Genetically engineered bacteria for vaccine production

Abstract

Vaccines are disclosed that include a tumor-homing bacterium genetically modified to express at least one cancer-associated antigen and a pharma- ceutically acceptable carrier. Uses thereof are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 150,681, filed February 18, 2021, the entire contents of which are incorporated herein by reference.

[0002] SEQUENCE LISTING STATEMENT A 567,458 byte ASCII file entitled "91248 SequenceListing.txt", dated February 15, 2022, which was submitted concurrently with the filing of this application, is hereby incorporated by reference.

[0003] The present invention, in some embodiments thereof, relates to bacterial vaccines that can be engineered to contain disease-associated antigens. [Background technology]

[0004] Advances in molecular biology, the ability to predict immunogenic neoantigens by next-generation sequencing and predictive algorithms, understanding the lifestyle of pathogenic bacteria, bacterial engineering, and synthetic biology tools have significantly accelerated the rational design of bacteria as antigen-delivery vectors. Being potent immunogens, bacteria can induce very large immune responses against themselves and, therefore, against the neoantigens they deliver. In fact, bacterial vectors carrying antigenic messages can also deliver strong danger signals mediated by pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides, lipoproteins, flagellin, and CpG. PAMPs derived from different classes of pathogens bind to a diverse family of pathogen recognition receptors (PRRs), including Toll-like receptors (TLRs), C-type lectin-like receptors (CLRs), retinoic acid-inducible gene (RIG)-like receptors (RLRs), and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). These interactions by each pathogen activate distinct signaling pathways to differentially activate antigen-presenting cells (APCs) and direct adaptive effector responses in a manner that is specifically tailored to the antigens carried by the microbe and thus the bacteria. Additionally, unique toxins used by bacteria for their own virulence can enhance effector or memory responses.

[0005] 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 September 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 is an example. Summary of the Invention

[0006] According to one aspect of the invention, there is provided a vaccine comprising a tumor-homing bacterium genetically modified to express at least one cancer-associated antigen and a pharma- ceutically acceptable carrier.

[0007] According to one aspect of the present invention, there is provided a method for treating cancer in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a vaccine described in any one of claims 1 to 14 to treat cancer.

[0008] According to one aspect of the present invention, there is provided a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of component (i) below, followed by administration of component (ii) below, thereby treating the cancer. (i) a first vaccine comprising a first bacterium genetically modified to express at least one cancer-associated antigen; and (ii) a second vaccine comprising a second bacterium genetically modified to express at least one cancer-associated antigen.

[0009] According to one aspect of the present invention, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vaccine described herein to treat cancer.

[0010] According to an embodiment of the present invention, the bacterium is a gram-positive bacterium.

[0011] According to an embodiment of the present invention, the bacterium is a gram-negative bacterium.

[0012] According to an embodiment of the invention, the bacteria is aerobic.

[0013] According to an embodiment of the present invention, the bacteria is anaerobic.

[0014] According to an embodiment of the invention, the bacteria is a live bacteria.

[0015] According to an embodiment of the invention, the bacterium is an attenuated bacterium.

[0016] According to an embodiment of the present invention, the bacterium is a bacterium of a species or genus listed in any one of Tables 1 to 3.

[0017] According to an embodiment of the present invention, the bacterial genome comprises a 16S rRNA sequence set forth in any one of SEQ ID NOs: 24-310.

[0018] According to an embodiment of the invention, the cancer associated antigen is a neoantigen.

[0019] According to an embodiment of the invention, the bacteria is genetically modified to express a therapeutic protein.

[0020] According to an embodiment of the invention, the therapeutic protein is a cytokine.

[0021] According to an embodiment of the invention, the vaccine is free of aluminium salts.

[0022] According to an embodiment of the invention, the carrier is adjuvant-free.

[0023] According to an embodiment of the invention, the first bacterium is a live bacterium and the second bacterium is a dead bacterium.

[0024] According to an embodiment of the invention, the first bacterium comprises a first bacterial strain genetically modified to express a cancer associated antigen, and the second bacterium comprises a second bacterial strain non-identical to the first bacterial strain, the second bacterium being genetically modified to express a cancer associated antigen.

[0025] According to an embodiment of the invention, the cancer is selected from the group consisting of breast cancer, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer, and brain cancer.

[0026] According to an embodiment of the invention, the brain cancer comprises glioblastoma.

[0027] According to an embodiment of the invention, at least one cancer-associated antigen of the first vaccine is identical to at least one cancer-associated antigen of the second vaccine.

[0028] According to an embodiment of the invention, at least one cancer-associated antigen of the first vaccine is non-identical to at least one cancer-associated antigen of the second vaccine.

[0029] According to an embodiment of the invention, the cancer is selected from the group consisting of breast cancer, melanoma, colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0030] According to an embodiment of the invention, the brain cancer comprises glioblastoma.

[0031] 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 this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this invention, exemplary methods and / or materials are described below. In case of conflict, the present patent specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not necessarily intended to be limiting.

[0032] Certain embodiments of the present invention are described herein, by way of example only, with reference to the accompanying drawings, in which: Figure 1 is a schematic diagram of a method for manufacturing a semiconductor device according to the present invention; Figure 2 is a schematic diagram of a semiconductor device according to the present invention; Figure 3 is a schematic diagram of a semiconductor device according to the present invention; Figure 4 is a schematic diagram of a semiconductor device according to the present invention; [Brief description of the drawings]

[0033] [Figure 1] 1A-1B. Long-term efficacy of vaccination with OVA-expressing bacteria against the B16-OVA tumor model. (A) Experimental timeline. (B) Tumor growth curves from the start of treatment. [Figure 2-1]2A-2G in Figure 2. Short-term efficacy and immunogenicity of vaccination with OVA-expressing bacteria against the B16-OVA tumor model. (A) Experimental timeline. (B) Tumor growth curves from the start of treatment. N=5 in all mouse cohorts. (C) Percentage of tumor volume at day 16 relative to day 0. P values ​​were determined by two-tailed Mann-Whitney test. The percentage of mice completely cured per cohort is shown below. (D) Mice from cohorts treated with anti-PD1 or PACMAN-OVA together with anti-PD1. Tumors in mice treated with PACMAN-OVA gradually disappeared, whereas tumors in mice treated with anti-PD1 alone continued to grow exponentially. (E) CFU enumeration of tumor and liver extracts. At day 16 post-vaccination, tumor- and liver-derived bacteria were inoculated onto LB plates for AMP resistance. CFU enumeration and tumor volume were obtained for each mouse. Of note, 4 out of 5 mice in the PACMAN-OVA cohort showed complete clearance of the bacteria. The mouse with the largest tumor had bacteria present, suggesting that the tumor tissue allows bacterial growth. (F) Serum from the mouse cohort was subjected to IFNg ELISA. The PACMAN-OVA cohort exerted the highest IFNg serum levels, indicating high systemic immune activity. The green dot represents mouse 836 in F. Mouse 836 was the only case in which bacteria were present in the liver, likely resulting in the highest serum levels of IFNg. (G) Quantification of SIINFEKL (SEQ ID NO: 11)-specific TCR by flow cytometry. To quantify neoantigen-specific T cell clones, splenocytes were incubated with tetramers presenting the OVA neoantigen (SIINFEKL-SEQ ID NO: 11)) and allowed to bind to a fluorescent dye. Thus, splenocytes positive for the tetramer dye bear TCRs capable of binding to the OVA neoantigen. Following FACS, the percentage of SIINFEKEL (SEQ ID NO: 11)-positive T cells in the CD3+CD8+ population was highest among mice vaccinated with PACMAN-OVA. P values ​​were obtained using two-tailed Mann-Whitney tests. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 3-1]3A-3D. Alternating vaccination with different bacterial vaccines overcomes adaptive immunity. (A) Timetable of the experiment. (B) Tumor growth curves. (C) Weight change (percentage of initial weight) over 24 days after the start of treatment. Mouse cohorts: STM3210+aPD1 (N=3), STM-pagC-SspH1-OVA+aPD1 (N=3), STM-SspH2-OVA+aPD1 (N=5). Dashed lines demarcate the days of bacterial challenge. (D) Weight change (percentage of initial weight) over 40 days after the start of treatment. Mouse cohorts: STM3210+aPD1 (N=3), STM-pagC-SspH1-OVA+aPD1 (N=3), STM-SspH2-OVA+aPD1 (N=5), CHA-OST. Dashed lines demarcate the days of bacterial challenge. [Figure 3-2] Same as above [Figure 3-3] Same as above [Diagram 3-4] Same as above [Figure 4] 4A-4B. Immune memory of vaccination with OVA-expressing bacteria against the B16-OVA tumor model. (A) Experimental timetable. (B) Tumor growth curve. [Figure 5-1]5A-5C. Long-term efficacy of vaccination with Adpgk-expressing bacteria against MC38 CRC tumor model. (A) Timetable of the experiment. (B) Tumor growth curve from the start of treatment. As shown here, tumor growth was significantly inhibited in the PACMAN-Adpgk cohort compared to other mouse cohorts. After two cycles of immunization, one mouse in the PACMAN-Adpgk cohort was completely cured. (C) To test the immune memory of mice vaccinated with PACMAN-Adpgk, mice that were completely cured after vaccination with PACKMAN-Adpgk or VNP20009 (without adpgk) were rechallenged with 105 MC38 cells and their tumor growth was compared to naive mice injected with the same amount of cells. While naive mice showed exponential tumor growth immediately after injection, rechallenged mice vaccinated with PACKMAN-Adpgk remained tumor-free, indicating the establishment of long-term immune memory against MC38 cells. Of note, mice that were completely cured after vaccination with VNP20009 alone showed tumor growth following rechallenge, indicating that immune memory was solely a consequence of Adpgk presentation by the bacteria. [Figure 5-2] Same as above [Figure 6] FIG. 6 is a graph showing tumor homing of attenuated (STM3120) Salmonella. [Figure 7] FIG. 7 is a graph comparing the toxicity of attenuated (STM3120) and parental (14028) Salmonella upon iv administration. [Figure 8-1] 8A-8B are graphs illustrating immune profiling of splenocytes following vaccination with OVA-expressing bacteria against the B16-OVA tumor model. 8A: Quantification of IFNg-positive CD8 T cells by FACS. 8B: Quantification of tumor-killing capacity of T cells. [Figure 8-2] Same as above [Figure 9-1]9A-9C illustrate the long-term efficacy of vaccination with Adpgk-expressing P. aeruginosa against the MC38 tumor model. 9A is the experimental timeline. 9B is the tumor growth curves. Mice treated with intravenous injection of PACMAN-ADPGK showed significantly delayed tumor growth. The number of mice completely cured per treatment is shown. 9C is the mean tumor growth curve for the mouse cohort in 9B. The whiskers indicate the standard error. [Figure 9-2] Same as above [Figure 10] 10A-10B illustrate the long-term efficacy of vaccination with Adpgk-expressing Bacillus Subtilis spores against the MC38 tumor model. 10A shows the experimental timeline. 10B shows the tumor growth curves. Mice treated with PACMAN-ADPGK spores orally or intravenously showed significantly delayed tumor growth. The number of mice completely cured per treatment is shown. [Figure 11] 11A-11B illustrate the long-term efficacy of vaccination with Adpgk-expressing attenuated Salmonella (STM3120) against the MC38 tumor model. 11A shows the experimental timeline. 11B shows the tumor growth curves. Mice treated with PACMAN-ADPGK showed significantly delayed tumor growth. The number of mice completely cured per treatment is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention, in some embodiments thereof, relates to bacterial vaccines that can be engineered to contain disease-associated antigens on their outer surface.

