Immunogenic sequences from a Phage Tail Length Tape Measure Protein, bacteria expressing the same and their use in treating a cancer

Abstract

The present invention relates to the field of probiotic adjuvantization of anticancer treatments. In particular, the present invention concerns immunogenic sequences from a prophage present in bacteria identified as efficient adjuvants of cancer treatments. The invention provides bacterial compositions expressing immunogenic sequences from this prophage, immunogenic compositions comprising such sequences and methods using sequences from this prophage, for increasing the anticancer armamentarium.

Claims

1. A bacterial composition comprising at least one naturally occurring or engineered bacterial strain for use in treating a cancer, in combination with an antineoplastic drug, wherein said at least one bacterial strain expresses the protein of SEQ ID No: 1 or a fragment thereof of at least 9, preferably at least 20 amino acids comprising at least one epitope selected from the group consisting of SEQ ID Nos: 13, 14, 53 to 188 and 209, with the proviso that said at least one bacterial strain is different from the Enterococcus hirae strain 13144 deposited on Nov. 7, 2013 at the CNCM under the number I-4815.

2. The bacterial composition of claim 1, for the use of claim 1, wherein said at least one bacterial strain comprises a prophage genome encoding a protein with at least 80, preferably at least 90 and more preferably at least 95% identity with the protein of SEQ ID No: 1.

3. The bacterial composition of claim 1 or claim 2, for the use of claim 1, wherein said composition comprises at least one strain harbouring a prophage genome with at least 80 and preferably at least 95% identity with the prophage of SEQ ID No: 2, so that the phage encoded by this prophage can in vivo infect the other strains of the composition and/or commensal bacteria of the gut microbiota of a subject in need thereof.

4. The bacterial composition of any of claims 1 to 3, for the use of claim 1, wherein said composition comprises bacteria selected from the group consisting of: (i) Enterococcus hirae strain CNCM I-4815, (ii) Enterococcus hirae strain IGR7 deposited on Aug. 31, 2017 at the CNCM under the number I-5224, (iii) Enterococcus hirae strain IGR11 deposited on Nov. 27, 2017, at the CNCM under the number I-5261, (iv) any other bacterial strain expressing a protein with at least 65, preferably at least 80 and more preferably at least 95% identity with a fragment of at least 20, preferably at least 30 and more preferably at least 40 nucleotides from the protein of SEQ ID No: 1, and (v) mixtures of at least two of the strains recited in (i) to (iv), preferably a mixture of the strains recited in (i) and (ii).

5. The bacterial composition of any of claims 1 to 4, for the use of claim 1, wherein said composition further comprises Enterococcus hirae strain IGR4 deposited on Nov. 27, 2017, at the CNCM under the number I-5260.

6. A method of increasing the immunogenicity of a bacterial strain, comprising in vitro introducing, into said strain, a nucleotide sequence encoding the protein of SEQ ID No: 1 or a fragment thereof comprising at least the peptides of SEQ ID Nos: 13 and 14, or a sequence encoding a peptide of at least 9, preferably at least 20 amino acids comprising at least one epitope selected from the group consisting of SEQ ID No: 53 to 187, or a sequence encoding a peptide of at least 9, preferably at least 20 amino acids comprising at least one epitope of SEQ ID No: 209.

7. The method of claim 6, comprising in vitro infecting bacteria of said strain with a bacteriophage encoding a protein with at least 80, preferably at least 90 and more preferably at least 95% identity with the protein of SEQ ID No: 1.

8. The method of claim 7, wherein said bacteriophage has a genome comprising a nucleotide sequence of SEQ ID NOs: 2 or a sequence having at least 90% or at least 95% identity thereto.

9. A bacterial composition comprising an engineered bacterial strain which has been obtained by the method according to any of claims 6 to 8, for use in treating a cancer.

10. The bacterial composition of claim 9, for the use of claim 9, wherein said bacterial strain is selected from the group consisting of Escherichia coli, Enterococcus gallinarum, Enterococcus faecalis and Enterococcus hirae.

11. The bacterial composition of claim 9 or claim 10, for the use of claim 9, for use in treating a cancer in a HLA-A*0201 patient.

12. A bacterial composition selected from the group consisting of a bacterial composition comprising the Enterococcus hirae CNCM I-4815 and the bacterial composition of claims 1 to 5 and 9 to 11, for use in treating a cancer, wherein said bacterial composition is used in combination with an antineoplastic drug capable of triggering the lytic cycle of a phage encoded by a prophage present in at least some bacteria of the composition.

13. The bacterial composition of claim 12, for the use of claim 12, wherein said bacterial composition is used in combination with a CDK4/6 inhibitor.

14. The bacterial composition of claim 12, for the use of claim 12 or 13, wherein said bacterial composition is used in combination with an immune checkpoint blocker.

15. An immunogenic composition comprising a polypeptide comprising a sequence of at least 9 consecutive amino acids from the protein of SEQ ID No: 1 or a sequence of SEQ ID No: 209, or a polynucleotide encoding the same, for use as an anticancer vaccine.

16. The immunogenic composition of claim 15, for the use of claim 15, wherein the sequence of at least 9 consecutive amino acids from the protein of SEQ ID No: 1 is selected from the group consisting of SEQ ID No: 53-187.

17. The immunogenic composition of claim 15 or claim 16, which comprises a peptide comprising sequence selected from the group consisting of SEQ ID No: 63, SEQ ID No: 188 and SEQ ID No: 209 or a polynucleotide encoding the same, for use as an anticancer vaccine in a HLA-A*0201 patient.

18. A cell composition comprising antigen presenting cells (APC) which have been pulsed ex vivo with a bacterial composition according to any of claims 1 to 5 and 9 to 12 or with an immunogenic composition according to any of claim 15 to 17.

19. An MHC multimer for isolating T-cells with high affinity for the protein of SEQ ID No: 1, characterized in that MHC molecules are bound to an epitope selected from the group consisting of SEQ ID No: 53-188 and SEQ ID No: 209.

20. The MHC multimer of claim 19, which is a HLA-A*0201 multimer loaded with an antigenic peptide comprising SEQ ID No: 63, SEQ ID No: 188 or another sequence of SEQ ID No: 209.

21. A bacteriophage composition for use in treating a cancer, wherein said bacteriophage expresses a protein having at least 80, preferably at least 90 and more preferably at least 95% identity with the protein of SEQ ID No: 1.

22. The bacteriophage composition of claim 21, for the use of claim 20, wherein said bacteriophage has a genome comprising a nucleotide sequence of SEQ ID No: 2 or a sequence having at least 90% or at least 95% identity thereto.

23. The immunogenic composition of any of claims 15 to 17, or the cell composition of claim 18, or T cells isolated with the MHC multimer of claim 19 or claim 20, or the bacteriophage composition of claim 21 or claim 22, for use in treating a cancer, wherein said composition is administered in combination with a drug blocking an immune checkpoint.

24. The bacterial composition of any of claims 1 to 5 and 9 to 12, or the immunogenic composition of any of claims 15 to 17, or the cell composition of claim 18, or T cells isolated with the MHC multimer of claim 19 or claim 20, or the bacteriophage composition of claim 21 or claim 22, for use in treating a tumor expressing mRNA levels of GPD1 L superior to levels expressed in paired normal tissues.

25. A screening method for identifying antineoplastic drugs, comprising using bacteria from the strain CNCM I-4815 or any other bacterial strain harbouring a prophage genome with at least 80 and preferably at least 90% or 95% identity with the prophage of SEQ ID No: 2, for assessing the ability of drug candidates to trigger the lytic cycle of the phage comprising the protein of SEQ ID No: 1.

26. A method of determining if a patient having a cancer is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, comprising assessing the presence, in a biological sample from said patient, of a sequence having at least 80% identity with the protein of SEQ ID No: 1, wherein if such a sequence is present in the sample, the patient is likely to respond to the treatment.

27. The method of claim 27, comprising the steps of (i) cultivating a stool sample from said patient in aerobic conditions in a permissive medium to allow isolation of enterococci colonies, (ii) performing a PCR in several cultivable isolated colonies with a pair of primers specific for a fragment of SEQ ID No: 1, and (iii) detecting the amplified fragment.

28. A method of determining if a patient is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, comprising measuring the levels of circulating CCR9.sup.+CXCR3.sup.+CD8.sup.+ T cells at diagnosis and/or during said treatment, wherein if said level is above a predetermined threshold, the patient is likely to respond to the treatment.

29. The method of claim 28, comprising using the MHC multimer of claim 19 or claim 20 to assess the presence, in a biological sample from the patient, of T cells specific to the protein of SEQ ID No: 1, wherein if such T cells are present in the sample, the patient is likely to respond to the treatment.

30. The method of claim 29, wherein the patient is HLA-A*0201 and the MHC multimer is according to claim 20.

31. The method of any of claims 26 to 30, further comprising assessing the level of GPD1 L mRNA or the level of GPD1L protein in the tumor, wherein if said level is above a predetermined threshold, the patient is likely to respond to the treatment.

32. A method of determining if a patient having a tumor is likely to be a good responder to a treatment by immune checkpoint blockade, comprising assessing the level of GPD1L mRNA or the level of GPD1L protein in the tumor, wherein if said level is above a predetermined threshold, the patient is likely to respond to the treatment.

33. The method of any of claims 26 to 32, wherein if the patient is not identified as likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, a bacterial composition according to any of claims 1 to 5 and 9 to 12 or an immunogenic composition according to any of claims 15 to 17 is administered to the patient to increase his/her response to the treatment by chemotherapy or immune checkpoint blockade.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1. Experimental setting. (A) Mice are treated with broad spectrum antibiotics (streptomycin, colistin, ampicillin and vancomycine) for 3 days before performing Fecal Microbiota Transplantation (FMT). 14 days post-FMT, MCA-205 sarcoma cell lines are inoculated in the right flank of mice (8.10.sup.5 cells per mice). Chemotherapy (Cyclophosphamide, CTX100 mg/kg) or saline solution (NaCl) is injected weekly ip, starting 5 days post tumor inoculation for a total of 3 injections. Oral gavage with E. hirae strain 13144 (1.10.sup.9 bacteria) is performed the day of CTX injection as well as the day after. (B) Non FMT-treated SPF mice are used as control.

[0028] FIG. 2. Dysbiosis by FMT BC patient 1: Effectiveness of E. hirae 13144 to restore CTX-mediated antitumor effects. Tumor growth curves of MCA-205 sarcoma in SPF mice (A) vs FMT-treated mice (B) injected with 3 cycles of CTX vs NaCl. (C) Tumor growth curves of MCA-205 sarcoma in FMT-treated mice after oral gavage with strain E. hirae 13144 or NaCl as control. 2 independent experiments are depicted. (D) Overall survival of FMT-treated, MCA-205 sarcoma bearing mice treated with CTX or NaCl after oral gavage with strain E. hirae 13144. A typical survival curve is depicted for six mice per group. (E) Concatenated data of tumor sizes at day 21 post-CTX from 2 independent experiments are shown. Anova & Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0029] FIG. 3. No dysbiosis by FMT BC patient 2: E. hirae 13144 ameliorates the CTX-mediated antitumor effects. Tumor growth curves of MCA-205 sarcoma in SPF mice (A) vs FMT-treated mice (B) injected with 3 cycles of CTX vs NaCl. (C) Tumor growth curves of MCA-205 sarcoma in FMT-treated mice after oral gavage with strain E. hirae 13144 or NaCl as control. 2 independent experiments are depicted. (D) Concatenated data of tumor sizes at day 21 post-CTX from 2 independent experiments are shown. Anova & Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0030] FIG. 4. E. hirae IGR1, IGR10 and 10815 failed to efficiently boost CTX tumoricidal activity in a dysbiotic FMT setting. Tumor growth curves of MCA-205 sarcoma in FMT-treated mice after oral gavage with strain E. hirae 13144 (A), IGR1 (B) or NaCl as control. A typical curve is depicted for six mice per group. (C) Concatenated data of tumor sizes at day 17 post-CTX from 1-2 independent experiments are shown. (D). Comparison of various strains of E. hirae for their capacity to control tumor growth. Identification and discovery of two novel strains EH IGR 4 and 7 which display efficacy comparable to EH 13144. EH10815 fails to work and that may be related to a mutation residing in TMP2 (see below FIG. 13B). Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0031] FIG. 5. The combination of EH13144+IGR4+IGR7 is superior to EH13144 leading compound. (A) Tumor surface of MCA-205 sarcoma in FMT-treated mice after oral gavage with strain E. hirae 13144, IGR4, IGR7, Actinomycetes spp (Ao) and injected with 3 cycles of CTX (C) vs NaCl (N). Each dot is one mouse/tumor. (B) Tumor growth curves of MCA-205 sarcoma in SPF mice of the best group, means+SEM of tumor sizes at each time point and tumor sizes at sacrifice (C: CTX). Each dot is one mouse/tumor. Anova statistics indicating significant differencees between CTX and the triple combination. (C) Tumor surface of MCA-205 sarcoma in FMT-treated mice after oral gavage with strains EH13144, IGR1, IGR11, Barnesiella or Akkermansia and injected with 3 cycles of CTX. Bacterial strains are used alive (V) or pasteurized (P). For pasteurization, bacteria are incubated 30 min at 70 C. and frozen at 80 C. at least 6 hours.

[0032] FIG. 6. Mouse E. hirae 13144 mediates its efficacy only when used alive. Tumor growth curves of MCA-205 sarcoma in FMT-treated mice after oral gavage with strain E. hirae 13144 alive (A), pasteurized (B) or NaCl as control. A typical curve is depicted for six mice per group. (C) Concatenated data of tumor sizes at day 17 post-CTX from 3 independent experiments are shown. Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0033] FIG. 7. Increased accumulation of CD8+ T cells in mLN and spleen post-oral gavages with E. hirae 13144. (A) Experimental setting. Mice are treated with broad spectrum antibiotics for 3 days before performing oral gavage with E. hirae (708 or 13144) and CTX ip injection. One day post-CTX, a second oral gavage with E. hirae is performed. 72 hours post-CTX, CD4+ and CD8+ T cells are isolated from mesenteric lymph node (mLN). Percentage of CD8+ (B) and CD4+ (C) T cells in mLN after oral gavage with E. hirae 708 or 13144 and CTX treatment. (D) Experimental setting. Mice are treated with broad spectrum antibiotics for 3 days before performing oral gavage with E. hirae (708 or 13144) every three days for a total of 4 gavages. 5 days after the first oral gavage, mice are treated with CTX. One week post-CTX, CD8+ T cells are isolated from spleen. Percentage of CD8+ T cells (E) and CCR9+CXCR3+CD8 T cells (F) in spleen after oral gavage with E. hirae 708 or 13144 and CTX treatment. Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0034] FIG. 8. Sustained accumulation of CD8+CCD9+CXCR3+ T cells in tumor beds post oral gavage with EH13144: considering CD8+CXCR3+CCR9+ as a tumor associated-biomarker of sustained anticancer responses with OncoBax. (A) SPF mice were treated with ATB for 3 days and then oral gavages with EH13144 before and after CTX systemic administration, every other week for 3 weeks. Mice were sacrificed 7, 14 and 21 day after the first CTX treatment to harvest spleen, tumor draining lymph node (dLN) and tumor (MCA205 (B) FMT-treated mice were subjected to oral gavages with EH13144 before and after CTX systemic administration, every other week for 3 weeks. Mice were sacrificed the day of CTX treatment and 72 hrs post-CTX 1, 2, 3 (day J0, J3, J7, J10, J14, J17) to harvest dLN and tumor (MCA205). Flow cytometry analyses of CD45+ cells gating on CD8+CD3+ analyzing the percentages of CCR9+CXCR3+ double positive cells are presented. A representative experiment of 3 groups comprising 5 mice/group. Anova statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0035] FIG. 9. Experimental immunization protocols to elucidate the immunogenicity of EH peptides. Mice are treated with broad spectrum antibiotics for 3 days before performing oral gavage with E. hirae (708 or 13144) every three days for a total of 4 gavages. 5 days after the first oral gavage, mice are treated with CTX. One week post-CTX, CD8+ T cells are isolated from spleen. These CD8+ T cells are incubated with dendritic cells (DCs) pulsed with 20 g/ml of peptides (during 1 hour) or with heat-inactivated bacteria (during 6 hours). Inactivation of bacteria consists in incubation at 65 C. during 2 hours. After 24 hours of culture, ELIspot of IFN is performed.

[0036] FIG. 10. Cross-reactivity between the two E. hirae 708 and 13144 species for CTL splenic reactivity. Number of IFN spot representing IFN-secreting CD8+ T cells after co-culture with DCs pre-incubated with or without heat-inactivated bacteria (708, 13144, EH17 and L. plantarum). The corresponding experimental setting is indicated in FIG. 9. Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0037] FIG. 11. Effects of EH on gut inflammation and role of CD8+ T cells in this inflammation: EH-specific CTL amplified in the mLN may return into the lamina propria. (A) Experimental setting. Mice are treated with broad spectrum antibiotics for 3 days. Then, we performed oral gavage with E. hirae (708 or 13144) and anti-CD8 injection (200 g per mice) every three days for a total of 4 gavages/injections. 5 days after the first oral gavage, mice are treated with CTX. One week post-CTX, colon is removed for Hematoxylin Eosin Staining (HES). (B) Extent of inflammatory infiltrates in colon of mice colonized with E. hirae and treated with anti-CD8 ablating Abs and CTX. Student t-test statistical analyses: *p<0.05, **p<0.01.

[0038] FIG. 12. Schematic overview of the filters used to select the MHC class I binding peptides from EH. From whole bacterial sequences of E. hirae (708, 13144 and EH17), we selected proteins with cell wall and extracellular localization with PSORT software. Then, proteins were divided in peptides of nine amino acids. These peptides were tested for their ability to bind MHC class I H-2K.sup.b with NetMHC software and only strong-binders were retained (1050<50 nM).

[0039] FIG. 13. Peptides from group 7 containing the TMP1 and TMP2 epitopes activate CD8+ T cells from spleen of mice immunized against E. hirae 13144. Number of IFN spot, representing IFN-secreting CD8+ T cells, after incubation with DCs pulsed with 13 different group of peptide (Table 6). Just group n 1 (A) and group n 7 (B) are shown. (C, E) Number of IFN spot, representing IFN-secreting CD8+ T cells, after incubation with DCs pulsed with 4 peptides belong to group n 7 (in table 7). The corresponding experimental setting is indicated in FIG. 9. (D) Sequence alignment of TMP1 and TMP2 of EH10815 compared to EH13144 at the level of immunogenic peptides. (E) In vitro reactivity of splenic CTL in a recall response to TMP1 and TMP2 as well as Group7 after in vivo immunization with other strains of EH. Anova statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0040] FIG. 14. TMP epitope 1 prophage 2 specific CD8+ T cells accumulate in dLN and spleen post oral gavage with EH13144 and IGR7. Mice are treated with broad spectrum antibiotics for 3 days before performing oral gavage with E. hirae 13144 (A,B) or 10815 or EH17 or IGR7 (C) and CTX ip injection. The corresponding experimental setting is indicated in FIG. 7A for mLN and FIG. 7D for spleen. TMP1 specific CD8+ T cells are isolated with tetramer from mesenteric lymph node (A) and spleen (B,C). Flow cytometry analysis of TMP1 prophage 2 specific CD8+ T cells and TMP1-specific CD8+ T among CCR9+ or CCR9 T cells in mLN and spleen. Each dot represents one mLN or spleen. 5-6 mice/group. Anova statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0041] FIG. 15. TMP epitope 1 prophage 2 specific CD8+ T cells accumulate in tumor beds. (A) SPF mice were treated with ATB for 3 days and then oral gavages with EH13144 before and after CTX systemic administration, every other week for 3 weeks. Mice were sacrificed 7, 14 and 21 day after the first CTX treatment to harvest tumor draining lymph node (dLN) and tumor (MCA205). (B) FMT-treated mice were treated with ATB for 3 days and then oral gavages with EH13144 before and after CTX systemic administration, every other week for 3 weeks. Mice were sacrificed the day of CTX treatment and 72 hrs post-CTX 1, 2, 3 (day J0, J3, J7, J10, J14, J17) to harvest dLN and tumor (MCA205). Flow cytometry analyses of TMP1-specific CD8 T cells and TMP1-specific CD8 T cells among CCR9+CXCR3+ double positive cells are presented. A representative experiment of 3 groups comprising 5 mice/group. Anova statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0042] FIG. 16. Protocol of immunization with DC pulsed with TMP peptides. After DCs differentiation (with GM-CSF and IL-4 during 6 days), we incubated them with poly I:C (10 g/ml) overnight before the addition of peptide (1 hour) or heat-inactivated bacteria (6 hours). These DCs are injected subcutaneous on right flank at day 0 and 10. One month after second injection, sarcoma (MCA205) or colon (MC38) tumor cells lines are injected subcutaneous on left flank (8.10.sup.5 and 1.10.sup.6 cells per mice respectively). We constitute 4 groups, one group without DCs, one group with DCs pulsed with 20 g/ml of irrelevant peptides (gr1) or TMP peptides or 13144 heat-inactivated bacteria. Mean tumor sizes over time (A), each mouse tumor kinetics for each group (B), and the detailed comparaison in between groups at the two last time points before sacrifice for MCA205 are shown (C), each dot representing one mouse. MC38 related data are not shown.

