Combination therapy for treating or preventing cancer
阅读说明：本技术 用于治疗或预防癌症的组合疗法 (Combination therapy for treating or preventing cancer ) 是由 亚历山大·史蒂文森 于 2019-01-18 设计创作，主要内容包括：本发明提供了一种包含细菌菌株的组合疗法,用于治疗或预防癌症。(The present invention provides a combination therapy comprising a bacterial strain for the treatment or prevention of cancer.)
1. A therapeutic combination for use in a method of treating or preventing cancer in a subject, wherein the therapeutic combination comprises:
(a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and
(b) pembrolizumab.
2. The therapeutic combination according to claim 1, wherein the composition is free of bacteria from any other species or comprises only a minor or biologically irrelevant amount of bacteria from another species.
3. The therapeutic combination according to any one of the preceding claims, wherein the therapeutic combination is for use in a method of treating or preventing lung cancer, breast cancer, kidney cancer, liver cancer, lymphoma, hepatoma, neuroendocrine cancer or colon cancer.
4. The therapeutic combination according to any one of the preceding claims, wherein the therapeutic combination is for use in a method of reducing tumor size, slowing tumor growth, preventing metastasis or preventing angiogenesis.
5. The therapeutic combination according to any one of the preceding claims, wherein the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO 2.
6. The therapeutic combination according to any one of the preceding claims, wherein the composition is for oral administration and/or wherein pembrolizumab is for intravenous administration.
7. The therapeutic combination of any one of the preceding claims, wherein the composition comprises one or more pharmaceutically acceptable excipients or carriers.
8. The therapeutic combination of any one of the preceding claims, wherein the bacterial strain is lyophilized.
9. The therapeutic combination of any one of the preceding claims, wherein the bacterial strain is capable of partial or complete colonization of the intestine.
10. The therapeutic combination according to any one of the preceding claims, wherein the composition comprises a single strain of enterococcus gallinarum.
11. The therapeutic combination according to any one of claims 1 to 9, wherein the composition comprises a bacterial strain of enterococcus gallinarum as part of a microbial consortium.
12. The therapeutic combination according to any one of the preceding claims, wherein the composition comprises the enterococcus gallinarum strain deposited under accession number NCIMB 42488.
13. The therapeutic combination of any one of the preceding claims, wherein the composition is comprised within a food product or a vaccine composition.
14. The therapeutic combination of any one of the preceding claims, wherein the composition is administered to the subject prior to the first administration of pembrolizumab to the subject.
15. The therapeutic combination of claim 14, wherein the composition is administered to the subject at least one, two, three or four weeks prior to the first pembrolizumab administration.
16. The therapeutic combination of any one of the preceding claims, wherein the composition is administered to the subject prior to and/or at least partially simultaneously with the first administration of pembrolizumab to the subject.
17. The therapeutic combination according to any one of the preceding claims, wherein the bacterial strain of the species enterococcus gallinarum and pembrolizumab are in separate compositions.
18. The therapeutic combination of any one of the preceding claims, wherein the subject is non-responsive to prior treatment with pembrolizumab alone.
19. A first composition comprising a bacterial strain of the species enterococcus gallinarum for use in combination with a second composition comprising pembrolizumab, for use in a method of treating or preventing cancer, optionally wherein the first composition is administered prior to and/or simultaneously with the first administration of the second composition.
20. A first composition comprising pembrolizumab for use in combination with a second composition comprising a bacterial strain of the species enterococcus gallinarum, in a method of treating or preventing cancer, optionally wherein the first composition is administered simultaneously with the administration of the second composition.
21. A composition comprising pembrolizumab for use in a method of treating or preventing cancer in a subject who has previously received administration of a composition comprising a bacterial strain of the species enterococcus gallinarum, preferably the strain deposited under accession number NCIMB 42488.
22. A composition comprising a bacterial strain of the species enterococcus gallinarum, preferably the strain deposited under accession number NCIMB42488, for use in a method of treating or preventing cancer in a subject diagnosed as in need of treatment with pembrolizumab.
23. A method of treating or preventing cancer in a subject in need thereof, comprising:
(a) administering to the subject a composition comprising a bacterial strain of the species enterococcus gallinarum; and
(b) administering pembrolizumab to said subject.
24. A kit, comprising:
(a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and
(b) a composition comprising pembrolizumab.
Technical Field
The present invention relates to the field of combination therapies for treating or preventing cancer: a combination of a composition comprising a bacterial strain and pembrolizumab (pembrolizumab) for the treatment or prevention of cancer.
Background
It is believed that the human intestine is sterile in utero, but is exposed to a wide variety of maternal and environmental microorganisms immediately after birth. Thereafter, a dynamic phase of microbial colonization and succession occurs, which is influenced by factors such as mode of labor, environment, diet, and host genotype, all of which affect the composition of the gut microbiota, particularly early in life. Subsequently, the microbiota stabilizes and becomes adult-like [1 ]. The human intestinal microbiota contains more than 500-1000 different evolutions, which essentially belong to two main bacterial classes, Bacteroidetes and Firmicutes [2 ]. The successful symbiotic relationship resulting from bacterial colonization of the human intestine has produced a wide variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activity of the colonizing intestines ensures degradation of otherwise indigestible dietary components with concomitant release of by-products, thereby providing an important source of nutrition for the host. Similarly, the immunological importance of the gut microbiota is well recognized and exemplified in sterile animals with compromised immune systems that functionally recover following introduction of commensal bacteria [3-5 ].
