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WO2023041574A1 - Compositions comprenant des souches bactériennes - Google Patents

Compositions comprenant des souches bactériennes Download PDF

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Publication number
WO2023041574A1
WO2023041574A1 PCT/EP2022/075514 EP2022075514W WO2023041574A1 WO 2023041574 A1 WO2023041574 A1 WO 2023041574A1 EP 2022075514 W EP2022075514 W EP 2022075514W WO 2023041574 A1 WO2023041574 A1 WO 2023041574A1
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Prior art keywords
level
cells
bacterial strain
predetermined threshold
intratumoural
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PCT/EP2022/075514
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English (en)
Inventor
Marsilio ADRIANI
Anna ETTORRE
Imke MULDER
Aurelie Couturier
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4D Pharma Research Limited
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Priority claimed from GBGB2204110.7A external-priority patent/GB202204110D0/en
Application filed by 4D Pharma Research Limited filed Critical 4D Pharma Research Limited
Publication of WO2023041574A1 publication Critical patent/WO2023041574A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7014—(Neo)vascularisation - Angiogenesis

Definitions

  • This invention is in the field of bacterial strains isolated from the mammalian digestive tract and the use of such strains, and compositions comprising these strains, in the treatment of disease.
  • the human intestine is thought to be sterile in utero, but it is exposed to a large variety of maternal and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host genotype, all of which impact upon the composition of the gut microbiota, particularly during early life.
  • the human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2],
  • the successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions.
  • the enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host.
  • the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5].
  • Reference [13] teaches that Enterococcus gallinarum treats cancer. Enterococcus gallinarum has been demonstrated to have anti-tumour efficacy in multiple cancer models and to induce activation of CD8 + T cells. Reference [14] teaches that Enterococcus hirae also enhances anticancer immune responses.
  • patients do not always respond to therapy. There is an urgent need to understand which patients are most likely to respond to therapy with Enterococcus strains in order to identify patients who are likely to derive benefit from the therapy, and thus to tailor the therapy to the patient.
  • the inventors have developed methods of identifying patients who are expected to respond to therapy with Enterococcus, for example, Enterococcus gallinarum or a related bacterium.
  • the invention is based on the inventors' unexpected finding that patients are more likely to respond to therapy with an Enterococcus strain if, prior to therapy, they have a certain minimum level of T regulatory cells (for example CD3 + Foxp3 + ) or proliferating T cells (for example CD3 + Ki67 + ) in the tumour tissue compartment (intratumoural level), and/or a certain maximum level of macrophages (for example CD68 + ) in either the total tumour region (comprising the intratumoural region and the stromal region) or the stromal region.
  • T regulatory cells for example CD3 + Foxp3 +
  • proliferating T cells for example CD3 + Ki67 +
  • macrophages for example CD68 +
  • bacterial strains of the genus Enterococcus for example strains having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, have immunotherapeutic effects in the treatment of cancer, in particular when the pre-treatment intratumoural levels of T regulatory cells and/or proliferating T cells exceed a given level, and/or when the macrophages in the intratumoural region or stromal region are below a given level.
  • tumour T regulatory cells are thought to drive resistance to a number of anticancer therapies including radiotherapy [16], adoptive cell therapy [17], anticancer vaccines [18] and hormone suppression therapy [19],
  • radiotherapy [16]
  • adoptive cell therapy [17]
  • anticancer vaccines [18]
  • hormone suppression therapy [19]
  • the inventors have now demonstrated that even in patients with an intratumoural level of T regulatory cells above a given threshold, treatment with an Enterococcus strain is particularly effective, despite the fact that high intratumoural T regulatory cell levels are expected to cause resistance to anticancer therapies.
  • the inventors have demonstrated that treatment with an Enterococcus strain is particularly effective in patients with a high intratumoural level of proliferating T cells. Again, this is unexpected given that high levels of intratumoural CD8 + Ki67 + T cells have been associated with higher tumour burden [20], The inventors' findings are therefore expected to be applicable to a wide range of patients with intratumoural levels of T regulatory cells and/or proliferating T cells above a given level, and/or intratumoural or stromal levels of macrophages below a given level, even if such patients have previously exhibited or developed resistance to one or more other anticancer therapies. Therapy with the bacterial strain can therefore be targeted to the patients most likely to benefit in accordance with the invention.
  • strains of the invention have an excellent safety and tolerability profile; typically where a patient exhibits resistance to cancer therapy, they will be moved onto other lines of therapy which may have increasingly unfavourable side effects.
  • the present invention provides clinicians and patients with a new and attractive treatment option.
  • the invention provides a bacterial strain of the genus Enterococcus for use in a method of treating cancer, wherein the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the invention also provides a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6 for use in a method of treating cancer, wherein the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the first predetermined threshold may be at least about 4 cells per square millimetre. References to the number of cells per square millimetre refer to the number of cells in a sample or section of the tumour obtained from the patient that is analysed microscopically, for example as described herein below. In some embodiments, the first predetermined threshold is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 cells per square millimetre. Alternatively, the first predetermined threshold may be calculated as described herein below.
  • the second predetermined threshold may be at least about 0.2 cells per square millimetre. In some embodiments, the second predetermined threshold is at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 cells per square millimetre. Alternatively, the second predetermined threshold may be calculated as described herein below.
  • the third predetermined threshold may be no more than about 30 cells per square millimetre. In some embodiments, the third predetermined threshold is no more than about 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 cells per square millimetre. Alternatively, the third predetermined threshold may be calculated as described herein below.
  • the bacterial strain may be administered to a patient with a cancer which is resistant or refractory to anticancer therapy, or to a patient who is in relapse after treatment with anticancer therapy.
  • strains of the invention have been clinically demonstrated as being well tolerated which is especially beneficial for treating patients who are resistant or refractory to prior lines of therapy.
  • cancer is defined as resistant or refractory to an anticancer therapy if the patient does not respond to the anticancer therapy after a period of 3 months or more, for example 6 months or more, 9 months or more, 12 months or more, 18 months or more, or 24 months or more.
  • cancer is defined as not having responded to the anticancer therapy if the patient has progressive disease (PD) after the defined treatment period.
  • PD progressive disease
  • cancer may be resistant to the anticancer therapy at the start of treatment with the anti cancer therapy, or it may become resistant during treatment.
  • a patient is described as being in relapse after treatment with an anticancer therapy if the patient's disease initially responded to therapy (e.g.
  • a subject who is non-responsive to therapy relates to a subject who has progressive disease after being treated with the therapy for a normal treatment cycle.
  • Response criteria are preferably defined according to the RECIST (Response Evaluation Criteria In Solid Tumours) criteria vl.l [21],
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the invention provides a method of treating or preventing cancer, comprising administering a bacterial strain of the genus Enterococcus, or a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, to a patient in need thereof, wherein the patient has, prior to administration of the bacterial strain:
  • the T regulatory cells express CD3 and Foxp3 (CD3 + Foxp3 + ). In some embodiments, alternatively or in addition to CD3 and Foxp3, the T regulatory cells express CD4 (CD4 + ). In some embodiments, alternatively or in addition to CD3 and Foxp3, the T regulatory cells express CD25 (CD25 + ). Alternatively or additionally, in some embodiments, the T regulatory cells do not express CD127, or express low levels of CD127 (CD127 -/lo ), for example as described in References [25] and [26], Alternatively or additionally, in some embodiments, the T regulatory cells express IL- 10 (IL-10 + ).
  • the proliferating T cells express CD3 and Ki67 (CD3 + Ki67 + ). In some embodiments, alternatively or in addition to CD3 and/or Ki67, the proliferating T cells express the minichromosome maintenance protein (MCM-2). In some embodiments, alternatively or in addition to CD3, Ki67 and/or MCM-2, the proliferating T cells express the proliferating cell nuclear antigen protein (PC AD) [22],
  • the macrophages express CD68 (CD68 + ). In some embodiments, alternatively or in addition to CD68, the macrophages express CD14 (CD14 + ). In some embodiments, alternatively or in addition to CD68, the macrophages express CD11b (CD11b + ). In some embodiments, the macrophages express the markers HLA-DR ⁇ , iNOS, CD11c, CD80, CD86 and/or pSTAT-1. For example, the macrophages may express HLA-DR ⁇ , iNOS, CD11c, CD80, CD86 and pSTAT-1. Preferably, in such embodiments, the macrophages also express CD68.
  • the macrophages express the markers CD 163, CD204, CD206, cMAF and/or VEGF.
  • the macrophages may express CD 163, CD204, CD206, cMAF and VEGF.
  • the macrophages also express CD68.
  • the level of T regulatory cells, proliferating T cells and/or macrophages in the sample of the tumour obtained from the patient may be assessed by multiplex immunofluorescence staining and image analysis.
  • the multiplex immunofluorescence staining and image analysis is preferably conducted using tissue samples that have been formalin-fixed and paraffin-embedded (FFPE).
  • the tissue samples are at least 10 mm x 2 mm in size.
  • the thickness of the tissue sample may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ⁇ m or more.
  • the tissue sample is 4 ⁇ m thick.
  • the tumour cells account for at least 10% of the cells in the biopsy specimen.
  • a minimum threshold of 100 malignant cells is present in the tissue sample.
  • Malignant cells may be identified by any suitable marker for the cancer from which the tissue sample is taken.
  • any necrotic areas in the tissue sample should be excluded from the analysis.
  • any mucus- secreting areas in the tissue sample should be excluded from the analysis.
  • image analysis is carried out using InForm 2.4.8 image analysis software (Akoya Biosciences).
  • multiplex immunofluorescence staining and image analysis may be carried out as described in Example 6.
  • multiplex immunofluorescence staining and image analysis with tyramide signal amplification may be carried out, for example according to the guidance provided in Reference [23] or Reference [24],
  • the level of T regulatory cells, proliferating T cells and/or macrophages is assessed by detecting cellular expression of particular markers associated with each cell type. For example:
  • the level of T regulatory cells may be assessed by detecting cellular expression of CD3 and Foxp3;
  • the level of proliferating T cells may be assessed by detecting cellular expression of CD3 and Ki67;
  • the level of macrophages may be assessed by detecting cellular expression of CD68.
  • any of the markers listed above for (a) T regulatory cells, (b) proliferating T cells and/or (c) macrophages may be substituted for, or used in addition to, (a) CD3 and Foxp3, (b) CD3 and Ki67, or (c) CD68, respectively.
  • the bacterial strain may have a chromosome with at least 95% sequence identity to SEQ ID NO: 3 across at least 90% of SEQ ID NO: 3.
  • the bacterial strain may have a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus.
  • the bacterial strain may have a 16s rRNA gene sequence that is at least 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:6.
  • the bacterial strain has a 16s rRNA gene sequence that is at least 99.5% or 99.9% identical to SEQ ID NO:2 or SEQ ID NO:6. Even more preferably, the bacterial strain has the 16s rRNA gene sequence of SEQ ID NO:2 or SEQ ID NO:6.
  • the bacterial strain is preferably of the genus Enterococcus. More preferably, the Enterococcus strain is of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae. Even more preferably, the Enterococcus strain is of the species Enterococcus gallinarum.
  • a preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 or NCIMB 42761.
  • the bacterial strain is flagellated (i.e. the cells have at least one flagellum).
  • Bacterial strains which are not of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae but which are closely related (e.g. a biotype strain) may also be used.
  • Such a bacterial strain may have a 16s rRNA that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae.
  • the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 1, 2 or 6.
  • the sequence identity is to SEQ ID NO:2 or SEQ ID NO:6, more preferably SEQ ID NO:2.
  • the bacterial strain has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to SEQ ID NO: 1 , 2 or 6.