[0043] FIG. 17. 13144 and TMP pulsed-DCs vaccination reduced tumor growth of MCA205. Tumor growth of MCA205 in mice vaccinated with DCs pulsed with gr1 peptides, TMP peptides and E. hirae 13144. (A) Mean+SEM of tumor sizes at different kinetics. (B) Tumor growth kinetics for each individual group of 5-10 mice. (C) Tumor sizes and statistical differences at time of sacrifice. Student t-test or ANOVA statistical analyses: *p<0.05, **p<0.1, ***p<0.001.

[0044] FIG. 18. The presence of a sarcoma at a distant site prevents the gut inflammatory lesions induced by oral gavages with E. hirae 13144. (A) Experimental setting. Mice are treated with broad spectrum antibiotics for 3 days before subcutaneous injection of MCA205 tumor cells in right flank. Then we performed oral gavage with E. hirae (708 or 13144) every three days for a total of 4 gavages. 5 days after the first oral gavage, mice were treated with CTX. One week post-CTX, colons are removed to be embedded in PPFE for Hematoxylin, Eosin and Safran staining (HES). (B) Extent of inflammatory infiltrates in colons of mice bearing MCA205 tumors or not and colonized with E. hirae and treated with CTX. Student t-test statistical analyses: **p<0.01.

[0045] FIG. 19. Comparative genomic analysis of E. hirae 13144 with four other E. hirae strains. (A) Pangenomic analysis of the five E. hirae genomes. (B) List of prophages regions in 13144 genome and homology to other phagefrom the web server PHASTER (PHAge Search Tool Enhanced Release), dedicated to the identification and annotation of prophage sequences within bacterial genomes and plasmids.

[0046] FIG. 20. Enterococcus hirae prophages comparative analysis. (A) Alignment of the sequences of several EH strains and location of the TMP sequences. A comparative analysis through a heatmap cluster based on a matrix of presence (black) and absence (white) of the 40.6-kb prophage (A) and 39.2-kb prophage (B) genes sequences and blast alignment parameter=80% identity and >=70% coverage.

[0047] FIG. 21. Homology between epitopes TMP of EH13144 and other E. hirae strains. (A) TMP from prophage 2 EH13144 is homologous to TMP from IGR7 (because both EH13144 and EH IGR7 sequences are identical). (B) Epitope TMP1 of EH13144 presents some homology with other E. hirae strains: 100% in IGR11 and 88.89% in IGR1 and 10815 (with 1 mutation indicated in (C). Epitope TMP2 of EH13144 presents 77% homology in IGR11 and 10815 (with 2 mutations indicated in (C).

[0048] FIG. 22. T cell responses against commensals in blood from breast cancer patients at diagnosis priori to chemotherapy. Autologous monocytes harvested from breast cancer patients were stimulated with distinct bacterial spp. and then incubated with autologous CD4+ T cells or CD8+ T cells to monitor IFN- and IL-10 release. (A) TH1/Tc1/Tr1 immune responses to E. hirae 13144. Less than 20% breast cancer (BC) patients at diagnosis exhibit a blood TH1/Tc1 immune response to E. hirae 13144 prior to chemotherapy. (B) TH1/Tc1/Tr1 immune responses to E. coli or TCR cross-linking. BC women display memory T cell responses against E. coli and respond to TCR cross-linking.

[0049] FIG. 23. Localization of the predicted peptides of TMP for each HLA haplotype to be tested in humans. Localization of predicted peptides of Tmp with significant binding potential for MHC Class I alleles (threshold of 50 nM) that was assessed using NetMHC software. For each peptide and allele, symbols represent the first amino acid of the identified sequence corresponding to 9 amino acid long peptides with their binding affinity to MHC Class I allele. Sequences of predicted peptides were resumed in table 9.

[0050] FIG. 24. Antigenicity of TMP peptides in human PBMCs from healthy volunteers. In vitro stimulation assays of recall responses either indirectly, after 2 rounds of stimulations by DCs pulsed with peptides (groups or individual peptides) to educate central memory CD8+ T cells from PBMCs using ELISPOT to reveal IFN release after 24 hr restimulation (A). Each peptide is HLA-A2 restricted (B) and 6 healthy volunteers HV (individualized with a 6 digit number) were selected on their HLA-A2.1 genotype (C). In (B), we underlined the peptides which are endowed with immunogenicity in the following graphs. We determined the threshold for each HV (D) and calculated the number of positive wells (above the threshold) and the pourcentages of responders for each peptide lisited in table B (E). When we represent these results in bar graphs, we observe that 5 peptides may be significantly associated with the immunogenicity of TMP in humans because they have been found to trigger Tc1 immune recall responses in at least 50% of HV (F). Arrows indicate the most significant epitopes (G). The list of peptides for each group and the most significant epitopes (2, 3, 9, 10, 13) are indicated in B.

[0051] FIG. 25. Protocol for phage excision by mitomycin C. Each step of the technical procedure is indicated.

[0052] FIG. 26. PCR detection of phage nucleic acids in the supernatants of E. hirae 13144. Supernatants of EH13144 cultivated at various temperatures (37 or 42 C.), at various concentrations of mitomycine C (0, 0.2 and 1 M), or active metabolite of CTX (mafosfamide25 g/ml) were treated to harvest the phage proteins encoding DNA with (B) or without (A) capside disruption. PCR was run to detect specific sequences of the prophage 1 or 2.

[0053] FIG. 27. Phage Tail Length Tape Measure Protein as the unique antigenic sequence in E. hirae 13144.

[0054] A and C. Experimental setting. Mice (naive (C), sarcoma bearers (A)) were treated with broad spectrum antibiotics (streptomycin, colistin, ampicillin, vancomycin) for 3 days before performing oral gavage with E. hirae strain 13144 (1.Math.10.sup.9 bacteria) before and after systemic administration of cyclophosphamide (ip CTX100 mg/kg) or saline solution (NaCl) at day 5 and 6 respectively, once (C) or three times on a weekly basis (A). One week later (C), purified CD8+ T cells splenocytes were restimulated ex vivo in a recall assay with bone marrow-derived DC loaded with saline or distinct heat killed bacteria strains. B. Tumor sizes at day 25 (sacrifice) of MCA-205 sarcoma in SPF C57BL/6 mice injected with 3 cycles of CTX vs NaCl after weekly oral gavages with various strains of E. hirae. D-E. Ex vivo recall assays. After in vivo exposure (C), splenic CD8+ T cells were restimulated with dendritic cells (DCs) pulsed with heat-inactivated bacteria (65 C. during 2 hours) (D) or peptides (E). IFN ELIspot was performed at 24 hours to enumerate IFN-secreting CD8+ T cells (spots) after co-culture. Each dot represents one mouse. F, G, H. Flow cytometry analyses of H-2K.sup.b/TSLARFANI tetramer binding CTL in spleens (F, H) or in tumor draining lymph nodes (G) at 72 hours post-therapy (regimen in A and C), in naive (F, H) or tumor bearers (G). The percentages of TMP1 prophage2-specific CD8+ T cells are depicted among splenic CD8+ T cells (F, left panel, G, H, top panel) or in the gate of CCR9.sup.+CXCR3.sup.+ T cells (F, right panel, G, bottom panel) or CCR9.sup.+ CTL (H, bottom panel). Each experiment involved one group of 10-15 mice from 2-3 independent experiments (B, D, E, F, H). A representative experiment out of 2 yielding similar results is shown in the kinetics study in G. Anova or Student t-test statistical analyses: *p<0.05, **p<0.01, ***p<0.001.

[0055] FIG. 28. Prophylactic and therapeutic immunization using Phage Tail Length Tape Measure Protein against sarcomas.

[0056] A-B. Prophylactic vaccinations. TLR3 ligand exposed DC were pulsed with peptides (irrelevant groups, individual TMP1 mutated or not in position 3) or heat-inactivated bacteria before inoculation sc in the right flank of naive mice, ten days apart. One month after second injection, sarcoma (MCA205) were implanted subcutaneous in the left flank. C-D. Therapeutic settings. Refer to FIG. 27A for the regimen where MCA205 tumor bearers were treated with CTX and gavaged with E. hirae 13144 or E. coli genetically modified to express TMP1 (TSLARFANI), TMP1 mut2 (TALARFANI), TMP1 mut3 (TSFARFANI) or EGFP sequence (as ctrl). Longitudinal tumor growth kinetics (A, C top panel) or cross-sectional tumor sizes (B, C bottom panel) of MCA205 are depicted as meansSEM of tumor sizes at different time points (A, C top panel) or at sacrifice (B, C bottom panel) for 12-18 animals (A), gathered from 2-3 independent experiments. D. Flow cytometry analyses of H-2K.sup.b/TSLARFANI tetramer binding CTL in spleens at sacrifice. The percentages of TMP1 prophage2-specific CD8+ T cells are depicted among splenic CD8+ T cells. Student t-test or ANOVA statistical analyses: *p<0.05, **p<0.1, ***p<0.001.

[0057] FIG. 29. Breadth of coverage (BOC) of the enterophage and clinical relevance of its molecular mimic GPD1-L in cancer patients.

[0058] A. BOC of the E. hirae and prophages genome in the MG reference catalog. 3027 metagenomes from 17 different datasets (referenced at the bottom, individual samples in columns) were screened for the presence of E. hirae strains and enterococcal phages genomes (featuring in rows). B-C. Percentages of stools with detectable E. hirae and/or E. faecalis colonies (B, top panel) and TMP in E. hirae and/or E. faecalis (B, bottom panel) colonies assessed by culturomics followed by PCR in 76 NSCLC and RCC bearing patients (cohort described in Routy et al. Science 2018) and corresponding Kaplan Meier curves indicating time to progression (C, top panel) or overall survival (C, bottom panel). Log-rank (Mantel-Cox) analysis with indicated p-value. D. Priming of naive CD8+ T cells from six HLA-A02*01 healthy volunteers with autologous monocyte-derived DC pulsed (or not) with 16 HLA-A02*01 binding TMP epitopes (Table 9, FIG. 24B and FIG. 37). Restimulation at day 7 with each of the 16 TMP peptides for IFN ELIspot assays and enumeration of positive spots. ANOVA statistical analyses: *p<0.05. E. Blast sequence alignment of immunogenic epitopes selected in D with the publicly available NCBI BLASTP suite and TOGA data set seaking >75% homology. Only epitope 10 (KLAKFASVV, SEQ ID No: 63) obtained a significant match (KLQKFASTV, SEQ ID No: 188) and was identified in the sequence of GPD1-L protein. F-H. Expression levels of GPD1-L gene product among bladder (F), lung adenocarcinoma (G), 530 renal cell cancer (H, according to HLA-A02*01 typing, bottom panel) patients from the TOGA data sets segregated according to the mean and Kaplan Meier curves of survival in univariate analysis. I-J. Time to progression following PD-1 blockade in second line therapy in 44 stage IIIC/IV NSCLC patients (CHUM validation cohort, I) validated with a second cohort of 62 stage IIIC/IV NSCLC patients (CGFL test cohort, J). Kaplan Meier curves for time to progression; patients were stratified according to the value value of the GPD1L expression. The cutoff was defined with an optimal cutoff strategy. K. Pearson correlations between GPD1-L expression and tumor immune infiltrates in all lung cancer type of TOGA assays (TOGA), lung adenocarcinoma (LUAD), lung squamous cells carcinoma (LUSC), CHUM and CGFL cohorts.

[0059] FIG. 30. Molecular mimicry between enterophage TMP and the oncogenic driver PSMB4 in mouse cancers.

[0060] A. Blast sequence alignment of the only one immunogenic epitope TSLARFANI selected in 27E with the publicly available TOGA data set seaking >70% homology. Only one hit (GSLARFRNI) obtained a significant match and was identified in the sequence of PSMB4 protein. B. Therapeutic settings comparing wild type versus knock in tumoral clones of MCA205. Id. as in FIG. 27A but mice were either inoculated with the WT MCA205 cell line or with distinct clones harboring a knock in mutation in position 3 of TSLARFANI, and then, were treated with CTX+/gavaged with E. hirae 13144 (or saline). Longitudinal tumor growth kinetics (B) or cross-sectional tumor sizes (B, right) of MCA205 are depicted as meansSEM of tumor sizes at different time points (top and bottom) or at sacrifice (right) for 6 animals/group, in a representative experiment out of 2 yielding similar conclusions. ANOVA statistical analyses: *p<0.05, **p<0.1, ***p<0.001. C-D. In vivo excision-infection cycle of the E. hirae siphoviridae phage. Gavage with E. hirae before and after CTX followed by harvesting of ileal content, for cultivation and isolation of bacterial colonies, MALDI-TOF identification and PCR using TMP specific probe sets (C). Graph depicting the proportions of colonies for each species in naive and gavaged animals, with colonies harboring the TMP sequence in PCR (D). Results of 5 mice/group and >70 colonies identified and scrutinized in PCR. Also refer to FIG. 39. E. Alignment of phage genome with E. gallinarum harboring TMP sequence in PCR after sequencing of this strain. F-G. Culture of organoid with E. hirae (10.sup.8) and E. gallinarum (10.sup.8) during 1 hour followed by mafosfamide treatment (25 g/ml). Six hours and 20 hours after mafosfamide treatment, supernatant were harvested for cultivation and isolation of bacterial colonies (F), MALDI-TOF identification and PCR using TMP specific probe sets (G). H. LEfSe analysis was performed on bacterial and viral species after MetaPhlAn2 analysis on localized breast cancer shotgun data, reporting the most discriminant ones (LDA score >2) in decreasing order for neoadjuvant palbociclib treatment on 10 patients from 83 included, the 73 others did not receive neoadjuvant CDK4/6 inhibitorss. Student t-test or ANOVA statistical analyses: *p<0.05, ** p<0.1, ***p<0.001.

[0061] FIG. 31. Clading and genomic analysis of E. hirae strains.

[0062] A. Dendrogramm of the various strains based on 16S sequence similarities. B. Phylogenomic tree of 20 E. hirae isolates based on SNPs alignment. C. Comparative genomic analysis of 13144 strain against five complete E. hirae genomes. From the center to the outside: GC skew, GC content, 13144, IGR7, IGR11, ATCC 9790, 708, 13344 strains. Prophages positions appear in black.

[0063] FIG. 32. Pre-identification of group 7 as the only immunogenic peptide group.

[0064] Naive mice were treated with broad spectrum antibiotics (streptomycin, colistin, ampicillin, vancomycin) for 3 days before performing oral gavage with E. hirae strain 13144 or 708 (1.10.sup.9 bacteria) before and after systemic administration of cyclophosphamide (ip CTX100 mg/kg) or saline solution (NaCl) at day 5 and 6 respectively. One week later, purified CD8+ T cells splenocytes were restimulated ex vivo in a recall assay with bone marrow-derived DC loaded with saline or distinct groups of peptides (refer to the list in Table 6). IFN ELIspot was performed at 24 hrs to enumerate IFN-secreting CD8.sup.+ cells (spots) after co-culture. Each dot represents one mouse. Statistical analyses revealed that only group 7 reached significant response: Anova test: *p<0.05, **p<0.1.

[0065] FIG. 33. Sequence of the TMP protein and comparative analysis of E. hirae 13144 prophage 2 protein sequence.

[0066] A. Whole TMP protein sequence of the 13144 prophage 2. B. Comparative analysis through a heatmap clustering based on a matrix of presence (black) and absence (white) of the 13144 prophages 2 protein sequences.

[0067] FIG. 34. Sequence alignment of the immunogenic epitope region within prophage 2 of E. hirae 13144.

[0068] The immunogenic peptide TSLARFANI from E. hirae 13144 was identified in experiments depicted and detailed in FIG. 27. The y axis presents the sequences of 6 other E. hirae strains in the same region tested in our models.

[0069] FIG. 35. Sub-cloning expression of part of the TMP gene in E. coli.

[0070] A. Amino acid sequences of TMP-FLAG, TMP-mut2-FLAG and TMP-mut3-FLAG expressed in E. coli DH5a. Note that only the N-terminal part of the TMP protein, including the indicated variants of the epitope (underlined), was expressed as fusion protein with a C-terminal FLAG tag (italics). B. Western blot analysis demonstrating expression of EGFP and TMP-FLAG in E. coli strains transformed with pDL28-P23-EGFP or pDL28-P23-TMP-FLAG, respectively.

[0071] FIG. 36. Sequence of the Phage Tail Length Tape Measure Protein in E. hirae. Genetic sequence of the whole TMP protein with binding areas for PCR primers indicated in italics.

[0072] FIG. 37. Localization of the HLA-A02*01 binding and immunogenic epitopes in the GPD1-L protein and their affinity (refer to FIG. 30A).

[0073] All the HLA-A02*01 binding and/or immunogenic epitopes found in Table 9 and depicted in FIG. 29D are located in a precise region of the whole TMP protein, as indicated by the color code and the amino acid sequence position, as a function of its binding affinity to the MHC class I allele.

[0074] FIG. 38. Generation of pmsb4-mutated MCA205 cell lines by means of the CRISPR/Cas9 technology.

[0075] A. Schematic diagrams of Psmb4 cDNA, and the designed mutation sites. The target site of sgRNA and point mutations are indicated. B. Representative sequence electropherograms for the validation of Pmsb4 mutation 2 and mutation 3 introduced by CRISPR/Cas9. Mutated amino acids are highlighted in grey.

[0076] FIG. 39. Identification of ileal bacterial colonies with or without treatment composed of CTX+oral gavage with 10.sup.9 cfu E. hirae 13144.

[0077] PCR amplification of the TMP sequence (refer to FIG. 36) in each colony growing after seeding of ileal content in aerobic conditions to isolate Gram+bacteria. A photograph of each agarose electrophoresis gel is shown for each animal. A depicts the results in 5 naive mice and B depicts the findings after CTX+oral gavage with the phage encoding bacterium. Each vertical lane corresponds to one bacterium identified in MALDI-TOF. Initials are detailed in the lower part of panel A. The positive control (Ctl+) represents the DNA of E. hirae 13144.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] In the present text, the following general definitions are used:

[0079] Gut Microbiota

[0080] The gut microbiota (formerly called gut flora or microflora) designates the population of microorganisms living in the intestine of any organism belonging to the animal kingdom (human, animal, insect, etc.). While each individual has a unique microbiota composition (60 to 80 bacterial species are shared by more than 50% of a sampled population on a total of 400-500 different bacterial species/individual), it always fulfils similar main physiological functions and has a direct impact on the individual's health: [0081] it contributes to the digestion of certain foods that the stomach and small intestine are not able to digest (mainly non-digestible fibers); [0082] it contributes to the production of some vitamins (B and K); [0083] it protects against aggressions from other microorganisms, maintaining the integrity of the intestinal mucosa; [0084] it plays an important role in the development of a proper immune system; [0085] a healthy, diverse and balanced gut microbiota is key to ensuring proper intestinal functioning.

[0086] Taking into account the major role gut microbiota plays in the normal functioning of the body and the different functions it accomplishes, it is nowadays considered as an organ. However, it is an acquired organ, as babies are born sterile; that is, intestine colonisation starts right after birth and evolves afterwards.

