WO2023244997A1 - Compositions and methods for inducing anticancer immunity - Google Patents

Compositions and methods for inducing anticancer immunity Download PDF

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Publication number
WO2023244997A1
WO2023244997A1 PCT/US2023/068336 US2023068336W WO2023244997A1 WO 2023244997 A1 WO2023244997 A1 WO 2023244997A1 US 2023068336 W US2023068336 W US 2023068336W WO 2023244997 A1 WO2023244997 A1 WO 2023244997A1
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tumor
cancer
vaccine
sequence
medicament
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PCT/US2023/068336
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French (fr)
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Leaf Huang
Menglin WANG
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The University Of North Carolina At Chapel Hill
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/34—Copper; Compounds thereof
    • 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
    • A61K39/114—Fusobacterium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure relates to methods, compositions, and systems or kits for treating cancer, promoting an anti-cancer response, and/or increasing anti-cancer immunity.
  • the disclosure relates to liposomal antibiotic formulations and compositions comprising one or more peptides comprising at least one tumor-associated bacterial epitope.
  • a liposomal antibiotic formulation comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
  • medicaments comprising a liposomal antibiotic formulation.
  • vaccine or medicaments comprising a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
  • methods of using the vaccines and medicaments for the treatment or prevention of cancer are also disclosed.
  • the antibiotic comprises a nitroimidazole antibiotic or a lincosamide antibiotic. In some embodiments, the antibiotic comprises a metal-tinidazole complex. In some embodiments, the antibiotic comprises a silver-tinidazole complex or a copper-tinidazole complex.
  • the at least one tumor-associated bacterial epitope comprises an amino acid sequence have at least 60% similarity with sequences in subject proteome.
  • the at least one tumor-associated bacterial epitope comprises: a major histocompatibility complex (MHC) class 1 epitope without sequence similarity with subject proteome, an MHC class II epitope without at least 60% sequence similarity with subject proteome, an MHC class I epitope with at least 60% sequence similarity with subject proteome, or a combination thereof.
  • MHC major histocompatibility complex
  • the at least one tumor-associated bacterial epitope is determined by a method comprising: identifying tumor- associated bacteria in a sample from the subject; and selecting the epitopes from tumor associated bacteria proteome based on MHC or human leukocyte antigen (HLA) presentation.
  • the at least one tumor-associated bacterial epitope is determined by a method further comprising: aligning sequences from bacterial proteome and subject proteome; and determining bacterial epitopes with sequence similarity to subject proteome.
  • the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-112. In some embodiments, the at least one tumor- associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-100. In some embodiments, the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 105-112.
  • the one or more peptides further comprise a signal sequence, major histocompatibility complex (MHC) transmembrane domain, an MHC cytoplasmic domain, or any combination thereof.
  • MHC major histocompatibility complex
  • the one or more nucleic acids comprise mRNA.
  • the one or more nucleic acids further comprise a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof.
  • the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
  • the one or more nucleic acids comprise DNA. In some embodiments, the one or more nucleic acids comprise circular RNA.
  • the one or more nucleic acids further comprise at least one chemically modified nucleotide.
  • the at least one chemically modified nucleotide comprises a modified uracil, a modified cytosine, or a combination thereof.
  • the one or more peptides are encoded on a single nucleic acid.
  • the single nucleic acid encodes two or more peptides.
  • the single nucleic acid is an mRNA.
  • the two or more peptides are each separated by a linker.
  • the linker comprises a cleavable linker, a flexible linker, or a combination thereof.
  • the composition further comprises an adjuvant, a delivery vehicle, or a combination thereof.
  • the adjuvant, the delivery vehicle or the combination thereof comprises a lipid.
  • the lipid comprises a cationic lipid, an ionizable lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.
  • the non-cationic lipid comprises a phospholipid.
  • the sterol comprises cholesterol or a modification or ester thereof.
  • the delivery vehicle comprises a lipid particle encapsulating the composition.
  • the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
  • the cancer is associated with tumor-associated bacteria.
  • the cancer comprises gastric cancer, colorectal cancer, or a combination thereof.
  • the subject was not administered a cytotoxic treatment.
  • the liposomal antibiotic formulation and the composition are administered simultaneously, separately, or consecutively, in any order separated by a time interval.
  • the liposomal antibiotic composition is administered prior to a resection.
  • FIGS. 1A-1I show F. nucleatum invaded into CT26(FL3) tumor cell in response to hypoxia.
  • FIG. 1 A is an exemplar)' illustration of F. nucleatum infection of CT26(FL3) cells in vitro.
  • FIG. ID is the three- dimensional re-construction of Z projection of stacked images of F.
  • FIG. IE is a Z-stack image of CT26(FL3) spheroid co-incubated with F. nucleatum.
  • F. nucleatum covalently labelled by CFSE mostly appeared in intra-spheroid.
  • the right panel is the bright field optical image of the spheroid.
  • Scale bar 100 pm.
  • FIG. 1G is TEM Images of CRC tumor sections.
  • FIG. II is fitting distribution of minimum distance of F. nucleatum to cell nucleus for each pixel. F. nucleatum invaded into three-dimensional spheroids in hypoxia.
  • FIGS. 2A-2H show the characterization of the pH-sensitive antibiotic liposomes.
  • FIG. 2A is an illustration of remotely loading by silver nitrate gradient and drug release in response to low pH.
  • FIG. 2B is the H NMR characterization of AgTNZ in neutral pH and weak acid.
  • FIG. 2C is images of LipoAg and Lipo AgTNZ by Cryo-EM.
  • FIG. 2F shows the pH-sensitive drug release of LipoAgTNZ in pH4.5 and pH7.
  • FIGS. 3A-3K show removing F. nucleatum in the tumor by liposomal antibiotics AgTNZ eradicated colorectal cancer.
  • FIG. 3A is a schematic timeline of F. nucleatum infected CT26(FL3) tumor study followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i. v.). The rechallenge study was performed at Day 60 by inoculating 10 6 CT26(FL3) cells subcutaneously.
  • FIG. 3A is a schematic timeline of F. nucleatum infected CT26(FL3) tumor study followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i. v.). The rechallenge study was performed at Day
  • FIG. 3F shows colony forming units of E.