  • Such bacterial strains will have comparable effects compared to Enterococcus gallinarum.
  • a comparable effect in this context means, for example, that the bacterial strain in combination with LPS can increase the expression of the inflammatory cytokine TNF- ⁇ in immature dendritic cells at least three fold, at least four fold or at least five fold, at least 10 fold, at least 20 fold, at least 50 fold or at least 100 fold when compared to a control experiment in the absence of the bacterial strain.
  • a comparable effect means that bacterial strain in combination with LPS can increase the expression of IL-6 in immature dendritic cells at least three fold, at least four fold, at least five fold, at least 10 fold, at least 20 fold, at least 50 fold or at least 100 fold when compared to a control experiment in the absence of the bacterial strain. Suitable assays for measuring this are known in the art and are also described in Example 4.
  • the inventors have shown that the bacterial strains described herein are useful for treating a range of cancers.
  • the invention is particularly useful in the treatment of cancer, preferably solid tumours.
  • suitable cancers which can be treated include renal cell carcinoma, melanoma, lung cancer, for example non-small cell lung cancer, bladder cancer, breast cancer and pancreatic cancer.
  • the Examples demonstrate that the bacterial strains described herein have a positive effect in such cancers, as discussed in Example 6.
  • the bacterial strain may be for use in a method of reducing tumour size, reducing tumour growth, preventing metastasis or preventing angiogenesis.
  • the bacterial strain may be for use in a method of treating cancer by decreasing the intratumoural level of T regulatory cells.
  • the bacterial strain may be included in a composition, which may optionally comprise one or more pharmaceutically acceptable excipients or carriers.
  • the composition may be for oral administration. Oral administration of the strains of the invention can be effective for treating cancer. Also, oral administration is convenient for patients and practitioners and if required, may optionally allow delivery to and / or partial or total colonisation of the intestine.
  • the bacterial strain is viable.
  • the bacterial strain is capable of partially or totally colonising the intestine.
  • the bacterial strain may be lyophilised.
  • it is the Enterococcus strain which is lyophilised.
  • a composition for use according to the invention may comprise a single strain of Enterococcus, such as a single strain of Enterococcus gallinarum, Enterococcus casseliflavus, or Enterococcus hirae.
  • the composition may comprise the bacterial strain as part of a microbial consortium.
  • the invention also provides a method of assessing the expected response of a patient having cancer to therapy with a bacterial strain of the genus Enterococcus, or a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, the method comprising:
  • the intratumoural level of T regulatory cells in the sample is greater than or equal to a first predetermined threshold
  • the intratumoural level of proliferating T cells is greater than or equal to a second predetermined threshold
  • the total tumour level of macrophages is less than or equal to a third predetermined threshold; wherein, if the intratumoural level of T regulatory cells in the sample is greater than or equal to a first predetermined threshold, the intratumoural level of proliferating T cells is greater than or equal to a second predetermined threshold, and/or the total tumour level of macrophages is less than or equal to a third predetermined threshold, the patient is assessed as being expected to respond to therapy with the bacterial strain.
  • the method may further comprise preparing the bacterial strain for administration to the patient having an intratumoural level of T regulatory cells greater than or equal to a first predetermined threshold, an intratumoural level of proliferating T cells greater than or equal to a second predetermined threshold, and/or a total tumour level of macrophages less than or equal to a third predetermined threshold.
  • the method comprises a step of administering the bacterial strain to the patient.
  • the therapeutic administration of the bacterial strain to the patient may comprise any of the features of the therapeutic uses described above.
  • the invention also provides a method of monitoring a patient, wherein said patient has cancer, said method comprising:
  • step (b) determining one or more of (i) the intratumoural level of T regulatory cells; (ii) the intratumoural level of proliferating T cells; and/or (iii) the total tumour level of macrophages in one or more biopsies obtained from a patient, optionally the one or more biopsies of step (a);
  • the first, second and third predetermined thresholds may be the same as the thresholds described above or may be calculated as described herein below.
  • the therapeutic administration of the bacterial strain to the patient may comprise any of the features of the therapeutic uses described above.
  • the invention further provides the bacterial strain or composition comprising a bacterial strain for use as described above, wherein the bacterial strain or the composition is administered to a patient identified by a method comprising:
  • administering the bacterial strain or the composition if: (i) the intratumoural level of T regulatory cells is determined to be greater than or equal to the first predetermined threshold; (ii) the intratumoural level of proliferating T cells is determined to be greater than or equal to the second predetermined threshold; and/or (iii) the total tumour level of macrophages is determined to be less than or equal to the third predetermined threshold.
  • the invention also provides the bacterial strain for use in the manufacture of a medicament for the treatment of cancer.
  • the invention provides a bacterial strain of the genus Enterococcus for use in the manufacture of a medicament for the treatment of cancer, wherein the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the invention also provides a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6 for use in the manufacture of a medicament for the treatment of cancer, wherein the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:
  • the Enterococcus strain may be of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae.
  • Figure 1 Mouse model of breast cancer - tumour volume.
  • Figure 2 Mouse model of lung cancer - tumour volume.
  • Figure 3 Mouse model of liver cancer - liver weight.
  • FIG. 4A Cytokine levels (pg/ml) in immature dendritic cells (No bacteria).
  • FIG. 4B Cytokine levels (pg/ml) in immature dendritic cells after the addition of LPS.
  • FIG. 4C Cytokine levels (pg/ml) in immature dendritic cells after the addition of NCIMB 42488.
  • Figure 4D Cytokine levels (pg/ml) in immature dendritic cells after the addition of NCIMB 42488 and LPS.
  • Figure 5A Cytokine levels in THP-1 cells (No bacteria).
  • Figure 5B Cytokine levels in THP-1 cells after addition of bacterial sediment.
  • FIG. 5C Cytokine levels in THP-1 cells after the addition of NCIMB 42488 alone or in combination with LPS.
  • FIG. 6 Cancer cell quantification and characterisation.
  • Tumour cells were identified by staining for expression of the cytokeratin (CK) marker.
  • the number of malignant cells (CK + ), proliferating tumour cells (CK + Ki67 + ) and tumour cells expressing PD-1 (CK + PD-1 + ) or PD-L1 (CK + PD-L1 + ) per mm 2 were evaluated in the tumour epithelial compartment (Tumour, i.e. the intratumoural level), the tumour stroma compartment (Stroma, i.e. the stromal level), or in the entire area scanned (Total, i.e. the total tumour level).
  • PR partial response
  • SD stable disease
  • PD progressive disease.
  • FIG. 7 T cell quantification and characterisation.
  • the number of T cells (CD3 + cells), proliferating T cells (CD3 + Ki67 + cells) and T cells expressing PD-1 (CD3 + PD1 + ), PD-L1 (CD3 + PDL1 + ) or both markers (CD3 + PD1 + PD-L1 + ) per mm 2 were evaluated in the tumour epithelial compartment (Tumour), the tumour stroma compartment (Stroma), or in the entire area scanned (Total). Mean +/- SEM; Mann-Whitney t-test.
  • Figure 8 T cell subset quantification and characterisation.
  • Figure 9 Macrophage quantification and characterisation.
  • the number of total macrophages (CD68 + cells), and macrophages expressing PD-1 (CD68 + PD-1 + ), PD-L1 (CD68 + PD-L1 + ) per mm 2 were evaluated in the tumour epithelial compartment (Tumour), the tumour stroma compartment (Stroma), or in the entire area scanned (Total).
  • FIG. 10 T cell quantification and characterisation.
  • the number of T cells (CD3 + cells), CD8 + cytotoxic T cells (CTL; CD3 + CD8 + cells), and proliferating T cells (CD3 + Ki67 + cells) per mm 2 were evaluated in the tumour epithelial compartment (Tumour) and the tumour stroma compartment (Stroma). Mean +/- SEM; Mann-Whitney t-test.
  • FIG 11 Flow cytometry of peripheral blood mononuclear cells (PBMCs) from paired on-treatment samples for renal cell carcinoma (RCC) pateints.
  • PBMCs peripheral blood mononuclear cells
  • RCC renal cell carcinoma
  • the bacterial strain may be administered to a patient having, in a sample of the tumour obtained from the patient prior to administration of the bacterial strain: (i) an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold; and/or (ii) an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold; and/or (iii) a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • tumours In addition to malignant cells, tumours contain many different cell types and noncellular factors.
  • the tumour stroma is an important part of the tumour microenvironment and affects tumour initiation, progression and metastasis.
  • the intratumoural region is defined as a group or nest of malignant cells found within the tumour.
  • the intratumoural region may comprise infiltrating lymphocytes, such as CD3 + Foxp3 + T regulatory cells (e.g. CD3 + Foxp3 + cells) and proliferating T cells (e.g. CD3 + Ki67 + cells).
  • the stromal region is defined as the stroma area between tumour cells.
  • the stroma comprises the basement membrane, fibroblasts, extracellular matrix, immune cells (including macrophages), and vasculature.
  • the total tumour region comprises both the intratumoural and stromal compartments.
  • Cells in contact with or within a group of malignant cells are consided as part of the intratumoural region, and cells between groups or nests of malignant cells are considered as a part of the stromal region, for example as described in References [23] and [24], As explained below and in reference [24], tumour markers may be used to assist in differentiating the intratumoural region from the stromal region.
  • tumor epithelium cells (including T regulatory cells, proliferating T cells and macrophages) that are in contact with or within the tumour epithelium are defined as "intratumoural ", whereas cells that are present in the interstitial space or in the stromal areas are defined as "stromal”.
  • the "total tumour” level of a particular type of cell refers to the combined total of the intratumoural and stromal cells.
  • the “total tumour level of macrophages” refers to the sum of the intratumoural and the stromal macrophages.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold. In an embodiment, the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold. In an embodiment, the bacterial strain is administered to a patient having a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold and an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold and a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold and a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold and an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold, and optionally a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold, an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold, and a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, a stromal level of macrophages that is below a fourth predetermined threshold, which may optionally be the same as the third predetermined threshold.
  • the bacterial strain is administered to a patient having, prior to administration of the bacterial strain, an intratumoural level of macrophages that is below a fifth predetermined threshold, which may optionally be the same as the third and/or fourth predetermined threshold.
  • the first predetermined threshold is at least about 4 cells, about 5 cells, about 6 cells, about 7 cells, about 8 cells, about 9 cells, about 10 cells, about 11 cells, about 12 cells, about 13 cells, or about 14 cells per square millimetre. In an embodiment, the first predetermined threshold is at least 4 cells per square millimetre, for example at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5 or 14 cells per square millimetre.
  • the first predetermined threshold is at least 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, or 5.9 cells per square millimetre. In an embodiment, the first predetermined threshold is at least about 5 or 5.5 cells per square millimetre. In an embodiment, the first predetermined threshold is at least about 9.5 or 10 cells per square millimetre.
  • the second predetermined threshold is at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 cells per square millimetre. In one embodiment, the second predetermined threshold is at least about 1 cell, about 2 cells, about 3 cells, about 4 cells, about 5 cells, about 6 cells, about 7 cells, about 8 cells, about 9 cells, or about 10 cells per square millimetre. In an embodiment, the second predetermined threshold is at least about 0.5 cells, 1 cell, 1.5 cells, 2 cells, or 2.5 cells per square millimetre.
