Compositions and methods for use in immunotherapy

Abstract

The present invention relates to a pharmaceutical combination comprising i) a stimulator of interferon genes (STING) activator and ii) an anti-regulatory T cell (Treg) agent and uses thereof, in particular for the treatment of cancer or of a STING-mediated disease or disorder.

Description

[Technical field]

[0001] The present invention relates to immunotherapy, in particular to compositions and methods for enhancing a host immune response, and more particularly to compositions and methods for inducing effector T cells and blocking the ability of regulatory T cells, preferably intratumoral regulatory T cells, to suppress the host immune response. [Background technology]

[0002] There are three main stages in tumor development. During the first stage, immune cells continuously eliminate the generation of transformed cells thanks to interferon-gamma (IFN-γ) production and lymphocyte effector functions. The second stage of tumor development is the equilibrium phase of dynamic balance, where IFN-γ production and lymphocyte effector functions impede tumor growth by relentlessly attacking tumor cells, but fail to eradicate transformed cells. This equilibrium stage allows the development of tumor heterogeneity and genetic instability in cells that escape elimination. The final stage of tumor development is escape. Tumor cell variants selected during the equilibrium phase are now able to grow unchecked even in the presence of a competent immune system (Dunn et al., 2004, Immunity, vol. 21, pp. 137-148).

[0003] The tumors express tumor-specific CD8 + Multiple mechanisms are utilized to circumvent immune clearance, including downregulation of positive signals to cytotoxic T cells (CTLs) and accumulation of regulatory T (Treg) cells within the tumor microenvironment (TME), i.e., in and around the tumor. Tregs are the effector CD4 + and CD8 + and CD4 endowed with regulatory properties that affect various immune cells such as natural killer (NK) cells but inhibit dendritic cell activation. +It is now well established that Tregs are a suppressive subset of T cells. Functionally, Tregs can inhibit the proliferation and lethal activity of CLTs by several mechanisms, such as secretion of cytokines (TGF-β1) and IL-10, metabolic subversion by CD39 and CD73 (Deaglio et al., 2007, J Exp Med., vol. 204(6):1257-65), or contact-dependent inhibition by programmed death-ligand 1 (PD-L1) signaling (Wu et al., 2018, Oncoimmunology, vol. 7, no. 11, e1500107). High tumor infiltration by Tregs and low ratios of effector T cells (Teff) to Tregs are associated with poor outcome in solid tumors.

[0004] Tregs are characterized by their expression of the high affinity IL-2 receptor, CD25, and the transcription factor forkhead box P3 (Foxp3) (Shimon Sakaguchi et al., 2008, Eur. J. Immunol. 38: 901-937). Foxp3 is a master regulator in Treg cells and is essential for their development and suppressive function (Maruyama et al., 2011, Semin. Immunol. 23(6): 418-23). ​​The Treg expression observed during tumor progression may be due to the expansion of naturally occurring Tregs (nTregs) or may be due to the upregulation of CD4 Tregs in the presence of IL-2 and TGF-b1. + CD25 - FoxP3 - CD4 on T cells + CD25 + FoxP3 +It may also result from conversion to Treg (iTreg). Although identical in terms of their suppressive function, these cells differ in terms of their instability with respect to Foxp3. In nTREG, Foxp3 expression is very stable and constitutively expressed, whereas in iTREG, such as those induced at tumor sites, Foxp3 expression is unstable (Floess et al., 2007, PLoS Biol. 2007 February;5(2): e38). Measurement of Foxp3 transcript levels in tumors does not provide clear evidence of the amount of Treg in the tumor microenvironment. Tumor heterogeneity is the first obstacle to the success of cancer immunotherapy.

[0005] In many types of cancer, upregulation of immune checkpoint molecules, such as programmed death 1 (PD-1) and other inhibitory receptors, such as T cell immunoglobulin mucin 3 (TIM-3), cytotoxic T lymphocyte antigen-4 (CTLA-4), glucocorticoid-inducible tumor necrosis factor receptor (GITR), and lymphocyte activation gene-3 (LAG-3), occurs in tumor-infiltrating Treg cells (Park et al., 2012, Cell Immunol., 278(1-2):76-83). Another immune checkpoint, T cell Ig and ITIM domain (TIGIT), is also present in Tregs (Kim et al., 2019, Journal for ImmunoTherapy of Cancer 7:339).

[0006] Tregs are one of the high barriers for the eradication of tumors by immune cells, so their therapeutic depletion using drugs and antibodies or their functional inactivation improves the response to cancer immunotherapy. Several receptors and enzymes expressed on Tregs have been identified as potential targets for enhancing antitumor immunity (Sundee et al., 2021, Eur. J. Immunol., 51:280-291). However, due to the expression of some of these receptors on other immune cells, including effector T cells and NK cells, the relevance of their use as an immunotherapeutic treatment of cancer is still under debate. Furthermore, it should be noted that the appropriate number and function of regulatory T cells (Tregs), especially intratumoral regulatory T cells, is essential for a balanced immune system: for example, an undersupply of these cells leads to autoimmunity, while an oversupply prevents an efficient immune response, with detrimental consequences for antitumor immunity.

[0007] Clinical studies exploring the use of vaccines in combination with daclizumab, a humanized IgG1 anti-human CD25 antibody, or denileukin diftitox, a recombinant fusion protein combining human IL-2 and a fragment of diphtheria toxin, or LMB-2, a fusion protein combining anti-human CD25 Fv and Pseudomonas endotoxin A (PE), have had variable effects on the number of circulating Tregs and vaccine-induced immunity (Luke et al., 2016, Journal for ImmunoTherapy of Cancer 4:35 / Jacobs et al., 2010, Clin Cancer Res 16:5067-5078 / Powell et al., 2007, J Immunol., 179(7): 4919-4928). Moreover, selective elimination or inactivation of Tregs in tumors using anti-CD25 antibodies remains a major challenge. Because these cells share the same cell marker (CD25) as activated conventional non-suppressive T cells, complete depletion of Tregs may severely impair self-tolerance mechanisms. Therefore, systemic depletion of Tregs may not be a suitable option for cancer treatment.

[0008] Another approach relies on the use of immune checkpoint blockade (ICB) antibodies to block the binding of inhibitory molecules and enhance antitumor immune responses. Currently, there are several FDA-approved ICB antibodies on the market against CTLA-4 (ipilimumab), PD-1 (pembrolizumab, nivolumab, and cemiplimab), and PD-L1 (atezolizumab, avelumab, and durvalumab). Despite great advances in immunotherapy, the clinical use of ICB antibodies is limited to a few cancer types (C. Lee Ventola, 2017, P&T®, Vol. 42, No. 8). Moreover, acquired resistance to ICB antibodies demonstrates the need for additional treatment.

[0009] The stimulator of interferon genes (STING) protein is a transmembrane receptor located in the endoplasmic reticulum that recognizes and binds to cyclic dinucleotides. Adjuvant formulations containing STING agonists capable of inducing an immune response in a subject are disclosed in WO2019136118. These formulations, when used as a means to induce antigen release from cells, enhance the immunogenicity of the antigens released from the cells during treatment. Another option for increasing immune activity in the treatment of cancer is the use of a combination of STING agonists and purinergic receptor agonists (WO2020 / 227159). Other STING agonists used in combination with checkpoint inhibitors, in particular compound number 14, produce beneficial effects for several days in some mouse synergistic tumor models exposed to the combination (WO2021 / 005541). However, conversely, recent studies suggest a potential inhibitory effect of STING activation on adaptive antitumor immune responses, particularly by inhibiting T cell proliferation and promoting their death. Furthermore, non-immune functions of STING have been described to promote the survival and growth of metastatic cancer cells (Zhu et al., 2019, Molecular Cancer, 18:152). [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2019136118 [Patent Document 2] WO2020 / 227159 [Patent Document 3] WO2021 / 005541 [Patent Document 4] International patent application WO2014 / 093936 [Patent Document 5] International patent application WO2014 / 189805 [Patent Document 6] International patent application WO2013 / 185052 [Patent Document 7] International patent application WO2015 / 077354 [Patent Document 8] International patent application WO2015 / 185565 [Patent Document 9] US Patent Application Publication No. 2014 / 0341976 [Patent Document 10] International patent application WO2011 / 138251 [Patent Document 11] EP 3430147 B1 [Patent Document 12] International Patent Application WO 2017 / 175147 [Patent Document 13] International Patent Application WO 2019 / 069270 [Patent Document 14] U.S. Patent No. 4,889,803 [Patent Document 15] U.S. Patent No. 5,047,335 [Patent Document 16] International patent application WO2018 / 200812 [Patent Document 17] U.S. Patent No. 5,500,161 [Non-patent literature]

[0011] [Non-Patent Document 1] Dunn et al., 2004, Immunity, 21, 137-148 [Non-Patent Document 2] Deaglio et al., 2007, J Exp Med., 204(6):1257-65 [Non-Patent Document 3] Wu et al., 2018, Oncoimmunology, Volume 7, Issue 11, e1500107 [Non-Patent Document 4] Shimon Sakaguchi et al., 2008, Eur. J. Immunol. 38: 901-937 [Non-Patent Document 5] Maruyama et al., 2011, Semin. Immunol., 23(6):418-23 [Non-Patent Document 6] Floess et al., 2007, PLoS Biol. 2007 February;5(2):e38 [Non-Patent Document 7] Park et al., 2012, Cell Immunol., 278(1-2):76-83 [Non-Patent Document 8] Kim et al., 2019, Journal for ImmunoTherapy of Cancer 7:339 [Non-Patent Document 9] Sundee et al., 2021, Eur. J. Immunol., 51:280-291 [Non-Patent Document 10] Luke et al., 2016, Journal for ImmunoTherapy of Cancer 4:35 [Non-Patent Document 11] Jacobs et al., 2010, Clin Cancer Res 16:5067-5078 [Non-Patent Document 12] Powell et al., 2007, J Immunol., 179(7): 4919-4928 [Non-Patent Document 13] C. Lee Ventola, 2017, P&T(R), Volume 42, Issue 8 [Non-Patent Document 14] Zhu et al., 2019, Molecular Cancer, 18:152 [Non-Patent Document 15] Corrales and Gajewski, 2015, Clin Cancer Res., 21(21):4774-4779 [Non-Patent Document 16] Feng et al., 2020, Drug Discovery Today, Vol. 25(1), pp. 230-237 [Non-Patent Document 17] Collison and Vignali, In Vitro Treg Suppression Assays, Chapter 2 in Regulatory T Cells: Methods and Protocols, Methods in Molecular Biology, Kassiotis and Liston, eds., Springer, 2011, 707:21-37. [Non-Patent Document 18] Workman et al., In Vivo Treg Suppression Assays, Chapter 9 in Regulatory T Cells: Methods and Protocols, Methods in Molecular Biology, Kassiotis and Liston, eds., Springer, 2011, pp. 119-156 [Non-Patent Document 19] Takahashi et al., Int. Immunol., 1998, 10:1969-1980. [Non-Patent Document 20] Thornton et al., J. Exp. Med., 1998, 188:287-296. [Non-Patent Document 21] Collison et al., J. Immunol., 2009, 182:6121-6128. [Non-Patent Document 22] Thornton and Shevach, J. Exp. Med., 1998, 188:287-296. [Non-Patent Document 23] Asseman et al., J. Exp. Med., 1999, 190:995-1004

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[0012] Thus, there is a strong need for therapeutic agents and methods, particularly for use in optimizing the manipulation of the immunosuppressive effects of Treg cells, and particularly for use in improving the treatment of cancer. [Means for solving the problem]

[0013] The present invention is based, at least in part, on the discovery that combining activation of the Stimulator of Interferon Genes (STING) pathway with modulation, preferably inhibition, of regulatory T cell (Treg) subpopulations, and even more preferably intratumoral regulatory T cells, allows for significantly and advantageously improving the treatment of cancer.

[0014] The present invention relates to a composition / combination comprising i) a STING (stimulator of interferon genes) activator, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, in other words a vectorized STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, selected from imatinib or a derivative or salt thereof; dasatinib or a derivative or salt thereof; an anti-CTLA-4 antibody having an IgG2 constant region or a mutated IgG1 constant region; an anti-LAG3 antibody; an anti-TIM-3 antibody; an anti-ICOS antibody; an anti-CD25 antibody; an anti-CCR4 antibody; an anti-CCR8 antibody; an anti-TNFR2 antibody; and any combination thereof.

[0015] The compositions / combinations described herein by the inventors are suitable for both in vitro for experimental purposes and in vivo for therapeutic purposes. The present invention particularly relates to a pharmaceutical composition / combination, such as a therapeutic pharmaceutical composition / combination, a vaccine or a veterinary composition / combination, comprising i) a STING (stimulator of interferon genes) activator, preferably in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, selected from imatinib or a derivative or salt thereof; dasatinib or a derivative or salt thereof; an anti-CTLA-4 antibody with an IgG2 constant region or a mutated IgG1 constant region; an anti-LAG3 antibody; an anti-TIM-3 antibody; an anti-ICOS antibody; an anti-CD25 antibody; an anti-CCR4 antibody; an anti-CCR8 antibody; an anti-TNFR2 antibody; and any combination thereof.

[0016] Preferably, the STING activator is selected from small molecules, in particular cyclic dinucleotides; nucleic acids encoding STING agonists; vectors or cells expressing STING or a STING agonist; antibody-conjugated STING agonists; and inhibitors of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1).

[0017] Preferably, the cyclic dinucleotide is cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic dimeric guanosine monophosphate (c-di-GMP), cyclic dimeric adenosine monophosphate (c-di-AMP), or a vectorized form thereof. More preferably, the cGAMP cyclic dinucleotide is 2'-3'-cyclic GMP-AMP or 3'-3'-cyclic GMP-AMP, or a mixture thereof.

[0018] Preferably, the vector comprising the STING activator is a vesicle, in particular a liposome; an exosome, such as exoSTING™; a virus, such as an adenovirus or an oncolytic virus; a virus-like particle (VLP); a polymer; or a hydrogel.

[0019] In certain aspects, the STING activator is a vectored dinucleotide, the vectored dinucleotide is a virus-like particle (VLP) that comprises a lipoprotein envelope that includes a viral fusogenic glycoprotein and contains cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), wherein the cGAMP is packaged within the virus-like particle.

[0020] In another embodiment, the STING activator is a small molecule, the small molecule being selected from the group consisting of dithio-(RP,RP)-[cyclic[A(20,50)pA(30,50)p]] (ML RR-S2 CDA, also known as ADU-S100); MK-1454; 5,6-dimethyl-xanthenone acetic acid (DMXAA); flavone-8-acetic acid (FAA); 10-carboxymethyl-9-acridanone (CMA); α-mangostin (xanthone); ML RR-S2 CDG; ML RR-S2 cGAMP; 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC); dispirodiketopiperazine (DSDP); 2'3'-cG S A S selected from a bisphosphorothioate analog of 2'3' cGAMP, named MP; a macrocycle-bridged STING agonist (MBSA); an MBSA derivative of ADU-S100, named E7766; a benzothiophene oxobutanoic acid (MSA-2) derivative; SB11285; a diamide benzimidazole (diABZI); IMSA-101, TAK-676; CRD5500; TTI-10001; and SEL312-4787.

[0021] In a preferred embodiment, the anti-Treg agent is an anti-CTLA-4 antibody with an IgG2 constant region, such as tremelimumab (CP-675,206).

[0022] In another preferred embodiment, the anti-regulatory T cell (Treg) agent is an anti-CTLA-4 antibody with a mutated IgG1 constant region, such as BMS 986249 (or also known as CTLA4-Probody), BMS-986288 (or also known as CTLA4-NF), zalifrelimab (or also known as AGEN1884), quavonlimab (or also known as MK-1308), HBM4003 (from Harbour BioMed) or ONC-392.

[0023] In another preferred embodiment, the anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, is an anti-CTLA-4 antibody in which the IgG1 constant region mutation is located in the CH2 domain of the (Fc) constant region.

[0024] In a further preferred embodiment, the anti-Treg agent, preferably the anti-intratumoral Treg agent, is an anti-CTLA-4 antibody, in which the IgG1 constant region mutation is located in the CH3 domain of the (Fc) constant region.

