GENETICALLY MODIFIED BACTERIA FOR GENERATING VACCINES

Abstract

A vaccine comprising tumor-homing bacteria which are genetically modified to express at least one cancer-associated antigen and a pharmaceutically acceptable carrier is disclosed. Uses thereof are also disclosed.

Description

SEQUENCE LISTING STATEMENT

The XML file, entitled 97463SequenceListing.xml, created on Aug. 17, 2023, comprising 678,858 bytes, submitted concurrently with the filing of this application is incorporated herein by reference. The sequence listing submitted herewith is identical to the sequence listing forming part of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to bacterial vaccines which may be manipulated to comprise disease-associated antigens.

Advances in the understanding of molecular biology, the ability to predict immunogenic neoantigens by next generation sequencing and prediction algorithms, the lifestyles of pathogenic bacteria, bacterial engineering and synthetic biology tools have significantly accelerated the rational design of bacteria as antigen delivery vectors. Being a strong immunogen, bacteria may trigger a vast immune response against itself and consequently against the delivered neoantigen. Indeed, bacterial vectors that deliver antigenic messages are also able to deliver a strong danger signal mediated by their pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, lipoproteins, flagellin and CpG. PAMPs derived from different classes of pathogens bind to diverse families of pathogen recognition receptors (PRRs) that include Toll-like receptors (TLRs), C-type lectin-like receptors (CLRs), retinoic acid-induciblegene(RIG)-like receptors (RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). These interactions according to each pathogen trigger distinct signaling pathways to differentially activate antigen presenting cells (APCs), thereby directing the adaptive effector response in a manner that is specifically adapted to the microbe and hence to the antigen delivered by the bacteria. Moreover, specialized toxins that bacteria use for their own virulence can reinforce effector or memory responses.

Background art includes US Patent Application Nos. 20200087703, 20200054739 and 20190365830, Gopalakrishnan V et al, Science. 2018 Jan. 5; 359(6371): 97-103; Geller et al., Science, Vol 357, Issue 6356 15 Sep. 2017; Riquelme E et al Cell. 2019 Aug. 8; 178(4):795-806.e12. doi: 10.1016/j.cell.2019.07.008; Straussman R et al., Nature. 2012 Jul. 26; 487(7408):500-4. doi: 10.1038/nature11183.

SUMMARY OF THE INVENTION

According to an aspect of the present invention there is provided a vaccine comprising tumor-homing bacteria which are genetically modified to express at least one cancer-associated antigen and a pharmaceutically acceptable carrier.

According to an aspect of the present invention there is provided a method of treating cancer of a subject in need thereof comprising administering to the subject a therapeutically effective amount of the vaccine of any one of claims 1-14, thereby treating the cancer.

According to an aspect of the present invention there is provided a method of treating cancer of a subject in need thereof comprising administering to the subject a therapeutically effective amount of:

• • (i) a first vaccine comprising a first bacteria which is genetically modified to express at least one cancer-associated antigen; and subsequently;
  • (ii) a second vaccine comprising a second bacteria which is genetically modified to express at least one cancer-associated antigen, thereby treating the cancer.

According to an aspect of the present invention there is provided a method of preventing cancer of a subject in need thereof the method comprising administering to the subject a prophylatically effective amount of the vaccine described herein, thereby preventing the cancer.

Accordance to embodiments of the present invention, the bacteria is a gram positive bacteria.

Accordance to embodiments of the present invention, the bacteria is a gram negative bacteria.

Accordance to embodiments of the present invention, the bacteria is aerobic.

Accordance to embodiments of the present invention, the bacteria is anaerobic.

Accordance to embodiments of the present invention, the bacteria are live bacteria.

Accordance to embodiments of the present invention, the bacteria are attenuated bacteria.

Accordance to embodiments of the present invention, the bacteria is of a species or genus set forth in any of Tables 1-3.

Accordance to embodiments of the present invention, the genome of the bacteria comprises a 16S rRNA sequence as set forth in any one of SEQ ID NOs: 24-310.

Accordance to embodiments of the present invention, the cancer-associated antigen is a neoantigen.

Accordance to embodiments of the present invention, the bacteria are genetically modified to express a therapeutic protein.

Accordance to embodiments of the present invention, the therapeutic protein is a cytokine.

Accordance to embodiments of the present invention, the vaccine is devoid of an aluminium salt.

Accordance to embodiments of the present invention, the carrier is devoid of adjuvant.

Accordance to embodiments of the present invention, the first bacteria are viable bacteria and the second bacteria are non-viable bacteria.

Accordance to embodiments of the present invention, the first bacteria comprises a first strain of bacteria that is genetically modified to express a cancer-associated antigen and the second bacteria comprises a second strain of bacteria that is non-identical to the first strain of bacteria, the second bacteria being genetically modified to express the cancer-associated antigen.

Accordance to embodiments of the present invention, the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

Accordance to embodiments of the present invention, the brain cancer comprises glioblastoma.

Accordance to embodiments of the present invention, the at least one cancer-associated antigen of the first vaccine is identical to the at least one cancer-associated antigen of the second vaccine.

Accordance to embodiments of the present invention, the at least one cancer-associated antigen of the first vaccine is non-identical to the at least one cancer-associated antigen of the second vaccine.

Accordance to embodiments of the present invention, the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

Accordance to embodiments of the present invention, the brain cancer comprises glioblastoma.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B. Long-term efficacy of vaccination by OVA expressing bacteria in B16-OVA tumor model. (A) Experiment time line. (B) Tumor growth curves from start of treatment.

FIGS. 2A-G: Short-term efficacy and immunogenicity of vaccination by OVA expressing bacteria in B16-OVA tumor model. (A) Experiment time line. (B) Tumor growth curves from start of treatment. N=5 for all mice cohorts. (C) Tumor volume percentage at day 16 relative to day 0. P-values were obtained by two sided Mann-Whitney test. Below are the percentage values of fully cured mice per cohort. (D) Mice from the cohort treated with Anti-PD1 or Anti-PD1 together with PACMAN-OVA. While the tumor of the mouse treated with PACMAN-OVA gradually disappears, the tumor of the mouse treated with anti-PD1 only continued to grow exponentially. (E) CFU count of tumor and liver extracts. Following 16 days from vaccination, bacteria from tumors and livers were seeded on LB plates with resistance to AMP. Per mouse, CFU count and tumor volumes are given. Of note, 4 out of 5 mice of the PACMAN-OVA cohort exhibited complete clearance of bacteria. Bacteria were present in the mouse with the biggest tumor, suggesting that the tumor tissue enables bacteria proliferation. (F) Sera of mice cohorts were subjected to IFNg ELISA. The PACMAN-OVA cohort exhibited the highest IFNg serum level indicating high systemic immune activation. Green dot refers to mouse 836 in F. Mouse 836 was the only case where bacteria were present in the liver, probably resulting in the highest serum level of IFNg. (G) Quantification of SIINFEKL (SEQ ID NO: 11) specific TCR by Flow Cytometry. To quantify neoantigen specific T cell clones, splenocytest were co-incubated with Tetramer presenting the OVA neoantigen (SIINFEKL—SEQ ID NO: 11)) and conjugated to a fluorescent dye. Thus, splenocytes which are positive to the Tetramer dye possess a TCR that can bind the OVA neoantigen. Following FACS, percentage of SIINFEKEL (SEQ ID NO: 11) positive T cells out of CD3+CD8+ population was the highest among mice vaccinated with the PACMAN-OVA. P-values were obtained by Two sided Mann-Whitney test.

FIGS. 3A-D. Alternate administration of different bacterial vaccines may overcome acquired immunity. (A) Experiment time table. (B) Tumor growth curves. (C) Weight change (percentage of initial weight) during the first 24 days of treatment. Mice cohorts are: STM3120 +aPD1(N=3), STM-pagC-SspH1-OVA+aPD1 (N=3), STM-SspH2-OVA+aPD1 (N=5). Dashed lines demarcate day of bacteria administration. (D) Weight change (percentage of initial weight) during the first 40 days of treatment. Mice cohorts are: STM3120+aPD1(N=3), STM-pagC-SspH1-OVA+aPD1 (N=3), STM-SspH2-OVA+aPD1 (N=5), CHA-OST. Dashed lines demarcate day of bacteria administration.

FIGS. 4A-B. Immune memory of vaccination by OVA expressing bacteria in B16-OVA tumor model. (A) Experiment time table. (B) Tumor growth curves.

FIGS. 5A-C. Long-term efficacy of vaccination by Adpgk expressing bacteria in MC38 CRC tumor model. (A) Experiment time line. (B) Tumor growth curves from treatment start. As shown, tumor growth was significantly inhibited in the PACMAN-Adpgk cohort relative to the other mice cohorts. Following two cycles of immunization, one mouse in the PACMAN-Adpgk cohort was fully cured. (C) To test the immune memory of mice vaccinated with PACMAN-Adpgk, the mice exhibiting full cure following vaccination with PACKMAN-Adpgk or VNP20009 (w/o adpgk) were re-challenged with 10⁵ MC38 cells and tumor growth was compared to naïve mice injected with the same amount of cells. While naïve mice exhibited exponentially growing tumors shortly after injection, re-challenged mouse which was vaccinated with PACKMAN-Adpgk remained tumor free, indicating the establishment of long term immune memory against MC38 cells. Of note, the fully cured mouse following vaccination only with the VNP20009, exhibited tumor growth following re-challenge indicating that the immune memory was a consequence of Adpgk presentation by the bacteria.

FIG. 6 is a graph illustrating tumor homing of attenuated (STM3120) Salmonella bacteria.

FIG. 7 is a graph illustrating toxicity of i.v. administration of attenuated (STM3120) vs parental (14028) Salmonella.

FIGS. 8A-B are graphs illustrating splenocytes immune profiling following vaccination by OVA expressing bacteria in B16-OVA tumor model. FIG. 8A—Quantification of IFNg positive CD8 T-cells by FACS. FIG. 8B—Quantification of T cells killing capacity.

FIGS. 9A-C illustrate long-term efficacy of vaccination by Adpgk expressing P. Aeruginosa in MC38 tumor model. FIG. 9A—Experiment timetable. FIG. 9B—Tumor growth curves. Mice treated with PACMAN-ADPGK i.v, exhibited a considerable delayed tumor growth. Per treatment, indicated number of fully cured mice. FIG. 9C—Average tumor growth curves of the mice cohorts in FIG. 9B. Whiskers indicate standard error.

FIGS. 10A-B illustrates long-term efficacy of vaccination by Adpgk expressing Bacillus Subtilis spores in MC38 tumor model. FIG. 10A—Experimental timetable. FIG. 10B. Tumor growth curves. Mice treated with PACMAN-ADPGK spores p.o or i.v, exhibited a considerable delayed tumor growth. Per treatment, indicated number of fully cured mice.

FIGS. 11A-B illustrates long-term efficacy of vaccination by Adpgk expressing attenuated Salmonella (STM3120) in MC38 tumor model. FIG. 11A. Experiment timetable. FIG. 11B—Tumor growth curves. Mice treated with PACMAN-ADPGK exhibited a considerable delayed tumor growth. Per treatment, indicated number of fully cured mice.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to bacterial vaccines which may be manipulated to contain disease-associated antigens on their outer surface.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

In vivo therapeutic cancer vaccine strategies based on bacterial vectors that directly deliver antigens or nucleic acids encoding antigens to the cytosol of APCs, have been developed in academic laboratories and pharmaceutical industry due to their ease of use. Typically, the bacteria is genetically modified to express (and even secrete) the disease antigen. Alternatively, the bacteria may be used to deliver plasmid cDNA which encode the disease antigen to the immune system.

The present inventors have now conceived of a novel vaccine which includes tumor-homing bacteria. The bacteria are genetically modified to express disease associated antigens. These vaccines are referred to herein as Personalized Anti-Cancer Microbiome-Assisted VaccinatioN (PACMAN).

As is illustrated herein under and in the examples section which follows, the present inventors show that it is possible to genetically modify bacteria to express tumor antigens. The genetically modified bacteria serve two purposes 1) as a targeting vehicle—homing to the tumor site and 2) as an adjuvant, stimulating the immune system.

The Inventors demonstrated the ability to produce effective vaccines using a number of different bacteria including Salmonella typhimurium (FIGS. 1A-B, 2A-G, 3A-D, 4A-B, 5A-C), P. aeruginosa (FIGS. 9A-B) and B. Subtilis (FIGS. 10A-B). The bacteria were genetically modified to express various tumor specific antigens including OVA (FIGS. 1A-B, 2A-G, 3A-D, 4A-B) and ADPGK (FIGS. 5A-C, 9A-B, 10A-B and 11A-B).

Whilst further reducing the present invention to practice, the present inventors showed that alternate administration of different bacterial vaccines can overcome acquired immunity (see FIGS. 3A-C).

Thus, according to an aspect of the present invention there is provided a vaccine comprising tumor-homing bacteria which are genetically modified to express at least one cancer-associated antigen and a pharmaceutically acceptable carrier.

As used herein, the term “vaccine” refers to a pharmaceutical preparation (pharmaceutical composition) that upon administration induces an immune response, in particular a cellular immune response, which recognizes and attacks a cancer cell. Preferably, the vaccine results in the formation of long-term immune memory towards the targeted antigen. The vaccine of the present invention preferably also includes a pharmaceutically acceptable carrier (e.g. a liquid composition which carries the bacteria). In one embodiment, the carrier is one which retains the viability of the bacteria.