[0087] The development of gut microbiota starts at birth. Sterile inside the uterus, the newborn's digestive tract is quickly colonized by microorganisms from the mother (vaginal, skin, breast, etc.), the environment in which the delivery takes place, the air, etc. From the third day, the composition of the intestinal microbiota is directly dependent on how the infant is fed: breastfed babies' gut microbiota, for example, is mainly dominated by Bifidobacteria, compared to babies nourished with infant formulas.

[0088] The composition of the gut microbiota evolves throughout the entire life, from birth to old age, and is the result of different environmental influences. Gut microbiota's balance can be affected during the ageing process and, consequently, the elderly have substantially different microbiota than younger adults.

[0089] While the general composition of the dominant intestinal microbiota is similar in most healthy people (4 main phyla, i.e., Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria), composition at a species level is highly personalised and largely determined by the individuals' genetic, environment and diet. The composition of gut microbiota may become accustomed to dietary components, either temporarily or permanently.

[0090] Dysbiosis

[0091] Although it can adapt to change and has a high resilience capacity, a loss of balance in gut microbiota composition may arise in some specific situations. This is called dysbiosis, a disequilibrium between potentially detrimental and beneficial bacteria in the gut or any deviation to what is considered a healthy microbiota in terms of main bacterial groups composition and diversity. Dysbiosis may be linked to health problems such as functional bowel disorders, inflammatory bowel diseases, allergies, obesity, diabetes and also cancer. It can also be the consequence of a treatment, such as a cytotoxic treatment or an antibiotic treatment.

[0092] Antineoplastic Treatments

[0093] Antineoplastic treatments herein designate any treatment for cancer except surgery. They include chemotherapy, hormonal and biological therapies, radiotherapy and targeted therapies (such as c-KIT, EGFR or HER2/HER3 or MET or ALK inhibitors . . . ).

[0094] Chemotherapy

[0095] Chemotherapy is defined herein as the treatment of cancer with one or more chemotherapeutic agents. Chemotherapeutic agents are chemical molecules which act by killing cells that divide rapidly, one of the main properties of most cancer cells. Several categories of chemical agents exist: [0096] alkylating agents; [0097] spindle poisons such as mebendazole, colchicine; [0098] mitotic inhibitors (including taxanes (paclitaxel (Taxol 0), docetaxel (Taxotere 0)) and vinca alkaloids (e.g.: vincristine, vinblastine, vinorelbine, vindesine)), [0099] cytotoxic/antitumor antibiotics: such as anthracyclines (e.g.: doxorubicin, daunorubicin, adriamycine, idarubicin, epirubicin and mitoxantrone, valrubicin), streptomyces (e.g.: actinomycin, bleomycin, mitomycin, plicamycin) [0100] anti-metabolites (such as pyrimidine analogues (e.g.: fluoropyrimidines analogs, 5-fluorouracil (5-FU), floxuridine (FUDR), Cytosine arabinoside (Cytarabine), Gemcitabine (Gemzar), capecitabine; purine analogues (e.g.: azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin, cladribine, capecitabine, clofarabine); folic acid analogues (e.g.: methotrexate, folic acid, pemetrexed, aminopterin, raltitrexed, trimethoprim, pyrimethamine), [0101] topoisomerase inhibitors (e.g.: camptothecins: irinotecan, topotecan, amsacrine, etoposide, etoposide phosphate, teniposide); [0102] DNA methyltransferase inhibitors: 2-deoxy-5-azacytidine (DAC), 5-azacytidine, 5-aza-2-deoxycytidine, 1-[beta]-D-arabinofuranosyl-5-azacytosine, dihydro-5-azacytidine; [0103] vascular disrupting agents, such as flavone acetic acid derivatives, 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and flavone acetic acid (FAA); [0104] also other chemotherapeutic drugs such as aprepitant, bortezomib (Velcade, Millenium Pharmaceuticals), imatinib mesylate (Gleevec 0), carmustine (BCNU), lomustine (CCNU), tamoxifen, gefitinib, erlotinib, carboxyamidotriazole, efaproxiral, tirapazamine, xcytrin, thymalfasin, vinflunine.

[0105] Immune Checkpoint Blockers

[0106] In the present text, a drug blocking an immune checkpoint, or immune checkpoint blocker (ICB) or immune checkpoint blockade drug designates any drug, molecule or composition which blocks an immune checkpoint of T lymphocytes. Such a drug reactivates the host immune system, and kills tumor cells indirectly by effector T lymphocytes. In particular, these terms encompass anti-CTLA-4 antibodies, anti-PD1 antibodies, anti-PD-L1 antibodies (such as Atezolizumab or Durvalumab) and anti-PD-L2 antibodies. More particularly, an ICB can be an anti-PD1 monoclonal antibody such as Nivolumab or Pembrolizumab. Other ICB include anti-Tim3, anti-BTLA, anti-VISTA, anti-CD38, anti-TIGIT, anti-GITR, anti-LAG3, anti-KIR antibodies, anti-OX40 antibodies, which also inhibit immune checkpoints.

[0107] Although the currently used drugs antagonizing CTLA-4, PD1, PD-L1,PD-L2, etc. are monoclonal antibodies, other molecules specifically binding to these could be used for the development of future ICB such as, for example, antibody fragments or specifically designed aptamers. Of course, the phrases drug blocking an immune checkpoint, or immune checkpoint blocker (ICB) or immune checkpoint blockade drug encompass any therapy with active molecules that antagonize and immune checkpoint such as CTLA-4, PD1, PD-L1, PD-L2, etc., such as oncolytic viruses recombinant for anti-CTLA4, anti-PD1 or PDL1 Abs.

[0108] Immune-Targeting Antibodies for Activating Receptors

[0109] In the present text, a drug activating an immunostimulatory receptor, designates any drug, molecule or composition which activates a T or NK cell receptor reactivating the host immune system, and killing tumor cells indirectly by effector T lymphocytes. In particular, it encompasses anti-ICOS antibodies, anti-OX40 antibodies, anti-CD137, anti-CD28 antibodies . . . .

[0110] CDK4/6 Inhibitors

[0111] In the present text, a CDK4/6 inhibitor designates a cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor, such as palbociclib, ribociclib, and abemaciclib. These drugs are currently used to treat patients with hormone receptor (HR)-positive, human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) advanced breast cancer, but could also be used to treat other cancers, such as ovarian cancer and acute myeloid leukaemia.

[0112] Probiotics

[0113] Probiotics are micro-organisms that have claimed health benefits when consumed. Probiotics are commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. Generally, probiotics help gut microbiota keep (or re-find) its balance, integrity and diversity. The effects of probiotics can be strain-dependent. Here we will use the phrase anticancer probiotics or the neologisms oncobax and oncomicrobiotics to designate any commensal composition that restores responsiveness to chemotherapy, PD1/PD-L1 blockade or combination of anti-CTLA4+anti-PD1 or PD-L1 Ab. In the context of the present invention, a probiotic composition is thus not limited to food or food supplements, but it generally designates any bacterial composition comprising microorganisms which are beneficial to the patients. Such probiotic compositions can hence be medicaments or drugs.

[0114] Cancer, Treatment, etc.

[0115] As used herein, cancer means all types of cancers. In particular, the cancers can be solid or non solid cancers. Non limitative examples of cancers are carcinomas or adenocarcinomas such as breast, prostate, ovary, lung, pancreas or colon cancer, sarcomas, lymphomas, melanomas, leukemias, germ cell cancers and blastomas.

[0116] The immune system plays a dual role against cancer: it prevents tumor cell outgrowth and also sculpts the immunogenicity of the tumor cells. Drugs blocking an immune checkpoint can hence be used to treat virtually any type of cancer. Thus, the methods according to the invention are potentially useful for patients having a cancer selected amongst adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancers (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancers (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancers (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adenocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancers (e.g. hemangioma, hepatic-adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancers (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancers (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancers (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). More particularly, the method according to the invention can be used for predicting and optimizing a patient's response to a medicament targeting an immune checkpoint, wherein the patient has a cancer selected from the group consisting of metastatic melanoma, non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), mesothelioma, bladder cancer, renal cell carcinoma, head and neck cancers, oesophageal and gastric cancers, rectal cancers, hepatocarcinoma, sarcoma, Wilm's tumor, Hodgkin lymphoma, ALK-neuroblastoma, (hormone refractory) prostate cancers and GIST.

[0117] Other definitions will be specified below, when necessary.

[0118] According to a first aspect, the present invention pertains to the use of a bacterial composition in a combined treatment for treating a cancer, wherein the bacterial composition comprises at least one bacterial strain which expresses the protein of SEQ ID No: 1 or a fragment thereof of at least 9, preferably at least 20 amino acids comprising at least one epitope selected from the group consisting of SEQ ID Nos: 13, 14, 53 to 188 and 209.

[0119] In the frame of this invention, the at least one bacterial strain present in the composition can be either a naturally occurring strain or an artificial, engineered strain obtained by gene technology or any other technique. Strains different from the Enterococcus hirae strain 13144 deposited on Nov. 7, 2013 at the CNCM under the number I-4815 can be used according to the invention.

[0120] In the present text, a bacterial composition designates any composition comprising bacteria, especially live bacteria. The composition can comprise a pure culture of one single strain, a mix of several cultured strains and/or a complex material such as fecal material for performing Fecal Microbiota Transplantation (FMT). The composition can be a liquid composition. Alternatively, the composition can comprise freeze dried or lyophilized materials, which can be formulated or manufactured into or as an edible or friable product, e.g., a biscuit-like product, which can be e.g., crushed into a powder to dissolve in a drink or to insert into a tablet or a capsule. Alternatively, the bacterial composition can be in the form of a dry lozenge or a chewing gum or equivalent. Compositions according to the present invention can also be prepared and/or formulated in a powdered form, or equivalent; these formulations can be useful for storage in e.g., a tablet or capsule, or in an ampoule to e.g., crack open and dissolve in a liquid for, e.g., insertion, mixing or injection into a channel of a colonoscope or a naso-enteric tube and the like; or as a powder in a bag ready to add, for example as a solution which can be infused into a nasogastric tube (or equivalent), or a colonoscope, or a gastroscope etc. Components possibly present in a bacterial composition according to the invention (apart from the bacteria) include salts, buffers, nutrients, water, pharmaceutically acceptable excipients, cryoprotectants, etc.

[0121] Using the bacterial composition in a combined treatment for treating a cancer means that the bacterial composition is used in combination with an antineoplastic drug in order to potentiate or increase the effects of this antineoplastic drug. Non-limitating example of drugs that can advantageously be administered in combination with the bacterial compositions according to the invention include chemotherapy, especially alkylating agents (e.g. cyclophosphamide), immune checkpoint blockers (e.g., drugs antagonizing CTLA-4, PD1, PD-L1 or PD-L2 etc., used alone or in combination), immune-targeting antibodies for activating receptors (e.g., anti-ICOS, anti-OX40, anti-CD137, anti-CD28 antibodies), CDK4/6 inhibitors, etc. The physician will chose, depending on the context, what drug is to be administered to the patient in combination with the bacterial composition, as well as the therapeutic protocol (i.e., the sequence of administration of the antineoplastic drug(s) and the bacterial composition).

[0122] According to a preferred embodiment of the composition according to the invention, the at least one bacterial strain comprises a prophage genome encoding a protein with at least 80, preferably at least 90 and more preferably at least 95% identity with the protein of SEQ ID No: 1.

[0123] In a particular embodiment of the invention, at least one strain in the bacterial composition harbours a prophage genome with at least 80 and preferably at least 90 or 95% identity with the prophage of SEQ ID No: 2 (prophage of E. hirae 13144 identified as prophage 2 in the experimental part below), so that the phage encoded by this prophage can in vivo infect the other strains of the composition and/or commensal bacteria of the gut microbiota of the patient to which the composition is administered.

[0124] The present invention also pertains to a bacterial composition comprising bacteria selected from the group consisting of:

[0125] (i) Enterococcus hirae strain 13144 deposited on Nov. 7, 2013 at the Collection Nationale de Cultures de Microorganismes (CNCM) under the number I-4815,

[0126] (ii) Enterococcus hirae strain IGR7 deposited on Aug. 31, 2017 at the CNCM under the number I-5224,

[0127] (iii) Enterococcus hirae strain IGR11 deposited on Nov. 27, 2017, at the CNCM under the number I-5261,

[0128] (iv) any other bacterial strain expressing a protein with at least 65, preferably at least 80 and more preferably at least 95% identity with a fragment of at least 20, preferably at least 30 and more preferably at least 40 nucleotides from the tail tape measure protein (TMP) of SEQ ID No: 1 (called the TMP of prophage 2 in the experimental part below), and

[0129] (v) mixtures of at least two of the strains recited in (i) to (iv).

[0130] According to one embodiment, this bacterial composition is used for treating a cancer.

[0131] The bacterial strains present in the composition according to the invention can belong to any bacterial family. Of course, since the bacteria are administered to a patient suffering from cancer, non pathogenic bacteria will be preferentially used. Hence, bacteria other than Enterococcus hirae strains can also be used according to the present invention.

[0132] As already mentioned, the bacterial composition is typically administered to a patient in need thereof in combination with an antineoplastic drug.

[0133] According to one embodiment, the bacterial composition of the invention further comprises Enterococcus hirae strain IGR4, deposited on Nov. 27, 2017, at the CNCM under the number I-5260.

[0134] According to one embodiment, illustrated in the experimental part below, the composition comprises Enterococcus hirae strain 13144 (CNCM I-4815), Enterococcus hirae strain IGR7 (CNCM I-5224) and Enterococcus hirae strain IGR4 (CNCM I-5260).

[0135] According to a particular embodiment of the invention, the bacterial composition is used in combination with an antineoplastic drug capable of triggering the lytic cycle of a phage encoded by the prophage. Non-(imitative examples of such a drug are mitomycin C, as illustrated in Example 8 below, as well as CDK4/6 inhibitors, as illustrated in Example 16.

[0136] According to one embodiment, the bacterial composition comprises at least one strain harbouring a prophage genome with at least 80 and preferably at least 95% identity with the prophage of SEQ ID No: 2, so that the phage encoded by this prophage can in vivo infect other strains of the composition and/or, possibly, other bacteria already present in the patient's gut microbiota.

[0137] Another aspect of the present invention is a method of increasing the immunogenicity of a bacterial strain of anticancer probiotic interest, by in vitro introducing into said bacterial strain a nucleotide sequence encoding the TMP of SEQ ID No: 1 of a fragment thereof comprising at least the peptides of SEQ ID Nos: 13 and 14, or a sequence encoding a peptide of at least 9, preferably at least 20 amino acids comprising at least one epitope selected from the group consisting of SEQ ID No: 53 to 187 or a sequence encoding a peptide of at least 9, preferably at least 20 amino acids comprising at least one epitope of SEQ ID No: 209. The peptides of SEQ ID Nos: 13 and 14 correspond to immunogenic epitopes binding to mouse H-2Kb, but they might also be immunogenic in humans harbouring appropriate HLA class I haplotypes capable of binding some of these sequences or broader sequences, shared across species. The sequences of SEQ ID No: 53 to 187 have been identified in silico as being epitopes from the TMP of SEQ ID No: 1 which are bound by human HLA molecules (see Table 9 below). SEQ ID No: 209 (KLX.sub.1KFASX.sub.2V with X.sub.1=A or Q and X.sub.2=V or T) corresponds to the TMP1 HLA-A*0201-restricted immunogenic epitope of sequence KLAKFASVV (SEQ ID No: 63), the human HLA-A*0201-restricted epitope from GPD1L of sequence KLQKFASTV (SEQ ID No: 188), as well as two hybrids of these two epitopes: KLAKFASTV (SEQ ID No: 210) and KLQKFASVV (SEQ ID No: 211). Advantageously, the bacterial strain of anticancer probiotic interest is transduced with a sequence comprising several epitopes, which can be presented by different HLA haplotypes, to that the resulting bacterial strain is immunogenic in patients of different HLA haplotypes.

[0138] According to one embodiment, the nucleotide sequence encodes at least KMVEILEEI (SEQ ID No: 55), RLLKYDVGV (SEQ ID No: 56), LLGIYQSYV (SEQ ID No: 62), KLAKFASVV (SEQ ID No: 63) or ILVAITTTI (SEQ ID No: 66), which are HLA-A0201-restricted epitopes, the immunogenicity of which has been experimentally confirmed in humans (see Example 7 and FIG. 24B below). In a particular embodiment, the nucleotide sequence encodes at least KLAKFASVV (SEQ ID No: 63), KLQKFASTV (SEQ ID No: 188) or any other epitope of sequence KLX.sub.1KFASX.sub.2V with X.sub.1=A or Q and X.sub.2=V or T (SEQ ID No: 209).

[0139] According to one embodiment, the bacterial strain is transduced with a nucleic acid (for example, a plasmid) encoding a protein having 80%, 90% or 95%, preferably 97.5% and more preferably 98.7% identity with the TMP of SEQ ID No: 1, or encoding a fragment of said protein including at least one epitope as above-described. The skilled artisan will chose appropriate sequences (promoter etc.) so that the resulting bacterial strain expresses the TMP or fragment thereof.

[0140] According to one embodiment, bacteria of the strain are infected with a bacteriophage encoding a TMP with at least 80%, preferably at least 90% and more preferably at least 95% identity with the TMP of SEQ ID No: 1.

[0141] Again, any bacterial strain of interest can be used in this method, provided it is not highly pathogenic. In particular, bacterial strains already known for their probiotic interest can be engineered by in vitro infection with a bacteriophage or by transduction with a plasmid encoding the TMP or fragment thereof. Non-limitative examples of such bacteria include Akkermansia muciniphila, Ruminococcacae, Faecalibacterium, Clostridium ramosum, Clostridium XVIII, Alistipes species (A. onderdonkii, A. finegoldii, A. shahii), Eubacterium species, Bacteroidales species, Methanobrevibacter smithii, Lactobacillus johnsonii, Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides salyersiae, Burkholderia cepacia, Burkholderia cenocepacia, Barnesiella intestinihominis, Erysipeloclostridia and Erysipelotrichaceae, Colinsella intestinalis, Collinsella takakaei, Eggerthella lenta/Coriobacteriaceae, Bifidobacteria (longum, breve, termophilus, adolescentis . . . ) and E. coli. More particularly, Escherichia coli, Enterococcus gallinarum, Enterococcus faecalis and Enterococcus hirae can advantageously be used as starting material to obtain a bacterial strain with increased immunogenicity through a method as above-described.

[0142] According to a particular embodiment of the above method, the bacteriophage has a genome comprising a nucleotide sequence of the SEQ ID Nos:2 or a sequence having at least 90% or at least 95% identity thereto. According to a preferred embodiment, the bacteriophage has a genome identical to the prophage of 39.2 kb present in Enterococcus hirae 13144 (CNCM I-4815).

[0143] According to a particular embodiment, the present invention pertains to a bacterial strain which has been obtained by a method as above-described. In particular, the invention pertains to a non-naturally occurring bacterial strain, obtained by in vitro infection of a probiotic strain with a phage encoding the TMP protein defined above, wherein the probiotic strain is not known to be infected by this bacteriophage in nature. According to another embodiment, the invention pertains to a non-naturally occurring bacterial strain obtained by transduction with a nucleic acid encoding the TMP protein defined above or a fragment thereof, by gene editing or by any other technology.

[0144] The bacterial strains according to the invention, engineered to have increased immunogenicity and improved anticancer properties, are advantageously used in the treatment of cancer.

[0145] These engineered bacterial strains, in particular those that express a protein, polypeptide or peptide comprising an epitope selected amongst SEQ ID Nos: 63, 188 and 209, can efficiently be used to treat cancer in a HLA-A*0201 patient.

[0146] As already mentioned, the bacterial compositions of the invention, including those comprising an engineered bacterial strain as above-described, are advantageously used in combination with an antineoplastic drug capable of triggering the lytic cycle of a phage encoded by a prophage present in bacteria and/or with an immune checkpoint blocker.

[0147] The present invention also relates to an immunogenic composition comprising a polypeptide comprising a sequence of at least 9 consecutive amino acids from the TMP of SEQ ID No: 1 or a sequence of SEQ ID No: 209 or a polynucleotide encoding the same. Such a composition can advantageously be used as an anticancer vaccine, either as a nucleotide sequence (mRNA or cDNA) or as a peptide stretch of at least 9 amino acids, for example between 9 aa (short peptides directly binding to the proper MHC class I grove) and 20 to 30 amino acids (long peptides cross presented by DC in their MHC class I and II molecules). Dendritic cells or artificial antigen presenting cells presenting this polypeptide or recombinant for this cDNA could serve as a platform to prime and amplify nave or effector memory T lymphocytes from a tumor or blood from a patient or a healthy volunteer.

