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WO2025024603A2 - Vaccins bactériens anticancéreux personnalisés - Google Patents

Vaccins bactériens anticancéreux personnalisés Download PDF

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WO2025024603A2
WO2025024603A2 PCT/US2024/039426 US2024039426W WO2025024603A2 WO 2025024603 A2 WO2025024603 A2 WO 2025024603A2 US 2024039426 W US2024039426 W US 2024039426W WO 2025024603 A2 WO2025024603 A2 WO 2025024603A2
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pbcvs
cancer
recombinant
neoantigens
patient
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WO2025024603A3 (fr
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Yiannis N. KAZNESSIS
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Elaine Tsiumas Kaznessis
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Elaine Tsiumas Kaznessis
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/07—Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer

Definitions

  • This invention presents Personalized Bacterial Cancer Vaccines (PBCVs) and related methods, a novel approach to cancer treatment.
  • a method involves collecting normal and cancer tissue samples from a patient, sequencing the samples to identify DNA sequence differences, discovering and selecting unique, patient-specific neoantigens.
  • This method includes isolating bacteria from patient stool samples, characterizing these bacteria, selecting ones that can safely recolonize the patient’s intestinal tract.
  • the method involves genetically engineering the selected bacterial isolates to express and secrete the discovered and selected neoantigens, thus creating a PBCV specific to the patient and their cancer.
  • the method includes administering the recombinant PBCV orally or rectally, the PBCVs colonizing the gastrointestinal tract and releasing neoantigens inside the gastrointestinal tract to interact with the patient's immune system.
  • the patient’s immune system responds by mounting an attack against cancer cells. This highly personalized and targeted approach enhances immunogenicity while differentiating cancer from healthy cells and allowing for local and systemic cancer cell killing.
  • the PBCVs are safe and effective against diverse cancer types, including solid cancers, hematological cancers and pediatric cancers, and can be engineered to stay ahead of cancer cell mutations.
  • Cancer characterized by uncontrolled cell growth and proliferation, remains a global health challenge with significant morbidity and mortality rates.
  • Conventional cancer treatment modalities such as surgery, chemotherapy, and radiation therapy, often exhibit limitations, including adverse side effects and incomplete eradication of tumors. Therefore, alternative therapeutic strategies are being explored to harness the power of the immune system for precise and effective cancer management.
  • cancer vaccines have emerged as a promising avenue in the field of immunotherapy. By stimulating the patient's immune system to recognize and attack cancer cells, vaccines have the potential to achieve targeted tumor destruction and long-lasting immune memory.
  • the development of cancer vaccines faces inherent complexities due to the heterogeneity and adaptability of cancer cells.
  • Personalized Bacterial Cancer Vaccines represent an innovative strategy that combines the unique characteristics of an individual's cancer with components of the patient’s microbiome and with the immunogenic potential of genetically engineered bacteria releasing neoantigens inside the intestinal tract of the patient.
  • This approach (schematically depicted in Figure 1) aims to elicit a robust and specific immune response against cancer cells while minimizing off-target effects.
  • PBCVs offer several distinct advantages.
  • PBCVs can be orally or rectally administered, reach and safely colonize the gastrointestinal tract of the patient.
  • the bacteria that become the organism basis for PBCVs can be selected for a) non-pathogenicity and non-virulence, b) survival through the stomach of the patient, and c) metabolic activity inside the Gl tract. These bacteria can also be amenable to genetic modifications and be amenable to manufacturing.
  • PBCVs capitalize on the vast immune system residing in the gastrointestinal (Gl) tract.
  • the administration of PBCVs orally or rectally allows for targeted delivery to the Gl tract, where a significant proportion of the immune system is localized. This targeting strategy takes advantage of the intricate interactions between the gut and the immune system, augmenting the immune response against cancer cells expressing neoantigens.
  • non-pathogenic bacteria such as probiotics
  • delivery vehicles for PBCVs offers an added layer of safety.
  • These bacteria are inherently designed to interact with the gut environment and are well-tolerated by the human host. Their ability to colonize the Gl tract and release neoantigens locally enhances the efficiency of antigen presentation and subsequent immune activation.
  • PBCVs may address the challenges posed by tumor heterogeneity and the development of resistance.
  • Neoantigen refers to a new protein that forms on cancer cells when certain mutations occur in tumor DNA. According to the US National Cancer Institute “Neoantigens may play an important role in helping the body make an immune response against cancer cells. Neoantigens used in vaccines and other types of immunotherapy are being studied in the treatment of many types of cancer.”
  • peptide and protein refers broadly to a polymer of two or more amino acids joined together by peptide bonds.
  • protein also includes molecules which contain more than one protein joined by a disulfide bond, or complexes of proteins that are joined together, covalently or noncovalently, as multimers (for example, dimers, trimers, tetramers).
  • peptide, oligopeptide, enzyme, subunit, and protein are all included within the definition of protein and these terms are used interchangeably. It should be understood that these terms do not connote a specific length of a polymer of amino acids, nor are they intended to imply or distinguish whether the protein is produced using recombinant techniques, chemical or enzymatic synthesis, or is naturally occurring.
  • neoantigen peptide vaccine refers to a peptide-based cancer vaccine consisting of patient-specific antigens, which are immunogenic and unique to the patient's tumor, with potential immunomodulating and antineoplastic activities.
  • CTL cytotoxic T-lymphocyte
  • polynucleotide refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double- and singlestranded RNA and DNA.
  • a polynucleotide can be obtained directly from a natural source, or can be prepared with the aid of recombinant, enzymatic, or chemical techniques.
  • a polynucleotide can be linear or circular in topology.
  • a polynucleotide may be, for example, a portion of a vector, such as an expression or cloning vector, or a fragment.
  • a polynucleotide may include nucleotide sequences having different functions, including, for instance, coding regions, and non-coding regions such as regulatory regions.
  • coding region As used herein, the terms “coding region,” “coding sequence,” and “open reading frame” are used interchangeably and refer to a nucleotide sequence that encodes a protein and, when placed under the control of appropriate regulatory sequences expresses the encoded protein.
  • the boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end.
  • a “regulatory sequence” is a nucleotide sequence that regulates expression of a coding sequence to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators.
  • operably linked refers to a juxtaposition of components such that they are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence is “operably linked” to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.
  • a “polycistronic mRNA” refers to a transcription product that includes two or more coding regions. Expression of the two or more coding regions is controlled by a single promoter, and the series of the two or more coding regions that are transcribed to produce a polycistronic mRNA is referred to as an operon.
  • probiotic refers to live microorganisms which when administered in adequate amounts confer a health benefit on the host. This is the definition offered by the Food and Agriculture Organization of the United Nations and the World Health Organization.
  • genetically modified bacterium refers to a bacterium that has been altered “by the hand of man.”
  • a genetically modified bacterium includes a bacterium into which an exogenous polynucleotide, for example, an expression vector, has been introduced.
  • an “exogenous protein” and “exogenous polynucleotide” refers to a protein and polynucleotide, respectively, which is not normally or naturally found in a microbe, and/or has been introduced into a microbe.
  • An exogenous polynucleotide may be separate from the genomic DNA of a cell (for example, it may be a vector, such as a plasmid), or an exogenous polynucleotide may be integrated into the genomic DNA of a cell.
  • a “heterologous” polynucleotide such as a heterologous promoter, refers to a polynucleotide that is not normally or naturally found in nature operably linked to another polynucleotide, such as a coding region.
  • a “heterologous” protein or “heterologous” amino acid refers to amino acids that are not normally or naturally found in nature flanking an amino acid sequence.
  • a pair-wise comparison analysis of amino acid sequences can be carried out using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., FEMS Microbiol. Lett., 1999; 174:247-250, and available on the National Center for Biotechnology Information (NCBI) website.
  • polypeptides may be compared using the BESTFIT algorithm in the GCG package (version 10.2, Madison Wl).
  • Conditions that are “suitable” for an event to occur such as expression of an exogenous polynucleotide in a cell to produce a protein, or production of a product, or “suitable” conditions are conditions that do not prevent such events from occurring. Thus, these conditions permit, enhance, facilitate, and/or are conducive to the event.
  • an “animal” includes members of the class Mammalia and members of the class Aves, such as human, avian, bovine, caprine, ovine, porcine, equine, canine, and feline.
  • the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
  • FIG. 1 is a schematic depiction of the process for selecting, developing, manufacturing and administering Personalized Bacterial Cancer Vaccines (PBCVs).
  • Tissue and cell samples are collected from a cancer patient.
  • the genomic material of tumor and normal cells is sequenced.
  • the DNA/RNA sequence differences between the tumor and normal cells are determined.
  • Patient-specific neoantigen peptides are discovered.
  • Selected neoantigens are encoded in recombinant DNA.
  • Bacteria are isolated from patient’s stool samples and characterized. These bacteria or other probiotic bacteria are genetically engineered for the expression and secretion of the neoantigen peptides.
  • the immunogenicity of secreted neoantigens is tested in laboratory in vitro and in vivo experiments.
  • the genetically modified probiotic is manufactured and formulated for oral administration.
  • the patient is administered PBCVs orally. Patient samples are collected to monitor the patient’s immune responses against cancer.
  • FIG. 2 is a schematic of plasmid pGP8001 , used to engineer Bacillus spp. for the expression of cancer neoantigens.
  • the plasmid contains the transcriptional units for thyA and a Tp53 neoantigen.
  • the neoantigen is the p53 sequence from amino acid number 165 to amino acid 185, with a single mutation, R175H.
  • thyA expression is driven by the p43 and the neoantigen peptide uses the AmyQ secretion tag and the R1 ribosome binding site.
  • the repB gene encodes the Bacillus replication protein that allows for a high plasmid copy number.
  • the plasmid also contains an ampicillin resistance gene and origin of replication for E. coli to allow for plasmid construction in an E. coli MC1061 F’.
  • FIG 4 is a schematic of plasmid pGP8002 (the sequence of which is set out in SEQ ID NO: 41), used to engineer E. coli for the expression of cancer neoantigens.
  • the plasmid contains the transcriptional units for dapA, adjuvant gene, and the Tp53 neoantigen.
  • the neoantigen is the p53 sequence from amino acid number 160 to amino acid 185, with a single mutation, R175H.
  • dapA expression is driven by the J23109 promoter and the neoantigen peptide and adjuvant proteins under the expression of the same J23106 promoter.
  • Figure 6 is a schematic of representative neoantigen peptide transcriptional units.
  • the adjuvant and p53_001 neoantigen genes are located on an operon expressed under the same J23106 promoter.
  • the second example (SEQ ID NO:
  • the neoantigen peptide is fused to the 3’ end of the adjuvant gene and expressed under the J23106 promoter as one fusion protein.
  • the ribosome-binding site (RBS) is BCD2 (SEQ ID NO:
  • Escherichia coli and Bacillus spp. provided herein are selected and genetically engineered to produce and secrete heterologous patientspecific neoantigen peptides. These genetically engineered strains can then be used to induce a targeted immune response against cancer cells anywhere in the patient’s body.
  • the present disclosure is based, at least in part, on unexpected findings showing that not all bacteria can be engineered to colonize the gut of animals or humans and maintain the metabolic activity for protein production.
  • E. coli and Bacillus isolates provided herein are thus selected based on localization and metabolic activity in intestinal tract conditions.
