WO2023072968A1

WO2023072968A1 - Compositions comprising bacterial strains

Info

Publication number: WO2023072968A1
Application number: PCT/EP2022/079840
Authority: WO
Inventors: Alexander Stevenson; Cullen Taniguchi
Original assignee: 4D Pharma Research Ltd
Priority date: 2021-10-25
Filing date: 2022-10-25
Publication date: 2023-05-04

Abstract

The invention relates to bacterial strains for treating cancer.

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 compositions 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. For example, certain Enterococcus species have been implicated in causing cancer [13],

Reference [14] teaches that Enterococcus gallinarum treats cancer but there is no disclosure in that document how neoadjuvant therapy can be improved. One of the challenges in treating cancer, in particular cancers like pancreatic cancer, is that neoadjuvant therapy used to treat the cancer can vary in effectiveness. For example, references [15] and [16] teach that the rate of a pathologic complete response (pCR) is merely 5% and 3.9%, respectively. Likewise, reference [17] teaches that only 3.1% of patients who received neoadjuvant therapy achieved a pathologic complete response (pCR), but a pCR is associated with a much more favourable outcome. The authors conclude that novel systemic therapies that improve clinical and pathologic response rates are urgently needed. Reference [18] is a retrospective analysis of patients who had undergone surgery for resectable pancreatic tumours which assessed the influence of certain factors on patient survival. This study excluded patients who had inoperable tumours after neoadjuvant therapy and also excluded patients with tumours with macroscopic-positive margins or unknown margin status. Reference [19] analyses the effects of preoperative radiotherapy on the patients’ outcome. It concluded that 61% of patients treated with preoperative radiotherapy were judged to have resectable tumours.

There is a need in the art to find improved methods for treating cancer.

SUMMARY OF THE INVENTION

The inventors have developed new therapies for treating diseases. These therapies improve the efficacy of neoadjuvant and/or adjuvant therapy. Most preferably, the therapy is neoadjuvant therapy.

The inventors have discovered that bacterial strains having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2 can increase sensitivity of a cancer cell to the cytotoxic effects of neoadjuvant and/or adjuvant therapy. Thus, the invention provides a bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 for use in treating cancer by neoadjuvant and/or adjuvant therapy, wherein the bacterial strain increases sensitivity of a cancer cell to the cytotoxic effects of the neoadjuvant and/or adjuvant therapy. In preferred embodiments, the bacterial strain is of the genus Enterococcus, for example Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae. In a most preferred embodiment, the bacterial strain is of the species Enterococcus gallinarum.

The inventors have shown particularly good results with neoadjuvant therapy which is particularly preferred.

The invention provides a bacterial strain of the genus Enterococcus for use in treating cancer by neoadjuvant and/or adjuvant therapy, wherein the bacterial strain increases sensitivity of a cancer cell to the cytotoxic effects of a the neoadjuvant and/or adjuvant therapy. The bacterial strain may be of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae. In a most preferred embodiment, the bacterial strain is of the species Enterococcus gallinarum.

The invention also provides abacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2 for use in treating cancer in a subject, wherein the treatment comprises subjecting the subject to neoadjuvant and/or adjuvant therapy.

Also provided is a bacterial strain of the genus Enterococcus for use in treating cancer, wherein the treatment comprises subjecting the subject to neoadjuvant and/or adjuvant therapy.

The examples demonstrate that the invention works particularly well for radiotherapy. Thus, in a preferred embodiment, the neoadjuvant and/or adjuvant therapy is radiotherapy. 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. 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 or 2. In some embodiments, the bacterial strain has a 16s rRNA sequence that is at least 97% identical to SEQ ID NO: 1 or 2. Preferably, the sequence identity is to SEQ ID NO: 2. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:2.

Most preferably, the Enterococcus strain is of the species Enterococcus gallinarum. When using this bacterial strain, the inventors have found that approximately 80% of the patients treated with this bacterial strain in addition to neoadjuvant therapy were judged to have operable tumours following treatment which is significantly higher than the rate reported in the literature for classic neoadjuvant therapy which is 61% (see reference 19). Additionally, the inventors also found that use of the strain of the invention in addition to neoadjuvant therapy resulted in the operable tumours to be resected with a margin status rated as RO, which is a significant improvement over results obtained in the art. A margin status of RO means that a microscopically margin-negative resection has been achieved, in which no gross or microscopic tumour remains in the primary tumour bed and is the most preferable of surgical outcomes. However, in pancreatic cancer this can usually be achieved only in 20% of cases (see for example reference 20).

A preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 or NCIMB 42761.

