Methods for diagnosing cancer-induced or antibiotic-induced dysbiosis and their use for improving immunotherapeutic cancer treatment - Patents.com

Abstract

The present invention relates to the field of anti-cancer immunotherapy. In particular, the present invention relates to the role of MAdCAM-1 / α4β7 axis gut flora in the efficacy of cancer treatment, and provides a method for determining whether a patient is likely to benefit from cancer treatment, more precisely from immuno-oncology (IO) therapy, such as a treatment comprising administering an antibody against immune checkpoint blockade PD1, PD-L1 or PD-L2, alone or together with CTLA4 and / or chemotherapy. The present invention also provides a method for improving the efficacy of such treatment in a patient in need thereof.

Description

[Technical field]

[0001] The present invention relates to the field of anti-cancer immunotherapy. In particular, the present invention relates to the role of MAdCAM-1 / α4β7 axis gut flora in the efficacy of cancer treatment, and provides a method for determining whether a patient is likely to benefit from cancer treatment, more precisely from immuno-oncology (IO) therapy, such as a treatment comprising administering an antibody against immune checkpoint blockade PD1, PD-L1 or PD-L2, alone or together with CTLA4. The present invention also provides a method for improving the efficacy of such treatment in a patient in need thereof. [Background technology]

[0002] Over the past decade, the gut microbiome has attracted significant attention in the evolving immuno-oncology context, with compelling evidence of a role for gut dysbiosis in primary resistance to immune checkpoint blockade across a broad range of advanced malignancies. This assertion was supported by numerous epidemiological studies pointing to the detrimental effects of broad-spectrum antibiotics on clinical outcomes in stage III and stage IV lung, kidney, and bladder cancers, as well as anti-PD-1 / anti-PDL-1 antibodies (Abs) in melanoma. Notably, reduced gut ecosystem abundance was associated with a tumor microenvironment poor in T cell infiltrates.

[0003] Increasing awareness of the potential contribution of intestinal dysbiosis in treatment failure has led many investigators to describe metagenomic-based intestinal blueprints associated with resistance to immunotherapy. However, it remains unclear to what extent cancer-associated dysbiosis precedes the tumorigenic process and is therefore causally related to neoplasia or simply a direct consequence of it.

[0004] β7 integrin-containing heterodimers play a central role in leukocyte homing to the intestine and retention at the epithelial surface. The β7 integrin subunit can form a heterodimer with the α4 (CD49d) integrin subunit to obtain the α4β7 integrin (also called "Lymphocyte Peyer's patch adhesion molecule-1" (LPAM-1)). Through interaction with its counterreceptor Mucosal Addressin Cellular Adhesion Molecule-1 (MAdCAM-1), the α4β7 integrin mediates lymphocyte adhesion and extravasation from the circulation across the vascular endothelial barrier into the gut-associated secondary lymphoid tissue (GALT) or lamina propria (LP). The transmembrane adhesion molecule MAdCAM-1 is constitutively expressed on LP venules and GALT high endothelial venules (HEVs) and is inducible by proinflammatory cytokines (Briskin et al., 1997; Gorfu et al., 2009; Ogawa et al., 2005). Retinoic acid (RA)-expressing CD103+ dendritic cells upregulate the expression of α4β7 integrin and CCR9 receptor in the GALT, allowing binding to MAdCAM-1 and homing of lymphocytes to the intestinal mucosa via the intestinal CCR9 ligand, CCL25. By preventing the influx and extravasation of inflammatory β7+ T cells from the circulation into the intestine, antibodies targeting α4β7 or MAdCAM-1 significantly reduced the severity of colitis in animal models and in patients suffering from inflammatory bowel disease ( Hassan-Zahraee et al., 2018 ; Reinisch et al., 2021 ).There is increasing recognition of the role that the gut microbiota plays in maintaining the regulatory function of intestinal Treg and T17 cells to ensure the integrity of the intestinal epithelial barrier ( Littman and Rudensky, 2010 ; Pandiyan et al., 2019 ).

[0005] Beyond their role in gut homeostasis, T17 also regulate extraintestinal inflammatory pathology (Krebs et al., 2016; Lee et al., 2011a; Magnuson et al., 2015; Morton et al., 2014; Wu et al., 2010). Importantly, T17 and their lineage-related FoxP3 + Regulatory cells blunt antitumor immune surveillance by restoring tolerance during periods of inflammation induced during chronic carcinogenesis (Cochaud et al., 2013; Fridman et al., 2012; Guery and Hugues, 2015; Langowski et al., 2006; Young, 2016). RORγt expression identified a phenotypically stable population of tumor-infiltrating Tregs in humans and mice induced by gut commensals (Sefik et al., 2015). Conditional ROR γ t knockout mice showed reduced tumor incidence or polyp formation through an indirect mechanism involving dendritic cells ( Blatner et al., 2012 ; Rizzo et al., 2018 ).

[0006] Immune checkpoint inhibitors (ICIs) have restored tumor immune surveillance and become standard of care for the clinical management of several histological types of cancer (Brahmer et al., 2015; Robert et al., 2011). Primary resistance to ICIs has been primarily attributed to low tumor mutational burden and poor intrinsic antigenicity of tumor cells (Riaz et al., 2016; Rizvi et al., 2015), defective antigen presentation (Spranger et al., 2015), intratumoral T cell exhaustion (Smyth et al., 2016), genomic defects in the interferon gamma (IFNγ) signaling pathway (Gao et al., 2016), CSF1-dependent tumor-associated macrophages (Neubert et al., 2018), and immunosuppressive metabolic cues (Smyth et al., 2016). In addition, several studies point to a deviated gut microbiome repertoire negatively impacting ICI efficacy, suggesting the critical importance of commensals in influencing cancer immune set points (Chen and Mellman, 2017). Retrospective and prospective studies analyzing the impact of antibiotics (ATB) on patient survival revealed their negative predictive impact on clinical outcomes during immune checkpoint blockade (Elkrief et al., 2019b; Mohiuddin et al., 2021; Routy et al., 2017; Tinsley et al., 2019; Derosa et al. 2022). A meta-analysis revealed that ATB uptake was more detrimental to clinical outcomes when administered before (rather than during) the first systemic dose of anti-PD1 / PDL-1 Ab, suggesting that recolonization after the ATB process rather than a direct iatrogenic effect may be detrimental (Derosa et al., 2021). Indeed, ATB-treated patients tended to be colonized by distinct species, such as Hungatella hathewayi (Derosa et al. 2022).These epidemiological findings led to the hypothesis that ATB, by influencing the composition of the intestinal ecosystem, may tilt the equilibrium between intratumoral effector and regulatory T cells by affecting the gut / tumor trafficking of intestinal homeostatic regulatory T cell subsets ( 38 ), thereby exacerbating cancer immunosuppression. Summary of the Invention

[0007] Recently, a meta-analysis performed by the inventors revealed that ATB uptake was more detrimental to clinical outcomes when administered before (rather than during) the first systemic administration of anti-PD1 / PDL-1 Ab, suggesting that recolonization after the ATB process may be detrimental rather than a direct iatrogenic effect (Derosa et al., Cancer Discovery, 2021).

[0008] In the experimental section below, we confirm this hypothesis and demonstrate a pathway by which the gut microbiota remotely influences the development of several types of cancer and patient response to immunotherapy.

[0009] Indeed, we have shown that ATB-induced dysbiosis (Routy et al., 2018; Vetizou et al., 2015) leads to the disruption of the interaction of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1) expressed by intestinal acting T cells with its receptor α4β7.

[0010] When the ATB process is halted, selected species from the bacterial genus Enterocloster nov. (such as Enterocloster clostridioformis nov., previously classified in the bacterial class Clostridia (Haas and Blanchard, 2020)) take over and downregulate ileal mucosal addressin cell adhesion molecule-1 (madcam1) gene and cell surface protein expression on the ileal lamina propria and high endothelial venules. Loss of MAdCAM-1 inhibits the expression of the gut-homing α4β7+ TH17 and ROR γ t + Treg CD4 + It induces exodus of T cells into the tumor bed, which results in a permissive cancer microenvironment and resistance to PD-1 blockade.

[0011] The present invention is based on these results and initially concerns a method for assessing in vitro whether an individual who also has cancer also has cancer-associated or antibiotic-associated dysbiosis based on downregulation of MAdCAM in serum or ileum and / or Gut OncoMicrobiome Signature (GOMS). This method can also be used to follow up the effect of a microbiota-centered intervention (MCI) in patients.

[0012] Diagnosing such cancer-associated and / or ATB-associated dysbiosis indicates that the subject requires compensatory microbiota-centered intervention (MCI), particularly before undergoing treatment with an immune checkpoint inhibitor (ICI) or another immuno-oncology (IO) therapy.

[0013] The use of such MCIs, including oral vancomycin antibiotics, phages and rare-cutting endonucleases capable of killing bacteria of the Enterocloster novae clade, Akkermansia spp., and / or Akkermansia muciniphila (possibly mixed with other beneficial bacteria), retinoic acid, fecal microbial transplants, and mixtures thereof, to restore susceptibility to IO therapy are also part of the present invention.

[0014] The present invention also relates to combination therapy with (i) anti-PD1 or anti-PDL1 antibodies and (ii) anti-IL-17A or anti-IL-17R antibodies, and / or recombinant IL-7 for cancer patients with low levels of serum soluble MAdCAM.

[0015] The present invention also relates to screening methods for identifying agents capable of normalizing MAdCAM-1 expression levels in endothelial cells of the lamina propria. [Brief description of the drawings]

[0016] [Figure 1-1] Broad-spectrum antibiotics (ATB) down-regulate MADCAM-1 expression in the ileal vasculature.

[0017] A–D. Spontaneous recolonization after successive broad-spectrum antibiotics (ATB: ampicillin, colistin, streptomycin) or 4–7 days after cessation of ATB (ATB RECO After RT-PCR (A), immunohistochemical staining of MAdCAM-1 in LP venules (upper photomicrograph) and high endothelial venules (HEV, lower photomicrograph) (B), and CD45 in LP - Relative transcript levels (A) and protein levels (B-D) of the Madcam1 gene product obtained by flow cytometry gating on cells (C) or ELISA (D) of ileal (A-D) or colonic (A) tissues in C57BL / 6J mice. Each dot represents one ileum or colon. E-F. Treatment with oral vancomycin (E) or broad-spectrum ATB for 8 days followed by 4-7 days of recolonization (ATB RECO(F) Quantitative RT-PCR of relative madcam1 gene expression in ileal mucosa of mice supplemented once with Enterocolobus clostridia by gavage before sacrifice 7 days later. Each graph illustrates a representative experiment (containing 5-6 mice / group) out of 2-3 yielding similar results. Panels A or E left panel pooled data from two experiments. Oral gavage was performed in C57BL / 6J mice housed in SPF conditions (without ATB pretreatment) as in GE but with the bacterial species aligned on the x-axis. [Figure 1-2]Broad-spectrum antibiotics (ATB) downregulate MADCAM-1 expression in the ileal vasculature. H. Left panel: Effect of fecal microbiota transplantation (FMT) in 3-day ATB-treated recipient mice with feces from NSCLC patients at the time of diagnosis (before PD-1 blockade) on ileal Madcam1 gene expression normalized to naive specific-pathogen free (SPF) mouse groups (log10 axis). Each experiment used a different FMT donor and included 5–6 animals / group. Each dot represents an ileum. Right panel: Non supervised hierarchical clustering of the taxonomic composition of donor feces (defined using shotgun MG sequencing), selecting for >25% high prevalence and clinically relevant bacteria. I. Same experiment as A-D, performing flow cytometry analysis of ileal lamina propria CD4+ T cell subsets (α4β7+ vs. CD25+ FoxP3+ Treg vs. RORγt+ CD4+ (TH17) cells) during continuous ATB or ATBRECO phase, each dot represents one ileum. A representative Facs analysis of two with similar results is shown. See also Figure 5H-I for qPCR related data on similar ileal mucosal tissue. As in JA, but Madcam-1 gene-deficient mice without ATB pretreatment (treated or not with anti-PD1 Ab, which did not affect ileal MADCAM-1 expression levels, not shown). Each dot represents qPCR data of one ileum. [Figure 1-3]Broad-spectrum antibiotics (ATB) downregulate MADCAM-1 expression in the ileal vasculature. K. RT-PCR-based transcript levels of human MADCAM-1, FOXP3, and RORC genes in intestinal biopsies collected during endoscopic interventions in nine control (ATB-untreated) and seven ATB-treated patients (Table 1). Each dot represents one biopsy from either the ileum, colon, and cecum, with a single patient represented one to three times. ANOVA statistical analysis (Kruskal-Wallis test): *p<0.05, **p<0.01, ***p<0.001. [Table 1]

[0018] [Figure 2-1] Transmigration of gut-acting IL-17A and IL-22-secreting α4β7+CD4+T and Treg cells into tumor beds induced by bacterial recolonization after ATB