  • FIG. 31 is a tumor growth curve of the rechallenge study. The survivor and control mice were rechallenged by the F.
  • FIGS. 4A-4J shows T cells from survivors suppressed the growth of both infected and uninfected tumors.
  • FIG. 4A shows an in vivo T cell proliferation assay. T cells were isolated from donor splenocytes and labelled with proliferation dye CFSE before injection into recipient mice. The labelled donor T cells were collected from the primary tumor of the recipient mice and analyzed by flow cytometry.
  • FIG. 4A shows an in vivo T cell proliferation assay. T cells were isolated from donor splenocytes and labelled with proliferation dye CFSE before injection into recipient mice. The labelled donor T cells were collected from the primary tumor of the recipient mice and analyzed by flow cytometry.
  • FIG. 4D is an illustration of the study. Mice with orthotopic CRC tumors with or without F. nucleatum infection received pan CD3 + T cells from either naive mice or long-term survivor mice from LipoAgTNZ treatment described in FIG. 2A. The donor survival mice were pulsed s.c. with 2xl0 6 CT26(FL3) tumor cells 2 days before their T cells were collected from the spleen.
  • FIG. 4G is representative flow cytometry analysis of CD3 + CD8 + T cells in tumors of the recipient mice after adoptive T cell transfer.
  • FIG. 4H shows donor T cells inhibited tumor growth in the recipient mice. At day 10 and day 15 post inoculation, the recipients with or without F. nucleatum infection received i.v.
  • FIG. 4J shows chaperone expression induced by killing intracellular bacteria treatment. CT 26(FL3) cells were infected by F. nucleatum under hypoxia condition followed by treatment with 5 pM drug.
  • FIGS. 5A-5F show the killing of tumor-associated bacteria promoted immune recognition of bacterial neoepitopes.
  • FIG. 5 A is topology analysis of the proteome and proteomics of F. nucleatum based on prediction of subcellular localization. The right panel is label-free quantitative proteomics mapped with predicted subcellular localization. The selected proteins are marked with enlarged black circles (KYLSNLGIL (SEQ ID NO: 113), LPMRHFHAF (SEQ ID NO: 114), SYFEWVQNI (SEQ ID NO: 115), RPMIGMHFF (SEQ ID NO: 117), and MPIYQTSTF (SEQ ID NO: 116)).
  • KYLSNLGIL SEQ ID NO: 113
  • LPMRHFHAF SEQ ID NO: 114
  • SYFEWVQNI SEQ ID NO: 115
  • RPMIGMHFF SEQ ID NO: 117
  • MPIYQTSTF SEQ ID NO: 116
  • FIG. 5B is a scatter plot showing the rank for total scores and peptide-MHC complex affinity.
  • FIG. 5C is the filters set for alignment of F. nucleatum and mouse genome to generate the homologous epitopes.
  • FIG. 5D shows information of the selected peptides (KYLSNLGIL (SEQ ID NO: 113), LPMRHFHAF (SEQ ID NO: 114), SYFEWVQNI (SEQ ID NO: 115), MPIYQTSTF (SEQ ID NO: 116), RPMIGMHFF (SEQ ID NO: 117), GVPQIEVTF (SEQ ID NO: 53), RQATKDAGTI (SEQ ID NO: 118), and LADDNFSIV (SEQ ID NO: 119) from IEDB database.
  • KYLSNLGIL SEQ ID NO: 113
  • LPMRHFHAF SEQ ID NO: 114
  • SYFEWVQNI SEQ ID NO: 115
  • MPIYQTSTF S
  • FIG. 5E is representative images of IFNy Elispot.
  • the bacteria epitopes in FIG. 5D were incubated with bone marrow derived dendritic cells, peptides and T cells isolated from CT26(FL3) tumor bearing Balb/C mice for 24 h.
  • FIGS. 6A-6I show F. nucleatum promoted colorectal cancer (CRC) growth and fostered immune-suppressive tumor microenvironments.
  • FIG. 6A is a schematic timeline of F. nucleatum infected CT26(FL3) tumor study, F. nucleatum colonized tumor following three oral gavages after tumor inoculation.
  • FIG. 6C is IVIS bioluminescence signal of luciferase assay for each mouse at Day 5, 12 and 19.
  • FIG. 6D is the bioluminescence images of FIG. 6B.
  • FIG. 6F shows the representative primary tumor (green dotted cycles) and metastasis (yellow dotted cycles) under photograph in situ.
  • FIG. 61 is representative flow cytometry analysis of CD3 + T cells, CD4 + CD44 + CD62L + T cells, CD3 + CD8 + T cells, CDllb + F4/80 + CD206 + macrophages, CDllc + MHCII + dendritic cells and CDllb + Grl + MDSC.
  • FIGS. 7A-7C show F. nucleatum colonized in the CRC tumor.
  • FIG. 7A shows Z-stack scanning of CT26(FL3) spheroid co-incubated with F. nucleatum.
  • F. nucleatum labelled by CSFE mostly appears in intra- spheroid layers.
  • FIG. 7B shows Four different tumor samples stained for fluorescence in situ hybridization (FISH) using an RNA probe visualizing F. nucleatum in frozen sections. The bars indicate 20 pm.
  • FIG. 7C is the distance map of the upright panel displaying the minimum distance between the red pixels indicating FISH signals to the blue pixels indicating DAPI staining, where the color from dark to green represents distance range from 0 to 4 pm.
  • FIGS. 8A-8E show E. coli Nissle infected colorectal cancer and tumor microenvironments.
  • FIG. 8A is stacking images of E. coli Nissle infected CT26(FL3) cells in hypoxia.
  • FIG. 8B is a schematic timeline of E. coli Nissle infected CT26(FL3) tumor study, F. nucleatum colonized tumor following three oral gavages after tumor inoculation.
  • FIG. 8D is E. coli Nissle biodistribution in the organs, tumor, and metastasis at Day 19.
  • FIGS. 9A-9H show the characterization of the pH-sensitive antibiotic liposomes.
  • FIG. 9A is the chemical structure of metal-tinidazole prodrug chelate.
  • FIG. 9B is the mass spectrum of AgTNZ.