Significant changes in microbiota composition have been documented in gastrointestinal disorders such as Inflammatory Bowel Disease (IBD). For example, in IBD patients, the levels of Clostridium (Clostridium) colonizing XIVa bacteria are reduced, while the number of escherichia coli (e.coli) increases, suggesting a shift in the balance of intestinal symbiont (symbiot) and pathogenic (pathobiot) [6-9 ]. Interestingly, this microbial dysbiosis is also associated with imbalances in T effector cell populations.
In view of the potential positive effects that certain bacterial strains may have on the intestine of animals, various strains have been proposed for the treatment of various diseases (see, e.g., [10-13 ]). Certain strains, including most strains of Lactobacillus and Bifidobacterium, have also been proposed for the treatment of various inflammatory and autoimmune diseases not directly associated with the intestine (for review see [14] and [15 ]). However, the relationship between different diseases and different bacterial strains, as well as the exact effect of a particular bacterial strain on the intestine and at the systemic level and on any particular type of disease, has not been fully characterized. For example, certain Enterococcus (Enterococcus) species are involved in the development of cancer [16 ]. In contrast, strains of the species enterococcus gallinarum (enterococcus gallinarum) have also been disclosed for the treatment and prevention of cancer [54 ].
Due to the various properties of cancer, different treatment modalities have been developed in order to treat different patient groups. One therapeutic modality that has proven effective is the use of Immune Checkpoint Inhibitors (ICI). ICI is a compound that inhibits the ability of cancer cells to prevent host immune cells from attacking cancer cells. ICI may be, for example, a therapeutic antibody that has been developed against the interaction between the transmembrane receptor programmed
While treatment of cancer patients with ICI when effective can produce a long-lasting and significant clinical effect, a significant percentage of patients still respond to ICI therapy, either only partially or not. Therefore, there is a need in the art for new and improved therapeutic modalities for the prevention and treatment of cancer, and in particular for therapeutic modalities that can improve the therapeutic effect of pembrolizumab.
Disclosure of Invention
The present invention relates to novel combination therapies for the treatment and prevention of cancer. In particular, the present invention relates to improved therapies wherein sequential and/or partial simultaneous administration of a bacterial strain of the species enterococcus gallinarum and pembrolizumab results in a more effective cancer treatment than treatment with the bacterial strain or pembrolizumab alone.
Compositions comprising bacterial strains of the species enterococcus gallinarum are generally effective in therapy, and are particularly effective in the treatment or prevention of cancer, as shown below and in [54 ]. The present invention is further based in part on the unexpected effects achieved after administration of both pembrolizumab and a composition comprising a bacterial strain of the species enterococcus gallinarum. As used herein, the terms "combination of the invention", "therapeutic combination of the invention" and "therapeutic combination" are used interchangeably and refer to the following therapeutic combinations: (a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and (b) pembrolizumab. It is to be understood that the term "combination" in the context of a therapeutic combination does not refer to the components (a) and (b) of the combination which are necessarily administered in the same composition and/or simultaneously. According to a preferred embodiment, the therapeutic combinations of (a) and (b) are in separate compositions. According to some embodiments, provided herein is a combination of the invention for use in a method of treating or preventing cancer in a subject. According to some embodiments, provided herein is a method for treating or preventing cancer in a subject comprising administering to the subject a therapeutic combination of the invention.
According to some embodiments, administration of the bacterial composition in the context of a therapeutic combination enables treatment of cancer patients who do not respond or show an inadequate response to treatment with an immune checkpoint inhibitor administered without the bacterial composition. According to some embodiments, a patient that is non-responsive or partially responsive to ICI therapy may be initially treated for ICI (i.e., it has not previously received ICI therapy) or it may become a non-responder or a partial responder after a previously successful administration of ICI.
Without wishing to be bound by theory or mechanism, this effect may be via modulation of mediators that improve the efficiency of pembrolizumab, such as via increasing CD8 infiltrating the tumor+T cells or increase tumor infiltrating CD8+Ratio of T cells to FoxP3+ cells.
According to one aspect, provided herein is a therapeutic combination for use in a method of treating or preventing cancer in a subject, wherein the therapeutic combination comprises:
(a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and
(b) pembrolizumab.
According to some embodiments, provided herein is a composition comprising a bacterial strain of the species enterococcus gallinarum for use in a method of treating or preventing cancer in a subject, wherein the composition is used in combination with pembrolizumab.
According to some embodiments, provided herein is a first composition comprising a bacterial strain of the species enterococcus gallinarum for use in a method of treating or preventing cancer in a subject in combination with a second composition comprising pembrolizumab, optionally wherein the first composition is administered prior to and/or concurrently with the first administration of the second composition, optionally wherein the subject is non-responsive to prior treatment with an immune checkpoint inhibitor alone.
According to another aspect, provided herein is a method of treating or preventing cancer in a subject in need thereof (also referred to herein as "the method of the invention"), the method comprising: (a) administering to the subject a composition comprising a bacterial strain of the species enterococcus gallinarum; and (b) administering pembrolizumab to the subject.
According to another aspect, provided herein is a kit comprising: (a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and (b) a composition comprising pembrolizumab.
According to some embodiments, the cancer is selected from the group consisting of: breast cancer, lung cancer, colon cancer, kidney cancer, liver cancer, lymphoma (such as non-hodgkin's lymphoma), hepatoma and neuroendocrine cancer. According to some embodiments, the therapeutic combination is for use in a method of treating or preventing lung cancer, breast cancer, kidney cancer, liver cancer, lymphoma, hepatoma, neuroendocrine cancer or colon cancer. According to some embodiments, the cancer is selected from the group consisting of: melanoma, non-small cell lung cancer, bladder cancer, and head and neck cancer. In certain embodiments, the therapeutic combinations or methods of the invention are used to reduce tumor size or prevent tumor growth in the treatment of cancer. According to some embodiments, the therapeutic combination or method of the invention is for at least one of: reducing tumor size, slowing tumor growth, preventing metastasis, or preventing angiogenesis.
