WO2023041203A1

WO2023041203A1 - Compositions comprising bacterial strains

Info

Publication number: WO2023041203A1
Application number: PCT/EP2022/056412
Authority: WO
Inventors: Marsilio ADRIANI; Anna ETTORRE; Imke MULDER; Aurelie Couturier
Original assignee: 4D Pharma Research Limited
Priority date: 2021-09-14
Filing date: 2022-03-11
Publication date: 2023-03-23
Also published as: TW202310861A

Abstract

The 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.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS

TECHNICAL FIELD

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.

BACKGROUND TO THE INVENTION

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. Subsequently, the microbiota stabilizes and becomes adult-like [1], 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. Similarly, 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],

In recognition of the potential positive effect that certain bacterial strains may have on the animal gut, various strains have been proposed for use in the treatment of various diseases (see, for example, [6- 9]). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [10] and [11] for reviews). Reference [12] teaches the use of Enterococcus gallinarum in the treatment of cancer. However, the relationship between different diseases and different bacterial strains, and the precise effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases, are poorly characterised.

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. However, due to the diverse nature of cancer, 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. SUMMARY OF THE INVENTION

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. In contrast, as demonstrated in the Examples disclosed herein, patients with intratumoural T regulatory cells or proliferating T cells below these levels, or with a number of macrophages above the maximum level in the total tumour or stromal region, are less likely to respond to therapy with an Enterococcus strain. The inventors have discovered that 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. The inventors’ findings are particularly surprising in view of the fact that patients with high tumour levels of CD3⁺ Foxp3⁺T regulatory cells appear less likely to respond to anticancer therapies, with tumour T regulatory cells appearing to drive resistance to therapy [15], Indeed, 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], 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. Similarly, 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.

This finding is particularly advantageous given that the applicant has demonstrated in a clinical study (clinicaltrials.gov identifier NCT03934827; reported in Example 7 below) that 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. By providing a benign and well-tolerated therapeutic which may be administered to patients who have responded poorly to prior lines of therapy, the present invention provides clinicians and patients with a new and attractive treatment option.

Accordingly, 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:

(a) an intratumoural level of T regulatory cells that is greater than or equal to a first predetermined threshold;

(b) an intratumoural level of proliferating T cells that is greater than or equal to a second predetermined threshold; and/or

(c) a total tumour level of macrophages that is less than or equal to a third predetermined threshold.

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. In some embodiments, 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. Advantageously, 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.

In these embodiments, 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. In these embodiments, cancer is defined as not having responded to the anticancer therapy if the patient has progressive disease (PD) after the defined treatment period. In these embodiments, cancer may be resistant to the anticancer therapy at the start of treatment with the anticancer 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. defined by the observance of a complete or partial remission, or stable disease) but, after a period of 6 months or more, stopped responding to the therapy (e.g. defined as the re-emergence of progressive disease). As used herein, 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 v1.1 [21],

In one embodiment, the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:

(a) an intratumoural level of T regulatory cells that is greater than or equal to the first predetermined threshold; and

(b) an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold.

(b) a total tumour level of macrophages that is less than or equal to the third predetermined threshold.

(a) an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold; and

(b) a total tumour level of macrophages that is less than or equal to the third predetermined threshold. In one embodiment, the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:

(a) an intratumoural level of T regulatory cells that is greater than or equal to the first predetermined threshold;

(b) an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold; and

(c) a total tumour level of macrophages that is less than or equal to the third predetermined threshold.

Additionally, 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:

(b) an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold; and/or

In some embodiments, 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 CD 127, or express low levels of CD 127 (CD127'^/10), for example as described in References [25] and [26], Alternatively or additionally, in some embodiments, the T regulatory cells express IL- 10 (IL-10⁺).

In some embodiments, 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 (PCAD) [22],

In some embodiments, 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 CDl lb (CDl lb⁺). In some embodiments, the macrophages express the markers HLA-DRa, iNOS, CDl lc, CD80, CD86 and/or pSTAT-1. For example, the macrophages may express HLA-DRa, iNOS, CDl lc, CD80, CD86 and pSTAT-1. Preferably, in such embodiments, the macrophages also express CD68. In some embodiments, the macrophages express the markers CD163, CD204, CD206, cMAF and/or VEGF. For example, the macrophages may express CD 163, CD204, CD206, cMAF and VEGF. Preferably, in such embodiments, 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. In this embodiment, the multiplex immunofluorescence staining and image analysis is preferably conducted using tissue samples that have been formalin-fixed and paraffin-embedded (FFPE). Preferably, 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 μm or more. Preferably, the tissue sample is 4 μm thick. Preferably, the tumour cells account for at least 10% of the cells in the biopsy specimen. Preferably, 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. Preferably, any necrotic areas in the tissue sample should be excluded from the analysis. Preferably, any mucussecreting areas in the tissue sample should be excluded from the analysis. In some embodiments, image analysis is carried out using InForm 2.4.8 image analysis software (Akoya Biosciences). In some embodiments, multiplex immunofluorescence staining and image analysis may be carried out as described in Example 6. In some embodiments, 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],

In some embodiments, 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:

(a) the level of T regulatory cells may be assessed by detecting cellular expression of CD3 and Foxp3;

(b) the level of proliferating T cells may be assessed by detecting cellular expression of CD3 and Ki67; and/or

(c) the level of macrophages may be assessed by detecting cellular expression of CD68.

In such methods, 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. Preferably, 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. In some embodiments, 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. Preferably, 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. Preferably, the sequence identity is to SEQ ID NO:2 or SEQ ID NO:6, more preferably SEQ ID NO:2. Most preferably, 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-α 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. In addition, or alternatively, 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. Thus, the invention is particularly useful in the treatment of cancer, preferably solid tumours. Examples of 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. Preferably, the bacterial strain is viable. Preferably, the bacterial strain is capable of partially or totally colonising the intestine.