[0148] According to a particular embodiment of the immunogenic composition according to the invention, the sequence of at least 9 consecutive amino acids from the TMP is a peptide which has been identified as likely to be presented by a MHC I human molecule, such as the peptides of SEQ ID No: 53-187. Specific immunogenic compositions according to the invention comprise the peptidic sequences of SEQ ID Nos: 55, 56, 62, 63 or 66, which have been demonstrated to be immunogenic in HLA-A2 individuals (example 7). According to another specific embodiment, the immunogenic composition comprises a peptide comprising a sequence of SEQ ID No: 63, SEQ ID No: 188 or SEQ ID No: 209 or a polynucleotide encoding the same. Such an immunogenic composition is particularly useful as an anticancer vaccine in a HLA-A*0201 patient, especially if this patient has a tumor exhibiting a high GPD1L expression level (measured at the mRNA or protein level).

[0149] According to a particular immunogenic composition according to the invention, the polypeptide is a short polypeptide (9-, 10- or 11-mer). When such short peptides are subcutaneously injected, they bind directly to MHC molecules of every cells present at the site of injection. According to this embodiment of the invention, a cocktail of peptides, comprising several TMP epitopes, can advantageously be used. It is to be noted that when several epitopes specific for the same HLA molecule are used together, the epitopes are in competition for the binding to the corresponding HLA molecule. Contrarily, by using a mix of different HLA-restricted epitopes (such as HLA-A*0201, HLA-A*2402, HLA-B*0702 or others), there will be no competition for HLA binding. Another advantage of such a cocktail of peptides is that it will be efficacious in a broader range of patients i.e., in individuals expressing any of the HLA molecules corresponding to the epitopes present in the cocktail.

[0150] According to another particular immunogenic composition according to the invention, the polypeptide is a long polypeptide of 20 to 50 amino acids, preferably 25 to 40 amino acids encompassing at least one 9-10 TMP immunogenic stretch selected amongst SEQ ID No: 53 to 187, flanked by 10-20 amino acids, which will be in vivo internalized and processed by antigen presenting cells. These cells will then present fragments thereof, thereby triggering an immunogenic response against TMP epitopes. Such long polypeptides are advantageously able to be cross-presented by local DC into not only MHC class I but also MHC class II molecules of the host. For example, chimeric polyepitope polypeptides, i.e., polypeptides comprising a concatenation of epitopes of the TMP, possibly separated by peptidic linkers, can be used. Of course, for the same reasons as mentioned in the above paragraph, a long polypeptide advantageously comprises different HLA-restricted epitopes. Such chimeric polyepitope polypeptides can also comprise epitopes from antigens different from the TMP, for example epitopes from tumor antigens.

[0151] The immunogenic compositions according to the invention also advantageously comprise appropriate adjuvants. The skilled artisan will chose, depending on the type of peptide and the type of application, the most appropriate adjuvant. Non-limitative adjuvant which can be used according to the present invention include Montanide, Flt3L, cyclophosphamide, DC and TLR or STING agonists.

[0152] According to one embodiment, the immunogenic composition comprises a polynucleotide encoding a polypeptide comprising a sequence of at least 9 consecutive amino acids from the TMP of SEQ ID No: 1. Such a composition can advantageously be used as an anticancer vaccine. Examples of polynucleotide compositions according to the invention include naked DNA, mRNAs encoding TMP, RNA loaded nanoparticles and recombinant viruses (such as lentiviruses, oncolytic vireuses adenoviruses, poxviruses etc.). Of course, the considerations mentioned above regarding the advantages of mixing epitopes specific for different HLA molecules in polypeptide compositions apply when designing the polynucleotides to be included in the compositions according to the invention. Alternatively, the epitopes can be selected and personalized according to the HLA haplotype of the patient (Table 9).

[0153] According to another aspect, the present invention pertains to a cell composition comprising antigen presenting cells (APC) which have been pulsed ex vivo with a bacterial composition as above described or with an immunogenic composition according to the invention. Such a cell composition can advantageously be used for cell therapy of cancer patients.

[0154] According to another of its aspects, the present invention relates to a MHC multimer, for isolating T-cells with high affinity for the TMP of SEQ ID No: 1. In such a MHC multimer, MHC molecules are preferably bound to a peptide selected from the group consisting of SEQ ID No: 53 to 187, for example to at least one peptide selected amongst those of SEQ ID Nos: 55, 56, 62, 63 and 66, or to at least one peptide of SEQ ID No: 188 or 209. MHC multimers according to the invention range in size from dimers to octamers (e.g.: tetra, penta, hexamers) or use even higher quantities of MHC per multimer (e.g., dextramers). Particular MHC multimers according to the invention are HLA-A*0201/KLAKFASVV multimers, HLA-A*0201/KLQKFASTV multi mers, HLA-A*0201/KLQKFASVV multimers and HLA-A*0201/KLAKFASTV multimers (e.g., tertramers or dextramers).

[0155] Another cell composition according to the present invention comprises CD4.sup.+ or CD8+ T cells specific for the TMP of SEQ ID No: 1. Cells comprised in such a composition can be obtained either by cell sorting (using, for example, a MHC multimer as above-described) followed by ex vivo expansion, or by transduction of T lymphocytes with a cDNA encoding a TCR with a high avidity for TMP. Examples of protocols that can be used to obtain such compositions are disclosed in examples 10 and 11 below.

[0156] As already mentioned, the immunogenic compositions and the cell compositions described above can be used for treating a cancer. In such a treatment, they can be used alone or combined with peptidic or nucleotidic vaccines as described above and/or combined with antineoplastic treatments such as chemotherapy, for example an alkylating agent such as cyclophosphamide, or immune checkpoint blockers, especially anti-PD1/PD-L1/PD-L2 antibodies.

[0157] The present invention also pertains to the use of a bacteriophage expressing a protein having at least 90, preferably at least 95-98.7% identity with the TMP of SEQ ID No: 1 for treating a cancer. Such a bacteriophage is preferably formulated in a pharmaceutically acceptable composition, which can be administered per os or intratumorally.

[0158] According to a particular embodiment of the bacteriophage composition of the invention, the bacteriophage has a genome comprising a nucleotide sequence of SEQ ID NOs: 2 or a sequence having at least 90% or 95% identity thereto.

[0159] When treating a cancer patient, a bacteriophage composition according to the invention can advantageously be administered in combination with a drug blocking an immune checkpoint, for example in combination with anti-PD1/PD-L1/PD-L2 antibodies.

[0160] As explained in the experimental part below, the inventors found that the HLA-0201-restricted epitope KLAKFASVV (SEQ ID No: 63), shares a 78% sequence homology with an epitope (KLQKFASTV, SEQ ID No: 188) of the glycerol-3 phosphate dehydrogenase 1 like protein (GPD1L, gene encoded on 3p22.3), and it is conceivable that cross-reactivities between TMP phage specific TCR and self tissues or tumor tissues overexpressing GPD1L account for the anticancer effectiveness of the phage delivered in the context of the invention. The bacterial compositions, the immunogenic compositions, the cell compositions and the bacteriophage compositions of the invention are thus particularly useful for treating a tumor overexpressing GPD1L, i.e., having mRNA levels of GPD1L superior to levels expressed in paired normal tissues (for example lung cancer versus surrounding healthy lung parenchyma). Tumors overexpressing GPD1L would indeed be considered as electively eligible for an oral therapy with E. hirae 13144 or EH IGR7 or EH IGR11 or a combination of all the 3 strains, or with any other bacterium recombinant for the TMP of SEQ ID No: 1 or a fragment thereof comprising SEQ ID No: 63, with or without intradermal boosts with TMP phage-related HLA restricted-peptides or nucleic acid or other vaccine modality. This applies to lung cancers, melanoma, tumors of the digestive tract, bladder cancer, RCC and breast cancer or any tumor expressing foetal antigens.

[0161] Another aspect of the present invention is a screening method for identifying antineoplastic drugs, comprising assessing the ability of drug candidates to trigger the lytic cycle of the phage comprising the TMP of SEQ ID No: 1 when they are incubated with bacteria from the strain CNCM I-4815 (or any other bacterial strain harbouring a prophage genome with at least 80 and preferably at least 90% or 95% identity with the prophage of SEQ ID No: 2). When performing this method, the skilled artisan can use different concentrations of the drug candidates and measure the phage excision at several time points of incubation.

[0162] The present invention also pertains to theranostic methods for determining if a patient is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, all based on the results disclosed below showing the importance of the phage TMP protein expressed by the strain CNCM I-4815 in the patient's response to the treatment. These methods are particularly useful to determine if a patient having a cancer such as NSCLC, RCC, bladder cancer, pancreas cancer, colorectal cancer and breast cancer is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade. More particularly, such a method can advantageously be used for a patient at diagnosis of a NSCLC or RCC prior to immunotherapy with an ICB.

[0163] According to one embodiment of this aspect of the invention, the theranostic method of the invention comprises assessing the presence, in a biological sample from said patient, of a sequence having at least 80%, 90% or 95% identity with the TMP of SEQ ID No: 1. According to this method, the patient is considered as likely to respond to the treatment if such a sequence is present in the sample. Non-(imitative examples of biological samples which can be used for performing this method are the tumor genome, intratumoral bacterial load, fecal phages, fecal bacteria containing the phages, feces samples, bronchoalveolar samples, buccal samples and sputum.

[0164] More specifically, this method can be performed by PCR amplification of specific sequences and comprise the following steps:

[0165] (i) cultivating a stool sample from the patient in aerobic conditions in a permissive medium to allow isolation of enterococci colonies,

[0166] (ii) performing a PCR on several cultivable isolated colonies with a pair of primers specific for a fragment of SEQ ID No: 1, and

[0167] (iii) detecting the amplified fragment.

[0168] More precisely, in step (ii), the PCR can be done within each E. gallinarum, E. hirae and E. faecalis isolated stool colony, or at least in 3 to 5 of them. For example, the PCR can be performed with the primers of SEQ ID Nos: 191 and 192, generating an amplicon of 1026 bp.

[0169] The present invention also pertains to a method of determining if a patient is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, comprising measuring, for example by flow cytometry or TMP tetramer-binding T lymphocytes, the levels of circulating CCR9+CXCR3+CD8+ T cells during said treatment, wherein if said level is above a predetermined threshold, the patient is likely to respond to the treatment.

[0170] According to a particular embodiment, a MHC multimer as above-described is used to assess the presence of T cells specific to the protein of SEQ ID No: 1, in a biological sample from a cancer patient. The presence of such T cells in the sample indicates that the patient is likely to respond to the treatment. More particularly, when this method is performed for assessing whether a HLA-A*0201 patient is likely to be a good responder to a treatment by chemotherapy or immune checkpoint blockade, a HLA-A*0201/SEQ ID No: 209 multimer can be used.

[0171] According to another embodiment, the theranostic method of the invention comprises assessing the level of GPD1L mRNA or the level of GPD1L protein in the tumor, wherein if said level is above a predetermined threshold, the patient is likely to respond to the treatment.

[0172] Of course, the physician or skilled technician can combine the above methods to refine the prognosis that a patient will respond to the treatment, for example by assessing both the GPD1L expression level in the tumor and the presence of T cells specific for KLAKFASVV (SEQ ID No: 63), or the GPD1L expression level and the presence of a sequence having a high percentage of identity with the TMP of SEQ ID No: 1.

[0173] If, when performing a theranostic method as above-described, the patient is not identified as likely to respond to a treatment by chemotherapy or immune checkpoint blockade, a probiotic treatment with a bacterial composition according to the present invention can advantageously be administered to the patient to increase his/her chances to respond to the treatment. Another preconditioning treatment to increase the patient's response is a vaccination with an immunogenic composition as above-described.

[0174] Other characteristics of the invention will also become apparent in the course of the description which follows of the biological assays which have been performed in the framework of the invention and which provide it with the required experimental support, without limiting its scope.

EXAMPLES

[0175] Some of the experiments illustrating the present invention are described two times in the following examples, showing the continued research effort made by the inventors in the past years, which led to a more precise knowledge of the mechanism explaining the effects of E. hirae strains 13144, IGR7 and IGR11 and to the design of newly engineered strains with anti-tumor properties.

[0176] Materials and Methods

[0177] For each experiment illustrated in FIGS. 1 to 26, the materials and methods used to perform the experiment are detailed in the figure legend.

[0178] The following table summarizes the E. hirae strains referred to in examples 1 to 11 below.

TABLE-US-00001 TABLE 1 Deposit Example Strain Origin number 1-9 13144 Murine-CTX-treated CNCM I-4815 1, 5, 9 IGR 1 stools of lung cancer CNCM I-5260 1, 9 IGR 4 patients CNCM I-5224 1, 3, 5, 9 IGR 7 CNCM I-5261 1, 9 IGR 10 1, 5, 9 IGR 11 2, 3, 5, 6 708 Human-Unknown 2, 3, 5 EH17 (13344) Human-Blood 1, 3, 5 10815 Type strain CIP 53.48.sup.T ATCC 9790 (ATCC 9790) 9 5348 Human-Unknown 7030 Human-Liver abscess 12607 Environmental- RiskManche project 13150 Environmental-Water 13152 Environmental-Water 13153 Environmental-Water 13155 Environmental- RiskManche project 13161 Environmental-Cockle 13343 Conservation liquid of kidney 13346 Human-Urine 13347 Blood culture Strains used in this study. Strains called IGR were isolated by U1015 INSERM at Gustave Roussy, Villejuif, in 2017, anonynously from non small cell lung cancer patients at diagnosis about to be treated with nivolumab as a second line therapy. 708 comes from INRA but was originally sent by a spanish collaborator more than 10 years ago. All the other strains come from Dr Vincent Cattoir, director of the reference center for commensal and pathogenic enterocci, CHU de RennesHpital Ponchaillou-Service de Bactriologie- Hygine hospitalire-2 rue Henri Le Guilloux 35033 RENNES Cedex.

[0179] The following materials and methods were also used for performing the experiments reported in Examples 12 to 16 below.

[0180] Cell Culture, Reagents and Tumor Cell Lines

[0181] MCA-205 WT were cultured at 37 C. with 5% CO.sub.2 in RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 UI/ml penicillin/streptomycin, 1 mM sodium pyruvate and MEM non-essential amino acids (henceforth referred to as complete RPM11640). All reagents were purchased from Gibco-lnvitrogen (Carlsbad, Calif., USA).

[0182] Mice

[0183] All animal experiments were carried out in compliance with French and European laws and regulations. The local institutional board approved all mouse experiments (permission numbers: 2016-109-7450). Experiments were performed in accordance with Government and institutional guidelines and regulations. Female C57BL/6 were purchased from Harlan (France). Mice were used at an age between 7 and 12 weeks of age. All mouse experiments were performed at the animal facility in Gustave Roussy Cancer Campus where animals were housed in specific pathogen-free conditions.

[0184] Antibiotic Treatments

[0185] Mice were treated during 3 days with an antibiotic solution (ATB) containing ampicillin (1 mg/ml), streptomycin (5 mg/ml), colistin (1 mg/ml) (Sigma-Aldrich) and vancomycin (0.25 mg/ml) added in the sterile drinking water of mice. Antibiotic activity was confirmed by cultivating fecal pellets resuspended in BHI+15% glycerol at 0.1 g/ml on COS (BD Columbia Agar with 5% Sheep Blood) plates for 48h at 37 C. in aerobic and anaerobic conditions. In the context of bacterial transplantation experiments, mice received 3 days of ATB before undergoing bacterial transplantation the next day by oral gavage using animal feeding needles.

[0186] Tumor Challenge and Treatment

[0187] Syngeneic C57BL/6 mice were implanted with 0.810.sup.6 MCA-205 WT sarcoma cells subcutaneously and treated intraperitoneally (i.p.) when tumors reached 20 to 35 mm.sup.2 in size with CTX (100 mg/kg). Depending on the experimental setting, mice were injected once or 3 times at 1-week interval. Tumor size was routinely monitored every 3 days by means of a caliper.

[0188] Gut Colonization with Dedicated Commensal Species

[0189] Enterococcus hirae 13144 were originally isolated from spleens of SPF mice treated with CTX in our laboratory. E. hirae 708 was provided by INRA (P. Langella), while E. hirae 13344, ATCC9790 were provided by Prof. Cattoir, CHU de Caen, France. L. plantarum was provided by Prof. Ivo Gomperts Boneca, Institut Pasteur strain repository, France. All E. hirae IGR strains were isolated from NSCLC patient stools in our laboratory, according to patient informed consent and local IRB approval (study Oncobiotics). All bacteria were grown on COS plates for 24h-48h at 37 C. in aerobic conditions. Colonization of ATBs pre-treated mice was performed by oral gavage with 100 l of suspension containing 110.sup.9 bacteria. For bacterial gavage: suspensions of 10.sup.8 CFU/mL were obtained using a fluorescence spectrophotometer (Eppendorf) at an optical density of 600 nm in PBS. Depending on the experimental setting, 2 or 6 bacterial gavages were performed for each mouse: the first, the same day as CTX injection, and then 24h after the injection of CTX. The efficacy of colonization was confirmed by culturing the feces 48h post-gavage. Fecal pellets were harvested and resuspended in BHI+15% glycerol at 0.1 g/ml. Serial dilutions of feces were plated onto COS plates and incubated for 48h at 37 C. in aerobic and anaerobic conditions. After 48h, the identification of specific bacteria was accomplished using a Matrix-Assisted Laser Desorption/Ionisation Time of Flight (MALDI-TOF) mass spectrometer (Andromas, Beckman Coulter, France).

[0190] Culture and Propagation of Bone Marrow-Derived Dendritic Cells

[0191] Bone marrow-derived dendritic cells (BM-DCs) were generated by flushing bone marrow precursors from the femurs and tibia of female C57131/6 WT mice aged of 8 to 12 weeks. Bones were collected in sterile PBS, washed in alcohol and Iscove's medium (IMDM, Sigma-Aldrich) baths, extremities of bones were cut and flushed using a 26G needle. After red blood cell lysis, cells were cultured in IMDM supplemented with 10% of FCS+2 mM L-glutamine+100 UI/ml penicillin/streptomycin +50 M 2-mercaptoethanol (Sigma-Aldrich) (referred herein as complete IMDM medium) at 0.510.sup.6/ml and treated with 40 ng/ml of GM-CSF (supernatant of GM-CSF transfected-cells J558) and 10 ng/ml of recombinant interleukin-4 (IL-4) for BM-DCs (from Peprotech). Cells were split at day 3 and used in experiments on day 7 or 8.

[0192] Test of Memory TC1 Immune Response and H2-Kb Restricted-Peptides on Splenic CD8+ T Cells by ELISpot IFN

[0193] IFN- ELISPOT assay was performed in 96-well PVDF bottomed sterile plates (Millipore MSIP S4510) by using a IFN- ELISPOT kit (Cell sciences, Newburyport, Etats-Unis) according to the manufacturer's instructions. After PVDF membrane activation with ethanol 35%, plates were coated overnight with capture antibody to IFN- and washed before incubation of blocking buffer during 2h. BM-DC cells (110.sup.5/well) were infected with heat-inactivated (2h at 65 C.) bacterial strains (E. hirae 13144, E. hirae 708, E. hirae 13344 and L. plantarum) at a multiplicity of infection 1:10 (MOI) or pulsed with peptides (20 g/ml) and were added together with CD8+ T cells (210.sup.5/well) and incubated for 20 h at 37 C. Cells were then removed and plates were developed with a detection antibody to IFN- (biotinylated) during 1h30 and streptavidin-alkaline phosphatase during 1 h. Finally, substrate of streptavidin (BCIP/NBT buffer) were incubated 5-20 min. Spots were counted by using CTL Immunospot Analyzer (Germany).

[0194] Mice Vaccination

[0195] After DCs differentiation, these DCs were activated with poly I:C (10 g/ml, Invivogen) overnight before infection with heat-inactivated (2 h at 65 C.) bacterial strains (MOI 10) or pulsed with peptides (20 g/ml, peptide 2.0). After 6 hours of incubation with bacteria or 1 hour of incubation with peptides, BM-DCs were washed 3 times with PBS before subcutaneous injection in the right flank of mice (1.510.sup.5 cells per mice). Mice were vaccinated twice at 10 days apart and challenged 4 weeks after the second vaccination with the minimal tumorigenic dose of MCA-205 tumor cells in left flank.

