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Definitions

• the present invention relates to the field of anticancer treatment.
• the present invention concerns the role of the microbiota in the efficacy of cancer treatments, and provides methods for determining if a patient is likely to benefit from a cancer treatment, as well as probiotics to improve the efficacy of such a treatment in patients in need thereof.
• Cancer chemotherapy is based on the use of drugs which kill replicating cells, hopefully faster than the agents kill the patient's normal cells.
• Surgery is used to reduce tumor bulk, but has little impact once the cancer has metastasized. Radiation is effective only in a localized area. All of these approaches pose significant drawbacks and added risks such as increased susceptibility to infection.
• a further approach to cancer therapy is to target the immune system ("immunotherapy") rather than or in addition to targeting the tumor itself.
• Anticancer chemotherapeutics often cause mucositis (a debilitating mucosal barrier injury associated with bacterial translocation) and neutropenia, two complications that require treatment with antibiotics, which in turn can result in dysbiosis (Ubeda et al, 2010; van Vliet et al., 2010).
• cyclophosphamide alters the composition of small intestinal microbiota in mice and provokes the translocation of selected species of Gram+ bacteria into secondary lymphoid organs. There, these bacteria stimulate the generation of a specific subset of "pathogenic" T helper 17 (pThl 7) cells and memory Thl immune responses.
• pThl 7 T helper 17
• the inventors also demonstrated that germ-free mice or hosts treated with antibiotics killing Gram+ bacteria exhibited reduced pThl7 responses and relative chemoresistance to CTX unless adoptively transferred with pThl7 cells.
• dysbiosis interfered with the activity of other anticancer chemotherapeutics (such as anthracyclines and oxaliplatine).
• the present invention provides a probiotic composition which can be used as an adjuvant to an antineoplastic treatment administered to a cancer patient, wherein said probiotic composition comprises bacteria selected amongst Enterococcus hirae, Lactobacillus johnsonii, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella, Holdemania and mixtures thereof.
• said probiotic composition comprises bacteria selected amongst Enterococcus hirae, Lactobacillus johnsonii, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella, Holdemania and mixtures thereof.
• Another aspect of the present invention is the use of a combination of a chemotherapeutic agent and of an antibiotic composition which decreases the firmicutes/bacteroidetes ratio or specifically augments SFB and/or Porphyromonadaceae and/or decreases Clostridium group IV in the gut microbiota of an individual when administered to said individual, for treating a patient having a cancer.
• the invention also relates to the use of an antibiotic composition such as those described above, for modulating the gut microbiota of a patient to potentiate the anticancer effects of a chemotherapeutic agent administered to said patient.
• An immunogenic composition comprising fragments of bacteria selected from the group consisting of Enterococcus hirae, Lactobacillus johnsonii, Enterococcus faecalis, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella, Holdemania and mixtures thereof is also part of the present invention, as well as its use as an adjuvant to an antineoplastic treatment administered to a cancer patient.
• a first cell composition comprises antigen presenting cells (APC) which have been pulsed ex vivo with a probiotic composition or with an immunogenic composition as described above
• a second cell composition comprises memory T cells obtained by a process comprising ex vivo contacting T cells from a cancer patient with a first cell composition as defined above.
• APC antigen presenting cells
• the present invention also provides an in vitro method of identifying a patient likely to be a good responder to a chemotherapy, comprising determining the functionality of TLR 4, NODI and NOD2 in said patient, wherein if said patient lacks a functional TLR 4 and/or NODI and/or NOD2, the patient is identified as a good responder to a chemotherapy.
• the present invention also provides a method for in vitro determining whether a cancer patient can benefit from an antineoplastic treatment, comprising the following steps:
• the present invention also provides a method for in vitro determining whether an antineoplastic treatment is to be continued or stopped for a cancer patient, comprising the following steps: (i) from a biological sample from said patient, such as a blood sample obtained 3 to 9 weeks, preferably 6-9 weeks after the beginning of said antineoplastic treatment, analyzing memory CD4 + T cell response directed against at least one commensal species of bacteria, for example against L. johnsonii, E. hirae and/or E. Faecalis;
• the antineoplastic treatment is continued, and in absence of such a response, the antineoplastic treatment is stopped.
• the classification of the response can be performed, for example, by comparing pre- and post-treatment secretion of cytokines in ex vivo restimulation assays.
• the present invention also pertains to a method for in vitro determining the biological effects of a neoadjuvant antineoplastic treatment which has been administered to a patient, comprising the following steps:
• the result indicates that the neoadjuvant antineoplastic treatment induced a T-bet/Thl local and systemic immune response.
• Another object of the present invention is a probiotic bacterial strain selected from the group consisting of Lactobacillus johnsonii (especially strain CNCM 1-4823), Enterococcus hirae (especially strain CNCM 1-4815) and Enterococcus faecalis, for use in combination with an antineoplastic agent for inducing a T-bet/Thl local and systemic immune response, as well as a composition comprising the same.
• the invention also pertains to adoptive cell transfer of a cell obtained by stimulating naive CD4+ T cells from a cancer patient in the presence of a mixture of IL- ⁇ ⁇ , IL-6 and IL23, in said cancer patient, in combination with an antineoplastic treatment, for treating cancer.
• FIG. 1 Cyclophosphamide disrupts gut mucosal integrity.
• A-B Hematoxilin-eosin staining of the small intestine epithelium at 48h post-NaCl (Co) or CTX or doxorubicin (Doxo) therapy in C57BL/6 na'ive mice
• C The numbers of inflammatory foci depicted/mm (B, left panel, indicated with arrowhead on A), thickness of the lamina limba reflecting edema (B, middle panel, indicated with # on A) and the reduced length of villi (B, right panel, indicated with arrowhead in A) were measured in 5 ilea on 100 villi/ile
• a representative microphotograph of an ileal villus containing typical mucin- containing goblet cells is shown in vehicle- and CTX or Doxo-treated mice (left panels). The number of goblet cells/villus was enumerated in the right panel for both chemotherapy agents.
• D Specific staining of Paneth cells is shown in two representative immunofluorescence microphotographs (D, left panels). The quantification of Paneth cells was performed measuring the average area of the lysozyme-positive clusters in 6 ilea harvested from mice treated with NaCl (Co) or CTX at 24-48 hours.
• E The quantification of Paneth cells was performed measuring the average area of the lysozyme-positive clusters in 6 ilea harvested from mice treated with NaCl (Co) or CTX at 24-48 hours.
• Quantitative PCR analyses of Lysozyme M and Regllly transcription levels in duodenum and ileum lamina intestinal cells from mice treated with CTX at 18 hours. Means ⁇ SEM of normalized deltaCT of 3-4 mice/group concatenated from three independent experiments.
• FIG. 1 Cyclophosphamide induces mucosa-associated microbial dysbiosis and bacterial translocation in secondary lymphoid organs.
• the microbial composition (genus level) was analyzed by 454 pyro sequencing of the 16S rRNA gene from ilea and caeca of na ' ive mice and B16F10 tumor bearers.
• Principal Component Analyses (PCA) highlighted specific clustering of mice microbiota (each dot represents one mouse) depending on the treatment (NaCl: Co, grey dots; CTX-treated, black dots).
• a Monte Carlo rank test was applied to assess the significance of these clusterings.
• D Quantitative PCR (qPCR) analyses of various bacterial groups associated with small intestine mucosa were performed on CTX or NaCl (Co)-treated, naive or MCA205 tumor-bearing mice.
• Absolute values were calculated for total bacteria, Lactobacilli, Enter ococci and Clostridium group IV and normalized by the dilution and weight of the sample. Standard curves were generated from serial dilutions of a known concentration of genomic DNA from each bacterial group and by plotting threshold cycles (Ct) vs. bacterial quantity (CFU). Points below the dotted lines were under the detection threshold. Data were analyzed with the linear model or generalized linear model. *, p ⁇ 0.5, **, p ⁇ 0.1, ***, pO.001, ns, non significant.
• FIG. 3 CTX-induced pThl7 effectors and memory Thl responses depend on gut microbiota.
• Each dot represents one mouse bearing no tumor (round dots), a B16F10 melanoma (diamond dots) or a MCA205 sarcoma (square dots), open dots featuring NaCl-treated mice and full dots indicating CTX-treated animals.
• C Intracellular analyses of splenocytes harvested from non-tumor-bearing mice after 7 days of either NaCl or CTX treatment, under ATB or water regimen as control. Means ⁇ SEM of percentages of IFNy + Thl7 cells, T-bet + cells among RORyt + CD4 + T cells and CXCR3 + cells among CCR6 + CD4 + T cells in 2 - 8 independent experiments, each dot representing one mouse.
• FIG. 1 Intracellular staining of total splenocytes harvested 7 days post-CTX treatment from naive mice orally-reconstituted with the indicated bacterial species after ATB treatment.
• E 7 days post CTX or NaCl (Co) treatment, splenic CD4 + T cells were restimulated ex vivo with bone-marrow dendritic cells (BM-DCs) loaded with decreasing amounts of bacteria for 24 hours. IFNy release, monitored by ELISA, is shown. The numbers of responder mice (based on the NaCl baseline threshold) out of the total number of mice tested is indicated (n). Statistical comparisons were based on the paired t-test. Data were either analyzed with beta regression or linear model and correlation analyses from modified Kendall tau. *, p ⁇ 0.05, ***, pO.001, ns, non significant.
• Figure 4 Vancomycin blunts CTX-induced pThl7 differentiation which is mandatory for the tumoricidal activity of chemotherapy.
• (A) After a 3 week-long pretreatment with broad-spectrum ATB, DBA2 mice were inoculated with P815 mastocytomas (day 0), treated at day 6 with CTX (arrow) and tumor growth was monitored. Tumor growth kinetics are shown in Fig. 13A and percentages of tumor-free mice at sacrifice are depicted for two experiments of 1 1-14 mice/group.
• B MCA205 sarcoma were inoculated at day 0 in specific pathogen-free (SPF) or germ-free (GF) mice that were optionally mono- associated with segmented filamentous bacteria (SFB), treated with CTX (arrow) and monitored for growth kinetics (means ⁇ SEM).
