How to Make a Vaccine

Abstract

Disclosed are vaccines comprising a pharma- ceutically acceptable carrier and a bacterium that presents at least one cancer-associated antigen, the bacterium not being genetically modified to express the at least one cancer-associated antigen, and uses thereof.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 150,692, filed February 18, 2021, the entire contents of which are incorporated herein by reference.

[0002] SEQUENCE LISTING STATEMENT A 567,434 byte ASCII file entitled "91250 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 on their outer surface. [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 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 pharma- ceutically acceptable carrier and a bacterium that presents at least one cancer-associated antigen, wherein the bacterium has not been genetically modified to express the at least one cancer-associated antigen.

[0007] According to one aspect of the invention, there is provided a method of making an antigenic composition, comprising the steps of: (a) incubating the bacteria in a culture medium containing a modified amino acid that is metabolized by the bacteria under conditions that allow the bacteria to incorporate the modified amino acid into the bacterial cell wall; (b) contacting the bacterium with at least one cancer associated antigen under conditions that allow binding of the cancer associated antigen to the modified amino acid to produce an antigenic composition; A method is provided that includes:

[0008] 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.

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

[0010] According to an embodiment of the invention, at least one cancer associated antigen is incorporated into the bacterial cell wall via modified amino acids contained in the bacteria.

[0011] According to embodiments of the invention, the cancer associated antigen comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, and a diazirine group.

[0012] According to an embodiment of the invention, the modified amino acid comprises D-alanine.

[0013] According to an embodiment of the present invention, the D-alanine is selected from the group consisting of D-alanine azide, D-alanine-D-alanine azide, D-alanine alkyne, D-alanine-D-alanine alkyne.

[0014] According to embodiments of the invention, the at least one cancer associated antigen comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocyclocutine (DIBO) group, a bicyclononyne (BCN) group, a transcyclooctene (TCO) group, and a strained-transcyclooctene (sTCO) group.

[0015] According to an embodiment of the present invention, the bacterium is a gram-positive bacterium.

[0016] According to an embodiment of the present invention, the bacterium is a gram-negative bacterium.

[0017] According to an embodiment of the present invention, the bacteria is an aerobic bacteria.

[0018] According to an embodiment of the present invention, the bacteria is a non-aerobic bacteria.

[0019] According to an embodiment of the invention, the bacteria is a live bacteria.

[0020] According to an embodiment of the invention, the bacterium is an attenuated bacterium.

[0021] According to an embodiment of the invention, at least one cancer associated antigen is conjugated to a modified amino acid via a click chemistry reaction.

[0022] According to an embodiment of the present invention, the bacterium is a bacterium belonging to a family, order, genus, or species listed in any of Tables 1 to 3.

[0023] 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.

[0024] According to an embodiment of the invention, the cancer associated antigen is a neoantigen.

[0025] According to an embodiment of the invention, the bacteria is genetically modified to express a therapeutic protein.

[0026] According to an embodiment of the invention, the therapeutic protein is a cytokine.

[0027] According to an embodiment of the invention, the vaccine is free of aluminium salts.

[0028] According to an embodiment of the invention, the carrier is adjuvant-free.

[0029] According to an embodiment of the invention, the modified amino acid comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, and a diazirine group.

[0030] According to an embodiment of the invention, the modified amino acid comprises D-alanine.

[0031] According to an embodiment of the present invention, the D-alanine is selected from the group consisting of D-alanine azide, D-alanine-D-alanine azide, D-alanine alkyne, D-alanine-D-alanine alkyne.

[0032] According to embodiments of the invention, the at least one cancer associated antigen comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocyclocutine (DIBO) group, a bicyclononyne (BCN) group, a transcyclooctene (TCO) group, and a strained-transcyclooctene (sTCO) group.

[0033] According to an embodiment of the present invention, steps (a) and (b) are carried out simultaneously.

[0034] According to an embodiment of the invention, the bacteria include Salmonella Typhimurium, Pseudomonas aeruginosa, and / or Bacillus Subtillis.

[0035] According to an embodiment of the invention, the cancer associated antigen is conjugated to a modified amino acid via a click chemistry reaction.

[0036] According to an embodiment of the invention, the cancer associated antigen is a neoantigen.

[0037] According to an embodiment of the invention, the bacteria is genetically modified to express a therapeutic protein.

[0038] According to an embodiment of the invention, the therapeutic protein is a cytokine.

[0039] According to an embodiment of the present invention, the bacterium is a bacterium of a family, order, genus, or species listed in any one of Tables 1 to 3.

[0040] 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.

[0041] According to an embodiment of the present invention, the vaccine is produced using the methods described herein.

[0042] According to an embodiment of the invention, the cancer is selected from the group consisting of breast cancer, lung cancer, gastric cancer, colorectal cancer, melanoma, pancreatic cancer, ovarian cancer, bone cancer, and brain cancer.

[0043] According to an embodiment of the invention, the brain cancer comprises glioblastoma.