[0196] Flow Cytometry Analyses

[0197] In experiments without tumor, spleens were harvested 7 days after the injection of CTX. In tumor growth experiments, spleens, tumors and tumor draining lymph node were harvested at different time points, 7, 14 and 21 days after the first injection of CTX into mice bearing MCA-205 tumors. Excised tumors were cut into small pieces and digested in RPMI medium containing Liberase TM at 25 g/mL and DNase1 at 150 UI/mL (Roche) for 30 minutes at 37 C. and then crushed and filtered twice using 100 and 40 m cell strainers (BD). Lymph nodes and spleen were crushed in RPMI medium and subsequently filtered through a 70 m cell strainer. Two million splenocytes, tumor cells or lymph node cells were pre-incubated with purified antimouse CD16/CD32 (clone 93; eBioscience) for 15 minutes at 4 C., before membrane staining. Dead cells were excluded using the Live/Dead Fixable Yellow dead cell stain kit (Life Technologies). Antimouse antibodies for CD3 (145-2C11), CD4 (GK1.5), CD8 (eBioH35-17.2), CXCR3 (CXCR3-173), CCR9 (CW-1.2), and TMP specific tetramer (BD, BioLegend, eBioscience and Cliniscience) were used. Stained samples were acquired on Canto II 7 colors cytometer (BD) and analyses were performed with FlowJo software (Tree Star, Ashland, Oreg., USA).

[0198] Human T Cell Responses to HLA-A02*01 Restricted-TMP Epitopes

[0199] Cytapheresis cones were collected from healthy volunteers (EFS, Etablissement franais du sang) and peripheral blood mononuclear cells (PBMC) were separated using a Ficoll Hypaque gradient. We selected only donors with HLA-A02*01 haplotype determined by flow cytometry with anti-HLA-A2 antibodies. PBMC were washed and resuspended in the separation medium (PBS, 1 mM EDTA, 2% human AB+ serum) for magnetic bead separation. CD14+ monocytic cells (human CD14 MicroBeads, Miltenyi) were enriched from 7510.sup.6 PBMC and cultured at 0.5106/ml in IMDM supplemented with 10% human AB+serum, 1% of 2 mmol/L glutamine (GIBCO Invitrogen), 1000 IU/ml GM-CSF and 1000 IU/ml IL-4 (Miltenyi). Cells were split at day 3 and used in experiments on day 6 or 7. Monocytes were seeded in 96-well plates at 110.sup.5 cells/well either alone or in the presence of peptides (20 g/ml), and incubated for 2 hour at 37 C., 5% CO.sub.2. The remaining autologous PBMC fractions were enriched for CD8+ T cells (CD8+ T Cell Isolation Kit, human, Miltenyi). The enriched CD8+ T cells were washed, counted and resuspended at 110.sup.5 cells/well in RPMI-1640 supplemented with 10% human AB+ serum, 1% 2 mMol/L glutamine, 1% penicillin/streptomycin (GIBCO Invitrogen) and 50 U/mL IL-2 (Proleukin). Monocyte-peptide/T cell co-cultures were incubated for one week at 37 C., 5% CO.sub.2 (medium was changed every 2 days). Then, the pools of cells were seeded in 96-well ELIspot plates at 210.sup.5 cells/well and restimulated with or without peptides (20 g/ml) or anti-CD3/anti-CD28 coated beads (1 L/mL, Dynabeads T-Activator, Invitrogen) as a positive control for 20 h at 37 C. IFN- ELISPOT assay were performed in 96-well PVDF bottomed sterile plates (Millipore MSIP S4510) by using a IFN- ELISPOT kit (Cell sciences, Newburyport, Etats-Unis) according to the manufacturer's instructions.

[0200] Stool Detection of Phage TMP Sequence by PCR

[0201] We cultivated the stools (of patients) or ileal material (mice) after several dilutions in aerobic conditions and permissive medium to allow isolation of enterococci colonies (according to a procedure described in (Samb-Ba et al., 2014)). We performed a PCR of the TMP sequence in each single cultivable Enterococcus colony. One colony was put in 100 l of nuclease-free water to release the bacterial DNA and PCR was performed with 5 l of DNA, 12.5 l of PCR master mix (Thermoscientific), 5 l of nuclease-free water and 1.25 l of each TMP primers (20 M). PCR products were separated on 1.5% agarose gel containing ethidium bromide and revealed by UV exposition. The sequence of primers are: forward 5-ACTGCAGCCGTAAAATGGGA-3 (SEQ ID No: 191) and reverse 5-TCCGTATCGTTTGCCAGCTT-3 (SEQ ID No: 192) (amplicon 1026 bp).

[0202] Generation of TMP-Expressing E. coli

[0203] A DNA fragment containing the P23 promoter sequence was generated by annealing two complementary primers (5-CAATAAAAAATCAGACCTAAGACTGATGACAAAAAGAGCAAATTTTGATAAAATAGT ATTAGAATTAAATTAAAAAGGGAGGCCAAATATAG-3 (SEQ ID No: 193) and 5-GATCCTATATTTGGCCTCCCTTTTTAATTTAATTCTAATACTATTTTATCAAAATTTG CTCTTTTTGTCATCAGTCTTAGGTCTGATTTTTTATTGCATG-3 (SEQ ID No: 194)). The sequence was then inserted into SphI/BamHI-digested vector pDL278 (Addgene 46882, gift from Gary Dunny (LeBlanc et al., 1992)) to generate vector pDL278-P23. A part of the TMP gene (N-terminal 1185 nucleotides of TMP, including the epitope TSLARFANI (SEQ ID No: 13), fused to a C-terminal FLAG-tag) was amplified from E. hirae 13144 genomic DNA (5-TCCGGATCCATGGCACAAAGTAAAACAGTCAAAGCG-3, (SEQ ID No: 195) 5-CAGGAATTCTTACTTGTCGTCATC GTCTTTGTAGTCACGTAGTAAACTATCACGTA ATCGAACTTC-3 (SEQ ID No: 196)) and inserted into BamHI/EcoRI-digested vector pDL278-P23 to generate vector pDL278-P23-TMP-FLAG. Mutations in the epitope were introduced using the QuikChange Lightning Kit (Agilent). Primers 5-AACGAGCTAAGGCAGTAGCAGCTGTATCTGCAGAC-3 (SEQ ID No: 197) and 5-GTCTGCAGATACAGCTGCTACTGCCTTAGCTCGTT-3 (SEQ ID No: 198) were used to mutate position 2 (S to A, pDL278-P23-TMP-mut2-FLAG), primers 5-ATTAGCAAAACGAGCGAAGGAAGTAGCAGCTGTATCTG-3 (SEQ ID No: 199) and 5-CAGATACAGCTGCTACTTCCTTCGCTCGTTTTGCTAAT-3 (SEQ ID No: 200) were used to mutate position 3 (L to F, pDL278-P23-TMP-mut3-FLAG). To generate the control plasmid pDL278-P23-EGFP, EGFP was amplified from pClB1(deltaNLS)-pmGFP (Addgene 28240, gift from Chandra Tucker (Kennedy et al., 2010)) using primers 5-CTTGGATCCATGGTGAGCAAGGGCGAG-3 (SEQ ID No: 201) and 5-CAGGAATTCCTACATAATTACACACTTTGTC-3 (SEQ ID No: 202) and inserted into BamHI/EcoRI-digested vector pDL278-P23. Plasmids were transformed into chemically competent E. coli DH5a (NEB) and the presence of plasmids with the correct insert was verified by sequence analysis (5-CCCAGTCACGACGTTGTAAAACG-3 (SEQ ID No: 203) and 5-GAGCGGATAACAATTTCACACAGG-3 (SEQ ID No: 204)). Expression of EGFP and TMP-FLAG in E. coli was verified by western blot analysis using antibodies targeting GFP (Cell Signaling, 2956) or FLAG (Sigma-Aldrich, F7425), respectively.

[0204] CRISPR/Cas9-Mediated Mutations of Mouse Psmb4 in MCA205 Cells

[0205] Wild type MCA205 cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA) and was maintained in RPMI-1640 medium (Thermo Fisher Scientifc, Inc., Waltham, Mass., USA) supplemented with 10% FBS (hermo Fisher Scientifc, Inc), 100 U/ml penicillin and 100 g/ml streptomycin (Thermo Fisher Scientifc, Inc.) at 3TC. For the CRISPR knock in mutations, we designed the gRNA (sequence AGATATTGCGGAAACGAGCC (SEQ ID No: 205)) by using the CRISPR design tool developed by the Zhang lab (http://crispr.mit.edu/). Oligonucleotides containing the designed sequence were synthesized (Sigma) and ligated into the pX458 backbone (Addgene #48138, (Ran et al., 2013)) containing the Cas9 gene (human codon-optimised and fused with 2A-GFP allowing for selection) under a CBh promoter and the cloned sgRNA under a U6 promoter. Homology templates (sequence attached) containing the mutation sites were synthesized by Invitrogen GeneArt Gene Synthesis (Thermo Fisher Scientifc, Inc.). The cloned pX458 plasmid and synthesized homology arms were cotransfected into MCA205 cells by means of lipofectamine 3000 (Thermo Fisher Scientifc, Inc.) following the manufacture's protocol. Forty-eight hours after transfection, GFP-positive cells were sorted to 96-well plates as single cells before surviving clones were expanded in duplicated conditions, one for frozen storage at 80 and the other for genomic DNA extraction. The targeted region in genomic DNA from clones was further amplified by PCR using the Phusion High-Fidelity PCR Master Mix (New England BioLabs; pswich, MA, USA) and primers 5CTCAGGGACCCTTTTCACGA 3 (SEQ ID No: 206) and 5CCCACTCCCTGTTCTACACA 3 (SEQ ID No: 207), and purified with the Monarch DNA Gel Extraction Kit (New England BioLabs) before being sent to Eurofins Genomics GmbH (BERSBERG GERMANY) for sequencing with the primer 5GGACCCTTTTCACGATTCAGG 3 (SEQ ID No: 208). According to sequence results, positive clones were expanded and subjected to DNA extraction for validating sequences. Transfected single cell clones that did not harbor the designed mutations were used as WT clones.

[0206] Genome Sequencing and Analysis

[0207] The whole genome sequence of 5 E. hirae (13144, 708, 13152, 13344 and EH-17) strains was determined with PacBio technology (GATC Biotech, Konstanz, Germany). Genomic DNA was isolated from 15 other E. hirae isolates using the using the Quick-DNA fungal/bacterial miniprep kit (Zymo Research, Irvine, Calif.) according to the manufacturer's recommendations. After DNA shearing, the DNA libraries were prepared using the NEBNext Ultra DNA library prep kit for Illumina (New England Biolabs, Ipswich, Mass.) and sequenced as paired-end reads (2300 bp) using an Illumina MiSeq platform and the MiSeq reagent kit version 3. The Illumina reads were trimmed using Trimmomatic (Bolger et al., 2014), quality filtered with the Fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/) and assembled using SPAdes (Bankevich et al., 2012). Protein sequences were predicted using prokka v1.11 software (Seemann, 2014). Prophage regions were detected using PHAST software. Predicted proteins were annotated using BLASTp against the National Center for Biotechnology Information (NCBI) non-redundant (NR) database.

[0208] Phylogenomic and Comparative Genomics

[0209] Single nucleotide polymorphism between 20 E. hirae genomes was investigated using the parsnp program (Treangen et al., 2014) and strain 13144 genome sequence as a reference. Phylogenetic analysis was performed by considering the 47,303 polymorphic sites retained in the core genome of the 20 genomes. Maximum likelihood phylogeny was constructed using Fastree (Guindon et al., 2010). Phylogenetic tree was visualized using figtree (http://tree.bio.ed.ac.uk/software/figtree/). Complete Proteome sequences of 20 E. hirae strains were compared using by BlastP and pairwise alignments using ClustalW. We clustered the E. hirae homologous genes using orthoMCL (Li et al., 2003) on the translated protein sequences of all predicted genes with a conservative parameter value of 70% amino acid sequence identity and 50% sequence coverage. The determination of the different unique core genomes was based on the homology clusters found by orthoMCL.

[0210] Statistical Analyses

[0211] Data analyses and representations were performed either with Prism 6 (Graph Pad, San Diego, Calif., USA). Tumor size differences were calculated either using Anova or dedicated software (https://kroemerlab.shinyapps.io/TumGrowth/). Briefly, tumor growth was subjected to a linear mixed effect modeling applied to log pre-processed tumor surfaces. P-values were calculated by testing jointly whether both tumor growth slopes and intercepts (on a log scale) were different between treatment groups of interests. Survival probabilities were estimated using the Kaplan-Meier method, best cutoffs for continuous variables were chosen using the median value or an optimal cutoff approach. Survival curves were evaluated using the log-rank test. All reported tests are two-tailed and were considered significant at P-values<0.05. The normalized gene expression values (FPKM-UQ) and the corresponding clinical data were downloaded from the TCGA data portal. For the survival analysis patients were grouped by the median expression of GPD1L across per cancer type datasets and by predicted HLA-alleles (Charoentong et al., 2017). Hazard ratio (HR) and 95% confidence interval (CI) for overall survival (OS) and progression-free survival (PFS) were calculated with Cox regression analysis. The tumor-infiltrating immune cell compositions were determined using CIBERTSORT (absolute mode) (Newman et al., 2015) and were compared with gene expression using Pearson correlation.

Example 1: Different Strains of E. hirae Exhibit Distinct Immunogenic Potential and Antitumor Effects

[0212] We first tested our E. hirae isolates in a more physiological setting of dysbiosis than the one caused by the 14 day-administration of a broad spectrum antibiotics. We indeed transferred feces (FMT) from two breast cancer patients exhibiting or not a deviated repertoire of the gut microbiome (and a distinct prognosis) into ATB-treated recipients and compensated their dysbiosis with different strains of adjunctive E. hirae (FIG. 1) attempting to restore full blown efficacy of CTX (as shown in eubiotic mice). While the FMT from a dysbiotic BC patient confered resistance to CTX compared with eubiotic littermates (FIGS. 2A-B), live EH13144 was very effective at restoring the efficacy (FIGS. 2C-E). In a second example of FMT from a different BC patient (exhibiting no overt dysbiosis (FIGS. 3A-B), the effects mediated by live EH13144 were also significant (FIG. 3C-D). However, in a similar setting, other strains of E. hirae (IGR1 and IGR11), cultivated from stools of lung cancer patients were not immunogenic (FIG. 4 A-C), although as effective as EH13144 to induce IL-16 and IL-12 production by BM-DC (not shown).

[0213] In addition, EH10815 did not mediate antitumor effects while E. hirae (IGR4 and IGR7), cultivated from stools of lung cancer patients were also immunogenic, as observed with 13144 (FIG. 4 D). Moreover, the combination of EH13144, IGR4 and IGR7 is superior to EH13144 leading compound (FIG. 5).

[0214] Interestingly, pasteurized EH13144 was far less effective than its live counterpart and failed to restore CTX tumoricidal activity in a dysbiotic FMT (FIG. 6).

[0215] We conclude that EH13144 have unique properties to boost CTX immune and tumoricidal activity against MCA205 sarcoma in not only ATB-but also FMT-induced patients' dysbiosis.

Example 2: E. hirae 13144 Amplifies CD8.SUP.+.Tc1 Cells in the Mesenteric Lymph Nodes and Spleens Cross-Reactive with EH708

[0216] EH13144 plays an adjuvant role with cyclophosphamide to boost antitumor immune responses and anticancer effects in a T cell- and IFN-dependent manner (Daillre et al., 2016). We dissected the mechanisms of the immunogenicity of EH13144 by analyzing the dynamics of the T cell immune responses in secondary lymphoid organs from the mesenteric lymph node (mLN) and the spleen (FIGS. 7A and 7D). EH13144 as well as E. hirae 708 were both capable of inducing the accumulation of CD8+ T cells in both, mLN after 1 week and spleen after 2 weeks (FIGS. 7B and 7E). However, while EH13144 induces an efficient anti-tumor immune response, E. hirae 708 fails to do so (FIG. 1D of Daillre et al., 2016). EH13144 also amplified the CD4+ T cell pool in the mLN (FIG. 7C). Moreover, EH13144 induce accumulation of CD8+CCR9+CXCR3+ in spleen (FIG. 7F) and in the tumor beds (FIG. 8).

[0217] To analyze the specificity of this CD8+ T cell expansion, we challenged splenic CD8.sup.+ T cells with bone marrow-derived dendritic cells (BM-DC) infected with various EH isolates (13144, EH708 and EH17 or L. plantarum). A commensal-specific memory CD8+ T cell response was monitored by an ELISPOT assay at 24 hrs by the enumeration of IFN positive spots (FIG. 9), pathognomonic of a Tc1 immune response. Interestingly, only EH708 and EH13144 could induce memory Tc1 immune responses with a cross-protection inbetween themselves (FIG. 10) while EH17 failed to do so, corroborating the poor protective antitumor effects of EH17 reported by Daillre et al. (Daillre et al., 2016).

[0218] We scrutinized colon mucosae for CD8+ T cell infiltrates by monitoring intestinal inflammatory lesions in the presence or absence of neutralizing anti-CD8 antibodies in immunohistochemistry (FIG. 11A). Hematoxylin eosin stained colon mucosae were more infiltrated after oral gavages with EH13144 or EH708 than without commensals post-CTX but these infiltrates were drastically reduced when animals were pretreated by ip administrations of neutralizing anti-CD8 Abs (FIG. 11 B). Of note, these CD8+ T cells primed in mLN or spleen have a tendency to home to the lamina propria in the absence of a distant tumor but not if a sarcoma deposit is introduced subcutaneously (FIG. 18).

[0219] Altogether, these findings suggest that oral gavages with EH13144 or EH708 in the context of cyclophosphamide trigger not only a splenic Th1 (Daillre et al., 2016) but also a systemic Tc1 immune response recognizing both EH708 and EH13144 (but not EH17) sequences with the potential to traffic back to the colon if no tumor deposit exist or to tumor beds when they develop.

Example 3: Screening EH Specific H-2K.SUP.b .Restricted Peptides: E. Hirae 13144 Genome Contains Immunogenic Peptide Sequences in the TMP Phage Protein

[0220] Whole genome differential analyses were performed on three E. hirae strains (13144, 708 and EH17) to identify potential 9-mer MHC class I-binding epitopes. The search focused on subcellular localization (enrichment for cell wall and extracellular proteins, via PSORT software), and on binding affinity to H2-K.sup.b (<50 nM binding affinity, NetMHC software) (FIG. 12, Tables 2-5).