  • the present disclosure is also based, at least in part, on unexpected findings showing that not all bacteria can be engineered to express and secrete neoantigen peptides because of specific intracellular and extracellular proteases encoded in the genome of the bacteria that enzymatically digest peptides. E. coli and Bacillus isolates provided herein are thus selected based on the absence of such proteases from their genome. [0050] The present disclosure is also based, at least in part, on unexpected findings showing that not all secretion systems can be used to secrete specific neoantigen peptides because of unpredictable biophysical interactions between neoantigen peptides and bacterial cell membranes and cell membrane proteins that secrete molecules out of the bacterial cell. E. coli and Bacillus isolates and secretion tags provided herein are thus selected based on the secretion strength of example neoantigen peptides.
  • E. coli and Bacillus isolates provided herein are selected based on the absence of virulence factors, other pathogenicity factors, and/or antibiotic resistance.
  • E. coli and Bacillus isolates selected herein are then genetically engineered to express neoantigen peptides.
  • Genetic constructs are engineered to contain polycistronic DNA constructs that consist of polynucleotides encoding a bacterial promoter, ribosome binding site, secretion tag, one or more neoantigen peptides, and terminator sequence.
  • the neoantigen peptides are specific to each patient.
  • E. coli and Bacillus isolates are transformed with libraries of the genetic constructs. Libraries of genetic constructs are screened for immunogenic activity in vitro to evaluate peptide production. Genetically engineered E. coli and Bacillus isolates are tested for in vitro immunogenic activity.
  • the host animal can be of small mammal of the order Rodentia.
  • the host animal can be of the species Mus musculus (mouse).
  • the cancers of interest can be solid cancers, e.g., colon cancer, breast cancer, and pancreas cancer, or hematological cancers, e.g., acute lymphoblastic leukemia, acute myeloid leukemia, and chronic lymphocytic leukemia, or pediatric cancers, e.g. leukemia, brain and spinal cord tumors, neuroblastoma, Wilms tumor, and lymphoma. Selection of Probiotic E. coli and Bacillus spp.
  • E. coli and Bacillus isolates can be commercially available, including, but not limited to E. coli Nissle 1917 (Sonnenborn U.,FEMS Microbiology Letters, Volume 363, Issue 19, October 2016, fnw212, doi: 10.1093/femsle/fnw212), Symbioflor 1 , Symbioflor 2, Bacillus subtilis DSM 29784 and DSM 17299, and Bacillus Licheniformis DSM 28710 (Lee et al. Food Sci Biotechnol. 2019 Oct; 28(5): 1297-1305. doi: 10.1007/s10068-019-00691-9).
  • E. coli and Bacillus spp. isolates can be proprietary strains including, but not limited to General Probiotics 673 (GP673), GP700, GP1191 (Kaznessis et a!., WO 2020/139852 A1 WO 2021/154872 A1), GP1252 and GP1336 (Kaznessis et al., PCT/US23/69266), GP8001, GP8002, GP8003, GP9001, GP9002, and GP9003.
  • Escherichia coli strain GP700 has been deposited with the American Type Culture Collection (ATCC) and assigned ATCC Accession No. PTA-126596.
  • Bacillus paralicheniformis strain GP1336 has been deposited with the American Type Culture Collection (ATCC) and assigned ATCC Accession No. PTA-127307.
  • E. coli and Bacillus spp. can be isolated from the stool samples of cancer patients as described, e.g., in Example 1.
  • Isolated E. coli and Bacillus spp. colonies can be tested using DNA fingerprinting colony PCR (cPCR) to identify unique isolates, as described for example in Example 2.
  • cPCR DNA fingerprinting colony PCR
  • E. coli and Bacillus spp. can be characterized using 16S ribosomal RNA sequencing, as described, e.g., in Example 3.
  • Bacillus spp. isolated according to the disclosure include, for example, Bacillus subtilis, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus coagulans, Bacillus pumilus, Bacillus clausii, or Bacillus amyloliquefaciens.
  • Isolated E. coli can be tested for the presence of virulence and pathogenic factors, including but not limited to, stx1 , stx2, estA, estB, LTI, LTI I, sigA, sepA, hylEtolC, and ibeA. E. coli are selected based on the absence of these factors.
  • Isolated Bacillus spp. can be tested for the presence of virulence and pathogenic factors, including but not limited to, T7SS, ALO, anthrax toxin, cerulide, certhrax, cytK, HBL, nhe, inhA, capsule, bacillibactin, hal, ilsA, petrobactin, atxA, and bslA. Bacillus spp. are selected based on the absence of these factors. [0065] E. coli and Bacillus spp. can be tested for neoantigen peptide inactivation (referred to as protease activity), as described, e.g., in Example 4. E. coli and Bacillus spp. are selected for low protease activity.
  • protease activity neoantigen peptide inactivation
  • E. coli and Bacillus spp. are tested for the presence of intracellular, membrane and extracellular proteases, including but not limited to, nprE, nprB, AprE, WprA, Vpr, Bpr, Epr, HtrA, and HtrB. E. coli and Bacillus spp. are selected for the absence of these proteases.
  • E. coli and Bacillus spp. can be tested for their susceptibility to antibiotics, including but not limited to, kanamycin, chloramphenicol, tetracycline, enrofloxacin, and amoxicillin.
  • E. coli and Bacillus spp. can be selected for being susceptible to antibiotics as described, e.g., in Example 5.
  • E. coli and Bacillus spp. can be tested for hemolytic activity.
  • E. coli and Bacillus spp. can be selected for not exhibiting hemolytic activity, as described, e.g., in Example 6.
  • the method of construction of synthetic DNA sequence involves utilizing standard molecular cloning techniques, such as restriction enzyme-based cloning or Gibson assembly, for engineering the expression of neoantigens in E. coli.
  • the neoantigen-encoding genes and any necessary genes encoding adjuvants obtained from genome sequencing data or gene synthesis, can be ligated into a suitable E. coli expression vector, such as pUC19 or pET-28a, at specific restriction enzyme sites or by seamless assembly using overlapping DNA fragments.
  • the recombinant plasmids can then be transformed into E. coli competent cells using established transformation protocols. This embodiment enables efficient cloning and expression of neoantigens in E. coli, facilitating their production for incorporation into Personalized Bacterial Cancer Vaccines.
  • the method includes employing Bacillus-specific cloning techniques for engineering the expression of neoantigens in Bacillus spp.
  • Bac/7/i/s-compatible vectors such as pHT01 or pUB110, can be used for cloning and expression of neoantigen genes in Bacillus species. These vectors typically contain suitable restriction enzyme sites or assembly sites for the insertion of neoantigen genes.
  • the cloning process can involve restriction enzyme digestion and ligation, or seamless assembly methods, to generate recombinant plasmids carrying the desired neoantigen sequences.
  • the resulting plasmids can be introduced into Bacillus competent cells using established transformation protocols. This embodiment enables efficient cloning and expression of neoantigens in Bacillus, facilitating their production for use in PBCVs.
  • E. coli and Bacillus spp. can be transformed with a DNA plasmid as described, e.g., in Example 7. See Example 8 for a description of exemplary plasmid designs.
  • the plasmids used in E. coli and Bacillus contain an origin of replication.
  • the plasmid can contain an E. coli origin of replication.
  • the plasmid can contain a Bacillus origin of replication such as the Bacillus origin of replication pUB110 or derivatives thereof.
  • the plasmids contain a selectable marker for the modified E. coli and Bacillus spp.
  • the selectable marker can be a kanamycin or a chloramphenicol resistance gene.
  • the selectable marker can be a functional dapA or thyA gene isolated from a E. coli or Bacillus spp. isolate, respectively.
  • the plasmids contain a promoter region linked to one or more ribosome binding sites which is/are linked to the secretion tag/neoantigen peptide gene fragment fusion (the coding region) which is linked to the terminator.
  • This polynucleotide comprises the neoantigen peptide transcriptional unit (TU).
  • the TU encompasses both the coding region and the regulatory sequence.
  • the TU can polycistronic and includes the secretion tag/neoantigen peptide along with an additional secretion tag/neoantigen peptides or other genes of interest.
  • the promoter used to express the secretion tag/neoantigen peptide fusion can be, for example, pLacI or variants thereof, PlacZ or variants thereof, GlnRS or variants thereof, and T7 or variants thereof.
  • the promoter used to express the secretion tag/neoantigen peptide fusion can be, for example, the Bacillus p43 constitutive promoter or variants thereof, the Bacillus pveg constitutive promoter or variants thereof, the Bacillus pgsiB promoter or variants thereof, the Bacillus pylB promoter or variants thereof, the Bacillus pxylA promoter or variants thereof, or the Bacillus plial promoter or variants thereof.
  • sigma factors The task of promoter recognition in bacteria is left to one of a few protein subunits called sigma factors. Each sigma factor binds to its cognate promoter and connects with the RNAP core enzyme, forming the fully functioning RNAP holoenzyme.
  • E. coli there are seven known sigma factors and each bind to DNA promoters under different conditions. For example, Sigma 70 binds to its cognate DNA promoters at all times. Sigma 38 binds to its DNA cognate promoters in stationary state.
  • expression of a gene of interest can be controlled by employing promoters that interact with sigma factors that are dominant under the desired expression condition.
  • promoters that interact with sigma factors that are dominant under the desired expression condition.
  • gene expression would be upregulated in stationary phase rather than in exponential phase.
  • Table 1 B Known sigma factors in Bacillus spp.
  • Table 2A provides a list of example promoters compatible with various E. coli.
  • Table 2B provides a list of example promoters compatible with various Bacillus spp.
  • Table 2A Examples of E. coli DNA promoters.
  • Promoters for E. coli used herein include but are not limited to, high, medium, and low expression constitutive promoters, promoters that respond to stress, nutrient limitations, varying pH, varying osmotic pressure, and promoters that activate in stationary state.
  • Constitutive promoters perform best in nutrient-rich environments of the Gl tract - their differences in strength of gene expression are also used as a way to produce neoantigen peptides, maturation factors and secretion machinery at the most optimal ratios.
  • the FNR promoter acts as a constitutive control in the most anerobic environments of the Gl tract, as it originates from a switch system in E. coli between aerobic and anaerobic metabolism, the FNR regulon.
  • GadA/B promoters are pH sensitive, which makes them useful for the highly acidic components of the Gl tract.
  • the rpoS promoters can be used.
  • anaerobically- inducible promoters can be used.
  • chloride-inducible promoters can be used.
  • stationary-phase promoters can be used.
  • promoter osmB is a stress-responsive rpoS promoter intended for nutrient-poor environments with a high salt/ion content (osmotic stress).
  • the pveg promoter in B. subtilis is a super constitutive promoter and is the strongest known promoter in Bacillus. It is an o A -dependent promoter that seems to be essential for the growth of germinating cells coming out of the sporulation stage.
  • Pveg is a popular promoter to use for heterologous protein production, both plasmid and genome-based, due to its strong activity.
  • PylB is another common auto-inducible promoter that is being explored for industry uses.
  • the promoter is most active during mid-exponential phase all the way through stationary phase.
  • the expression level of pylB is directly correlated with the cell density.
  • pylB has been shown to produce more of a target protein during exponential and stationary phase than the constitutive promoter p43.
  • Pxyla is a promoter that is induced by xylose.
  • the promoter is accompanied with a gene called xylR which acts as the repressor on the promoter. In the presence of xylose, the repressor is released and the promoter is activated through derepression.
  • the Hal promoter is regulated by the LiaRS two-component system that responds to presence of bacitracin in sub-lethal amounts. It has a very low level of basal activity and is activated in a concentration-dependent manner.