[0044] According to an embodiment of the invention, the cancer is selected from the group consisting of breast cancer, melanoma, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0045] According to an embodiment of the invention, the brain cancer comprises glioblastoma.

[0046] 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.

[0047] 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]

[0048] [Figure 1] Figure 1A-C shows click-treated bacteria as a platform for neoantigen delivery. (A) Schematic of click-treated bacteria. (B) Flow cytometric validation of click reaction. Bacterial fractions clicked with OVA were incubated with avidin-Cy5 and analyzed by flow cytometry. Bacteria not incubated with D-ala served as negative control. (C) In vivo imaging validation of click reaction and tumor homing. Cy5-clicked bacteria were injected iv (tail vein) into tumor-bearing C57BL / 6 mice. [Figure 2-1] Figure 2, A-E, shows the long-term efficacy and immunogenicity of vaccination with OVA click-conjugated bacteria in the B16-OVA tumor model. (A) Timetable of the experiment. (B) Tumor growth curves. All treated mice showed delayed tumor growth. Mouse 814 was completely cured. (C) Representative mice of the anti-PD1 treatment cohort and mouse 814 treated with anti-PD1 together with PACMAN-CLICK-OVA showed complete cure. (D) Expanded tumor growth curves of mice vaccinated with PACMAN-CLICK-OVA (mice: 839, 814, 824, 801, 802) in tumor volumes ranging from 0 to 600 mm3. The completely cured mouse (mouse no. 814) showed a reduction in tumor volume from day 2. (E) Quantification of SIINFEKL (SEQ ID NO: 11)-specific TCR by flow cytometry. To quantify neoantigen-specific T cell clones, splenocytes were co-incubated with tetramers of the OVA neoantigen (SIINFEKL, SEQ ID NO: 11). The percentage of SIINFEKEL (SEQ ID NO: 11) positive T cells in the CD3 / CD8 population was highest in mice vaccinated with PACMNA-CLICK-OVA compared to untreated mice. Notably, mouse 814 (orange dots) showed the highest percentage of SIINFEKL (SEQ ID NO: 11) specific T cells. [Figure 2-2] Same as above [Figure 2-3] Same as above [Diagram 3] FIG. 3 is a graph showing tumor homing of attenuated (STM3120) Salmonella. [Figure 4] FIG. 4 is a graph comparing the toxicity of attenuated (STM3120) and parental (14028) Salmonella upon iv administration. [Diagram 5] Figure 5. FACS readout showing the generation of Staphylococcus pasteuri bacteria clicked with OVA using an NHS-based anchor. The clicked bacterial fraction is marked. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] The present invention, in some embodiments thereof, relates to bacterial vaccines that can be engineered to contain disease-associated antigens on their outer surface.

[0050] 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.

[0051] 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.

[0052] The inventors have conceived of a novel vaccine in which bacteria are engineered to display disease-associated antigens on their outer surface without genetic modification.

[0053] As illustrated in the following description and the following Examples section, the present inventors demonstrate that it is possible to label bacteria with neoantigens without genetic modification. First, bacteria were incubated with a modified amino acid (alkyne-D-alanine-D-alanine (D-Ala)) to incorporate the modified amino acid into the peptidoglycan bacterial cell wall. Next, as shown in FIG. 1A, OVA neoantigens containing an azide residue at the N-terminus were click-reacted with the bacteria. As shown in FIG. 1B, the presence of bacteria click-bound to OVA neoantigens was confirmed.

[0054] While further putting the invention into practice, the inventors demonstrated that the clicked bacteria could reach the tumor site after iv injection (FIG. 1C) and provide a therapeutic effect (FIGS. 2B and 2D).

[0055] As a result, the present teachings suggest that other modified cell wall components (e.g., MurNAc, teichoic acid, and lipopolysaccharides) can be taken up by bacteria from the culture medium and incorporated into the bacterial cell wall, paving the way for an easy and cost-effective method for the production of vaccines for the treatment of cancer. Furthermore, non-genetic engineering of bacteria for optimal neoantigen presentation solves major challenges due to biosafety and regulatory constraints associated with genetically engineered bacteria. While any genetic engineering of bacteria requires a lengthy and costly approval process, the disclosed strategy allows for a rapid and inexpensive strategy. Furthermore, the disclosed method allows for the use of bacteria that are difficult to genetically modify as a setup for neoantigen presentation.

[0056] As a result, bacteria genetically engineered to display tumor neoantigens have the potential to become tiebreakers in the field of personalized anti-cancer vaccines.

[0057] Thus, according to one aspect of the invention there is provided a vaccine comprising a pharma- ceutically acceptable carrier and a bacterium that presents at least one cancer-associated antigen, wherein the bacterium has not been genetically modified to express the at least one cancer-associated antigen.

[0058] 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 also preferably includes a pharma- ceutical acceptable carrier (i.e., a liquid in which the bacteria are held). The carrier may be one that does not affect the viability of the bacteria.