TABLE-US-00002 TABLE2 H-2brestricted-EH13144peptidesnotpresentintheotherstrains Hirae sequence namesoftheproteins SEQIDNo: 13144 SAFPYEQEL C3familyADP-ribosyltransferase 3 13144 YNYSKSYPV hypotheticalprotein 4 13144 VSFSHYRPG hypotheticalprotein 5 13144 VTFLGYNAF cellsurfaceprotein 6 13144 TVYTFHVNI cellsurfaceprotein 7 13144 TSYSPLFLL cellsurfaceprotein(putative) 8 13144 TNYIYPNIL 2,3-cyclic-nucleotide2- 9 phosphodiesterase 13144 VVPILFLGL FmtBprotein 10 13144 KNYKAYVEL hypotheticalprotein 11 13144 SAMKYGIPL hypotheticalprotein 12 13144 TSLARFANI Phagetaillengthtape-measureprotein 13 13144 AMIEFIQGL Phagetaillengthtape-measureprotein 14 13144 VAITFGGPL Phagetaillengthtape-measureprotein 15 13144 VSTNHYGLL hypotheticalprotein 16 13144 VMFGLFITI cellsurfaceproteinprecursor 17 13144 TVFSLVSLL Chitinase 18 13144 SIYNLEKPL IgA1protease 19 13144 YTIIRYGNL IgA1protease 20 13144 SNGLLYTPM IgA1protease 21 13144 NNYHYVGGL IgA1protease 22 13144 SMFLNCNNL hypotheticalprotein 23 13144 IAFQGYSSL hypotheticalprotein 24 13144 QVTNFFNMF hypotheticalprotein 25 13144 IMLGLFMTM cellsurfaceproteinprecursor 26

TABLE-US-00003 TABLE3 H-2brestricted-EH708peptidesnotpresentintheotherstrains Hirae sequence namesoftheproteins SEQIDNo: 708 INAKFSSQL Membraneproteinsrelatedto 27 metalloendopeptidases 708 YIYNHYKDM Membraneproteinsrelatedto 28 metalloendopeptidases 708 YVYGKSRTM Membraneproteinsrelatedto 29 metalloendopeptidases 708 IAFLSYKLF cellsurfaceproteinprecursor 30 708 IMYEYMYPV hypotheticalprotein 31 708 SSMEYFLKV Phagetaillengthtape-measureprotein 32 708 ISFFQENQL Collagenadhesin 33 708 TNLLFMTSL extracellularprotein 34 708 KIFSIFMLL Phosphatidylinositol-specificphospholipaseC 35 708 LNIFKFNRF Chitinase 36 708 MTYDYRGGF Chitinase 37 708 PSYMFRTSF Chitinase 38 708 QSYTYYMTA cellwallsurfaceanchorfamilyprotein 39 708 ITFSHYEPT cellwallsurfaceanchorfamilyprotein 40

TABLE-US-00004 TABLE4 H-2brestricted-EH17peptidesnotpresentintheotherstrains Hirae sequence namesoftheproteins SEQIDNo: EH17 MSFTFFSST hypotheticalprotein 41 EH17 IAFQNFVNL Chitinase 42 EH17 SMFIAFQNF Chitinase 43 EH17 LNYDYGNRI Chitinase 44 EH17 AGICFFTGV PeptidoglycanN-acetylglucosamine 45 deacetylase EH17 VEYTYFPTL Membraneproteinsrelatedto 46 metalloendopeptidases EH17 AAYVFEMNF Membraneproteinsrelatedto 47 metalloendopeptidases EH17 EMYRKLSTL Membraneproteinsrelatedto 48 metalloendopeptidases EH17 YNYGYKSVL enhancinfamilyprotein 49 EH17 VIHELYNSL bacteriocinimmunityprotein 50

TABLE-US-00005 TABLE5 H-2brestricted-EH13144/EH708commonpeptides Hirae sequence namesoftheproteins SEQIDNo: 708-13144 TNYVKLRPL hypotheticalprotein 51 708-13144 QAVNHFTGI Portalproteinphageassociated 52

[0221] CD8.sup.+ T cell splenocytes isolated from CTX-treated mice were restimulated with pooled 9-mer peptides (group 1-13, Table 6) to identify potentially immunogenic epitopes in vivo. Using the same protocol as the one outlined in FIG. 9, we found that the immunogenicity of 13144 relied upon group 7 associated peptides (FIG. 13B) contrary to other group of peptides (data not shown) such as group 1 (FIG. 13A). Next, we split the group 7 into the four H-2K.sup.b peptides (Table 7). Immunogenicity of E. hirae 13144 could reside within the Phage Tail Length Tape Measure Protein (Tmp) (Table 7). Indeed, we unveiled that only TMP1 and TMP2 peptides were highly recognized by memory EH13144 (but not 708 and EH17)-induced Tc1 cells (FIG. 13C). Of note, the strain EH10815, which harbored the two same prophages as EH13144 but mutations in position 3 in TMP1 and in positions 2 and 7 of TMP2 (FIG. 13D), does not induce recall responses (FIG. 13E) and might explain why this bacterium failed to mediate significant antitumor effects. In contrast, results obtained with EH clone IGR7 which shares a very high sequence homology with EH13144, induce recall responses (FIG. 13E).

TABLE-US-00006 TABLE6 H-2brestrictedE.hiraepeptidesbygroupsusedininvivoexperiments SEQ Group n Hirae sequence namesoftheproteins IDNo: 1 1 708 INAKFSSQL Membraneproteinsrelatedto 27 metalloendopeptidases 2 708 YIYNHYKDM Membraneproteinsrelatedto 28 metalloendopeptidases 3 708 YVYGKSRTM Membraneproteinsrelatedto 29 metalloendopeptidases 4 708 IAFLSYKLF cellsurfaceproteinprecursor 30 2 5 708 IMYEYMYPV hypotheticalprotein 31 6 708 SSMEYFLKV Phagetaillengthtape-measureprotein 32 7 708 ISFFQENQL Collagenadhesin 33 8 708 TNLLFMTSL extracellularprotein 34 3 9 708 KIFSIFMLL Phosphatidylinositol-specificphospholipaseC 35 10 708 LNIFKFNRF Chitinase 36 11 708 MTYDYRGGF Chitinase 37 12 708 PSYMFRTSF Chitinase 38 4 13 708 QSYTYYMTA cellwallsurfaceanchorfamilyprotein 39 14 708 ITFSHYEPT cellwallsurfaceanchorfamilyprotein 40 15 13144 SAFPYEQEL C3familyADP-ribosyltransferase 3 16 13144 YNYSKSYPV hypotheticalprotein 4 5 17 13144 VSFSHYRPG hypotheticalprotein 5 18 13144 VTFLGYNAF cellsurfaceprotein 6 19 13144 TVYTFHVNI cellsurfaceprotein 7 20 13144 TSYSPLFLL cellsurfaceprotein(putative) 8 6 21 13144 TNYIYPNIL 2,3-cyclic-nucleotide2-phosphodiesterase 9 22 13144 VVPILFLGL protein 10 23 13144 KNYKAYVEL hypotheticalprotein 11 24 13144 SAMKYGIPL hypotheticalprotein 12 7 25 13144 TSLARFANI Phagetaillengthtape-measureprotein 13 26 13144 AMIEFIQGL Phagetaillengthtape-measureprotein 14 27 13144 VAITFGGPL Phagetaillengthtape-measureprotein 15 28 13144 VSTNHYGLL hypotheticalprotein 16 8 29 13144 VMFGLFITI cellsurfaceproteinprecursor 17 30 13144 TVFSLVSLL Chitinase 18 31 13144 SIYNLEKPL IgA1protease 19 32 13144 YTIIRYGNL IgA1protease 20 9 33 13144 SNGLLYTPM IgA1protease 21 34 13144 NNYHYVGGL IgA1protease 22 35 13144 SMFLNCNNL hypotheticalprotein 23 36 13144 IAFQGYSSL hypotheticalprotein 24 10 37 13144 QVTNFFNMF hypotheticalprotein 25 38 13144 IMLGLFMTM cellsurfaceproteinprecursor 26 39 EH17 MSFTFFSST hypotheticalprotein 41 40 EH17 IAFQNFVNL Chitinase 42 11 41 EH17 SMFIAFQNF Chitinase 43 42 EH17 LNYDYGNRI Chitinase 44 43 EH17 AGICFFTGV PeptidoglycanN-acetylglucosamine 45 deacetylase 44 EH17 VEYTYFPTL Membraneproteinsrelatedto 46 metalloendopeptidases 12 45 EH17 AAYVFEMNF Membraneproteinsrelatedto 47 metalloendopeptidases 46 EH17 EMYRKLSTL Membraneproteinsrelatedto 48 metalloendopeptidases 47 EH17 YNYGYKSVL enhancinfamilyprotein 49 48 EH17 VIHELYNSL bacteriocinimmunityprotein 50 13 49 708- TNYVKLRPL hypotheticalprotein 51 13144 50 708- QAVNHFTGI Portalproteinphageassociated 52 13144

TABLE-US-00007 TABLE7 Proteinscorrespondingtotheimmunogenic EHpeptidesequences SEQ namesofthe ID Group n Hirae sequence proteins No: 7 1 13144 TSLARFANI Phagetaillength 13 tape-measure protein 2 13144 AMIEFIQGL Phagetaillength 14 tape-measure protein 3 13144 VAITFGGPL Phagetaillength 15 tape-measure protein 4 13144 VSTNHYGLL hypotheticalprotein 16

[0222] The manufacturing of TMP1 specific tetramers allowed us to monitor TMP1-specific T cells in the mLN (FIG. 14A) and the spleen (FIG. 14B-C) in mice without tumor and post-oral gavages with EH13144, EH17, 10815 or IGR7 and CTX treatment. We observed that only T cells from the gut (CCR9+ T cells) are TMP1-specific T cells for EH13144 and IGR7 (FIG. 14). Moreover, in tumor bearing mice, TMP-specific tetramer stainings in tumor draining lymph node and tumor show that TMP epitope 1 prophage 2 specific CD8+ T cells accumulate in tumor beds and are contained in the CCR9+CXCR3+CTLs (FIG. 15).

[0223] Temperate bacteriophages are bacterial viruses that transfer virulence, antimicrobial resistance genes, and immunogenic sequences to new bacterial hosts via transduction (Weinbauer, 2004). The Phage Tail Length Tape Measure Protein (Tmp) is highly conserved in a large number of phages and prophages, most importantly in Siphoviridae family of phages, containing a variable number of tandem repeats with highly conserved tryptophan and phenylalanine aminoacids at fixed positions. Phages belonging to the Siphoviridae family contain several motifs in their Tmp (Belcaid et al., 2011; Piuri and Hatfull, 2006), among which peptidoglycan hydrolases facilitating their infectivity of surrounding bacteria and containing rescuscitationpromoting factors (Rpfs). Rpfs have not only been implicated in the reactivation of dormant bacteria but also modulate innate responses to Mycobacterium tuberculosis (Russell-Goldman et al., 2008) as well as cognate long-term immune responses (Commandeur et al., 2011). In fact, M. tuberculosis Rpfs T cell epitopes were reported to be key immunogens in the human immune responses to M. tuberculosis (Commandeur et al., 2011).

Example 4: Immunization with E. hirae 13144 Tmp Peptides or Live Bacteria Confer Protection Against Tumor Challenge

[0224] We performed a subcutaneous vaccination using 1.5.10.sup.5 BM-DC activated with poly I:C followed by exposition to TMP peptides or live E. hirae 13144 to immunize twice, 10 days apart, nave C57BL/6 animals against MCA205 sarcomas or syngeneic MC38 colon cancers (FIG. 16). While unpulsed BM-DC or BM-DC exposed to Group 1 peptides were not efficient at mediating protection against a lethal challenge with MCA205, BM-DC exposed to TMP1-TMP2 markedly conveyed protective effects against MCA205 (FIG. 17) as efficiently as live bacteria.

[0225] We hypothesized that oral gavages with E. hirae 13144 trigger an immune response in the mLN and spleen that can either traffick back to intestinal mucosae or in the tumor microenvironment. To link gut and tumor immunosurveillance, we compared the relative immune infiltrates induced by sequential oral gavages with E. hirae 13144 in naive versus tumor bearing mice. Indeed, we found that E. hirae 13144-induced CD8+ T cell-dependent colon inflammatory lesions (FIG. 11) were markedly reduced in tumor bearers, supporting the competition between the gut and tumor microenvironment during oral administration of the bacteria (FIG. 18).

Example 5: E. hirae 13144 Genome Encodes Two 40.6-Kb and 39.2-Kb Prophage Sequences of the Siphoviridae Family

[0226] Strain EH13144 and four other E. hirae genomes (708, 10815, 13152 and EH17) were annotated using Prokka program. The homology relationships between genes of each different strain were assessed using BLASTP program and Roary software with >80% amino acid identity cutoff. Core genome alignment of the five E. hirae genomes was performed using PRANK program. Prophage regions were predicted using PHASTER online program.

[0227] Comparative analysis of E. hirae strains yielded a pangenome of 12,748 genes. The core genome (the set of genes shared by all strains) was composed of 2,036 orthologous genes (59%) and the accessory genome (the set of genes present in some but not all the species) was composed of 570 orthologous genes and 923 unique genes (unique genes to individual species). Strain EH13144 encoded 196 unique genes while strains 13144 and 708 shared 27 orthologous proteins (FIG. 19A).

[0228] A particularity in the genome of E. hirae 13144 is that it encodes two intact prophages regions (40.6-kb and a 39.2-kb) showing sequence homology to Enterococcus phage phiEf11 and Staphylococcus phage CNPx, respectively (FIG. 19B). The 40.6-kb prophage encoded 57 genes, including 12 shared between the five genomes, 16 shared between strains 13144 and 708 and 8 unique to E. hirae 13144 (FIG. 20A). The 39.2-kb prophage encoded 65 genes, including 7 shared between the five genomes, 3 shared between strains 13144 and 708 and 22 unique to E. hirae 13144 (FIG. 20B). Genomic structures of both prophages suggested that they belonged to the Siphoviridae family. Indeed, the two prophages harbor genes encoding capsid, portal and tail structure, a characteristic of Siphoviridae phages. The two prophages encoded a tail tape measure protein (TMP) with a 38% amino acid homology. The TMP of the 40.6-kb prophage presented sequence homology with E. hirae, Enterococcus villorum and Enterococcus faecium prophage TMPs with 98.8%, 96.7%, and 97.7% amino acid identity, respectively. The TMP of the 39.2-kb prophage showed sequence homology with only EH clone IGR7 (CNCM I-5224) with 100% homology (FIG. 21, Table 8) and to a lesser extent with E. faecalis prophage TMP with 89.2% amino acid identity. Despite sequence homologies with other phage genes, the whole two prophages were uniquely encoded in the strain 13144. Finally, both TMP1 and TMP2 peptides are part of the same phage tail tape measure protein (TMP) gene of the second prophage (SEQ ID No: 2). TMP1 of 13144 presented 100% homology with IGR7 and IGR11 but 88.89% homology with 10815 and IGR1 (1 mutation in position 3). TMP2 of 13144 presented 100% homology only with IGR7 and 77.78% homology with 10815 and IGR11 (2 mutations in position 2 and 7 or 2 and 4 respectively) (FIG. 21, Table 8).

TABLE-US-00008 TABLE 8 summary of the characteristics of the strains Homology with Epitope TMP1 Epitope TMP2 TMP Protein TMP Protein Anti-tumor (Prophage (Prophage (prophage (prophage Strains effect no 2) no 2) no 1) no 2) 13144 Yes 708 No No homology No homology 98.72% No homology EH17 (13344) No No homology No homology 25.52% No homology 10815 (ATCC9790) No 88.8889% 77.778% 24.03% 66.38% (1 mutation) (2 mutations) IGR 1 No 88.8889% No homology 99.04% 74.77% (1 mutation) IGR 4 Yes No homology No homology 99.15% No homology IGR 7 Yes 100% 100% 100% 100% IGR 10 No No homology No homology No homology No homology IGR 11 Yes 100% 77.778% No homology 66.52% (2 mutations)

Example 6: E. hirae 13144 Bacterial and Phage Sequences have been Occasionally Found in the MG Reference Catalog Containing >3,000 Metagenomes in Humans

[0229] A total of 3027 metagenomes from 17 different datasets were screened (FIG. 29A). They werefor the large majoritygut microbiomes (stools) but also oral cavity, skin, vagina, and sputum. Most of the datasets are publicly available covering all continents, including several non-westernized populations. We also included some of Nicola Segata's unpublished data (most notably of 25 paired mother/infant subjects followed longitudinally). We assessed the breadth of coverage (BOC) of the E. hirae genome and its phages in each of the sample. The BOC measures the fraction of the genome that is covered by the reads in the metagenomes. A BOC of 1.0 means that the whole genome of the reference strain is found in the metagenome. Based on this score, we could confidently conclude that E. hirae is present for sure in two samples. One is from a mongolian subject, the other from a Swedish one (BOC of 0.9 and 0.75 respectively). There are other 5 samples with a BOC between 0.1 and 0.37 that are probably indicative of E. hirae presence. Below a BOC of 0.1, we fail to characterize the presence of a E. hirae strain. As for the phages sequences, it seems that the mongolian subject positive for E. hirae also has prophage 2 from 13144 of SEQ ID No: 2, whereas the Swedish one has prophage 2 from 708. The phages seem to be present in more samples than the bacterium genome. One interesting case is the presence at 0.66 BOC of the Phage 13144 in three samples from an infant at 1, 3, and 7 days of life (samples CA_C100061S2084FE_t1M15,CA_C100061S2087FE_t2M15,CA_C100061S2 091FE_t3M15). These three samples do not have E. hirae, but seem to have another strain from another Enterococcus species. This might suggest that this phage is not specific of E. hirae only.

Example 7: E. hirae 13144 is Immunogenic not Only in Mice but Also in Humans (Normal Volunteers and Breast Cancer Patients): Defining TMP-Specific Peptides of High Immunogenicity in Humans

[0230] Since E. hirae 13144 is a mouse strain but its sequence has been recovered in study alignment with human stool metagenomics, we postulated that humans (normal volunteers or breast cancer patients) could develop immune responses against some of the epitopes presented by this strain, as reported in (Daillre et al., 2016). FIG. 22A shows that 12 to 19% of CD4 and CD8+ T cells can display memory IFN-geared T cell responses to E. hirae 13144, suggesting that indeed, this EH strain can be recognized by human cells. FIG. 22A also highlights that 32% of BC patients have CD4+ T cells exhibiting no reactivity to monocytes pulsed with EH13144 while 52% developed IL-10 producing memory CD4+ T cell responses. This is in sharp contrast with the reactivity of the memory CD4+ TH1 cells to E. coli and TCR cross-linking (FIG. 22B). The predicted peptides of Tmp to be tested in human PBMC or TILs are listed in Table 9 below, and their localization has been described in FIG. 23. Are indicated their binding potential for MHC class I alleles (threshold set at <50 nM, Y axis) assessed using NetMHC software. For each peptide and allele, symbols represent the first amino acid of the identified sequence corresponding to 9 amino acid long (mer) peptides. Next, we tested all HLA-A2-restricted Tmp epitopes on PBMC by in vitro stimulation assays of recall responses (FIG. 24A) from six HLA-A2+ healthy volunteers (HV) (FIG. 24C) and found that 5 peptides (indicated in FIG. 24B) exhibited significant reactivities at least in 50% of HV (FIG. 24 D-E). Arrows indicate the most significant epitopes (FIG. 24F-G).