Bacterial strains which are not of the species Enterococcus gallinarum 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. 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 or 2. Preferably, the sequence identity is to 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 or 2. 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 TNFα 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. 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 of the invention are useful for enhancing the effects of neoadjuvant therapy. Thus, the invention is particularly useful in the treatment of cancer, preferably solid tumours. Examples of suitable cancers which can be treated include pancreatic cancer, lung cancer, breast cancer, liver cancer or colon cancer. The examples demonstrate that the bacterial strains of the invention have a positive effect in these cancers when used in combination with neoadjuvant therapies and so it can be expected that the combination with neoadjuvant therapy will have the beneficial effects described herein, in view of the results discussed in example 6. Pancreatic cancer (e.g. resectable pancreatic cancer) is particularly preferred as the inventors have shown that Enterococcus gallinarum works well for enhancing the effects of neoadjuvant therapy in pancreatic cancer.

In certain embodiments, the bacterial strain is for use in a method of reducing tumour size or preventing tumour growth in the treatment of cancer. Bacterial strains of the invention may be particularly effective for reducing tumour size or preventing tumour growth in the treatment of cancer.

The neoadjuvant and adjuvant therapy for use in the invention may be those commonly used in the art. The therapy may be chemotherapy, radiation therapy, or biological therapy. Examples of suitable biological therapies include adoptive cell transfer, angiogenesis inhibitors, bacillus calmette-guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint inhibitors, immunoconjugates, (monoclonal) antibodies, and oncolytic virus therapy. Most preferably, the neoadjuvant therapy is radiation therapy. The inventors have demonstrated good results with radiation therapy (such as hypofractionated preoperative radiation therapy), as shown in the examples.

In certain embodiments, the invention provides a composition comprising a bacterial strain(s) of the invention. The composition 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 allows delivery to and / or partial or total colonisation of the intestine.

In certain embodiments, the bacterial strain that has been lyophilised. Preferably it is the Enterococcus strain which is lyophilised. Most preferably it is the Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae strain of the invention which is lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

In certain embodiments, the invention provides a food product comprising the composition as described above.

In certain embodiments, the invention provides a vaccine composition comprising the composition as described above. Additionally, the invention provides a method of treating or preventing cancer, comprising administering a composition comprising a bacterial strain of the genus Enterococcus in combination with neoadjuvant and/or adjuvant therapy. 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 (pg/ml) in immature dendritic cells (No bacteria).

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

Figure 4C: Cytokine levels (pg/ml) in immature dendritic cells after the addition ofNCIMB 42488.

Figure 4D: Cytokine levels (pg/ml) in immature dendritic cells after the addition of NCIMB 42488 and LPS.

Figure 5A: Cytokine levels in THP-1 cells (No bacteria).

Figure 5B: Cytokine levels in THP-1 cells after addition of bacterial sediment.

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

PREFERRED EMBODIMENTS

Bacterial strains

The invention provides a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2 for use in a method of increasing sensitivity of a cancer cell to cytotoxic effects of a neoadjuvant and/or adjuvant therapy. Preferably, the bacterial strain is of the genus Enterococcus. 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.

The invention provides a bacterial strain of the genus Enterococcus for use in treating cancer by neoadjuvant and/or adjuvant therapy, wherein the bacterial strain increases sensitivity of a cancer cell to the cytotoxic effects of the neoadjuvant and/or adjuvant therapy. In a most preferred embodiment, the bacterial strain is of the species Enterococcus gallinarum.

Also provided is a bacterial strain of the genus Enterococcus for use in treating cancer by neoadjuvant and/or adjuvant therapy, wherein the treatment comprises subjecting the subject to neoadjuvant and/or adjuvant therapy. It is to be understood that the increased sensitivity is relative to a group of patients (the reference group) who have not received a bacterial strain according to the invention. The cytotoxic effects of a particular neoadjuvant and/or adjuvant therapy may be reported in the literature and so the cytotoxic effect in the control group does not need to be established every time. For example, reference [19] reports that 61% of patients with pancreatic cancer were judged to have operable tumours when treated with classic neoadjuvant therapy. Thus, in this example, an increased sensitivity of a cancer cell to cytotoxic effects of a neoadjuvant therapy would be achieved where >61%, for example more than 65% of patients treated according to the invention were judged to have operable tumours.

For the purposes of the present invention, treatment with a bacterial strain of the invention by itself is not considered neoadjuvant or adjuvant therapy in this context. Rather, the invention uses an adjuvant or neoadjuvant agent in addition to treatment with a bacterial strain as described herein.

The invention provides a bacterial strain having a 16s rRNA sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or at least 99.9% identical to SEQ ID NO:2 for use in treating cancer in a subject, wherein the treatment comprises treating the subject with neoadjuvant and/or adjuvant therapy. Preferably, the bacterial strain is of the genus Enterococcus. 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.

The bacterial strains of the invention are of the genus Enterococcus; most preferably of the species Enterococcus gallinarum. The examples demonstrate that bacteria of this species significantly enhance the effects of radiation therapy. The invention can also be practised using, for example, bacterial strains of the species Enterococcus casseliflavus or Enterococcus hirae.