In any of the embodiments described herein, the bacterial strain may be lyophilised. Preferably it is the Enterococcus strain which is lyophilised. Most preferably it is the Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae strain for use according to the invention which is lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

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. Alternatively, 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:

(a) optionally obtaining a tumour sample from the patient prior to administration of the bacterial strain;

(b) measuring one or more of the following in a tumour sample obtained from the patient prior to administration of the bacterial strain:

(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;

(c) determining whether:

(i) the intratumoural level of T regulatory cells in the sample is greater than or equal to a first predetermined threshold;

(ii) the intratumoural level of proliferating T cells is greater than or equal to a second predetermined threshold; and/or

(iii) 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. In some embodiments, 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:

(a) optionally obtaining one or more biopsies of one or more of the patient’s tumours;

(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);

(c) comparing (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 to a first, second, and/or third predetermined threshold, respectively; and

(d) 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, if: (i) the level of intratumoural T regulatory cells is determined to be greater than or equal to the first predetermined threshold; (ii) the level of intratumoural proliferating T cells is determined to be greater than or equal to the second predetermined threshold; and/or (iii) the level of intratumoural macrophages is determined to be less than or equal to the third predetermined threshold.

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:

(a) 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;

(b) comparing (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 to a first, second, and/or third predetermined threshold, respectively; and

(c) 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. For example, 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:

(c) a total tumour level of macrophages that is less than or equal to a third predetermined threshold. In any of the above embodiments, the Enterococcus strain may be of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae.

BRIEF DESCRIPTION OF DRAWINGS

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.

Figure 4A: Cytokine levels (μg/ml) in immature dendritic cells (No bacteria).

Figure 4B: Cytokine levels (μg/ml) in immature dendritic cells after the addition of LPS.

Figure 4C: Cytokine levels (μg/ml) in immature dendritic cells after the addition of NCIMB 42488.

Figure 4D: Cytokine levels (μg/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.

Figure 5C: Cytokine levels in THP-1 cells after the addition of NCIMB 42488 alone or in combination with LPS.

Figure 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 mm² 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). Mean +/- SEM; Mann-Whitney t-test. PR = partial response; SD = stable disease; PD = progressive disease.

Figure 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 mm² 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. The number of CD8⁺ cytotoxic T cells (CTL; CD3⁺ CD8⁺ cells), proliferating CTL cells (CD3⁺ CD8⁺ Ki67⁺ cells), CTL cells expressing PD-1 (CD3⁺ CD8⁺ PD-1⁺) or PD-L1 (CD3⁺ CD8⁺ PD-L1⁺), and the number of T-regulatory (Treg) cells (CD3⁺ CD8' FOXP3⁺) per mm² 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 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 mm² 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.

DETAILED DESCRIPTION

Threshold T cell levels

In the present invention, 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. 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.

As used herein, 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. Thus, by way of example, the “total tumour level of macrophages” refers to the sum of the intratumoural and the stromal macrophages.

In an embodiment, 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.

In an embodiment, 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. 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 and a total tumour level of macrophages that is less than or equal to a third 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 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. In an embodiment, 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.

In an embodiment, 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.

Alternatively or additionally, 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. Alternatively or additionally, 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.

In one embodiment, 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. In an embodiment, 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.

In one embodiment, 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. In an embodiment, 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, 1.9, or 2 cells per square millimetre. In one embodiment, 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,

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 one embodiment, 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,

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 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. In an embodiment, the fourth predetermined threshold is the same as the third predetermined threshold.

In one embodiment, 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.

As used herein, “about” may refer to any value which is within +/- 0.5 of the given integer. For example, 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. Alternatively, when the value is specified to one decimal place, “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). Preferably, 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 μm. Preferably, the tissue sample is 4 μm thick. Preferably, the tumour cells account for at least 10% of the cells in the biopsy specimen. Preferably, 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. Preferably, any necrotic areas in the tissue sample should be excluded from the analysis. Preferably, 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. For example, each region of the tissue sample that is assessed may be about 931 x 698 μm in size. The resolution used may be 20X. In a more preferred embodiment, 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. In an embodiment, the average is the mean, median, or modal value. Preferably, 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. Preferably, 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. In some embodiments, multiplex immunofluorescence staining and image analysis may be carried out as described in Example 6. In some embodiments, 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. Preferably, 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. Preferably, fluorescent markers are used. Each label may be specifically attached to one cell marker, for example CD3 or Foxp3. Preferably, each cell marker is associated with a different label. For example, 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. For example, 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. Further 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.

In some embodiments, 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 CD 127, or express low levels of CD 127 (CD127μm), 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 (CD49dμm), for example as described in Reference [27].

In some embodiments, 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 CDl lb (CDl lb⁺). In some embodiments, the macrophages express the markers HLA-DRa, iNOS, CDllc, CD80, CD86 and/or pSTAT-1. For example, the macrophages may express HLA-DRa, iNOS, CDl lc, CD80, CD86 and pSTAT-1. Preferably, in such embodiments, the macrophages also express CD68. In some embodiments, the macrophages express the markers CD163, CD204, CD206, cMAF and/or VEGF. For example, the macrophages may express CD 163, CD204, CD206, cMAF and VEGF. Preferably, in such embodiments, the macrophages also express CD68.

Thus, in some embodiments, 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], In some embodiments, 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.

In some embodiments, 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 PCAD. In some embodiments, cells that express one or more, two or more, three or more, or all of CD3, Ki67, MCM-2 and PCAD above a background level may be classified as proliferating T cells. In some embodiments, the proliferating T cells express CD4. In some embodiments, the proliferating T cells express CD8. In some embodiments, the proliferating T cells do not express CD8.

In some embodiments, 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, CDl lb, HLA-DRa, iNOS, CDl lc, CD80, CD86, pSTAT-1, CD163, CD204, CD206, cMAF and/or VEGF.

In some embodiments, 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. However, marker expression may also be detected by other methods known in the art. For example, 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 (lOxGenomics). For techniques which involve the quantification of specific cell types of interest (e.g. T regulatory cells, proliferating T cells and/or macrophages), in a in a tissue section having a substantial thickness, 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 μm). A volumetric threshold may similarly be used for flow cytometry based techniques, based on the volume of the sample that is processed. In such embodiments, the thresholds defined herein in square millimetres (e.g. for a tissue section of 4 μm thickness) 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. For example, in immunofluorescence analysis, 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. When optimising the assay, 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.