The isolated bacteria of this aspect of the present invention may be gram positive or gram negative bacteria or may be a combination of both.

The bacteria may be aerobic or anaerobic bacteria.

As mentioned, the bacteria are capable of homing to a tumor site.

In another embodiment, the bacteria which are capable of homing to a tumor are present in a tumor microbiome of the subject.

The term “tumor microbiome” refers to the totality of microbes (bacteria, fungae, protists), their genetic elements (genomes) in a defined environment, e.g. within the tumor of a host. In a particular embodiment, the microbiome refers only to the totality of bacteria in a defined environment, e.g. within the tumor of a host. The tumor may be a primary tumor or a secondary tumor (i.e. metastasized tumor).

Examples of bacteria known to be present in a breast tumor microbiome are set forth in Table 1, herein below. Such bacteria may be particular relevant for use in vaccines for treating breast cancer. The sequence provided refers to the 16S rRNA sequence for each bacteria.

TABLE 1
kingdom	phylum	class	order	family	genus	species	SEQ ID
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae	Trueperella		24
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Bogoriellaceae	Georgenia		25
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Cellulomonadaceae	Cellulomonas		26
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Cellulomonadaceae	Oerskovia		27
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Corynebacterium	29
						tuberculostearicum
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Corynebacterium	31
						tuberculostearicum
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Corynebacterium	32
						variabile
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium		33
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium		35
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium		36
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium		37
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Dermabacteraceae	Dermabacter		38
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Dermacoccaceae	Dermacoccus		39
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Dermacoccaceae	Dermacoccus		40
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Dietziaceae	Dietzia		41
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Geodermatophilaceae	Blastococcus		42
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Intrasporangiaceae	Janibacter		43
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Intrasporangiaceae	Ornithinimicrobium		44
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Agrococcus		45
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Agrococcus		46
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Microbacterium		47
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Microbacterium		48
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Microbacterium		49
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Microbacterium		50
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Microbacterium		51
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Arthrobacter	Arthrobacter	52
						aurescens
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	53
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	54
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	55
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	56
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	57
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	58
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	59
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	60
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	61
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	62
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	63
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	64
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	65
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	66
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	67
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	68
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	69
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	70
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus	Micrococcus	71
						luteus
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Arthrobacter		72
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Kocuria		73
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Microbispora		74
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Microbispora		75
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus		76
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus		77
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus		78
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Micrococcus		79
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Mycobacteriaceae	Mycobacterium		80
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Nocardiaceae	Rhodococcus	Rhodococcus	81
						erythropolis
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	82
						acnes
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	83
						acnes
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	84
						acnes
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	85
						avidum
Bacteria	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	86
						avidum
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	Bacillus	87
						flexus
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	Bacillus	88
						flexus
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	Bacillus	89
						muralis
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus		90
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 1	Bacillus	Bacillus	91
						subtilis
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 1	Bacillus	Bacillus	92
						subtilis
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 1	Bacillus	Bacillus	93
						subtilis
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 1	Bacillus	Bacillus	94
						foraminis
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 1	Bacillus	Bacillus	95
						nealsonii
Bacteria	Firmicutes	Bacilli	Bacillales	Bacillaceae 2	Terribacillus	Chryseomicrobium	96
Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Chryseomicrobium	imtechense	97
Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Chryseomicrobium		98
Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Sporosarcina		99
Bacteria	Firmicutes	Bacilli	Bacillales	Planococcaceae	Sporosarcina		100
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	101
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	102
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	103
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	104
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	105
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	106
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	107
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	108
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	109
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	110
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	111
						epidermidis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	112
						haemolyticus
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	113
						hominis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	114
						hominis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	115
						hominis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	116
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	117
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	118
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	119
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	120
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	121
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	122
						lugdunensis
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	123
						succinus
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	124
						succinus
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	125
						succinus
Bacteria	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus		126
Bacteria	Firmicutes	Bacilli	Bacillales	Unknown	Exiguobacterium	Exiguobacterium	127
				species		mexicanum
Bacteria	Firmicutes	Bacilli	Bacillales	Unknown	Exiguobacterium	Exiguobacterium	128
				species		profundum
Bacteria	Firmicutes	Bacilli	Bacillales	Unknown	Exiguobacterium		129
				species
Bacteria	Firmicutes	Bacilli	Lactobacillales	Aerococcaceae	Aerococcus	Aerococcus	130
						viridans
Bacteria	Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	Enterococcus	131
						faecalis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	132
						infantis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	133
						infantis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	134
						infantis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	135
						infantis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	136
						oralis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	137
						pneumoniae
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	138
						pneumoniae
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	139
						sanguinis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	140
						vestibularis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	141
						vestibularis
Bacteria	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Assigned	142
						species1791
Bacteria	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus	Paracoccus	143
						aminovorans
Bacteria	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus		144
Bacteria	Proteobacteria	Alphaproteobacteria	Rhodospirillales	Acetobacteraceae	Roseomonas	Roseomonas	145
						mucosa
Bacteria	Proteobacteria	Alphaproteobacteria	Rhodospirillales	Acetobacteraceae	Roseomonas		146
Bacteria	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	Sphingomonas	147
						desiccabilis
Bacteria	Proteobacteria	Betaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia		148
Bacteria	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Neisseria	Neisseria	149
						macacae
Bacteria	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Neisseria	Neisseria	150
						subflava
Bacteria	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Neisseria	Neisseria	151
						subflava
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	152
						cloacae
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Proteus	Proteus	153
						mirabilis
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Proteus	Proteus	154
						mirabilis
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Proteus	Proteus	155
						mirabilis
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Erwinia		156
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Erwinia		157
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Erwinia		158
Bacteria	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Erwinia		159
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	Acinetobacter	160
						radioresistens
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Enhydrobacter	Enhydrobacter	161
						aerosaccus
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Enhydrobacter	Enhydrobacter	162
						aerosaccus
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Enhydrobacter	Enhydrobacter	163
						aerosaccus
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Enhydrobacter		164
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas		165
Bacteria	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas		166
Fungi	Ascomycota	Eurotiomycetes	Eurotiales	Trichocomaceae	Aspergillus	Aspergillus	167
						kawachii
Fungi	Ascomycota	Eurotiomycetes	Eurotiales	Trichocomaceae	Aspergillus	Aspergillus	168
						niger
Fungi	Ascomycota	Eurotiomycetes	Eurotiales	Trichocomaceae	Aspergillus	Aspergillus	169
						pseudoglaucus
Fungi	Ascomycota	Saccharomycetes	Saccharomycetales	Saccharomycetaceae	Saccharomyces	Saccharomyces	170
						cerevisiae

Table 2 includes bacterial taxa that may be particular relevant for use in a vaccine for treating breast, lung or ovarian cancers. Bacteria are sorted according to their p-values (lowest to highest) for enrichment per tumor type.

TABLE 2
							Tumor	SEQ
bact_ID	phylum	class	order	family	genus	species	type	ID NO:
12873	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingom	Sphingomonas	Unknown	Breast	171
				onadaceae		species602
13620	Proteobacteria	Betaproteobacteria	Burkholderiales	Comamonadaceae	Tepidimonas	Unknown	Breast	172
						species11
32080	Proteobacteria	Betaproteobacteria	Burkholderiales	Comamonadaceae	Tepidimonas		Breast
11657	Proteobacteria	Alphaproteobacteria	Rhizobiales	Methylobacteriaceae	Methylobacterium	Methylobacterium	Breast	173
						organophilum
11656	Proteobacteria	Alphaproteobacteria	Rhizobiales	Methylobacteriaceae	Methylobacterium	Methylobacterium	Breast	174
						mesophilicum
30362	Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella		Breast
50030	Bacteroidetes	Bacteroidia	Bacteroidales				Breast
30867	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus		Breast
50075	Firmicutes	Bacilli	Bacillales				Breast
50148	Proteobacteria	Gammaproteobacteria	Pseudomonadales				Breast
31477	Firmicutes	Clostridia	Clostridiales	Tissierellaceae	Finegoldia		Breast
40195	Firmicutes	Clostridia	Clostridiales	Tissierellaceae			Breast
70016	Firmicutes						Breast
30663	Cyanobacteria	Chloroplast	Streptophyta	Unknown	Unknown		Breast
				family	genus116
4523	Cyanobacteria	Chloroplast	Streptophyta	Unknown	Unknown	Unknown	Breast	175
				family	genus116	species19
40168	Firmicutes	Bacilli	Bacillales	Staphylococcaceae			Breast
9900	Firmicutes	Clostridia	Clostridiales	Tissierellaceae	Finegoldia	Unknown	Breast	176
						species11
5302	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Unknown	Breast	178
						species8
5324	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	Breast	179
						haemolyticus
15324	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	Acinetobacter	Breast	180
						ursingii
30817	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus		Breast
30858	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus		Breast
31799	Proteobacteria	Alphaproteobacteria	Rhizobiales	Methylobacteriaceae	Methylobacterium		Breast
40245	Proteobacteria	Alphaproteobacteria	Rhizobiales	Methylobacteriaceae			Breast
60052	Firmicutes	Bacilli					Breast
60078	Proteobacteria	Betaproteobacteria					Breast
13182	Proteobacteria	Betaproteobacteria	Burkholderiales	Burkholderiaceae	Ralstonia	Ralstonia	Breast	181
						mannitolilytica
31786	Proteobacteria	Alphaproteobacteria	Rhizobiales	Hyphomicrobiaceae	Devosia		Breast
40181	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae			Breast
40330	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae			Breast
969	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Corynebacterium	Breast	182
						stationis
32410	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas		Breast
230	Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae	Actinomyces	Actinomycesoris	Breast	183
5687	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	Lactobacillus	Breast	184
						iners
40176	Firmicutes	Bacilli	Lactobacillales	Aerococcaceae			Breast
50079	Firmicutes	Clostridia	Clostridiales				Breast
40179	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae			Breast
3941	Bacteroidetes	Flavobacteriia	Flavobacteriales	Weeksellaceae	Wautersiella	Unknown	Breast	187
						species18
30113	Actinobacteria	Actinobacteria	Actinomycetales	Cellulomonadaceae	Cellulomonas		Breast
6166	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	Breast	188
						cristatus
15055	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Klebsiella	Klebsiella	Breast	189
						pneumoniae
30866	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Lactococcus		Breast
40144	Cyanobacteria	Chloroplast	Streptophyta	Unknown			Breast
				family
40276	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae			Breast
50085	Fusobacteria	Fusobacteriia	Fusobacteriales				Breast
40046	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae			Breast
50077	Firmicutes	Bacilli	Lactobacillales				Breast
60081	Proteobacteria	Gammaproteobacteria					Breast
1021	Actinobacteria	Actinobacteria	Actinomycetales	Geodermatophilaceae	Blastococcus	Unknown	Breast	190
						species13
4719	Firmicutes	Bacilli	Bacillales	Bacillaceae	Anoxybacillus	Anoxybacillus	Breast	191
						kestanbolensis
40042	Actinobacteria	Actinobacteria	Actinomycetales	Nocardiaceae			Breast
2556	Bacteroidetes	Bacteroidia	Bacteroidales	Paraprevotellaceae	Prevotella	Prevotella	Breast	192
						tannerae
30099	Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae	Actinomyces		Breast
9261	Firmicutes	Clostridia	Clostridiales	Ruminococcaceae	Faecalibacterium	Faecalibacterium	Breast	195
						prausnitzii
4521	Cyanobacteria	Chloroplast	Streptophyta	Unknown	Unknown	Unknown	Breast	197
				family	genus116	species17
30190	Actinobacteria	Actinobacteria	Actinomycetales	Mycobacteriaceae	Mycobacterium		Breast
30225	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium		Breast
32336	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter		Breast
40021	Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae			Breast
40038	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae			Breast
40193	Firmicutes	Clostridia	Clostridiales	Ruminococcaceae			Breast
40198	Firmicutes	Clostridia	Clostridiales	Veillonellaceae			Breast
40278	Proteobacteria	Betaproteobacteria	Rhodocyclales	Rhodocyclaceae			Breast
30845	Firmicutes	Bacilli	Lactobacillales	Aerococcaceae	Alloiococcus		Breast
9771	Firmicutes	Clostridia	Clostridiales	Tissierellaceae	1-68	1-68 Unknown	Breast	198
5687	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	Lactobacillus	Lung	199
						iners
40260	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Erythrobacteraceae			Lung

Table 3 summarizes the different bacterial species that are prevalent in specific tumor types.

