HK1254166B - Compositions comprising bacterial strains - Google Patents

Description

Compositions comprising bacterial strains

Field of the Invention

The present invention is in the field of compositions comprising bacterial strains isolated from the digestive tract of mammals and the use of such compositions for treating diseases.

Background of the Invention

Although the human intestine is considered sterile in utero, it is immediately exposed to a multitude of maternal and environmental microorganisms after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as mode of delivery, environment, diet and host genotype, all of which influence the composition of the intestinal microbiota, especially during childhood. Subsequently, the microbiota stabilizes and becomes adult-like [1]. The human intestinal microbiota contains more than 500-1000 different phylotypes belonging to two major bacterial taxa, Bacteroidetes and Firmicutes [2]. The successful symbiotic relationship resulting from bacterial colonization of the human intestine results in a variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activity of the colonized intestine ensures the degradation of dietary components that would otherwise be ingested, while releasing by-products that provide an important source of nutrients for the host. Similarly, the immunological importance of the intestinal microbiota is well recognized and has been exemplified in germ-free animals with weakened immune systems, whose immune systems are functionally restored after the introduction of commensal bacteria [3-5].

Significant changes in the composition of the microbiota have been demonstrated in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, levels of Clostridium XIVa cluster bacteria are reduced in IBD patients, while Escherichia coli numbers are increased, indicating a shift in the balance between intestinal commensals and pathogenic commensals [6-9]. Interestingly, this microbial dysbiosis is also associated with an imbalance in effector T cell populations.

Recognizing that certain bacterial strains may have potentially positive effects on the animal intestine, various strains have been proposed for use in treating various diseases (see, for example, [10-13]). Certain strains, including most Lactobacillus and Bifidobacterium strains, have also been proposed for use in treating various inflammatory and autoimmune diseases that are not directly related to the intestine (for review, see [14] and [15]). However, the relationship between different diseases and different bacterial strains and the precise effects of specific bacterial strains on the intestine and at the systemic level and on any particular type of disease are not well characterized. For example, certain enterococcal species have been linked to causing cancer [16].

There is a need in the art for new methods of treating disease.There is also a need to characterize the potential roles of enteric bacteria so that new therapies using enteric bacteria can be developed.

Summary of the Invention

The present inventors have developed novel therapies for treating and preventing diseases. Specifically, the present inventors have developed novel therapies for treating and preventing cancer. Specifically, the present inventors have determined that bacterial strains of the species Enterococcus gallinarum can effectively treat and prevent cancer. As described in the Examples, oral administration of a composition comprising Enterococcus gallinarum can reduce tumor size in a mouse model of cancer.

In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum for use in a method for treating or preventing cancer, such as breast cancer, lung cancer, or liver cancer. The inventors have determined that treatment with a composition comprising a bacterial strain of the species Enterococcus gallinarum can reduce tumor growth in mouse models of breast cancer, lung cancer, and liver cancer. In certain embodiments, the composition is used in a method for reducing tumor size or preventing tumor growth in cancer treatment. Compositions using Enterococcus gallinarum can be particularly effective for reducing tumor size or preventing tumor growth in cancer treatment.

In a preferred embodiment of the present invention, the bacterial strain in the composition belongs to Enterococcus gallinarum. Closely related strains may also be used, for example bacterial strains having 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 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. Preferably, the bacterial strain used in the present invention has a 16s rRNA sequence represented by SEQ ID NO: 2.

Therefore, the present invention also provides a composition comprising a bacterial strain having a 16s rRNA sequence that is at least 95% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus gallinarum, for use in a method of treating or preventing cancer. Specifically, the present invention provides a composition comprising 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 treating or preventing cancer. In some embodiments, the bacterial strain in the composition does not belong to Enterococcus gallinarum. In some embodiments, the bacterial strain in the composition does not belong to Enterococcus gallinarum, but is a closely related strain.

In certain embodiments, the compositions of the present invention are for oral administration. Oral administration of the strains of the present invention can effectively treat cancer. In addition, oral administration is convenient for patients and practitioners and allows for delivery to the intestine and/or partial or complete colonization of the intestine.

In certain embodiments, the compositions of the present invention comprise one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the compositions of the present invention comprise lyophilized bacterial strains. Lyophilization is an efficient and convenient technique for preparing stable compositions that allow for the delivery of bacteria.

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

In certain embodiments, the present invention provides a vaccine composition comprising the composition described above.

Additionally, the present invention provides a method for treating or preventing cancer, comprising administering a composition comprising a bacterial strain of the species Enterococcus gallinarum.

In developing the above invention, the inventors have identified and characterized bacterial strains that are particularly suitable for therapy. The Enterococcus gallinarum strain of the present invention has been shown to be effective in treating cancer. Therefore, in another aspect, the present invention provides a cell of an Enterococcus gallinarum strain deposited under the accession number NCIMB 42488 or a derivative thereof. The present invention also provides a composition comprising such cells or a biologically pure culture of such cells. The present invention also provides a cell of an Enterococcus gallinarum strain deposited under the accession number NCIMB42488 or a derivative thereof, which is used in therapy, in particular for cancer. Similarly, the present invention provides a cell of a bacterial strain or a derivative thereof having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2. The present invention also provides a composition comprising such cells or a biologically pure culture of such cells. The present invention also provides a cell of a bacterial strain or a derivative thereof having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2, which is used in therapy, in particular for treating or preventing cancer.

Simple illustration

Figure 1: Mouse model of breast cancer - tumor volume.

Figure 2: Mouse model of lung cancer - tumor volume.

Figure 3: Mouse model of liver cancer - liver weight.

FIG4A : Cytokine levels (pg/ml) in immature dendritic cells (without bacteria).

FIG4B : Cytokine levels (pg/ml) in immature dendritic cells after addition of LPS.

FIG4C : Cytokine levels (pg/ml) in immature dendritic cells after addition of MRX518.

FIG4D : Cytokine levels (pg/ml) in immature dendritic cells after addition of MRX518 and LPS.

Figure 5A: Cytokine levels in THP-1 cells (without bacteria).

FIG5B : Cytokine levels in THP-1 cells after addition of bacterial sediment.

FIG5C : Cytokine levels in THP-1 cells after addition of MRX518 alone or in combination with LPS.

Detailed Description of the Invention

bacterial strains

The composition of the present invention comprises a bacterial strain of the species Enterococcus gallinarum. The examples demonstrate that bacteria of this species can be used to treat or prevent cancer.

The present invention also provides a composition comprising a bacterial strain having a 16s rRNA sequence that is at least 95% identical to the 16s rRNA sequence of a bacterial strain of Enterococcus gallinarum, for use in therapy, such as in a method for treating or preventing cancer. Specifically, the present invention also provides a composition comprising a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:2, for use in therapy, such as in a method for treating or preventing cancer. In some embodiments, the bacterial strain in the composition is not Enterococcus gallinarum, but is a closely related strain.

The present invention provides an Enterococcus gallinarum for use in therapy, e.g., for treating or preventing cancer. Similarly, the present invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum for use in therapy, e.g., for treating or preventing cancer. In certain embodiments, the composition of the present invention comprises a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2, e.g., an Enterococcus gallinarum, and does not contain any other bacterial species. In certain embodiments, the composition of the present invention comprises a single strain of a bacterial strain having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2, e.g., an Enterococcus gallinarum, and does not contain any other bacterial strains or species.

Enterococcus gallinarum forms spherical cells, mostly in pairs or short chains. It is immobile and colonies on blood agar or nutrient agar are round and smooth. Enterococcus gallinarum reacts with Lancefield group D antiserum. The type strain of Enterococcus gallinarum is F87/276 = PB21 = ATCC 49573 = CCUG 18658 = CIP 103013 = JCM 8728 = LMG13129 = NBRC 100675 = NCIMB 702313 (formerly NCDO 2313) = NCTC12359 [17]. The GenBank accession number for the 16S rRNA gene sequence of Enterococcus gallinarum is AF039900 (disclosed herein as SEQ ID NO: 1). An exemplary strain of Enterococcus gallinarum is described in [17].

Enterococcus gallinarum bacteria deposited under accession number NCIMB 42488 were tested in the examples and are also referred to herein as strain MRX518. References to MRX518 and MRx0518 are used interchangeably. The 16S rRNA sequence of the tested MRX518 strain is provided in SEQ ID NO: 2. Strain MRX518 was deposited by 4D Pharma Research Ltd. (LifeSciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) under the genus Enterococcus sp. on November 16, 2015, with NCIMB International Depository Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) and assigned accession number NCIMB 42488.

The genome of strain MRX518 comprises a chromosome and a plasmid. The chromosome sequence of strain MRX518 is provided in SEQ ID NO: 3. The plasmid sequence of strain MRX518 is provided in SEQ ID NO: 4. These sequences were generated using the PacBio RS II platform.