[0025] In another preferred embodiment, the anti-Treg agent, preferably the anti-intratumoral Treg agent, is an anti-CTLA-4 antibody, in which the IgG1 constant region mutation is located in the CH1 domain of the Fab region.

[0026] In one embodiment, the anti-intratumoral anti-regulatory T cell (Treg) agent is imatinib or a derivative or salt thereof.

[0027] In another embodiment, the anti-intratumoral anti-regulatory T cell (Treg) agent is dasatinib or a derivative or salt thereof.

[0028] In certain embodiments, VLPs containing a STING activator, imatinib, and an immune checkpoint inhibitor are used in combination, particularly in the compositions of the invention.

[0029] In another embodiment, a VLP containing a STING activator, an anti-CTLA4 inhibitor with an IgG2 constant region or a mutated IgG1 constant region, and an immune checkpoint inhibitor are used in combination, particularly in the compositions of the invention.

[0030] In yet another particular embodiment, VLPs containing a STING activator, anti-CD25 antibodies, particularly anti-CD25 inhibitors with an IgG2 constant region or a mutated IgG1 constant region, and immune checkpoint inhibitors are used in combination, particularly in the compositions of the invention.

[0031] The present invention also relates to pharmaceutical compositions / combinations as described herein for use as a medicament, in particular a therapeutic, vaccine or veterinary composition / combination, The present invention particularly relates to such compositions / combinations for use in the prevention or treatment of cancer or of a STING-mediated disease or disorder, preferably cancer, in a subject.

[0032] The present invention also relates to a method for treating cancer or a STING-mediated disease or disorder, particularly cancer, in a subject, or for preventing cancer or a STING-mediated disease or disorder in a subject, particularly for preventing cancer relapse. The method comprises administering a therapeutically effective amount of a composition, particularly a pharmaceutical composition, a vaccine composition or a veterinary composition, as disclosed herein, to a subject in need thereof. The present invention particularly relates to the use of a composition, particularly a pharmaceutical composition, a vaccine composition or a veterinary composition, as disclosed herein, for the manufacture of a medicament or vaccine for preventing or treating cancer in a subject. The present invention also relates to a pharmaceutical composition / combination, a vaccine composition / combination or a veterinary composition / combination, as disclosed herein, for use in preventing or treating cancer, particularly for preventing cancer relapse.

[0033] In another aspect, the present invention relates to a method for inducing or stimulating a therapeutic immune effect in a subject in need thereof, the method comprising the steps of: i) administering to a subject in need thereof a pharmaceutical composition comprising a STING (stimulator of interferon genes) activator, preferably in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as described herein, thereby reducing or inhibiting the immunosuppressive activity of Treg cells, wherein reducing or inhibiting the activity of Treg cells, in particular intratumoral Treg cells, induces a therapeutic effect in the subject against a disease, typically cancer, from which the subject suffers.

[0034] In a further aspect, the present invention relates to a method of reducing or inhibiting immunosuppressive function in a subject, the method comprising the steps of: administering to a subject in need thereof a pharmaceutical composition comprising i) a STING (stimulator of interferon genes) activator, preferably in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent as described herein, preferably an anti-intratumoral Treg agent, thereby reducing or inhibiting the immunosuppressive activity of Treg cells, whereby reducing or inhibiting the activity of Treg cells reduces or inhibits the immunosuppressive function in the subject.

[0035] Preferably, the STING activator will be administered to the subject by a systemic route, in particular by a subcutaneous (sc), oral, intraperitoneal (ip), intramuscular (im), intradermal (id) or intravenous (iv) route, preferably by a subcutaneous route.

[0036] Preferably, the anti-Treg agent will be administered by a systemic route, in particular by intraperitoneal, oral, subcutaneous or intravenous route.

[0037] The present invention also relates to a kit comprising at least two parts, a first part comprising a STING (stimulator of interferon genes) activator, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and a second part comprising an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, wherein the first and second parts of the kit are preferably in separate compartments or containers.

[0038] In a particular embodiment, a first part of the kit comprises a STING (stimulator of interferon genes) activator in a vectorized form, that is a virus-like particle (VLP), in other words a vector comprising (containing or expressing) a STING activator, and a second part of the kit comprises an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, which is imatinib or a derivative or salt thereof or an anti-CTLA-4 with an IgG2 constant region or a mutated IgG1 constant region.

[0039] The present invention also relates to the use of a composition or of a kit as described herein for the manufacture of a medicament for preventing or treating cancer or a STING-mediated disease or disorder, preferably cancer, in a subject. [Brief description of the drawings]

[0040] [Figure 1] FIG. 1 is a schematic diagram of the experimental design to study the effect of cGAMP-VLP in combination with imatinib chemotherapy in the MCA-OVA tumor model. Six days after MCA-OVA tumor cell injection (sc), mice were randomly assigned to four treatment groups: (1) PBS+PBS; (2) PBS+imatinib 10 mg / kg; (3) cGAMP-VLP (50 ng cGAMP)+PBS, and (4) cGAMP-VLP (50 ng cGAMP)+imatinib 10 mg / kg. cGAMP-VLP was administered by the subcutaneous (sc) route, and imatinib was administered by the intraperitoneal (ip) route. [Diagram 2]Serum inflammatory cytokine levels (pg / mL) in MCA-OVA tumor-bearing mice treated with cGAMP-VLP or vehicle (PBS) on day 6. Serum samples were collected from separate groups of mice after sc injection 3 hours after treatment on day 6. [Diagram 3] Specific CD8 and CD4 T cell responses. Ten days after the first sc injection of cGAMP-VLP, peripheral blood mononuclear cells (PBMCs) were stimulated with OVA and p15 peptide and assessed by IFN-γ ELISPOT. [Figure 4] FIG. 1 shows the mean tumor growth (cm3) over time for the different treatments indicated. [Diagram 5] FIG. 1 shows the mean tumor growth (cm3) on day 21 for the different treatments indicated in the figure. [Figure 6] Survival curves for all groups. Death events are defined as tumor size >2 cm3. Statistics were calculated using the log-rank (Mantel-Cox) test. [Figure 7] FIG. 1: Percentage of weight loss in mice during imatinib treatment. [Figure 8] FIG. 1 is a schematic of the experimental design to study the effect of cGAMP-VLP in combination with the anti-Treg agent CTLA-4-mIgG2a in the MCA-OVA tumor model. Six days after MCA-OVA tumor cell injection (sc), mice were randomly assigned to four treatment groups: (1) PBS + anti-IgG2a isotype; (2) PBS + anti-CTLA4-mIgG2a; (3) cGAMP-VLP (50 ng cGAMP) + anti-IgG2a isotype, and (4) cGAMP-VLP (50 ng cGAMP) + anti-CTLA4-mIgG2a. cGAMP-VLP was administered by the subcutaneous (sc) route and antibodies were administered by the intraperitoneal (ip) route. [Figure 9]Serum inflammatory cytokine levels (pg / mL) in MCA-OVA tumor-bearing mice treated with cGAMP-VLP and / or anti-CTLA4-mIgG2a or vehicle (PBS) on day 6. Serum samples were collected from separate groups of mice after sc injection 3 hours after treatment on day 6. [Figure 10] Representative plots of the results of flow cytometric determination of the percentage and number of Tregs in MCA-OVA tumors, blood and spleen following ip injection of anti-CTLA4-mIgG2a and isotype. [Figure 11] Representative plots of the results of flow cytometric determination of the percentage and number of Tregs in MCA-OVA tumors, blood and spleen following ip injection of anti-CTLA4-mIgG2a and isotype. [Figure 12] FIG. 11: Percentage and number of FOXP3+ Treg cells relative to total CD45+TCRb+CD4+ T cells and CD4+ / Treg and CD8+ / Treg cell ratios in tumor samples (MCA-OVA tumors) 48 hours after ip injection of the antibody. [Figure 13] FIG. 11: Percentage of FOXP3+ Treg cells relative to total CD45+TCRb+CD4+ T cells and CD4+ / Treg and CD8+ / Treg cell ratios in blood 48 hours after ip antibody injection. [Figure 14] FIG. 11: Percentage of FOXP3+ Treg cells relative to total CD45+TCRb+CD4+ T cells and CD4+ / Treg and CD8+ / Treg cell ratios in the spleen 48 hours after ip antibody injection. [Figure 15] Specific CD8 and CD4 T cell responses. Ten days after the first sc injection of cGAMP-VLP with or without anti-CTLA4-mIgG2a, peripheral blood mononuclear cells (PBMCs) were stimulated with OVA and p15 peptide and assessed by IFN-γ ELISPOT. [Figure 16]Specific CD8 and CD4 T cell responses. Ten days after the first sc injection of cGAMP-VLP with or without anti-CTLA4-mIgG2a, peripheral blood mononuclear cells (PBMCs) were stimulated with OVA and p15 peptide and assessed by IFN-γ ELISPOT. [Figure 17] Caliper measurements of tumor size (cm3). Every line represents an individual C57BL / 6J mouse. [Figure 18] FIG. 1 shows the mean tumor growth over time for the different treatments shown in the figure and in Example 2. [Figure 19] Survival curves for all groups. Death events are defined as tumor size >2 cm3. Statistics were calculated using the log-rank (Mantel-Cox) test. [Figure 20] Tumor volumes in tumor-bearing and tumor-free mice after MCA-OVA re-challenge (sc) on day 95. CR, complete responder mice. [Figure 21] FIG. 1 is a schematic of the experimental design to study the effect of cGAMP-VLP in combination with the anti-Treg agent CD25-mIgG2a (KLC) in the MCA-OVA tumor model. Six days after MCA-OVA tumor cell injection (sc), mice were randomly assigned to four treatment groups: (1) PBS + anti-IgG2a isotype; (2) PBS + anti-CD25-mIgG2a (KLC); (3) cGAMP-VLP (50 ng cGAMP) + anti-IgG2a isotype, and (4) cGAMP-VLP (50 ng cGAMP) + anti-CD25-mIgG2a (KLC). cGAMP-VLP was administered by the subcutaneous (sc) route and the antibody was administered by the intraperitoneal (ip) route. [Figure 22] FIG. 11. Percentage of FOXP3+ cells relative to total TCRb+CD4+ T cells in splenocyte and tumor samples 48 hours after ip injection of antibody. [Figure 23]Specific CD4 and CD8 T cell responses. Ten days after the first sc injection of cGAMP-VLP with or without anti-CD25-mIgG2a, peripheral blood mononuclear cells (PBMCs) were stimulated with two OVAs and assessed by IFN-γ ELISPOT. [Figure 24] FIG. 1 shows the mean tumor growth (cm3) over time for the different treatments shown in the figure and in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The inventors have developed compositions / combinations that can be used to prevent, alleviate or treat cancer or STING-mediated diseases or disorders, more generally diseases in which regulatory T cells block the immune response of effector T cells (e.g., in certain cancers). In particular, the compositions described herein for the first time by the inventors allow complete tumor regression and induce systemic anti-tumor immunity in treated subjects.

[0042] In a first aspect, the present invention relates to a composition / combination, typically a pharmaceutical composition / combination, such as a therapeutic, vaccine or veterinary composition / combination, comprising i) a STING (stimulator of interferon genes) activator, preferably in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, selected from imatinib or a derivative or salt thereof; dasatinib or a derivative or salt thereof; an anti-CTLA-4 antibody with an IgG2 constant region or a mutated IgG1 constant region; an anti-LAG3 antibody; an anti-TIM-3 antibody; an anti-ICOS antibody; an anti-CD25 antibody; an anti-CCR4 antibody; an anti-CCR8 antibody; an anti-TNFR2 antibody; and any combination thereof.

[0043] For example, a composition / combination of the invention comprises i) a STING (stimulator of interferon genes) activator, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) imatinib or a derivative or salt thereof, an anti-CTLA-4 antibody with an IgG2 constant region or a mutated IgG1 constant region, and / or an anti-CD25 antibody, or any other combination of anti-regulatory T cell (Treg) agents described in the previous paragraph.

[0044] The compositions / combinations described herein are also considered "medications" herein.

[0045] The expressions "STING (Stimulator of Interferon Genes)" and "Stimulator of Interferon Genes (STING)" refer to an adaptor transmembrane protein located in the endoplasmic reticulum. STING, also known as TMEM173, MITA, ERIS and MPYS, is a central signaling molecule in the innate immune response to cytosolic nucleic acids. Human and mouse STING amino acid sequences can be found at NCBI Locus NP_938023 and NP_082537, respectively.

[0046] STING is activated when double-stranded DNA enters the cytosol. Besides its role in sensing the presence of infectious agents (viruses, bacteria, parasites, and fungi), the STING pathway is also directly involved in sensing mammalian DNA. Cytosolic DNA is detected when it binds to the sensor cyclic GMP-AMP synthase (cGAS, MB21D1), which catalyzes the synthesis of cyclic GMP-AMP (cGAMP) from guanosine triphosphate (GTP) and adenosine triphosphate (ATP). cGAMP acts as a second messenger that binds to and activates STING. Upon binding of cGAMP, STING undergoes a conformational change, which triggers its transport from the endoplasmic reticulum (ER) to the Golgi and then to perinuclear endosomes. As a result, STING recruits tank-binding kinase 1 (TBK1), which then phosphorylates it and thereby makes it available for binding the transcription factor interferon regulatory factor 3 (IRF3). TBK1 then phosphorylates IRF3, which translocates to the nucleus and drives transcription of IFN-β and other genes (Corrales and Gajewski, 2015, Clin Cancer Res., 21(21):4774-4779).

[0047] "STING activator" refers to any natural or synthetic compound that upon incubation with human PBMCs binds to STING and acts as an inducer, agonist or enhancer to induce or stimulate the expression of type 1 interferons and other cytokines. This binding involves the cGAS-STING signaling pathway. Compounds that induce or stimulate the expression of human interferons may therefore be useful in the prevention or treatment of various diseases or disorders, such as precancerous conditions and cancer. For example, STING activators activate STING within the tumor microenvironment (TME), i.e., in or around the tumor, thereby upregulating CD8 + This results in efficient cross-priming of tumor-specific antigens to T cells and aids in the recruitment of effector T cells by inducing essential chemokines, such as interferon-γ (IFN-γ).

[0048] STING activator activity may be determined by one or more STING agonist assays selected from an interferon stimulation assay, an hSTING wt assay, a THP1-Dual assay, a TANK-binding kinase 1 (TBK1) assay, and an interferon-gamma inducible protein 10 (IP-10) secretion assay.

[0049] STING activators, also known as STING agonists, can be divided into three classes based on their chemical scaffolding and their ability to enter and bind to specific amino acids within the ligand-binding pocket of the cytosolic domain of STING (Feng et al., 2020, Drug Discovery Today, vol. 25(1), pp. 230-237). Cyclic dinucleotides (CDNs) were identified as the first class of molecules that can bind to and activate STING. The second class of molecules that directly connect with the ligand-binding domain (LBD) of STING and activate the cGAS-STING pathway are flavonoids and xanthone derivatives. The third class of STING agonists includes diaminobenzimidazoles (diABZIs). Together with CDN-independent agents that activate the cGAS-STING pathway, the third class of compounds of STING agonists can be broadly classified, and are referred to herein as "small molecules."

[0050] In certain aspects, the STING activator is selected from small molecules, particularly cyclic dinucleotides; nucleic acids encoding STING agonists; vectors or cells comprising / containing / expressing STING or a STING agonist; antibody-conjugated STING agonists, such as, for example, SB11325 / 11396; and inhibitors of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), such as, for example, MAVU-104.

[0051] The term "cyclic dinucleotide" refers to a small molecule second messenger that can directly bind to the endoplasmic reticulum resident receptor STING (stimulator of interferon genes) and activate a signal transduction pathway that induces the expression of type I interferons and also nuclear factor-κB (MFkB)-dependent inflammatory cytokines. Natural cyclic dinucleotides include cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), more specifically c[G(2',5')pA(3',5')p] (CAS number: 1441190-66-4) or c[G(3',5')pA(3',5')p] (CAS number: 849214-04-6), cyclic dimeric guanosine monophosphate (c-di-GMP), more specifically bis-( 3'-5')-cyclic dimeric guanosine monophosphate (3',5'-cyclic diguanylic acid, cyclic di-GMP, c-di-GMP), or cyclic dimeric adenosine monophosphate (c-di-AMP), more specifically, bis-(3'-5')-cyclic dimeric adenosine monophosphate (3',5'-cyclic diadenylic acid, cyclic-di-AMP, c-di-AMP).

