SUPERNATANT FROM A COCULTURE OF MACROPHAGES AND IRRADIATED LEUKOCYTES, FOR CONTROLLING TUMOUR PROGRESSION OR RESTORING ANTI-TUMOR IMMUNITY

Abstract

The invention relates to a method for the resolution of pro-tumour inflammation by means of a pharmaceutical preparation. Leukocytes are isolated from the buffy coat obtained from whole blood and a portion of the leukocytes is placed in a first pouch receiving a differentiation factor and stored in conditions that preserve cellular viability for several days, before a defined, fresh culture medium is supplied, after which said leukocyte portion is left to rest for a further several days, which results in the production of macrophages. A further portion of the leukocytes is placed in a second pouch and irradiated therein. The two pouches are then mixed and the supernatant is recovered to serve as pharmaceutical preparation. The invention also relates to the use in the treatment of cancers, either alone or in combination with other therapies, in human and veterinary medicine.

Claims

1. Pharmaceutical preparation used for controlling tumor progression or for restoring anti-tumor immunity, comprising a supernatant obtained from a co-culture between macrophages and irradiated leucocytes.

2. The pharmaceutical preparation according to claim 1, wherein said macrophages are leucocytes.

3. The pharmaceutical preparation according to claim 2, wherein said leucocytes are isolated from the buffy coat resulting from whole blood.

4. The pharmaceutical preparation according to claim 3, wherein a portion of said leucocytes is washed, resuspended in a defined culture medium, then placed in a first pocket receiving a differentiation factor and preserved in conditions which preserve cellular viability for several days, prior to again receiving fresh defined culture medium, then left as it is again for several days, resulting in the production of macrophages.

5. The pharmaceutical preparation according to claim 3, wherein another portion of said leucocytes is placed in a second pocket kept at a temperature below +5° C.

6. The pharmaceutical preparation according to claim 5, wherein said second pocket, after having been freed from its irradiated medium, is freed from its medium, then put in a secretion medium in conditions that preserve cellular viability.

7. The pharmaceutical preparation according to claim 6, wherein said second pocket is transferred into the said first pocket, previously freed from its medium, the set of two pockets being stored in conditions that preserve cellular viability for forming the co-culture between the leucocytes for a period leading to the production of a conditioned supernatant.

8. The pharmaceutical preparation according to claim 7, wherein the resulting supernatant of said co-culture is collected in a new pocket, filtered and kept at a very low temperature.

9. The pharmaceutical preparation according to claim 1, for use in the treatment of cancers, either alone or in addition to other anti-cancer therapies.

10. The pharmaceutical preparation according to claim 9, wherein said cancers are selected from the group consisting of: lymphomas, leukemias, sarcomas, carcinomas, T cell lymphomas, B-cell lymphomas, melanomas and colon carcinomas.

11. The pharmaceutical preparation for use according to claim 9, wherein the preparation is the result of a mixture of supernatants from several leucocyte sources.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In a more precise manner, in vivo models of tumor growth were used. The results obtained are shown in the following figures:

[0022] FIG. 1: biological and activity comparison of the new method of production resulting in the supernatant named Resolvix. The new method of production makes it possible to obtain a greater quantity of pro-resolution factors such as the anti-inflammatory cytokine TGF-beta, quantified by the ELISA test (a), and a biological superiority (b) evaluated by the biological test inhibiting the production of TNF in monocytes stimulated by phytohaemagglutinin. Data from 35 samples of the old method and 89 of the new method, expressed as a % of the inhibition of TNF production, represented by the mean+/−standard error of the mean. ***=p<0.001, ****=p<0.0001, unpaired Student's t-test. In this Figure, the old method relates to the method resulting in the supernatant described in the aforementioned patent EP 2 941 257.