TABLE 3
								Prevalence
Tumor								in specific	SEQ
type	bact_ID	phylum	class	order	family	genus	species	tumor type	ID NO:
Breast	1824	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	38%	201
							granulosum
Breast	1346	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Rothia	Rothia	37%	202
							mucilaginosa
Breast	5687	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	Lactobacillus	37%	203
							iners
Breast	6175	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	36%	204
							infantis
Breast	10545	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Veillonella	Veillonella	36%	205
							dispar
Breast	1344	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Rothia	Rothia	32%	206
							dentocariosa
Breast	587	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Unknown	28%	207
							species1715
Breast	5330	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	28%	208
							pasteuri
Breast	3046	Bacteroidetes	Bacteroidia	Bacteroidales	Prevotellaceae	Prevotella	Prevotella	27%	209
							melaninogenica
Breast	10726	Fusobacteria	Fusobacteriia	Fusobacteriales	Fusobacteriaceae	Fusobacterium	Fusobacterium	24%	210
							nucleatum
Breast	4523	Cyanobacteria	Chloroplast	Streptophyta	Unknown	Unknown	Unknown	23%	211
					family	genus116	species 19
Breast	9900	Firmicutes	Clostridia	Clostridiales	Tissierellaceae	Finegoldia	Unknown	23%	212
							species 11
Breast	15324	Proteobacteria	Gamma	Pseudomonadales	Moraxellaceae	Acinetobacter	Acinetobacter	23%	213
			proteobacteria				ursingii
Breast	6184	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	22%	214
							pneumoniae
Breast	2555	Bacteroidetes	Bacteroidia	Bacteroidales	Paraprevotellaceae	Prevotella	Prevotella	22%	215
							Unknown
Breast	5286	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Unknown	22%	216
							species691
Breast	10546	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Veillonella	Veillonella	22%	217
							parvula
Breast	12106	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus	Paracoccus	21%	218
							chinensis
Breast	13904	Proteobacteria	Betaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia	Massilia	21%	219
							timonae
Breast	12109	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus	Paracoccus	20%	220
							marcusii
Lung	1824	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	19%	221
							granulosum
Lung	12551	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Kaistobacter	Kaistobacter	16%	222
							Unknown
Lung	10545	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Veillonella	Veillonelladispar	16%	223
Lung	587	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Unknown	16%	224
							species1715
Lung	1346	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Rothia	Rothia	16%	225
							mucilaginosa
Lung	2555	Bacteroidetes	Bacteroidia	Bacteroidales	Paraprevotellaceae	Prevotella	Prevotella	14%	226
							Unknown
Lung	5687	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	Lactobacillus	14%	227
							iners
Lung	12937	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	Sphingomonas	13%	228
							yunnanensis
Lung	1766	Actin	Actinobacteria	Actinomycetales	Propionibacteriaceae	Unknown	Unknown	12%	229
		Proteobacteria				genus24	species 1
Lung	12109	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus	Paracoccus	11%	230
							marcusii
Lung	12289	Proteobacteria	Alphaproteobacteria	Rhodospirillales	Acetobacteraceae	Roseomonas	Roseomonas	10%	231
							mucosa
Lung	15666	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Pseudomonas	9%	232
							baetica
Lung	1815	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Unknown	8%	233
							species 18
Lung	12808	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	Unknown	8%	234
							species 45
Lung	5338	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	7%	235
							warneri
Lung	13018	Proteobacteria	Betaproteobacteria	Burkholderiales	Alcaligenaceae	Alcaligenes	Alcaligenes	7%	236
							faecalis
Lung	13194	Proteobacteria	Betaproteobacteria	Burkholderiales	Comamonadaceae	Acidovorax	Acidovorax	7%	237
							temperans
Lung	545	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Unknown	7%	238
							species1626
Lung	6066	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Unknown	7%	239
							species346
Lung	9900	Firmicutes	Clostridia	Clostridiales	Tissierellaceae	Finegoldia	Unknown	7%	240
							species 11
Melanoma	12109	Proteobacteria	Alphaproteobacteria	Rhodobacterales	Rhodobacteraceae	Paracoccus	Paracoccus	20%	241
							marcusii
Melanoma	5315	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	14%	242
							aureus
Melanoma	2495	Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	Bacteroides	10%	243
							dorei
Melanoma	15607	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Unknown	5%	244
							species632
Melanoma	10444	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Selenomonas	Unknown	5%	245
							species 18
Melanoma	15733	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Pseudomonas	4%	246
							viridiflava
Melanoma	4886	Firmicutes	Bacilli	Bacillales	Bacillaceae	Geobacillus	Unknown	4%	247
							species208
Melanoma	15043	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Klebsiella	Unknown	4%	248
							species 25
Melanoma	15608	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Unknown	4%	249
							species643
Melanoma	4619	Cyanobacteria	Chloroplast	Streptophyta	Unknown	Unknown	Unknown	3%	250
					family	genus39	species 8
Melanoma	15956	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Xanthomonas	Xanthomonas	3%	251
							arboricola
Melanoma	436	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Unknown	2%	252
							species1061
Melanoma	6291	Firmicutes	Clostridia	Clostridiales	Clostridiaceae	Clostridium	Unknown	2%	253
							species 19
Melanoma	15368	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	Unknown	2%	25
							species 31
Melanoma	13810	Proteobacteria	Betaproteobacteria	Burkholderiales	Oxalobacteraceae	Massilia	Unknown	2%	255
							species177
Melanoma	67031	Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Unknown	Unknown	2%	256
						genus3008	species 1
Melanoma	4087	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Eikenella	Eikenella	2%	257
							corrodens
Melanoma	2437	Bacteroidetes	Bacteroidia	Bacteroidales	Bacteroidaceae	Bacteroides	Unknown	2%	258
							species388
Melanoma	10458	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Selenomonas	Unknown	2%	259
							species208
Melanoma	8343	Firmicutes	Clostridia	Clostridiales	Lachnospiraceae	Lachnoanaerobaculum	Eubacterium	1%	260
							saburreum
Melanoma	10504	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Selenomonas	Unknown	1%	261
							species 59
Pancreas	14775	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Citrobacter	Citrobacter	45%	262
							freundii
Pancreas	15055	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Klebsiella	Klebsiella	42%	263
							pneumoniae
Pancreas	14847	Proteobacteri	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	33%	264
							asburiae
Pancreas	10545	Firmicutes	Clostridia	Clostridiales	Veillonellaceae	Veillonella	Veillonella	19%	265
							dispar
Pancreas	10726	Fusobacteria	Fusobacteriia	Fusobacteriales	Fusobacteriaceae	Fusobacterium	Fusobacterium	18%	266
							nucleatum
Pancreas	14849	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	18%	267
							cloacae
Pancreas	15054	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Klebsiella	Klebsiella	15%	268
							oxytoca
Pancreas	14846	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	13%	269
							aerogenes
Pancreas	6161	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Streptococcus	13%	270
							anginosus
Pancreas	15695	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Pseudomonas	12%	271
							mendocina
Pancreas	1346	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Rothia	Rothia	10%	272
							mucilaginosa
Pancreas	14680	Proteobacteria	Gammaproteobacteria	Alteromonadales	Shewanellaceae	Shewanella	Shewanella	10%	273
							decolorationis
Pancreas	5583	Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	Enterococcus	10%	274
							gallinarum
Pancreas	5552	Firmicutes	Bacilli	Lactobacillales	Carnobacteriaceae	Granulicatella	Granulicatella	9%	275
							adiacens
Pancreas	986	Actinobacteria	Actinobacteria	Actinomycetales	Dermabacteraceae	Brachybacterium	Brachybacterium	7%	276
							conglomeratum
Pancreas	14174	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Neisseria	Neisseria	7%	277
							subflava
Pancreas	2555	Bacteroidetes	Bacteroidia	Bacteroidales	Paraprevotellaceae	Prevotella	Prevotella	6%	278
							Unknown
Pancreas	5582	Firmicutes	Bacilli	Lactobacillales	Enterococcaceae	Enterococcus	Enterococcus	6%	279
							faecium
Pancreas	1344	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Rothia	Rothia	4%	280
							dentocariosa
Pancreas	10757	Fusobacteria	Fusobacteriia	Fusobacteriales	Leptotrichiaceae	Leptotrichia	Unknown	4%	281
							species235
Ovary	12289	Proteobacteria	Alphaproteobacteria	Rhodospirillales	Acetobacteraceae	Roseomonas	Roseomonas	20%	282
							mucosa
Ovary	12873	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingomonas	Unknown	20%	283
							species602
Ovary	5319	Firmicutes	Bacilli	Bacillales	Staphylococcaceae	Staphylococcus	Staphylococcus	9%	284
							cohnii
Ovary	16227	Thermi	Deinococci	Deinococcales	Deinococcaceae	Deinococcus	Unknown	7%	285
							species124
Ovary	5627	Firmicutes	Bacilli	Lactobacillales	Lactobacillaceae	Lactobacillus	Unknown	5%	286
							species479
Bone	12674	Proteobacteria	Alphaproteobacteria	Sphingomonadales	Sphingomonadaceae	Sphingobium	Sphingomonas	36%	287
							yanoikuyae
Bone	1824	Actinobacteria	Actinobacteria	Actinomycetales	Propionibacteriaceae	Propionibacterium	Propionibacterium	28%	288
							granulosum
Bone	225	Actinobacteria	Actinobacteria	Actinomycetales	Actinomycetaceae	Actinomyces	Actinomyces	18%	289
							massiliensis
Bone	15662	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Pseudomonas	13%	290
							argentinensis
Bone	14847	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	10%	291
							asburiae
Bone	15568	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Pseudomonadaceae	Pseudomonas	Unknown	10%	292
							species 39
Bone	5968	Firmicutes	Bacilli	Lactobacillales	Streptococcaceae	Streptococcus	Unknown	8%	293
							species2029
Bone	16119	TM7	TM7-3	CW040	Unknown	Unknown	Unknown	5%	294
					family	genus3	species 7
Bone	16025	Spirochaetes	Spirochaetes	Spirochaetales	Spirochaetaceae	Treponema	Treponema	5%	295
							socranskii
Bone	4868	Firmicutes	Bacilli	Bacillales	Bacillaceae	Bacillus	Bacillus	5%	296
							clausii
Bone	527	Actinobacteria	Actinobacteria	Actinomycetales	Corynebacteriaceae	Corynebacterium	Unknown	5%	297
							species1534
GBM	14849	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Enterobacter	10%	298
							cloacae
GBM	14169	Proteobacteria	Betaproteobacteria	Neisseriales	Neisseriaceae	Neisseria	Neisseria	8%	299
							macacae
GBM	1311	Actinobacteria	Actinobacteria	Actinomycetales	Micrococcaceae	Kocuria	Kocuria	8%	300
							atrinae
GBM	15409	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	Unknown	8%	301
							species424
GBM	14934	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Escherichia/	Unknown	8%	302
						Shigella	species231
GBM	14795	Proteobacteria	Gammaproteobacteria	Enterobacteriales	Enterobacteriaceae	Enterobacter	Unknown	8%	303
							species196
GBM	1129	Actinobacteria	Actinobacteria	Actinomycetales	Microbacteriaceae	Agromyces	Agromyces	5%	304
							mediolanus
GBM	11842	Proteobacteria	Alphaproteobacteria	Rhizobiales	Rhizobiaceae	Agrobacterium	Unknown	5%	305
							species298
GBM	15853	Proteobacteria	Gammaproteobacteria	Xanthomonadales	Xanthomonadaceae	Luteimonas	Unknown	5%	306
							species 76
GBM	5106	Firmicutes	Bacilli	Bacillales	Planococcaceae	Lysinibacillus	Lysinibacillus	5%	307
							boronitolerans
GBM	5009	Firmicutes	Bacilli	Bacillales	Exiguobacteraceae	Exiguobacterium	Unknown	5%	308
							species 29
GBM	15333	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Acinetobacter	Unknown	5%	309
							species127
GBM	15494	Proteobacteria	Gammaproteobacteria	Pseudomonadales	Moraxellaceae	Psychrobacter	Unknown	5%	310
							species 28

According to a particular embodiment, the bacteria is Salmonella typhimurium—e.g. the Salmonella typhimurium attenuated strain VNP20009, Salmonella typhimurium 14028 strain STM3120, Salmonella typhimurium 14028 strain STM1414, Pseudomonas aeruginosa (strain CHA-OST) and/or Bacillus subtillis (strain PY79).

The term “isolated” or ‘enriched’ encompasses bacteria that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated microbes may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is ‘pure’ if it is substantially free of other components. The terms ‘purify,’ ‘purifying’ and ‘purified’ refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microbe or a microbial population may be considered purified if it is isolated at, or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of microbial compositions provided herein, the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions and the microbial components thereof are generally purified from residual habitat products.

In certain embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the bacteria in the vaccine are of a genus, species or strain listed in Tables 1-3.

According to a specific embodiment, the genome of the bacteria comprises a 16S rRNA sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95% identical to any one of the sequences as set forth in SEQ ID NO: 24-310.

As used herein, “percent homology”, “percent identity”, “sequence identity” or “identity” or grammatical equivalents as used herein in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9].

Percent identity can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

Other exemplary sequence alignment programs that may be used to determine % homology or identity between two sequences include, but are not limited to, the FASTA package (including rigorous (SSEARCH, LALIGN, GGSEARCH and GLSEARCH) and heuristic (FASTA, FASTX/Y, TFASTX/Y and FASTS/M/F) algorithms, the EMBOSS package (Needle, stretcher, water and matcher), the BLAST programs (including, but not limited to BLASTN, BLASTX, TBLASTX, BLASTP, TBLASTN), megablast and BLAT. In some embodiments, the sequence alignment program is BLASTN. For example, 95% homology refers to 95% sequence identity determined by BLASTN, by combining all non-overlapping alignment segments (BLAST HSPs), summing their numbers of identical matches and dividing this sum with the length of the shorter sequence.