  • the second predetermined threshold is any non-zero value of cells per square millimetre, for example at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cell per square millimetre. In an embodiment, the second predetermined threshold is at least about 1 cell per square millimetre, for example at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,
  • the third predetermined threshold is no more than about 30 cells per square millimetre. In an embodiment, the third predetermined threshold is no more than about 30, 25, 20, 15,
  • the third predetermined threshold is no more than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12,
  • the fourth predetermined threshold is no more than about 35 cells per square millimetre. In an embodiment, the third predetermined threshold is no more than about 35, 30, 25, 20,
  • the third predetermined threshold is no more than about 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 cells per square millimetre.
  • the fourth predetermined threshold is the same as the third predetermined threshold.
  • the fifth predetermined threshold is no more than about 30 cells per square millimetre. In an embodiment, the third predetermined threshold is no more than about 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 cells per square millimetre. In an embodiment, the third predetermined threshold is no more than about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 cells per square millimetre. In an embodiment, the fifth predetermined threshold is the same as the third and/or fourth predetermined thresholds.
  • about may refer to any value which is within +/- 0.5 of the given integer.
  • about 1 cell may be 0.5 to 1.5 cells, about 2 cells may be 1.5 to 2.5 cells, and so on.
  • “about” may refer to any value which is within +/- 0.05 of the given value, for example about 0.5 cells may be 0.45 to 0.55 cells, about 1.5 cells may be 1.45 to 1.55 cells, and so on.
  • the level of T regulatory cells, proliferating T cells, and/or macrophages in a sample of the tumour may be determined by staining the cells and analysing the image to count the number of cells in at least one region of the sample.
  • the number of cells in each tumour region may be assessed by multiplex immunofluorescence staining followed by image analysis.
  • the multiplex immunofluorescence staining and image analysis is preferably conducted using tissue samples that have been formalin-fixed and paraffin-embedded (FFPE).
  • the tissue samples are at least 10 mm x 2 mm in size.
  • the thickness of the tissue sample may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more ⁇ m.
  • the tissue sample is 4 ⁇ m thick.
  • the tumour cells account for at least 10% of the cells in the biopsy specimen.
  • a minimum threshold of 100 malignant cells is present in the tissue sample.
  • Malignant cells may be identified by any suitable marker for the cancer from which the tissue sample is taken.
  • any necrotic areas in the tissue sample should be excluded from the analysis.
  • any mucus-secreting areas in the tissue sample is excluded from the analysis.
  • Each region of the tissue sample that is assessed may be at least 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 square millimetres in size.
  • each region of the tissue sample that is assessed may be about 931 x 698 ⁇ m in size.
  • the resolution used may be 20X.
  • the number of cells in a sample is determined by counting the number of cells in at least two regions of the sample and averaging the number of cells to obtain the average number of cells per square millimetre.
  • the average is the mean, median, or modal value.
  • the average is the mean value.
  • the level of cells in a sample may be determined by counting the number of cells in at least three, four, five six, seven, eight, nine, or ten regions of the sample.
  • the number of cells in at least five regions is counted.
  • the regions of the sample may be overlapping but preferably do not not overlap, or may not substantially overlap. For example the area of overlap may be no more than half the total area of each region.
  • multiplex immunofluorescence staining and image analysis may be carried out as described in Example 6.
  • multiplex immunofluorescence staining and image analysis with tyramide signal amplification may be carried out, for example according to the guidance provided in Reference [23] or Reference [24],
  • Cells may be counted manually or automatically. For example, cells may be counted automatically using a computer programme.
  • the InForm image analysis software from Akoya Biosciences is used.
  • Tumour samples may be stained with one or more labels, for example an optically active dye label, a fluorophore, a quantum dot, an enzymatic label, or an isotope marker.
  • fluorescent markers are used.
  • Each label may be specifically attached to one cell marker, for example CD3 or Foxp3.
  • each cell marker is associated with a different label.
  • CD3 may be labelled with a red fluorescent label and Foxp3 may be labelled with a green fluorescent label, such that a cell labelled with both red and green labels may be categorised as a CD3 + Foxp3 + T regulatory cell.
  • Samples may additionally be stained with a label which is specific for a tumour cell marker, to verify that the sample is from a tumour and/or to differentiate the intratumoural and stromal regions.
  • cytokeratin may be used as a marker for epithelial tumours; glial fibrillary acidic protein may be used as a marker for glioblastoma; SOX10/S100 may be used as a marker for melanoma; and vimentin may be used as a marker for sarcoma.
  • markers for other types of cancer are known, or can be readily identified by characterising the markers expressed in the tumour cells of the tissue sample. Cells in contact with or within a group of malignant cells are consided as part of the intratumoural region, and cells between tumour nests are considered as a part of the stromal region, for example as described in References [23] and [24], Most preferably, levels of cells are determined using the methods described in Example 6.
  • the T regulatory cells express CD3 and Foxp3 (CD3 + Foxp3 + ). In some embodiments, alternatively or in addition to CD3 and Foxp3, the T regulatory cells express CD4 (CD4 + ). In some embodiments, alternatively or in addition to CD3 and Foxp3, the T regulatory cells express CD25 (CD25 + ). Alternatively or additionally, in some embodiments, the T regulatory cells do not express CD127, or express low levels of CD127 (CD127 -/lo ), for example as described in References [25] and [26], Alternatively or additionally, in some embodiments, the T regulatory cells express IL- 10 (IL-10 + ). Alternatively or additionally, in some embodiments, the T regulatory cells do not express CD49d, or express low levels of CD49d (CD49d -/lo ), for example as described in Reference [27],
  • the proliferating T cells express CD3 and Ki67 (CD3 + Ki67 + ). In some embodiments, alternatively or in addition to CD3 and/or Ki67, the proliferating T cells express the minichromosome maintenance protein (MCM-2). In some embodiments, alternatively or in addition to CD3, Ki67 and/or MCM-2, the proliferating T cells express the proliferating cell nuclear antigen protein (PC AD) [22],
  • the macrophages express CD68 (CD68 + ). In some embodiments, alternatively or in addition to CD68, the macrophages express CD14 (CD14 + ). In some embodiments, alternatively or in addition to CD68, the macrophages express CD11b (CD11b + ). In some embodiments, the macrophages express the markers HLA-DR ⁇ , iNOS, CD11c, CD80, CD86 and/or pSTAT-1. For example, the macrophages may express HLA-DR ⁇ , iNOS, CD11c, CD80, CD86 and pSTAT-1. Preferably, in such embodiments, the macrophages also express CD68.
  • the macrophages express the markers CD 163, CD204, CD206, cMAF and/or VEGF.
  • the macrophages may express CD 163, CD204, CD206, cMAF and VEGF.
  • the macrophages also express CD68.
  • a T regulatory cell may be defined as a cell which expresses CD3 and Foxp3 and/or one or more further T regulatory cell markers selected from the group consisting of CD4, CD25, and/or IL-10, and/or which does not express CD127 or expresses low levels of CD127, for example as described in Reference [25] or [26], and/or which does not express CD49d or expresses low levels of CD49d, for example as described in Reference [27],
  • cells that express one or more, two or more, three or more, four or more, or all of CD3, Foxp3, CD4, CD25 and/or IL- 10 above a background level may be classified as T regulatory cells.
  • a proliferating T cell may be defined as a cell which expresses CD3 and Ki67 and/or one or more further proliferating T cell markers selected from the group consisting of MCM-2 and PC AD.
  • cells that express one or more, two or more, three or more, or all of CD3, Ki67, MCM-2 and PC AD above a background level may be classified as proliferating T cells.
  • the proliferating T cells express CD4.
  • the proliferating T cells express CD8.
  • the proliferating T cells do not express CD8.
  • a macrophage may be defined as a cell which expresses CD68 and/or one or more further macrophage markers selected from the group consisting of CD14, CD11b, HLA-DR ⁇ , iNOS, CD11c, CD80, CD86, pSTAT-1, CD163, CD204, CD206, cMAF and/or VEGF.
  • the level of T regulatory cells, proliferating T cells and/or macrophages is assessed by detecting cellular expression of particular markers associated with each cell type.
  • Detecting cellular expression of a marker is preferably by immunohistochemistry, for example immunofluorescence staining, in particular multiplex immunofluorescence staining, for example as described above.
  • marker expression may also be detected by other methods known in the art.
  • cellular expression of a marker may be detected by fluorescence activated cell sorting (FACS), RNAscope and digital spatial profiling (DSP) technologies such GeoMx (Nanostring) and Visium (10xGenomics).
  • FACS fluorescence activated cell sorting
  • DSP digital spatial profiling
  • the thresholds described herein in cells per square millimetre can be converted to a volumetric threshold by multiplying the area in square millimetres by the thickness of the tissue section (preferably 4 ⁇ m).
  • a volumetric threshold may similarly be used for flow cytometry based techniques, based on the volume of the sample that is processed.
  • the thresholds defined herein in square millimetres may be directly compared with the equivalent volumetric threshold.
  • a background level of any of the cell markers disclosed herein may be defined with reference to a cell population that does not express the marker in question.
  • endogenous and exogenous autofluorescence can be excluded during the image analysis by comparison with a control sample from the same tissue type which is stained in parallel: either with all the antibodies but no fluorophores; or with all the fluorophores but no antibodies; or without antibodies or fluorophores.
  • samples from the tumour of interest can be used to determine the background level of fluorescence for each antibody and the dynamic range of its fluorophore expression.
  • the first, second, third, fourth, and/or fifth predetermined thresholds may be determined based on a reference population comprising patients that have been treated with the bacterial strain as described herein.
  • the first predetermined threshold may be determined by:
  • pre-treatment sample (b) determining the intratumoural level of T regulatory cells in a sample of the tumour taken from each patient prior to administration of the bacterial strain ("pre-treatment sample");
  • the reference population may comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients.
  • the average may be the mean, median, or modal level of cells, and is preferably the mean level.
  • the method for determining the first predetermined threshold may include a further step between steps (d) and (e) of determining the average intratumoural level of T regulatory cells in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • the determination of the first predetermined threshold according to step (e) may optionally comprise determining a threshold that is greater than the average pre-treatment level of intratumoural T regulatory cells in the patients who did not respond to therapy with the bacterial strain, but less than the average level of intratumoural T regulatory cells in the patients who did respond to therapy with the bacterial strain.
  • the threshold is determined as being the mid-point between the average pre-treatment level of intratumoural T regulatory cells in the patients who did not respond to therapy with the bacterial strain, and the average level of intratumoural T regulatory cells in the patients who did respond to therapy with the bacterial strain, wherein the mid-point is the mean of the two averages.
  • step (d) comprises determining the average intratumoural level of T regulatory cells in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • step (e) comprises determining a first predetermined threshold that is less than the average level of intratumoural T regulatory cells in the patients who did respond to therapy with the bacterial strain, for example wherein the first predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre less than the average level of intratumoural T regulatory cells in the patients who did respond to therapy with the bacterial strain.
  • the second predetermined threshold may be determined by:
  • pre-treatment sample (b) determining the intratumoural level of proliferating T cells in a sample of the tumour taken from each patient prior to administration of the bacterial strain ("pre-treatment sample");
  • determining a second predetermined threshold that is greater than the average level of intratumoural proliferating T cells in the patients who did not respond to therapy with the bacterial strain for example wherein the second predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre greater than the average level of intratumoural proliferating T cells in the patients who did not respond to therapy with the bacterial strain.
  • the reference population may comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients.
  • the average may be the mean, median, or modal level of cells, and is preferably the mean level.
  • the method for determining the second predetermined threshold may include a further step between steps (d) and (e) of determining the average intratumoural level of + proliferating T cells in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • the determination of the second predetermined threshold according to step (e) may optionally comprise determining a threshold that is greater than the average pre-treatment level of intratumoural proliferating T cells in the patients who did not respond to therapy with the bacterial strain, but less than the average level of intratumoural proliferating T cells in the patients who did respond to therapy with the bacterial strain.