  • the ribosome binding site can be, for example, RO and variants thereof, R1 and variants thereof, R2 and variants thereof, R3 and variants thereof, R4 and variants thereof, R5 and variants thereof, R6 and variants thereof, and/or R7 and variants thereof.
  • RBS ribosome binding site
  • Guiziou et al. developed multiple libraries of ribosomal binding sites and characterized their relative gene expression in Bacillus subtilis 168.
  • Table 3 provides a list of exemplary RBSs and their sequences as described by Guiziou et al, supra. These RBSs and derivatives thereof can be used in various Bacillus spp. provided herein to control translation of genes of interest.
  • Table 3 Exemplary RBSs used in Bacillus spp.
  • the DNA plasmids include a gene encoding for at least one neoantigen protein.
  • a nucleotide sequence encoding a neoantigen peptide may be predicted based on reference to the standard genetic code. When a neoantigen peptide is to be expressed in a particular microbe, a nucleotide sequence encoding the neoantigen peptide may be produced with reference to preferred codon usage for the particular microbe.
  • the method involves utilizing secretion systems in E. coli for efficient secretion of neoantigens.
  • secretion systems suitable for E. coli include:
  • the Sec secretion system is a widely used pathway in E. coli for protein translocation across the cytoplasmic membrane. By incorporating the appropriate signal peptide sequence upstream of the neoantigen gene in the expression plasmid, the neoantigen can be targeted for secretion via the Sec pathway.
  • TAT System The Twin-Arginine Translocation (TAT) secretion system is another secretion pathway in E. coli that allows the translocation of folded proteins across the cytoplasmic membrane.
  • TAT Twin-Arginine Translocation
  • the method includes utilizing secretion systems in Bacillus spp. for efficient secretion of neoantigens.
  • secretion systems suitable for Bacillus include:
  • Tat System The Tat secretion system, including TatA, TatB, and TatC proteins, is functional in Bacillus subtilis. By incorporating a Tat signal peptide, the neoantigen can be directed for secretion through the Tat pathway in Bacillus strains.
  • the secretion tag sequence is fused to the mature neoantigen peptide sequence.
  • the secretion tag sequence can be derived from Bacillus spp. such as Bacillus subtilis, Bacillus licheniformis, Bacillus paralicheniformis, Bacillus amyloliquefaciens, Bacillus coagulins.
  • the secretion tag can be a sec-dependent or tat-dependent secretion tag.
  • the sec-dependent secretion tag can be the AmyQ secretion tag, the LipA secretion tag, the Mdr secretion tag, the AprE secretion tag, the AbnB secretion tag, the EglS secretion tag, the PhrC secretion tag or the LipA secretion tag.
  • the tat-dependent secretion tag can be the PhoD secretion tag or the YwbN (EfeB) secretion tag.
  • the secretion tag can be a variant of the aforenoted secretion tags.
  • a coding sequence encoding a neoantigen peptide may further include nucleotides encoding a secretion signaling protein, such that the neoantigen peptide and the secretion signaling protein are fused and expressed as a single protein.
  • a secretion signaling protein, or secretion tag targets a protein for secretion out of the cell, and is usually present at the aminoterminal end of a protein.
  • Secretion signaling proteins useful in prokaryotic microbes are known in the art and routinely used.
  • secretion tags or ‘secretion signaling proteins’ are protein sequences that are directly upstream of the mature protein that would be secreted out of the cell.
  • secretion tags for heterologous gene expression in Bacillus, refer to Fu et al. [Biotechnology Advances, 25, 1-12 (2007)] and Simonen and Palva [Microbiological Reviews, 57, 109-137 (1993)].
  • the Sec and Tat secretion pathways are common routes for secretion from Bacillus spp.
  • the regions of a secretion tag that uses the Sec pathway include a positively charged NH2 terminus followed by a hydrophobic region in the middle termed the H region and ends with a polar C region where the cleavage site is located.
  • the consensus cleavage motif for a Secdependent secretion tag is typically A-X-A or V-X-A but this is not always the case.
  • the secretion tags can serve many purposes such as preventing premature folding of protein and the degradation of the protein in the cell. Most importantly the secretion tags guide the protein to the correct secretion machinery at the membrane.
  • SPase type I signal peptidases
  • a signal recognition particle forms a ribonucleoprotein complex with the signal peptide.
  • the SRP consists of a small condition RNA (scRNA) with a Ffh (GTPase) protein and two molecules of the Hbsu protein. After a successful interaction, the SRP guides the complex to a membrane protein called FtsY where precursor protein is then passed along to the Sec translocases.
  • the motor component of the translocation machinery for the Sec-dependent pathway is the SecA protein providing the energy via ATP hydrolysis domain for the translocation.
  • the SecA is a transmembrane protein essential for targeting and translocation the desired secreted protein.
  • the SecY, SecE, SecG, and SecDF proteins are integral proteins involved in the translocation of the unfolded mature target proteins across the membrane. In addition to the translocation, there are also several chaperone proteins located in the cytoplasm and extracellular space that aid in protein folding and ensure there is minimal aggregation of protein in the cell. [0114] E. coli is favorable for engineering PBCVs because of the available tools for heterologous protein production and because of the available tools for manufacturing.
  • Bacillus spp. are favorable for production of heterologous protein due to absence of an outer membrane which would theoretically simplify translocation and secretion. Additionally, Bacillus spp. are known to efficiently secrete a large number of proteins, the majority of which use a general secretion pathway.
  • T able 4 shows the different exemplary Sec and Tat-dependent secretion tags that were fused to mature peptides as described in the Examples. Included with each secretion tag is the amino acid sequence that ends with the cleavage site where the tag is cleaved from the mature peptide. The cleavage site is generally recognized by the A-X-A or V-X-A motif (italicized) though this is not the case for every Sec-dependent secretion tag. Also listed is the function of the protein that the secretion tag is originally attached to in B. subtilis 168’s genome (exception is AmyQ which is found in B. amyloliquefacien’s genome).
  • Table 4 Examples of Sec and Tat-dependent secretion tags for Bacillus spp.
  • the secretion tags derived from Bacillus subtilis such as Bacillus 168 can be inserted upstream of a neoantigen peptide gene fragment to generate a secretion tag library, e.g., as described in Example 9.
  • the secretion tag library with the neoantigen peptide is transformed into a desired Bacillus spp. isolate.
  • E. coll and Bacillus spp. provided herein are made competent and transformed with the engineered plasmid, e.g., as described in Example 7.
  • E. coll and Bacillus spp. provided can be made rifampicin resistant, e.g., as described in Example 10.
  • An essential gene (ex. dapA or thyA) can be knocked out of the genome of provided E. coli or Bacillus spp. yielding an auxotroph, e.g., as described in Example 11.
  • Exemplary genetically engineered E. coli isolates GP_8001, GP_8002, and GP_8003 and Bacillus isolates GP_9001 , GP_9002, and GP_9003 are provided herein.
  • E. coli and Bacillus spp. can be tested in intestinal tract-mimicking environments (ex. low pH, presence of bile salts); or in intestinal contents (ex. stomach/gizzard contents, duodenum contents, jejunum contents, ileum contents, large intestine contents, ceca contents). Growth of modified or unmodified E. coli and Bacillus spp. are tested in intestinal tract-mimicking environments (ex. minimal medium, rich medium); or in intestinal contents (ex.
  • stomach/gizzard contents duodenum contents, jejunum contents, ileum contents, large intestine contents, ceca contents, mucus from various animal hosts, or combinations of intestinal contents and laboratory-derived medium), e.g., as described in Example 12.
  • Tissue and cell samples from cancer patients can be obtained through tissue biopsy procedures.
  • Tissue biopsies are widely used and involve the surgical removal of a small portion of the cancerous tissue for diagnostic and research purposes.
  • Common techniques for tissue biopsy include: a) Needle Biopsy: Utilizing fine-needle aspiration or core needle biopsy techniques, a needle is inserted into the tumor site to extract tissue samples. This minimally invasive approach allows for sampling of solid tumors in various locations, b) Endoscopic Biopsy: this method involves performing endoscopic biopsies to collect tissue samples from internal organs or cavities. Endoscopic procedures allow for direct visualization and targeted biopsy sampling.
  • Bronchoscopy In this procedure, a flexible tube with a camera (bronchoscope) is inserted through the nose or mouth to collect tissue samples from the lungs or airways, ii) Colonoscopy: This procedure involves the insertion of a flexible tube with a camera (colonoscope) through the rectum to collect tissue samples from the colon or rectum, c) Surgical Biopsy: In cases where a larger tissue sample is required, open surgical biopsy procedures may be performed. These include excisional biopsy, incisional biopsy, or punch biopsy, involving the removal of the entire tumor or a representative portion of it.
  • Blood samples can be obtained from cancer patients. Blood samples provide access to circulating cancer cells, tumor-derived DNA, and other components that can aid in the identification of neoantigens. Common methods for blood sample collection include: a) Venipuncture: This widely used method involves the insertion of a needle into a vein, typically in the arm, to draw blood. The collected blood can be processed to isolate different components, such as white blood cells or cell-free DNA. b) Liquid Biopsy: Liquid biopsies are non-invasive methods that involve the collection of blood or other bodily fluids, such as plasma or serum, to detect tumor-derived genetic material or circulating tumor cells. Techniques like circulating tumor DNA (ctDNA) analysis or circulating tumor cell (CTC) isolation can be employed. Whole-Genome Sequencing and Identification of DNA Sequence Differences.
  • ctDNA circulating tumor DNA
  • CTC circulating tumor cell isolation
  • WGS wholegenome sequencing
  • Examples of WGS technologies include, but are not limited to, A) Illumina sequencing: Illumina sequencing, also known as sequencing by synthesis (SBS), is one of the most widely used WGS technologies. It utilizes reversible terminators and fluorescently labeled nucleotides to sequence DNA.
  • WES whole-exome sequencing
  • SNVs single-nucleotide variants
  • Indels small insertions/deletions
  • Examples of WES technologies include, but are not limited to, A) Agilent SureSelect: Agilent SureSelect employs hybrid capture technology, where biotinylated RNA probes are used to capture and enrich the exonic regions of interest. The captured DNA fragments are then sequenced using next-generation sequencing (NGS) platforms.
  • NGS next-generation sequencing
  • Twist Bioscience offers an exome enrichment panel that utilizes their proprietary target capture technology. The panel consists of custom-designed, single-stranded DNA probes that hybridize to the exonic regions. The captured DNA is then sequenced using NGS platforms.
  • Targeted panel sequencing involves selectively sequencing a panel of genes known to be involved in cancer development and progression. This approach offers high sequencing depth and sensitivity, allowing for the detection of low-frequency mutations. Targeted panel sequencing can be customized to include genes relevant to the specific cancer type, facilitating the identification of neoantigens associated with the patient's cancer.
  • RNA-Seq involves the sequencing of the transcriptome, providing insights into gene expression patterns and identifying differentially expressed genes between normal and cancerous tissues.
  • RNA-Seq analysis aids in the identification of fusion genes, alternative splicing events, and the quantification of gene expression levels. This information contributes to the identification of neoantigens and their subsequent incorporation into Personalized Bacterial Cancer Vaccines (PBCVs).
  • PBCVs Personalized Bacterial Cancer Vaccines
  • the cancer and normal tissue/cell samples can be subjected to single-cell sequencing techniques.
  • Single-cell sequencing allows for the analysis of individual cells, enabling the characterization of intra-tumoral heterogeneity and the identification of rare cell populations.
