of maturation. This maturation is characterized by an increase on the surface of dendritic cells of molecules involved in the activation of T lymphocytes (such as CD40, CD54, CD58, CD86 and MHC class I / O), the production of proinflarnmatory cytokines (IL-6 ) and chemokines (such as TNFα, E -8, MCP-1 and MlP-la) and lose their ability to process the antigen. Chemokines recruit inflammatory cells and promote the initiation of an immune response. In particular, IL-8, MCP-1 and MlP-1α attract not only inflammatory cells but also naive and memory T lymphocytes. Thus, dendritic cells by secreting chemokines increase their chances of coming into contact with T lymphocytes. The proinflammatory cytokines will also act by directly activating CD. These mature dendritic cells present the antigens to the T lymphocytes and initiate the specific T responses very efficiently, the costimulation molecules being expressed in large quantities on these cells. As cells mature, they lose their ability to capture and process the antigen. In the thymo-dependent areas of the lymphoid organs, the migrated dendritic cells have acquired powerful immunostimulatory properties and will therefore very effectively activate the circulating naive T lymphocytes.
La fraction membranaire de K. pneumoniae 1145 entre dans la composition d'une préparation pharmaceutique prévenant la survenue et la récidive d'infections respiratoires d'origine bactérienne et utilisée chez l'homme depuis 20 ans. A ce titre, il existe un recul de non toxicité du produit.The membrane fraction of K. pneumoniae 1145 is used in the composition of a pharmaceutical preparation preventing the occurrence and recurrence of respiratory infections of bacterial origin and used in humans for 20 years. As such, there is a decline in non-toxicity of the product.
Dans la demande FR9903154 il a été montré que la FMKp ou l'un de ses constituants majeurs, la protéine de membrane externe OmpA, dénommée P40 était capable d'induire la production notamment par les monocytes de TNF-α et d'IL-12, cytokines impliquées dans la réponse immunitaire anti-tumorale.In application FR9903154 it was shown that FMKp or one of its major constituents, the outer membrane protein OmpA, called P40, was capable of inducing the production in particular by monocytes of TNF-α and IL-12 , cytokines involved in the anti-tumor immune response.
Dans la demande FR9903153, il a été montré que la ladite fraction membranaire FMKp associée à un antigène avait non seulement la capacité de réorienter la réponse cytokinique vers un profil Thl/Th2 et ce quel que soit le mode d'administration desdites fractions membranaires. Les auteurs de la présente invention ont maintenant mis en évidence qu'une fraction membranaire de bactéries à gram négatif, préférentiellement de Klebsiella pneumoniae induit la maturation des cellules dendritiques humaines permettant ainsi le développement d'une réponse lymphocytaire T spécifique.In application FR9903153, it has been shown that the said FMKp membrane fraction associated with an antigen not only has the capacity to redirect the cytokinic response towards a Th1 / Th2 profile and this regardless of the mode of administration of said membrane fractions. The authors of the present invention have now demonstrated that a membrane fraction of gram-negative bacteria, preferably of Klebsiella pneumoniae induces the maturation of human dendritic cells thus allowing the development of a specific T lymphocyte response.
La présente invention concerne ainsi l'utilisation d'au moins une fraction membranaire de bactéries à gram négatif, préférentiellement de Klebsiella pneumoniae pour induire la maturation des cellules dendritiques.The present invention thus relates to the use of at least one membrane fraction of gram negative bacteria, preferably of Klebsiella pneumoniae to induce the maturation of dendritic cells.
La présente invention concerne aussi l'utilisation d'au moins une fraction membranaire de bactéries à gram négatif, notamment de Klebsiella pneumoniae pour la fabrication d'un médicament destiné à induire la maturation des cellules dendritiques.
Par fraction membranaire de bactérie, on entend désigner dans la présente invention toute fraction ou extrait membranaire purifié ou partiellement purifié obtenu à partir d'une culture de ladite bactérie et dont le procédé de préparation comprend au moins une étape de lyse des bactéries obtenues après culture et une étape de séparation de la fraction contenant les membranes desdites bactéries du lysat total obtenu après l'étape de lyse, notamment par centrifugation ou filtration. Ainsi, au sens de la présente invention, une fraction membranaire comprend au moins des protéoglycanes membranaires. Ainsi, au sens de la présente invention, une fraction membranaire peut aussi être définie comme un une fraction comprenant au moins des protéines membranaires associées aux LPS (lipopolysaccharides), connus de l'homme du métier.The present invention also relates to the use of at least one membrane fraction of gram-negative bacteria, in particular of Klebsiella pneumoniae for the manufacture of a medicament intended to induce the maturation of dendritic cells. The term “bacterial membrane fraction” is intended to denote in the present invention any purified or partially purified membrane fraction or extract obtained from a culture of said bacteria and the preparation process of which comprises at least one step of lysis of the bacteria obtained after culture. and a step of separating the fraction containing the membranes of said bacteria from the total lysate obtained after the lysis step, in particular by centrifugation or filtration. Thus, within the meaning of the present invention, a membrane fraction comprises at least membrane proteoglycans. Thus, within the meaning of the present invention, a membrane fraction can also be defined as a fraction comprising at least membrane proteins associated with LPS (lipopolysaccharides), known to those skilled in the art.
Le titre en protéoglycane des fractions membranaires selon l'invention est représenté par la somme des teneurs en galactose et en protéines, est de préférence compris :The proteoglycan titer of the membrane fractions according to the invention is represented by the sum of the galactose and protein contents, is preferably understood:
- pour le galactose : entre 1,2 g/1 et 3,4 g/1 ; - pour les protéines : entre 7,5 g/1 et 14,9 g/1.- for galactose: between 1.2 g / 1 and 3.4 g / 1; - for proteins: between 7.5 g / 1 and 14.9 g / 1.
De manière plus préférée, ce titre sera :More preferably, this title will be:
- pour le galactose : entre 1,8 g/1 et 2,6 g/1 ;- for galactose: between 1.8 g / 1 and 2.6 g / 1;
- pour les protéines : entre 9,3 g/1 et 11,7 g/1.- for proteins: between 9.3 g / 1 and 11.7 g / 1.
Les fractions membranaires selon l'invention sont susceptibles d'être obtenues par les procédés décrits ci après ou par tout autre procédé équivalent, notamment ceux décrits dans les demandes de brevet FR9903154 et FR9903153.The membrane fractions according to the invention are capable of being obtained by the methods described below or by any other equivalent method, in particular those described in patent applications FR9903154 and FR9903153.
Ces fractions membranaires pourront être utilisées à des concentrations de 0,1 à 100, préférentiellement de 1 à 50, et plus préférentiellement de 1 à 20 microgrammes par millilitre de milieu de culture. Dans un mode de réalisation préféré de l'invention, les bactéries à gram négatif sont choisies parmi les bactéries de genre Klebsiella et préférentiellement d'espèce pneumoniae.These membrane fractions can be used at concentrations of 0.1 to 100, preferably from 1 to 50, and more preferably from 1 to 20 micrograms per milliliter of culture medium. In a preferred embodiment of the invention, the gram-negative bacteria are chosen from bacteria of the genus Klebsiella and preferably of pneumoniae species.
Dans un mode de réalisation préféré de l'invention, les fractions membranaires selon l'invention peuvent servir à favoriser la maturation des cellules dendritiques in vitro, les cellules matures pouvant ensuite être réinjectées in vivo.
Dans un mode de réalisation encore plus préféré de l'invention ce médicament peut servir à favoriser la génération et la maturation des cellules dendritiques in vitro, après mise en contact avec un agent biologique. Ainsi, l'invention concerne l'utilisation d'une fraction membranaire selon l'invention et en outre d'au moins un agent biologique pour la fabrication d'un médicament. Ce dernier pourra par exemple augmenter la réponse irnmunologique vis-à-vis de cet agent biologique.In a preferred embodiment of the invention, the membrane fractions according to the invention can serve to promote the maturation of dendritic cells in vitro, the mature cells then being able to be reinjected in vivo. In an even more preferred embodiment of the invention, this medicament can be used to promote the generation and maturation of dendritic cells in vitro, after contact with a biological agent. Thus, the invention relates to the use of a membrane fraction according to the invention and in addition to at least one biological agent for the manufacture of a medicament. The latter may for example increase the immunological response vis-à-vis this biological agent.
Cet agent biologique peut être choisi parmi les acides nucléiques, les protéines, les lipides, les lipopeptides ou les polysaccharides.This biological agent can be chosen from nucleic acids, proteins, lipids, lipopeptides or polysaccharides.