[0052] In a preferred embodiment, the cGAMP cyclic dinucleotide used in the compositions / combinations of the present invention is 2'-3'-cyclic GMP-AMP. In another preferred embodiment, the cGAMP cyclic dinucleotide used in the compositions / combinations of the present invention is 3'-3'-cyclic GMP-AMP.

[0053] Other cyclic dinucleotides suitable for incorporation into the compositions / combinations of the present invention include derivatives of natural CDNs. ADU-S100 (also known as MIW815 or ML RR-S2 CDA), a dithio derivative of the natural CDN 2'-3'cAMP, and E7766, a macrocyclic bridge derivative of AD-S100, were the first synthetic CDNs to be studied. Other non-natural CDNs, such as MK1454, SB11285 and BMS-986301, are currently under development.

[0054] International Patent Applications WO2014 / 093936, WO2014 / 189805, WO2013 / 185052, WO2015 / 077354, WO2015 / 185565 and US Patent Application Publication No. 2014 / 0341976 provide examples of cyclic dinucleotides. STING activators, such as cyclic dinucleotides, can be used in the context of the present invention in vectorized form.

[0055] The vector, e.g., a vector containing a cyclic dinucleotide or a vector expressing a STING activator or agonist, can be a vesicle, in particular a liposome; an exosome; a virus, e.g., an adenovirus or an oncolytic virus; a virus-like particle (VLP); a polymer; or a hydrogel. Preferably, the vector is a virus or a virus-like particle (VLP), even more preferably a VLP.

[0056] A commercially available exosome suitable in the context of the present invention may be, for example, exoSTING™ (CODIAK, Cambridge, MA 02140). exoSTING™ is composed of exosomes engineered to express high levels of PTGFRN and exosomal proteins (on the surface of the exosome to facilitate specific uptake into tumor-resident antigen-presenting cells) and loaded with a STING agonist (located in the lumen of the exosome).

[0057] Along with liposomes, polymers and hydrogels are the main delivery systems currently used to deliver STING agonists.

[0058] Polymers, and more particularly polymeric nanoparticles, are suitable nanocarriers for STING agonists due to their favorable properties, including in vivo hydrolysis, controlled drug loading and release kinetics, and overall safety. Polymers that can be used in the context of the present invention can be selected from the group including poly(beta-amino ester) (PBAE), poly(ethylene glycol)-block-[(2-diethylaminoethyl methacrylate)-co-(butyl methacrylate)-co-(pyridyl disulfide ethyl methacrylate)] (PEG-DBP), and acetylated dextran (Ace-DEX).

[0059] Hydrogels are highly hydrophilic polymer networks that facilitate localized and controlled drug release leading to the recruitment of tumor-toxic immune cells. Hydrogels that can be used in the context of the present invention can be selected from the group including linear polyethylenimine (LPEI) / hyaluronic acid (HA), HA hydrogel scaffolds, Matrigel and STINGel.

[0060] Viruses that can be used as vectors in the context of the present invention can be adenoviruses or oncolytic viruses, such as those derived from Herpes Simplex-1 virus, Vesicular Stomatitis Virus (VSV) or Newcastle Disease Virus (NDV).

[0061] In a preferred embodiment, the STING activator used in the composition of the invention is a vectored dinucleotide, the vectored dinucleotide is a virus-like particle (VLP), the dinucleotide is packaged in said virus-like particle, and the virus-like particle comprises a lipoprotein envelope comprising a viral fusogenic glycoprotein. In a particular embodiment, the VLP comprises a lipoprotein envelope comprising a viral fusogenic glycoprotein and contains cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), and the cGAMP is packaged in said virus-like particle.

[0062] VLPs or virus-like particles resemble viruses but are non-infectious. They do not contain any wild-type viral genetic material, and more preferably, do not contain any viral infectious genetic material. Expression of viral structural proteins, such as envelope or capsid, leads to self-assembly of the VLP. The VLP may be a virosome (i.e., a lipoprotein envelope lacking a capsid) or a VLP that contains both a capsid and a lipoprotein envelope. The VLP may further comprise an epitope, an antigen, or any other protein or nucleic acid of interest, preferably a tumor-associated antigen, or a combination thereof.

[0063] The enveloped VLPs may comprise several, in particular two or more, different epitopes / antigens, which may be selected from (a) different virus strains of the same virus, (b) different serotypes of the same virus, and / or (c) different virus strains specific for different hosts, for example different strains of influenza virus, such as influenza virus A strains H1N1, H5N1, H9N1, H1N2, H2N2, H3N2 and / or H9N2, influenza virus B and / or influenza virus C. Different serotypes are for example different serotypes of human papillomavirus (HPV), such as serotypes 6, 11, 16, 18, 31, 33, 35, 39, 45, 48, 52, 58 62, 66, 68, 70, 73 and / or 82, but also serotypes of proto-oncogenic type HPV 5, 8, 14, 17, 20 and / or 47, or serotypes of papilloma-associated type HPV 6, 11, 13, 26, 28, 32 and / or 60.

[0064] The lipoprotein envelope of the VLP may contain a fusion protein. The term "fusion protein" or "fusogenic glycoprotein" as used herein refers to viral type I transmembrane proteins that are classified into three classes. Class I viral fusion proteins are trimers with a large globular head region and a long α-helical coiled-coil stalk region. Examples of class I viral fusion proteins include, but are not limited to, influenza HA, respiratory syncytial virus F, and HIV gp4. Class II fusion proteins are trimers that are essentially composed of β-sheets. Examples of class II viral fusion proteins include, but are not limited to, tick-borne encephalitis virus E, Semliki Forest virus E1, and Rift Valley fever virus Gc. Class III viral fusion proteins are five-domain molecules that are composed of both α-helices and β-strands as secondary structural elements. Examples of class III viral fusion proteins include, but are not limited to, vesicular stomatitis virus G, herpes simplex virus gB, or baculovirus gp64. Preferably, the lipoprotein envelope of the VLPs used in the compositions of the invention comprises a class III viral fusion protein.

[0065] The viral fusion or fusogenic protein may be a glycoprotein or a combination of several glycoproteins from the Retroviridae (including lentiviruses and retroviruses, e.g., alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses, epsilonretroviruses), Herpesviridae, Poxviridae, Hepadnaviridae, Flaviviridae, Togaviridae, Coronaviridae, Hepatitis D virus, Orthomyxoviridae, Paramyxoviridae, Filoviridae, Rhabdoviridae, Bunyaviridae, or Orthopoxviridae (e.g., smallpox). In a preferred embodiment, the viral fusogenic glycoprotein is from the Flaviviridae, Retroviridae, Orthomyxoviridae, Paramyxoviridae, Bunyaviridae, or Hepadnaviridae. In a specific embodiment, the viral fusogenic glycoprotein is from an orthomyxovirus, a rhabdovirus, or a retrovirus. In a more preferred embodiment, the viral fusogenic glycoprotein is a glycoprotein from HIV (human immunodeficiency virus), including HIV-1 and HIV-2, influenza, including influenza A (e.g., H5N1 and H1N1 subtypes) and influenza B, Thogotovirus, or VSV (vesicular stomatitis virus).

[0066] The virus-like particle preferably further comprises a capsid. Preferably, the capsid is from the Retroviridae family. The Retroviridae family includes lentiviruses and retroviruses, such as alpharetroviruses, betaretroviruses, gammaretroviruses, deltaretroviruses and epsilonretroviruses. For example, the capsid is from Human Immunodeficiency Virus (HIV), including HIV-1 and HIV-2, Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), Puma Lentivirus, Bovine Immunodeficiency Virus (BIV), Caprine Arthritis-Encephalitis Virus, Feline Leukemia Virus (FeLV), Murine Leukemia Virus (MLV), Bovine Leukemia Virus (BLV), Human T-Lymphotropic Virus (HTLV, e.g., HTLV-1, -2, -3 or -4), Rous Sarcoma Virus (RSV), Avian Sarcoma-Leukemia Virus, Equine Infectious Anemia Virus, Moloney Murine Leukemia Virus (MMLV). More preferably, the retroviral capsid is from HIV or MLV. Preferably, the capsid is from a lentivirus or a retrovirus.

[0067] Optionally, the viral glycoprotein can be fused or covalently linked to an antigen of interest or any other protein or nucleic acid of interest, preferably a tumor-associated antigen, or a combination thereof. In addition to the viral glycoproteins and capsid proteins, a non-exhaustive list of antigens that can be further included in the VLP is disclosed below. More specifically, the VLPs are capable of expressing tumor associated antigens such as Her2 / neu, CEA (carcinoembryonic antigen), HER2 / neu, MAGE2 and MAGE3 (melanoma associated antigens), RAS, mesothelin or p53, from HIV, such as Vpr, Vpx, Vpu, Vif and Env, from bacteria, such as C. albicans SAP2 (secreted aspartyl proteinase 2), from Clostridium difficile, parasites, such as Plasmodium falciparum proteins, such as CSP (circumsporozoite protein), AMA-1 (apical membrane antigen-1), TRAP / SSP2 (sporozoite surface protein 2, LSA (liver stage antigen), Pf Exp1 (Pf exported protein 1), and the like. 1), SALSA (Pf antigen 2 sporozoite and liver stage antigen), STARP (sporozoite threonine and asparagine rich protein) or any of the proteins disclosed in International Patent Application WO2011 / 138251.

[0068] The composition of VLPs and methods for their production are disclosed in detail in EP 3430147 B1.

[0069] As mentioned above, in certain embodiments described herein, the STING activator is a small molecule. The term "small molecule" herein refers to any compound, natural or synthetic, that targets STING, binds to STING, and activates the cGAS-STING pathway, thereby promoting IKK-related kinase TANK-binding kinase 1 (TBK1) signaling and activating nuclear factor-kappa B (NF-kB) and interferon regulatory factor 3 (IRF3) in immune cells in the tumor microenvironment (TME). This leads to the production of inflammatory cytokines, including interferon (IFN), and more specifically, the production / expression of IFN-beta (IFN-β). The binding of small molecules to STING results in a cytotoxic T lymphocyte (CTL)-mediated immune response against tumor cells, causing tumor cell lysis. Due to their discrepancies in chemical structure, and in the context of the present invention, small molecules encompass CDNs, including both natural and synthetic CDNs, as well as STING agonists that are CDN-unrelated agents, such as flavonoids and xanthone derivatives, and diaminobenzimidazole (diABZI).

[0070] Among flavonoids, flavone acetic acid (FAA) was the first STING agonist identified. In an attempt to improve efficacy, various modifications were introduced into the molecular structure of FAA, resulting in a series of derivatives, including 5,6-dimethylxanthenone-4-acetic acid (DMXAA, also known as ASA404 or vadimezan).

[0071] α-Mangostin is a xanthone derivative with substituted allyl and hydroxyl groups within the xanthone scaffold. Despite its weaker potency in inducing type I IFN in reporter assays compared to cGAMP, α-Mangostin is an interesting STING agonist that can be used in conjunction with the therapeutic uses described herein.

[0072] FAA and dimethyloxoxanthenyl acetate (DMXAA) are specific mouse (m)STING agonists, and α-mangostin shows higher affinity for human (h)STING than for mSTING.

[0073] Diaminobenzimidazoles (also known as diABZI or amidobenzimidazole dimers) show enhanced binding to STING and cellular function compared to cGAMP. Furthermore, diABZI induces STING-dependent activation of type I IFN and inflammatory cytokines. Such compounds, along with bispyrazole diacids or bisphenol dimers, are disclosed in International Patent Applications WO 2017 / 175147 and WO 2019 / 069270.

[0074] E7766 belongs to a novel class of macrocycle-bridged STING agonists (MBSAs). E7766, an MBSA derivative of ADU S100, is the only STING agonist currently being tested as a stand-alone intravenous intervention in cancer patients. The tolerability, safety and preliminary activity of this molecule are being investigated in patients with advanced solid tumors or lymphomas (NCT04144140), as well as in individuals with non-muscle invasive bladder cancer (NCT04109092).

[0075] Other STING to small molecules are currently in development, such as MK-2118, GSK3745417, IMSA-101, TAK-676, CDR5500, TTI-10001 and SEL312-4787.

[0076] Preferably, the STING activator is dithio-(RP,RP)-[cyclic[A(20,50)pA(30,50)p]] (ML RR-S2 CDA, also known as ADU-S100); MK-1454; 5,6-dimethyl-xanthenone acetic acid (DMXAA); flavone-8-acetic acid (FAA); 10-carboxymethyl-9-acridanone (CMA); α-mangostin (xanthone); ML RR-S2 CDG; ML RR-S2 cGAMP; 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC); dispirodiketopiperazine (DSDP); 2'3'-cG S A S A small molecule selected from MP (bisphosphorothioate analog of 2'3'-cGAMP); macrocycle-bridged STING agonist (MBSA); E7766 (MBSA derivative of ADU-S100); benzothiophene oxobutanoic acid (MSA-2) derivatives; SB11285; diamide benzimidazole (diABZI); IMSA-101, TAK-676; CRD5500; TTI-10001; and SEL312-4787.

[0077] The expression "nucleic acid encoding a STING activator" refers to a polynucleotide that directly specifies the nucleic acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame beginning with a start codon, such as ATG, GTG, or TTG, and ending with a stop codon, such as TAA, TAG, or TGA. The coding sequence can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0078] The above-mentioned nucleic acid encoding the STING agonist can be packaged into a vector that optimizes its transcription and translation. The term "vector" refers to a linear or circular DNA molecule that contains a polynucleotide encoding the STING agonist of the present invention and is operably linked to a control sequence that allows its expression. The expression vector comprises an expression cassette that is suitable for expressing the STING agonist of the present invention. Expression vectors that can be used in the present invention include, non-exhaustively, eukaryotic expression vectors, in particular mammalian expression vectors, virus-based expression vectors, baculovirus expression vectors, plant expression vectors, and plasmid expression vectors. Suitable expression vectors can be derived from viruses, such as baculoviruses, papovaviruses, such as SV40, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses, or retroviruses, in particular lentiviruses.

[0079] Vectors can also be of viral origin, such as adenoviruses producing the bacterial STING agonist c-di-GMP. Alternatively, the coding sequence of the STING agonist can be integrated into the cell, either into the cell's chromosome or into its cytoplasm. Any eukaryotic cell can be used in the method. For example, cells used for production include mammalian cells, such as COS-1 cells, CHO (Chinese Hamster Ovary) cells (U.S. Pat. No. 4,889,803; U.S. Pat. No. 5,047,335), HEK (human embryonic kidney) cells, cell lines, such as cells from the 293, 293T, HL-116, Vero and BHK (baby hamster kidney) cell lines; plant cells (e.g., N. bethamiana); insect cells, such as Spodoptera frugiperda (5 / J derived cells, e.g., Sf-9 cells), SF21, Hi-5, Express Sf + , and S2 Schneider cells, particularly in the baculovirus-insect expression system; or avian cells.

[0080] In another embodiment, the STING activator may be an antibody conjugated to a STING agonist whose specific target remains unknown as of the filing date of this application, such as SB11325 / 11396. The terms "immunoconjugate" and "antibody conjugate" are used interchangeably herein. Antibody conjugates comprising agonists of STING are disclosed in International Patent Application WO2018 / 200812.

[0081] Hydrolysis of 2'3'-cGAMP by phosphodiesterases may be a barrier to the activation of the STING signaling pathway. Recently, several inhibitors of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), such as MAVU-104, MV626, and SR8314, have been designed and developed to indirectly activate STING.

[0082] In another embodiment, the STING activator can be an inhibitor of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), e.g., MAVU-104.

[0083] The compositions / combinations of the present invention also comprise (i.e. possibly some) anti-regulatory T cell (Treg) agents, preferably intratumoral Treg agents as disclosed herein. The expression "regulatory T cells" refers to a subpopulation of T cells, particularly T cells located within tumors, expressing markers such as IL2ra (CD25), Ikzf2 (Helios), Ikzf4 (Eos), IL2rb (CD122), Socs2, Nrp1, Ebi3 or CTLA. The immunosuppressive activity of Treg cells is well known to those skilled in the art, as expressed by CD25-CD4 + T and CD8 + This refers to the ability of Tregs to suppress T cell proliferation. T cell proliferation assays are the gold standard for evaluating Treg immunosuppressive activity. Constitutive and inducible, CD4 + and CD8 + , FoxP3 + and FoxP3 - Several subpopulations of Tregs have been described in association with malignancies. Most studies have focused on the expression of CD4+ CD25 + FoxP3 + The presence of Tregs has been associated with poor prognosis.