  • Methods such as single-cell RNA sequencing (scRNA-Seq) and single-cell DNA sequencing (scDNA-Seq) can provide insights into gene expression profiles, somatic mutations, and clonal evolution within the tumor. These findings can contribute to the identification of patient-specific neoantigens for the development of PBCVs.
  • the cancer and normal tissue/cell samples can be subjected to other sequencing techniques, such as methylome sequencing, chromatin immunoprecipitation sequencing (ChlP- Seq), or other emerging technologies.
  • sequencing techniques such as methylome sequencing, chromatin immunoprecipitation sequencing (ChlP- Seq), or other emerging technologies.
  • the choice of sequencing method can be based on the specific requirements of the cancer type, the level of genomic information desired, and the available resources.
  • a method for neoantigen discovery by comparing sequences of cancer and normal cells can involve aligning the sequences obtained from cancer cells and normal cells to a reference genome, followed by variant calling. This process compares the differences between the two sequences at the nucleotide level, identifying single-nucleotide variants (SNVs), small insertions/deletions (indels), and other genomic alterations. By focusing on the variants unique to the cancer cells, neoantigens can be identified as those derived from mutated genes or gene regions.
  • a method for neoantigen discovery by comparing sequences of cancer and normal cells can utilize prediction algorithms to identify neoantigens based on the identified genetic variants.
  • Various computational tools and algorithms are available that predict neoantigens based on factors such as peptide binding affinity to major histocompatibility complex (MHC) molecules and the likelihood of immunogenicity.
  • MHC major histocompatibility complex
  • These algorithms analyze the genetic alterations identified in the cancer cells and predict the potential neoantigens that can elicit an immune response [Brown SD et al., Oncoimmunology. 2018 Dec 22;8(3): 1556080. doi: 10.1080/2162402X.2018.1556080],
  • Methods for neoantigen discovery and selection can utilize machine learning and artificial intelligence algorithms. These methods include the following steps: 1. Training Datasets: Curating large-scale datasets comprising genomic, transcriptomic, and proteomic data from cancer patients, including information on mutations, gene expression profiles, and protein sequences. 2. Machine Learning Models: Training machine learning models, such as deep neural networks, using the curated datasets to identify features and patterns associated with immunogenic neoantigens. The models learn to predict the immunogenicity and antigenicity of neoantigens based on input features. 3.
  • Neoantigen Prioritization Utilizing the trained machine learning models to prioritize neoantigens from the identified pool based on their predicted immunogenicity, binding affinity to MHC molecules, and potential to elicit a robust immune response. 4. Incorporating Patient-specific Data: Integrating patient-specific data, such as HLA typing and individual immune profiles, into the Al models to further refine the selection process and ensure personalized neoantigen prioritization. 5. Iterative Optimization: Employing iterative optimization processes where the Al models continuously learn and adapt based on experimental and clinical data. Feedback from immune response evaluations and clinical outcomes is used to refine the models' predictions and enhance the selection of neoantigens.
  • Methods for neoantigen discovery and selection can include deep learning algorithms, as described in Cai et al., Front Oncol. 2023 Jan 9;12:1054231. doi: 10.3389/fonc.2022.1054231 and references therein. These algorithms include the following steps: 1. Deep Learning Architectures: Designing and training deep neural network architectures, such as convolutional neural networks (CNNs) or recurrent neural networks (RNNs), specifically tailored for neoantigen prediction. These models learn complex patterns and relationships between genetic variations, gene expression data, and immunogenicity. 2. Integration of Multiple Data Sources: Incorporating diverse data sources, including genomic sequencing data, transcriptomic profiles, and proteomic data, into the deep learning models.
  • CNNs convolutional neural networks
  • RNNs recurrent neural networks
  • Feature Extraction Utilizing the deep learning models to extract relevant features and representations from the input data that are indicative of neoantigen immunogenicity. This can include identifying mutation patterns, splicing events, gene expression levels, and protein structural characteristics.
  • Predictive Scoring Assigning predictive scores to neoantigens based on the output of the deep learning models, reflecting their likelihood of eliciting an immune response. The scoring can be used for ranking and selecting the most promising neoantigens for inclusion in PBCVs.
  • Methods for neoantigen discovery and selection can involve isolating and sequencing peptides from cancer cells and normal cells using mass spectrometry techniques. This approach allows for the direct identification of peptides derived from proteins present in the cancer cells. By comparing the peptide sequences from cancer and normal cells, neoantigens can be identified as those that are specific to the cancer cells and absent in the normal cells.
  • Methods for neoantigen selection can incorporate immunogenicity assays to evaluate the potential of identified neoantigens to elicit an immune response. These assays can include in vitro tests, such as T-cell activation assays, to assess the recognition and activation of T cells specific to neoantigens. For example, a plethora of immunogenicity assays can be used for specific gene mutations, as described for mutations of Tp53 in Malekzadeh, et al., J Clin Invest. 2019;129(3):e123791. doi.org/10.1 172/JCI123791 and references therein.
  • Methods for neoantigen selection can utilize computational algorithms and bioinformatics tools to predict neoantigens based on the genomic sequences of cancer and normal tissue samples. By applying algorithms that analyze somatic mutations, gene expression data, and binding affinity prediction models, potential neoantigens can be identified. Other models take into account various factors, including antigen processing and presentation, T-cell receptor recognition, and immune system activation. By assessing the immunogenic potential of neoantigens, the computational approach facilitates the ranking and prioritization of candidate neoantigens for incorporation into PBCVs to stimulate an immune response against cancer cells.
  • Methods for neoantigen selection can include employing computational tools to predict epitopes derived from cancer-associated mutations and analyzing their binding affinity to human leukocyte antigen (HLA) molecules.
  • HLA human leukocyte antigen
  • algorithms can identify neoantigens with high affinity for patient-specific HLA alleles. This embodiment enables the selection of neoantigens that have a higher likelihood of generating an effective immune response when included in PBCVs.
  • Methods for neoantigen selection can involve integrating transcriptomic data with genomic information. By comparing gene expression profiles between cancer and normal tissues, computational analysis can identify genes that are overexpressed or aberrantly expressed in cancer cells. The identified genes can serve as targets for neoantigen discovery, as their differential expression indicates the potential presence of cancer-specific antigens.
  • the described examples illustrate a range of methods for neoantigen discovery by comparing sequences of cancer and normal cells. It should be noted that these examples are not exhaustive, and other approaches, such as epitope prediction algorithms, functional assays, or combination strategies, can also be employed within the scope of the patent. The choice of methods can be based on factors such as the available resources, the desired sensitivity and specificity, and the specific characteristics of the cancer type being targeted.
  • Methods for neoantigen selection can focus on genes that are commonly mutated in various types of cancers. Examples include but are not limited to: 1. **TP53**: TP53 is a tumor suppressor gene that regulates cell division and prevents the formation of tumors. Mutations in TP53 are one of the most frequent alterations found in multiple cancer types, including lung, breast, colorectal, ovarian, and pancreatic cancers. 2. **KRAS**: KRAS is an oncogene that plays a critical role in cell signaling pathways regulating cell growth and division. Mutations in the KRAS gene are commonly found in several cancers, including colorectal, lung, and pancreatic cancers. 3.
  • EGFR** Epidermal Growth Factor Receptor
  • EGFR Epidermal Growth Factor Receptor
  • HER2 Human Epidermal Growth Factor Receptor 2
  • HER2 amplification or overexpression is often seen in breast and gastric cancers.
  • BRCA1 and BRCA2** BRCA1 and BRCA2 are tumor suppressor genes that play a crucial role in DNA repair. Mutations in these genes increase the risk of breast and ovarian cancers, as well as other types of cancer. 7. **PTEN**: PTEN is a tumor suppressor gene that regulates cell growth and division by inhibiting cell survival pathways. Mutations in PTEN are commonly found in several cancers, including breast, prostate, and endometrial cancers. 8. **ALK**: Anaplastic Lymphoma Kinase (ALK) is a gene that encodes a protein involved in cell signaling. ALK rearrangements or fusions are frequently seen in specific types of lung cancer, such as non-small cell lung cancer (NSCLC). 9.
  • ALK Anaplastic Lymphoma Kinase
  • RET is a gene that encodes a receptor tyrosine kinase involved in cell growth and division. RET mutations and fusions are observed in multiple cancers, including thyroid cancer and certain types of lung cancer. 10. **CDKN2A**: CDKN2A is a tumor suppressor gene that regulates the cell cycle. Mutations in CDKN2A are commonly associated with melanoma and pancreatic cancer.
  • Methods for neoantigen selection can focus on inherited genes that are associated with an increased risk of developing certain types of cancer. Examples include but are not limited to:
  • BRCA1 and BRCA2 Mutation Detection This method involves detecting mutations in the BRCA1 and BRCA2 genes, which are associated with an increased risk of breast, ovarian, and other cancers. By analyzing the genomic sequences of BRCA1 and BRCA2 obtained from patient samples, the presence of pathogenic mutations can be identified. These inherited mutations can be utilized for neoantigen selection in the development of Personalized Bacterial Cancer Vaccines (PBCVs) tailored to patients carrying these genetic alterations.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • Lynch Syndrome Gene Detection This method focuses on detecting mutations in genes associated with Lynch syndrome, such as MLH1 , MSH2, MSH6, and PMS2. Lynch syndrome increases the risk of colorectal, endometrial, and other cancers. Through genomic analysis of these genes, the presence of pathogenic mutations can be determined, providing a basis for selecting neoantigens for incorporation into PBCVs to target the specific cancer associated with Lynch syndrome.
  • APC Gene Mutation Detection This method includes identifying mutations in the APC gene, commonly associated with familial adenomatous polyposis (FAP) and an increased risk of colorectal cancer. By analyzing the genomic sequences of APC, mutations can be identified, and corresponding neoantigens can be selected for inclusion in PBCVs. These personalized vaccines target the specific cancer risk associated with APC mutations in individuals with FAP.
  • FAP familial adenomatous polyposis
  • PTEN Mutation Detection This method involves detecting mutations in the PTEN gene, associated with Cowden syndrome and an increased risk of breast, thyroid, and other cancers. Genomic analysis of PTEN can reveal pathogenic mutations, enabling the selection of neoantigens for incorporation into PBCVs. These personalized vaccines target the specific cancer risk associated with PTEN mutations in individuals with Cowden syndrome.
  • TP53 Germline Mutation Detection This method focuses on identifying germline mutations in the TP53 gene, associated with Li-Fraumeni syndrome and an increased risk of multiple cancer types. By examining the genomic sequences of TP53, germline mutations can be detected, serving as sources of neoantigens for PBCV development. These personalized vaccines target the specific cancer risks associated with TP53 germline mutations in individuals with Li-Fraumeni syndrome.
  • genes to target can depend on factors such as the prevalence of mutations, the specific hereditary cancer syndrome, and the available genetic information for the patient population.
  • Methods for growing engineered PBCV and testing for the expression and secretion of neoantigen peptides can involve species-specific procedures.
  • Example methods for growing E. coli can involve the following steps: 1. Start by preparing a suitable culture medium, such as Luria-Bertani (LB) broth or agar, supplemented with appropriate antibiotics if necessary. 2. Inoculate a small volume of the medium with the engineered E. coli strain containing the plasmid carrying the neoantigen and any adjuvant genes. 3. Incubate the culture at the optimal temperature and conditions for E. coli growth, typically 37°C with shaking. 4. Monitor the growth of the culture by measuring optical density (OD) at a specific wavelength or by plating serial dilutions on agar plates and counting colonyforming units (CFUs). 5. At a desired time point, harvest a portion of the culture to assess neoantigen expression and secretion.