[0019] A-B. Flow cytometric determination of photoconverted (A) or CFSE-labeled (B) intestinal origin cells in secondary lymphoid organs (mesenteric LN, spleen, tdLN: tumor-draining LN) assessed in Kaede mice after illumination according to the schematic diagram (A, left panel) or by injection of CFSE into the mLN of wild-type mice (B, left panel). The left panel shows a schematic diagram of the experimental setup, and the right panel shows the relative accumulation (log2 fold change (Fold Change: FC)) of photoconverted (PC) cells vs. resident (non photoconverted (NPC)) cells (A) or CFSE-labeled cells vs. resident (non-labeled) cells (B) in each subset detailed in the lineage using a color gradient, 24 h after tissue UV-A irradiation or CFSE injection in the mLN of the various secondary lymphoid organs. Statistics: Mann-Whitney test with p-value adjusted by the Benjamini-Hochberg method, * p<0.05, ** p<0.01, *** p<0.001. [Figure 2-2]Transmigration of gut-acting IL-17A and IL-22-secreting α4β7+ CD4+ T and Treg cells to the tumor bed induced by bacterial recolonization after ATB. C. Effect of neutralization of MAdCAM-1 with αMAdCAM-1 mAb on recirculation of gut-derived cells to tdLNs in Kaede (left) or mLN-CFSE-injected WT mice (middle panel) or to contralateral (control) LNs (right panel). Each dot represents one mouse. A representative experiment of two is shown, reaching similar conclusions. D. Volcano plot illustrating differential gene transcripts in RNA sequencing of α4β7high vs. α4β7negCD4+ lymphocytes cell-sorted from mLNs in five MCA205 tumor-bearing animals. For each gene product expressed in each cell type for five mice, a volcano plot was generated by computing (i) the log2 of the fold ratio (FR) between the mean relative abundance of the transcript after normalization in α4β7high vs. α4β7neg CD4+ lymphocytes (x-axis); (ii) the co-log10 of the p-value derived from the Mann-Whitney U test (y-axis) calculated based on the absolute relative abundance. Black and grey dots are considered significant (p<0.05), whereas back dots are not significant (p>0.05). E. Experimental setup (left panel) and flow cytometric evaluation of IL-17A+ secreting α4β7+ Treg (middle panel) or α4β7+ T-transduced (right panel) CD4+ T cells within CFSE+ (derived from mLNs, grey dots) or CFSE+ cells (tdLN resident cells, black dots) reaching tdLNs during the ATBRECO phase after ATB pretreatment (ampicillin, colistin, streptomycin). Each graph illustrates two independent pooled experiments (including 5-6 mice / group). F. Flow cytometric determination of CFSE+ Treg cells reaching tdLNs or contralateral LNs in MCA205 bearing wild type versus Madcamt1− / − mice (not treated with ATB) after CFSE injection in mLNs. Each dot represents one mouse and a representative experiment out of two that yielded similar results is illustrated. [Figure 2-3]Transmigration of gut-acting IL-17A and IL-22-secreting α4β7+ CD4+ T and Treg cells to the tumor bed induced by bacterial recolonization after ATB. G. As above. As in E, but in Kaede mice treated or not with anti-MADCAM-1 Ab, flow cytometry could identify Tregs using membrane staining for CD25 and CD127. H. Experimental setup to evaluate the short-term (4-7 days) effect of bacterial recolonization after stopping the ATB process on the recirculation of gut-acting CD4+ T cells with or without oral gavage with Enterocloster species (left panel). Flow cytometric phenotyping of TH17 cells expressing high levels of CD25 (Tr17-like, middle panel) or TH17 transformation (right panel) in Kaede mice within PC (derived from mLN) or NPC cells (tdLN-resident cells) that reach the tdLN during the ATBRECO phase (G). Each dot represents one mouse and the graph illustrates two pooled experiments. ANOVA statistical analysis (Kruskal-Wallis test) or Wilcoxon paired signed rank test: raw p values ​​are indicated. [Figure 2-4]Transmigration of gut-acting IL-17A and IL-22-secreting α4β7+ CD4+ T and Treg cells into the tumor bed induced by bacterial recolonization after ATB. I. Single-cell transcriptomics analysis of CFSE+ mLN-derived CD4+ T cells transmigrating into tdLNs in MCA205 tumor-bearing bodies treated with water (group 1), ATB treatment for 7 days with continued ATB (group 2), or ATB stopped for 4 days (ATBRECO) (group 3), or replaced by oral gavage with E. clostridioformis for 4 days (group 4). This experiment contains 5 animals / group. UMAP gene patterns in 4 subsets of transmigrating CFSE+ CD4+ T cells in plate-based full-length single-cell RNA-seq data with unsupervised clustering, overlaid with 4 groups of mice (I, left panel). Volcano plots of differential gene expression patterns associated with each specific cell type by Log2 p-value, and fold ratio of the proportion of each subtype compared to the other three: Tregs (I, center panel) and proliferating cells (I, right panel). See Figure 7 for cell type gene patterns from each of the four experimental groups. [Figure 2-5] Transmigration of gut-acting IL-17A and IL-22-secreting α4β7+ CD4+ T and Treg cells into the tumor bed induced by bacterial recolonization after ATB. UMAP gene patterns in four subsets of transmigrating CFSE+ CD4+ T cells in plate-based full-length single-cell RNA-seq data by unsupervised clustering, overlaid with four groups of mice (I, left panel). Volcano plots of differential gene expression patterns associated with each specific cell type by Log2 p-value, and fold ratio of the proportion of each subtype compared to the other three (Tregs (I, middle panel) and proliferating cells (I, right panel). See Figure 7 for cell type gene patterns from each of the four experimental groups.

[0020] [Figure 3-1] Disruption of the MAdCAM-1 / α4β7 axis induced a maladaptive response to αPD-1-based immunotherapy in mice.

[0021] A. Tumor growth kinetics of subcutaneous MCA205 (syngeneic C57BL / 6J) implanted in wild type (wt) versus Itgb7 or Madcam1 gene-deficient mice. Mean + SEM of tumor size across 5-6 mice / group over time in the two treatment groups (anti-PD1 vs. isotype control Ab). B-D. Tumor growth kinetics or tumor luminescence following implantation of subcutaneous MCA205 (B), mammary 4T1 (syngeneic BALB / c, C), and lung orthotopic TC1-luc (syngeneic C57BL / 6J, D) tumors in animals treated with isotype control, anti-α4β7 mAb, or -αMAdCAM-1 mAb while receiving anti-PD1 therapeutic antibody (or isotype control Ab). For lung orthotopic TC1 tumors, whole body luminescence with an IVIS imaging system was used to quantify luciferase-expressing TC1 kinetics in the lung. Ratios were calculated before and after PD-1 blockade with isotype control mAb, αα4β7 mAb, or αMAdCAM-1 mAb. E. Flow cytometry analysis of α4β7 expression on CD4+ T cells splenocytes and tumor infiltrating lymphocytes (TILs) in wild type (wt) versus Madcam1 gene-deficient mice. Each dot represents one mouse. Graphs are pooled from two independent experiments with 5 mice / group. Student's t-test. [Figure 3-2] Disruption of the MAdCAM-1 / α4β7 axis induced a maladaptive response to αPD-1-based immunotherapy in mice. F. Unsupervised hierarchical clustering represented as heatmaps showing the relative proportions of different immune α4β7+CD4+ T cell subsets (determined by flow cytometric analysis of TILs dissociated from established subcutaneous MCA205) in mice treated with neutralizing αMAdCAM-1 mAb or isotype control mAb. [Figure 3-3]Disruption of the MAdCAM-1 / α4β7 axis induced a maladaptive response to αPD-1-based immunotherapy in mice. G-I. Intracellular flow cytometry analysis of Rorγt expression in α4β7+ Treg TILs in wild-type (wt) versus Madcam1 gene-deficient mice (G) or in wild-type MCA205 tumor-bearing mice undergoing ATB pretreatment regimen (4 days after ATB cessation (ATBRECO-S) or 12 days after ATB cessation (ATBRECO-L)) with or without treatment with anti-PD1 Ab (H,I). Each dot represents one mouse. A representative experiment out of two independent experiments with similar results and including 5 mice / group is shown (G,I). Student's t-test (G). [Diagram 3-4] Disruption of the MAdCAM-1 / α4β7 axis induced a maladaptive response to αPD-1-based immunotherapy in mice. J. Flow cytometry analysis of tumor infiltrating cells (TILs) for CCR5+CD8+ effector T cells in subcutaneous MCA205 tumor-bearing mice treated with isotype control mAb, αα4β7 mAb, or αMAdCAM-1 mAb in the setting of PD-1 blockade. K. Schematic diagram of the experimental set-up (left panel) and cross-sectional study of tumor size (mean ± SEM) of subcutaneous 4T1 wild-type (left) or 4T1 IL-22Rα1 knockout (right) tumor cell lines at the time of sacrifice after treatment with αPD-1 mAb during the ATBRECO phase and after systemic neutralization of IL-17A with αIL-17A mAb (ip) in parallel with immunotherapy. In each experiment, four αPD-1 intraperitoneal (ip) injections were administered every 3 days during the ATBRECO phase. Each experiment contained 6 mice / group. A representative experiment out of 2-3 is shown. ANOVA statistical analysis (Kruskal-Wallis test): * indicates raw p-value.

[0022] [Figure 4-1] Serum soluble MADCAM-1 is a predictor of clinical benefit to PD1 blockade in patients with NSCLC and a measure of intestinal dysbiosis.

[0023] A. Spearman correlation between RT-PCR-determined ileal Madcam1 gene expression levels and serum soluble MADCAM-1 (determined by ELISA) in 65 MCA205 tumor-bearing mice (each dot represents one animal). B. ELISA monitoring of serum levels of soluble MADCAM-1 in 301 NSCLC patients belonging to two independent cohorts (C01, C02, left panels) according to recent ATB uptake history (right panel) compared to serum levels in healthy volunteers (HV, n=70). Each dot represents one patient serum. C-D. Alpha and beta diversity of taxonomic content of gut microbiota according to serum sMADCAM-1 levels in NSCLC patients. MGS diversity-based abundance assessment in shotgun MG sequencing according to median sMADCAM-1 in the entire population of 95 NSCLC patients (C) and inter-individual variability of composition in both groups (D). E. Kaplan-Meier survival curves and Cox regression linear analysis of predictive values ​​of serum levels of sMADCAM-1, divided according to the median of each cohort, in two independent (left and right columns) cohorts of stage IV NSCLC treated with αPD-1 mAb immunotherapy for PFS (E) and OS (F) (see Tables 5-6 for patient descriptions and multivariate analyses). [Figure 4-2] Serum soluble MADCAM-1 is a predictor of clinical benefit to PD1 blockade in NSCLC patients and a measure of intestinal dysbiosis. G. Supervised hierarchical clustering (spread >10%) of MGS according to sMADCAM-1 serum levels (<(low) or >(high) median), color codes characterize the relative abundance of each significant species aligned in the bar graph in the left column using different pipelines and algorithms. H. Relative abundance of E. clostridioformis in proportion among all MGS species in fecal material of 95 patients divided into two groups according to sMADCAM-1 serum levels (<(low) or >(high) median). Each dot represents one stool / serum / patient. Student's t' test.

[0024] [Figure 5-1]ATB-induced dysbiosis affects the transcriptional program of immune gene products in the intestinal lamina propria.

[0025] A. Heatmaps of the log2 fold change ratios between ATB-treated and non-treated (water) ileum, colon, and tumor beds for chemokine (left panel) and cytokine / transcription factor (right panel) expression profiles in ELISA (left panel) and RT-PCR (right panel) of tissue lysates. Statistics: Mann-Whitney test with p-value adjusted by Benjamini-Hochberg method. * p<0.05, ** p<0.01, *** p<0.001. [Figure 5-2] ATB-induced dysbiosis affects the transcriptional program of immune gene products in the intestinal lamina propria. B. Evaluation of CD31 expression on ileal HEV by IHC in ATB-treated mice. C. RT-PCR evaluation of the effect of 7 days of pretreatment or after ATBRECO with different antibiotic regimens on the relative transcription levels of the Madcam1 gene in ileal tissues (left) or in mesenteric lymph nodes (right). Sequential (c.), Ampic:c. ampicillin, Colistc:c. colistin, Streptoc:c. streptomycin, Vancoc:c. vancomycin, Erythrc:c. vancomycin. Each dot represents one ileum. Graphs are pooled from 2-3 experiments with 5 mice / group. D. RT-PCR evaluation of the relative expression of the Madcam1 (D, E) and VCAM1 (E) genes in Peyer's patches (PP) (D). Each dot represents one ileum. The graph is pooled from two experiments with 5 mice / group. [Figure 5-3]ATB-induced dysbiosis affects the transcriptional program of immune gene products in the intestinal lamina propria. E. RT-PCR evaluation of the relative expression of Madcam1 (D, E) and VCAM1 (E) genes in different lymph nodes (LN) (mLN: mesenteric LN, sk: cutaneous LN, td: tumor-draining LN) (E). Each dot represents one ileum. The graph is a compilation of two experiments with 5 mice / group. F. Culturomics under aerobic and anaerobic conditions of ileal material taken from mice treated with different ATB regimens (see C) and sacrificed 4 days after cessation of treatment. [Figure 5-4] ATB-induced dysbiosis affects the transcriptional program of immune gene products in the intestinal lamina propria. G. Qualitative identification by mass spectrometry of cultured species in each condition. H-I. RT-PCR evaluation of mouse Foxp3, RORc, and IL17a genes in ileal tissues of mice treated or not with ATB (H) and Spearman correlation of expression levels of ileal Madcam1 with Rorc or Foxp3 in mice (I). Each dot represents one animal. Concatenated data from three independent experiments. J. Spearman correlation between RT-PCR-based transcription levels of human FOXP3 (left) and IL-17A (right) genes with Madcam-1 in intestinal biopsies collected during endoscopic interventions in nine control (no ATB) and seven ATB-treated patients (Table 1). Each dot represents one biopsy from either the ileum, colon, and cecum, with a single patient represented one to three times. ANOVA statistical analysis (Kruskal-Wallis test): *p<0.05, **p<0.01, ***p<0.001.

[0026] [Figure 6-1] Profiling of gut-origin cells in different compartments.

[0027] A. Schematic of the experimental setup of UV-A irradiation of the intestine (ileum, cecum, mLN) in tumor-bearing Kaede mice and flow cytometry gating strategy of photoconverted cells (PCs) in tdLN (upper panel) and mLN (lower panel). PC frequency in target organs before, 5 min, and 24 h after laparotomy and UV-A irradiation of the intestinal compartment is indicated. B. As above. As in A, with detailed percentages of PC cells in mLN, spleen, tdLN, and tumor bed at 24 h, each dot represents one mouse. A typical experiment out of three with similar results is illustrated. C. Experimental setup (left panel) and flow cytometry determination of CFSE+ cells 24 h after CFSE injection in mLN in different tissues (x-axis), each dot represents one mouse. Data from three independent experiments are pooled. D. As above. As in B, the recruitment of gut-active cells towards the tdLN or tumor bed was monitored following UV-A irradiation of the "gut tract" (ileum + cecum + mLN) versus the ileum only, each dot represents one mouse. A representative experiment of two with similar results is illustrated. [Figure 6-2] Profiling of intestinal origin cells in different compartments. E-F. Flow cytometric assessment of intestinal integrin α4β7 expression by resident (NPC) or recirculating (PC) CD4+ T cells in the spleen, tdLN or tumor bed in Kaede mice or wild type mice injected with CFSE in mLN (F) depending on UV-A irradiated area (ileum vs. "gut tract" (ileum+cecum+mLN). G. Flow cytometric assessment of different CD4+ Treg cell types in tdLN (varying expression of α4β7, IL-17, IL-22) followed by CFSE labeling in animals treated or not with ATB and bearing subcutaneous MCA205 and subjected to CFSE injection in mLN 24 h prior. ANOVA statistical analysis (Kruskal-Wallis test): *note raw p-value. [Figure 6-3]H. Flow cytometric assessment of different CD4+ Treg cell types (variable expression of α4β7, IL-17, IL-22) in tdLNs followed by CFSE labeling in animals treated or not with ATB and bearing subcutaneous MCA205 and subjected to CFSE injection in mLNs 24 h prior. ANOVA statistical analysis (Kruskal-Wallis test): *note raw p-value.

[0028] [Figure 7-1] Recolonization-induced gene expression profiling.