  • FIG. 9C is the characterization of size by nanoparticle tracking system.
  • FIG. 9D is the characterization of zeta potential by nanoparticle tracking system.
  • FIG. 9E is the cryo-EM images of LipoCu and LipoCuTNZ. The bar stands for 100 pm.
  • FIG. 9F is the mass spectrum of CuTNZ.
  • FIG. 9G is the characterization of size of LipoCuTNZ by nanoparticle tracking system.
  • FIG. 9H is the characterization of zeta potential of LipoCuTNZ by nanoparticle tracking system.
  • FIGS. 10A-10G show LipoAgTNZ predominantly accumulated in the tumor.
  • FIG. 10C is the pharmacokinetic parameters of TNZ and Ag delivered as LipoAgTNZ or free TNZ.
  • FIG. 10D is IVIS images of DiD fluorescence in major organs and tumors at 24 h after i.v. injection.
  • FIG. 10D is IVIS images of DiD fluorescence in major organs and tumors at 24 h after i.v. injection.
  • FIG. 10D is IVIS images of DiD fluorescence in major organs and tumors at 24 h after i.v. injection.
  • FIG. 10E is fluorescence quantification of the data in FIG. 10D.
  • n 3 per group.
  • Data are mean ⁇ SD.
  • FIGS. 11A-11F show LipoAgTNZ showed low toxicity in vitro and in vivo.
  • FIGS. 12A-12E show Liposomal AgTNZ eradicated F. nucleatum infected CRC tumor.
  • the orthotopic F. nucleatum infected CRC model is described in FIG. 3 A.
  • FIG. 12A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 18, 33 and 42.
  • FIG. 12B shows CT26(FL3) orthotopic tumor growth with F. nucleatum infection.
  • FIG. 12D shows oral polymyxin B decreased viable F. nucleatum in the feces.
  • the orthotopic CRC model is described in FIG. 3A but without F. nucleatum infection.
  • FIGS. 13A and 13B show LipoAgTNZ inhibited tumor growth and induced long-term survival in F. nucleatum infected MC38 tumors orthotopically inoculated in C57BL/6 mice.
  • FIG. 13A is a schematic timeline of F. nucleatum infected MC38 tumor study followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.). The bottom panel shows the images of orthotopic CRC tumor. One mouse was euthanized every 7 days for tumor growth monitoring.
  • FIGS. 14A-14G show removing F. nucleatum in the tumor by LipoAgTNZ improved immune surveillance of the tumor.
  • FIG. 14B is representative flow cytometry analysis of CD3+ T cells, CD4 + CD44 + CD62L + T cells and CD3 + CD8 + T cells.
  • FIG. 14D is representative flow cytometry analysis of CDllb + F4/80 + CD206 + macrophages in the tumor.
  • FIG. 14B is representative flow cytometry analysis of CD3+ T cells, CD4 + CD44 + CD62L + T
  • FIG. 14F is representative images of the rechallenged and primary tumors at the endpoint of anti-CD8 and CD4 study. The upper panel shows the rechallenged subcutaneous tumors of the survivor mice (blue dotted cycles) grew when CD8 + or CD4 + T cells were compromised. The bottom panel shows the primary tumor inoculated on the cecum (yellow dotted rectangles) were already cured at Day 75 after inoculation.
  • FIGS. 15A-15G show LipoAgTNZ inhibited the tumor growth of F. nucleatum. infected liver metastasis.
  • FIG. 15 A is a scheme of F. nucleatum infected liver metastasis model.
  • the CT26(FL3) cells were pre-infected by F. nucleatum and inoculated to the liver by hemi-splenic injection, followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.).
  • FIG. 15 A is a scheme of F. nucleatum infected liver metastasis model.
  • the CT26(FL3) cells were pre-infected by F. nucleatum and inoculated to the liver by hemi-splenic injection, followed by three
  • FIG. 15C is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 4, 10 and 15.
  • FIG. 15D is the tumor growth in liver metastasis in each mouse with F. nucleatum infection.
  • FIG. 15G is FISH imaging visualizing F. nucleatum 16s rRNA, immunofluorescent staining of CD206+ macrophages and CD8+ T cells in the frozen sections of liver containing metastasis.
  • FIGS. 16A-16C show T cell from survivor suppress both infected and uninfected tumor. Lymphocytes were isolated from survivor or naive mice and incubated with CT26(FL3) cells for 24 h in vitro.
  • FIG. 16A is representative flow cytometry data of CD8+IFNy+ T cells after incubation. Tumor growth after adoptive T cell transfer. The adoptive T cell transfer is illustrated in FIG. 5H.
  • FIG. 16B is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 3, 9, 15 and 23.
  • FIG. 16C is CT26(FL3) orthotopic colorectal tumor growth in each mouse with or without F. nucleatum infection with T cells adoptively transferred from naive or survivor mice.
  • FIG. 17 is a schematic of an exemplary overall approach for epitope discovery and vaccine development.
  • FIGS. 18A-18D show bacterial epitope prediction and in vivo confirmation of MHC-I restricted T cells.
  • FIG. 18A shows epitope sequences selected from F. nucleatum for antigen presentation by MHC-I of C57BL6 mice.
  • FIG. 18B is tetramer staining carried out by using flow cytometry.
  • FIG. 18D is representative images of IFNy Elispot assay. The bacteria epitopes in FIG.
  • SVFVFYPADF SEQ ID NO: 108
  • ISLPFITM SEQ ID NO: 109
  • AAIQGGVLM SEQ ID NO: 110
  • LADDNFSTI SEQ ID NO: 105
  • RGVPQIEVTF SEQ ID NO: 106
  • VITAFSNI SEQ ID NO: 111
  • SVFVFYPADFTF SEQ ID NO: 112
  • FIGS. 19A and 19B show bacteria-derived peptides inhibited tumor growth and induced long-term survival in F. nucleatum infected MC38 tumors orthotopically inoculated in C57BL/6 mice.
  • FIG. 19A is a schematic timeline of F. nucleatum infected MC38 tumor study followed by two doses of DOTAP-peptide treatment.