• SPPF pathogen-free
• GF germ-free mice
• mice received an adenovirus expressing the Cre recombinase (AdCre) by intranasal instillation to initiate lung adenocarcinoma (dO).
• AdCre adenovirus expressing the Cre recombinase
• dO lung adenocarcinoma
• Vancomycin was started for a subgroup of mice ("Chemo + Vanco") on d77 post- AdCre, One week after the start of vancomycin, CTX-based chemotherapy was applied i.p. to mice that only received chemotherapy ("Chemo") or those that received in parallel vancomycin (“Chemo + Vanco”). Mice received chemotherapy on d84, d91 and d98. A control group was left untreated ("Co").
• Figure 5 Lack of dysbiosis 24 or 48 h post-CTX.
• A Overall composition of the gut microbiota as assessed by high- throughput 454 pyrosequencing of the 16S rRNA gene at various time points (0, 24, 48 hours post-CTX). Each column represents data from one mouse small intestine mucosal microbiota, tO (before CTX injection), t24 and t48 (24 and 48 hours post-CTX). The positive gradient of representativity of distinct genera (heatmap of the Logio- tranformation) is indicated.
• Statistical analyses ns between tO and t24 or t48 hrs. (B-C). Detailed example of the pyrosequencing data for Clostridium sp.
• FIG. 6 Distribution of bacterial genera in the ileum of mice treated with CTX. Heatmap of the Logio-transformation of relative abundance of genus in the small intestine from NaCl (Co) and CTX-treated animals. Prior to CTX therapy, tumors were inoculated in a subgroup of animals (TB+). Only bacterial genera representing more than 0.05% of the whole microbiota are presented. The applied logio- transformation on relative abundances data has been explained in the microbiota Materials and Methods section. No specific clustering method has been applied for heatmap construction. Average delta of percentages between Co and CTX for each genus was calculated to re-order bacterial genera.
• CTX induced a reduction of bacterial groups from the Firmicutes phylum distributed within four genera and groups ⁇ Clostridium cluster XlVa, Roseburia, unclassified Lachnospiraceae, Coprococcus, Table 2) in the mucosa of CTX-treated animals.
• some bacteria were either overrepresented (such as Lactobacillus reuteri) or underrepresented (such as Clostridium sp.
• Figure 7 Loss of CD103 + CDllb + and Thl7 cells in the duodenal lamina limbal polarization of splenocytes correlating with small intestine bacterial species.
• A Dendritic cell (DC) subsets in LP of the small intestine. Flow cytometry analyses and quantification of various DC subsets residing in small and large intestine LP at day 0, day 3 and day 7 post-CTX injection. The graph depicts means+SEM of the percentages of DC in 7 mice/time point in two concatenated experiments. Large intestine DC subsets were not affected by CTX (not shown). Data were analyzed with the Mann Whitney t-test. (B-C). Modulations of Thl 7 cells seven days post-CTX. (B) Flow cytometry analyses of lymphocytes separated from the LP of duodenum and ileum, harvested from NaCl versus CTX-treated mice.
• the graphs depict the concatenated data from eight independent experiments, each dot representing one experiment. Statistical comparisons were based on the Wilcoxon test.
• C Left panel: a micrograph picture of immunofluorescence staining of ileum in NaCl versus CTX- treated mice, ⁇ TCR + cells were stained in green (Alexa Fluor 488) using an anti- ⁇ TCR Ab and CD3 + T cells were stained in blue (Alexa Fluor 647) using anti-CD3 Ab.
• Right panel the enumeration of positive cells was performed on 100 villi in three ilea by two independent researchers.
• D Thl polarization of splenocytes at day 7 post-CTX injection.
• Figure 8 Doxorubicin failed to induce pThl7 cells in the spleen and does not require gut commensals for reducing tumor growth.
• A-B Failure of doxorubicin (Doxo) to induce splenic IL-17 producing CD4 + T cells.
• Doxo was injected i.p. into mice at the indicated doses (A) or at a fixed dose of 50 ⁇ at 2 mM (being 3 mg/kg for a mouse weighing 20g) (B), and splenocytes were recovered 7 days later to evaluate the production of IL-17 in response to 48 hours anti-CD3/anti-CD28 cross-linking (A, B) or the frequency of cells with a CD4 + T-bet + RORyt + phenotype was determined by flow cytometry (B).
• Cyclophosphamide (CTX) used at a dose of 100 mg/kg was used as a positive control.
• regulatory T cells were depleted by injections (250 g, 1 and 3 days before Doxo administration) of anti-CD25 Ab and an irrelevant isotype-matched control Ab was used as control.
• C Antitumor effects of doxorubicin against established MCA205 in specific pathogen-free (SPF), antibiotic (ATB)-treated and germ-free mice.
• Kinetics of tumor growth (mean size ⁇ SEM) are depicted in 2 to 3 pooled experiments including 4-6 animals/group. Data were analyzed with the t-test, linear model or generalized linear model. *, p ⁇ 0.05, **, pO.Ol, ***, p ⁇ 0.001, ns, not significant.
• Figure 9 Efficacy of broad spectrum ATB in bacterial depletion from the feces of naive or tumor-bearing mice.
• Feces were freshly harvested from mice that were left untreated or were treated with broad spectrum ATB at various time points and plated onto blood agar plates for aerobic and anaerobic conditions, as well as onto DCO agar plates (BioMerieux) for the specific growth of enterococci. After 48h of culture, isolated colonies were enumerated. All the mice of each distinct experiment have been monitored and scored in this manner. One representative monitoring is shown.
• FIG. 3C Flow cytometry analyses of lymphocytes harvested from NaCl versus CTX-treated WT (as in Fig. 3C) or Nod2 '/" versus Myd88 ⁇ /' mice restimulated 4 hours with PMA/ionomycin (using intra-and extra-cellular stainings with anti- CD3, CD8, IFNy and IL-17 Abs).
• the graph depicts the mean percentages of IFNy + positive cells among IL-17 + CD4 + T cells from two independent experiments, each dot representing one mouse.
• B Nodi and Nod2 are dispensable for tumor growth reduction induced by CTX.
• MCA205 tumors were established in WT or Nodl ⁇ / ⁇ Nod2 ' ⁇ mice before administration, at day 5 and 12, of CTX.
• the tumor growth kinetics (means ⁇ SEM) were monitored in 5 animals/group. Two independent experiments yielded similar results. Data were analyzed with the t-test, linear model or generalized linear model. ***, pO.001, ns, not significant
• FIG. 11 Immunization against commensal bacteria post-CTX. (A-C). Recovery of CBir Tg T cells in congenic mice after CTX.
• One million naive B6.CD45.1 + CBirl TCR Tg CD4 + T cells were adoptively transferred i.v. in naive CD45.2 WT recipient congenic mice that were treated, one day later, with NaCl or CTX and sacrificed 7 days later for FACS analysis of splenocytes and ex vivo restimulation with CBirl specific peptides.
• Figure 12 Translocated bacteria processed and presented by dendritic cells lead to the polarization of naive CD4 + T cells in vitro. Ex vivo differentiation of Thl7/Thl cells with translocated bacteria. Cross-talk between BMDCs loaded with various bacteria and naive CD4 + T for 4 days. Monitoring of IL-17 (left) or IFNy (right) cytokine concentrations by commercial ELISA. Each dot represents one in vitro experiment performed in triplicate wells. Eleven experiments were performed and are depicted, t-test: *, p ⁇ 0.5, **, p ⁇ 0.01, ns, non significant.
• FIG. 13 Gut microbiota affects chemotherapy efficacy.
• Figure 14 Parabacteroides distasonis and chemoresistance.
• mice Conventionally reared mice were treated for 3-4 weeks with ATB, implanted 4 days with MCA205 and then orally inoculated with P. distasonis that monocolonized feces. At day 6 post-tumor inoculation, mice were treated with doxorubicin. The tumor growth kinetics between P. distasonis reconstituted or unreconstituted ATB treated-mice post-doxorubicin (meansiSEM) were monitored in 8-12 mice/group. Data were analyzed with the linear model or generalized linear model. *p ⁇ 0.05, ***p ⁇ 0.001.
• FIG. 15 Transcriptional profiling of ex vivo generated Thl7 and pThl7 compared with CTX-induced spleen CD4 + T cells.
• Naive T cells were stimulated with plate-bound antibodies against anti-CD3 and anti-CD28 Abs in the absence (ThO) or presence of either recombinant mouse IL-ip (10 ng/ml)+ IL-6 (10 ng/ml)+ IL-23 (20 ng/ml) (as for " P Thl7"cells) or with rTGF- ⁇ (2.5 ng/ml)+IL-6 (as for "Thl7” cells).
• Figure 16 Vancomycin-resistant microbial microbiota.
• FIG. 18 Primary cellular Thl and Tel immune responses against chicken OVA are not affected by antibiotics regimen in wild type naive mice.
• C57B1/6 mice were pre-treated for 8-10 days with various antibiotic regimens, including large spectrum antibiotics (ATB), colistin (Coli) or vancomycin (Vanco), monitored by culturing feces at various time points and then, immunized in the footpad with 1 mg of OVA admixed with 50 ⁇ g of Poly (I:C) three days after i.p. CTX administration.
• ATB large spectrum antibiotics
• Coli colistin
• Vanco vancomycin
• mice received an adenovirus expressing Cre recombinase (Ad-cre) by intranasal instillation to initiate lung adenocarcinoma (dO).
• Mice were either left untreated ( « Co») or received chemotherapy (d84, d91 and d98) in absence ( « Chemo ») or presence of 0.25mg/ml vancomycin ( « Chemo + Vanco ») (mixed into drinking water starting on d77 post Ad-cre and until the end of the experiment ; antibiotic-containing water was replaced biweekly).
• Figure 22 Random forest analysis : Main discriminative genera between patients receiving or not chemotherapy and bearing a colon cancer.
• Figure 23 Main genera that are significantly different between controls (no chemotherapy) versus post-neoadjuvant chemotherapy.
