Methods and compositions for modifying macrophage polarization into pro-inflammatory cells to treat cancer

Abstract

To provide treatment of cancer by modifying the macrophage polarization into pro-inflammatory cells.SOLUTION: Disclosed is a pharmaceutical composition for treating cancer except SIRPa positive acute myeloid leukemia, comprising an anti-SIRPa compound selected from the group consisting of antibodies or antigen-binding moieties, and an immune checkpoint blocker selected from the group consisting of anti-PDL1 blocking antibodies, anti-PD1 blocking antibodies and anti-CTLA-4 blocking antibodies, or a second therapeutic agent selected from the group consisting of anti-CD137 agonist antibodies.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of immunotherapy. More specifically, the present invention provides a method for inhibiting M2-type macrophage polarization to induce a pro-inflammatory environment, resulting in a proper immune response in cancer, infectious diseases, vaccination, trauma and chronic inflammatory diseases.

[0002] The present invention particularly relates to the use of anti-SIRPa compounds capable of inhibiting the polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages. In a preferred embodiment, such compounds are used to treat cancer. Interestingly, the present invention allows the treatment of cancer via an indirect route involving the immune system. [Background technology]

[0003] Cancers resulting from uncontrolled cell proliferation form a diverse group of diseases. Surgery and radiotherapy are not able to treat all cancers, especially in the metastatic stage. A more effective treatment is required to reach all organs of the patient: this is the case of modern chemotherapy, which is able to induce the death of tumor cells. However, the cytotoxic effect of drugs remains a major obstacle for chemotherapy.

[0004] The advent of molecular biology and genetics has allowed us to understand the mechanisms that lead to cancer cell development and "targeted therapies". These treatments, combined with chemotherapy, attack tumor cells while sparing healthy cells. However, although these therapies significantly extend the life expectancy of patients, they still do not provide a cure. Today, it is well established that human tumor cells become resistant to treatment and evade surveillance by the immune system. Therefore, combination therapies are essential to cure patients, but they have the disadvantage of increasing the problem of side effects.

[0005] Early work in the field of cancer immunotherapy aimed to "boost" immune system effector cells and make them more aggressive against tumors, a strategy that has met with some success.

[0006] A new generation of immunotherapy molecules that will revolutionize cancer treatment blocks the inhibitory mechanisms of these cells, allowing effector T cells (Teff) to exert their action: the concept of "inhibiting the inhibitor". Anti-CTLA-4 antibody (Yervoy®) is the first molecule to treat metastatic forms of melanoma, prolonging the median survival of patients by 6-10 months, with a quarter of patients still alive after 2 years. Unfortunately, these results, while impressive in themselves, still do not cure most patients.

[0007] The present invention provides a novel method that relies on an "inhibit the inhibitor" approach and is useful in immunotherapy. More specifically, the present invention relates to a method of modifying macrophage polarization to induce a pro-inflammatory environment. This method consists in the use of anti-SIRPa compounds that are able to inhibit the polarization of anti-inflammatory M2 macrophages and / or not inhibit pro-inflammatory M1 macrophages, in order to inhibit the anti-inflammatory signals provided by M2 macrophages and in favor of the pro-inflammatory signals provided by M1 macrophages. This approach makes it possible to reconstruct an inflammatory environment that favors the action of T effector cells, especially in eliminating cancer cells.

[0008] Macrophage plasticity and polarization Macrophages are the most plastic cells in the hematopoietic system. They are involved in both innate immunity (phagocytosis tolerance) and adaptive immunity (cell polarization), but also in ontogeny, homeostasis, and tissue repair (Non-Patent Document 1; Non-Patent Document 2). Macrophages are present in all tissues. Macrophages have a wide phenotypic and functional diversity. During ontogeny, macrophage cells also show a diverse origin, which persists into adulthood. In tissues, monocytes-macrophages respond to environmental stimuli (products of microbial infection, cellular injury, and lymphocyte activation) and acquire distinct phenotypes. Previously, these cells have been classified according to their function in a dualistic manner in relation to inflammatory conditions.

[0009] Depending on the stimuli they receive, monocytes-macrophages reprogram their transcriptomes to yield distinct functional and phenotypic spectra. Macrophages are simply classified into two subpopulations or polarization (or activation) states: the classical activation phenotype M1 and the alternative activation phenotype M2 (Non-Patent Document 3). The M1 classification is associated in vitro with the use of IFNg factors alone or in combination with microbial factors such as LPS or inflammatory cytokines such as TNF-α and GM-CSF. Polarized M2 is rather associated with IL4 or IL13 (Non-Patent Document 4). Other cytokines are also identified as inducing M2-type polarization, such as Arg1 (arginase 1), which plays a role in inflammation, IL33, which induces overexpression of CCL24 or CCL17. IL21, and more generally CSF1, play a major role in macrophage polarization. Macrophages can also acquire an "M2-like" state that shares the general characteristics of M2. Indeed, many stimuli, such as LPS, IL-1, glucocorticoids, TGF-beta, Wnt5a and IL10-associated immune complexes, result in an "M2-like" functional phenotype.

[0010] Similarly, in vivo studies have revealed the existence of M1, M2 and M2-like macrophages, which represent only the extremes of a continuum of functional states that must be integrated into the environmental complex system.

[0011] In general, M1 macrophages are IL12 high , IL23 high and IL10 low M1 macrophages exhibit a phenotype that produces reactive oxygen species (ROS), nitric oxide (NO) intermediates, and molecular effectors such as proinflammatory cytokines (IL1b, TNF-α, IL-6). M1 macrophages are involved in Th1 responses, play a role in resistance to intracellular parasites, and are important effectors in the elimination of tumor cells. In contrast, M2 macrophages produce IL12 low , IL‐23 low and IL10high M2 macrophages have a phenotype and vary in the production of inflammatory cytokines depending on the stimuli present in the environment. M2 cells strongly express scavenger, mannose and galactose type receptors on their surface. Arginine metabolism is shifted to ornithine and polyamine metabolism. M2 macrophages are generally associated with Th2 type responses, parasite clearance, reduced inflammation, promoted tissue repair, angiogenesis, tumor growth and immune regulation.

[0012] M1 and M2 also have distinct chemokine expression profiles: M1 macrophages express CXCL9 and CXCL10 chemokines known to attract Th1, whereas M2 macrophages express CCL17, CCL22, and CCL24. Chemokines such as CCL2 and CXCL4 can also polarize macrophages towards an M2-like phenotype.

[0013] Depending on their polarization state, these macrophages have different characteristics in terms of iron, folate and glucose metabolism. For example, M1 expresses proteins involved in iron storage such as ferritin in large amounts, but only weakly expresses ferroportin, which is involved in iron transport to the extracellular medium. Conversely, M2 macrophages express low levels of ferritin, but high levels of ferroportin. This difference may have functional consequences, such as the bacteriostatic effect of M1 (defense against infection), and the tissue repair promoting effect of M2 macrophages, which also promotes tumor growth, as observed in several studies. The management of iron by macrophages according to these polarizations is a key factor that highlights the importance of controlling macrophage polarization according to the individual's condition.

[0014] Similarly, macrophages face an oxygen gradient in tissues under normal or pathological conditions. Macrophages or monocytes adapt to this gradient by modifying their glycolytic metabolism. HIF1 and 2 are transcription factor initiators of these modifications, which also include the expression of chemokines or chemokine receptors CXCR4 or CXCL12 and VEGF (angiogenic factors). Macrophages are involved in tissue responses to hypoxia.

[0015] Polyamines present in the cellular environment have been shown to be type 2 macrophage polarization factors.

[0016] The present invention aims to modulate macrophage polarization to inhibit anti-inflammatory M2 type macrophages and / or in favor of pro-inflammatory M1 type macrophages.

[0017] Tumor microenvironment Different defensive cells exist in the tumor environment, which is an a priori contradiction, since their presence would mean that the tumor is attacked. In reality, many of these immune cells are maintained in an inactive state and rendered inoperative by the presence of regulatory cells. Instead of fighting the tumor, these regulatory cells promote the development of the tumor by helping it overcome barriers, allowing it to spread and form secondary tumors, i.e. metastases.

[0018] Tumor immune evasion and immunosuppression mechanisms Cellular and molecular effectors of inflammation play an important role in the tumor microenvironment. Indeed, since the 90s, this interrelationship between inflammation and tumors has been the subject of numerous studies (Non-Patent Document 5; Non-Patent Document 6). To avoid the immune system, tumors implement an escape mechanism in three stages: elimination-balance-escape. In the first stage, they eliminate the immune mechanisms that recognize tumor cells. Then, they establish an equilibrium between tumor cells and the immune: killing and survival. This equilibrium can last for several years without tumor progression. During this period, tumor cells are genetically unstable and eventually escape and induce an appropriate immune response or inhibit the anti-tumor immune response by inducing the suppressive mechanisms mentioned above, i.e., blocking the anti-tumor response. Afterwards, the cells are recognized as normal.