[0084] The term "Treg" or "regulatory T cells" refers to CD4 + CD25 - and CD8 + CD4 suppresses T cell proliferation and / or effector function or otherwise downregulates the immune response + It refers to T cells. Notably, Tregs can downregulate immune responses mediated by natural killer (NK) cells, natural killer T cells, as well as other immune cells. In a preferred embodiment, the Tregs of the present invention are Foxp3 + It is.

[0085] The term "regulatory T cell function" or "Treg function" refers to the function of CD4 + CD25 - or CD8 + It is used interchangeably to refer to any biological function of Tregs that results in reduced T cell proliferation or reduced effector T cell-mediated immune responses. Treg function can be measured by techniques established in the art. Non-limiting examples of in vitro assays useful for measuring Treg function include the transwell suppression assay, more generally, target conventional T cells (Tconv) and Tregs (or antigen presenting cells (APCs), such as irradiated splenocytes or purified dendritic cells (DCs) or irradiated PBMCs) purified from human peripheral blood or umbilical cord blood (or mouse spleen or lymph nodes) are optionally stimulated with anti-CD3 +In vitro assays include activation with anti-CD28 coated beads followed by in vitro detection of conventional T cell proliferation (e.g., by measuring radioactive nucleotides (e.g., [H]-thymine, etc.) or fluorescent nucleotides, or by Cayman Chemical's MTT Cell Proliferation Assay Kit, or by monitoring the dilution of green fluorescent dye esters CFSE or Seminaphtharhodafluor (SNARF-1) dyes by flow cytometry). Other common assays measure T cell cytokine responses. Useful in vivo assays of Treg function include animal models of diseases in which Tregs play an important role, such as (1) homeostatic models (naive homeostatically expanded CD4 T cells as target cells primarily suppressed by Tregs) and (2) mouse models of disease in which Tregs play an important role, such as (3) mouse models of disease in which Tregs play an important role, such as (4) mouse models of disease in which Tregs play an important role, such as (5) mouse models of disease in which Tregs play an important role, such as (6) mouse models of disease in which Tregs play an important role, such as (7) mouse models of disease in which Tregs play an important role, such as (8) mouse models of disease in which Tregs play an important role, such as (9) mouse models of disease in which Tregs play an important role, such as (10) mouse models of disease in which Tregs play an important role, such as (11) mouse models of disease in which Tregs play an important role, such as (12) mouse models of disease in which Tregs play an important role, such as (13) mouse models of disease in which Tregs play an important role, such as (14) mouse models of disease in which Tregs play an important role, such as (15) mouse models of disease in which Tregs play an important role, such as (16) mouse models of disease in which Tregs play an important role, such as (17) mouse models of disease in which Tregs play an important role, such as (18) mouse models of disease in which Treg + (2) an inflammatory bowel disease (IBD) recovery model (using Th1 T cells (Th17) as target cells primarily suppressed by Tregs); (3) an experimental autoimmune encephalomyelitis (EAE) model (using Th17 and Th1 T cells as target cells primarily suppressed by Tregs); (4) a B16 melanoma model (suppression of antitumor immunity) (using CD8 T cells as target cells primarily suppressed by Tregs); + T cells), (5) naive CD4 + CD45 + RBhi Tconv cells express Rag1 - / - (5) suppression of colon inflammation in adoptive transfer colitis transfected mice, and (6) assays in a Foxp3 rescue model (using lymphocytes as target cells that are primarily suppressed by Tregs). According to one protocol, all of the models require the use of Rag1 for the recipient as well as mice for the donor T cell population. - / -or Foxp3 mice are also required. For further details regarding various useful assays, see, for example, Collison and Vignali, In Vitro Treg Suppression Assays, Chapter 2: Methods and Protocols, in Regulatory T Cells, Methods in Molecular Biology, Kassiotis and Liston, eds., Springer, 2011, 707:21-37; Workman et al., In Vivo Treg Suppression Assays, Chapter 9: Methods and Protocols, in Regulatory T Cells, Methods in Molecular Biology, Kassiotis and Liston, eds., Springer, 2011, 119-156; Takahashi et al., Int. Immunol., 1998, 10:1969-1980; Thornton et al., J. Exp. Med., 1998, 188:287-296; Collison et al., J. Immunol., 1998, 133:141-144; Immunol., 2009, 182:6121-6128; Thornton and Shevach, J. Exp. Med., 1998, 188:287-296; Asseman et al., J. Exp. Med., 1999, 190:995-1004; Dieckmann et al., J. Exp. Med., 2001, 193:1303-1310; Belkaid, Nature Reviews, 2007, 7:875-888; Tang and Bluestone, Nature Immunology, 2008, 9:239-244; Bettini and Vignali, Curr. Opin. Immunol., 2009, 21:612-618; Dannull et al., J Clin See Invest, 2005, 115(12):3623-33; Tsaknaridis et al., J Neurosci Res., 2003, 74:296-308.

[0086] The expression "anti-regulatory T cell (Treg) agent" or "anti-intratumoral regulatory T cell (Treg) agent" refers to a compound capable of modulating different subpopulations of Tregs, in particular those present in a tumor, either by reducing the number of Tregs or by modulating their phenotypic signature. In a preferred embodiment, the anti-regulatory T cell (Treg) agent used in the composition / combination of the present invention is a compound capable of modulating the CD45 + TCRb + CD4 + Tregs, especially CD25 + FoxP3 + and FoxP3 + Ki67 + Subpopulations of Tregs are specifically targeted, particularly those subpopulations of Tregs located within tumors.

[0087] In certain embodiments described herein, the compounds of the invention do not include purinergic receptor agonists. In particular, the anti-regulatory T cell (Treg) agent, preferably the anti-intratumoral Treg agent, is not a purinergic receptor agonist.

[0088] In certain embodiments described herein, the anti-regulatory T cell (Treg) agent, preferably the anti-intratumoral Treg agent, is selected from imatinib or a derivative or salt thereof; dasatinib or a derivative or salt thereof; an anti-CTLA-4 antibody having an IgG2 constant region or a mutated IgG1 constant region; an anti-LAG3 antibody; an anti-TIM-3 antibody; an anti-ICOS antibody; an anti-CD25 antibody; an anti-CCR4 antibody; an anti-CCR8 antibody; an anti-TNFR2 antibody; and any combination thereof. For example, the anti-regulatory T cell (Treg) agent, preferably the anti-intratumoral Treg agent, is selected from imatinib or a derivative or salt thereof, an anti-CTLA-4 antibody having an IgG2 constant region or a mutated IgG1 constant region; an anti-CD25 antibody; and any combination thereof.

[0089] CTLA4, LAG3, TIM-3 and ICOS are immune checkpoints. Agonists of these immune checkpoints, especially antibodies, act as immune checkpoint inhibitors. The term "immune checkpoint inhibitor" refers to any drug that blocks proteins called checkpoints made by some types of immune cells, such as T cells, and cancer cells. These checkpoints help prevent immune responses from becoming too strong and can sometimes prevent T cells from killing cancer cells. When these checkpoints are blocked, T cells can kill cancer cells more efficiently. Examples of checkpoint proteins found on T cells or cancer cells include, but are not limited to, LAG3 / MHC-II and CTLA-4 / B7-1 / B7-2. In the context of the present invention, the immune checkpoints described herein that are of interest are those that are ubiquitous on Tregs.

[0090] Preferably, the anti-regulatory T cell (Treg) agent is an anti-CTLA-4 with an IgG2 constant region (also known as CD152), such as tremelimumab (CP-675,206), or an anti-CTLA-4 with a mutated IgG1 constant region, such as BMS 986249 (or alternatively known as CTLA4-probody), BMS-986288 (or alternatively known as CTLA4-NF), zalifrelimab (or alternatively known as AGEN1884), quabonlimab (or alternatively known as MK-1308), HBM4003 (Harbour from BioMed), ONC-392, or, for example, those described herein below; anti-LAG3 monoclonal antibodies, such as, for example, Sym-2011, MK-4280, TSR-033, IMP321, leratolimab (BMS986016), LAG525, REGN3767, MGD013, FS118, INCAGN02385 or EOC202; anti-TIM-3 monoclonal antibodies, such as, for example, Sym-023, BMS-986258, LY3321367, SHR-1702 or covolimab (TSR-022); anti-ICOS monoclonal antibodies, such as, for example, JTX-2011, GSK3359609, BMS-986226, KY1044 or MEDI-570. More preferably, the anti-LAG3, anti-TIM-3 or anti-ICOS antibody has an IgG2 constant region or a mutated IgG1 constant region (as further defined herein below).

[0091] CD25, CCR4, CCR8 and TNFR2 are receptors present on the surface of T cells, particularly Tregs. Preferably, the anti-regulatory T cell (Treg) agent is selected from anti-CD25 antibodies, such as daclizumab, RG-6292 (RO7296682) or camidalumab tesirin (ADCT-301); anti-CD25 antibodies coupled with a toxin, such as RFT5-dgA immunotoxin (IMTOX25); anti-CCR4 antibodies, such as mogamulizumab; anti-CCR8 antibodies, such as BMS-986340, JTX-1811, SRF114, HBM1022, MAB1429, FPA157; anti-TNFR2 antibodies. More preferably, the anti-CD25, anti-CCR4, anti-CCR8 or anti-TNFR2 antibody has an IgG2 constant region or a mutated IgG1 constant region (as further defined herein below).

[0092] More preferably, the anti-Treg agent, preferably the intratumoral Treg agent, is an anti-CTLA-4 antibody of IgG1 isotype engineered, typically mutated, in its Fc constant region and / or in the Fab region, preferably at the specific positions described herein. The engineering of the CH1, CH2 and / or CH3 constant regions corresponds to deliberate human engineering of the gene sequence. The mutations are preferably the replacement of one amino acid by another. The mutations defined herein below for the Fc constant region of an anti-CTLA-4 antibody of IgG1 subtype can also be transposed to the Fc constant region of an anti-LAG3, anti-TIM-3, anti-ICOS, anti-CD25, anti-CCR4, anti-CCR8 or anti-TNFR2 antibody.

[0093] In a preferred embodiment, the anti-intratumoral regulatory T cell (Treg) agent is an anti-CTLA-4 antibody in which the IgG1 constant region mutation is located in the CH2 domain (of the (Fc) constant region).

[0094] In a further preferred embodiment, the anti-intratumoral Treg agent is an anti-CTLA-4 antibody in which the IgG1 constant region mutation is located in the CH3 domain (of the (Fc) constant region).

[0095] In another preferred embodiment, the anti-intratumoral Treg agent is an anti-CTLA-4 antibody in which the IgG1 constant region mutation is located in the CH1 domain (in the Fab region).

[0096] In a further preferred embodiment, the anti-intratumoral regulatory T cell (Treg) agent is an anti-CTLA-4 antibody comprising at least two mutations located in the CH1, CH2 and / or CH3 domains.

[0097] The preferred amino acid substitutions made in the Fc region result in enhanced effector functions of anti-CTLA-4 antibodies of the IgG1 isotype, in particular enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. The Fc constant region may be modified to increase ADCC and / or to increase affinity for the Fcy receptor (FcyR) by modifying one or more amino acids at the following positions in the CH2 (corresponding to amino acids 113-223 of SEQ ID NO:1) or CH3 domain (corresponding to amino acids 224-329 of SEQ ID NO:1): 233, 234, 235, 236, 237, 238, 239, 243, 247, 256, 262, 267, 268, 270, 271, 280, 290, 292, 298, 300, 305, 324, 326, 328, 330, 332, 333, 334, 339 or 396 (the latter numbering is that of the EU index or the equivalent in the Kabat scheme). The numbering of the amino acid positions results from the IGHG1 amino acid translation of sequence J00228 (SEQ ID NO: 1), which is now replaced in the database by sequence AH007035. J00228 corresponds to the IGHG1*01 allele (allelic alignment: human IGHG1) and to the G1m1, 17 chain (G1m allotype). The EU gamma 1 chain is encoded by the IGHG1*03 allele (CH1 K120>R, CH3 D12>E and L14>M), which is the G1m3 chain (G1m allotype). The EU numbering is defined by Edelman et al. (Proc. Natl. Acad. USA, 63, 78-85 (1969)). Kabat numbering is disclosed in Kabat et al., Sequences of proteins of immunological interest. 5th ed. - US Department of Health and Human Services, NIH Publication No. 91-3242, pp. 662, 680, 689 (1991).

[0098] Preferably, the IgG1 mutations are in the CH2 and / or CH3 domain.

[0099] In one embodiment, the first CH2 domain of the Fc constant region of the anti-CTLA4 antibody comprises at least one mutation at an amino acid position selected from 234, 235, 236, 239, 268, 270, 298, preferably a L234Y / L235Q / G236W / S239M / H268D / D270E / S298A substitution, and the second CH2 domain of the Fc constant region of the anti-CTLA4 antibody comprises at least one mutation at an amino acid position selected from 270, 326, 330 and 334, preferably a D270E / K326D / A330M / K334E substitution.

[0100] In another embodiment, each of the two CH2 domains of the anti-CTLA4 antibody Fc constant region comprises at least one mutation at an amino acid position selected from 239, 330 and 332, preferably a S239D / A330L / I332E substitution.

[0101] In another embodiment, at least one CH2 domain of the anti-CTLA4 antibody Fc constant region comprises a mutation at amino acid position 236, 238, 239, 267 and / or 328, preferably a substitution selected from G236D, P238D, S239D, S267E, L328F, L328E, and any combination thereof.

[0102] In another embodiment, at least one CH2 domain of the anti-CTLA4 antibody Fc constant region comprises the substitution P238D and one or more substitutions selected from the following group: - E233D, G237D, H268D, P271G, and A330R; - V262E, S267E, and L328F; and - V264E, S267E and L328F.

[0103] In another embodiment, at least one CH2 domain of the anti-CTLA4 antibody Fc constant region comprises at least one mutation at an amino acid position selected from 236, 239, 267, 268, 324, 332, preferably at least one substitution selected from 236A, 239D, 239E, 267E, 268D, 268E, 268F, 324T, 332D and 332E.

[0104] In another aspect, at least one CH2 and / or CH3 domain of the anti-CTLA4 antibody Fc constant region comprises at least one mutation at an amino acid position selected from 236, 239, 243, 256, 290, 292, 298, 300, 305, 330, 332, 333, 334, 339, 396, preferably at least one substitution selected from G236A, S239D, F243L, T256A, K290A, R292P, S298A, Y300L, V305I, A330L, I332E, E333A, K334A, A339T and P396L.

[0105] In another aspect, at least one CH2 and / or CH3 domain of the anti-CTLA4 antibody Fc constant region comprises at least one mutation at an amino acid position selected from 243, 247, 280, 290, 292, 298, 300, 305, 326, 333, 334, 339, 396, preferably at least one substitution selected from 243L, 247I, 280H, 290S, 292P, 298A, 298D, 298V, 300L, 305I, 326A, 333A, 334A, 339D, 339Q and 396L.

[0106] More preferably, the IgG1 mutation is present in the CH1 domain (of the Fab region) (corresponding to amino acids 1 to 97 of SEQ ID NO: 1) and / or in the CH2 domain (of the Fc region).

[0107] In one aspect, at least one CH1 and / or CH2 domain of the anti-CTLA4 antibody comprises at least one mutation at an amino acid position selected from 135, 137, 139, 181, 216, 217, preferably at least one substitution selected from M135Y, S137T, T139E, S181A, E216A and K217A.

[0108] Another suitable anti-CTLA-4 antibody that can be used in the compositions of the invention is tremelimumab (CP-675,206), an IgG2 antibody that binds well to FCGRIIA but not to FCGRIIIA.

[0109] The anti-CTLA-4 antibody used in the compositions of the invention does not have a wild-type (i.e., non-mutated / engineered) IgG1 constant region. In particular, the anti-CTLA-4 antibody used in the compositions of the invention is not ipilimumab.