  • a suitable culture medium such as Luria-Bertani (LB) broth or agar, supplemented with appropriate antibiotics if necessary. 2. Inoculate a small volume of the medium with the engineered E. coli
  • Example methods for testing expression and secretion of neoantigens in E. coli can include the following examples: 1. Perform cell lysis: Collect a fraction of the bacterial culture and perform cell lysis using appropriate methods such as sonication or freeze-thaw cycles to release cellular contents. 2. Analyze protein expression: Use techniques like SDS-PAGE or Western blotting to detect and analyze the expression of neoantigens in the lysate. This can involve probing with specific antibodies or antigen-specific probes. 3. Assess secretion: Collect the remaining culture supernatant and concentrate it using methods like ultrafiltration or precipitation. Analyze the concentrated supernatant using SDS-PAGE, Western blotting, or other immunological assays to detect and assess the presence of secreted neoantigens.
  • Example methods for growing Bacillus spp. can involve the following steps: 1. Prepare a suitable culture medium specific to the Bacillus species being used, such as nutrient agar or broth supplemented with appropriate antibiotics if required. 2. Inoculate a small volume of the medium with the engineered Bacillus strain harboring the plasmid carrying the neoantigen gene. 3. Incubate the culture at the optimal temperature and conditions for Bacillus growth, typically 30-37°C. 4. Monitor the growth of the culture by measuring optical density (OD) or by plating serial dilutions on agar plates and counting colony-forming units (CFUs). 5. Harvest a portion of the culture at the desired time point for further analysis of neoantigen expression and secretion.
  • a suitable culture medium specific to the Bacillus species being used such as nutrient agar or broth supplemented with appropriate antibiotics if required.
  • Example methods for testing expression and secretion of neoantigens in Bacillus spp. can include: 1. Perform cell lysis: Collect a fraction of the bacterial culture and perform cell lysis using methods suitable for Bacillus spp., such as enzymatic lysis or sonication. 2. Analyze protein expression: Employ techniques like SDS-PAGE, Western blotting, or mass spectrometry to detect and analyze the expression of neoantigens in the lysate. Use specific antibodies or antigen-specific probes for detection. 3. Assess secretion: Collect the remaining culture supernatant and concentrate it using techniques like ultrafiltration or precipitation. Analyze the concentrated supernatant for the presence of secreted neoantigens using SDS-PAGE, Western blotting, or immunological assays.
  • a method for evaluating the immunogenicity of expressed/secreted neoantigens using tumor cell lines can comprise a. Cultivating tumor cell lines representative of the cancer type being targeted by the PBCV. b. Treating the tumor cell lines with the expressed/secreted neoantigens derived from the engineered bacteria, c. Assessing the immune response by measuring the activation and proliferation of immune cells, such as T cells, natural killer (NK) cells, or antigen-presenting cells (APCs). d. Analyzing the production of immune-related molecules, including cytokines, chemokines, or cytotoxic effector molecules, e. Monitoring the specific recognition and killing of tumor cells expressing the neoantigens by immune cells using techniques such as flow cytometry, cytotoxicity assays, or ELISPOT assays.
  • a method for examining the immunogenicity of expressed/secreted neoantigens using dendritic cells comprising: a. Cultivating DCs from peripheral blood monocytes or other suitable sources, b. Co-culturing the DCs with the expressed/secreted neoantigens derived from the engineered bacteria, c. Evaluating the maturation and activation of DCs by assessing surface marker expression, cytokine production, or antigen presentation capability, d. Assessing the ability of DCs to prime and stimulate T cells by measuring T cell activation, proliferation, or cytokine secretion, e. Monitoring the interaction between DCs and T cells, such as antigenspecific T cell recognition or antigen-specific T cell receptor (TCR) signaling.
  • DCs dendritic cells
  • a method for evaluating the immunogenicity of expressed/secreted neoantigens using lymphocyte populations comprising: a. Isolating lymphocytes, including peripheral blood mononuclear cells (PBMCs), from blood samples of cancer patients or healthy donors, b. Stimulating the lymphocytes with the expressed/secreted neoantigens derived from the engineered bacteria, c. Assessing lymphocyte activation, proliferation, or cytokine production as indicators of immune response, d. Analyzing the differentiation and expansion of antigenspecific T cell populations using techniques such as flow cytometry, ELISPOT assays, or TCR sequencing, e.
  • PBMCs peripheral blood mononuclear cells
  • cytotoxicity assays such as the chromium release assay or granzyme/perforin assays.
  • cytotoxicity assays such as the chromium release assay or granzyme/perforin assays.
  • a method for evaluating the expression of Interferon-gamma (IFN-y) in response to expressed/secreted neoantigens comprising a. Stimulating immune cells, such as T cells or lymphocytes, with the expressed/secreted neoantigens derived from the engineered bacteria, b. Incubating the stimulated cells for a designated period, c. Harvesting the cell culture supernatant, d. Quantifying the expression of IFN-y using techniques such as enzyme-linked immunosorbent assay (ELISA) or flow cytometry, e. Analyzing the levels of IFN-y production as an indicator of immune activation and the potential immunogenicity of neoantigens when expressed and selected from PBCVs.
  • IFN-y Interferon-gamma
  • a method for assessing the production of Tumor Necrosis Factor-alpha (TNF-a) in response to expressed/secreted neoantigens comprising a. Treating immune cells, such as dendritic cells (DCs) or macrophages, with the expressed/secreted neoantigens derived from the engineered bacteria, b. Incubating the treated cells for a specified duration, c. Collecting the cell culture supernatant, d. Quantifying the production of TNF-a using techniques such as ELISA or multiplex cytokine assays, e. Analyzing the levels of TNF-a production as an indicator of immune activation and the potential immunogenicity of the selected neoantigens when expressed and secreted from PBCVs.
  • TNF-a Tumor Necrosis Factor-alpha
  • a method for analyzing the secretion of lnterleukin-2 (IL-2) in response to expressed/secreted neoantigens comprising a. Co-culturing immune cells, such as T cells or lymphocytes, with the expressed/secreted neoantigens derived from the engineered bacteria, b. Allowing the co-cultured cells to incubate for a designated time period, c. Collecting the cell culture supernatant, d. Detecting the secretion of IL-2 using techniques like ELISA or cytokine bead arrays, e. Assessing the levels of IL-2 secretion as an indication of T cell activation and the immunogenic potential of the neoantigens when expressed and selected from PBCVs.
  • IL-2 lnterleukin-2
  • IFN-y Interferon-gamma
  • TNF-a Tumor Necrosis Factor-alpha
  • IL-2 lnterleukin-2
  • ELISA cytokine quantification
  • a method for evaluating the efficacy of Personalized Bacterial Cancer Vaccines (PBCVs) using mouse xenograft models comprising a. Establishing tumor xenografts in immunocompromised mice by injecting human cancer cells, b. Administering the recombinant PBCVs orally to the mice. c. Monitoring tumor growth over time through caliper measurements or imaging techniques, d. Assessing the impact of the PBCVs on tumor growth inhibition or regression compared to control groups, e. Analyzing the immune response by examining infiltrating immune cells within the tumor microenvironment using techniques such as immunohistochemistry or flow cytometry, f. Measuring the systemic immune response by evaluating immune cell subsets and cytokine production in peripheral blood or lymphoid organs, g. Evaluating the overall survival and long-term effects of the PBCVs on tumor burden and metastasis.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • a method for evaluating the immunogenicity of Personalized Bacterial Cancer Vaccines (PBCVs) using syngeneic animal models comprising a. Establishing syngeneic tumor models in immunocompetent mice by implanting murine cancer cells, b. Administering the recombinant PBCVs orally or via other suitable routes to the mice. c. Monitoring tumor growth kinetics using caliper measurements or imaging techniques, d. Assessing the immune response by analyzing immune cell infiltration within the tumor, cytokine production, and antibody responses using techniques such as flow cytometry, ELISA, or immunohistochemistry, e. Evaluating the activation and expansion of tumor-specific T cells using techniques like tetramer staining or TCR sequencing, f. Assessing the impact of the PBCVs on tumor regression, overall survival, and the establishment of immune memory.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • a method for evaluating the systemic immune response induced by Personalized Bacterial Cancer Vaccines (PBCVs) using animal models comprising a. Administering the recombinant PBCVs orally or via other suitable routes to immunocompetent animals, b. Assessing peripheral immune cell populations, including T cells, B cells, natural killer (NK) cells, and antigen-presenting cells (APCs), by flow cytometry or other suitable techniques, c. Analyzing the production of immune-related molecules, such as cytokines, chemokines, or immunoglobulins, in serum or other biological fluids using techniques like ELISA or multiplex assays, d.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • PBCVs Personalized Bacterial Cancer Vaccines
  • a method for GMP manufacturing of Personalized Bacterial Cancer Vaccines (PBCVs) in the form of pills for oral delivery can comprise the following steps:
  • Step 1 Master Cell Bank (MCB) and Working Cell Bank (WCB) Generation a. Generating a Master Cell Bank (MCB) from the engineered bacterial strain expressing the neoantigens, following established GMP guidelines and documentation. b. Establishing a Working Cell Bank (WCB) from the MCB to ensure a consistent and renewable source of the engineered bacteria for production.
  • MCB Master Cell Bank
  • WB Working Cell Bank
  • Step 2 Fermentation and Culture a. Scaling up the production of the engineered bacteria in a controlled fermentation system using suitable growth media and conditions. b. Monitoring and controlling critical parameters such as temperature, pH, dissolved oxygen, and agitation to achieve optimal growth and productivity. c. Harvesting the bacterial culture at the desired growth phase.
  • Step 3 Purification and Concentration a. Employing purification techniques, such as filtration, centrifugation, or chromatography, to separate and purify the bacterial cells from the fermentation broth. b. Concentrating the bacterial cells to obtain a high-density bacterial suspension.
  • Step 4 Formulation and Encapsulation a. Formulating the concentrated bacterial suspension with suitable excipients, stabilizers, and additives to ensure stability and oral delivery compatibility. b. Encapsulating the formulated PBCVs into pills using pharmaceutical-grade encapsulation equipment, ensuring uniformity and consistent dosing.
  • Step 5 Quality Assurance/Quality Control (QA/QC) Documentation a. Implementing comprehensive QA/QC measures throughout the manufacturing process to ensure product quality, consistency, and compliance with GMP regulations. b. Performing rigorous testing of raw materials, in-process samples, and final PBCV products for identity, purity, potency, sterility, endotoxin levels, and other relevant specifications. c. Documenting and maintaining detailed records of manufacturing procedures, batch records, testing results, and any deviations or corrective actions taken. d. Conducting stability studies to assess the shelf life and storage conditions of the PBCV pills. e. Complying with regulatory requirements and submitting appropriate documentation for regulatory approval and compliance.
  • QA/QC Quality Assurance/Quality Control
  • This example embodiment describes the GMP manufacturing process of Personalized Bacterial Cancer Vaccines (PBCVs) in the form of pills for oral delivery.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • the process involves generating cell banks, fermentation and culture, purification and concentration, formulation and encapsulation, and comprehensive QA/QC documentation.
  • Adhering to GMP guidelines and implementing stringent quality control measures ensures the production of high-quality, safe, and effective PBCV pills for oral administration to patients.