[0019] In this suppressive mechanism, inflammatory cells and molecules play a key role. Adaptive immune responses are suppressed / blocked by activating multiple pathways that lead to inhibition of dendritic cell differentiation and activation (through the presence in tumor microenvironment factors such as IL10 and VEGF). Also, an increase in regulatory T cells (Treg) in peripheral blood and lymph nodes inhibits innate and adaptive responses. The presence of tumor suppressor cells in the microenvironment, such as MDSC (myeloid-derived suppressor cells) and TAM (tumor-associated macrophages or M2), influences tumor development with poor prognosis by secreting cytokines, growth factors, enzymes that degrade the extracellular matrix, and proteases (Non-Patent Document 7).

[0020] Although immunotherapy is safe, in some cancers its efficacy is moderate, in part due to the presence of immunosuppressive cells in the peripheral blood, lymphoid organs, and in the tumor environment that interfere with immunotherapy treatment. Several strategies have been implemented or are currently being tested to improve the efficacy of immunotherapy by acting on suppressive cells such as MDSCs, Tregs, and TAMs, which are increased in most cancer patients. It is becoming clear that these populations contribute to the impaired antitumor responses frequently observed in cancer patients.

[0021] Therefore, combating immune suppression by modulating these cell types is a key to increasing the efficacy of immunotherapy and should improve the prognosis of cancer patients.The present invention aims to alter the M1 / M2 balance of macrophage populations in favor of M1-type macrophages to provide a favorable immune environment for immunotherapy.

[0022] Macrophages and Cancer Macrophages are found in large numbers in tumors. Initially, this cell population was thought to be involved in antitumor responses, but many experimental and clinical studies have shown that macrophages are involved in tumor initiation and progression, as well as in the metastatic process. During the tumor process, macrophages secrete proinflammatory cytokines such as IFNγ, TNF-α, and IL6, and attract other immune cells that form chronic inflammation and cause initiation and tumor progression. However, upon tumor induction, tumor macrophages (TAMs) adopt an immunosuppressive cell profile and become less active, leading to tumor growth and malignancy. TAMs are involved in tumor cell migration, extravasation, and invasion (metastasis) and are involved in tumor angiogenesis (Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10).

[0023] Monocyte Ly6C reaches tumors + (CD14 + ) / CD11b high TAMs undergo phenotypic changes such as the reduction of Ly6C and CD11b markers and the expression of MHC class II (MHCII), VCAM and CD11c. However, the differentiation and distribution of TAMs depends on the anatomical localization of the tumor and its developmental stage. The definition "TAM function" was previously based on antitumor M1 macrophage type (iNOS-inducible) and M2 tumor-promoting ARG-positive macrophage type. This rather simplistic dichotomy must be placed in the context that highly plastic TAMs are in a cytokine and chemokine milieu that favors their suppressive function and allows the recruitment of Tregs involved in resistance to tumor cells (Non-Patent Document 11; Non-Patent Document 2).

[0024] The present invention relates to the inhibition of TAMs to reduce or prevent tumor processes, including the metastatic process.

[0025] CD47-SIRPa pathway Signal regulatory protein alpha, also called CD172a or SHPS-1 and referred to herein as "SIRPa", was first identified as a membrane protein present primarily in macrophages and myeloid cells that associates with the Src homology region 2 (SH2) domain--phosphatase containing--SHP-1 and SHP-2. SIRPa is the prototypic member of the SIRP-paired receptor family of closely related SIRP proteins. Engagement of CD47 with SIRPa results in a downregulatory signal that inhibits phagocytosis of host cells, thus CD47 functions as a "don't eat me" signal.

[0026] SIRPa is expressed on monocytes, which make up the majority of tissue macrophage subpopulations, granulocytes, a subset of dendritic cells (DCs) in tissues, several bone marrow progenitor cells, at various levels on neurons, and at particularly high levels within synapse-rich regions of the brain such as the granular layer of the cerebellum and hippocampus (Non-Patent Document 12; Non-Patent Document 13; Non-Patent Document 14).

[0027] SIRPa interaction with CD47 has been described primarily, which results in a downregulatory signal that inhibits host cell phagocytosis (see Non-Patent Document 15). Both CD47 and SIRPa are also involved in other interactions. Researchers have suggested that the lung surface proteins SP-A and SP-D regulate inflammatory responses in the lung through interactions with SIRPa (Non-Patent Document 16).

[0028] One of the best-characterized physiological functions of CD47-SIRPα interaction is its role in the homeostasis of hematopoietic cells, especially erythrocytes and platelets. Because CD47 acts as a "don't-eat-me" signal and is a key determinant of host cell phagocytosis by macrophages, the possibility that CD47-SIRPα interaction contributes to cancer cell elimination has been actively explored in recent years.

[0029] Currently, the SIRPA / CD47 pathway is also the subject of various pharmaceutical developments, all directed at enhancing macrophage phagocytosis. Indeed, like infected cells, cancer cells carry abnormal cargo, such as unknown proteins or abnormal levels of normal proteins, and these cells often subvert innate immune control mechanisms by simultaneously overexpressing immune regulatory molecules. It is gradually becoming clear that one such mechanism is CD47 (Non-Patent Document 17), a "self" protein expressed on normal cells. CD47 interacts with several different ligands, including SIRPa. This specific interaction is known to send a "don't eat me" signal to phagocytic macrophages, after which the target cell remains unaffected (Non-Patent Document 18). Overexpression of CD47 by cancer cells renders them resistant to macrophages, even when the cancer cells are coated with therapeutic antibodies (Non-Patent Document 19), and correlates with poor clinical outcomes in a number of solid and hematological cancers (Non-Patent Document 20; Non-Patent Document 21). In experimental models, particularly human tumor xenograft models in immunodeficient mice, blocking the CD47 / SIRPa pathway was highly effective in promoting tumor elimination by macrophages and reducing cancer cell spread and metastasis formation (Non-Patent Document 22; Non-Patent Document 23; Non-Patent Document 24; Non-Patent Document 25). In these studies, TAM function or phenotype was not investigated. It has been reported that blocking the CD47 / SIRPa pathway acts synergistically with depleting therapeutic anti-cancer antibodies such as trastuzumab (anti-Her2), cetuximab (anti-EGFR), rituximab (anti-CD20) and alemtuzumab (anti-CD52) by enhancing antibody-dependent phagocytosis by macrophages (Non-Patent Document 26).

[0030] From the above, it can be seen that SIRPa has been reported to be involved in the phagocytic function of myeloid cells, antigen presentation and cytokine secretion of dendritic cells, and trafficking of mature granulocytes. However, the function of SIRPa on macrophage polarization and their potent suppressive function during tumorigenesis have not been reported so far.

[0031] US Patent No. 5,999,663 discloses a method for treating hematological cancers, comprising modulating the interaction between human SIRPa and CD47. This document shows that blocking SIRPa-CD47 induces activation of the innate immune system via the phagocytic pathway. The transplantation model of human leukemia described in this patent application uses bone marrow cells, and the transplant was rejected when the animals were treated with a CD47 antagonist. The results suggest increased phagocytosis upon treatment with anti-CD47, but do not suggest any modification of the polarization state of macrophages or their pro-inflammatory function.

[0032] A method for inhibiting cell function for use in anti-inflammatory and anti-tumor therapy is described in US Pat. No. 5,399,633. The method comprises administering a drug comprising a substance that specifically recognizes the extracellular domain of SIRPa and inhibits the function of pathological myeloid cells. The inventors of this patent application claim that anti-SIRPa antibodies specific for the extracellular domain have properties that block inflammation and inhibit macrophage phagocytosis (referred to herein as the function of pathological myeloid cells). This effect is completely contradicted by the results disclosed below, which do not suggest any effect of anti-SIRPa antibodies on macrophage polarization or pro-inflammatory function.

[0033] CD47-Fc and CD47 extended fusobody molecules that bind to SIRPa have been studied and claimed in patent application WO 2005 / 023363. These molecules are alleged to be capable of inhibiting immune complex-stimulated cell cytokine release (e.g. release of IL-6, IL10, IL12p70, IL23, IL8 and TNF-α) from peripheral blood monocytes, DCs and / or monocyte-derived DCs stimulated with pansorbin or soluble CD40L, and IFNγ. The activity of these molecules is completely different from that of the anti-SIRPa antibodies disclosed in this application.

[0034] Patent document 4 describes the use of an antibody anti-human SIRPa (called 29AM4-5, corresponding to SIRP-29 named by the inventors) in a model of SIRPa-positive AML, i.e. the xenografting of primary human AML cells expressing human SIRPa bound by said antibody anti-human SIRPa into immunodeficient mice. This approach therefore consists in the use of an anti-human SIRPa antibody acting on tumor cells expressing human SIRPa. Thus, the treatment is directed at the tumor.

[0035] Alblas J. et al. (Non-Patent Document 27) observed that an anti-SIRPa antibody (ED9) was unable to induce the production of inflammatory cytokines, especially TNF-α and IL-6 (Figure 1A). However, the inventors demonstrated that, conversely, this particular antibody was able to repolarize M2 anti-inflammatory macrophages into M1 pro-inflammatory macrophages (Figure 14B in the experimental part). The difference in results could be explained by the fact that the authors of Alblas et al. used a macrophage cell line (rat NR8383 cell line) (which no longer expresses SIRPa), whereas the inventors used a fresh preparation of bone marrow-derived rat macrophages in the course of the experiment.