In an embodiment, 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. For example, the first predetermined threshold may be determined by:

(a) identifying a reference population to be treated with the bacterial strain;

(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”);

(c) after therapy with the bacterial strain, determining patients who did not respond to therapy with the bacterial strain;

(d) determining the average intratumoural level of T regulatory cells in the pre-treatment samples taken from the patients who did not respond to therapy with the bacterial strain;

(e) determining a first predetermined threshold that is greater than the average level of intratumoural T regulatory cells in the patients who did not 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 greater than the average level of intratumoural T regulatory 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 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. In this embodiment, 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. In one embodiment, 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. Alternatively, 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. In this embodiment, 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:

(a) identifying a reference population to be treated with the bacterial strain;

(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”);

(d) determining the average intratumoural level of proliferating T cells in the pre-treatment samples taken from the patients who did not respond to therapy with the bacterial strain;

(e) 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. In this embodiment, 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. In one embodiment, 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. Alternatively, 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. In this embodiment, 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:

(a) identifying a reference population to be treated with the bacterial strain;

(b) determining the total tumour level of macrophages in a sample of the tumour taken from each patient prior to administration of the bacterial strain (“pre-treatment sample”);

(d) determining the average total tumour level of macrophages in the pre-treatment samples taken from the patients who did not respond to therapy with the bacterial strain;

(e) 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. In this embodiment, the determination of the third predetermined threshold according to step (e) may optionally comprise determining a threshold that is less than the average pretreatment 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. In one embodiment, 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. Alternatively, 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. In this embodiment, 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.

In one embodiment, 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. Response criteria are preferably defined according to the RECIST (Response Evaluation Criteria In Solid Tumours) criteria vl .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:

(a) identifying a reference population to be treated with the bacterial strain;

(b) determining the stromal level of macrophages in a sample of the tumour taken from each patient prior to administration of the bacterial strain (“pre-treatment sample”);

(d) determining the average stromal level of macrophages in the pre-treatment samples taken from the patients who did not respond to therapy with the bacterial strain;

(e) 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, 7.5, 8, 8.5, 9, 9.5 or 10 cells per square millimetre less than the average level of total tumour level of stromal 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 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. In this embodiment, the determination of the fourth predetermined threshold according to step (e) may optionally comprise determining a threshold that is less than the average pretreatment 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. In one embodiment, the threshold is determined as being the midpoint 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. Alternatively, 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. In this embodiment, 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,

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 stromal macrophages in the patients who did respond to therapy with the bacterial strain.

The fifth predetermined threshold may be determined by:

(a) identifying a reference population to be treated with the bacterial strain;

(b) determining the intratumoural level of macrophages in a sample of the tumour taken from each patient prior to administration of the bacterial strain (“pre-treatment sample”);

(d) determining the average intratumoural level of macrophages in the pre-treatment samples taken from the patients who did not respond to therapy with the bacterial strain;

(e) 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. In this embodiment, 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. In one embodiment, the threshold is determined as being the midpoint 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. Alternatively, 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. In this embodiment, 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.

Alternatively or additionally, in other embodiments 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.

In an embodiment, 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. In an embodiment, 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. In an embodiment, 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. Methods of monitoring a patient

In one embodiment, the present invention provides a method of monitoring a patient, wherein said patient has cancer, said method comprising:

(d) 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, 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:

In one embodiment, 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). In one embodiment, 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). Alternatively, 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. In some embodiments, 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.

In an embodiment, 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.

Bacterial strains

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. Preferably, 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). Preferably, 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. Thus, without being bound by theory, 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. As described above, it is surprising that 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.

Preferably, 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.

Preferably, 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. In some embodiments, the bacterial strain is flagellated (i.e. the cells have at least one flagellum). The Examples demonstrate that bacteria of this species are particularly effective in certain patient sub-populations, for example where the patients have (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.

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. The type strain of Enterococcus gallinarum is F87/276 = PB21 = ATCC 49573 = CCUG 18658 = CIP 103013 = JCM 8728 = LMG 13129 = NBRC 100675 = NCIMB 702313 (formerly NCDO 2313) = NCTC 12359 [31], The 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.

The genome of 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. "N¹ 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

Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing cancer. In certain embodiments, 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. Preferably, 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. Preferably, the sequence identity is to SEQ ID NO:2 or 6. Preferably, 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). For example, in some embodiments, 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. Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)₅, 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. In some embodiments, 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. In some embodiments, 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. In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO: 3. In preferred embodiments, 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. For example, 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 SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 98% identity to SEQ ID NO:3 across 95% of SEQ ID NO:3, or at least 98% sequence identity to SEQ ID NO:3 across 100% of SEQ ID NO:3, or at least 99.5% sequence identity to SEQ ID NO:3 across 90% of SEQ ID NO:3, or at least 99.5% identity to SEQ ID NO:3 across 95% of SEQ ID NO:3, or at least 99.5% identity to SEQ ID NO:3 across 98% of SEQ ID NO:3, or at least 99.5% sequence identity to SEQ ID NO:3 across 100% of SEQ ID NO:3.

In certain embodiments, the bacterial strain for use in the invention has a plasmid with sequence identity to SEQ ID NO: 4. In preferred embodiments, 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 NON across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NON. For example, the bacterial strain for use in the invention may have a plasmid with at least 90% sequence identity to SEQ ID NON across 70% of SEQ ID NON, or at least 90% sequence identity to SEQ ID NON across 80% of SEQ ID NON, or at least 90% sequence identity to SEQ ID NON across 90% of SEQ ID NON, or at least 90% sequence identity to SEQ ID NON across 100% of SEQ ID NON, or at least 95% sequence identity to SEQ ID NON across 70% of SEQ ID NON, or at least 95% sequence identity to SEQ ID NON across 80% of SEQ ID NON, or at least 95% sequence identity to SEQ ID NON across 90% of SEQ ID NON, or at least 95% sequence identity to SEQ ID NON across 100% of SEQ ID NON, or at least 98% sequence identity to SEQ ID NON across 70% of SEQ ID NON, or at least 98% sequence identity to SEQ ID NON across 80% of SEQ ID NON, or at least 98% sequence identity to SEQ ID NON across 90% of SEQ ID NON, or at least 98% sequence identity to SEQ ID NON across 100% of SEQ ID NON.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NON and a plasmid with sequence identity to SEQ ID NON. In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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. Alternatively, 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 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Enterococcus gallinarum strains.