[0059] The isolated bacteria of this aspect of the invention may be a gram-positive or gram-negative bacterium, or a combination of both.

[0060] The isolated bacteria can be aerobic or non-aerobic.

[0061] In one embodiment, the bacteria are capable of homing to the tumor site.

[0062] In another embodiment, the bacteria is present in the tumor microbiome.

[0063] 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).

[0064] Examples of bacteria known to be present in the microbiome of breast tumors are listed herein below in Table 1. Such bacteria may be particularly suitable for use in vaccines to treat breast cancer.

[0065] [Table 1] TIFF2024506955000003.tif242164TIFF2024506955000004.tif234162TIFF2024506955000005.tif238164TIFF2024506955000006.tif189163

[0066] 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.

[0067] [Table 2] TIFF2024506955000008.tif238161TIFF2024506955000009.tif174164

[0068] Table 3 summarizes the different bacterial species that predominate in specific tumor types.

[0069] [Table 3] TIFF2024506955000011.tif255167TIFF2024506955000012.tif252165TIFF2024506955000013.tif253168TIFF2024506955000014.tif254100

[0070] 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.

[0071] 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 above.

[0072] 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.

[0073] 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].

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] The method for producing bacteria can include three major process steps: organism banking, organism production, and storage.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 bacteria of the vaccine formulated as provided herein.

[0085] In certain aspects, a vaccine (i.e., a bacterial composition) is provided for administration to a subject. In some embodiments, the bacteria of the vaccine are 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.

[0086] 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).

[0087] In another embodiment, the bacteria present in the vaccine are non-viable.

[0088] In yet another embodiment, the bacteria is attenuated so that it cannot cause disease.

[0089] As mentioned, the bacteria of the vaccines disclosed herein present at least one cancer-associated antigen.

[0090] 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.

[0091] Peptide sequences can be synthesized by methods known to those of skill in the art, for example, peptide synthesis using an automated peptide synthesizer (such as those available from Applied Biosystems, Inc., Foster City, Calif.). Longer peptides or polypeptides can also be prepared, for example, by recombinant methods.

[0092] 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.

[0093] 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).

[0094] 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).

[0095] 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).

[0096] 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).

[0097] 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).

[0098] In yet another embodiment, the cancer associated antigen is derived from a stimulator of prostate cancer specific T cells-SPAS-1.

[0099] In yet another embodiment, the cancer associated antigen is derived from human telomerase reverse transcriptase (hTERT) or hTRT (human telomerase reverse transcriptase).

[0100] In yet another embodiment, the cancer associated antigen is ovalbumin (OVA), e.g., OVA 257-264MHCI 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 R175H The peptide antigen is of the sequence EVVRHCPHHE (SEQ ID NO: 5), or is derived from the BRAF-V600E peptide (GDFGLATEKSRWSGS (SEQ ID NO: 13)).

[0101] According to a particular embodiment, the cancer associated antigen is set forth in SEQ ID NO:11.

[0102] 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).

[0103] 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).

[0104] 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.

[0105] 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.

[0106] 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).

[0107] 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).

[0108] In another embodiment, the cancer associated antigen is a tumor neoantigen.

[0109] 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.

[0110] An example of an APC mutant antigen is QATEAERSF (SEQ ID NO:3).

[0111] Examples of BRCA mutant epitopes are the YIHTHTFYV (SEQ ID NO: 22) and SQIWNLNPV (SEQ ID NO: 23) HLA-A*02:01 restricted neoepitopes.

[0112] 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.

[0113] Another contemplated cancer-associated neoantigen is the GL261 neoantigen (mImp3 D81N, sequence AALLNKLYA-SEQ ID NO:6).

[0114] As mentioned above, cancer antigens are displayed on the outer surface of bacteria.

[0115] In some embodiments, the bacteria included in the vaccine are linked to the cancer associated antigen by a cross-linking agent. As used herein, the term "cross-linking agent" refers broadly to compositions that can be used to link various molecules together, including proteins. Examples of cross-linking agents include 1,5-difluoro-2,4-dinitrobenzene, 3,3'-dithiobis(succinimidyl propionate), bis(2-succinimidooxycarbonyloxy)ethyl)sulfone, bis(sulfosuccinimidyl)suberate, dimethyl 3,3'-dithiobispropionimidate, dimethyl adipimidate, dimethyl pimelimidate, dimethyl suberimidate, disuccinimidyl glutarate, disuccinimidyl suberate, disuccinimidyl tartrate, dithiobis(succinimidyl propionate), ethylene glycol(ethylene glycosl)bis(succinimidyl succinate), ethylene glycol(ethylene glycosl) bis(sulfosuccinimidyl succinate), PEGylated bis(sulfosuccinimidyl) suberate (containing PEGS), PEGylated bis(sulfosuccinimidyl) suberate (containing PEGS), and tris-(succinimidyl)aminotriacetate.