Il peut être plus particulièrement choisi parmi les antigènes vaccinants et/ou les antigènes de bactéries, de virus, de levures, de parasites, de champignons.It can more particularly be chosen from vaccinating antigens and / or antigens from bacteria, viruses, yeasts, parasites, fungi.
Plus préférentiellement, l'agent biologique est un antigène tumoral. Au sens de la présente invention, un antigène tumoral est défini comme une protéine ou un peptide tumoral, et notamment comme un épitope, notamment un épitope CTL (séquences peptidiques interagissant avec les molécules de classe I et présentés aux lymphocytes T CD8+) ou comme la séquence nucléique codant pour de tels protéines, peptides ou épitopes. On peut citer à titre non limitatif les antigènes tumoraux suivants : MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens ON-TL3 et MON 18, TUAΝ, alpha-feto protein (AFP), OFP, CA-125, CA-50, CA-19-9, rénal tumor-associated antigen G250, EGP-40 (ou EpCAM), S100 (malignant melanoma-associated antigen), p53, prostate tumor-associated antigens (e.g., PSA and PSMA), et p21ras.More preferably, the biological agent is a tumor antigen. For the purposes of the present invention, a tumor antigen is defined as a tumor protein or peptide, and in particular as an epitope, in particular a CTL epitope (peptide sequences interacting with class I molecules and presented to CD8 + T lymphocytes) or as the nucleic acid sequence encoding such proteins, peptides or epitopes. The following tumor antigens may be mentioned without limitation: MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, GD2, carcinoembryonic antigen (CEA), TAG-72, ovarian-associated antigens ON- TL3 and MON 18, TUAΝ, alpha-feto protein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associated antigen G250, EGP-40 (or EpCAM), S100 (malignant melanoma -associated antigen), p53, prostate tumor-associated antigens (eg, PSA and PSMA), and p21ras.
L'agent biologique peut encore être choisi parmi un lysat de cellules tumorales autologues et/ou heterologues. Au sens de la présente invention on entend par cellules tumorales autologues, des cellules de tumeurs appartenant au sujet qui va recevoir les compositions selon l'invention. Par cellules tumorales heterologues, il faut comprendre des cellules issues de tumeurs provenant d'un individu différent de celui à qui la composition selon l'invention est destinée. L'utilisation de cellules heterologues permet d'obtenir des compositions pharmaceutiques permettant notamment de traiter des patients atteints de cancer chez qui un prélèvement de cellules tumorales n'est pas possible. L'utilisation de cellules heterologues permet aussi d'obtenir des compositions selon
l'invention standardisées comprenant des antigènes retrouvés dans de nombreux types de cancer et ainsi utilisables chez une majorité de patients.The biological agent can also be chosen from 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 compositions according to the invention standardized comprising antigens found in many types of cancer and thus usable in a majority of patients.
Les cellules tumorales peuvent être obtenues suite à un prélèvement de tissus cancéreux, par exemple suite à une biopsie ou résection chirurgicale. Ces cellules peuvent ensuite être utilisées telles qu'elles ou être mises en culture avant d'être lysées. Un lysat cellulaire peut être défini au sens de la présente invention comme un mélange d'antigènes intra-cellulaires et/ou membranaires, préférentiellement intra et membranaires. Ledit lysat de cellules tumorales autologues et/ou heterologues selon l'invention peut-être obtenu par une lyse mécanique, chiinique ou enzymatique de cellules tumorales. Pour lyser les cellules de façon mécanique on peut notamment citer les techniques connues de l'homme de métier, à savoir notamment la sonication, l'ultrasonication ou la congélation décongélation. On préfère tout particulièrement la congélation/décongélation, et tout particulièrement l'utilisation de plusieurs cycles de congélation/décongélation. On peut aussi lyser les cellules en utilisant des composés chimiques ou des enzymes, comme par exemple un tampon de lyse à digitonine, du triton X-100 ou du Nonidet P40. Toute méthode permettant de rompre la membrane cellulaire des cellules de tumeurs pourra être utilisée afin d'obtenir un lysat.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 they are or can be cultured before being lysed. A cell lysate can be defined within the meaning of the present invention as a mixture of intra-cellular and / or membrane antigens, preferably intra and membrane. Said autologous and / or heterologous tumor cell lysate according to the invention can be obtained by mechanical, chemical or enzymatic lysis of tumor cells. 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 and 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.
Les cellules dendritiques sont ainsi modifiées en utilisant ces antigènes ou lysats cellulaires de sorte qu'elles expriment des antigènes tumoraux. Ainsi, ledit médicament pourra être injecté en même temps que les cellules dendritiques ou de façon préférée ledit médicament pourra être mis en oeuvre in vitro, afin de favoriser la maturation des cellules dendritiques avant de les réinjecter aux patients.The dendritic cells are thus modified using these antigens or cell lysates so that they express tumor antigens. Thus, said drug can be injected at the same time as the dendritic cells or, preferably, said drug can be used in vitro, in order to promote the maturation of the dendritic cells before reinjecting them into patients.
L'administration d'une fraction membranaire de bactéries à gram négatif en même temps que l'antigène permettra d'augmenter la présentation de l'antigène par les cellules dendritiques et par là même l'efficacité du traitement thérapeutique.The administration of a membrane fraction of gram negative bacteria at the same time as the antigen will make it possible to increase the presentation of the antigen by the dendritic cells and thereby the effectiveness of the therapeutic treatment.
Ainsi, grâce à leur efficacité à présenter les antigènes et à stimuler le système immunitaire, les cellules dendritiques en présence d'une fraction membranaire de bactéries à gram négatif pourront être utilisées pour générer des réponses CTL anticancéreuses (Nestlé F.O. et al., 1998, Nat. Med., 4, 328-332). Cette approche, dénommée "ex vivo" consiste donc à charger les cellules dendritiques ex vivo avec l'antigène d'intérêt (peptides ou lysat cellulaire) en présence d'une fraction membranaire de bactéries à gram
négatif et à réimplanter ces cellules chez le patient. Une étape de lavage des cellules dendritiques de manière à supprimer la fraction membranaire de bactéries à gram négatif du milieu contenant les cellules dendritiques pourra être effectuée avant de réimplanter ces cellules chez le patient. Une autre approche selon l'invention consiste à transfecter ex vivo les cellules dendritiques avec le gène codant pour l'antigène d'intérêt et notamment avec un gène codant pour un antigènes de bactérie, de virus, de levure, de parasite, de champignon, ou pour un antigène tumoral, tels que ceux décrits ci-dessus, et/ou avec un gène codant pour une cytokine ou un facteur de croissance, tels que ceux décrits ci-dessous, et de mettre les cellules dendritiques, avant et/ou pendant et/ou après la transfection, en présence d'une fraction membranaire de bactéries à gram négatif et à réinjecter ces cellules transfectées (Gilboa E. et al., 1998, Cancer Immunol. Immunother., 46, 82-87). Une étape de lavage des cellules dendritiques de manière à supprimer la fraction membranaire de bactéries à gram négatif du milieu contenant les cellules dendritiques pourra être effectuée avant de réimplanter ces cellules chez le patient. De telles approches ont été utilisées avec succès chez la souris et chez l'homme (Hsu F.J. et al., 1996, Nat. Med., 2, 52-58).Thus, thanks to their efficiency in presenting antigens and stimulating the immune system, dendritic cells in the presence of a membrane fraction of gram-negative bacteria can be used to generate CTL anticancer responses (Nestlé FO et al., 1998, Nat. Med., 4, 328-332). This approach, called "ex vivo" therefore consists in loading the dendritic cells ex vivo with the antigen of interest (peptides or cell lysate) in the presence of a membrane fraction of bacteria with gram negative and to re-implant these cells in the patient. A step of washing the dendritic cells so as to remove the membrane fraction of gram-negative bacteria from the medium containing the dendritic cells can be carried out before re-implanting these cells in the patient. Another approach according to the invention consists in transfecting ex vivo the dendritic cells with the gene coding for the antigen of interest and in particular with a gene coding for a bacteria, virus, yeast, parasite, fungus antigens, or for a tumor antigen, such as those described above, and / or with a gene encoding a cytokine or a growth factor, such as those described below, and to put the dendritic cells, before and / or during and / or after transfection, in the presence of a membrane fraction of gram-negative bacteria and to reinject these transfected cells (Gilboa E. et al., 1998, Cancer Immunol. Immunother., 46, 82-87). A step of washing the dendritic cells so as to remove the membrane fraction of gram-negative bacteria from the medium containing the dendritic cells can be carried out before re-implanting these cells in the patient. Such approaches have been successfully used in mice and in humans (Hsu FJ et al., 1996, Nat. Med., 2, 52-58).