  • the threshold is determined as being the mid-point between the average pre-treatment level of intratumoural proliferating T cells in the patients who did not respond to therapy with the bacterial strain, and the average level of intratumoural proliferating T cells in the patients who did respond to therapy with the bacterial strain, wherein the mid-point is the mean of the two averages.
  • step (d) comprises determining the average level of intratumoural proliferating T cells in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • step (e) comprises determining a second predetermined threshold that is less than the average level of intratumoural proliferating T cells in the patients who did respond to therapy with the bacterial strain, for example wherein the second predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre less than the average level of intratumoural proliferating T cells in the patients who did respond to therapy with the bacterial strain.
  • the third predetermined threshold may be determined by:
  • pre-treatment sample determining the total tumour level of macrophages in a sample of the tumour taken from each patient prior to administration of the bacterial strain
  • determining a third predetermined threshold that is less than the average level of total tumour macrophages in the patients who did not respond to therapy with the bacterial strain for example wherein the third predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre less than the average level of total tumour level of macrophages in the patients who did not respond to therapy with the bacterial strain.
  • the reference population may comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients.
  • the average may be the mean, median, or modal level of cells, and is preferably the mean level.
  • the method for determining the third predetermined threshold may include a further step between steps (d) and (e) of determining the average total tumour level of macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • the determination of the third predetermined threshold according to step (e) may optionally comprise determining a threshold that is less than the average pre- treatment level of total tumour macrophages in the patients who did not respond to therapy with the bacterial strain, but greater than the average level of total tumour macrophages in the patients who did respond to therapy with the bacterial strain.
  • the threshold is determined as being the mid-point between the average pre-treatment level of total tumour macrophages in the patients who did not respond to therapy with the bacterial strain, and the average level of total tumour macrophages in the patients who did respond to therapy with the bacterial strain, wherein the mid-point is the mean of the two averages.
  • step (d) comprises determining the average level of total tumour macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • step (e) comprises determining a third predetermined threshold that is greater than the average level of total tumour macrophages in the patients who did respond to therapy with the bacterial strain, for example wherein the third predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre greater than the average level of total tumour macrophages in the patients who did respond to therapy with the bacterial strain.
  • a patient is defined as not having responded to therapy if, at least 6 months after the start of the therapy, the patient is classified as having progressive disease (PD). In one embodiment, a patient is defined as having responded to therapy if, at least 6 months after the start of therapy, the patient is classed as having a complete remission (CR), partial remission (PR), or stable disease (SD). In some embodiments, the patient is classified as having responded to therapy if the patient has any response to the therapy other than progressive disease.
  • PD progressive disease
  • CR complete remission
  • PR partial remission
  • SD stable disease
  • the patient is classified as having responded to therapy if the patient has any response to the therapy other than progressive disease.
  • Response criteria are preferably defined according to the RECIST (Response Evaluation Criteria In Solid Tumours) criteria v1.1 [21], but may also be defined according to the irRECIST (immune-related Response Evaluation Criteria In Solid Tumours) criteria [28], or other criteria appropriate for the cancer in question.
  • the fourth predetermined threshold may be determined by:
  • determining a fourth predetermined threshold that is less than the average level of stromal macrophages in the patients who did not respond to therapy with the bacterial strain for example wherein the fourth predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7,
  • the reference population may comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients.
  • the average may be the mean, median, or modal level of cells, and is preferably the mean level.
  • the method for determining the fourth predetermined threshold may include a further step between steps (d) and (e) of determining the average stromal level of macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • the determination of the fourth predetermined threshold according to step (e) may optionally comprise determining a threshold that is less than the average pre- treatment level of stromal macrophages in the patients who did not respond to therapy with the bacterial strain, but greater than the average level of stromal macrophages in the patients who did respond to therapy with the bacterial strain.
  • the threshold is determined as being the mid- point between the average pre-treatment level of stromal macrophages in the patients who did not respond to therapy with the bacterial strain, and the average level of stromal macrophages in the patients who did respond to therapy with the bacterial strain, wherein the mid-point is the mean of the two averages.
  • step (d) comprises determining the average level of stromal macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • step (e) comprises determining a fourth predetermined threshold that is greater than the average level of stromal macrophages in the patients who did respond to therapy with the bacterial strain, for example wherein the fourth predetermined threshold is at least 0.5, 1, 1.5, 2,
  • the fifth predetermined threshold may be determined by:
  • pre-treatment sample determining the intratumoural level of macrophages in a sample of the tumour taken from each patient prior to administration of the bacterial strain
  • determining a fifth predetermined threshold that is less than the average level of intratumoural macrophages in the patients who did not respond to therapy with the bacterial strain for example wherein the fifth predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre less than the average level of total tumour level of intratumoural macrophages in the patients who did not respond to therapy with the bacterial strain.
  • the reference population may comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more patients.
  • the average may be the mean, median, or modal level of cells, and is preferably the mean level.
  • the method for determining the fifth predetermined threshold may include a further step between steps (d) and (e) of determining the average intratumoural level of macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • the determination of the fifth predetermined threshold according to step (e) may optionally comprise determining a threshold that is less than the average pre-treatment level of intratumoural macrophages in the patients who did not respond to therapy with the bacterial strain, but greater than the average level of intratumoural macrophages in the patients who did respond to therapy with the bacterial strain.
  • the threshold is determined as being the mid- point between the average pre-treatment level of intratumoural macrophages in the patients who did not respond to therapy with the bacterial strain, and the average level of intratumoural macrophages in the patients who did respond to therapy with the bacterial strain, wherein the mid-point is the mean of the two averages.
  • step (d) comprises determining the average level of intratumoural macrophages in the pre-treatment samples taken from the patients who did respond to therapy with the bacterial strain.
  • step (e) comprises determining a fifth predetermined threshold that is greater than the average level of intratumoural macrophages in the patients who did respond to therapy with the bacterial strain, for example wherein the fifth predetermined threshold is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre greater than the average level of intratumoural macrophages in the patients who did respond to therapy with the bacterial strain.
  • the bacterial strain is administered to a patient having, in a sample of the tumour obtained from the patient prior to administration of the bacterial strain: (i) an intratumoural level of T cells (e.g. CD3 + cells) greater than or equal to a sixth predetermined threshold; (ii) an intratumoural level of CD8-expressing T cells greater than or equal to a seventh predetermined threshold (e.g. CD3 + CD8 + cells); and/or (iii) an intratumoural level of proliferating CD8-expressing T cells greater than or equal to an eighth predetermined threshold (e.g. CD3 + CD8 + Ki67 + cells.
  • the sixth, seventh and/or eighth predetermined thresholds may be determined based on a reference population in substantially the same manner described above in relation to the first, second, third, and/or fourth predetermined thresholds.
  • the sixth predetermined threshold is at least about 30 cells per square millimetre, such as at least about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 cells per square millimetre.
  • the seventh predetermined threshold is at least about 8 cells per square millimetre, such as at least about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, or 50 cells per square millimetre.
  • the eighth predetermined threshold is at least about 0.2 cells per square millimetre, such as at least about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5 cells per square millimetre.
  • the bacterial strain may be administered to a patient having, in a sample of peripheral blood mononuclear cells (PBMCs) obtained from the patient prior to administration of the bacterial strain, circulating CD4 + CTLA-4 + cells at a frequency of no more than 0.25, as measured by flow cytometry.
  • the frequency of circulating CD4 + CTLA-4 + cells is no more than 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01, as measured by flow cytometry.
  • the frequency of circulating CD4 + CTLA-4 + cells is about 1.8, as measured by flow cytometry.
  • the sample of PBMCs is a whole blood sample.
  • the present invention provides a method of monitoring a patient, wherein said patient has cancer, said method comprising:
  • step (b) determining one or more of (i) the intratumoural level of T regulatory cells; (ii) the intratumoural level of proliferating T cells; and/or (iii) the total tumour level of macrophages in one or more biopsies obtained from a patient, optionally the one or more biopsies of step (a);
  • bacterial strain of the genus Enterococcus or a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, if: (i) the intratumoural level of T regulatory cells is determined to be greater than or equal to the first predetermined threshold; (ii) the intratumoural level of T cells is determined to be greater than or equal to the second predetermined threshold; and/or (iii) the total tumour level of macrophages is determined to be less than or equal to the third predetermined threshold.
  • the invention also encompasses the bacterial strain for use in a method of treating cancer in a patient identified by a method comprising:
  • step (b) determining one or more of (i) the intratumoural level of T regulatory cells; (ii) the intratumoural level of proliferating T cells; and/or (iii) the total tumour level of macrophages in one or more biopsies obtained from a patient, optionally the one or more biopsies of step (a);
  • a patient is defined as not responsive to therapy if, at least 6 months after the start of the therapy, the patient is classified as having progressive disease (PD).
  • a patient is defined as responsive to therapy if, at least 6 months after the start of therapy, the patient is classed as having a complete remission (CR), partial remission (PR), or stable disease (SD). This corresponds to the disease control rate (DCR).
  • the patient may be defined as responsive to therapy if, at least 6 months after the start of therapy, the patient is classed as having an objective response (OR), which includes CR or PR.
  • the patient is classified as responsive to therapy if the patient has any response to the therapy other than progressive disease.
  • Response criteria are preferably defined according to the RECIST (Response Evaluation Criteria In Solid Tumours) criteria v1.1 [21], but may also be defined according to the irRECIST (immune-related Response Evaluation Criteria In Solid Tumours) criteria [29], or other criteria appropriate for the cancer in question.
  • steps (d) to (f) may additionally or alternatively comprise determining whether: (i) the level of stromal macrophages is below a fourth predetermined threshold; (ii) the level of total tumour macrophages is below a fifth predetermined threshold; (iii) the level of intratumoural T cells (e.g. CD3 + cells) is above a sixth predetermined threshold; (iv) the level of intratumoural CD8- expressing T cells (e.g. CD3 + CD8 + cells) is above a seventh predetermined threshold; and/or (v) the level of intratumoural CD8-expressing, proliferating T cells (e.g. CD3 + CD8 + Ki67 + cells) is above an eighth predetermined threshold.
  • the level of stromal macrophages is below a fourth predetermined threshold
  • the level of total tumour macrophages is below a fifth predetermined threshold
  • the level of intratumoural T cells e.g. CD3 + cells
  • the invention provides a bacterial strain of the genus Enterococcus, or a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO: 6 for use in a method of treating cancer, said method comprising administering the bacterial strain to a patient having, prior to administration of the bacterial strain: (i) an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold; and/or (ii) an intratumoural level of proliferating T cells that is above a second predetermined threshold; and/or (iii) a total tumour level of macrophages that is less than or equal to a third predetermined threshold, as described herein.
  • the bacterial strain is of the genus Enterococcus. More preferably, the Enterococcus strain is of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae. Even more preferably, the Enterococcus strain is of the species Enterococcus gallinarum.
  • Preferred strains for use in the invention include the Enterococcus gallinarum strains deposited under accession number NCIMB 42488 or NCIMB 42761 [30], In some embodiments, the bacterial strain is flagellated (i.e. the cells have at least one flagellum).
  • the bacterial strain has a 16s rRNA sequence that is at least 99%, at least 99.5% or 99.99% identical to SEQ ID NO:2 or SEQ ID NO: 6.
  • the Examples demonstrate that patients responded well to treatment with a bacterial strain as described herein, in particular if they had an intratumoural level of T regulatory cells greater than or equal to a first predetermined threshold as described herein; and/or an intratumoural level of proliferating T cells greater than or equal to a second predetermined threshold as described herein; and/or a total tumour level of macrophages less than or equal to a third predetermined threshold as described herein.