[0036] Patent Document 5 describes an anti-human SIRPa antibody that binds to human SIRPa and blocks the interaction between CD47 expressed on target cells and SIRPa expressed on phagocytes. This document does not suggest or express evidence of the effect of anti-SIRPa on macrophage polarization and increased pro-inflammatory function of macrophages. Interestingly, the inventors named in Patent Document 5 presented an abstract at the 56th ASH Annual Meeting (Non-Patent Document 28). In this abstract after the filing of Patent Document 5, they state that anti-human SIRPa antibodies that block the interaction between CD47 and SIRPa are not sufficient to induce human macrophage phagocytosis. Overall, these publications suggest that the mechanism of action of anti-human SIRPa in vitro is different from that of anti-human CD47, where the in vivo phagocytic effect has been extensively described in the literature.

[0037] From the above, it is clear that the various blocking SIRPa-CD47 interaction strategies, targeting CD47 or SIRPa by antibodies or fusion proteins, show distinct results and different efficiencies, indicating distinct roles for each target. Indeed, antibodies directed against the CD47 antigen block the interaction of CD47 with all its ligands. Using SIRPa-Fc proteins that bind to cells via their Fc and block the endogenous CD47 pathway, preventing its activation, does not allow blocking the activity of endogenous SIRa, as opposed to anti-SIRPa antibodies. The direct role of the SIRPa pathway in regulating the immune system has been underestimated so far compared to the CD47 pathway. None of the prior art studies have suggested or explained the function of the SIRPa pathway in macrophage polarization (either at the phenotypic or functional level), which plays a key role in tumor escape mechanisms.

[0038] In this context, the inventors provide new insights into the use of anti-SIRPa compounds, since modulation of the immune environment represents a major achievement in the treatment of many diseases, especially cancer. [Prior art documents] [Patent documents]

[0039] [Patent Document 1] International Publication No. 2010 / 130053 [Patent Document 2] WO00 / 66159 [Patent Document 3] International Publication No. 2012 / 172521 [Patent Document 4] International Publication No. 2013 / 056352 [Patent Document 5] International Publication No. 2015 / 138600 [Non-patent literature]

[0040] [Non-Patent Document 1] Mantovani, A., Biswas, SK, Galdiero, MR, Sica, A., and Locati, M. (2013). Macrophage plasticity and polarization in tissue repair and remodelling. J. Pathol. 229, 176–185. [Non-Patent Document 2] Wynn, T. A., Chawla, A., and Pollard, J. W. (2013). Macrophage biology in development, homeostasis and disease. Nature 496, 445-455. [Non-Patent Document 3] Gabrilovich, DI, Ostrand‐Rosenberg, S., and Bronte, V. (2012). Coordinated regulation of myeloid cells by tumours. Nat.Rev.Immunol.12, 253–268. [Non-Patent Document 4] Stein, M., Keshav, S., Harris, N., and Gordon, S. (1992). Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation. J.Exp.Med.176,287~292. [Non-Patent Document 5] Mittal, D., Gubin, MM, Schreiber, RD, and Smyth, MJ (2014). New insights into cancer immunoediting and its three component phases--elimination, equilibrium and escape. Curr. Opin. Immunol. 27, 16-25. [Non-Patent Document 6] Teng, M.W.L., Galon, J., Fridman, W.-H., and Smyth, M.J. (2015). From mice to humans: developments in cancer immunoediting. J. Clin. Invest. 125, 3338 - 3346.

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[0041] As described in the experimental part below, the inventors have now identified SIRPa as a new checkpoint inhibitor and demonstrated its role in macrophage polarization. The inventors have indeed shown that anti-SIRPa compounds as defined herein induce the pro-inflammatory function of macrophages associated with type 1 macrophages (M1 pro-inflammatory = M(IFNg)) and inhibit the suppressive activity of M2 macrophages in tumors, since the pro-inflammatory profile of macrophages is obtained at the expense of type 2 macrophages (M2 type high phagocytic activity = M(IL4)). This effect is obtained by targeting SIRPa, but not CD47, which is involved in phagocytic function. One advantage of the present invention is that the use of anti-SIRPa compounds can result in fewer side effects than the use of anti-CD47 compounds. Indeed, CD47 is expressed by a wide range of cells, not just tumor cells, and interacts with several ligands. The more restricted expression of SIRPa makes it easier to target the therapeutic effect to the tumor microenvironment. Thus, the use of anti-SIRPa compounds is less toxic and harmful than the use of anti-CD47 compounds. Furthermore, because the therapy is directed to macrophages and not to tumor cells, no selective pressure is placed on tumor cells, preventing tumor escape and the development of tumor resistance to the therapy. [Means for solving the problem]

[0042] The invention thus relates to the use of anti-SIRPa compounds, such as anti-SIRPa antibodies, capable of inhibiting the polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages, to alter macrophage polarization. The method of the invention thus consists in the use of anti-SIRPa compounds, which inhibit the polarization of M2-type macrophages and / or favor pro-inflammatory M1-type macrophages.

[0043] In certain embodiments, the anti-SIRPa compound may be selected from the group consisting of anti-SIRPa antibodies, in particular anti-SIRPa antagonist antibodies, nucleic acids encoding such compounds, and compounds capable of inhibiting the expression of SIRPa protein, in particular siRNAs.

[0044] Therefore, anti-SIRPa compounds, particularly anti-SIRPa specific antibodies, can be used to treat a variety of conditions that are likely to be ameliorated or prevented by proinflammatory macrophages, such as cancer, infectious diseases, trauma, autoimmune diseases, vaccinations, neurological diseases, brain and neurological disorders, polycythemia, hemochromatosis, and chronic inflammatory diseases. Cancer is a preferred therapeutic indication.

[0045] In a particular embodiment, the present invention relates to anti-SIRPa compounds capable of inhibiting the polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages, for use in the treatment of cancer other than SIRPa-positive acute myeloid leukemia and / or SIRPa-positive non-acute myeloid leukemia and / or SIRPa-positive non-Hodgkin's leukemia or SIRPa-positive hematological cancers.

[0046] An important aspect of the present invention is a therapeutic approach that aims to target SIRPa on macrophages in order to modulate polarization and recreate an immune environment detrimental to tumor development and survival. Essentially, successful antitumor therapy is based on an indirect pathway and does not require tumor cells to be sensitive to anti-SIRPa compounds. Thus, in a particular embodiment, the present invention relates to the use of anti-SIRPa compounds as defined herein, i.e. capable of inhibiting the polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages in the treatment of cancer, which compounds are administered to patients presenting with SIRPa-negative tumors.

[0047] The anti-SIRPa compounds defined herein can be used in monotherapy, as described in the experimental part.

[0048] Combinations of anti-SIRPa compounds as defined herein with other therapeutic agents, in particular agents that block other immune checkpoints, are also part of the present invention, since synergistic effects have been demonstrated by the inventors.

[0049] Another aspect of the present invention is a method for obtaining pro-inflammatory M1 type macrophages in vitro by incubating macrophages with an anti-SIRPa compound as defined herein.

[0050] The present invention also relates to a method for selecting a patient likely to benefit from treatment with an anti-SIRPa compound as defined herein, by determining the presence of M2-type macrophages in a sample obtained from the patient.

[0051] A method for monitoring treatment with an anti-SIRPa compound as defined herein to assess its effectiveness by measuring the presence of pro-inflammatory M1-type macrophages and / or anti-inflammatory M2-type macrophages in samples obtained from individuals treated with said compound is also part of the invention. [Brief description of the drawings]