  • Embodiment Oral Administration with 0.01 Billion, 0.1 Billion, 1 Billion, 2 Billion, 3 Billion, 4 Billion, 5 Billion, 6 Billion, 7 Billion, 8 Billion, 9 Billions or 10 Billion colony forming units (CFUs) of PBCV once, twice or thrice per day, for one, two, three, four, five, six or seven days a week, for one, two, three, four, five, six, seven, eight or more weeks.
  • the PBCV pills containing the engineered bacteria are administered orally to the patient at a prescribed dose.
  • This embodiment aims to provide an appropriate dose and treatment course intended to enhance the exposure of the immune system to the neoantigens and potentially improve the immune response against the cancer cells.
  • Embodiment Rectal Administration with 0.01 Billion, 0.1 Billion, 1 Billion, 2 Billion, 3 Billion, 4 Billion, 5 Billion, 6 Billion, 7 Billion, 8 Billion, 9 Billions or 10 Billion colony forming units (CFUs) of PBCV once, twice or thrice per day, for one, two, three, four, five, six or seven days a week, for one, two, three, four, five, six, seven, eight or more weeks
  • the patient is instructed to insert PBCV suppositories into the rectum, allowing for localized delivery of the vaccine to the lower gastrointestinal tract.
  • the suppositories contain the engineered bacteria expressing the neoantigens.
  • the dosage and composition of the aims to target the immune-rich environment of the lower gastrointestinal tract, allowing direct interaction between the neoantigens and the immune cells present in the local tissue. This localized delivery may enhance the immune response against the cancer cells expressing the neoantigens while minimizing systemic exposure and potential side effects.
  • Embodiment Fecal Analysis for PBCV Detection.
  • stool samples from the patient are collected and analyzed to validate the presence of PBCVs in the gut.
  • the analysis may involve techniques such as quantitative polymerase chain reaction (qPCR) or DNA sequencing to detect and quantify the specific genetic markers or sequences unique to the engineered bacteria used in the PBCVs. Positive results indicate the colonization and persistence of PBCVs in the gastrointestinal tract.
  • qPCR quantitative polymerase chain reaction
  • Embodiment Serological Assays for Immune Response Assessment. This embodiment involves conducting serological assays on blood samples from the patient to assess the immune response triggered by the PBCVs.
  • the serological assays may include enzyme-linked immunosorbent assays (ELISAs) or multiplex immunoassays to measure the levels of specific antibodies targeting the neoantigens expressed by the PBCVs. An increase in neoantigen-specific antibody titers over time indicates an immune response against the cancer cells expressing the neoantigens.
  • ELISAs enzyme-linked immunosorbent assays
  • multiplex immunoassays to measure the levels of specific antibodies targeting the neoantigens expressed by the PBCVs.
  • An increase in neoantigen-specific antibody titers over time indicates an immune response against the cancer cells expressing the neoantigens.
  • Embodiment Flow Cytometry Analysis of Peripheral Blood Mononuclear Cells (PBMCs).
  • peripheral blood samples are collected, and the immune cells, particularly peripheral blood mononuclear cells (PBMCs), are isolated.
  • PBMCs peripheral blood mononuclear cells
  • Flow cytometry analysis is then performed to assess the activation and phenotype of immune cells, such as T cells and natural killer (NK) cells, in response to the PBCVs. Markers of immune cell activation, cytokine production, and expression of immune checkpoint receptors can be measured, indicating an ongoing immune response against the cancer cells expressing the neoantigens.
  • Embodiment Prostate-Specific Antigen (PSA) Test for Prostate Cancer.
  • PSA Prostate-Specific Antigen
  • the PSA test is a widely used blood test to measure the levels of prostate-specific antigen, a protein produced by the prostate gland. Elevated PSA levels can indicate the presence of prostate cancer or other prostate-related conditions.
  • This embodiment involves performing the PSA test to assess prostate cancer antigen levels in the patient's blood, providing valuable information for prostate cancer diagnosis and monitoring.
  • Embodiment CA-125 Test for Ovarian Cancer.
  • the CA-125 test measures the levels of cancer antigen 125 in the blood.
  • CA-125 is a protein that is often elevated in ovarian cancer. This embodiment involves conducting the CA-125 test to assess the levels of CA-125 in the patient's blood, aiding in the diagnosis and monitoring of ovarian cancer.
  • Embodiment HER2/neu Testing for Breast Cancer.
  • HER2/neu testing is performed to determine the overexpression or amplification of the HER2/neu gene in breast cancer cells. This testing helps identify breast cancer patients who may benefit from targeted therapies that specifically target the HER2 protein.
  • the embodiment involves performing HER2/neu testing on tumor samples obtained from the patient to determine the HER2/neu status and guide treatment decisions.
  • Embodiment Alpha-Fetoprotein (AFP) Test for Liver Cancer.
  • the AFP test measures the levels of alpha-fetoprotein in the blood, which can be elevated in patients with liver cancer or other liver-related conditions. This embodiment includes conducting the AFP test to assess AFP levels in the patient's blood, aiding in the diagnosis and monitoring of liver cancer.
  • Embodiment Carcinoembryonic Antigen (CEA) Test for Colon Cancer.
  • CEA Carcinoembryonic Antigen
  • the CEA test is commonly used for the detection and monitoring of colon cancer.
  • CEA is a glycoprotein that is often elevated in individuals with colon cancer.
  • This embodiment involves performing the CEA test to measure the levels of CEA in the patient's blood. Elevated CEA levels can indicate the presence of colon cancer and help in monitoring treatment response and disease progression.
  • Embodiment CD20 Testing for B-cell Lymphoma.
  • CD20 is a cell surface marker expressed on B-cells, and it is often associated with B-cell lymphomas such as non-Hodgkin lymphoma.
  • This embodiment includes conducting CD20 testing on tumor samples or peripheral blood to determine the presence and levels of CD20-expressing cells. CD20 testing helps in the diagnosis, classification, and monitoring of B-cell lymphomas and aids in the selection of appropriate targeted therapies.
  • Embodiment Philadelphia Chromosome Testing for Chronic Myeloid Leukemia (CML).
  • the Philadelphia chromosome is a genetic abnormality commonly found in patients with chronic myeloid leukemia (CML).
  • This embodiment involves performing Philadelphia chromosome testing, such as fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR), on blood or bone marrow samples to detect the presence of the Philadelphia chromosome. This testing is crucial for confirming the diagnosis of CML and guiding treatment decisions, including the use of targeted therapies.
  • FISH fluorescence in situ hybridization
  • PCR polymerase chain reaction
  • This testing is crucial for confirming the diagnosis of CML and guiding treatment decisions, including the use of targeted therapies.
  • Embodiment Genetic Testing for Pediatric Cancers. Genetic testing plays a significant role in the diagnosis and management of pediatric cancers.
  • This embodiment encompasses various genetic tests, such as chromosomal analysis, next-generation sequencing, or specific gene mutation testing, performed on tumor samples or blood samples from pediatric cancer patients. These tests help identify genetic abnormalities, gene mutations, or inherited cancer predisposition syndromes, enabling personalized treatment approaches and genetic counseling for the patient and their family.
  • the PBCVs are designed for the treatment, control, and prevention of breast cancer.
  • Breast cancer is a prevalent malignancy, and the PBCVs can be tailored to target patient-specific neoantigens derived from genetic alterations observed in breast cancer cells.
  • the vaccines can be administered orally to target the gastrointestinal tract, where a significant portion of the immune system resides, thereby eliciting a specific immune response against breast cancer cells expressing neoantigens.
  • the PBCVs are formulated to combat lung cancer.
  • Lung cancer poses significant challenges due to its diverse subtypes and genetic heterogeneity.
  • the PBCVs can be personalized to incorporate neoantigens specific to lung cancer patients, enabling the immune system to recognize and destroy cancer cells in the lungs.
  • Oral administration of PBCVs allows for targeted delivery to the gastrointestinal tract, augmenting the immune response and enhancing therapeutic outcomes.
  • the PBCVs are developed for the treatment, control, and prevention of colorectal cancer.
  • Colorectal cancer is a common malignancy, and PBCVs can be designed to target patient-specific neoantigens found in colorectal cancer cells. By colonizing the gastrointestinal tract and releasing neoantigens, the PBCVs enhance immune recognition and subsequent immune-mediated elimination of colorectal cancer cells, both locally and systemically.
  • the PBCVs are engineered to address prostate cancer.
  • Prostate cancer is a significant health concern, and PBCVs can be personalized to incorporate patient-specific neoantigens derived from genetic alterations observed in prostate cancer cells.
  • the oral administration of PBCVs allows for targeted delivery to the gastrointestinal tract, where a substantial portion of the immune system resides, thereby triggering a specific immune response against prostate cancer cells expressing neoantigens.
  • the PBCVs are developed to target pediatric cancers.
  • Pediatric cancers present unique challenges, and PBCVs offer a personalized approach to address these malignancies.
  • the PBCVs can trigger a robust and specific immune response against pediatric cancer cells.
  • the oral administration of PBCVs allows for targeted delivery to the gastrointestinal tract, enhancing the efficacy of the immune response in pediatric cancer patients.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • PBCVs can be customized to target various other cancer types, including but not limited to ovarian cancer, pancreatic cancer, melanoma, lymphoma, leukemia, and others.
  • the tailored approach of PBCVs holds promise for personalized cancer immunotherapy across a wide spectrum of malignancies.
  • PBCVs Bacterial Cancer Vaccines
  • PBCVs represent a paradigm shift towards personalized medicine in cancer treatment.
  • PBCVs can identify neoantigens, which are specific to the individual and their tumor.
  • organisms that are safe and can colonize the patient’s gastrointestinal tract can be identified.
  • PBCVs have the ability to activate and enhance the patient's immune response against cancer cells. By expressing and secreting neoantigens, PBCVs effectively present these tumor-specific targets to the immune system, triggering a robust anti-tumor immune response. This immune activation can lead to the destruction of cancer cells locally and systemically, offering the potential for long-lasting and durable responses. PBCVs hold the promise of overcoming immune tolerance mechanisms that often hinder conventional cancer treatments, such as immune checkpoint inhibitors.
  • PBCVs Broad Applicability and Versatility.
  • One of the remarkable features of PBCVs is their potential applicability across various types of cancers.
  • PBCVs have shown promise in pediatric cancers, where conventional treatment options are often limited. This versatility makes PBCVs a potential therapeutic option for a broad spectrum of cancer patients, increasing the reach and impact of this novel approach.
  • PBCVs when administered orally, can colonize the gut and interact with the gut microbiome, thereby modulating the local immune environment. This targeted approach takes advantage of the gut microbiome-immune axis to potentiate the immune response against cancer cells.
  • the unique interplay between PBCVs, the gut microbiome, and the immune system holds tremendous potential for harnessing the power of the body's own defenses against cancer.
  • the present invention provides a comprehensive and innovative approach for the development and utilization of Personalized Bacterial Cancer Vaccines (PBCVs) in the treatment of various cancers.
  • PBCVs Personalized Bacterial Cancer Vaccines
  • PBCVs The versatility of PBCVs is evident in their applicability to different types of cancers, including solid tumors, blood cancers, and pediatric cancers. Furthermore, by targeting the gastrointestinal tract and utilizing the gut microbiome-immune axis, PBCVs tap into a critical immune hub, enhancing the body's defense mechanisms against cancer.
  • the invention also encompasses the use of computational bioinformatics and artificial intelligence techniques to optimize the selection of neoantigens, ensuring the highest immunogenicity and therapeutic efficacy. Additionally, the patent describes robust manufacturing processes adhering to Good Manufacturing Practices (GMP) standards, ensuring the production of safe and quality PBCVs for clinical use.