Le médicament selon l'invention peut comprendre en outre une cytokine ou un facteur de croissance, notamment l'interféron alpha ou gamma, le TNFα, le GM-CSF, 1TL- 2, 1"IL-12, ΓIL-4, ΓIL-6 et IL-IS, une HSP (Heat Shock Protein) telle que par exemple l'hsp70, l'hsp90, l'hsp96, qui permet de potentialiser la réponse immunitaire; et/ou des fibroblastes modifiés génétiquement de façon à relarguer une cytokine ou un facteur de croissance. On peut citer les fibroblastes exprimant du GM-CSF commercialisés par la société Immune Response Corporation. L'utilisation d'une fraction membranaire de bactéries à gram négatif avec au moins un agent biologique associé au TNF alpha peut être utilisé pour la fabrication d'un médicament destiné à augmenter la prolifération des lymphocytes T.The medicament according to the invention may also comprise a cytokine or a growth factor, in particular interferon alpha or gamma, TNFα, GM-CSF, 1TL- 2, 1 "IL-12, ΓIL-4, ΓIL- 6 and IL-IS, an HSP (Heat Shock Protein) such as for example hsp70, hsp90, hsp96, which makes it possible to potentiate the immune response; and / or fibroblasts genetically modified so as to release a cytokine or a growth factor. Mention may be made of GM-CSF-expressing fibroblasts sold by the company Immune Response Corporation. The use of a membrane fraction of gram-negative bacteria with at least one biological agent associated with TNF alpha can be used for the manufacture of a medicament intended to increase the proliferation of T lymphocytes
Le médicament selon l'invention peut comprendre en outre un adjuvant permettant d'augmenter la réponse immunitaire, notamment choisi dans le groupe d'adjuvant comprenant le MPL-A, le Quil-A, l'ISCOM, les CpG, Leif, la CT (toxine cholérique) ou la LT (LT pour « Heat labile enterotoxin » entérotoxine labile à la chaleur), tout comme les versions détoxifiées de la CT ou la LT, ou des protéines membranaires
bactériennes telles que les OMPC de Neisseria meningitidis (Nella et al., Infect. Immun. 60, 1992, 4977-4983), TraT d'Escherichia coli (Croft et al, J. Immunol. 146, 1991, 793- 798) ou PorB de Neisseria meningitidis (Fusco et al, J. Infect. Dis. 175, 1997, 364-372), et préférentiellement une OmpA d'une bactérie du genre Klebsiella, protéine majeure de la membrane externe baptisée P40, présentant une activité adjuvante pour des antigènes sous- unitaires peptidiques (WO 95/27787 et WO 96/14415 ; Haeuw et al, Eur. J. Biochem. 255 ,1998, 446-454 ; Plornicky-Gilquin étal, J. Virol. 73, 1999, 5637-5645).The medicament according to the invention may also comprise an adjuvant making it possible to increase the immune response, in particular chosen from the group of adjuvant comprising MPL-A, Quil-A, ISCOM, CpG, Leif, CT (cholera toxin) or LT (Heat labile enterotoxin), just like the detoxified versions of CT or LT, or membrane proteins bacteria such as OMPC of Neisseria meningitidis (Nella et al., Infect. Immun. 60, 1992, 4977-4983), TraT of Escherichia coli (Croft et al, J. Immunol. 146, 1991, 793- 798) or PorB of Neisseria meningitidis (Fusco et al, J. Infect. Dis. 175, 1997, 364-372), and preferably an OmpA of a bacterium of the genus Klebsiella, a major protein of the outer membrane called P40, exhibiting an adjuvant activity for peptide subunit antigens (WO 95/27787 and WO 96/14415; Haeuw et al, Eur. J. Biochem. 255, 1998, 446-454; Plornicky-Gilquin et al, J. Virol. 73, 1999, 5637- 5645).
Le médicament selon l'invention peut encore comprendre un milieu pharmaceutiquement acceptable. Au sens de la présente invention, le milieu pharmaceutiquement acceptable est le milieu dans lequel les composés de l'invention sont administrés, préférentiellement un milieu injectable chez l'homme. Il peut être constitué d'eau, d'une solution aqueuse saline ou d'une solution aqueuse à base de dextrose et/ou de glycérol.The medicament according to the invention can also comprise a pharmaceutically acceptable medium. 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 consist of water, an aqueous saline solution or an aqueous solution based on dextrose and / or glycerol.
Le médicament selon l'invention peut en outre être véhiculé sous une forme permettant d'améliorer sa stabilité et/ou son immunogénicité; ainsi, il peut être véhiculé sous forme de liposomes, virosomes, nanosphères, microsphères ou microcapsules. Le médicament ou l'association d'une fraction membranaire de bactéries à gram négatif- agent biologique pourra aussi se présenter sous une forme facilement administrable telle qu'une pommade, une lotion, une solution ou encore sous forme d'une composition adhésive : emplâtre, « patch ».The medicament according to the invention can also be conveyed in a form making it possible to improve its stability and / or its immunogenicity; thus, it can be conveyed in the form of liposomes, virosomes, nanospheres, microspheres or microcapsules. The medicament or the association of a membrane fraction of gram-negative bacteria-biological agent may also be in an easily administered form such as an ointment, a lotion, a solution or even in the form of an adhesive composition: plaster , "Patch".
Le médicament selon l'invention pourra être utilisé notamment pour le traitement :The medicament according to the invention may be used in particular for the treatment:
- des infections microbiennes, en particulier les infections de type chronique associées au développement d'une réponse immune spécifique inefficace (virus : par exemple le virus de l'immunodéficience humaine (VIH), les virus hépatiques, en particulier les virus hépatiques A, B, C et D et le parainfluenza virus, bactéries, parasites et levures),- microbial infections, in particular chronic infections associated with the development of an ineffective specific immune response (virus: for example the human immunodeficiency virus (HIV), hepatic viruses, in particular hepatic viruses A, B , C and D and parainfluenza virus, bacteria, parasites and yeasts),
- des cancers, en particulier chez les sujets porteurs du VIH et/ou atteints de myélomes, lymphomes, leucémies, mélanomes, carcinomes, du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon, de la remise, par exemple.
Comme décrit ci dessus, l'invention concerne particulièrement l'immunothérapie cellulaire utilisant des vaccins anti-cancéreux et anti-infectieux en particulier, en générant in vitro des cellules dendritiques autologues, en y introduisant un antigène tumoral et en les réinjectant après une étape de lavage facultative. L'exposition des cellules dendritiques in vitro à la fraction membranaire de bactéries à gram négatif permettra d'augmenter la maturation des cellules dendritiques et donc d'augmenter l'efficacité du vaccin.- cancers, in particular in subjects carrying HIV and / or suffering from myelomas, lymphomas, leukemias, melanomas, carcinomas, of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, the discount, for example. As described above, the invention particularly relates to cellular immunotherapy using anti-cancer and anti-infectious vaccines in particular, by generating autologous dendritic cells in vitro, by introducing a tumor antigen there and by reinjecting them after a step of optional washing. Exposing dendritic cells in vitro to the membrane fraction of gram-negative bacteria will increase the maturation of dendritic cells and therefore increase the effectiveness of the vaccine.
Aussi, la présente invention concerne particulièrement l'utilisation d'une fraction membranaire de bactéries à gram négatif, et plus particulièrement de la fraction membranaire de Klebsiella pneumoniae pour la préparation d'un médicament pour augmenter la réponse immunologique vis-à-vis d'un agent biologique tel que défini ci dessus. Ce médicament peut être un vaccin pour le traitement ou la prévention des maladies infectieuses d'origine virale -comme notamment le NIH, les virus hépatiques et le parainfluenza virus-, bactérienne, fongique, ou provoquées par une levure ou un parasite.Also, the present invention relates particularly to the use of a membrane fraction of gram-negative bacteria, and more particularly of the membrane fraction of Klebsiella pneumoniae for the preparation of a medicament for increasing the immunological response vis-à-vis a biological agent as defined above. This medicine can be a vaccine for the treatment or prevention of infectious diseases of viral origin - like in particular the NIH, the hepatic viruses and the parainfluenza virus -, bacterial, fungal, or caused by a yeast or a parasite.
La présente invention concerne encore l'utilisation d'une fraction membranaire de bactéries à gram négatif avec au moins un agent biologique pour la fabrication d'un médicament pour le traitement ou la prévention des cancers et particulièrement des cancers parmi les myélomes, lymphomes, leucémies, carcinomes du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon, et pour la fabrication d'un médicament pour le traitement ou la prévention des cancers cutanés choisi parmi les kératinomes et les carcinomes.The present invention also relates to the use of a membrane fraction of gram-negative bacteria with at least one biological agent for the manufacture of a medicament for the treatment or prevention of cancers and particularly cancers among myelomas, lymphomas, leukemias , carcinomas of the kidney, brain, prostate, rectum, pancreas, ovaries, lung, and for the manufacture of a medicament for the treatment or prevention of skin cancers chosen from keratinomas and carcinomas.