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 [21], 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 [21],

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 is also particularly useful for use in the invention. It 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 22 May 2017 as "Enterococcus sp" and was assigned accession number NCIMB 42761. The genome sequence of this bacterium is disclosed herein as SEQ ID NO:5. The genome sequence was assembled from multiple contigs. Ns in the sequence represent gaps between the contigs. "N' may represent an A, G, C or T nucleotide. A 16S rRNA gene sequence for the NCIMB 42761 strain is provided in SEQ ID NO:6. SEQ ID NO:6 represents the full length sequence present in the assembly, rather than a consensus of the five 16S genes present in NCIMB 42761.

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 also expected to be effective for treating or preventing cancer. 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, (GTGfy or REP or [22], 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 42761and 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. 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 42488or 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 NO:4 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:4. For example, the bacterial strain for use in the invention may have a plasmid with at least 90% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO: 3 and a plasmid with sequence identity to SEQ ID NON.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NON, 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, more preferably which comprises the 16S rRNA sequence of SEQ ID NON, and optionally comprises a plasmid with sequence identity to SEQ ID NON, as described above.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NON, for example as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NON, 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 NON, 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 NON, 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 (for example, which comprises the 16S rRNA sequence of SEQ ID NON) and a chromosome with at least 95% sequence identity to SEQ ID NON across at least 90% of SEQ ID NON, and optionally comprises a plasmid with sequence identity to SEQ ID NON, 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 (for example, which comprises the 16S rRNA sequence of SEQ ID NON) and a chromosome with at least 98% sequence identity (e.g. at least 99% or at least 99.5% sequence identity) to SEQ ID NON across at least 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NON, and optionally comprises a plasmid with sequence identity to SEQ ID NON, 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 aEnterococcus gallinarum strain which 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 (for example, which comprises the 16S rRNA sequence of SEQ ID NO:2) 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 23 s 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, [23]). 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 of 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, P-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/μl (for example, 6.1-6.6 ng/μl or 6.2-6.5 ng/μl or 6.33 ± 0.10 ng/μl) 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 (Schenk et al., 2011), promotion of intestinal Thl7 cell differentiation (Atarashi etal., 2008) and induction of secretion of the pro-inflammatory mediator IL-1β by activating the NLRP3 inflammasome (Karmarkar et al., 2016). 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 FIG00632333: 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 FIG00632333: hypothetical protein; Xylose ABC transporter, permease component and FIG00632333: hypothetical protein; or Mobile element protein, Xylose ABC transporter, permease component, and FIG00632333: 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 effective for improving the effects of neoadjuvant and/or adjuvant therapy. 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 42488. This is the exemplary strain tested in the examples and shown to be effective for improving the effects of neoadjuvant and/or adjuvant therapy. 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 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 the Examples. 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 bacterial strains of the invention are for use in treating cancer. The examples demonstrate that administration of the compositions of the invention leads to improved efficacy when a patient is treated with neoadjuvant and/or adjuvant therapy.

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 compositions of the invention 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. In certain embodiments, the compositions of the invention are for use in patients with solid tumours. In certain embodiments, the compositions of the invention are for use in reducing or preventing angiogenesis in the treatment of cancer. The bacterial strains of the invention may have an effect on the immune or inflammatory systems, which have central roles in angiogenesis. In certain embodiments, the bacterial strains of the invention are for use in preventing metastasis. The bacterial strains of the invention may improve the tumour’s margin rating. For example, the margin may change from positive to negative. The bacterial strains of the invention may improve the operability and / or resectability of a tumour. In preferred embodiments, treatment with a bacterial strain according to the invention results in a pCR response.

Preferably, in certain embodiments, the compositions of the invention are for use in treating pancreatic cancer. The examples demonstrate that the compositions of the invention may be effective for treating pancreatic cancer in combination with neoadjuvant and/or adjuvant therapy. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of pancreatic cancer.

In certain embodiments, the compositions of the invention are for use in treating or preventing colon cancer. The examples demonstrate that the compositions of the invention have an effect on colon cancer cells and may be effective for treating colon cancer. Furthermore, in certain embodiments, the compositions of the invention are 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.

Preferably, in certain embodiments, the compositions of the invention are for use in treating cervical cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of cervical cancer.

In certain embodiments, the compositions of the invention are for use in treating or preventing breast cancer. The examples demonstrate that the compositions of the invention may be effective for treating breast cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, the compositions of the invention are for use in treating or preventing lung cancer. The examples demonstrate that the compositions of the invention may be effective for treating lung cancer. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer. In preferred embodiments the cancer is lung carcinoma.

In certain embodiments, the compositions of the invention are for use in treating or preventing liver cancer. The examples demonstrate that the compositions of the invention may be effective for treating liver cancer. In certain embodiments, the compositions of the invention are 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 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 certain embodiments, the compositions of the invention are for use in treating or preventing carcinoma. The examples demonstrate that the compositions of the invention may be effective for treating numerous types of carcinoma. In certain embodiments, the compositions of the invention are for use in treating or preventing non-immunogenic cancer. The examples demonstrate that the compositions of the invention may be effective for treating non-immunogenic cancers.

