Compositions used in combination with immune modulatory treatments for treating cancers

Abstract

The present invention relates to the field of anticancer treatments. In particular, the present invention concerns therapeutic compositions for use in combination with immune modulatory treatments (IMTs) for treating cancers. The present invention also concerns kits of parts comprising said compositions and immune modulatory treatments. The present invention further concerns in vitro methods for screening said compositions.

Description

[0001] COMPOSITIONS USED IN COMBINATION WITH IMMUNE MODULATORY TREATMENTS FOR TREATING CANCERS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of anticancer treatments. In particular, the present invention concerns therapeutic compositions for use in combination with immune modulatory treatments (IMTs) for treating cancers. The present invention also concerns kits of parts comprising said compositions and immune modulatory treatments. The present invention further concerns in vitro methods for screening said compositions.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancer initiation, development and progression result from a complex relationship between genetically diseased cells, tumor microenvironment (TME), immune system and microbiota. Current therapeutic approaches comprise surgery, small molecules usage such as chemotherapy, radiation-based therapies, cellular therapies, vaccines and / or targeted therapies such as immunotherapy. Immune Checkpoint Inhibitors (ICI) are a novel class of immunotherapy drugs that have revolutionized patient care and significantly improved outcomes of patients with solid tumors. They became the standard of care for several solid tumors, including metastatic melanoma, Non-Small Cell Lung Cancer (NSCLC), Renal Cell Cancer (RCC) or colorectal cancer. Despite this therapeutic breakthrough, not all patients respond to ICIs and, in RCC and NSCLC, primary resistance rates range from 35 % to 44 % respectively. In advanced melanoma, 40 % to 65 % of patients progress despite the use of ICI (Elkrief et al. 2019 - Antibiotics are associated with decreased progression-free survival of advanced melanoma patients treated with immune checkpoint inhibitors, Oncoimmunology, 2019 Feb 18;8(4):e1568812).

[0006] To overcome immunotherapeutic treatment resistance, modulating immune system locally and systemically appears to be pivotal. Indeed, studies on TME have enlightened that the generation and regulation of anti tumor defences reside not only in primary lymphoid organs (bone marrow and thymus) and secondary lymphoid organs (lymph nodes, spleen, Peyer's patches, mucosal tissues, the nasal associated lymphoid tissue, adenoids, and tonsils) but also directly at the tumor site within small organized immune structures. Those cellular aggregates resembling Secondary Lymphoid Organs (SLO) are called Tertiary Lymphoid Structures (TLS) (Dieu-Nosjean et al. 2016 - Tertiary lymphoid structures, drivers of the anti-tumor responses in human cancers, Immunol Rev, 2016 May;271 (1 ):260-75). TLSs are composed of a large variety of organized cell types such as naive B cells, memory B cells, plasma cells, CD4+ and CD8+ T cells, T helper 1 , T follicular helper (TFH) cells and regulatory T (Treg) cells (intracellular forkhead box P3 + (FOXP3+) especially), follicular dendritic cells (FDC), dendritic cells (DC), neutrophils, macrophages and high endothelial venules (Sautes- Fridman et al, 2019 - Tertiary lymphoid structures in the era of cancer immunotherapy, Nat Rev Cancer, 2019 Jun;19(6):307-325). Maturation of TLS is a progressive phenomenon that has been described in the literature (Fridman et al, 2023 - Tertiary lymphoid structures and B cells: An intratumoral immunity cycle, Immunity, 2023 Oct 10;56(10):2254-2269). Two easily identifiable classes of TLS have been characterized. Immature TLS are aggregates of T and B cells with few DCs. In such structures, there is no evidence of induction of efficient immune reactions, and they are rather associated with T-cell-exhausted, inflamed, and / or immunosuppressive TME. In mature TLS (mTLS), mature DCs are in contact with T cells, and CD4+ programmed death 1 (PD-1) + C-X-C chemokine receptor type 5 (CXCR5) + TFH cells are in contact with B cells. The B cell zone contains a network of FDCs expressing CD21 in primary follicles as well as CD23 in secondary follicles (Fridman et al., 2023). In addition, naive or memory B cells can be activated by tumor-associated antigens presented by FDC, further triggered by signals delivered by TFH cells and mature into antibody-producing plasma cells (PC) or memory B cells. PC can also come from extrafollicular response zones as described in inflamed tissues. PCs travel into tumor beds in contrast to germinal center B cells and plasmablasts that are retained in TLS. This is also the case for memory T and B cells that can also migrate to peripheral lymphoid organs and to the bone marrow where they can be reactivated by a subsequent tumor recurrence or metastasis. Inside tumors, B cells can internalize, process and present tumor-associated antigens to CD4+ T cells through MHCII molecules upon recognition via their B cell receptor. Antigen presentation via MHCI may also be relevant: B cells in ovarian cancer can present antigenic peptides to CD8+ T cells via MHCI molecules. Based on the correlations between antigen-specific B cells in the tumor and IgG bound to tumor cells, it is appealing to postulate that when generated in response to tumor-associated antigens, PCs could produce antibodies that will bind tumor cells therefore inducing apoptosis upon macrophage activation by tumor bound IgG. The resulting immune complexes can be endocytosed by DCs, which will present MHCI- and MHCH-associated peptides to CD8+ and CD4+ T cells, respectively, thereby amplifying T cell immunity and diminishing the threshold for stimulation or reinvigoration by immunotherapies. Immune complexes can also be taken up by FDC that will present the released antigens to B cells in the germinal center of TLS, maintaining and amplifying B cell responses. It may also allow epitope spreading to other tumorspecific and self-antigens, sustaining the autoimmune responses often associated with efficient immunotherapies. Altogether, these mechanisms demonstrate that mature TLS might sustain an intra-tumoral immune loop capable of enhancing sensitivity of tumors to immune modulatory treatments (Fridman et al., 2023).

[0007] Accumulating evidences indicate that TLS play a key role in modulating tumoral invasion and metastasis. As an example, TLS density has a favourable impact on overall survival and disease-free survival of patients irrespective of the detection method on sections of tumoral tissues such as lung cancer, colorectal cancer, pancreatic cancer, oral squamous cell carcinoma and invasive breast cancer. In addition, presence of TLSs is correlated with a good response to immunotherapies and especially Immune Checkpoint Inhibitors (ICIs). TLSs and in particular B cell maturation have been associated to overall response to anti-PD1 . Furthermore, it has been suggested that TLS activation could improve immunotherapy and survival in melanoma patients (Cabrita et al, 2020 - Tertiary lymphoid structures improve immunotherapy and survival in melanoma, Nature, 2020 Jan;577(7791):561-565). Impact of some therapeutic modalities has been reported on TLS modulation. For example, anti-CD40 therapy in glioma revealing that immunotherapies can modulate TLS formation in the brain of a mouse model (Van Hooren et al, 2021 - Agonistic CD40 therapy induces tertiary lymphoid structures but impairs responses to checkpoint blockade in glioma, Nat Commun, 2021 Jul 5; 12(1 ):4127). Radiation therapy has also been reported to modulate TLS structures (Boivin et al, 2018 - Cellular Composition and Contribution of Tertiary Lymphoid Structures to Tumor Immune Infiltration and Modulation by Radiation Therapy, Front Oncol, 2018 Jul 9:8:256).

[0008] So far, only works led by Overacre-Delgoffe and colleagues demonstrated a maturation of TLS induced by a microorganism, a single strain of bacteria (Overacre- Delgoffe et aL, 2021 - Microbiota-specific T follicular helper cells drive tertiary lymphoid structures and anti-tumor immunity against colorectal cancer, Immunity, 2021 Dec 14;54(12):2812-2824. e4). By administering Helicobacter hepaticus (Hhep) to mice bearing tumors, they observed a better survival and a maturation of TLS. Nonetheless, this intestinal bacterium is known to induce local immune responses varying broadly depending on the immune status of the host. (Chai et aL, 2017 - Helicobacter species are potent drivers of colonic T cell responses in homeostasis and inflammation, Sci Immunol, 2017 Jul 21 ;2(13):eaal5068; Xu et aL, 2018 - c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont, Nature, 2018 Feb 15;554(7692):373-377). In healthy individuals, Hhep colonization triggers CD4+ T cells differentiation into Treg or TFH states, but under immunodeficient settings the presence of Hhep drives the differentiation of inflammatory Hhep-specific T helper 1 and Th 17 cells and the development of colitis (Kullberg et aL, 2003 - Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope, Proc Natl Acad Sci U S A, 2003 Dec 23;100(26):15830-5; Xu et aL, 2018 - c-MAF-dependent regulatory T cells mediate immunological tolerance to a gut pathobiont, Nature, 2018 Feb 15;554(7692):373-377). Examples of bacteria acting differently depending on their environment are numerous in the literature. Thus, using a single species of microorganism as a therapeutic agent in combination with immune modulatory treatments (IMTs) appears risky as patients receiving IMTs generally have already been treated with chemotherapy leading to an immunosuppressed status. In addition, many patients with solid tumors display an altered composition of gut microbiota, called dysbiosis, caused by several factors, including the disease itself and / or administered treatments such as antibiotics (Chaput et aL, 2017 - Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab, Ann Oncol, 2017 Jun 1 ;28(6):1368-1379; Nakatsu et aL 2015 - Gut mucosal microbiome across stages of colorectal carcinogenesis, Nat Commun, 2015 Oct 30:6:8727; Zitvogel L. et aL 2017 - Anticancer effects of the microbiome and its products, Nat Rev Microbiol, 2017 Aug;15(8):465-478). Dysbiosis has been reported in patients with solid tumors such as metastatic melanoma and colorectal cancer (Chaput et aL, 2017 - Baseline gut microbiota predicts clinical response and colitis in metastatic melanoma patients treated with ipilimumab, Ann Oncol, 2017 Jun 1 ;28(6):1368-1379; Nakatsu et aL 2015 - Gut mucosal microbiome across stages of colorectal carcinogenesis, Nat Commun, 2015 Oct 30:6:8727). Furthermore, dysbiosis often leads to intestinal colonization by pathogenic microorganisms leading to infections in this patient population.

[0009] Activating TLS maturation appears pivotal to increase the intra-tumoral immune loop and enhance sensitivity of tumors to IMTs. Nonetheless, patients in need for TLS maturation display an immunosuppressed state and most of the time dysbiosis. A complementary strategy to improve tumor sensitivity to IMTs would be to trigger the formation of TLS exclusively in the tumor microenvironment. There is therefore a compelling need for the development of safe therapeutic approaches that trigger TLS maturation, or even TLS formation in tumor microenvironment in subjects suffering from dysbiosis and / or presenting an immunosuppressed status. This, to increase overall immune response toward tumoral cells and improve both clinical outcome and safety of IMT treatments such as ICIs.

[0010] SUMMARY OF THE INVENTION

[0011] In this context, the inventors have shaped therapeutic microbial compositions composed of populations of microorganisms and demonstrated their ability to trigger TLS maturation alone or in combination with an IMT in in vitro or in vivo models of TLS. Advantageously, the therapeutic microbial compositions according to the invention are able to trigger TLS formation in the tumor microenvironment specifically. The therapeutic compositions according to the invention are able to trigger TLS formation, and maturation (i.e. induce B cell differentiation into plasmablast) via microbial antigen presentation by both dendritic cells and B cells to T follicular helper cells and subsequent activation of B cells. Furthermore, the inventors have surprisingly found that such therapeutic microbial compositions increase IMT efficacy in activating TLS maturation.

[0012] According to a first aspect, the present invention relates to a pharmaceutical composition comprising Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Faecalibacterium prausnitzii, Flavonifractor plautii, Ruminococcus bromii, Coprococcus comes, Roseburia intestinalis, Dysosmobacter sp. BX15 and Phocaeicola vulgatus, for use in combination with an immune checkpoint inhibitor (I Cl ) for treating a cancer in a patient, wherein the cancer is a solid tumor comprising a tertiary lymphoid structure (TLS) in its microenvironment.