In certain embodiments, 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]). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42488. In some embodiments, the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMerieux). In some embodiments, 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

(ii) intermediate for fermentation of at least one of (e.g. at least 2, 3, 4 or all of): D-mannitol, Methyl-aD-gly copyranoside, D-lactose, starch, and L-fucose; preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMerieux).

Other 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. In particular, 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. In particular, 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.

In some embodiments, the bacterial strain used in the invention is:

(i) Positive for at least one of (e.g. at least 2, 3, 4, 5, 6, 7 or all of): mannose fermentation, glutamic acid decarboxylase, arginine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, histidine arylamidase and serine arylamidase; and/or

(ii) Intermediate for at least one of (e.g. at least 2 or all of): P-galactosidase-6-phosphate, β-glucosidase and N-acetyl-P-glucosaminidase; and/or

(iii) Negative for at least one of (e.g. at least 2, 3, 4, 5, 6 or all of): Raffinose fermentation, Proline arylamidase, Leucyl glycine arylamidase, Leucine arylamidase, Alanine arylamidase, Glycine arylamidase and Glutamyl glutamic acid arylamidase, preferably as determined by an assay of carbohydrate, amino acid and nitrate metabolism, and optionally an assay of alkaline phosphatase activity, more preferably as determined by Rapid ID 32A analysis (preferably using the Rapid ID 32A system from bioMerieux).

In some embodiments, the bacterial strain used in the invention is:

(i) 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 bioMerieux); 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 bioMerieux).

In some embodiments, the bacterial strain used in the invention is an extracellular ATP producer, for example one which produces 6-6.7 ng/pl (for example, 6.1-6.6 ng/pl or 6.2-6.5 ng/pl or 6.33 ± 0.10 ng/pl) 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 Thl7 cell differentiation [35] and induction of secretion of the pro-inflammatory mediator IL-ip by activating the NLRP3 inflammasome [36], Accordingly, a bacterial strain which is an extracellular ATP producer is useful for treating or preventing cancer.

In some embodiments, 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 FIGOO632333: hypothetical protein. For example, in certain embodiments, the bacterial strain for use in the invention comprises genes encoding Mobile element protein and Xylose ABC transporter, permease component; Mobile element protein and FIGOO632333: hypothetical protein; Xylose ABC transporter, permease component and FIGOO632333: hypothetical protein; or Mobile element protein, Xylose ABC transporter, permease component, and FIGOO632333: 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. In preferred embodiments of the invention, 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. In preferred embodiments of the invention, 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. In particular, 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. In particular, 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.

In preferred embodiments, the bacterial strains in the compositions of the invention are viable and/or capable of partially or totally colonising the intestine.

Treating cancer

The Examples 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. Therefore, 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.

In certain embodiments, treatment with the bacterial strains of the invention results in a reduction in tumour size or a reduction in tumour growth. In certain embodiments, 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.

In certain embodiments, 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. In preferred embodiments, treatment with a bacterial strain according to the invention results in a pathologic complete response (pCR).

In preferred embodiments, 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. In preferred embodiments, the renal cancer is renal cell carcinoma. In preferred embodiments, the lung cancer is non-small cell lung cancer. In preferred embodiments, 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.

In certain embodiments, 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. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer. In preferred embodiments the breast cancer is triple negative breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, 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. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer. In some embodiments the cancer is lung carcinoma. In preferred embodiments the cancer is non-small cell lung cancer.

In certain embodiments, 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. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of liver cancer. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma).

In certain embodiments, 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. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of colon cancer. In preferred embodiments the cancer is colorectal adenocarcinoma.

In certain embodiments, the bacterial strain is for use in treating or preventing 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. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of renal cancer. In preferred embodiments the cancer is renal cell carcinoma or transitional cell carcinoma.

In certain embodiments, the bacterial strain is for use in treating or preventing melanoma. According to some embodiments, the bacterial strain has an effect on melanocytes and may be effective for treating melanoma. In certain embodiments, the bacterial strain is for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of melanoma.

In some embodiments, 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.

In some embodiments, the cancer is not of the head and/or neck.

In certain embodiments, 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. In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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 TNFα 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 IFNy-inducible genes (IL-32) may indicate that the compositions for use according to the invention elicit an IFNy-type response. IFNy is a potent macrophage-activating factor that can stimulate tumirocidal activity [39], and CXCL9 and CXCL10, for example, also have anti-cancer effects [40-42], Therefore, in certain embodiments, the bacterial strains are for use in promoting inflammation in the treatment of cancer. In preferred embodiments, the bacterial strains are for use in promoting Thl inflammation in the treatment of cancer. Thl cells produce IFNy and have potent anti-cancer effects [37], In certain embodiments, 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. Promoting inflammation may be more effective against early-stage cancers [37], In certain embodiments, the treatment or prevention of cancer comprises increasing the level of expression of one or more cytokines. For example, in certain embodiments, the treatment or prevention of cancer comprises increasing the level of expression of one or more of IL-1β, IL-6 and TNF-α , for example, IL-1β and IL-6, IL-1β and TNF-α , IL-6 and TNF-α or all three of IL-1β, IL-6 and TNF-α . Increases in levels of expression of any of IL-1β, IL-6 and TNF-α are known to be indicative of efficacy in treatment of cancer.

In some embodiments, 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.

In certain embodiments, 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.

In certain embodiments, the bacterial strains are for use in treating hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.

In certain embodiments, the bacterial strains are for use in treating invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.

In further embodiments, 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, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumour, gastrointestinal stromal tumour (GIST), germ cell tumour, glioma, childhood visual pathway and hypothalamic glioma, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

In some embodiments, 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. In some embodiments, the bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition administered in accordance with the invention.

Modes of administration

Administration of the bacterial strain may be carried out as follows. Preferably, 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. Generally, 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.

In certain embodiments, the bacterial strain may be administered as a foam, as a spray or a gel.

In certain embodiments, 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.

In certain embodiments, the bacterial strain is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jej unostomy 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. In certain embodiments, the bacterial strains or compositions for use according to the invention are to be administered daily. In certain embodiments, the bacterial strains or compositions for use according to the invention are administered twice daily or more. In certain embodiments, 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. Preferably, the bacterial strains or compositions for use according to the invention are administered twice daily.

In certain embodiments, 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.

In certain embodiments, 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 bom.

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 develoμment 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. For example, 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.