[0023] FIG. 2: tracking tumor growth. The bioluminescence emitted by the tumoral line EL4-Luc+ (after injection of luciferase) has been quantified in mice C57B1/6 with leukemia, treated or not treated with Resolvix on D0 or D7 after injection of leukemia cells (a). Data resulting from a representative experience of two with 5 animals per group, expressed as a group mean+/−standard error of the mean. *=p<0.05, ****=p<0.0001, vs vehicle, 2way ANOVA test. Representative images of bio luminescence of 5 untreated mice (vehicle) and 5 mice who have received Resolvix on D0, acquired 21 days after the injection of tumor cells, are shown (b). The intensity of the luminescence emitted (from dark blue to yellow) is associated proportionally with the number of tumor cells that have proliferated.

[0024] FIG. 3: evolution of tumors after treatment with Resolvix. The volume of the tumors (in mm3) was evaluated from the injection of tumor line B16-OVA (a) or from tumor line EL-4 (b) subcutaneously into the right abdominal flank of mice C57B1/6 over time. Resolvix was injected either on the date of injection of cancer cells (J0), or 7 days later (J7). Data from 2 experiments (a and b) representative of 6 with 5 animals per group, expressed as a mean+/−standard error of the mean.

[0025] *=p<0.05, **=p<0.01 & ***=p<0.001 vs vehicle, 2way ANOVA test.

[0026] FIG. 4: evolution of in vitro cell proliferation of the cancerous cell line EL-4 in the presence of Resolvix. The cellular proliferation was evaluated by a cell count every day for 3 days. The data are from a representative experiment of two, expressed as an mean of triplicates+/−standard error of the mean.*=p<0.05, ****=p<0.0001 vs Resolvix, 2way ANOVA test.

[0027] FIG. 5: Resolvix treatment of solid tumors in immunodeficient mice. The volume (in mm3) of tumors induced by the cancer line EL-4 subcutaneously into the right abdominal flank of immunodeficient mice RAG-γ/c, was determined at different times. Resolvix was injected either on the date of injection of cancer cells (D0) or 7 days later (D7). The data are from an experiment with 5 animals per group, expressed as the mean+/−standard error of the mean.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Said data demonstrate in an interesting manner that the injection of Resolvix in mice carrying T lymphoma leukemia (tumor line EL4-luciferase+ enabling tracking of the tumor growth by bio-imaging) results in a regression of tumor growth (FIG. 1).

[0029] These same observations were made in a second model, with solid tumors (tumor lines EL-4 or B16-OVA injected subcutaneously), in which the injection of Resolvix makes it possible to reduce the size of tumors (FIG. 2).

[0030] These results suggest that the Resolvix treatment could directly influence the growth of the tumor line in vivo, by means of a direct cytotoxic effect. In order to evaluate this possibility, additional experiments in vitro have been performed and have shown that the culture of tumor cells EL-4 in the presence of Resolvix encourages rather the growth of the line (FIG. 3). These data show that the inhibition of the tumor growth observed in vivo after treatment with Resolvix is not linked to a direct cytotoxic effect of the drug on the tumor cells.

[0031] Furthermore, the fact that the effect of the treatment is not directly on the tumor cells suggests that the restoration of anti-tumor immunity is due to the treatment. Indeed, in the absence of the immune system, i.e. in immunodeficient mice C57B1/6 RAG-γ/c with tumors, the Resolvix treatment has no effect on the in vivo tumor growth (FIG. 4).

[0032] The injection of Resolvix in the tumor models is performed at the time of grafting tumor cells (D0) or 7 days afterwards (D7). By thus controlling the inflammation at these different times, it is possible to control the tumor growth and reestablish anti-tumor immunity.

[0033] Thus, from the above it appears that the use of Resolvix according to the present invention makes it possible to restore anti-tumor immunity and/or stop tumor progression. More generally, the pharmaceutical preparation according to the invention can be used to target, control, inhibit, resolve inflammation associated with cancer, thus making it possible as just mentioned to restore anti-tumor immunity and/or to stop tumor progression.

[0034] The supernatant according to the invention can be used in particular in the treatment of cancers, either alone or along with other therapies, in human or veterinary medicine. The cancers are in particular those represented by lymphomas, leukemias, sarcomas and carcinomas, and more precisely T-cell lymphomas, B-cell lymphomas, melanomas and colon carcinomas.