[0116] In some embodiments, the bacteria included in the vaccine are linked to the cancer associated antigen via a nucleic acid linker. For example, in some embodiments, the bacteria described herein display a first nucleic acid oligonucleotide (e.g., an oligonucleotide at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and / or up to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length) on the surface of the bacteria to bind to a specific nucleic acid oligonucleotide for the first nucleic acid oligonucleotide. The second nucleic acid oligonucleotide may comprise a second nucleic acid oligonucleotide (e.g., an oligonucleotide at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and / or up to 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides in length) that hybridizes to and / or provides a binding site for a reagent linked to the second nucleic acid oligonucleotide. Methods for binding oligonucleotides to the surface of bacterial cells are known in the art, see, for example, Twite AA, et al., Adv. Mater., 2012, 24(18):2380-5, which is incorporated herein by reference. In some embodiments, the first oligonucleotide has a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the second oligonucleotide. Exemplary methods for linking agents to oligonucleotides are provided, for example, in David A. Rusling & Keith R. Fox, Small Molecule-Oligonucleotide Conjugates, DNA Conjugates and Sensors, 2012, Ch3, 75-102, which is incorporated herein by reference.In some embodiments, the cancer therapeutic agent is covalently linked to a single stranded nucleic acid oligonucleotide that specifically hybridizes to a single stranded nucleic acid oligonucleotide displayed on the cell surface of the bacteria described herein. The hybridized oligonucleotide hybridizes and the resulting double stranded nucleic acid duplex is stable for several days. In some embodiments, the stability of the duplex is improved by incorporating phosphorothioate linkages (e.g., 1, 2, 3, 4, 5, 6, or more than 7 phosphorothioate linkages) at the 5' and / or 3' ends of one or both oligonucleotides.

[0117] In some embodiments, the bacteria of the vaccines described herein are linked to the cancer associated antigen via a biotin / streptavidin interaction.

[0118] In some embodiments, the bacteria described herein are linked to biotin or cancer-associated antigens using amine-reactive N-hydroxysuccinimide (NHS) esters or N-hydroxysulfosuccinimide (sulfo-NHS) esters (adding PEG to NHS-esters can serve to keep antigens extracellular). NHS esters or sulfo-NHS esters (Life Technologies) can be made from virtually any carboxyl-containing molecule of interest by mixing NHS or sulfo-NHS with the carboxyl-containing molecule of interest and a dehydrating agent (e.g., carbodiimide EDC) using methods available in the art. Exemplary methods for labeling bacteria using NHS esters are provided in Bradburne JA, et al., AppL Environ. Microbiol, 1993, 59(3):663-8, which is incorporated herein by reference.

[0119] The NHS ester binds directly to the cell wall, and when conjugated to an alkyne group, it can be coupled to any peptide bearing an azide residue by standard click chemistry.

[0120] Examples of cross-linker reactive groups for protein conjugation are summarized in Table 4 herein below.

[0121] [Table 4]

[0122] According to certain embodiments, the cancer associated antigen peptide is made to have an NHS-ester at the C-terminus. The NHS-ester can be attached to the free amine present on the N-terminus or lysine of any protein. To prevent the NHS-azide of one peptide from attaching to the free amine on another peptide, the N-terminus of the peptide may be modified (e.g., by acetylation) so that it does not contain a free amine. Alternatively, the lysines in the peptide may be protected so that their free amines are not exposed and are not reactive. This protection may be removed after the peptide is bound to the bacteria.

[0123] According to another embodiment, a hydrazine group can be added to the cancer associated antigen peptide. This group can be attached to the C-terminus of proteins and aldehyde-containing molecules such as the side chains of the amino acids aspartic acid and glutamic acid. The C-terminus of the cancer associated antigen peptide is typically protected. This method is preferred for peptides that do not contain glutamic acid or aspartic acid.

[0124] In some embodiments, the bacteria of the vaccine described herein are linked to cancer-associated antigens via sequence-specific DNA hybridization interactions.For example, the molecule of interest is covalently linked to a single-stranded DNA oligonucleotide and then binds to a bacterial cell that displays a complementary single-stranded DNA oligonucleotide on the cell surface.The two complementary oligonucleotides hybridize, and the resulting double-stranded DNA duplex is stable for several days.The stability and nuclease resistance of the DNA duplex are further improved by incorporating four phosphorothioate bonds at the 5' and 3' ends of both oligonucleotides.