TABLE-US-00009 TABLE9 PredictedpeptidesofTmpforeachHLAhaplotype SEQ ID Start Stop HLA Peptide Affinity(nM) No: 968 976 HLA-A0101 YTDYSNQLK 35.98 53 357 365 HLA-A0201 AMIEFIQGL 4.88 54 1462 1470 HLA-A0201 KMVEILEEI 7.8 55 1397 1405 HLA-A0201 RLLKYDVGV 11.55 56 765 773 HLA-A0201 TLVGVTFAI 16.94 57 1374 1382 HLA-A0201 AMQNLVAAV 17.32 58 793 801 HLA-A0201 AIMAIANGV 20.56 59 862 870 HLA-A0201 AMSMNMEEV 24.83 60 504 512 HLA-A0201 KVFGKMTSV 26.84 61 1130 1138 HLA-A0201 LLGIYQSYV 29.4 62 631 639 HLA-A0201 KLAKFASVV 29.89 63 1176 1184 HLA-A0201 KLWANMSKA 30.99 64 692 700 HLA-A0201 MLSNPITAI 32.68 65 700 708 HLA-A0201 ILVAITTTI 36.32 66 491 499 HLA-A0201 KMAALAASA 46.27 67 691 699 HLA-A0201 AMLSNPITA 49.58 68 473 481 HLA-A0201 NMAEAFASA 49.85 69 835 843 HLA-A0301 TMFSDSALK 20.82 70 573 581 HLA-A0301 LLTRFTTLK 23.32 71 727 735 HLA-A0301 KTAFSGIVK 32.3 72 515 523 HLA-A0301 KTISTMFEK 47.18 73 505 513 HLA-A2402 VFGKMTSVF 40.33 74 904 912 HLA-A2403 AYFNHTLDL 4.83 75 714 722 HLA-A2403 AWKSNFMNI 5.04 76 427 435 HLA-A2403 RYGTTESQL 5.97 77 1136 1144 HLA-A2403 SYVNNGASI 10.38 78 556 564 HLA-A2403 KYKSNLAGL 13.47 79 505 513 HLA-A2403 VFGKMTSVF 14.88 80 729 737 HLA-A2403 AFSGIVKSF 19.19 81 967 975 HLA-A2403 VYTDYSNQL 23.45 82 1074 1082 HLA-A2403 AFQNQITQL 45.36 83 639 647 HLA-A3101 VINPIGSLR 19.59 84 1389 1397 HLA-A3101 KAKIKSPSR 23.3 85 515 523 HLA-A3101 KTISTMFEK 24.29 86 667 675 HLA-A3101 ASKAGGGFR 27.79 87 675 683 HLA-A3101 RTFAATGIR 28.02 88 611 619 HLA-A3101 GNKVTNFFR 35.72 89 1437 1445 HLA-A3101 ITGSRLIKR 39.31 90 401 409 HLA-A3301 DVFEGAVKR 33.42 91 476 484 HLA-A6801 EAFASADPK 6.97 92 401 409 HLA-A6801 DVFEGAVKR 7.96 93 835 843 HLA-A6801 TMFSDSALK 14.72 94 94 102 HLA-A6801 ESAFTGVKK 15.34 95 1419 1427 HLA-A6801 TSVAVQSAK 15.68 96 175 183 HLA-A6801 DTAATSLAR 15.91 97 358 366 HLA-A6801 MIEFIQGLK 17.45 98 1148 1156 HLA-A6801 MALLAGMLR 20.5 99 675 683 HLA-A6801 RTFAATGIR 28.54 100 730 738 HLA-A6801 FSGIVKSFK 29.62 101 252 260 HLA-A6801 EAGGSAFSR 29.95 102 1350 1358 HLA-A6801 TSVGSNMAK 31.53 103 515 523 HLA-A6801 KTISTMFEK 38.32 104 968 976 HLA-A6801 YTDYSNQLK 42.02 105 1463 1471 HLA-A6801 MVEILEEIR 42.29 106 639 647 HLA-A6801 VINPIGSLR 42.9 107 333 341 HLA-A6801 EDFANVTGR 43.39 108 773 781 HLA-A6801 IAGFVDGLR 47.53 109 1458 1466 HLA-B0702 TPMGKMVEI 24.41 110 572 580 HLA-B0801 NLLTRFTTL 4.7 111 1441 1449 HLA-B0801 RLIKRSNAI 16.31 112 182 190 HLA-B2705 ARFANITQM 30.73 113 674 682 HLA-B2705 FRTFAATGI 30.97 114 780 788 HLA-B2705 LRAIITVGK 47.66 115 182 190 HLA-B2720 ARFANITQM 14.78 116 1146 1154 HLA-B2720 QQMALLAGM 32.65 117 1441 1449 HLA-B2720 RLIKRSNAI 42.54 118 353 361 HLA-B3501 NPSQAMIEF 7.99 119 946 954 HLA-B3501 FANASTEYM 9.08 120 250 258 HLA-B3501 EAEAGGSAF 25.17 121 492 500 HLA-B3501 MAALAASAG 32.95 122 240 248 HLA-B3501 FAAALSSVG 33.38 123 495 503 HLA-B3501 LAASAGPVL 42.72 124 89 97 HLA-B3501 AAVKWESAF 48.3 125 1182 1190 HLA-B3901 SKADIVNTL 26.44 126 539 547 HLA-B3901 IKNGSSSAL 36.65 127 1018 1026 HLA-B3901 NKLNNNQAL 47.23 128 902 910 HLA-B4001 VEAYFNHTL 6.41 129 385 393 HLA-B4001 TEVRLRDSL 30.39 130 1092 1100 HLA-B4001 SELEQGAQL 39.74 131 385 393 HLA-B4002 TEVRLRDSL 42.24 132 143 151 HLA-B4402 AEAAGQLGI 18.68 133 752 760 HLA-B5801 KGLGNIFKW 6.09 134 797 805 HLA-B5801 IANGVKGLW 6.74 135 523 531 HLA-B5801 KAGNIDSKW 9.31 136 351 359 HLA-B5801 KSNPSQAMI 12.06 137 619 627 HLA-B5801 RSFSASLQL 13.45 138 395 403 HLA-B5801 RAANASDVF 37.43 139 898 906 HLA-B5801 STAGVEAYF 41.32 140 693 701 HLA-B5801 LSNPITAIL 42.57 141 627 635 HLA-B5801 LSNSKLAKF 48.23 142 946 954 HLA-00303 FANASTEYM 3.56 143 350 358 HLA-00303 FKSNPSQAM 4.75 144 495 503 HLA-00303 LAASAGPVL 5.93 145 324 332 HLA-00303 EASKASGSL 6.31 146 445 453 HLA-00303 VAITFGGPL 9.21 147 459 467 HLA-00303 SAISAAKPM 9.9 148 412 420 HLA-00303 EAFNENTAL 10.33 149 1328 1336 HLA-00303 SANNAGREL 10.61 150 652 660 HLA-00303 AAGKSGTVL 11.56 151 844 852 HLA-00303 KAAKSTEEL 12.11 152 635 643 HLA-00303 FASVVINPI 12.38 153 677 685 HLA-00303 FAATGIRSI 13.26 154 395 403 HLA-00303 RAANASDVF 15.87 155 1142 1150 HLA-00303 ASIDQQMAL 19.24 156 463 471 HLA-00303 AAKPMIEAL 21.86 157 1284 1292 HLA-00303 NAKQKGAEL 23.31 158 789 797 HLA-00303 TAVNAIMAI 23.51 159 480 488 HLA-00303 SADPKTQEF 23.9 160 35 43 HLA-00303 NASDIPSNL 25.71 161 173 181 HLA-00303 SADTAATSL 26.05 162 478 486 HLA-00303 FASADPKTQ 31.85 163 250 258 HLA-00303 EAEAGGSAF 31.9 164 553 561 HLA-00303 FVSKYKSNL 33.06 165 89 97 HLA-00303 AAVKWESAF 36.44 166 685 693 HLA-00303 IASLTGAML 40.14 167 651 659 HLA-00303 SAAGKSGTV 41.97 168 449 457 HLA-00303 FGGPLVAAL 44.33 169 539 547 HLA-00303 IKNGSSSAL 45.59 170 480 488 HLA-00501 SADPKTQEF 18.54 171 107 115 HLA-00501 MVDSNGKVI 25.04 172 173 181 HLA-00501 SADTAATSL 29.14 173 580 588 HLA-00501 LKDTIVGLF 33.18 174 182 190 HLA-00602 ARFANITQM 34.48 175 182 190 HLA-00701 ARFANITQM 28.58 176 677 685 HLA-C1203 FAATGIRSI 7.86 177 635 643 HLA-C1203 FASVVINPI 15.83 178 1012 1020 HLA-C1203 FVEAGVNKL 40.2 179 946 954 HLA-C1203 FANASTEYM 41.29 180 1475 1483 HLA-C1203 VVMDTGQVV 45.03 181 504 512 HLA-C1203 KVFGKMTSV 47.93 182 904 912 HLA-C1402 AYFNHTLDL 7.99 183 1136 1144 HLA-C1402 SYVNNGASI 15.64 184 967 975 HLA-C1402 VYTDYSNQL 25.25 185 617 625 HLA-C1402 FFRSFSASL 31.08 186 619 627 HLA-C1502 RSFSASLQL 14.65 187

Example 8: Phage Excision in EH13144 In Vitro

[0231] Using a classical procedure to excise a phage and harvest it in the supernatants of the E. hirae (FIG. 25) (Duerkop et al., 2014), we could observe prophage 1 and 2 excision after incubation of EH13144 with mitomycin C (1 g/mI) but not CTX (FIG. 26A), at different temperatures (37 C. and 42 C.) regardless of expansion phase of the bacterium. After capside lysis of the supernatants (augmenting the sensitivity of phage DNA detection), prophages DNA were visualized in spontaneous conditions, suggesting that phages can also be released without excision stresses (FIG. 26B).

Example 9: Identification of Novel Strains Harboring the Gene Encoding the TMP of SEQ ID No: 1

[0232] The genomes of twenty different E. hirae strains (Table 10 below) were sequenced and aligned. Remarkably, 3 prophage regions, of which 2 regions are intact, were identified in the genome of the strain IGR7, which proved immunogenic (see example 1 above), and it was found that this genome encode the same TMP as the TMP of SEQ ID No: 1 encoded by E. hirae 13144 (Table 9). This strain was deposited at the CNCM on 12 Oct. 2017, under the number I-5224. Moreover, IGR11 which proved efficient to mediate some tumoricidal activity with CTX have also the same TMP1 epitope encoded by 13144 (Table 9). This strain was deposited at the CNCM on Nov. 27, 2017, at the CNCM under the number I-5261.

TABLE-US-00010 TABLE 10 No of No of Strain scaffolds Length GC % CDS 5348 4 2773687 36.84 2541 7030 2 2839464 36.72 2665 12607 3 2981483 36.62 2662 13150 3 2881518 36.76 2692 13153 1 2732232 36.84 2479 13155 9 2956728 36.56 2688 13161 2 2814115 36.90 2696 13343 2 2748768 37.01 2555 13346 3 2758418 36.98 2511 13347 3 2876247 36.71 2668 IGR1 2 2874823 36.73 2681 IGR4 3 2958768 36.71 2649 IGR7 2 2705237 36.84 2543 IGR10 2 2921736 36.70 2589 IGR11 3 2836600 36.91 2600 13144 1 2735684 36.96 2542 13152 1 2835470 36.92 2486 13344 1 2711996 37.12 2472 ATCC9790 1 2827741 36.94 2670 708 1 2951512 36.89 2613

Example 10: Cell Sorting and Expansion of TIL or Blood T Cells Specific for the TMP

[0233] Several protocols can be used to obtain TILs or T cells specific for the TMP of SEQ ID No: 1.

[0234] Protocol 1: Immunomagnetic Cell Sorting and Expansion of T Cell Sorted Populations

[0235] HLA-A*0201/TMP monomers (20 g/ml) are incubated for 1 h at room temperature with 6.7.Math.10.sup.6 streptavidin-coated beads (Dynabeads M-280 streptavidin, DYNAL, Compiegne, France) and washed in PBS/0.1% BSA. 5.10.sup.6 PBMC are rotated for 4 h at 4 C. with monomer-coated beads (Bodinier et al., 2000). After ten washes, bead coated cells are expanded using a polyclonal T cell stimulation protocol (Jotereau et al., 1991). Subsequently, cells are incubated with sheep anti-mouse IgG coated Dynabeads (Dynal Biotec, Compiegne, France) at a 1:1 ratio for 4 h at 4 C. with gentle rotation. The cell/bead suspension are incubated in culture medium in 6-well plates overnight at 37 C. to allow beads to detach. After overnight incubation, beads are extracted by the magnet, and sorted lymphocytes are transferred on feeder cells, as previously described (Jotereau et al., 1991). Briefly, 2000 bead-coated T cells/well are distributed in 96-well plates mixed with irradiated feeder cells [LAZ EBV-B cells (2.10.sup.4/well) and allogeneic PBMC (10.sup.5/well)], in 150 l of culture medium supplemented with IL-2 (150 U/ml) and PHA (15 g/ml).

[0236] Protocol 2

[0237] Tumor samples from cancer specimens are cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs are stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells (as described in Meng et al., 2016).

Example 11: Obtention of T Cells Specific for the TMP by Transduction of T Lymphocytes

[0238] Autologous or allogeneic T cells are placed in an expanding phase using anti-CD3/CD28 coated beads or low dose IL-2 (and IL-7, IL-15, IL-21) before being exposed to retroviral vectors or lentiviral vectors engineered to express the beta chain of the high avidity TCR encoding cDNA (for the TMP prophage 2 epitope corresponding to HLA-A2 or other haplotypes) and/or the alpha chain of the high avidity TCR encoding cDNA (for the TMP prophage 2 epitope corresponding to HLA-A2 or other haplotypes) and the CDR3 region of this TCR encoding cDNA (for the TMP prophage 2 epitope corresponding to HLA-A2 or other haplotypes). Polyclonal T cells are then cloned and tested for their specificity (IFNg or TN Fa release upon exposure to TMP epitopes) in 96 well plates. Cytokine-producing T cells are then selected and reexpanded with T cell growth factors (IL-2; IL-7, IL-15, IL-21) on a weekly basis for two to three weeks until expansion to 10.sup.10 to 10.sup.12 cells in GMP cell factories.

Discussion

[0239] Our findings revealed that the antigenicity of mouse EH 13144 and the newly cloned human EH IGR7 (CNCM I-5224) or EH IGR11 (both 100% homologous in their sequence) relies on the Phage Tail Length Tape Measure Protein (Tmp) of the Siphoviridae phages (lactococcal bacteriophage tail tape measure protein TP901 family) mainly in the 39.2-kb prophage encoded 65 genes, including 7 shared between the five genomes, 3 shared between strains 13144 and 708 and 22 unique to E. hirae 13144. Transfer of genetic materials inbetween enterococci species have been reported (Mazaheri Nezhad Fard et al., 2010, 2011). Of note, phages that infect Gram positive bacteria often contain peptidoglycan-hydrolysing motifs corresponding to peptidase and transglycosylase activities localized within tape measure proteins of Siphoviridae phages (Piuri and Hatfull, 2006). We will now attempt to restore immunogenicity of non-immunogenic E. hirae strains (EH17, 10815) by transducing these strains with bacteriophages belonging to the family of Siphoviridae, or with genetically modified plasmids encoding the whole Phage Tail Length Tape Measure Protein (Tmp) or epitopes selected from TMP prophage 2, and with negative controls (proteins or epitopes mutated in the MHC binding groove). Of note, M. tuberculosis Rpfs T cell antigens were reported to be important targets in the human immune responses to M. tuberculosis (Commandeur et al., 2011).

[0240] These mouse data have a clinical relevance. Based on in silico prediction (sequence alignment of the several strains of E. hirae, protein subcellular localization and algorithms of prediction of MHC binding affinities), a list of candidate epitopes harbouring a putative immunogenicity has been established in the first step of the selection (FIG. 23). In a second step, in vitro stimulation assays using peripheral blood mononuclear cells from HLA-A0201+ normal volunteers with 4 groups of 4 nine mer-TMP specific epitopes followed by a recall response using single 9 mer epitopes performed in 6 healthy volunteers revealed that 5 epitopes could be considered immunogenic (FIG. 24). Third step, a bioinformatic screening of the normal human proteome and gene expression in >350 tumors cell lines investigating sequence homologies between these 5 human immunogenic TMP epitopes and such proteomes concluded that one of this HLA-0201 epitope KLAKFASVV (SEQ ID No: 63), shares a 78% sequence homology with the glycerol-3 phosphate dehydrogenase 1 like protein (GPD1L, gene encoded on 3p22.3). GPD1L is a cytosolic protein, associated at the plasma membrane with a sodium channel, voltage-gated, type V alpha subunit (SCNSA). GPD1L is a hypoxia-associated protein negatively regulated by miR-210, overexpressed in many tissues (brain, during embryo- and foeto-genesis), and contributes to the proteosomal degradation of hypoxia inducible factor 1-alpha (HIF-1a). Indeed, MiR-210 represses levels of the glycerol-3-phosphate dehydrogenase 1-like (GPD1L) enzyme, contributing to suppression of prolyl hydroxylase (PHD) activity. Under normal physiological conditions, PHD hydroxylates prolines in hypoxia inducible factor 1-alpha (HIF-1), leading to its degradation by the proteasome. When PHD activity is suppressed due to downregulation of GPD1L by miR-210, HIF-1 is not degraded by the proteasome and translocates to the nucleus where it forms a heterodimer with hypoxia-inducible factor 1-beta (HIF-1); dimerization of HIF-1 and HIF-1 activates transcriptional responses that contribute to cancer metastasis (Costales et al., 2017). Hence, higher mRNA levels and/or protein expressions of GPD1L in head and neck squamous cell carcinoma compared with healthy surrounding tissues was an independent prognostic parameter and a favorable predictor of longer time to local recurrence and distant metastases in multivariate Cox regression analyses (Feng et al., 2014).

[0241] Since the binding affinity for HLA-A0201 of this phage peptide is similar to the one of normal tissue peptide, the phage peptide could be viewed as an immunogenic molecular mimick of the normal peptide. It is therefore conceivable that cross-reactivities between TMP phage specific TCR and self tissues or tumor tissues (overexpressing GPD1L), mainly foetal tissues or stem cells, could account for the anticancer effectiveness of the phage delivered in the context of this EH bacterium. Of note, tumor cell lines can express variable levels of GPD1L protein, as appears from gene expression atlas available on the web.

[0242] HLA-A0201 restricted-epitope TMP 2 (KMVEILEEI, SEQ ID No: 55), epitope 3 (RLLKYDVGV, SEQ ID No: 56), epitope 9 (LLGIYQSYV, SEQ ID No: 62), epitope 10 (KLAKFASVV, SEQ ID No: 63, homologous to sequences from GPD1L) and epitope TMP 13 (ILVAITTTI, SEQ ID No: 66) have been found so far (FIG. 24), enabling tetramer manufacturing, T cell capture in tumor bearers (human patients or mice) and structural and functional characterization. Reactivity of E. hirae-specific CTL clones from tumor infiltrating lymphocytes can be tested against a variety of syngeneic tumor cell lines to bring up the proof of principle of molecular mimicry between microbial and tumor antigens.

[0243] This asset accounts for the quasi-unique antigenicity of E. hirae 13144 exploitable for cancer vaccines and T cell transfer and prompts to the use of this phage to infect other bacterial strains for use as anticancer probiotics or OncoBax in combination with any drug identical or different from cyclophosphamide capable of enabling the niching of the dedicated species and/or stressing the excision of this phage. For instance, cyclophosphamide could allow the overrepresentation of E. hirae over its competitor E. gallinarum while the competition between these two strains promoted phage excision per se.

Example 12: Phage Tail Length Tape Measure Protein as the Unique Antigenic Sequence in E. hirae 13144

[0244] Unleashing immune responses against tumor-associated antigens through chemotherapy, radiotherapy, targeted therapies or immune checkpoint inhibitors has become the mainstay of successful cancer treatments (Galluzzi et al., 2015; Sharma and Allison, 2015). The recent discovery that the gut microbiota determines the cancer-immune set point, thus influencing the clinical outcome of anticancer therapies, has rekindled the concept that microbes or their products modulate not only intestinal but also systemic immunity (Zitvogel et al., 2018). Indeed, memory IFN producing CD4+(TH1) and CD8+(TC1) T cell responses directed against Enterococcus hirae, Bacteroides fragilis, and Akkermansia muciniphila are associated with favorable clinical outcome in cancer patients (Daillre et al., 2016; Rong et al., 2017; Routy et al., 2018; Vtizou et al., 2015), suggesting that pre-existing microbe-specific T cells may contribute to anticancer immune responses. However, the question how microbes may affect the development of systemic autoimmune disease or local intestinal chronic inflammation has not been resolved (Rose, 2017). The theory of molecular mimicry posits that T cells elicited by bacteria or viruses may accidentally recognize autoantigens as they escaped from self-tolerance inducing mechanisms (such as clonal deletion or inactivation). While MHC class I and class II binding epitopes from bacterial genomes have been identified to mediate immunogenicity in vitro or in vivo (Chai et al., 2017; Perez-Munoz et al., 2015; Rubio-Godoy et al., 2002; Vujanovic et al., 2007; Yang et al., 2014), very few reports have unarguably demonstrated the functional relevance of microbe-specific CD4+ or CD8+ T lymphocytes for immune responses against normal or neoplastic tissues (Balachandran et al., 2017; Bradley et al., 2017; Ji et al., 2010).

[0245] Cyclophosphamide (CTX) induces the translocation of E. hirae from the gut lumen to the mesenteric and splenic immune tissues, thereby eliciting specific CD4+ and CD8+ T lymphocytes producing IL-17 and IFN, correlating with therapeutically effective anticancer immune responses (Daillre et al., 2016; Viaud et al., 2013). Broad-spectrum antibiotics abolished the therapeutic efficacy of CTX unless E. hirae was supplied by oral gavage (Daillre et al., 2016). When comparing a panel of E. hirae strains (Table 11, FIG. 31A) for their capacity to restore the antibiotic-perturbed anticancer effects of CTX, we found that only a few E. hirae isolates (such as 13144, IGR7, and IGR11) were efficient (FIG. 27A-B, (Daillre et al., 2016)). Given that the therapeutic efficacy of the combination of CTX and E. hirae 13144 is abrogated by the depletion of CD8.sup.+ T cells or the neutralization of IFN (Daillre et al., 2016), we screened the differential capacity of E. hirae strains to elicit memory TC1 immune responses after a prime-boost exposure of the host (FIG. 27C), and ex vivo restimulation of splenic CD8.sup.+ T cells with various E. hirae strains loaded onto dendritic cells (DC). While E. hirae 13144 triggered specific TC1 immune responses (that were not cross-reactive against irrelevant Enterococci), E. hirae 708 (a prototypic inefficient strain) failed to do so (FIG. 27D). Pan genomic analysis of twenty E. hirae strains yielded a core genome of 1,677 orthologous genes (59%) and the accessory genome was composed of 946 and 477 orthologous and unique genes respectively (FIG. 31B). This phylogenomic analysis showed that the strain 13144 is 100% homologous to strain IGR7 with 86 orthologous unique genes (FIG. 31B).