  • the cell markers and thresholds described herein are thought to identify the patients most likely to respond to therapy with a bacterial strain as described herein.
  • the bacterial strain is able to improve therapeutic outcomes in these particular groups of patients given that high levels of intratumoural T regulatory cells and proliferating T cells are often associated with a poor response to therapy and/or poor prognosis [ 14]-[20] .
  • Bacterial strains useful in accordance with the invention are of the genus Enterococcus and/or have a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6.
  • the bacterial strain has a 16s rRNA sequence that is at least 99%, at least 99.5% or 99.99% identical to SEQ ID NO:2 or SEQ ID NO:6.
  • the strain is of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae. Most preferably, the strain is of the species Enterococcus gallinarum.
  • a preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488.
  • Another preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42761.
  • the bacterial strain is flagellated (i.e. the cells have at least one flagellum).
  • Enterococcus gallinarum forms coccoid cells, mostly in pairs or short chains. It is non-motile and colonies on blood agar or nutrient agar are circular and smooth. Enterococcus gallinarum reacts with Lancefield group D antisera.
  • GenBank accession number for a 16S rRNA gene sequence of Enterococcus gallinarum is AF039900 (disclosed herein as SEQ ID NO: 1).
  • An exemplary Enterococcus gallinarum strain is described in [31],
  • the Enterococcus gallinarum bacterium deposited under accession number NCIMB 42488 was tested in the Examples.
  • a 16S rRNA sequence for the deposited strain that was tested is provided in SEQ ID NO:2.
  • the strain was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16th November 2015 as “Enterococcus sp” and was assigned accession number NCIMB 42488.
  • strain NCIMB 42488 comprises a chromosome and plasmid.
  • a chromosome sequence for strain NCIMB 42488 is provided in SEQ ID NO:3.
  • a plasmid sequence for strain NCIMB 42488 is provided in SEQ ID NO:4. These sequences were generated using the PacBio RS II platform.
  • the Enterococcus gallinarum bacterium deposited under accession number NCIMB 42761 was also tested in the examples. It was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB2 1 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 22 May 2017 as "Enterococcus sp" and was assigned accession number NCIMB 42761.
  • the genome sequence of this bacterium is disclosed herein as SEQ ID NO: 5.
  • the genome sequence was assembled from multiple contigs. Ns in the sequence represent gaps between the contigs. "N' may represent an A, G, C or T nucleotide.
  • a 16S rRNA gene sequence for the NCIMB 42761 strain is provided in SEQ ID NO:6.
  • SEQ ID NO:6 represents the full length sequence present in the assembly, rather than a consensus of the five 16S genes present in NCIMB 42761
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus gallinarum.
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO: 1, 2 or 6.
  • the sequence identity is to SEQ ID NO:2 or 6.
  • the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:2 or 6.
  • Bacterial strains that are biotypes of the bacterium deposited under accession number 42488 or NCIMB 42761 are effective for treating or preventing cancer and for the medical uses disclosed herein.
  • a biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.
  • Strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 or NCIMB 42761 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42488 or NCIMB 42761. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome).
  • a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome.
  • suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTGfy or REP or [32], Biotype strains may have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42488 or NCIMB 42761.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain NCIMB 42488 or NCIMB 42761 and comprises a 16S rRNA sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:2 or SEQ ID NO: 6, respectively.
  • a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain NCIMB 42488 or NCIMB 42761 and has the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO: 6, respectively.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO: 3.
  • the bacterial strain for use in the invention has a chromosome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO: 3 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:3.
  • the bacterial strain for use in the invention may have a chromosome with at least 90% sequence identity to SEQ ID NO: 3 across 70% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 90% sequence identity to SEQ ID NO:3 across 100% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 95% sequence identity to SEQ ID NO:3 across 100% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 70% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 80% of SEQ ID NO:3, or at least 98% sequence identity to
  • the bacterial strain for use in the invention has a plasmid with sequence identity to SEQ ID NO:4.
  • the bacterial strain for use in the invention has a plasmid with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:4 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:4.
  • the bacterial strain for use in the invention may have a plasmid with at least 90% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 98% sequence identity to
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 and a plasmid with sequence identity to SEQ ID NO:4.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3, for example as described above, and a 16S rRNA sequence with sequence identity to any of SEQ ID NO: 1 or 2, for example as described above, preferably with a 16s rRNA sequence that is at least 99% identical to SEQ ID NO:2 or SEQ ID NO:6, more preferably which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4, as described above.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3, for example as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4, as described above, and is effective for treating cancer in accordance with the invention.
  • the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3, for example as described above, and a 16S rRNA sequence with sequence identity to any of SEQ ID NOs: 1 or 2, for example as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NO:4, as described above, and is effective for treating cancer in accordance with the invention.
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:2 or SEQ ID NO:6 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6) and a chromosome with at least 95% sequence identity to SEQ ID NO:3 across at least 90% of SEQ ID NO: 3, and optionally comprises a plasmid with sequence identity to SEQ ID NO: 4, as described above, and which is effective for treating cancer in accordance with the invention.
  • SEQ ID NO:2 or SEQ ID NO:6 for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6
  • the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:2 or SEQ ID NO:6 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6) and a chromosome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NO:3 across at least 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NO: 3, and optionally comprises a plasmid with sequence identity to SEQ ID NO: 4, as described above, and which is effective for treating cancer in accordance with the invention.
  • SEQ ID NO:2 or SEQ ID NO:6 for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6
  • a chromosome with at least 98% sequence identity e.g. at least 99%
  • the bacterial strain for use in the invention is a Enterococcus gallinarum and has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:2 or SEQ ID NO:6 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6) and a chromosome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NO:3 across at least 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NO:3, and optionally comprises a plasmid with sequence identity to SEQ ID NO: 4, as described above, and which is effective for treating or preventing cancer.
  • SEQ ID NO:2 or SEQ ID NO:6 for example, which comprises the 16S rRNA sequence of SEQ ID NO:2 or SEQ ID NO:6
  • a chromosome with at least 98% sequence identity
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42488 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23 s rDNA sequencing.
  • FAFLP fluorescent amplified fragment length polymorphism
  • rep repetitive DNA element
  • protein profiling or partial 16S or 23 s rDNA sequencing.
  • such techniques may be used to identify other Enterococcus gallinarum strains.
  • strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention are strains that provide the same pattern as the bacterium deposited under accession number NCIMB 42488 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [33]).
  • ARDRA amplified ribosomal DNA restriction analysis
  • biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42488.
  • the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMérieux).
  • the bacterial strain used in the invention is: (i) positive for fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or all of): L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D- fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D- maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and potassium gluconate; and/or
  • Enterococcus strains that are useful in the compositions and methods for use according to the invention, such as biotypes of the bacterium deposited under accession number NCIMB 42488, may be identified using any appropriate method or strategy, including the assays described in the Examples.
  • bacterial strains that have similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42488 may be useful in the invention.
  • a useful strain will have comparable immune modulatory activity to the NCIMB 42488 strain.
  • a biotype strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
  • the bacterial strain used in the invention is:
  • the bacterial strain used in the invention is:
  • Negative for at least one of (e.g. at least 2, 3, or all 4 of) glycine arylamidase, raffinose fermentation, proline arylamidase, and leucine arylamidase for example, as determined by an assay of carbohydrate, amino acid and nitrate metabolism, preferably as determined by Rapid ID 32A analysis (preferably using the Rapid ID 32A system from bioMérieux); and/or (ii) Intermediate positive for fermentation of L-fucose, preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMérieux).
  • the bacterial strain used in the invention is an extracellular ATP producer, for example one which produces 6-6.7 ng/ ⁇ l (for example, 6.1-6.6 ng/ ⁇ l or 6.2-6.5 ng/ ⁇ l or 6.33 ⁇ 0.10 ng/ ⁇ l) of ATP as measured using the ATP Assay Kit (Sigma- Aldrich, MAKI 90).
  • Bacterial extracellular ATP can have pleiotropic effects including activation of T cell-receptor mediated signalling [34], promotion of intestinal Th17 cell differentiation [35] and induction of secretion of the pro-inflammatory mediator IL-1 ⁇ by activating the NLRP3 inflammasome [36], Accordingly, a bacterial strain which is an extracellular ATP producer is useful for treating or preventing cancer.
  • the bacterial strain for use in the invention comprises one or more of the following three genes: Mobile element protein; Xylose ABC transporter, permease component; and FIG00632333: hypothetical protein.
  • the bacterial strain for use in the invention comprises genes encoding Mobile element protein and Xylose ABC transporter, permease component; Mobile element protein and FIG00632333: hypothetical protein; Xylose ABC transporter, permease component and FIG00632333: hypothetical protein; or Mobile element protein, Xylose ABC transporter, permease component, and FiI00632333: hypothetical protein.
  • a particularly preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488. This is the exemplary strain tested in the Examples and shown to be particularly effective in patients with high intratumoural T regulatory cell levels, high intratumoural proliferating T cell levels, and/or low total tumour macrophage levels.
  • the bacterial strain is the bacterial strain deposited under accession number NCIMB 42488, or a derivative thereof.
  • a derivative of the strain deposited under accession number NCIMB 42488 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original.
  • a preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42761. This is the exemplary strain tested in the examples and shown to be particularly effective in patients with high intratumoural T regulatory cell levels, high intratumoural proliferating T cell levels, and/or low total tumour macrophage levels.
  • the bacterial strain is the bacterial strain deposited under accession number NCIMB 42761, or a derivative thereof.
  • a derivative of the strain deposited under accession number NCIMB 42761 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original
  • a derivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity.
  • a derivative strain of the invention is therapeutically active.
  • a derivative strain will have comparable activity to the original NCIMB 42488 or NCIMB 42761 strain.
  • a derivative strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in Examples 1-5.
  • a derivative of the NCIMB 42488 or NCIMB 42761 strain will generally be a biotype of the NCIMB 42488 or NCIMB 42761 strain, respectively.
  • the bacterial strains in the compositions of the invention are viable and/or capable of partially or totally colonising the intestine.
  • Example 6 demonstrate that administration of the bacterial strains described herein can lead to improved anticancer efficacy when administered to the patient subgroups as defined herein.
  • the patients of Example 6 received co-therapy with an immune checkpoint inhibitor (ICI). However, the patients of Example 6 had previously developed resistance to ICIs, indicating that the benefit in Example 6 is derived from the administration of the bacterial strain.
  • Examples 1-5 demonstrate that the bacterial strains described herein have anticancer efficacy when administered alone, and Example 7 demonstrates that the bacterial strains of the invention are well-tolerated when administered alone.
  • Example 6 the improved efficacy observed in the clinical trial of Example 6 is similarly to be expected when administering a bacterial strain as described herein as a monotherapy to patients with (i) an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold; and/or (ii) an intratumoural level of proliferating T cells that is above a second predetermined threshold; and/or (iii) a total tumour level of macrophages that is less than or equal to a third predetermined threshold.
  • treatment with the bacterial strains of the invention results in a reduction in tumour size or a reduction in tumour growth.
  • the bacterial strains are for use in reducing tumour size or reducing tumour growth.
  • the bacterial strains of the invention may be effective for reducing tumour size or growth. Any references to a bacterial strain for use in therapy also refer to a composition comprising said bacterial strain for the same therapeutic use. References to a bacterial strain for use in therapy also refer to said bacterial strain for use in the manufacture of a medicament for the same therapeutic use.