En effet, dans les cancers de la peau tels que les mélanomes et les kératinomes, les cellules cancéreuses sont en contact direct avec les cellules de Langerhans, situées dans l'épidémie, l'utilisation selon la présente invention par application cutanée permet d'agir directement, localement sur les cellules de Langerhans. Ainsi, le médicament selon l'invention pourra être appliqué au niveau de la peau notamment par voie cutanée, sous- cutanée, transdermique, intra-épidermique ou au niveau des muqueuses, il agit alors de façon systémique par la maturation des cellules dendritiques et localement par la maturation des cellules de Langerhans.Indeed, in skin cancers such as melanomas and keratinomas, the cancer cells are in direct contact with the Langerhans cells, located in the epidemic, the use according to the present invention by skin application makes it possible to act directly, locally on Langerhans cells. Thus, the medicament according to the invention may be applied to the skin, in particular by the cutaneous, subcutaneous, transdermal, intra-epidermal route or to the mucous membranes, it then acts systemically by the maturation of dendritic cells and locally by the maturation of Langerhans cells.
Comme décrit ci dessus le traitement de ces cancers, peut aussi être envisagé en injectant des cellules dendritiques autologues, et notamment des cellules dendritiques
autologues qui ont été modifiées afin d'exprimer des antigènes tumoraux. La maturation des cellules dendritiques sera assurée par la fraction membranaire de bactéries à gram négatif.As described above, the treatment of these cancers can also be envisaged by injecting autologous dendritic cells, and in particular dendritic cells. autologous which have been modified to express tumor antigens. The maturation of dendritic cells will be ensured by the membrane fraction of gram negative bacteria.
L'invention a encore pour objet un dispositif pour la maturation des cellules dendritiques, tel que par exemple un kit, comprenant au moins la fraction membranaire de bactéries à gram négatif.The invention also relates to a device for the maturation of dendritic cells, such as for example a kit, comprising at least the membrane fraction of gram negative bacteria.
Ce kit pourra être adapté pour la maturation des cellules dendritiques in vitro et pourra être utilisé par exemple dans les laboratoires de recherche. Ce kit peut contenir en outre de la fraction membranaire de bactéries à gram négatif des cellules dendritiques immatures et/ou les moyens nécessaires à isoler des cellules dendritiques immatures, tels que par exemple des moyens de purification de cellules mononucléaires sanguines. Il pourra ainsi comprendre par exemple différents milieux de culture, des solutions de lavage, des plaques de culture, des réactifs, des contrôles tels que par exemple des anticorps, et une notice explicative pour la mise en oeuvre de la méthode selon l'invention de maturation des cellules dendritiques.This kit can be adapted for the maturation of dendritic cells in vitro and can be used for example in research laboratories. This kit may also contain the membrane fraction of gram-negative bacteria from immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means for purifying blood mononuclear cells. It may thus include, for example, different culture media, washing solutions, culture plates, reagents, controls such as, for example, antibodies, and an explanatory notice for the implementation of the method according to the invention. maturation of dendritic cells.
Un kit selon l'invention pourra aussi être adapté à la mise en oeuvre de la méthode thérapeutique ci-dessus mentionnée. Ce kit peut contenir en outre de la fraction membranaire de bactéries à gram négatif des cellules dendritiques immatures etfou les moyens nécessaires à isoler des cellules dendritiques immatures, tels que par exemple des moyens de purification de cellules mononucléaires sanguines. Il pourra ainsi comprendre par exemple différents milieux de culture, des solutions de lavage, des plaques de culture, des réactifs, des contrôles, et une notice explicative pour la mise en œuvre de la méthode thérapeutique ci-dessus mentionnée. Le cas échéant il pourra encore contenir des antigènes heterologues ou des moyens pour obtenir un lysat de cellules autologues. Les légendes des figures et exemples qui suivent sont destinés à illustrer l'invention sans aucunement en limiter la portée. Ces exemples démontrent l'action d'une fraction membranaire de bactéries à gram négatif sur les cellules dendritiques : la fraction membranaire de bactéries à gram négatif induit la maturation des cellules dendritiques humaines immatures et leur confère de puissantes propriétés stimulatrices et présentatrice d'antigène.
Dans ces exemples, on se référera à la figure 1 annexée qui illustre le fait que :A kit according to the invention can also be adapted to the implementation of the therapeutic method mentioned above. This kit can also contain the membrane fraction of gram-negative bacteria from immature dendritic cells and / or the means necessary to isolate immature dendritic cells, such as for example means for purifying blood mononuclear cells. It may thus include, for example, different culture media, washing solutions, culture plates, reagents, controls, and an explanatory note for the implementation of the therapeutic method mentioned above. If necessary, it may also contain heterologous antigens or means for obtaining a lysate of autologous cells. The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope. These examples demonstrate the action of a membrane fraction of gram negative bacteria on dendritic cells: the membrane fraction of gram negative bacteria induces the maturation of immature human dendritic cells and gives them powerful stimulatory and antigen presenting properties. In these examples, reference will be made to the appended FIG. 1 which illustrates the fact that:
i La fraction membranaire de Klebsiella Pneumonia induit l'expression de la molécule CD83, augmente l'expression CD86 ainsi que la production d'IL-8.i The membrane fraction of Klebsiella Pneumonia induces the expression of the CD83 molecule, increases CD86 expression as well as the production of IL-8.
Les résultats d'analyse par FACS des molécules de surface sont exprimés en MFIThe results of FACS analysis of the surface molecules are expressed in MFI
(moyenne d'intensité de fluorescence) et sont représentatifs d'une expérience parmi 3.(average fluorescence intensity) and are representative of one of 3 experiments.
Concernant CD83 et CD86, les résultats sont également exprimés en pourcentage de cellules positives. Les résultats du dosage d'IL-8 sont exprimés en ng/ml et sont exprimés en moyenne ± s.d. de 4 expériences.For CD83 and CD86, the results are also expressed as a percentage of positive cells. The results of the IL-8 assay are expressed in ng / ml and are expressed as an average ± n.d. of 4 experiments.
if) : La fraction membranaire de Klebsiella Pneumonia augmente les propriétés costimulatrices des lymphocytes T (voir 4ème colonne MLR).if): The membrane fraction of Klebsiella Pneumonia increases costimulatory properties for T lymphocytes (see MLR 4th column).
Des CD immatures ont été ou non traitées pendant 24 heures avec de la fraction membranaire de Klebsiella Pneumonia ou du LPS. Puis elles ont été irradiées et cultivées avec des lymphocytes T allogénique de 2 donneurs différents. La prolifération des lymphocytes T a été mesurée au bout de 5 jours. Les résultats sont exprimés en coup par minute (cpm) et représente la moyenne de 5 valeurs et sont représentatif d'une expérience parmi 3.Immature DCs were treated or not for 24 hours with membrane fraction of Klebsiella Pneumonia or LPS. Then they were irradiated and cultured with allogeneic T lymphocytes from 2 different donors. The proliferation of T lymphocytes was measured after 5 days. The results are expressed in counts per minute (cpm) and represent the average of 5 values and are representative of one of 3 experiments.
EXEMPLESEXAMPLES
Exemple 1 : Obtention d'une fraction membranaire de K. pneumomaeExample 1: Obtaining a membrane fraction of K. pneumomae
Après décongélation à + 4°C pendant 48 h minimum, 1 kg de cellules sèches de K. pneumoniae est remis en suspension à 5 % cellules sèches. La DNase est ajoutée à 5 mg/1. On procède ensuite au broyage en boucle au Manton Gaulin pendant 30 min puis à une clarification sur SHARPLES à 50 1/h, suivie d'une précipitation à l'acide acétique à pH = 4,2 + 0,1 pendant 30 min. Le culot est éliminé (SHARPLES à 25 1/h) et le surnageant est neutralisé, dilué à 2 fois le volume initial avec de l'eau osmosée. Une dialyse à volume constant est alors effectuée sur PUF 100 jusqu'à 800 Ωcm, suivie d'une concentration de la suspension membranaire (SM) ainsi obtenue, à 11 1/kg de cellules
sèches. On procède alors à l'autoclavage de la SM à + 121 °C durant 35 min que l'on peut conserver à + 4°C pendant 6 semaines.After thawing at + 4 ° C for 48 hours minimum, 1 kg of dry cells of K. pneumoniae is resuspended in 5% dry cells. DNase is added to 5 mg / 1. Next, loop grinding with Manton Gaulin is carried out for 30 min and then clarification on SHARPLES at 50 l / h, followed by precipitation with acetic acid at pH = 4.2 + 0.1 for 30 min. The pellet is eliminated (SHARPLES at 25 l / h) and the supernatant is neutralized, diluted to 2 times the initial volume with reverse osmosis water. A constant volume dialysis is then carried out on PUF 100 up to 800 Ωcm, followed by a concentration of the membrane suspension (SM) thus obtained, at 11 1 / kg of cells. dry. The SM is then autoclaved at + 121 ° C for 35 min which can be stored at + 4 ° C for 6 weeks.