[0110] In a preferred embodiment, the anti-CTLA-4 antibody used in the compositions of the invention is an IgG2, particularly an IgG2A (isotype 2A) anti-CTLA-4 antibody.

[0111] Preferably, the anti-CTLA-4 antibody, or fragment thereof, is administered at a concentration of about 10 -8 M, preferably about 10 -9 M, more preferably about 10 -10 M, more preferably 10 -11 M has a binding affinity for CTLA-4.

[0112] In certain aspects described herein, the anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, is imatinib or a derivative or salt thereof.

[0113] The term "imatinib" refers to a compound (CAS number 152459-95-5) that binds to a specific enzyme called Bcr-Abl tyrosine kinase, which is involved in increasing the production of white blood cells. It also inhibits several other TK receptors: Kit, the SCF (stem cell factor) receptor encoded by the oncogene c-Kit, the discoidin domain receptors (DDR1 and DDR2), the CSF-1R (colony stimulating factor receptor) and the PDGF (platelet-derived growth factor: PDGFR-alpha and PDGFR-beta) receptors. Imatinib also inhibits cellular processes mediated by the activation of these receptor kinases. A commercially available imatinib is GLIVEC™.

[0114] In another particular embodiment, the anti-regulatory T cell (Treg) agent, preferably the anti-intratumoral Treg agent, is dasatinib or a derivative or salt thereof.

[0115] The term "dasatinib" refers to a dual BCR / ABL and Src family tyrosine kinase inhibitor (CAS number 302962-49-8). The primary targets of dasatinib are breakpoint cluster region Abelson (BCRABL), SRC, ephrin and growth factor (GFR). The commercially available dasatinib is SPRYCEL™.

[0116] In another specific embodiment, a combination of separate anti-regulatory Treg agents is used in the compositions of the invention.

[0117] In another particular embodiment, the STING activator is a VLP, which may be used together with an anti-CTLA4 antibody having an IgG2 constant region or a mutated IgG1 constant region, and together with another distinct anti-Treg agent as described herein, in particular together with another distinct immune checkpoint inhibitor.

[0118] The present invention also relates to a pharmaceutical composition / combination as described herein for use as a medicament, in particular a therapeutic, vaccine or veterinary composition.

[0119] The compositions / combinations of the invention can be used as drugs / medicines, e.g. as vaccines or vaccine adjuvants.The invention therefore also relates to pharmaceutical compositions / combinations, e.g. therapeutic pharmaceutical compositions / combinations, vaccine or veterinary compositions, comprising i) a STING (stimulator of interferon genes) activator as described herein, preferably in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as described herein.

[0120] In a particular aspect, the present invention also relates to a pharmaceutical composition / combination, such as a therapeutic pharmaceutical composition / combination, a vaccine or a veterinary composition, comprising i) a STING (stimulator of interferon genes) activator as described herein, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-intratumoral regulatory T cell (Treg) agent as described herein.

[0121] The composition may further comprise an adjuvant. The composition may also comprise or be administered in combination with one or more further active substances. Described herein in particular is a pharmaceutical, vaccine or veterinary composition comprising i) a STING (stimulator of interferon genes) activator, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral regulatory T cell (Treg) agent, and a pharma- ceutically acceptable carrier or excipient.

[0122] The pharmaceutical compositions / combinations of the present invention may in fact include pharma- ceutically acceptable carriers, supports or excipients, which, as used herein, may or may not include solvents, dispersion media, diluents or other liquid vehicles, dispersing or dispersing aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, AR Gennaro (Lippincott, Williams and Wilkins, Baltimore, MD, 2006) describes various excipients used in the formulation of pharmaceutical compositions, and known techniques for their preparation. Use of any conventional excipient vehicle is contemplated within the scope of the present invention, except insofar as it is incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition. The pharma- ceutically acceptable excipient may be a preservative, an inert diluent, a dispersing agent, a surfactant and / or emulsifier, a buffer, and the like. Suitable excipients include, for example, water, saline, dextrose, sucrose, trehalose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the composition. In some aspects, the (pharmaceutical) compositions described herein include one or more preservatives. In some aspects, the (pharmaceutical) compositions described herein do not include a preservative.

[0123] The (pharmaceutical) compositions / combinations disclosed herein may include an adjuvant. Any adjuvant may be used in accordance with the present invention. Numerous adjuvants are known. A useful list of many such compounds is provided by the National Institutes of Health and can be found by the skilled artisan (www.niaid.nih.gov / daids / vaccine / pdf / compendium.pdf). See also Allison (1998, Dev. Biol. Stand., 92:3-11; incorporated herein by reference), Unkeless et al. (1998, Annu. Rev. Immunol., 6:251-281; ​​incorporated herein by reference), and Phillips et al. (1992, Vaccine, 10:151-158; incorporated herein by reference). Hundreds of different adjuvants are known in the art and may be utilized in the practice of the present invention. Exemplary adjuvants that can be utilized in connection with the present invention include, but are not limited to, cytokines, gel-type adjuvants (e.g., aluminum hydroxide, aluminum phosphate, calcium phosphate, etc.); microbial adjuvants (e.g., immunomodulatory DNA sequences containing CpG motifs; endotoxins, e.g., monophosphoryl lipid A; exotoxins, e.g., cholera toxin, E. coli heat labile toxin, and pertussis toxin; muramyl dipeptides, etc.); oil emulsions and emulsifier-based adjuvants (e.g., Freund's adjuvant, MF59 [Novartis], SAF, etc.); particulate adjuvants (e.g., liposomes, biodegradable microspheres, saponins, etc.); synthetic adjuvants (e.g., non-ionic block copolymers, muramyl peptide analogs, polyphosphazenes, synthetic polynucleotides, etc.); and / or combinations thereof. Other exemplary adjuvants include certain polymers (eg, the polyphosphazenes described in US Pat. No. 5,500,161), Q57, QS21, squalene, tetrachlorodecaoxide, and the like.

[0124] The present invention particularly relates to the compositions / combinations described herein for use in the prevention or treatment of cancer or a STING-mediated disease or disorder, particularly a STING-mediated cancer, in a subject.

[0125] The pharmaceutical compositions / combinations disclosed herein are useful for inducing or stimulating / enhancing an immune response / effect in a subject in need thereof. The present invention relates to a method for inducing or enhancing an immune response in a subject in need thereof, comprising the step of reducing or inhibiting the immunosuppressive activity of Treg cells, preferably Treg cells present in a tumor, by administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition disclosed herein comprising i) a STING (stimulator of interferon genes) activator, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as described herein, thereby inducing a therapeutic effect in the subject against a disease, particularly cancer, suffered by the subject, by reducing or inhibiting the activity of Treg cells, preferably Treg cells present in and around the tumor (i.e. within the tumor microenvironment). The immune response may refer to cellular immunity, may refer to humoral immunity, or may include both. The immune response may also be limited to a part of the immune system. For example, in certain aspects, the immunogenic compositions described herein can induce an increased IFNy response. In certain aspects, the immunogenic compositions described herein can induce a mucosal IgA response (e.g., as measured in nasal and / or rectal washes). In certain aspects, the immunogenic compositions described herein can induce a systemic IgG response (e.g., as measured in serum). In certain aspects, the immunogenic compositions described herein can induce virus-neutralizing antibodies, or neutralizing antibody responses. In certain aspects, the immunogenic compositions described herein can induce a CTL response.

[0126] Also described herein is a method of reducing or inhibiting immunosuppressive function in a subject, the method comprising the steps of: i) administering to a subject in need thereof a pharmaceutical composition comprising a STING (stimulator of interferon genes) activator, preferably a vectorized form of STING activator, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as described herein, thereby reducing or inhibiting the immunosuppressive activity of Treg cells, preferably Treg cells present within a tumor, whereby the immunosuppressive function in the subject is reduced or inhibited by reducing or inhibiting the activity of Treg cells, preferably Treg cells present within and around the tumor (i.e. within the tumor microenvironment).

[0127] Therefore, the pharmaceutical composition / combination of the present invention is useful as a vaccine or as a vaccine adjuvant.The composition of the present invention can be used to treat various diseases in patients.The disease can be cancerous or non-cancerous.Cancerous diseases can include cancers that produce tumors as well as cancers that do not produce tumors, such as hematological malignancies.

[0128] In one embodiment, the STING activator and anti-regulatory T cell (Treg) agent are administered in combination. In another embodiment, the STING activator and anti-regulatory T cell (Treg) agent are administered in combination, either sequentially or simultaneously.

[0129] In a preferred embodiment, the STING activator and the anti-intratumor regulatory T cell (Treg) agent are administered in combination. In another preferred embodiment, the STING activator and the anti-intratumor regulatory T cell (Treg) agent are administered in combination, either sequentially or simultaneously.

[0130] In one aspect, the present invention relates to a method for treating cancer or a STING-mediated disease or disorder, preferably cancer, or for preventing cancer or a STING-mediated disease or disorder, in particular for preventing cancer relapse, in a subject in need thereof. The method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition / combination, in particular a pharmaceutical composition, a vaccine composition or a veterinary composition, as described herein. The present invention relates in particular to the use of a composition / combination, in particular a pharmaceutical composition, a vaccine composition or a veterinary composition, as disclosed herein, for the manufacture of a medicament, such as a vaccine, for treating cancer in a subject. The present invention also relates to a pharmaceutical composition / combination as disclosed herein for use in treating cancer or preventing cancer relapse.

[0131] "Method of treating" refers to a process aimed at producing a beneficial change in the condition of an individual, e.g., a mammal, particularly a human. Both human and veterinary treatments are contemplated. The beneficial change may include one or more of the following: restoration of function, alleviation of symptoms, limiting or slowing down a disease, disorder or condition, or preventing, limiting or slowing down the deterioration of a patient's condition, disease or disorder; in particular, as used herein, the term "treatment" (also "treat" or "treating") refers to any administration of an immunogenic composition that partially or completely alleviates, relieves, alleviates, suppresses, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition or predisposition to a disease. Such treatment may be treatment of subjects who do not show signs of the relevant disease, disorder, and / or condition, and / or treatment of subjects who show only early signs of the disease, disorder, and / or condition. Alternatively, or in addition, such treatment may be treatment of a subject that exhibits one or more established symptoms of the relevant disease, disorder, and / or condition. In certain embodiments, the term "treating" refers to vaccination of a patient.

[0132] The term "cancer" encompasses diseases or disorders such as cancer, precancerous conditions, and tumor metastasis. Cancer may be a solid cancer or a liquid cancer. Preferably, the cancer is a solid cancer. Examples of cancer diseases and conditions for which the compositions of the present invention have a beneficial anti-tumor effect include cancer of the lung, bone, pancreas, skin, head, neck, uterus, ovary, stomach, rectum, heart, esophagus, small intestine, intestine, endocrine system, thyroid, parathyroid, adrenal gland, urethra, prostate, penis, testes, ureter, bladder, kidney, or liver; rectal cancer; anal cancer; carcinoma of the fallopian tube, endometrium, cervix, vagina, vulva, renal pelvis, renal cell; sarcoma of soft tissue; myxoma; rhabdomyosarcoma; fibroma; lipoma. ; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; hepatocellular carcinoma; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; CNS neoplasms; spinal axis tumors; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brain stem glioma; pituitary adenoma; bronchial adenoma; chondroitin hamartoma; mesothelioma; Hodgkin's disease or a combination of one or more of the foregoing cancers.

[0133] When used to treat solid tumors or prevent solid tumor relapse, the pharmaceutical compositions / combinations disclosed herein advantageously deplete Treg cells present within the tumor and its microenvironment.

[0134] The pharmaceutical compositions / combinations disclosed herein may further be used in combination with one or more second therapeutic agents, particularly chemotherapeutic agents (i.e., cancer therapeutic agents), such as aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, buserelin, busulfan, campotecan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, cyclophosphamide ... , epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medicamentosyltransferase Roxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procabazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, taxol, temozolomide, teniposide, testosterone, thioguanine, thiotepa, dichloride Examples of such agents include, but are not limited to, titanocene hydrochloride, topotecan, trastuzumab, tretinoin, trimetrexate, vinblastine, vincristine, vindesine, vinorelbine, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphamide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), and methotrexate (MTX).

[0135] Patients exposed to the pharmaceutical compositions disclosed herein may, for example, be further exposed to radiation therapy and / or treated with surgery, hormone therapy, or bone marrow transplantation, depending on the type of tumor, the condition of the patient, and other healthy tissues.

[0136] As used herein, the term "vaccination" refers to the administration of a composition for the purpose of generating an immune response, for example to a disease-causing agent (e.g., a virus). In the present invention, vaccination can be performed before, during, and / or after exposure to the disease-causing agent, and in certain embodiments, before, during, and / or immediately after exposure to the agent. In some embodiments, vaccination includes multiple administrations of a vaccination composition, appropriately spaced apart in time. As used herein, the term "therapeutically effective amount" refers to an amount (or dose) sufficient to produce a therapeutic effect in the treated subject, with a reasonable benefit-risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject gives an indication or feeling of an effect). In particular, a "therapeutically effective amount" refers to an amount of the composition of the present invention effective to prevent, ameliorate or treat a desired disease or condition, or to exhibit a detectable therapeutic or prophylactic effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or further reducing the severity or frequency of symptoms of the disease. Therapeutically effective amounts are typically administered in a dosing regimen that may include multiple unit doses. For any particular immunogenic composition, the therapeutically effective amount (and / or the appropriate dose in an effective dosing regimen) may vary, for example, depending on the route of administration, depending on the combination with other pharmaceutical agents. Additionally, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder to be treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, weight, general health, sex, and diet of the patient; the number of doses, route of administration, and / or excretion or metabolic rate of the specific immunogenic composition employed; and similar factors well known in the medical arts.The amount of a given compound that would correspond to such an amount will vary depending on factors such as the particular compound [e.g., the potency (pIC50), efficacy (EC50), and biological half-life of the particular compound], the disease state and its severity, the identity of the patient requiring treatment (e.g., age, size, and weight), etc., but can nevertheless be routinely determined by one of ordinary skill in the art. Similarly, the duration of treatment and the time of administration of the compound (the period between doses and the timing of dosing, e.g., before / with / after a meal) will vary according to the identity of the subject requiring treatment (e.g., body weight), the particular compound and its characteristics (e.g., pharmacological properties), the disease or disorder and its severity, and the specific composition and method to be used, but can nevertheless be routinely determined by one of ordinary skill in the art.

[0137] The term "dose" is used hereinafter with reference to a "unit" dose of the composition of the invention, i.e., a dose that can be administered to a subject in need of treatment, typically a subject suffering from cancer, or a STING-mediated disease or disorder, particularly cancer, or a subject in whom cancer or a STING-mediated disease or disorder, particularly cancer, is to be prevented. As explained above, the terms "dose" and "amount" are synonymous.

[0138] In certain embodiments, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of imatinib present in the composition of the invention ranges from about 1 mg to about 1 g.

[0139] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-CTLA-4 antibody present in the composition of the invention ranges from about 10 mg to about 1 g.

[0140] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of dasatinib present in the composition of the invention ranges from about 10 mg to about 1 g.

[0141] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-LAG3 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0142] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-TIM-3 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0143] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-ICOS antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0144] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-CD25 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0145] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-CCR4 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0146] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-CCR8 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0147] In another specific embodiment, the dose of cyclic dinucleotide (preferably packaged in a virus-like particle), e.g., cGAMP, present in the composition of the invention ranges from about 1 ng to about 100 mg, and the dose of anti-TNFR2 antibody present in the composition of the invention ranges from about 1 mg to about 1 g.

[0148] As used herein, the term "amelioration" or "improvement" refers to the prevention, relief, or temporary amelioration of a subject's condition, or the improvement of a subject's condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease, disorder, or condition. The term "prevention" refers to delaying the onset of a disease, disorder, or condition. Prevention can be considered complete when the onset of a disease, disorder, or condition is delayed for a predefined period of time. As used herein, the terms "dosage form" and "unit dosage form" refer to a physically separate unit of therapeutic agent for a patient to be treated. Each unit contains a predetermined amount of active material calculated to produce a desired therapeutic effect. However, it will be understood that the total dosage of the composition will be determined by the attending physician within the scope of sound medical judgment. A "dosage regimen" (or "treatment regimen"), as this term is used herein, is a set of unit doses (typically more than one) that are administered individually to a subject, typically spaced apart. In some embodiments, a given therapeutic agent has a recommended dosing regimen that may include one or more doses. In some embodiments, the dosing regimen includes multiple doses, each of which is separated by the same length of time, and in some embodiments, the dosing regimen includes multiple doses and at least two different time periods separating the individual doses.