[0125] In some embodiments, cancer associated antigens are linked to bacteria using unnatural amino acids containing ketone, azide, alkyne or other functional groups that are incorporated into surface-expressed proteins of the bacteria described herein. Unnatural amino acids containing ketone, azide, alkyne or other functional groups known to those skilled in the art can be incorporated into proteins of interest in a residue-specific manner using auxotrophic bacterial strains, for example, as described in Marquis H., et al., Infect. Immun., 1993, 61(9):3756-60, which is incorporated herein by reference. For example, methionine-requiring bacterial strains can be grown in the presence of the unnatural amino acid azidohomoalanine, which serves as a surrogate for methionine and is incorporated in place of methionine during protein biosynthesis, to label the surface of the cells. This allows wild-type proteins on the bacterial surface, which normally contain a surface-exposed methionine, to be functionalized with surface-exposed azide groups that can be modified with molecules of interest containing alkyne groups (e.g., alkyne-derived small molecule drugs or alkyne-derived proteins) using click chemistry as described in Link AJ & Tirrell DA, Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2]cycloaddition, J. Am. Chem. Soc., 2003, 125(37):11164-5, which is incorporated herein by reference. Once incorporated into surface-expressed proteins, these functional groups can serve as moieties to which small molecules of interest are added, for example, using the methods described in Prescher JA & Bertozzi CR, Nat. Chem. Biol, 2005, 1(1):13-21, which is incorporated herein by reference. In another embodiment, wild-type bacteria are cultured with modified D-alanine, such as D-alanine azide, D-alanine-D-alanine azide, D-alanine alkyne, or D-alanine-D-alanine alkyne, to add an azide or alkyne group to the neoantigen.In another embodiment, a copper-free click chemistry reaction (eg, using DB CO-amine) is performed to attach the cancer associated antigen to the bacteria.

[0126] In some embodiments, the bacteria described herein are Gram-negative bacteria, and the cancer-associated antigen is linked to the surface-bound glycan. The cancer-associated antigen can be linked to the surface-bound glycan, for example, using a two-step metabolic / chemical labeling protocol. In the first step, the Gram-negative bacteria are metabolically labeled with a chemically modified monosaccharide containing an azide functional group that is incorporated into the macromolecular structure on the bacterial surface to modify the surface-associated sugar polymer. In the second step, the cancer-associated antigen is selectively linked to the modified polymer on the bacterial cell surface using click chemistry, for example, as described in Dumont A., et al., Angew. Chem. Int. Ed. Engl., 2012, 51(13):3143-6, which is incorporated herein by reference.

[0127] In some embodiments, the cancer associated antigen is linked to the peptidoglycan (PG) of the bacterial cell wall. This linkage may be appropriate for Gram-positive or Gram-negative bacteria. However, the cell wall of Gram-positive bacteria contains many interconnected layers of peptidoglycan (PG), whereas the cell wall of Gram-negative bacteria contains only one or two layers of peptidoglycan. Thus, linkage to the bacterial PG may be more appropriate for Gram-positive bacteria.

[0128] A two-step metabolic / chemical labeling approach can be used to add exogenously added molecules of interest to PG. Gram-positive bacterial cells are first metabolically labeled by growing in the presence of an alkyne-functionalized D-alanine analogue that is incorporated into the nascent PG layer during cell wall biosynthesis. With the incorporation of the alkyne group, PG can then be labeled with an azide-functionalized molecule of interest using a copper-catalyzed click reaction, for example, as described in Siegrist MS, et al., ACS Chem. Biol., 2013, 8(3):500-5, which is incorporated herein by reference. In some embodiments, Gram-positive bacterial cells are grown in a medium containing a cyclooctyne-functionalized D-alanine analogue (e.g., exobcnDala or endobcnDala), which is incorporated into the PG of the growing cells. The cells are washed with fresh medium and incubated with a cancer-associated antigen derivatized with an azido-PEG3 group to add the molecule of interest to the PG in a copper-free reaction, for example as described in Shieh P., et al., Proc. Natl. Acad. Sci. USA, 2014, 111(15):5456-61, which is incorporated herein by reference. In some embodiments, Gram-positive bacterial cells are grown in a medium containing an unnatural D-amino acid with a norbornene (NB) group (e.g., D-Lys-NB-OH, D-Dap-NB-OH, D-Dap-NB-NH2). The unnatural amino acid is metabolically incorporated into the PG of the growing bacterial cells, providing the bacterial cell surface with an alkene functional group with increased reactivity due to the strained alkene in the norbornene ring. The cells are then incubated with a tetrazine derivative of a cancer-associated antigen to allow ligation of the cancer-associated antigen to PG, as described in Pidgeon SE & Pires MM, Chem. Commun. (Camb). 2015, 51(51):10330-3, which is incorporated herein by reference.

[0129] In some embodiments, the cancer-associated antigen is incorporated into the PG layer of the Gram-negative bacteria described herein. Methods for incorporating molecules into the PG layer of Gram-negative bacteria are provided, for example, in Liechti GW, et al., Nature, 2014, 506(7489):507-10, which is incorporated herein by reference. In some embodiments, the Gram-negative bacteria are grown in the presence of the D-amino acid dipeptide EDA-DA (ethynyl-D-alanine-D-alanine) or DA-EDA (D-alanine-ethynyl-D-alanine). EDA-DA or DA-EDA is incorporated into the PG layer of the actively growing bacteria, providing the PG with surface-exposed alkyne groups. Using copper-catalyzed click chemistry, the cancer-associated antigen containing a terminal azide group is added to the newly introduced alkyne group of the PG layer. In some embodiments, a D-amino acid derivative of a cancer associated antigen is incorporated directly into the PG layer of growing bacteria, for example, using the methods described in Kuru E., et al., Nat. Protoc., 2015, 10(1):33-52, which is incorporated herein by reference.