[0013] The pharmaceutical composition of the invention may comprise a population of microorganisms that includes bacteria, viruses, archaea, bacteriophages, fungi or mixtures thereof. Preferably, the pharmaceutical composition of the invention comprises a population of microorganisms that includes bacteria and fungi.

[0014] The pharmaceutical composition of the invention may comprise a stool derived microorganism. Preferentially, the pharmaceutical composition of the invention comprises microorganisms that are exclusively stool derived microorganisms.

[0015] The pharmaceutical composition of the invention may advantageously trigger the maturation of tertiary lymphoid structures (TLS) in the tumor microenvironment. Still advantageously, the pharmaceutical composition of the invention may trigger the formation of tertiary lymphoid structure (TLS) in the tumor microenvironment.

[0016] The pharmaceutical composition of the invention may trigger an efficient IgG production by B cells in tertiary lymphoid structures (TLS). Said efficient IgG production by B cells may be observed in in vitro or in vivo models of tertiary lymphoid structures (TLS), preferentially a human tonsil-based in vitro model.

[0017] The pharmaceutical composition of the invention may induce expansion of at least one of the following cell populations: T cells specific to the microorganisms present within the pharmaceutical composition, tumor specific T cells, mature dendritic cells, ILC3 T cells, plasmablasts and / or T follicular helper (TFH).

[0018] The expansion of at least one cell populations as previously defined may be observed in in vitro or in vivo models of tertiary lymphoid structures (TLS), preferentially a human tonsil-based in vitro model.

[0019] The pharmaceutical composition of the invention may induce B cell differentiation into plasmablast via microbial antigen presentation by both dendritic cells and B cells to T follicular helper cells and subsequent activation of B cells.

[0020] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0021] - between 25% and 75% of Bacteroidetes,

[0022] - between 20% and 60% of Firmicutes,

[0023] - between 0.01% and 15% of Actinobacteria,

[0024] - between 0.01% and 10% of Proteo bacteria.

[0025] Preferentially, Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes represent between 60% and 100% of the bacterial population of the pharmaceutical composition as previously defined.

[0026] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0027] - between 2% and 60% of Bacteroidaceae,

[0028] - between 1% and 35% of Ruminococcaceae,

[0029] - between 1% and 25% of Lachnospiraceae,

[0030] - between 0.01% and 5% of Clostridiaceae,

[0031] - at least 7 bacterial subpopulations selected from Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae.

[0032] Preferentially, Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae represent between 40% and 95% of the bacterial population of the pharmaceutical composition according to the above embodiment.

[0033] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0034] - between 2% and 60% of Bacteroides,

[0035] - between 0.01% and 20% of Phocaeicola,

[0036] - between 0.01% and 20% of Faecalibacterium, - between 0.01 % and 5% of Blautia,

[0037] - between 0.01 % and 5% of Flavonifractor,

[0038] - between 0.01 % and 5% of Gemmiger,

[0039] - between 0.01 % and 5% of Dorea,

[0040] - at least 12 bacterial subpopulations selected from: Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania.

[0041] Preferentially, Phocaeicola, Faecalibacterium, Bacteroides, Flavonifractor, Blautia, Dorea, Gemmiger, Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania represent between 40% and 95% of the bacterial population of the pharmaceutical composition as previously defined.

[0042] Preferably, the immune checkpoint inhibitor (ICI) to be used with the pharmaceutical composition of the invention may be a drug blocking one of the following targets: PD-1 , PD-L1 , PD-L2, CD27, CD28, CD40, CD122, CD137, 0X40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3 (such as Relatlimab), NOX2, TIM- 3, VISTA, SIGLEC7 or TIGIT.

[0043] More preferably, the immune checkpoint inhibitor (ICI) to be used with the pharmaceutical composition of the invention may be a drug blocking one of the following targets: PD-1 , PD-L1 , PD-L2, CTLA-4, TIM-3 or TIGIT.

[0044] Still more preferably, the immune checkpoint inhibitor (ICI) to be used with the pharmaceutical composition of the invention may be an antibody targeting PD-1.

[0045] Preferably, the pharmaceutical composition for use according to the invention may have followed at least a manufacturing step of homogeneization with a cryoprotectant and a manufacturing step of freezing below 40°.

[0046] Preferably, the pharmaceutical composition for use according to the invention may be used in combination with an ICI for treating a lung cancer or a skin cancer.

[0047] Preferably, the pharmaceutical composition for use according to the invention may be administered 1 , 2, 3 or 4 times during an administration cycle of at least 5 days. The treatment may comprise one or several administration cycle.

[0048] More preferably, one administration cycle treatment may last 5 days.

[0049] According to some alternatives, one administration cycle may last 10, 15, 20, 25 or 30 days, and comprise 1 , 2, 3 or 4 administrations of the pharmaceutical composition. Still preferably, the pharmaceutical composition for use according to the invention may be administered less than 5 times per administration cycle.

[0050] Still more preferably, the pharmaceutical composition for use according to the invention may be administered before the immune checkpoint inhibitor (I Cl ).

[0051] According to another aspect, the invention pertains to a kit of parts comprising a pharmaceutical composition according to the invention and an immune modulatory treatment for simultaneous or sequential administration to a patient.

[0052] According to another aspect, the invention concerns an in vitro method for screening a composition comprising a population of microorganisms, the method comprising steps of administering said composition to a tertiary lymphoid structure (TLS) in vitro model, optionally with an immune modulatory treatment (IMT), and assessing IgG production by B cells within said TLS in vitro model.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 : TLS maturation assessment in bulk tonsil-derived immune cell assay and TFH / B cell co-culture assay

[0055] TLS maturation has been evaluated in two in vitro models of TLS based on human tonsils. In Figure 1A, the whole immune cell population was isolated from tonsils and exposed to Composition 5 - Cult with or without Nivolumab. In this model, T Cells, B cells, DCs and many other immune cell populations are present. IgG production by differentiated B cells (plasmablasts) was assessed as a marker of TLS maturation. Whatever the composition dilution (1 / 1000 or 1 / 100) IgG production is observed. In addition, combination of Nivolumab and the compositions 1 / 1000 and 1 / 100 allows to increase Nivolumab efficiency. Significance was reached for the 1 / 100 condition. These results demonstrate a TLS maturation triggered by the composition of the present invention. In Figure 1B, solely TFH cells and B cells were isolated from tonsils and they were exposed to Composition 5 - Cult with or without Nivolumab. As for the bulk model (Figure 1A), IgG production is assessed as a marker of TLS maturation. Maturation of TLS is also triggered in each condition but in a lesser extent. Significance was reached for the 1 / 100 condition. DCs are missing in this setup, and are not able to present microbial antigens to activate TFH cells. Nonetheless, memory B cells are present in the B cell population isolated from tonsils and observed IgG production corresponds to preprimed memory B cells that are capable of antigen presentation directly to TFH cells with no need of DCs. These two experiments allow to demonstrate that composition of the invention is able to trigger TLS maturation via two different mechanisms, B cell differentiation into plasmablast either via microbial antigen presentation by DCs to TFH cells and subsequent activation of naive B cells or via microbial antigen presentation by pre-primed memory B cells to TFH cells and subsequent activation of pre-primed memory B cells. Control experiments with SEB are not shown.

[0056] Figure 2: TLS formation and maturation assessment in a murine lung tumor model

[0057] TLS formation and maturation was assessed in a mouse lung tumor model of which the set-up is displayed on Figure 2. 2 days before tumoral cells administration (TC-1-luc cells), mice were treated with antibiotics mix to make room in their guts. After antibiotics treatment, mice were split in 4 groups:

[0058] G1 : 5 treatments with a PBS solution (once a day during 5 days) as for the group 3. This condition was used as a negative control,

[0059] - G2: A single treatment with Composition 2 - Pool

[0060] G3: 5 treatments with Composition 2 - Pool (once a day during 5 days) G4: 5 treatments with LPS without administration of TC-1 tumoral cells. This goup was used a positive control of TLS formation and maturation.

[0061] Animal weight was followed from D2 to the end of the experiment at least twice a week, tumoral growth was analyzed from D5 to the end of the experiment at least twice a week, and mice were sacrificed at D15.

[0062] Figure 3: Tumoral growth in vivo imaging

[0063] Figure 3 displays images of tumoral TC-1 luc cells present in different groups of mice. Solely the group 4 is not represented as no TC-1 cells were administrered in this condition. A In-Vivo Imaging System (MS) was used to analyze TC-1 luciferin production. Tumoral cells are detected in mice lungs in each condition with a stronger presence in the G3. T umoral cells are also observed at the TC-1 cells injection site.

[0064] Figure 4: TLS formation and maturation analysis

[0065] Figure 4 displays tumoral tissue sections stained by ImmunoHistoChemistry (IHC). 3 panels display staining of two immune markers present in TLS: B220 and CD3 and the last panel is a Hematoxylin I Eosin coloration. (B220 is a marker of B lymphocytes including pro-, mature and activated B cells and CD3 is a marker of T lymphocytes). TLS formation was observed for each group but was sparse in G1. In addition, fewer TLS structures were observed in G1 compared to G2 or G3. Besides, the main differences between G2 and G3 were the quasi-systematic presence of TLS distant to tumoral nodules (TN) and an elevated number of TLS structures in G3, whereas in G2, TLS were solely localized around TNs and were fewer than in G3. 1 to several TLS around the same TN could be observed in both G2 and G3.

[0066] DETAILED DESCRIPTION

[0067] The disclosure provides a pharmaceutical composition comprising a population of microorganisms, for use in combination with an immune modulatory treatment (IMT) for treating a cancer in a patient, wherein the cancer is a solid tumor comprising a tertiary lymphoid structure (TLS) in its microenvironment.

[0068] The inventors provide experimental evidences that the therapeutic microbial composition of the invention can trigger tertiary lymphoid structure maturation. Accordingly, the inventors provide experimental support demonstrating that TLS maturation is triggered by the therapeutic microbial composition of the invention either via microbial antigen presentation by DCs to TFH cells and subsequent activation of naive B cells, or via microbial antigen presentation by pre-primed memory B cells to TFH cells and subsequent activation of pre-primed memory B cells. Hence, the use of therapeutic microbial composition of the invention able to trigger maturation of TLS is validated as a robust strategy to increase tumor sensitivity to immune modulatory treatment and improve clinical outcome and safety of said I MT treatments.

[0069] By “Tertiary Lymphoid Structures” (TLS), it is preferably referred to small organized immune structures resembling secondary lymphoid organs (Dieu-Nosjean et al. 2016). Those cellular aggregates resembling SLO are called Tertiary Lymphoid Structures (TLS) (Dieu-Nosjean et al. 2016). TLSs are most commonly found in the regions associated with chronic inflammation, infectious diseases, autoimmune diseases, transplanted organs, and tumor sites. TLSs are composed of a large variety of organized cell types such as naive B cells, memory B cells, plasma cells, CD4+ and CD8+ T cells, T helper 1 , T follicular helper (TFH) cells and regulatory T (Treg) cells (intracellular forkhead box P3 + (FOXP3+) especially), follicular dendritic cells (FDC), dendritic cells (DC), neutrophils, macrophages and high endothelial venules (Sautes- Fridman et al, 2019). Cell types present within the TLS and their activation status depends on maturation stage. For example, two easily identifiable classes of TLS have been characterized. Immature TLS (iTLS) are aggregates of T and B cells with few DCs. In such structures, there is no evidence of induction of efficient immune reactions, and they are rather associated with T-cell-exhausted, inflamed, and / or immunosuppressive TME. In mature TLS (mTLS), mature DCs are in contact with T cells, and CD4+ programmed death 1 (PD-1 ) + C-X-C chemokine receptor type 5 (CXCR5) + TFH cells are in contact with B cells. The B cell zone contains a network of FDCs expressing CD21 in primary follicles as well as CD23 in secondary follicles (Fridman et aL, 2023).

