Supernatants from co-cultures of macrophages and irradiated leukocytes for controlling tumor progression or restoring anti-tumor immunity

Abstract

The present invention relates to a method for addressing tumor-promoting inflammation by means of a pharmaceutical preparation. Leukocytes are separated from a buffy coat obtained from whole blood, and a portion of the leukocytes are placed in a first bag receiving differentiation factors and stored for several days under conditions that maintain cell viability before providing a fresh medium with defined composition, after which the leukocyte portion is left to rest for several more days, resulting in the production of macrophages. Another portion of the leukocytes is placed in a second bag and irradiated therein. The two bags are then mixed, and the supernatant is recovered for use as a pharmaceutical preparation. The present invention also relates to the use of the leukocytes alone or in combination with other therapies for the treatment of cancer in human and veterinary medicine.

Description

Technical Field

[0001] The present invention relates to methods of addressing tumor-promoting inflammation via pharmaceutical formulations. Background Art

[0002] Inflammation is a biological process that is part of the body's natural defense response but is also associated with a variety of pathological responses.

[0003] Inflammation, in particular, causes inflammatory diseases such as inflammatory diseases of the intestine, for example Crohn's disease and rheumatoid arthritis, but it is also involved in the pathogenesis of obesity, diabetes and neurological disorders such as depression. Proinflammatory factors, such as proinflammatory cytokines, participate in and support inflammation during obesity, tumorigenesis and cancer development. In fact, chronic inflammation plays an important role in promoting the failure of the immune system to treat tumors and maintaining the development of tumors. Inflammation remains an acute risk factor for cancer.

[0004] Chronic inflammation may be associated with a partial or incomplete resolution of the inflammatory response and may be promoted by genetic, environmental or infectious factors. Chronic inflammation is particularly involved in the progression of cancers caused by viral infections, such as that associated with papillomavirus (HPV), which increases the risk of cervical cancer, with hepatitis B and C viruses and the emergence of liver cancer, or with Helicobacter pylori and the development of adenocarcinomas or lymphomas. Chronic inflammation in autoimmune diseases, such as Crohn's disease or ulcerative colitis, is also associated with an increased risk of colorectal cancer. Finally, inflammation caused by exposure to irritants or obesity also promotes tumor-promoting activities.

[0005] Therefore, during the attack of the organism that caused the inflammation, it is important to trigger the resolution of the inflammation. If the inflammation is not fully resolved, it may become chronic, leading to the pathologies mentioned above.

[0006] If inflammatory mediators are essential for antitumor surveillance, especially during the stage of oncogenic immunoediting, allowing the establishment of an adaptive antitumor immune response, the latter can play a pro-tumor role, especially TNF (tumor necrosis factor). In fact, the tumor inflammatory microenvironment can promote tumor growth, angiogenesis, invasion and formation of metastases. The development of tumors is the result of an increased proliferation of cancer cells, their resistance to death by apoptosis and angiogenesis in the tumor microenvironment, which leads to tumor cell invasion.

[0007] For example, TNF promotes the attraction of regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs) in tumors, further attenuating antitumor responses. Paradoxically, while cytotoxic therapies that induce immunogenic death of tumor cells have demonstrated a major role for the immune system in the selection of conventional anticancer treatments, the same means will now be described that link the dead cells killed by these therapies to secondary pro-tumor effects that stimulate tumor growth.

[0008] In chronic inflammatory diseases, unwinding the resolution of inflammation also remains a therapeutic option to reactivate the natural resolution of inflammation, i.e. allowing the inflammatory response to be stopped. Currently, targeted therapeutic approaches that neutralize or destroy inflammatory factors have been shown to have limitations. Furthermore, it seems illusory to be able to control complex pathologies such as Crohn's disease simply by neutralizing inflammatory factors, which are simply a consequence of the pathology. However, resolving inflammation by re-establishing an effective resolution may make it possible to treat the core of the inflammatory response.