In some embodiments, the sequence alignment program is a basic local alignment program, e.g., BLAST. In some embodiments, the sequence alignment program is a pairwise global alignment program. In some embodiments, the pairwise global alignment program is used for protein-protein alignments. In some embodiments, the pairwise global alignment program is Needle. In some embodiments, the sequence alignment program is a multiple alignment program. In some embodiments, the multiple alignment program is MAFFT. In some embodiments, the sequence alignment program is a whole genome alignment program. In some embodiments, the whole genome alignment is performed using BLASTN. In some embodiments, BLASTN is utilized without any changes to the default parameters.

According to some embodiments of the invention, the identity is a global identity, i.e., an identity over the entire nucleic acid sequences of the invention and not over portions thereof.

Methods of qualifying which bacteria are present in a tumor microbiome are described herein below. Care should be taken to take a sufficient number of measurements when analyzing which microbes are present in the microbiome to minimize and control for contaminations.

In some embodiments, determining a presence of one or more bacteria or components or products thereof comprises determining a level or set of levels of one or more DNA sequences. In some embodiments, one or more DNA sequences comprises any DNA sequence that can be used to differentiate between different bacterial types. In certain embodiments, one or more DNA sequences comprises 16S rRNA gene sequences. In certain embodiments, one or more DNA sequences comprises 18S rRNA gene sequences. In some embodiments, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 1,000, 5,000 or more sequences are amplified.

In some embodiments, a microbiota sample (e.g. tumor sample) is directly assayed for a presence, a level or set of levels of one or more DNA sequences. In some embodiments, DNA is isolated from a tumor microbiota sample and isolated DNA is assayed for a level or set of levels of one or more DNA sequences. Methods of isolating microbial DNA are well known in the art. Examples include but are not limited to phenol-chloroform extraction and a wide variety of commercially available kits, including QIAamp DNA Stool Mini Kit (Qiagen, Valencia, Calif.).

In some embodiments, a presence, a level or set of levels of one or more DNA sequences is determined by amplifying DNA sequences using PCR (e.g., standard PCR, semi-quantitative, or quantitative PCR). In some embodiments, a level or set of levels of one or more DNA sequences is determined by amplifying DNA sequences using quantitative PCR. These and other basic DNA amplification procedures are well known to practitioners in the art and are described in Ausebel et al. (Ausubel F M, Brent R, Kingston R E, Moore D, Seidman J G, Smith J A, Struhl K (eds). 1998. Current Protocols in Molecular Biology. Wiley: New York).

In some embodiments, DNA sequences are amplified using primers specific for one or more sequence that differentiate(s) individual microbial types from other, different microbial types. In some embodiments, 16S rRNA gene sequences or fragments thereof are amplified using primers specific for 16S rRNA gene sequences. In some embodiments, 18S DNA sequences are amplified using primers specific for 18S DNA sequences.

In some embodiments, a presence, a level or set of levels of one or more 16S rRNA gene sequences is determined using phylochip technology. Use of phylochips is well known in the art and is described in Hazen et al. (“Deep-sea oil plume enriches indigenous oil-degrading bacteria.” Science, 330, 204-208, 2010), the entirety of which is incorporated by reference. Briefly, 16S rRNA genes sequences are amplified and labeled from DNA extracted from a microbiota sample. Amplified DNA is then hybridized to an array containing probes for microbial 16S rRNA genes. Level of binding to each probe is then quantified providing a sample level of microbial type corresponding to 16S rRNA gene sequence probed. In some embodiments, phylochip analysis is performed by a commercial vendor. Examples include but are not limited to Second Genome Inc. (San Francisco, Calif.).

In some embodiments, determining a presence, a level or set of levels of one or more types of microbes or components or products thereof comprises determining a presence, a level or set of levels of one or more microbial RNA molecules (e.g., transcripts). Methods of quantifying levels of RNA transcripts are well known in the art and include but are not limited to northern analysis, semi-quantitative reverse transcriptase PCR, quantitative reverse transcriptase PCR, and microarray analysis.

In some embodiments, determining a presence, a level or set of levels of one or more types of microbes or components or products thereof comprises determining a presence, a level or set of levels of one or more microbial polypeptides. Methods of quantifying polypeptide levels are well known in the art and include but are not limited to Western analysis and mass spectrometry. These and all other basic polypeptide detection procedures are described in Ausebel et al.

In some embodiments, determining a presence, a level or set of levels of one or more types of microbes or components or products thereof comprises determining a presence, a level or set of levels of one or more microbial metabolites. In some embodiments, levels of metabolites are determined by mass spectrometry. In some embodiments, levels of metabolites are determined by nuclear magnetic resonance spectroscopy. In some embodiments, levels of metabolites are determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, levels of metabolites are determined by colorimetry. In some embodiments, levels of metabolites are determined by spectrophotometry.

In certain embodiments, the vaccine comprises at least 1×10³ colony forming units (CFUs), 1×10⁴ colony forming units (CFUs), 1×10⁵ colony forming units (CFUs), 1×10⁶ colony forming units (CFUs), 1×10⁷ colony forming units (CFUs), 1×10⁸ colony forming units (CFUs), 1×109 colony forming units (CFUs), 1×10¹⁰ colony forming units (CFUs) of bacteria of a family/genus/species/strain listed in Tables 1-3.

Methods for producing bacteria may include three main processing steps. The steps are: organism banking, organism production, and preservation.

For banking, the strains included in the bacteria may be (1) isolated directly from a specimen or taken from a banked stock, (2) optionally cultured on a nutrient agar or broth that supports growth to generate viable biomass, and (3) the biomass optionally preserved in multiple aliquots in long-term storage.

In embodiments using a culturing step, the agar or broth may contain nutrients that provide essential elements and specific factors that enable growth. An example would be a medium composed of 20 g/L glucose, 10 g/L yeast extract, 10 g/L soy peptone, 2 g/L citric acid, 1.5 g/L sodium phosphate monobasic, 100 mg/L ferric ammonium citrate, 80 mg/L magnesium sulfate, 10 mg/L hemin chloride, 2 mg/L calcium chloride, 1 mg/L menadione. Another examples would be a medium composed of 10 g/L beef extract, 10 g/L peptone, 5 g/L sodium chloride, 5 g/L dextrose, 3 g/L yeast extract, 3 g/L sodium acetate, 1 g/L soluble starch, and 0.5 g/L L-cysteine HCl, at pH 6.8. A variety of microbiological media and variations are well known in the art (e.g., R. M. Atlas, Handbook of Microbiological Media (2010) CRC Press). Culture media can be added to the culture at the start, may be added during the culture, or may be intermittently/continuously flowed through the culture. The strains in the vaccine may be cultivated alone, as a subset of the microbial composition, or as an entire collection comprising the microbial composition. As an example, a first strain may be cultivated together with a second strain in a mixed continuous culture, at a dilution rate lower than the maximum growth rate of either cell to prevent the culture from washing out of the cultivation.

The inoculated culture is incubated under favorable conditions for a time sufficient to build biomass. For microbial compositions for human use this is often at 37° C. temperature, pH, and other parameter with values similar to the normal human niche. The environment may be actively controlled, passively controlled (e.g., via buffers), or allowed to drift. For example, for anaerobic bacterial compositions, an anoxic/reducing environment may be employed. This can be accomplished by addition of reducing agents such as cysteine to the broth, and/or stripping it of oxygen. As an example, a culture of a bacterial composition may be grown at 37° C., pH 7, in the medium above, pre-reduced with 1 g/L cysteine-HCl.

When the culture has generated sufficient biomass, it may be preserved for banking. The organisms may be placed into a chemical milieu that protects from freezing (adding ‘cryoprotectants’), drying (‘lyoprotectants’), and/or osmotic shock (‘osmoprotectants’), dispensing into multiple (optionally identical) containers to create a uniform bank, and then treating the culture for preservation. Containers are generally impermeable and have closures that assure isolation from the environment. Cryopreservation treatment is accomplished by freezing a liquid at ultra-low temperatures (e.g., at or below −80° C.). Dried preservation removes water from the culture by evaporation (in the case of spray drying or ‘cool drying’) or by sublimation (e.g., for freeze drying, spray freeze drying). Removal of water improves long-term microbial composition storage stability at temperatures elevated above cryogenic. If the microbial composition comprises, for example, spore forming species and results in the production of spores, the final composition may be purified by additional means such as density gradient centrifugation preserved using the techniques described above. Microbial composition banking may be done by culturing and preserving the strains individually, or by mixing the strains together to create a combined bank. As an example of cryopreservation, a microbial composition culture may be harvested by centrifugation to pellet the cells from the culture medium, the supernatant decanted and replaced with fresh culture broth containing 15% glycerol. The culture can then be aliquoted into 1 mL cryotubes, sealed, and placed at −80° C. for long-term viability retention. This procedure achieves acceptable viability upon recovery from frozen storage.

Microbial production may be conducted using similar culture steps to banking, including medium composition and culture conditions. It may be conducted at larger scales of operation, especially for clinical development or commercial production. At larger scales, there may be several subcultivations of the microbial composition prior to the final cultivation. At the end of cultivation, the culture is harvested to enable further formulation into a dosage form for administration. This can involve concentration, removal of undesirable medium components, and/or introduction into a chemical milieu that preserves the microbial composition and renders it acceptable for administration via the chosen route. After drying, the powder may be blended to an appropriate potency, and mixed with other cultures and/or a filler such as microcrystalline cellulose for consistency and ease of handling, and the bacterial composition formulated as provided herein.

In certain aspects, provided are vaccines (i.e. bacterial compositions) for administration to subjects. In some embodiments, the bacteria are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.

The bacteria present in the vaccine may be viable (e.g. capable of propagating when cultured in the appropriate medium, or inside the body, following administration).

In another embodiment, the bacteria present in the vaccine are non-viable.

In still another embodiment, the bacteria are attenuated such that they are not capable of causing disease.

As mentioned, the bacteria of the vaccine disclosed herein express at least one cancer associated antigen.

Cancer-associated antigens are typically short peptides corresponding to one or more antigenic determinants of a protein. The cancer-associated antigen typically binds to a class I or II MHC receptor thus forming a ternary complex that can be recognized by a T-cell bearing a matching T-cell receptor binding to the MHC/peptide complex with appropriate affinity. Peptides binding to MHC class I molecules are typically about 8-14 amino acids in length. T-cell epitopes that bind to MHC class II molecules are typically about 12-30 amino acids in length. In the case of peptides that bind to MHC class II molecules, the same peptide and corresponding T cell epitope may share a common core segment, but differ in the overall length due to flanking sequences of differing lengths upstream of the amino-terminus of the core sequence and downstream of its carboxy terminus, respectively. A T-cell epitope may be classified as an antigen if it elicits an immune response.

The antigens for cancers can be antigens from testicular cancer, ovarian cancer, brain cancer such as glioblastoma, pancreatic cancer, melanoma, lung cancer, prostate cancer, hepatic cancer, breast cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, renal cancer, sarcoma, neuroblastoma, Hodgkins and non-Hodgkins lymphoma and leukemia.

In one embodiment, the cancer-associated antigen is a cancer testis antigen (e.g. a member of the melanoma antigen protein (MAGE) family, Squamous Cell Carcinoma-1 (NY-ESO-1), BAGE (B melanoma antigen), LAGE-1 antigen, Brother of the Regulator of Imprinted Sites (BORIS) and members of the GAGE family).

In another embodiment, the cancer-associated antigen is derived from MART-1/Melan-A protein e.g. (MART1 MHC class I peptides (Melan-A:26-35(L27), ELAGIGILTV; SEQ ID NO: 1) and MHC class II peptides (Melan-A:51-73(RR-23) RNGYRALMDKSLHVGTQCALTRR; SEQ ID NO: 2).

In another embodiment, the cancer-associated antigen is derived from glycoprotein 70, glycoprotein 100 (gp100:25-33 (MHC class I (EGSRNQDWL—SEQ ID NO: 7)) or gp100:44-59 MHC class II (WNRQLYPEWTEAQRLD—SEQ ID NO: 8) peptides).

In still another embodiment, the cancer-associated antigen is derived from tyrosinase, tyrosinase-related protein 1 (TRP1), tyrosinase-related protein 2 (TRP-2) or TRP-2/INT2 (TRP-2/intron2).

In still another embodiment, the cancer-associated antigen comprises MUT30 (mutation in Kinesin family member 18B, Kif18b—PSKPSFQEFVDWENVSPELNSTDQPFL—SEQ ID NO: 9) or MUT44 (cleavage and polyadenylation specific factor 3-like, Cpsf31—EFKHIKAFDRTFANNPGPMVVFATPGM—SEQ ID NO: 10).

In still another embodiment, the cancer-associated antigen is derived from stimulator of prostatic adenocarcinoma-specific T cells-SPAS-1.

In still another embodiment, the cancer-associated antigen is derived from human telomerase reverse transcriptase (hTERT) or hTRT (human telomerase reverse transcriptase).