• Figure 24 Distribution of lactobacilli, Bifidobacterium and Clostridium group IV. In the ileum among colon cancer patients treated or not with chemotherapy.
• FIG. 25 TH1 and pTH17 immune responses following mono- association with EMrae.
• C57BL/6 mice were treated with vancomycine, streptomycine, ampicilline and colistine (broad spectrum ATB regimen) for 14 days, followed by one ip injection of lOOmg/kg of CTX on day 15 and oral feeding with 10 9 bacteria (as illustrated on the graph) on day 16 prior to flow cytometric analyses of the splenocytes at day 22.
• A-C Flow cytometric analyses of pTH17 cells.
• C-F Tumor growth kinetics in the presence of oral gavage with E.hirae, or L. johnsonii or both of them (cocktail).
• F Concatanated data from 3 experiments including 5 mice/group. Anova statistical analyses : *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
• Figure 27 E.hirae induces OVA-specific antitumor-immune responses.
• CD45.1 C57BL/6 mice were treated with vancomycine, streptomycine, ampicilline and colistine (broad spectrum ATB regimen) for 14 days, then inoculated sc with MCA205-OVA sarcoma, then treated with one ip injection of lOOmg/kg of CTX on day 21 and oral feeding with 10 9 E.hirae (as illustrated on the graph A) on day 22.
• 10 6 CD45.2 + OTII transgenic T cells were transferred iv and mice were sacrifized 8 days later for flow cytometric analyses of recipient (CD45.1, B) or donor (CD45.2) CD4 + T lymphocytes.
• FIG. 28 E.hirae induces E7-spccific antitumor-immune responses.
• D Monitoring of D b -E73 -47 tetramer binding CD8 + T cells in the spleens. Results from two experiments are presented. Anova test for statistical analyses. *p ⁇ 0.05.
• Figure 29 Pulsed field gel electrophoresis of a series of E.hirae isolates from different public libraries. Non supervised hierarchical clustering of the sequence similarities among these clones. Clone 13144-13147 have been isolated in the Gustave Roussy animal facility from mice splenocytes after different kind of therapies (CTLA4 blockade, CTX). The one isolate that has been used henceforth or above is noted "13144 VillejuiP in this Figure, and corresponds to the Enterocococcus hirae strain deposited on November 7, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number 1-4815.
• CCM Collection Nationale de Cultures de Microorganismes
• Figure 30 Differential immunogenicity of various E.hirae isolates in vivo, C57BL/6 mice were treated with vancomycine, streptomycine, ampicilline and colistine (broad spectrum ATB regimen) for 14 days, followed by one ip. injection of 100 mg/kg of CTX on day 15 and oral feeding with 10 9 bacteria (clone 708 versus clone CNCM 1-4815) on day 16 prior to flow cytometric analyses of the splenocytes at day 22. The positive controls are represented by mice treated with CTX without prior ATB. A-C. Flow cytometric analyses of TH1, Tel or pTH17 cells.
• CD4 + TH1 cells expressing IFNy or CXCR3 (A, left and right panel), CD8+ Tel expressing IFNy (B) or CD4 + pTH17 cells expressing CXCR3 in the gate of live CCR6+ CD4+ T splenocytes (C).
• Figure 31 Triggering of NOD receptors hampered bacterial translocation and priming of pTH17 cells by promoting the release of antimicrobial peptides.
• A Enumeration of bacterial colonies in splenocytes 48 hrs post-CTX therapy in various mouse backgrounds. Splenic cells (left panel) and mesenteric LN (middle panel) harvested from C57BL/6 WT or NODI 7" xNOD2 - " mice were cultured in anaerobic conditions for 48 hours. Bacterial outgrowth was enumerated (number of colonies/plate and frequencies of positive plates/animal (right panel)) and eventually characterized by mass spectrometry for bacterial identification. B. Flow cytometric analyses of pTH17 cells.
• FIG. 33 Relative efficacy of CTX against sarcomas growing in various gene deficient- hosts.
• CTX was administered every other 7 days at 1 OOmg/kg ip. in WT (A), NODI (B), NOD2 (C, left panel), CARD9 (C, middle panel), RIP2 (C, right panel), NODlxNOD2 (D, left panel and right panel)-deficient mice.
• Each graph depicts the means of 5 tumors/group in one representative growth kinetics. Student t'test : *p ⁇ 0.05.
• FIG. 34 Inhibitory effects of TLR4 agonists in the anticancer probiotic activity of the association of E.hirae+L.johnsonii.
• C57BL/6 mice were treated with vancomycine, streptomycine, ampicilline and colistine (broad spectrum ATB regimen) for 14 days, then inoculated sc. with MCA205 sarcoma, then treated with one ip. injection of 100 mg/kg of CTX on day 21 (and day 29) and oral feeding with LPS (A) 500ug/mouse or 10 9 bacteria ⁇ E.coli) (B) on day 22.
• Tumor growth kinetics were monitored biweekly for 1 month. The positive (CTX without ATB) and negative (PBS in ATB) controls are indicated.
• Figure 36 Principle component analysis of the pyrosequencing of 16srRNA of gene amplicons of biofilms of small intestines from WT versus NODlxNOD2 deficient mice treated or not with CTX.
• B Heat map representation of the diversity and differences between the 4 groups highlighting the enrichment in Clostridiaceae (mostly SFB, Table 4) at the expense of Erysipelotrichaceae with CTX therapy as shown in statistical analyses presented in Table 1.
• Figure 37 Combination of the commensal E.hirae and the pathobiont Clostridium perfringens. Same experimental setting as in Fig. 26 but C. perfringens has been introduced by oral gavage as well. Tumor growth kinetics were monitored biweekly for 1 month. A. Tumor growth for the two groups E.hirae versus C.perfringens. B. Tumor growth kinetics in the presence of oral gavage with E.hirae+ L. plantarum versus E.hirae + Clostridium perfringens. Two experiments including 5 mice/group are shown. Anova statistical analyses : *p ⁇ 0.05.
• ATB regimen (Zhang Y et al. Toxicology and Applied Pharmacology 277 (2014) 138- 145) were administered for 15 days prior to tumor inoculation and CTX therapy every other 13 days. Tumor outgrowth was monitored with a caliper twice a week. Protocols reported to reduce Firmicutes, most specifically Clostridiae eventually decreasing the Firmicutes/ Bacteroides ratio (such as the combination of neomycine+cephalothin or vancomycine+imipenem) (panel C-D) could improve the CTX-induced antitumor effects while cifloxacin (which, in contrast, induced a marked suppression of Bacteroidetes) was not efficient (panel B). Of note, the combination of neomycine+cephalothin could augment SFB representativity while vanco+imipenem increased that of Porphyromonas. Each experiment contains several groups of 5 mice. .*Anova test: p ⁇ 0.05.
• gut microbiota (formerly called gut flora or microflora) designates the population of microorganisms living in the intestine of any organism belonging to the animal kingdom (human, animal, insect, etc.). While each individual has a unique microbiota composition (60 to 80 bacterial species are shared by more than 50% of a sampled population on a total of 400-500 different bacterial species/individual), it always fulfils similar main physiological functions and has a direct impact on the individual's health:
• gut microbiota • a healthy, diverse and balanced gut microbiota is key to ensuring proper intestinal functioning. Taking into account the major role gut microbiota plays in the normal functioning of the body and the different functions it accomplishes, it is nowadays considered as an "organ”. However, it is an "acquired" organ, as babies are born sterile; that is, intestine colonisation starts right after birth and evolves afterwards.
• gut microbiota starts at birth. Sterile inside the uterus, the newborn's digestive tract is quickly colonized by microorganisms from the mother (vaginal, skin, breast, etc.), the environment in which the delivery takes place, the air, etc. From the third day, the composition of the intestinal microbiota is directly dependent on how the infant is fed: breastfed babies' gut microbiota, for example, is mainly dominated by Bifidobacteria, compared to babies nourished with infant formulas.
• the composition of the gut microbiota evolves throughout the entire life, from birth to old age, and is the result of different environmental influences. Gut microbiota' s balance can be affected during the ageing process and, consequently, the elderly have substantially different microbiota than younger adults.
• composition at a species level is highly personalised and largely determined by the individuals' genetic, environment and diet.
• the composition of gut microbiota may become accustomed to dietary components, either temporarily or permanently.
• Japanese people, for example, can digest seaweeds (part of their daily diet) thanks to specific enzymes that their microbiota has acquired from marine bacteria.
• Dysbiosis a disequilibrium between potentially “detrimental” and known “beneficial” bacteria in the gut or any deviation to what is considered a “healthy” microbiota in terms of main bacterial groups composition and diversity.
• Dysbiosis may be linked to health problems such as functional bowel disorders, inflammatory bowel diseases, allergies, obesity and diabetes. It can also be the consequence of a treatment, such as a cytotoxic treatment or an antibiotic treatment.
• a specific dysbiosis can be highlighted depending on the pathogenic condition. For instance, patients with Crohn's disease, a chronic inflammatory bowel disease, present a microbiota with reduced percentages and diversity of bacteria belonging to the Firmicutes phylum, and mostly from the Clostridium Upturn (cluster IV) group (Manichanh et al., 2006; Sokol et al., 2006). Generally, decreased percentages of bacteria from the Lachnospiraceae family can be observed.
• mucosa- associated microbiota of these patients is depleted in bacteria from the Bifidobacterium and Lactobacillus genera toward increased levels of potentially pathogenic bacteria such as specific strains of Escherichia coli with adherent and invasive phenotypes (AIEC) (Darfeuille-Michaud et al. 2011, 2004; Joossens et al., 2011).
• AIEC adherent and invasive phenotypes
• gut microbial dysbiosis relates to enrichment in bacterial species from the Bacteroides genus and decrease of Faecalibacterium and Roseburia genera belonging species (Sobhani et al., 2011; Wu et al., 2013). Specifically, Fusobacterium and Campylobacter genera were found to be consistently increased in both feces and mucosa of CRC patients.