[0029] A. Experimental design for scRNA-seq data collection. Four experimental groups of mice containing five MCA205 tumor-bearing animals (water, continuous ATB, ATB RECO A, E. clostridioformis), single-cell transcriptomics was performed on CFSE+ CD4+ T cells harvested from tdLNs 24 h after CFSE inoculation in the mLNs. B. Absolute numbers of CFSE+ cells recovered in the tdLNs for each condition after quality control and sequencing. [Figure 7-2] Gene expression profiling induced by recolonization. C. Volcano plot illustrating differential gene transcription in RNA sequencing of CFSE+CD4+ T cells belonging to Treg subset cells in ATBRECO group versus water group (control). For each gene product expressed in each cell type for five mice, (i) the log2 of the fold ratio (FR) between the mean relative abundance of the transcript after normalization in group 3 vs. group 1 (x-axis); (ii) the co-log10 of the p-value derived from the Mann-Whitney U test (y-axis) calculated based on the absolute relative abundance. Blue (downregulated gene products) and red (upregulated gene products) dots are considered significant (p<0.05), whereas back dots are not significant (p>0.05). [Figure 7-3]Recolonization-induced gene expression profiling. Similar to DCs, gene transcripts in Tregs are compared with other T cell subsets within ATBRECO (group 3, left panel) or ATB E. clostridioformis (group 4). [Figure 7-4] Recolonization-induced gene expression profiling. Similar to ED, but comparing gene transcripts of proliferating T cell subsets with other T cell subsets in the negative control group (water, group 1, left panel) and ATBRECO (group 3, right panel). [Figure 7-5] Gene expression profiling induced by recolonization. Similar to FE, but comparing gene transcripts of cells belonging to cluster 1 (UMAP, Figure 2I) in two mouse groups (Water, group 1 vs. ATBRECO (group 3) in the left panel, then comparing the transcriptional profile of these cluster 1 cell subsets with other clusters in ATBRECO (group 3) (right panel). [Figure 7-6] Recolonization-induced gene expression profiling. Similar to GE, but comparing gene transcripts in cells belonging to cluster 0 (UMAP, Figure 2I) in two mouse groups (water, group 1 vs. ATBRECO (group 3)).

[0030] [Figure 8-1] αPD-1 mAb aggravates the accumulation of gut-acting α4β7+RORγt+Tregs or IL-17A+IL-22+Tregs in tumor lesions caused by ATB-induced dysbiosis.

[0031] A. Spearman correlation between ileal Madcam1 and tumor size in MCA205 tumor-bearing (anti-PD1 or isotype control Ab treated) mice. Each dot represents one mouse. ANOVA statistical analysis (Kruskal-Wallis test): * Note raw p-values. B. α4β7 in subcutaneous MCA205 tumors + or α4β7 - CD4 + ROR in the TIL fraction γt+ Treg(T r Flow cytometric evaluation of PD1 blockade in mLN tumors (D) or subcutaneous MCA205 tumors (E). Each dot represents one tumor and mouse. C. Experimental setup of PD1 blockade during ATB intervals of various durations in tumor-bearing subjects. D-E. E ) in Tregs, RORγt + Flow cytometric assessment of IL-17A+IL-22+ cells. [Figure 8-2] αPD-1 mAb exacerbates the accumulation of gut-acting α4β7+RORγt+Treg or IL-17A+IL-22+Treg into tumor lesions caused by ATB-induced dysbiosis. F. Flow cytometric assessment of RORγt+ cells or IL-17A+IL-22+ cells within Treg in 4T1 tumors (F). G. Flow cytometric determination of IL-17A+IL-22+secreting Treg cells (left) and α4β7+Treg cells (right) in subcutaneous (sc) MCA205 tumors treated by PD1 blockade after ATB withdrawal and by spontaneous ATBRECOL+ / - forced gut colonization with E. clostridioformis or L. reuteri bacteria during αPD-1-based therapy. Each dot represents the tumor size of each animal at the time of sacrifice in a typical experiment containing 6 mice / group out of 2 with similar results. See Table 4 for detailed phenotypes. ANOVA statistical analysis (Kruskal-Wallis test): *note raw p-values.

[0032] [Figure 9-1] Akkermansia muciniphila upregulated ileal Madcam-1 gene expression and prevented the recirculation of gut-acting T cells to the tumor bed in a MADCAM-1-dependent manner.

[0033] A. Tumor size at sacrifice in MCA205 tumor-bearing mice treated with ATB for 3 days with or without oral gavage supplementation of live or pasteurized A. muciniphila SGB9228 (A. muc, left panel) and intestinal humanized with FMT from a PD1-refractory NSCLC patient (FMTNR). + Flow cytometric assessment of α4β7 and / or CCR9 expression by T cells (middle and right panels). B. Effect of anti-MADCAM-1 neutralizing antibody on the compensatory effect of pasteurized A. muciniphila SGB9228 on the response to PD1 blockade illustrated as the ratio between tumor sizes of anti-PD1 treated FMT NR avatar mice compensated with bacteria + / - anti-MADCAM-1 Ab. C-D. Flow cytometric assessment of IL-17A and IL-22 cytokine secretion by CD4+ T cells and Treg cells in tdLNs of mice colonized with FMT NR and supplemented with A. muciniphila (A.muc) by oral gavage with or without αMAdCAM-1 mAb treatment and with treatment with αPD-1 mAb (C). IL-17+IL-22+Treg or total CD4 reach tdLN in anti-PD1 treated FMT NR avatar mice compensated with bacteria + / - anti-MADCAM-1 Ab + Effect of anti-MADCAM-1 neutralizing antibodies on the protective efficacy of pasteurized A. muciniphila SGB9228 to prevent extraintestinal exodus of Tr17, depicted as the ratio between T cells and MADCAM-1 neutralizing antibodies (D). ANOVA statistical analysis (Kruskal-Wallis test) or Wilcoxon paired signed rank test: * Raw p-values ​​are noted. [Figure 9-2]Akkermansia muciniphila upregulated ileal Madcam-1 gene expression and prevented the recirculation of gut-acting T cells to the tumor bed in a MADCAM-1-dependent manner. Diagrammatic overview illustrating the recirculation of gut-primed IL-17A+IL-22+RORγt+α4β7+Treg (Tr17) cells to the tumor microenvironment when there is disruption of MADCAM-1 expression in ileal HEV in the context of E.ATB-induced dysbiosis.

[0034] [Figure 10-1] Soluble MADCAM-1 serum levels are a robust predictor of clinical benefit to PD-1 blockade.

[0035] A. Cox linear regression analysis assessing the hazard ratio (HR) for risk of progression or death during PD1 blockade and serum sMADCAM-1 (monitored by ELISA) in each of cohorts C01 and C02 listed in Table 5, using a restricted cubic spline (RCS) with 4 knots. The number of knots was selected according to Akaike's information criterion. Left panel (C01): N=115 NSCLC patients, 62 deaths, median OS=15 months (95%CI: 11.2-25 months), HR=0.92 (95%CI: 0.88-0.97, p=0.001) Right panel (C02): N=186 NSCLC patients, 83 deaths, median OS=12 months (95%CI: 9-17 months), HR=0.97 (95%CI: 0.94-0.99, p=0.02). B. Prevalung study: serum sMADCAM-1 levels in CVD smokers prior to lung cancer incidence in patients diagnosed with lung cancer within 2 years of follow-up (N=9) vs. patients not diagnosed with lung cancer (N=56 controls). C. Spearman correlation between serm sMADCAM-1 and circulating α4β7+Rorγt Tregs. E. ANOVA statistical analysis of taxonomic MGS-based clustering and separation with median sMADCAM-1. [Figure 10-2]Soluble MADCAM-1 serum levels are a robust predictor of clinical benefit to PD-1 blockade. D. Unsupervised hierarchical clustering of the taxonomic composition based on shotgun MG sequencing of feces of NSCLC patients with sMADCAM-1 paired serum levels.

[0036] [Figure 11] Kaplan-Meier overall survival curves and Cox regression univariate analysis with log-rank statistical test of 212 RCC patients according to the median serum sMAdCAM-1 levels. RCC in second-line immunotherapy based on anti-PD1 Ab after failure of tyrosine kinase inhibitors.

[0037] [Figure 12] Kaplan-Meir progression-free survival curves and Cox regression univariate analysis with log-rank statistical test of 212 RCC patients according to the median serum sMAdCAM-1 levels. RCC in second-line immunotherapy based on anti-PD1 Ab after failure of tyrosine kinase inhibitors.

[0038] [Figure 13] Validity of the biomarker sMAdCAM-1 to predict clinical benefit to anti-PDL-1 Ab durvalumab in pretreated second-line metastatic bladder cancer patients.

[0039] A. Each dot represents one time point and patient, each patient is represented in triplicate. Concentration of sMAdCAM_1 in the serum of patients at baseline before durvalumab and during 1 and 4 months of treatment in responders (elite patients, PFS>5 months) and non-responders (progressors, OS>6 months). B-C. Log-rank univariate analysis of the predictive value of sMAdCAM_1 and Kaplan-Meier curves for survival (OD, B) and PFS (C) in patients separated according to the median MAdCAM_1 value within the entire cohort. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] According to a first aspect, the present invention relates to a method for diagnosing cancer-associated or antibiotic-associated intestinal dysbiosis in an individual having cancer in vitro comprising measuring soluble MAdCAM-1 in a serum sample from said individual (e.g. by ELISA), wherein a reduced level of serum soluble MAdCAM-1 is indicative of the individual having cancer-associated or antibiotic-associated intestinal dysbiosis.

[0041] Alternatively, the above method can be performed by assessing expression of MAdCAM-1 in ileal lamina propria (LP) venules or in high endothelial venules (HEV) in ileal biopsies of the patient, where reduced MAdCAM-1 expression in LP or HEV indicates that the individual has cancer-associated ileopathy / dysbiosis. In such cases, expression of MAdCAM-1 can be measured by RT-PCR or by ELISA or flow cytometry or immunohistochemistry performed on the biopsy.

[0042] Thus, the present invention relates to the use of serum soluble MAdCAM-1 levels as a marker for cancer-associated or antibiotic-associated dysbiosis, wherein a reduction in the level of serum soluble MAdCAM-1 is a marker for cancer-associated or antibiotic-associated dysbiosis.

[0043] As shown in the experimental section below, the inventors have demonstrated that the level of soluble MAdCAM-1 in an individual's serum is also a marker of resistance or sensitivity to immuno-oncology (IO) therapy, where reduced levels of serum soluble MAdCAM-1 are a marker of resistance to IO therapy.

[0044] Thus, in accordance with the present invention, the level of soluble MAdCAM-1 in an individual's serum can be used to assess whether an individual is likely to be resistant to immuno-oncology (IO) therapy (low levels). Normal (or elevated) levels of serum soluble MAdCAM-1 are a marker of clinical benefit.

[0045] In the present context, the phrase "IO therapy" includes immune checkpoint inhibitors (ICI), as well as CAR-T cells, adoptive TIL transfer, and combinations thereof. In the context of the present invention, "IO therapy" also includes combination therapy, including one of the above IO agents, and other antineoplastic treatments, such as chemotherapy, particularly immune checkpoint inhibitors (ICI), in any combination with taxanes, permetrexed, cisplatin, and / or oxaliplatinum, or EGFR inhibitors.

[0046] In the context of the present invention, "ICI" includes other molecules that perform the same function(s), such as anti-PD1 antibodies (Abs), anti-PDL-1 Abs, anti-CTLA4 Abs, anti-Lag3 Abs, anti-Tim3 Abs, anti-TIGIT Abs, anti-OX40 Abs, anti-41 BB Abs, anti-VISTA Abs, bispecific antibodies targeting PD1 and Lag3, and non-Ab molecules that block any of the above immune checkpoints. According to certain embodiments, the IO therapy includes anti-PD1 / PDL-1 Abs, such as monoclonal Abs that block PD1 or PDL-1.

[0047] It has been shown that in certain cases of treatment resistance, ICI drugs are not only useless, but can even have harmful effects and can lead to rapid tumor progression (i.e., hyperprogressive disease or HPD).Therefore, it is very important to identify patients who are likely to resist IO treatment in order to decide not to administer IO treatment to these patients, at least not without compensatory or combined treatment to avoid HPD development.

[0048] The present invention is particularly useful for assessing the resistance status of individuals who have taken broad-spectrum antibiotics during a period ranging from 60 days before to 42 days after the first dose of IO therapy / ICI. Indeed, as shown in the experimental section below, broad-spectrum antibiotics increase the risk of dysbiosis associated with IO resistance.

[0049] When carrying out the above method, the level of serum soluble MAdCAM-1 is considered to be reduced when it is lower than a certain threshold. This threshold can be, for example, the median level of soluble MAdCAM-1 in a representative cohort, such as a cohort of individuals suffering from cancer, preferably a cohort of individuals suffering from the same cancer as the individual tested. Even more preferably, the representative cohort is a cohort of individuals suffering from the same cancer as the individual tested and receiving IO therapy / ICI in the same treatment scheme (particularly distinguishing at least 1L vs. 2L or more therapy as the same treatment line). Those skilled in the art can refine the threshold by measuring the median level of soluble MAdCAM-1 in a cohort of patients who share the same treatment history and receive the same IO therapy. In the experimental results disclosed below, the median values ​​were 177.1 ng / mL and 233.3 ng / mL, respectively, in a discovery and validation cohort of patients with advanced non-small cell lung cancer (NSCLC) treated with anti-PD1 / L-1 antibodies with or without chemotherapy (Example 1), whereas they were 88.8 ng / mL in a cohort of patients with kidney cancer treated with nivolumab in second-line therapy (Example 2.1), and 158.8 ng / mL in a cohort of patients with pre-treated metastatic bladder cancer treated with durvalumab (Example 2.2).

[0050] Alternatively, the predetermined threshold can be calculated from a cohort of individuals not affected by cancer. For example, the level of serum soluble MAdCAM-1 can be considered reduced if it is in the bottom tertile of a cohort of individuals not affected by cancer.

[0051] The above methods are of particular interest for patients suffering from cancer for which immunotherapy alone or in combination with chemotherapy or tyrosine kinase inhibitors or hormonal therapy (androgen or estrogen deprivation, or LHRH antagonists) is applicable.

[0052] Examples of such cancers include breast cancer, chronic myelomonocytic leukemia (CMML), colorectal cancer, renal cancer, lung cancer (e.g., NSCLC), urothelial cancer, melanoma, ovarian cancer, gastric and esophageal cancer, mesothelioma, hepatocellular carcinoma, prostate cancer, and any mismatch repair insufficiency (MSI) high tumor with non-visible histology that is FDA approved for anti-PD1 Ab. According to certain embodiments, the individual is afflicted with a cancer selected from the group consisting of NSCLC, melanoma, breast cancer, renal cancer, bladder cancer, and colorectal cancer.

[0053] The present invention also relates to a theranostic method for determining whether an individual having cancer requires compensatory microbiota-centered intervention (MCI) prior to administration of a cancer immuno- (IO) therapy, comprising assessing whether the individual has a cancer-associated or antibiotic-associated dysbiosis according to said method, wherein if the individual has a cancer-associated or antibiotic-associated dysbiosis, then the individual requires MCI prior to administration of treatment with an IO therapy.

[0054] In certain cases, such as lung cancer, MCI can be combined with another treatment, for example chemotherapy, to avoid HPD.

[0055] In this context, the phrase "microbiota-centered intervention (MCI)" refers to any treatment that has a direct or indirect effect on the gut microbiota composition.