[0052] [Figure 1] Monocyte type 1 polarization in the GM-CSF+M-CSF protocol: effect of SIRPa blockade. Human monocytes were cultured with growth factors M-CSF and GM-CSF to induce macrophages, and treated with Ctrl Ab or anti-Sirp antibodies (anti-a or ab or b isotypes), or CD47-Fc protein, or several anti-CD47 antibodies (B6H12 or CC2C6), or left untreated. Type 1 macrophage phenotype and function were analyzed by FACS and ELISA. A. Cell surface marker analysis: CD86 and CCR7. B. Secreted cytokines / chemokines: IL6, IL12p40, CCL-2 and TNF-α. IL6, p12p40 and CCL-2 were significantly increased when cells were treated with anti-SIRPa antibodies. [Diagram 2]Monocyte type 2 polarization in the GM-CSF+M-CSF protocol: Effect of SIRPa blockade. Human monocytes were cultured with growth factors M-CSF and GM-CSF to induce macrophages, and treated with Ctrl Ab or anti-Sirp antibodies (anti-a or ab or b isotypes), or CD47-Fc protein, or several anti-CD47 antibodies (B6H12 or CC2C6), or left untreated, and type 2 macrophage phenotypic and functional analysis was performed by FACS and ELISA. A. shows cell surface marker expression: CD206, CD200R, CD11b, B. shows secreted chemokine: CCL-17. [Diagram 3] Monocyte type 1 polarization with M-CSF and IFNγ protocol: Effect of SIRPa blockade. Human monocytes were cultured with M-CSF for 5-6 days, then with IFNγ and with or without Ctrl Ab or anti-Sirp antibodies (anti-a or ab or b Sirp isotypes) or CD47-Fc protein, or several anti-CD47 antibodies (B6H12 or CC2C6) for 2 days, and M1 secreted cytokines: IL6 and IL12p40 were analyzed and statistical analysis was performed. [Figure 4] Monocyte type 1 polarization with M-CSF+IFNγ and LPS protocol: Effect of SIRPa blockade on iNOS expression. Human monocytes were cultured with M-CSF for 5-6 days, then with IFNγ+LPS and with or without antibodies against SIRPa:SE7C2 (A.) or SIRPa / b:SE5A5 (B.) or CD47:B6H12 (C.) for 2 days, and iNOS expression was then analyzed by FACS. Ctrl Ab is represented by dotted lines and monocytes before polarization are represented by open grey lines in the three panels. [Diagram 5]Monocyte type 2 polarization in the M-CSF+IL4 protocol: effect of antibody-mediated SIRPa blockade. Human monocytes were cultured for 5-6 days with M-CSF and then with IL4 and with or without the addition of Ctrl Ab or anti-Sirp antibodies (anti-Sirp a or ab or b) or CD47-Fc fusion protein or several anti-CD47 antibodies (B6H12 or CC2C6). A. Expression of cell surface markers: CD206, CD200R and CD11b; B. Release of IL6 cytokine in the supernatant. [Figure 6] Monocyte type 2 polarization in M-CSF+IL4 protocol: Effect of SIRPa blockade at mRNA level by siRNA knockdown assay. Human monocytes were cultured with M-CSF for 5-6 days, then cultured with IL4 for 2 days and cells were transfected with null-siRNA or SIRPa siRNA: A. represents the expression of cell surface markers: CD206 and CD200R; B. represents IL6 cytokine release in the supernatant, anti-SIRPa SiRNA was obtained from ThermoFisher scientific. [Figure 7] Effect of SIRPa blockade on M2 repolarization after M-CSF+IL4 treatment. Human monocytes were cultured with M-CSF for 5-6 days and then with IL4 for 2 days, after which M2 macrophages were treated with Ctrl Ab or anti-Sirp antibodies (anti-Sirp a or ab or b), or CD47-Fc fusion protein, or several anti-CD47 antibodies (B6H12 or CC2C6), or left untreated, and then cytokine IL6 and TNF-α release was measured in the supernatants. [Figure 8] FIG. 1 shows the effect of anti-SIRPa + anti-CD137 combination treatment on an in vivo model of hepatocellular carcinoma. One week after tumor inoculation, animals were treated with 3G8 isotype control antibody (Iso control: black squares: n=33) or anti-SIRPa antibody (clone p84: grey squares; n=33) or anti-CD137 antibody (4-1BB mAb: black triangles; n=8) or combination treatment (anti-SIRPa + anti-CD137: grey diamonds; n=8) three times a week for 4 weeks, and then overall survival was analyzed. [Figure 9] Figure 1 shows the effect of anti-SIRPa + anti-PD-L1 combination treatment on an in vivo model of hepatocellular carcinoma. One week after tumor inoculation, animals were treated with 3G8 isotype control antibody (Iso control: black squares: n=5) or anti-SIRPa antibody (clone p84: grey squares; n=5) or anti-PD-L1 antibody (10F-9G2 mAb: black triangles; n=8) or combination treatment (anti-SIRPa + anti-PD-L1: grey diamonds; n=5) three times a week for 4 weeks, and then overall survival was analyzed. [Figure 10] Effect of anti-SIRPa + anti-PD-L1 combination treatment on an in vivo model of melanoma. Animals were treated with isotype control antibody (Iso control: stars; n=5), anti-SIRPa antibody (clone p84: squares; n=5), anti-PD-L1 antibody (10F-9G2 mAb: triangles; n=8) or combination treatment (anti-SIRPa + anti-PD-L1: circles; n=5) simultaneously with tumor inoculation. A. Results analyzed for overall survival. B. Results obtained by sacrificing some animals 2 weeks after the first inoculation to analyze macrophage infiltration using the CMH class II / CD11b marker. [Figure 11] FIG. 1 shows the effect of anti-SIRPA antibodies on mouse macrophage polarization. Mouse M2 macrophage polarization phenotype is inhibited by anti-SIRPa mAb but not by anti-CD47 mAb, as shown by quantification of the expression of M2 markers CD206, CD11b, and PD-L1; in this experiment, anti-SIRPa mAb corresponds to the p84 clone, and anti-CD47 mAb corresponds to the MIAP310 clone. [Figure 12] FIG. 1 shows the effect of anti-SIRPa antibody on human M2 macrophage polarization. The human M2 macrophage polarization phenotype is inhibited by anti-SIRPa mAb but not by anti-CD47 mAb. A. is represented by detection of M2 surface markers (CD200R, CD80), and B. is at the functional level, represented by assessment of cytokine secretion (IL-6). In this experiment, anti-SIRPa mAb corresponds to SE7C2 or SIRP29 clones, and anti-CD47 mAb corresponds to B6H12 clone. [Figure 13] FIG. 1 shows the effect of anti-SIRPa antibodies on human M1 phagocytosis. Human macrophage phagocytosis of CD47+ tumor cells (Raji) is increased by CD47 targeting agents, but not by SIRPa targeting agents. Analysis of phagocytosis was assessed by flow cytometry with gating on M1 fluorescent macrophages and analysis of target (Raji) cell fluorescence to M1 macrophages. In this experiment, CD47 targeting agents correspond to anti-CD47 mAb B6H12 or CC2C6 clone, and SIRPa targeting agents correspond to anti-SIRPa clone SE7C2 or CD47-Fc protein. [Figure 14] FIG. 1 shows the effect of anti-SIRPA antibody on rat M1 / M2 polarization. A. Rat M2 macrophages become pro-inflammatory and secrete inflammatory cytokines (IL-6 and TNF-α) in the presence of anti-SIRPa mAb but not anti-CD47 mAb. B. M1 rat macrophage polarization is not altered by anti-SIRPa or anti-CD47 mAb. The anti-SIRPa mAb used in this experiment corresponds to the ED9 clone, and the anti-CD47 mAb corresponds to the OX101 clone. [Figure 15] FIG. 1 shows the effect of anti-SIRPa antibodies on a mouse model of breast tumors. Anti-SIRPa mAb monotherapy inhibits tumor growth in the mouse 4T1 breast cancer model, and the anti-SIRPa mAb used in this experiment corresponds to clone p84. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The following definitions are used throughout this document:

[0054] Macrophage polarization The term "polarization" is used herein to designate the phenotypic and functional characteristics of macrophages. The phenotype can be defined via the surface markers they express. The functionality can be defined, for example, based on the nature and amount of chemokines and / or cytokines they express, and in particular secrete. Indeed, macrophages exhibit different phenotypic and functional characteristics depending on their status as either pro-inflammatory M1 type macrophages or anti-inflammatory M2 type macrophages. M2 type macrophages are characterized by the expression of surface markers such as CD206, CD11b, PD-L1 and CD200R, followed by the secretion of cytokines such as CCL17. M1 type macrophages can be defined by the expression of surface markers such as CD86 and CCR7, and the secretion of cytokines such as IL-6, TNF-α and IL12p40. In the context of the present invention, anti-SIPRa compounds allow for modulation of the polarization of macrophage populations by inhibiting M2 type macrophages and / or favoring M1 type macrophages.

[0055] Perturbation of macrophages by exogenous substances encompasses the commonly used meaning of the term "activation." Macrophages change their polarization state in response to external stimuli such as growth factors (CSF-1 and GM-CSF), microorganisms, microbial products and nucleotide derivatives, antibody-Fc receptor stimulation, glucocorticoids and phagocytosis.

[0056] anti-SIRPa compounds As used herein, "anti-SIRPa compound" refers to a compound that specifically binds to the extracellular domain of SIRPa or to a nucleic acid encoding such a compound, as well as a compound that can inhibit the expression of SIRPa protein. Such compounds can inhibit the polarization of anti-inflammatory M2-type macrophages and / or favor pro-inflammatory M1-type macrophages.

[0057] The anti-SIRPa compound can be a compound that specifically binds to the extracellular domain of signal regulatory protein alpha (SIRPa). The anti-SIRPa compound can also be a SIRPa blocking compound. Preferably, the anti-SIRPa compound is an antagonist peptide or an anti-SIRPa antibody, in particular an anti-SIRPa antagonist antibody.

[0058] As used herein, an antibody refers to a polyclonal antibody, a monoclonal antibody or a recombinant antibody. The monoclonal antibody of the present invention includes recombinant antibodies, such as chimeric antibodies, CDR-grafted antibodies and humanized antibodies, but also includes antigen-binding portions. As used herein, the term "antigen-binding portion" of an antibody (or simply "antibody portion") refers to one or more fragments of an antibody of the present invention, where the fragment or fragments still have the binding affinity defined above. According to the term "antigen-binding portion" of an antibody, examples of binding fragments include Fab fragments, F(ab')2 fragments, Fv fragments and single chain Fv (ScFv). Other forms of single chain antibodies, such as "bispecific antibodies", are also included here.

[0059] As used herein, an antagonist peptide refers to a peptide capable of inhibiting the interaction of SIRPa with one of its ligands, in particular CD47, or capable of blocking or reducing the SIRPa signaling pathway.