[0035] Before describing 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 detailed description or illustrated by the examples, as the invention is capable of other embodiments or of being practiced or carried out in various ways.

[0036] The strategy of in vivo therapeutic cancer vaccine based on bacterial vectors that directly deliver antigen or nucleic acid encoding antigen to the cytosol of APC has been developed in academic laboratories and pharmaceutical industry due to their ease of use.Typically, bacteria are genetically modified to express (and even secrete) disease antigen.Alternatively, bacteria can be used to deliver plasmid cDNA encoding disease antigen to immune system.

[0037] The inventors have now conceived a novel vaccine comprising tumor-homing bacteria that have been genetically modified to express disease-associated antigens, and these vaccines are referred to herein as Personalized Anti-Cancer Microbiome-Assisted VaccinatioN (PACMAN).

[0038] As described below and illustrated in the Examples that follow, we show that it is possible to genetically modify bacteria to express tumor antigens. The genetically modified bacteria serve two purposes: 1) homing to tumor sites as a targeting vehicle, and 2) boosting the immune system as an adjuvant.

[0039] The inventors have demonstrated the ability to produce effective vaccines using a number of different bacteria, including Salmonella Typhimurium (1A-1B in FIG. 1, 2A-2G in FIG. 2, 3A-3D in FIG. 3, 4A-4B in FIG. 4, 5A-5C in FIG. 5), P. aeruginosa (9A-9B in FIG. 9) and B. subtilis (10A-10C in FIG. 10). The bacteria have been genetically modified to express a variety of tumor-specific antigens, including OVA (1A-1B in FIG. 1, 2A-2G in FIG. 2, 3A-3D in FIG. 3, 4A-4B in FIG. 4) and ADPGK (5A-5C in FIG. 5, 9A-9B in FIG. 9, 10A-10C in FIG. 10 and 11A-11B in FIG. 11).

[0040] While putting the present invention into practical use, the present inventors demonstrated that alternating administration of different bacterial vaccines can overcome adaptive immunity (see Figures 3A-3C).

[0041] Thus, according to one aspect of the present invention there is provided a vaccine comprising a tumor-homing bacterium genetically modified to express at least one cancer-associated antigen and a pharma- ceutically acceptable carrier.

[0042] As used herein, the term "vaccine" refers to a pharmaceutical preparation (composition) that, when administered, induces an immune response, particularly a cellular immune response that recognizes and attacks cancer cells. Preferably, the vaccine results in the formation of long-term immune memory against the target antigen. The vaccine of the present invention is also preferably administered in a pharma- ceutical acceptable carrier (e.g., a liquid composition that holds the bacteria). In one embodiment, the carrier maintains the viability of the bacteria.

[0043] The bacteria may be gram-positive or gram-negative bacteria, or a combination of the two.

[0044] The bacteria may be aerobic or anaerobic.

[0045] As already mentioned, the bacteria are capable of homing to tumor sites.

[0046] In other embodiments, the bacteria capable of homing to tumors are present within the tumor microbiome of the subject.

[0047] The term "tumor microbiome" refers to the totality of microorganisms (bacteria, fungi, protozoa) and their genetic components in a defined environment, e.g., a host tumor. In certain embodiments, the microbiome refers to only the totality of bacteria in a defined environment, e.g., a host tumor. The tumor may be either a primary tumor or a secondary tumor (i.e., a metastatic tumor).

[0048] Examples of bacteria known to be present in the microbiome of breast tumors are listed in Table 1 herein below. Such bacteria may be particularly suitable for use in vaccines to treat breast cancer. The sequences provided correspond to the 16S rRNA of each bacterium.

[0049] [Table 1] TIFF2024506954000003.tif196150TIFF2024506954000004.tif189153TIFF2024506954000005.tif193154TIFF2024506954000006.tif154149

[0050] Table 2 includes bacterial taxa that may be particularly suitable for use in vaccines to treat breast, lung, or ovarian cancer. Bacteria were sorted according to their p-values ​​(lowest to highest) for enrichment per tumor type.

[0051] [Table 2] TIFF2024506954000008.tif192147TIFF2024506954000009.tif140149

[0052] Table 3 summarizes the different bacterial species that predominate in specific tumor types.

[0053] [Table 3] TIFF2024506954000011.tif107170TIFF2024506954000012.tif107170TIFF2024506954000013.tif106170TIFF2024506954000014.tif63170

[0054] According to certain embodiments, the bacterium is Salmonella Typhimurium (e.g., attenuated strain VNP20009 of Salmonella Typhimurium, strain STM3120 of Salmonella Typhimurium 14028, strain STM1414 of Salmonella Typhimurium 14028), Pseudomonas aeruginosa (strain CHA-OST) and / or Bacillus Subtillis (strain PY79).

[0055] The terms "isolated" or "enriched" encompass bacteria that are (1) separated from at least some of the components with which they were originally associated when produced (whether in nature or in an experimental setting) and / or (2) artificially produced, prepared, purified, and / or manufactured. Isolated microorganisms can 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 originally associated. In some embodiments, isolated microorganisms are greater 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 greater 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 microorganism or other substance that has been separated from at least some of the components with which it was associated either when it was first produced or created (e.g., in nature or in an experimental environment) or for any time after its initial production. A microorganism or population of microorganisms can be considered purified if it has been isolated, e.g., from the materials or environment that contain the microorganism or population of microorganisms at or after production, and a purified microorganism or population of microorganisms can contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% other materials and still be considered "isolated". In some embodiments, a purified microorganism or population of microorganisms is 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 case of the microbial compositions provided herein, one or more microbial types present in the composition can be purified away from one or more other microorganisms produced and / or present in the material or environment containing such microbial type. Microbial compositions and their microbial components are generally purified from imported production from the environment.

[0056] 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 herein.

[0057] According to certain embodiments, the bacterial genome comprises a 16S rRNA sequence that is 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 set forth in SEQ ID NOs: 24-310.

[0058] As used herein, "percent homology", "percent identity", "sequence identity" or "identity", or grammatical equivalents, when used herein with respect to two nucleic acid or polypeptide sequences, include reference to residues in the two sequences that are the same when aligned. When percentages of sequence identity are used with reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), so that the functional properties of the molecule are unchanged. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ in such conservative substitutions are considered to have "sequence similarity" or "similarity". Techniques for making this adjustment are well known to those of skill in the art. Typically, this adjustment involves scoring conservative substitutions as partial mismatches rather than complete mismatches to increase the percentage of sequence identity. Thus, for example, where identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions are given a score of 0 to 1. Scoring of conservative substitutions is calculated, for example, according to the algorithm of Henikoff S and Henikoff JG [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 1992, 89(22): 10915-9].

[0059] Percent identity can be determined using any homology comparison software, including, for example, the BlastN software from the National Center of Biotechnology Information (NCBI), for example, by utilizing default parameters.

[0060] Other exemplary sequence alignment programs that can be used to determine the percent homology or identity between two sequences include, but are not limited to, the FASTA package (including precise (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), 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 as determined by BLASTN by combining all non-overlapping aligned segments (BLAST HSP), summing their number of identical matches, and dividing this sum by the length of the shorter sequence.

[0061] In some embodiments, the sequence alignment program is a basic local alignment program, such as BLAST. In some embodiments, the sequence alignment program is a pairwise global alignment program. In some embodiments, a pairwise global alignment program is used for protein-protein alignment. In some embodiments, the pairwise global alignment program is Needle. In some embodiments, the sequence alignment program is a multiple alignment program. In some aspects, 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.

[0062] According to some embodiments of the invention, the identity is a global identity, ie, identity over the entire nucleic acid sequence of the invention, and not over a portion thereof.

[0063] Methods for investigating the bacteria present in the tumor microbiome are discussed below. Care must be taken to measure a sufficient number of bacteria when characterizing which bacteria are present in the microbiome in order to minimize and control for contamination.

[0064] In some embodiments, determining the presence of one or more bacteria or components or products thereof comprises determining the level or a combination of levels of one or more DNA sequences. In some embodiments, the one or more DNA sequences include any DNA sequence that can be used to distinguish different types of bacteria. In some embodiments, the one or more DNA sequences include a 16S rRNA gene sequence. In some embodiments, the one or more DNA sequences include an 18S DNA gene sequence. In some embodiments, 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 1,000, 5,000 or more sequences are amplified.

[0065] In some embodiments, a microbiota sample (e.g., a tumor sample) is directly analyzed for the presence, level, or combination of levels of one or more DNA sequences. In some embodiments, DNA is isolated from the tumor microbiota sample and the isolated DNA is analyzed for the presence, level, or combination of levels of one or more DNA sequences. Methods for isolating microbial DNA are well known in the art, including, but not limited to, phenol-chloroform extraction and various commercially available kits, including the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA).