Caractéristiques de la fraction membranaire obtenue :Characteristics of the membrane fraction obtained:
Par définition, le titre en protéoglycanne, est égal à la somme des teneurs en galactose et en protéines.By definition, the proteoglycan titer is equal to the sum of the galactose and protein contents.
- Galactose : en moyenne 2,2 g /l- Galactose: on average 2.2 g / l
- Protéines : en moyenne 10,5 g/1- Proteins: on average 10.5 g / 1
Exemple 2 ; La fraction membranaire de Klebsiella Pneumonia augmente l'expression de CD 83 A. MéthodologieExample 2; The membrane fraction of Klebsiella Pneumonia increases the expression of CD 83 A. Methodology
A.1. Génération in vitro de CD humaines immatures.A.1. In vitro generation of immature human CD.
Les cellules dendritiques humaines sont générées à partir de monocytes isolés du sang périphérique. Le sang est prélevé par leucophérèse en présence d'anticoagulant comme par exemple Phéparinate de lithium. Les cellules mononucléées (CMN) sont isolées de sujets sains par centrifugation sur un gradient de Ficoll-Hypaque (densité=l,077) (Amersham Pharmacia Biotech, Uppsala, Suède). Les cellules du sang sont centrifugées à 1500 rpm pendant 30 minutes à température ambiante. Les CMN, localisées à l'interface Ficoll-plasma, sont récupérées et lavées deux fois en présence de milieu RPMI 1640 (Life technologies, Cergy Pontoise, France). Les monocytes sont purifiés par sélection positive en utilisant un séparateur magnétique de cellules (MACS™; Miltenyi Biotex, Bergisch Gladbach, Allemagne) en accord avec les instructions du fabriquant. Les CMN sont incubées pendant 20 minutes à 4°C avec des billes magnétiques sur lesquelles sont fixées un anticorps monoclonal anti-CD 14 humain. Après lavage, la suspension cellulaire plus billes est déposée sur une colonne et soumise à un champ magnétique. Après trois lavages, la colonne n'est plus soumise au champ magnétique et les monocytes sont collectés par gravitation. La pureté des monocytes est évaluée par cytofluorométrie (cytofluoromètre FACScan ; Becton Dickinson, Erembodegem, Belgique) sur la base des paramètres taille- granulosité des cellules. La pureté est supérieure à 98%. Les monocytes sont ensuite mis en culture à la concentration de 5x106 cellules/ml dans le milieu suivant (dénommé par la suite milieu de culture complet) : milieu RPMI 1640 supplémenté de 10% de sérum de
veau foetal (chauffage à 56°C pendant 30 minutes), 2 mM de L-glutamine, 50 U/ml de pénicilline et 50 ug/ml de streptomycine (Life technologies) dans des plaques de culture 6 puits (Nunc, Roskilde, Danemark) à raison de 5 ml de milieu par puits. Les cellules sont activées avec 20 ng/ml d'IL-4 humaine recombinante et 20 ng/ml de GM-CSF humain recombinant (R&D Systems, Abingdon, Royaume Uni). Après 5 à 7 jours de culture (37°C, 5% CO2 en atmosphère humide), le phénotype des cellules est défini par cytofluorométrie. Brièvement, une aliquote de la suspension cellulaire est prélevée. Les cellules sont lavées dans du tampon FACS (tampon phosphate 10 mM pH 7,4 contenant 1 % de sérum albumine bovine et 0,01% d'azide de sodium) puis réparties dans des puits d'une plaque de culture 96 puits à fond conique (Nunc) à raison de 2x105 cellules dans un volume de 50 ml de tampon FACS. Dans chaque puits est ajouté soit un anticorps anti-CDla humain marqué à la fluorescéine (Becton Dickinson) soit un anticorps anti-CD83 humain non marqué révélé par un anticorps anti-immunoglobuline de souris marqué à la fluorescéine (Becton Dickinson). Après 20 minutes d'incubation à 4°C, les cellules sont lavées trois fois avec 200 μl de tampon FACS puis sont remises en suspension dans 200 μl de ce même tampon. L'analyse de l'expression de CDla versus CD83 est évaluée par FACS. Seules les cellules dendritiques immatures caractérisées par une expression de la molécules CDla (intensité moyenne de fluorescence (IMF)>100) et l'absence d'expression de la molécule CD83 ont été utilisées.Human dendritic cells are generated from monocytes isolated from peripheral blood. The blood is taken by leukopheresis in the presence of an anticoagulant such as, for example, lithium heparinate. Mononuclear cells (CMN) are isolated from healthy subjects by centrifugation on a Ficoll-Hypaque gradient (density = 1.077) (Amersham Pharmacia Biotech, Uppsala, Sweden). The blood cells are centrifuged at 1500 rpm for 30 minutes at room temperature. The CMNs, located at the Ficoll-plasma interface, are recovered and washed twice in the presence of RPMI 1640 medium (Life technologies, Cergy Pontoise, France). The monocytes are purified by positive selection using a magnetic cell separator (MACS ™; Miltenyi Biotex, Bergisch Gladbach, Germany) in accordance with the manufacturer's instructions. CMNs are incubated for 20 minutes at 4 ° C with magnetic beads on which are fixed an anti-human CD 14 monoclonal antibody. After washing, the cell suspension plus beads is placed on a column and subjected to a magnetic field. After three washes, the column is no longer subjected to the magnetic field and the monocytes are collected by gravitation. The purity of the monocytes is evaluated by cytofluorometry (FACScan cytofluorometer; Becton Dickinson, Erembodegem, Belgium) on the basis of the size-granulosity parameters of the cells. The purity is greater than 98%. The monocytes are then cultured at a concentration of 5 × 10 6 cells / ml in the following medium (hereinafter referred to as complete culture medium): RPMI 1640 medium supplemented with 10% serum fetal calf (heating at 56 ° C for 30 minutes), 2 mM L-glutamine, 50 U / ml of penicillin and 50 ug / ml of streptomycin (Life technologies) in 6-well culture plates (Nunc, Roskilde, Denmark ) at a rate of 5 ml of medium per well. The cells are activated with 20 ng / ml of recombinant human IL-4 and 20 ng / ml of recombinant human GM-CSF (R&D Systems, Abingdon, United Kingdom). After 5 to 7 days of culture (37 ° C, 5% CO 2 in a humid atmosphere), the phenotype of the cells is defined by cytofluorometry. Briefly, an aliquot of the cell suspension is taken. The cells are washed in FACS buffer (10 mM phosphate buffer pH 7.4 containing 1% bovine serum albumin and 0.01% sodium azide) and then distributed in wells of a 96-well culture plate at the bottom conical (Nunc) at the rate of 2 × 10 5 cells in a volume of 50 ml of FACS buffer. To each well is added either an anti-human CDla antibody labeled with fluorescein (Becton Dickinson) or an unlabeled human anti-CD83 antibody revealed by an anti-mouse immunoglobulin antibody labeled with fluorescein (Becton Dickinson). After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 μl of FACS buffer and then are resuspended in 200 μl of this same buffer. The analysis of the expression of CDla versus CD83 is evaluated by FACS. Only immature dendritic cells characterized by expression of the CDla molecule (mean fluorescence intensity (IMF)> 100) and the absence of expression of the CD83 molecule were used.
A.2. Analyse par cytofluorométrie de l'expression des marqueurs de différenciation induite par la fraction membranaire de Klebsiella pneumoniae sur des cellules dendritiques humaines.A.2. Analysis by cytofluorometry of the expression of markers of differentiation induced by the membrane fraction of Klebsiella pneumoniae on human dendritic cells.