[0060] Anti-SIRPa compounds can also be compounds that can inhibit the expression of SIRPa protein, such as antisense oligonucleotides, or interfering RNA, such as shRNA or siRNA. Examples of siRNAs that can regulate macrophage polarization according to the present invention are provided in the experimental part. Moreover, methods for identifying such compounds are known to those skilled in the art.

[0061] In a particular embodiment, anti-SIRPa capable of inhibiting the polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages can be identified by applying one of the protocols described in the experimental part below.

[0062] For example, identification of an anti-SIRPa X compound can be performed as follows:

[0063] Monocytes are cultured with M-CSF (100 μg / mL) for 5-6 days to obtain M0 macrophages, and the cells are then cultured in vitro for 2 days with recombinant hIL4 (20 ng / mL) in the presence or absence of compound X. As a positive control for anti-SIRPa activity, an anti-SIRPa mAb with known activity can be used. Any compound that does not target the extracellular domain of SIRPa can be used as a negative control (e.g., anti-SIRPb antibody or CD47-Fc). Secretion of various cytokines, such as IL-6, TNF-α and IL12, as well as the chemokine CCL17, can be measured by ELISA.

[0064] Compound X can be classified as an "anti-SIRPa compound" if it (i) inhibits indicative of M2 macrophage characteristics, in particular overexpression of surface markers such as CD206, CD11b, PD-L1 and / or CD200R and secretion of certain cytokines such as CCL17, and / or (ii) increases indicative of M1 macrophage characteristics, in particular expression of surface markers such as CD86 and CCR7 and secretion of cytokines such as IL-6, TNF-α, IL12-p40 and / or IL12, as efficiently as or more efficiently than a positive control mAb.

[0065] In certain embodiments, the efficacy of the anti-SIRPa compound can be evaluated in comparison with the efficacy of a specific antibody SE7C2 (Santa Cruz sc-23863) used at a concentration of 10 μg / mL. If the results of the test show that it is as effective as SE7C2 or more effective than SE7C2, compound X is classified as an "anti-SIRPa compound."

[0066] Based on this protocol, a comparison can be made with any standard anti-SIRPa compound as a positive control.

[0067] Cancer, treatment, etc. As used herein, "cancer" refers to all types of cancer. In particular, cancer may be a solid or non-solid cancer. Non-limiting examples of cancer are carcinomas or adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, liver cancer, lung cancer, bladder cancer, pancreatic cancer or colon cancer, sarcoma, lymphoma, melanoma, leukemia, germ cell carcinoma and blastoma.

[0068] As used herein, the terms "treat", "treatment" and "treating" refer to the reduction or amelioration of the progression, severity and / or duration of cancer, particularly solid tumors; for example, in liver cancer, the alleviation of one or more symptoms thereof resulting from the administration of one or more therapies.

[0069] Treatment As used herein, a "therapeutic procedure" refers to any effective force or substance capable of producing an effect. Thus, therapeutic procedures include radiation, surgery, probiotics, as well as drugs of any kind.

[0070] Immune checkpoint blockers and stimulators In this document, "drugs that block immune checkpoints" or "immune checkpoint blockade agents" or "immune checkpoint blocking drugs" or "immune checkpoint inhibitors" refer to any drug, molecule or composition that blocks immune checkpoints. In particular, it encompasses anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies and anti-SIRPa antibodies. Furthermore, two or more immune checkpoint inhibitors can be used in combination to target both adaptive and innate immune cells. This approach is of particular interest in the treatment of cancer. For this purpose, anti-SIRPa compounds can be used in combination with anti-PD-L1 compounds. Such combinations can be used simultaneously, individually or sequentially, particularly in the treatment of cancer.

[0071] In contrast, an "immune checkpoint stimulator" refers to any drug, molecule, or composition that activates an immune checkpoint. An example of such a molecule or drug is an anti-CD137 antibody that stimulates costimulatory molecules leading to target cell activation.

[0072] Where necessary, other definitions are specified below.

[0073] A first aspect of the invention is the use of anti-SIRPa compounds capable of modifying macrophage polarization, in particular inhibiting the polarization of anti-inflammatory M2 macrophages and / or favoring pro-inflammatory M1 macrophages, in an individual (e.g., a human) in need thereof. As described above, macrophages mature in tissues and are activated in dynamic response to combinations of stimuli, acquiring specialized functional phenotypes, in certain cases detrimental to the individual. For lymphoid systems, a binary classification of macrophage activation has been proposed: classical (M1) vs. alternative (M2). Although several intermediate functional states have been observed, M1 and M2 subtypes remain suitable to describe the extreme forms of a continuum of macrophage states, with M1 being the most pro-inflammatory state and M2 being more associated with reduced inflammation.

[0074] By "modifying macrophage polarization" it is meant herein that the compound modifies the balance between different subtypes of macrophages in the treated individual, at least at the phenotypic and / or functional level. Thus, according to the present invention, treatment of an individual with an anti-SIRPa compound modifies the profile of surface markers expressed by the individual's macrophages, including a decrease in CD206, CD11b, PD-L1 and / or CD200R expression, and an increase in CD86 and CCR7 expression, as well as the profile of chemokines and cytokines expressed and / or secreted by the individual's macrophages, including a decrease in CCL-17 expression, and an increase in IL6, TNF-α and IL12p40 expression.

[0075] According to certain embodiments of the present invention, the modification of macrophage polarization by anti-SIRPa compounds includes the inhibition of M2-type macrophage polarization and / or the increase of pro-inflammatory M1-type macrophage polarization. In particular, it may include the inhibition of M2 phenotype polarization of macrophages, which reduces the proportion of macrophages that overexpress cell markers such as CD206, CD11b, PD-L1 and / or CD200R and / or produce cytokines such as CCL17. At the same time, anti-SIRPa compounds can induce the appearance of more macrophages that show M1 phenotype polarization, as well as an increase in the proportion of macrophages that overexpress cell markers such as CD86 and CCR7 and / or produce cytokines such as IL-6, IL-12p40 and / or TNF-α. Thus, anti-SIRPa compounds can modulate macrophage polarization at both the phenotypic level (expression of cell surface markers) and the functional level (production of chemokines and cytokines).

[0076] According to a preferred embodiment of the present invention, the compound used to modify macrophage polarization is an anti-SIRPa compound. Exemplary tests for determining whether a given compound is an anti-SIRPa compound within the meaning of the present application are described above.

[0077] Compounds that can be used in accordance with the present invention include small chemical molecules, polypeptides, antagonist peptides, antibodies and fragments thereof, in particular anti-SIRP antibodies as used in the experiments described below, any other antibodies selected from the large number of anti-SIRPa commercially available antibodies, or any other (novel) anti-SIRPa antagonist antibodies, antibody fragments, aptamers targeting SIRPa, etc.

[0078] The present invention also relates to the use of nucleic acids (mRNA or DNA) encoding anti-SIRPa compounds to modify macrophage polarization in favor of M1 pro-inflammatory macrophages in individuals in need thereof. Indeed, administering a nucleic acid that results in the expression of an anti-SIRPa compound by the patient's cells as described above is one option to obtain the desired effect on macrophage polarization. The skilled artisan is free to choose any expression cassette with any regulatory element and any vector (lipid vectors such as polymers, cations and / or liposomes, or viral vectors such as adenoviruses, lentiviruses, adeno-associated viruses (aav)) to express the anti-SIRPa compound at an appropriate level in an appropriate number of patient cells. In the following description of the detailed embodiments of the present invention, it will not be repeated that a nucleic acid that allows the expression of anti-SIRPa can be used instead of the anti-SIRPa compound itself. In this specification, the term "anti-SIRPa compound" is considered to encompass a nucleic acid that allows the in vivo expression of such a compound.

[0079] In particular, the anti-SIRPa compound is selected from the group consisting of antagonist peptides, anti-SIRPa antibodies, in particular anti-SIRPa antagonist antibodies, nucleic acids encoding such compounds, compounds capable of inhibiting the expression of SIRPa protein, in particular siRNAs. Preferably, the anti-SIRPa compound is an antagonist peptide or an anti-SIRPa antibody, in particular an anti-SIRPa antagonist antibody.

[0080] Modification of macrophage polarization in favor of M1 proinflammatory cells may be useful in many disease states or situations. As described above, this modification is particularly useful in the context of cancer to restore the antitumor activity of macrophages and / or block the tumor-promoting activity of M2 macrophages. Indeed, immune responses due to excessive M2 macrophage polarization also occur in infections, vaccinations, trauma, and chronic inflammatory diseases.

[0081] Macrophages are thought to interact with stem or progenitor cells to regulate repair and remodeling functions. Cells derived from the macrophage lineage exhibit several neuroprotective effects. Mesenchymal cells (MSCs) are targeted to promote tissue repair and immune regulation. Injection of MSCs was associated with benefits for functional recovery of spinal cord injury, such as axonal preservation and reduced fear formation. The neuroprotective effect was attributed to a shift in polarization from M2 to M1 macrophages by MSCs (Nakajima et al., 2012), indicating that any molecule that enables this shift, such as anti-SIRPa compounds, may exert a neuroprotective effect.