[0149] As used herein, the term "subject", "individual", or "patient" refers to a mammal, and in particular to a human or non-human mammalian subject, whatever its age or sex. The individual to be treated (also called a "patient" or "subject") is an individual (fetus, infant, child, adolescent, or adult) suffering from cancer. In some aspects, the subject is a human. In some other aspects described herein, the subject is an animal, in particular a pet (e.g., dog and cat), livestock (e.g., cow, pig, sheep, rabbit, hog, fish, poultry), or horse.

[0150] The pharmaceutical composition / combination of the present invention may, in certain embodiments, be administered by any systemic or local administration route or be suitable for administration by any systemic or local administration route.For example, the route may be parenteral, such as intraperitoneal, subcutaneous, intramuscular, intravenous, intradermal, intrathecal, intraarterial, intraarticular and intramedullary; or enteral, such as oral and mucosal (e.g. sublingual, intranasal, rectal, vaginal or intrabronchial) route.Preferred administration routes are intraperitoneal, subcutaneous, intravenous and oral.

[0151] For example, the pharmaceutical compositions provided herein may be provided in a sterile injectable form (e.g., suitable for subcutaneous or intravenous injection). For example, in some embodiments, the pharmaceutical compositions are provided in a liquid dosage form suitable for injection / infusion. In some embodiments, the pharmaceutical compositions are provided as a powder (e.g., lyophilized and / or sterilized), optionally under vacuum, to be reconstituted with an aqueous diluent (e.g., water, buffer, saline solution, etc.) prior to injection. In some embodiments, the pharmaceutical compositions are diluted and / or reconstituted with water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder should be mixed gently with the aqueous diluent (e.g., should not be shaken).

[0152] In certain embodiments, the STING activator will be administered to the subject by a systemic route, such as a subcutaneous, oral, intraperitoneal, intramuscular, transdermal or intravenous route, preferably by a subcutaneous route.

[0153] In certain embodiments, when the STING activator is a virus-like particle (VLP), the VLP will be administered to the subject by a systemic route, such as a subcutaneous, oral, intraperitoneal or intravenous route, preferably by a subcutaneous route.

[0154] In certain embodiments, the anti-Treg agent will be administered to the subject by a systemic route, such as an intraperitoneal, oral, subcutaneous or intravenous route, preferably by an intraperitoneal route.

[0155] The pharmaceutical compositions and parts of the kits described herein can be provided in a form that can be refrigerated and / or frozen. Alternatively, they can be provided in a form that cannot be refrigerated and / or frozen. If necessary, the reconstituted solution and / or liquid dosage form can be stored for a certain period of time (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, or longer) after freezing.

[0156] The formulations of the pharmaceutical compositions and parts of the kits described herein can be prepared by any method known or hereafter developed in the pharmacological art. Such methods for preparation include the step of bringing into association the active ingredient with one or more excipients and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into the desired single or multiple dosage units. Pharmaceutical compositions according to the invention can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses.

[0157] The relative amounts of active ingredient, pharma- ceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the invention may vary depending on the identity, size, and / or condition of the subject being treated and / or depending on the route by which the composition is to be administered. By way of example, the composition may contain between 0.1 percent and 100 percent (w / w) active ingredient.

[0158] The compositions described herein will generally be administered in such an amount and for such a time as is necessary or sufficient to induce an immune response. The dosing regimen may consist of a single dose or multiple doses over a period of time. The exact amount of the immunogenic composition (e.g., a combination of i) a stimulator of interferon genes (STING) activator and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent) to be administered may vary from subject to subject and may depend on several factors. It will therefore be understood that, in general, the exact dose used will be as determined by the attending physician and will depend not only on the subject's weight and route of administration, but also on the subject's age, and the severity of symptoms and / or risk of infection. In a first aspect, a particular amount of the pharmaceutical composition is administered as a single dose. Alternatively, a particular amount of the pharmaceutical composition is administered as more than one dose (e.g., 1-3 doses spaced 1-12 months apart). Alternatively, a particular amount of the pharmaceutical composition is administered as a unit dose over several times (e.g., 1-3 doses spaced 1-12 months apart). The pharmaceutical composition may be administered in an initial dose and at least one booster dose.

[0159] The present invention also relates to a kit of parts comprising at least two parts, a first part comprising a STING (stimulator of interferon genes) activator as disclosed herein, preferably a STING activator in vectorized form, i.e. a vector comprising (containing or expressing) a STING activator, and a second part comprising an anti-regulatory T cell (Treg) agent as disclosed herein, preferably an anti-intratumoral Treg agent, wherein the first and second parts of the kit are preferably in separate compartments.

[0160] In a particular embodiment, a first part of the kit comprises a STING (stimulator of interferon genes) activator that is a virus-like particle (VLP) and a second part of the kit comprises an anti-intratumor regulatory T cell (Treg) agent that is imatinib or a derivative or salt thereof.

[0161] In another particular embodiment, a first part of the kit comprises a STING (stimulator of interferon genes) activator that is a virus-like particle (VLP) and a second part of the kit comprises an anti-intratumor regulatory T cell (Treg) agent that is dasatinib or a derivative or salt thereof.

[0162] In another specific embodiment, a first part of the kit comprises a STING (stimulator of interferon genes) activator that is a virus-like particle (VLP) and a second part of the kit comprises an anti-intratumor regulatory T cell (Treg) agent that is an anti-CTLA-4 antibody with an IgG2 constant region or a mutated IgG1 constant region as described herein.

[0163] In another particular embodiment, a first part of the kit comprises a STING (stimulator of interferon genes) activator that is a virus-like particle (VLP) and a second part of the kit comprises an anti-intratumor regulatory T cell (Treg) agent that is an anti-CD25 antibody, particularly an anti-CD25 antibody with an IgG2 constant region or a mutated IgG1 constant region, as described herein.

[0164] In one embodiment, the part of the kit that includes the STING activator, preferably in vectorized form, is also in a form adapted for oral, intraperitoneal, intravenous or subcutaneous route, preferably for the subcutaneous route. The oral route of the composition of the invention is expected to elicit a well adapted immune response in the treatment of non-solid tumors or hematological malignancies.

[0165] In one aspect, the part of the kit comprising the anti-regulatory T cell agent, preferably an anti-intratumoral Treg cell agent, is in a form suitable for oral, intraperitoneal or intravenous route, preferably the intraperitoneal route.

[0166] Depending on the indication to be treated, the first part of the kit comprising a STING activator, preferably a vectorized STING activator, and the second part of the kit comprising an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg cell agent, are co-administered. In one embodiment, the first part of the kit comprising a STING activator agent, preferably a vectorized STING activator agent, and the second part of the kit comprising an anti-regulatory T cell (Treg) agent, in particular an anti-intratumoral Treg cell agent, are co-administered sequentially or simultaneously.

[0167] The present invention also relates to the kit of the present invention disclosed herein for use in treating cancer or STING-related disease or disorder as described herein above. In one aspect, "treat", "treating" or "treatment" in relation to cancer refers to alleviating cancer, eliminating or reducing one or more symptoms of cancer, slowing or eliminating the progression of cancer, and delaying the recurrence of the condition in a previously affected or diagnosed patient or subject. In another aspect, "treat", "treating" or "treatment" in relation to infectious disease refers to alleviating pain, fever, viral load, reducing biofilm formation, etc.

[0168] Also disclosed herein is the use of a kit of the present invention for the manufacture of a medicament for treating cancer or a STING-mediated disease or disorder, in particular a STING-mediated cancer, in a subject.

[0169] In another aspect, the present invention relates to a method for stimulating a therapeutic immune effect in a living mammalian subject, the method comprising the steps of reducing the immunosuppressive effect of Treg cells, preferably Treg cells present in a tumor, by administering to the living mammalian subject a therapeutic composition comprising i) a STING (stimulator of interferon genes) activator, preferably a vectorized STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as disclosed herein, thereby inducing a therapeutic effect against a disease in the living mammalian subject by reducing and / or inhibiting the activity of Treg cells.

[0170] In a further aspect, the present invention relates to a method of inhibiting or reducing immunosuppressive function in a subject, the method comprising the steps of: inhibiting or reducing the immunosuppressive activity of Treg cells, preferably Treg cells present in a tumor, by administering to a subject in need thereof a therapeutic composition comprising: i) a STING (stimulator of interferon genes) activator, preferably a vectorized form of STING activator, i.e. a vector comprising (containing or expressing) a STING activator, and ii) an anti-regulatory T cell (Treg) agent, preferably an anti-intratumoral Treg agent, as disclosed herein, whereby reducing or inhibiting the activity of Treg cells, preferably Treg cells within the TME, i.e. in and around the tumor, reduces or inhibits the immunosuppressive function in the subject.

[0171] "Suppression of immune tolerance" as referred to herein relates to suppressing the ability of the autoimmune system to tolerate the presence of disease antigens, including any natural tolerance and / or suppression of tumor evasion by the subject's immune system.

[0172] The method of the invention comprises administering a composition of the invention to a subject in need thereof, where administration of the composition may suppress tolerance of the disease and / or antigens of the disease by the immune system of the subject. Administration of the compositions described herein to a subject allows deactivating tumor evasion mechanisms in the subject, leading to better tumor eradication. Preferably, administration of the composition is inducible by CD4 + CD25 + FoxP3 + As referred to herein, "Treg" cells are cells that inhibit the activity of Treg cells, which are CD4 + CD25 + FoxP3 + and includes both nTregs and iTregs. Naive T cells as referred to herein are CD4 + CD25 - FoxP3 - These are T cells.

[0173] The method of the present invention includes suppressing Treg cells, particularly Treg cells present in tumors, in various ways. Suppressing the activity of Treg cells can be, for example, by inhibiting the conversion of naive T cells to iTreg cells. The method can include administering a composition as described herein that interferes with the conversion of naive cells to Treg cells, for example, by modulating the activity of FoxP3. FoxP3 is a transcription factor that is a marker of cells that can cause immunosuppressive activity. The absence or reversal of FoxP3 in a cell is an indication that the cell does not or no longer performs suppressive function.

[0174] The methods disclosed herein can be used in veterinary applications, such as canine and feline applications. If desired, the methods described herein can also be used for livestock, such as ovine, avian, bovine, porcine and equine breeding.

[0175] The following examples are provided to demonstrate and further illustrate certain preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Experimental section EXAMPLES

[0176] Materials and Methods cell line 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FBS, GIBCO) and 1% penicillin-streptomycin (GIBCO). THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). The cells used for the in vivo experiments were the mouse fibrosarcoma cell line MCA-OVA, originally purchased from ATCC. Tumor cells were grown in monolayer in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS, Biosera), 1% penicillin-streptomycin, and 1 mM β-mercaptoethanol (GIBCO) at 37°C and 5% CO2. Before reaching confluence, tumor cells were harvested with 0.05% trypsin, washed, and suspended in Hank's balanced salt solution (HBSS, GIBCO) for injection. The cell lines tested negative for several pathogens, including Mycoplasma ssp., by PCR.

[0177] mouse All animals were used according to protocols approved by the Institut Curie Animal Committee and were maintained under pathogen-free conditions in a barrier facility. C57BL / 6J mice were purchased from Charles River Laboratories. Mice were acclimated to the housing facility for at least 3 days. All experiments were initiated using female mice between 6 and 8 weeks of age.

[0178] cGAMP-VLP production before purification 7,500,000 293T cells in 150cm 2Cell culture flasks are seeded and incubated overnight. The next day, each flask is transfected with 13 μg mouse cGAS (pVAX1-cGAS), 8.1 μg HIV-1 GAGPOL (psPAX2), 3.3 μg VSVG (pVAX1-VSVG-INDIANA2), and 50 μL PEIpro (Ozyme, reference POL115-010) according to the manufacturer's instructions. The transfection mix was made in Opti-MEM (GIBCO). The morning after transfection, 52 mL of warm VLP production medium (293T culture medium supplemented with 10 mM HEPES and 50 μg / mL gentamicin) was added to the medium and the cells were incubated at 37 °C and 5% CO2 until the next day.

[0179] cGAMP-VLP recovery and purification through sucrose cushion The cGAMP-VLP-containing medium was harvested from the cells, centrifuged for 10 min at 200g at 4°C, and 0.45 μm filtered. 39 mL of the cGAMP-VLP-containing medium was gently overlaid on 6 mL of cold 20% sterile filtered endotoxin-free sucrose in six Ultra-Clear tubes (Beckman Coulter, ref 344058) and centrifuged for 1 h 30 min at 100,000g at 4°C. The medium and sucrose were gently aspirated, the pellet resuspended in cold PBS, transferred to one Ultra-Clear 13.2 mL (Beckman Coulter, ref 344059) and centrifuged again for 1 h 30 min at 100,000g at 4°C. The PBS was gently poured off and the pellet resuspended in an appropriate volume of cold PBS, typically 320 μL.

[0180] THP-1 activation and SIGLEC-1 expression measurements 50,000 THP-1 cells were seeded in 100 μL of medium in round-bottom 96-well plates and stimulated with 100 μL of cGAMP-VLP and soluble 2'3' cGAMP dilutions. Cells were incubated for 18-24 hours, stained with anti-human SIGLEC-1 (Miltenyi, ref 130-098-645), fixed with PFA 1%, and data were acquired using a BD FACS Verse cytometer.

[0181] Tumor growth Inject 5 x 10 cells in 100 µL of PBS into the right lower flank of female C57BL / 6J mice. 5 MCA-OVA cells were inoculated subcutaneously. Mice were monitored daily for weight loss, morbidity, and mortality. Tumors were monitored two or three times weekly. Tumors were monitored when ulceration occurred or when tumor volumes reached 2000 mm3. 3 Mice were humanely euthanized when tumor size reached 100 μg / kg. Tumor size was measured using digital calipers and tumor volume was calculated using the formula (length × width). 2 After tumor implantation, mice were randomly assigned to treatment groups using the Randommice software provided by Stimunity.

[0182] In vivo immunotherapy STING systemic (sc) therapy consisted of injecting cGAMP-VLPs (50 ng cGAMP) in 50 μL of PBS buffer. 3 Once the tumor had grown to between 0.5 and 1 cm, SC injections were started. A U-100 insulin syringe or equivalent: 0.33 mm (29G) x 12.7 mm (0.5 mL) was filled with the composition and all air bubbles were removed. The mouse was anesthetized with isoflurane. Beveling the needle towards the skin, the needle was shallowly inserted into the area adjacent to the tumor and the needle was moved subcutaneously until it reached the back side of the tumor (1 cm). The composition was slowly injected into this area close to the tumor. The needle was then gently removed to avoid backflow.

[0183] In vivo antibody Treg depletion For Treg depletion studies, MCA-OVA tumor-bearing mice were treated with 10 mg / kg imatinib chemotherapy (imatinib mesylate - SIGMA) by intraperitoneal route (ip) or received PBS on days 6, 7, 8, 9 and 10 after MCA-OVA melanoma implantation. Treg depletion was confirmed by FACS using tumors and spleens 72 hours after the last imatinib injection.

[0184] Ex vivo stimulation assay T cell responses were assessed by IFN-γ ELISPOT 10 days after the first sc injection of cGAMP-VLP or PBS. Mice were bled from the retro-orbital sinus and PBMCs were isolated from whole blood by lysing red blood cells. 2 × 10 5 PBMCs were seeded per well in RPMI medium containing 1% penicillin-streptomycin and stimulated overnight with medium as a negative control, 10 μg / mL p15 peptide (KSPWFTTL, SEQ ID NO: 2), 10 μg / mL OVA 257-264 peptide (OVA-1) (SIINFEKL, SEQ ID NO: 3), or 40 μg / mL OVA 265-280 peptide (OVA-2) (TEWTSSNVMEERKIKV, SEQ ID NO: 4). Spots were developed using mouse IFN-γ ELISPOT antibody (Diaclone) according to the manufacturer's instructions, and the number of spots was counted using an ImmunoSpot analyzer.