[0066] In some embodiments, the presence, level, or combination of levels of one or more DNA sequences are determined by amplifying the DNA sequences using PCR (e.g., standard PCR, semi-quantitative or quantitative PCR). In some embodiments, the presence, level, or combination of levels of one or more DNA sequences are determined by amplifying the 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 FM, Brent R, Kingston RE, Moore D, Seidman JG, Smith JA, Struhl K (eds). 1998. Current Protocols in Molecular Biology. Wiley: New York).

[0067] In some embodiments, DNA sequences are amplified using primers specific for one or more sequences that differentiate a particular type of microorganism from other different types of microorganisms. 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 gene sequences or fragments thereof are amplified using primers specific for 18S DNA gene sequences.

[0068] In some embodiments, the presence, level, or combination of levels of one or more 16S rRNA gene sequences is determined using phylochip technology. The use of Phylochip is well known in the art and described in Hazen et al. ("Deep-sea oil plume enriches indigenous oil-degrading bacteria." Science, 330, 204-208, 2010), the entire contents of which are incorporated herein by reference. In summary, 16S rRNA gene sequences are amplified and labeled from DNA extracts of microbiota samples. The amplified DNA is then hybridized to an array containing probes for microbial 16S rRNA genes. The binding level for each probe is then quantified to provide the sample level of the type of microorganism that corresponds to the 16S rRNA gene sequence probed. In some embodiments, phylochip analysis is performed by a commercial vendor. One non-limiting example is Second Genome Inc. (San Francisco, CA).

[0069] In some embodiments, determining the presence, level or combination of levels of one or more types of microorganisms, or components or products thereof, comprises determining the presence, level or combination of levels of one or more microbial RNA molecules (e.g., transcripts). Methods for 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.

[0070] In some embodiments, determining the presence, level or combination of levels of one or more types of microorganisms, or components or products thereof, comprises determining the presence, level or combination of levels of one or more microbial polypeptides. Methods for 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 techniques are described in Ausebel et al.

[0071] In some embodiments, determining the presence, level or combination of levels of one or more types of microorganisms, or components or products thereof, comprises determining the presence, level or combination of levels of one or more microbial metabolites. In some embodiments, the level of the metabolite is determined by mass spectrometry. In some embodiments, the level of the metabolite is determined by nuclear magnetic resonance. In some embodiments, the level of the metabolite is determined by enzyme-linked immunosorbent assay (ELISA). In some embodiments, the level of the metabolite is determined by colorimetry. In some embodiments, the level of the metabolite is determined by spectrophotometry.

[0072] In certain embodiments, the vaccine comprises at least 1×10 3 Colony forming units (CFU), 1 × 10 4 Colony forming units (CFU), 1 × 10 5 Colony forming units (CFU), 1 × 10 6 Colony forming units (CFU), 1 × 10 7 Colony forming units (CFU), 1 × 10 8 Colony forming units (CFU), 1 x 109 Colony forming units (CFU), 1 x 10 10 Contains colony forming units (CFU).

[0073] The method for producing bacteria can include three major process steps: organism banking, organism production, and storage.

[0074] For banking, the bacteria contained in the strain can be either (1) directly isolated from a specimen or removed from a banked stock, (2) optionally cultured on growth-supporting nutrient agar or broth to generate a viable biomass, or (3) optionally, biomass stored in multiple aliquots for long-term storage.

[0075] In embodiments using a culture process, the agar or broth may contain nutrients that provide the necessary elements and specific factors that allow growth. One example is 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 dihydrogen phosphate, 100 mg / L ferric ammonium citrate, 80 mg / L magnesium sulfate, 10 mg / L hemin chloride, 2 mg / L calcium chloride, and 1 mg / L menadione. Another example is 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. Various microbial media and variations are well known in the art (e.g., RM Atlas, Handbook of Microbiological Media (2010) CRC Press). Culture medium can be added to the culture at the beginning, may be added during the culture, or may be intermittently / continuously flowed into the culture. The strains in the vaccine can be cultured alone, as a subset of the microbial composition, or as a whole collection that includes the microbial composition. For example, a first strain and a second strain may be cultured together in a mixed continuous culture at a dilution rate that is lower than the maximum growth rate of either microorganism to prevent washout of the cultured microorganism from the culture.

[0076] The inoculated culture is incubated under favorable conditions for a time sufficient to allow biomass to form. For microbial compositions for human use, such incubation is often performed at a temperature of 37° C., at a pH and other parameters having values ​​similar to those of the normal human niche. The environment may be actively controlled, passively controlled (e.g., via a buffer), or drifted. For example, for anaerobic bacterial compositions, an anoxic / reducing environment may be used. Such an environment may be obtained by adding a reducing agent, such as cysteine, to the broth and / or by removing oxygen from the broth. As an example, a culture of the bacterial composition may be grown at 37° C. and pH 7 in the above medium pre-reduced with 1 g / L cysteine-HCl.

[0077] Once the culture has produced sufficient biomass, such biomass can be preserved for banking. The target organisms may be placed in a chemical environment that protects them from freezing (addition of a "cryoprotectant"), from drying ("lyoprotectant"), and / or from osmotic shock ("osmoprotectant"), distributed into multiple (optionally identical) containers to create a homogenous bank, and then the cultures may be processed for storage. The containers are generally impermeable and have a closed nature that ensures isolation from the environment. Cryopreservation processing is done by freezing the liquid at ultra-low temperatures (e.g., -80°C or lower). Dry storage removes water from the culture by evaporation (in the case of spray drying or "cool drying") or sublimation (in the case of freeze drying, spray freeze drying, for example). Removal of water improves the long-term storage stability of the microbial composition at temperatures higher than cryogenic temperatures. If the microbial composition contains, for example, a spore-forming microbial species, resulting in the production of spores, the final composition may be purified by additional means such as density gradient centrifugation, which is preserved using the techniques described above. Banking of microbial compositions can be done by culturing and storing strains individually or by mixing strains together to make a combinatorial bank. As an example of cryopreservation, the microbial composition culture can be harvested by pelleting the microbial cells from the culture medium by centrifugation, decanting the supernatant and replacing it with fresh culture broth containing 15% glycerol, and then dispensing the culture into 1 mL cryotubes, sealing them, and placing them at -80°C for long-term viability. This procedure results in acceptable viability upon recovery from cryopreservation.

[0078] Microorganism production may be performed using a culture process similar to banking, including media composition and culture conditions. Such production may also be performed in larger scale operations, particularly for clinical development or commercial production. At large scale, there may be multiple subcultures of the microbial composition before the final culture. At the end of the culture, the culture is harvested for further formulation into a dosage form for administration. This may include concentration, removal of undesirable media components, and / or introduction into a chemical environment that preserves the microbial composition and makes it acceptable for administration by the selected route. After drying, the powder can be blended to an appropriate potency and mixed with other cultures and / or bulking agents, for example, microcrystalline cellulose for uniformity and ease of handling, and the bacterial composition formulated as provided herein.

[0079] In certain aspects, a vaccine (i.e., a bacterial composition) is provided for administration to a subject. In some embodiments, the bacteria is combined with additional active and / or inactive materials to create a final product that can be a single dose unit or in a multi-dose format.

[0080] The bacteria present in the vaccine may be viable (eg capable of growing when cultured in an appropriate medium or in the body after administration).

[0081] In another embodiment, the bacteria present in the vaccine are non-viable.

[0082] In yet another embodiment, the bacteria is attenuated so that it cannot cause disease.

[0083] As mentioned, the bacteria of the vaccines disclosed herein express at least one cancer-associated antigen.

[0084] Cancer-associated antigens are typically short peptides that correspond to one or more antigenic determinants of a protein. Cancer-associated antigens typically bind to class I or II MHC receptors to form a trimeric complex. This trimeric complex can be recognized by T cells bearing a T cell receptor suitable for binding the MHC / peptide complex with appropriate affinity. Peptides that bind 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 the corresponding T cell epitope may share a common core portion, but may differ in overall length due to different lengths of the flanking sequences 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.

[0085] Antigens against cancer can be antigens from testicular cancer, ovarian cancer, brain cancer such as glioblastoma, pancreatic cancer, melanoma, lung cancer, prostate cancer, liver cancer, breast cancer, rectal cancer, colon cancer, esophageal cancer, gastric cancer, renal cancer, sarcoma, glioblastoma, Hodgkin's and non-Hodgkin's lymphoma, and leukemia.

[0086] 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 a member of the GAGE ​​family).

[0087] In another embodiment, the cancer associated antigen is derived from the MART-1 / Melan-A protein, e.g., the MART1 MHC class I peptide (Melan-A:26-35(L27),ELAGIGILTV; SEQ ID NO:1) and the MHC class II peptide (Melan-A:51-73(RR-23)RNGYRALMDKSLHVGTQCALTRR; SEQ ID NO:2).

[0088] 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) peptide).

[0089] In yet 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 / intron 2).

[0090] In yet another embodiment, the cancer associated antigen comprises MUT30 (mutations in kinesin family member 18B, Kif18b-PSKPSFQEFVDWENVSPELNSTDQPFL-SEQ ID NO: 9) or MUT44 (cleavage and polyadenylation specific factor 3-like, Cpsf3l-EFKHIKAFDRTFANNPGPMVVFATPGM-SEQ ID NO: 10).

[0091] In yet another embodiment, the cancer associated antigen is derived from a stimulator of prostate cancer specific T cells-SPAS-1.

[0092] In yet another embodiment, the cancer associated antigen is derived from human telomerase reverse transcriptase (hTERT) or hTRT (human telomerase reverse transcriptase).