[0082] Macrophages are also involved in some iron deficiency disorders, such as hemochromatosis, where iron homeostasis is clearly impaired. Polycythemia vera or primary erythrocytosis is a myeloproliferative disorder characterized by erythrocytosis (a large increase in the number of red blood cells) and an increase in total cell mass. The red blood cells then enter the blood and may progress to myeloproliferative syndrome. Patients treated with anti-CD47 exhibit anemia, indicating that blocking CD47 in the context of the present invention is not as safe as expected; targeting SIRPa via anti-SIRPa compounds can avoid this side effect.

[0083] Thus, the present invention relates to the use of anti-SIRPa compounds to alter macrophage polarization in favor of M1 pro-inflammatory macrophages in individuals suffering from cancer, infectious diseases, trauma, autoimmune diseases, neurological diseases brain injury, neurological injury, polycythemia, hemochromatosis or chronic inflammatory diseases, as well as in the setting of vaccination of individuals.

[0084] According to certain embodiments, the anti-SIRPa compound is used to treat an individual with a cancer selected from the group consisting of lung cancer, ovarian cancer, liver cancer, bladder cancer, brain cancer, breast cancer, colon cancer, thymoma, glioma, melanoma and hematological cancer.

[0085] In a particular embodiment, the anti-SIRPa compound is used to treat any cancer except SIRPa-positive acute myeloid leukemia (AML). Thus, in fact, the treatment of tumor cells involved in AML expressing SIRPa is performed by targeting the tumor cells, i.e. by directly targeting the tumor. This treatment strategy is therefore different from the indirect approach proposed in the present invention. In a more particular embodiment, the anti-SIRPa compound is used to treat any cancer except SIRPa-positive acute myeloid leukemia and SIRPa-positive non-acute myeloid leukemia. In another particular embodiment, the anti-SIRPa compound is used to treat any cancer except SIRPa-positive non-Hodgkin's lymphoma or non-Hodgkin's lymphoma. In a further particular embodiment, the anti-SIRPa compound is used to treat any cancer except SIRPa-positive acute myeloid leukemia and / or SIRa-positive non-acute myeloid leukemia and / or non-Hodgkin's lymphoma or hematological cancer. In a further embodiment, the anti-SIRPa compound is used to treat any cancer except i) SIRPa positive acute myeloid leukemia or acute myeloid leukemia, and / or ii) SIRPa positive non-acute myeloid leukemia or non-acute myeloid leukemia, and / or iii) SIRPa positive non-Hodgkin's lymphoma or non-Hodgkin's lymphoma, or iv) SIRPa positive hematological cancer or hematological cancer. In a further embodiment, the anti-SIRPa compound is used to treat a cancer with SIRPa negative tumor cells, as described herein below.

[0086] In another particular embodiment, the anti-SIRPa compound is used as a monotherapy, particularly in the treatment of breast cancer, hepatocellular carcinoma or melanoma.

[0087] Regulatory macrophage polarization is a very attractive approach for the treatment of cancer, especially for combination cancer therapy. Antonia et al. (Antonia et al., 2014) defined immuno-oncological combinations that could be interesting for cancer treatment using checkpoint inhibitor approaches. Immuno-oncology is an evolving therapeutic approach that includes immunotherapies designed to harness the patient's own immune system.

[0088] In this context, in the context of the present invention, anti-SIRPα compounds are an important complement to several other possible strategies to overcome tumor immune evasion mechanisms with drugs in clinical development or already on the market: 1 - Abrogation of inhibition of adaptive immunity (blocking T cell checkpoint pathways), for example by using anti-CTLA4, anti-PD1 or PD-L1 molecules; 2-Switching adaptive immunity (enhancing T cell costimulatory receptor signaling with agonistic antibodies), for example by using anti-CD137 molecules; 3-Improvement of innate immune cell function; 4- Activation of the immune system (enhancing immune cell effector functions), e.g. through vaccine-based strategies It can be used in combination with (see Table 1 in (Antonia et al., 2014)).

[0089] Recently, Zitvogel et al. have emphasized the importance of gut microbes in optimal therapeutic immune modulation (Viaud et al., 2014; WO2015 / 075688). In the framework of the present invention, anti-SIRPa compounds can also be used in combination with microbial modulation strategies to improve anti-cancer immune responses.

[0090] Therefore, another aspect of the present invention is the use of an anti-SIRPα compound as defined above in combination with a second therapeutic agent to treat an individual in need thereof, particularly a cancer patient. Such combinations can be used simultaneously, separately or sequentially, particularly in the treatment of cancer.

[0091] According to a preferred embodiment of this aspect of the invention, the second therapeutic agent is selected from the group consisting of chemotherapy, radiotherapy, surgery, immunotherapy, antibiotics and probiotics. In particular, the second therapeutic agent can be advantageously selected from the group consisting of therapeutic vaccines, immune checkpoint blockers, such as anti-PDL1, anti-PD1 and anti-CTLA4, and immune checkpoint activators, such as anti-CD137. As illustrated in the experimental part below, these combinations produce synergistic effects. In particular, one aspect of the invention is the use of an anti-SIRPa compound in combination with a second immune checkpoint modulator in the treatment of cancer selected from hepatocellular carcinoma or melanoma. In a preferred embodiment, an anti-SIRPa mAb is combined with an anti-CD137 mAb in the treatment of hepatocellular carcinoma. In another aspect of the invention, an anti-SIRPa mAb is combined with an anti-PD-L1 mAb in the treatment of melanoma.

[0092] Another aspect of the present invention is a method for obtaining proinflammatory M1-type macrophages ex vivo, comprising incubating macrophages with an anti-SIRPa compound as described above. This method may be particularly useful in cancer patients, for example in cell therapy. This document also reports a method for treating cancer patients, comprising obtaining macrophages from said patients, then modifying their polarization in favor of M1-related proinflammatory functions by incubation with an anti-SIRPa compound, and re-administering the obtained proinflammatory cells to the patient. Of course, such methods can include additional steps, such as expanding the cells and selecting for cells exhibiting an M1-type phenotype and / or counterselecting for cells exhibiting an M2-type phenotype.

[0093] According to another of its aspects, the present invention relates to a method for determining in vivo the efficacy of a treatment with an anti-SIRPa compound as defined above, comprising the step of determining the presence of pro-inflammatory M1 type macrophages and / or the presence of M2 type macrophages in a sample from an individual treated with said compound. When carrying out said method, the presence of pro-inflammatory macrophages can be determined for example by measuring the levels of IL6, TNF-α and / or IL12p40 secreted by macrophages present in the sample. The presence of M2 type macrophages can be determined for example by measuring the expression of CD206, CD11b, PD-L1 and / or CD200R on the macrophage surface.

[0094] In order to avoid unnecessary treatment with anti-SIRPa compounds, it is of paramount importance to accurately identify patients who are likely to benefit from such treatment, i.e., patients for whom this treatment is effective. Patients who exhibit high levels of M2-type macrophages, especially cancer patients with abundant tumor-infiltrating M2-type macrophages, are the patients most likely to respond to treatment with anti-SIRPa according to the present invention.

[0095] One aspect of the present invention is to propose the use of anti-SIRPa compounds, alone or in combination, to treat patients presenting with SIRPa-negative tumors, i.e., patients whose tumor cells do not express SIRPa. This therapeutic approach was not previously envisaged, as the prior art teaches that the treatment of cancer relies on the inhibition of SIRPa-CD47 interaction at the tumor cell level (in particular by inducing phagocytosis). In contrast, the inventors have demonstrated that anti-SIRPa mAbs do not induce phagocytosis, but act on a separate pathway. They have demonstrated that anti-SIRPa compounds target M2-type macrophages and repolarize this macrophage population to M1-type macrophages. Thus, the proposed invention also addresses a "new clinical situation" not previously reported. As a further differentiation between the claimed invention and the teachings of the prior art, the anti-SIRPα compounds of the present invention treat cancer by an indirect approach targeting the innate immune system, in particular by blocking the inhibition of inflammation in the tumor environment, more specifically by inhibiting anti-inflammatory M2-type macrophages.

[0096] Thus, the object of the present invention is the use of anti-SIRPa compounds in cancer treatment, in which the compounds are administered to patients presenting with SIRPa-negative tumors.As used herein, "SIRPa-negative tumors" correspond to tumors that contain a SIRPa-negative cell population.However, taking into account the heterogeneity of tumors, this term encompasses both tumors that are composed of SIRPa-negative cells and tumors that may contain a mixed population of SIRPa-positive tumor cells and SIRPa-negative tumor cells.In certain embodiments, the present invention also relates to the use of the above compounds to treat cancers in which the patient's tumor contains a mixed population of tumor cells that includes both SIRPa-positive and SIRPa-negative cells.

[0097] The present invention also provides a method for treating cancer comprising administering to a patient in need thereof an anti-SIRPa compound capable of inhibiting polarization of anti-inflammatory M2-type macrophages and / or favoring pro-inflammatory M1-type macrophages.