[0070] Advantageously, in the present invention, TLS maturation is characterized by an increase in IgG production by B cells present within TLS after treatment with the pharmaceutical composition of the present invention in combination with the IMT. It can also be characterized by the presence of T cells specific for microorganisms present in the composition of the present invention and / or an increase in mature DCs, ILC3 T cells and / or TFH in the TLS.

[0071] By “Immune modulatory treatment” (IMT) it is preferably referred to a therapeutic treatment aimed at modulating immune system activity. In the case of solid tumors, IMTs are used to increase host immune response to cancer and control inflammation, mainly via immune cell regulation. A wide variety of approaches have been developed and IMTs according to the invention include, but are not limited to, hormonal therapy, monoclonal antibodies, bispecific antibodies, nanobodies, aptamers, molecules targeting neoantigens, immune checkpoint inhibitors, immuno-conjugates, cytokines (including interferons such as IFNa; interleukins such as IL-2, IL-11 , G-CSM, GM-CSF), immunomodulatory small molecules targeting STING or IDO, nucleotides, polynucleotides, cancer-killing viruses, therapeutic vaccines (e.g., Sipuleucel-T (Provenge®)), adoptive cell transfers (T-cells, natural killer cells, macrophages), modified dendritic cell transfers and T-cell engagers. Combination of different IMTs is also covered within the present invention.

[0072] The immune modulatory treatment used in combination with the pharmaceutical composition according to the invention may be selected from immune checkpoint inhibitors (ICIs) such as an anti-PD1 antibody, immuno-conjugates, cytokines, immunomodulatory small molecules targeting STING or IDO, nucleotides, polynucleotides, therapeutic vaccines, allogenic or autologous adoptive cell transfers, modified dendritic cell transfers, T-cell engagers, and combination thereof, preferably from immune checkpoint inhibitors (ICIs) such as an anti-PD1 antibody. Advantageously, the immune modulatory treatment may be an ICI, preferentially an anti- PD-1 antibody.

[0073] The pharmaceutical composition of the invention may increase tumor sensitivity to an Immune Modulatory Treatment (IMT).

[0074] The pharmaceutical composition of the invention may comprise a population of microorganisms that includes bacteria, viruses, archaea, bacteriophages, fungi or mixtures thereof. Preferably, the pharmaceutical composition of the invention may comprise a population of microorganisms that includes bacteria and fungi.

[0075] The pharmaceutical composition of the invention may comprise a population of microorganisms that comprises a stool derived microorganism. Preferably, the pharmaceutical composition of the invention may comprise a population of microorganisms that is exclusively stool derived microorganisms.

[0076] The pharmaceutical composition may comprise a population of microorganisms that does not contain detectable levels of Helicobacter hepaticus. Preferably, detection level of Helicobacter hepaticus is made using shotgun sequencing data and an appropriate analysis pipeline. A potential approach to be used for such analysis may be the following:

[0077] (a) DNA isolation and shotgun metagenomic sequencing. Genomic DNA may be extracted from the fecal samples using the Machery Nagel NucleoSpin Soil kit. A shotgun sequencing library may be constructed for each DNA sample and libraries are then sequenced in a 2 x 150bp NovaSeq2500 run (Illumina). Positive and negative controls may be added throughout the process to validate the successful completion of each step.

[0078] (b) Shotgun bioinformatics analyses performed on an appropriate pipeline. A potential pipeline example may be: quality filtering using Trimmomatic, host sequence decontamination using Bowtie2. To ensure comparability, all samples may be rarefied to the same sequencing depth. Taxonomic profiling may be performed with Kraken2 and the RefSeq genomic database (2020 release). A taxon can be considered as detected if its relative abundance is greater than 0.05%. Alternatively, a taxon can be considered as detected if its relative abundance is greater than 0.01 %.

[0079] Microorganisms present in the pharmaceutical compositions of the invention may be capable of producing some or all of the following metabolites: acetate, butyrate, propionate, valerate, indole, indole 3-acetic acid, indole 3-propionic acid, indole-3-carboxaldehyde, indole-3-lactic acid, cholic acid, hyocholic acid, beta- Muricholic acid, chenodeoxycholic acid, deoxycholic acid, 7-Ketodeoxycholic acid, omega-Muricholic acid, ursocholic acid, ursodeoxycholic acid, 7-Ketolithocholic acid, 12- Ketolithocholic acid, hyodeoxycholic acid, 3-Oxocholic acid, dioxolithocholic acid, lithocholic acid, nordeoxycholic acid, norursodeoxycholic acid, trimethylamine (TMA), lysine, choline, y-Aminobutyric acid (GABA), lactate, adenosine, kynurenine, tryptophan, serotonin, inosine, nicotinic acid, and combination thereof. Metabolite detection is preferentially performed by mass spectrometry e.g. LC-MRM / MS.

[0080] Microorganisms presents in the pharmaceutical composition of the invention may be able to modulate the concentration levels of some of the following molecules: TNF-a, IL-13, IL-4, IL-10, IL-6, IL-2, TNF-p, IFN-y, IL-17A, IL-12p70, APRIL, BAFF and sCD40L in vitro and / or in vivo. In vivo, this modulation may occur locally in the TME and / or within the TLS. APRIL, BAFF and CD40L are markers of activation and survival of B cells. IL-10 and TGF-beta1 are produced by regulatory B cells. TNF-alpha, TNF-beta, IFN-gamma and IL-12p70 are produced by Be1 effector cells. IL-2, IL-4, IL-6, IL-13 and TNF-alpha are produced by Be2 effector cells. All this panel of molecules allows to characterize some of the members of the B cell population. Preferentially, modulation of concentration levels of above-mentioned molecules occurs locally in the TME, and more preferentially within the TLS.

[0081] The pharmaceutical composition of the invention may trigger an efficient IgG production by B cells in an in vitro model of tertiary lymphoid structures (TLS) such as a human tonsil-based in vitro model.

[0082] The pharmaceutical composition of the invention may also induce the expansion of at least one of the following cell populations:

[0083] T cells specific to the microorganisms present within said pharmaceutical composition, tumor specific T cells, mature dendritic cells, ILC3 T cells, plasmablasts and / or T follicular helper (TFH).

[0084] The pharmaceutical composition of the invention may induce B cell differentiation into plasmablast via microbial antigen presentation by both dendritic cells and B cells to T follicular helper cells and subsequent activation of memory B cells.

[0085] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0086] - between 25% and 75% of Bacteroidetes,

[0087] - between 20% and 60% of Firmicutes,

[0088] - between 0.01% and 15% of Actinobacteria,

[0089] - between 0.01% and 10% of Proteo bacteria.

[0090] Preferentially, Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes may represent between 60% and 100% of the bacterial population of the pharmaceutical composition according to the above aspect.

[0091] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0092] - between 2% and 60% of Bacteroidaceae,

[0093] - between 1% and 35% of Ruminococcaceae,

[0094] - between 1% and 25% of Lachnospiraceae,

[0095] - between 0.01% and 5% of Clostridiaceae,

[0096] - at least 7 bacterial subpopulations selected from Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae.

[0097] Preferentially, Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae represent between 40% and 95% of the bacterial population of the pharmaceutical composition as previously defined.

[0098] The pharmaceutical composition of the invention may comprise a bacterial population that includes:

[0099] - between 2% and 60% of Bacteroides,

[0100] - between 0.01 % and 20% of Phocaeicola,

[0101] - between 0.01% and 20% of Faecalibacterium,

[0102] - between 0.01 % and 5% of Blautia,

[0103] - between 0.01 % and 5% of Flavonifractor,

[0104] - between 0.01 % and 5% of Gemmiger,

[0105] - between 0.01 % and 5% of Dorea,

[0106] - at least 12 bacterial subpopulation selected from: Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hu ng atelia and Holdemania.

[0107] Preferentially, Phocaeicola, Faecalibacterium, Bacteroides, Flavonifractor, Blautia, Dorea, Gemmiger, Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania may represent between 40% and 95% of the bacterial population of the pharmaceutical composition as previously defined.

[0108] According to another aspect, the invention pertains to a kit of parts comprising a pharmaceutical composition according to the invention and an immune modulatory treatment for simultaneous or sequential administration to a patient.

[0109] Also provided are kits which includes a pharmaceutical composition comprising a population of microorganisms as described herein, and an immune modulatory treatment (IMT). Optionally, the kits can also include instructions, e.g., comprising administration schedules, to allow a practitioner (e.g., a physician, nurse, or patient) to administer the composition as described herein to a patient having cancer (e.g., a solid tumor). Optionally, the kits may include multiple packages of the singledose pharmaceutical compositions each containing an effective amount of the pharmaceutical composition comprising a population of microorganisms as described herein and / or an immune modulatory treatment for a single administration in accordance with the methods or uses provided according to the present invention. Microbial composition:

[0110] The pharmaceutical composition (also called microbial composition or therapeutic microbial composition) to be used in combination with the I MT according to the invention may comprise at least a microorganism derived from stool, stool being preferably of mammalian origin, more preferably of human origin. The microbial composition may include exclusively stool derived microorganisms. These stool derived microorganisms can be bacteria, viruses, archaea, bacteriophages, fungi (comprising yeasts) or mixtures thereof. Stool derived microorganisms according to the invention include cultivated microorganisms, non-cultivated microorganisms and mixtures thereof. Furthermore, they include also isolated microorganisms, non-isolated microorganisms and mixtures thereof.

[0111] The pharmaceutical composition (also called microbial composition or therapeutic microbial composition) of the invention may comprise a population (or ecosystem) of microorganisms comprising at least a stool derived microorganism. This microorganism may be isolated alone or isolated as a fraction of microorganisms from stools e.g. spores and vegetative forms. The stool derived microorganism can further have been cultivated or not in a bioreactor between stool donation and pharmaceutical product formulation steps. Mixing cultivated and non-cultivated forms of the stool derived microorganism is also part of the invention. The stool derived microorganism may be added to a composition comprising microorganisms from non-fecal origin such as food, vegetal, environmental, skin and other animal mucosae. The term “animal” means human and non-human animals in this context. Microorganisms from non-fecal origin can be cultivated or not in a bioreactor between collection and mixing with the stool derived microorganism. Non fecal microorganisms and the stool derived microorganism can be cultivated together or not after mixing. Those microorganisms from non-fecal origin can be isolated or not before mixing with the stool derived microorganism. Such compositions according to the invention comprising non fecal microorganisms may comprise from one to several hundreds of distinct individual species. The stool derived microorganism may be frozen and banked alone or part of a composition comprising microorganisms from non-fecal origin, in a stool sample library.

[0112] The microbial composition of the invention may include exclusively stool derived microorganisms. Microorganisms can be obtained from stool donations with techniques known by the skilled person such as filtration, centrifugation and isolation. Those microorganisms can be planktonic, can form biofilms or can be in a vegetative form such as a spore. Mixtures of microorganisms thereof are also encompassed according to the present invention. Microorganisms can come from a single donor or from multiple donors. In this later case, stools from different donors are pooled together. Microorganisms can also come from several donations of the same donor, pooled together. Microorganisms can be cultivated or not in a bioreactor between stool donation and pharmaceutical product formulation steps. Microorganisms can have been isolated or not before product formulation. Combination of isolation and cultivation steps to generate the composition to be formulated can also be covered by the present invention. Stool derived microorganisms may be frozen and banked alone or part of a composition comprising multiple individual species, in a stool sample library.