[0185] LEGENDplex™ Assays Serum samples collected 3 hours after the first (sc) injection were analyzed for inflammatory cytokines (IFN-α, IFN-β, TNF-α, IL-6, MCP-1, and IL-1β) using the Mouse Inflammation LEGENDplex™ Kit (BioLegend) according to the manufacturer's instructions. Data were acquired on a FACS Verse cytometer (BD Biosciences) and analyzed with BioLegend's LEGENDplex Data Analysis Software. Standard curve regression was used to calculate the concentration of each target cytokine.

[0186] Quantification and statistical analysis Data were analyzed with GraphPad Prism 8 software. All data are presented as mean ± standard error (SEM). Data for multiple groups and mean tumor volumes were analyzed by one-way ANOVA and Tukey's multiple comparison test. Survival curves were analyzed by the log-rank (Mantel-Cox) test. p ≤ 0.05 was considered significant.

[0187] result To optimize the immune response to subcutaneous (sc) administration of cGAMP-VLPs, the effect of imatinib on T cells and tumor growth was investigated.

[0188] Timeline for MCA-OVA tumor model Mice bearing single MCA-OVA melanoma flank tumors were treated subcutaneously with 50 ng of cGAMP-VLPs injected three times over 10 days, with or without daily intraperitoneal injections of imatinib (10 mg / kg) for 5 days, beginning on day 6 after tumor engraftment (Figure 1).

[0189] Synthesis of inflammatory cytokines Three hours after the first injection of cGAMP-VLP, the production of inflammatory cytokines was measured in serum by LEGENDplex (Figure 2). Serum from mice treated with cGAMP-VLP + imatinib showed a significant increase compared to imatinib alone, and elevated cytokine levels (IFN-α, IFN-β, IL-6) compared to cGAMP-VLP alone.

[0190] CD8 and CD4 T cell responses Four days after the third injection of cGAMP-VLPs, + / - The imatinib-treated group of mice received PBS + / -Compared with the group treated with imatinib, the group showed a significant increase in tumor-specific T cell response in peripheral blood (Figure 3). Imatinib treatment did not induce tumor-specific T cell response compared with the PBS group. In addition, the group treated with cGAMP-VLP + imatinib showed a higher T cell response than cGAMP-VLP alone, which was significant for the OVA-2 peptide.

[0191] Tumor growth monitoring The effect of treatment on tumor growth was monitored. Imatinib alone had no effect on tumors compared to the control group (Figure 4). cGAMP-VLPs injected alone by the sc route stabilized and delayed tumor growth for a short period of time compared to the control group. The combination of imatinib treatment with cGAMP-VLPs showed a significant increase in antitumor response compared to cGAMP-VLPs alone or imatinib alone, suggesting a synergistic mechanism of action of cGAMP-VLPs and imatinib (Figure 5).

[0192] Toxicity and survival of treated mice Mice treated with imatinib alone did not show a survival benefit compared to PBS treatment (Figure 6). cGAMP-VLP treatment significantly improved survival compared to PBS treatment. cGAMP-VLP + imatinib treatment significantly extended survival compared to cGAMP-VLP treatment alone. These results are consistent with a synergistic effect of cGAMP-VLP and imatinib on protection from tumor-induced death. Mice treated with imatinib did not show weight loss during all experiments, confirming the absence of toxicity from this treatment (Figure 7). We conclude that imatinib treatment demonstrates unexpected synergy with cGAMP-VLPs in generating systemic tumor-specific T cell responses and antitumor effects. EXAMPLES

[0193] Materials and Methods cell line 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FBS, GIBCO) and 1% penicillin-streptomycin (GIBCO). THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). The cells used for the in vivo experiments were the mouse fibrosarcoma cell line MCA-OVA, originally purchased from ATCC. Tumor cells were grown in monolayer in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS, Biosera), 1% penicillin-streptomycin, and 1 mM β-mercaptoethanol (GIBCO) at 37°C and 5% CO2. Before reaching confluence, tumor cells were harvested with 0.05% trypsin, washed, and suspended in Hank's Balanced Salt Solution (HBSS, GIBCO) for injection. The cell lines tested negative for several pathogens, including Mycoplasma species, by PCR.

[0194] mouse All animals were used according to protocols approved by the Institut Curie Animal Committee and were maintained under pathogen-free conditions in a barrier facility. C57BL / 6J mice were purchased from Charles River Laboratories. Mice were acclimated to the housing facility for at least 3 days. All experiments were initiated using female mice between 6 and 8 weeks of age.

[0195] cGAMP-VLP production before purification 7,500,000 293T cells in 150cm 2Cell culture flasks are seeded and incubated overnight. The next day, each flask is transfected with 13 μg mouse cGAS (pVAX1-cGAS), 8.1 μg HIV-1 GAGPOL (psPAX2), 3.3 μg VSVG (pVAX1-VSVG-INDIANA2), and 50 μL PEIpro (Ozyme, reference POL115-010) according to the manufacturer's instructions. The transfection mix was made in Opti-MEM (GIBCO). The morning after transfection, 52 mL of warm VLP production medium (293T culture medium supplemented with 10 mM HEPES and 50 μg / mL gentamicin) was added to the medium and the cells were incubated at 37 °C and 5% CO2 until the next day.

[0196] cGAMP-VLP recovery and purification through sucrose cushion The cGAMP-VLP-containing medium was harvested from the cells, centrifuged for 10 min at 200g at 4°C, and 0.45 μm filtered. 39 mL of the cGAMP-VLP-containing medium was gently overlaid on 6 mL of cold 20% sterile filtered endotoxin-free sucrose in six Ultra-Clear tubes (Beckman Coulter, ref 344058) and centrifuged for 1 h 30 min at 100,000g at 4°C. The medium and sucrose were gently aspirated, the pellet resuspended in cold PBS, transferred to one Ultra-Clear 13.2 mL (Beckman Coulter, ref 344059) and centrifuged again for 1 h 30 min at 100,000g at 4°C. The PBS was gently poured off and the pellet resuspended in an appropriate volume of cold PBS, typically 320 μL.

[0197] 2'3' cGAMP ELISA After extraction of cGAMP with methanol, an ELISA kit was used for quantification of 2'3'-cGAMP in VLPs according to the manufacturer's instructions (Cayman kit).

[0198] THP-1 activation and SIGLEC-1 expression measurements 50,000 THP-1 cells were seeded in 100 μL of medium in round-bottom 96-well plates and stimulated with 100 μL of cGAMP-VLP and soluble 2'3' cGAMP dilutions. Cells were incubated for 18-24 hours, stained with anti-human SIGLEC-1 (Miltenyi, ref 130-098-645), fixed with PFA 1%, and data were acquired using a BD FACS Verse cytometer.

[0199] LEGENDplex™ Assays Serum samples collected 3 hours after the first sc injection were analyzed for inflammatory cytokines (IFN-α, IFN-β, TNF-α, IL-6, MCP-1, and IL-1β) using the Mouse Inflammation LEGENDplex™ kit (BioLegend) according to the manufacturer's instructions. Data were acquired on a FACS Verse cytometer (BD Biosciences) and analyzed with BioLegend's LEGENDplex Data Analysis Software. Standard curve regression was used to calculate the concentration of each target cytokine.

[0200] Tumor growth Inject 5 x 10 cells in 100 µL of PBS into the right lower flank of female C57BL / 6J mice. 5 MCA-OVA cells were inoculated subcutaneously. Mice were monitored daily for morbidity and mortality. Tumors were monitored two or three times weekly. Tumors were monitored when ulceration occurred or when tumor volume exceeded 2000 mm. 3 Mice were humanely euthanized when tumor size reached 100 μg / kg. Tumor size was measured using digital calipers and tumor volume was calculated using the formula (length × width). 2 ) / 2. After tumor implantation, mice were randomly assigned to treatment groups using Randommice software provided by Stimunity. A few weeks after primary tumor relapse, tumor-free surviving mice were rechallenged with tumor cells in the contralateral uninjected flank. Age-matched naive mice were used as controls.

[0201] In vivo immunotherapy STING systemic (sc) therapy consisted of injecting cGAMP-VLPs (50 ng cGAMP per dose) in 50 μL of PBS buffer. 3 Once the tumor had grown to between 0.5 and 1 cm, SC injections were started. A U-100 insulin syringe or equivalent: 0.33 mm (29G) x 12.7 mm (0.5 mL) was filled with the composition and all air bubbles were removed. The mouse was anesthetized with isoflurane. Beveling the needle towards the skin, the needle was shallowly inserted into the area adjacent to the tumor and the needle was moved subcutaneously until it reached the back side of the tumor (1 cm). The composition was slowly injected into this area close to the tumor. The needle was then gently removed to avoid backflow.

[0202] In vivo antibody Treg depletion For Treg depletion studies, MCA-OVA tumor-bearing mice were treated with 200 μg of anti-CTLA4 monoclonal antibody (anti-mCTLA4-mIgG2a InvivoFit, Invivogen) or 200 μg of isotype control antibody (mouse IgG2a, BioXcell) by intraperitoneal route (ip) on days 6, 9 and 12 after MCA-OVA melanoma implantation. Treg depletion was confirmed by FACS using tumor, spleen, draining lymph node and blood samples 48 hours after the last antibody injection.

[0203] Ex vivo stimulation assay T cell responses were assessed by IFN-γ ELISPOT 10 days after the first sc injection of cGAMP-VLP or PBS. Mice were bled from the retro-orbital sinus and PBMCs were isolated from whole blood by lysing red blood cells. 2 × 10 5PBMCs were seeded per well in RPMI medium containing 1% penicillin-streptomycin and stimulated overnight with medium as a negative control, 10 μg / mL p15 peptide (KSPWFTTL, SEQ ID NO: 2), 10 μg / mL OVA 257-264 peptide (OVA-1) (SIINFEKL, SEQ ID NO: 3), or 40 μg / mL OVA 265-280 peptide (OVA-2) (TEWTSSNVMEERKIKV, SEQ ID NO: 4). Spots were developed using mouse IFN-γ ELISPOT antibody (Diaclone) according to the manufacturer's instructions, and the number of spots was counted using an ImmunoSpot analyzer.

[0204] Quantification and statistical analysis Data were analyzed with GraphPad Prism 8 software. All data are presented as mean ± standard error (SEM). Data for multiple groups and mean tumor volumes were analyzed by one-way ANOVA and Tukey's multiple comparison test. Survival curves were analyzed by the log-rank (Mantel-Cox) test. p ≤ 0.05 was considered significant.

[0205] result To study Treg depletion in tumors and to optimize the immune response to subcutaneous (sc) administration of cGAMP-VLPs, the effect of anti-CTLA4-mIgG2a (an isotype that specifically depletes / inhibits Tregs within the tumor microenvironment ("TME"), i.e., in and around the tumor, but not outside this area, or in other words, does not systemically deplete Tregs, e.g., does not deplete Tregs in the blood or non-draining lymphoid organs) on T cells and tumor growth was investigated.

[0206] Timeline for MCA-OVA tumor model Mice bearing single MCA-OVA melanoma flank tumors were treated subcutaneously with cGAMP-VLPs (50 ng cGAMP per dose) injected three times over 12 days with or without intraperitoneal injection of mouse IgG2a anti-CTLA4 (200 μg / mouse) starting on day 6 after tumor engraftment (FIG. 8).

[0207] Synthesis of inflammatory cytokines Three hours after the first injection of cGAMP-VLP, the production of inflammatory cytokines was measured in serum by LEGENDplex (Figure 9). Serum from mice treated with cGAMP-VLP showed a significant increase in cytokine levels (IL-6 and TNF-α in the mIg2a isotype control and anti-CTLA4-mIgG2 groups; IFN-α and MCP-1 in the mIgG2a isotype control group only) compared to the group that received PBS+ / -anti-CTLA4-mIgG2a.

[0208] Treg depletion Based on evidence demonstrating the contribution of intratumoral Treg cell depletion to the activity of immunomodulatory antibodies, we compared the effect of anti-CTLA4-mIgG2a on the frequency of Treg cells in the blood, spleen, and tumors of tumor-established mice (Figures 10 and 11). Administration of 200 μg of anti-CTLA4-mIgG2a on days 6, 9, and 12 after tumor challenge significantly increased the frequency of tumor-infiltrating Tregs (CD4 + Foxp3 + CD25 + ) cell frequency, as well as a decrease in the frequency of CD8 + T cells and CD4 + FoxP3 + T cell ratio and CD4 + FoxP3 - and CD4 + FoxP3 + Anti-CTLA4-mIgG2a resulted in an increase in the ratio of Treg cells to Tregs in the blood (FIG. 13) and spleen (FIG. 14). However, anti-CTLA4-mIgG2a failed to deplete Treg cells in the blood (FIG. 13) and spleen (FIG. 14). Their frequency remained comparable to that of untreated mice.

[0209] CD8 and CD4 T cell responses Four days after the third sc injection of cGAMP-VLP, the groups of mice treated with cGAMP-VLP and / or anti-CTLA4-mIgG2a showed a significant increase in OVA tumor antigen-specific T cell responses in peripheral blood compared to the PBS-treated group (Figure 15). Mice treated with cGAMP-VLP alone or anti-CTLA4-mIgG2a alone did not induce a significant response against the p15 tumor antigen. On the other hand, combining cGAMP-VLP with anti-CTLA4-mIgG2a induced a significant level of blood T cell response against p15 (Figure 16).

[0210] Tumor growth monitoring Next, we monitored the potential of anti-CTLA4-mIgG2a in the treatment of tumors. Anti-CTLA4-mIgG2a alone was able to induce stabilization or reduction of tumor growth compared to the control group (Figure 17). cGAMP-VLP injected alone by sc route stabilized and delayed tumor growth for a short period of time compared to the control group. However, cGAMP-VLP combined with anti-CTLA4-mIgG2a induced a strong anti-tumor response and complete tumor regression compared to cGAMP-VLP alone or anti-CTLA4-mIgG2a alone, indicating a synergistic mechanism of action of cGAMP-VLP and anti-CTLA4-mIgG2a (Figure 18).

[0211] Survival time of treated mice Treatment consisting of cGAMP-VLP and anti-CTLA4-mIgG2a eradicated established MCA-OVA tumors in 100% of the mice, resulting in long-term survival of over 90 days (FIG. 19).

[0212] memory immunity Full responder mice developed memory T cell responses capable of rejecting a second MCA-OVA challenge (FIG. 20).

[0213] We conclude that anti-CTLA4-mIgG2a, when used in combination with cGAMP-VLP, results in a synergistic anti-tumor effect due to selective depletion of Tregs from the tumor and its microenvironment (i.e., from the TME). This combination generates robust systemic tumor-specific T cell responses and sustained anti-tumor and memory immunity. EXAMPLES

[0214] Materials and Methods cell line 293T cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (FBS, GIBCO) and 1% penicillin-streptomycin (GIBCO). THP-1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS (GIBCO) and 1% penicillin-streptomycin (GIBCO). The cells used for the in vivo experiments were the mouse fibrosarcoma cell line MCA-OVA, originally purchased from ATCC. Tumor cells were grown in monolayer in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS, Biosera), 1% penicillin-streptomycin, and 1 mM β-mercaptoethanol (GIBCO) at 37°C and 5% CO2. Before reaching confluence, tumor cells were harvested with 0.05% trypsin, washed, and suspended in Hank's Balanced Salt Solution (HBSS, GIBCO) for injection. The cell lines tested negative for several pathogens, including Mycoplasma species, by PCR.

[0215] mouse All animals were used according to protocols approved by the Institut Curie Animal Committee and were maintained under pathogen-free conditions in a barrier facility. C57BL / 6J mice were purchased from Charles River Laboratories. Mice were acclimated to the housing facility for at least 3 days. All experiments were initiated using female mice between 6 and 8 weeks of age.

[0216] cGAMP-VLP production before purification 7,500,000 293T cells in 150cm 2 Cell culture flasks are seeded and incubated overnight. The next day, each flask is transfected with 13 μg mouse cGAS (pVAX1-cGAS), 8.1 μg HIV-1 GAGPOL (psPAX2), 3.3 μg VSVG (pVAX1-VSVG-INDIANA2), and 50 μL PEIpro (Ozyme, reference POL115-010) according to the manufacturer's instructions. The transfection mix was made in Opti-MEM (GIBCO). The morning after transfection, 52 mL of warm VLP production medium (293T culture medium supplemented with 10 mM HEPES and 50 μg / mL gentamicin) was added to the medium and the cells were incubated at 37 °C and 5% CO2 until the next day.