[0098] In certain embodiments, the present invention also provides a method for the treatment of cancer. at least one anti-SIRPa compound capable of inhibiting the polarization of anti-inflammatory M2 macrophages and / or favoring pro-inflammatory M1 macrophages, in particular said anti-SIRPa compound being selected from the group of antagonist peptides, anti-SIRPa antibodies, in particular anti-SIRPa antagonist antibodies, nucleic acids encoding such compounds and compounds capable of inhibiting the expression of the SIRPa protein, in particular siRNA; and - a second therapeutic agent which is another immune checkpoint compound, in particular selected from the group consisting of anti-PDL1, anti-PD1, anti-CTLA4 and anti-CD137; In a particular embodiment, the hybrid product is used to treat cancer other than SIRPa positive acute myeloid leukemia.

[0099] The preferred embodiments are as defined above.

[0100] The present invention therefore also relates to a method for determining in vivo whether an individual is likely to be a good responder to a treatment with an anti-SIRPa compound within the meaning of the present invention, said method comprising the step of determining the presence of M2-type macrophages in a sample from said individual, e.g. by measuring the expression of CD206, CD11b, PD-L1 and / or CD200R on the surface of macrophages present in the sample.

[0101] When carrying out the above methods, the sample used to assess whether an individual is likely to be a good responder to treatment with an anti-SIRPa compound or to determine the effectiveness of such treatment can be a blood sample, a tissue sample, a tumor-derived sample, or a synovial fluid sample. EXAMPLES

[0102] Throughout the experimental part disclosed below, the terminology used follows the scientific community studying macrophages (Murray et al., 2014). The experimental results were obtained using the materials and methods shown below.

[0103] Human blood mononuclear cells isolation PBMCs (peripheral blood mononuclear cells) were isolated and purified using centrifugal countercurrent washing (Clinical Transfer Facility CICBT0503, Dr. M. Gregoire, Nantes) according to the protocol used and reported by Coulais et al. (Coulais et al., 2012).

[0104] Blocking antibodies All blocking molecules tested in the experiments were used at 10 μg / mL. SIRPa mAbs: SE7C2 (23863, Santa Cruz) or clone p84 (Merck Millipore); Sirp α / β mAbs: SE5A5 (BLE323802, BioLegend); anti-CD47: B6H12 (eBioscience 14-0479-82) or CC2C6 (BLE323102, BioLegend); CD47-Fc: SinoBiological 12283-H02H; anti-CD137 antibody (in-house clone 3H3) and anti-PD-L1 antibody (BioXCell 10F-9G2).

[0105] In vitro M-CSF+ (IFNg or IL4) macrophage polarization assay (M1 or M2 type) Monocytes were cultured in 24-well plates in complete RPMI at 4.10 5Cells / well were cultured in M-CSF (100 μg / mL - R&D Systems) for 5–6 days to induce M0 macrophages using conventional differentiation methods (Zajac et al., 2013). M2 polarization was induced with rhIL-4 (20 ng / mL - CellGenix) to generate M2 anti-inflammatory macrophages or with rhIFNg + / - LPS (20 ng / mL - R&D Systems; 100 ng / mL - Sigma-Aldrich, respectively) to generate M1 pro-inflammatory macrophages over 2 days, after which cells were harvested and analyzed by flow cytometry using antibodies from BD Bioscience.

[0106] In vitro CSF+GM-CSF macrophage polarization assay (M1 and M2 types) M1 / M2 differentiation in the same well was performed at 4.10 °C in complete RPMI containing GM-CSF (10 ng / mL, CellGenix). 5 This was performed by culturing monocytes at 10 cells / well in 24-well plates for 3 days and then culturing with GM-CSF and M-CSF (2 ng / ml and 10 ng / ml, respectively, R&D systems) for an additional 3 days (Haegel et al., 2013). Antibodies were added on days 0 and 3. On day 6, cells were harvested and analyzed by flow cytometry using antibodies from BD Bioscience. Secreted cytokines were titrated by ELISA (BD Bioscience and R&D systems).

[0107] Macrophage phenotyping by flow cytometry In vitro mouse macrophage differentiation was performed using complete RPMI supplemented with mouse M‐CSF (100 μg / mL, PeproTech) at 0, 5.10 6This was performed by culturing bone marrow-derived cells at 1000 cells / mL in 24-well plates for 4 days to induce M0 macrophages. M2 polarization was induced with mouse IL-4 (20 ng / mL - PeproTech) for 24 hours to generate M2 anti-inflammatory macrophages. Analysis of phenotypic changes was performed by flow cytometry staining with antibodies from BD Bioscience. Macrophage expression markers were analyzed by flow cytometry using the fluorescent dyes shown in the table below.

[0108] [Table 1]

[0109] iNOS measurement using multiple nanoflares iNOS expression was revealed by SmartFlare™ technology (Prigodich et al., 2012). Briefly, monocytes or macrophages were harvested, incubated with iNOS probe (final concentration 1 nM) for 2 h at 4°C, washed, and analyzed on an LSR II (BD).

[0110] siRNA experiments Macrophages (M0 macrophages pre-polarized with M-CSF) were transfected with siRNAs encoding endogenous SIRPa. Three sequences of siRNAs (reference numbers: 112328, 112327 and 109944, ThermoFisher Scientific) were selected and pooled to downregulate SIRPa expression. In a 24-well plate, 90 pmol of siRNA-SIRPa (3 x 30 pmol of each siRNA-SIRPa) was diluted in 100 μl Opti-MEM medium without serum and mixed gently. Then, 1 μl / well of Lipofectamine RNAiMAX (reference number 13778-150, ThermoFisher Scientific) was mixed and incubated at room temperature for 20 min. M2 were seeded at 100,000 cells / ml in 500 μl of complete growth medium containing IL4 (20 ng / ml) + / - 100 μl of siRNA-Lipofectamine complex. Null-siRNA was used as a transfection control. Cells were incubated at 37°C, 5% CO2 for 48 hours.

[0111] Functional assay by Elisa Cytokines and chemokines released in the supernatants were analyzed by Elisa using materials from BD Biosciences and R&D systems (reference numbers below). Supernatants were diluted 1 / 200.

[0112] [Table 2]

[0113] As isotype control antibodies, PK136 anti-NK1.1 (mouse mAb IgG2a) and SF1-1.1 anti-H2Kd (mouse mAb IgG1) were used.

[0114] Assessment of phagocytic activity of human macrophages To address the ability of anti-SIRPa antibodies to induce phagocytosis, human M1 proinflammatory macrophages were generated and stained with a fluorescent dye as described previously. CD47-expressing Raji cells were stained with another fluorescent dye and incubated with M1 stained macrophages for 2 h at 37°C. Cells were fixed with paraformaldehyde and analysis of phagocytosis was assessed by flow cytometry by gating on M1 fluorescent macrophages and analysis of target (Raji) cell fluorescence to M1 macrophages.

[0115] In vivo mouse hepatocellular carcinoma model Eight-week-old C57Bl / 6J male mice were inoculated with 2.5 × 10 IgG in 100 μL via the portal vein as described above. 6 Hepa1.6 mouse hepatoma cells were inoculated into the mice (Gauttier et al., 2014). Four and eight days after tumor inoculation, the mice were intraperitoneally injected with 100 μg of rat anti-4-1BB mAb (in-house clone 3H3), or 300 μg of anti-mouse SIRPa monoclonal antibody (Merck Millipore clone P84), or both, or an irrelevant control antibody (clone 3G8), three times a week for 4 weeks, or with 200 μg of anti-PD-L1 mAb (BioXCell clone 10F-9G2), or both antibodies (anti-Sirpa + anti-PDL1) for 4 weeks.

[0116] In vivo mouse melanoma model 2 × 10 6 B16-Ova mouse melanoma cells were subcutaneously injected. Starting on day 0 after tumor inoculation, mice were intraperitoneally treated with either 300 μg of an irrelevant control antibody (clone 3G8) or anti-mouse SIRPa monoclonal antibody (clone P84) three times a week, or 200 μg of anti-PD-L1 mAb (BioXCell clone 10F-9G2) twice a week, or both antibodies (anti-Sirpa and anti-PD-L1 antibodies) for 4 weeks. Two weeks after tumor inoculation, some animals were sacrificed and tumor leukocyte infiltration was characterized by flow cytometry. Overall survival was analyzed.

[0117] In vivo mouse breast cancer model 0, 25 × 10 6 50 μL of 4T1 (breast cancer) cells were injected into the mice. Starting 4 days after tumor inoculation, mice were intraperitoneally administered 300 μg of an irrelevant control antibody (clone 3G8) or an anti-mouse SIRPa blocking antibody (clone P84) three times a week for 4 weeks. Mice were euthanized 6 weeks after tumor inoculation. Tumors were measured every 2-3 days and tumor volumes were calculated: length × width × Pi / 6 (mm 3 ) was calculated according to

[0118] Example 1: In vitro study of macrophage polarization (M1 and M2) and blockade of the SIRPa pathway 1.1. Selective blockade of Sirpα inhibits type 2 (M2) but not type 1 (M1) polarization of human macrophages Figure 1A shows that antibodies against SIRP molecules or CD47 did not block GM-CSF+M-CSF-induced M1 macrophage polarization, since the expression of M1 cell surface markers (CD86 and CCR7) was not altered compared to control conditions. In contrast, overexpression of CD206, CD200R, CD11b and PD-L1 (indicative of an M2 macrophage phenotype) was significantly inhibited by selective anti-SIRPα mAbs (Figures 2A and 11), but not by control antibodies (anti-SIRPa / b or Sirpb), CD47-Fc recombinant protein or anti-CD47 mAbs (clones B6H12 and CC2C6). In particular, Figure 11 shows that blockade of SIRPa with monoclonal antibodies prevented the acquisition of the M2 macrophage phenotype induced by IL-4, and in fact did not increase the expression of M2 markers (CD206, CD11b, and PD-L1) whereas an increase was observed in the isotype control condition. This blockade of the anti-inflammatory state of macrophages was not observed when the cognate ligand of SIRPa (CD47) was blocked with monoclonal antibodies.