• "beneficial or "favorable” bacteria are essentially Lactobacillus and Bifidobacterium
• “detrimental” or “unfavorable” bacteria are essentially the species Parabacteroides distasonis and Faecalibacterium prausnitzH, the genera Gemmiger, Alistipes and Clostridium Cluster IV. (Clostridium leptum group,).
• Antineoplastic treatments herein designate any treatment for cancer except surgery. They include chemotherapy, hormonal and biological therapies, and radiotherapy.
• Chemotherapeutic agents are chemical molecules which act by killing cells that divide rapidly, one of the main properties of most cancer cells.
• chemotherapeutic agents are chemical molecules which act by killing cells that divide rapidly, one of the main properties of most cancer cells.
• - spindle poisons such as mebendazole, colchicine
• mitotic inhibitors including taxanes (paclitaxel (Taxol ®), docetaxel (Taxotere ®)) and vinca alkaloids ⁇ e.g.-. vincristine, vinblastine, vinorelbine, vindesine)
• taxanes paclitaxel (Taxol ®), docetaxel (Taxotere ®)
• vinca alkaloids e.g.-. vincristine, vinblastine, vinorelbine, vindesine
• cytotoxic/antitumor antibiotics such as anthracyclines (e.g.: doxorubicin, daunorubicin, adriamycine, idarubicin, epirubicin and mitoxantrone, valrubicin), streptomyces ⁇ e.g.: actinomycin, bleomycin, mitomycin, plicamycin)
• anthracyclines e.g.: doxorubicin, daunorubicin, adriamycine, idarubicin, epirubicin and mitoxantrone, valrubicin
• streptomyces ⁇ e.g.: actinomycin, bleomycin, mitomycin, plicamycin
• anti-metabolites such as pyrimidine analogues (e.g.: fluoropyrimidines analogs, 5-fluorouracil (5-FU), floxuridine (FUDR), Cytosine arabinoside (Cytarabine), Gemcitabine (Gemzar ®), capecitabine; purine analogues (e.g.: azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, cladribine, capecitabine, clofarabine); folic acid analogues (e.g.: methotrexate, folic acid, pemetrexed, aminopterin, raltitrexed, trimethoprim, pyrimethamine),
• pyrimidine analogues e.g.: fluoropyrimidines analogs, 5-fluorouracil (5-FU), floxuridine (FUDR), Cytosine arabinoside (Cytarabine),
• topoisomerase inhibitors e.g.: camptothecins: irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide
• camptothecins irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide
• DAC 2'-deoxy-5-azacytidine
• 5- azacytidine 5-aza-2'- deoxycytidine
• vascular disrupting agents such as flavone acetic acid derivatives, 5,6-dimethylxanthenone-4- acetic acid (DMXAA) and flavone acetic acid (FAA);
• chemotherapeutic drugs such as aprepitant, bortezomib (Velcade ®, Millenium Pharmaceuticals), imatinib mesylate (Gleevec ®), carmustine (BCNU), lomustine (CCNU), tamoxifen, gefitinib, erlotinib, carboxyamidotriazole, efaproxiral, tirapazamine, xcytrin, thymalfasin, vinflunine.
• aprepitant bortezomib (Velcade ®, Millenium Pharmaceuticals)
• imatinib mesylate Galeevec ®
• carmustine BCNU
• lomustine CCNU
• tamoxifen gefitinib
• erlotinib carboxyamidotriazole
• efaproxiral efaproxiral
• tirapazamine xcytrin
• Alkylating agents are so named because of their ability to alkylate many molecules, including proteins, RNA and DNA. This ability to bind covalently to DNA via their alkyl group is the primary cause for their anti-cancer effects, since it provokes cell apoptosis. Alkylating agents are cell cycle-independent drugs, and their effects are usually dose dependent.
• alkylating agents are the nitrogen mustards, nitrosoureas, tetrazines, aziridines, and non-classical alkylating agents.
• Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan.
• Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin.
• Tetrazines include dacarbazine, mitozolomide and temozolomide
• Aziridines include thiotepa, mytomycin and diaziquone (AZQ).
• Non-classical alkylating agents include procarbazine and hexamethylmelamine.
• alkylating-like agents which are platinum-based chemotherapeutic drugs (also termed “platinum analogues”) and act in a similar manner as alkylating agents, will be included in the category of "alkylating agents”. These agents do not have an alkyl group, but nevertheless damage DNA. They permanently coordinate to DNA to interfere with DNA repair.
• alkylating agents as herein defined are platinum, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin and triplatin tetranitrate.
• Anti cancer “biological therapies” involve the use of living organisms, substances derived from living organisms, or laboratory-produced versions of such substances to treat cancer, by targeting either the cancer cells directly, or by stimulating the body's immune system to act against cancer cells (“immunotherapy")-
• Biological therapies include monoclonal antibodies (including those targeting cancer cell surface, e.g.
• anti-CTLA4 Mabs such as ipilimumab
• targeting growth factors e.g.: bevacizumab, cetuximab, panitumumab and trastuzumab
• anti-PD- 1 Mabs e.g.: bevacizumab, cetuximab, panitumumab and trastuzumab
• anti-PD- 1 Mabs e.g.: bevacizumab, cetuximab, panitumumab and trastuzumab
• anti-PD- 1 Mabs e.g.: anti-PD- 1 Mabs
• anti-Tin 3 Mabs anti-ICOS Mabs
• immunoconjugates e.g.: 90 Y-ibritumomab tiuxetan, 131 I-tositumomab, and ado -trastuzumab emtansine
• cytokines including interferons such as IFNa
• Prebiotics are non-digestible food ingredients that stimulate the growth and/or activity of bacteria in the digestive system in ways claimed to be beneficial to health. They usually are selectively fermented ingredients that allow specific changes, both in the composition and/or activity of the gut microbiota.
• Probiotics are micro-organisms that have claimed health benefits when consumed. Probiotics are commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. Generally, probiotics help gut microbiota keep (or re-find) its balance, integrity and diversity. The effects of probiotics are usually strain-dependent.
• “Synbiotics” refer to nutritional supplements combining probiotics and prebiotics in a form of synergism, hence synbiotics. Using prebiotics and probiotics in combination is often described as synbiotic, but the United Nations Food & Agriculture Organization (FAO) recommends that the term “synbiotic” be used only if the net health benefit is synergistic.
• FEO Food & Agriculture Organization
• cancer means all types of cancers.
• the cancers can be solid or non solid cancers.
• Non limitative examples of cancers are carcinomas or adenocarcinomas such as breast, prostate, ovary, lung, pancreas or colon cancer, sarcomas, lymphomas, melanomas, leukemias, germ cell cancers and blastomas.
• treat refers to any reduction or amelioration of the progression, severity, and/or duration of cancer, particularly a solid tumor; for example in a breast cancer, reduction of one or more symptoms thereof that results from the administration of one or more therapies.
• the present invention pertains to a probiotic composition
• a probiotic composition comprising bacteria selected from the group consisting of Enterococcus hirae, Lactobacillus johnsonii, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella, Holdemania and mixtures thereof, for use as an adjuvant to an antineoplastic treatment administered to a cancer patient.
• said composition comprises Enterococcus hirae and at least one strain selected amongst Porphyromonas, Barnesiella and Holdemania.
• a preferred strain of Enterocococcus hirae for the above compositions is the strain deposited on November 7, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number 1-4815.
• Such a composition can advantageously further comprise a Lactobacillus johnsonii strain such as Lactobacillus johnsonii strain LJFS001B, deposited on November 15, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number I- 4823.
• compositions can advantageously be formulated for oral administration and administered either as food supplements or as functional food.
• food supplements e.g., pills, tablets and the like
• functional food e.g., drinks, fermented yoghurts, etc.
• the probiotic composition according to the invention is administered to a patient in need thereof after the administration of an antineoplastic treatment, for example a chemotherapeutic agent such as cyclophosphamide (CTX) to said patient.
• an antineoplastic treatment for example a chemotherapeutic agent such as cyclophosphamide (CTX)
• CTX cyclophosphamide
• the probiotic composition can be administered the same day as a CTX dose, or after a few days of treatment.
• the probiotic composition can be administered daily, after each CTX uptake or even at the same time.
• the probiotic composition according to the invention is administered to a patient in need thereof before the administration of an antineoplastic treatment.
• chemotherapeutic agents especially CTX
• CTX chemotherapeutic agent
• a particularly useful application of the probiotic compositions according to the present invention is their use in combination with such a chemotherapeutic agent, for further increasing the efficacy of cancer vaccination.
• a method for treating a cancer patient comprising administering a probiotic bacterial composition such as above-described, prior to and/or after administering a chemotherapeutic agent, either alone or combined to an anticancer vaccine, to said patient, is also part of the present invention.
• compositions can be appropriately administered to any patient treated with and antineoplastic treatment such as chemotherapy (alone or in combination with an antitumor vaccine), they are particularly useful for patients who have a dysbiosis with an under-representation of species present in said probiotic composition.
• Another aspect of the present invention is the use of a combination of a chemotherapeutic agent and of an antibiotic composition which decreases the firmicutes/bacteroidetes ratio, specifically augments SFB and/or Porphyromonadaceae and/or decreases Clostridium group IV in the gut microbiota of an individual when administered to said individual, for treating a cancer.
• the antibiotic composition comprises or consists of a combination of vancomycin and imipenem.
• the antibiotic composition comprises or consists of a combination of neomycin and cephalothin.
• the chemotherapeutic agent used in combination with an antibiotic composition as described above is cyclophosphamide (CTX).
• the term “combination” refers to the use of more than one agent (e.g., vancomycin + imipenem and CTX).
• the use of the term “combination” does not restrict the order in which the therapeutic agents are administered to the patient, although it is preferable to administer the antibiotic cocktail prior to or simultaneously with the chemotherapeutic agent.
• vancomycin and imipenem can be administered prior to CTX (e.g., 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks before), either punctually or several times (for example, each day), preferably for 3 to 7 days before the antineoplastic treatment is administered.
• the antibiotic composition is administered before administration of a chemotherapeutic drug, in order to modulate the patient's gut microbiota to optimize the effect of said chemotherapeutic drug (such as CTX).