  • GMP Good Manufacturing Practices
  • PBCVs provide a comprehensive roadmap for the development, manufacturing, and administration of PBCVs as a personalized and potent therapeutic approach for cancer treatment.
  • PBCVs have the capacity to transform the landscape of oncology, providing new hope for patients and revolutionizing the field of cancer immunotherapy.
  • the present invention represents a significant advancement in the field of cancer treatment, bringing us closer to the realization of personalized, effective, and curative strategies for combating this devastating disease.
  • Example 1 Isolation of E. coli and Bacillus spp. from Patient Stool Samples
  • the stool samples of a patient can be stored in the -20°C freezer until use. The samples can then be thawed at room temperature, separated into segments weighing 2 grams each, placed into tubes along with 2 grams of phosphate buffered saline at a 1 :1 ratio and shaken to mix the contents.
  • the sample is then centrifuged at 3000 ref for 2 minutes and 50 uL of the pellet is plated on another MacConkey agar plate. When necessary, 10 and 100x dilutions are performed prior to plating to obtain single colonies. The plates are incubated overnight (-20 hours) at 37°C. Colonies exhibiting lactose-fermenting E. coli morphology on MacConkey agar are saved for further evaluation.
  • Various techniques are used to isolate the Bacillus spp. spores which included boiling, ethanol shock, or a combination of both.
  • the procedure for boiling includes diluting the stool sample in phosphate buffered saline at a 1 :1 ratio followed by heating at 80°C for 20 minutes.
  • the procedure for ethanol shock includes mixing the intestinal contents with 95% ethanol at a 1 :1 ratio and incubating at room temperature for 1 hour. If both selection methods are used, the ethanol shock is done first followed by the boiling.
  • ⁇ 0.5ml_ of the treated stool contents are plated on various microbiological media such as Brain Heart Infusion agar, Luria-Bertani agar, Mueller-Hinton agar, and Nutrient Agar. Plates are incubated overnight at 37°C and individual colonies are isolated and transferred for subsequent testing and preservation.
  • E. coli and Bacillus spp. after isolation were cultivated aerobically in Luria-Beranti broth at 37°C using a rotary shaker (200rpm) for 18-20 hours.
  • antibiotics were supplemented into the media for engineered derivatives and included chloramphenicol (20ug/mL) and kanamycin (75ug/mL) for E. coli and kanamycin (20ug/mL) for Bacillus spp.
  • PCRs colony Polymerase Chain Reaction
  • E. coli Nissle 1917 as well as another verified E. coli isolate are used as positive controls. Detection of one or both PCRs with the expected band size signifies positive E. coli identity. Five positive isolates from each segment of the patient’s stools are grown overnight in lysogeny broth and saved as freezer stocks at -80°C.
  • the PCR was performed using Promega GoTaq Green Mastermix according to the manufacturer’s protocol.
  • the PCR reaction mixture consisted of 12.5 uL of GoTaq Green Mastermix, 2 uL of the BOXA1 R primer (10 pM solution), and 10.5 uL of autoclaved distilled water.
  • a colony of Bacillus was swabbed with a sterile tip and placed into the reaction mixtures. The tip was then ground into the reaction tube to dislodge cells and release their DNA.
  • thermocycler settings were as follows:
  • Bacillus spp. isolates were sent for 16S rRNA sequencing to identify the species of the isolate.
  • the 16S rRNA gene was amplified using polymerase chain reaction (PCR) with the universal primers, 27F (5’AGAGTTTGATCMTGGCTCAG-3’, SEQ ID NO: 38) and 1492R (5’- GGYTACCTTGTTACGACTT-3’, SEQ ID NO: 39).
  • PCR polymerase chain reaction
  • GP1336 was initially identified as Bacillus licheniformis and GP1252 was identified as Bacillus oleronius. GP1336 (ATCC Accession No. PTA-127307) was later discovered to a Bacillus paralicheniformis strain after the whole genome was sequenced.
  • the PCR was performed using Promega GoTaq Green Mastermix.
  • the PCR reaction mixture consisted of 12.5 uL of GoTaq Green mastermix, 1 uL of each primer (10 pM solution), and 10.5 uL of autoclaved distilled water.
  • a colony of Bacillus was swabbed with a sterile tip and placed into the reaction mixtures. The tip was then ground into the reaction tube to dislodge cells and release their DNA.
  • thermocycler settings were as follows:
  • Bacillus subtilis supernatant was then incubated with the peptides of interest and peptide presence was tested via electrophoresis. It was found that incubation with B. subtilis 168 supernatant for just 3 hours resulted in complete elimination of peptide activity.
  • B. subtilis A8-22 was tested with several genetic constructs to produce peptides. However, no production was observed. Supernatant protease activity assays were performed with this strain. Despite the elimination of all detectable protease genes, rapid peptide inactivation was still observed. Thus, the heterologous production of peptides was unable to be attained using traditional methods previously known to those skilled in the art.
  • the peptide of interest can be produced using various strains of bacteria known to produce high quantities of the peptides. 50 mL cultures of the producer strains were grown in BHI at 37°C for ⁇ 20 hr. Cultures were then centrifuged to remove cells and supernatant was sterilized by boiling for 10 minutes at 100°C. The act of boiling at this time and temperature has been found to eliminate any protease activity from the producer supernatant without impacting peptide activity. This supernatant will be referred to as the “peptide stock.”
  • 500 uL of Bacillus supernatant was mixed 500 uL of the peptide stock and incubated at 37°C for 3 hours.
  • 500 uL of plain BHI was mixed with 500 uL of the peptide stock as a negative control for protease activity.
  • 500 uL of plain B. subtilis 168 supernatant was mixed with 500 uL of peptide stock as a positive control for protease activity.
  • 180 uL of the mixed supernatant was placed into sterile PCR tubes and boiled in the thermocycler (100°C, 10min) to inactivate protease activity.
  • a Bacillus isolate was considered to exhibit “favorable protease activity” if peptide detection techniques (e.g. mass spectrometry or protein gels) identified the peptide in the peptide/supernatant mixture. If no peptide could be observed, the isolate was considered unfavorable and was discounted for further assessment.
  • peptide detection techniques e.g. mass spectrometry or protein gels
  • antibiotics tested and their concentrations are spectinomycin (100ug/mL), kanamycin (20ug/mL) and erythromycin (0.5ug/ml_).
  • Isolates are tested for hemolytic activity by streaking fresh patches of each isolate on to Blood Agar medium (Thermo Scientific TSA with Sheep blood, part number R01201). Plates arre incubated overnight at 37°C. The following morning, plates are analyzed for signs of betahemolysis which includes a clear halo containing lysed red blood cells surrounding the bacterial growth. Isolates are selected when they show no signs of beta-hemolysis.
  • E. coli PBCVs DNA constructs are first generated in standard cloning strains (ex. E. coli JM109 and E. coli MC1061 F’). Constructs are then isolated, sequence-verified, and transformed into the desired delivery host.
  • E. coli isolated from patient stool samples are made electrocompetent using standard methods. Briefly, 100 mL of lysogeny broth (LB) is inoculated with 400 uL of an overnight culture of E. coli then incubated at 37C under agitation until the culture reaches an OD600 of ⁇ 0.4. The cells are pelleted down by centrifugation at 4000xg for 10 minutes and washed three times in ice cold deionized water. The final pellet is resuspended in 400 uL ice cold 10% glycerol in deionized water.
  • LB lysogeny broth
  • Neoantigen peptide plasmid design is described in detail in Example 10.
  • Bacillus PBCVa are transformed with the plasmid of interest via electroporation.
  • Cells were made competent using the protocol described by Lu et al. [Letters in Applied Microbiology, 55, 9-14 (2012)]. This protocol was originally intended for transformation of Bacillus subtilis 168 derivatives but was found to be effective for various isolates of Bacillus.
  • Bacillus PBCVs are struck out on lysogeny broth (LB) plates and incubated overnight at 37°C. The next day, a colony is selected from each of the plates and is inoculated into a 3 mL LB culture and incubated overnight shaking at 37°C. The following day, 50 uL of the overnight culture is inoculated into 3mL of LB and incubated shaking at 37°C for 17 hrs. After the 17 hr incubation, 1 mL of the culture is inoculated into a 100 mL Erlenmeyer flask containing 40 mL of LB + 0.5 M sorbitol. The flask culture is incubated shaking at 37°C until the 600 nm optical density (OD600) reaches - 0.8.
  • OD600 600 nm optical density
  • the cultures are placed into 50 mL conical tubes and incubated on ice for 5 minutes. After the 5-minute incubation, the cultures are centrifuged at 5,000xg for 10 min at 4°C to collect the cells. After the cells are pelleted, 15 mL of ice-cold Bacillus electroporation buffer (0.5M sorbitol, 0.5M trehalose, and 0.5M mannitol in 10% glycerol) is dispensed into the tubes and the pellets are resuspended via shaking.
  • Bacillus electroporation buffer 0.5M sorbitol, 0.5M trehalose, and 0.5M mannitol in 10% glycerol
  • the cells are recovered in 1mL of BHI containing 0.5M sorbitol and 0.38M mannitol shaking for 3 hours. After the 3-hour incubation, the cells are pelleted (5000xg, 5 minutes) and resuspended in -100 uL of media. The cells are then plated on BHI plus kanamycin (20 ug/mL) and incubated overnight at 37°C.
  • Plasmids containing the neoantigen of interest along with any additional adjuvant genes were constructed using a seamless assembly method that employs digestions along with subsequent ligations in the same reaction.
  • the method utilizes plasmids containing separate building blocks of a transcriptional unit (i.e. promoters, RBSs, genes, and terminators) that are isolated via digestion and ligated into a new plasmid backbone forming a functional transcriptional unit
  • a transcriptional unit i.e. promoters, RBSs, genes, and terminators
  • Plasmid pGP8001 in Figure 2 also includes an example of the transcriptional unit for the neoantigen peptide production as well as an example of the transcriptional unit for the auxotrophy-selection gene (thyA in this case).
  • thyA auxotrophy-selection gene
  • a kanamycin resistance transcriptional unit is used in place of the thyA transcriptional unit.
  • Figure 3 shows examples of possible neoantigen peptide transcriptional units.
  • the promoter regulating the neoantigen peptide transcriptional unit is the Bacillus- derived constitutive promoter, p43.
  • the peptide of interest was a Tp53 neoantigen with a single missense mutation (R175H or G524A).
  • Figure 3 shows the use of the RO, R4, or R5 ribosomal binding sites and B0015 terminator sequence (SEQ ID NO: 5 [parts. igem.org/Part:BBa_B0015]) in the neoantigen peptide transcriptional unit.
  • B0015 terminator sequence SEQ ID NO: 5 [parts. igem.org/Part:BBa_B0015]
  • These components provide additional critical parameters to tune peptide expression.
  • Guiziou et al., supra demonstrate that gene expression can be changed by orders of magnitude by altering the ribosomal binding sites.
  • Plasmids used to modify E. coli for the production of NAPs contained at minimum, the following components: 1) a plasmid backbone consisting an E. coli- compatible origin of replication and a selection marker (either antibiotic-resistance or auxotrophy-dependent); 2) one or more neoantigen peptides fused to the 3’ end of a gene encoding an adjuvant protein or expressed on its own 3) promoter region; 4) ribosomal binding site; and 5) terminator.