[0056] Examples of MCIs according to the present invention include: - oral vancomycin antibiotic (e.g., same protocol as for treating C. difficile infection), -Bacteria-killing phages of the Enterocloster nov. clade, - Rare-cutting endonucleases such as Crispr Cas9 engineered to kill bacteria from the Enterocloster new clade; - Akkermansia spp. and / or Akkermansia muciniphila (possibly mixed with other beneficial bacteria), -retinoic acid, - fecal microbial transplantation (FMT), in particular with the aid of the product, which has been shown to increase the level of MAdCAM-1 expression in endothelial cells of the lamina propria, for example by the method described below, and - mixtures of the above therapeutic agents, Includes:

[0057] In the above, the phages and endonucleases are considered to be "killing bacteria of the Enteroclosteria neoclave" if they are capable of killing at least one bacterium of the Enteroclosteria species corresponding to the clading taxonomy reported in Haas and Blanchard, Int J Syst Evol Microbiol 2020;70:23-34.

[0058] This list includes: Clostridium / Enterocloster asparagiformis Clostridium / Enterocloster lavalense Clostridium / Enterocloster boltae Clostridium / Enterocloster clostridioforme Clostridium / Enterocloster citroniae Clostridium / Enterocloster aldenense Clostridium / Enterocloster symbosium Hungatella hathewayi Hungatella effluvia

[0059] The present invention also relates to the use of the above listed agents for MCI to treat cancer in combination with IO therapy in individuals with cancer-induced dysbiosis.

[0060] Another object of the present invention is a method for following up the success of MCI comprising measuring the level of serum soluble MAdCAM-1, wherein normalized levels of serum soluble MAdCAM-1 indicate that MCI has successfully restored intestinal eubiosis or at least partially corrected dysbiosis.

[0061] When practicing this method, the level of serum soluble MAdCAM-1 can be considered "normalized" if it is above a predetermined threshold (as described above), or alternatively, can be considered "normalized" or at least "partially normalized" if it is higher than before MCI.

[0062] The present invention also relates to the use of serum soluble MAdCAM-1 levels as a biomarker to predict early NSCLC incidence in high-risk heavy smokers (HRHSCV) experiencing cardiovascular events, wherein a reduction in serum soluble MAdCAM-1 is a marker to predict the development of NSCLC.

[0063] In the following experimental section, we demonstrated that in the setting of intestinal dysbiosis, PD1 blockade promotes tumor expansion and homing of TH17 / Tr17 in the GALT. We also showed that in such a situation, blocking IL-17A bioactivity can avoid the deleterious effects of dysbiosis.

[0064] Therefore, combining IL-17A and PD1 blockade is of great clinical interest for patients identified by one of the above approaches as potentially resistant to treatment with anti-PD1 Abs.

[0065] Moreover, IL-7 regulates α4β7 integrin expression, imprints gut-homing specificity on T cells, and acts on CD127, which is highly expressed on transmigrated Tr17 in inflammatory bowel disease (Belarif et al. J. Cin Invest May 2019, vol 129, numero 5).

[0066] As a result, combining recombinant IL-7 with PD1 blockade represents another option of great clinical interest for patients identified by one of the above methods as potentially resistant to treatment with anti-PD1 Abs.

[0067] Thus, another aspect of the invention is a combination therapy comprising (i) an anti-PD1 antibody or an anti-PDL1 antibody and (ii) an anti-IL-17A antibody or an anti-IL-17R antibody and / or recombinant IL-7 for the treatment of cancer in patients with low serum levels of soluble MAdCAM.

[0068] In the above combination therapy, the two or more agents can be administered together or separately, beginning with blocking IL-17R or IL-17, and then inhibiting PD1 or PDL-1 / PDL-2.

[0069] Another aspect of the invention relates to methods and tools for identifying agents capable of normalizing MAdCAM-1 expression levels in endothelial cells of the lamina propria.

[0070] Such methods include (i) contacting in vitro cells that express MAdCAM-1 or that have been engineered to express a reporter gene under the control of the MAdCAM-1 promoter with the compounds to be tested, and (ii) assessing whether any of the compounds induce expression of MAdCAM-1 or the expression of the reporter gene expressed under the control of the MAdCAM-1 promoter.

[0071] Examples of cells that can be used to practice this method include human umbilical vein endothelial cells (HUVEC), transformed sinusoidal endothelial cells (TSEC), bEnd.3 cells, and their derivatives, particularly engineered cells derived from these cell lines by stable integration of a reporter gene under the control of MAdCAM-1 regulatory elements.

[0072] According to another method, the compound to be tested is administered to gut-humanized avatar mice and the expression of MAdCAM-1 in endothelial cells of the mice is measured.

[0073] The two methods can also be combined.

[0074] In such combined screening methods, the in vitro results are completed by a step of validation in avatar mice, where the avatar mice are gut-humanized mice pretreated with broad-spectrum ATB for 8-10 days and then recolonized with the human FMT product by oral gavage, where (i) the FMT product has been identified as one that induces expression of MAdCAM-1 in cells that express MAdCAM-1 or has been engineered to express a reporter gene under the control of the MAdCAM-1 promoter; or (ii) the FMT product is identified as not inducing expression of MAdCAM-1 in such cells and is enriched with another agent identified as inducing expression of MAdCAM-1 in these cells; or (iii) FMT has a taxonomic composition associated with eubiotic or highly soluble MADCAM-1 (in our database or as a result of any previous experiments).

[0075] The method according to the invention also comprises the steps of: (i) Up to 8 days after gavage, a portion of the intestinal humanized avatar mice will be sacrificed to perform MAdCAM-1 and Foxp3-based ileal PCR (and, optionally, IHC staining) in FMT recipients and compare them to controls (continuous ATB); (ii) in the case of significant upregulation of MAdCAM-1 in gut-humanized avatar mice, inoculate at least three mice in each group with subcutaneous sarcoma and treat them with anti-PD1 Ab to ensure that this FMT could mediate therapeutic efficacy compared to ATB-treated mice; (iii) predicting that if the gut-humanized avatar mice respond to treatment with anti-PD1 Ab, the FMT product or non-FMT agent used to enrich it can normalize MAdCAM-1 expression levels in lamina propria endothelial cells; may include.

[0076] A screening platform for carrying out the above method is also part of the present invention. Such a platform includes cells expressing a reporter gene under the control of the MAdCAM-1 promoter (e.g., HUVEC, TSEC, or bEnd.3 cells, or cells derived therefrom), and a robot configured to pick drugs in the product bank, deliver them to the cultured cell line, and evaluate the expression of the reporter gene (e.g., by measuring fluorescence if the reporter gene expresses a fluorescent protein).

[0077] As mentioned above, low levels of soluble MAdCAM in the serum of cancer patients are a marker of intestinal dysbiosis in these patients and are likely to induce resistance to IO therapy, therefore these patients require a compensatory preparation prior to IO therapy and advantageously comprise the following steps: (i) administering an MCI selected from those described above; (ii) administering an IO therapy selected from those described above; The present invention can advantageously benefit from treatment including

[0078] Measuring the levels of soluble MAdCAM in the serum of a cancer patient before initiating or continuing IO therapy (e.g., by the methods described above) can help determine whether the patient requires combination treatment (MCI+IO).

[0079] Patients for whom this measurement is particularly useful are those who have taken broad-spectrum antibiotics during the period ranging from 60 days before to 42 days after the first dose of an ICI, especially an anti-PD1 / PDL-1 Ab, as these patients are more likely to have severe intestinal dysbiosis.

[0080] Advantageously, a new measurement of serum levels of soluble MAdCAM is performed prior to step (ii) above to check whether the MCI has reduced cancer-associated dybiosis.

[0081] When implementing the above combination treatment (MCI+IO), individuals who received broad-spectrum antibiotics less than 60 days prior may advantageously receive a fecal microbial transplant that may be enriched with pasteurized Akkermansia spp. and / or Akkermansia muciniphila, and individuals who did not receive broad-spectrum antibiotics less than 60 days prior may advantageously receive live Akkermansia spp. and / or Akkermansia muciniphila.

[0082] Other features of the invention will also become apparent in the course of the following description of biological assays which are carried out within the framework of the invention and which provide the necessary experimental support without limiting its scope. EXAMPLES

[0083] Example 1: Antibiotics disrupt the ileal MAdCAM-1 / α4β7 axis and impair tumor immune surveillance during PD-1 blockade

[0084] Materials and Methods Medical Center and Regulatory Approval for Translational Research.

[0085] Fecal material. For fecal collection, a substudy was performed at the Institut Gustave Roussy / France, following ethical guidelines and approval of the local CCPPRB. The study name was "Oncobiotics", B2M ethical protocol number PP:15-013. Written informed consent was obtained from all patients according to the Declaration of Helsinki. For collection of endoscopy and blood samples, a clinical study "Einfluss von Antibiotika auf das Darm-Chemokinnetzwerk bei Patienten mit soliden Tumoren" was performed at the University Clinics Heidelberg / Germany, following ethical guidelines and approval of the Regierungsprasisium Karlsruhe. Eligible patients had stage IIIA-stage IV non-small cell lung cancer (NSCLC) with squamous or non-squamous histology and documented recurrence or progression after at least one previous line of treatment. The participating site was the Gustave Roussy Cancer Campus (Villejuif, France). Patients with NSCLC received the anti-PD-1 mAb, nivolumab, as part of a treatment modality approved by the European Medicines Agency (EMA). Tumor response was assessed using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST1.1). Computer tomography (CT) scans were performed at baseline and every 8-12 weeks for the first year, then every 12-15 weeks until disease progression. Feces were collected according to the International Human Microbiome Standards (IHMS) guidelines (SOP 03 V1) before the first injection (T0). Briefly, patients were given a collection kit containing an anaerobic generator (Biomerieux).Samples were collected by patients at home and frozen after 4-24 hours at -80°C in plastic tubes (1000-plastic containers by Sarstedt) with or without BHI + 2% glycerol at the Gustave Roussy Cancer Campus.

[0086] Collection of endoscopic and blood samples. Eligible patients underwent ileo-colonoscopy according to clinical standard protocols for non-study related indications between July 2018 and November 2019 (Table 1). When feasible, endoscopic biopsies of the mucosa of the terminal ileum, cecum, and left and right colon were performed in each patient. Tissue samples were either snap frozen in liquid nitrogen and stored at -80 °C or immersed in 2% PFA for histology. In addition, two blood samples (10 mL EDTA tubes) were collected before ileo-colonoscopy. All included patients responded to a questionnaire to assess dietary history, and clinical baseline data were retrieved from the local clinical information system.

[0087] Cell culture, reagents, and tumor cell lines. MCA-205 fibrosarcoma cells, MC38, and RET melanoma cells (i.e., melanomas generated by transgene-enhanced expression of the Ret proto-oncogene under the control of the metallothionein-1 promoter driving spontaneous melanoma formation, kindly provided by Professor Viktor Umansky) (all syngeneic for C57BL / 6 mice) were cultured at 37° C. in the presence of 5% CO2 in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 Ul / mL penicillin / streptomycin, 1 mM sodium pyruvate, and MEM non-essential amino acids (hereafter referred to as complete RPMI 1640). Luciferase-transfected TC-1 cell line (syngeneic of C57bl6 mice, kindly provided by Professor Eric Deutsch (Institut Gustave Roussy, France)) was cultured at 37° C. in the presence of 5% CO2 in complete RPMI 1640 and 1 mM Hepes buffer. Cell lines were routinely tested for mycoplasma contamination and were not used after more than 10 passages.

[0088] Mice. All animal experiments were performed in accordance with French and European laws and regulations. The local institutional animal ethics board and the French Ministere de la Recherche approved all mouse experiments (permit numbers: 2016-049-4646, 2017_049_99741, 2019_036_21124). Experiments were performed in accordance with governmental and institutional guidelines and regulations. Female C57BL / 6 were purchased from Harlan (France). Mice were used between 7 and 12 weeks of age. MAdCAM-1-KO and ITGB 7-KO mice were kindly provided by Angela Schippers, University hospital Aachen, Aachen, Germany. MAdCAM-1-KO and ITGB7-KO mice, as well as control littermates, were backcrossed on a C57BL / 6 background and obtained from the in-house breeding of the Regional Animal Care Facility of the University Hospital Aachen. CCR5-KO mice were kindly provided by Christophe Combadiere, Hôpital Salpetriere, Paris, France. CCR9-KO mice were kindly provided by Forster, University hospital Hannover, Hannover, Germany. Both CCR5-KO and CCR9-KO mice were maintained on a C57BL / 6 background. All mouse experiments were performed in the animal facilities of the Gustave Roussy Cancer Campus, where animals were housed under specific pathogen-free conditions.

[0089] Antibiotic treatment. Unless otherwise indicated, mice were treated with broad-spectrum antibiotic solution (ATB) (Sigma-Aldrich) containing ampicillin (1 mg / mL), streptomycin (5 mg / mL), and colistin (1 mg / mL) added to their sterile drinking water. In experiments with single antibiotics, the concentrations were ampicillin (1 mg / mL), streptomycin (5 mg / mL), colistin (1 mg / mL), erythromycin (1 mg / mL), or ciprofloxacin (0.1 mg / mL), respectively. Solutions and bottles were changed twice a week. In experiments with antibiotic mixtures, antibiotic activity was confirmed by weekly incubation of fecal pellets resuspended in 0.1 g / mL BHI + 15% glycerol on COS (Columbia agar with 5% sheep blood) plates for 48 h at 37°C in aerobic and anaerobic conditions. The duration of ATB treatment varied slightly based on the experimental setup (7-14 days prior to αPD-1 treatment) and is indicated in the results of each experiment. Briefly, to impair the efficacy of anti-PD-1 mAb, mice were treated with ATB 1-2 weeks prior to tumor implantation and continued ATB throughout the experiment or discontinued on the day anti-PD-1 treatment started as indicated in individual experiments. In the context of fecal microbial transplantation experiments, mice received 3 days of ATB before receiving fecal microbial transplantation the following day by oral gavage using an animal feed needle. In the TC-1 model, ATB treatment was started 3 days prior to tumor injection and discontinued 1 day prior to the first bacterial gavage.

[0090] Subcutaneous models of MCA-205 sarcoma, MC38, and RET melanoma. Syngeneic C57BL / 6 mice were injected with 0.8 × 10 6 MCA-205 sarcoma, 1.0 × 10 6 0.5×106 MC38 or 0.5×106 RET melanoma cells were subcutaneously implanted, and the tumors were 20–40 mm 2When the tumors reached a size of 100 μg / mouse, they were treated intraperitoneally (ip) with anti-PD-1 mAb (250 μg / mouse; clone RMP1-14), or isotype control (clone 2A3). Mice were injected with anti-PD-1 mAb 4 times at 3-day intervals. Tumor length and width were routinely monitored 3 times a week by caliper. In experiments with anti-a4b7 mAb (DATK32, 200 μg per mouse) or anti-MAdCAM mAb (MECA-367, 200 μg per mouse), the mAbs (or their isotype controls, clone 2A3 in both cases) were injected intraperitoneally every 3 days starting from day 0 until the final anti-PD-1 injection. All antibodies were purchased from BioXcell, NH, USA.