In still another embodiment, the cancer-associated antigen is derived from ovalbumin (OVA) for example OVA_257-264 MHCI H-2Kb (SIINFEKL—SEQ ID NO: 11) and OVA_323-339 MHCII I-A(d) (ISQAVHAAHAEINEAGR SEQ ID NO: 12), a RAS mutation, mutant oncogenic forms of p53 (TP53) (p53mut (peptide antigen of mouse mutated p53_R172H sequence VVRHCPHHER—SEQ ID NO: 4 (human mutated p53_R175H sequence EVVRHCPHHE—SEQ ID NO: 5)), or from BRAF-V600E peptide (GDFGLATEKSRWSGS—SEQ ID NO: 13).

According to a particular embodiment, the cancer associated antigen is set forth in SEQ ID NO: 11.

In still another embodiment, the cancer-associated antigen is a breast cancer associated disease antigen including but not limited to α-Lactalbumin (α-Lac), Her2/neu, BRCA-2 or BRCA-1 (RNF53), KNG1K438-R457 (kininogen-1 peptide) and C3fS1304-R1320 (peptides that distinguish BRCA1 mutated from other BC and non-cancer mutated BRCA1).

In still another embodiment, the cancer-associated antigen is a colorectal cancer associated disease antigen including but not limited to MUC1, KRAS, CEA (CAP-1-6-D [Asp6]; YLSGADLNL—SEQ ID NO: 14) and Adpgk_R304M MC38 (MHCI-Adpgk: ASMTNMELM SEQ ID NO: 15; MHCII-Adpgk: GIPVHLELASMTNMELMSSIVHQQVFPT SEQ ID NO: 16).

In still another embodiment, the cancer-associated antigen is a pancreatic cancer associated disease antigen including but not limited to CEA, CA 19-9, MUC1, KRAS, p53mut (peptide antigen of mouse mutated p53_R172H sequence VVRHCPHHER—SEQ ID NO: 4 (human mutated p53_R175H sequence EVVRHCPHHE—SEQ ID NO: 5)) and MUC4 or MUC13, MUC3A or CEACAM5, KRAS peptides (e.g. KRAS-G12R, KRAS-G13D, p5-21 sequence KLVVVGAGGVGKSALTI (SEQ ID NO: 17), p5-21 G12D sequence KLVVVGADGVGKSALTI (SEQ ID NO: 18), p17-31 sequence SALTIQLIQNHFVDE (SEQ ID NO: 19), p78-92 sequence FLCVFAINNTKSFED (SEQ ID NO: 20), p156-170 sequence FYTLVREIRKHKEKM (SEQ ID NO: 21), NRAS (e.g. NRAS-Q61R), PI3K (e.g. PIK3CA-H1047R), C-Kit-D816V, and BRCA mutated epitopes YIHTHTFYV (SEQ ID NO: 22) and SQIWNLNPV (SEQ ID NO: 23) HLA-A*02:01 restricted neoepitopes.

In still another embodiment, the cancer-associated antigen is a lung cancer associated disease antigen including but not limited to Sperm Protein 17 (SP17), A-kinase anchor protein 4 (AKAP4) and Pituitary Tumor Transforming Gene 1 (PTTG1), Aurora kinase A, HER2/neu, and p53mut.

In still another embodiment, the cancer-associated antigen is a prostate cancer associated disease antigen such as prostate cancer antigen (PCA), prostate-specific antigen (PSA) or prostate-specific membrane antigen (PSMA).

In still another embodiment, the cancer-associated antigen is a brain cancer, specifically glioblastoma cancer associated disease antigen such as GL261 neoantigen (mImp3 D81N AALLNKLYA—SEQ ID NO: 6).

In another embodiment, the cancer-associated antigen is a neoantigen.

As used herein the term “neoantigen” is an epitope that has at least one alteration that makes it distinct from the corresponding wild-type, parental antigen, e.g., via mutation in a tumor cell or post-translational modification specific to a tumor cell. A neoantigen can include a polypeptide sequence or a nucleotide sequence. A mutation can include a frameshift or nonframeshift indel, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF. A mutation can also include a splice variant. Post-translational modifications specific to a tumor cell can include aberrant phosphorylation. Post-translational modifications specific to a tumor cell can also include a proteasome-generated spliced antigen.

An example of a mutant APC antigen is QATEAERSF (SEQ ID NO: 3).

Examples of BRCA mutated epitopes are YIHTHTFYV (SEQ ID NO: 22) and SQIWNLNPV (SEQ ID NO: 23) HLA-A*02:01 restricted neoepitopes.

An examples of a universal HLA-DR-binding T helper synthetic epitope (AKFVAAWTLKAAA, SEQ ID NO: 311) is the pan DR-biding epitope (PADRE), which is a 13 amino acid peptide that activates CD4+ T cells.

Another contemplated cancer-associated neoantigen is the GL261 neoantigen (mImp3 D81N, sequence AALLNKLYA—SEQ ID NO: 6).

The bacteria described herein are genetically modified to express the cancer associated antigen, intracellularly and/or on the bacterial surface (i.e., genetic surface display). In another embodiment, the bacteria are genetically modified to secrete the cancer associated antigen.

For example, in some embodiments, the bacteria comprises a nucleic acid encoding the cancer-associated antigen operably linked to transcriptional regulatory elements, such as a bacterial promotor. The transcriptional regulatory element can further comprise a secretion signal. In some embodiments, the cancer-associated antigen is constitutively expressed by the bacteria. In some embodiments, the cancer-associated antigen is inducibly expressed by the bacteria (e.g., it is expressed upon exposure to a sugar or an environmental stimulus like low pH or an anaerobic environment). In some embodiments, the bacteria comprises a plurality of nucleic acid sequences that encode for multiple different cancer-associated antigens that can be expressed by the same bacterial cell.

In some embodiments, the bacteria displays a recombinantly produced cancer-associated antigen on its surface using a bacterial surface display system. Examples of bacterial surface display systems include outer membrane protein systems (e.g., LamB, FhuA, Ompl, OmpA, OmpC, OmpT, eCPX derived from OmpX, OprF, and PgsA), surface appendage systems (e.g., F pillin, FimH, FimA, FliC, and FliD), lipoprotein systems (e.g., INP, Lpp-OmpA, PAL, Tat-dependent, and TraT), and virulence factor-based systems (e.g., AIDA-1, EaeA, EstA, EspP, MSP1 a, and invasin). Exemplary surface display systems are described, for example, in van Bloois, E., et al., Trends in Biotechnology, 2011, 29:79-86, which is hereby incorporated by reference.

Examples of bacterial promoters include but are not limited to STM1787 promoter, pepT promoter, pflE promoter, ansB promoter, vhb promoter, FF+20* promoter or p(luxI) promoter.

In some embodiments, the genetically modified bacteria described herein comprise a cancer therapeutic (e.g., the cancer therapeutic is loaded into the bacteria prior to administration to a subject, or is genetically modified to express the cancer therapeutic).

In some embodiments, the cancer therapeutic is loaded into the bacteria by growing the bacteria in a medium that contains a high concentration (e.g., at least 1 mM) of the cancer therapeutic, which leads to either uptake of the cancer therapeutic during cell growth or binding of the cancer therapeutic to the outside of the bacteria. The cancer therapeutic can be taken up passively (e.g. by diffusion and/or partitioning into the lipophilic cell membrane) or actively through membrane channels or transporters. In some embodiments, drug loading is improved by adding additional substances to the growth medium that either increase uptake of the molecule of interest (e.g., Pluronic F-127) or prevent extrusion of the molecules after uptake by the bacterium (e.g., efflux pump inhibitors like Verapamil, Reserpine, Carsonic acid, or Piperine). In some embodiments, the bacteria is loaded with the cancer therapeutic by mixing the bacteria with the cancer therapeutic and then subjecting the mixture to electroporation, for example, as described in Sustarsic M., et al., Cell Biol., 2014, 142(1):113-24, which is hereby incorporated by reference. In some embodiments, the cells can also be treated with an efflux pump inhibitor (see above) after the electroporation to prevent extrusion of the loaded molecules.

In still further embodiments, the bacteria is genetically modified to express the cancer therapeutic.

In some embodiments the bacteria of the vaccine comprise an inhibitory antibody or small molecule directed against the immune checkpoint protein—e.g. anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1 and anti-PDL1.

The present inventors further contemplate that the bacteria of the vaccine may comprise therapeutic agents attached to the outside of the bacteria using an attachment method such as CLICK chemistry. Such methods are further described in US Patent Application No. 20200087703 and US Patent Application No. 20200054739, the contents of which are incorporated herein by reference.