[0119] Measurement of cytokine and chemokine secretion showed that anti-SIRPa mAb blocked CCL-17 secretion (a chemokine indicative of M2 secretion) while increasing proinflammatory cytokines (IL-6, IL12p40, TNF-α) and chemokine (CCL-2) secreted by M1 macrophages (Figs. 1B and 2B). Thus, anti-SIRPa may play a role in blocking M2 polarization and in the proinflammatory function of macrophages. When monocytes were differentiated exclusively into M2 (but not M1) macrophages according to the M-CSF+IL-4 protocol, selective inhibition of M2 macrophage polarization by anti-SIRa mAb alone was confirmed, whereas M1 polarization was not altered when monocytes were treated with M-CSF+IFNγ (data not shown). Again, only SIRPa mAb (but not other anti-SIRP molecule mAbs or anti-CD47 mAb) blocked the overexpression of M2 (CD206 and CD200R) specific surface markers and concomitantly increased IL-6 proinflammatory cytokine secretion (Figure 5B). We next investigated the role of SIRPa on M2 polarization at the transcriptional level with SIRPa inhibition using siRNA cocktails. The results shown in Figure 6A confirmed that inhibition of SIRPa inhibited M2 polarization (CD206 and CD200R expression) of cells.

[0120] Taken together, these results demonstrate that blocking the SIPRa-CD47 interaction with anti-SIRPa but not anti-CD47 antibodies can inhibit M2 macrophages. Furthermore, inhibition of the M2 phenotype is observed at both the phenotypic level (inhibition of expression of M2-specific surface markers) and the functional level (inhibition of M2-specific cytokine secretion).

[0121] 1.2. Selective blockade of SIRPa increases secretion of pro-inflammatory factors, characteristic of M1 macrophages We observed that selective anti-SIRPa mAbs increased inflammatory cytokine secretion (IL6, IL12p40, CCL2 and TNFα) under M1+M2 polarization with GM-CSF+M-CSF (Figure 1B). This property was confirmed in a conventional M1 (only) macrophage polarization assay induced by M-CSF+IFNg+ / -LPS (Figure 3), showing an increase in IL6 and IL12p40 in the supernatant. Maximal / high M1 polarization was achieved with M-CSF+IFNg+LPS, resulting in all indices of M1 macrophages, especially iNOS expression. Figure 4 shows the expression of iNOS in M1 polarized cells treated or not with various blocking antibodies. As shown in this figure, blocking SIRPa with antibodies increased the expression of iNOS. However, anti-SIRPβ or anti-CD47 mAbs did not induce any alterations in the iNOS expression profile. These results confirmed that blockade of SIRPa altered macrophage function in pro-inflammatory conditions, including M1 type, although M1 surface markers tested were not altered compared to controls.

[0122] 1.3. Selective blockade of SIRPa repolarizes human M2 macrophages in inflammatory cells The phenotype and function of polarized M1 / M2 macrophages are to some extent constrained in vitro or in vivo due to the plasticity of these cells (Sica and Mantovani, 2012). We addressed the issue of M2 repolarization to the pro-inflammatory M1 type by blocking SIRPa. To do so, we cultured monocytes with M-CSF+IL-4, which induces M2 hyperpolarization. Then, we treated M2 cells with different blocking antibodies. The results shown in Figure 7 indicate that anti-SIRPa mAb can repolarize M2 macrophages to M1 pro-inflammatory macrophages, since IL6 and TNF-α cytokines (indicative cytokines of M1 macrophages) were induced. This effect was not observed when cells were treated with anti-Sirpβ, CD47-Fc or anti-CD47 antibodies. As explained above, cell surface markers were not modified. Similarly, the results in FIG. 12 show that two different anti-SIRPa mAbs make it possible to induce macrophage polarization in favor of M2 macrophages both at the phenotypic level with the expression of surface markers CD200R and CD80 (A) and at the functional level with the secretion of IL-6 (B). Furthermore, the results in FIG. 14 show that selective blockade of SIRPa but not CD47 prevents M2 polarization (B) and does not affect M1 polarization (A). Indeed, during the polarization of pro-inflammatory M1 macrophages, the secretion of pro-inflammatory cytokines (IL-6 and TNF-a) is not affected by the addition of specific monoclonal antibodies against rat SIRPa or anti-CD47. In contrast, specific blockade of SIRPa (but not by anti-CD47) during M2 polarization of rat macrophages switches the cytokine profile of the macrophages to an inflammatory profile (like M1 macrophages) with the secretion of IL-6 and TNF-a.

[0123] These results indicate that SIRPa is important for M2 polarization of monocytes and that blocking this pathway represents an opportunity to generate macrophages with a pro-inflammatory profile to treat pathologies that require it, such as cancer or infections, where macrophages are blocked in the M2 state.

[0124] Taken together, all these results indicate that all anti-SIRPα antibodies tested in these experiments (regardless of whether they are directed against human, mouse or rat SIRPa) are able to modulate macrophage polarization by inhibiting the M2 macrophage phenotype. Conversely, anti-CD47 antibodies have no effect on macrophage polarization.

[0125] 1.4 Anti-SIRPα antibodies do not increase human macrophage phagocytosis on tumor cells expressing CD47 The ability of anti-SIRPa antibody (clone SE7C2) to induce phagocytosis was evaluated on human macrophages. Figure 13 shows that two different anti-CD47 mAbs increase the phagocytosis of CD47+ tumor cells (Raji) by M1 macrophages, as described in the literature. In contrast, selective blockade of SIRPa by anti-SIRpa mAb or recombinant CD47-Fc fusion protein does not affect the increase in phagocytic activity of M1 macrophages. These results suggest that phagocytosis of CD47+ tumor cells is not induced by SIRPa / CD47 interaction, but by an ADCP (antibody-dependent cellular phagocytosis) mechanism. Furthermore, these results confirm that anti-SIRPa compounds are not able to induce phagocytosis of tumor cells.

[0126] Example 2: In vivo testing of SIRPa blocking effects 2.1. Effect of SIRPa blockade in an in vivo model of hepatocellular carcinoma Figure 8 shows the overall survival rate of animals inoculated with hepatocellular carcinoma and treated with anti-CD137, anti-SIRPa, or both for 4 weeks. 20% of animals treated with anti-Sirpa monotherapy survived for more than 25 days, and 25% of animals treated with anti-CD137 monotherapy survived for more than 30 days. However, 100% of animals treated with the combination of anti-Sirp+anti-CD137 were still alive after 80 days, indicating a synergistic effect of the two molecules compared to other conditions.

[0127] Figure 9 shows the overall survival rate of animals inoculated with hepatocellular carcinoma and treated for 4 weeks with anti-PD-L1, anti-SIRPa, or both. The results showed very interesting survival rates when animals were treated with both molecules compared to each molecule alone (20% survival after 20 days with anti-SIRPa compared to 12% survival with anti-PD-L1 treatment). This result indicates a synergistic effect of anti-SIRPa and anti-PD-L1 antibodies in this cancer model.

[0128] 2.2. Effect of SIRPa blockade in in vivo models of melanoma Figure 10 shows the overall survival of animals inoculated with melanoma cells and treated with anti-PD-L1, anti-SIRPa, or both for 4 weeks. The efficacy of the combination was better than that of either molecule alone. Figure 10B shows macrophage infiltration in animals treated with anti-SIRPa, demonstrating an increase in the number of macrophages infiltrating the tumor.

[0129] In vivo experiments in two different cancer models indicate that SIRPa represents an interesting target for cancer therapy, especially when combined with other immunotherapies, and suggest that Sirp represents a novel checkpoint inhibitor that may be important to block in order to reconstitute a pro-inflammatory tumor environment.

[0130] 2.3. Effect of SIRPa blockade in in vivo models of breast cancer Figure 15 shows that monotherapy with anti-SIRPa mAb (clone P84) inhibits tumor formation in a syngeneic and orthotopic triple-negative breast model in mice. Tumor volumes are significantly reduced in anti-SIRPa treated mice from 2 weeks after tumor inoculation until the end of the experiment.

Claims

1. A pharmaceutical composition for treating cancer, except for SIRPa-positive acute myeloid leukemia, comprising: an anti-SIRPa compound selected from the group consisting of an antibody or an antigen-binding portion thereof; a second therapeutic agent selected from the group consisting of an immune checkpoint blockade agent selected from the group consisting of an anti-PDL1 blocking antibody, an anti-PD1 blocking antibody and an anti-CTLA-4 blocking antibody, or an anti-CD137 agonist antibody.

13. A pharmaceutical composition comprising:

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