The therapeutic effects of the compositions of the invention on 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 [24] and pro-inflammatory cytokines such as TNFα are being investigated as cancer therapies [25], The up-regulation of genes such as TNF shown in the examples may indicate that the compositions of the invention may be useful for treating cancer via a similar mechanism. The up-regulation of CXCR3 ligands (CXCL9, CXCL10) and IFNγ-inducible genes (IL- 32) may indicate that the compositions of the invention elicit an IFNγ-type response. IFNγ is a potent macrophage-activating factor that can stimulate tumirocidal activity [26], and CXCL9 and CXCL10, for example, also have anti-cancer effects [27-29], Therefore, in certain embodiments, the compositions of the invention are for use in promoting inflammation in the treatment of cancer. In preferred embodiments, the compositions of the invention are for use in promoting Thl inflammation in the treatment of cancer. Thl cells produce IFNγ and have potent anti-cancer effects [24], In certain embodiments, the compositions of the invention 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 [24], In certain embodiments, the compositions of the invention are for use in promoting inflammation to enhance the effect of a second anti-cancer agent. 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 TNFα , for example, IL-1β and IL-6, IL-1β and TNFα , IL-6 and TNFα or all three of IL-1β, IL-6 and TNFα . Increases in levels of expression of any of IL-1β, IL-6 and TNFα are known to be indicative of efficacy in treatment of cancer.

Examples 4 and 5 demonstrate that when a bacterial strain as described herein is used in combination with lipopolysaccharide (LPS), there is a synergistic increase in IL-1β. LPS is known to elicit a pro- inflammatory effect. Thus, in certain embodiments, the treatment or prevention comprises using a bacterial strain as described herein in combination with an agent that upregulates IL-1β. In certain embodiments, the treatment or prevention comprises using a bacterial strain as described herein in combination with LPS. Accordingly, a composition of the invention may additionally comprise an agent that upregulates IL-1β. Accordingly, a composition of the invention may additionally comprise LPS.

In certain embodiments, the compositions of the invention are for use in treating non-small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating small-cell lung carcinoma. In certain embodiments, the compositions of the invention are for use in treating squamous-cell carcinoma. In certain embodiments, the compositions of the invention are for use in treating adenocarcinoma. In certain embodiments, the compositions of the invention are for use in treating glandular tumours, carcinoid tumours, or undifferentiated carcinomas.

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

In certain embodiments, the compositions of the invention are for use in treating invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.

In further embodiments, the compositions of the invention 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, 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. The treatment of pancreatic cancer is particularly preferred.

The inventors have shown that the invention works particularly well in the treatment of pancreatic cancer. Thus, in a particularly preferred embodiment the cancer is pancreatic cancer. Neoadjuvant therapy refers to the systemic treatment of cancer prior to definitive surgical therapy with an agent in addition to the bacterial strain of the invention. Examples of neoadjuvant therapy include chemotherapy, biological therapy, radiation therapy, and hormone therapy. Examples of definitive surgical therapy include tumour resection.

Adjuvant therapy refers to the systemic treatment of cancer following definitive surgical therapy with an agent in addition to the bacterial strain of the invention. Examples of adjuvant therapy include chemotherapy, biological therapy, radiation therapy, and hormone therapy. Examples of definitive surgical therapy include tumour resection.

Radiotherapy is preferred for the invention as the inventors have demonstrated particularly good results. Radiotherapy may comprise a cumulative external irradiation of a patient in a dose of 1 to 100 Gy. A preferred range of the irradiation dose is 1 to 60 Gy.

In certain embodiments, the dose of radiation therapy is less than 90 Gy, such as less than 80 Gy, such as less than 70 Gy, such as less than 60 Gy, such as less than 50 Gy, PU66280 such as less than 40 Gy, such as less than 30 Gy, such as less than 20 Gy. In certain embodiments the dose or radiation therapy is between about 10 to 100 Gy, such as from about 20 to 80 Gy, such as about 30 to 70 Gy, such as about 40 to 60 Gy. In certain embodiments, the irradiation dose is selected from 5-25 Gy, such as from 10-20 Gy.

Radiation therapy may be stereotactic body radiotherapy (SBRT). Stereotactic radiotherapy uses essentially the same approach as stereotactic radiosurgery to deliver radiation to the target tissue; however, stereotactic radiotherapy generally uses multiple small fractions of radiation as opposed to one large dose, but certain applications of SBRT may still be accomplished with a single fraction.

Suitable types of radiotherapy include conventional external beam radiotherapy, stereotactic radiation therapy (e.g., Axesse, Cyberknife, Gamma Knife, Novalis, Primatom, Synergy, X- Knife, TomoTherapy or Trilogy), Intensity-Modulated Radiation Therapy, particle therapy (e.g., proton therapy), brachytherapy, delivery of radioisotopes, intraoperative radiotherapy, Auger therapy, Volumetric modulated arc therapy (VMAT), Virtual simulation, 3 -dimensional conformal radiation therapy, and intensity-modulated radiation therapy, etc.