Examples of therapeutic agents include immune modulatory proteins, such as a cytokine. Examples of immune modulating proteins include, but are not limited to, B lymphocyte chemoattractant (“BLC”), C-C motif chemokine 11 (“Eotaxin-1”), Eosinophil chemotactic protein 2 (“Eotaxin-2”), Granulocyte colony-stimulating factor (“G-CSF”), Granulocyte macrophage colony-stimulating factor (“GM-CSF”), 1-309, Intercellular Adhesion Molecule 1 (“ICAM-1”), Interferon gamma (“IFN-gamma”), Interlukin-1 alpha (“IL-1 alpha”), Interlukin-1 beta (“IL-1 beta”), Interleukin 1 receptor antagonist (“IL-1ra”), Interleukin-2 (“IL-2”), Interleukin-4 (“IL-4”), Interleukin-5 (“IL-5”), Interleukin-6 (“IL-6”), Interleukin-6 soluble receptor (“IL-6 sR”), Interleukin-7 (“IL-7”), Interleukin-8 (“IL-8”), Interleukin-10 (“IL-10”), Interleukin-11 (“IL-11”), Subunit beta of Interleukin-12 (“IL-12 p40” or “IL-12 p70”), Interleukin-13 (“IL-13”), Interleukin-15 (“IL-15”), Interleukin-16 (“IL-16”), Interleukin-17 (“IL-17”), Chemokine (C-C motif) Ligand 2 (“MCP-1”), Macrophage colony-stimulating factor (“M-CSF”), Monokine induced by gamma interferon (“MIG”), Chemokine (C-C motif) ligand 2 (“MIP-1 alpha”), Chemokine (C-C motif) ligand 4 (“MIP-1 beta”), Macrophage inflammatory protein-1-delta (“MIP-1 delta”), Platelet-derived growth factor subunit B (“PDGF-BB”), Chemokine (C-C motif) ligand 5, Regulated on Activation, Normal T cell Expressed and Secreted (“RANTES”), TIMP metallopeptidase inhibitor 1 (“TIMP-1”), TIMP metallopeptidase inhibitor 2 (“TIMP-2”), Tumor necrosis factor, lymphotoxin-alpha (“TNF alpha”), Tumor necrosis factor, lymphotoxin-beta (“TNF beta”), Soluble TNF receptor type 1 (“sTNFRI”), sTNFRIIAR, Brain-derived neurotrophic factor (“BDNF”), Basic fibroblast growth factor (“bFGF”), Bone morphogenetic protein 4 (“BMP-4”), Bone morphogenetic protein 5 (“BMP-5”), Bone morphogenetic protein 7 (“BMP-7”), Nerve growth factor (“b-NGF”), Epidermal growth factor (“EGF”), Epidermal growth factor receptor (“EGFR”), Endocrine-gland-derived vascular endothelial growth factor (“EG-VEGF”), Fibroblast growth factor 4 (“FGF-4”), Keratinocyte growth factor (“FGF-7”), Growth differentiation factor 15 (“GDF-15”), Glial cell-derived neurotrophic factor (“GDNF”), Growth Hormone, Heparin-binding EGF-like growth factor (“HB-EGF”), Hepatocyte growth factor (“HGF”), Insulin-like growth factor binding protein 1 (“IGFBP-1”), Insulin-like growth factor binding protein 2 (“IGFBP-2”), Insulin-like growth factor binding protein 3 (“IGFBP-3”), Insulin-like growth factor binding protein 4 (“IGFBP-4”), Insulin-like growth factor binding protein 6 (“IGFBP-6”), Insulin-like growth factor 1 (“IGF-1”), Insulin, Macrophage colony-stimulating factor (“M-CSF R”), Nerve growth factor receptor (“NGF R”), Neurotrophin-3 (“NT-3”), Neurotrophin-4 (“NT-4”), Osteoclastogenesis inhibitory factor (“Osteoprotegerin”), Platelet-derived growth factor receptors (“PDGF-AA”), Phosphatidylinositol-glycan biosynthesis (“PIGF”), Skp, Cullin, F-box containing comples (“SCF”), Stem cell factor receptor (“SCF R”), Transforming growth factor alpha (“TGFalpha”), Transforming growth factor beta-1 (“TGF beta 1”), Transforming growth factor beta-3 (“TGF beta 3”), Vascular endothelial growth factor (“VEGF”), Vascular endothelial growth factor receptor 2 (“VEGFR2”), Vascular endothelial growth factor receptor 3 (“VEGFR3”), VEGF-D 6Ckine, Tyrosine-protein kinase receptor UFO (“Axl”), Betacellulin (“BTC”), Mucosae-associated epithelial chemokine (“CCL28”), Chemokine (C-C motif) ligand 27 (“CTACK”), Chemokine (C-X-C motif) ligand 16 (“CXCL16”), C-X-C motif chemokine 5 (“ENA-78”), Chemokine (C-C motif) ligand 26 (“Eotaxin-3”), Granulocyte chemotactic protein 2 (“GCP-2”), GRO, Chemokine (C-C motif) ligand 14 (“HCC-1”), Chemokine (C-C motif) ligand 16 (“HCC-4”), Interleukin-9 (“IL-9”), Interleukin-17 F (“IL-17F”), Interleukin-18-binding protein (“IL-18 BPa”), Interleukin-28 A (“IL-28A”), Interleukin 29 (“IL-29”), Interleukin 31 (“IL-31”), C-X-C motif chemokine 10 (“IP-10”), Chemokine receptor CXCR3 (“I-TAC”), Leukemia inhibitory factor (“LIF”), Light, Chemokine (C motif) ligand (“Lymphotactin”), Monocyte chemoattractant protein 2 (“MCP-2”), Monocyte chemoattractant protein 3 (“MCP-3”), Monocyte chemoattractant protein 4 (“MCP-4”), Macrophage-derived chemokine (“MDC”), Macrophage migration inhibitory factor (“MIF”), Chemokine (C-C motif) ligand 20 (“MIP-3 alpha”), C-C motif chemokine 19 (“MIP-3 beta”), Chemokine (C-C motif) ligand 23 (“MPIF-1”), Macrophage stimulating protein alpha chain (“MSPalpha”), Nucleosome assembly protein 1-like 4 (“NAP-2”), Secreted phosphoprotein 1 (“Osteopontin”), Pulmonary and activation-regulated cytokine (“PARC”), Platelet factor 4 (“PF4”), Stroma cell-derived factor-1 alpha (“SDF-1 alpha”), Chemokine (C-C motif) ligand 17 (“TARC”), Thymus-expressed chemokine (“TECK”), Thymic stromal lymphopoietin (“TSLP 4-IBB”), CD 166 antigen (“ALCAM”), Cluster of Differentiation 80 (“B7-1”), Tumor necrosis factor receptor superfamily member 17 (“BCMA”), Cluster of Differentiation 14 (“CD14”), Cluster of Differentiation 30 (“CD30”), Cluster of Differentiation 40 (“CD40 Ligand”), Carcinoembryonic antigen-related cell adhesion molecule 1 (biliary glycoprotein) (“CEACAM-1”), Death Receptor 6 (“DR6”), Deoxythymidine kinase (“Dtk”), Type 1 membrane glycoprotein (“Endoglin”), Receptor tyrosine-protein kinase erbB-3 (“ErbB3”), Endothelial-leukocyte adhesion molecule 1 (“E-Selectin”), Apoptosis antigen 1 (“Fas”), Fms-like tyrosine kinase 3 (“Flt-3L”), Tumor necrosis factor receptor superfamily member 1 (“GITR”), Tumor necrosis factor receptor superfamily member 14 (“HVEM”), Intercellular adhesion molecule 3 (“ICAM-3”), IL-1 R4, IL-1 RI, IL-10 Rbeta, IL-17R, IL-2Rgamma, IL-21R, Lysosome membrane protein 2 (“LIMPII”), Neutrophil gelatinase-associated lipocalin (“Lipocalin-2”), CD62L (“L-Selectin”), Lymphatic endothelium (“LYVE-1”), MEC class I polypeptide-related sequence A (“MICA”), MEC class I polypeptide-related sequence B (“MICB”), NRG1-beta1, Beta-type platelet-derived growth factor receptor (“PDGF Rbeta”), Platelet endothelial cell adhesion molecule (“PECAM-1”), RAGE, Hepatitis A virus cellular receptor 1 (“TIM-1”), Tumor necrosis factor receptor superfamily member IOC (“TRAIL R3”), Trappin protein transglutaminase binding domain (“Trappin-2”), Urokinase receptor (“uPAR”), Vascular cell adhesion protein 1 (“VCAM-1”), XEDARActivin A, Agouti-related protein (“AgRP”), Ribonuclease 5 (“Angiogenin”), Angiopoietin 1, Angiostatin, Catheprin S, CD40, Cryptic family protein IB (“Cripto-1”), DAN, Dickkopf-related protein 1 (“DKK-1”), E-Cadherin, Epithelial cell adhesion molecule (“EpCAM”), Fas Ligand (FasL or CD95L), Fcg RIIB/C, FoUistatin, Galectin-7, Intercellular adhesion molecule 2 (“ICAM-2”), IL-13 R1, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, Neuronal cell adhesion molecule (“NrCAM”), Plasminogen activator inhibitor-1 (“PAI-1”), Platelet derived growth factor receptors (“PDGF-AB”), Resistin, stromal cell-derived factor 1 (“SDF-1 beta”), sgp130, Secreted frizzled-related protein 2 (“ShhN”), Sialic acid-binding immunoglobulin-type lectins (“Siglec-5”), ST2, Transforming growth factor-beta 2 (“TGF beta 2”), Tie-2, Thrombopoietin (“TPO”), Tumor necrosis factor receptor superfamily member 10D (“TRAIL R4”), Triggering receptor expressed on myeloid cells 1 (“TREM-1”), Vascular endothelial growth factor C (“VEGF-C”), VEGFR1Adiponectin, Adipsin (“AND”), Alpha-fetoprotein (“AFP”), Angiopoietin-like 4 (“ANGPTL4”), Beta-2-microglobulin (“B2M”), Basal cell adhesion molecule (“BCAM”), Carbohydrate antigen 125 (“CA125”), Cancer Antigen 15-3 (“CA15-3”), Carcinoembryonic antigen (“CEA”), cAMP receptor protein (“CRP”), Human Epidermal Growth Factor Receptor 2 (“ErbB2”), Follistatin, Follicle-stimulating hormone (“FSH”), Chemokine (C-X-C motif) ligand 1 (“GRO alpha”), human chorionic gonadotropin (“beta HCG”), Insulin-like growth factor 1 receptor (“IGF-1 sR”), IL-1 sRII, IL-3, IL-18 Rb, IL-21, Leptin, Matrix metalloproteinase-1 (“MMP-1”), Matrix metalloproteinase-2 (“MMP-2”), Matrix metalloproteinase-3 (“MMP-3”), Matrix metalloproteinase-8 (“MMP-8”), Matrix metalloproteinase-9 (“MMP-9”), Matrix metalloproteinase-10 (“MMP-10”), Matrix metalloproteinase-13 (“MMP-13”), Neural Cell Adhesion Molecule (“NCAM-1”), Entactin (“Nidogen-1”), Neuron specific enolase (“NSE”), Oncostatin M (“OSM”), Procalcitonin, Prolactin, Prostate specific antigen (“PSA”), Sialic acid-binding Ig-like lectin 9 (“Siglec-9”), ADAM 17 endopeptidase (“TACE”), Thyroglobulin, Metalloproteinase inhibitor 4 (“TIMP-4”), TSH2B4, Disintegrin and metalloproteinase domain-containing protein 9 (“ADAM-9”), Angiopoietin 2, Tumor necrosis factor ligand superfamily member 13/Acidic leucine-rich nuclear phosphoprotein 32 family member B (“APRIL”), Bone morphogenetic protein 2 (“BMP-2”), Bone morphogenetic protein 9 (“BMP-9”), Complement component 5a (“C5a”), Cathepsin L, CD200, CD97, Chemerin, Tumor necrosis factor receptor superfamily member 6B (“DcR3”), Fatty acid-binding protein 2 (“FABP2”), Fibroblast activation protein, alpha (“FAP”), Fibroblast growth factor 19 (“FGF-19”), Galectin-3, Hepatocyte growth factor receptor (“HGF R”), IFN-gammalpha/beta R2, Insulin-like growth factor 2 (“IGF-2”), Insulin-like growth factor 2 receptor (“IGF-2 R”), Interleukin-1 receptor 6 (“IL-1R6”), Interleukin 24 (“IL-24”), Interleukin 33 (“IL-33”, Kallikrein 14, Asparaginyl endopeptidase (“Legumain”), Oxidized low-density lipoprotein receptor 1 (“LOX-1”), Mannose-binding lectin (“MBL”), Neprilysin (“NEP”), Notch homolog 1, translocation-associated (Drosophila) (“Notch-1”), Nephroblastoma overexpressed (“NOV”), Osteoactivin, Programmed cell death protein 1 (“PD-1”), N-acetylmuramoyl-L-alanine amidase (“PGRP-5”), Serpin A4, Secreted frizzled related protein 3 (“sFRP-3”), Thrombomodulin, Tolllike receptor 2 (“TLR2”), Tumor necrosis factor receptor superfamily member 10A (“TRAIL R1”), Transferrin (“TRF”), WIF-1ACE-2, Albumin, AMICA, Angiopoietin 4, B-cell activating factor (“BAFF”), Carbohydrate antigen 19-9 (“CA19-9”), CD 163, Clusterin, CRT AM, Chemokine (C-X-C motif) ligand 14 (“CXCL14”), Cystatin C, Decorin (“DCN”), Dickkopf-related protein 3 (“Dkk-3”), Delta-like protein 1 (“DLL1”), Fetuin A, Heparin-binding growth factor 1 (“aFGF”), Folate receptor alpha (“FOLR1”), Furin, GPCR-associated sorting protein 1 (“GASP-1”), GPCR-associated sorting protein 2 (“GASP-2”), Granulocyte colony-stimulating factor receptor (“GCSF R”), Serine protease hepsin (“HAI-2”), Interleukin-17B Receptor (“IL-17B R”), Interleukin 27 (“IL-27”), Lymphocyte-activation gene 3 (“LAG-3”), Apolipoprotein A-V (“LDL R”), Pepsinogen I, Retinol binding protein 4 (“RBP4”), SOST, Heparan sulfate proteoglycan (“Syndecan-1”), Tumor necrosis factor receptor superfamily member 13B (“TACI”), Tissue factor pathway inhibitor (“TFPI”), TSP-1, Tumor necrosis factor receptor superfamily, member 10b (“TRAIL R2”), TRANCE, Troponin I, Urokinase Plasminogen Activator (“uPA”), Cadherin 5, type 2 or VE-cadherin (vascular endothelial) also known as CD144 (“VE-Cadherin”), WNT1-inducible-signaling pathway protein 1 (“WISP-1”), and Receptor Activator of Nuclear Factor .kappa. B (“RANK”). The immune modulatory protein can be made recombinantly using methods known to one skilled in the art. The immune modulatory protein can be presented on the surface of a bacterium using bacterial surface display, where the bacterium expresses a genetically engineered protein-protein fusion of e.g., a membrane protein and the immune modulatory protein.

The bacteria of the vaccine of the present invention may serve as an adjuvant, thereby rendering the use of additional adjuvant not relevant.

In one embodiment, the vaccine is devoid of adjuvant (other than the bacteria itself).

In another embodiment, the vaccine comprises an adjuvant additional to the bacteria.

Adjuvants are substance that can be added to an immunogen or to a vaccine formulation to enhance the immune-stimulating properties of the immunogenic moiety. Examples of adjuvants or agents that may add to the effectiveness of proteinaceous immunogens include aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, and oil-in-water emulsions. A particular type of adjuvant is muramyl dipeptide (MDP) and various MDP derivatives and formulations, e.g., N-acetyl-D-glucosaminyl-(.beta.1-4)-N-acetylmuramyl-L-alanyl-D-isoglutami-ne (GMDP) (Hornung, R L et al. Ther Immunol 1995 2:7-14) or ISAF-1 (5% squalene, 2.5% pluronic L121, 0.2% Tween 80 in phosphate-buffered solution with 0.4 mg of threonyl-muramyl dipeptide; see Kwak, L W et al. (1992) N. Engl. J. Med., 327:1209-1238). Other useful adjuvants are, or are based on, cholera toxin, bacterial endotoxin, lipid X, whole organisms or subcellular fractions of the bacteria Propionobacterium acnes or Bordetella pertussis, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponin and saponin derivatives such as QS21 (White, A. C. et al. (1991) Adv. Exp. Med. Biol., 303:207-210) which is now in use in the clinic (Helling, F et al. (1995) Cancer Res., 55:2783-2788; Davis, T A et al. (1997) Blood, 90: 509), levamisole, DEAE-dextran, blocked copolymers or other synthetic adjuvants. A number of adjuvants are available commercially from various sources, for example, Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.), Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Aluminum is approved for human use.

As mentioned, the vaccines described herein may be used to treat and/or prevent cancer.

As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition.

According to a particular embodiment, the term preventing refers to substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

Particular subjects which are treated are mammalian subjects—e.g. humans.

According to a particular embodiment, the subject has been diagnosed as having cancer.

Cancer

The term “cancer” as used herein refers to an uncontrolled, abnormal growth of a host's own cells which may lead to invasion of surrounding tissue and potentially tissue distal to the initial site of abnormal cell growth in the host. Major classes include carcinomas which are cancers of the epithelial tissue (e.g., skin, squamous cells); sarcomas which are cancers of the connective tissue (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias which are cancers of blood forming tissue (e.g., bone marrow tissue); lymphomas and myelomas which are cancers of immune cells; and central nervous system cancers which include cancers from brain and spinal tissue. “Cancer(s),” “neoplasm(s),” and “tumor(s)” are used herein interchangeably. As used herein, “cancer” refers to all types of cancer or neoplasm or malignant tumors including leukemias, carcinomas and sarcomas, whether new or recurring.