  • the bacterial strains are for use in patients with solid tumours. In certain embodiments, the bacterial strains are for use in reducing or preventing angiogenesis in the treatment of cancer.
  • the bacterial strains may have an effect on the immune or inflammatory systems, which have central roles in angiogenesis. In certain embodiments, the bacterial strains are for use in preventing metastasis.
  • treatment with a bacterial strain according to the invention results in a pathologic complete response (pCR).
  • the bacterial strain is for use in treating renal cancer, melanoma, lung cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, urethral cancer, or pancreatic cancer.
  • the renal cancer is renal cell carcinoma.
  • the lung cancer is non-small cell lung cancer.
  • the breast cancer is triple negative breast cancer.
  • the bacterial strain may also be for use in treating head and/or neck cancer, for example head and neck squamous cell carcinoma.
  • the bacterial strain may also be for use in treating microsatellite unstable cancer.
  • the bacterial strain is for use in treating or preventing breast cancer.
  • the examples demonstrate that the bacterial strains described herein may be effective for treating breast cancer.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer.
  • the breast cancer is triple negative breast cancer.
  • the cancer is mammary carcinoma.
  • the cancer is stage IV breast cancer.
  • the bacterial strain is for use in treating or preventing lung cancer.
  • the examples demonstrate that the bacterial strains described herein may be effective for treating lung cancer.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer.
  • the cancer is lung carcinoma.
  • the cancer is non-small cell lung cancer.
  • the bacterial strain is for use in treating or preventing liver cancer.
  • the examples demonstrate that the bacterial strains described herein may be effective for treating liver cancer.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of liver cancer.
  • the cancer is hepatoma (hepatocellular carcinoma).
  • the bacterial strain is for use in treating or preventing colon cancer.
  • the examples demonstrate that the bacterial strains described herein have an effect on colon cancer cells and may be effective for treating colon cancer.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colon cancer.
  • the cancer is colorectal adenocarcinoma.
  • the bacterial strain is for use in treating or preventing kidney cancer (also referred to herein as renal cancer).
  • kidney cancer also referred to herein as renal cancer.
  • the examples demonstrate that the bacterial strains described herein have an effect on renal cancer cells and may be effective for treating renal cancer.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of renal cancer.
  • the cancer is renal cell carcinoma or transitional cell carcinoma.
  • the bacterial strain is for use in treating or preventing melanoma.
  • the bacterial strain has an effect on melanocytes and may be effective for treating melanoma.
  • the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of melanoma.
  • the cancer is of the intestine. In some embodiments, the cancer is of a part of the body which is not the intestine. In some embodiments, the cancer is not cancer of the intestine. In some embodiments, the cancer is not colorectal cancer. In some embodiments, the cancer is not cancer of the small intestine. In some embodiments, the treating or preventing occurs at a site other than at the intestine. In some embodiments, the treating or preventing occurs at the intestine and also at a site other than at the intestine.
  • the cancer is not of the head and/or neck.
  • the bacterial strain is for use in treating or preventing carcinoma.
  • the examples demonstrate that the bacterial strains described herein may be effective for treating numerous types of carcinoma.
  • the compositions for use according to the invention are for use in treating or preventing non-immunogenic cancer.
  • the examples demonstrate that the bacterial strains described herein may be effective for treating non-immunogenic cancers.
  • the bacterial strain is for use in treating or preventing bladder cancer. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of bladder cancer.
  • the bacterial strain is for use in treating or preventing pancreatic cancer. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of pancreatic cancer.
  • the therapeutic effects of the bacterial strains against cancer may be mediated by a pro-inflammatory mechanism.
  • Examples 2, 4 and 5 demonstrate that the expression of a number of pro-inflammatory cytokines may be increased following administration of NCIMB 42488. Inflammation can have a cancer-suppressive effect [37] and pro-inflammatory cytokines such as TNF- ⁇ are being investigated as cancer therapies [38],
  • the up-regulation of genes such as TNF shown in the examples may indicate that the bacterial strains may be useful for treating cancer via a similar mechanism.
  • the up-regulation of CXCR3 ligands (CXCL9, CXCL10) and IFN ⁇ -inducible genes (IL-32) may indicate that the compositions for use according to the invention elicit an IFN ⁇ -type response.
  • the bacterial strains are for use in promoting inflammation in the treatment of cancer.
  • the bacterial strains are for use in promoting Thl inflammation in the treatment of cancer.
  • Thl cells produce IFN ⁇ and have potent anti-cancer effects [37],
  • the bacterial strains are for use in treating an early-stage cancer, such as a cancer that has not metastasized, or a stage 0 or stage 1 cancer.
  • the treatment or prevention of cancer comprises increasing the level of expression of one or more cytokines.
  • the treatment or prevention of cancer comprises increasing the level of expression of one or more of IL-1 ⁇ , IL-6 and TNF- ⁇ , for example, IL-1 ⁇ and IL-6, IL-1 ⁇ and TNF- ⁇ , IL-6 and TNF- ⁇ or all three of IL-1 ⁇ , IL-6 and TNF- ⁇ .
  • Increases in levels of expression of any of IL-1 ⁇ , IL-6 and TNF- ⁇ are known to be indicative of efficacy in treatment of cancer.
  • the bacterial strain for use as described herein is used to treat a cancer patient who was non responsive to a prior treatment with an anticancer therapy, for example an immunotherapy, chemotherapy, radiotherapy, adoptive cell therapy, anticancer vaccine and/or hormone suppression therapy.
  • an anticancer therapy for example an immunotherapy, chemotherapy, radiotherapy, adoptive cell therapy, anticancer vaccine and/or hormone suppression therapy.
  • the bacterial strains are for use in treating non-small-cell lung carcinoma. In certain embodiments, the bacterial strains are for use in treating small-cell lung carcinoma. In certain embodiments, the bacterial strains are for use in treating squamous-cell carcinoma. In certain embodiments, the bacterial strains are for use in treating adenocarcinoma. In certain embodiments, the bacterial strains are for use in treating glandular tumours, carcinoid tumours, or undifferentiated carcinomas.
  • the bacterial strains are for use in treating hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.
  • the bacterial strains are for use in treating invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.
  • the bacterial strains are for use in treating or preventing acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumour, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumour, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumours, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumour, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophage
  • ALL acute
  • the one or more bacterial strains having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, for example which is an Enterococcus gallinarum is/are the only therapeutically active agent(s) in a composition administered in accordance with the invention.
  • the bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition administered in accordance with the invention.
  • the bacterial strain is to be administered to the gastrointestinal tract in order to enable delivery to and/or partial or total colonisation of the intestine with the bacterial strain of the invention.
  • the compositions for use according to the invention comprising the bacterial strain are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.
  • the bacterial strain may be administered as a foam, as a spray or a gel.
  • the bacterial strain may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • a rectal suppository for example in the form of a theobroma oil (coa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
  • the bacterial strain is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • a tube such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.
  • the bacterial strain may be administered once, or they may be administered sequentially as part of a treatment regimen.
  • the bacterial strains or compositions for use according to the invention are to be administered daily.
  • the bacterial strains or compositions for use according to the invention are administered twice daily or more.
  • the bacterial strains or compositions for use according to the invention are administered once every two days, once every three days, once every four days, once every five days, once every six days, or once a week.
  • the bacterial strains or compositions for use according to the invention are administered twice daily.
  • treatment according to the invention is accompanied by assessment of the patient's gut microbiota. Treatment may be repeated if delivery of and / or partial or total colonisation with the strain of the invention is not achieved such that efficacy is not observed, or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed. According to some embodiments, the subject's gut microbiota is assessed after administration of the bacterial strain.
  • the bacterial strain may be administered to a pregnant animal, for example a mammal such as a human in order to reduce the likelihood of cancer developing in her child in utero and/or after it is born.
  • the bacterial strain may be administered to a patient that has been diagnosed with cancer, or that has been identified as being at risk of a cancer.
  • the bacterial strain may also be administered as a prophylactic measure to prevent the development of cancer in a healthy patient.
  • the bacterial strain may be administered to a patient that has been identified as having an abnormal gut microbiota.
  • the patient may have reduced or absent colonisation by Enterococcus gallinarum.
  • the bacterial strain may be administered as a food product, such as a nutritional supplement.
  • the bacterial strain is for the treatment of humans, although they may be used to treat animals including monogastric mammals such as poultry, pigs, cats, dogs, horses or rabbits.
  • the bacterial strains and compositions for use in the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.
  • compositions comprising the bacterial strain
  • the bacterial strain may be administered as part of a composition.
  • the composition is formulated in freeze-dried form.
  • the composition may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain as described herein.
  • the composition comprising the bacterial strain for use according to the invention comprises lyophilised bacteria.
  • Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [43-45],
  • composition may comprise a live, active bacterial culture.
  • compositions disclosed herein are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the intestine with the bacterial strain of the invention.
  • the bacteria may have colonised some or all of the gastrointestinal tract and / or such colonisation may be transient or permanent.
  • the "total colonisation of the intestine” means that bacteria have colonised all parts of the intestine (i.e. the small intestine, large intestine and rectum). Additionally or alternatively, the term “total colonisation” means that the bacteria engraft permanently in the some or all parts of the intestine.
  • partial colonisation of the intestine means that bacteria have colonised some but not all parts of the intestine. Additionally or alternatively, the term “partial colonisation” means that the bacteria engraft transiently in some or all parts of the intestine.
  • the bacterial strain in the composition may not have been inactivated, for example, may not have been heat-inactivated.
  • the bacterial strain in the composition for use according to the invention may not have been killed, for example, not been heat-killed.
  • the bacterial strain in the composition for use according to the invention may not have been attenuated, for example, not been heat- attenuated.
  • the bacterial strain in the composition for use according to the invention may not have been killed, inactivated and/or attenuated.
  • the bacterial strain in the composition for use according to the invention is live.
  • the bacterial strain in the composition for use according to the invention is viable.
  • the bacterial strain in the composition for use according to the invention is capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition may be viable and capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition for use according to the invention may be live and capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition for use according to the invention may be live and viable.
  • the bacterial strain in the composition for use according to the invention may be live, viable and capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition has not been inactivated, for example, has not been heat-inactivated. In some embodiments, the bacterial strain in the composition for use according to the invention has not been killed, for example, has not been heat-killed. In some embodiments, the bacterial strain in the composition for use according to the invention has not been attenuated, for example, has not been heat- attenuated. For example, in some embodiments, the bacterial strain in the composition for use according to the invention has not been killed, inactivated and/or attenuated. For example, in some embodiments, the bacterial strain in the composition for use according to the invention is live. For example, in some embodiments, the bacterial strain in the composition for use according to the invention is viable.
  • the bacterial strain in the composition for use according to the invention is capable of partially or totally colonising the intestine.
  • the bacterial strain in the composition for use according to the invention is viable and capable of partially or totally colonising the intestine.
  • the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.
  • the composition comprising the bacterial strain is encapsulated to enable delivery of the bacterial strain to the intestine.
  • Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule.
  • compositions for use according to the invention are available in, for example, references [46] and [47],
  • the composition may be administered orally and may be in the form of a tablet, capsule or powder.
  • Encapsulated products are preferred because Enterococcus gallinarum are anaerobes.
  • Other ingredients such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo.
  • the probiotic composition for use according to the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.
  • composition may be formulated as a probiotic.
  • a composition comprising the bacterial strain for use according to the invention includes a therapeutically effective amount of a bacterial strain as described herein.
  • a therapeutically effective amount of a bacterial strain is sufficient to exert a beneficial effect upon a patient.
  • a therapeutically effective amount of a bacterial strain may be sufficient to result in delivery to and / or partial or total colonisation of the patient's intestine.