[0113] Microorganisms present in the pharmaceutical compositions of the invention may include bacteria, viruses, archaea, bacteriophages, fungi (including yeasts) or mixtures thereof. Microorganism populations or ecosystems comprised in composition according to the present invention may be complex and may gather a wide diversity of individual species to mimic as closely as possible a healthy gut flora and bring metabolic and immune functions missing in each individual subject. Compositions of the invention may comprise at least bacteria and fungi.

[0114] Stool derived microorganisms may include cultivated microorganisms, non-cultivated microorganisms and mixtures thereof. Cultivated microorganisms can be grown in a single bioreactor or several bioreactors. The term “bioreactor” preferably means every way of cultivating microorganisms known by the skilled person such as solid, liquid cultivation or liquid cultivation with particles for microorganism adhesion. If multiple bioreactors are needed, they can be used in parallel or in series. Microorganisms cultivated in multiple bioreactors can be similar in terms of taxonomy or different. The different bioreactors can comprise from one to several hundreds of distinct individual species of microorganisms at the same time. Pooling of compositions arising from all these cultivation approaches, whatever the moment in the manufacturing process, may also be covered by the present invention. Non cultivated microorganisms may be directly obtained from stool donations with techniques known by the skilled person such as filtration, centrifugation and isolation. Those microorganisms can be planktonic, can form biofilms or can be in a vegetative form such as a spore. Mixtures of microorganisms thereof may also be encompassed according to the present invention. Cultivated or not, microorganisms of the present invention can come from a single donor or from multiple donors. In this later case, stools from different donors may be pooled together and microorganisms may be extracted from such pool. Microorganisms can also come from several donations of the same donor, pooled together. The composition of the invention can also comprise mixes of cultivated and non-cultivated microorganisms. Microorganisms may be frozen and banked alone or part of a composition comprising multiple individual species, in a stool sample library.

[0115] Stool derived microorganisms may include isolated microorganisms, nonisolated microorganisms and mixtures thereof. Isolated microorganism preferably refers to a microorganism that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and / or produced, prepared, purified, and / or manufactured. Isolated microorganisms may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which it was initially associated. Isolated microorganisms may be more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. The term "pure" preferably means that the complex community of microorganisms is substantially free of other components. When referring to a microorganism, the terms "purify," "purifying" and "purified" preferably refer to a microorganism that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microorganism may be considered purified if it is isolated at or after production, such as from a material or environment containing the microorganism, and a purified microorganism may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered "isolated." Preferably, the microorganism is more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. The microorganism can be independently purified from one or more complex community of microorganisms produced and / or can be present in the material or environment containing the microorganism. The microorganism may be purified from residual habitat products. Non limiting examples of purification or isolation methods comprise, but are not limited to, filtration, centrifugation, dilution, solid or liquid cultivation. Non isolated microorganisms may be directly obtained from stool donations with techniques known by the skilled person such as filtration, centrifugation, isolation. Those microorganisms can be planktonic, can form biofilms or can be in a vegetative form such as a spore. The composition of the invention can also comprise mixes of isolated and non-isolated microorganisms. Microorganisms may be frozen and banked alone or as part of a composition comprising multiple individual species in a stool sample library.

[0116] Microorganisms from the microbial composition of the invention that engraft in subjects’ gut may be different from a subject to another. Microorganism selection is advantageously operated by subject’s gut depending on its needs in microorganisms and missing functions to be recovered to restore a symbiotic microbiota. One or several different microorganisms can advantageously engraft within subject’s gut. Advantageously, while each individual has a unique microbiota composition, similar main physiological functions are fulfilled thanks to the capacity of different microorganisms to perform the same chemical reaction. Thus, needs in microorganisms may differ from a subject to another. Therapeutic microbial compositions of the present invention may comprise a rich and diverse equilibrium of microorganism composed of different species, in quantities reflecting those of a healthy gut flora, allowing subject’s gut to pick up species I functions it needs for recovering a healthy gut status. Advantageously, the therapeutic microbial composition of the present invention allows subject’s gut to select microorganisms able to perform functions that are missing in preexisting flora. In addition, advantageously, quantities of microorganisms present in the composition correspond to those found in healthy gut consortia allowing a controlled colonization of subject’s gut. Advantageously, at least 10 different species not present within subject’s gut before treatment with the therapeutic microbial composition may engraft in subject’s gut. Preferably, between 10 and 400 different species not present within subject’s gut before treatment with the therapeutic microbial composition may engraft in subject’s gut.

[0117] Microorganisms present in the composition of the invention may be capable of producing some or all of the following metabolites (as shown in Table 1 below): Acetate, butyrate, propionate, valerate, indole, indole 3-acetic acid, indole 3- propionic acid, indole-3-carboxaldehyde, indole-3-lactic acid, cholic acid, hyocholic acid, beta-Muricholic acid, chenodeoxycholic acid, deoxycholic acid, 7-Ketodeoxycholic acid, omega-Muricholic acid, ursocholic acid, ursodeoxycholic acid, 7-Ketolithocholic acid, 12- Ketolithocholic acid, hyodeoxycholic acid, 3-Oxocholic acid, dioxolithocholic acid, lithocholic acid, nordeoxycholic acid, norursodeoxycholic acid, trimethylamine (TMA), lysine, choline, y-Aminobutyric acid (GABA), lactate, adenosine, kynurenine, tryptophan, serotonin, inosine and / or nicotinic acid. Table 1 : Production in metabolites of interest of a therapeutic microbial composition. The analysis exhibited in table 1 was performed on conditioned medium collected during manufacturing of a specific cultivated product (Composition 5 - Cult). With respect to table 1 , metabolite production by microbiota was quantified by LC- MRM / MS. Results exhibited in table 1 represent mean values ± SEM of 6 different cultivated products. These metabolites act on different metabolic pathways of interest in gut homeostasis and immuno-oncology. The first category, Short Chain Fatty Acids (SCFA), designates a group of metabolites known to possess anti-inflammatory properties. This category includes acetate, butyrate, propionate and valerate (pentanoate). Metabolites belonging to the indole pathway are also known to be involved in gut homeostasis such as indole, indole 3-acetic acid, indole 3-propionic acid, indole-3-carboxaldehyde, indole- 3-lactic acid. Primary (cholic acid, hyocholic acid, beta-Muricholic acid, chenodeoxycholic acid) and secondary (deoxycholic acid, 7-Ketodeoxycholic acid, omega-Muricholic acid, ursocholic acid, ursodeoxycholic acid, 7-Ketolithocholic acid, 12- Ketolithocholic acid, hyodeoxycholic acid, 3-Oxocholic acid, dioxolithocholic acid, lithocholic acid, nordeoxycholic acid and norursodeoxycholic acid) bile acids are also of interest because of their potential role in immune response activation. Another pathway of interest is the kynurenine pathway. Indeed, it is correlated to a favorable patient outcome when activated. Kynurenine and tryptophan are the main metabolites of this pathway. Other metabolites involved in the control of gut homeostasis and immune response are also produced by microorganisms of the composition such as trimethylamine (TMA), lysine, choline, y-Aminobutyric acid (GABA), lactate, adenosine, kynurenine, tryptophan, serotonin, inosine and / or nicotinic acid.

[0118] The therapeutic microbial composition of the invention to be used in combination with an immune modulatory treatment, comprising a population of microorganisms, may be advantageously able to trigger an efficient IgG production by B cells in an in vitro model of TLS. Indeed, the therapeutic microbial composition is able to reproduce in vitro one of the various effects it has on TLS in vivo. IgG production by B cells is one of the markers of TLS activation showing that the synapse between TFH cells and B cells is functional and that B cell differentiation into plasmablast is activated. The in vitro TLS model may be a human tonsil-based model. Tonsils are SLOs that closely resemble TLS (Aoyama et al. 2021). The main difference between both is the location within the body which is ectopic for TLS. In addition, tonsils are easily accessible compared to TLS and contain important and diverse population of immune cells comparable to TLS. The therapeutic microbial composition of the invention may be able to activate the synapse between TFH cells and B cells, and B cell differentiation into plasmablasts in the in vitro human tonsil model.

[0119] The therapeutic microbial composition of the invention may be able to induce some or all of the following changes within the TLS: (a) induction of T cells specific to microorganisms present within the composition, (b) induction of tumor specific T cells and / or (c) increase the quantity of mature DCs, ILC3 T cells, plasmablasts and / or TFH cells. Microorganisms included in the composition may be able to dialogue with immune cells present locally in the gut, leading to generation of a repertoire of specific T cells located within TLS, helping in regulating anti-tumor defense mechanisms, either inflammation control or tumor targeting. Microorganisms of the invention may also allow to induce an increase in tumor specific T cells e.g. tumor infiltration by T cells. An increase in different cell types can also be triggered by the composition. For example, increase in mature DCs, allowing to educate locally naive immune cells to increase immune reaction pace and efficacy; in ILC3 T cells, participating in lymphoid neogenesis, vasculature modeling, recruitment of leucocytes in TME; in plasmablasts that are mature B cells capable of producing antibodies; in TFH cells, interacting with B cells and forming networks with follicular dendritic cells to activate T cells and B cells immunity and leading to the production of low affinity antibodies.

[0120] The pharmaceutical composition of the present invention may correspond to compositions 1 , 2, or 5 as described below. It is herein described the following detailed composition, Composition 1 (also called General composition), which corresponds to criteria able to cover the two types of compositions described below (Compositions 2 and 5). The content in bacteria of such composition may be characterized at different taxonomic levels.

[0121] At the phylum level, the composition of the present invention may comprise between 25% and 75% of Bacteroidetes, between 20% and 60% of Firmicutes, between 0.01 % and 15% of Actinobacteria, between 0.01 % and 10% of Proteobacteria, and Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes as a whole may represent between 60% and 100% of the bacterial population.

[0122] At the family level, the composition of the present invention may comprise between 2% and 60% of Bacteroidaceae, between 1% and 35% of Ruminococcaceae, between 1 % and 25% of Lachnospiraceae, between 0.01% and 5% of Clostridiaceae, and at least 7 of the following families: Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae,

[0123] Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae,

[0124] Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae or Selenomonadaceae. Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae,

[0125] Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae as a whole may represent between 40% and 95% of the bacterial population.

[0126] At the genus level, the composition of the present invention may comprise between 2% and 60% of Bacteroides, between 0.01% and 20% of Phocaeicola, between 0.01% and 20% of Faecalibacterium, between 0.01% and 5% of Blautia, between 0.01% and 5% of Flavonifractor, between 0.01 % and 5% of Gemmiger, between 0.01 % and 5% of Dorea, and at least 12 of the following genera: Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania . Phocaeicola, Faecalibacterium, Bacteroides, Flavonifractor, Blautia, Dorea, Gemmiger, Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania as a whole may represent between 40% and 95%, preferably between 30% and 90% of the bacterial population. The therapeutic microbial composition may comprise all of the following species: Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Faecalibacterium prausnitzii, Flavonifractor plautii, Ruminococcus bromii, Coprococcus comes, Roseburia intestinalis, Dysosmobacter sp. BX15 and Phocaeicola vulgatus.

[0127] Percentages of the different taxonomic levels preferably refer to relative abundance within the population. The term “relative abundance” is preferably defined as the number of studied microorganism type (e.g. bacteria or fungi) of a particular taxonomic level (from phylum to species) as a percentage of the total number of microorganism type of that level in a therapeutic microbial composition. Relative abundance can be assessed, for example, by measuring the proportion of each phylum, family or genus via Shotgun sequencing analysis on a suitable pipeline as previously defined. Taxonomic identifications of the microorganisms listed here may have been obtained from specific databases. These microorganisms may have a different taxonomic annotation if the sequencing data are analyzed with a different bioinformatics pipeline or with different databases.