In some embodiments, the neoadjuvant and/or adjuvant therapy is a biological therapy. Examples of suitable biological therapies include adoptive cell transfer, angiogenesis inhibitors, bacillus calmette- guerin therapy, biochemotherapy, cancer vaccines, chimeric antigen receptor (CAR) T-cell therapy, cytokine therapy, gene therapy, immune checkpoint inhibitors, immunoconjugates, monoclonal antibodies, hormone therapy and oncolytic virus therapy.

Hormone therapy is a treatment that adds, blocks, or removes hormones. For example, hormones may be given to adjust low hormone levels. Synthetic hormones or other drugs may be given to block the body's natural hormones to slow or stop the growth of certain cancers (such as prostate and breast cancer).

Examples of hormone therapy include selective estrogen receptor modulators (SERMs), such as tamoxifen, raloxifene, endoxifene, toremifene, lasofoxifene, pipendoxifene, bazedoxifene, and ospemifene, aromatase inhibitors, such anastrozole, letrozole, exemestane, formestane, fadrozole, aminoglutethimide, and testolactone, a HER2 intervention drug, such as a HER2 inhibitor, such as Herceptin (trastuzumab), pertuzumab, and lapatinib, and estrogen-receptor downregulators, such as fulvestrant (ICI 182,780).

In preferred embodiments, the composition comprises an anti-cancer agent selected from the group consisting of: Yervoy (ipilimumab, BMS); Keytruda (pembrolizumab, Merck); Opdivo (nivolumab, BMS); MEDI4736 (AZ/Medlmmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/Medlmmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/Medlmmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/Medlmmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varhlumab; CellDex Therapeutics); TRX- 518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (Jounce Therapeutics); ARGX-115 (arGEN-X); NLG-9189 (indoximod, NewLink Genetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ); NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (Eh Lilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics); Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299 (Eh Lilly); EW-7197 (Ewha Women's University, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre); Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto (blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva (obinutuzumab, Roche/Biogen); Kadcyla (ado- trastuzumab emtansine, Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris (brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK); Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-1131, GSK); Zevalin (ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab, Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab (Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Elotuzumab (Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C (Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38 (Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affimed); AFM11 (Affimed); AGS-16C3F (Agensys); AGS- 16M8F (Agensys); AGS-22ME (Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab, Alexion); ALT-836 (Aitor Bioscience); ALT-801 (Aitor Bioscience); ALT-803 (Aitor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232 (adecatumumab, Amgen); AMG211 (Amgen/Medlmmune); BAY20-10112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen); MEDI575 (AZ/Medlmmune); MEDI3617 (AZ/Medlmmune); MEDI6383 (AZ/Medlmmune); MEDI551 (AZ/Medlmmune); Moxetumomab pasudotox (AZ/Medlmmune); MEDI565 (AZ/Medlmmune); MEDI0639 (AZ/Medlmmune); MEDI0680 (AZ/Medlmmune); MEDI562 (AZ/Medlmmune); AV-380 (AVEO); AV203 (AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer); vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (Boehringer Ingleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioCiin); BIIB015 (Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206 (Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-OlOt (Biosceptre); MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB); CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U3-1565 (Daiichi Sankyo); patritumab (Daiichi Sankyo); tigatuzumab (Daiichi Sankyo); nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (Daiichi Sankyo); DS- 5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai); MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358 (Eli Lilly); LY2812176 (Eli Lilly); LY3012217(Eli Lilly); LY2495655 (Eh Lilly); LY3012212 (Eli Lilly); LY3012211 (Eh Lilly); LY3009806 (Eh Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eh Lilly); IMC-TR1 (Eh Lilly); Ramucirumab (Eh Lilly); Tabalumab (Eh Lilly); Zanolimumab (Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five Prime Therapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five Prime Therapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed); IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745 (Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab (GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen); IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumab-dox (Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics); IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab (Immunomedics); Clivatuzumab (Immunomedics); IPH41 (Innate Immunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab, Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios); mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science); Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab (Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646 (dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono); DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574 (Morphosys); BPC-1C (ensituximab, Precision Biologies); TAS266 (Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031 (Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355 (Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK); OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumab ozogamicin, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer); PSMA-ADC (Progenies); REGN1400 (Regeneron); REGN910 (nesvacumab, Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221, RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636, RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160 (imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab), RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi); SAR650984 (Sanofi); SAR153192 (Sanofi); SAR3419 (Sanofi); SAR256212 (Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (Seattle Genetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A (Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (Seattle Genetics); ibritumomab tiuxetan (Spectrum); MLN0264 (Takeda); ganitumab (Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845 (Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YM Biosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10 TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics); KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (Bellicum Pharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (Unum Therapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101 (Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences); Colo-Adi (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102 (Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, Jennerex Biotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene); HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologies); OrienXOlO ( OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SW-001 (Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eh Lilly), cisplatin, oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide, 5- fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xalkori (Pfizer), Iressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma), Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib (Chugai), LDK378 (Novartis), Genetespib (Synta Pharma), Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib (ArQule); Cytoxan (BMS); Oncovin (Eh Lilly); Adriamycin (Pfizer); Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene); Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132 (Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis); Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (Taiho Pharmaceuticals); and TS-1 (Taiho Pharmaceuticals).