[0093] In yet another embodiment, the cancer associated antigen is ovalbumin (OVA), e.g., OVA 257-264 MHCI H-2Kb (SIINFEKL-SEQ ID NO: 11) and OVA 323-339 MHCII IA(d) (ISQAVHAAHAEINEAGR - SEQ ID NO: 12), RAS mutation, oncogenic mutation of p53 (TP53) (p53mut (mouse mutant p53 R172H The sequence VVRHCPHHER-SEQ ID NO:4 (human mutant p53 R175HThe peptide antigen is of the sequence EVVRHCPHHE (SEQ ID NO: 5), or is derived from the BRAF-V600E peptide (GDFGLATEKSRWSGS (SEQ ID NO: 13)).

[0094] According to a particular embodiment, the cancer associated antigen is set forth in SEQ ID NO:11.

[0095] In yet another embodiment, the cancer associated antigen is a breast cancer related disease antigen, including but not limited to alpha-lactalbumin (alpha-Lac), Her2 / neu, BRCA-2 or BRCA-1 (RNF53), KNG1K438-R457 (kininogen-1 peptide) and C3fS1304-R1320 (a peptide that distinguishes mutant BRCA1 from other BC and non-oncogenic mutant BRCA1).

[0096] In yet another embodiment, the cancer associated antigens are MUC1, KRAS, CEA (CAP-1-6-D[Asp6]; YLSGADLNL-SEQ ID NO: 14) and Adpgk R304M Colorectal cancer associated disease antigens including, but not limited to, MC38 (MHCI-Adpgk: ASMTNMELM - SEQ ID NO: 15; MHCII-Adpgk: GIPVHLELASMTNMELMSSIVHQQVFPT - SEQ ID NO: 16).

[0097] In yet another embodiment, the cancer associated antigen is CEA, CA 19-9, MUC1, KRAS, p53mut (mouse p53 R172H Peptide antigen of the mutant sequence VVRHCPHHER-SEQ ID NO: 4 (human p53 R175HMutant sequence EVVRHCPHHE-SEQ ID NO:5) and MUC4 or MUC13, MUC3A or CEACAM5, KRAS peptide (e.g., KRAS-G12R, KRAS-G13D, p5-21 sequence KLVVVGAGGVGKSALTI (SEQ ID NO:17), p5-21 Pancreatic cancer-associated disease antigens include, but are not limited to, the G12D sequence KLVVVGADGVGKSALTI (SEQ ID NO: 18), the p17-31 sequence SALTIQLIQNHFVDE (SEQ ID NO: 19), the p78-92 sequence FLCVFAINNTKSFED (SEQ ID NO: 20), the p156-170 sequence FYTLVREIRKHKEKM (SEQ ID NO: 21), NRAS (e.g., NRAS-Q61R), PI3K (e.g., PIK3CA-H1047R), C-Kit-D816V, and the BRCA mutated epitopes YIHTHTFYV (SEQ ID NO: 22) and SQIWNLNPV (SEQ ID NO: 23) HLA-A*02:01 restricted neoepitopes.

[0098] In yet another embodiment, the cancer associated antigen is a lung cancer related disease antigen, including, but not limited to, sperm protein 17 (SP17), A-kinase anchoring protein 4 (AKAP4) and pituitary tumor transforming gene 1 (PTTG1), aurora kinase A, HER2 / neu, and p53mut.

[0099] In yet 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).

[0100] In yet another embodiment, the cancer associated antigen is a brain cancer, particularly a glioblastoma cancer associated disease antigen, such as the GL261 neoantigen (mImp3 D81N AALLNKLYA - SEQ ID NO: 6).

[0101] In another embodiment, the cancer associated antigen is a neoantigen.

[0102] As used herein, the term "neoantigen" refers to an epitope that has at least one alteration that makes it different from the corresponding wild-type parent antigen, for example, due to a mutation in a tumor cell or a tumor cell-specific post-translational modification. A neoantigen may include a polypeptide sequence or a nucleotide sequence. The alteration may include a frameshift or non-frameshift indel, a missense or nonsense substitution, a splice site alteration, a genomic rearrangement or gene fusion, or any genomic or expression alteration that results in a neo-ORF. The alteration may also include a splice variant. The tumor cell-specific post-translational modification may include aberrant phosphorylation. The tumor cell-specific post-translational modification may also include a spliced ​​antigen generated by the proteasome.

[0103] An example of an APC mutant antigen is QATEAERSF (SEQ ID NO:3).

[0104] Examples of BRCA mutant epitopes are the YIHTHTFYV (SEQ ID NO: 22) and SQIWNLNPV (SEQ ID NO: 23) HLA-A*02:01 restricted neoepitopes.

[0105] An example of a universal HLA-DR binding helper T cell synthetic epitope (AKFVAAWTLKAAA, SEQ ID NO: 311) is the CD4 + The pan DR-binding epitope (PADRE) is a 13 amino acid peptide that activates T cells.

[0106] Another contemplated cancer-associated neoantigen is the GL261 neoantigen (mImp3 D81N, sequence AALLNKLYA-SEQ ID NO:6).

[0107] 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 other embodiments, the bacteria are genetically modified to secrete the cancer associated antigen.

[0108] For example, in some embodiments, the bacterium comprises a nucleic acid encoding a cancer associated antigen operably linked to a transcriptional regulatory element, such as a bacterial promoter. The transcriptional regulatory element may further comprise a secretion signal. In some embodiments, the cancer associated antigen is constitutively expressed by the bacterium. In some embodiments, the cancer associated antigen is inducibly expressed in the bacterium (e.g., expressed upon exposure to an environmental cue, such as sugar or a low pH or anaerobic environment). In some embodiments, the bacterium comprises multiple nucleic acid sequences encoding multiple different cancer associated antigens that can be expressed by the same bacterial cell.

[0109] In some embodiments, the bacteria display the recombinantly produced cancer therapeutic on the surface by a bacterial surface display system. Examples of bacterial surface display systems include outer membrane protein systems (e.g., LamB, FhuA, OmpI, OmpA, OmpC, OmpT, eCPX derived from OmpX, OprF, and PgsA), surface accessory 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, MSP1a, 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 incorporated herein by reference.

[0110] 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.

[0111] In some embodiments, the genetically modified bacteria described herein comprise a cancer therapeutic agent (e.g., the cancer therapeutic agent is introduced into the bacteria prior to administration to a subject, or the bacteria is genetically modified to express a cancer therapeutic agent).

[0112] In some embodiments, the cancer therapeutic is loaded into the bacteria by growing the bacteria in medium containing the cancer therapeutic at a high concentration (e.g., at least 1 mM) that results in either incorporation of the cancer therapeutic during bacterial cell growth or binding of the cancer therapeutic to the outside of the bacteria. The cancer therapeutic can be incorporated passively (e.g., by diffusion and / or partitioning into the lipophilic cell membrane) or actively via membrane channels or transporters. In some embodiments, drug loading is improved by adding additional substances to the growth medium that either increase incorporation of the molecule of interest (e.g., Pluronic F-127) or prevent release of the molecule after incorporation by the bacteria (e.g., efflux pump inhibitors such as verapamil, reserpine, Carsonic acid, or piperine). In some embodiments, the bacteria are loaded with a cancer therapeutic agent by mixing the bacteria with the cancer therapeutic agent 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 incorporated herein by reference.In some embodiments, the cells can be treated with an efflux pump inhibitor (see above) after electroporation to prevent the release of the loaded molecule.

[0113] In yet another embodiment, the bacteria is genetically modified to express a cancer therapeutic agent.

[0114] In some embodiments, the vaccine component comprises an inhibitory antibody or small molecule against immune checkpoint proteins such as anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, and anti-PDL1.

[0115] The inventors further contemplate that the vaccine bacteria may include a therapeutic agent attached to the outside of the bacteria using attachment methods such as CLICK chemistry, as further described in U.S. Patent Application Nos. 20200087703 and 20200054739, which are incorporated herein by reference.