Les cellules dendritiques immatures ont été collectées, lavées puis remises en culture en milieu complet à la concentration de 105 cellules dans un volume de 200 μl dans des plaques de culture 96 puits à fond plat (Costar, Cabridge, USA). Les cellules sont activées avec de la fraction membranaire de Klebsiella pneumoniae à des concentrations de 10 microgrammes, 20 microgrammes et 50 microgrammes par millilitre de milieu final. 24 heures après stimulation, l'expression des molécules CD83 et CD86 est évaluée par cytofluorométrie à l'aide d'anticorps monoclonaux spécifiques marqués à la fluorescéine (Becton Dickinson). Les anticorps isotypiques contrôles utilisés proviennent de Becton
Dickinson. Les cellules sont lavées dans du tampon FACS puis réparties dans des puits d'une plaque de culture 96 puits à fond conique à raison de 2xl05 cellules dans un volume de 50 μl de tampon FACS. Dans chaque puits est ajouté un anticorps. Après 20 minutes d'incubation à 4°C, les cellules sont lavées trois fois avec 200 μl de tampon FACS puis sont remises en suspension dans 200 μl de ce même tampon. L'analyse de l'expression des marqueurs de surface est évaluée par FACS.Immature dendritic cells were collected, washed and then re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 μl in 96-well flat-bottomed culture plates (Costar, Cabridge, USA). The cells are activated with the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms, 20 micrograms and 50 micrograms per milliliter of final medium. 24 hours after stimulation, the expression of the CD83 and CD86 molecules is evaluated by cytofluorometry using specific monoclonal antibodies labeled with fluorescein (Becton Dickinson). The control isotypic antibodies used come from Becton Dickinson. The cells are washed in FACS buffer and then distributed in wells of a 96-well culture plate with a conical bottom at the rate of 2 × 10 5 cells in a volume of 50 μl of FACS buffer. An antibody is added to each well. After 20 minutes of incubation at 4 ° C, the cells are washed three times with 200 μl of FACS buffer and then are resuspended in 200 μl of this same buffer. The analysis of the expression of the surface markers is evaluated by FACS.
B. RésultatsB. Results
Les résultats présentés dans la figure 1 montrent que la fraction membranaire de Klebsiella pneumoniae augmente l'expression par les cellules dendritiques immatures de molécules de surface impliquées dans Pactivation des lymphocytes T :The results presented in FIG. 1 show that the membrane fraction of Klebsiella pneumoniae increases the expression by immature dendritic cells of surface molecules involved in the activation of T lymphocytes:
- Comme cela a été préalablement démontré la majorité de la population de cellules dendritiques immatures n'exprime pas de CD83. La fraction membranaire de Klebsiella pneumoniae induit l'expression de la molécule de costimulation.- As previously demonstrated, the majority of the population of immature dendritic cells do not express CD83. The membrane fraction of Klebsiella pneumoniae induces the expression of the costimulation molecule.
- La fraction membranaire de Klebsiella pneumoniae augmente aussi l'expression d'autres molécules impliqués dans Pactivation des lymphocytes T, telle que CD86.- The membrane fraction of Klebsiella pneumoniae also increases the expression of other molecules involved in the activation of T lymphocytes, such as CD86.
Exemple LU. : La fraction membranaire de Klebsiella Pneumonia induit l'expression d'IL-8 par les CD A. Méthodologie : Des cellules dendritiques humaines immatures ont été générées comme décrit dans l'exemple 1. Après 5 à 7 jours de culture, les cellules sont remises en culture en milieu complet à la concentration de 105 cellules dans un volume de 200 ml dans des plaques de culture 96 puits à fond plat. Les cellules sont activées avec de la fraction membranaire de Klebsiella pneumoniae à des concentrations de 10 microgrammes et 50 microgrammes par millilitre de milieu de culture et après 24 heures de culture, les surnageants de culture sont centrifugés à 10000 rpm pendant 15 minutes à 4°C et l'IL-8 est dosée à l'aide de kits de dosage commercial type ELIS A (R&D Systems) selon les instructions du fabricant. B. Résultats : Les résultats présentés dans la figure 1 montrent que la fraction membranaire de Klebsiella pneumoniae induit l'expression dTL-8. Il peut ainsi être conclu que les fractions membranaires de bactéries à gram négatif activent les cellules dendritiques immatures.
Exemple IV. La fraction membranaire de Klebsiella pneumoniae augmente les propriétés costimulatrices des cellules dendritiques A. Méthodolo ie : A.1. Réaction lymphocytaire mixte.Example LU. : The membrane fraction of Klebsiella Pneumonia induces the expression of IL-8 by the CD A. Methodology: Immature human dendritic cells were generated as described in Example 1. After 5 to 7 days of culture, the cells are re-cultured in complete medium at the concentration of 10 5 cells in a volume of 200 ml in 96-well flat-bottomed culture plates. The cells are activated with the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms and 50 micrograms per milliliter of culture medium and after 24 hours of culture, the culture supernatants are centrifuged at 10,000 rpm for 15 minutes at 4 ° C. and the IL-8 is assayed using commercial assay kits type ELIS A (R&D Systems) according to the manufacturer's instructions. B. Results: The results presented in FIG. 1 show that the membrane fraction of Klebsiella pneumoniae induces the expression dTL-8. It can thus be concluded that the membrane fractions of gram negative bacteria activate immature dendritic cells. Example IV. The membrane fraction of Klebsiella pneumoniae increases the costimulatory properties of dendritic cells A. Methodolo ie: A.1. Mixed lymphocyte reaction.
Les cellules dendritiques humaines immatures sont générées comme décrit dans l'exemple 1. Après 5 à 7 jours de culture, les cellules sont lavées en milieu RPMI 1640 puis remises en culture dans le milieu complet à la concentration de 2,5x105 cellules/puits dans une plaque de culture 6 puits (5 ml/puits). Les cellules ne sont pas stimulées ou sont stimulées en présence de la fraction membranaire de Klebsiella pneumoniae à des concentrations de 10 microgrammes et 50 microgrammes ou 10 ng/ml LPS. Après 24 heures de culture, les cellules sont collectées et lavées trois fois en milieu RPMI 1640. Les cellules sont ensuite irradiées à 3000 rad. Les lymphocytes T humains fraîchement isolés du sang périphérique ont été préparés par la technique des rosettes avec des érythrocytes de mouton. Brièvement, les cellules mononucléées sont isolées sur gradient de Ficoll- Hypaque, comme décrit dans l'exemple 1. Les cellules mononucléées sont remises en suspension dans du milieu complet à la concentration de 200x106 cellules/ml et mélangées avec 1 ml d'une suspension à 50% d' érythrocytes de mouton (BioMérieux, Marcy l'Etoile, France). La suspension cellulaire est incubée à 4°C pendant une nuit. Après remise en suspension douce, les cellules T sont isolées par centrifugation sur un gradient de Ficoll- Hypaque (1500 rpm pendant 30 minutes à température ambiante). Les complexes cellules T/érythrocytes sont collectés au fond du tube. Les globules rouges sont lysés par deux chocs hypotoniques successifs. La pureté des cellules ainsi isolées est évaluée par cytofluorométrie à l 'aide d'un anticorps anti-CD3 humain marqué à la fluorescéine (Becton Dickinson). La pureté est >95%. Après lavage, les cellules sont remises en suspension dans le milieu de culture complet à la concentration de 2,5x105 cellules/ml.Immature human dendritic cells are generated as described in Example 1. After 5 to 7 days of culture, the cells are washed in RPMI 1640 medium and then re-cultured in complete medium at a concentration of 2.5 × 10 5 cells / well in a 6-well culture plate (5 ml / well). The cells are not stimulated or are stimulated in the presence of the membrane fraction of Klebsiella pneumoniae at concentrations of 10 micrograms and 50 micrograms or 10 ng / ml LPS. After 24 hours of culture, the cells are collected and washed three times in RPMI 1640 medium. The cells are then irradiated at 3000 rad. Human T cells freshly isolated from peripheral blood were prepared by the rosette technique with sheep erythrocytes. Briefly, the mononuclear cells are isolated on a Ficoll-Hypaque gradient, as described in Example 1. The mononuclear cells are resuspended in complete medium at the concentration of 200 × 10 6 cells / ml and mixed with 1 ml of a 50% suspension of sheep erythrocytes (BioMérieux, Marcy l'Etoile, France). The cell suspension is incubated at 4 ° C overnight. After gentle resuspension, the T cells are isolated by centrifugation on a Ficoll-Hypaque gradient (1500 rpm for 30 minutes at room temperature). The T cell / erythrocyte complexes are collected at the bottom of the tube. The red blood cells are lysed by two successive hypotonic shocks. The purity of the cells thus isolated is evaluated by cytofluorometry using a human anti-CD3 antibody labeled with fluorescein (Becton Dickinson). Purity is> 95%. After washing, the cells are resuspended in the complete culture medium at a concentration of 2.5 × 10 5 cells / ml.