[0128] It is herein described the following detailed composition, Composition 2 (also called Pool composition). The content in bacteria of such composition may be characterized at different taxonomic levels (for example, see table 2A below for the phylum level, see table 2B below at the family level and see table 2C at the genus level).

[0129] At the phylum level, the composition of the present invention may comprise between 30% and 75% of Bacteroidetes, between 15% and 45% of Firmicutes, between 0.01 % and 10% of Proteobacteria, between 0.01% and 5% of Actinobacteria, and Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes as a whole may represent between 60% and 100% of the bacterial population.

[0130] At the family level, the composition of the present invention may comprise between 2% and 40% of Bacteroidaceae, between 5% and 25% of Ruminococcaceae, between 0.01% and 20% of Rikenellaceae, between 1% and 20% of Lachnospiraceae, between 0.01% and 10% of Tannerellaceae, between 0.01 % and 5% of Oscillospiraceae, between 0.01% and 5% of Barnesiellaceae, between 0.01 % and 5% of Clostridiaceae, between 0.01 % and 5% of Odoribacteraceae, between 0.01% and 5% of Sutterellaceae, between 0.01% and 5% of Desulfovibrionaceae, and 6 of the following families: Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae,

[0131] Bifidobacteriaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae or Selenomonadaceae. Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae as a whole may represent between 40% and 80% of the bacterial population.

[0132] At the genus level, the composition of the present invention may comprise between 2% and 36% of Bacteroides, between 0.01% and 20% of Alistipes, between 0.01% and 20% of Phocaeicola, between 2% and 20% of Faecalibacterium, between 0.01 % and 11% of Roseburia, between 0.01% and 10% of Ruminococcus, between 0.01 % and 10% of Parabacteroides, between 0.01 % and 5% of Subdoligranulum, between 0.01% and 5% of Coprococcus, between 0.01% and 5% of Barnesiella, between 0.01% and 5% of Clostridium, between 0.01% and 5% of Oscillibacter, between 0.01 % and 5% of Dysosmobacter, between 0.01% and 3% of Blautia, between 0.01% and 3% of Gemmiger, between 0.01 % and 2% of Flavonifractor, between 0.01 % and 2% of Odoribacter, between 0.01% and 2% of Bilophila, between 0.01 % and 2% of Dorea, and at least 10 of the following genera: Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania. Phocaeicola, Faecalibacterium, Bacteroides, Flavonifractor, Blautia, Dorea, Gemmiger, Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania as a whole may represent between 40% and 80% of the bacterial population.

[0133] The therapeutic microbial composition may comprise all of the following species: Bacteroides uniformis, Bacteroides fragilis, Faecalibacterium prausnitzii, Ruminococcus bromii, Phocaeicola vulgatus, Flavonifractor plautii, Coprococcus comes, Odoribacter splanchnicus, Barnesiella intestinihominis, Blautia wexlerae, Alistipes shahii, Parabacteroides merdae, Bacteroides caccae, Bilophila wadsworthia, Bacteroides thetaiotaomicron, Bacteroides ovatus, Gemmiger formicilis, Roseburia inulinivorans, Alistipes communis, Dorea longicatena, Oscillibacter sp. ER4, Blautia obeum, Subdoligranulum sp. APC924 / 74, Roseburia intestinalis, Alistipes putredinis and Dysosmobacter sp. BX15.

[0134] Preferably, composition 2 is defined in tables 2A to 2C below and corresponds to criteria covering products comprising pools of 3 to 8 stool donations, homogenized with a diluent comprising a cryoprotectant, and frozen.

[0135] Table 2: Analysis of the content in bacteria of Composition 2 - Pool at different taxonomic levels.

[0136] 2A: Analysis at the phylum level

[0137] 2B: Analysis at the family level

[0138]

[0139] 2C: Analysis at the genus level

[0140] The analysis exhibited in Tables 2A, 2B and 2C above is based on Shotgun sequencing data and has been performed on 201 products comprising microorganisms derived from pools of 3 to 8 different stool donors. Each stool has been processed separately, homogenized with a diluent comprising a cryoprotectant and mixed in defined ratios with other stool samples to form a pool. This product has then been frozen for preservation. Every manufacturing step has been led under GMP conditions.

[0141] It is herein described the following detailed composition, Composition 5 (also called composition Cult). The content in bacteria of such composition may be characterized at different taxonomic levels (for example, see table 3A below for the phylum level, see table 3B below at the family level and see table 3C at the genus level). At the phylum level, the composition of the present invention may comprise between 25% and 75% of Bacteroidetes, between 20% and 60% of Firmicutes, between 0.01% and 15% of Actinobacteria, between 0.01 % and 10% of Proteobacteria, and Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes as a whole may represent between 70% and 100% of the detectable bacteria of the bacterial population.

[0142] At the family level, the composition of the present invention may comprise between 20% and 60% of Bacteroidaceae, between 1 % and 35% of Ruminococcaceae, between 5% and 25% of Lachnospiraceae, between 0.01% and 10% of Erysipelotrichaceae, between 0.01% and 10% of Bifidobacteriaceae, between 0.01% and 5% of Clostridiaceae, between 0.01 % and 5% of Peptostreptococcaceae, and at least 4 of the following families: Streptococcaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae or Selenomonadaceae. Bacteroidaceae, Ruminococcaceae, Lachnospiraceae, Clostridiaceae, Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae as a whole may represent between 65% and 95% of the bacterial population.

[0143] At the genus level, the composition of the present invention may comprise between 20% and 60% of Bacteroides, between 1 % and 30% of Ruminococcus, between 1% and 20% of Eisenbergiella, between 0.01 % and 10% of Bifidobacterium, between 0.01% and 10% of Erysipelatoclostridium, between 0.01% and 5% of Holdemania, between 0.01% and 5% of Faecalibacterium, between 0.01% and 5% of Phocaeicola, between 0.01% and 5% of Dorea, between 0.01 % and 5% of Blautia, between 0.01% and 5% of Gemmiger, between 0.01% and 5% of Flavonifractor, between 0.01 % and 5% of Enterocloster, and at least 9 of the following genera: Lachnoclostridium, Ruthenibacterium, Hungatella, Mediterraneibacter, Clostridium, Parabacteroides, Collinsella, Coprococcus, Anaerostipes, Escherichia, Lachnospira, Dysosmobacter, Intestinimonas, Eubacterium, Phascolarctobacterium, Dialister, Anaerobutyricum, Bilophila, Roseburia, Subdoligranulum, Sutterella, Faecalicatena, Fusicatenibacter, Oscillibacter, Prevotella, Akkermansia, Alistipes, Barnesiella, Butyricimonas, Odoribacter and Paraprevotella. Phocaeicola, Faecalibacterium, Bacteroides, Flavonifractor, Blautia, Dorea, Gemmiger, Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister, Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania as a whole may represent between 55% and 95% of the bacterial population.

[0144] The therapeutic microbial composition of the invention may comprise all of the following species: Bacteroides uniformis, Bacteroides ovatus, Bacteroides fragilis, Phocaeicola vulgatus, Faecalibacterium prausnitzii, Ruminococcus bromii, Flavonifractor plautii, Bacteroides xylanisolvens, Bacteroides thetaiotaomicron, Bacteroides intestinalis, Ruminococcus sp. AM31-32, Ruminococcus sp. AF37-3AC, Eisenbergiella tayi and [Clostridium] innocuum. Furthermore, the therapeutic composition of the invention may comprise additionally at least 35 species of the following list: Dysosmobacter sp. BX15, Prevotella copri, Holdemania massiliensis, Ruminococcus sp. AF43-11 , [Clostridium] symbiosum, Escherichia coli, Anaerotignum lactatifermentans, Phocaeicola massiliensis, Bacteroides cellulosilyticus, Ruminococcus sp. AF16-40, Bacteroides finegoldii, Collinsella aerofaciens, Faecalibacterium sp. Marseille-P9312, Bacteroides oleiciplenus, Mediterraneibacter massiliensis, Porphyromonas somerae, Bacteroides sp. 14(A), Roseburia intestinalis, Bacteroides sp. 3_1_40A, Bacteroides sp. D55t1_190419_B8, Bacteroides sp. L5, Ruminococcus sp. AM36-18, Bacteroides stercorirosoris, Eisenbergiella massiliensis, Emergencia timonensis, Bacteroides timonensis, Longicatena caecimuris, Bifidobacterium adolescentis, Negativicoccus succinicivorans, Bifidobacterium dentium, Bifidobacterium longum, Porphyromonas sp. HMSC077F02, Bifidobacterium pseudocatenulatum, Roseburia hominis, Campylobacter ureolyticus, Clostridium sp. AF34-10BH, Ruminococcus sp. AF17-11 , Clostridium sp. AF36-18BH, Ruminococcus sp. AF42-10, Clostridium sp. AM34-9AC, Ruminococcus sp. AM28-29LB, Clostridium sp. KLE 1755, Clostridium sp. TF08-15 and Coprococcus comes.

[0145] Preferably, the composition 5 is defined in tables 3A to 3C below and corresponds to criteria covering products comprising cultivated microorganisms starting from a single stool derived sample or a pool of stool derived samples, homogenized with a diluent comprising a cryoprotectant, lyophilized and frozen for preservation.

[0146] Table 3: Analysis of the content in bacteria of Composition 5 - Cult at different taxonomic levels.

[0147] 3A: Analysis at the phylum level 3B: Analysis at the family level

[0148] 3C: Analysis at the genus level

[0149] The analysis exhibited in tables 3A, 3B and 3C above is based on Shotgun sequencing data and has been performed on 6 products comprising microorganisms derived from pools of 3 to 8 different stool donors. Each stool has been processed separately, homogenized with a diluent comprising a cryoprotectant, lyophilized and then frozen for preservation. Every manufacturing step has been led under GMP conditions.

[0150] The composition of the invention may also further comprise fungi (as exhibited for example in table 4 below). The content in fungi may be characterized by a dominance in Ascomycota and Basidiomycota within the fungal population. Preferably, Ascomycota may compulsorily be present in the composition and Mucoromycota and / or Mortierellomycota may be potentially present as minor members of the fungal population. More preferably, at least two of the following families may be present within the composition: Aspergillaceae, Saccharomycetaceae, Cladosporiaceae, Dipodascaceae, Debaryomycetaceae, Saccharomycetales_fam_lncertae_sedis, Pleosporaceae and Sporidiobolaceae. Preferably, at least two of the following genera may be present within the composition: Saccharomyces, Penicillium, Cladosporium, Geotrichum, Debaryomyces, Aspergillus, Candida, Dipodascus and Alternaria. With respect to table 4, DNA isolation and ITS2 sequencing were performed as follows: Genomic DNA was extracted from fecal samples using the NucleoSpin Soil kit (Macherey Nagel). A sequencing library targeting the ITS2 region was constructed for each sample. Libraries were then sequenced in paired-end (2 x 300 bp) MiSeq runs (Illumina). Positive and negative controls were added throughout the process to validate the successful completion of each step. ITS bioinformatics analyses were performed with an appropriate analysis pipeline. In brief, after amplicon merging using FLASH, reads were quality-filtered using Trimmomatic. Amplicons were then clustered and a taxonomical annotation was assigned to each output using VSEARCH and the UNITE+INSD database (10.05.2021 release). Taxonomic identifications of the microorganisms listed here have been obtained from specific databases. These microorganisms may have a different taxonomic annotation if the sequencing data are analyzed with a different bioinformatics pipeline or with different databases.

[0151] The following table 6A exhibits the analysis of 115 products comprising microorganisms derived from pools of 4 to 6 stools from different donors. Each stool has been processed separately, homogenized with a diluent comprising a cryoprotectant and mixed in defined ratios with other stool samples to form a pool. This product has then been frozen for preservation. Every manufacturing step has been led under GMP conditions.