It is also expected that bacterial strains closely related to the strains tested in the examples are effective in treating or preventing cancer. In certain embodiments, the bacterial strains used in the present invention 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 gallinarum. Preferably, the bacterial strains used in the present invention have 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. Preferably, the bacterial strains used in the present invention have a 16s rRNA sequence represented by SEQ ID NO: 2.

Bacterial strains that are biotypes of the bacterium deposited under Accession No. 42488 are also expected to be effective in treating or preventing cancer. Biotypes are closely related strains that have the same or very similar physiological and biochemical properties.

Strains suitable for use in the present invention that are biotypes of the bacterium deposited under accession number NCIMB 42488 can be identified by sequencing other nucleotide sequences of the bacterium deposited under accession number NCIMB 42488. For example, substantially the entire genome can be sequenced, and the biotype strain used in the present invention can have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity over at least 80% of its entire genome (e.g., over at least 85%, 90%, 95% or 99% or over its entire genome). For example, in some embodiments, the biotype strain has at least 98% sequence identity over at least 98% of its genome or at least 99% sequence identity over 99% of its genome. Other suitable sequences for identifying biotype strains can include hsp60 or repetitive sequences, such as BOX, ERIC, (GTG) ₅ or REP or [18]. The biotype strain can have a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to the corresponding sequence of the bacterium deposited as NCIMB 42488. In some embodiments, the biotype strain has a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to the corresponding sequence of strain MRX518 deposited as NCIMB 42488 and comprises a 16S rRNA sequence that is at least 99% identical (e.g., at least 99.5% or at least 99.9% identical) to SEQ ID NO: 2. In some embodiments, the biotype strain has a sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to the corresponding sequence of strain MRX518 deposited as NCIMB 42488 and has a 16S rRNA sequence of SEQ ID NO:2.

In certain embodiments, the bacterial strains used in the present invention have a chromosome that has sequence identity to SEQ ID NO: 3. In preferred embodiments, the bacterial strains used in the present invention have a chromosome that is at least 90% identical (e.g., at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO: 3 over 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, bacterial strains for use in the present invention may have at least 90% sequence identity to SEQ ID NO: 3 over 70% of SEQ ID NO: 3, or at least 90% sequence identity to SEQ ID NO: 3 over 80% of SEQ ID NO: 3, or at least 90% sequence identity to SEQ ID NO: 3 over 90% of SEQ ID NO: 3, or at least 90% sequence identity to SEQ ID NO: 3 over 100% of SEQ ID NO: 3, or at least 95% sequence identity to SEQ ID NO: 3 over 70% of SEQ ID NO: 3, or at least 95% sequence identity to SEQ ID NO: 3 over 80% of SEQ ID NO: 3, or at least 95% sequence identity to SEQ ID NO: 3 over 90% of SEQ ID NO: 3, or at least 95% sequence identity to SEQ ID NO: 3 over 100% of SEQ ID NO: 3, or at least 98% sequence identity to SEQ ID NO: 3 over 70% of SEQ ID NO: 3, or at least 98% sequence identity to SEQ ID NO: 3 over 80% of SEQ ID NO: 3. NO:3 at least 98% sequence identity, or at least 98% sequence identity with SEQ ID NO:3 at 90% of SEQ ID NO:3, or at least 98% sequence identity with SEQ ID NO:3 at 95% of SEQ ID NO:3, or at least 98% sequence identity with SEQ ID NO:3 at 100% of SEQ ID NO:3, or at least 99.5% sequence identity with SEQ ID NO:3 at 90% of SEQ ID NO:3, or at least 99.5% sequence identity with SEQ ID NO:3 at 95% of SEQ ID NO:3, or at least 99.5% sequence identity with SEQ ID NO:3 at 98% of SEQ ID NO:3, or at least 99.5% sequence identity with SEQ ID NO:3 at 100% of SEQ ID NO:3.

In certain embodiments, the bacterial strains used in the present invention have a plasmid that has sequence identity to SEQ ID NO: 4. In preferred embodiments, the bacterial strains used in the present invention have a plasmid that has 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 over 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, bacterial strains for use in the present invention may have at least 90% sequence identity to SEQ ID NO: 4 over 70% of SEQ ID NO: 4, or at least 90% sequence identity to SEQ ID NO: 4 over 80% of SEQ ID NO: 4, or at least 90% sequence identity to SEQ ID NO: 4 over 90% of SEQ ID NO: 4, or at least 90% sequence identity to SEQ ID NO: 4 over 100% of SEQ ID NO: 4, or at least 95% sequence identity to SEQ ID NO: 4 over 70% of SEQ ID NO: 4, or at least 95% sequence identity to SEQ ID NO: 4 over 80% of SEQ ID NO: 4, or at least 95% sequence identity to SEQ ID NO: 4 over 90% of SEQ ID NO: 4, or at least 95% sequence identity to SEQ ID NO: 4 over 100% of SEQ ID NO: 4, or at least 98% sequence identity to SEQ ID NO: 4 over 70% of SEQ ID NO: 4, or at least 98% sequence identity to SEQ ID NO: 4 over 80% of SEQ ID NO: 4. A plasmid having at least 98% sequence identity to SEQ ID NO: 4, or having at least 98% sequence identity to SEQ ID NO: 4 over 90% of SEQ ID NO: 4, or having at least 98% sequence identity to SEQ ID NO: 4 over 100% of SEQ ID NO: 4.

In certain embodiments, the bacterial strain used in the present invention has a chromosome with a sequence identity to SEQ ID NO:3 and a plasmid with a sequence identity to SEQ ID NO:4.

In certain embodiments, the bacterial strains used in the present invention have a chromosome with a sequence identity to SEQ ID NO: 3, e.g., as described above, and a 16S rRNA sequence with a sequence identity to either SEQ ID NO: 1 or 2, e.g., as described above, preferably a 16s rRNA sequence at least 99% identical to SEQ ID NO: 2, more preferably comprising a 16S rRNA sequence of SEQ ID NO: 2, and optionally comprising a plasmid with a sequence identity to SEQ ID NO: 4, as described above.

In certain embodiments, the bacterial strain used in the present invention has a chromosome with sequence identity to SEQ ID NO: 3, e.g., as described above, and optionally comprises a plasmid with sequence identity to SEQ ID NO: 4, as described above, and is effective for treating or preventing cancer.

In certain embodiments, the bacterial strains used in the present invention have a chromosome with sequence identity to SEQ ID NO: 3, e.g., as described above, and a 16S rRNA sequence with sequence identity to either SEQ ID NO: 1 or 2, e.g., as described above, and optionally comprise a plasmid with sequence identity to SEQ ID NO: 4, as described above, and are effective for treating or preventing cancer.

In certain embodiments, the bacterial strain used in the present 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 (e.g., it comprises the 16S rRNA sequence of SEQ ID NO: 2), and a chromosome that is at least 90% identical to SEQ ID NO: 3 at least 95% identical to SEQ ID NO: 3, and optionally comprises a plasmid having a sequence identity to SEQ ID NO: 4, as described above, and is effective in treating or preventing cancer.

In certain embodiments, the bacterial strain used in the present 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 (e.g., it comprises the 16S rRNA sequence of SEQ ID NO: 2), and a chromosome that is at least 98% identical to SEQ ID NO: 3 (e.g., at least 99% or at least 99.5%), and optionally comprises a plasmid having a sequence identity to SEQ ID NO: 4, as described above, and is effective in treating or preventing cancer.

In certain embodiments, the bacterial strain used in the present invention is Enterococcus gallinarum, and has a 16s rRNA sequence that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ ID NO: 2 (e.g., it comprises the 16S rRNA sequence of SEQ ID NO: 2), and a chromosome that is at least 98% identical to SEQ ID NO: 3 (e.g., at least 99% or at least 99.5% identical to SEQ ID NO: 3), and optionally comprises a plasmid with a sequence identity to SEQ ID NO: 4, as described above, and is effective in treating or preventing cancer.

Alternatively, strains suitable for use in the present invention that are biotypes of the bacterium deposited under accession number NCIMB 42488 can be identified by using the deposited material under accession number NCIMB 42488 and restriction fragment analysis and/or PCR analysis, for example, by using fluorescence enhanced fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting or protein profiling or partial 16S or 23s rDNA sequencing. In a preferred embodiment, such techniques can be used to identify other Enterococcus gallinarum strains.

In certain embodiments, a strain that is a biotype of the bacterium deposited under accession number NCIMB 42488 and is suitable for use in the present invention is a strain that provides the same pattern as the bacterium deposited under accession number NCIMB 42488 when analyzed by enhanced ribosomal DNA restriction analysis (ARDRA), for example when using the Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [19]). Alternatively, a biotype strain is determined to be a strain that has the same carbohydrate fermentation pattern as the bacterium deposited under accession number NCIMB 42488. In some embodiments, the carbohydrate fermentation pattern is determined using the API 50CHL panel (bioMérieux). In some embodiments, preferably as determined by the API 50CHL analysis (preferably using the API 50CHL panel from bioMérieux), the bacterial strain for use in the present invention:

(i) positive for fermentation of at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all) of the following: L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdaloids, arbutin, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose, and potassium gluconate; and/or

(ii) intermediately positive for fermentation of at least one (e.g., at least 2, 3, 4, or all) of the following: D-mannitol, methyl-αD-glucopyranoside, D-lactose, starch, and L-fucose.