  • a suitable daily dose of the bacteria may be from about 1 x 10 3 to about 1 x 10 11 colony forming units (CFU); for example, from about 1 x 10 7 to about 1 x 10 10 CFU; in another example from about 1 x 10 6 to about 1 x 10 10 CFU.
  • CFU colony forming units
  • the composition contains the bacterial strain in an amount of from about 1 x 10 6 to about 1 x 10 11 CFU/g, respect to the weight of the composition; for example, from about 1 x 10 8 to about 1 x 10 10 CFU/g.
  • the dose may be, for example, 1 g, 3g, 5g, and 10g.
  • a probiotic such as the composition comprising the bacterial strain for use in accordance with the invention, is optionally combined with at least one suitable prebiotic compound.
  • a prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract.
  • Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.
  • the probiotic composition includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight).
  • Carbohydrates may be selected from the group consisting of: fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo- oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers.
  • the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.
  • the bacterial compositions may comprise pharmaceutically acceptable excipients or carriers.
  • excipients may be found in the reference [48], Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [49], Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavouring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • compositions comprising the bacterial strain may be formulated as a food product.
  • a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement.
  • a food product may be formulated to enhance the taste of the composition for use according to the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition.
  • the composition is formulated as a milk-based product.
  • milk-based product means any liquid or semi-solid milk- or whey- based product having a varying fat content.
  • the milk-based product can be, e.g., cow's milk, goafs milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products.
  • milk beverages such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.
  • the compositions contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.
  • the invention provides a composition comprising one or more strains from the genus Enterococcus, preferably from the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae, which does not contain bacteria from any other genus or species or which comprises only de minimis or biologically irrelevant amounts of bacteria from another species for use in therapy.
  • compositions for use in the invention comprise more than one bacterial strain or species.
  • the compositions for use in the invention comprise more than one bacterial strain (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or 45 strains). These bacterial strain(s) may all be from the same bacterial species and may, optionally, not contain bacteria from any other species.
  • the compositions for use in the invention comprise fewer than 50 bacterial strains (e.g. fewer than 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 strains).
  • the compositions for use in the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6- 30, 6-15, 16-25, or 31-50 strains and, optionally, do not contain bacteria from any other species.
  • compositions for use in the invention comprise more than one species from within the same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not contain bacteria from any other genus.
  • the compositions for use in the invention comprise less than 50 species from within the same genus (e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species), and, optionally, do not contain bacteria from any other genus.
  • compositions for use in the invention comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2- 20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species from within the same genus and, optionally, do not contain bacteria from any other genus.
  • the invention comprises the use of any combination of the foregoing.
  • the composition comprises a microbial consortium.
  • the composition comprises the bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, for example, which is an Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae, as part of a microbial consortium.
  • the bacterial strain is present in combination with one or more (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) other bacterial strains.
  • these bacterial strains may from other genera with which the bacterial strain of the invention can live symbiotically in vivo in the intestine.
  • the composition comprises a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, for example, which is an Enterococcus gallinarum, in combination with a bacterial strain from a different genus.
  • the microbial consortium comprises two or more bacterial strains obtained from a faeces sample of a single organism, e.g. a human. In some embodiments, the microbial consortium is not found together in nature.
  • the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms.
  • the two different organisms are from the same species, e.g. two different humans, e.g. two different human infants.
  • the two different organisms are an infant human and an adult human.
  • the two different organisms are a human and a non-human mammal.
  • the composition for use in the invention additionally comprises abacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 42488, but which is not the strain deposited as NCIMB 42488, or which is not an Enterococcus gallinarum.
  • the composition for use in the invention additionally comprises abacterial strain that has the same safety and therapeutic efficacy characteristics as strain NCIMB 42761, but which is not the strain deposited as NCIMB 42761, or which is not an Enterococcus gallinarum.
  • the composition for use in the invention comprises more than one bacterial strain, species or genus
  • the individual bacterial strains, species or genera may be for separate, simultaneous or sequential administration.
  • the composition may comprise all of the more than one bacterial strain, species or genera, or the bacterial strains, species or genera may be stored separately and be administered separately, simultaneously or sequentially.
  • the more than one bacterial strains, species or genera are stored separately but are mixed together prior to use.
  • the bacterial strain for use in the invention is obtained from human infant faeces. In some embodiments in which the composition for use according to the invention comprises more than one bacterial strain, all of the bacterial strains are obtained from human infant faeces or if other bacterial strains are present they are present only in de minimis amounts.
  • the bacteria may have been cultured subsequent to being obtained from the human infant faeces and being used in a composition for use according to the invention.
  • the the one or more bacterial strains having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:6, for example which is an Enterococcus gallinarum is/are the only therapeutically active agent(s) in the bacterial composition.
  • the bacterial strain(s) in the bacterial composition is/are the only therapeutically active agent(s) in the composition.
  • compositions for use in accordance with the invention may or may not require marketing approval.
  • the above pharmaceutical composition is provided for use in accordance with the invention, wherein said bacterial strain is lyophilised. In certain embodiments, the above pharmaceutical composition is provided for use in accordance with the invention, wherein said bacterial strain is spray dried. In certain embodiments, the above pharmaceutical composition is provided for use in accordance with the invention, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the above pharmaceutical composition is provided for use in accordance with the invention, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable.
  • the above pharmaceutical composition is provided for use in accordance with the invention, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of partially or totally colonising the intestine. In certain embodiments, the above pharmaceutical composition is provided for use in accordance with the invention, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partially or totally colonising the intestine.
  • the lyophilised or spray dried bacterial strain is reconstituted prior to administration.
  • the reconstitution is by use of a diluent described herein.
  • compositions can comprise pharmaceutically acceptable excipients, diluents or carriers.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is pancreatic cancer, for example resectable pancreatic cancer.
  • the cancer is pancreatic adenocarcinoma.
  • the pharmaceutical composition comprises: a bacterial strain as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is breast cancer.
  • the cancer is triple negative breast cancer.
  • the cancer is mammary carcinoma.
  • the cancer is stage IV breast cancer.
  • the pharmaceutical composition comprises: a bacterial strain as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is lung cancer.
  • the cancer is lung carcinoma.
  • the cancer is non small cell lung cancer.
  • the pharmaceutical composition comprises: a bacterial strain as used in the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is liver cancer.
  • the cancer is hepatoma (hepatocellular carcinoma).
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is colon cancer.
  • the cancer is colorectal adenocarcinoma.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is carcinoma.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is a non-immunogenic cancer.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is selected from the group consisting of non-small-cell lung carcinoma, small-cell lung carcinoma, squamous-cell carcinoma, adenocarcinoma, glandular tumours, carcinoid tumours undifferentiated carcinomas.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is selected from the group consisting of hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is selected from the group consisting of invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.
  • the pharmaceutical composition comprises: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder; and wherein the disorder is selected from the group consisting of acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumour, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumour, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumours, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumour, cervical cancer, chronic lymphocytic leukemia, chronic myelogen
  • ALL acute
  • the amount of the bacterial strain in the bacterial composition is from about 1 x 10 3 to about 1 x 10 11 colony forming units per gram with respect to a weight of the composition.
  • the pharmaceutical composition is administered at a dose of 1 g, 3 g, 5 g or 10 g. In certain embodiments, the pharmaceutical composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.
  • the pharmaceutical composition comprises a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.
  • the pharmaceutical composition comprises a diluent selected from the group consisting of ethanol, glycerol and water.
  • the pharmaceutical composition comprises an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.
  • the pharmaceutical composition further comprises at least one of a preservative, an antioxidant and a stabilizer.
  • the pharmaceutical composition comprises a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • the pharmaceutical composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.
  • the composition is provided in a sealed container.
  • the sealed container is a sachet or bottle.
  • the composition is provided in a syringe.
  • the bacterial composition may, in some embodiments, be provided as a pharmaceutical formulation.
  • the composition may be provided as a unit dosage form, for example as a tablet or capsule.
  • the capsule is a gelatine capsule ("gel-cap").
  • each unit dosage form e.g. tablet or capsule
  • the unit dosage form comprises about 1x10 8 , about 1x10 9 or about 1x10 10 to about 1x10 11 CFU of the bacterial strain.
  • the bacterial compositions for use according to the invention are administered orally.
  • Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.
  • compositions suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
  • solid plugs solid microparticulates, semi-solid and liquid (including multiple phases or dispersed systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids (e.g. aqueous solutions), emulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.
  • the pharmaceutical formulation is an enteric formulation, i.e. a gastro-resistant formulation (for example, resistant to gastric pH) that is suitable for delivery of the composition of the invention to the intestine by oral administration.
  • Enteric formulations may be particularly useful when the bacteria or another component of the composition is acid-sensitive, e.g. prone to degradation under gastric conditions.
  • the enteric formulation comprises an enteric coating.
  • the formulation is an enteric-coated dosage form.
  • the formulation may be an enteric- coated tablet or an enteric-coated capsule, or the like.
  • the enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery.
  • the formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.
  • the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an enteric coating.
  • the formulation is an enteric formulation that does not comprise an enteric coating.
  • the formulation is a capsule made from a thermogelling material.
  • the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC).
  • the capsule comprises a shell that does not contain any film forming polymer.
  • the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [50 ]).
  • the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).
  • the formulation is a soft capsule.
  • Soft capsules are capsules which may, owing to additions of softeners, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, present in the capsule shell, have a certain elasticity and softness.
  • Soft capsules can be produced, for example, on the basis of gelatine or starch. Gelatine-based soft capsules are commercially available from various suppliers.
  • soft capsules can have various shapes, they can be, for example, round, oval, oblong or torpedo-shaped.
  • Soft capsules can be produced by conventional processes, such as, for example, by the Scherer process, the Accogel process or the droplet or blowing process.
  • the bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in, for example, references [51-53],
  • the solid or liquid medium used for culture may be YCFA agar or YCFA medium.
  • YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO 3 (0.4 g), cysteine (0.1 g), K 2 HPO 4 (0.045 g), KH 2 PO 4 (0.045 g), NaCl (0.09 g), (NH 4 ) 2 SO 4 (0.09 g), MgSO 4 • 7H 2 O (0.009 g), CaCl 2 (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), p-aminobenzoic acid (3 pg), folic acid (5 pg), and pyridoxamine (15 pg).
  • the bacterial strains of the invention are useful for treating or preventing cancer. This is likely to be a result of the effect that the bacterial strains of the invention have on the host immune system. Therefore, the compositions described herein may also be useful for preventing cancer, when administered as vaccine compositions in accordance with the invention.
  • the bacterial strains are viable.
  • the bacterial strains are capable of partially or totally colonising the intestine.
  • the bacterial strains are viable and capable of partially or totally colonising the intestine.
  • the bacterial strains may be killed, inactivated or attenuated.
  • the compositions may comprise a vaccine adjuvant.
  • the compositions are for administration via injection, such as via subcutaneous injection.
  • composition comprising X may consist exclusively of X or may include something additional e.g. X + Y.
  • references to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [62], A preferred alignment is determined by the Smith-Waterman homology search algorithm using the DNA full substitution matrix and an affine gap search with a gap open penalty of 5 and a gap extension penalty of 2.
  • the Smith- Waterman homology search algorithm is disclosed in ref. [63], Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.
  • Example 1 Efficacy of bacterial inocula in mouse models of cancer
  • Test substance Bacterial strain NCIMB 42488.
  • Reference substance - Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: Syrian Hamster IgGl, Bioxcell).
  • Test and reference substances vehicles - Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each day of injection to mice, antibody was diluted with PBS (ref: BE14- 516F, Lonza, France).