[0152] Table 4: Analysis of the content in fungi of Composition 2 - Pool at different the phylum level.

[0153] Microorganisms of the therapeutic microbial composition engrafted in subjects’ gut may be different from a patient to another. Microorganism selection may be operated by subject gut depending on its needs in microorganisms and missing functions to be recovered to restore a symbiotic microbiota. One or several different microorganisms can engraft within subject’s gut.

[0154] The therapeutic microbial compositions of the invention are advantageously useful to promote maturation of TLS in a subject and enhance immune response toward tumoral cells.

[0155] As previously defined, the composition according to the invention is used in combination with an immune modulatory treatment in a subject in need thereof (I MT). Such composition may comprise a population of stool derived microorganisms able to trigger an efficient IgG production by B cells in an in vitro model of TLS. Preferably, the in vitro model is a human tonsil model. The composition may be able to induce some or all of the following changes within the TLS: (a) formation or expansion of T cells specific to microorganisms present within the composition, (b) formation or expansion of tumor specific T cells and / or (c) expansion of mature DCs, ILC3 T cells, plasmablasts and / or TFH cells.

[0156] Immune Modulatory Treatment:

[0157] The immune modulatory treatment (I MT) used in combination with the microbial composition may comprise immune checkpoint inhibitors, immuno-conjugates, cytokines, immunomodulatory small molecules targeting STING or IDO, nucleotides, polynucleotides, therapeutic vaccines, adoptive cell transfers, modified dendritic cell transfers or T-cell engagers.

[0158] The IMT may be an ICI . Preferably, ICIs, also known as drug blocking an immune checkpoint, immune checkpoint blocker or immune checkpoint blockade drug, designates molecule or composition which blocks one or several immune checkpoints. Preferably, it encompasses blockers of the following targets: PD-1 , PD-L1 , PD-L2, CD27, CD28, CD40, CD122, CD137, 0X40, GITR, IGOS, A2AR, B7-H3 (such as 1311- Omburtamab), B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3 (such as Opdualag - Relatlimab), NOX2, TIM-3, VISTA, SIGLEC7 and TIGIT (such as Tiragolumab). More particularly, it can be anti-CTLA-4, anti-PD-L1 or anti-PD-1 antibodies such as Cadonilimab, PUYOUHENG (Pucotenlimab), IMJUDO (Tremelimumab), AIBINING (Geptanolimab), KEYTRUDA (Pembrolizumab), OPDIVO (Nivolumab), YERVOY (Ipilimumab) or LIBTAYO (cemiplimab), Penpulimab, Socazolimab, Adebrelimab, Tagitanlimab, Toripalimab, Tislelizumab, Retifanlimab, Sintilimab, Cosibelimab, Camrelizumab, Sugemalimab, IBI310, Nofazinlimab, Erfonrilimab. Preferably, the molecule or composition which blocks PD-1 is a monoclonal antibody targeting PD-1. The term “monoclonal antibody” as used herein preferably refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modified “monoclonal” preferably indicates the character of the antibody as not being a mixture of antibodies with different epitope specificities. Such a monoclonal antibody may typically include an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence may also be a monoclonal antibody of this disclosure. In contrast to polyclonal antibody preparations, which typically include several different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0159] Method of treatment and therapeutic use:

[0160] As previously indicated, the invention also concerns a pharmaceutical composition comprising a population of microorganisms as previously defined, for use in combination with an immune modulatory treatment (I MT) for treating a cancer in a patient, wherein the cancer is a solid tumor comprising a tertiary lymphoid structure (TLS) in its microenvironment.

[0161] According to another aspect, the invention also provides a method for treating cancer in a patient comprising administering a composition comprising a population of microorganisms as previously defined, in combination with an immune modulatory treatment (I MT), wherein the cancer is a solid tumor comprising a tertiary lymphoid structure (TLS) in its microenvironment.

[0162] According to a further aspect, provided is a method for promoting maturation of tertiary lymphoid structure (TLS), comprising administering to a patient a pharmaceutical composition comprising a population of microorganisms as previously defined and an immune modulatory treatment (I MT). Advantageously, a method for promoting maturation of tertiary lymphoid structures (TLS) aims to enhance immune response toward tumoral cells in a patient in need thereof. A method for promoting maturation of TLS according to the invention comprises administering to a subject a combination of a composition comprising a population of microorganisms as previously defined, and an immune modulatory treatment (IMT). TLS maturation can be characterized by an increase in IgG production by B cells present within TLS after treatment with such a composition. TLS maturation can also be characterized by the presence of T cells specific for microorganisms present in the composition and / or an increase in mature DCs, ILC3 T cells, plasmablasts and / or TFH in the TLS. The composition may induce B cell differentiation into plasmablast both via microbial antigen presentation by both DCs and B cells to TFH cells and subsequent activation of memory B cells.

[0163] According to a further aspect, provided is the use of a composition comprising a population of microorganisms as previously defined, for the manufacture of a medicament for the treatment of cancer, in combination with an IMT.

[0164] As used herein, the terms “tumor”, “solid tumor” or “cancer” preferably mean all type of solid tumors comprising a TLS in its microenvironment (TME). As the immune system is involved in cancer development and progression, drugs modulating it, immune modulatory treatments, can hence be used to treat virtually all type of solid tumors comprising a TLS. Thus, the methods according to the invention can be potentially useful for patients having a cancer selected amongst adrenal cortical cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain cancer, central nervous system cancer, breast cancer, cancer of the peritoneum, cervical cancer, choriocarcinoma, colon cancer, rectal cancer, connective tissue cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma, hepatic carcinoma, hepatoma, intra-epithelial neoplasm, kidney cancer, renal cancer, larynx cancer, liver cancer, lung cancer, melanoma, neuroblastoma, oral cavity cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma; cancer of the respiratory system, salivary gland carcinoma, sarcoma, skin cancer, squamous cell cancer, stomach cancer, testicular cancer, thyroid cancer, urothelial cancer, uterine cancer, endometrial cancer, cancer of the urinary system and vulval cancer.

[0165] The subject or patient may have one of the following cancers: bladder cancer, breast cancer, head & neck cancer, kidney cancer, hepatocellular carcinoma, mismatch repair deficiency (dMMR) or microsatellite instability-high (MSI-H), cervical cancer, nasopharyngeal cancer (such as nasopharyngeal carcinoma), esophageal cancer (such as esophageal squamous cell carcinoma), lymphoma (such as peripheral T-cell lymphoma), metastatic cancers (such as metastatic nasopharyngeal carcinoma), anal cancer (carcinoma of the anal canal), brain cancer (such as CNS / leptomeningeal metastasis from neuroblastoma), skin cancer and lung cancer. This list of cancers corresponds to those that are currently treated with IC Is. The subject or patient may have a cancer that is resistant, has not responded, has relapsed and / or progressed despite (e.g. during or following) treatment (e.g. a first line treatment). Besides, the term “subject”, preferably refers to an individual organism such as a human or an animal. The subject may be a mammal (e.g., a human, a non-human primate, or a non-human mammal), a vertebrate, a laboratory animal, a domesticated animal, an agricultural animal, or a companion animal. The subject may be a human (e.g., a human patient). The subject may be a rodent, a mouse, a rat, a hamster, a rabbit, a dog, a cat, a cow, a goat, a sheep, a turkey, a chicken, or a pig. The subject, e.g., a human, may be refractory and / or poorly responsive and / or non-responsive to an IMT.

[0166] The subject may have a dysbiosis and / or presents a depressed immune system in addition to its solid tumor. Subjects suffering from cancer may often display an imbalance in the microbiota with potential gut colonization by deleterious microorganisms and a weaken immune system, generally caused by antibiotics and anticancer treatments usage. Compositions of the present invention advantageously allow a synergic action. On the one hand, microorganisms of the compositions of the present invention are advantageously able to eliminate deleterious microorganisms having populated the intestinal mucosa, and provide bacterial equilibrium and homeostasis in the gut. They can modulate also locally and systemically the immune system by calming inflammation and redirecting immune cells to TME. On the other hand, microorganisms of the composition of the present invention can act on TLS maturation, preferably to trigger TLS maturation. Both effects combined advantageously allow to increase sensitivity of tumors to IMTs.

[0167] The subject suffering from cancer may be poorly or non-responsive to IMT. Subjects suffering from cancer having already received an anti-cancer therapy, such as an IMT, most often experience relapse or treatment inefficacy. TLS maturation can thus be particularly important for such patient population because it allows to render them receptive to the anticancer treatment. The microbial composition of the present invention allows to mature TLS to diminish relapse rate and enhance IMT efficacy.

[0168] Administering the therapeutic microbial composition may maintain or induce responsiveness of a tumor of the subject to the IMT. Induce responsiveness of a tumor of the subject to the IMT may refer to the ability of a therapeutic microbial composition or method using the same to increase beneficial and desired effects of an immune modulatory therapeutic agent, i.e., killing cancer cells, reducing tumor size, and / or simulating an immune response against a cancer cell or tumor. The microbial composition of the present invention advantageously allows to mature TLS and act on tumor responsiveness to I MT.

[0169] As used herein, “treating” preferably refers to completely or partially inhibiting a disease, disorder or condition, for example, arresting its development; completely or partially relieving a disease, disorder or condition, for example, causing regression of the disease, disorder and / or condition; or completely or partially preventing a disease, disorder or condition from occurring in a patient that may be predisposed to the disease, disorder and / or condition, but has not yet been diagnosed as having it. Similarly, "treatment" preferably refers to both therapeutic treatment and prophylactic or preventative measures.

[0170] The therapeutic microbial composition may be administered to the patient orally, nasogastrically, by enema or by coloscopy. Rectal enema may be a liquid solution formulated in 150 mL oxygen resistant bags containing at least 30g of feces. They may be stored at -80°C, and after thawing may be ready to use. Oral formulation may be a lyophilized composition filled in a gastro-resistant capsule with a targeted delivery. They may be stored at -5°C and may be ready to use. Preferably, administration of the therapeutic microbial composition may be performed orally. Oral formulation may be a lyophilized composition filled in a gastro-resistant capsule. This capsule may allow a targeted delivery within the gut. A delivery in the ileo-colic region may be contemplated to optimize product efficiency. Preferably, the capsule content in dry matter of microorganisms may be comprised between 1 % and 60%. The capsule content in dry matter of cryoprotectant may be comprised between 5% and 95%. The capsule content in excipients other than cryoprotectant may be comprised between 0% and 90%. More preferably, the oral formulation may comprise an excipient and a ratio cryoprotectant I biomass may be comprised between 0.5 and 16. A suitable excipient may be mannitol for example. The therapeutic microbial composition of the invention may also comprise a pharmaceutically acceptable carrier.

[0171] As used herein, the term “combination” preferably refers to the use of more than one agent (e.g., a therapeutic microbial composition and an anti-PD-1 , possibly associated to radiotherapy). The use of the term "combination" does not restrict the order in which therapies are administered to the patient, although it is preferable to administer the therapeutic microbial composition prior to or simultaneously with the immune modulatory treatment, such as an immune checkpoint blocker. For example, the therapeutic microbial composition can be administered prior to the I MT (e. g., 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks or 26 weeks before), either punctually or several times (for example, each day) before the I MT is administered. Advantageously, the therapeutic microbial composition may be administered before administration of the IMT in order to modulate the subject's gut microbiota to optimize the effect of the IMT (such as those defined above). Furthermore, the therapeutic microbial composition can also be administered after the IMT treatment to maintain subject’s gut microbiota and TLS maturation ensuring longer efficacy of IMTs (e. g., 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, 14 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks or 26 weeks after). According to an advantageous embodiment, the treatment regimen may comprise one or several administration cycles. Each administration cycle may last at least 5 days, for examples 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 days. One administration cycle may comprise 1 , 2, 3 or 4 administrations of the pharmaceutical composition (less than 5 administration).