Other Enterococcus gallinarum strains suitable for use in the compositions and methods of the present invention, such as the biotype of the bacteria deposited under the registration number NCIMB 42488, can be identified using any appropriate method or strategy, including the analytical identification described in the examples. For example, the strains used in the present invention can be identified by culturing in anaerobic YCFA and/or administering the bacteria to a type II collagen-induced arthritis mouse model and then evaluating cytokine levels. Specifically, bacterial strains with growth patterns, metabolic types, and/or surface antigens similar to those of the bacteria deposited under the registration number NCIMB 42488 can be used in the present invention. Applicable strains will have immunomodulatory activity comparable to that of the NCIMB 42488 strain. Specifically, the biotype strains will cause effects on cancer disease models comparable to those shown in the examples, which can be identified using the culture and administration protocols described in the examples.

In some embodiments, preferably as determined by analysis of carbohydrate, amino acid and nitrate metabolism and optionally analysis of alkaline phosphatase activity, more preferably as determined by Rapid ID 32A analysis (preferably using the Rapid ID 32A system from bioMérieux), the bacterial strains used in the present invention:

(i) positive for at least one (e.g., at least 2, 3, 4, 5, 6, 7, or all) of the following: mannose fermentation, glutamate decarboxylase, arginine arylamidase, phenylalanine arylamidase, pyroglutamate arylamidase, tyrosine arylamidase, histidine arylamidase, and serine arylamidase; and/or

(ii) intermediately positive for at least one (e.g., at least two or all) of the following: β-galactosidase-6-phosphate, β-glucosidase, and N-acetyl-β-glucosaminidase; and/or

(iii) negative for at least one (e.g., at least 2, 3, 4, 5, 6, or all) of the following: raffinose fermentation, proline arylamidase, leucylglycine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, and glutamylglutamate arylamidase.

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

(i) negative for at least one (e.g., at least 2, 3, or all 4) of glycine arylamidase, raffinose fermentation, proline arylamidase, and leucine arylamidase, e.g., as determined by an assay of carbohydrate, amino acid, and nitrate metabolism, optionally as determined by a Rapid ID 32A assay (preferably using the Rapid ID 32A system from bioMérieux); and/or

(ii) preferably intermediately positive for fermentation towards L-fucose as determined by API 50 CHL assay (preferably using the API 50 CHL panel from bioMérieux).

In some embodiments, the bacterial strains used in the present invention are extracellular ATP generators, such as those measured using an ATP assay kit (Sigma-Aldrich, MAK190), producing 6-6.7 ng/μl (e.g., 6.1-6.6 ng/μl or 6.2-6.5 ng/μl or 6.33 ± 0.10 ng/μl) of ATP. Extracellular ATP from bacteria can have pleiotropic effects, including activation of T cell receptor-mediated signaling (Schenk et al., 2011), promotion of intestinal Th17 cell differentiation (Atarashi et al., 2008), and induction of pro-inflammatory mediator IL-1β secretion by activating the NLRP3 inflammasome (Karmarkar et al., 2016). Therefore, bacterial strains that are extracellular ATP generators can be used to treat or prevent cancer.

In some embodiments, the bacterial strains used in the present invention comprise one or more of the following three genes: a mobile element protein; a xylose ABC transporter permease component; and FIG00632333: a hypothetical protein. For example, in certain embodiments, the bacterial strains used in the present invention comprise genes encoding a mobile element protein and a xylose ABC transporter permease component; a mobile element protein and FIG00632333: a hypothetical protein; a xylose ABC transporter permease component and FIG00632333: a hypothetical protein; or a mobile element protein, a xylose ABC transporter permease component, and FIG00632333: a hypothetical protein.

A particularly preferred strain of the present invention is the Enterococcus gallinarum strain deposited under the registration number NCIMB 42488. It is the exemplary MRX518 strain tested in the examples and shown to be effective in treating diseases. Therefore, the present invention provides cells of the Enterococcus gallinarum strain deposited under the registration number NCIMB 42488 or its derivatives, such as isolated cells. The present invention also provides a composition comprising cells of the Enterococcus gallinarum strain deposited under the registration number NCIMB 42488 or its derivatives. The present invention also provides a biologically pure culture of the Enterococcus gallinarum strain deposited under the registration number NCIMB 42488. The present invention also provides cells of the Enterococcus gallinarum strain deposited under the registration number NCIMB 42488 or its derivatives for use in treatment, in particular for the diseases described herein. Derivatives of the strain deposited under the registration number NCIMB 42488 can be daughter strains (offspring) or strains derived from the original culture (subclones).

Derivatives of the strains of the present invention can be modified, for example, at the genetic level, without abolishing biological activity. Specifically, the derivative strains of the present invention are therapeutically active. The derivative strains will have immunomodulatory activity comparable to that of the original NCIMB 42488 strain. Specifically, the derivative strains will elicit effects comparable to those shown in the Examples in cancer disease models, which can be identified using the culture and administration protocols described in the Examples. Derivatives of the NCIMB 42488 strain will generally be biotypes of the NCIMB 42488 strain.

Reference to cells of the Enterococcus gallinarum strain deposited under Accession No. NCIMB 42488 encompasses any cells having the same safety and therapeutic efficacy profile as the strain deposited under Accession No. NCIMB 42488, and the present invention encompasses such cells. Thus, in some embodiments, reference to cells of the Enterococcus gallinarum strain deposited under Accession No. NCIMB 42488 refers only to the MRX518 strain deposited with NCIMB 42488 and not to bacterial strains not deposited with NCIMB 42488. In some embodiments, reference to cells of the Enterococcus gallinarum strain deposited under Accession No. NCIMB 42488 refers to any cells having the same safety and therapeutic efficacy profile as the strain deposited under Accession No. NCIMB 42488, but not the strain deposited with NCIMB 42488.

In preferred embodiments, the bacterial strains in the compositions of the present invention are viable and capable of partially or fully colonizing the intestine.

Treating cancer

In a preferred embodiment, the compositions of the present invention are used to treat or prevent cancer. The examples demonstrate that administration of the compositions of the present invention can reduce tumor growth in various tumor models.

In certain embodiments, treatment with the compositions of the present invention reduces tumor size or reduces tumor growth. In certain embodiments, the compositions of the present invention are used to reduce tumor size or reduce tumor growth. The compositions of the present invention can effectively reduce tumor size or growth. In certain embodiments, the compositions of the present invention are used in patients with solid tumors. In certain embodiments, the compositions of the present invention are used to reduce or prevent angiogenesis in cancer treatment. The compositions of the present invention can have an effect on the immune or inflammatory systems that play an important role in angiogenesis. In certain embodiments, the compositions of the present invention are used to prevent metastasis.

In a preferred embodiment, the compositions of the present invention are used to treat or prevent breast cancer. The examples demonstrate that the compositions of the present invention can be effective in treating cancer. In certain embodiments, the compositions of the present invention are used to reduce tumor size, reduce tumor growth, or reduce angiogenesis in the treatment of breast cancer. In a preferred embodiment, the cancer is breast cancer. In a preferred embodiment, the cancer is stage IV breast cancer.

In certain embodiments, the compositions of the present invention are used to treat or prevent lung cancer. The examples demonstrate that the compositions of the present invention are effective in treating lung cancer. In certain embodiments, the compositions of the present invention are used to reduce tumor size, reduce tumor growth, or reduce angiogenesis in the treatment of lung cancer. In preferred embodiments, the cancer is lung cancer.

In certain embodiments, the compositions of the present invention are used to treat or prevent liver cancer. The examples demonstrate that the compositions of the present invention can effectively treat liver cancer. In certain embodiments, the compositions of the present invention are used to reduce tumor size, reduce tumor growth, or reduce angiogenesis in the treatment of liver cancer. In a preferred embodiment, the cancer is liver cancer (hepatocellular carcinoma).

In certain embodiments, the compositions of the present invention are used to treat or prevent colon cancer. The examples demonstrate that the compositions of the present invention have an effect on colon cancer cells and can be effective in treating colon cancer. In certain embodiments, the compositions of the present invention are used to reduce tumor size, reduce tumor growth, or reduce angiogenesis in the treatment of colon cancer. In preferred embodiments, the cancer is colorectal adenocarcinoma.