  • Figure 4 depicts an example of a typical plasmid (pGP8002 (109_dapA) (SEQ ID NO: 41) used to produce a neoantigen peptide (in this case a Tp53-derived neoantigen, a peptide extended to 26 amino acids to include the methionine as the start codon (amino acids 160-185 of the native p53 with a single mutation R175H).
  • the neoantigen peptide is codon-optimized for expression in E. coli and a part of an operon that also contains a gene encoding an adjuvant protein under the expression of the J23106 promoter.
  • the plasmid contains an E.
  • E. coli- compatible chloramphenicol or kanamycin resistance genes can be utilized.
  • the sequence of the entire plasmid is SEQ ID: 40 And utilizes the heat habile enterotoxin subunit B (eltB) as the adjuvant gene.
  • Plasmid pGP8003 in Figure 5 contains the transcriptional unit that consists of a gene encoding the 21 amino acid segment of Tp53-derived neoantigen (amino acids 165-185 with a single mutation R175H) fused to the 3’ prime end of the adjuvant gene. This fusion protein is expressed as one protein under the J23106 promoter.
  • the sequence provided for the plasmid utilizes the eltB gene as the adjuvant.
  • the ori and selective pressure of pGP8003 are identical to pGP8002 utilizing the dapA transcriptional unit to retain the plasmid. For the E.
  • BCD2 RBS (SEQ ID NO: 45) gggcccaagttcacttaaaaggagatcaacaatgaaagcaattttcgtactgaaacatcttaatcatgctaaggaggttttct
  • Example 9 Generation of a Bacillus spp. isolate Secretion Tag Library
  • the mature peptide of interest (lacking the secretion tag) is amplified and inserted into the Ndel restriction site of pBE-S using standard molecular cloning techniques.
  • the p43 promoter, R1 , and a restriction site are inserted upstream of the peptide of interest using Gibson assembly.
  • the p43 promoter and R1 RBS are selected because we had previously found them to be operational in various Bacillus spp. isolates.
  • the assembly is transformed into E. coli MC1061 F’ and resulting colonies are verified using colony PCR and Sanger sequencing. A transformant containing the correctly assembled plasmid is selected and cultured at 37°C overnight. The final assembled plasmid is then isolated using a standard miniprep protocol.
  • the plasmid miniprep is digested using M lul and Eagl which removed a fragment directly upstream of the mature neoantigen peptide gene which would serve as the insertion location for the library of Bacillus secretion tags.
  • the digest is run on a 0.8% agarose gel, excised, then gel purified.
  • the digested plasmid is then placed in a ligation reaction with the secretion tag mix (Takara Bio) using the In-Fusion HD Enzyme Mix (Takara Bio).
  • the reaction uses a 2:1 insert to vector ratio and is incubated at 50°C for 15 minutes. After incubation, the ligation product is transformed in E. coll HST08 using heat shock. Transformants are plated on LB with ampicillin (100 ug/mL) and incubated at 37°C overnight.
  • transformant colonies are resuspended with ⁇ 5mL of LB and miniprepped.
  • the plasmid secretion tag library is transformed into the competent Bacillus isolates and then plated on BHI with kanamycin (20 ug/mL) and incubated overnight.
  • the producer strains are marked with resistance to rifampicin. This is done through selection of spontaneous mutants on growth agar containing the antibiotic of interest at the concentration of interest.
  • a Bacillus PBCV strain is struck out on a BHI agar and is incubated overnight at 37°C.
  • a fresh colony is inoculated in a 3mL BHI culture and incubated overnight under aerobic conditions at 37°C.
  • the next day the culture is centrifuged (16,000xg, 30 sec) and resuspended in ⁇ 100uL of media.
  • the bacterial resuspension is spread on BHI + rifampicin (150ug/mL) and incubated overnight at 37°C.
  • Example 11 Construction of ThyA and DapA Knockouts in E. co// and Bacillus spp.
  • plasmids are inherently unstable and are lost from the engineered strain in the absence of pressure.
  • plasmids typically contain an antibiotic resistance gene and are maintained in the engineered bacteria by applying the designated antibiotic to the growth medium. Thus, only cells that have maintained the plasmid are able to propagate.
  • gRNA guide RNA
  • Cas9 plasmid contains the flanking regions of the target gene, either the dapA or thyA gene locus, as well as the gRNA for the target gene, ampicillin resistance gene, and pMB-1 derived origin of replication for selection and replication in E. coli.
  • the Cas9 plasmid contains the genes encoding the Cas9 protein and the A-red recombinase system under the control of an arabinose-inducible promoter as well as the gRNA to target the ori of the gRNA plasmid under the control of the lac promoter.
  • the plasmid uses the kanamycin resistance marker and the pSC101 temperature sensitive origin of replication.
  • E. coli cells were transformed with the pCas plasmid and subsequently plated on LB + Kan (50ug/mL) and incubated overnight at 30°C. The next day the presence of the plasmid was confirmed using colony PCR and selected colonies used to make electrocompetent cells for the introduction of the gRNA plasmid.
  • Electrocompetent E. coli.pCas cells were prepared in a similar fashion to the previous E. coli cells in Example 7 with two key differences. Firstly, the cells were grown in the presence of kanamycin (50ug/mL) and at 30°C. Secondly, prior to harvesting at the desired OD600, the cells inoculating with arabinose solution (50mM final concentration) and incubated for 15 minutes at 250rpm to induce the A-red recombinase system. After harvesting, the cells were transformed with the gRNA plasmid containing the flanking regions for either the thyA and dapA gene.
  • the transformations were plated on LB supplemented with kanamycin (50ug/mL) and ampicillin (100ug/mL) along with the essential nutrient (diaminopimolic acid (DAP) or thymindine (Thy) (50ug/mL)) and incubated at 30°C overnight. The next day the colonies were screened for the correct deletion of the target gene.
  • DAP diaminopimolic acid
  • Thy thymindine
  • gRNA plasmid Upon verification of the knockout, curation of the gRNA plasmid was initiated by inducing expression of the gRNA scaffold on the pCas that would target the ori of the gRNA plasmid.
  • LB cultures were prepared and supplemented with kanamycin and either DAP or Thy along with IPTG (1mM final concentration) and incubated shaking for 17 hours at 30°C. After incubation, dilutions were plated on LB, LB + Kan+ Amp+ nutrient, and LB + Kan+ nutrient and incubated overnight at 30°C. Colonies from the Kan + nutrient plate were tested for the presence of the gRNA via cPCR and colonies confirmed to no longer contain the gRNA plasmid proceeded to the pCas curation step.
  • a plasmid was assembled to contain the pKS1 backbone, a spectinomycin resistance gene, and flanking regions targeting the gene of interest.
  • the pKS1 backbone contains a temperature sensitive origin of replication compatible with both E. coli and Bacillus.
  • the plasmid was assembled and transformed in MC1061 F’. The strain was grown at 30°C under aerobic conditions in BHI +spectinomycin (100ug/mL). Note that cells were grown at 30°C rather than 37°C to enable plasmid replication.
  • the plasmid was miniprepped and transformed into Bacillus (see Example 9 for protocol). After confirmation of the presence of the plasmid in the given strain, a colony was selected and grown in BHI + spectinomycin at 37°C shaking for 24 hours. The increase of temperature would inhibit replication of plasmid but the presence of spectinomycin would force the whole plasmid to integrate into the target gene locus. This is the first recombination step at the thyA gene locus.
  • the culture was serially diluted, and the 100-104 dilutions were plated on BHI + thymidine (50ug/ml_) and trimethoprim (10ug/ml_) for thyA knockouts and incubated at 37°C overnight.
  • trimethoprim assists in eliminating cells that revert back to the wild-type genotype.
  • Example 12 Viability of PBCV Isolates in Various Intestinal Tract-Mimicking Environments
  • PBCV isolates are tested in rich media typically used in laboratory assays as well as media generated from intestinal contents from mice. As such, it is crucial that the modified strain be metabolically active given the nutrient in that region.
  • isolates are struck out from freezers stocks onto BHI agar plates and incubated overnight at 37°C. The next day overnight cultures are made of the strains in 3 mL of BHI and incubated overnight shaking at 37°C.
  • BHI nutrient-rich media
  • Tween 80 concentration help decrease the coaggregation and the cells do not seem to be strongly affected by the presence of the Tween 80 in the nutrient rich media. However, the Tween 80 seems to negatively impact the growth of the cells in the jejunal contents.
  • Example 13 Testing and screening of PBCVs in stomach-like environments
  • PBCVs For PBCVs to be orally administered and delivered inside the gastrointestinal tract, it is beneficial that PBCVs survive passage through the acidic conditions of the stomach. PBCVs are thus tested for their sensitivity to stomach contents using survival assays in vitro. For these assays, contents are removed from the stomachs of healthy mice and are mixed with water ( ⁇ 1 :1 ratio). The mixture is then centrifuged for 1 minute at 3500 ref to remove large solids. The pH is measured for each experiment (pH ⁇ 2). Stomach contents are then inoculated with ⁇ 10 A 7 CFU/mL of each probiotic. The cultures are then incubated anaerobically at 37°C for 120 minutes. This time was selected as a conservative estimate of the residence time inside a patient’s stomach.
  • the PBCV is enumerated at 0 hours, 1 hour and at 2 hours minutes by dilution plating on selective agar (BHI or LB+150 ug/mL rifampicin). Plates are incubated overnight at 37°C and the CFU/mL are determined for all time points. The survival ratio of each strain is then calculated as follows:
  • PBCVs are thus tested for their sensitivity to bile using survival assays in vitro. For these assays, contents are removed from the duodenums of healthy mice centrifuged for 1 minute at 3500 ref to remove large solids. Duodenum contents are then inoculated with ⁇ 107 CFU/mL of each probiotic. The PBCV cultures are then incubated anaerobically at 37°C for 3 hours. This time was selected as a conservative estimate of the residence time in a patient’s duodenum.
  • the PBCV is enumerated at 0 hours and at 3 hours by dilution plating on selective agar (BHI or LB+150 ug/mL rifampicin). Plates arere incubated overnight at 37°C and the CFU/mL arere determined for both time points. The survival ratio of each strain is then calculated as follows:
  • PBCVs are preferably metabolically active inside the patient’s intestinal tract.
  • mucus from the jejunum and ileum of healthy mice is isolated.
  • the mucus is then diluted 10x in M9 minimal medium and sterile filtered to generate the biomatrix growth medium.
  • the PBCV is first grown overnight in lysogeny broth (LB). 2.5 uL of the overnight culture is then added to 250 uL of the biomatrix growth medium in a sterile 96 well plate. The plate is then incubated at 37°C with agitation in a plate reader. Growth is monitored continuously for 20 hours based on optical density at 600 nm.
  • LB lysogeny broth

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Abstract

L'invention concerne des vaccins bactériens anticancéreux personnalisés (PBCV) et des procédés associés, comprenant des procédés d'isolement de bactéries à partir du microbiome intestinal d'un patient, l'ingénierie génétique de bactéries sélectionnées, les voies d'administration et le dosage, la fabrication, l'approbation réglementaire, les tests de validation et l'évaluation de la réponse immunitaire et de l'efficacité thérapeutique.
PCT/US2024/039426 2023-07-25 2024-07-24 Vaccins bactériens anticancéreux personnalisés Pending WO2025024603A2 (fr)

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CA2914195C (fr) * 2013-06-03 2020-06-02 Nanoblate Corporation Procedes et dispositifs de stimulation d'une reponse immunitaire a l'aide de champs electriques pulses de tres courte duree (nanosecondes)
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