[0172] According to an example, a treatment regimen may comprise one administration cycle of 5 days, the administration cycle comprising an administration of the pharmaceutical composition at day 1. Optionally, the immune checkpoint inhibitor may be administered after the administration of the pharmaceutical composition, for example at day 5 of the administration cycle.

[0173] According to another advantageous embodiment, one administration of the pharmaceutical composition may be administering an amount of between 1 108and 9 101° bacteria.

[0174] TLS maturation:

[0175] Tertiary lymphoid structure (TLS) maturation may be characterized by an increase in IgG production by B cells present within TLS after treatment with the microbial composition (e.g. a therapeutic microbial composition according to the invention). An increase in IgG production by B cells is a marker of an effective synapse between T follicular helper (TFH) cells and B cells leading to B cell differentiation into plasmablasts and a maturated TLS. As shown in Figure 1 , compositions of the invention are advantageously able to trigger IgG production in an in vitro human tonsil model. In addition, combination of the compositions with an immune checkpoint inhibitor (ICI) advantageously allows to increase ICI efficiency. This IgG production may be mediated via two complementary mechanisms triggering B cell differentiation into plasmablast. The first one is the captation of microbial antigens by dendritic cells (DCs) and their presentation to TFH cells, leading to the activation of TFH cells and to the primo- activation of naive B cells and their differentiation into IgG-producing plasmablasts. The second is the captation of microbial antigens by pre-primed memory B cells and their presentation to TFH cells, leading to the subsequent activation of B cells. Like dendritic cells, B cells constitutively express MHC class II molecules. Upon antigen engagement via the BCR, B cells can initiate the expression of various cytokines, including interleukin (I L)-6, IL-10, and tumor necrosis factor alpha (TNFa). These cytokines affect CD4 + T cell activation and differentiation. CD4 + T cells, once activated and differentiated into a specific T helper (Th) cell phenotype, cross-activate B cells via cytokines and costimulatory molecules, including cluster of differentiation (CD)40-CD40 ligand and CD80 / CD86-CD28. Pre-primed memory B cells were originally naive B cells that received a first signal of activation through activated TFH cells. Activated B cells will then either differentiate into IgG-producing plasmablasts or memory B cells capable of being reactivated upon stimulation with specific antigens. Indeed, it is documented in the literature that whatever the maturation stage of TLS, B cells at all maturation stages are present in TLS including naive and memory B cells (Meylan et aL, 2022). Such phenomenon is exhibited in Figure 1 B.

[0176] TLS maturation may be characterized by the presence of T-cells specific for microorganisms present in the composition of the invention within TLS, and / or an increase or expansion in mature DCs, ILC3 T cells, plasmablasts and / or TFH in the TLS. Microorganisms included in the composition of the invention are advantageously able to dialogue with immune cells locally present in the gut leading to generation of a repertoire of specific T cells located within TLS, helping in regulating anti-tumor defense mechanisms, either inflammation control or tumor targeting by immune cells e.g. tumor infiltration by T cells. An increase in different cell types can be observed in mature TLS: mature DCs, ILC3 T cells, plasmablasts and / or TFH. Mature DCs increased presence in TLS allows to educate locally naive immune cells to increase immune reaction pace and efficacy. ILC3 T cells contribute to TLS maturation by their multiple capabilities such as lymphoid neogenesis, vasculature modeling, recruitment of leucocytes in TME. Plasmablasts are B cell that maturated and are capable of producing antibodies. TFH cells interact with B cells and form networks with follicular dendritic cells where T cells immunity is activated, and low affinity antibodies are produced by plasmablasts. Other cell population can also be analyzed to identify TLS maturation. Overall, cells of interest can be analyzed thanks to the following markers: B cells by surface expression of CD20 or CD19; some germinal centre B cells intracellularly express activation-induced deaminase (AID) and the proliferation marker Ki67, follicular dendritic cells (FDCs) located inside the germinal by CD21 (also known as CR2), CD35 (also known as CR1) and CD23 (also known as FCERII), all of which are also present on B cells, plasma cells with CD138 (weak expression on late-stage germinal centre B cells) and CD269 (also present on germinal centre B cells), T cells with CD3, CD8 or CD4, Dendritic cells (DCs) with DC-lysosome-associated membrane glycoprotein (DC-LAMP; mature DCs), CD83 or CD86 (activated DCs), Neutrophils with CD66b or myeloperoxidase, Macrophages with CD68, High endothelial venules (HEVs): peripheral node addressin (PNAd) and MECA79 (Sautes-Fridman et al., 2019).

[0177] TLS maturation may be triggered by modulation of concentration levels of some or all of the following molecules: TNF-a, IL-13, IL-4, IL-10, IL-6, IL-2, TNF- , IFN- y, IL-17A, IL-12p70, APRIL, BAFF and SCD40L in vitro and in vivo. These molecules can either be produced by microorganisms present in the composition of the invention or be produced by cells activated by microorganisms present in such composition. Modulation of concentration levels can be observed in in vitro models or in in vivo models such as rodent models. Modulations can also be observed in patients either systemically or locally depending on the analysis method used. Modulation of concentration levels may occur locally in the TME and / or within TLS. APRIL, BAFF and CD40L can be markers of activation and survival of B cells. IL-10 and TGF-beta1 are produced by regulatory B cells. TNF-alpha, TNF-beta, IFN-gamma and IL-12p70 are produced by Be1 effector cells. IL-2, IL-4, IL-6, IL- 13 and TNF-alpha are produced by Be2 effector cells. All this panel of molecules allows to characterize some of the members of the B cell population.

[0178] According to another aspect, the invention relates to an in vitro method for promoting maturation of a tertiary lymphoid structure (TLS), comprising administering to a TLS in vitro model a microbial composition as described herein. The TLS in vitro model may be a human tonsil derived model.

[0179] According to another aspect of the invention, provided is an in vitro method for screening a composition (e.g. a therapeutic microbial composition) the method comprising steps of administering said composition to a tertiary lymphoid structure (TLS), optionally with an Immune Modulatory Treatment (IMT), and assessing IgG production by B cells within said TLS in vitro model. Preferably, the tertiary lymphoid structure may be an in vitro human tonsil derived model. Such in vitro method advantageously allows to assess whether the tested composition trigger maturation of TLS, based on the assessment of IgG production by B cells in an in vitro human tonsil derived model. This method advantageously allows to screen rapidly and economically compositions comprising microorganisms. Indeed, tonsils can be easily accessible compared to TLS and contain important and diverse population of immune cells comparable to TLS. Measuring IgG release in a bulk derived immune cell assay or a TFH / B cell co-culture assay may be used as in vitro human tonsil model. Whatever the chosen model, the assay may be performed in presence or absence of an I MT. The screening method may be performed with conditioned media or heat inactivated bacteria. The term “conditioned media” preferably refers to supernatants of therapeutic microbial compositions to be tested in the screening method. These supernatants may not contain viable cells but otherwise comprise metabolites, nucleic acids, microorganisms cell membrane fragments such as extracellular vesicles. The term “heat inactivated bacteria” preferably refers to therapeutic microbial compositions to be tested in the screening method that have been heated to 95°C for 5 minutes to kill microorganisms. Resulting composition preferably comprises metabolites, nucleic acids, microorganisms cell membrane fragments and many other compounds present intracellularly within microorganisms.

[0180] Other definitions:

[0181] In addition to all terms already defined above, some terms used in the text may be defined in the following manner:

[0182] Substantially preferably refers to modify a quality, generally allows certain degree of variation without that quality being lost. For example, in certain aspects such degree of variation can be less than 0.1 %, about 0.1 %, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, between 1-2%, between 2-3%, between 3-4%, between 4-5%, or greater than 5%.

[0183] The terms "about" and "approximately" as used herein when referring to a measurable value such as a percentage, density, volume and the like, can encompass variations of 20%, 10%, 5%, 1 %, 0.5%, or even 0.1% of the specified amount.

[0184] The terms “lower”, “reduced”, “reduction”, “decrease”, or “inhibit” as used herein generally may mean a decrease by a statistically significant amount. However, for avoidance of doubt, “lower”, ’’reduced”, “reduction”, “decrease,” or “inhibit” may mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10- 100% as compared to a reference level.

[0185] The terms “increased”, ’’increase”, “enhance”, or “activate” as used herein may mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase”, “enhance”, or “activate” may mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4- fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0186] The term “comprising”, which is synonymous with “including”, “containing,” or “characterized by,” may be inclusive or open-ended and does not exclude additional, unrecited elements or method steps. The phrase “consisting of’ may exclude any element, step, or ingredient not specified. The phrase “consisting essentially of’ may limit the scope of described subject matter to the specified materials or steps and those that do not materially affect its basic and novel characteristics. The term may permit the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention. With respect to pharmaceutical compositions, the term “consisting essentially of’ includes the active ingredients recited, excludes any other active ingredients, but does not exclude any pharmaceutical excipients or other components that are not therapeutically active. It is contemplated that embodiments described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0187] The term “consisting of’ may refer to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0188] As used in this specification and the appended claims, the singular forms "a," "an" and "the" may include plural referents unless the context clearly dictates otherwise. Unless specifically stated or obvious from context, as used herein, the term "or" may be understood to be inclusive and covers both "or" and "and".

[0189] As used herein, the terms “or” and “and / or” may be utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z”, “(x and y) or z”, “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment.

[0190] A stated range may be understood to be any value between and at the limits of the stated range. As examples, a range between 1 and 5 may include 1 , 2, 3, 4, and 5; a range between 1 and 10 may include 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10; and a range between 1 and 100 may include 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,

[0191] 19 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 ,

[0192] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64,

[0193] 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87,

[0194] 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100.

[0195] Unless defined otherwise, all technical and scientific terms used herein may have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains. Although other compositions, methods, or equivalent, to those described herein can be used in the practice of the present subject matter, the preferred materials and methods are described herein. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein. Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.

[0196] EXAMPLES

[0197] Example 1:

[0198] Principle

[0199] To evaluate the ability of therapeutic microbial compositions of the invention to promote a B-cell response through type G immunoglobulins (IgG) release, bulk human tonsil-derived immune cell assay and Tfh / B-cell co-culture assay were performed in the absence or presence of Staphylococcal Enterotoxin B (SEB) and in the absence or presence of Nivolumab as a reference immune checkpoint inhibitor (anti PD- 1 ). SEB, a known superantigen that can trigger polyclonal T cell activation and massive cytokine release, was used as a positive control in this study. Heat-inactivated bacteria of Composition 5 - Cult and their respective controls at 2 dilutions (1 / 100 and 1 / 1000) were assessed for their ability to promote activation of human tonsil-derived immune cells through their secretion of IgG in the supernatant over a 7-day period. Tested items were applied in the absence or presence of SEB at one dose (0,1 ng / mL), in the absence or the presence of Nivolumab at one dose (1 pg / mL) and in the presence of both.

[0200] Test compounds preparation

[0201] Heat inactivated bacteria and their respective controls were prepared according to the following steps:

[0202] Thawing 5 minutes at 37°C the cryotube containing live microbiota in trehalose and maltodextrin media,

[0203] Diluting the content of the cryotube at 1 / 5 in cell culture medium, Incubating the diluted sample during 5 minutes at 95°C, Incubating the sample during 5 minutes on ice,

[0204] Aliquoting the obtained Bacterial lysate and storing at -80°C until their use.