In some embodiments, the cancer is in the intestine. In some embodiments, the cancer is in a part of the body other than the intestine. In some embodiments, the cancer is not in the intestine. In some embodiments, the cancer is not colorectal cancer. In some embodiments, the cancer is not small intestine cancer. In some embodiments, the treatment or prevention is performed in a location other than the intestine. In some embodiments, the treatment or prevention is performed in the intestine and in a location other than the intestine.

In certain embodiments, the compositions of the present invention are used to treat or prevent cancer. The examples demonstrate that the compositions of the present invention are effective in treating various types of cancer. In certain embodiments, the compositions of the present invention are used to treat or prevent non-immunogenic cancers. The examples demonstrate that the compositions of the present invention are effective in treating non-immunogenic cancers.

The therapeutic effects of the compositions of the present invention on cancer may be mediated by pro-inflammatory mechanisms. Examples 2, 4, and 5 demonstrate that the expression of a number of pro-inflammatory cytokines may be increased after administration of MRX518. Inflammation can have cancer-suppressing effects [20] and pro-inflammatory cytokines such as TNFα are being studied as cancer therapies [21]. The upregulation of genes such as TNF shown in the examples may indicate that the compositions of the present invention can be used to treat cancer through similar mechanisms. The upregulation of CXCR3 ligands (CXCL9, CXCL10) and IFNγ-inducible genes (IL-32) may indicate that the compositions of the present invention elicit an IFNγ-type response. IFNγ is a potent macrophage activating factor that can stimulate tumoricidal activity [22], and CXCL9 and CXCL10, for example, also have anti-cancer effects [23-25]. Therefore, in certain embodiments, the compositions of the present invention are used to promote inflammation in cancer treatment. In a preferred embodiment, the compositions of the present invention are used to promote Th1 inflammation in cancer treatment. Th1 cells produce IFNγ and have potent anti-cancer effects [20]. In certain embodiments, the compositions of the present invention are used to treat early-stage cancer, such as cancer that has not yet metastasized or stage 0 or stage 1 cancer. Promoting inflammation can more effectively target early-stage cancer [20]. In certain embodiments, the compositions of the present invention are used to promote inflammation to enhance the effect of a second anticancer agent. In certain embodiments, the treatment or prevention of cancer includes increasing the expression level of one or more cytokines. For example, in certain embodiments, the treatment or prevention of cancer includes increasing the expression level of one or more of IL-1β, IL-6, and TNF-α, such as IL-1β and IL-6, IL-1β and TNF-α, IL-6 and TNF-α, or all three of IL-1β, IL-6, and TNF-α. It is known that an increase in the expression level of any one of IL-1β, IL-6, and TNF-α indicates the efficacy of cancer treatment.

Examples 4 and 5 demonstrate that when the bacterial strains described herein are used in combination with lipopolysaccharide (LPS), IL-1β increases synergistically. LPS is known to cause pro-inflammatory effects. Therefore, in certain embodiments, treatment or prevention comprises the use of a bacterial strain as described herein in combination with an agent that upregulates IL-1β. In certain embodiments, treatment or prevention comprises the use of a bacterial strain as described herein in combination with LPS. Therefore, the compositions of the present invention may additionally comprise an agent that upregulates IL-1β. Therefore, the compositions of the present invention may additionally comprise LPS.

In certain embodiments, the compositions of the present invention are used to treat patients who have previously received chemotherapy. In certain embodiments, the compositions of the present invention are used to treat patients who are no longer tolerant to chemotherapy. The compositions of the present invention may be particularly suitable for such patients.

In certain embodiments, the compositions of the present invention are used to prevent recurrence. The compositions of the present invention may be suitable for long-term administration. In certain embodiments, the compositions of the present invention are used to prevent cancer progression.

In certain embodiments, the compositions of the present invention are used to treat non-small cell lung cancer. In certain embodiments, the compositions of the present invention are used to treat small cell lung cancer. In certain embodiments, the compositions of the present invention are used to treat squamous cell carcinoma. In certain embodiments, the compositions of the present invention are used to treat adenocarcinoma. In certain embodiments, the compositions of the present invention are used to treat glandular tumors, carcinoid tumors, or undifferentiated carcinomas.

In certain embodiments, the composition of the present invention is used to treat hepatoblastoma, hepatobiliary carcinoma, cholangiocarcinoma, or liver cancer caused by viral infection.

In certain embodiments, the compositions of the invention are used to treat invasive ductal carcinoma, ductal carcinoma in situ, or invasive lobular carcinoma.

In other embodiments, the compositions of the present invention are used to treat or prevent acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, breast cancer, bronchial adenoma/carcinoid tumor, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid disease, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, sarcoma), intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, childhood visual pathway and hypothalamic glioma, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, renal cell carcinoma, laryngeal cancer, leukemia, lymphoma, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacytoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

The compositions of the present invention are particularly effective when used in combination with other therapeutic agents. The immunomodulatory effects of the compositions of the present invention are effective when combined with more direct anticancer agents. Therefore, in certain embodiments, the present invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum and an anticancer agent. In preferred embodiments, the anticancer agent is an immune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cell growth inhibitory drug. In preferred embodiments, the composition comprises an anticancer agent selected from the group consisting of: Yervoy (ipilimumab, BMS); Keytruda (pembrolizumab, Merck); Opdivo (nivolumab, BMS); MEDI4736 (AZ/MedImmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/MedImmune); CT- 011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/MedImmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/MedImmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; 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); Genetics); Anti-VISTA (JnJ); Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (Eli Lilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics); Bavituximab (Peregrine Pharmaceuticals); CC90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women's University, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Center); 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, GlaxoSmithKline); Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-I131, GlaxoSmithKline); Zevalin (ibritumomab tiuxetanumab) tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, 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 (Actinium) estafenatox) (ActiveBiotech); AFM13 (Affimed); AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS -22ME (Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab, Alexion); ALT-836 (Altor Bioscience); ALT-801 (Altor Bioscience); ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232 (adalimumab, Amgen); AMG211 (Amgen/MedImmune); BAY20-10112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen); MEDI575 (AZ/MedImmune); MEDI3617 (AZ/MedImmune); MEDI6383 (AZ/MedImmune); MEDI551 (AZ/MedImmune); Moxetumomab pasudotox)(AZ/MedImmune); MEDI565(AZ/MedImmune); MEDI0639(AZ/MedImmune); MEDI0680(AZ/MedImmune); MEDI562(AZ/MedImmune); 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 (BioClin); BIIB015 (Biogen); onutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206 (Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest); BIL-010t (Biosceptre); MDX-1203 (BMS); MDX-1204 (BMS); nesitumomab (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) 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) Lilly); LY2495655 (Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806 (Eli Lilly); cixutumumab (Eli Lilly); flanvotumab (Eli Lilly); IMC-TR1 (Eli Lilly); ramucirumab (Eli Lilly); tabalumab (Eli 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-doxorubicin (Immunomedics); IMMU-115 (Immunomedi cs); 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 Biologics); TAS266 (Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031 (Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355 (Novartis); OMP-21M18 (densizumab, OncoMed); OMP52M51 (Oncomed/GSK); OMP-59R5 (Oncomed/GSK); ventuzumab (Oncomed/Bayer); CMC-544 (inotuzumab ozogamicin (Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer); PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacumab, Regeneron/Sanofi); REGN421 (enoticumab, Regeneron/Sanofi); RG7221, RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636, RG7450, RG7593, RG7596, DCDS34 10A, 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-10TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (JunoTherapeutics); KTE-C19CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (Bellicum Pharmaceuticals); BPX-601 (Bellicum Pharmaceuticals) Pharmaceuticals); ATTCK20 (UnumTherapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101 (ShanghaiSunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences); Colo-Ad1 (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, VirttuBiologics); OrienX010 (OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SVV-001 (Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin, oxaliplatin, irinotecan, folinic acid 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 (Eli Lilly); Adriamycin (Pfizer); Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene); Trelstar (Debiopharm); Taxotere (Sanofi); Pharmaceuticals); Camptosar (Pfizer); UFT (Taiho Pharmaceuticals); and TS-1 (Taiho Pharmaceuticals).

In some embodiments, one or more bacterial strains having a 16s rRNA sequence at least 95% identical to SEQ ID NO: 2, such as Enterococcus gallinarum, are the sole therapeutically active agent in the compositions of the invention. In some embodiments, the bacterial strains in the compositions are the sole therapeutically active agent in the compositions of the invention.

Mode of administration

Preferably, the composition of the present invention is to be administered to the gastrointestinal tract to enable passage to the intestine and/or partial or complete colonization of the intestine by the bacterial strain of the present invention. Generally, although the composition of the present invention is administered orally, it may be administered rectally, intranasally, or by the buccal or sublingual route.

In certain embodiments, the compositions of the present invention may be applied in the form of a foam, spray, or gel.