[0009] Therefore, promoting resolution of inflammation and improving elimination of tumor cells killed by anticancer therapies has become a major challenge in cancer care. Targeting resolution of inflammation in the same manner may make it possible to control the underlying causes of inflammatory pathology. Summary of the invention

[0010] The invention relates in particular to a method for preventing cancer by administering a combination of pro-resolutive factors, i.e., triggering the resolution of inflammation, by inhibiting tumor escape from immune surveillance and its development and by reestablishing effective anti-tumor immunity.

[0011] Patent EP 2 941 257 describes a pharmaceutical preparation for preventing or treating a pathological immune response. The pharmaceutical preparation comprises a supernatant obtainable from a co-culture of phagocytes and apoptotic cells. In the patent, the pharmaceutical preparation is used to treat conditions such as autoimmune diseases, such as rheumatoid arthritis (RA) or chronic inflammatory diseases of the intestine (chronic inflammatory bowel disease-CIBD). This pharmaceutical preparation is also used to treat graft-versus-host disease (GVHD).

[0012] The present invention has a different object, namely a method for resolving pro-tumor inflammation by means of a supernatant obtained from a co-culture of irradiated macrophages and leukocytes; this supernatant has, in an unexpected manner, a number of advantages compared to the supernatant described in patent EP 2 941 257. In particular, it provides a greater number of pro-resolving factors ( Figure 1 a); it also has biological advantages ( Figure 1 b).

[0013] The pharmaceutical preparation according to the invention can be used in case of resolution failure to re-establish effective resolution and allow arrest of the inflammatory response. Therefore, the object of the present invention is in particular to resolve chronic inflammation and to resolve inflammation that promotes tumor growth by means of the above-mentioned pharmaceutical preparation.

[0014] The present invention is based firstly on the fact that targeting the resolution of pro-tumor inflammation allows the restoration of anti-tumor immune responses and the cessation of chronic anti-inflammatory reactions.

[0015] It should be remembered at this stage that inflammation is beneficial when tumors are first forming, but has negative effects thereafter that promote treatment failure, which is what the present invention is intended to control.

[0016] The method according to the present invention comprises using a supernatant obtained by producing monocyte-derived macrophages from leukocytes and storing it together with leukocytes that have been previously irradiated so that they can be co-cultured, and collecting factors contained in the supernatant produced by the cells.

[0017] In the following description, the supernatant of the pharmaceutical preparation according to the present invention aimed at resolving inflammation will be referred to as "Resolvix".

[0018] The infusion of these factors made it possible to resolve active inflammatory responses in various experimental models (arthritis, inflammatory bowel disease-IBD, colitis, experimental autoimmune encephalomyelitis) and also to promote tissue repair. In an interesting way, this approach also made it possible to restore anti-cancer immunity, thus promoting tumor regression.

[0019] As a non-limiting example, a method for producing a supernatant (Resolvix) according to the invention will now be described.