[0130] To perform click chemistry, the cancer associated antigen may comprise at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocycloctine (DIBO) group, a bicyclononyne (BCN) group, a transcyclooctene (TCO) group, and a strained-transcyclooctene (sTCO) group.

[0131] Methods for attaching cancer associated antigens to reactive groups are known in the art and are described in Johansson and Pedersen, European Journal of Organic Chemistry, Volume 2012, Issue 23, August 2012, pages 4267-4281.

[0132] After the reactive group is added to the amino acid, the peptide can be synthesized by techniques known to those skilled in the art of peptide synthesis. For solid phase peptide synthesis, overviews of many techniques can be found in JM Stewart and JD Young, Solid Phase Peptide Synthesis, WH Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For classical solution synthesis, see G. Schroder and K. Lupke, The Peptides, vol. 1, Academic Press (New York), 1965.

[0133] In general, these methods involve the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then be added to an inert solid support or can be utilized in solution by adding the next amino acid in the sequence with a suitably protected complementary (amino or carboxyl) group under conditions suitable to form an amide bond. The protecting group is then removed from this newly added amino acid residue, and then the next amino acid (suitably protected) is added, and so on. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or simultaneously to obtain the final peptide compound. By simple modifications of this general procedure, it is possible to add more than one amino acid at a time to a growing chain, such as by coupling a protected tripeptide with a suitably protected dipeptide (under conditions that do not racemize the chiral centers) to form a pentapeptide after deprotection. Further description of peptide synthesis is given in U.S. Pat. No. 6,472,505.

[0134] A preferred method for preparing the peptide compounds of some embodiments of the present invention involves solid phase peptide synthesis.

[0135] Large scale polypeptide synthesis is described in Andersson Biopolymers 2000;55(3):227-50.

[0136] The inventors further contemplate that the bacteria of the vaccine may contain therapeutic agents other than the cancer associated antigens described herein above. Such therapeutic agents may be attached to the outside of the bacteria using the conjugation methods described herein above. Alternatively, the bacteria may be genetically modified to express the therapeutic agent.

[0137] For example, in some embodiments, the bacterium comprises a nucleic acid encoding a therapeutic agent 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 therapeutic agent is constitutively expressed by the bacterium. In some embodiments, the therapeutic 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 therapeutic agents that can be expressed by the same bacterial cell.

[0138] 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.

[0139] 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 and Secreted), 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 4 ("FGF-4"), keratinocyte growth factor ("FGF-7"), growth differentiation factor 15 ("GDF-15"), glial cell line-derived neurotrophic factor ("GDN"), F"), 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-D6Ckine, tyrosine protein kinase receptor UFO ("Axl"), betacellulin ("BTC"), mucosa-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"), and the like. 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 4-IBB ("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"), type 1 membrane glycoprotein ("PDG1"), and IL-1 receptor agonist ("IL-1 receptor agonist) ... ("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 cell receptor 1 ("TIM-1"), tumor necrosis factor receptor superfamily member IOC ("TRAIL-1R"), and / or IL-2R. R3), trappin protein transglutaminase binding domain ("Trappin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDARActivin A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, 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), FcgRIIB / 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 oncolytic enzyme activator 1 ("TREM-1"), and sgp130 ("ShhN"). myeloid cells 1), 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-18Rb, 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.

[0140] In some embodiments, the bacteria described herein are engineered to express a therapeutic protein (e.g., a cancer therapeutic protein) within the bacteria and / or on the bacterial surface (i.e., surface display by genetic engineering). For example, in some embodiments, the bacteria comprises a nucleic acid encoding the cancer therapeutic protein operably linked to a transcriptional regulatory element, such as a promoter. In some embodiments, the protein is constitutively expressed by the bacteria. In some embodiments, the protein is inducibly expressed in the bacteria (e.g., expressed upon exposure to an environmental cue, such as sugar or a low pH or anaerobic environment). In some embodiments, the bacteria comprise multiple nucleic acid sequences encoding different recombinant proteins that can be expressed by the same bacterial cell.

[0141] 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.

[0142] In some embodiments, the bacteria described herein include a cancer therapeutic (e.g., the cancer therapeutic is loaded into the bacteria prior to administration to a subject). 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 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 the 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.

[0143] 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.

[0144] The bacteria of the vaccine of the present invention can act as an adjuvant and therefore the use of additional adjuvants is not relevant.

[0145] In one embodiment, the vaccine does not contain an adjuvant (other than the bacterium itself).

[0146] In another embodiment, the vaccine comprises an adjuvant in addition to the bacterium.

[0147] 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.

[0148] As mentioned, the vaccines described herein can be used to treat and / or prevent cancer.