In certain embodiments, the compositions of the invention may be administered in the form of suppositories, eg, rectal suppositories, such as cocoa butter, synthetic hard fats (eg, suppocire, witepsol), glyceryl-gelatin, polyethylene glycol, or soap glycerin compositions.

In certain embodiments, the compositions of the invention are administered to the gastrointestinal tract via a tube, e.g., a nasogastric tube, an orogastric tube, a gastric tube, a jejunostomy tube (J-tube), a percutaneous endoscopic gastrostomy (PEG), or a port, e.g., a chest wall port leading to the stomach, jejunum, and other suitable access ports.

The compositions of the present invention can be administered once, or they can be administered continuously as part of a treatment regimen. In certain embodiments, the compositions of the present invention will be administered daily.

In certain embodiments of the present invention, treatment according to the present invention is accompanied by an assessment of the patient's intestinal microbiota. If delivery and/or partial or complete colonization of the strain of the present invention is not achieved, such that efficacy is not observed, the treatment can be repeated. If delivery and/or partial or complete colonization is successful and efficacy is observed, the treatment can be stopped.

In certain embodiments, the compositions of the invention can be administered to a pregnant animal, such as a mammal, such as a human, to reduce the likelihood that cancer will develop in their offspring in utero and/or after birth.

The compositions of the present invention can be administered to patients diagnosed with cancer or identified as being at risk for cancer. The compositions can also be administered as a preventative measure to prevent the development of cancer in healthy patients.

The compositions of the present invention can be administered to a patient who has been identified as having an abnormal intestinal microflora. For example, the patient may have reduced or absent colonization with Enterococcus gallinarum.

The composition of the present invention can be administered as a food, for example, a nutritional supplement.

Typically, the compositions of the present invention are used to treat humans, but they can also be used to treat animals, including monogastric mammals such as poultry, pigs, cats, dogs, horses, or rabbits. The compositions of the present invention can be used to enhance the growth and performance of animals. If administered to animals, oral gavage can be used.

Composition

Typically, the composition of the present invention comprises bacteria. In a preferred embodiment of the present invention, the composition is formulated in a lyophilized form. For example, the composition of the present invention may comprise granules or gelatin capsules, such as hard gelatin capsules, containing the bacterial strain of the present invention.

Preferably, the composition of the invention comprises freeze-dried bacteria. Freeze-drying of bacteria is a well-known procedure and relevant guidance can be found in, for example, references [26-28].

Alternatively, the compositions of the invention may comprise live active bacterial cultures.

In some embodiments, the bacterial strains in the compositions of the present invention have not been inactivated, e.g., have not been heat-inactivated. In some embodiments, the bacterial strains in the compositions of the present invention have not been killed, e.g., have not been heat-killed. In some embodiments, the bacterial strains in the compositions of the present invention have not been attenuated, e.g., have not been heat-attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention have not been killed, inactivated, and/or attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable. For example, in some embodiments, the bacterial strains in the compositions of the present invention are capable of partially or completely colonizing the intestine. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable and are capable of partially or completely colonizing the intestine.

In some embodiments, the composition comprises a mixture of live bacterial strains and killed bacterial strains.

In a preferred embodiment, the composition of the present invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from degradation until delivery at the target site by rupture, for example, by chemical or physical stimulation, such as pressure, enzymatic activity, or physical disintegration (which may be triggered by a change in pH). Any suitable encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption to a solid support surface, self-aggregation by flocculation or with a cross-linking agent, and mechanical inclusion behind a microporous membrane or microcapsule. Guidance on encapsulation that can be used to prepare the composition of the present invention can be found, for example, in references [29] and [30].

The composition can be administered orally and can be in the form of tablets, capsules, or powders. Encapsulated products are preferred because Enterococcus gallinarum is an anaerobic bacterium. Other ingredients (e.g., vitamin C) can be included as oxygen scavengers and prebiotic matrices to improve in vivo delivery and/or partial or complete colonization and survival. Alternatively, the probiotic composition of the present invention can be administered orally as a food or nutritional product, such as a fermented dairy product based on milk or whey, or as a pharmaceutical product.

The composition may be formulated as a probiotic.

The compositions of the present invention include a therapeutically effective amount of the bacterial strain of the present invention. The therapeutically effective amount of the bacterial strain is sufficient to exert a beneficial effect on the patient. The therapeutically effective amount of the bacterial strain is sufficient to deliver to the patient's intestine and/or partially or completely colonize the patient's intestine.

For example, a daily dose of bacteria suitable for an adult can be about 1×10 ³ to about 1×10 ¹¹ colony forming units (CFU); for example, about 1×10 ⁷ to about 1×10 ¹⁰ CFU; in another example, about 1×10 ⁶ to about 1×10 ¹⁰ CFU.

In certain embodiments, the composition contains the bacterial strain in an amount of about 1×10 ⁶ to about 1×10 ¹¹ CFU/g, such as about 1×10 ⁸ to about 1×10 ¹⁰ CFU/g, relative to the weight of the composition. The dosage can be, for example, 1 g, 3 g, 5 g, and 10 g.

Typically, probiotics, such as the compositions of the present invention, are optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are typically non-digestible carbohydrates, such as oligosaccharides or polysaccharides, or sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known probiotics include commercial products such as inulin and trans-galacto-oligosaccharides.

In certain embodiments, the probiotic composition of the present invention includes a probiotic compound in an amount of about 1 to about 30% by weight (e.g., 5 to 20% by weight) relative to the total weight of the composition. The carbohydrate may be selected from the group consisting of fructooligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomaltooligosaccharides, pectin, xylo-oligosaccharides (or XOS), chito-oligosaccharides (or COS), β-glucans, modified and resistant starches of gum arabic, polydextrose, D-tagatose, gum arabic fiber, carob, oat, and citrus fiber. In one aspect, the probiotic is a short-chain fructooligosaccharide (hereinafter shown as FOSs-c.c for simplicity); the FOSs-c.c is a non-digestible carbohydrate generally obtained by converting beet sugar and comprising a sucrose molecule bonded to three glucose molecules.

The compositions of the present invention may comprise a pharmaceutically acceptable excipient or carrier. Examples of such suitable excipients can be found in reference [31]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [32]. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, 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 based on the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise any suitable binder, lubricant, suspending agent, coating agent, solubilizing agent as a carrier, excipient, or diluent or may comprise such a substance in addition to a carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars (e.g., glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners), natural and synthetic gums (e.g., gum arabic, gum 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 flavorings can be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and parahydroxybenzoates. Antioxidants and suspending agents can also be used.

The compositions of the present invention can be formulated as foods. For example, in addition to the therapeutic effects of the present invention, foods can provide nutritional benefits, such as in nutritional supplements. Similarly, foods can be formulated to enhance the flavor of the compositions of the present invention, or to make the compositions more appealing to eat by making them more similar to common foods rather than pharmaceutical compositions. In certain embodiments, the compositions of the present invention are formulated as milk-based products. The term "milk-based product" refers to any liquid or semisolid product based on milk or whey with varying fat content. Milk-based products can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk or processed products in which milk powder and whey are reconstituted without any processing, such as yogurt, curd, curd, sour milk, sour whole milk, buttermilk and other sour milk products. Another important group includes dairy beverages, such as whey beverages, fermented milk, condensed milk, infant or baby milk; flavored milk, ice cream; and foods containing milk, such as sweets.

In certain embodiments, the compositions of the present invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may only contain minimal amounts or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures that are substantially free of other organism species. Therefore, in some embodiments, the invention provides a composition comprising one or more bacterial strains from an Enterococcus gallinarum species that does not contain bacteria from any other species or only contains minimal amounts or biologically irrelevant amounts of bacteria from another species, and is used for treatment.

In some embodiments, the composition of the present invention comprises and is more than a bacterial strain or species.For example, in some embodiments, the composition of the present invention comprises and is more than one bacterial strain (for example more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 bacterial strain) from same species and optionally does not contain the bacterium from any other species.In some embodiments, the composition of the present invention comprises and is less than 50 bacterial strains (for example less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 bacterial strain) from same species and optionally does not contain the bacterium from any other species. 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 from the same species and optionally contain no bacteria from any other species. In some embodiments, the compositions of the invention comprise more than one species from 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 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 present 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 the same genus and optionally do not contain bacteria from any other genus. The present invention comprises any combination of the above bacteria.

In some embodiments, the composition comprises a microbial flora. For example, in some embodiments, the composition comprises a 16s rRNA sequence having at least 95% consistency with SEQ ID NO: 2, such as a bacterial strain of Enterococcus gallinarum as part of the microbial flora. For example, in some embodiments, the bacterial strain is present in combination with one or more (e.g., at least 2, 3, 4, 5, 10, 15, or 20) other bacterial strains from other genera that can coexist in the intestine together. For example, in some embodiments, the composition comprises a 16s rRNA sequence having at least 95% consistency with SEQ ID NO: 2, such as a bacterial strain of Enterococcus gallinarum, combined with bacterial strains from different genera. In some embodiments, the microbial flora comprises two or more bacterial strains obtained from fecal samples of a single organism such as a human. In some embodiments, the microbial flora is not found together in nature. For example, in some embodiments, the microbial flora comprises bacterial strains obtained from fecal 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 a human infant and an adult. In some embodiments, the two different organisms are a human and a non-human mammal.