Specific examples of cancers that may be treated using the bacteria described herein include, but are not limited to adrenocortical carcinoma, hereditary; bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptibility to; breast cancer, type 4; breast cancer, type 4; breast cancer-1; breast cancer-3; breast-ovarian cancer; triple negative breast cancer, Burkitt's lymphoma; cervical carcinoma; colorectal adenoma; colorectal cancer; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis, type 3; colorectal cancer, hereditary nonpolyposis, type 6; colorectal cancer, hereditary nonpolyposis, type 7; dermatofibrosarcoma protuberans; endometrial carcinoma; esophageal cancer; gastric cancer, fibrosarcoma, glioblastoma multiforme; glomus tumors, multiple; hepatoblastoma; hepatocellular cancer; hepatocellular carcinoma; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, acute myeloid, with eosinophilia; leukemia, acute nonlymphocytic; leukemia, chronic myeloid; Li-Fraumeni syndrome; liposarcoma, lung cancer; lung cancer, small cell; lymphoma, non-Hodgkin's; lynch cancer family syndrome II; male germ cell tumor; mast cell leukemia; medullary thyroid; medulloblastoma; melanoma, malignant melanoma, meningioma; multiple endocrine neoplasia; multiple myeloma, myeloid malignancy, predisposition to; myxosarcoma, neuroblastoma; osteosarcoma; osteocarcinoma, ovarian cancer; ovarian cancer, serous; ovarian carcinoma; ovarian sex cord tumors; pancreatic cancer; pancreatic endocrine tumors; paraganglioma, familial nonchromaffin; pilomatricoma; pituitary tumor, invasive; prostate adenocarcinoma; prostate cancer; renal cell carcinoma, papillary, familial and sporadic; retinoblastoma; rhabdoid predisposition syndrome, familial; rhabdoid tumors; rhabdomyosarcoma; small-cell cancer of lung; soft tissue sarcoma, squamous cell carcinoma, basal cell carcinoma, head and neck; T-cell acute lymphoblastic leukemia; Turcot syndrome with glioblastoma; tylosis with esophageal cancer; uterine cervix carcinoma, Wilms' tumor, type 2; and Wilms' tumor, type 1, and the like.

According to a particular embodiment, the cancer is cancer is selected from the group consisting of breast, melanoma, pancreatic cancer, ovarian cancer, bone cancer and brain cancer (e.g. glioblastoma).

According to another embodiment, the cancer is melanoma.

Malignant melanomas are clinically recognized based on the ABCD(E) system, where A stands for asymmetry, B for border irregularity, C for color variation, D for diameter >5 mm, and E for evolving. Further, an excision biopsy can be performed in order to corroborate a diagnosis using microscopic evaluation. Infiltrative malignant melanoma is traditionally divided into four principal histopathological subgroups: superficial spreading melanoma (SSM), nodular malignant melanoma (NMM), lentigo maligna melanoma (LMM), and acral lentiginous melanoma (ALM). Other rare types also exists, such as desmoplastic malignant melanoma. A substantial subset of malignant melanomas appear to arise from melanocytic nevi and features of dysplastic nevi are often found in the vicinity of infiltrative melanomas. Melanoma is thought to arise through stages of progression from normal melanocytes or nevus cells through a dysplastic nevus stage and further to an in situ stage before becoming invasive. Some of the subtypes evolve through different phases of tumor progression, which are called radial growth phase (RGP) and vertical growth phase (VGP).

In a particualar embodiment, the melanoma is resistant to treatment with inhibitors of BRAF and/or MEK.

The tumor may be a primary tumor or a secondary tumor (i.e. metastasized tumor).

The compositions may be administered using any route such as for example oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT), subtumoral (ST), peritumoral (PT), and subcutaneous (SC) administration. The pharmaceutical compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intra-arterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In preferred embodiments, the pharmaceutical compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.

According to another aspect of the present invention there is provided a method of treating cancer of a subject in need thereof comprising administering to the subject a therapeutically effective amount of:

• • (i) a first vaccine comprising a first bacteria which is genetically modified to express at least one cancer-associated antigen; and subsequently;
  • (ii) a second vaccine comprising a second bacteria which is genetically modified to express at least one cancer-associated antigen, thereby treating the cancer.

The present invention contemplates at least 2 different vaccination cycles for the treatment of cancer, wherein at least one of the vaccination cycles includes one strain of genetically modified bacteria and at least another of the vaccination cycles includes a second (non-identical) genetically modified strain of bacteria. The two strains of bacteria may be genetically modified to express the same cancer associated antigens or different cancer associated antigens. Additionally, or alternatively, the present inventors contemplate at least one of the vaccination cycles includes viable bacteria (e,g, the first vaccination) and at least another of the vaccination cycles (e.g. a subsequent vaccination) includes attenuated (or dead) bacteria.

The vaccine of the present invention may be administered with additional anti-cancer agents.

In some embodiments the additional anti-cancer agent is an inhibitory antibody or small molecule directed against the immune checkpoint protein—e.g. anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1 and anti-PDL1.

Other contemplated anti-cancer agents which may be administered to the subject in combination with the bacteria described herein include, but are not limited to Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Taxol; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofuirin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Additional antineoplastic agents include those disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner), and the introduction thereto, 1202-1263, of Goodman and Gilman's “The Pharmacological Basis of Therapeutics”, Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division).

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, C T (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, C A (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Materials and Methods

Plasmids:

To generate backbone plasmids for Salmonella typhimurium strains, Ssph2 promoter and secretion signal (aa:1-200), or the pagC promoter and Ssph1 secretion signal (aa:1-208) were amplified from the Salmonella typhimurium attenuated strain VNP20009. Ssph2 and pagC-Ssph1 were inserted into pQE60 by NEBbuilder cloning kit (cat. E5520S).

Proteins of interest were fused with either Ssph1 or Ssph2. To generate a backbone plasmid for Pseudomonas aeruginosa, proteins of interest were fused with the N-terminal 54 amino acids of ExoS in plasmid pEAI3-S54 (a courtesy of Bertrand Toussaint, PMID: 17010670). To generate a backbone plasmid for Bacillus Subtilis spores, proteins of interest were fused with CotC (amplified from Bacillus Subtilis 168) and cloned into pDG364 plasmid. In addition, 6His tag element was inserted to allow detection of the protein product.

Neoantigens:

To obtain a neoantigen of B16-OVA tumors, the C-terminal of Ovalbumin (aa 252-386) was amplified from pcDNA-OVA (Addgene 64599). The amplified oligo contains the sequence which corresponds to SIINFEKL (SEQ ID NO: 11), the epitope of Ovalbumin.

To obtain a neoantigen of MC38 tumors, a section of Adpgk (aa 289-421) was amplified from cDNA of MC38 cells. The amplified oligo contains a sequence which corresponds to a validated neoantigen of MC38, based on Yadav et al. (PMID: 25428506).

Both neoantigens were inserted to the backbone plasmids by NEBuilder cloning kit.

Bacteria:

The attenuated Salmonella typhimurium strains VNP20009 (also named YS1646, ATCC, cat. 202165) and STM3120 were transformed with the relevant plasmids by electroporation. Briefly, bacteria were cultured to OD of 0.6-0.8, washed 3 times with Hepes 1 mM and suspended in 10% glycerol in DDW. Suspension was electroporated with 0.2 cm, cuivette (BioRad, EC2) and moved to 1 ml cold SOC. Following 1 hour incubation in 37° C., bacteria were seeded on LB agar plate containing ampicilin. Selected colonies were verified by Sanger sequencing and Western blot using anti-6His tag (Cell Signalling, 2365S).

The attenuated Pseudomonas aeruginosa (CHA-OST) was transformed as described by Diver et al. PMID: 2126169. The Bacillus Subtilis strain PY79 was transformed following incubation in minimal medium and 0.01M MGSO₄ in DDW (MC: 80 mM K₂HPO₄, 30 mM KH₂PO₄, 2% Glucose, 30 mM Trisodium citrate, 22 μg/ml Ferric ammonium citrate, 0.1% Casein Hydrolysate (CAA), 0.2% potassium glutamate) for 3 hours to induce competent bacteria. Next, plasmid pDG364 which contains an antigen fused to CotC protein was cut with Xba and incubated with competent bacteria for 3 hours. Upon integration into the amylase gene, colonies were selected by resistance to chloramphenicol 5 μg/ml.

In all transformations, selected colonies were verified by Sanger sequencing and Western blot using anti-6His tag (Cell Signalling, 2365S).

Freezing Working Stocks of Salmonella typhimurium:

Exponentially growing culture (OD 0.6-0.8), was washed twice in cold PBS. Bacteria pellet was suspended in 25% glycerol in PBS. A sample from the bacterial stock was serially diluted and seeded on LB agar plate, while the rest of the pool was aliquoted and stored in −80° C. To verify viability of bacteria, a frozen aliquot was defrosted and seeded on LB agar plate. Recovery rate following freezing was quantified by calculating the ratio of frozen/fresh CFU count. Calculation of bacteria dosage in mice experiment was based on the CFU count of the frozen culture.

Sporulation of Bacillus Subtilis PY79:

PY79 were grown in LB, at 37° C. to OD 0.8. LB medium was removed and replaced by half volume of DSM exhaustion medium. Culture was incubated at 37° C., whilst shaking for 60 hrs. Finally, bacteria were washed twice in cold water. To quantify spores, and sporulation rate, a sample from the washed sample was seeded on LB agar plate pre- and post 1 hour heating at 65° C. The ration of heated/non heated CFU count is indicative of sporulation rate. Exhaustion medium preparation (per 1 liter): dissolve 8g Difco nutrient broth (BD, cat. 234000), 1 g KCl and 1 mM MgSO₄ in DDW. Titrate with NaOH to PH7.6 and autoclave. Before usage, add 10 μM MnCl₂, 1 mM Ca(NO₃)₂ and 1 mM FeSO₄.

Mice Models:

B16-OVA mouse melanoma cell line (10⁶) or MC38 mouse CRC cell line (10⁵) were injected s.c. to the right flank of 7 weeks C57BL/6 females. Tumor volume was calculated as width{circumflex over ( )}2*length/2.

Immune Profiling of Splenocytes by FACS:

Freshly resected spleens were mashed on a 70 micron strainer into cold PBS. To lyse red blood cells, the splenocytes were incubated with ACK lysis buffer (Quality Biological, cat. 118-156-101), then washed thoroughly in PBS and suspended in FACS labeling buffer. 100 μl of splenocytes were incubated for 1 hour at 4° C. with a mixture containing Fc blocker (BD, cat. 553142, 1:100), SIINFEKL (SEQ ID NO: 11) Tetramer (NIH Tetramer Core Facility, 1:500), anti-CD4 (BioLegend, cat. 100438, 1:800), anti-CD8 (Invitrogen, cat. 2021-05-05, 1:400), anti CD3 (Invitrogen, cat. 2023-07-31, 1:1000) and Brilliant Buffer (BD, cat. 566349, 1:5). Next, cells were washed twice in labeling buffer and fixed with CytoFix/CytoPerm solution (BD, cat. 51-2090KZ) for 20 mins at 4° C. Finally, cells were washed twice in Perm/Wash buffer (BD, cat. 51-2091KZ, diluted in DDW 1:10) suspended in labeling buffer and subjected to FACS.

Quantification of Activated CD8 T Cells by Peptide Stimulation

Splenocytes were produced as described above. Next, splenocytes were incubated with OVA peptide (final conc. 2.5 μg/ml) for 2 hours at 37° C. Next, Brafeldin A (BD, 51-2301kz) was added to the cells and incubated for additional 4 hours at 4° C. FACS staining for CD3, CD8 and INFg were preformed the next day as described above.

Ex Vivo Killing Assay

MC38 or B16-OVA cells were seeded on 48 well plate. Cells were stained with CFSE (5 uM) for 20 min at 37° C., then quenched with culture medium (RPMI with 10% FCS) for 10 min at 37° C. and washed twice with culture medium.

The next day, spleens were resected as described above and cells were counted. Next, 10⁵ splenocytes were co-cultured with the tumor cells and incubated for 72 hours at 37° C.

Following incubation, FACS staining for DEAD/LIVE (Invitrogen, L34962) and CFSE positive (tumor cells) were preformed the next day as described.

IFNg Quantification by ELISA:

To quantify serum level of IFNg, mice were bled into Eppendorf tube containing 20 μl Heparin (10 mg/ml). Following centrifugation for 10 mins, 10,000 g, sera were transferred to new tubes for long term storage at −20° C. ELISA was performed according to manufacturer instructions (R&D, cat. DY485) using sera diluted 1:4.

Bacteria Quantification in Liver and Tumor:

Slices of tumors and livers were suspended in sterile tubes containing LB and metal beads. Following vortex for 10 minutes at max speed, 200 μl of sup, were seeded on LB plates with the relevant antibiotics and incubated over night at 37° C.