[0149] 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.

[0150] According to certain embodiments, the term prevent refers to substantially preventing the appearance of clinical or cosmetic symptoms of the condition.

[0151] Particular subjects to be treated are mammalian subjects, for example, human subjects.

[0152] According to certain embodiments, the subject has been diagnosed with cancer.

[0153] 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 tissues. "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.

[0154] 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.

[0155] 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).

[0156] According to another embodiment, the cancer is melanoma.

[0157] 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).

[0158] In certain embodiments, the melanoma is resistant to treatment with an inhibitor of BRAF and / or MEK.

[0159] The tumor can be a primary tumor or a secondary tumor (ie, a metastatic tumor).

[0160] 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.

[0161] The present invention contemplates at least two different vaccination cycles for the treatment of cancer, where at least one of the vaccination cycles includes one strain of bacteria and at least another of the vaccination cycles includes a second strain (of a non-identical bacterium). Additionally or alternatively, the inventors contemplate that at least one of the vaccination cycles includes a live bacterium and at least another of the vaccination cycles (e.g., a subsequent vaccination) includes an attenuated (or killed) bacterium.

[0162] The vaccine of the present invention may be administered together with an additional anti-cancer agent.

[0163] 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.

[0164] 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.

[0165] As used herein, the term "about" refers to ±10%.

[0166] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including but not limited to."

[0167] The term "consisting of" means "including and limited to."

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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

[0175] 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.

[0176] 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.

[0177] Materials and Methods Neoantigens: To obtain neoantigens for B16-OVA tumors, the c-terminus (aa 252-386) of ovalbumin was amplified from pcDNA-OVA (Addgene 64599). The amplified oligo contains a sequence corresponding to the ovalbumin epitope SIINFEKL (SEQ ID NO: 11).

[0178] 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).

[0179] Both neoantigens were inserted into the plasmid backbone by the NEBuilder cloning kit.

[0180] Bacteria: Attenuated Salmonella Typhimurium strains VNP20009 (also named YS1646, ATCC, catalogue number 202165) and STM3210 (PMID: 20231149) were used. 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.

[0181] Bacterial Click Reaction: To conjugate the OVA neoantigen to the bacterial cell wall by click chemistry, bacteria were incubated overnight with 1.25 mM concentration of azido-D-alanine (VNP20009) or ethynyl-D-alanine-D-alanine (STM3210). Fresh starters were inoculated from overnight cultures and grown to an OD of 0.6–0.8 by adding 1.25 mM of D-alanine derivatives. After washing with PBS, bacteria were incubated for 1 h at room temperature in click solution (sodium ascorbate: 2.5 mM, CuSO4: 50 μM, BTTAA: 300 μM) and 50 μM of azido-SIINFEKL-biotin or alkyne-SIINFEKL-biotin, respectively.

[0182] To verify the click reaction by FACS, the bacterial fraction was washed with FACS labeling buffer (1% FBS in PBS) after incubation with D-alanine derivatives. Bacteria not incubated with D-alanine served as a negative control. After the click reaction, as detailed above, the bacteria were washed in labeling buffer and incubated with 2ug / mL neutral avidin-Cy5 (Southern Biotech, 7200-15) for 30 minutes at 4°C. The bacteria were then washed with labeling buffer and resuspended in 2% PFA for 45 minutes at room temperature. Finally, the bacteria were resuspended in FACS labeling buffer and analyzed by flow cytometry.

[0183] To enhance the click-mediated binding to bacterial cell walls, the NHS-alkyne anchor was used. The D-ala-alkyne is incorporated into the newly formed cell wall, whereas the NHS-alkyne anchor binds to all exposed primary amines. In contrast to the D-ala-alkyne anchor, the NHS-anchor does not require preincubation as in the D-ala-alkyne. Exponentially growing Staphylococcus pasteuri were incubated with the NHS-alkyne. The OVA neoantigen, which contains an azide residue at the N-terminus, was then clicked with the bacteria. For validation purposes, the biotin residue at the C-terminus of the neoantigen was used for biotin-avidin reaction with the fluorophore Cy5. The neoantigen obtained by the click reaction was azido-SIINFEKL-biotin. After incubation, the bacteria were fixed and cy5 was used for FACS quantification (see Figure 5).

[0184] 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.

[0185] 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.

[0186] 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).

[0187] 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.

[0188] result To generate a bacterial vaccine click-conjugated with OVA neoantigen, STM3210 bacteria were incubated overnight with alkyne-D-alanine-D-alanine (D-ala) to incorporate D-ala into the bacterial cell wall. OVA neoantigen containing an azide residue at the N-terminus was then click-reacted with the bacteria, as shown in Figure 1A. For validation purposes, the C-terminus of the neoantigen contained a biotin residue for biotin-avidin reaction with the fluorophore Cy5. The click reaction yielded azido-SIINFEKL-biotin as the neoantigen.