• a chemotherapeutic drug such as CTX
• the present invention hence provides a method for treating a cancer patient, comprising administering an antibiotic composition which decreases the firmicutes/bacteroidetes ratio, specifically augments SFB and/or Porphyromonadaceae and/or decreases Clostridium group IV in the gut microbiota of an individual when administered to said individual, prior to administering a chemotherapeutic drug (either alone or in combination with an anticancer vaccine).
• the present invention also pertains to the use of an antibiotic composition which decreases the firmicutes/bacteroidetes ratio, specifically augments SFB and/or Porphyromonadaceae and/or decreases Clostridium group TV in the gut microbiota of an individual when administered to said individual, as an adjuvant therapy to potentiate the anticancer effects of a chemotherapeutic agent administered to said patient.
• an antibiotic composition which decreases the firmicutes/bacteroidetes ratio, specifically augments SFB and/or Porphyromonadaceae and/or decreases Clostridium group TV in the gut microbiota of an individual when administered to said individual, as an adjuvant therapy to potentiate the anticancer effects of a chemotherapeutic agent administered to said patient.
• a chemotherapeutic agent such as, for example, CTX.
• Antibiotic compositions such as vancomycin + imipenem and neomycin + cephalothin are particularly useful to this aim.
• Another object of the present invention is an immunogenic composition
• an immunogenic composition comprising fragments of bacteria selected from the group consisting of Enterococcus hirae, Lactobacillus johnsonii, Enterococcus faecalis, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella, Holdemania and mixtures thereof, for use as an adjuvant to an antineoplastic treatment administered to a cancer patient.
• the immunogenic composition comprises fragments of Enterococcus hirae, more preferably fragments of the strain CNCM 1-4815, together with fragments of at least one strain selected from the group consisting of Porphyromonas, Barnesiella and Holdemania.
• the immunogenic compositions according to the invention are preferably formulated for subcutaneous or intramuscular administration. They can advantageously be administered before, at the same time or after administration of a chemotherapeutic agent such as CTX, in order to indice an immune response which will have an adjuvant effect to the treatment.
• a chemotherapeutic agent such as CTX
• a first cell composition according to the invention is a cell composition comprising antigen presenting cells (APC) such as dendritic cells (DC) which have been pulsed ex vivo with a probiotic composition or with an immunogenic composition as above-described.
• APC antigen presenting cells
• DC dendritic cells
• the antigen presenting cells present in the cell composition have also been pulsed ex vivo with a tumor antigen.
• the cell compositions according to the present invention are particularly useful for treating a cancer, by combining adoptive cell transfer with an antineoplastic treatment. Depending on the clinical context, the physician will decide how to administer such a cell composition. In particular, these compositions can be administered by intra-nodal injection, intravenous injection or subcutaneous injection.
• the above APC compositions can be used to ex vivo "educate" T cells obtained from the patient, before re-injecting these educated T cells, especially memory T cells, to the patient.
• a cell composition comprising memory T cells obtained by a process comprising ex vivo contacting T cells from a cancer patient with an APC composition as above-described, is hence also part of the present invention.
• Such a T cell composition can advantageously be used in adoptive cell transfer as an adjuvant to potentiate the effects of an antineoplastic treatment such as a chemotherapy (especially CTX administration), administered alone or in combination with an antitumoral vaccination.
• the T cells acan be administered by intra-nodal injection, intravenous injection or subcutaneous injection, depending on the cinical context.
• the present invention also relates to a method for ex vivo obtaining T cells able to improve the anticancer activity of a chemotherapeutic drug, comprising ex vivo expanding a polyclonal T cell line or bulk autologous T cells with dendritic cells (DC) presenting peptides from Enterococcus hirae, Lactobacillus johnsonii, segmented filamentous bacteria (SFB), Porphyromonas, Barnesiella and/or Holdemania.
• DC dendritic cells
• Another aspect of the present invention is an in vitro method of identifying a patient likely to be a good responder to a chemotherapy, comprising determining the functionality of TLR 4, NODI and NOD2 in said patient, wherein if said patient lacks a functional TLR 4 and/or NOD1/CARD4 (rs2006847, rs2066844, rs2066845, rs2066842, ND(l)+32656, rs2075820, Among and/or NOD2/CARD 15 (such as p.R702W, p.G908R, p.Leul007fsX1008), the patient is identified as a good responder to a chemotherapy (all except anthracyclines and oxaplatin and radiotherapy).
• SNPs single nucleotide polymorphisms
• Asp299Gly and Thr399Ile Two cosegregating single nucleotide polymorphisms (SNPs)— Asp299Gly and Thr399Ile — have been identified within the gene encoding TLR4. These SNPs are present in approximately 10% of white individuals, and have been found to be positively correlated with several infectious diseases. In a particular embodiment of this method, the presence or absence of one or both of these SNPs is determined, for example by PCR or by any other method known by the skilled artisan.
• the present invention pertains to a method for in vitro determining whether a cancer patient can benefit from an antineoplastic treatment, comprising the following steps:
• the "unfavorable" bacteria from the taxons recited in step (i) indicates that the patient will not be a good responder to antineoplastic treatment.
• the "relative abundance” is defined as the number of bacteria of a particular taxonomic level (from phylum to species) as a percentage of the total number of bacteria in the biological sample. This relative abundance can be assessed, for example, by measuring the percentage of 16S rRNA gene sequences present in the sample which are assigned to these bacteria. It can be measured by any appropriate technique known by the skilled artisan, such as 454 pyrosequencing and quantitative PCR of these specific bacterial 16S rRNA gene markers, as described in the experimental part below, or quantitative PCR of any gene specific for a bacterial group.
• a "good responder to a treatment” also called a “responder” or “responsive” patient or in other words a patient who "benefits from” this treatment, refers to a patient who is affected with a cancer and who shows or will show a clinically significant relief in the cancer after receiving this treatment.
• the disease clinical data may be assessed according to the standards recognized in the art, such as immune-related response criteria (irRC), WHO or RECIST criteria.
• the biological sample is a biofilm of a biopsy (preferably of a large biopsy) of duodenum or ileum mucosae obtained from the patient.
• this biopsy can have been obtained during a specific surgery in pancreatic, stomach, biliary tract or colon cancers.
• the biological sample is a sample of feces obtained from the patient. This sample can have been collected at diagnosis, for example, or at any moment before deciding the beginning of the treatment.
• the present invention pertains to a method for in vitro determining whether an antineoplastic treatment is to be continued or stopped for a cancer patient, comprising the following steps:
• the antineoplastic treatment is continued, and in absence of such a response, the antineoplastic treatment is stopped or compensated with appropriate probiotics (see below).
• This pharmacodynamic assay is particularly useful to predict, after 3-9 weeks of a chemotherapy (1-3 cycles of chemotherapy), preferably after 6-9 weeks (2-3 cycles) of chemotherapy, whether this chemotherapy is likely to trigger an adjuvant immune response and a clinical benefit.
• the secretions of IL-2, TNFa, IFNy and IL-10 are measured in ex vivo restimulation assays.
• a first assay is done before the beginning of the treatment, in order to compare the cytokine secretion profile after a few weeks of treatment to that observed pre-treatment.
• These assays can be performed, for example, using patients' autologous monocytes loaded with defined bacteria and incubated with CD4 + CD45RO + T cells purified from autologous blood.
• the response will be classified in the third (favourable) category if it is of a Thl phenotype, i.e., if restimulation triggers a significant secretion of IL-2, TNFa and IFNy, and a low secretion of IL-10, especially when comparing the results obtained post- to pre-treatment.
• a Thl phenotype i.e., if restimulation triggers a significant secretion of IL-2, TNFa and IFNy, and a low secretion of IL-10, especially when comparing the results obtained post- to pre-treatment.
• at least a 2-fold increase of IFNy secretion is observed post-treatment (compared to pre-treatment).
• the first category corresponds to the absence of significant cytokine secretion in restimulation assays post-treatment
• the second category corresponds to a response in which the IL- 10 secretion in a restimulation assay post-treatment is superior to that observed pre- treatment.
• the memory CD4 + T cell responses directed against at least two species selected amongst Lactobacillus johnsonii, Enterococcus hirae and Enterococcus faecalis are analyzed.
• the responses directed against 2 of these, and more preferably against all of these, are assessed.
• the present invention pertains to a method for in vitro determining the biological effects of a neoadjuvant antineoplastic treatment which has been administered to a patient, comprising the following steps:
• the result indicates that the neoadjuvant antineoplastic treatment induced a T-bet/Thl local and systemic immune response.
• the "favorable” bacteria can be those from a group comprising or consisting of the genera Lactobacillus and Bifidobacterium, and the “unfavorable” bacteria can be those from a group comprising or consisting of the species Parabacteroides distasonis and Faecalibacterium prausnitzii and the genera Gemmiger, Alistipes and Clostridium Cluster IV ⁇ Clostridium leptum group).
• the skilled artisan will determine the appropriate threshold depending on the technique which is used to determine the relative abundance of bacteria from each group (for example, pyrosequencing or quantitative PCR) and depending on the definition of each group of patients. Indeed, a unique threshold cannot be determined for all cancer patients, and the ratio must be appreciated having regard to several factors, including the patient's health and food habits.
• the biological sample preferably is a biofilm from a biopsy (preferably from a large biopsy) of duodenum or ileum mucosae obtained from the patient.
• this biopsy can have been obtained during a specific surgery in pancreatic, stomach, biliary tract or colon cancers.
• the methods described above can be performed to prognosticate or diagnose the responsiveness of a cancer patient to any antineoplastic treatment as defined above, including chemotherapies, biological therapies, radiotherapies, hormone therapies, etc.
• these methods can be advantageously used to assess the (potential) benefit, for a cancer patient, of a chemotherapy, more particularly with an alkylating agent or a platinum salt such as any of those cited above, and/or an anti-tumor vaccine.
• the experimental data below clearly describe the role of microbiota on the immune response induced by cyclophosphamide (Examples 1 , 3 and 4), doxorubicine (see at least Fig. 14) and oxaliplatine (Example 2).