[0020] Leukocytes are separated from whole blood at D0 by density gradient, washed, resuspended in a defined culture medium commonly used by those skilled in the art, with a salt, amino acid and vitamin base, such as, for example, a medium called RPMI 1640, and then 60% is distributed to the first bag and 40% to the second bag. The first bag then receives a differentiation factor, which may be M-CSF (macrophage colony stimulating factor), and is stored under conditions that maintain cell viability, such as at 37° C., 5% CO2 for several days, preferably 3 days, and then receives fresh defined culture medium again. The first bag is then kept intact, and after several days, preferably 4 days, under conditions that maintain cell viability, such as at 37° C., 5% CO2, macrophages are generated. The second bag is again stored at low temperature, preferably at a temperature below +10° C., and more preferably below +5° C. On day 7, the second bag is irradiated, such as by X-rays, and then the first bag and the second bag are freed of their culture medium. The second bag is then placed in a secretion medium with a salt, amino acid and vitamin base known to those skilled in the art, such as one known as MEM, and transferred to the first bag for storage under conditions that maintain cell viability, such as at 37°C, 5% CO2. During this storage period, co-culture occurs resulting in the production of a conditioned supernatant, which may take several days, preferably 2 days. In this specification, a "conditioned supernatant" is a supernatant containing factors derived from cells. After this co-culture period, the supernatant is collected in a new bag, filtered, and then stored at a very low temperature, preferably within the range of -80°C. The supernatant is then advantageously mixed with the supernatant of the buffy coat produced from other blood donors according to the above method, and the mixture is then divided into unit doses, lyophilized or not, and then stored. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0022] Figure 1: Biological and activity comparison of a new production process for producing a supernatant called Resolvix. This new production process makes it possible to obtain a greater amount of pro-resolving factors, such as the anti-inflammatory cytokine TGF-β quantified by an ELISA test (a) and a biological advantage in inhibiting the production of TNF in monocytes stimulated by lectin, evaluated by a biological test (b). The data from 35 samples of the old method and 89 samples of the new method are expressed as percentages of inhibition of TNF production, expressed as mean + / - standard error of the mean. ***=p<0.001, ****=p<0.0001, unpaired Student's t-test (test t de Student non apparié). In the figure, the old method relates to the method for producing the supernatant described in the aforementioned patent EP 2 941 257.

[0023] Figure 2 : Tracking of tumor growth. The bioluminescence emitted by the tumor cell line EL4-Luc+ (after injection of luciferase) has been quantified in leukemic mice C57B1 / 6, treated or untreated with Resolvix at D0 or D7 after injection of leukemic cells (a). Data derived from a representative experience of two groups of 5 animals each, expressed as group mean + / - standard error of the mean. *=p<0.05, ****=p<0.0001, test 2way AOVA vs. vehicle. Representative images of the bioluminescence of 5 untreated mice (vehicle) and 5 mice that received Resolvix at D0, obtained 21 days after injection of tumor cells, are shown (b). The intensity of the emitted fluorescence (from dark blue to yellow) is proportional to the number of tumor cells that have proliferated.

[0024] Figure 3 : Tumor evolution after Resolvix treatment. Tumor volume (in mm 3 = (a) or (b) assessed by injecting the tumor line B16-OVA (a) or the tumor line EL-4 (b) into the right flank of mice C57B1 / 6 over time. Resolvix was injected on the day of cancer cell injection (J0) or 7 days later (J7). Data are representative of 2 experiments (a and b) from 6 groups of 5 animals per group and are expressed as mean + / - standard error of the mean. * = p < 0.05, ** = p < 0.001, and *** = p < 0.001, relative to vehicle, two-way ANOVA test.

[0025] Figure 4: Evolution of in vitro cell proliferation of the cancer cell line EL-4 in the presence of Resolvix. Cell proliferation was assessed by daily cell counting over 3 days. Data are from two representative experiments and are expressed as mean + / - standard error of the mean of triplicates. * = p < 0.05, **** = p < 0.000l vs. Resolvix, two-way ANOVA test.

[0026] Figure 5 : Resolvix treatment of solid tumors in immunodeficient mice. The tumor volume (in mm) of the tumors injected subcutaneously into the right flank of immunodeficient mice RAG-γ / c by the cancer cell line EL-4 was determined at different times. 3 Resolvix was injected on the day of cancer cell injection (D0) or 7 days later (D7). Data from experiments with 5 animals per group are presented as mean + / - standard error of the mean. DETAILED DESCRIPTION

[0027] The data demonstrate in an interesting manner that injection of Resolvix in mice bearing T lymphoma leukemia (the tumor cell line EL4-luciferase+ enables tracking of tumor growth by bioimaging) leads to regression of tumor growth ( Figure 1 ).

[0028] The same observations were obtained in a second model of solid tumors (subcutaneous injection of tumor cell lines EL-4 or B16-OVA), where the injection of Resolvix made it possible to reduce the size of the tumors ( Figure 2 ).