  • FIG. 19B is animal survival curves following the dosing of timeline from day 0 to day 75 (SVFVFYPADFTF (SEQ ID NO: 112) and RGVPQIEVTF (SEQ ID NO: 106)).
  • n 5 per group. Data are mean ⁇ SD. *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 20 is a schematic of a poly -bacterial-epitope RNA for cancer vaccine.
  • the MHC class I and MHC class II restricted bacterial epitopes without sequence similarity to host proteome are designed to induce cytotoxic T cell responses and helper T cell responses respectively towards neoantigens from cancer microbiota.
  • the MHC class I epitopes with sequence overlapped with host proteome are designed to induce cross reactive anticancer effect.
  • FIG. 21 is a schematic of the filters set for alignment of F. nucleatum and human genome to generate the homologous epitopes.
  • FIGS. 22A-22C show that liposomal AgTNZ eradicated F. nucleatum infected CT26 CRC tumor.
  • FIG. 22B is a graph of the number of metastases of FIG. 22A.
  • FIG. 22C is a graph of the tumor weight including primary site and metastases of (E). *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIGS. 23A-23C show that targeting F.
  • FIG. 23A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 14, 23 and 33.
  • F. nucleatum infected CT26FL3(Luc/RFP) tumors were established following the schedule shown in FIG. 3A.
  • Three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.) in combination with anti-CD8 and CD4 antibodies (200 pg/mouse, i.p.).
  • FIG. 23C is a schematic timeline of the F. nucleatum infected CT26FL3(Luc/RFP) tumor study followed by three doses of LipoAgTNZ treatment or antibiotic cocktails in the drinking water from Day 12 to Day 21.
  • FIGS. 24A and 24B show the characterization of the pH-sensitive antibiotic liposomes.
  • FIG. 24A is a graph showing the release of LipoAgTNZ in PBS buffer at pH 7.
  • FIG. 24B is graphs of the size, zeta, and PDI of LipoAgTNZ dispersed in 5% glucose solution and PBS buffer at pH 7.
  • FIGS. 25A-25C show that removing F. nucleatum in the tumor by liposomal antibiotics AgTNZ eradicated colorectal cancer.
  • FIG. 25A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 14, 23 and 33. F.
  • FIG. 25C is a schematic timeline of the F. nucleatum infected CT26FL3(Luc/RFP) tumor study followed by three doses of LipoAgTNZ treatment in combination with anti-CD8 or anti-CD4 antibody.
  • compositions, and systems or kits for treating cancer promoting an anti-cancer response, and/or increasing anti-cancer immunity.
  • Pre-resection liposomal antibiotic formulations targeting anaerobic bacteria significantly improved the disease- free survival rate of colorectal cancer (CRC) patients by 25%.
  • Silver-tinidazole complexes encapsulated in liposomes (LipoAgTNZ) eliminated tumor-associated bacteria in the primary tumor and liver metastasis without causing gut microbiome dysbiosis.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors.
  • Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.
  • chemotherapeutic agents include, but are not limited to: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, carboplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, everolimus, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, erlotinib, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib
  • nucleic acid or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)).
  • the present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like.
  • the polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced.
  • the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double- stranded form, including homoduplex, heteroduplex, and hybrid states.
  • a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat.
  • LNA locked nucleic acid
  • cyclohexenyl nucleic acids see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000), and/or a ribozyme.
  • nucleic acid or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand.
  • nucleic acid refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds.
  • Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies.
  • the terms “polypeptide” and “protein” are used interchangeably herein.
  • sequence identity refers to the extent that sequences are identical on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys,
  • sequence similarity or sequence identity between sequences can be performed as follows.
  • the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence.
  • amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST can be used.
  • the terms “providing,” “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanocomplexes and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization to a desired site.
  • a “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non- mammals include, but are not limited to, birds, fish, and the like.
  • the mammal is a human.
  • “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease.
  • “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
  • the present disclosure provides methods for treating cancer, promoting an anti-cancer response, or increasing anti-cancer immunity in a subject in need thereof.
  • the subject is a human.
  • the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
  • the cancer may be a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma.
  • the cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus.
  • the cancer is associated with tumor- associated bacteria e.g., gastric (Helicobacter pylori) and colorectal (Fusobacterium nucleatum and Bacteroides fragilis) cancer.
  • the cancer comprises gastric cancer, colorectal cancer, or a combination thereof.
  • the cancer comprises a solid tumor.
  • the cancer is metastatic cancer.
  • the methods result in decreased tumor growth.
  • the methods result in tumor regression.
  • the methods result in decreased numbers of tumor.
  • the methods prevent tumor recurrence.
  • the methods result in increases in overall subject survival.
  • the methods include administering to the subject an effective amount of one or both of: a liposomal antibiotic formulation; and a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
  • an “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which is effective for inducing a response in the subject. This amount varies depending upon the health and physical condition of the subject to be treated, the respective formulations of the compositions and antibiotics, the relative stage of the cancer, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by one of skill in the art through routine trials.
  • compositions, polypeptides, polynucleotides (e.g., mRNA), vaccines, or medicaments disclosed herein can be administered in a wide variety of therapeutic dosage forms.
  • the route and regimen of administration will vary depending upon the population and is to be determined by the skilled practitioner.
  • the formulations, compositions, vaccines, or medicaments disclosed herein may be administered parentally, e.g., intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form.
  • the administration may comprise an initial immunization or dose and at least one subsequent immunization or booster dose, following known standard immunization protocols.
  • the boosting doses will be adequately spaced at such times where the levels of circulating antibody fall below a desired level.
  • Boosting doses may consist of the compositions or vaccines disclosed herein and may comprise alternative carriers and/or adjuvants.
  • the booster dosage levels may be the same or different that those of the initial immunization dosage.
  • administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof.
  • administration includes parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections).
  • the administration is intratumoral or adjacent to the site of cancer.
  • the specific dose level may depend upon a variety of factors including the activity of the polynucleotides (e.g., mRNA), polypeptides, compositions, formulations, vaccine, or medicament, the age, body weight, general health, and diet of the subject, time of administration, and route of administration.
  • the amount in each dose is an amount which induces an immunoprotective response without significant adverse side effects.