Generally, 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.

In preferred embodiments, the composition is formulated in freeze-dried form. For example, the composition may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain as described herein.

Preferably, 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],

Alternatively, the composition may comprise a live, active bacterial culture.

Preferably, the 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. In other words, the bacteria may have colonised some or all of the gastrointestinal tract and / or such colonisation may be transient or permanent.

More specifically, in some embodiments, 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.

In some embodiments, “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. For example, the bacterial strain in the composition for use according to the invention may not have been killed, inactivated and/or attenuated. For example, the bacterial strain in the composition for use according to the invention is live. For example, the bacterial strain in the composition for use according to the invention is viable. For example, 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.

In some embodiments, 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. For example, in some embodiments, the bacterial strain in the composition for use according to the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition for use according to the invention is viable and capable of partially or totally colonising the intestine. In some embodiments, the composition comprises a mixture of live bacterial strains and bacterial strains that have been killed.

In preferred embodiments, 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 entraμment 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. Guidance on encapsulation that may be useful for preparing compositions for use according to the invention is 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. Alternatively, 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.

The 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, for example for an adult human, may be from about 1 x 10³ to about 1 x 10¹¹ colony forming units (CFU); for example, from about 1 x 10⁷ to about 1 x 10¹⁰ CFU; in another example from about 1 x 10⁶ to about 1 x 10¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 10⁶to about 1 x 10¹¹ CFU/g, respect to the weight of the composition; for example, from about 1 x 10⁸ to about 1 x 10¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

Typically, 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.

In certain embodiments, 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, xylooligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, 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. Examples of such suitable 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). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of 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. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

The compositions comprising the bacterial strain may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement. Similarly, 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. In certain embodiments, the composition is formulated as a milk-based product. The term "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, goa s 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. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

In certain embodiments, 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. Thus, in some embodiments, 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.

In some embodiments, the compositions for use in the invention comprise more than one bacterial strain or species. For example, in some embodiments, 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.

In some embodiments, 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). In some embodiments, 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.

In some embodiments, the 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. In some embodiments, 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. In some embodiments, the 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.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, 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 o Enterococcus hirae, as part of a microbial consortium. For example, in some embodiments, 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. Optionally, these bacterial strains may from other genera with which the bacterial strain of the invention can live symbiotically in vivo in the intestine. For example, in some embodiments, 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. In some embodiments, 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. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organisms. In some embodiments, the two different organisms are from the same species, e.g. two different humans, e.g. two different human infants. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the composition for use in the invention additionally comprises a bacterial 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.

In some embodiments, the composition for use in the invention additionally comprises a bacterial 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.

In some embodiments in which 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. For example, 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. In some embodiments, the more than one bacterial strains, species or genera are stored separately but are mixed together prior to use.

In some embodiments, 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.

As mentioned above, in some embodiments, 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. In some embodiments, the bacterial strain(s) in the bacterial composition is/are the only therapeutically active agent(s) in the composition.

The compositions for use in accordance with the invention may or may not require marketing approval.

In certain embodiments, 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. 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 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.

In some cases, the lyophilised or spray dried bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The compositions can comprise pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides 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. In preferred embodiments the cancer is pancreatic adenocarcinoma.

In certain embodiments, 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. In preferred embodiments, the cancer is triple negative breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, 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. In preferred embodiments the cancer is lung carcinoma. In preferred embodiments, the cancer is non small cell lung cancer. In certain embodiments, 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. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma).

In certain embodiments, 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. In preferred embodiments the cancer is colorectal adenocarcinoma.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, 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 myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumour, gastrointestinal stromal tumour (GIST), germ cell tumour, glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

In certain embodiments, the amount of the bacterial strain in the bacterial composition is from about 1 x 10³ to about 1 x 10¹¹ colony forming units per gram with respect to a weight of the composition.

In certain embodiments, 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.

In certain embodiments, the pharmaceutical composition comprises a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol and sorbitol.

In certain embodiments, the pharmaceutical composition comprises a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, 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.

In certain embodiments, the pharmaceutical composition further comprises at least one of a preservative, an antioxidant and a stabilizer.

In certain embodiments, the pharmaceutical composition comprises a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.

In certain embodiments, 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. In some embodiments, the composition is provided in a sealed container. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition is provided in a syringe.

The bacterial composition may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a unit dosage form, for example as a tablet or capsule. In some embodiments, the capsule is a gelatine capsule (“gel-cap”). Preferably, each unit dosage form (e.g. tablet or capsule) contains at least about 1x10³, at least about 1 x 10⁵, at least about 1x10⁶, at least about 1x10⁷, at least about 1x10⁸ or at least about 1x10⁹ CFU of the bacterial strain. In embodiments, the unit dosage form comprises about 1x10⁸, about 1x10⁹ or about 1x10¹⁰to about 1x10¹¹ CFU of the bacterial strain.

In some embodiments, 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.

Pharmaceutical formulations 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.

In some embodiments 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.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, 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.

In some embodiments, the enteric formulation is intrinsically enteric, for example, gastro-resistant without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulosic material, such as methylcellulose, hydroxymethylcellulose or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any fdm forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [50 ]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).

In some embodiments, 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. Depending on the method of administration, such as, for example, orally or rectally, 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.

Culturing methods

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), NaHCO₃ (0.4 g), cysteine (0.1 g), K₂HPO4 (0.045 g), KH₂PO4 (0.045 g), NaCl (0.09 g), (NH₄)₂SO4 (0.09 g), MgSO₄ • 7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folic acid (5 μg), and pyridoxamine (15 μg).

Bacterial strains for use in vaccine compositions

The inventors have identified that 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. In certain such embodiments, the bacterial strains are viable. In certain such embodiments, the bacterial strains are capable of partially or totally colonising the intestine. In certain such embodiments, the bacterial strains are viable and capable of partially or totally colonising the intestine. In other certain such embodiments, the bacterial strains may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subcutaneous injection.

General

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., references [54] and [55- 61], etc. The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

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.

Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combined with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE INVENTION

Example 1 - Efficacy of bacterial inocula in mouse models of cancer

Summary

This study tested the efficacy of compositions comprising bacterial strains according to the invention in four tumour models.

Materials

Test substance - Bacterial strain NCIMB 42488.