In some embodiments, the compositions of the invention further comprise a bacterial strain having the same safety and therapeutic efficacy profile as strain MRX518, but which is not MRX518 deposited as NCIMB 42488 or which is not Enterococcus gallinarum.

In some embodiments where the compositions of the present invention comprise more than one bacterial strain, species or species, the individual bacterial strains, species or species can be administered separately, simultaneously or continuously. For example, the composition can comprise all of more than one bacterial strain, species or species, or the bacterial strains, species or species can be stored separately and administered separately, simultaneously or continuously. In some embodiments, more than one bacterial strain, species or species are stored separately but mixed together before use.

In some embodiments, the bacterial strains used in the present invention are obtained from human infant feces. In some embodiments where the compositions of the present invention comprise more than one bacterial strain, all bacterial strains are obtained from human infant feces, or if other bacterial strains are present, they are only present in minimal amounts. The bacteria can be cultured after being obtained from human infant feces and used in the compositions of the present invention.

As mentioned above, in some embodiments, one or more bacterial strains having a 16s rRNA sequence that is at least 95% identical to SEQ ID NO: 2, such as Enterococcus gallinarum, are the sole therapeutically active agent in the compositions of the invention. In some embodiments, the bacterial strain in the composition is the sole therapeutically active agent in the compositions of the invention.

Compositions for use according to the present invention may or may not require marketing approval.

In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is freeze-dried. In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is spray-dried. In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is freeze-dried or spray-dried and wherein it is viable. In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is freeze-dried or spray-dried and wherein it is viable. In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is freeze-dried or spray-dried and wherein it is capable of partially or completely colonizing the intestine. In certain embodiments, the present invention provides the above pharmaceutical compositions, wherein the bacterial strain is freeze-dried or spray-dried and wherein it is viable and capable of partially or completely colonizing the intestine.

In some cases, the lyophilized or spray-dried bacterial strain is reconstituted prior to administration. In some cases, reconstitution is by using a diluent described herein.

The compositions of the present invention may comprise a pharmaceutically acceptable excipient, diluent or carrier.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain, when administered to a subject in need thereof, is in an amount sufficient to treat a condition; and wherein the condition is breast cancer. In a preferred embodiment, the cancer is breast cancer. In a preferred embodiment, the cancer is stage IV breast cancer.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain is administered to a subject in need thereof in an amount sufficient to treat a condition; and wherein the condition is lung cancer. In a preferred embodiment, the cancer is lung cancer.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain is administered to a subject in need thereof in an amount sufficient to treat a condition; and wherein the condition is liver cancer. In a preferred embodiment, the cancer is liver cancer (hepatocellular carcinoma).

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain, when administered to a subject in need thereof, is in an amount sufficient to treat a condition; and wherein the condition is colon cancer. In a preferred embodiment, the cancer is colorectal adenocarcinoma.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain is in an amount sufficient to treat a condition when administered to a subject in need thereof; and wherein the condition is a cancer.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain is in an amount sufficient to treat a condition when administered to a subject in need thereof; and wherein the condition is a non-immunogenic cancer.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is administered to a subject in need thereof in an amount sufficient to treat a condition; and wherein the condition is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, adenocarcinoma, glandular tumor, carcinoid tumor, and undifferentiated carcinoma.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is administered to a subject in need thereof in an amount sufficient to treat a condition; and wherein the condition is selected from the group consisting of hepatoblastoma, hepatobiliary carcinoma, cholangiocarcinoma or liver cancer caused by a viral infection.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the bacterial strain is in an amount sufficient to treat a condition when administered to a subject in need thereof; and wherein the condition is selected from the group consisting of invasive ductal carcinoma, ductal carcinoma in situ, or invasive lobular carcinoma.

In certain embodiments, the present invention provides a pharmaceutical composition comprising: a bacterial strain as used in the present invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is administered to a subject in need thereof in an amount sufficient to treat a condition; and wherein the condition 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 tumor, osteosarcoma/malignant fibrous histiocytoma, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, breast cancer, bronchial adenoma/carcinoid tumor, Burkitt's lymphoma cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloid disease, colon cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors, childhood visual pathway and hypothalamic gliomas, Hodgkin's lymphoma, melanoma, islet cell carcinoma, Kaposi's sarcoma, renal cell carcinoma, laryngeal cancer, leukemia, lymphoma, mesothelioma, neuroblastoma, non-Hodgkin's lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasmacytoma, prostate cancer, renal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.

In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is about 1×10 ³ to about 1×10 ¹¹ colony forming units per gram relative to the weight of the composition.

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

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

In certain embodiments, the present invention provides the above pharmaceutical composition, which comprises an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweetener, gum arabic, gum tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate and sodium chloride.

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

In certain embodiments, the present invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and parabens.

In certain embodiments, the present invention provides the above pharmaceutical composition, wherein at least 80% of the bacterial strain remains as measured in colony forming units 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 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 with 50% relative humidity.

In some embodiments, the compositions of the present invention are provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a pouch or bottle. In some embodiments, the compositions of the present invention are provided in a syringe comprising a composition as described herein.

In some embodiments, the compositions of the present invention can be provided as a pharmaceutical formulation. For example, the compositions can be provided as a tablet or a capsule. In some embodiments, the capsule is a gelatin capsule ("gelatin capsule").

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, whereby the compound enters the bloodstream directly from the mouth.

Pharmaceutical formulations suitable for oral administration include solid plugs, solid particulates, semisolids, and liquids (including multiphase or dispersed systems), such as tablets; soft or hard capsules containing microparticles or nanoparticles, liquids (e.g., aqueous solutions), emulsions, or powders; lozenges (including liquid-filled); chewable tablets; gels; rapidly dispersing dosage forms; films; ovoids; sprays; and buccal/mucoadhesive patches.

In some embodiments, the pharmaceutical formulation is an enteric formulation, i.e., a gastro-resistant formulation (e.g., resistant to gastric pH) suitable for delivering 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 can be an enteric-coated tablet or an enteric-coated capsule, etc. The enteric coating can be a conventional enteric coating, such as a conventional coating used for tablets, capsules, etc. for oral delivery. The formulation can comprise a film coating, such as a film layer of an enteric polymer, such as an acid-insoluble polymer.

In some embodiments, the enteric formulation is enteric in nature, e.g., resistant to the stomach, and does not require an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not include an enteric coating. In some embodiments, the formulation is a capsule made of a thermogelling material. In some embodiments, the thermogelling material is a cellulosic material, e.g., 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 contain any film-forming polymer (e.g., see [33]). In some embodiments, the formulation is an enteric capsule (e.g., from Capsugel).

In some embodiments, preparation is a soft capsule.Soft capsule is a capsule with certain elasticity and softness due to the addition of a softener such as glycerol, sorbitol, maltitol and polyethylene glycol present in the capsule shell.Soft capsule can, for example, be produced according to gelatin or starch.Soft capsules based on gelatin are purchased from multiple suppliers.Depending on the method of administration, for example, oral or rectal, soft capsule can have various shapes, which can, for example, be circular, oval, rectangular or torpedo-shaped.Soft capsule can be produced by conventional methods, for example, by Scherer process, Accogel process or droplet or blowing method.

Cultivation methods

The bacterial strains used in the present invention can be cultured using standard microbiological techniques as described in detail in, for example, references [34-36].

The solid or liquid medium used for culturing can be YCFA agar or YCFA medium. YCFA medium may include (per 100 ml, approximate values): tyrosine (1.0 g), yeast extract (0.25 g), NaHCO ₃ (0.4 g), cysteine (0.1 g), K ₂ HPO ₄ (0.045 g), KH ₂ PO ₄ (0.045 g), NaCl (0.09 g), (NH ₄ ) ₂ SO ₄ (0.09 g), MgSO ₄ ·7H ₂ O (0.009 g), CaCl ₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 μg), cobalamin (1 μg), p-aminobenzoic acid (3 μg), folic acid (5 μg) and pyridoxamine (15 μg).

Bacterial strains used in vaccine compositions

The present inventors have determined that the bacterial strains of the present invention can be used to treat or prevent cancer. This may be the result of the bacterial strains of the present invention acting on the host immune system. Therefore, when administered as a vaccine composition, the compositions of the present invention can also be used to prevent cancer. In certain such embodiments, the bacterial strains of the present invention are viable. In certain such embodiments, the bacterial strains of the present invention are capable of partially or completely colonizing the intestine. In certain such embodiments, the bacterial strains of the present invention are viable and are capable of partially or completely colonizing the intestine. In other such embodiments, the bacterial strains of the present invention may be killed, inactivated, or attenuated. In certain such embodiments, the composition may comprise a vaccine adjuvant. In certain embodiments, the composition is for administration by injection, for example, by subcutaneous injection.