According to some embodiments, the terms "composition", "bacterial composition" and "composition of the invention" are used interchangeably and refer to a composition comprising a bacterial strain of the species enterococcus gallinarum, included in the therapeutic combination of the invention. According to some embodiments, the composition comprising a bacterial strain of the species enterococcus gallinarum does not comprise bacteria from any other species or comprises only a minor or biologically irrelevant amount of bacteria from another species. According to some embodiments, closely related strains of enterococcus gallinarum may also be used as part of the therapeutic combination, such as 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
Thus, a therapeutic combination of the invention may comprise 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 (optionally with SEQ ID NO:2), for use in a method of treating or preventing cancer. Enterococcus gallinarum in some embodiments, the bacterial strain in the composition is not of 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 invention is effective for the treatment of cancer, particularly when administered as part of the therapeutic combination of the invention. Furthermore, oral administration is convenient for patients and practitioners and allows delivery to and/or partial or complete colonization of the intestine. According to some embodiments, pembrolizumab used as part of a therapeutic combination of the present invention is administered intravenously. According to some embodiments, the therapeutically combined bacterial composition and pembrolizumab are each present in a separate composition, each of which may comprise a carrier and/or excipient suitable for its mode of administration. In certain embodiments, the compositions of the present invention comprise one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, pembrolizumab is in a composition comprising one or more pharmaceutically acceptable excipients or carriers.
In certain embodiments, the bacterial compositions of the present invention comprise a bacterial strain that has been lyophilized. Freeze-drying is an efficient and suitable technique for preparing stable compositions that allow for the delivery of bacteria. According to some embodiments, the bacterial strain in the composition is capable of partial or complete colonization of the intestine.
In certain embodiments, the bacterial composition is contained within a food product. In certain embodiments, the bacterial composition is contained within a vaccine.
According to some embodiments, the bacterial composition comprises a single strain of enterococcus gallinarum. According to some embodiments, the bacterial composition comprises an enterococcus gallinarum bacterial strain as part of a microbial consortium (consortium). Preferably, the bacterial composition comprises the enterococcus gallinarum strain deposited under accession number NCIMB 42488.
According to some embodiments of the methods of the invention, the bacterial composition is administered to the subject prior to the first administration of pembrolizumab to the subject. According to some embodiments of the methods of the invention, the bacterial composition is administered to the subject at least one, two, three or four weeks prior to the first administration of pembrolizumab. It is to be understood that in the context of the methods of the present invention, the first administration of pembrolizumab refers to the first administration as part of the therapeutic combination of the present invention. Before administering the therapeutic combination of the present invention, pembrolizumab may have been administered to the subject without administering the bacterial composition of the present invention during/before pembrolizumab administration. According to some embodiments, at least one, two, three, or four weeks elapse between administration of the therapeutic combination of the present invention and prior administration of pembrolizumab alone or the bacterial composition alone.
According to some embodiments of the methods of the invention, the bacterial composition is administered to the subject at least partially concurrently with the administration of pembrolizumab to the subject. In the context of the time of administration of the bacterial composition and pembrolizumab, at least partially simultaneous administration means administration that may be completely (e.g., both components are administered over the course of 12 months) or partially (e.g., one component is administered over the course of 12 months and a second component is administered over the course of 8 months, which may completely or partially overlap with the 12 month period). It will be understood that the simultaneous administration of the two components does not mean that the two components must be administered using the same dosing regimen. According to some embodiments of the methods of the invention, the bacterial composition is administered to the subject prior to and/or at least partially simultaneously with the first administration of pembrolizumab to the subject. According to certain embodiments, the bacterial composition is administered to the subject at least one, two, three or four weeks prior to the first administration of pembrolizumab, followed by at least partial concurrent administration of the bacterial composition with pembrolizumab for at least two, four or six weeks.
According to some embodiments, the bacterial strain of the species enterococcus gallinarum and pembrolizumab are in separate compositions, preferably wherein the bacterial composition is formulated for oral administration and pembrolizumab is in a formulation formulated for intravenous administration.
According to some embodiments, the therapeutic combination of the invention is for use in the treatment or prevention of cancer in a subject that is non-responsive to prior treatment with an immune checkpoint inhibitor alone. As used herein, a subject that is non-responsive to treatment with an immune checkpoint inhibitor refers to a subject that is non-responsive according to recist (Response Evaluation criterion In Solid tumors) guidelines or according to irRECIST (animal-related Evaluation criterion In Solid tumors) guidelines.
According to some embodiments, the therapeutic combination of the present invention is for treating or preventing cancer in a subject, wherein pembrolizumab or the bacterial composition alone is unable to provide an effective cancer treatment or prevention in a subject. According to some embodiments, the effective treatment of cancer in the subject comprises at least one of: reducing tumor size, slowing tumor growth, and/or preventing metastasis to an extent that will result in complete or partial remission of the cancer in the subject.
According to some embodiments, the therapeutic combination of the present invention is capable of reducing tumor size and/or slowing tumor growth and/or preventing metastasis and/or preventing angiogenesis to a greater extent than pembrolizumab or bacterial composition alone.