[0091] Orthotopic luciferase engineering TC-1. C57BL / 6 mice were anesthetized with isoflurane. Under sterile conditions, a lateral incision was made in the chest wall of each mouse and 6 × 10 5 TC-1-Luc cells were injected into the lungs. The skin incision was closed with surgical skin clips. Tumor growth was monitored twice weekly with an I VIS Imaging System 50 series (Caliper Life Sciences / Xenogen). Starting on day 3, mice were injected with anti-PD-1 mAb or the recommended isotype according to the same dose and schedule as above.

[0092] FMT experiments. Fecal microbiota transplantation (FMT) was performed by thawing fecal material. Mice were placed in new cages. 200 μL of the suspension was then transferred by oral gavage into each germ-free or ATB-pretreated (3 days) recipient. In addition, an additional 100 μL was applied to the fur of each animal. Two weeks after FMT, tumor cells were injected subcutaneously or orthotopically, and mice were treated with anti-PD-1 or isotype control as described above. Oral bacterial gavage was performed on the same day as αPD-1 treatment, as described below.

[0093] Oral bacterial gavage with commensal species. Akkermansia muciniphila CSUR P2261 and A. indistictus CSUR P723 were provided by the Institut hospitalo-universitaire Mediterranee Infection, Marseille, France. Enterococcus hirae 13144 isolates were originally isolated from the spleen or mesenteric lymph nodes of SPF mice treated with CTX at the Gustave Roussy Cancer Campus. A. muciniphila was grown for at least 72 h at 37 °C on COS plates in an anaerobic atmosphere created with three anaerobic generators (Biomerieux). E. hirae 13144 was grown for 24 h at 37 °C in aerobic conditions on 5% sheep blood enriched Columbia agar. Colonization of ATB-pretreated or GF C57BL / 6 mice was performed using 1 × 10 8 Oral gavage was performed using 100 μL of a suspension containing 10 bacteria. For bacterial gavage: Using a fluorescence spectrophotometer (Eppendorf), the optical density at 600 nm in PBS was measured at 10 9A suspension of CFU / mL was obtained. For each mouse, bacterial gavage was performed 24 hours before the first injection of anti-PD-1 mAb, followed by four times on the same day as anti-PD-1 mAb injection. Efficacy of colonization by E. hirae 13144 was confirmed by culturing feces 48 hours after gavage. Fecal pellets were collected and resuspended in 0.1 g / mL BHI + 15% glycerol. Serial dilutions of feces were plated on 5% sheep blood enriched Columbia agar and incubated at 37°C for 48 hours in aerobic and anaerobic conditions. After 48 hours, single colonies were isolated and Gram stained. Identification of specific bacteria was achieved using a Matrix-Assisted Laser Desorption / Ionisation Time of Flight (MALDI-TOF) mass spectrometer (Andromas, Beckman Coulter, France).

[0094] Flow cytometry analysis. Tumors, mesenteric lymph nodes (mLN), draining lymph nodes (tdLN), and spleens were harvested at different time points as indicated in each experiment. Resected tumors were cut into small pieces and digested in RPMI medium containing 25 μg / mL Liberase™ (Roche) and 150 UI / mL DNase1 (Roche) for 30 min at 37°C, then crushed and filtered twice using 100 μm and 70 μm cell strainers (Becton & Dickinson). Lymph nodes and spleens were crushed in RPMI medium, followed by filtering twice using 100 μm cell strainers. Four million tumor cells, lymph node cells, or splenocytes were preincubated with purified anti-mouse CD16 / CD32 (clone 93; eBioscience) for 30 min at 4°C, followed by membrane staining. For intracellular staining, a Foxp3 staining kit (eBioscience) was used. Dead cells were excluded using the Live / Dead Fixable Yellow dead cell stain kit (Life Technologies) or the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit. CD3 (145-2C11), CD4 (GK1.5 or RM4-5), CD8 (53-6.7), CD25 (3C7), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD127 (A7R34), Foxp3 (150D), ROR γCells were stained with anti-mouse antibodies against t(B2D), CXCR3(FAB1685P), CXCR5(J252D4), CCR2(SA203G11), CCR4(2G12), CCR5(HM-CCR5), CCR6(29-2L17), CCR7(4B12), CCR9(CW-1.2), beta7(FIB504), a4b7 / LPAM-1(DATK32 and REA457), MadCAM-1(MECA367) (all purchased from Miltenyi, BioLegend, and eBioscience). Stained samples were acquired on a CytoFLEX S 13-color (Beckman Coulter) or BD Facs CANTO II (BD), and analysis was performed with Kaluza software 1.5 (Beckmann Coulter). T central memory (TCM) gating: after gating on viable CD3+, CD4+ were selected, and TCM were then identified as either CD62L+. Effector memory T (TEM) cells were selected as CD62L and CD44+. Th17 gating was dependent on the mouse model used. Kaede fluorochrome does not retain its photoconversion state upon fixation with PFA. Th17 gating: after gating on viable CD3+, CD4+ were selected, and Th17 were then identified as either CD62L+ or CD44+. γ For fixed cells, Th17 were identified as CXCR3- and CCR6+. Treg gating: after gating on viable CD3+, CD4+ were selected and for fixed cells, Treg were identified as FoxP3+ CD25+. For non-fixed cells, Treg were identified as CD127-CD25+. Fluorescence minus one (FMO) controls were used as appropriate to define the cut-off values.

[0095] Immunohistochemistry. Formalin-fixed, paraffin-embedded mouse ileum and colon sections, 3 μm thick, were prepared as "swiss rolls" and mounted on poly-L-lysine-coated slides, deparaffinized, and hydrated through a graded alcohol to water sequence. Antigen retrieval was performed by heating the sections at 98° C. for 30 min in buffer (0.01 M sodium citrate buffer, pH 6.0 for staining of CD3 and 1 mM EDTA, pH 8.0 for staining of FoxP3, respectively). Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase (DAKO) for 10 min, followed by saturation with PowerVision IHC / ISH Super Blocking Solution (Leica Biosystems, #PV6122) for 20 min. Without washing, primary antibodies Rabbit anti-human CD3 polyclonal Ab (off-the-shelf, DAKO, #IS503) and Rabbit anti-mouse Foxp3 polyclonal Ab (2 μg / mL, Invitrogen, #PA1-46126) were applied and incubated for 1 h, followed by secondary Ab PowerVision poly-horseradish peroxidase-conjugated anti-rabbit Ab (Leica Biosystems #PV6119). Peroxidase was detected by Di Amino Benzidine (DAB)-peroxidase substrate kit (DAKO) and sections were counterstained with Mayer's hematoxylin. Immunohistochemical staining of CD4 was performed with an automated immunostainer (BenchMark ULTRA, Ventana, IGR). Heat-induced antigen retrieval was performed in EDTA buffer (pH 8.0) at 95°C for 32 min. Primary monoclonal anti-mouse CD4 antibody (1,246 μg / mL, #ab183685, Abcam) was diluted 1:500 in antibody diluent (Zytomed) and slides were incubated for 1 h at 37° C. Detection technology was applied using a biotin-free peroxidase system with DAB as chromogen (Kit ultraView Universal DAB Detection kit, Ventana).Slides were also counterstained with a hematoxylin kit (Ventana).

[0096] Kaede experiments. Kaede mice were kindly provided by Michio Tomura, Kyoto University, Kyoto, Japan, backcrossed, and maintained on a C57BL / 6 background. Kaede transgenic mice were anesthetized with 2–2.5% isoflurane and administered buprenorphine (0.01 mg / kg) intraperitoneally for analgesia. For photoconversion of the ileum, the abdominal skin and peritoneum were cut at the midline to access the intraperitoneal terminal ileum. For photoconversion of the ileum including the colon, ileum, or mesenteric lymph nodes, the cecal pole was identified, and the cecal pole including the terminal ileum, mesenteric lymph nodes, and proximal colon was gently mobilized through a mid-abdominal incision onto a sterile plastic-coated surgical drape. Nontarget structures were covered with aluminum foil. The ventral and dorsal parts of the target structures were exposed to violet light emitted from a 395 nm wavelength light-emitting diode (Winzwon) light for 30 s each. After irradiation, the tissue was moistened with sterile isotonic sodium chloride and gently replaced into the peritoneal cavity. The peritoneum was closed with a continuous stitch of 5-0 monofilament nylon suture (Ethicon). The skin was closed with two 9 mm wound clips (EZ Clip Kit).

[0097] Tracking leukocyte migration from mesenteric and tumor-draining lymph nodes with CFSE. C57Bl6 mice were anesthetized with 2-2.5% isoflurane and administered buprenorphine (0.01 mg / kg) intraperitoneally for analgesia. The abdominal skin and peritoneum were cut in the midline to access the mesenteric lymph nodes. The mesenteric lymph nodes were gently mobilized onto a sterile plastic-coated surgical drape through a mid-abdominal incision. The ileal-draining mesenteric lymph nodes were visually identified according to their vasculature. The two most prominent mesenteric lymph nodes were injected with 100 μM CFSE diluted in 5 μL of PBS using a 30 G insulin syringe. After repositioning of the mesenteric lymph nodes, the peritoneum was closed by a continuous stitch with 5-0 monofilament nylon suture (Ethicon). For the tumor-draining lymph nodes, the abdominal skin was incised in the midline, leaving the peritoneum. Tumor-draining lymph nodes were visualized by gently separating the abdominal skin and peritoneum with scissors. Tumor-draining lymph nodes were injected with 100 μM CFSE diluted in 5 μL of PBS using a 30G insulin syringe. The skin was closed with two 9 mm wound clips (EZ Clip Kit).

[0098] RNA extraction and rtPCR. Lysis and extraction protocols were identical for human and mouse samples. Tumor or intestinal samples were flash frozen in liquid nitrogen in RLT Plus buffer containing 0.1% beta-mercaptoethanol. On the day of extraction, samples were thawed at 4°C and homogenized in RNA-free glass bead tubes (Dutscher) with a microtube homogenizer (Benchmark Scientific). Total RNA extraction and genomic DNA removal were performed using the RNeasy Mini kit (Qiagen) according to the manufacturer's recommendations. Up to 1 μg of RNA, measured using a NanoDrop™ spectrophotometer (Thermo Fischer Scientific), was reverse transcribed into cDNA using a mixture consisting of SuperScript III Reverse Transcriptase (Life Technologies), RNaseOUT™ recombinant ribonuclease inhibitor (Life Technologies), Random primers (Promega), and Deoxynucleoside Triphosphate Set, PCR grade (Roche Diagnostics).

[0099] Single-cell RNA sequencing by Rhapsody. After isolating CFSE+CD4+T cells by flow cytometry, cells were washed at 10.000 cells in cold PBS, loaded onto a BD Rhapsody™ cartridge, and processed according to the manufacturer's instructions for targeted single-cell RNA-seq using a pre-designed immune response panel (mouse). Libraries were clustered at 1.75 pM on a NextSeq500 system (Illumina) to generate approximately 40.000 paired-end (2 × 75 bp) reads per cell using High Output v2 chemistry. Sequencing single-cell data was demultiplexed using bcl2fastq2 v2.20.

[0100] Quantitative Gene Expression Assays. Expression of B2M, FoxP3, IFNγ, IL-10, IL-17, MadCAM, Ppia, RORc, TNFα (all from Life Technologies) was analyzed by TaqMan® Gene Expression Assay using Universal Master Mix II on a StepOnePlus™ Real-Time PCR System (Life Technologies). Amplification was performed using the following ramping profile: 1 cycle at 95°C for 10 min, followed by 45 cycles at 95°C for 30 s and 60°C for 1 min. Quantitative RT-PCR data was analyzed using the 2-ΔCt method over a 10 min period. 6 The results were normalized to the expression levels of the housekeeping genes β2M or Ppia, as indicated in each figure, by multiplying by x.

[0101] Tissue lysis and chemokine analysis. Intestinal and tumor samples were flash frozen in liquid nitrogen in non-denaturing cell lysis buffer containing 50 mM Tris HCl pH 7.4, 150 mM NaCL, 300 mM sucrose, 10 mM EDTA, and 0.1% Triton 100X. For subsequent lysis, samples were thawed at 4°C and lysed on a tube homogenizer (Precellys) in ceramic bead lysis tubes (Precellys). Tissue homogenates were centrifuged at 4000 g for 5 min. The supernatant was used for subsequent analysis. Chemokine concentrations in tissue lysates were determined using CCL2, CCL3, CCL4, CCL5, CCL25, and MadCAM Duoset ELISA kits (RnD) or using the Legendplex Mouse Proinflammatory Chemokine Panel (Biolegend) with cytometric analysis performed on a CytoFLEX S (Beckmann coulter) according to the manufacturer's recommendations.

[0102] Statistics. Mice. Data analysis was performed in either the statistical environment R (http: / / www.R-project.org / ) or Prism 6 (GraphPad, San Diego, CA, USA). Differences in tumor size were calculated using non-parametric t-tests. All reported tests were two-sided and considered significant at p<0.05. Patients. MADCAM-1 as a predictor of response to PD1 blockade in NSCLC patients. Restricted cubic splines (RCS) with 4 knots were used to study the prognostic role of sMadCAM1 levels. The number of knots was selected according to Akaike's information criterion. We tested for nonlinearity of biomarker effects. If the linearity hypothesis was not rejected, linear coding was used to estimate the biomarker effects. result 1.1. ATB downregulates ileal expression of MAdCAM-1 in mice and patients

[0103] We reported that intestinal sterilization of mice with a broad-spectrum ATB cocktail (ampicillin, colistin, and streptomycin) blunted the anticancer efficacy of PD-1 blockade (Routy et al., 2018a). We first analyzed ATB-induced changes in the expression of chemokines and integrin ligands in the intestinal tract of MCA205 tumor-bearing mice treated with anti-PD-1 mAb. ATB induced a significant loss of expression of most ileal chemokines and Madcam1 gene products (Figure 5A, left panel and right panel, Figure 1A left panel), while failing to affect colon and tumor chemokine and Madcam1 gene patterns (Figure 1A right panel, Figure 5A right panel). The reduction in ileal Madcam1 gene expression was seen not only at the transcriptional but also protein level, as measured by immunohistochemistry of ileal tissue (Fig. 1B), flow cytometry of ileal CD45'LP cells (Fig. 1C), and immunoenzymatic assay of ileal lysates (Fig. 1D). +Integrity of the intestinal architecture and vasculature was preserved under administration of ATB, as shown by the stabilization of blood vessel numbers (Figure 5B). The decrease in Madcam1 gene expression began by day 3 of ATB administration and did not recover by day 12 after ATB cessation during spontaneous recolonization (RECO) when ATB was fed for 7–14 days (Figure 1D). Apart from broad-spectrum ATB, other ATB regimens downregulated Madcam1 gene expression, such as streptomycin, not only in ileal LP but also in mesenteric lymph nodes (mLN), where Madcam1 gene expression was higher than in LP (Figure 5C, left and right panels) as well as Peyer's patches (PPs) (Figure 5D). In contrast to ampicillin or erythromycin, vancomycin failed to downregulate mLN Madcam1 expression and even increased Madcam1 transcription in ileal LP (Figure 5C, right and left panels, respectively). Of note, Madcam1 (but not Vcam1) gene expression levels were 10-fold lower in tumor-draining LNs than in mLNs (Figure 5E). Interested in the potent inhibitory effect of ATB on GALT Madcam1, we performed aerobic and anaerobic cultures of ileal contents from animals treated with different ATB regimens. Mass spectrometry identification of ileal bacterial colonies revealed several species (bacteria) belonging to the Enterocloster new genus clade of bacteria (e.g., Enterocloster clostrigioformis novel combination, Enterocloster volteae novel combination (Haas and Blanchard, 2020)) that became predominant 4 days after ATB cessation but not in other experimental conditions (Figure 5F). These Enterocloster species were previously identified in the stool of kidney and lung cancer patients resistant to PD1 blockade (Derosa et al., 2020), (Derosa et al. 2022). Vancomycin (which kills Enterocloster species) upregulated ileal Madcam1 gene transcripts (Fig. 5C, left panel), whereas oral supplementation with Enterocloster clostridiaformis abolished this effect and further affected addressin expression (Fig. 1E).Similarly, oral gavage with Enterocloster clostridiaformis after ATB cessation during the spontaneous recolonization phase exacerbated the loss of Madcam1 expression (Figure 1F). In contrast, oral gavage with immunogenic commensals such as Akkermansia muciniphila (Routy et al., 2018a), (Derosa et al., 2022) or Enterococcus hirae (Daillere et al., 2016; Goubet et al., 2021) was able to further increase basal Madcam1 expression in ileal tissues of eubiotic mice kept under specific pathogen-free (SPF) conditions (Figure 1G).