General

Unless otherwise indicated, the practice of the present invention will employ conventional methods of chemistry, biochemistry, molecular biology, immunology, and pharmacology within the skill of the art. Such techniques are fully explained in the literature. See, for example, references [37] and [38-44].

The term "comprising" encompasses "including" as well as "consisting of," eg, a composition "comprising" X may consist solely of X, or may include something else, eg, X+Y.

The term "about" with respect to a value x is optional and means, for example, x ± 10%.

The word "substantially" does not exclude "completely", for example a composition "substantially free" of Y may be completely free of Y. If necessary, the word "substantially" may be omitted from the definition of the present invention.

Reference to the percent sequence identity between two nucleotide sequences means that when the two sequences are aligned, the percent of nucleotides that are identical is compared. This alignment and homology or percent sequence identity can be determined using software programs known in the art, such as the software program described in section 7.7.18 of reference [45]. Preferably, the alignment is determined using an affine gap search (wherein the open gap penalty is 12 and the gap extension penalty is 2, BLOSUM 62 matrix) by the Smith-Waterman homology search algorithm. The Smith-Waterman homology search algorithm is disclosed in reference [46].

Unless otherwise stated, a process or method comprising multiple steps may include other steps at the beginning or end of the method, or may include other intervening steps. In addition, steps may be combined, omitted, or performed in an alternative order when appropriate.

A plurality of embodiments of the present invention are described herein. It should be understood that the features described in each embodiment can be combined with other described features to provide other embodiments. Specifically, the embodiments emphasized as suitable, typical or preferred herein can be combined with each other (unless they are mutually exclusive).

Mode for Carrying Out the Invention

Example 1 - Efficacy of bacterial inoculum in a mouse cancer model

Overview

This study tested the efficacy of a composition comprising a bacterial strain according to the invention in four tumor models.

Material

Test substance - bacterial strain #MRX518.

Reference material - anti-CTLA-4 antibody (clone: 9H10, catalog number: BE0131, isotype: golden hamster IgG1, Bioxcell).

Test and reference substances Vehicle—bacterial culture medium (yeast extract, casein, fatty acid medium (YCFA)). Antibodies were diluted in PBS (Cat. No. BE14-516F, Lonza, France) on each day of injection into mice.

Treatment dose - bacteria: 2 x ¹⁰⁸ in 200 μL. Anti-CTLA-4 was injected at 10 mg/kg per injection. Anti-CTLA-4 was administered at a dose volume of 10 mL/kg per administration based on the mouse's recent body weight (i.e., a mouse weighing 20 g would receive 200 μL of test substance).

Administration Route - Bacterial inoculum was administered by oral gavage (PO) via a cannula. The cannula was decontaminated daily. Anti-CTLA-4 was injected into the peritoneal cavity of mice (intraperitoneally, IP).

Culture conditions for bacterial strains - The culture conditions for bacterial strains are as follows:

Pipette 10 mL of YCFA (prepared from a 10 mL E&O laboratory bottle) into a Hungate tube

The tube was sealed and flushed with _CO2 using a syringe input and exhaust system

Autoclave the Hungate tube

When cool, inoculate the Hungate tube with 1 mL of glycerol stock solution.

• Place the tube in a static 37°C incubator for approximately 16 hours.

The next day, take 1 mL of this subculture and inoculate 10 mL of YCFA (again, pre-warmed rinsed Hungate tubes, both in duplicate)

Place in a static 37°C incubator for 5 to 6 hours

Cancer cell lines and culture conditions

The cell lines used are detailed in the table below:

cell lines type Mouse strains source EMT-6 Breast cancer BALB/c ATCC LL/2(LLC1) lung cancer C57BL/6 ATCC CRL1642 Hepa1-6 Hepatocellular carcinoma C57BL/6 IPSEN INNOVATION

The EMT-6 cell line was established from a transplantable murine mammary carcinoma generated in BALB/cCRGL mice following implantation of hyperplastic mammary alveolar nodules [47].

The LL/2 (LLC1) cell line was established from the lungs of C57BL mice bearing tumors arising from implanted primary Lewis lung carcinomas [48].

The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatocarcinoma generated in C57/L mice [49].

Cell culture conditions - All cell lines were grown as monolayers at 37°C in a humidified atmosphere (5% _CO2 , 95% air). Media and supplements are indicated in the following table:

For experimental use, adherent tumor cells were detached from the culture flasks by treatment with trypsin-versene (Cat. No. BE17-161E, Lonza) in Hanks' medium (Cat. No. BE10-543F, Lonza) without calcium or magnesium for 5 minutes and neutralized by adding complete culture medium. Cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion analysis.

Animal Use

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

Healthy female C57BL/6 (C57BL16J) mice matched for weight and age were obtained from CHARLES RIVER (L'Arbresles) for LL/2 (LLC1) and Hepa1-6 model experiments.

Animals were maintained in SPF health conditions according to FELASA guidelines, and animal housing and experimental procedures were followed according to French and European regulations and the NRC Guide for the Care and Use of Laboratory Animals [50,51]. Animals were maintained in a housing room under controlled environmental conditions: temperature: 22 ± 2°C, humidity 55 ± 10%, photoperiod (12 h light/12 h dark), HEPA-filtered air, 15 air changes per hour, no recirculation. Animal enclosures were sterile and adequately spaced with bedding material, food, and water. Environmental and social enrichment (housing groups) were as follows: 900 ^cm2 cages in ventilated racks (Art. No.: green, Tecniplast), Epicea bedding (SAFE), 10 kGy irradiated diet (A04-10, SAFE), refined diet for immunocompetent rodents - R/MH Extrudate, and water from water bottles.

Experimental design and treatment

Antitumor activity, EMT6 model

Treatment time course - the first dose was considered as day 0. On day 0, non-transplanted mice were randomized to 9/8 groups according to their individual body weight using Vivo software (Biosystemes, Couternon, France). On day 0, mice received vehicle (culture medium) or bacterial strain. On day 14, all mice were transplanted with EMT-6 tumor cells as described below. On day 24, mice from the positive control group received anti-CTLA-4 antibody treatment.

The processing timeline is summarized in the following table:

Animal monitoring was performed as described below.

Induction of EMT6 Tumors in Animals - On day 14, tumors were induced by subcutaneous injection of 1×10 ⁶ 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 from the start of dosing.

Antitumor activity, LL/2 (LLC1) model

Treatment Schedule - The first dose was considered to be day 0. On day 0, untransplanted mice were randomized into 7 groups of 9/8 according to their individual body weight using Vivo software (Biosystemes, Couternon, France). On day 0, mice received either vehicle (culture medium) or bacterial strain. On day 14, all mice were transplanted with LL/2 tumor cells as described below. On day 27, mice from the positive control group received anti-CTLA-4 antibody treatment.

The processing timeline is summarized in the following table:

Animal monitoring was performed as described below.

Induction of LL/2 (LLC1) Tumors in Animals - On day 14, tumors were induced by subcutaneous injection of 1 x ¹⁰⁶ LL/2 (LLC1) 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 from the start of dosing.

Antitumor activity, Hepa1-6 model

Treatment Schedule - The first dose was considered to be day 0. On day 0, untransplanted mice were randomized into 7 of 9 groups based on their individual body weight using Vivo software (Biosystemes, Couternon, France). On day 0, mice received either vehicle (culture medium) or bacterial strain. On day 14, all mice were transplanted with Hepa 1-6 tumor cells as described below. On day 16, mice from the positive control group received anti-CTLA-4 antibody treatment.

The processing timeline is summarized in the following table:

Animal monitoring was performed as described below.

Orthotopic Induction of Hepa 1-6 Tumor Cells in Animals by Intrasplenic Injection - On day 14, one million (1×10 ⁶ ) Hepa 1-6 tumor cells in 50 μL of RPMI 1640 medium were implanted into mice by intrasplenic injection. Briefly, a small left flank incision was made below the ribs and the spleen was removed from the abdomen. The spleen was exposed on a sterile gauze pad and the cell suspension was injected with a 27-gauge needle under visual control. Following cell inoculation, the spleen was excised.

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

Assessment of Tumor Burden at Euthanasia - At termination, livers were harvested and weighed.

Animal monitoring

Clinical monitoring - Tumor length and width were measured twice weekly with calipers and tumor volume was estimated by the formula [52]:

Humane endpoints[53]: signs of pain, distress, or distress: painful posture, facial expression, or behavior; tumor exceeding 10% of normal body weight but not exceeding 2000 mm ³ ; tumor interfering with ambulation or nutrition; tumor ulceration or tissue erosion; sustained weight loss of 20% for 3 consecutive days; poor physical condition, emaciated, cachexia, or dehydration; prolonged lack of voluntary response to external stimuli; rapid, labored breathing, anemia, or heavy bleeding; neurological signs: circling, convulsions, or paralysis; a persistent decrease in body temperature; or a distended abdomen.