  • a wide range of second therapies may be used in conjunction with the polynucleotides, peptides, formulations, compositions, vaccines, medicaments, and methods of the present disclosure.
  • the second therapy may be administration of a therapeutic agent or may be a second therapy not connected to administration of another agent.
  • Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, or a chemotherapeutic or anti-cancer agent.
  • the second therapy e.g., an immunotherapy
  • the second therapy may be administered at the same time as the initial therapy.
  • the second therapy may be administered in the same composition or in a separate composition administered at substantially the same time as the initial therapy.
  • the second therapy may precede or follow administration of the disclosed polynucleotides, peptides, formulations, compositions, vaccines, medicaments, and methods by time intervals ranging from hours to months.
  • the second therapy includes immunotherapy.
  • Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, or administration of antibodies (e.g., monoclonal antibodies).
  • the subject was not administered a cytotoxic treatment (e.g., a chemotherapeutic or radiation) prior to the disclosed methods.
  • the methods further comprise full or partial resection of a tumor or cancerous tissue or organ.
  • the liposomal antibiotic composition and/or the composition e.g., one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof
  • the liposomal antibiotic composition and/or the composition is administered prior to the resection.
  • the liposomal antibiotic composition and/or the composition e.g., one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof
  • the liposomal antibiotic formulation and the composition may be administered simultaneously, separately, or consecutively, in any order separated by a time interval (e.g., minutes, hours, days, or weeks).
  • the liposomal antibiotic formulation and the composition may be administered at the same time using different methods of administration or the same method of administration.
  • liposomal antibiotic formulations e.g., liposomal antibiotic formulations suitable for use in the present methods.
  • the antibiotic targets anaerobic bacteria.
  • the antibiotic comprises a nitroimidazole antibiotic, a lincosamide antibiotic, or a combination thereof.
  • the antibiotic comprises a nitroimidazole antibiotic.
  • Nitroimidazole antibiotics include those that have a nitro group appended to an imidazole ring. Nitroimidazole antibiotics include, but are not limited to, metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, omidazole, megazol, azanidazole, and benznidazole.
  • the antibiotic comprises a metal-tinidazole complex.
  • Metal- tinidazole complexes include, but are not limited to, cobalt-tinidazole, copper-tinidazole, zinc- tinidazole, and silver-tinidazole.
  • the antibiotic comprises a copper- tinidazole complex.
  • the antibiotic comprises a silver-tinidazole complex.
  • the antibiotic comprises a lincosamide antibiotic. Lincosamides consist of a pyrrolidine ring linked to a pyranose moiety (methylthio-lincos amide) via an amide bond. Lincosamide antibiotics include, but are not limited to, lincomycin and clindamycin.
  • the disclosed compounds are incorporated into liposomal compositions comprising one or more vesicle forming lipids.
  • Methods of making liposomal compositions include, for example, lipid film hydration, optionally coupled with sonication or extrusion, solvent evaporation (e.g., ethanol injection, ether injection, or reverse phase evaporation) or detergent removal methods.
  • the disclosed compounds can be combined with the lipid(s) before formation of the vesicles (passive loading) or after vesicle formation (active loading).
  • the liposome compositions may prolong circulation time in vivo, increase stability of the compound, and prevent degradation in the bloodstream.
  • the liposomal composition may increase the distribution of the compounds within the lung, breast, pancreas, and spleen.
  • the one or more vesicle forming lipids may be selected from di-aliphatic chain lipids, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids such as sphingomyelin or glycosphingolipid; steroidal lipids; hydrophilic polymer derivatized lipids; or mixtures thereof.
  • the liposomes may contain other non-vesicle forming lipids or other moieties, including but not limited to amphiphilic polymers, polyanions, sterols, and surfactants.
  • the liposomes contained in the liposome composition can also be targeting liposomes, e.g., liposomes containing one or more targeting moieties or biodistribution modifiers on the surface of the liposomes.
  • a targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target and are generally known in the art, for example ligands such as folic acid, proteins, antibody or antibody fragments, and the like).
  • the liposomes can have any liposome structure, e.g., structures having an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilic central part where the membrane sequesters an interior.
  • the liposome may be a unilamellar liposome, having a single lipid layer.
  • the disclosed compounds may be completely or partially located in the interior space of the liposome or completely or partially within the bilayer membrane of the liposome.
  • the lipids for a micelle may be completely or partially located in the interior space of the liposome or completely or partially within the bilayer membrane of the liposome.
  • the liposomal antibiotic formulations are pH-sensitive. pH- sensitive or pH-responsive liposomes can be employed to effect site-specific release of their cargo (e.g., antibiotics or antibiotic formulations). For example, pH changes can trigger changes in permeability of the liposomal membrane by protonation/deprotonation of functional groups that induce morphological changes of the lipid bilayers.
  • pH-responsive liposomes are formulated using a natural phospholipid component that is sensitive to pH changes that may be encountered as they traffic to or are internalized by target cells. Additionally, inclusion of negatively charged moieties into pH-sensitive liposome compositions is a common strategy for destabilizing the liposome structure through the phase conversion, to enable rapid release of the cargo.
  • the liposomal antibiotic formulations may comprise pH sensitive lipids including, but are not limited to phosphatidylethanolamine, palmitoylhomocysteine or analogs or derivatives thereof, such as dioleoylphosphatidylethanolamine, and/or pH sensitive polymers including a pH sensitive moiety, such as, but not limited to alklyacrylic acid, ethylacrylic acid, propylacrylic acid, butylacrylic acid, and methylacrylic acid.
  • pH sensitive lipids including, but are not limited to phosphatidylethanolamine, palmitoylhomocysteine or analogs or derivatives thereof, such as dioleoylphosphatidylethanolamine, and/or pH sensitive polymers including a pH sensitive moiety, such as, but not limited to alklyacrylic acid, ethylacrylic acid, propylacrylic acid, butylacrylic acid, and methylacrylic acid.
  • compositions comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
  • the composition may comprise a single peptide comprising one or more tumor-associated bacterial epitopes, multiple peptides (e.g., two or more) each comprising a tumor-associated bacterial epitope, multiple peptides (e.g., two or more) each comprising multiple (e.g., two or more) tumor- associated bacterial epitopes, or a combination thereof, or one or more nucleic acids encoding any of those listed.