[0104] Fecal microbiota transplantation (FMT) of stool from melanoma patients who benefited from PD1 blockade was able to circumvent primary resistance to ICIs in one-third of metastatic melanoma recipients (Baruch et al., 2021; Davar et al., 2021). Indeed, different donor FMT failed to transfer clinical benefit to the recipients (Table 1). Therefore, we tested whether random FMT from lung or kidney cancer patients into avatar mice treated with ATB (Routy et al., 2018a, 2018b) (as reported above) could downregulate ileal Madcam1 gene expression in recipient mice kept in SPF conditions. This was the case for three of the six FMTs (Figure 1H, left panel), corresponding to a unique taxonomic composition, defined by the over-representation of Enterocloster species, including E. clostridioformis (or the ATB-associated Hungatella hathewayi (Derosa et al., 2020) and sharing the same clade as Enterocloster species (Haas and Blanchard, 2020) in a shotgun metagenomics-based analysis (Figure 1H, right panel).

[0105] Oral supplementation of FMT recipients with various commensals, including the proinflammatory Atopobium parvulum and L. reuteri but not H. hashewayi, could further affect Madcam1 ileal gene expression (Figure 5G).

[0106] The ATB-induced Madcam1 gene downregulation in the ileum was parallel and correlated with the downregulation of regulatory cytokines and transcription factors (Il17a, Il22, Foxp3, RORc, etc.) (Fig. 5A, left panel; Fig. 5H-I). These PCR results showed that ATB upregulated the mucosal CD25 + FoxP3 + CD4 + T cells (Treg) and RORγt + CD4 + This was supported by flow cytometry analysis showing that ATB induced a significant loss of the T cell (TH17) population (Figure 1I). Indeed, ATB phenocopied the ileal immunomodulatory effects of Madcam1 gene deletion or antibody neutralization of MADCAM1 in the ileal LP (Figure 1J).

[0107] As shown in mice, we confirmed the coordinated inhibitory effect of ATB on the intestinal expression of Madcam1, Foxp3, and Rorc in 16 patients who received ATB and underwent intestinal endoscopy and biopsy for various indications (Figure 1K, Figure 5J, Table 2). [Table 2]

[0108] In summary, broad-spectrum ATB regimens induced sustained downregulation of the ileal mucosal addressin MADCAM1 in mice and humans, which significantly correlated with reduced ileal Foxp3, Il17a, and RORc expression.

[0109] 1.2. Gut-acting α4β7 from the gut to tumor-draining lymph nodes + CD4 +ATB-induced exodus of T cells We hypothesized that the loss of MADCAM-1 molecules may affect the homing or retention of intestinal active T cells expressing its α4β7 receptor in the GALT. To track ileal T cell migration in tumor-bearing hosts, we utilized two complementary experimental strategies. First, we used the transgenic Kaede mouse model, which allows cells to be irreversibly labeled without destroying their integrity. This model utilizes reporter mice that ubiquitously express the coral-derived photoconvertible fluorescent protein Kaede (Tomura et al., 2008), which can be photoconverted from green to red after exposure to ultraviolet light with wavelengths of 350-400 nm. Kaede mice track the homing or retention of migratory intestinal T cells to inflamed peripheral organs. HPhotoconversion is a powerful tool for tracking GALT cells (Krebs et al., 2016; Magnuson et al., 2015; Morton et al., 2014). To track the fate of GALT cells in MCA205-bearing mice, the ileum, cecum, and mesenteric LNs were photoconverted on day 10 (D10) after subcutaneous tumor implantation, and mice were sacrificed 24 hours later for detection of photoconverted cells in various organs by flow cytometry (Figure 2A, Figure 6A). We achieved a photoconversion rate of approximately 60% in the mesenteric LNs 5 minutes after irradiation (Figure 6A). At 24 h, only 22.8 ± 2.6% of gut photoconverted (PC) leukocytes remained in the mLN, but a significant proportion of GALT-emitted PC cells could be detected at distant sites in the spleen (5.1 ± 0.5%) and tumor-draining lymph nodes (tdLNs) (4.0 ± 0.3%) (Figure 2A, Figure 6A-6B). Using a second approach, we directly tracked carboxyfluorescein succinimidyl ester (CFSE)-labeled cells after direct CFSE labeling of the mLNs by surgical procedures (Singh et al., 2016) (Figure 2B, Figure 6C, left panels). Within 24 hours after CFSE injection, up to 1.0 ± 0.2% of splenocytes, 0.8 ± 0.1% of tdLN cells, and 0.2 ± 0.02% of tumor-resident leukocytes were replaced by mLN-derived leukocytes (Figure 6C, right panel). Ileum irradiation was less efficient than GALT-targeted irradiation in recruiting exodus of intestinal leukocytes to tdLN or sc tumors (Figure 6D). However, both local and GALT irradiation significantly increased the number of photoconverted (PC) (gut-derived)-CD4 cells in the spleen, tdLN, and sc MCA205 at 24 hours. + α4β7 in T cells + The tumor microenvironment (TME) induced selective recruitment of the α4β7 fraction, whereas other PC cell subsets were not enriched compared to tissue-resident (non-PC) cells (Figures 2A-B and 6E). Again, the α4β7 + CD4 + The majority of T cells express CFSE after CFSE mLN labeling. + (Fig. 2B, Fig. 6F). CD4+ Confirming the molecular involvement of α4β7 integrin in the extraintestinal exodus of T cells, we showed that photoconversion or CFSE labeling of cells in the tdLNs (but not the contralateral LNs) was significantly increased in tumor-bearing mice treated with neutralizing anti-MAdCAM-1 Ab (Figure 2C, left, middle, and right panels).

[0110] α4β7 egressing from mesenteric LN + CD4 + To decipher the phenotype of T cells, we used purified α4β7 - CD4 + Compared with T cells, α4β7 high CD4 + Bulk RNA sequencing of T cells was performed. high CD4 + The transcriptional profiles of T cells revealed not only their intestinal signature, Itga4 (encoding α4β7 integrin), but also the expression of gene products associated with Treg function, such as Icos, Ctla4, Cd74, Mki67, P2rx7 (Daniel et al., 2010), and T H We revealed a significant increase in T cell-specific IL-17 (Il17re, Tnfsf11, Il22) polarizing genes, whereas the Tnfrsf9 T cell costimulatory gene product (4-1BB) was significantly downregulated in this subset (Figure 2D).

[0111] Next, we investigated how ATB-induced dysbiosis modulates gut-acting α4β7 expression using both imaging approaches combined with intracellular flow cytometry. + CD4 + We investigated whether α4β7 affects T cell exodus to tumor-draining LNs (Figures 2E-F). + Foxp3 + CD4 + Promoted the homing of IL-17A secreting T cells (gut-derived Tr17) but not α4β7 - Foxp3 + CD4 +Homing of T cells (tdLN-resident Tr17) was also regulated by IL-17A. + α4β7 + Foxp3CD4 + ATB also did not promote homing of T cells to tdLNs (Fig. 2E, Fig. 6G). ATB phenocopied madcam1 gene deletion (Fig. 2F). In fact, ATB bearly increased the bona fide Treg pool in tdLNs, which is largely composed of locally expanded (extraintestinal, CFSE-negative) cells (Fig. 6H, left panel). + CFSE + The intestinal extravasated fraction represented 0.2±0.1% of total CD4+ in the arriving tdLN per 24 h during recolonization after ATB (Fig. 6H, middle panel). Notably, intestinal-acting Tr17 extravasated during recolonization produced not only IL-17 but also IL-22 (Fig. 6H, right panel).

[0112] Neutralizing anti-MADCAM-1 antibodies also inhibited CD25 high α4β7 + CD4 + The ATB process promoted the transmigration of T. enterocolobus (Fig. 2G). Recolonization after the ATB process was accompanied by the appearance of Enterocloster species (Fig. 5F), which we attempted to mimic by oral gavage with E. clostridioformis. Indeed, this bacterium promoted the transmigration of PC (but not non-PC) TH 17 CD25 high (CD25 - (not) α4β7 + Tr17-like CD4 + promoted the egress of T cells from the intestine ( Fig. 2H ).

[0113] Because photoconversion does not allow for accurate intracellular staining and high-dimensional phenotyping, we performed a 4-day ATB course (or no ATB) in which E. clostridioformis was or was not supplemented, and then CFSE reaching the tdLNs 24 h after CFSE injection of the mLNs. + CD4 +We investigated a deeper characterization of intestinal transmigrating cells by performing Rhapsody-based single-cell RNA sequencing of T cells (Figures 7A-B). We collected a total of 451,246 individual CFSEs, each distributed in each of the four groups. + CD4 + tdLN T cells were profiled (Figures 7A-7B) and gene signatures associated with ATB-induced dysbiosis were characterized. + CD4 + Unsupervised clustering of T cells divided the data into four cell clusters (Figure 2H, left panel), which we visualized using stochastic neighborhood embedding and post-hoc labeling with expression of reported marker genes (Cano-Gamez et al., 2020). Each cluster was associated with a distinct phenotype. The small cluster featured a prototypical pan-tissue effector Treg expression pattern defined by Foxp3, Nrp1, Il2ra, Tnfsf4, Ctlα4, and gut-specific gene expression imprinting (Tnfsf18, Rgs1), accompanied by downregulation of Tcf7 and Cd52 (Sefik et al., 2015) (Figure 2H, middle panel). This Treg subset overexpressed the negative immune regulator Pik3ip1, involved in tumor immune suppression (Chen et al., 2019), and upregulated CD4 during the recolonization phase compared to the steady state. +It downregulated Fosb (Figure 7C), a member of the transcription factor AP1 involved in CD95L-mediated apoptosis in T cells (Baumann et al., 2003), and genes related to the Tr17 blueprint during recolonization or oral gavage with E. clostridioformis (Sefik et al. al., 2015) (which contrasted with all other transmigrated cells by overexpression of Iksf2 (also called Helios) and Lrrc32 (also called TGFβ activator GARP) (Figure 7D, left and right panels). In contrast, another cluster with proliferation features (Pcna, Myc, Mapk1, Fyn, Hif1a) was dominated by Irf8, as well as Pou2af1 (also called OBF1, acting as a transcriptional coactivator for the transcription factors OCT1 or OCT2) and Fas, both of which promoted the TH17 program by suppressing the expression of IL-2 or IFNγ, respectively (Yosef et al. al., 2013) (Figure 2H, right panel). This proliferative T17 subset also shared archetypal markers with pan-tissue Tregs (II2rb, Ctla4, Icos), intestinal Tregs (Bcl2a1a, Bcl6, etc.), and skin Tregs (Lgals3). The caspase 1 gene, described as an inflammasome-independent T cell-intrinsic gene essential for optimal priming of pathogen-specific Th17 responses during ATB arrest-induced recolonization and recruitment with E. clostridioformis (Gao et al., 2013). al., 2020) were upregulated in conjunction with TNFRSF and cytokine / chemokine transduction and NFkb activation pathways (Nfkb1, Cxcr3, Cxcr5, Eomes, Tnfrsf8, Tnfrsf9, Tnf, Fasl, Fas, Tigit, Icam1, Runx3, Jak2) (Figure 7E, Table 3). The remaining two dominant cell clusters shared expression of archetypes of II7r and Pik3ip1 genes characteristic of intestinal inflammatory activation and / or suppression (Belarif et al., 2019; Chen et al., 2019).They differ slightly in other fingerprints such as Egr1, Foxo1, Stat6, Ikbkb transcription factors versus Btla, Cnot2 and Dusp2 during recolonization (Figures 7F-G), yet all converge on tumor immunosuppressive properties (Dan Lu et al., 2020; Kim et al., 2019; Li et al., 2012).

[0114] Therefore, we demonstrate that gut-acting α4β7, which exhibits an immunosuppressive fingerprint potentially detrimental to tumor immune surveillance, from the GALT to tumor-draining lymph nodes by two independent tracing methods under conditions in which the MAdCAM-1 / α4β7 axis is compromised by either anti-MAdCAM-1 mAb or ATB-induced recolonization. + CD4 + , most specifically T r 17 Cell egress was demonstrated. [Table 3-1] [Table 3-2] [Table 3-3]

[0115] 1.3. Anticancer efficacy of PD1 blockade depends on MAdCAM-1 / α4β7 axis Given the reported immunosuppressive role of Tr17 cells during cancer immunosurveillance (Blatner et al., 2012; Rizzo et al., 2018; Voigt et al., 2017) and the ability of the healthy intestine to harbor these T cell subsets, we predicted that genetic defects in the MADCAM-1 addressin or β7 integrin, or host neutralization of MADCAM-1 or the α4β7 heterodimer by specific antibodies, might interfere with the immune stimulatory capacity of therapeutic anti-PD-1 mAbs.