According to some embodiments, the therapeutic combination of the present invention is for treating cancer in a subject such that the cancer in the subject is completely alleviated, preferably within a shorter period of time than achieved with treatment with pembrolizumab or bacterial composition alone.
The present invention also provides a composition comprising pembrolizumab for use in a method of treating or preventing cancer in a subject who has previously received administration of a composition comprising a bacterial strain of the species enterococcus gallinarum, preferably the strain deposited under accession number NCIMB 42488.
The invention also provides a composition comprising a bacterial strain of the species enterococcus gallinarum, preferably the strain deposited under accession number NCIMB42488, for use in a method of treating or preventing cancer in a subject diagnosed as in need of treatment with pembrolizumab.
Drawings
FIG. 1A: mouse model of breast cancer-tumor volume.
FIG. 1B: the upper diagram: necrotic regions in EMT6 tumors (untreated n-6, vehicle n-6, MRx0518 n-8). The following figures: percentage of dividing cells in EMT6 tumors. P is 0.019 (untreated n is 4, total number of cells counted is 37201, vector n is 6, total number of cells counted is 64297, MRx0518 n is 6, total number of cells counted is 33539).
FIG. 1C: mouse model of breast cancer-immune cell infiltration. Scatter plots show the cell counts of different immune markers from individual animals of each treatment group.
FIG. 1D: mouse model of breast cancer-cytokine production in tumor lysates. Bars represent the average of total protein from each treatment group in pg/mL. P <0.05, between groups, using one-way ANOVA followed by Dunnett's multiple comparison test.
FIG. 1E: mouse model of breast cancer-cytokine production in plasma. Bars represent mean values pg/mL (+/-SEM) from each treatment group.
FIG. 1F: representative images of ileal cryosections from vehicle, MRx0518 and CTLA-4 treated mice, immunolabeled with an antibody against CD8 a (lower panel) and stained with DAPI contrast (upper panel).
FIG. 1G: quantification of animals with more than 3 CD8 α + cells per field of view a plot of a subset of the study taken from ileal crypt tract of mice treated with vehicle, MRx0518 or CTLA-4.
FIG. 2: mouse model of lung cancer-tumor volume.
FIG. 3A: mouse model of liver cancer-liver weight.
FIG. 3B: mouse model of renal cancer-tumor volume.
FIG. 4A: cytokine levels (pg/ml) in immature dendritic cells (without bacteria).
FIG. 4B: cytokine levels (pg/ml) in immature dendritic cells after addition of LPS.
FIG. 4C: cytokine levels (pg/ml) in immature dendritic cells after addition of MRX 518.
FIG. 4D: cytokine levels (pg/ml) in immature dendritic cells after addition of MRX518 and LPS.
FIG. 5A: cytokine levels in THP-1 cells (no bacteria).
FIG. 5B: cytokine levels in THP-1 cells after addition of bacterial deposits.
FIG. 5C: cytokine levels in THP-1 cells after addition of MRX518 alone or in combination with LPS.
FIG. 6: bar graphs depicting the percentage of proliferating CD8+ cells after various treatments (NCD-no cell division, 1 RCD-one cell division, 2 RCD-two cell divisions, 3 RCD-three cell divisions, 4 RCD-four cell divisions).
FIG. 7A: schematic representation of the different groups of treatment regimens used in example 6 described below.
FIG. 7B: mean tumor volume in mice bearing tumors formed by EMT-6 cells. Mice were untreated or treated with: YCFA Vehicle (Vehicle), MRx518 bacteria in YCFA medium (MRx518), anti-PD 1 antibody and YCFA medium (anti-PD 1), anti-CTLA-4 antibody and YCFA medium (anti-CTLA-4), a combination of MRx518 and anti-PD 1 antibody or a combination of MRx518 and anti-CTLA-4 antibody.
Detailed Description
Bacterial strains
The composition of the invention comprises a bacterial strain of the species enterococcus gallinarum. These examples demonstrate that therapeutic combinations comprising bacteria of this species are useful for treating or preventing cancer.
According to some embodiments, provided herein is a therapeutic combination for use in a method of treating or preventing cancer in a subject, wherein the therapeutic combination comprises:
(a) a composition comprising a bacterial strain of the species enterococcus gallinarum; and
(b) pembrolizumab
According to some embodiments, compositions 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 may be used in the therapeutic combinations and methods of the present invention. According to certain embodiments, the present invention also provides a composition comprising a bacterial strain having a 16s rRNA sequence that is at least 95% identical to
In certain embodiments, the compositions of the invention comprise a bacterial strain having a 16 srna sequence that is at least 95% identical to SEQ ID No. 2, e.g., it is enterococcus gallinarum, and does not contain any other bacteria. In certain embodiments, the compositions of the invention comprise 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., it is enterococcus gallinarum and is free of any other bacterial strain or species.
Enterococcus gallinarum forms coccal cells, mostly in pairs or short chains. It is immobile and the colonies on blood agar or nutrient agar are circular and smooth. Enterococcus gallinarum reacted with the Lancefield group D antiserum. The model strain of enterococcus gallinarum is F87/276 ═ PB21 ═ ATCC 49573 ═ CCUG 18658 ═ CIP 103013 ═ JCM 8728 ═ LMG13129 ═ NBRC 100675 ═ NCIMB 702313 (old name NCDO 2313) ═ NCTC 12359[17 ]. GenBank accession number AF039900 (disclosed herein as SEQ ID NO:1) of the 16S rRNA gene sequence of enterococcus gallinarum. Exemplary enterococcus gallinarum strains are described in [17 ].