  • multiple epitopes may be arranged with a linker between the epitopes or directly to one another without a linker between the epitopes in a single peptide.
  • the two or more epitopes are each separated by a linker.
  • the linker is preferably a polypeptide linker of 1-20 amino acids.
  • the linker may comprise any amino acid sequence.
  • the linker may be flexible such that it does not constrain the epitopes.
  • the linkers may essentially act as a spacer.
  • the linker is glycine-rich.
  • the linker may comprise glycine-serine polymers, glycine-alanine polymers, alanine- serine polymers and/or other flexible linkers known in the art.
  • the linker may be a cleavable linker.
  • the linker may comprise an amino acid sequence configured to be cleaved enzymatically or chemically (e.g., at certain pHs, reduction/oxidation conditions or presence of reducing/oxidizing agents, temperatures, etc.).
  • the cleavable linker comprises a self-cleaving peptide-based cleavage site.
  • the at least one bacterial epitope comprises: a major histocompatibility complex (MHC) class I epitope without sequence similarly with subject proteome, an MHC class II epitope without sequence similarly with subject proteome, an MHC class I epitope with sequence similarly with subject proteome, or a combination thereof.
  • MHC major histocompatibility complex
  • the at least one bacterial epitope comprises an amino acid sequence have at least 60% similarity (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more similarity) with sequences in subject proteome (e.g., the cancer cell genome).
  • the epitopes are neoepitopes.
  • a neo-epitope is a new (or neo) epitope (e.g., antigenic determinant) that has not been previously recognized by the immune system.
  • Neo-epitopes can encompass epitopes on a neoantigen, which is a new antigen.
  • the at least one bacterial epitope is determined by a method comprising identifying tumor- associated bacteria in a sample from the subject and selecting epitopes from the tumor-associated bacteria based on MHC or human leukocyte antigen (HLA) presentation.
  • the epitopes do not have sequence similarity with a peptide from the subject.
  • the sample may be obtained from any suitable source, such as, a physiological fluid including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, feces, and the like.
  • a physiological fluid including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, feces, and the like.
  • the sample is blood or blood products. Blood products are any therapeutic substance prepared from human blood.
  • the sample from the subject may comprise a tumor sample, a tissue sample, a blood sample, or any sample that comprises tumor-associated bacteria or tumor-associated bacterial nucleic acids.
  • the sample is a fecal sample.
  • the methods further comprise aligning sequences from bacterial proteome and subject proteome and determining bacterial epitopes with sequence similarity to subject proteome.
  • the epitopes have sequence similarity with a peptide from the subject.
  • the present invention relates to methods for identifying and/or detecting epitopes.
  • the invention provides methods of identifying and/or detecting tumor specific epitopes that are useful for increasing an anti-cancer immune response in a subject.
  • Some of these epitopes are capable of activating anti-tumor CD8 T-cells, e.g., bind to MHC class I polypeptides. Others bind to MHC class II polypeptides and activate CD4+ T helper cells.
  • the epitope comprises a sequence selected from those shown in Tables 1-4, or a sequence comprising one, two, or three substitutions in reference to a sequence from Tables 1-4. In some embodiments, the epitope comprises a sequence selected from any of SEQ ID NOs: 1-112. In some embodiments, the epitope comprises a sequence comprising one, two, or three substitutions in reference to any of SEQ ID NOs: 1-112.
  • the epitope comprises a sequence selected from any of SEQ ID NOs: 43, 46, and 51-100. In some embodiments, the epitope comprises a sequence comprising one, two, or three substitutions in reference to any of SEQ ID NOs: 43, 46, and 51-100.
  • the epitope comprises a sequence selected from: IINEPTAA (SEQ ID NO: 101), PQIEVTF (SEQ ID NO: 102), RQRVWGV (SEQ ID NO: 103), DIDANGI (SEQ ID NO: 104), GAAIQGGVL (SEQ ID NO: 40), QDPLAELVKI (SEQ ID NO: 45), YTHFTSPIR (SEQ ID NO: 50), IDANGIVHV (SEQ ID NO: 54), NTYGTGCFL (SEQ ID NO: 55), KQRVAIARA (SEQ ID NO: 57), DAFGTAHRA (SEQ ID NO: 63), SQRQATKDA (SEQ ID NO: 65), NAGGVTVSY (SEQ ID NO: 67), GLDPAVYEL (SEQ ID NO: 74), YLAEGFYKA (SEQ ID NO: 75), KLGGKVVAV (SEQ ID NO: 76), KLVEIIAGL (SEQ ID NO: 101), PQI
  • the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 105-112.
  • amino acid “substitution” or “replacement” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence.
  • Amino acids are broadly grouped as “aromatic” or “aliphatic.”
  • An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp).
  • Non- aromatic amino acids are broadly grouped as “aliphatic.”
  • “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
  • the amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative.
  • the phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
  • Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained.
  • “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group.
  • substitution of aspartic acid for asparagine, or asparagine for lysine involves amino acids within the same group, but different sub-groups. “Non- conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
  • the one or more nucleic acids may be DNA, RNA, or a combination thereof. In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids are RNA. In some embodiments, the one or more nucleic acids are circular RNA. In some embodiments, the one or more nucleic acids are mRNA.
  • the one or more nucleic acids are optimized for enhanced expression, productive co-translational folding, increased stability, or a combination thereof.
  • the one or more peptides are encoded on a single nucleic acid.
  • a single nucleic acid encodes two or more peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.).
  • the composition can comprise one or more polycistronic mRNAs. “Polycistronic” refers to an mRNA encoding more than peptide.
  • the one or more nucleic acids or mRNA are codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs
  • the one or more nucleic acids may further comprise or encode other structures and sequences necessary for proper functionality or stability, including but not limited to a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, a signal sequence, major histocompatibility complex (MHC) class transmembrane and cytoplasmic domain(s), or any combination thereof.
  • UTR untranslated region
  • MHC major histocompatibility complex
  • the disclosure provides a mRNA comprising: a 5’ UTR, an open reading frame encoding the disclosed peptides, and a 3’ UTR.