[0189] The OVA-clicked bacterial fraction was incubated with avidin-Cy5 and analyzed by flow cytometry. Bacteria that were not incubated with D-ala were used as a negative control. Indeed, as shown in Figure 1B, the clicked bacteria were enriched in Cy5-positive cells, confirming the click reaction.

[0190] Cy5-clicked bacteria were injected iv (tail vein) into tumor-bearing C57BL / 6 mice. As expected, bacteria were observed in tumor tissues 24 and 48 hours after injection by IVIS imaging, as shown in Figure 1C. Of note, MC38 tumors were used in this experiment because the dark color of melanocyte-rich B16 tumor tissue masks the Cy5 fluorescent signal.

[0191] To demonstrate the efficacy of the click-linked bacteria-based PACMAN vaccine, attenuated Salmonella Typhimurium STM3210 was click-reacted with OVA neoantigen (PACMAN-CLICK-OVA). 6 B16 OVA expressing cells were injected into the right flank. Tumors grew to approximately 100 mm 3 When the volume reached 100 mg / mL, mice were inoculated with PACMAN-CLICK-OVA (10 6CFU, i.v.), followed by weekly anti-PD1 (75 μg, i.p.). Spleens were harvested on day 55 and subjected to immune profiling.

[0192] As shown in Figure 2B, all treated mice showed delayed tumor growth. Mouse 814 was completely cured. As shown in Figure 2C, the tumors of mice treated with PACMAN-CLICK-OVA gradually disappeared, while the tumors of mice treated with anti-PD1 alone continued to grow logarithmically. The completely cured mouse (mouse #814) showed a decrease in tumor volume from day 2, as shown in Figure 2D. To quantify neoantigen-specific T cell clones, splenocytes were co-incubated with OVA neoantigen tetramers (SIINFEKL-SEQ ID NO: 11). The percentage of SIINFEKEL (SEQ ID NO: 11) positive T cells among the CD3 / CD8 population was highest in mice vaccinated with PACMNA-CLICK-OVA compared to untreated mice. Notably, mouse 814 (orange dots) showed the highest percentage of SIINFEKL (SEQ ID NO: 11) specific T cells (Figure 2E).

[0193] 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 3, the bacteria selectively homed to the tumor compared to the liver and spleen.

[0194] 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. As shown in Figure 4, STM3120 1 × 10 6At all doses except for , all mice in a cohort (N = 4–5) died (indicated by an X).

[0195] 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.

[0196] 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 shall not be construed as an admission that such reference is available as prior art to the present invention.

[0197] To the extent that section headings are used, they should not be construed as necessarily limiting. Additionally, any priority document of this application is incorporated herein by reference in its entirety. [Sequence List Free Text]

[0198] 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. 1. A vaccine comprising a pharma- ceutically acceptable carrier and a bacterium presenting at least one cancer-associated antigen, wherein the at least one cancer-associated antigen is incorporated into the cell wall of the bacterium via a modified amino acid contained in the bacterium.

2. 2. The vaccine of claim 1, wherein the at least one cancer associated antigen comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, and a diazirine group.

3. The vaccine of claim 1 or 2, wherein the modified amino acid comprises D-alanine.

4. 4. The vaccine of claim 3, wherein the D-alanine is selected from the group consisting of D-alanine azide, D-alanine-D-alanine azide, D-alanine alkyne, and D-alanine-D-alanine alkyne.

5. 5. The vaccine of any one of claims 1 to 4, wherein the at least one cancer associated antigen comprises at least one reactive group selected from the group consisting of an alkene group, an alkyne group, an azide group, a cyclopropenyl group, a tetrazine group, a dibenzocyclooctyl (DBCO) group, a dibenzocyclocutine (DIBO) group, a bicyclononyne (BCN) group, a transcyclooctene (TCO) group, and a strained-transcyclooctene (sTCO) group.

6. The vaccine of any one of claims 1 to 5, wherein the bacterium is a gram-positive bacterium.

7. The vaccine according to any one of claims 1 to 5, wherein the bacterium is a gram-negative bacterium.

8. The vaccine according to any one of claims 1 to 7, wherein the bacterium is an aerobic bacterium.

9. The vaccine of any one of claims 1 to 7, wherein the bacterium is a non-aerobic bacterium.

10. The vaccine of any one of claims 1 to 7, wherein the bacterium is a live bacterium.

11. The vaccine according to any one of claims 1 to 7, wherein the bacterium is an attenuated bacterium.

12. The vaccine of any one of claims 1 to 11, wherein the at least one cancer associated antigen is linked to the modified amino acid via a click chemistry reaction.

13. The vaccine according to any one of claims 1 to 12, wherein the bacterium is a bacterium of a family, order, genus or species listed in any one of Tables 1 to 3.

14. The vaccine of any one of claims 1 to 13, wherein the bacterial genome comprises a 16S rRNA sequence according to any one of SEQ ID NOs: 24 to 310.

15. The vaccine of any one of claims 1 to 14, wherein the at least one cancer associated antigen is a neoantigen.

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