Enterococcus gallinarum bacteria deposited under accession number NCIMB42488 were tested in the examples and are also referred to herein as strain MRX 518. MRX518 and MRX0518 may be used interchangeably. The 16S rRNA sequence of the tested MRX518 strain is provided in
The genome of strain MRX518 contains chromosomes and plasmids. The chromosomal sequence of strain MRX518 is provided in SEQ ID NO. 3 of WO 2017/085520. The plasmid sequence of strain MRX518 is provided in SEQ ID NO. 4 of WO 2017/085520. These sequences were generated using the PacBio RS II platform.
Bacterial strains closely related to the strains tested in the examples are also expected to be effective for cancer treatment or prevention in the therapeutic combination of the invention. In certain embodiments, the bacterial strain used in the therapeutic combination of the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to the 16s rRNA sequence of a bacterial strain of enterococcus gallinarum. Preferably, the bacterial strains used in the therapeutic combination of the invention have a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to
Bacterial strains that are biotypes of the bacteria deposited under accession number 42488 are also expected to be effective for treating or preventing cancer in the context of the therapeutic combination of the invention. Biotypes are closely related strains with identical or very similar physiological and biochemical characteristics.
Strains that are biotypes of the bacteria deposited under accession number NCIMB42488 and that are suitable for use in the therapeutic combinations of the invention may be identified by sequencing other nucleotide sequences for the bacteria deposited under accession number NCIMB 42488. For example, a strain of a biotype that can be sequenced for substantially the entire genome and used in a therapeutic combination of the 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, a 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 use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC, (GTG)5Or REP or [18]. The biotype strain may have a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42488. In some embodiments, the biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the corresponding sequence of strain MRX518 deposited under accession number NCIMB42488 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 with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRX518 deposited under accession number NCIMB42488 and has the 16S rRNA sequence of SEQ id No. 2.
In certain embodiments, the bacterial strain used in the therapeutic combination of the present invention has a chromosome that shares sequence identity with SEQ ID No. 3 of WO 2017/085520. In a preferred embodiment, the bacterial strain used in the therapeutic combination of the present invention has a chromosome that shares at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with SEQ ID No. 3 of WO2017/085520 on at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID No. 3 of WO 2017/085520. For example, the bacterial strains used in the therapeutic combinations of the present invention may have the following chromosomes: has at least 90% sequence identity to SEQ ID NO:3 of WO2017/085520 at 70% of SEQ ID NO:3 of WO2017/085520, or has at least 90% sequence identity to SEQ ID NO:3 of WO2017/085520 at 80% of SEQ ID NO:3 of WO2017/085520, or has at least 90% sequence identity to SEQ ID NO:3 of WO2017/085520 at 90% of SEQ ID NO:3 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:3 of WO2017/085520 at 100% of SEQ ID NO:3 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:3 of WO2017/085520 at 70% of SEQ ID NO:3 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:3 of WO2017/085520 at 80% of WO 7/085520, Or at least 95% sequence identity with SEQ ID NO:3 of WO2017/085520 at 90% of SEQ ID NO:3 of WO2017/085520, or at least 95% sequence identity with SEQ ID NO:3 of WO2017/085520 at 100% of SEQ ID NO:3 of WO2017/085520, or at least 98% sequence identity with SEQ ID NO:3 of WO2017/085520 at 70% of SEQ ID NO:3 of WO2017/085520, or at least 98% sequence identity with SEQ ID NO:3 of WO2017/085520 at 80% of SEQ ID NO:3 of WO2017/085520, or at least 98% sequence identity with SEQ ID NO: 2013 of WO2017/085520 at 90% of SEQ ID NO:3 of WO2017/085520, or at least 98% sequence identity with SEQ ID NO:3 of WO2017/085520 at 95% of WO 7/085520, Or at least 98% sequence identity with SEQ ID No. 3 of WO2017/085520 at 100% of SEQ ID No. 3 of WO2017/085520, or at least 99.5% sequence identity with SEQ ID No. 3 of WO2017/085520 at 90% of SEQ ID No. 3 of WO2017/085520, or at least 99.5% sequence identity with SEQ ID No. 3 of WO2017/085520 at 95% of SEQ ID No. 3 of WO2017/085520, or at least 99.5% sequence identity with SEQ ID No. 3 of WO2017/085520 at 98% of SEQ ID No. 3 of WO2017/085520, or at least 99.5% sequence identity with SEQ ID No. 3 of WO2017/085520 at 100% of SEQ ID No. 3 of WO 2017/085520.
In certain embodiments, the bacterial strain used in the therapeutic combination of the present invention has a plasmid possessing sequence identity with SEQ ID No. 4 of WO 2017/085520. In a preferred embodiment, the bacterial strain used in the therapeutic combination of the present invention has a plasmid that shares at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with SEQ ID No. 4 of WO2017/085520 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 of WO 2017/085520. For example, the bacterial strains used in the therapeutic combinations of the present invention may have the following plasmids: has at least 90% sequence identity to SEQ ID NO:4 of WO2017/085520 at 70% of SEQ ID NO:4 of WO2017/085520, or has at least 90% sequence identity to SEQ ID NO:4 of WO2017/085520 at 80% of SEQ ID NO:4 of WO2017/085520, or has at least 90% sequence identity to SEQ ID NO:4 of WO2017/085520 at 90% of SEQ ID NO:4 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:4 of WO2017/085520 at 100% of SEQ ID NO:4 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:4 of WO2017/085520 at 70% of SEQ ID NO:4 of WO2017/085520, or has at least 95% sequence identity to SEQ ID NO:4 of WO2017/085520 at 80% of WO 7/085520, Or with SEQ ID NO of WO 2017/085520: 4 in 90% of the SEQ ID NO of WO 2017/085520: 4, or at least 95% sequence identity to SEQ ID NO of WO 2017/085520: 4 in 100% of the SEQ ID NO of WO 2017/085520: 4, or at least 95% sequence identity to SEQ ID NO of WO 2017/085520: 4 in 70% of the sequence of SEQ ID NO of WO 2017/085520: 4, or a sequence having at least 98% sequence identity to SEQ ID NO of WO 2017/085520: 4 in 80% of the SEQ ID NO of WO 2017/085520: 4, or at least 98% sequence identity to SEQ id no:4 in 90% of the SEQ ID NO of WO 2017/085520: 4, or a sequence having at least 98% sequence identity to SEQ ID NO of WO 2017/085520: 4 in 100% of the SEQ ID NO of WO 2017/085520: 4 have at least 98% sequence identity.