In some embodiments, the neoadjuvant therapy and/or adjuvant therapy is chemotherapy. For example, the chemotherapy agent may be a small molecule, for example an anthracycline such as doxorubicin and / or epirubicin, a taxane for example paclitaxel and / or docetaxel, 5 -fluorouracil, capeci tabine, cyclophosphamide, carboplatin, oxaliplatin, cisplatin, irinotecan, topotecan, folfirinox, vinorelbine, gemcitabine, ixabepilone and / or eribulin.

In some embodiments, the use of the bacterial strains of the invention involves a step of treating the subject with one or more of the chemotherapeutics selected from the group consisting of Gemcitabine, Abraxane and Folfirinox, for example a subject may be treated with a combination of Gemcitabine and Abraxane. The bacterial strains of the invention may be particularly effective when used in combination with further therapeutic agents. The immune-modulatory effects of the compositions of the invention may be effective when combined with more direct anti-cancer agents. Therefore, in certain embodiments, the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum and an anticancer agent, as described herein.

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, for example which is an Enterococcus gallinarum, is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition of the invention.

Modes of administration

Preferably, the bacterial strains of the invention 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. Generally, the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.

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

In certain embodiments, the bacterial strain of the invention 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 of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopic gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jejunum and other suitable access ports.

The bacterial strain of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily.

The bacterial strain of the invention may be administered prior to the commencement of neoadjuvant therapy. For example, a patient may first receive a bacterial strain according to the invention at least 1 week, at least 2 weeks, at least 3 weeks or at least 4 weeks before the beginning of neoadjuvant therapy. In some embodiments, the treatment with a bacterial strain of the invention may stop before neoadjuvant therapy starts. The patient may also continue to receive a bacterial strain of the invention following the start of neoadjuvant therapy.

Additionally or alternatively, administration of the bacterial strain of the invention and / or the neoajuvant therapy may commence at least one week prior to the definitive surgical therapy, at least two weeks prior to the definitive surgical therapy, at least three weeks prior to the definitive surgical therapy, at least four weeks prior to the definitive surgical therapy, at least five weeks prior to the definitive surgical therapy, at least six weeks prior to the definitive surgical therapy, at least eight weeks prior to the definitive surgical therapy, at least ten weeks prior to the definitive surgical therapy, or at least twelve weeks prior to the definitive surgical therapy. In embodiments, administration of the bacterial strain of the invention and / or the neoadjuvant therapy may commence six months or less prior to the definitive surgical therapy, five months or less prior to the definitive surgical therapy, four months or less prior to the definitive surgical therapy or three months or less prior to the definitive surgical therapy.

Where a patient is treated with adjuvant therapy, the patient may receive a bacterial strain and/or the adjuvant therapy according to the invention within 1, 2, 3, 4, 5, 6, or 7 days after definitive surgical therapy, or within 1 week, 2 weeks, 3 weeks, 6 weeks or 12 weeks after definitive surgical therapy. In certain embodiments of the invention, 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.

In certain embodiments, the bacterial strain of the invention 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 of the invention 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 of the invention 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 of the invention may be administered as a food product, such as a nutritional supplement.

Generally, the bacterial strain of the invention 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 compositions of the invention may be useful for enhancing the growth and performance of animals. If administered to animals, oral gavage may be used.

Compositions

The bacterial strain of the invention may be administered as part of a composition. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. For example, the composition of the invention may comprise granules or capsules (e.g. gelatin or cellulose-based such as HPMC) comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [30-32],

Alternatively, the composition of the invention 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 of the invention may not have been inactivated, for example, may not have been heat-inactivated. The bacterial strain in the composition of the invention may not have been killed, for example, not been heat-killed. The bacterial strain in the composition of the invention may not have been attenuated, for example, not been heat-attenuated. For example, the bacterial strain in the composition of the invention may not have been killed, inactivated and/or attenuated. For example, the bacterial strain in the composition of the invention is live. For example, the bacterial strain in the composition of the invention is viable. For example, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine.

The bacterial strain in the composition of the invention may be viable and capable of partially or totally colonising the intestine. The bacterial strain in the composition of the invention may be live and capable of partially or totally colonising the intestine. The bacterial strain in the composition of the invention may be live and viable. The bacterial strain in the composition of the invention may be live, viable and capable of partially or totally colonising the intestine.