[0205] Bulk tonsil-derived immune cell assay

[0206] Experiments were performed on human tonsil-derived immune cells originating from one human donor. Cell isolation from tonsil was previously performed according to provider’s protocols based on tissue dissociation and digestion (GentleMACS Octo Dossociator) and immune cells were cryopreserved (using CryoStor® CS10, StemCell). In brief, 105human tonsil-derived immune cells per well (96-well plate) were plated in 250pL (IMDM - 10%FBS) in the presence and absence of the tested item and their respective controls, assessed at 2 doses (1 / 100 and 1 / 1000 dilutions) plus the negative control, in the absence or presence of SEB (0,1 ng / mL), in the presence or absence of Nivolumab (1 pg / mL) and in the presence of both Nivolumab and SEB. Cells were cultivated for 7 days.

[0207] The following conditions were included as controls:

[0208] - Untreated as negative control, - Cryoprotectant (at 1 / 100 and 1 / 1000 dilutions) as heat inactivated bacteria control,

[0209] - lgG4 (1 pg / mL) as Nivolumab control,

[0210] - CD40L, IL21 , IL4 cocktail as positive control for B cells activation.

[0211] Each experimental condition was performed in triplicate and in one experiment. At day 7 post cell seeding and treatment, cell culture supernatants were collected and stored at -80°C until IgG quantification using dedicated HTRF kit.

[0212] This assay allowed to demonstrate that the compositions of the invention trigger maturation of TLS through an increase in IgG production. In presence of the compositions of the invention, B cells mature and differentiate into plasmablasts capable of secreting IgG. This observation was performed with the two different dilutions of the compositions. Furthermore, IgG production is amplified when the compositions of the invention are combined with Nivolumab, compared to Nivolumab alone, whatever compositions dilutions.

[0213] Tfh / B cell co-culture assay

[0214] This assay was conducted on an autologous co-culture of human tonsil- derived Tfh (CD4+CXCR5+) and memory B cells (CD19+CD27+), sourced from either one or two donors, according to the number of cells that were isolated. Tfh (CD4+CXCR5+) and memory B cells (CD19+CD27+) were isolated from cryopreserved tonsil-derived immune cells. After flow-cytometry-based purity evaluation, cells were plated in 96-well plate in 250 pL of IMDM supplemented with 10% FBS, in the presence or absence of each of the tested item (heat-inactivated bacteria) and its respective controls, all assessed at 2 doses (1 / 100 and 1 / 1000), and a negative control, in the absence or presence of SEB (0,1 ng / mL), in the presence or absence of Nivolumab (1 pg / mL) and in the presence of both Nivolumab and SEB. Cells were cultivated for 7 days.

[0215] The following conditions were included as controls:

[0216] - Untreated as negative control,

[0217] - Cryoprotectant (at 1 / 100 and 1 / 1000 dilutions) as heat inactivated bacteria control,

[0218] - lgG4 (1 pg / mL) as Nivolumab control,

[0219] - CD40L, IL21 , IL4 cocktail as positive control for B cells activation (applied on B cells only).

[0220] Each experimental condition was performed in triplicate and in one experiment. At day 7 post cell seeding and treatment, cell culture supernatants were collected and stored at -80°C until IgG quantification using dedicated HTRF kit.

[0221] This experiment allowed to demonstrate that the compositions of the invention trigger maturation of TLS through an increase in IgG production. Such maturation was observed whatever the dilution of the compositions. In addition, IgG production is amplified when the compositions of the invention are combined with Nivolumab, compared to Nivolumab alone, whatever the compositions dilutions. Nonetheless, activation of TFH cells is lower than in the presence of the whole immune population. Indeed, in absence of DCs, there is less presentation of microbial antigens to TFH cells, and solely memory B cells are capable of activating TFH cells present in this model. These two above mentioned experiments show a dual mechanism of maturation of B cells, involving DCs and memory B cells. The microbial compositions of the invention induce B cell differentiation into plasmablast either via microbial antigen presentation by DCs to TFH cells and subsequent activation of naive B cells or via microbial antigen presentation by pre-primed memory B cells to TFH cells and subsequent activation of pre-primed memory B cells.

[0222] Example 2:

[0223] Principle

[0224] The aim of this experiment was to evaluate the ability of therapeutic microbial compositions of the invention to promote TLS formation and maturation before administration of an ICI. A murine lung tumor model was treated with Composition 2 - Pool and several physiological parameters were studied: mouse weight, tumor growth, TLS formation and TLS maturation.

[0225] Experimental set-up

[0226] As presented on Figure 2, C57BL / 6JRJ mice were treated, 2 days before tumoral cells administration, with antibiotics for 6 days to make room in their bowels. A mix of ampicillin, colistin and streptomycin was administered by the drinking water. Tumoral cells were administered intravenously in animal tails at DO. 4 groups were used: G1 , the negative control, treated with NaCL 5 times (the same way as for Composition 2 - Pool); G2, the group treated once with Composition 2 - Pool; G3, the group treated 5 times with Composition 2 - Pool; G4, the positive control, treated 5 times (once a day for 5 days) with LipoPolySaccharides (LPS) but without TC-1 cells. Tumoral cells were TC- 1 cells that is a well-established cancer cell line derived from lung epithelial cells that have been transformed with oncogenic human papillomavirus (HPV) E6 and E7 genes. TC-1 cells are commonly used in preclinical studies of lung cancer. Composition 2 - Pool was defrosted rapidly at 37°C and 1.108bacteria per mouse per administration were administered by oral gavage. LPS was administered intra-nasaly. It is used as positive control of TLS formation. Animal weight and tumoral growth were followed at least 2 times a week. Tumoral growth was followed by in-vivo imaging. After sacrifice, lungs were processed to be analyzed by immunohistochemistry to study TLS presence and maturation.

[0227] Results

[0228] As presented on Figure 3, TC-1 cells were detected in each condition. Indeed, G1 and G2 groups experienced a fewer cancer cell engraftment in the lung compared to G3, especially for G2. Regarding animal weight, mice from G 1 and G2 knew a comparable evolution with few weight variations. Instead, in G3, mice experienced a sensitive weight decrease across the gavage period (5 administrations within 5 days). Besides, stricking differences between G2 and G3 were observed. First, the number of TLS structures in total, identified here by the presence of immune cell clusters containing both B and T cells (identified by B220 and CD3 markers, respectively), which was more elevated in G3 than in G2. Secondly, the presence of numerous TLS distant to tumoral sites in G3 compared to presence of TLS around the tumors solely, in G2. In addition, when comparing tumor size between groups, there was a strong trend of being more important in G3. In G1 , some TLS around tumors were observed but less frequently than in G2 or G3.

[0229] All these different elements demonstrated that administration of Composition 2 - Pool triggered TLS formation and maturation around tumors at low dose in vivo, and that it is not impacting host organism in terms of weight. It seems additionally that it prevented tumoral cell engraftment as fewer and smaller tumors were encountered in the G2 group compared to G1 . Nonetheless, a threshold in terms of product quantity must not be passed off. Indeed, multiple product administrations, 5 administrations within 5 days in this experimental set-up, led to ectopic formation of TLS impacting host weight and tumoral growth negatively. This is coherent with observations made in Example 1 where the dose 1 / 100 gave better results than 1 / 1000.

Claims

CLAIMS1 . A pharmaceutical composition comprising Bacteroides fragilis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Faecalibacterium prausnitzii, Flavonifractor plautii, Ruminococcus bromii, Coprococcus comes, Roseburia intestinalis, Dysosmobacter sp. BX15 and Phocaeicola vulgatus, for use in combination with an immune checkpoint inhibitor (I Cl ) for treating a cancer, wherein the cancer is a solid tumor comprising a tertiary lymphoid structure (TLS) in its microenvironment.

2. The pharmaceutical composition for use according to claim 1 , wherein the composition comprises a bacterial population that includes:- between 25% and 75% of Bacteroidetes,- between 20% and 60% of Firmicutes,- between 0.01% and 15% of Actinobacteria,- between 0.01% and 10% of Proteo bacteria.

3. The pharmaceutical composition for use according to any one of claims 1 or 2, wherein the composition comprises a bacterial population that includes:- between 2% and 60% of Bacteroidaceae,- between 1 % and 35% of Ruminococcaceae,- between 1 % and 25% of Lachnospiraceae,- between 0.01 % and 5% of Clostridiaceae,- at least 7 bacterial subpopulations selected from Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae, Bifidobacteriaceae, Rikenellaceae, Odoribacteraceae, Barnesiellaceae, Tannerellaceae, Oscillospiraceae, Sutterellaceae, Desulfovibrionaceae, Acidaminococcaceae, Eubacteriaceae, Prevotellaceae, Akkermansiaceae, Coriobacteriaceae, Veillonellaceae, Clostridiales Family XIII. Incertae Sedis, Enterobacteriaceae and Selenomonadaceae.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the composition comprises a bacterial population that includes:- between 2% and 60% of Bacteroides,- between 0.01 % and 20% of Phocaeicola,- between 0.01% and 20% of Faecalibacterium,- between 0.01 % and 5% of Blautia,- between 0.01 % and 5% of Flavonifractor,- between 0.01 % and 5% of Gemmiger,- between 0.01 % and 5% of Dorea,- at least 12 bacterial subpopulation selected from: Alistipes, Odoribacter, Parabacteroides, Coprococcus, Barnesiella, Roseburia, Bilophila, Ruminococcus, Clostridium, Subdoligranulum, Dysosmobacter, Oscillibacter, Lachnospira, Eubacterium, Mediterraneibacter, Anaerostipes, Anaerobutyricum, Paraprevotella, Sutterella, Phascolarctobacterium, Bifidobacterium, Akkermansia, Butyricimonas, Collinsella, Fusicatenibacter, Faecalicatena, Prevotella, Dialister,Ruthenibacterium, Escherichia, Enterocloster, Eisenbergiella, Erysipelatoclostridium, Lachnoclostridium, Hungatella and Holdemania.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the ICI is a drug blocking one of the following targets: PD-1 , PD-L1, PD-L2, CD27, CD28, CD40, CD122, CD137, 0X40, GITR, ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3 (such as Relatlimab), NOX2, TIM-3, VISTA, SIGLEC7 or TIGIT.

6. The pharmaceutical composition for use according to any one of the claims 1 to 5, wherein the ICI is a drug blocking one of the following targets: PD-1 , PD-L1 , PD-L2, CTLA-4, TIM-3 or TIGIT.

7. The pharmaceutical composition for use according to any one of the claims 1 to 6, wherein the ICI is an antibody targeting PD-1.

8. The pharmaceutical composition for use according to any one of claims 1 to 7, wherein the composition includes bacteria, viruses, archaea, bacteriophages, fungi or mixtures thereof, preferably bacteria and fungi.

9. The pharmaceutical composition for use according to any one of claims 1 to 8, wherein the composition comprises a stool derived microorganism, preferably is a population of stool derived microorganisms.

10. The pharmaceutical composition for use according to any one of the claims 1 to 9, wherein the composition has followed at least a manufacturing step of homogeneization with a cryoprotectant and a manufacturing step of freezing below 40°.

11. The pharmaceutical composition for use according to any one of claims 1 to 10, wherein the composition triggers an efficient IgG production by B cells in an in vitro model of tertiary lymphoid structures (TLS) such as a human tonsil-based model.

12. The pharmaceutical composition for use according to any one of claims 1 to 12, wherein the cancer is a lung or a skin cancer.

13. The pharmaceutical composition for use according to claim 1 to 12, wherein the pharmaceutical composition is administered 1 , 2, 3 or 4 times during an administration cycle of at least 5 days.

14. The pharmaceutical composition for use according to claim 1 to 13, wherein the product is administered before the ICI.

15. A kit of parts comprising a pharmaceutical composition as defined in any one of claims 1 to 13 and an immune modulatory treatment for simultaneous or sequential administration to a patient.

Images