In certain embodiments, the bacterial strains used in the therapeutic combinations of the present invention may have a chromosome possessing sequence identity with SEQ ID No. 3 of WO2017/085520 and a plasmid possessing sequence identity with SEQ ID No. 4 of WO 2017/085520.
In certain embodiments, the bacterial strain used in the therapeutic combination of the invention has a chromosome possessing sequence identity to SEQ ID NO:3 of WO2017/085520 (e.g., as described above), and a 16S rRNA sequence possessing sequence identity to either of
In certain embodiments, the bacterial strain used in the therapeutic combination of the present invention has a chromosome possessing sequence identity to SEQ ID NO:3 of WO2017/085520 (e.g., as described above), and optionally comprises a plasmid possessing sequence identity to SEQ ID NO:4 of WO2017/085520 (as described above), and is effective for treating or preventing cancer.
In certain embodiments, the bacterial strains used in the therapeutic combinations of the present invention have chromosomes possessing sequence identity to SEQ ID NO:3 of WO2017/085520 (e.g., as described above), and 16S rRNA sequences possessing sequence identity to either of
In certain embodiments, the bacterial strains used in the therapeutic combinations of the present invention have a 16S rRNA sequence (e.g., which comprises the 16S rRNA sequence of SEQ ID NO:2) that is at least 99%, 99.5%, or 99.9% identical to the 16S rRNA sequence represented by SEQ ID NO:2 and a chromosome that shares at least 95% sequence identity with SEQ ID NO:3 of WO2017/085520 on at least 90% of SEQ ID NO:3 of WO2017/085520, and optionally comprise a plasmid that shares sequence identity with SEQ ID NO:4 of WO2017/085520 (as described above), and are effective for treating or preventing cancer.
In certain embodiments, the bacterial strain used in the therapeutic combination of the invention has a 16S rRNA sequence (e.g., which comprises the 16S rRNA sequence of SEQ ID NO:2) that is at least 99%, 99.5%, or 99.9% identical to the 16S rRNA sequence represented by SEQ ID NO:2 and a chromosome that shares at least 98% sequence identity (e.g., at least 99% or at least 99.5%) with SEQ ID NO:3 of WO2017/085520 on at least 98% (e.g., at least 99% or at least 99.5%) of SEQ ID NO:3 of WO2017/085520, and optionally comprises a plasmid that shares sequence identity with SEQ ID NO:4 of WO2017/085520 (as described above), and is effective for treating or preventing cancer.
In certain embodiments, the bacterial strain used in the therapeutic combination of the 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., which comprises the 16S rRNA sequence of SEQ ID NO:2) and a chromosome that shares at least 98% sequence identity (e.g., at least 99% or at least 99.5% sequence identity) with SEQ ID NO:3 of WO2017/085520 on at least 98% (e.g., at least 99% or at least 99.5%) of SEQ ID NO:3 of WO2017/085520, and optionally comprises a plasmid that shares sequence identity with SEQ ID NO:4 of WO2017/085520 (as described above), and is effective for treating or preventing cancer.
Alternatively, strains that are biotypes of the bacteria deposited under accession number NCIMB42488 and that are suitable for use in the therapeutic combinations of the invention may be identified by using the accession number NCIMB42488 deposit and restriction fragment analysis and/or PCR analysis, for example by using Fluorescent Amplified Fragment Length Polymorphism (FAFLP) and repetitive DNA element (rep) -PCR fingerprinting, or protein mass spectrometry, or partial 16S or 23S rDNA sequencing. In preferred embodiments, such techniques may be used to identify other enterococcus gallinarum strains.
In certain embodiments, a strain that is a biotype of the bacteria deposited under accession number NCIMB42488 and that is suitable for use in the therapeutic combinations of the invention is a strain that provides the same pattern as the bacteria deposited under accession number NCIMB42488, when analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA), for example when using Sau3AI restriction enzyme (see e.g., [19] for exemplary methods and guidance). Alternatively, the biotype strain is identified as a strain having the same carbohydrate fermentation pattern as the bacterium deposited under accession number NCIMB 42488. In some embodiments, the carbohydrate fermentation mode is determined using API50CHL plates (biomeririeux). In some embodiments, the bacterial strains used in the therapeutic combinations of the present invention are:
(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: l-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and potassium gluconate; and/or
(ii) As an intermediate in the fermentation of at least one (e.g., at least 2, 3, 4, or all) of: d-mannitol, methyl-alpha D-glucopyranoside, D-lactose, starch and L-trehalose;
preferably as determined by API50CHL analysis (preferably using API50CHL plates from biomerieux).