  • the one or more nucleic acids may comprise a 5’ and 3’ untranslated region (UTR). Sequence elements within the UTRs affect translational efficiency and RNA stability but do not encode a polypeptide.
  • the 3 ’ UTR relates to the region located at the 3 ’ end of the mRNA, downstream of the termination codon of a protein-encoding region, which is transcribed but not translated into an amino acid sequence.
  • the 5’UTR refers to the region directly upstream from the initiation codon.
  • Eukaryotic 5’ UTRs contain the Kozak consensus sequence (ACCAUGG), which contains the initiation codon.
  • the eukaryotic 5’ UTR may also contain As- acting regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs) and termination codons, which can impact translation regulation.
  • uORFs upstream open reading frames
  • uAUGs upstream AUGs
  • termination codons termination codons
  • the 5’ UTR, 3’ UTR, or both is heterologous to the sequence or open read frame encoding the disclosed peptide.
  • 5’ -cap refers to the structure found on the 5 ’ -end of an mRNA which ordinarily consists of a guanosine nucleotide connected to the mRNA via a 5 ’ to 5 ’ triphosphate linkage.
  • the guanosine may be methylated at the 7-position creating a 7-methylguanosine cap (m 7 G).
  • the 5’ cap may be a naturally occurring 5’ cap.
  • the 5’ cap is a 5’ cap analog or a modified 5’ cap structure which is non-naturally occurring (e.g., phosphorothioate-cap-analogs).
  • the one or more nucleic acids may comprise a 3’ tailing sequence.
  • the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
  • the one or more nucleic acids may encode a signal sequence.
  • Signal sequences are generally about 16-30 amino acids (aa) in length, at the N-terminus of a recently synthesized protein that directs the sequence to the secretory pathway (e.g., for routing to the endoplasmic reticulum).
  • the signal sequence may be N- or C-terminal to the one or more peptides.
  • the one or more nucleic acids may further encode transmembrane and cytoplasmic domains of an MHC polypeptide.
  • the presence of these domains in the peptide facilitates improved presentation of the epitopes, for example by improving intracellular signaling and trafficking to the plasma membrane.
  • the transmembrane and cytoplasmic domains are from an MHC class II polypeptide.
  • the transmembrane and cytoplasmic domains are from an MHC class I polypeptide.
  • the one or more nucleic acids further comprises a triple helix forming sequence.
  • a tiple helix is formed after the binding of a third strand to the major groove of a duplex nucleic acid through Hoogsteen base pairing (e.g., hydrogen bonds) while maintaining the duplex structure of two strands making the major groove.
  • Pyrimidine-rich and purine-rich sequences e.g., two pyrimidine tracts and one purine tract or vice versa
  • can form stable triplex structures as a consequence of the formation of triplets e.g., A-U-A and C-G-C).
  • the triple helix sequence is derived from the 3’ terminal triple helix sequences of triple helix terminators from a long non-coding RNAs (IncRNAs), e.g., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).
  • the one or more nucleic acids further comprises a tRNA-like sequence.
  • the tRNA-like sequences are those sequences which form similar overall secondary and tertiary structure to tRNA.
  • the tRNA-like sequence is derived from a long non-coding RNAs (IncRNAs), e.g., MALAT1.
  • the tRNA-like sequence derived from IncRNAs may be truncated or modified as long as they retain the clover secondary structure.
  • the MALAT1 sequences for the triple helix or the tRNA-like sequence can be from any species.
  • the MALAT1 sequences are from a human.
  • the M AL ATI sequences are from a mouse.
  • the MALAT1 sequences are from a non- human primate.
  • the 3’ poly(A) sequence of mRNA is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA).
  • the poly (A) tail is a segment of RNA at the 3’ end of the molecule that has only adenine bases.
  • a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • the poly(A) tail may contain two segments of only adenine bases separated by a linker.
  • the one or more nucleic acids comprise at least one chemical modification or chemically modified base, nucleoside, or nucleotide.
  • the chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally- occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides.
  • A adenosine
  • G guanosine
  • U uridine
  • C cytidine
  • a single nucleic acid or mRNA may include both naturally-occurring and non-naturally-occurring modifications.
  • nucleic acid or mRNA molecule may contain any percentage of modified nucleosides (1-100%).
  • every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine).
  • at least 80% of any single nucleotide (e.g., uracil) in the of the nucleic acid or mRNA is chemically modified.
  • a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl-pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (See mods.ma.albany.edu/home).
  • the at least one chemical modification comprises a modified uridine residue.
  • modified uridine residues include, but are not limited to, pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methyluridine, 2- thio-1 -methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5 -aza-uridine, 2-thio- dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5- aza- uridine, dihydropseudouridine, 5 -methoxy uridine and 2’-0-methyl
  • the at least one chemical modification comprises a modified cytosine residue.
  • exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl- cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl
  • the at least one chemical modification comprises a modified adenine residue.
  • exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl- adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl- adenosine, 2-methylthio-N6-isopentenyl-
  • the at least one chemical modification comprises a modified guanine residue.
  • exemplary nucleosides having a modified guanine include inosine, 1-methyl- inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxy wybutosine, hydroxy wybutosine, undermodified hydroxy wybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza- guanosine, 7-aminomethyl-7-deaza- guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza- guanosine, 6-thio-7--deaza- gu
  • the disclosure also provides polynucleotide segments encoding the one or more nucleic acids, vectors containing these segments, and cells containing the vectors.
  • the vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment (e.g., an expression vector).
  • an expression vector The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
  • the composition further comprises an adjuvant or a polynucleotide encoding thereof, a delivery vehicle, or a combination thereof.
  • Adjuvants are compounds or compositions that either directly or indirectly stimulate the immune system’s response to a co- administered antigen. Suitable adjuvants are commercially available as, for example, Glucopyranosyl Lipid Adjuvant (GLA); Pam3CSK4; Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), A1K(SO 4 ) 2 , AlNa(SO 4 ) 2 , A1NH 4 (SO 4 ), and A1(OH) 3 ; salts of calcium (e.g., Ca 3 (PO 4 ) 2 ), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or
  • adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants.