[0116] First, loss of ileal Madcam1 expression correlated with increased tumor size in MCA205 tumor-bearing animals, regardless of PD1 blockade (Figure 8A). Second, we found that loss of Itgb7 expression correlated with increased tumor size in MCA205 tumor-bearing animals, regardless of PD1 blockade (Figure 8B). Second, we found that loss of Itgb7 expression correlated with increased tumor size in MCA205 tumor-bearing animals, regardless of PD1 blockade (Figure 8C). - / - Mouse or Madcam1 - / - We observed a marked reduction in the anticancer efficacy of PD-1 blockade in mice (Figure 3A). Similarly, MCA205 fibrosarcoma, orthotopic TC1 lung carcinoma, and syngeneic 4T1 breast carcinoma from BALB / c mice grew in vivo despite PD1 blockade in the presence of neutralizing anti-MAdCAM-1 or anti-α4β7 heterodimer antibodies, whereas they responded to ICIs when the MADCAM1 / α4β7 axis was unaffected (Figure 3B-D). - / - In mice, α4β7 in the spleen (Figure 3E, left panel) and tumors + There was a constitutive increase in recirculating CD4+ T cells, where they represented up to 3% of TILs (Figure 3E, right panel), as previously described (Denning et al., 2005). Flow cytometric analysis demonstrated that blockade of the MAdCAM-1 addressin / α4β7 integrin axis with a neutralizing anti-MADCAM-1 Ab during spontaneous tumor progression remodeled the TME and enhanced ROR. γ t + Treg (Foxp3 + CD25 + )T r In the MCA205 TME without any manipulation, the accumulation of α4β7 cells was found to be approximately three-fold increased (Figure 3F-G). + CD4 + Up to 76±1.5% of TILs were associated with ROR γ t + Treg, α4β7' CD4 + In subcutaneous tumors, Tregs were 2-3 times less abundant (17 ± 0.9%) than TILs (Figure 8B). + CD4 + Representing 12.1±1.0% of TILs, of these Tregs, 30.4±4.5% were ROR γ t+ (Table 4) [Table 4]

[0117] Recolonization 4 days after ATB cessation phenocopies madcam-1 gene deletion and ROR in the tumor bed γ t + ATB induces a 3- to 5-fold increase in the proportion of Treg (Tr17) (Figure 3H). Notably, this Tr17 intratumoral homing was transient and was no longer observed by 12 days after cessation of ATB, except when anti-PD1 Ab was administered (Figures 3H-3I, Figure 8C). Indeed, anti-PD1 Ab promoted the priming and / or expansion of Tr17 in mesenteric LNs of MCA205 tumor-bearing mice (Figure 8D). Anti-PD1 Ab promoted the priming and / or expansion of Rorγt in MCA205 tumor-bearing mice. + Treg or IL-17 + IL-22 + Tr17, defined as Tregs (Fig. 3I, Fig. 8E), and contributed to the maintenance and / or expansion of intratumoral accumulation of 4T1 tumor-bearing cells (Fig. 8F, left and right panels). In this context, intratumoral Tregs contributed to the maintenance and / or expansion of intratumoral accumulation of α4β7, accounting for 18.7±3.4% of the Tregs. + CD4 + Among these Tregs, 56.7 ± 8.3% were ROR. γ t + (Table 4)

[0118] The Tr17 recruitment promoted by PD1 inhibition was further increased when E. clostridioformis was supplemented by oral gavage after ATB withdrawal, whereas oral Lactobacillus reuteri failed to do so under similar conditions (Figure 8G, right and left panels). Not surprisingly, blockade of the MAdCAM-1 / α4β7 axis during anti-PD-1 mAb therapy reduced the expression of the effector CCR5 + CD8 + It dramatically impaired TIL infiltration ( Fig. 3J ).

[0119] Given that IL-17 and IL-22 have direct and indirect proangiogenic and protumorigenic effects (Lim and Savan, 2014; Voigt et al., 2017), neutralization of either one or both cytokines may avoid the deleterious effects of recolonization after ATB during PD1 inhibition. We used tumorigenic doses of wild-type or IL-22Rα1 - / - Mice seeded with deficient mammary 4T1 tumor cells were treated by ip injection of anti-PD1 Ab in combination with an anti-IL-17A neutralizing antibody (Figure 3K, left panel). As expected, IL-22Rα1 - / - IL-17A-deficient mammary glands 4T1 grew slower than their wild-type counterparts. Interestingly, anti-IL-17A antibodies did not inhibit either wild-type or IL-22Rα1 - / - Both deficient and 4T1 were able to avoid the deleterious effects of ATB to a similar extent ( Fig. 3K ), suggesting that tumor cell reactivity to IL-22 is not an essential mechanism underlying the immunosuppressive effects of ATB.

[0120] Therefore, we conclude that PD1 blockade promotes TH17 / Tr17 amplification in the GALT and homing to tumors in the setting of intestinal dysbiosis.

[0121] Re-establishment of the compatibility of the α4β7 / MAdCAM-1 axis by exogenous manipulation may restore resistance to PD-1 blockade in tumor-bearers treated with ATB or suffering from overt intestinal dysbiosis. Restoring intestinal eubiosis before initiating PD1 blockade may be a reasonable option. Short-term ATB, followed by fecal microbial transplantation from patients who relapsed despite αPD-1 Ab, caused ileal Madcam1 downregulation (Figure 1G) and inefficiency of PD-1 blockade unless the host was compensated with oral Akkermansia muciniphila ((Routy et al., 2017), Figure 1F, Figure 9A). The compensatory action of exogenous A. muciniphila depended on MAdCAM-1 function, as neutralizing antibodies prevented the beneficial effect of this commensal drug (Figure 9B). A. muciniphila downregulates α4β7 in a MADCAM-1-dependent manner.+ and CCR9 + Single or double positive CD4 + We were able to prevent extraintestinal exodus of T cells and their recirculation into the TME recirculating to the tdLN (Figure 9A, right panel), as well as α4β7+CD4+IL-17+IL-22+ and Tr17 (Figures 9C-D).

[0122] Therefore, restoration of intestinal eubiosis or neutralization of IL-17A are potential options to counteract ATB-induced resistance to PD1 blockade. 1.4. Soluble MAdCAM-1 is a predictive biomarker of resistance to PD1 blockade in patients with advanced NSCLC

[0123] Since it is difficult to obtain intestinal tissue biopsies, useful blood assays such as sMAdCAM-1 ELISA have been developed to study the follow-up of patients suffering from IBD and treated with anti-heterodimeric α4β7 antibodies (such as vedolizumab) (Holmer et al., 2020). However, soluble MAdCAM-1 levels have also never been monitored in mice. We found a strong correlation between ileal madcam1 gene expression and serum soluble MAdCAM-1 (Figure 4A). Next, we analyzed the clinical significance of serum soluble MAdCAM-1 at diagnosis in two independent cohorts of advanced non-small cell lung cancer (NSCLC) patients treated with anti-PD1 / L-1 antibodies with or without chemotherapy in France (N=186) and in France and Canada (N=115) (Table 5). First, NSCLC patients had reduced circulating serum sMAdCAM-1 concentrations in both cohorts compared to healthy volunteers, even more so in those who took ATB (Figure 4B, left and right panels). Indeed, patients taking ATB showed sMAdCAM-1 serum levels in the same range as non-ATB individuals in the bottom tertile of sMAdCAM-1 expression (Figure 4B). Patients exhibiting high sMAdCAM-1 serum levels above the median for the entire cohort had prolonged progression-free and overall survival compared to the other half in both cohorts (Figure 4G). There was a linear relationship between increasing absolute sMAdCAM-1 serum concentrations and reduced risk of death, with an 8-10% risk reduction for every +10 U / mL increase in biomarker for each cohort (Figure 10A, left and right panels). ATB-treated patients showed a significantly higher increase in MAdCAM-1 serum levels compared to the other half in both cohorts (Figure 10C). lowThe subgroup presented the worst PFS (Figure 4F). Furthermore, multivariate Cox regression analysis, taking into account ECOG-PS, age, sex, PD-L1 expression, and treatment options in the model, concluded that sMAdCAM-1 was an independent predictor of clinical benefit to PD1 blockade in NSCLC patients (Table 6). We extended the clinical relevance of this biomarker beyond PD1 inhibition as a biomarker predictive of early NSCLC incidence in high-risk heavy smokers (HRHSCV) experiencing cardiovascular events (PREVALUNG study, Table 7). Indeed, sMAdCAM-1 serum levels were significantly lower in 13 HRHSCV individuals found to have developed lung cancer nodules 6-12 months prior to the diagnosis of NSCLC compared with 99 matched controls who did not (Figure 10B). Soluble MAdCAM-1 levels were significantly higher than circulating α4β7 in the entire cohort. + Rorγt + CD4 + correlated with T cells (Fig. 10C).

[0124] Finally, to directly demonstrate the relationship between soluble MAdCAM-1 serum levels and intestinal dysbiosis in advanced NSCLC patients, we performed unsupervised and supervised hierarchical clustering of metagenomic species (MGS) to define the taxonomic composition of the intestinal microbiota according to the median sMAdCAM-1 serum levels using shotgun metagenomics (MG) analysis in n=95 patients providing all required MG and serum information. low There was a reduction in MGS abundance in patients (separated based on the median of all 112 NSCLC patients) (Figure 4C). Moreover, this different alpha diversity was accompanied by a significantly different beta diversity, meaning that the taxonomic gut composition was not similar in both subsets (Figure 5F). Unsupervised MGS analysis indeed revealed two clusters that also separated patients based on MAdCAM-1 median (Fisher's exact test, p<0.05, Figures 10D-E).

[0125] Finally, selecting only the list of MGS found in mice (Figure 1G, right panel) and clinically associated with poor prognosis in a large series of NSCLC patients (Tsay et al., 2021; Zitvogel and Kroemer, 2021) (Derosa et al. 2022), we conclude and confirm that patients with high intestinal E. clostridioformis and V. parvula spread are those who present with low levels of sMAdCAM-1.

[0126] Collectively, these findings indicate that soluble MAdCAM-1 is a surrogate marker of intestinal dysbiosis that predicts resistance to PD1 blockade in cancer patients.

[0127] [Table 5] [Table 6] [Table 7]

[0128] Consideration Our findings suggest that GALT mediates the expression of gut-tropic immunosuppressive T H 22 / T H 17 (of which, up to 20% are Foxp3 + CD25 + T reg This study reveals the crucial role of the ileum in a host attacked by the oncogenic process, which keeps in check the transmigration of enterocytes (the IL-1 and IL-2 T cells). Both the intestine and the TME are rich in cell attraction and homing molecules that compete for circulating lymphocytes expressing their receptors or ligand pairs. With tools that allow fate mapping from the GALT to the tumor bed, we reveal that the fate decision of intestinal-acting T cells between intestinal residence and chemoattraction to tumor deposits is at least in part controlled by MAdCAM-1 addressin / α4β7 integrin molecular interactions.

[0129] First, ATB downregulated MAdCAM-1 expression in LP ileal venules, as well as HEVs in mucosa and mLNs, favoring ileal exodus of α4β7+Th17 / Tr17 cells to extraintestinal lesions. Second, deficiency or neutralization of MAdCAM phenocopied the effects of ATB. Mice lacking MAdCAM-1 or β7 integrin genes, or animals receiving neutralizing Abs targeting these molecules, were unable to respond to ICIs by accumulating gut-derived α4β7+Tr17 / TH17 cells in the TME. Third, manipulations aimed at restoring MAdCAM-1 expression on ileal HEVs (such as A. muciniphila) or blocking IL-17A bioactivity compensated for the inhibitory effects of ATB in tumor immune surveillance. Fourth, oral gavage with Enterocloster species (such as E. clostridioformis) exacerbated the exodus of gut-acting Tr17 to tdLNs. Finally, PD1 blockade tended to exacerbate the expansion and / or priming of α4β7+Tr17 cells in mLNs and home back to tumors, impairing cancer immune surveillance. All these lines of evidence support epidemiological studies pointing to the adverse effects of ATB before ICI administration, as opposed to during or after ICI initiation (Derosa et al. 2022). As a result, we show that serum soluble MADCAM-1 is a biological surrogate for ileal madcam1 gene expression and a reliable predictor of response to PD1 blockade, at least in NSCLC patients. Low sMADCAM-1 serum levels reflect intestinal dysbiosis dominated by pro-TH17 bacteria (e.g., Veilonella parvula) or species from the Enterocloster genus that are associated with physiopathological disorders (Ghosh et al., 2020; Tsay et al., 2021; Zitvogel and Kroemer, 2021) (Derosa et al., 2022).

[0130] This demonstration is consistent with gut-derived T HThis is in line with previous evidence showing that ROR 17 also regulates extraintestinal autoimmunity (Magnuson et al., 2015; Morton et al., 2014; Wu et al., 2010) (Krebs et al., 2016; Lee et al., 2011b) or inflammatory / ischemic pathology (Benakis et al., 2016; Liesz et al., 2009). γ t + Tregs have an exacerbated immunosuppressive phenotype compared to their lineage-related Tregs, with overexpression of gene products (e.g., Ctlα4, Icos, Havcr2 (Sefik Science 2015)) that we also found to be upregulated in the setting of ATB-induced intestinal dysbiosis. Furthermore, we identified gut-specific Treg features in our Tr17 fingerprint, a TH17-related blueprint (Yosef et al., 2013) previously described in landmark papers (Miragaia et al., 2019; Munoz-Rojas and Mathis, 2021), as well as immunosuppressive traits functionally related to tumor immune surveillance (e.g., Dusp2 / PCA1 (Dan Lu et al., 2020), PIK3ip1 (Uche et al., 2018)).

[0131] This example in a cancer carrier shows that extraintestinal α4β7 + T reg and central memory T cells or CCR6 + CD4 +Increased T lymphocyte recirculation has been reported, consistent with previous studies conducted in patients or macaques suffering from inflammatory bowel disease (Calenda et al., 2018; D'Haens et al., 2018; Fischer et al., 2016). Our findings have important clinical implications. So far, vedolizumab was considered a safer alternative to TNFα inhibition in cancer patients suffering from ICI-induced autoimmune colitis due to its gut-restricted mode of action (Sandborn et al., 2016). However, prospective studies are required to assess microbiome composition, gut, or soluble serum MAdCAM-1 expression, as well as CCR9 α4β7 expression, to correlate these novel parameters with clinical outcomes and toxicity profiles in patients treated with ICIs. + T reg , Tr17, and T h 22 Recirculation of cell subsets should be monitored. Finally, the field of fecal microbial transplantation may evolve toward guiding the selection of donor feces based on their ability to normalize MADCAM-1 levels in cancer-bearing recipients.

[0132] These findings support the notion that cancer-bearing patients may exacerbate their cancer-associated ileal injury by taking antibiotics that alter one of the most powerful gut immune checkpoints, MADCAM-1 (Yonekura et al., 2022). We speculate that ATB-induced dysbiosis, most specifically the recolonization process after the ATB process, is a key regulator of the gut immune checkpoint MADCAM-1. Several molecular clues may link gut dysbiosis to changes in GALT MADCAM-1, such as perturbation of bacteria-induced metabolism of bile salts (Campbell et al., 2020; Song et al., 2020) and activation of the sympathetic nervous system (Schiller et al., 2021; Yan et al., 2021), which merit further investigation.