TABLE-US-00011 TABLE 11 Description of E. hirae strains Species Samples Origin Cancer Patient outcome Enterococcus hirae 13144 MurineCTX-treated Enterococcus hirae 708 Human-Unknown Enterococcus hirae 13344 Human-Blood Enterococcus hirae Type strain CIP 53.48T ATCC9790 Enterococcus hirae IGR1 MAT-HE Human (stool) Lung Responder Enterococcus hirae IGR2 AND-CL Human (stool) Lung Responder Enterococcus hirae IGR3 BLO-VA Human (stool) Lung Responder Enterococcus hirae IGR4 SAI-GE Human (stool) Lung Complete Responder (HR selon RECIST) Enterococcus hirae IGR5 CAR-RO Human (stool) Lung Responder Enterococcus hirae IGR6 BOU-MO Human (stool) Lung Responder Enterococcus hirae IGR7 BOU-MO Human (stool) Lung Responder Enterococcus hirae IGR8 AND-CL Human (stool) Lung Responder Enterococcus hirae IGR9 AND-CL Human (stool) Lung Responder Enterococcus hirae ADO-EL Human (stool) Lung Responder IGR10 Enterococcus hirae ADO-EL Human (stool) Lung Responder IGR11 Enterococcus hirae TROJA Human (stool) Kidney Responder IGR12 Enterococcus hirae LOUNO Human (stool) Kidney Responder IGR13 Enterococcus hirae BOIAR Human (stool) CRC Responder IGR14 Enterococcus hirae NAIFE Human (stool) CRC Responder IGR15 Enterococcus hirae GIRAL Human (stool) CRC Responder IGR16 Enterococcus hirae LEZPH Human (Tumor juice) Melanoma Responder IGR17 Enterococcus hirae PRIMI Human (Tumor juice) Melanoma Responder IGR18 Enterococcus hirae NONRO Human (Tumor juice) Melanoma NA IGR19 Enterococcus hirae Lyon No 1 Human (Tumor juice) Melanoma NA IGR20 (CAR) Enterococcus hirae PRUPA Human (Skin swab) IGR21

[0246] Next, we performed sequence alignment of bacterial genes encoding cell wall and secreted proteins (PSORT software) for immunogenic (13144) versus non-immunogenic (708 and 13344) bacterial strains followed by the identification of nonapeptides with strong binding affinities for the MHC class I H-2K.sup.b protein (<50 nM binding affinity, NetMHC software) (Table 6 in Example 3). Subsequently, we recovered splenic CD8.sup.+ T cells from mice that had been repeatedly exposed to E. hirae 13144 and CTX (FIG. 27C). These T cells were restimulated in vitro with pools of potentially immunogenic nonapeptides from E. hirae 13144 (Table 6) to measure IFN production (FIG. 32), then splitting the most efficient group 7 into individual peptides (FIG. 27E). This approach led to the identification of one dominant epitope (one-letter amino acid code: TSLARFANI (SEQ ID No: 25), abbreviation TMP1) in position 197 to 187 of the amino acid sequence of the phage tail length tape measure protein (TMP) from a prophage of 39.2 kb (1506 aa) of E. hirae 13144 (FIG. 33A). Indeed, a particular genomic trait of E. hirae 13144 is that it encodes two intact prophages regions (40.6-kb and a 39.2-kb) showing weak sequence homologies with the most common Enterococcus phages phiEf11 vB EfaS IME197 (14% and 11% of shared genes respectively) (FIG. 31C, Table 12). Comparative analysis of the 39.2-kb prophage of E. hirae 13144 with 19 other sequenced E. hirae genomes showed 100% protein identity with IGR7 prophage proteins. The 39.2-kb prophage encodes 65 genes, including 1 shared between all genomes and 38 unique to E. hirae 13144 and IGR7 (FIG. 33B), encoding capsid, portal and tail structure, a characteristic of Siphoviridae phages. Importantly, the TMP1 epitope of the 39.2-kb prophage from E. hirae 13144, E. hirae IGR7 and E. hirae IGR11 showed 100% sequence homology (FIG. 34). As a matter of fact, E. hirae IGR7 and E. hirae IGR11 were as efficient as E. hirae 13144 in reducing the growth of MCA205 fibrosarcomas treated with CTX (FIG. 27B). In contrast, no homologies (observed in E. hirae 708 and 13344) and a mutation in position 3 of the TSLARFANI peptide (L.fwdarw.F observed in E. hirae ATCC9790, FIG. 34) correlated with a reduced anticancer effect of these E. hirae strains (FIG. 27B and (Daillre et al., 2016). Elispot assays designed to detect peptide-specific IFN-producing T cells revealed that mice gavaged with E. hirae 13144 (or strain E. hirae IGR7 and IGR11) mounted a TC1 response against TMP1 (but not the control peptides TMP2 and TMP3), while E. hirae strains devoid of anticancer activity and lacking TMP1 (strains 708, 13344, ATCC9790) were unable to do so (FIG. 27E). We used a fluorescent H-2K.sup.b/TSLARFANI tetrameric complex to detect the frequency and distribution of TMP1-specific cytotoxic T lymphocytes (CTLs) in naive and MCA205 fibrosarcoma bearing C57BL/6 mice. Up to 1% of splenic CD8.sup.+ T recognized the TMP1 peptide at day 12 following a CTXIE.hirae 13144 regimen (FIG. 27F, left panel) with a 3-5 fold enrichment of TMP1-specific CTLs in the TC1 subset harboring the ileal chemokine receptor CCR9 in spleens from naive (FIG. 27F, right panel) and tumor bearers, as well as in tumor draining lymph nodes (FIG. 27G). Of note, H-2K.sup.b/TSLARFANI tetramers also revealed a high proportion of splenic TMP1-specific CTLs after immunization with E. hirae strains IGR7 or IGR11 but not with ATCC9790 nor E. hirae 13344 (FIG. 27H).

TABLE-US-00012 TABLE 12 Seeking prophage sequences in E. hirae 13144 genomes Region # Total Region Region Length Completeness Score Proteins Position Most Common Phage GC % 1 40.6 Kb intact 150 58 481066- PHAGE_Entero_phiEf11_NC_013696(9) 33.79% 521729 2 39.2 Kb intact 140 59 2123983- PHAGE_Entero_vB_IME197_NC_028671(6) 34.95% 2163272

Example 13: Prophylactic and Therapeutic Immunization Using Phage Tail Length Tape Measure Protein in a Mouse Model

[0247] To explore the capacity of TMP1-specific H-2K.sup.b restricted TC1 cells to control the growth of MCA205 cancers, we subcutaneously (s.c.) immunized naive C57BL/6 mice with dendritic cells (DC) loaded with heat-inactivated E. hirae (positive control), the naturally occurring TSLARFANI peptide from 13144, IGR7 or IGR11, its L.fwdarw.F mutant from E. hirae ATCC9790 (mut3, FIG. 34) or other non-immunogenic bacterial peptides (group 1, FIG. 32). In this prophylactic setting, DC pulsed with TSLARFANI (but not mut3 TSFARFANI) were as efficient as the whole E. hirae extract in preventing or restraining tumor outgrowth (FIG. 28A-B). Next, we explored whether the TMP1 peptide would be able to confer immunogenicity to the usually inefficient bacterium Escherichia coli strain DH5a in the therapeutic setting, in which antibiotic treatment is followed by gavage with different bacterial strains and CTX-based chemotherapy (FIG. 27A, (Daillre et al., 2016)). E. coli engineered to express the TSLARFANI (SEQ ID No: 13) peptide (FIG. 35) was as efficient as E. hirae 13144 in restraining MCA205 tumor growth (FIG. 28C) and eliciting the generation of tetramer binding CTL in the spleen (FIG. 28D). In contrast, E. coli expressing an irrelevant sequence (encoding mouse EGFP protein) or a mutant peptide bearing a S.fwdarw.A exchange in the anchor position 2 (mut2, TALARFANI) or the rnut3 TMP1 peptide failed to induce such a cancer-protective immune response (FIG. 28C-D).

Example 14: Clinical Relevance of the Enterophage's Effects in Cancer Patients

[0248] We next explored the possible pathophysiological relevance of these findings. We screened a total of 3027 adult and mother-infant metagenomes (mostly from human stools but also from various mucosae) from 17 publicly available datasets to assess the breadth of coverage (BOC) of the E. hirae genome and its phages (FIG. 29A). E. hirae was present with 100% confidence in 13 samples from disparate geography, age and datasets. In other 40 cases, strains closely related to E. hirae were detectable. In 90% of the samples in which E. hirae was found, one of the three phages (from E. hirae 13144, 708 or 13344) sequence were inserted in the genome in a mutually exclusive fashion (FIG. 29A). However, the E. hirae 13144 phage was detectable in many samples lacking the presence of the E. hirae core genome, suggesting that other bacteria than E. hirae can host this phage as well. We could detect the presence of the phage 13144 at 0.66 BOC in three mother-infant paired stool specimens and in the infant at 1, 3, and 7 days after birth. Contrasting with metagenomics analyses that are unable to detect low-abundant species, culturomics followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) provides a technology for detecting even rare E. hirae colonies in the stool of healthy individuals (Samb-Ba et al., 2014) or cancer patients (Routy et al., 2018). PCR analyses of each single cultivatable enterococcal colony (up to 5 per species and individual, representing colonies from E. hirae, E. gallinarum, E. durans, E. faecium, E. faecalis, E. casseliflavus or E. avium) from 76 cancer patients led to the detection of the TMP sequence encompassing the TMP1 peptide (FIG. 36) in 34% of the patients, mostly inside E. faecalis (FIG. 29B). Renal and lung cancer patients with detectable fecal TMP at diagnosis exhibited a prolonged overall survival after therapy with immune checkpoint inhibitors (FIG. 29C). Therefore, we screened sixteen TMP-derived nonapeptides predicted to bind the human MHC class I HLA-A02*01 with high affinity (SEQ ID Nos: 54-69) for their ability to prime naive CD8.sup.+ T cells from six healthy volunteers in vitro. We found 6 out of 16 epitopes capable of triggering significant peptide-specific IFN release that were located in two distinct regions of the TMP protein (504-708 and 1397-1462, FIG. 29D, FIG. 37, FIG. 24B, Table 9). Using the NCBI BLASTP suite, we searched the human peptidome associated with significant expression levels in human cancer biopsies (TOGA database) for a high degree of homology with these 6 HLA-A02*01-restricted immunogenic nonapeptides. We found that only peptide KLAKFASVV (631-639, SEQ ID No: 63) shared an 78% homology (7 out of nine 9 positions, with identical amino acids at the MHC anchoring positions 2 and 9) with a peptide contained in the protein glycerol-3-phosphate dehydrogenase 1-like GPD1-L (FIG. 29E). GPD1-L reportedly counteracts the oncogenic HIF1-dependent adaptation to hypoxia and is associated with favorable prognosis in head and neck squamous cell carcinomas (Feng et al., 2014; Kelly et al., 2011; Liu et al., 2014). To our knowledge, no mutation in the GPD-1L gene has been reported in cancers. The TOGA transcriptomics database unveiled that high expression of GPD1-L is associated with improved overall survival in bladder, lung adenocarcinoma and in a large cohort of >500 kidney cancers (with a trend towards a worse prognosis in tumors with low GPD-1L that do not express HLA-02*01) (FIG. 29F-H). Moreover, high expression of GPD1-L mRNA by tumors at diagnosis was associated with improved progression-free survival in two independent cohorts of non-small cell lung cancer (NSCLC) patients treated with anti-PD1/PDL-1 Abs (FIG. 291, J). Interestingly, expression levels of GPD-1L correlated with the cytotoxic lymphocytes, myeloid dendritic cells, neutrophils and endothelial cells gene signature in TOGA dataset of lung cancer (LUAD, LUSC and TOGA) and in the two cohorts of NSCLC patients (CHUM and CGFL) (FIG. 29K).

Example 15: Molecular Mimicry Between Enterophade TMP and the Oncogenic Driver PSMB4 in Mouse Cancers

[0249] In order to identify the mechanism by which TMP1 exerted its anticancer activity in the mouse model (MCA205 tumors in C57/B6 mice, FIG. 27-28), we investigated whether there exist any H-2Kb-restricted mouse tumor antigens with high homology to the TMP1 peptide (TSLARFANI, SEQ ID No: 13). Using the NCBI BLASTP suite we found that the peptide (GSLARFRNI, SEQ ID No: 189) belonging to the proteasome subunit beta type-4 (PSMB4) between amino acid positions 76-84 shared an 78% homology (7 out of 9 amino acid, with identical amino acids at the MHC anchoring positions 2 and 9) (FIG. 30A). We queried the potential neoepitopes of MCA205 but found no significant homology that would explain our results, hence our focus on the non-mutated PSMB4 peptide. PSMB4 is an oncogenic driver involved in proliferation and invasion (Lee et al., 2014) in a variety of malignancies such as glioblastoma (Cheng et al., 2018), melanoma (Zhang et al., 2017) and breast cancers (Wang et al., 2018), associated with dismal prognosis (Cheng et al., 2018; Lee et al., 2014; Wang et al., 2018). CRISPR/Cas9-mediated genomic knock-in of the PSMB4 sequence replacing GSLARFRNI (SEQ ID No: 189) by GSFARFRNI (SEQ ID No: 190) (with an L.fwdarw.F exchange in position 3 equivalent to mut 3 of TSLARFANI) in MCA205 cells (FIG. 38) slowed down spontaneous tumor growth kinetics but drastically blunted the anticancer effects of E. hirae 13144 while not interfering with the CTX treatment alone (FIG. 30B). These results support the idea that the TSLARFANI TMP1 peptide encoded by E. hirae 13144 indeed induces a therapy-relevant response against the PSMB4-derived GSLARFRNI peptide.

Example 16: Capacity of E. hirae 13144 to Contagiously Disseminate the Phage TMP Sequence

[0250] Temperate bacteriophages are bacterial viruses that transfer virulence, antimicrobial resistance genes, and immunogenic sequences to new bacterial hosts (Weinbauer, 2004). The TMP protein, which contains a variable number of tandem repeats with highly conserved tryptophan and phenylalanine residues at fixed positions is encoded by the genome of Siphoviridae phages (Belcaid et al., 2011; Piuri and Hatfull, 2006). To analyze the capacity of E. hirae 13144 to contagiously disseminate the phage TMP sequence, we performed culturomic analyses of the ileal content from C57BL/6 mice subjected to oral gavage with E. hirae 13144 and systemic CTX therapy (FIG. 30C). We tested 7 to 18 bacterial colonies from each animal to discover that, out of 76 colonies, E. gallinarum was the only by-stander Enterococci transduced by the E. hirae-born phage in vivo, as confirmed by sequencing of the phage genome (FIG. 30D, E and FIG. 39). Of note, none of the 90 colonies (mostly of E. gallinarum) isolated from naive mice harbored the TMP sequence (FIG. 39). Moreover, CTX plays a role in eliminating the E. gallinarum, allowing the niching and/or colonization of E. hirae in the small intestine, as shown in vitro in enteroid systems or in PCR of the ileal content (FIG. 30G and not shown). Importantly, incubation of small intestine enteroids with a balanced 1:1 ratio of E. hirae or E. gallinarum harbouring or not the phage respectively, lead to the transmission of the infectious/lytic phage to E. gallinarum by E. hirae in 9% of colonies at 6 hrs and 26% at 20 hrs (FIG. 30G).

[0251] In contrast to cyclophosphamide, CDK4/6 inhibitors (such as palbociclib) appear to trigger phage excision in breast cancer patients. Indeed, shot gun metagenomics analyses of patients stools harvested prior to surgery in 10 patients treated by 3 week-palbociclib, versus 73 non treated patients revealed a drastic enrichment and over-representation of phages sequences (from Lactococcus, Salmonella, Sodalis, Escherichia, enterobacteria) as shown in the LefSe diagramm (FIG. 30H).

[0252] This observation suggests that the peptide encoding phage exhibiting this molecular mimicry with cancer antigens is infectious, in line with its detection in E. faecalis in humans.

[0253] To the best of our knowledge, these results represent the first demonstration that an enterococcal phage codes for an MHC class I-restricted antigen, TMP1, that induces a memory TC1 immune response, which then cross-reacts with cancer antigens, following three major lines of evidence. First, naturally occurring (mut3) or artificial mutations (mut2 or mut3) introduced into the MHC class I-binding TMP1 epitope suppressed the tumor-prophylactic and therapeutic potential of the phage-bearing E. hirae strains. Second, transfer of the TMP1-encoding gene into E. coli conferred immunogenic capacity to this proteobacterium, which acquired the same antitumor properties as TMP1-expressing E. hirae. Third, when cancer cells were genetically modified to remove the TMP1-crossreactive peptide within the PSMB4 protein, they formed tumors that could no longer be controlled upon oral gavage with TMP1-expressing E. hirae.

[0254] Discussion 2

[0255] Phages are among the most abundant biological entities on earth. Their numbers have been estimated to reach as high as 10.sup.31 particles with the potential of 10.sup.25 phage infections occurring every second (Pedulla et al., 2003; Wommack and Colwell, 2000). The antigenicity of the enterophage studied here resides in hot spots of the TMP protein. Beyond their structural role in determining the length of the phage tail, TMP encoded by phages from the Siphoviridae family contains several functional domains, one of which has peptidoglycan hydrolase activity, facilitating efficient infection of bacteria, and another one with lysozyme activity, acting as resuscitation-promoting factor, Rpf (Duerkop et al., 2014). Rpfs have not only been implicated in the reactivation of dormant bacteria but also modulate innate responses to Mycobacterium tuberculosis (Russell-Goldman et al., 2008) and cognate long-term immune responses (Commandeur et al., 2011). In fact, T cell epitopes contained in M. tuberculosis Rpfs are key for the human immune response against this pathogen (Commandeur et al., 2011). Beyond their specific antigenic properties, phages convey broad adjuvanticity to DNA vaccines through filamentous bacteriophage coat protein III domain I (Cuesta et al., 2006; Larsen et al., 2008). Thus, the perspective opens that bacteriophages may enrich the therapeutic armamentarium for stimulating anticancer immune responses.

Abbreviations Used in this Text

ATB: Antibiotic

[0256] BHI: Brain heart infusion
BM-DC: Bone marrow dendritic cell
cDNA: complementary deoxyribonucleic acid

CGFL: Centre Georges Francois Leclerc

CHUM: Centre Hospitalier Universitaire de Montreal

CTL: Cytotoxic Tcell

Ctrl: Control

CTX: Cyclophosphomide

[0257] DC: Dendritic cell
DNA: Deoxyribonucleic acid
GM-CSF: Granulocyte-macrophage colony-stimulating factor
GPD1-L: Glycerol-3-phosphate dehydrogenase 1-like
IFN: Interferon gamma

IMDM: Iscove's Modified Dulbecco's

Medium

Ip: Intraperitoneal

[0258] MALDI-TOF: Matrix-assisted laser desorption ionization time-of-flight
MHC: Major histocompatibility complex
NaCl: Sodium chloride
NSCLC: Non-small cell lung cancer
PBMC: Peripheral blood mononuclear cell
PCR: Polymerase chain reaction
PSMB4: Proteasome subunit beta type-4
PVDF: Polyvinylidene difluoride
RCC: Renal cell carcinoma
Rpf: Resuscitation-promoting factor

RPMI: Roswell Park Memorial Institute

Sc: Subcutaneous

[0259] SEM: Standard error of mean
Tc1: T cytotoxic cell type 1
Th1: T helper cell type 1
TLR3: Toll-like receptor 3
TMP: Tape-measure protein

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