Bacterial strain NCIMB 42761

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: 2x10⁸ in 200 μL. 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 μL of test substance will be administered) according to the most recent body weight of mice.

Routes of administration - Bacterial inoculum was administered by oral gavage (per os, PO) via a cannula. Cannulas were decontaminated every day. Anti-CTLA-4 was injected into the peritoneal cavity of mice (Intraperitoneally, IP).

Culture conditions of bacterial strain - The culture conditions for the bacterial strain were as follows:

• Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles) into Hungate tubes

• Seal the tubes and flush with CO2 using a syringe input and exhaust system

• Autoclave the Hungate tubes

• When cooled, inoculate the Hungate tubes with 1 mL of the glycerol stocks

• Place the tubes in a static 37°C incubator for about 16 hours.

• The following day, take 1 mL of this subculture and inoculate 10 mL of YCFA (pre- warmed flushed Hungate tubes again, all in duplicate)

• Place them in a static 37°C incubator for 5 to 6h

Cancer cell line and culture conditions -

The cell lines that were used are detailed in the table below:

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 a tumour 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], Cell culture conditions - All cell lines were grown as monolayer at 37°C in a humidified atmosphere (5% CO2, 95% air). The culture medium and supplement are indicated in the table below:

For experimental use, 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.

Use of animals -

Healthy female Balb/C (BALB/cByJ) mice, of matching weight and age, were obtained from CHARLES RIVER (L'Arbresles) for the EMT6 model experiments.

Healthy female C57BL/6 (C57BL16J) mice, of matching weight and age, were obtained from CHARLES RIVER (L'Arbresles) for the LL/2(LLC1) and the Hepal-6 model experiments.

Animals were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals were followed [67,68], Animals were maintained in housing rooms under controlled environmental conditions: Temperature: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod (12h light/12h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm² 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.

Experimental design and treatments

Antitumour activity, EMT6 model Treatment schedule - The start of first dosing was considered as D0. On D0, non-engrafted mice were randomized according to their individual body weight into groups of 9/8 using Vivo manager® software (Biosystemes, Coutemon, France). On D0, the mice received vehicle (culture medium) or bacterial strain. On D14, all mice were engrafted with EMT-6 tumour cells as described below. On D24, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule for NCIMB 42488 is summarized in the table below:

The treatment schedule for NCIMB 42761 is summarised in the table below:

The monitoring of animals was performed as described below. Induction of EMT6 tumours in animals - On DI 4, tumours were induced by subcutaneous injection of 1x10⁶ EMT-6 cells in 200 μL 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.

Antitumour activity, LL/2 (LLC1) model Treatment schedule - The start of first dosing was considered as D0. On D0, non-engrafted mice were randomized according to their individual body weight into 7 groups of 9/8 using Vivo manager® software (Biosystemes, Coutemon, France). On D0, the mice will received vehicle (culture medium) or bacterial strain. On D14, all mice were engrafted with LL/2 tumour cells as described below. On D27, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

The monitoring of animals was performed as described below.

Induction of LL/2 (LLC1) tumours in animals - On DI 4, tumours were induced by subcutaneous injection of 1x10⁶ LL/2 (LLC1) cells in 200 μL RPMI 1640 into the right flank of mice.

Antitumour activity, Hepal-6 model

Treatment schedule - The start of first dosing was considered as D0. On D0, non-engrafted mice were randomized according to their individual body weight into 7 groups of 9 using Vivo manager® software (Biosystemes, Coutemon, France). On D0, the mice received vehicle (culture medium) or bacterial strain. On D14, all mice were engrafted with Hepa 1-6 tumour cells as described below. On D16, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

The monitoring of animals was performed as described below. Orthotopic induction of Hepa 1-6 tumour cells in animals by intrasplenic injection - On D14, one million (1x10⁶) Hepa 1 -6 tumour cells in 50 μL 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.

Euthanasia - Each mouse was euthanized when it reached a humane endpoint as described in section below, or after a maximum of 6 weeks post start of dosing.

Evaluation of tumour burden at euthanasia - At the time of termination, livers were collected and weighed.

Animal monitoring

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 mm³; 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 - Isoflurane 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. Results

Antitumour activity, EMT6 model

The results are shown in Figure 1. Treatment with the bacterial strain of the invention led to a clear reduction in tumour volume relative to both the negative controls. The positive control also led to a reduction in tumour volume, as would be expected. This result was seen with both of the tested strains.

Antitumour activity, LL/2 (LLC1) model

The results are shown in Figure 2. Treatment with the bacterial strain of the invention led to a clear reduction in tumour volume relative to both the negative controls.

Antitumour activity, Hepal-6 model

The results are shown in Figure 3. The untreated negative control does not appear as would be expected, because liver weight was lower in this group than the other groups. However, the vehicle negative control and the positive control groups both appear as would be expected, because mice treated with vehicle alone had larger livers than mice treated with anti-CTLA4 antibodies, reflecting a greater tumour burden in the vehicle negative control group. Treatment with the bacterial strain of the invention led to a clear reduction in liver weight (and therefore tumour burden) relative to the mice in the vehicle negative control group.

These data indicate that strain NCIMB 42488 may be useful for treating or preventing cancer, and in particular for reducing tumour volume in breast, lung and liver cancers.

Example 2 - PCR gene analysis

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:

These data appear to show two gene expression signatures - CXCR1/2 ligands (CXCL3, CXCL2, CXCL1, IL-8), which is associated with pro-inflammatory cell migration, and CXCR3 ligands (CXCL9,CXCL10), which is more specifically indicative of IFN-γ-type responses, also supported by IL-32, which is IFN-γ-inducible.

Example 3 - Stability testing

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

Summary

This study tested the effect of the bacterial strain NCIMB 42488 alone and in combination with lipopolysaccharide (LPS) on cytokine production in immature dendritic cells.

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 10⁷/ml, with the optional addition of LPS at a final concentration of lOOng/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 lOOng/ml. The cytokine content of the cells was then analysed.

Results

The results of these experiments can be seen in Figures 4a-d. The addition of NCIMB 42488 alone leads to a substantial increase in the level of cytokines IL-6 and TNF-α compared to the negative control (Figure 4a and c). The addition of LPS (positive control) leads to an increase in the level of IL-6 and TNF-α compared to the negative control but not IL-1β (Figure 4b). A combination of NCIMB 42488 and LPS led to a synergistic increase in the level of IL- 10 produced (Figure 4d).