In some embodiments, the bacterial strain in the composition of the invention has not been inactivated, for example, has not been heat-inactivated. In some embodiments, the bacterial strain in the composition of the invention has not been killed, for example, has not been heat-killed. In some embodiments, the bacterial strain in the composition of the invention has not been attenuated, for example, has not been heat- attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention has not been killed, inactivated and/or attenuated. For example, in some embodiments, the bacterial strain in the composition of the invention is live. For example, in some embodiments, the bacterial strain in the composition of the invention is viable. For example, in some embodiments, the bacterial strain in the composition of the invention is capable of partially or totally colonising the intestine. For example, in some embodiments, the bacterial strain in the composition of 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 of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation until delivery at the target location through, for example, rupturing with chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [33] and [34],

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 of 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 of the invention includes a therapeutically effective amount of a bacterial strain of the invention. 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. The bacterial strain in the composition has a therapeutic effect as discussed

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1x10 ³ to about 1x10 ¹¹ colony forming units (CFU); for example, from about 1x10 ⁷ to about 1x10 ¹⁰ CFU; in another example from about 1x10 ⁶ to about 1x10 ¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 10⁶to about 1x10 ¹¹ CFU/g, respect to the weight of the composition; for example, from about 1x10 ⁸ to about 1x10 ¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g. Typically, a probiotic, such as the composition of 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 of the present invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt- oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), betaglucans, 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 compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [35], Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [36], 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 of the invention 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 of 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 of the invention 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 milkbased product can be, e.g., cow's milk, goafs milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. 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 of the invention 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 species Enterococcus gallinarum, which does not contain bacteria from any other 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 of the invention comprise more than one bacterial strain or species. For example, in some embodiments, the compositions of 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 of 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 of 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 of 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 of 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 of 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 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, for example, which is an Enterococcus gallinarum, 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, 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 of 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 of 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 of 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 of 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 of the invention.

As mentioned above, in some embodiments, the bacterial strain of the invention is the only therapeutically active agent(s) in a composition of the invention.

The compositions for use in accordance with the invention may or may not require marketing approval. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised. In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In certain embodiments, the invention provides the above pharmaceutical composition, 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 invention provides the above pharmaceutical composition, 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 of the invention 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 invention provides a pharmaceutical composition comprising: 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 when administered to a subject in need thereof; and wherein the disorder is breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer.

In certain embodiments, the invention provides a pharmaceutical composition comprising: 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 when administered to a subject in need thereof; and wherein the disorder is lung cancer. In preferred embodiments the cancer is lung carcinoma.

In certain embodiments, the invention provides a pharmaceutical composition comprising: 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 when administered to a subject in need thereof; and wherein the disorder is liver cancer. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma). 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 colon cancer. In preferred embodiments the cancer is colorectal adenocarcinoma.

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 carcinoma.

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 a non-immunogenic cancer.

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 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 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 selected from the group consisting of hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma or liver cancer resulting from a viral infection.

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 selected from the group consisting of invasive ductal carcinoma, ductal carcinoma in situ or invasive lobular carcinoma.

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 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 invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1x10 ³ to about 1x10 ¹¹ colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

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

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising 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 invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the 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 of the invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein.

The 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 1x10 ⁵, 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 lxl0¹⁰to about 1x10¹¹ CFU of the bacterial strain. In other embodiments, the capsule may be cellulose-based, e.g. formed of hydroxypropylmethylcellulose as the sole component or blended with functional ingredients.

In some embodiments, the compositions of 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 film 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 [37]). 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 [38-40],

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 of the invention may also be useful for preventing cancer, when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention are viable. In certain such embodiments, the bacterial strains of the invention are capable of partially or totally colonising the intestine. In certain such embodiments, the bacterial strains of the invention are viable and capable of partially or totally colonising the intestine. In other certain such embodiments, the bacterial strains of the invention 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 inj ection, such as via subcutaneous inj ection.

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 [41] and [42-48], 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. [49], 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. [50],

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: 2xl0⁸ 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 [51],

The LL/2 (LLC1) cell line was established from the lung of a C57BL mouse bearing atumour resulting from an implantation of primary Lewis lung carcinoma [52],

The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [53].

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 [54,55], 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 D14, 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 D14, 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 DI 6, 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 [56]:

Humane endpoints [57]: 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 - Isofl urane gas anesthesia were used for all procedures: surgery or tumour inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthesia were used for stereotaxia surgical procedure. Analgesia - Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severity of surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally, pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinarian.

Euthanasia - Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

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 100ng/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 TNFα 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 TNFα 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-1β produced (Figure 4d).

Conclusion

NCIMB 42488 has the ability to induce higher IL-6 and TNFα 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-1β, IL-6 and TNFα , 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 MO medium for 48h with 5ng/mL phorbol-12-myristate- 13 -acetate (PMA). These cells were subsequently incubated with NCIMB 42488 at a final concentration of 10⁸/ml, with or without the addition of LPS at a final concentration of 100ng/ml. The bacteria were then washed off and the cells allowed to incubate under normal growing conditions for 24 h. The cells were then spun down and the resulting supernatant was analysed for cytokine content.

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 TNFα 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-1β, IL-6 and TNFα , 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 - effect of E. gallinarum on neoadjuvant therapy The effect of E. gallinarum in combination with preoperative hypofractionated radiation was assessed in a single center, open-label, phase I study on patients with pancreatic cancer. The patients took E. gallinarum daily for one week prior to the start of radiation therapy, throughout radiation and until surgical resection of the tumour or withdrawal.

Results taken at an interim timepoint following the treatment of 13 patients are shown in the table below.