[0116] Examples of therapeutic agents include immunomodulatory proteins such as cytokines. Examples of immunomodulatory proteins include B lymphocyte chemoattractant ("BLC"), CC motif chemokine 11 ("eotaxin-1"), eosinophil chemotactic protein 2 ("eotaxin-2"), granulocyte-colony stimulating factor ("G-CSF"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), I-309, intercellular adhesion molecule 1 ("ICAM-1"), interferon gamma ("IFN-γ"), interleukin-1 alpha ("IL-1α"), interleukin-1 beta ("IL-1β"), 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”), interleukin-12 subunit beta (“IL-12 p40” or “IL-12 p70"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 ("IL-17"), chemokine (CC motif) ligand 2 ("MCP-1"), macrophage colony-stimulating factor ("M-CSF"), monokine induced by gamma interferon ("MIG"), chemokine (CC motif) ligand 2 ("MIP-1α"), chemokine (CC motif) ligand 4 ("MIP-1β"), macrophage inflammatory protein 1-δ ("MIP-1δ"), platelet-derived growth factor subunit B ("PDGF-BB"), chemokine (CC motif) ligand 5, RANTES (Regulated on Activation, Normal T cell Expressed andSecreted), TIMP metallopeptidase inhibitor 1 ("TIMP-1"), TIMP metallopeptidase inhibitor 2 ("TIMP-2"), tumor necrosis factor, lymphotoxin alpha ("TNFα"), tumor necrosis factor, lymphotoxin beta ("TNFβ"), 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-derived vascular endothelial growth factor ("EG-VEGF"), fibroblast growth factor receptor ("EGFR"), endocrine-derived vascular endothelial growth factor ("EG-VEGF"), fibroblast growth factor receptor ("EGFR"), endocrine-derived vascular endothelial growth factor ("EG-VEGF"), endocrine-derived vascular endothelial growth factor receptor ("EGFR ... FGF-4, Keratinocyte Growth Factor ("FGF-7"), Growth Differentiation Factor 15 ("GDF-15"), Glial Cell Line-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 receptor ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp, cullin, F-box-containing complex ("SCF"), stem cell factor receptor ("SCF R"), transforming growth factor alpha ("TGFα"), transforming growth factor beta-1 ("TGFβ1"), transforming growth factor beta-3 ("TGFβ3"), 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"), mucosal-associated epithelial chemokine ("CCL28"), chemokine (CC motif) ligand 27 ("CTACK"), chemokine (CXC motif) ligand 16 ("CXCL16"), CXC motif chemokine 5 ("ENA-78"), chemokine (CC motif) ligand 26 ("eotaxin-3"), granulocyte chemoattractant protein 2 ("GCP-2"), GRO, chemokine (CC motif) ligand 14 ("HCC-1"), chemokine (CC motif) ligand 16 ("HCC-4"), interleukin-9 ("IL-9"), interleukin-17F ("IL-17F"), interleukin-18 binding protein ("IL-18 BPa"), interleukin 28A ("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 ("M CP-3"), monocyte chemotactic protein 4 ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibitory factor ("MIF"), chemokine (CC motif) ligand 20 ("MIP-3α"), CC motif chemokine 19 ("MIP-3β"), chemokine (CC motif) ligand 23 ("MPIF-1"), macrophage stimulating protein alpha chain ("MSPα"), NAP-2 (Nucleosome stimulating protein alpha chain), assembly protein 1-like 4), secreted phosphoprotein 1 ("osteopontin"), PARC (Pulmonary and activation-regulated cytokine), platelet factor 4 ("PF4"), stromal cell-derived factor 1 alpha ("SDF-1α"), chemokine (CC motif) ligand 17 ("TARC"), thymic expressed chemokine ("TECK"), thymic stromal lymphopoietin (TSLP)4-IBB"), CD166 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"), 1 type I 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 R1, IL-10 Rβ, IL-17R, IL-2Rγ, IL-21R, lysosomal membrane protein 2 ("LIMPII"), neutrophil gelatinase-associated lipocalin ("lipocalin-2"), CD62L ("L-selectin"), lymphatic endothelial ("LYVE-1"), MEC class I polypeptide-related sequence A ("MICA"), MEC class I polypeptide-related sequence B ("MICB"), NRG1-β1, platelet-derived growth factor receptor beta ("PDGF Rβ"), 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"), XENOACTIN ("XENOACTIN"), and VEGF receptor 1 ("VEGF-1"). A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, cathepsin S, CD40, cryptic family protein 1B ("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, neural cell adhesion molecule ("NrCAM"), plasminogen activator inhibitor-1 ("PAI-1"), platelet-derived growth factor receptor ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1β"), sgp130, secreted Frizzled-related protein 2 ("ShhN"), sialic acid-binding immunoglobulin-like lectin ("Siglec-5"), ST2, transforming growth factor beta 2 ("TGFβ2"), Tie-2, thrombopoietin ("TPO"), tumor necrosis factor receptor superfamily member 10D ("TRAIL R4"), TREM-1 (Triggering receptor expressed on myeloid cells1), Vascular Endothelial Growth Factor C ("VEGF-C"), VEGFR1, Adiponectin, 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 (CXC Motif) Ligand 1 ("GROα"), Human Chorionic Gonadotropin ("βHCG"), 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-1") 3"), 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"), ADAM17 endopeptidase ("TACE"), thyroglobulin, inhibitor of metalloproteinase 4 ("TIMP-4"), TSH2B4, Disintegrin And Metalloprotease-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-gamma Alpha / Beta R2, insulin-like growth factor 2 ("IGF-2"), insulin-like growth factor 2 receptor ("IGF-2R"), 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), nephroblastoma overexpressed protein ("NOV"), osteoactivin, programmed cell death protein 1 ("PD-1"), N-acetylglucosamine-L-alanine amidase ("PGRP-5"), serpin A4, secreted frizzled-related protein 3 ("sFRP-3"), thrombomodulin, Toll-like 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"), CD163, clusterin, CRTAM, chemokine (CXC 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"), GASP-2 (GPCR-associated sorting protein 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 AV ("LDL Examples of immunomodulatory proteins include, but are not limited to, cadherin 5 type 2 or VE-cadherin (vascular endothelial), also known as CD144, ("VE-cadherin"), WISP-1 (WNT1-inducible-signaling pathway protein 1), and receptor for activator of nuclear factor kappa B ("RANK"). Immunomodulatory proteins can be recombinantly produced using methods known to those of skill in the art. Immunomodulatory proteins can be displayed on the surface of bacteria using bacterial surface display. The bacteria express genetically engineered protein-protein fusions, for example fusion proteins between a membrane protein and an immunomodulatory protein.

[0117] The bacteria of the vaccine of the present invention can act as an adjuvant and therefore the use of additional adjuvants is not relevant.

[0118] In one embodiment, the vaccine does not contain an adjuvant (other than the bacteria itself).

[0119] In another embodiment, the vaccine comprises an adjuvant in addition to the bacterium.

[0120] Adjuvants are substances that can be added to an immunogen or vaccine formulation to enhance the immunostimulatory properties of the immunogenic moiety. Examples of adjuvants or agents that can increase the effectiveness of an immunogen protein include aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oil emulsions, and oil-in-water emulsions. A specific type of adjuvant is muramyl dipeptide (MDP) and various MDP derivatives and formulations, such as N-acetyl-D-glucosaminyl-(β1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP) (Hornung, RL 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 containing 0.4 mg of threonyl-muramyl dipeptide; see Kwak, LW et al. (1992) N. Engl. J. Med., 327:1209-1238). Other useful adjuvants are cholera toxin, bacterial endotoxin, lipid X, the bacteria Propionobacterium acnes or Bordetella These adjuvants may be or are based on those currently in clinical use (Helling, F et al. (1995) Cancer Res., 55:2783-2788; Davis, TA et al. (1997) Blood, 90: 509), such as B. pertussis whole cells or subcellular fractions, polyribonucleotides, sodium alginate, lanolin, lysolecithin, vitamin A, saponins and saponin derivatives such as QS21 (White, AC et al. (1991) Adv. Exp. Med. Biol., 303:207-210), levamisole, DEAE-dextran, block copolymers or other synthetic adjuvants.Many adjuvants are commercially available from a variety of sources, such as Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ) or incomplete / complete Freund's adjuvant (Difco Laboratories, Detroit, MI), Amphigen (oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of Amphigen and Alhydrogel. Aluminum is approved for human use.

[0121] As mentioned, the vaccines described herein can be used to treat and / or prevent cancer.

[0122] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing, or reversing the progression of a condition, and substantially ameliorating the clinical or cosmetic symptoms of a condition.

[0123] According to certain embodiments, the term prevent refers to substantially preventing the appearance of clinical or cosmetic symptoms of the condition.

[0124] Particular subjects to be treated are mammalian subjects, for example, human subjects.

[0125] According to certain embodiments, the subject has been diagnosed with cancer.

[0126] cancer As used herein, the term "cancer" refers to the uncontrolled abnormal proliferation of the host's own cells, which may result in the invasion of tissues surrounding and potentially distal to the primary site of abnormal cell proliferation in the host. Major classes include carcinomas, which are cancers of epithelial tissues (e.g., skin, squamous cells); sarcomas, which are cancers of connective tissues (e.g., bone, cartilage, fat, muscle, blood vessels, etc.); leukemias, which are cancers of blood-forming tissues (e.g., bone marrow tissue); lymphomas and myelomas, which are cancers of immune cells; and cancers of the central nervous system, including cancers originating from brain and spinal cord tissue. "Cancer," "neoplasm," and "tumor" are used interchangeably herein. As used herein, "cancer" refers to all types of cancers or neoplasms or malignant tumors, including leukemias, carcinomas, and sarcomas, whether primary or recurrent.

[0127] Specific examples of cancers that may be treated using the bacteria described herein include adrenal cortical tumors, hereditary; bladder cancer; breast cancer; breast cancer, ductal; breast cancer, invasive intraductal; breast cancer, sporadic; breast cancer, susceptible; breast cancer, type 4; breast cancer, type 4 breast cancer-1; breast cancer-3; breast cancer-ovarian cancer; triple negative breast cancer, Burkitt's lymphoma; cervical cancer; colorectal adenoma; colorectal cancer; colorectal cancer, hereditary nonpolyposis, type 1; colorectal cancer, hereditary nonpolyposis, type 2; colorectal cancer, hereditary nonpolyposis polyposis, type 3;colorectal cancer, hereditary nonpolyposis, type 6;colorectal cancer, hereditary nonpolyposis, type 7;dermatofibrosarcoma protuberans;endometrial cancer;esophageal cancer;gastric cancer;fibrosarcoma, glioblastoma multiforme;glomus tumor, multiple;hepatoblastoma;hepatocellular carcinoma;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; Lynch cancer family syndrome II; male germ cell tumors; mast cell leukemia; medullary thyroid cancer; medulloblastoma; melanoma, malignant melanoma, meningioma; multiple endocrine neoplasia; multiple myeloma, myeloid malignancies, predisposition to);myxosarcoma, neuroblastoma;osteosarcoma;bone cancer, ovarian cancer;ovarian cancer, serous;ovarian cancer;ovarian sex cord tumors;pancreatic cancer;pancreatic endocrine tumors;paraganglioma, familial nonchromaffin;pilomatrixoma;pituitary tumors, invasive;prostate adenocarcinoma;prostate cancer;renal cell carcinoma, papillary, familial and sporadic;retinoblastoma;rhabdoid predisposition syndrome, familial;rhabdoid tumor;rhabdomyosarcoma;small cell carcinoma of the lung;soft tissue sarcoma, squamous cell carcinoma, basal cell carcinoma, head and neck;T-cell acute lymphoblastic leukemia;Turcot's syndrome with glioblastoma;callosoma with esophageal cancer;cervical cancer, Wilms tumor, type 2;Wilms tumor, type 1, and the like.

[0128] According to certain embodiments, the cancer is a cancer selected from the group consisting of breast cancer, melanoma, pancreatic cancer, ovarian cancer, bone cancer, and brain cancer (e.g., glioblastoma).

[0129] According to another embodiment, the cancer is melanoma.