Les cellules dendritiques activées et irradiées sont mises en culture à la concentration de 104 cellules/200 microlitres dans une plaque de culture 96 puits en présence ou non de 5x104 lymphocytes allogéniques. Après 5 jours de culture, la prolifération des cellules T est évaluée par mesure de l'incorporation de thymidine tritiée (3H-Thy) (Amersham, Amersham, Royaume Uni). Brièvement, 0.25 mCi de 3H-Thy sont
ajoutés dans chaque puits de culture. L'incorporation de 3H-Thy est mesurée par un compteur à scintillation liquide (Packard Instruments, Australie). Les résultats sont présentés en coups par minute (cpm). Les réactions lymphocytaires mixtes sont réalisées avec les lymphocytes T provenant de deux donneurs sains différents. B. RésultatsThe activated and irradiated dendritic cells are cultured at the concentration of 10 4 cells / 200 microliters in a 96-well culture plate in the presence or not of 5 × 10 4 allogenic lymphocytes. After 5 days of culture, the proliferation of T cells is evaluated by measuring the incorporation of tritiated thymidine ( 3 H-Thy) (Amersham, Amersham, United Kingdom). Briefly, 0.25 mCi of 3 H-Thy are added to each culture well. The incorporation of 3 H-Thy is measured by a liquid scintillation counter (Packard Instruments, Australia). The results are presented in counts per minute (cpm). Mixed lymphocyte reactions are performed with T lymphocytes from two different healthy donors. B. Results
Les résultats présentés dans la figure 1 montrent que les cellules dendritiques stimulées par la fraction membranaire de Klebsiella pneumoniae présentent une capacité accrue à fournir des signaux de costimulation aux lymphocytes T et induisent une prolifération de lymphocytes T plus importante que les CD non stimulées.The results presented in FIG. 1 show that the dendritic cells stimulated by the membrane fraction of Klebsiella pneumoniae have an increased capacity to supply costimulation signals to the T lymphocytes and induce a greater proliferation of T lymphocytes than the unstimulated CD.
Exemple V. KpOmpA agit en synergie avec le LPS pour induire la maturation des cellules dendritiquesExample V. KpOmpA acts in synergy with LPS to induce the maturation of dendritic cells
Parmi les protéines membranaires contenues dans la FMKp (fraction membranaire) figure la KpOmpA dont la capacité à induire la maturation des cellules dendritiques humaines a déjà été montrée (Jeannin et al. , 2000, Nat hnmunol, 1 (6), 502-9).Among the membrane proteins contained in the FMKp (membrane fraction) is KpOmpA, the ability of which to induce the maturation of human dendritic cells has already been shown (Jeannin et al., 2000, Nat hnmunol, 1 (6), 502-9) .
Toutefois, la FMKp, contenant un taux de protéines compris entre 1,2 g/1 et 3,4 g/1 et de galactose compris entre 7,5 et 14,9 g/1, est beaucoup plus efficace pour induire k maturation des cellules dendritiques que la protéine seule, ainsi que le démontre le tableau π, où la maturation des cellules dendritiques humaines est objectivée par la production dTL-12.However, FMKp, containing a protein level between 1.2 g / 1 and 3.4 g / 1 and galactose between 7.5 and 14.9 g / 1, is much more effective in inducing the maturation of dendritic cells as protein alone, as shown in Table π, where the maturation of human dendritic cells is objectified by the production of dTL-12.
Ainsi, la FMKp induit la production de 134 pg/ml dTL-12 à 0,1 μg de protéines/ml alors que parmi ces protéines totales KpOmpA ne représente qu'un faible pourcentage (environ 10%).Thus, FMKp induces the production of 134 pg / ml dTL-12 at 0.1 μg of proteins / ml whereas among these total proteins KpOmpA represents only a small percentage (approximately 10%).
En revanche, même à forte concentration (20 μg/ml) KpOmpA seule, ne déclenche la production que de 30 pg/ml dTL-12 (valeurs extraites de Jeannin et al, op. cit.).On the other hand, even at high concentration (20 μg / ml) KpOmpA alone, triggers the production of only 30 pg / ml dTL-12 (values extracted from Jeannin et al, op. Cit.).
Des cellules dendritiques à 10 x 106/ml préparées comme décrit précédemment ont été stimulées avec des doses croissantes de FMKp ou KpOmpA. Après 24 heures, l'IL-12, indicatrice de maturation des cellules dendritiques a été dosée dans les surnageants par ELISA (R&D Systems).
Dendritic cells at 10 x 10 6 / ml prepared as described above were stimulated with increasing doses of FMKp or KpOmpA. After 24 hours, IL-12, an indicator of maturation of the dendritic cells, was assayed in the supernatants by ELISA (R&D Systems).
Tableau II: maturation de cellules dendritiques humaines en présence de KpOmpA ou de FMKPTable II: Maturation of Human Dendritic Cells in the Presence of KpOmpA or FMKP
Sans être lié par cette théorie, le Déposant pense que l'efficacité de la FMKp à induire la maturation des cellules dendritiques humaines peut s'expliquer par un effet synergique entre KpOmpA et le LPS contenu dans la FMKp.Without being bound by this theory, the Applicant believes that the effectiveness of FMKp in inducing the maturation of human dendritic cells can be explained by a synergistic effect between KpOmpA and the LPS contained in FMKp.
Les résultats présentés dans le tableau HI permettent d'étayer cette hypothèse. Des cellules dendritiques à 10 x 106/ml préparées comme décrit précédemment ont été stimulées avec des doses croissantes de LPS en présence ou en absence de kpOmpA (lOμg/ml). Après 24 heures, l'IL-8 a été dosée dans les surnageants par ELISA (R&D Systems). Les résultats présentés sont exprimés en ng/ml et sont représentatifs d'une expérience parmi trois.The results presented in Table HI support this hypothesis. Dendritic cells at 10 x 10 6 / ml prepared as described above were stimulated with increasing doses of LPS in the presence or absence of kpOmpA (10 μg / ml). After 24 hours, IL-8 was assayed in the supernatants by ELISA (R&D Systems). The results presented are expressed in ng / ml and are representative of one of three experiments.
Tableau HL. Maturation des cellules dendritiques humaines avec du LPS. en présence ou absence de KpOmpA.
Table HL. Maturation of human dendritic cells with LPS. in the presence or absence of KpOmpA.
Claims
1. Utilisation d'au moins une fraction membranaire de bactéries à gram négatif, préférentiellement de Klebsiella pneumoniae, comprenant au moins des protéines membranaires associées aux LPS (lipopolysaccharides), pour induire la maturation des cellules dendritiques.1. Use of at least one membrane fraction of gram-negative bacteria, preferably Klebsiella pneumoniae, comprising at least membrane proteins associated with LPS (lipopolysaccharides), to induce the maturation of dendritic cells.
2. Utilisation d'au moins une fraction membranaire de bactéries à gram négatif, et préférentiellement de Klebsiella Pneumonia, comprenant au moins des protéines membranaires associées aux LPS (lipopolysaccharides), pour la fabrication d'un médicament destiné à induire la maturation des cellules dendritiques.2. Use of at least one membrane fraction of gram-negative bacteria, and preferably Klebsiella Pneumonia, comprising at least membrane proteins associated with LPS (lipopolysaccharides), for the manufacture of a medicament intended to induce the maturation of dendritic cells .
3. Utilisation selon la revendication 1 ou 2, caractérisée en ce que ladite fraction membranaire présente un titre en protéoglycane représenté par la somme des teneurs en galactose et en protéines, entre 1,2 g/1 et 3,4 g/1 pour le galactose et entre 7,5 g/1 et 14,9 g/1 pour les protéines3. Use according to claim 1 or 2, characterized in that said membrane fraction has a proteoglycan titer represented by the sum of the galactose and protein contents, between 1.2 g / 1 and 3.4 g / 1 for the galactose and between 7.5 g / 1 and 14.9 g / 1 for proteins
4. Utilisation selon l'une des revendications 1 à 3, caractérisée en ce que la génération et la maturation des cellules dendritiques est effectuée in vitro.4. Use according to one of claims 1 to 3, characterized in that the generation and maturation of dendritic cells is carried out in vitro.
5. Utilisation selon l'une des revendications 2 à 4, caractérisée en ce que la génération et la maturation des cellules dendritiques est effectuée in vitro, les cellules matures étant ensuite réinjectées in vivo.5. Use according to one of claims 2 to 4, characterized in that the generation and maturation of dendritic cells is carried out in vitro, the mature cells then being reinjected in vivo.
6. Utilisation selon l'une des revendications 2 à 5 et en outre d'au moins un agent biologique pour la fabrication d'un médicament.6. Use according to one of claims 2 to 5 and in addition of at least one biological agent for the manufacture of a medicament.