Conclusion

NCIMB 42488 has the ability to induce higher IL-6 and TNF-α cytokine production in immature dendritic cells. The combination LPS and NCIMB 42488 can increase the levels of cytokines IL-1β in immature dendritic cells. These data indicate that NCIMB 42488 alone or in combination with LPS can increase inflammatory cytokines IL- 10, IL-6 and TNF-α , 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 5 - cytokine production in THP-1 cells induced by NCIMB 42488 compared to NCIMB 42488 + LPS

Summary

This study tested the effect of bacterial strain NCIMB 42488 alone and in combination with LPS on cytokine production in THP-1 cells, a model cell line for monocytes and macrophages.

THF-1 cells were differentiated in M0 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 10⁸/ml, with or without the addition of LPS at a final concentration of lOOng/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.

Results

The results of these experiments can be seen in Figures 5a-c. The addition of NCIMB 42488 without LPS leads to an increase in the cytokine levels of IL-1β, IL-6 and TNF-α compared to the no bacterial and the bacterial sediment controls. The addition of LPS and NCIMB 42488 leads to a synergistic increase in the production of cytokines.

Conclusion

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- 10, IL-6 and TNF-α , 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. Pre-clinical in vivo studies demonstrated that 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. These observations suggest that NCIMB 42488 may switch a less T-cell-inflamed “cold” tumour to an immune-responsive, more T-cell-inflamed “hot” tumour, and thereby render the tumour more sensitive to immunotherapy approaches.

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. As part of this, the adaptive and innate immune cells in pre-treatment tumour tissues obtained from anti-PD-(L)l secondary resistant patients were quantified.

Methods

Patients received NCIMB 42488 1 capsule orally, twice daily (PO BID) (1x10¹⁰ to 1x10¹¹ CFU) and pembrolizumab (200mg Q3 W) for up to 2 years or until progression. Eligible patients have experienced clinical benefit from a prior ICI before eventually progressing. That is, the cancer has developed resistance to the ICI. Tumour response was assessed every 9 weeks by RECIST vl .1. Responders were classed as patients who experienced CR, PR or SD >6 months. To date, baseline (i.e. pre-treatment) 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.

Multiplex Immunofluorescence staining and image analysis.

Multiplex immunofluorescence (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). All the markers were stained in sequence using their respective fluorophore containing in the Opal 7 kit (catalogue #NEL797001KT; Akoya Biosciences, Waltham, MA) and the individual tyramide signal amplification fluorophores Opal Polaris 480 (catalog #FP1500001KT) and Opal Polaris 780 kit (catalog #FP1501001KT, Akoya Biosciences), [71], The slides were scanned using the Vectra/Polaris 3.0.3 (Akoya Biosciences) at low magnification, lOx (1.0 μm/pixel) through the full emission spectrum and using positive tonsil controls from the run staining to calibrate the spectral image scanner protocol [72], A pathologist selected at least 5 regions of interest (ROIs) for scanning in high magnification using the Phenochart Software image viewer 1.0.12 (931 x 698 μm size at resolution 20x) in order to capture various elements of tissue heterogeneity. 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/mm² [72], All the data was consolidated using the R studio 3.5.3 (Phenopter 0.2.2 packet, Akoya Biosciences).

Statistical analysis

Data are presented as mean ± standard error in Figures 6 to 9. Statistical significance was determined by the Student t test using GraphPad Prism 9 (Graph- Pad Software). P values <0.05 were considered significant.

Evaluation of target legions

Complete Response (CR): Disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm.

Partial Response (PR): At least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline (i.e. pre-treatment) sum diameters.

Progressive Disease (PD): At least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression).

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum of diameters while on study.

Results

Response to therapy with the bacterial strain was associated with higher baseline densities of intratumoural Treg (CD3⁺CD8 FOXP3⁺) (p=0.0381) and proliferating total T-cells (CD3⁺KI67⁺) (p=0.0048) at baseline, whereas higher densities of macrophages (CD68⁺ cells, p=0.0303) were detected in the stroma and total tumour tissue compartment of non-responders.

Understanding mechanisms of resistance to anticancer therapies is key in identifying patients who may respond to subsequent therapies. This analysis, together with the observations of increased CD8⁺ T cell activation and reduced induction of Treg cells in the presence of tolerogenic cytokines in vitro and in preclinical models, provides insight for further investigation into the potential for NCIMB 42488 and related bacterial strains to overcome Treg-mediated resistance to cancer treatment. As demonstrated herein, novel biomarkers have been provided which assist in identifying the patients most likely to respond to therapy based on Enterococcus strains including NCIMB 42488.

Example 7 - Assessment of safety and tolerability of the administration of NCIMB 42488 as a monotherapy

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.

Sequences

SEQ ID NO:1 (Enterococcus gallinarum 16S rRNA gene - AF039900)

1 taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag

61 agtggcgaac gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg

121 aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgctt

181 ttgcgtcact gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc

241 aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac tgagacacgg

301 cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa agtctgaccg

361 agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt tagagaagaa

421 caaggatgag agtagaacgt tcatcccttg acggtatcta accagaaagc cacggctaac

481 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt

541 aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg

601 gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgtgtagcgg

661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact

721 gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc

781 gtaaacgatg agtgctaagt gttggagggt ttccgccctt cagtgctgca gcaaacgcat

841 taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc

901 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc

961 ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg

1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca

1081 acccttattg ttagttgcca tcatttagtt gggcactcta gcgagactgc cggtgacaaa

1141 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg

1201 tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc

1261 ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat

1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac

1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgcctaag

1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg

1501 atcacc SEQ ID NO:2 (consensus 16S rRNA sequence for Enterococcus gallinarum strain NCIMB 42488)

TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGGTGA GTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTC CGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTA ACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAG GTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCCTTGACGGTATCTAA CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGC GTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGG GAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGT GGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGG TAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCA CTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTG GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTT CGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC GCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGG GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAA GTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGA ATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGA GAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTG

SEQ ID NO:3 (strain NCIMB 42488 chromosome sequence) - see electronic sequence listing.

SEQ ID NO:4 (strain NCIMB 42488 plasmid sequence) - see electronic sequence listing.