[0217] cGAMP-VLP recovery and purification through sucrose cushion The cGAMP-VLP-containing medium was harvested from the cells, centrifuged for 10 min at 200g at 4°C, and 0.45 μm filtered. 39 mL of the cGAMP-VLP-containing medium was gently overlaid on 6 mL of cold 20% sterile filtered endotoxin-free sucrose in six Ultra-Clear tubes (Beckman Coulter, ref 344058) and centrifuged for 1 h 30 min at 100,000g at 4°C. The medium and sucrose were gently aspirated, the pellet resuspended in cold PBS, transferred to one Ultra-Clear 13.2 mL (Beckman Coulter, ref 344059) and centrifuged again for 1 h 30 min at 100,000g at 4°C. The PBS was gently poured off and the pellet resuspended in an appropriate volume of cold PBS, typically 320 μL.

[0218] THP-1 activation and SIGLEC-1 expression measurements 50,000 THP-1 cells were seeded in 100 μL of medium in round-bottom 96-well plates and stimulated with 100 μL of cGAMP-VLP and soluble 2'3' cGAMP dilutions. Cells were incubated for 18-24 hours, stained with anti-human SIGLEC-1 (Miltenyi, ref 130-098-645), fixed with PFA 1%, and data were acquired using a BD FACS Verse cytometer.

[0219] Tumor growth Inject 5 x 10 cells in 100 µL of PBS into the right lower flank of female C57BL / 6J mice. 5 MCA-OVA cells were inoculated subcutaneously. Mice were monitored daily for weight loss, morbidity, and mortality. Tumors were monitored two or three times weekly. Tumors were monitored when ulceration occurred or when tumor volumes reached 2000 mm3. 3 Mice were humanely euthanized when tumor size reached 100 μg / kg. Tumor size was measured using digital calipers and tumor volume was calculated using the formula (length × width). 2 After tumor implantation, mice were randomly assigned to treatment groups using the Randommice software provided by Stimunity.

[0220] In vivo immunotherapy STING systemic (subcutaneous, sc) therapy consisted of injecting cGAMP-VLPs (50 ng cGAMP) in 50 μL of PBS buffer. 3 Once the tumor had grown to between 100 and 150 mm, SC injections were started. A U-100 insulin syringe or equivalent: 0.33 mm (29G) x 12.7 mm (0.5 mL) was filled with the composition and all air bubbles were removed. The mouse was anesthetized with isoflurane. With an angle towards the skin, the needle was shallowly inserted into the area adjacent to the tumor and the needle was moved subcutaneously until it was close to the tumor (1 cm). The composition was slowly injected into this area close to the tumor. The needle was then gently removed to avoid backflow.

[0221] In vivo antibody Treg depletion For Treg depletion studies, MCA-OVA tumor-bearing mice were treated with 150 μg of anti-CD25 IgG2a (KLC) monoclonal antibody (mIgG2a KLC anti-mCD25, Invivogen) or 150 μg of isotype control antibody (mouse anti-b-Gal-mIgG2a, Invivogen) by intraperitoneal route (ip) on days 6, 9 and 12 after MCA-OVA melanoma implantation. Treg depletion was confirmed by FACS using tumor and spleen samples 48 h after the final antibody injection.

[0222] Ex vivo stimulation assay T cell responses were assessed by IFN-γ ELISPOT 10 days after the first sc injection of cGAMP-VLP or PBS. Mice were bled from the retro-orbital sinus and PBMCs were isolated from whole blood by lysing red blood cells. 2 × 10 5 PBMCs were seeded per well in RPMI medium containing 1% penicillin-streptomycin and stimulated overnight with medium as a negative control, 10 μg / mL OVA 257-264 peptide (OVA-1) (SIINFEKL, SEQ ID NO: 3), or 40 μg / mL OVA 265-280 peptide (OVA-2) (TEWTSSNVMEERKIKV, SEQ ID NO: 4). Spots were developed using mouse IFN-γ ELISPOT antibody (Diaclone) according to the manufacturer's instructions, and the number of spots was counted using an ImmunoSpot analyzer.

[0223] Quantification and statistical analysis Data were analyzed with GraphPad Prism 8 software. All data are presented as mean ± standard error (SEM). Data for multiple groups and mean tumor volumes were analyzed by one-way ANOVA and Tukey's multiple comparison test. Survival curves were analyzed by the log-rank (Mantel-Cox) test. p ≤ 0.05 was considered significant.

[0224] result To study Treg depletion in tumors and to optimize the immune response to subcutaneous (sc) administration of cGAMP-VLPs, the effect of anti-CD25-mIgG2a (an isotype that specifically depletes / inhibits Tregs within the tumor microenvironment ("TME"), i.e., in and around the tumor, but not outside this area, or in other words, does not systemically deplete Tregs, e.g., does not deplete Tregs in the blood or non-draining lymphoid organs) on T cells and tumor growth was investigated.

[0225] Timeline for MCA-OVA tumor model Mice bearing single MCA-OVA melanoma flank tumors were treated subcutaneously with cGAMP-VLPs (50 ng cGAMP per dose) injected three times over 12 days with or without intraperitoneal injection of mouse IgG2a anti-CD25 (150 μg / mouse) starting on day 6 from tumor engraftment (FIG. 21).

[0226] Treg depletion Based on evidence demonstrating the contribution of intratumoral Treg cell depletion to the activity of immunomodulatory antibodies, we compared the effect of anti-CD25-mIgG2a on the frequency of Treg cells in the spleen and tumors of tumor-established mice (Figure 22). Administration of 150 μg of anti-CD25-mIgG2a on days 6, 9, and 12 after tumor challenge reduced the expression of tumor-infiltrating FOXP3. + Frequency of cells (total TCRb + CD4 + Anti-CD25-mIgG2a resulted in a statistically significant decrease in FOXP3 expression in spleen cells. + The cells were slightly and non-significantly depleted, and their frequency remained comparable to that of untreated mice.

[0227] CD8 and CD4 T cell responses Four days after the third sc injection of cGAMP-VLP, the group of mice treated with cGAMP-VLP showed a significant increase in OVA tumor antigen-specific T cell responses in peripheral blood compared to the PBS-treated group (Figure 23). Mice treated with anti-CD25-mIgG2a alone did not induce a significant response to the OVA 265-280 tumor antigen. On the other hand, combining cGAMP-VLP with anti-CD25-mIgG2a induced a significant level of blood CD4 T cell responses to the OVA peptide.

[0228] Tumor growth monitoring Next, we monitored the potential of anti-CD25-mIgG2a in the treatment of tumors. Anti-CD25-mIgG2a alone was able to induce stabilization or reduction of tumor growth compared to the control group (Figure 24). cGAMP-VLP injected alone by the sc route stabilized and delayed tumor growth for a short period of time compared to the control group. However, cGAMP-VLP in combination with anti-CD25-mIgG2a induced a significant anti-tumor response and tumor growth delay compared to the PBS control group, indicating a synergistic mechanism of action of cGAMP-VLP and anti-CD25-mIgG2a. Treatment consisting of cGAMP-VLP in combination with anti-CD25-mIgG2a declined established MCA-OVA tumors in mice, resulting in a delay of mouse death by more than 10 days compared to the control group.

[0229] We conclude that anti-CD25-mIgG2a, when used in combination with cGAMP-VLP, results in a synergistic anti-tumor effect due to selective depletion / inhibition of Tregs from the tumor and its microenvironment (i.e., from the TME). This combination results in a robust systemic tumor-specific T cell response and anti-tumor. [Explanation of symbols]

[0230] 5-FU 5-fluorouracil 5-FUdR 5-Fluorodeoxyuridine Ace-DEX Acetylated dextran ADCC antibody-dependent cytotoxicity AMA-1 Apical membrane antigen-1 APC antigen presenting cells ATP Adenosine Triphosphate BCRABL breakpoint cluster region Abelson BHK Baby Hamster Kidney BIV Bovine immunodeficiency virus BLV Bovine Leukemia Virus BNBC 6-Bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide c-di-AMP cyclic dimer adenosine monophosphate c-di-GMP cyclic dimer guanosine monophosphate CA Cytarabine CDN Cyclic dinucleotide CEA Carcinoembryonic antigen cGAMP cyclic guanosine monophosphate-adenosine monophosphate cGAS cyclic GMP-AMP synthase CHO Chinese hamster ovary CMA 10-Carboxymethyl-9-acridanone CTL cytotoxic T cells CTLA-4 Cytotoxic T lymphocyte antigen-4 CR Complete responder mice CSF-1R Colony-stimulating factor receptor CSP circumsporozoite protein DC dendritic cells DDR discoidin domain receptor DMXAA 5,6-Dimethyl-xanthenone acetic acid or 5,6-Dimethylxanthenone-4-acetic acid or Dimethyloxoxanthenyl acetic acid diABZI Diaminobenzimidazole or diaminobenzimidazole DMEM Dulbecco's Modified Eagle's Medium DSDP Dispirodiketopiperazine EAE Experimental autoimmune encephalomyelitis ENPP1 Ectonucleotide pyrophosphatase phosphodiesterase 1 ER endoplasmic reticulum FAA Flavone-8-acetic acid or flavone acetic acid FBS Fetal Bovine Serum FcyR Fcy receptor FeLV feline leukemia virus Foxp3 Fork Head Box P3 FIV Feline immunodeficiency virus GITR Glucocorticoid-inducible tumor necrosis factor receptor GFR Growth Factor GTP Guanosine triphosphate IBD inflammatory bowel disease ICB Immune Checkpoint Blockade HA Hyaluronic Acid HBSS Hanks Balanced Salt Solution HEK Human Embryonic Kidney HIV Human Immunodeficiency Virus HTLV human T-lymphotropic virus HPV Human papillomavirus id intradermal im intramuscular ip intraperitoneal iv IFN Interferon IRF3 Interferon regulatory factor 3 IP-10 Interferon-γ-inducible protein 10 LAG-3 Lymphocyte activation gene-3 LBD Ligand Binding Domain LSA Liver Stage Antigen LPEI Linear Polyethylenimine MBSA macrocycle-bridged STING agonist MLV Murine Leukemia Virus MMLV Moloney murine leukemia virus MSA-2 Benzothiophene oxobutanoic acid MTX methotrexate NDV Newcastle disease virus NFkB nuclear factor-κB NK Natural Killer nTreg Naturally occurring Treg OVA-1 OVA 257-264 peptide OVA-2 OVA 265-280 peptide PBAE Poly(beta-amino ester) PD-1 Program Death 1 PD-L1 Programmed Death Ligand 1 PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PE Pseudomonas endotoxin A Pf Exp1 Pf export protein 1 PEG-DBP Poly(ethylene glycol)-block-[(2-diethylaminoethyl methacrylate)-co-(butyl methacrylate)-co-(pyridyl disulfide ethyl methacrylate)] RPMI Roswell Park Memorial Institute RSV Rous sarcoma virus sc subcutaneous SALSA Pf antigen 2 sporozoite and liver stage antigen SAP2 secreted aspartyl proteinase 2 SCF Stem Cell Factor SIV simian immunodeficiency virus SSP2 sporozoite surface protein 2 STARP sporozoite threonine and asparagine-rich protein STING Stimulator of Interferon Genes Tconv conventional T cells TBK1 TANK-binding kinase 1 TIGIT T cell Ig and ITIM domains TIM-3 T-cell immunoglobulin mucin 3 TME Tumor Microenvironment Teff effector T cells Treg regulatory T cells VLP virus-like particle VSV Vesicular stomatitis virus

Claims

1. A pharmaceutical combination comprising i) a vector containing a stimulator of interferon genes (STING) activator and ii) an anti-regulatory T cell (Treg) agent selected from imatinib or a derivative or salt thereof; dasatinib or a derivative or salt thereof; an anti-CTLA-4 antibody having an IgG2 constant region or a mutated IgG1 constant region; an anti-LAG3 antibody; an anti-TIM-3 antibody; an anti-ICOS antibody; an anti-CD25 antibody; an anti-CCR4 antibody; an anti-CCR8 antibody; an anti-TNFR2 antibody; and any combination thereof.

2. The combination of claim 1, wherein the STING activator is selected from small molecules, in particular cyclic dinucleotides; nucleic acids encoding STING agonists; vectors or cells expressing STING or STING agonists; antibody-conjugated STING agonists; and inhibitors of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1).

3. The combination of claim 2, wherein the small molecule is a cyclic dinucleotide, such as cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), cyclic dimeric guanosine monophosphate (c-di-GMP) or cyclic dimeric adenosine monophosphate (c-di-AMP).

4. The combination according to any one of claims 1 to 3, wherein the vector is a vesicle, in particular a liposome; an exosome; a virus, such as an adenovirus or an oncolytic virus; a virus-like particle (VLP); a polymer; or a hydrogel.

5. The combination of claim 4, wherein the virus-like particle (VLP) comprises a lipoprotein envelope containing a viral fusogenic glycoprotein and contains cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), wherein the cGAMP is packaged in the virus-like particle.

6. The STING activator is selected from the group consisting of dithio-(RP,RP)-[cyclic[A(20,50)pA(30,50)p]] (ML RR-S2 CDA, also known as ADU-S100); MK-1454; 5,6-dimethyl-xanthenone acetic acid (DMXAA); flavone-8-acetic acid (FAA); 10-carboxymethyl-9-acridanone (CMA); α-mangostin (xanthone); ML RR-S2 CDG; ML RR-S2 cGAMP; 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC); dispirodiketopiperazine (DSDP); 2'3'-cG S A S The combination of claim 2, wherein the small molecule is selected from MP; macrocycle-bridged STING agonist (MBSA); E7766; benzothiophene oxobutanoic acid (MSA-2) derivatives; SB11285; diamide benzimidazole (diABZI); IMSA-101, TAK-676; CRD5500; TTI-10001; and SEL312-4787.

7. A combination according to any one of claims 1 to 3, wherein the mutation in the IgG1 constant region of the anti-CTLA4 antibody is located in the CH2 domain of the (Fc) constant region.

8. 8. The combination of claim 7, wherein the mutation is present at an amino acid position selected from 233, 234, 235, 236, 237, 238, 239, 243, 247, 256, 262, 267, 268, 270, 271, 280, 290, 292, 298, 300, 305, 324, 326, 328, 330, 332, 333, 334, 339 or 396 (EU index).

9. 9. The combination of claim 8, wherein each of the two CH2 domains of the Fc constant region comprises at least one mutation at an amino acid position selected from 239, 330 and 332, preferably an S239D, A330L and / or I332E substitution.

10. The first CH of the Fc constant region 2 The domain comprises at least one mutation at an amino acid position selected from 234, 235, 236, 239, 268, 270, 298, preferably a substitution selected from L234Y, L235Q, G236W, S239M, H268D, D270E and S298A, and the second CH of the Fc constant region 2 9. The combination of claim 8, wherein the domain comprises at least one mutation at an amino acid position selected from 270, 326, 330 and 334, preferably a substitution selected from D270E, K326D, A330M and K334E.

11. 4. A pharmaceutical combination according to any one of claims 1 to 3 for use in the prevention or treatment of cancer in a subject.

12. 12. The pharmaceutical combination for use according to claim 11, wherein the STING activator is to be administered to the subject by subcutaneous, oral, intraperitoneal, intramuscular, intradermal or intravenous route.

13. 12. The pharmaceutical combination for use according to claim 11, wherein said anti-Treg agent is to be administered to said subject by intraperitoneal, oral, subcutaneous or intravenous route.

14. 1. A kit comprising at least two parts, a first part comprising a vector comprising a Stimulator of Interferon Genes (STING) activator and a second part comprising an anti-regulatory T cell (Treg) agent, said first and second parts of the kit being preferably in separate compartments or containers.

15. The kit of claim 14, wherein the stimulator of interferon genes (STING) activator is a virus-like particle (VLP) of claim 5 and the anti-regulatory T cell (Treg) agent is either imatinib or a derivative or salt thereof, or an anti-CTLA-4 antibody having an IgG2 constant region or a mutated IgG1 constant region.

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