[0029] These results suggest that Resolvix treatment can directly affect the in vivo growth of tumor cell lines through direct cytotoxic effects. To evaluate this possibility, other in vitro experiments have been performed and have shown that the cultivation of tumor cells EL-4 in the presence of Resolvix instead promotes the growth of the line ( Figure 3 ). These data suggest that the in vivo tumor growth inhibition observed after treatment with Resolvix is ​​not related to direct cytotoxic effects of the drug on tumor cells.

[0030] Furthermore, the fact that the effects of the treatment were not directly on tumor cells suggests that the restoration of antitumor immunity was due to the treatment. Indeed, in the absence of an immune system, i.e., in immunodeficient C57B1 / 6RAG-γ / c tumor-bearing mice, Resolvix treatment had no effect on tumor growth in vivo ( Figure 4 ).

[0031] Resolvix was injected into the tumor model either at the time of tumor cell transplantation (D0) or 7 days after transplantation (D7). Therefore, by controlling inflammation at these different times, it is possible to control tumor growth and re-establish anti-tumor immunity.

[0032] Therefore, the above shows that the use of Resolvix according to the present invention appears to make it possible to restore anti-tumor immunity and / or stop tumor progression. More generally, the pharmaceutical preparation according to the present invention can be used to target, control, inhibit, resolve inflammation associated with cancer, thus making it possible to restore anti-tumor immunity and / or stop tumor progression as just mentioned.

[0033] The supernatant according to the invention can be used in particular, alone or in combination with other therapies, for the treatment of cancer in human or veterinary medicine, in particular cancers represented by lymphomas, leukemias, sarcomas and carcinomas, more precisely T-cell lymphomas, B-cell lymphomas, melanomas and colon cancer.

Claims

1. A pharmaceutical preparation for controlling tumor progression or restoring anti-tumor immunity, the pharmaceutical preparation comprising a supernatant obtained from a co-culture of leukocytes, wherein the co-culture of leukocytes comprises 60% of a first part and 40% of a second part, wherein the first part comprises macrophages and the second part comprises irradiated leukocytes, wherein the leukocytes in the co-culture are isolated from the buffy coat generated from whole blood, The first part of the co-culture of leukocytes is washed, resuspended in a defined medium RPMI 1640, and then placed in a first bag of differentiation factors receiving macrophage colony stimulating factor and stored at 37°C and 5% CO2 for 3 days to maintain cell viability, and then again received fresh defined medium RPMI 1640, and then allowed to stand at 37°C and 5% CO2 for 4 days to maintain cell viability, thereby resulting in the generation of macrophages, wherein a second portion of the co-culture of leukocytes is placed in a second bag, which is maintained at a temperature below +5°C, and wherein the second portion is irradiated in the second bag on day 7, wherein the second bag, after removing the irradiated culture medium, is placed in a MEM secretion culture medium containing salt, amino acids and vitamins under the condition of maintaining cell viability, wherein the second portion in the second bag is transferred into the first portion in the first bag, the first bag having its culture medium previously removed, to form a co-culture, the co-culture being stored for 2 days under conditions of 37° C. and 5% CO2 to maintain cell viability, to form a co-culture between the leukocytes, thereby resulting in the production of a conditioned supernatant, wherein the resulting supernatant of the co-culture is collected in a new bag, filtered and kept at about -80°C, and The pharmaceutical preparation increases TGF-β concentration and inhibits TNF production in lectin-stimulated monocytes, and controls tumor progression in vivo.

2. Use of the pharmaceutical preparation according to claim 1 alone or together with other anticancer therapies in the preparation of a medicament for treating cancer.

3. The use according to claim 2, wherein the cancer is lymphoma, leukemia, sarcoma or carcinoma.

4. The use according to claim 2, wherein the cancer is T-cell lymphoma, B-cell lymphoma, melanoma or colon cancer.

5. Use according to any one of claims 2 to 4, wherein the medicament is the result of a mixture of supernatants obtained from a source of several leukocytes according to claim 1.

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