13 patients to date have participated in the study, of which 10 (i.e., approximately 77%) were judged to have operable tumours following treatment with E. gallinarum in accordance with the study protocol and therefore proceeded to surgery. This is an improvement over conventional therapy where only 60% of patients treated with neoadjuvant radiotherapy are deemed to have operable tumours (with the other 40% of patients progressing to a stage at which they are untreatable). In total, 7 patients underwent full pancreatectomy. Surprisingly, all 7 of these patients were scored a margin status of RO, which means that a macroscopically complete tumour removal with negative microscopic surgical margins was possible. This finding was surprising and unexpected given that, in pancreatic cancer a margin status of RO is usually only observed in 20% of cases (see for example reference 20).

The histologic responses measured by Treatment Effect Score (TES) rates for these 7 patients are shown in the table above. Several measures of histologic response are used globally. TES is the measure employed by The University of Texas MD Anderson Cancer Center where the study participants were treated. A TES of I indicates 0% residual tumour cells in the resected specimen (pCR). A TES of II indicates 1 to <5% residual tumour cells in the resected specimen. A TES of III indicates ≥5% residual tumour cells in the resected specimen.

Sequences

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

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

SEQ ID NO: 3 (strain NCIMB 42488 chromosome sequence) - 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)

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PCT

(Original in Electronic Form)

(This sheet is not part of and does not count as a sheet of the international application)

FOR RECEIVING OFFICE USE ONLY

PCT

(Original in Electronic Form)

FOR INTERNATIONAL BUREAU USE ONLY

Claims

1. A bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 for use in treating cancer, wherein the bacterial strain increases sensitivity of a cancer cell to the cytotoxic effects of a neoadjuvant therapy and/or an adjuvant therapy.

2. A bacterial strain having a 16s rRNA gene sequence that is at least 95% identical to SEQ ID NO:2 for use in treating cancer in a subject, wherein the treatment comprises subjecting the subject to neoadjuvant therapy and/or an adjuvant therapy.

3. A bacterial strain of the genus Enterococcus for use in treating cancer, wherein the bacterial strain increases sensitivity of a cancer cell to the cytotoxic effects of a neoadjuvant therapy and/or an adjuvant therapy.

4. A bacterial strain of the genus Enterococcus for in treating cancer in a subject, wherein the treatment comprises subjecting the subject to neoadjuvant therapy and/or adjuvant therapy.

5. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain has a chromosome with at least 95% sequence identity to SEQ ID NO:3 across at least 90% of SEQ ID NO: 3.

6. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain 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.

7. The bacterial strain of claim 4 for the use of claim 4, wherein 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 wherein the bacterial strain has a 16s rRNA gene sequence according to SEQ ID NO: 2.

8. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain is of the genus Enterococcus.

9. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain is of the species Enterococcus gallinarum, Enterococcus casseliflavus or Enterococcus hirae.

10. The bacterial strain of claim 7 for the use of claim 7, wherein the bacterial strain is the Enterococcus gallinarum strain deposited under accession number NCIMB 42488.

11. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain is for use in the treatment of a solid tumour.

12. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the composition is for use in a method of treating or preventing pancreatic cancer, lung cancer, breast cancer, liver cancer or colon cancer.

13. The bacterial strain of claim 10 for the use of claim 10, wherein the cancer is pancreatic cancer, optionally resectable pancreatic cancer.

14. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the neoadjuvant therapy and/or adjuvant therapy is selected from radiation therapy, biological therapy chemotherapy, hormone therapy, and combinations thereof.

15. The bacterial strain of claim 12 for the use of claim 12, wherein the neoadjuvant therapy and/or adjuvant therapy is radiation therapy, optionally hypofractionated preoperative radiation therapy.

16. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the subject has been treated with chemotherapy prior to administration of the bacterial strain, optionally wherein the chemotherapy is selected from gemcitabine; nab-paclitaxel (Abraxane®); a combination of folinic acid, 5 -fluorouracil, irinotecan and oxaliplatin (FOLFIRINOX); and combinations thereof.

17. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the cancer cell or cancer is surgically resected following administration of the bacterial strain and neoadjuvant therapy.

18. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the composition is for use in a method of reducing tumour size, reducing tumour growth, preventing metastasis or preventing angiogenesis.

19. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain is lyophilised.

20. The bacterial strain of any preceding claim for the use of any preceding claim, wherein the bacterial strain is viable and optionally capable of partially or totally colonising the intestine.

21. A composition comprising the bacterial strain of any preceding claim for the use of any preceding claim, wherein the composition is for oral administration.

22. The composition of claim 19 for the use claim 19, wherein the composition comprises one or more pharmaceutically acceptable excipients or carriers.

23. The composition of any one of claims 19-20 for the use of any one of claims 19-20, wherein the composition comprises a single strain of Enterococcus gallinarum.

24. The composition of any one of claims 19-21 for the use of any one of claims 19-21, wherein the composition comprises the Enterococcus gallinarum bacterial strain as part of a microbial consortium.