• mice can be extrapolated to humans.
• the experimental data show that a "beneficial" microbiota also has a positive impact on the efficiency of a treatment by anthracyclins (Fig. 8) and obviously, if bacterium species having an immunomodulatory role, such as Faec libacterium prausnitzii, are too abundant in the gut microbiota, these bacteria will negatively impact the drug efficiency.
• the present invention also relates to a probiotic bacterial strain selected from the group consisting of Lactobacillus johnsonii, Enterococcus hirae and Enterococcus faecalis, for use in combination with an antineoplastic agent for inducing a T-bet/Thl local and systemic immune response, for treating a cancer.
• a probiotic bacterial strain selected from the group consisting of Lactobacillus johnsonii, Enterococcus hirae and Enterococcus faecalis, for use in combination with an antineoplastic agent for inducing a T-bet/Thl local and systemic immune response, for treating a cancer.
• probiotics are the Lactobacillus johnsonii strain LJFS001B, deposited on November 15, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number I- 4823, and the Enterocococcus hirae strain EHFS001, deposited on November 7, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number I- 4815.
• the probiotic bacterial strain according to the invention is formulated for oral administration.
• the skilled artisan knows a variety of formulas which can encompass living or killed microorganisms and which can present as food supplements (e.g., pills, tablets and the like) or as functional food such as drinks, fermented yoghurts, etc.
• the present invention still relates to the use of such probiotics, in combination with an antineoplastic treatment, for treating a cancer patient.
• the term "in combination” refers to the use of more than one agents (e.g., a probiotic strain and a chemotherapeutic drug).
• the use of the term “in combination” does not restrict the order in which therapies are administered to the patient, although it is preferable to administer the probiotic strain prior to or simultaneously with the antineoplastic treatment.
• the probiotic strain can be administered prior to the antineoplastic agent (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, or 12 weeks before), either punctually or several times (for example, each day) before the antineoplastic treatment is administered.
• the probiotic bacterial strain according to the invention is used in combination with a chemotherapeutic agent or an biological immunotherapy, for example in combination with a treatment by an alkylating agent or by immunotherapy.
• a composition comprising at least the Lactobacillus johnsonii strain LJFS001B, (Fig. 26A) deposited on November 15, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number 1-4823, and/or the Enterocococcus hirae strain EHFS001 (Fig. 26B), deposited on November 7, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM), under the number I- 4815, is also part of the present invention.
• the compositions according to the invention can be in the form of food supplements (e.g., pills, tablets, syrups and the like) or in the form of functional food such as drinks, fermented yoghurts, etc. The probiotics are preferentially alive in these compositions.
• the present invention relates to adoptive cell transfer of "pathogenic" Thl7 (pThl7) cells derived from CD4+ T cells from a cancer patient, preferentially in combination with an antineoplastic treatment such as a chemotherapy (e.g., with an alkylating agent) or an immunotherapy (e.g., antitumor vaccine, ...), for treating said patient.
• pThl7 Thl7
• CD4+ naive T cells can be obtained from blood, then amplified and stimulated ex vivo in the presence of cytokines favouring the pThl7 phenotype (for example in the presence of IL- ⁇ , IL-6, IL-21 and IL-23 and optionally IL-lb+IL-9) as well as TCR cross-linking (such as beads coated with anti-CD3/anti-CD28 Ab).
• cytokines favouring the pThl7 phenotype for example in the presence of IL- ⁇ , IL-6, IL-21 and IL-23 and optionally IL-lb+IL-9
• TCR cross-linking such as beads coated with anti-CD3/anti-CD28 Ab.
• pThl 7 cells share hallmarks of Thl cells (nuclear expression of the transcription factor T-bet, cytoplasmic expression of IFNy and surface exposure of the chemokine receptor CXCR3) and Thl 7 cells (expression of RORyt, IL-17 and C
• EXAMPLE 1 THE INTESTINAL MICROBIOTA MODULATES THE ANTICANCER IMMUNE EFFECTS OF CYCLOPHOSPHAMIDE - MOUSE STUDY
• MCA205, B16F10 (syngeneic from C57BL/6J mice) and P815 (syngeneic from DBA2/J mice) were cultured at 37°C under 5% C0 2 in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate and MEM non-essential amino acids (Invitrogen).
• 0.5 - 1 10 6 MCA205, 0.3 x 10 6 B16F10 or 0.8 x 10 6 P815 tumor cells were inoculated s.c. in the right flank.
• Chemotherapy was performed by intratumoral injection of doxorubicin (Doxo) (2 mM, 50 ⁇ ) or intraperitoneal inoculation of CTX (100 mg/kg of body weight) when tumors reached 35-60 mm 2 .
• Doxo doxorubicin
• CTX intraperitoneal inoculation of CTX
• mice were either left untreated or received chemotherapy (d84, d91 and d98) in absence or presence of 0.25mg/ml vancomycin (mixed into drinking water starting on d77 and until the end of the experiment; antibiotic-containing water was replaced biweekly).
• Tumor volumes were quantified on d73 and 100 (equivalent of 'pre' and 'post' chemotherapy) in anesthetized mice by noninvasive imaging as described before (Cortez-Retamozo et al., 2013). Data show absolute changes in total lung tumor volumes (means ⁇ SEM) between the two time points.
• Cyclophosphamide (CTX) (Endoxan, Baxter) was provided by Institut Gustave Roussy.
• CTX Cyclophosphamide
• D1515 Doxorubicin hydrochloride
• FITC-dextran Fluorescein isothiocyanate-dextran
• CD3s CXCR3 (CXCR3-173), CD4 (GK1.5), CD8ot (53-6.7), ⁇ TCR (GL-3), IL-17 (eBiol7B7), IFNy (XMG1.2), T-bet (4B10), RORyt (AFKJS-9), CD45, CCR6 (140706) were obtained from BioLegend, eBioscience and R&D. LIVE/DEAD fixable yellow stain fluorescence for viability staining was purchased from Invitrogen/Molecular Probes. All cells were analyzed on a Cyan (Beckman Coulter) or a FACSCANTO II (BD) flow cytometer with FloJo (Tree Star) software.
• mice were treated with antibiotics 2-3 weeks before tumor implantation and continued until the end of the experiment.
• a mix of ampicillin (1 mg/ml) + streptomycin (5 mg/ml) + colistin (1 mg/ml) (Sigma- Aldrich) or vancomycin (0.25 mg/ml) or colistin alone (2.10 3 U/ml) were added in sterile drinking water. Solutions and bottles were changed 2-3 times a week.
• Antibiotic activity was analyzed by macroscopic changes observed at the level of caecum (dilatation) and by cultivating the fecal pellets resuspended in BHI+1 % glycerol on blood agar and anaerobic blood agar plates for 48h at 37°C with 5% C0 2 for aerobic conditions or in anaerobic conditions respectively.
• vancomycin biased the repertoire of commensal bacteria towards distinct commensal species (such as E. coli and different species of Clostridium, Fig. 16) whereas colistin promoted the outgrowth of E. faecalis.
• E. distasonis used in the experiments was isolated from feces of SPF mice treated with prolonged broad spectrum ATB and identified as described above.
• E. hirae, E. faecalis and E. coli were grown in BHI medium (Fluka analytical), while L. johnsonii, L. plantarum and L. murinus in MRS broth (BD) at 37°C until they reach an OD 6 oo-l when the growth was exponential.
• L. reuteri was grown in anaerobic conditions onto COS agar plates for 48h at 37°C. Serial dilutions of bacteria preparations were plated so that the administered doses could be assessed.
• E. coli MCI 061 E.
• the identification of bacteria was done by MALDI-TOF analysis and 16S rRNA gene sequencing.
• the MALDI-TOF MS analysis was done on prepared cells as follows. Strains were grown overnight at 37°C on MRS agar. About 5 to 10 mg of cells were resuspended in 300 ⁇ of sterile ultrapure water and 900 ⁇ of absolute ethanol, homogenized by flicking the tubes, centrifuged for 2 min at 13000 g and the supernatant was discarded. Subsequently, 50 ⁇ of formic acid was added to the pellet and mixed before the addition of 50 ⁇ acetonitrile. The mixture was centrifuged again at 13000 g for 2 min.
• Spectra were recorded in the positive linear mode (laser frequency, 200Hz, ion source I, voltage at 20kV; ion source 2, voltage at 18.4 kV; lens voltage, 9.1 kV; mass range, 2000- 20 000 Da).
• laser frequency 200Hz
• ion source I voltage at 20kV
• ion source 2 voltage at 18.4 kV
• lens voltage 9.1 kV
• mass range 2000- 20 000 Da
• the 16S rRNA gene from the strains studied was amplified by PCR using the universal primers A, 5 ' -AGAGTTTGATC ATGGCTC AG-3 ' (SEQ ID No: 1) (position 8 to 27, Escherichia coli numbering) and H, 5'-AAGGAGGTGATCCAACCGCA-3 ' (SEQ ID No: 2) (position 1541 to 1522) (Bottger, 1989), in a GeneAmp® thermal cycler (Perkin- Elmer, Wellesley, MA) and the following parameters: 4 min at 94 °C, 25 cycles of 1 min at 94 °C, 25 of 1 min at 57 °C, 25 of 2 min at 72 °C with a final extension step at 72 °C for 5 min.
• the longitudinal sections were counterstained with hematoxilin and eosin.
• inflammatory foci, altered villi and the thickness of lamina propria were scored for each section, while the number of goblet cells was counted for each villus.
• paneth cells enumeration the longitudinal sections were permeabilized with 0.5% triton for 15 min, and were blocked with a solution of 0.1% triton, 5% serum and 1% BSA for lh.
• ⁇ TCR + and ⁇ TCR " T cell quantification transversal sections were blocked with a solution of 0.1% triton and 10% goat normal serum for lh. Then, the sections were immunostained with hamster anti- ⁇ TCR (10 ⁇ g/ml O/N 4°C, BD Pharmingen) and with goat anti - hamster A488 (7.5 ⁇ g/ml for 45 min, Jackson ImmunoResearch) as secondary antibody, or with hamster anti-mouse CD3 A647 (5 g/ml for 2h, Biolegend). For each section, the number of total cells, ⁇ TCR + CD3 + and CD3 + cells were counted to determine the percentage of ⁇ TCR + and ⁇ TCR " T cells.