Other enterococcus gallinarum strains suitable for use in the compositions and methods of the present invention, such as the biotype of the bacterium deposited under accession number NCIMB42488, may be identified using any suitable method or strategy, including the assays described in the examples. For example, strains for use in the therapeutic combination of the invention can be identified by: bacteria were cultured in anaerobic YCFA and/or administered to a mouse model of collagen II-induced arthritis, followed by assessment of cytokine levels. In particular, bacterial strains having a similar growth pattern, metabolic type and/or surface antigen as the bacteria deposited under accession number NCIMB42488 may be suitable for use in the therapeutic combination of the invention. Useful strains will have comparable immunomodulatory activity to the NCIMB42488 strain. In particular, the biotype strain will elicit effects on cancer disease models comparable to those shown in the examples, which can be identified by using the culture and administration protocols described in the examples. According to some embodiments, the biotype strain useful in the therapeutic combination of the invention is a strain capable of eliciting the same effect on cancer disease models as shown in the examples when administered in the therapeutic combination or method of the invention.
In some embodiments, the bacterial strains used in the therapeutic combinations of the present invention are:
(i) positive for at least one (e.g., at least 2, 3, 4, 5, 6, 7, or all) of: mannose fermentation, glutamate decarboxylase, arginine arylamidase, phenylalanine arylamidase, pyroglutamate arylamidase, tyrosine arylamidase, histidine arylamidase, and serine arylamidase; and/or
(ii) As an intermediate to at least one (e.g., at least 2 or all) of the following: beta-galactosidase-6-phosphate, beta-glucosidase, and N-acetyl-beta-glucosaminidase; and/or
(iii) Negative for at least one (e.g., at least 2, 3, 4, 5, 6, or all) of: raffinose fermentation, proline arylamidase, leucyl glycine arylamidase, leucine arylamidase, alanine arylamidase, glycine arylamidase, and glutamyl glutamic acid arylamidase,
preferably as determined by assays of carbohydrate, amino acid and nitrate metabolism and optionally alkaline phosphatase activity, more preferably as determined by Rapid ID32A analysis (preferably using Rapid ID32A system from biomeririeux).
In some embodiments, the bacterial strains used in the therapeutic combinations of the present invention are:
(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, for example, as determined by assays of carbohydrate, amino acid and nitrate metabolism, preferably as determined by Rapid ID32A analysis (preferably using Rapid ID32A system from biomeririeux); and/or
(ii) Intermediates which are positive for fermentation of L-trehalose are preferably determined as by API50CHL analysis (preferably using API50CHL plates from bioMerieux).
In some embodiments, the bacterial strain used in the therapeutic combination of the invention is an extracellular ATP producer, e.g., a strain that produces 6-6.7ng/μ Ι (e.g., 6.1-6.6ng/μ Ι or 6.2-6.5ng/μ Ι or 6.33 ± 0.10ng/μ Ι) ATP, as measured using an ATP assay kit (Sigma-Aldrich, MAK 190). Extracellular ATP of bacteria may 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 the secretion of the proinflammatory mediator IL-1 β by activation of NLRP3 inflammatory body (karprkar et al, 2016). Thus, bacterial strains that are extracellular ATP producers are useful in the context of the therapeutic combinations and methods of the present invention for treating or preventing cancer.
In some embodiments, the bacterial strain used in the therapeutic combination of the present invention comprises one or more of the following three genes: a movement factor protein; xylose ABC transporter, i.e. permease component; and FIG 00632333: a protein is hypothesized. For example, in certain embodiments, the bacterial strains used in the therapeutic combinations of the present invention comprise genes encoding: a mobile factor protein and a xylose ABC transporter, i.e. permease component; movement factor protein and FIG 00632333: a hypothetical protein; xylose ABC transporter, permease component and FIG 00632333: a hypothetical protein; or a movement factor protein, a xylose ABC transporter, i.e. permease component and FIG 00632333: a protein is hypothesized.
A particularly preferred strain of the therapeutic combination of the invention is the enterococcus gallinarum strain deposited under accession number NCIMB 42488. This is an exemplary MRX518 strain tested in the examples and shown to be effective for treating disease. According to some embodiments, the present invention provides a bacterial composition comprising cells of the enterococcus gallinarum strain or a derivative thereof deposited under accession number NCIMB42488 as part of the therapeutic combination of the present invention. The derivative of the strain deposited under accession number NCIMB42488 may be a daughter strain (progeny) or a strain grown from the original (subcloned).
Derivatives of the strains of the compositions comprised in the therapeutic combination of the invention may be modified, for example at the gene level, without abolishing the biological activity. In particular, the derivative strains of the therapeutic combinations of the invention are therapeutically active. The derivative strain will have comparable immunomodulatory activity to the original NCIMB42488 strain. In particular, the derivative strain, when combined with pembrolizumab, will elicit an effect on cancer disease models comparable to the effect shown in the examples, which can be identified by using the culture and administration protocols described in the examples. Derivatives of the NCIMB42488 strain will typically be biotypes of the NCIMB42488 strain.
Reference to cells of the enterococcus gallinarum strain deposited under accession number NCIMB42488 encompasses any cell having the same safety and therapeutic efficacy characteristics as the strain deposited under accession number NCIMB42488, and such cells are encompassed by the therapeutic combination of the invention. Thus, in some embodiments, reference to cells of the enterococcus gallinarum strain deposited under accession number NCIMB42488 refers only to the MRX518 strain deposited under NCIMB42488 and does not refer to bacterial strains not deposited under NCIMB 42488. In some embodiments, reference to cells of the enterococcus gallinarum strain deposited under accession number NCIMB42488 refers to cells having the same safety and therapeutic efficacy characteristics as the strain deposited under accession number NCIMB42488, but which are not the strain deposited under NCIMB 42488.