7. Utilisation selon la revendication 6, caractérisée en ce que l'agent biologique est choisi parmi les antigènes de bactérie, de virus, de levure, de parasite, de champignon, les antigènes tumoraux, et les lysats de cellules tumorales autologues et/ou heterologues.7. Use according to claim 6, characterized in that the biological agent is chosen from bacteria, virus, yeast, parasite, fungus antigens, tumor antigens, and lysates of autologous tumor cells and / or heterologous.
8. Utilisation selon l'une des revendications 2 à 5, caractérisée en ce que les cellules idendritiques sont transfectées ex vivo avec un gène codant pour un antigène et/ou une cytokine ou un facteur de croissance. 8. Use according to one of claims 2 to 5, characterized in that the idendritic cells are transfected ex vivo with a gene coding for an antigen and / or a cytokine or a growth factor.
9. Utilisation selon l'une des revendications 2 à 8, caractérisée en ce que le médicament contient en outre une cytokine ou un facteur de croissance, préférentiellement l'interféron alpha ou gamma, le TNFα, le GM-CSF, l'JL-2, 1TL-4, l'JL-6 et IL-18 ou une HSP. 9. Use according to one of claims 2 to 8, characterized in that the medicament also contains a cytokine or a growth factor, preferably interferon alpha or gamma, TNFα, GM-CSF, JL- 2, 1TL-4, the JL-6 and IL-18 or an HSP.
10. Utilisation selon l'une des revendications 1 à 9 pour la fabrication d'un médicament pour le traitement ou la prévention des maladies infectieuses d'origine virale, bactérienne, fongique ou provoquée par une levure, ou un parasite.10. Use according to one of claims 1 to 9 for the manufacture of a medicament for the treatment or prevention of infectious diseases of viral, bacterial, fungal origin or caused by a yeast, or a parasite.
11. Utilisation selon l'une des revendications 1 à 9 pour la fabrication d'un médicament pour lutter contre un virus choisi parmi le VIH, les virus hépatiques et le parainfluenza virus.11. Use according to one of claims 1 to 9 for the manufacture of a medicament for combating a virus chosen from HIV, hepatic viruses and parainfluenza virus.
12. Utilisation selon l'une des revendications 1 à 9 pour la fabrication d'un médicament pour le traitement ou la prévention des cancers.12. Use according to one of claims 1 to 9 for the manufacture of a medicament for the treatment or prevention of cancers.
13. Utilisation selon l'une des revendications 8 ou 9 pour la fabrication d'un médicament pour le traitement ou la prévention d'un cancer parmi les myélomes, lymphomes, leucémies, carcinomes du rein, du cerveau, de la prostate, du rectum, du pancréas, des ovaires, du poumon, les kératinomes et les carcinomes.13. Use according to one of claims 8 or 9 for the manufacture of a medicament for the treatment or prevention of cancer among myelomas, lymphomas, leukemias, carcinomas of the kidney, brain, prostate, rectum , pancreas, ovaries, lung, keratinomas and carcinomas.
14. Dispositif pour la maturation des cellules dendritiques caractérisé en ce qu'il comprend au moins une fraction membranaire de bactéries à gram négatif, et préférentiellement de Klebsiella Pneumonia, comprenant au moins des protéines membranaires associées aux LPS (lipopolysaccharides).14. Device for the maturation of dendritic cells, characterized in that it comprises at least one membrane fraction of gram-negative bacteria, and preferably of Klebsiella Pneumonia, comprising at least membrane proteins associated with LPS (lipopolysaccharides).
15. Dispositif selon la revendication 14, caractérisé en ce que ladite fraction membranaire présente un titre en proteoglycane représenté par la somme des teneurs en galactose et en protéines, entre 1,2 g/1 et 3,4 g/1 pour le galactose et entre 7,5 g/1 et 14,9 g/1 pour les protéines. 15. Device according to claim 14, characterized in that said membrane fraction has a proteoglycan titer represented by the sum of the galactose and protein contents, between 1.2 g / 1 and 3.4 g / 1 for galactose and between 7.5 g / 1 and 14.9 g / 1 for proteins.
16. Dispositif selon la revendication 14 ou 15, caractérisé en ce qu'il comprend en outre des antigènes heterologues ou des moyens pour obtenir un lysat de cellules autologues. 16. Device according to claim 14 or 15, characterized in that it further comprises heterologous antigens or means for obtaining a lysate of autologous cells.
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| FR0103518A FR2822071B1 (en) | 2001-03-15 | 2001-03-15 | USE OF A MEMBRANE FRACTION OF GRAM NEGATIVE BACTERIA TO INDUCE THE MATURATION OF DENDRITIC CELLS |
| FR0103518 | 2001-03-15 | ||
| PCT/FR2002/000906 WO2002074939A1 (en) | 2001-03-15 | 2002-03-14 | Use of gram-negative bacterial membrane fraction for inducing the maturation of dendritic cells |
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| EP (1) | EP1368457A1 (en) |
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| CA (1) | CA2460779A1 (en) |
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| EP1352052B1 (en) | 2001-01-15 | 2006-02-08 | I.D.M. Immuno-Designed Molecules | Method for preparing submicron particle suspensions of pharmaceutical agents |
| ATE530194T1 (en) * | 2003-04-30 | 2011-11-15 | Medi Service S R L | IMMUNOMODULATING COMPOSITION CONTAINING A PARTICLE FRACTION OF BACTERIAL MECHANICAL LYSATES |
| US9107864B2 (en) | 2004-06-07 | 2015-08-18 | Qu Biologics Inc. | Tissue targeted antigenic activation of the immune response to treat cancers |
| US8501198B2 (en) | 2004-06-07 | 2013-08-06 | Qu Biologics Inc. | Tissue targeted antigenic activation of the immune response to treat cancers |
| US8034359B2 (en) * | 2004-06-07 | 2011-10-11 | Qu Biologics Inc. | Tissue targeted antigenic activation of the immune response to cancers |
| DK176322B1 (en) * | 2004-09-21 | 2007-08-06 | Broendum V As | Detention device for seating and berths in buses |
| CN101854820B (en) * | 2007-11-13 | 2013-06-19 | 纳幕尔杜邦公司 | Breathable garment having a fluid drainage layer |
| NZ702796A (en) | 2010-07-26 | 2017-03-31 | Qu Biologics Inc | Immunogenic anti-inflammatory compositions |
| EP2662441A1 (en) * | 2012-05-12 | 2013-11-13 | Universiteit Maastricht | Method for the in vitro maturation of dendritic cells |
| CA2947631A1 (en) | 2014-05-02 | 2015-11-05 | Qu Biologics Inc. | Anti-microbial immunomodulation |
| MX2021003551A (en) * | 2018-09-27 | 2021-08-16 | Decoy Biosystems Inc | Methods of treatment of infections using bacteria. |
| JP2022520999A (en) * | 2019-02-22 | 2022-04-04 | エヴェロ バイオサイエンシズ，インコーポレーテッド | Bacterial membrane preparation |
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| FR2785542B1 (en) * | 1998-11-06 | 2001-02-09 | Pf Medicament | USE OF AN ENTEROMACTERY OmpA PROTEIN FOR THE SPECIFIC TARGETING OF A BIOLOGICALLY ACTIVE SUBSTANCE THAT IS ASSOCIATED WITH CELLS PRESENTING ANTIGENS SUCH AS HUMAN DENDRITIC CELLS |
| FR2790960B1 (en) * | 1999-03-15 | 2002-10-31 | Pf Medicament | USE OF BACTERIAL MEMBRANE FRACTIONS WITH IMMUNOSTIMULANT ACTIVITY IN THE TREATMENT OF CANCERS, THEIR PREPARATION METHODS AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| FR2790959B1 (en) * | 1999-03-15 | 2003-06-27 | Pf Medicament | USE OF BACTERIAL MEMBRANARY FRACTIONS WITH ADJUVANT EFFECT, THEIR PREPARATION METHODS AND PHARMACEUTICAL COMPOSITION CONTAINING THEM |
| FR2819263B1 (en) * | 2001-01-10 | 2003-04-11 | Lco Sante | PROCESS FOR MATURATION OF DENDRITIC CELLS AND ACTIVATION OF MACROPHAGES WITH RU 41740 |
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| US8697154B2 (en) | 2007-03-05 | 2014-04-15 | Om Pharma Sa | Bacterial extract for respiratory disorders and process for its preparation |
| US9463209B2 (en) | 2007-03-05 | 2016-10-11 | Om Pharma Sa | Bacterial extract for respiratory disorders and process for its preparation |
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| FR2822071A1 (en) | 2002-09-20 |
| AU2002247813B2 (en) | 2007-06-28 |
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| FR2822071B1 (en) | 2005-07-01 |
| CA2460779A1 (en) | 2002-09-26 |
| JP2004531496A (en) | 2004-10-14 |
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