• Isolation of lamina intestinal cells from small intestine Whole duodenum and ileum were harvested, Peyer's patches were removed, as well as all fat residues and fecal content. Small fragments were obtained by cutting them first longitudinally along the length and then transversally into pieces of 1-2 cm length. After removing the intra-epithelial lymphocytes (IELs), the gut pieces were further cut and incubated with 0.25 mg/ml collagenase VIII and 10 U/ml DNase I for 40 min at 37 °C under shaking to isolate lamina intestinal cells (LPCs). After digestion, intestinal pieces were mashed on a cell strainer. For FACS analysis, cell suspensions were subjected to a percoll gradient for 20 min at 2100 RPM, while for RNA extraction, cells were directly lysed in RLT buffer (Qiagen) and frozen at -80°C.
• RLT buffer Qiagen
• CD1 lc N418), CD1 lb (Ml/70), Ly6c (HK1.4), MHC class II (M5/114.15.2), CD24 (Ml/69), CD64 (X54-5/7.1), CD317 (ebio927), CD45 (30- Fl l), F4/80 (C1 :A3-1), CD8a (53-6.7).
• DAPI was used for dead cell exclusion.
• Antibodies were purchased from eBiosciences, BD Biosciences or BioLegend respectively.
• CD103 + DC CD45 + CDl lc + MHC-II + CD 103 + CD24 +
• CDl lb + CD103 + CD45 + CD1 lc + MHC-If CD103 + CD1 lb + CD24 +
• CD1 lb + CD45 + CD1 lc + MHC- ⁇ CDl lb + CD24 +
• inflammatory DC CD45 + CDl lc + MHC-II + CDl lb + CD64 + Ly6c*
• large intestine CD103 + DC (CD45 + CDl lc + MHC-II + CD103 + CD24 + ), CDl lb + (CD45 + CDl lc + MHC-II + CDl lb + CD24 + ), inflammatory DC (CD45 + CDl lc + MHC-II + CDl lb + CD64 + Ly6c + ).
• Microbiota reconstitution For inoculation of GF mice with SFB, fecal pellets were collected from SFB-monocolonized mice with sterilized test tubes. Colonization was performed by oral gavage with 200 ⁇ of suspension obtained by homogenizing the fecal pellets in water. Efficient colonization was first checked before tumor inoculation.
• E. hirae, L. johnsonii and L. plantarum were grown in BHI (Fluka analytical) and MRS (BD) broth, respectively, overnight at 37°C. Bacteria were centrifuged, washed once and resuspended in sterile PBS at an OD(600nm) of 1, which corresponds approximately to lxl 0 9 colony-forming units (CFU)/ml. Equal volume of each bacteria suspension was mixed to give a suspension of equal proportion of each type of bacteria at lxl 0 9 bacteria/ml, L. reuteri was grown in anaerobic conditions onto COS agar plates for 48h at 37°C. For P.
• mice were treated with a mix of ampicillin/streptomycin/colistin (ATB) for 4 weeks and orally inoculated with 10 9 CFU in 200 ⁇ of PBS 4 days post MCA205 inoculation.
• ATB ampicillin/streptomycin/colistin
• mice were orally gavaged with 10 9 CFU of E. hirae + L. johnsonii or L. plantarum or L.reuteri one day after CTX administration and 0 to 3 days post treatment suspension.
• TCR and T cell assays were incubated in MaxiSorp plates (Nunc) precoated with anti-CD3s mAb (145-2C1 1) (0.5 ⁇ g per well ; eBioscience) and/or anti- CD28 mAb (37.51) (2 ⁇ g/ml; BD). The supernatants were assayed at 48h by ELISA for mouse IL-17A (eBioscience) and IFNy (BD). For TIL analyses, tumors were removed, cut into small pieces and digested in Liberase TM (Roche) and DNase I for 30 min at 37°C.
• Single-cell suspensions were obtained by crushing the digested tissue with a syringe plunger and filtering through a 100 ⁇ cell strainer.
• cells were incubated for 4h at 37°C with 50 ng/ml of PMA, 1 ⁇ g/ml of ionomycin and BD Golgi STOPTM. After membrane staining, cells were stained with anti-IL-17A, IFNy, T-bet and RORyt using eBioscience FoxP3/Transcription factor staining buffer set.
• T cell polarization and propagation in vitro Adoptive transfer ofThl ? cells (pathogenic or regulatory TM 7).
• Naive CD4 + T cells (CD4 + CD62L hi ) were obtained from spleens and lymph nodes of C57BL/6 WT mice. Cells were then sorted by flow cytometry (BD ARIA III with FACSDiva Software) accordingly. The purity of isolated T cell populations routinely exceeded 95%.
• Naive T cells were stimulated with plate-bound antibodies against CD3e (145-2C11, 2 ⁇ g/ml) and CD28 (PV-1, 2 ⁇ g/ml) in the presence of either recombinant mouse IL- ⁇ (10 ng/ml), IL-6 (10 ng/ml), and IL-23 (20 ng/ml) (pThl 7) or TGF- ⁇ (2.5 ng/ml) and IL-6 (Thl 7) (Miltenyi).
• Thl7 Regulatory Thl7 (Thl 7) resulted from a differentiation in TGF- ⁇ (2,5 ng/ml) and IL-6 while pathogenic Thl 7 (pThl7) resulted from incubation in IL- ⁇ ⁇ , IL-6 and IL-23. Mice were intravenously injected with 3 ⁇ 10 6 T cells. Priming of T cells in vitro.
• BMDCs Bone marrow- derived dendritic cells
• Iscove's medium Sigma-Aldrich
• J558 supernatant containing 40 ng/ml of GM-CSF
• 10% FCS 100 IU/ml penicillin/streptomycin
• 2 mM L-glutamin 50 ⁇ 2-mercaptoethanol
• BMDCs were infected with the isolated bacterial strains at a MOI (multiplicity of infection) 1 :50 for 1 h at 37°C in the appropriate medium without antibiotics.
• BMDCs were cultured together with naive CD4 + CD62L + T cells, purified from spleen and lymph nodes (Miltenyi), at the ratio 1 : 1 for 4 days. Culture supernatants were then assayed for IL-17 and IFNy by ELISA. CD4 + T cell memory response.
• BMDCs were infected with different doses of bacteria (ratio cells: bacteria 1 :2, 1 : 10 and 1 :50) as described above and after 24h were cultured 1 : 1 with CD4 + T cells, purified from spleens (Miltenyi) of CTX- or NaCI-treated C57BL/6 mice. After 24h culture supernatants were assayed for IL-17 and IFNy by ELISA.
• B6.CBirl TCR transgenic mice (Cong et al., 2009) were generated and bred in the Animal Facility at the University of Alabama at Birmingham. All experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
• CD4 + T cells were isolated from B6.CBirl TCR Tg mice using anti- mouse CD4 magnetic beads. Briefly, splenic cells were washed twice and incubated with anti-CD4 magnetic beads at 4°C for 30 min and then separated by magnetic field. When checked by flow cytometry, over 95% of the cells were CD4 + T cells.
• CD45.1 + CBirl Tg T cells
• CD45.2 + NaCl treated- naive congenic mice
• Flow cytometry analyses gated on CD45.1 + cells to appreciate percentages of intracellular IL-17 + or IFNy + cells after PMA/ionomycin 5h restimulation in the presence of monensin.
• splenocytes were incubated in triplicate in 24 well flat bottom plates at 1.0 million/ml, cultured without or with CBirl- peptide 455-475 (DM ATEMVKYSN ANILS QAGQ) at 1 ⁇ g/ml and supernatants were analysed using anti-IFNy specific commercial ELISA.
• RNA extraction and genomic DNA removal were performed with the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.
• Total RNA extraction and genomic DNA removal of ilea or duodena were performed with the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.
• Expression of Regllly (Mm00441127_ml) and LysM (MmOl 61274 l_ml) -related genes was analyzed with TaqMan ® Gene Expression Assays using the Universal Master Mix II on a StepOnePlusTM Real-Time PCR System (Life Technologies, France). Quantitative RT-PCR data were invariably normalized to the expression levels of the housekeeping gene peptidylprolyl isomerase A (Ppia) by means of the 2 _ACt method.
• Microbiota composition was assessed by 454 pyrosequencing (GS)
• V3fwd 5 ' TACGGRAGGCAGC AG3 ' , SEQ ID No: 5; V4rev: 5 ' GGACTACC AGGGTATCTAAT3 ' , SEQ ID No: 6
• Sequences were trimmed for barcodes, PCR primers, and binned for a minimal sequence length of 300pb, a minimal base quality threshold of 27, a maximum homopolymers length of 6. Resulting sequences were assigned to the different taxonomic levels, from phylum to genus using the RDP database (release 10, update 31) (Cole et al., 2009).
• OTUs Orthogonal Taxonomic Units or phylotypes
• QIIME Caporaso et al., 2010
• cdhit Li and Godzik, 2006
• OTUs were assigned to closest taxonomic neighbors and relative bacterial species using Seqmatch (RDP) and Blastall (NCBI).
• RDP Seqmatch
• NCBI Blastall
• Relative abundance of each OTUs and other taxonomic levels was calculated for each sample to account for different levels of sampling across multiple individuals. After trimming, the number of sequences clustered within each OTUs (or other taxonomic levels) was converted to a fraction representing the relative contribution of each feature to each of the individuals.
• Targeted qPCR systems were applied using either Taqman technology (for systems targeting All Bacteria domain, Clostridium Upturn group (Mayeur et al., 2013) or SybrGreen (for systems targeting Lactobacillus/Leuconoctoc/Pediococcus group (Mayeur et al., 2013), Enterococcus group (Furet et al., 2009), SFB (Yin et al., 2013) and TM7 (Hugenholtz et al., 2001)). No CTX-specific modulations of the relative amounts of SFB and TM7 or Clostridium group XIV was observed at day 7 post-CTX (not shown).