In a preferred embodiment, the bacterial strains in the composition of the invention are viable and capable of partial or complete colonization of the intestine.
Treating cancer
In a preferred embodiment, the therapeutic combination of the invention is for the treatment or prevention of cancer. These examples demonstrate that administration of a therapeutic combination of the present invention results in a reduction in tumor growth.
In certain embodiments, treatment with the therapeutic combination of the present invention results in a reduction in tumor size or a slowing of tumor growth. In certain embodiments, the therapeutic combination of the present invention is for reducing tumor size or slowing tumor growth. The therapeutic combination of the present invention is effective for reducing the size of a tumor or slowing its growth. In certain embodiments, the therapeutic combination of the present invention is for use in a patient having a solid tumor. In certain embodiments, the therapeutic combinations of the present invention are used to reduce or prevent angiogenesis in the treatment of cancer. The therapeutic combination of the present invention may have an effect on immune or inflammatory systems, which play an important role in angiogenesis. In certain embodiments, the therapeutic combination of the present invention is for preventing metastasis.
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of breast cancer. These examples demonstrate that the therapeutic combinations of the present invention are effective in treating breast cancer. In certain embodiments, the therapeutic combinations of the present invention are used in the treatment of breast cancer to reduce tumor size, slow tumor growth, or reduce angiogenesis. In a preferred embodiment, the cancer is breast cancer. In a preferred embodiment, the cancer is stage IV breast cancer.
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of lung cancer. These examples demonstrate that the therapeutic combinations of the present invention are effective for treating lung cancer. In certain embodiments, the therapeutic combinations of the present invention are used in the treatment of lung cancer to reduce tumor size, slow tumor growth, or reduce angiogenesis. In a preferred embodiment, the cancer is lung cancer.
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of liver cancer. These examples demonstrate that the therapeutic combinations of the present invention are effective for treating liver cancer. In certain embodiments, the therapeutic combinations of the present invention are used in the treatment of liver cancer to reduce tumor size, slow tumor growth, or reduce angiogenesis. In a preferred embodiment, the cancer is hepatoma (hepatocellular carcinoma).
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of colon cancer. These examples demonstrate that the therapeutic combination of the present invention has an effect on colon cancer cells and is effective in the treatment of colon cancer. In certain embodiments, the therapeutic combinations of the present invention are used to reduce tumor size, slow tumor growth, or reduce angiogenesis in the treatment of colon cancer. In a preferred embodiment, the cancer is colorectal adenocarcinoma.
In certain embodiments, the therapeutic combination of the invention is for the treatment or prevention of kidney cancer (also referred to herein as "renal cancer"). These examples demonstrate that the therapeutic combination of the present invention has an effect on renal cancer cells and can be effectively used for the treatment of renal cancer. In certain embodiments, the therapeutic combinations of the present invention are used in the treatment of renal cancer to reduce tumor size, slow tumor growth, or reduce angiogenesis. In a preferred embodiment, the cancer is renal cell carcinoma or transitional cell carcinoma.
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of melanoma. According to some embodiments, the therapeutic combination of the present invention has an effect on melanocytes and may be effectively used for the treatment of melanoma. In certain embodiments, the therapeutic combination of the present invention is used in the treatment of melanoma to reduce tumor size, slow tumor growth, or reduce angiogenesis.
In some embodiments, the cancer is intestinal. In some embodiments, the cancer is a part of the body that is not the intestine. In some embodiments, the cancer is not an intestinal cancer. In some embodiments, the cancer is not colorectal cancer. In some embodiments, the cancer is not small bowel cancer. In some embodiments, the treatment or prevention occurs at a site other than the intestine. In some embodiments, the treatment or prevention occurs at the intestine and at sites other than the intestine.
In certain embodiments, the therapeutic combination of the present invention is for the treatment or prevention of cancer. These examples demonstrate that the therapeutic combinations of the present invention are effective in treating many types of cancer. In certain embodiments, the therapeutic combination of the invention is for the treatment or prevention of a non-immunogenic cancer. These examples demonstrate that the therapeutic combinations of the present invention are effective in treating non-immunogenic cancers.
In the context of the therapeutic combination of the present invention, the therapeutic effect of the bacterial composition 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 can be increased following administration of MRX 518. Inflammation may have a cancer-inhibiting effect [20] and proinflammatory cytokines such as TNF α have been investigated as cancer therapies [21 ]. Upregulation of genes such as TNF as shown in the examples may indicate that the bacterial compositions of the invention may be useful for the treatment of cancer via a similar mechanism. Upregulation of CXCR3 ligands (CXCL9, CXCL10) and the IFN γ inducible gene (IL-32) can indicate that the bacterial compositions of the invention elicit an IFN γ type response. IFN gamma can stimulate the activity of tumor killing effective macrophage activation factor [22], and CXCL9 and CXCL10 for example also have anti-tumor effects [23-25 ]. Thus, in certain embodiments, the bacterial compositions of the present invention, when used in the context of the therapeutic combination of the present invention, are useful for promoting inflammation in the treatment of cancer. In a preferred embodiment, the composition of the invention, when used in the context of the therapeutic combination of the invention, is for promoting Th1 inflammation in the treatment of cancer. Th1 cells produce IFN γ and have potent anti-cancer effects [20 ]. In certain embodiments, the compositions of the invention, when used in the context of the therapeutic combinations of the invention, are for the treatment of early stage cancer, such as non-metastatic cancer or
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