Results

To demonstrate the efficacy of the Personalized Anti-Cancer Microbiome-Assisted VaccinatioN (PACMAN) vaccine, bacteria expressing the Ovalbumin known neoantigen SIINFEKL (SEQ ID NO: 11) were administered to mice bearing the B16 melanoma tumors which express the Ovalbumin protein (B16-OVA). To generate the OVA expressing bacteria vaccine, the OVA neoantigen SIINFEKL (SEQ ID NO: 11) was fused to Ssph2 secretion signal of Salmonella typhimurium. The resulted oligomer was transformed into the attenuated Salmonella typhimurium strain VNP20009 (VNP-OVA). C57BL/6 mice were injected with 10⁶ B16 OVA expressing cells in the right flank. When tumors reached a volume of −100 mm³, mice were shuffled into the following treatment cohorts: No treatment control, mice receiving the checkpoint inhibitor, anti-PD1 (75 μg per mouse, i.p, once a week), and mice receiving anti-PD1 together with PACMAN-OVA (10⁶ CFU, tail vein). The experiment time line is shown in FIG. 1A. Tumor growth curves from treatment start are shown in FIG. 1B. Tumor growth was completely stopped for 20 days in the PACMAN-OVA cohort versus the exponential growth observed in the other mice cohorts. Following two cycles of immunization, all mice in the VNP-OVA cohort survived significantly longer than the mice in the other cohorts.

To demonstrate the immunogenicity of the vaccine, splenocytes were profiled from mice bearing the B16-OVA tumor following the administration of the PACMAN vaccine. The PACMAN-OVA contained the OVA neoantigen SIINFEKL (SEQ ID NO: 11) fused to Ssph2 secretion signal of Salmonella typhimurium in the attenuated strain STM3120. For a negative control the OVA neoantigen was replaced by the MC38 neoantigen, Adpgk (PACMAN-Adpgk), which is not present in B16-OVA cells.

C57BL/6 mice were injected with 10⁶ B16 OVA expressing cells in the right flank. When tumors reached a volume of ˜100 mm³, mice were shuffled into the following treatment cohorts: No treatment control, mice receiving the checkpoint inhibitor, anti-PD1 (75 μg per mouse, i.p, once a week), mice receiving anti-PD1 together with PACMAN-OVA (10⁶ CFU, tail vein) and mice receiving anti-PD1 with PACMAN-Adpgk (10⁶ CFU, tail vein). Sixteen days post immunization spleens and liver were harvested for further analysis. The results are illustrated in FIGS. 2A-D.

To test the effect of alternate administration of PACMAN-OVA which is based on different attenuated bacteria, mice bearing B16-Ova tumor were vaccinated consecutively with two attenuated bacteria expressing the OVA neoantigen. The first bacteria is the Salmonella attenuated strain STM3120 expressing Ova neoantigen fused to either SshpH2 secretion signal under its endogenous promoter or to Ssph1 secretion signal under pagC promoter which is induced upon phagocytosis by macrophages (STM-OVA). The second bacteria is the Pseudomonas aeruginosa attenuated strain, CHA-OST, expressing Ova neoantigen fused to the secretion signal of ExoS, a toxin of the type-three secretion system (TTSS). ExoS promoter is activated by the TTSS regulator ExsA, following induction by IPTG (CHA-OST-OVA).

C57BL/6 mice were injected with 10⁶ B16 OVA expressing cells in the right flank. When tumors reached a volume of ˜100 mm³, mice were shuffled into the following treatment cohorts: No treatment control, mice receiving the checkpoint inhibitor, anti-PD1 (75 μg per mouse, i.p, once a week), mice receiving anti-PD1 together with STM-SspH2-OVA and mice receiving anti-PD1 together with STM-pagC-SspH1-OVA. The vaccinated mice were treated with 3 doses of STM-OVA (10⁶ CFU, tail vein), followed by anti-PD1 (75 μg per mouse, i.p, once a week). Two weeks since the last STM-OVA vaccine, the mice were treated with 2 doses of CHA-OST-OVA (107 CFU, tail vein, following 3 hours incubation with IPTG 0.5 mM). As illustrated in FIG. 3B, tumor growth was significantly delayed in the mice which were vaccinated with STM-OVA compared to non-vaccinated mice. The majority of tumors in the vaccinated mice regained growth 20-30 days post vaccination, suggesting that the additional injections of the same bacteria did not contribute enough to anti-tumor immunity. Strikingly, vaccinating the mice with CHA-OST-OVA slowed down tumor growth and in some cases even caused exponential decay. As illustrated in FIG. 3C and FIG. 3D, weight decrease is observed following each bacteria administration, however, weight loss is less pronounced after additional vaccination with the same bacteria, further supporting the hypothesis that the mice develop immunity towards the bacteria resulting in fast clearance and thus less effect on body weight.

To test the immune memory of mice vaccinated with PACMAN-OVA, fully cured mice from the experiment described in FIG. 3A were re-challenged with 10⁶ B16-Ova cells and tumor growth was compared to naïve mice injected with the same amount of cells. As illustrated in FIG. 4B, while naïve mice exhibited exponentially growing tumors shortly after injection, re-challenged mice remained tumor free, indicating the establishment of long term immune memory against B16-Ova cells.

To demonstrate the efficacy of the PACMAN vaccine with naturally occurring neoantigen in another mouse model, the effect of bacteria expressing the Adpgk neoantigen of MC38 model was tested on mice bearing the MC38 CRC tumors. To generate the Adpgk expressing bacteria vaccine, the Adpgk neoantigen was fused to Ssph1 secretion signal of Salmonella typhimurium under pagC promoter which is induced upon phagocytosis by macrophages. Next, the oligomer was transformed into the attenuated Salmonella typhimurium strain VNP20009 (PACMAN-Adpgk). C57BL/6 mice were injected with 10⁵ MC38 cells in the right flank. When tumors reached a volume of ˜100 mm³, mice were shuffled into the following treatment cohorts: mice receiving the checkpoint inhibitor, anti-PD1 (75 μg per mouse, i.p, once a week), mice receiving anti-PD1 together with VNP20009 and mice receiving anti-PD1 together with PACMAN-Adpgk (10⁶ CFU, tail vein).

To test the immune memory of mice vaccinated with PACMAN-Adpgk, the mice exhibiting full cure following vaccination with PACKMAN-Adpgk or VNP20009 (w/o adpgk) were re-challenged with 10⁵ MC38 cells and tumor growth was compared to naïve mice injected with the same amount of cells. While naïve mice exhibited exponentially growing tumors shortly after injection, re-challenged mouse which was vaccinated with PACKMAN-Adpgk remained tumor free, indicating the establishment of long term immune memory against MC38 cells. Of note, the fully cured mouse following vaccination only with the VNP20009, exhibited tumor growth following re-challenge indicating that the immune memory was a consequence of Adpgk presentation by the bacteria (FIG. 5C).

To demonstrate selective homing of Salmonella to MC38 tumors, attenuated Salmonella (STM3120) was injected to the tail vein of mice bearing the MC38 CRC tumors at the indicated numbers. After 9 days, tumors, livers and spleens were resected and vigorously shaken in 1 ml LB and a metal ball. Supernatant was seeded on LB plates and colonies were counted following 24 hrs incubation at 37° C. CFU was normalized to the dilution factor and tissue mass. For 1×10⁶, 1×10⁵ N=4, for 1×10⁴ N=3. As illustrated in FIG. 6, the bacteria selectively homed to the tumors as compared to livers and spleens.

To compare the maximal tolerable dose of attenuated Salmonella (STM3120) vs parental Salmonella (14028), Salmonella were injected to the tail vein at various concentrations and body weight was monitored. As illustrated in FIG. 7, in all doses but STM3120 1e6, all mice in the cohort (N=4-5) died (indicated by X).

In order to quantify the amount of active T cells following PACMAN vaccination, splenocytes were harvested from the following cohorts: naïve mice (N=3), B16-OVA tumor bearing mice (N=5), mice injected with attenuated Salmonella STM3120 (N=4), B16 OVA tumor bearing mice injected with STM3120 expressing the unrelated neoantigen ADPGK (N=3), B16-OVA tumor bearing mice injected with STM3120 expressing the OVA neoantigen (N=5). In all cases, 1e6 bacteria were injected to the tail vein. Sixteen days post injection, splenocytes were harvested. FIG. 8A illustrates the increase in IFNg positive CD8 T-cells following vaccination with the appropriate neoantigen.

In a further experiment to quantify T cell killing capacity, MC38 or B16-OVA tumor cells were pre-incubated with CFSE (green) to distinguish them from immune cells. Harvested splenocytes were co-cultured with tumor cells. Following 72 hours, dead tumor cells (CFSE positive) were quantified by FACS using Live/dead staining. Significant B16-OVA specific killing was observed in splenocytes originating from mice vaccinated with STM3120 expressing the OVA neoantigen (Two-tail t-test, Pval <0.001).

To demonstrate the immune mediated efficacy of P. aeruginosa based PACMAN vaccine in MC38 colorectal cancer model, the attenuated P. aeruginosa, CHA-OST either naïve or expressing Adpgk neoantigen was injected to the tail vein, followed by anti PD1 treatment. C57BL/6 mice were injected with 1×10⁵ MC38 colorectal cancer cells in the right flank. When tumors reached a volume of ˜100 mm³, mice were injected with CHA-OST naïve or PACMAN-ADPGK (1×10⁶ CFU, i.v) followed by weekly administration of 150 μg anti-PD1, i.p. FIG. 9A is a graphic representation of the treatment protocol. As illustrated in FIG. 9B, only mice which where injected with the PACMAN-ADPGK vaccine showed a full cure. Of note, the cured mouse was re-challenged with MC38 cells, however no tumor growth was observed.

To demonstrate the immune mediated efficacy of Bacillus Subtilis based PACMAN vaccine in MC38 colorectal cancer model, the spores of the lab strain PY79 expressing Adpgk neoantigen were injected to the tail vein or administered orally (os), followed by aPD1 treatment. C57BL/6 mice were injected with 5×10⁵ MC38 colorectal cancer cells in the right flank. When tumors reached a volume of ˜100 mm³, mice were injected with bacillus spores of PACMAN-ADPGK (5×10⁸-1×10⁹ CFU, i.v) or given per os (5×10⁹ CFU, p.o) followed by weekly administration of 150 μg anti-PD1, i.p. FIG. 10A is a graphic representation of the treatment protocol. As illustrated in FIG. 10B, mice which where injected with the PACMAN-ADPGK vaccine showed a full cure.

To demonstrate the immune mediated efficacy of Salmonella based PACMAN vaccine (which has previously been frozen) in MC38 colorectal cancer model, the attenuated Salmonella Typhimurium STM3120 expressing related (Adpgk) and unrelated (OVA) neoantigen was injected to the tail vein followed by aPD1 treatment. C57BL/6 mice were injected with 10⁶ B16 OVA expressing cells in the right flank. When tumors reached a volume of −100 mm³, mice were injected with PACMAN-ADPGK or PACMAN-OVA (3×10⁶ CFU, i.v) followed by weekly administration of 75/150 μg anti-PD1, i.p. FIG. 11A is a graphic representation of the treatment protocol. As illustrated in FIG. 11B, mice treated with PACMAN-ADPGK exhibited a considerable delayed tumor growth.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

1. A method of treating cancer of a subject in need thereof comprising administering by injection to a subject a therapeutically effective amount of a vaccine comprising: i. tumor-homing bacteria being genetically modified to constitutively express at least one neoantigen; andii. a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein said tumor-homing bacteria comprise Salmonella Typhimurium, Pseudomonas aeruginosa or Bacillus Subtillis.

3. The method of claim 1, wherein said tumor-homing bacteria are of a species or genus set forth in any of Tables 1-3.

4. The method of claim 1, wherein a genome of the bacteria comprises a 16S rRNA sequence as set forth in any one of SEQ ID NOs: 24-310.

5. The method of claim 2, wherein said bacteria are an attenuated strain Salmonella Typhimurium 14028 strain STM3120, Salmonella typhimurium 14028 strain STM1414, Pseudomonas aeruginosa strain CHA-OST and/or Bacillus Subtillis strain PY79.

6. The method of claim 2, wherein said bacteria are attenuated VNP20009, Salmonella typhimurium 14028 strain STM3120.

7. The method of claim 2, wherein said bacteria are genetically modified to express a therapeutic protein.

8. The method of claim 7, wherein the therapeutic protein is an immune modulatory protein.

9. The method of claim 8, wherein said therapeutic protein is a cytokine.

10. The method of claim 1, further comprising administering to the subject an additional anti-cancer agent.

11. The method of claim 10, wherein the additional anti-cancer agent is an inhibitory antibody or small molecule directed against an immune checkpoint protein.

12. The method of claim 11, wherein the cancer is selected from a group consisting of breast cancer, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

13. The method of claim 12, wherein said brain cancer comprises glioblastoma.

14. A vaccine comprising tumor-homing bacteria which are genetically modified to constitutively express at least one neoantigen, and a pharmaceutically acceptable carrier.

15. The vaccine of claim 14, wherein said tumor-homing bacteria comprise Salmonella Typhimurium, Pseudomonas aeruginosa or Bacillus Subtillis.

16. The vaccine of claim 15, wherein said bacteria are an attenuated strain selected from Salmonella typhimurium 14028 strain STM3120, Salmonella typhimurium 14028 strain STM1414, Pseudomonas aeruginosa strain CHA-OST, and/or Bacillus subtillis strain PY79.

17. The vaccine of claim 15, wherein said bacteria are live bacteria.

18. The vaccine of claim 15, wherein said bacteria comprise attenuated Salmonella Typhimurium 14028 strain STM3120.

19. The vaccine of claim 15, wherein said bacteria are genetically modified to express a therapeutic protein.

20. The vaccine of claim 19, wherein the therapeutic protein is an immune modulatory protein.

21. The vaccine of claim 20, wherein said immune modulatory protein is a cytokine.