• Quantitative PCR was performed using an ABI 7000 SequenceDetection System with software version 1.2.3 (Applied-Biosystems). Amplification and detection were carried out with either TaqMan Universal PCR 2_MasterMix (Applied- Biosystems) or SYBR-Green PCR 2_Master Mix (Applied-Biosystems) in duplicate in a final volume of 25 ⁇ with 10 ⁇ of appropriate dilutions of DNA samples as previously described. Amplifications were carried out using the following ramping profile: 1 cycle at 95 °C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min. For SYBR-Green amplification, a melting step was added (Yin et al., 2013).
• Standard curves were generated from serial dilutions of a known concentration of genomic DNA from a representative of each group. Standard curves were generated by plotting threshold cycles (Ct) vs. bacterial quantity (CFU). The total number of bacteria (CFU) was interpolated from the averaged standard curves.
• RNA from T cells was extracted with Trizol (Invitrogen). 100 to 300 ng of RNA were reverse- transcribed into cDNA by M-MLV reverse transcriptase, Random Primers, and RNaseOUT inhibitor (Invitrogen). cDNA were quantified by realtime PCR with a SYBR Green Real-time PCR kit (Applied Biosystems) on a Fast7500 detection system (Applied Biosystems, France). Relative mRNA levels were determined with the ACt method. Values were expressed relative to cyclophilin A. The sequences of the oligonucleotides used are described below.
• Table 1 oligonucleotides used for characterizing Thl 7 cells expression profiles
• FITC fluorescein isothiocyanate
• CTX failed to cause a major dysbiosis at early time points (24-48h, Fig. 5), CTX significantly altered the microbial composition of the small intestine (but not of the caecum) in mice bearing subcutaneous cancers (namely metastasizing B16F10 melanomas and non-metastasizing MCA205 sarcomas) one week after its administration (Fig. 2C, Fig. 5).
• CTX induced a reduction of bacterial species from the Firmicutes phylum (Fig. 5) distributed within four genera and groups ⁇ Clostridium cluster XlVa, Roseburia, unclassified Lachnospiraceae, Coprococcus, Table 2) in the mucosa of CTX-treated animals.
• Table 2 CTX-induced mucosal microbiota dysbiosis at a species level
• CTX treated mice CTX treated mice
• Co NaCl-treated mice
• Taxonomic affiliation of these sequences is also added (phylum and genus levels).
• OTU phylotype centroids are assigned to their closest relative isolate (RDP Seqmatch database). All animals were compared together and tumor bearers were further distinguished.
• AN NCBI Accession Number
• Sab_Score RDP similarity score between the centroid sequence and the referent isolate. Wilcoxon test p-values.
• Thl7 cells which produce interleukin-17, IL-17
• strong links between gut-residing and systemic Th 17 responses have been established in the context of autoimmune diseases affecting joints, the brain or the pancreas (Ghiringhelli et al., 2004; Lee et al., 201 1 ; Wu et al., 2010).
• CTX induced the polarization of splenic CD4 + T cells towards a Thl (interferon- ⁇ [IFNy] -producing) and Thl7 pattern (Fig. 3A, Fig. 7D).
• CTX increased the frequency of "pathogenic" Thl 7 (pThl7) cells, which share hallmarks of Thl cells (nuclear expression of the transcription factor T-bet, cytoplasmic expression of IFNy and surface exposure of the chemokine receptor CXCR3) and Thl7 cells (expression of RORyt, IL-17 and CCR6) (Ghoreschi et al dislike 2010; Lee et al., 2012), within the spleen (Fig. 7F, Fig. 3C). Again, this response depended on the gut microbiota (Fig. 3C). Moreover, the increase in pThl7 cells required expression of myeloid differentiation primary response gene 88 (MyD88), which signals downstream of toll-like receptors (Fig.
• MyD88 myeloid differentiation primary response gene 88
• the inventors analyzed the impact of vancomycin on the microenvironment of suchthonous non-small cell lung cancers resulting from oncogenic activation of K-Ras and P53 and treated with CTX-based chemotherapy. They analyzed the impact of vancomycin on the infiltration of chemotherapy-treated tumor beds by ⁇ 17 cells, which are known to be crucial for the recruitment of antitumor CTLs post- chemotherapy (Ma et al., 2011). In vancomycin- or broad-spectrum ATB-treated mice, tumor beds were devoid of ⁇ 17 post-therapy in contrast to water-treated chemotherapy recipients (Fig. 17).
• EXAMPLE 2 THE INTESTINAL MICROBIOTA MODULATES THE ANTICANCER IMMUNE EFFECTS OF CYCLOPHOSPHAMIDE - RESULTS ON A PRECLINICAL MODEL MIMICKING HUMAN TUMORIGENESIS
• a transgenic tumor model of autochthonous NSCLC driven by oncogenic K-Ras coupled to a conditional P53 deletion was used to test the inhibitory role of vancomycin-based antibiotherapy on the anticancer efficacy of a combination of oxaliplatin plus CTX.
• the concept that the eradication of Gram-positive bacteria by vancomycin compromised the efficacy of CTX-based chemotherapy was validated (Fig. 19A and Fig. 4D), correlating with a reduced intratumoral CD8 + T effector/Foxp3 + regulatory T cell ratio (Fig. 19B).
• Gram-positive bacteria appear to be necessary for the optimal efficacy of the CTX-induced anticancer immune response and tumor mass reduction.
• EXAMPLE 3 HUMAN RESULTS: CYCLOPHOSPHAMIDE INDUCES THI AND THIQ IMMUNE RESPONSES DIRECTED AGAINST COMMENSAL BACTERIA IN CANCER PATIENTS
• CTX cyclophosphamide
• the inventors anticipate that only pattern 3 will be proned to benefit from chemotherapy, and they now correlate this anti-commensal bacterial immune response with clinical outcome. This pharmacodynamic assay is useful to predict, after 3-6 weeks (1-2 cycles of chemotherapy) whether such a CTX-based chemotherapy would trigger an adjuvant immune response and a clinical benefit.
• EXAMPLE 4 HUMAN RESULTS: OXALIPLATINE-BASED CHEMOTHERAPY INDUCES A CHANGE IN THE DISTRIBUTION OF BACTERIAL SPECIES IN GUT MICROBIOTA AND AN INCREASE OF T-BET TRANSCRIPTION BY THE GUT MICROBIOTA
• mucosal samples can be scratched and harvested (for 16S rRNA gene pyrosequencing analyses and description of the mucosal microbiota composition at the different taxonomic levels as described above), as well as mucosa that can be kept frozen (in RNAzol for qRT-PCR) or in paraffin-embedded tissues (for immunohistochemistry analyses).
• This surgery can be performed either before chemotherapy (adjuvant chemotherapy) or after chemotherapy (neoadjuvant chemotherapy).
• ileal mucosa from patients operated for a right colon cancer (6 patients in neoadjuvant oxaliplatine-based chemotherapy and 7 patients prior to therapy) were analyzed to compare the composition of ileal microbiota and the relative loss or gain of representativity of distinct genera and species (isolates) in cases of adjuvant versus neoadjuvant chemotherapy, meaning in colon cancer bearing patients that already received ( « chemo ») or did not receive ( « controls ») chemotherapy.
• the inventors also investigated, in parallel to pyrosequencing analyses of 16SrRNA of gut microbiota of ileum, the transcriptional profiling of cytokines and transcription factors detectable in mucosae of patients receiving or not chemotherapy. This investigation was done by qRT-PCR from ileal mucosa from the same patients. While RORyt and IL-17 were not very different in both groups, T-bet was upregulated post-chemotherapy and in two patients that had high levels of Bifidobacterium and Lactobacilli post-chemotherapy, T-bet transcripts were rather high compared with the other patients, suggesting that a pThl7 T cell response had been elicited by the treatment.
• E.HIRAE IS A PTH17 TH1 BACTERIA, ALONE OR IN CONJUNCTION WITH LACTOBACILLI (L. JOHNSONII) INDUCING PROBIOTIC ANTITUMOR EFFECTS
• OVA ovalbumine antigen
• clone 708 exhibited greater TH1 and Tel potential than the clone CNCM 1-4815 (Fig. 30). However, clone 708 did not perform better than the CNCM 1-4815 clone for the anticancer probiotic effect in CTX- treated established MCA205 sarcomas (not shown). Sequence and functional differences inbetween various E.hirae isolates were corroborated in an in vitro assay aimed at monitoring LDH release (featuring cell death) and human ⁇ 2 defensin secretion from the Caco-2 epithelial intestinal cell line exposed to CTX +/- various clones of E.hirae or E.coli.
• the inventors investigated the role of major pattern recognition receptors regulating intestinal homeostasis in the elicitation of splenic pTH17 cells and in tumor control promoted by CTX in MCA205-breaing C57BL/6 mice.
• the inventors phenocopied these effects using pharmacomimetics, i.e., ligands for NODI and NOD2, the peptidoglycans myramyl dipeptide and TriDAP, which acted locally by promoting the release of the antimicrobial peptide lipocalin-2 in stools (Fig. 3 ID), thereby reducing CTX-mediated pTH17 in the spleen (Fig. 31C).
• pharmacomimetics i.e., ligands for NODI and NOD2
• the peptidoglycans myramyl dipeptide and TriDAP which acted locally by promoting the release of the antimicrobial peptide lipocalin-2 in stools (Fig. 3 ID), thereby reducing CTX-mediated pTH17 in the spleen (Fig. 31C).
• HLA-DR NK cell levels correlate with disease outcome. Oncoimmunology, 2, e23080.
• Faecal D/L lactate ratio is a metabolic signature of microbiota imbalance in patients with short bowel syndrome.
• Sobhani, I., et al. (201 1). Microbial dysbiosis in colorectal cancer (CRC) patients.
• CRC colorectal cancer
• Cyclophosphamide induces differentiation of Thl7 cells in cancer patients. Cancer Res. 71(3), 661-665.

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