[0133] Example 2: Soluble MAdCAM-1 is a predictive biomarker of resistance to PD1 blockade in several solid cancers 2.1. Soluble MADCAM-1 is a predictive biomarker for resistance to PD1 blockade in patients with kidney cancer In Example 1, we show that serum soluble MAdCAM-1 levels are a marker of resistance or sensitivity to immuno-oncology (IO) therapy in two independent cohorts of advanced non-small cell lung cancer patients (NSCLC), where reduced levels of serum soluble MAdCAM-1 are a marker of resistance to IO therapy.

[0134] Here, we extend this finding to another cancer type: kidney cancer treated with nivolumab in the second line (after failure of tyrosine kinase inhibitors) (Table 8). [Table 8]

[0135] Overall, 212 patients from the NIVOREIN cohort were enrolled to receive anti-PD1 mAb (nivolumab, BMS). Thirty-one were antibiotic users (days -60 to +42 after treatment initiation) and 176 did not receive antibiotics. Monitoring by ELISA of serum soluble MAdCAM-1 at baseline revealed a median (min, max) of 88.8 ng / mL (19.1, 174.7) (and mean ± SD of 88.4 ± 27.7 ng / mL) for these 212 metastatic clear cell renal cell carcinoma (RCC) patients. As shown for NSCLC patients, antibiotic uptake (ATB) tended to decrease concentrations of sMAdCAM-1 (from a median of 88.7 ng / mL in patients who refrained from ATB to 76.3 ng / mL in patients who received ATB). Of note, this is an underestimate, as the majority of these ATB+ patients (23 / 31) received ATB after initiation of anti-PD1 Ab (less clinically significant).

[0136] Overall survival (OS) was analyzed according to the median baseline sMAdCAM-1 levels just before anti-PD1 Ab initiation for the entire cohort of 212 patients. Kaplan-Meier OS curves of RCC patients show that those with sMAdCAM-1 serum levels below 88.8 ng / mL died in 72 / 106 cases, whereas those with sMAdCAM-1 serum concentrations above 88.8 ng / mL had a mortality rate of 36 / 106 (p<10e-4) (Figure 11). This prognostic parameter was analyzed in a multivariate analysis including all clinical factors influencing survival in stage IV RCC, such as age, IM DC score, line of treatment, and metastatic location, as well as hypoalbuminemia. Multivariate analysis revealed that sMAdCAM-1 < 88.8 ng / mL was associated with a higher IMDC score for IMDC severity with a hazard ratio of 2.40 (1.52-3.80, p=0.0002) for sMAdCAM-1 versus HR=2.29 (1.11-4.76, p=0.08) for IMDC severity (Table 9). [Table 9]

[0137] Progression-free survival (PFS) was analyzed according to the median sMAdCAM-1 baseline level immediately before anti-PD1 Ab initiation for the entire cohort of 212 patients. Kaplan-Meier PFS curves for RCC show that patients presenting sMAdCAM-1 serum levels <88.8ng / mL progressed in 99 / 106 cases, whereas in patients with sMAdCAM-1 serum concentrations ≥88.8ng / mL, the progression rate was 86 / 106 (p<10e-4) (Figure 12). This prognostic parameter was analyzed in a multivariate analysis including all clinical factors influencing survival in stage IV RCC, such as age, IMDC score, line of therapy, and metastatic location, as well as hypoalbuminemia. Multivariate analysis revealed that sMAdCAM-1 <88.8ng / mL was as good as IMDC score (Table 10, HR=1.55, p=0.0071). [Table 10]

[0138] Regarding objective response rate (ORR), the NIVOREN study (N=729 RCC patients) recorded an objective response rate of 20.8% (1.2% complete response, 19.6% partial response, 79.2% progressive disease (PD)). Interestingly, the subgroup of 212 RCC patients for whom sMAdCAM-1 serum levels were available was subdivided according to presence / absence of ATB uptake and sMAdCAM-1 (≧88.8 vs. <88.8 ng / mL). First, subgroup analysis of best objective response by sMAdCAM-1 revealed that the rate of PD was 36.7% vs. 62.6% in patients with sMAdCAM-1 ≧88.8 vs. <88.8 ng / mL, respectively (p=0.0003) (Table 10). Secondly, when we considered only patients who did not take ATB, the same subgroup analysis of best response by sMAdCAM-1 revealed that the rate of PD was 38.6% vs. 60.5% in patients with sMAdCAM-1 ≧88.8 vs. <88.8ng / mL, respectively (p=0.0044). Thirdly, when we considered RCC patients who took ATB, this subgroup analysis of best response rate by sMAdCAM-1 revealed that the rate of PD was 36.4% vs. 76.9% in patients with sMAdCAM-1 ≧88.8 vs. <88.8ng / mL, respectively (p=0.0446). In the ATB-treated subgroup, the rate of complete responders was 18.2% vs. 7.7% in patients with sMAdCAM-1 ≧88.8 vs. <88.8ng / mL, respectively (Table 11 below). [Table 11]

[0139] Taken together, these findings demonstrate that high levels of serum soluble MAdCAM-1 are associated with an increased response to PD1 blockade in second-line immunotherapy of RCC, more so in patients who received antibiotics. The threshold of 88 ng / mL is valid for second-line but does not represent first-line patients. For routine use, the median value of the 1L or 2L therapy cohort must be considered.

[0140] 2.2. Soluble MADCAM-1 is a predictive biomarker for resistance to PD1 blockade in bladder cancer patients Finally, we attempted to triple the predictive value of sMAdCAM-1 in a subcohort of a phase 3 study enrolling pretreated metastatic bladder cancer patients to receive a fixed dose of 1500 mg durvalumab iv (anti-PD-L1 Abs) / 4 weeks. In this powered study, only 79 patients were analyzed, with a median overall OS of 5.79 months and a median PFS of 2.79 months. We selected an extremely powered cohort, 30 elite patients (OS>6 months, PFS>5 months, PR+CR only), and rapid diseasers (n=49, OS<6 months and PFS<5 months on first CT scan, and PD). In this cohort, the median MAdCAM-1 (min, max) was 158.8 ng / mL (53.41, 244.36). In Figure 13A, we show that the median sMAdCAM-1 ± SEM dramatically decreased in progressors, and OS was significantly better in the first half of patients with baseline levels of sMAdCAM-1 above 158.8 ng / mL compared to the other half (Figure 13B, p = 0.03). PFS tended to be better in patients with high MAdCAM-1 levels (Figure 13C, ns).

[0141] 2.3. Conclusion Collectively, these findings demonstrate that high levels of serum soluble MAdCAM-1 are associated with increased responses to PD1 / PDL-1 blockade in second-line immunotherapy of RCC, second-line metastatic bladder tumors, and 1L and 2L NSCLC treated with pembrolizumab, independent of ATB uptake.

[0142] Abbreviation: α4β7: alpha 4 beta 7, Ab: antibody, ACS: ampicillin, colistin, streptomycin, ATB: antibiotic, ATRA: all-trans retinoic acid, CCL: chemokine ligand, CCR: chemokine receptor, CD: cluster of differentiation, cDC1: classical dendritic cell type 1, CFSE: carboxyfluorescein succinimidyl ester, cLN: contralateral lymph node, CSF-1: colony-stimulating factor 1, CTL: cytotoxic T lymphocyte, FMT: fecal mucosal transfer, FoxP3: perforated box P3, GALT: Gut-associated lymphoid tissue, HEV: high endothelial venules, IBD: inflammatory bowel disease, ICI: immune checkpoint inhibitors, IFNγ: interferon gamma, LP: lamina propria, mAB: monoclonal antibody, MAdCAM-1: mucosal addressin cell adhesion molecule-1, mLN: mesenteric lymph node, PP: Peyer's patch, RA: retinoic acid, RORC: RAR-related orphan receptor C, SLO: secondary lymphoid organ, tdLN: tumor-draining lymph node, T H 17: T-helper 17-CD4-T cells, TME: tumor microenvironment, TNFα: tumor necrosis factor alpha, T reg :Regulatory T cell, Tr17:ROR γ t+T reg -CD4-T cells. 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Claims

1. Use of serum soluble MAdCAM-1 levels as a marker of resistance or sensitivity to immuno-oncology (I-O) therapy, wherein a reduction in the level of serum soluble MAdCAM-1 is a marker of resistance to said I-O therapy.

2. Use of serum soluble MAdCAM-1 levels as a marker of cancer-related or antibiotic-related dysbiosis, wherein a reduction in the level of serum soluble MAdCAM-1 is a marker of cancer-related or antibiotic-related dysbiosis.

3. A method for in vitro diagnosis of cancer-related or antibiotic-related intestinal dysbiosis in an individual having cancer, comprising measuring serum soluble MAdCAM-1, wherein a reduction in the level of serum soluble MAdCAM-1 indicates that the individual has cancer-related or antibiotic-related intestinal dysbiosis.

4. The use according to claim 1 or claim 2, or the method according to claim 3, wherein the level of said serum soluble MAdCAM-1 is considered reduced if it is lower than the median level of said soluble MAdCAM-1 in a representative cohort.

5. A seranostic method for determining whether an individual having cancer requires a microbiota-centered intervention (MCI) prior to administration of cancer immunotherapy (I-O) therapy, comprising evaluating whether said individual has cancer-related or antibiotic-related dysbiosis using the method according to claim 3, and wherein if said individual has cancer-related or antibiotic-related dysbiosis, said individual requires MCI prior to administration of treatment with I-O therapy.

6. The seranostic method according to claim 5, wherein said treatment with I-O therapy is selected from the group consisting of anti-PD1 antibody (Ab), anti-PDL-1 Ab, anti-CTLA4 Ab, anti-Lag3 Ab, anti-Tim3 Ab, anti-TIGIT Ab, anti-OX40 Ab, anti-41BB Ab, anti-VISTA Ab, bispecific antibodies targeting PD1 and Lag3, CAR-T cells, adoptive TIL transfer, and any combination thereof, administered alone or in combination with another anti-cancer agent, particularly any combination of an immune checkpoint inhibitor (ICI) with a taxane, cisplatin, and / or oxaliplatin.

7. Said MCI is as follows: - Oral vancomycin antibiotic, - A phage that kills bacteria of the Enterocloster new genus branch group, - A rare-cut endonuclease such as CRISPR Cas9 engineered to kill bacteria of the Enterocloster new genus branch group, - Akkermansia species and / or Akkermansia muciniphila (which may be mixed with other beneficial bacteria), - Retinoic acid, - Fecal microbiota transplantation, and - A mixture thereof, The seranostic method according to claim 5, selected from the group consisting of.

8. A medicament for MCI for treating cancer in combination with I-O therapy in an individual having cancer-induced dysbiosis, said medicament comprising the following: - Oral vancomycin antibiotic, - A phage that kills bacteria of the Enterocloster new genus branch group, - A rare-cut endonuclease such as CRISPR Cas9 engineered to kill bacteria of the Enterocloster new genus branch group, - Akkermansia species and / or Akkermansia muciniphila (which may be mixed with other beneficial bacteria), - A fecal microbiota composition, and - A mixture thereof, A medicament for MCI selected from the group consisting of.

9. A method for following up the success of MCI, comprising measuring serum soluble MAdCAM-1, wherein a normalized level of serum soluble MAdCAM-1 indicates that the MCI has successfully restored eubiosis of the intestine.

10. Use of serum soluble MAdCAM-1 levels as a biomarker for predicting the incidence of early NSCLC in heavy smokers with a high risk of experiencing cardiovascular events (HRHS-CV), wherein a reduction in the level of serum soluble MAdCAM-1 is a marker for predicting the onset of NSCLC.

11. A combination therapy for use in the treatment of cancer in patients having low serum levels of soluble MAdCAM, comprising (i) an anti-PD1 antibody or an anti-PDL1 antibody, and (ii) an anti-IL17A antibody or an anti-IL17R antibody and / or recombinant IL-7.

12. A screening method for identifying an agent capable of normalizing the expression level of MAdCAM-1 in endothelial cells of the lamina propria mucosae, comprising: (i) contacting in vitro a cultured cell that expresses MAdCAM-1 or is engineered to express a reporter gene under the control of the MAdCAM-1 promoter with a test compound; and (ii) evaluating which of said compounds induces the expression of MAdCAM-1 or the reporter gene expressed under the control of the MAdCAM-1 promoter.

13. A screening method for identifying an agent capable of normalizing the expression level of MAdCAM-1 in endothelial cells of the lamina propria mucosae, comprising administering a test compound to an intestinal humanized avatar mouse.

14. Including the step of verifying the results obtained from cultured cells or other experiments in avatar mice, wherein the avatar mice are intestinal humanized mice pretreated with a broad-spectrum ATB for 8 to 10 days and then recolonized with human FMT products by oral gavage, (i) whether the FMT product is a compound identified as inducing the expression of MAdCAM-1 in the cultured cells, or (ii) whether the FMT product is identified as not inducing the expression of MAdCAM-1 in the cultured cells and is enriched with another compound identified as inducing the expression of MAdCAM-1 in HUVEC cells; or (iii) the FMT has a taxonomic composition associated with eubiotic or highly soluble MADCAM-1, The method according to claim 12.

15. Including the step of verifying the results obtained from cultured cells or other experiments in avatar mice, wherein the avatar mice are intestinal humanized mice pretreated with a broad-spectrum ATB for 8 to 10 days and then recolonized with human FMT products by oral gavage, (i) whether the FMT product is a compound identified as inducing the expression of MAdCAM-1 in the cultured cells, or (ii) the FMT product has been identified as not inducing the expression of the MADCAM-1 in the cultured cells and enriched with another compound identified as inducing the expression of the MADCAM-1 in HUVEC cells; or (iii) the FMT has a taxonomic composition associated with eu-biosis or highly soluble MADCAM-1, The method according to claim 13.

16. as follows: (i) By 8 days after the enteral nutrition method, sacrificing a part of the intestinal humanized avatar mice, performing MADCAM-1 and Foxp3-based ileum PCR in the FMT recipient, and comparing them with the control; (ii) In the case of significant upregulation of MADCAM-1 in the intestinal humanized avatar mice, inoculating subcutaneous sarcomas into at least three mice in each group and treating them with anti-PD1 Ab to ensure that this FMT can mediate therapeutic efficacy compared to the ATB-treated mice; (iii) When the intestinal humanized avatar mice respond to the treatment with anti-PD1 Ab, presuming that the FMT product or the non-FMT compound used to enrich it can normalize the MADCAM-1 expression level in the endothelial cells of the lamina propria mucosae, The method according to any one of claims 13 to 15, comprising the above.

17. A screening platform for implementing the method according to any one of claims 12 to 15, comprising cells expressing a reporter gene under the control of the MADCAM-1 promoter, a robot configured to pick drugs from a product bank, deliver them to the cultured cells, and evaluate the expression of the reporter gene.

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