  • Treatment doses - Bacteria 2x10 8 in 200 ⁇ L.
  • the a-CTLA-4 was injected at 10 mg/kg/inj.
  • Anti- CTLA-4 was administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse weighing 20 g, 200 ⁇ L of test substance will be administered) according to the most recent body weight of mice.
  • the EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule [64],
  • the LL/2 (LLC1) cell line was established from the lung of a C57BL mouse bearing atumour resulting from an implantation of primary Lewis lung carcinoma [65],
  • the Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [66],
  • adherent tumour cells were detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.
  • mice Healthy female Balb/C mice, of matching weight and age, were obtained from CHARLES RIVER (L'Arbresles) for the EMT6 model experiments.
  • Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents - R/M-H Extrudate, water from water bottles.
  • group housing 900 cm 2 cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immuno-competent rodents - R/M-H Extrudate, water from water bottles.
  • DO Treatment schedule - The start of first dosing was considered as DO.
  • DO non-engrafted mice were randomized according to their individual body weight into groups of 9/8 using Vivo manager® software (Biosystemes, Coutemon, France).
  • DO the mice received vehicle (culture medium) or bacterial strain.
  • D14 all mice were engrafted with EMT-6 tumour cells as described below.
  • D24 mice from the positive control group received anti-CTLA-4 antibody treatments.
  • tumours were induced by subcutaneous injection of 1x10 6 EMT-6 cells in 200 ⁇ L RPMI 1640 into the right flank of mice. Euthanasia - Each mouse was euthanized when it reached a humane endpoint as described below, or after a maximum of 6 weeks post start of dosing.
  • DO Treatment schedule - The start of first dosing was considered as DO.
  • DO non-engrafted mice were randomized according to their individual body weight into 7 groups of 9/8 using Vivo manager® software (Biosystemes, Coutemon, France).
  • DO the mice will received vehicle (culture medium) or bacterial strain.
  • DI 4 all mice were engrafted with LL/2 tumour cells as described below.
  • D27 mice from the positive control group received anti-CTLA-4 antibody treatments.
  • DO Treatment schedule - The start of first dosing was considered as DO.
  • DO non-engrafted mice were randomized according to their individual body weight into 7 groups of 9 using Vivo manager® software (Biosystemes, Coutemon, France).
  • DO the mice received vehicle (culture medium) or bacterial strain.
  • DI 4 all mice were engrafted with Hepa 1-6 tumour cells as described below.
  • DI 6 mice from the positive control group received anti-CTLA-4 antibody treatments.
  • Hepa 1-6 tumour cells in animals by intrasplenic injection On DI 4, one million (1x10 6 ) Hepa 1 -6 tumour cells in 50 ⁇ L RPMI 1640 medium were transplanted via intra-splenic injection into mice. Briefly, a small left subcostal flank incision was made and the spleen was exteriorized. The spleen was exposed on a sterile gauze pad, and injected under visual control with the cell suspension with a 27-gauge needle. After the cell inoculation, the spleen was excised.
  • Clinical monitoring The length and width of the tumour was measured twice a week with callipers and the volume of the tumour was estimated by this formula [69]: Humane endpoints [70]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumour exceeding 10% of normal body weight, but non-exceeding 2000 mm 3 ; Tumours interfering with ambulation or nutrition; Ulcerated tumour or tissue erosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, emaciation, cachexia, dehydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleeding; Neurologic signs: circling, convulsion, paralysis; Sustained decrease in body temperature; Abdominal distension.
  • Anaesthesia - Isofl urane gas anesthesia were used for all procedures: surgery or tumour inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthesia were used for stereotaxia surgical procedure.
  • Analgesia - Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severity of surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally, pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinarian.
  • Euthanasia - Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.
  • NCIMB 42488 A pure culture of bacteria NCIMB 42488 was studied in a PCR gene analysis. There were two arms to the experiment: 1) NCIMB 42488 was co-cultured with human colonic cells (CaCo2) to investigate the effects of the bacteria on the host, and 2) NCIMB 42488 was co-cultured on CaCo2 cells that were stimulated with IL1 to mimic the effect of the bacteria in an inflammatory environment. The effects in both scenarios were evaluated through gene expression analysis. The results are shown below:
  • CXCR1/2 ligands CXCL3, CXCL2, CXCL1, IL-8
  • CXCR3 ligands CXCL9,CXCL10
  • a composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25°C or 4°C and the container is placed in an atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.
  • Example 4 - cytokine production in immature dendritic cells induced by NCIMB 42488 compared to NCIMB 42488 + LPS
  • a monocyte population was isolated from peripheral blood mononuclear cells (PBMCs).
  • the monocyte cells were subsequently differentiated into immature dendritic cells.
  • the immature dendritic cells were plated out at 200,000 cells/well and incubated with NCIMB 42488 at a final concentration of 10 7 /ml, with the optional addition of LPS at a final concentration of 100ng/ml.
  • the negative control involved incubating the cells with RPMI media alone and positive controls incubated the cells with LPS at a final concentration of 100ng/ml.
  • the cytokine content of the cells was then analysed.
  • NCIMB 42488 has the ability to induce higher IL-6 and TNF- ⁇ cytokine production in immature dendritic cells.
  • the combination LPS and NCIMB 42488 can increase the levels of cytokines IL-1 ⁇ in immature dendritic cells.
  • NCIMB 42488 alone or in combination with LPS can increase inflammatory cytokines IL-1 ⁇ , IL-6 and TNF- ⁇ , which promotes inflammation that can suppress cancer.
  • Treatment with NCIMB 42488 alone or in combination with LPS can induce cytokines that can limit tumour growth.
  • THF-1 cells were differentiated in MO medium for 48h with 5ng/mL phorbol-12-myristate- 13 -acetate (PMA). These cells were subsequently incubated with NCIMB 42488 at a final concentration of 10 8 /ml, with or without the addition of LPS at a final concentration of 100ng/ml. The bacteria were then washed off and the cells allowed to incubate under normal growing conditions for 24 h. The cells were then spun down and the resulting supernatant was analysed for cytokine content.
  • PMA phorbol-12-myristate- 13 -acetate
  • NCIMB 42488 has the ability to induce cytokine production in THP-1 cells, which can be synergistically increased with the addition of LPS. These data indicate that NCIMB 42488 alone or in combination with LPS can increase inflammatory cytokines IL- 1 ⁇ , IL-6 and TNF- ⁇ , which promotes inflammation that can suppress cancer. Treatment with NCIMB 42488 alone or in combination with LPS can induce cytokines that can limit tumour growth.
  • Example 6 Identification of a predictive TME immune signature associated with response to NCIMB 42488 therapy in secondary resistant solid tumour patients
  • Enterococcus gallinarum is a commensal bacterium that is naturally present in the human gastrointestinal tract in up to 25% of the healthy population.
  • NCIMB 42488 is a specific, proprietary strain of E. gallinarum, able to directly stimulate immune cells in vitro inducing the production of pro- inflammatory cytokines/chemokines previously reported to have anti-tumoural activity and to facilitate chemotactic recruitment of tumour-infiltrating lymphocyte (TILs) to the tumour site.
  • TILs tumour-infiltrating lymphocyte
  • NCIMB 42488 alters the tumour microenvironment (TME) of a range of tumour models with varying degrees of immunogenicity by increasing the infiltration of immune cells to enhance immunogenicity and inducing pro-inflammatory cytokines and chemokines.
  • TAE tumour microenvironment
  • This study is designed to evaluate the safety and anti-tumour effect of NCIMB 42488 in participants having second line resistance to immune checkpoint inhibitors.
  • the adaptive and innate immune cells in pre-treatment tumour tissues obtained from anti-PD-(L)1 secondary resistant patients were quantified.
  • FFPE tumour biopsies from 12 patients, 4 responders and 8 non responders, were immunoprofiled using a mIF panel against CD3, CD8, PD-1, FOXP3, Ki-67, PD-L1, CD68, and cytokeratin antibodies in tumour and stroma compartments from the tissue using image analysis.
  • mIF staining was performed using methods similar to those that have been previously described and optimized [71], Briefly, four micrometre-thick formalin fixed, paraffin embedded sample sections were stained using a mIF panel containing antibodies against: cytokeratin (CK; clone AE1/AE3, Dako), CD3 (cat#IS503, Dako), CD8 (clone C8/144B, Thermo Fisher Scientific), FOXP3 (clone D2W8E, Cell Signaling Technology), PD-1 (clone [EPR4877(2)], ABCAM), CD68 (clone [PG-M1 (M)], DAKO), PD-L1 (clone E1L3N, Cell Signaling Technology) and KI67 (clone MIB-1, DAKO).
  • CK cytokeratin
  • CD3 catalog#IS503, Dako
  • CD8 clone C8/144B, Thermo Fisher Scientific
  • FOXP3 clone D2W8E
  • Each ROI was analyzed by a pathologist using InForm 2.4.8 image analysis software (Akoya Biosciences).
  • the different ROIs were divided according to the expression of CK or not in tumour compartment (groups or nests of malignant cells) and stroma compartment (represented by the stroma area between tumour cells), respectively [72], Marker co-localization was used to identify specific cell phenotypes in the different compartments. Densities of each cell phenotype were quantified, and the final data was expressed as number of cells/mm 2 [72], All the data was consolidated using the R studio 3.5.3 (Phenopter 0.2.2 packet, Akoya Biosciences).
  • CR Complete Response
  • Partial Response At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline (i.e. pre-treatment) sum diameters.
  • PD Progressive Disease
  • SD Stable Disease
  • NCIMB 42488 A phase la study in seventeen patients to assess, amongst other things, the safety and efficacy of NCIMB 42488 as a neoadjuvant monotherapy in patients undergoing surgical resection of solid tumors. Patients enrolled had been diagnosed with resectable tumors and NCIMB42488 was then dosed as a monotherapy for two to four weeks prior to resection. In all patients, treatment with NCIMB 42488 was well tolerated and no drug-related serious adverse events were observed.
  • Example 8 Identification of a predictive TME immune signature associated with response to NCIMB 42488 therapy in secondary resistant solid tumour patients
  • Example 9 Association between positive outcomes with NCIMB 42488 and higher baseline densities of total T cells (CD3+) and subsets (CD8+, and Ki67+ — a marker of proliferation)
  • tumour biopsy samples In addition to the analysis of tumour biopsy samples (as detailed in Example 6), patient blood, serum, and plasma samples were collected and analysed. For each patient, samples were collected and stored as follows:
  • PBMC peripheral blood mononuclear cell
  • PBMCs were analysed using an LSRFortessa X-20 flow cytometer. Flow panels used are detailed in the tables below:
  • SEQ ID NO:1 Enterococcus gallinarum 16S rRNA gene - AF039900
  • SEQ ID NO:2 (consensus 16S rRNA sequence for Enterococcus gallinarum strain NCIMB 42488)
  • SEQ ID NO: 3 strain NCIMB 42488 chromosome sequence
  • SEQ ID NO:4 strain NCIMB 42488 plasmid sequence
  • SEQ ID NO: 5 see electronic sequence listing. Strain NCIMB 42761 genome sequence. Ns in the sequence represent gaps between contigs.
  • SEQ ID NO:6 (16S rRNA gene for Enterococcus gallinarum strain NCIMB 42761)

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Abstract

L'invention se rapporte au domaine des souches bactériennes isolées du tube digestif de mammifère et l'utilisation de telles souches, et de compositions comprenant ces souches, dans le traitement d'une maladie.
PCT/EP2022/075514 2021-09-14 2022-09-14 Compositions comprenant des souches bactériennes WO2023041574A1 (fr)

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