[0130] Malignant melanoma is clinically recognized based on the ABCD(E) system, where A represents asymmetric shape, B represents irregular border, C represents variegated color, D represents diameter >5mm, and E represents change in course. Additionally, excision biopsy may be performed to confirm the diagnosis using microscopic evaluation. Invasive malignant melanoma is traditionally divided into four major histopathological subgroups: superficial spreading melanoma (SSM), nodular malignant melanoma (NMM), lentigo maligna melanoma (LMM), and acral lentigo melanoma (ALM). Other rare types such as desmoplastic malignant melanoma also exist. A substantial subclassification of malignant melanoma appears to arise from melanocytic nevi, and features of dysplastic nevi are often found in the vicinity of invasive melanoma. Melanoma is thought to arise in multiple stages, progressing from normal melanocytes or nevus cells, through a dysplastic nevus stage, and then to a pre-invasive in situ stage. Some subtypes develop through distinct tumor progression stages called the horizontal growth phase (RGP) and vertical growth phase (VGP).

[0131] In certain embodiments, the melanoma is resistant to treatment with an inhibitor of BRAF and / or MEK.

[0132] The tumor can be a primary tumor or a secondary tumor (ie, a metastatic tumor).

[0133] The compositions can be administered using any route, such as, for example, oral administration, rectal administration, topical administration, inhalation (intranasal), or injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT), subtumoral (ST), peritumor (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, ocular, (intranasal), local, parenteral such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., vaginal and perivaginal), intravesical, intrapulmonary, intraduodenal, intragastric, and intrabronchial. In preferred embodiments, the pharmaceutical compositions described herein are administered orally, rectally, intratumorally, topically, intravesically, by injection into or near a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously.

[0134] According to another aspect of the present invention, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of component (i) below, followed by administration of component (ii) below, thereby treating the cancer. (i) a first vaccine comprising a first bacterium genetically modified to express at least one cancer-associated antigen; and (ii) a second vaccine comprising a second bacterium genetically modified to express at least one cancer-associated antigen.

[0135] The present invention contemplates at least two different vaccination cycles for the treatment of cancer, where at least one of the vaccination cycles comprises one strain of genetically modified bacteria and at least another of the vaccination cycles comprises a second (non-identical) strain of genetically modified bacteria. The two strains of bacteria may be genetically modified to express the same or different cancer-associated antigens. Additionally or alternatively, the inventors contemplate that at least one of the vaccination cycles comprises live bacteria (e.g., a first vaccination) and at least another of the vaccination cycles (e.g., a subsequent vaccination) comprises attenuated (or killed) bacteria.

[0136] The vaccine of the present invention may be administered together with an additional anti-cancer agent.

[0137] In some embodiments, the additional anti-cancer agent is an inhibitory antibody or small molecule against immune checkpoint proteins such as anti-CTLA4, anti-CD40, anti-41BB, anti-OX40, anti-PD1, and anti-PDL1.

[0138] Other contemplated anti-cancer drugs that may be administered to a subject in combination with the bacteria described herein include Acivicin, Aclarubicin, Acodazole hydrochloride, Acronine, Adriamycin, Adzelesin, Aldesleukin, Altretamine, Ambomycin, Amethanthrone 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, Castellon, Caracemide, Carbetimer, Carbopro, latin, carmustine, carrubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexomaplatin, dezaguanine, dezaguanine mesylate, diaziquone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, zuazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride Hydrochloride), estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone; fostriecin sodium, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interferon alpha-2a, interferon alpha-2b, interferon alpha-n1, interferon alpha-n3, interferon beta-1a, interferon gamma-1b, 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, metholedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisla , paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxatrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurine, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium, spar Somycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, taxol, tecogalan sodium, tegafur, teroxantrone hydrochloride, temoporfin, teniposide, teroxylon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofuirin, tirapazamine, topotecan hydrochloride, toremifene citrate, trestron acetate, triciribine phosphate Additional anti-neoplastic agents include, but are not limited to, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrocidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride.Goodman and Gilman, "The Pharmacological Basis of Therapeutics", Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions Division), Chapter 52, Antineoplastic Agents (Paul Calabresi and Bruce A. Chabner) and Introduction, pp. 1202-1263.

[0139] As used herein, the term "about" refers to ±10%.

[0140] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including but not limited to."

[0141] The term "consisting of" means "including and limited to."

[0142] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or moieties, but only if the additional components, steps, and / or moieties do not materially change the basic and novel characteristics of the claimed composition, method, or structure.

[0143] As used herein, the singular forms "a," "an," and "the" include the plural references unless the context clearly indicates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds and may also include mixtures thereof.

[0144] Throughout this application, various embodiments of the 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 of the scope of the invention. Thus, the description of a range should be considered to specifically disclose all of the possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose not only the subranges 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., but also the individual numerical values ​​within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0145] Whenever a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the given range. The phrases "range between" a first indicated number and a second indicated number and "range from" a first indicated number to a second indicated number are used interchangeably herein and are intended to include the first indicated number and the second indicated number, and all fractional and integer numbers between the first indicated number and the second indicated number.

[0146] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry, and medicine or that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0147] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, several features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination, or with respect to any other described embodiment of the invention, as appropriate. A particular feature described in the context of various embodiments should not be considered an essential feature of that embodiment, unless the embodiment is inoperable without that element.

[0148] 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

[0149] 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.

[0150] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are fully explained in the literature, e.g., "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, 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), “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, JE, ed. (1994), “Culture of Animal Cells - A Manual of Basic Technique” by Freshney, Wiley-Liss, NY (1994), Third Edition, “Current Protocols in Immunology” Volumes I-III Coligan JE, ed. (1994), Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Reference: されたい. Usability is available for use with the U.S. Patent No. 3,791,932 Specifications, the same Specifications No. 3,839,153, the same Specifications No. 3,850,752, and the same Specifications No. 3, Specification No. 850,578, same as Specification No. 3,853,987, same as Specification No. 3,867,517, same as Specification No. 3,879,262, same as Specification No. 3,901,654 Same as Specification No. 3,935,074, same as Specification No. 3,984,533, same as Specification No. 3,996,345, same as Specification No. 4,034,074, same as Specification No. 4,098, Specification No. 876, Specification No. 4,879,219, Specification No. 5,011,771, and Specification No. 5,281,521, "Oligonucleotide Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, eds. (1984), "Animal Cell Culture" Freshney, RI, 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, CA (1990), Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated by reference in their entireties as if set forth herein. Other general references are provided throughout the specification. The procedures in these publications are believed to be well known in the art, but are provided for the convenience of the reader. All information contained in the above publications is incorporated herein by reference.

[0151] Materials and Methods Plasmids: To generate the basic plasmids for Salmonella Typhimurium strains, the Ssph2 promoter and secretion signal (aa: 1–200) or the pagC promoter and Ssph1 secretion signal (aa: 1–208) were amplified from the attenuated Salmonella Typhimurium strain VNP20009. Ssph2 and pagC-Ssph1 were inserted into pQE60 using the NEBuilder cloning kit (cat. E5520S).

[0152] The protein of interest was fused to either Ssph1 or Ssph2. For the generation of the base plasmid for Pseudomonas aeruginosa, the protein of interest was fused to the N-terminal 54 amino acids of ExoS of the plasmid pEAI3-S54 (gift of Bertrand Toussaint, PMID: 17010670). For the generation of the base plasmid for Bacillus Subtilis spores, the protein of interest was fused to CotC (amplified from Bacillus Subtilis 168) and cloned into the pDG364 plasmid. In addition, a 6His tag element was inserted to allow detection of the protein product.

[0153] Neoantigens: To obtain neoantigens for B16-OVA tumors, the C-terminus of ovalbumin (aa 252-386) was amplified from pcDNA-OVA (Addgene 64599). The amplified oligo contains a sequence corresponding to the ovalbumin epitope SIINFEKL (SEQ ID NO: 11).

[0154] To obtain neoantigens from MC38 tumors, the Adpgk (aa289-421) portion was amplified from cDNA of MC38 cells. The amplified oligos contain sequences corresponding to the MC38 neoantigens verified according to Yadav et al. (PMID: 25428506).

[0155] Both neoantigens were inserted into the plasmid backbone by the NEBuilder cloning kit.

[0156] Bacteria: Attenuated Salmonella Typhimurium strains VNP20009 (also named YS1646, ATCC, cat. 202165) and STM3210 were transformed with the appropriate plasmids by electroporation. Briefly, bacteria were grown to an OD of 0.6–0.8, washed three times with 1 mM Hepes, and suspended in 10% glycerol in DDW. Electroporation of the suspension was performed in a 0.2 cm cuvette (BioRad, EC2) and transferred to 1 ml of cold SOC. After 1 h of incubation at 37°C, bacteria were plated on LB agar plates containing ampicillin. Selected colonies were verified by Sanger sequencing and Western blot with anti-6His tag (Cell Signalling, 2365S).

[0157] Attenuated pseudomonas aeruginosa (CHA-OST) was developed by Diver et al. PMID: 2126169 Transformation was performed as described in. Bacillus subtilis strain PY79 was transformed after 3 hours of incubation with 0.01 M MGSO4 in minimal medium and DDW (MC: 80 mM K2HPO4, 30 mM KH2PO4, 2% glucose, 30 mM trisodium citrate, 22 μg / ml ammonium ferric citrate, 0.1% casein hydrolysate (CAA), 0.2% potassium glutamate) to induce competent cells. Plasmid pDG364, containing the antigen fused to the CotC protein, was then cut with Xba and incubated with competent bacteria for 3 hours. Upon introduction of the amylase gene, colonies were selected by resistance to 5 μg / ml chloramphenicol.

[0158] In all transformations, selected colonies were verified by Sanger sequencing and Western blot with anti-6His tag (Cell Signalling, 2365S).

[0159] Frozen working stock of Salmonella typhimurium: Cultures in logarithmic growth phase (OD 0.6–0.8) were washed twice with cold PBS. Bacterial pellets were suspended in 25% glycerol in PBS. Samples from bacterial stocks were serially diluted, a portion inoculated onto LB agar plates, and the remainder of the pool was aliquoted and stored at -80°C. To confirm bacterial survival, frozen aliquots were thawed and inoculated onto LB agar plates. Recovery after freezing was quantified by calculating the frozen / fresh CFU count ratio. Bacterial doses in mouse experiments were calculated based on CFU counts of frozen cultures.