Anesthesia - Isoflurane gas anesthesia was used for all procedures: surgery or tumor inoculation, intravenous injections, and blood collection. Ketamine and xylazine anesthesia were used for parenchymal procedures.

Analgesia - Carprofen or polymodal carprofen/buprenorphine analgesia regimen adjusted for the severity of the surgical procedure. Non-pharmacological care was provided for all pain procedures. In addition, pharmacological care that did not interfere with the study (subject handling) was provided at the discretion of the attending veterinarian.

Euthanasia - Animals were euthanized by gas anesthetic overdose (isoflurane) followed by cervical displacement or exsanguination.

result

Antitumor activity, EMT6 model

The results are shown in Figure 1. Treatment with the bacterial strain of the present invention significantly reduced tumor volume relative to both negative controls. As would be expected, the positive control also reduced tumor volume.

Antitumor activity, LL/2 (LLC1) model

The results are shown in Figure 2. Treatment with the bacterial strain of the invention resulted in a significant reduction in tumor volume relative to the two negative controls.

Antitumor activity, Hepa1-6 model

The results are shown in Figure 3. The untreated negative control did not perform as expected because the liver weight was lower in this group than in the other group. However, both the vehicle negative control and positive control groups performed as expected because the livers of mice treated with vehicle alone were larger than those of mice treated with anti-CTLA4 antibodies, reflecting the greater tumor burden in the vehicle negative control group. Treatment with the bacterial strain of the present invention significantly reduced liver weight (and therefore tumor burden) relative to mice in the vehicle negative control group.

These data suggest that strain MRX518 may be used to treat or prevent cancer, and in particular to reduce tumor volume in breast, lung, and liver cancers.

Example 2-PCR gene analysis

Pure cultures of the bacterium MRX518 were studied in PCR-based gene analysis. Two experiments were conducted: 1) MRX518 was co-cultured with human colon cells (CaCo2) to investigate the bacterium's effects on the host, and 2) MRX518 was co-cultured on CaCo2 cells stimulated with IL-1 to mimic the effects of the bacterium in an inflammatory environment. The effects in both cases were assessed by gene expression analysis. The results are shown below:

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

Example 3 - Stability Test

The compositions described herein containing at least one bacterial strain described herein are stored in a sealed container at 25° C. or 4° C. and the container is placed in an atmosphere with a relative humidity of 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95%. 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 should remain, as measured by colony forming units determined by a standard protocol.

Example 4 - Cytokine production in immature dendritic cells induced by MRX518 compared to MRX518+LPS

Overview

This study tested the effects of bacterial strain MRX518 alone and in combination with lipopolysaccharide (LPS) on cytokine production in immature dendritic cells.

Monocyte populations were isolated from peripheral blood mononuclear cells (PBMCs). The monocytes were then differentiated into immature dendritic cells. Immature dendritic cells were plated at 200,000 cells/well and incubated with MRX518 at a final concentration of 10 ⁷ /ml, optionally supplemented with LPS at a final concentration of 100 ng/ml. Negative controls included incubation of cells with RPMI medium alone, and positive controls included incubation of cells with LPS at a final concentration of 100 ng/ml. The cells were then analyzed for cytokine content.

result

The results of these experiments can be seen in Figures 4a-d. The addition of MRX518 alone resulted in a significant increase in the levels of the cytokines IL-6 and TNF-α compared to the negative control (Figures 4a and c). The addition of LPS (positive control) resulted in an increase in the levels of IL-6 and TNF-α, but not IL-1β, compared to the negative control (Figure 4b). The combination of MRX518 and LPS resulted in a synergistic increase in the levels of IL-1β produced (Figure 4d).

in conclusion

MRX518 can induce higher production of IL-6 and TNF-α cytokines in immature dendritic cells. The combination of LPS and MRX518 can increase levels of the cytokine IL-1β in immature dendritic cells. These data suggest that MRX518 alone or in combination with LPS can increase the production of the inflammatory cytokines IL-1β, IL-6, and TNF-α, promoting inflammation that can suppress cancer. Treatment with MRX518 alone or in combination can induce cytokines that can limit tumor growth.

Example 5 - Cytokine production in THP-1 cells induced by MRX518 compared to MRX518+LPS

Overview

This study tested the effects of bacterial strain MRX518 alone and in combination with LPS on cytokine production in THP-1 cells, a model cell line of monocytes and macrophages.

THF-1 cells were differentiated to MO medium with 5 ng/mL phorbol-12-myristate-13-acetate (PMA) for 48 hours. These cells were then incubated with MRX518 at a final concentration of 10 ⁸ /mL, with or without the addition of LPS at a final concentration of 100 ng/mL. The bacteria were then washed away and the cells were incubated under normal growth conditions for 24 hours. The cells were then centrifuged at low speed, and the resulting supernatant was analyzed for cytokine content.

result

The results of these experiments can be seen in Figures 5a-c. Addition of MRX518 in the absence of LPS resulted in increased cytokine levels of IL-1β, IL-6, and TNF-α compared to the no bacteria and bacterial sediment controls. Addition of LPS and MRX518 resulted in a synergistic increase in cytokine production.

in conclusion

MRX518 can induce cytokine production in THP-1 cells, which is synergistically increased with the addition of LPS. These data suggest that MRX518 alone or in combination with LPS can increase the production of inflammatory cytokines IL-1β, IL-6, and TNF-α, a boost that may suppress cancer inflammation. Treatment with MRX518 alone or in combination can induce cytokines that may limit tumor growth.

sequence

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

SEQ ID NO: 2 (consensus 16S rRNA sequence of Enterococcus gallinarum strain MRX518)

SEQ ID NO: 3 (strain MRX518 chromosomal sequence) - see electronic sequence listing.

SEQ ID NO: 4 (strain MRX518 plasmid sequence) - see electronic sequence listing.

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Claims (20)

1. Use of a composition containing a bacterial strain of the species Enterococcus gallinarum in the preparation of a medicament for the treatment or prevention of solid tumors. 2. The use as claimed in claim 1, wherein the composition does not contain bacteria from any other species, or contains only a minimum amount or biologically incoherent amount of bacteria from another species. 3. The use as described in claim 1 or 2, wherein the solid tumor is lung cancer, breast cancer, liver cancer, or colon cancer. 4. The use as described in claim 1 or 2, wherein the bacterial strain has a 16S rRNA sequence represented by SEQ ID NO:2. 5. The use as described in claim 1 or 2, wherein the drug is for oral administration, wherein the drug comprises one or more pharmaceutically acceptable excipients or carriers, and/or wherein the bacterial strain is lyophilized. 6. The use as described in claim 1 or 2, wherein the bacterial strain is capable of partially or completely colonizing the intestine. 7. The use as described in claim 1 or 2, wherein the composition comprises a single strain of Enterococcus quati. 8. The use as described in claim 1 or 2, wherein the composition comprises a strain of Enterococcus quatilis as part of the microbial flora. 9. The use as described in claim 1 or 2, wherein the bacterial strain of the Enterococcus quatilis species is deposited with accession number NCIMB42488. 10. Use of a composition containing a bacterial strain of the species Enterococcus quatilis in the preparation of a vaccine composition for the treatment or prevention of solid tumors. 11. The use as claimed in claim 10, wherein the composition contains no bacteria of any other species, or contains only a minimum amount or biologically incoherent amount of bacteria of another species. 12. The use as described in claim 10 or 11, wherein the solid tumor is lung cancer, breast cancer, liver cancer, or colon cancer. 13. The use as claimed in claim 10 or 11, wherein the bacterial strain has a 16S rRNA sequence represented by SEQ ID NO:2. 14. The use as claimed in claim 10 or 11, wherein the vaccine composition is for oral administration, wherein the vaccine composition comprises one or more pharmaceutically acceptable excipients or carriers, and/or wherein the bacterial strain is lyophilized. 15. The use as described in claim 10 or 11, wherein the bacterial strain is capable of partially or completely colonizing the intestine. 16. The use as described in claim 10 or 11, wherein the composition comprises a single strain of Enterococcus quati. 17. The use as claimed in claim 10 or 11, wherein the composition comprises a strain of Enterococcus quatilis as part of the microbial flora. 18. The use as described in claim 10 or 11, wherein the bacterial strain of the Enterococcus quatilis species is deposited with accession number NCIMB 42488. 19. A cell or biological pure culture of a strain of Enterococcus quati deposited under accession number NCIMB 42488. 20. A composition comprising the cells of claim 19, optionally comprising a pharmaceutically acceptable carrier or excipient.