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)

TAATACATGCAAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGGTGAG TAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTCC GCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAA CGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACT CCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGG TTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCCTTGACGGTATCTAAC CAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCG TAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGG AGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTG GCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGT AGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCAC TCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGG TTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTC GGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGG GATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAG

TCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAA

TCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAG

AGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAA GTCGTAACAAGGTAGCCGTATCGGAAGGTGCGGCTGGATCACC

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Claims

1. 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, or 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:

2. The bacterial strain for use according to claim 1, wherein the first predetermined threshold is at least about 4 cells per square millimetre for example wherein the first predetermined threshold is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 cells per square millimetre.

3. The bacterial strain for use according to claim 1 or claim 2, wherein the second predetermined threshold is at least about 0.2 cells per square millimetre, for example wherein 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.

4. The bacterial strain for use according to any one of the preceding claims, wherein the third predetermined threshold is no more than about 30 cells per square millimetre, for example wherein 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.

5. The bacterial strain for use according to any one of the preceding claims, wherein the bacterial strain is administered to a patient having, prior to administration of the bacterial strain:

(a) an intratumoural level of T regulatory cells that is greater than or equal to the first predetermined threshold and an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold;

(b) an intratumoural level of T regulatory cells that is greater than or equal to the first predetermined threshold and a total tumour level of macrophages that is less than or equal to the third predetermined threshold;

(c) an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold and a total tumour level of macrophages that is less than or equal to the third predetermined threshold; or (d) an intratumoural level of T regulatory cells that is greater than or equal to the first predetermined threshold, an intratumoural level of proliferating T cells that is greater than or equal to the second predetermined threshold and a total tumour level of macrophages that is less than or equal to the third predetermined threshold.

6. The bacterial strain for use according to any one of the preceding claims, wherein:

(a) the T regulatory cells express CD3 and Foxp3 (CD3⁺ Foxp3⁺);

(b) the proliferating T cells express CD3 and Ki67 (CD3⁺ Ki67⁺); and/or

(c) the macrophages express CD68 (CD68⁺); and/or

(d) the level of T regulatory cells is assessed by detecting cellular expression of CD3 and Foxp3;

(e) the level of proliferating T cells is assessed by detecting cellular expression of CD3 and Ki67; and/or

(f) the level of macrophages is assessed by detecting cellular expression of CD68; and/or

(g) the level of T regulatory cells, proliferating T cells and/or macrophages is assessed by multiplex immunofluorescence staining and image analysis.

7. The bacterial strain for use according to any one of the preceding claims, wherein the bacterial strain:

(a) has a chromosome with at least 95% sequence identity to SEQ ID NO:3 across at least 90% of SEQ ID NO: 3;

(b) has 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;

(c) 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;

(d) has a 16s rRNA gene sequence according to SEQ ID NO:2 or SEQ ID NO:6;

(e) is of the genus Enterococcus,' and/or

(f) is flagellated;

(g) is of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae.

8. The bacterial strain for use according to any one of the preceding claims, wherein the bacterial strain is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488.

9. The bacterial strain for use according to any one of the preceding claims, wherein the cancer is: (a) a solid tumour; and/or

(b) renal cancer, for example renal cell carcinoma, melanoma, lung cancer, for example nonsmall cell lung cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, urethral cancer, or pancreatic cancer.

10. The bacterial strain for use according to any one of the preceding claims, wherein the bacterial strain is:

(a) for use in a method of reducing tumour size, reducing tumour growth, preventing metastasis or preventing angiogenesis;

(b) lyophilised;

(c) viable; and/or

(d) viable and capable of partially or totally colonising the intestine.

11. A composition comprising the bacterial strain of any one of the preceding claims for use according to any one of the preceding claims.

12. The composition of claim 11 for use according to claim 11, wherein the composition:

(a) is for oral administration;

(b) comprises one or more pharmaceutically acceptable excipients or carriers;

(c) comprises a single bacterial strain; and/or

(d) comprises the bacterial strain as part of a microbial consortium.

13. A method of assessing the expected response of a patient having cancer to therapy with 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:

(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;

(c) determining whether:

(ii) the intratumoural level of proliferating T cells is greater than or equal to a second predetermined threshold; and/or (iii) 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 the 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.

14. The method of claim 13, wherein:

(a) the first predetermined threshold is according to claim 2, the second predetermined threshold is according to claim 3, and/or the third predetermined threshold is according to claim 4;

(b) the method further comprises preparing the bacterial strain for administration to the patient having an intratumoural level of T regulatory cells greater than or equal to the first predetermined threshold, an intratumoural level of proliferating T cells greater than or equal to the second predetermined threshold, and/or a total tumour level of macrophages less than or equal to the third predetermined threshold;

(c) the patient having an intratumoural level of T regulatory cells greater than or equal to the first predetermined threshold, an intratumoural level of proliferating T cells greater than or equal to the second predetermined threshold, and/or a total tumour level of macrophages less than or equal to the third predetermined threshold is treated according to any one of claims 1 to 12.

15. A method of monitoring a patient, wherein said patient has cancer, said method comprising:

(d) administering 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 level of intratumoural T regulatory cells is determined to be greater than or equal to the first predetermined threshold; (ii) the level of intratumoural proliferating T cells is determined to be greater than or equal to the second predetermined threshold; and/or (iii) the level of intratumoural macrophages is determined to be less than or equal to the third predetermined threshold.

16. The method of claim 15, wherein the patient is treated according to any one of claims 1-12.

17. The bacterial strain for use according to any one of claims 1-10, or the composition for use according to claim 11 or claim 12, wherein the bacterial strain or composition is administered to a patient identified by a method comprising:

(a) 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 the patient;

(b) comparing (i) the intratumoural level of T regulatory cells; (ii) the intratumoural level of T cells; and/or (iii) the total tumour level of macrophages to a first, second, and/or third predetermined threshold, respectively; and (c) administering the bacterial strain or composition if: (i) the level of intratumoural T regulatory cells is determined to be greater than or equal to the first predetermined threshold; (ii) the level of intratumoural proliferating T cells is determined to be greater than or equal to the second predetermined threshold; and/or (iii) the level of intratumoural macrophages is determined to be less than or equal to the third predetermined threshold.