[0160] Sporulation of Bacillus Subtilis PY79: PY79 was grown in LB at 37°C to an OD of 0.8. The LB medium was removed and replaced with half the volume of DSM release medium. The culture was incubated at 37°C under shaking for 60 hours. Finally the bacteria were washed twice with cold water. To quantify the spores and sporulation rate, samples taken from the washed samples were inoculated onto LB agar plates before and 1 hour after heating at 65°C. The heated / unheated CFU count ratio is an indication of the sporulation rate. Release medium preparation (per liter): Dissolve 8 g Difco Nutrient Broth (BD, cat. 234000), 1 g KCl and 1 mM MgSO4 in DDW. Titrate to pH 7.6 with NaOH and autoclave. Add 10 μM MnCl2, 1 mM Ca(NO3)2 and 1 mM FeSO4 before use.

[0161] Mouse models: B16-OVA mouse melanoma cell line (10 6 ) or MC38 mouse CRC cell line (10 5 ) was subcutaneously injected into the right flank of 7-week-old female C57BL / 6 mice. 2 Calculated as ×length / 2.

[0162] Immune profiling of splenocytes by FACS: Freshly excised spleens were mashed in cold PBS through a 70 micron strainer. To lyse red blood cells, splenocytes were incubated in ACK lysis buffer (Quality Biological, Catalog No. 118-156-101), then washed extensively in PBS and suspended in FACS labeling buffer. 100 μL of splenocytes were incubated with a mixture containing Fc blocker (BD, Catalog No. 553142, 1:100), SIINFEKL (SEQ ID NO: 11) tetramer (NIH Tetramer Core Facility, 1:500), anti-CD4 (BioLegend, Catalog No. 100438, 1:800), anti-CD8 (Invitrogen, Catalog No. 2021-05-05, 1:400), anti-CD3 (Invitrogen, Catalog No. 2023-07-31, 1:1000), and Brilliant Buffer (BD, Catalog No. 566349, 1:5) for 1 hour at 4° C. The cells were then washed twice with labeling buffer and fixed with CytoFix / CytoPerm solution (BD, Catalog No. 51-2090KZ) for 20 minutes at 4° C. Finally, the cells were washed twice with Perm / Wash buffer (BD, Cat. No. 51-2091KZ (diluted 1:10 in DDW)), suspended in labeling buffer and subjected to FACS.

[0163] Quantification of activated CD8 T cells by peptide stimulation Splenocytes were prepared as described above. Splenocytes were then incubated with OVA peptide (final concentration 2.5 μg / ml) for 2 hours at 37° C. Brafeldin A (BD, 51-2301kz) was then added to the cells and further incubated for 4 hours at 4° C. The following day, FACS staining for CD3, CD8 and INFg was performed as described above.

[0164] Ex vivo killing assay MC38 cells or B16-OVA cells were seeded in 48-well plates. The cells were stained with CFSE (5 μM) for 20 min at 37°C, then the reaction was stopped with culture medium (RPMI containing 10% FCS) for 10 min at 37°C, and washed twice with culture medium.

[0165] The next day, splenocytes were removed and cells were counted as described above. 5 Splenocytes were co-cultured with tumor cells and incubated at 37° C. for 72 hours.

[0166] Following incubation, FACS staining for DEAD / LIVE (Invitrogen, L34962) and CFSE positivity (tumor cells) was performed.

[0167] Quantification of IFNg by ELISA: To quantify serum IFNg levels, mice were bled into Eppendorf tubes containing 20 μL of heparin (10 mg / ml). After centrifugation at 10,000 g for 10 min, serum was transferred to new tubes for long-term storage at -20°C. ELISA was performed with serum diluted 1:4 according to the manufacturer's instructions (R&D, Cat. No. DY485).

[0168] Quantification of bacteria in liver and tumors: Tumor and liver slices were suspended in a sterile tube containing LB and metal beads. After vortexing at maximum speed for 10 min, 200 μL of the supernatant was plated onto an LB plate containing the appropriate antibiotic and incubated overnight at 37°C.

[0169] result To demonstrate vaccine efficacy for personalized anti-cancer microbiome-assisted vaccination (PACMAN), bacteria expressing ovalbumin, a known neoantigen, SIINFEKL (SEQ ID NO: 11), were administered to mice bearing B16 melanoma expressing ovalbumin protein (B16-OVA). To generate an OVA-expressing bacterial vaccine, the OVA neoantigen SIINFEKL (SEQ ID NO: 11) was fused to the Ssph2 secretion signal of Salmonella Typhimurium. The resulting oligomers were transformed into the attenuated VNP20009 strain of Salmonella Typhimurium (VNP-OVA). C57BL / 6 mice were injected with 10 6B16 OVA-expressing cells were injected into the mice. Tumors with a volume of approximately 100 mm 3 Once the mice reached 10 μg / mouse, they were assigned to the following treatment cohorts: untreated controls, mice receiving the checkpoint inhibitor anti-PD1 (75 μg per mouse, ip, once weekly), and mice receiving anti-PD1 together with PACMAN-OVA (10 6 CFU, tail vein) in mice. The experimental timeline is shown in Figure 1A. Tumor growth curves from the start of treatment are shown in Figure 1B. Tumor growth ceased for 20 days in the PACMAN-OVA cohort, whereas logarithmic growth was observed in the other mouse cohorts. Following two cycles of immunization, all mice in the VNP-OVA cohort survived significantly longer than mice in the other cohorts.

[0170] To demonstrate vaccine immunogenicity, splenocytes were profiled from mice bearing B16-OVA tumors after administration of the PACMAN vaccine. PACMAN-OVA contained the OVA neoantigen SIINFEKL (SEQ ID NO: 11) fused to the Ssph2 secretion signal of the attenuated STM3210 strain of Salmonella Typhimurium. As a negative control, the OVA neoantigen was replaced with Adpgk (PACMAN-Adpgk), an MC38 neoantigen not present in B16-OVA cells.

[0171] C57BL / 6 mice were injected with 10 6 B16 OVA-expressing cells were injected into the mice. Tumors with a volume of approximately 100 mm 3 Once the mice reached 10 μg / mouse, they were assigned to the following treatment cohorts: untreated controls, mice receiving the checkpoint inhibitor anti-PD1 (75 μg per mouse, ip, once weekly), and mice receiving PACMAN-Adpgk (10 6 CFU, tail vein) in mice. Spleens and livers were harvested 16 days after immunization for further analysis. The results are shown in Figure 2, 2A-2D.

[0172] To test the effect of alternating administration of PACMAN-OVA based on different attenuated bacteria, mice bearing B16-Ova tumors were sequentially vaccinated with two attenuated bacteria expressing OVA neoantigens. The first bacterium was the attenuated Salmonella strain STM3210 (STM-OVA) expressing either the Ova neoantigen fused to the SshpH2 secretion signal under the endogenous promoter or the Ova neoantigen fused to the Ssph1 secretion signal under the pagC promoter induced upon phagocytosis by macrophages. The second bacterium was the attenuated Pseudomonas aeruginosa strain CHA-OST expressing the Ova neoantigen fused to the secretion signal of ExoS, a toxin of the type 3 secretion system (TTSS). The ExoS promoter was activated by the TTSS regulator ExsA following induction with IPTG (CHA-OST-OVA).

[0173] C57BL / 6 mice were injected with 10 6 B16 OVA-expressing cells were injected into the mice. Tumors with a volume of approximately 100 mm 3 Once the mice reached 100 mg / mouse, they were assigned to the following treatment cohorts: untreated control, mice receiving the checkpoint inhibitor anti-PD1 (75 μg per mouse, ip, once a week), mice receiving STM-SspH2-OVA with anti-PD1, and mice receiving STM-pagC-SspH1-OVA with anti-PD1. Vaccinated mice were vaccinated with three doses of STM-OVA (10 6 CFU, tail vein), followed by anti-PD1 (75 μg per mouse, i.p., once a week). Two weeks after the last STM-OVA vaccination, mice were vaccinated with two doses of CHA-OST-OVA (10 7CFU, tail vein, followed by 0.5 mM IPTG and 3 h incubation). As illustrated in Figure 3B, tumor growth was significantly delayed in mice vaccinated with STM-OVA compared to unvaccinated mice. Most of the tumors in vaccinated mice grew again 20-30 days after vaccination, suggesting that booster injections of the same bacteria do not contribute sufficiently to antitumor immunity. Surprisingly, vaccination of mice with CHA-OST-OVA slowed tumor growth rates, even to logarithmic resolution in some cases. As shown in Figure 3C and 3D, weight loss was observed following each bacterial challenge, but was less after booster vaccinations with the same bacteria, supporting the hypothesis that mice develop immunity to the bacteria, resulting in faster clearance and less impact on body weight.

[0174] To test the immune memory of mice vaccinated with PACMAN-OVA, mice that were completely cured in the experiment shown in Figure 3A were given 10 6 The mice were re-challenged with 16-Ova cells and the tumor growth was compared to that of naive mice injected with the same amount of cells. As shown in FIG. 4B, the naive mice showed logarithmic tumor growth shortly after injection, whereas the re-challenged mice remained tumor-free, indicating the establishment of long-term immune memory against the B16-Ova cells.

[0175] To demonstrate efficacy of the PACMAN vaccine with natural neoantigens in other mouse models, bacteria expressing the Adpgk neoantigen in the MC38 model were tested in mice bearing MC38 CRC tumors. To generate an Adpgk-expressing bacterial vaccine, the Adpgk neoantigen was fused to the Ssph1 secretion signal of Salmonella Typhimurium under the pagC promoter, which is induced upon phagocytosis by macrophages. Attenuated Salmonella Typhimurium strain VNP20009 was then transformed with the oligomer (PACMAN-Adpgk). C57BL / 6 mice were injected with 10 ng / mL of the Adpgk neoantigen into the right flank. 5MC38 cells were injected. The tumor volume was approximately 100 mm 3 Upon reaching 10 μg / mouse, mice were assigned to the following treatment cohorts: mice receiving the checkpoint inhibitors anti-PD1 (75 μg per mouse, i.p., once weekly), mice receiving VNP20009 together with anti-PD1, and mice receiving PACMAN-Adpgk (10 6 CFU, tail vein) in mice.

[0176] To test the immune memory of mice vaccinated with PACMAN-Adpgk, mice that were completely cured following vaccination with PACKMAN-Adpgk or VNP20009 (no adpgk) were given 10 5 Mice were rechallenged with MC38 cells and their tumor growth was compared to naive mice injected with the same amount of cells. While naive mice showed logarithmic tumor growth shortly after injection, mice rechallenged with PACKMAN-Adpgk remained tumor-free, indicating the establishment of long-term immune memory to MC38 cells. Of note, mice that were completely cured following vaccination with VNP20009 alone showed tumor growth following rechallenge, indicating that immune memory was the result of Adpgk presentation by the bacteria (Figure 5C).

[0177] To demonstrate selective homing of Salmonella to MC38 tumors, attenuated Salmonella (STM3120) was injected into the tail vein of mice bearing MC38 CRC tumors at the indicated numbers. After 9 days, tumors, livers and spleens were excised and vigorously shaken in 1 ml of LB and metal bowls. Supernatants were plated on LB plates and colonies were counted after 24 h incubation at 37°C. CFUs were normalized to dilution factor and tissue mass. 1 × 10 6 , 1×10 5 is N=4, and 1×10 4 N = 3. As shown in Figure 6, the bacteria selectively homed to the tumor compared to the liver and spleen.

[0178] To compare the maximum tolerated dose of attenuated Salmonella (STM3120) versus parental Salmonella (14028), Salmonella was injected into the tail vein at various concentrations and body weight was monitored. 6 At all doses except for , all mice in the cohort (N = 4 - Figure 5) died (indicated by an X).

[0179] To measure the amount of activated T cells following PACMAN vaccination, splenocytes were harvested from the following cohorts: naive mice (N=3), mice bearing B16-OVA tumors (N=5), mice injected with attenuated Salmonella STM3120 (N=4), mice bearing B16-OVA tumors injected with STM3120 expressing the unrelated neoantigen ADPGK (N=3), and mice bearing B16-OVA tumors injected with STM3120 expressing the OVA neoantigen (N=5). 6 (1e6) bacteria were injected into the tail vein. Splenocytes were harvested 16 days after injection. Figure 8A shows the increase in IFNg positive CD8 T cells following vaccination with the appropriate neoantigen.

[0180] In further experiments to quantify the tumor-killing ability of T cells, MC38 or B16-OVA tumor cells were preincubated with CFSE (green) to distinguish them from immune cells. Harvested splenocytes were co-cultured with tumor cells. After 72 hours, dead tumor cells (CFSE positive) were quantified by FACS using live / dead staining. Significant B16-OVA-specific killing was observed in splenocytes from mice vaccinated with STM3120 expressing the OVA neoantigen (two-tailed t-test, Pval<0.001).

[0181] To demonstrate immune-mediated efficacy of the P. aeruginosa-based PACMAN vaccine in the MC38 colorectal cancer model, attenuated P. aeruginosa, naive CHA-OST, or CHA-OST expressing the Adpgk neoantigen were injected into the tail vein followed by anti-PD1 treatment. C57BL / 6 mice were injected with 1x10 5MC38 colorectal cancer cells were injected into the tumors. The tumor volume was approximately 100 mm 3 Once the incubation period reached 10 days, mice were inoculated with CHA-OST naïve or PACMAN-ADPGK (1x10 6 CFU, iv) followed by weekly ip administration of 150 μg of anti-PD1. Figure 9A illustrates the treatment protocol. As illustrated in Figure 9B, only mice injected with the PACMAN-ADPGK vaccine showed complete cure. Of note, rechallenge of cured mice with MC38 cells did not result in tumor growth.

[0182] To demonstrate immune-mediated efficacy of the Bacillus Subtilis-based PACMAN vaccine in the MC38 colorectal cancer model, spores of the experimental strain PY79 expressing the Adpgk neoantigen were injected into the tail vein or orally (os) followed by aPD1 treatment. C57BL / 6 mice were injected with 5x10 5 MC38 colorectal cancer cells were injected into the tumors. The tumor volume was approximately 100 mm 3 Once the mice reached 100 μg / mL, mice were inoculated with PACMAN-ADPGK Bacillus spores (5x10 8 ~1x10 9 CFU, iv) or orally administered (5x10 9 CFU, po) followed by weekly ip administration of 150 μg of anti-PD1. Figure 10, 10A illustrates the treatment protocol. As illustrated in Figure 10, 10B, only mice injected with the PACMAN-ADPGK vaccine showed complete cure.

[0183] To demonstrate immune-mediated efficacy of the Salmonella-based PACMAN vaccine (frozen) in the MC38 colorectal cancer model, attenuated Salmonella Typhimurium STM3210 strain expressing relevant (Adpgk) and irrelevant (OVA) neoantigens was injected into the tail vein followed by aPD1 treatment. C57BL / 6 mice were injected with 10 mL of the vaccine into the right flank. 6 B16 OVA-expressing cells were injected into the mice. Tumors with a volume of approximately 100 mm 3Once the incubation period reached 10 days, mice were inoculated with either PACMAN-ADPGK or PACMAN-OVA (3x10 6 CFU, iv) followed by weekly ip administration of 75μg / 150μg anti-PD1. Figure 11, 11A illustrates the treatment protocol. As illustrated in Figure 11, 11B, mice treated with the PACMAN-ADPGK vaccine showed a significant delay in tumor growth.

[0184] While the present 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 as fall within the spirit and broad scope of the appended claims.

[0185] It is the intention of the applicants that all publications, patents, and patent applications mentioned herein are incorporated herein by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should 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, the headings should not be construed as necessarily limiting. Additionally, any priority documents of this application are incorporated herein by reference in their entirety. [Sequence List Free Text]

[0186] SEQ ID NO:1: Cancer associated antigen SEQ ID NO:2: Cancer associated antigen SEQ ID NO: 3: Example of a mutant APC antigen SEQ ID NO: 4: Cancer associated antigen SEQ ID NO: 5: Cancer associated antigen SEQ ID NO: 6: Cancer associated antigen SEQ ID NO: 7: Cancer associated antigen SEQ ID NO: 8: Cancer associated antigen SEQ ID NO: 9: Cancer associated antigen SEQ ID NO: 10: Cancer associated antigen SEQ ID NO: 11: OVA neoantigen SEQ ID NO: 12: Cancer associated antigen SEQ ID NO: 13: Cancer associated antigen SEQ ID NO: 14: Cancer associated antigen SEQ ID NO: 15: Cancer associated antigen SEQ ID NO: 16: Cancer associated antigen SEQ ID NO: 17: Cancer associated antigen SEQ ID NO: 18: Cancer associated antigen SEQ ID NO: 19: Cancer associated antigen SEQ ID NO: 20: Cancer associated antigen SEQ ID NO: 21: Cancer associated antigen SEQ ID NO: 22: Example of a BRCA mutation epitope SEQ ID NO: 23: Example of a BRCA mutation epitope SEQ ID NO:175: 16S ribosomal RNA of unknown bacteria SEQ ID NO:197: 16S ribosomal RNA of unknown bacteria SEQ ID NO:198: 16S ribosomal RNA of unknown bacteria SEQ ID NO:211: 16S ribosomal RNA of unknown bacteria SEQ ID NO: 229: 16S ribosomal RNA of unknown bacteria SEQ ID NO:250: 16S ribosomal RNA of unknown bacteria SEQ ID NO: 256: 16S ribosomal RNA of unknown bacteria SEQ ID NO:294: 16S ribosomal RNA of unknown bacteria SEQ ID NO: 311: Example of a universal HLA-DR binding T helper synthetic epitope

Claims

1. A vaccine for treating or preventing cancer, comprising a tumor-homing bacterium genetically modified to constitutively express at least one neoantigen, and a pharma- ceutical acceptable carrier.

2. 2. The vaccine of claim 1, wherein the tumor-homing bacterium is any one of Salmonella Typhimurium, Pseudomonas aeruginosa, or Bacillus Subtillis.

3. The vaccine described in claim 2, wherein the tumor-homing bacterium is Salmonella Typhimurium 14028 STM3120 strain, Salmonella Typhimurium 14028 STM1414 strain), Pseudomonas aeruginosa (CHA-OST strain) and / or Bacillus Subtillis (PY79 strain).

4. The vaccine described in claim 3, wherein the tumor-homing bacterium is the STM3120 strain of Salmonella Typhimurium 14028.

5. The vaccine of claim 1, wherein the tumor-homing bacterium is a bacterium of a species or genus listed in any one of Tables 1 to 3, and / or the genome of the tumor-homing bacterium comprises a 16S rRNA sequence listed in any one of SEQ ID NOs: 24 to 310.

6. The vaccine of any one of claims 1 to 5, wherein the tumor-homing bacterium has a genetic modification to express a therapeutic protein.

7. The vaccine of claim 6 , wherein the therapeutic protein is a cytokine.

8. A vaccine comprising a tumor-homing bacterium genetically modified to constitutively express at least one neoantigen, and a pharma- ceutically acceptable carrier.

9. The vaccine of claim 8, wherein the tumor-homing bacteria comprises Salmonella Typhimurium, Pseudomonas aeruginosa or Bacillus Subtillis.

10. The vaccine according to claim 9, wherein the tumor-homing bacterium is an attenuated strain selected from Salmonella Typhimurium 14028 STM3120 strain, Salmonella Typhimurium 14028 STM1414 strain), Pseudomonas aeruginosa (CHA-OST strain) and / or Bacillus Subtillis (PY79 strain).

11. The vaccine described in claim 10, wherein the tumor-homing bacterium is a bacterium of a species or genus listed in any of Tables 1 to 3, and / or the genome of the tumor-homing bacterium comprises a 16S rRNA sequence listed in any one of SEQ ID NOs: 24 to 310.

12. A vaccine described in any one of claims 8 to 11, wherein the tumor-homing bacterium has a genetic modification for expressing a therapeutic protein.

13. The vaccine of claim 12, wherein the therapeutic protein is an immunomodulatory protein.

14. The vaccine of claim 13, wherein the therapeutic protein is a cytokine.

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