  • cytokines e.g., GM-CSF or interleukin-2, -7, or -12
  • interferons e.g., interferon-associated antigen, tumor necrosis factor
  • tumor necrosis factor e.g., tumor necrosis factor
  • Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule.
  • immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4’, 4-diaminodiphenylmethane-3, 3’ -dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids, or carbohydrates.
  • non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4’, 4-diaminodiphenylmethane-3, 3’ -dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1
  • the adjuvantive polypeptide comprises immune activator proteins, such as CD70, CD40 ligand, and constitutively active TLR4, or polycationic peptides (e.g., protamine).
  • the adjuvantive polypeptide is a flagellin polypeptide.
  • mRNA encoding adjuvantive polypeptides are available, for example, as TriMix (See Bonehill, A. et al. Mol. Ther. 16, 1170-1180 (2008), incorporated herein by reference).
  • the adjuvant, the delivery vehicle or the combination thereof comprises a lipid.
  • the lipid may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols.
  • the lipid comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.
  • Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8.
  • This class includes lipid molecules which remain neutral at physiological pH, but are protonated at low pH, making them positively charged.
  • the polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan.
  • amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan.
  • Cationic lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O- octadecenyl-3 -trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl (dimethyl)ammonium bromide (DDAB), l,2-dioleyloxypropyl-3-dimethyl -hydroxyethyl ammonium bromide (DORIE), 3[T[N-(N ⁇ N’-dimethylamino-ethane)carbamoyl]cholesterol (DC-
  • Ionizable lipids include, but are not limited to, l,2-dioleyloxy-3 -dimethylamino-propane (DODMA).
  • the cationic lipid is DOTAP or R-DOTAP.
  • the sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
  • a neutral or non-cationic lipid may include one or more phospholipids.
  • Phospholipids include a phospholipid moiety and one or more fatty acid moieties.
  • a phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin.
  • a fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
  • Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2-dioleoyl- sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1 ,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecan
  • the delivery vehicle comprises a lipid particle encapsulation the composition.
  • Lipid particle compositions of the disclosure may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, and/or sterols, as described.
  • the lipid particle comprises a cationic lipid and/or ionizable lipid, a neutral or non- cationic lipid, and cholesterol.
  • the lipid particle further comprises a polyethylene glycol (PEG)- lipid conjugate.
  • PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.
  • a PEG lipid may be PEG-DMG (l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol), PEG-c-DOMG (R-3-[(cu-methoxy poly(ethylene glycol)2000)carbamoyl)]-l ,2-dimyristyloxlpropyl-3 -amine), PEG-DMA (PEG-dimethacrylate), PEG-DLPE (l,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- 1,2- dimyristoyl-sn-glycero-3 -phosphoethanolamine), PEG-DPPC (PEG-dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2-distearoyl-sn- glycero-3 -phosphoethanolamine
  • the lipid particles may also include other components typically used in the formation of vesicles (e.g., for stabilization).
  • other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
  • compositions may further comprise excipients or pharmaceutically acceptable carriers.
  • excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.
  • the compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
  • compositions may be formulated for any particular mode of administration including for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral).
  • systemic administration e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral.
  • compositions and formulations described herein may be used to prepare vaccines or another medicament.
  • the vaccines or medicaments of the present disclosure may also contain other compounds, which may be biologically active or inactive.
  • one or more immunogenic and antigenic portions of polypeptides or polynucleotides encoding immunogenic polypeptides, or nucleic acid(s) encoding thereof may be present within the vaccines or medicaments.
  • the vaccines or medicaments may generally be used for prophylactic and therapeutic purposes.
  • the vaccines or medicaments may be formulated for any appropriate manner of administration, and thus administered, including for example, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, intratumoral, subcutaneous, intramuscular administration, or via inhalation.
  • the vaccines or medicaments may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose or dextrans
  • mannitol proteins
  • proteins polypeptides
  • amino acids such as glycine
  • antioxidants e.g., antioxidants, bacteriostats, chelating agents such as EDTA or gluta
  • compositions, vaccines, or medicaments may be prepared, packaged, or sold in a form suitable for bolus administration or sold in unit dosage forms, such as in ampules or multi- dose containers.
  • the compositions and vaccines contain a preservative.
  • compositions, formulations, nucleic acids, vaccines, or medicaments disclosed herein are also disclosed herein.
  • kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components.
  • the kits can also comprise instructions for using the components of the kit.
  • the instructions are relevant materials or methodologies pertaining to the kit.
  • the materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents.
  • Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
  • kits can be employed in connection with the disclosed methods.
  • the kit may further contain containers or devices for use with the methods or compositions disclosed herein.
  • kits provided herein are in suitable packaging.
  • suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
  • Clinical Data Sources The clinical study was performed by analyzing the French Cancer Cohort from the cancer institute data platform of the French National Cancer Institute (INCa). This prospective cohort is an extraction from the National Health Data System (SNDS) and includes all people diagnosed, treated, or followed up for a cancer in France since 2010. Briefly, health insurance funds were requested by French legislation to develop a national health insurance information system, which encompasses the whole population, in order to more precisely determine and evaluate health care utilization and the health care expenditure of beneficiaries. This dataset includes all drug consumption related to hospitalization care or outpatient care.
  • SNDS National Health Data System
  • the French cancer cohort protocol was approved by a national committee (Comite Consultatif sur le Traitement de T Information en Matiere deInstitut dans le Domaine de la Sante) and authorized by the French Data Protection Agency (Commissionêt de 1’situation et des libertes — Cnil, number 2019-082). Confidentiality in the cohort is guaranteed for all participants with regards to any personal information, as all data are pseudonymized.
  • 3-DFS Statistical Analysis for Clinical Data 3-DFS was defined as the time elapsed between the date of surgery (time origin) and the date of recurrence, death, or the end of 3-year follow-up, whichever occurred first.
  • the 3-DFS has been modelled using Cox models to study the link with antibiotics intake adjusted for all covariates.
  • the survival curves were represented using the Kaplan-Meier method and compared with the log-rank test.
  • Hazard ratios (HR) were estimated by Cox proportional regression models. As this study was conducted in the overall population (not a sample) statistical tests for descriptive comparisons were not considered relevant.
  • Antibiotic exposure before TO was considered a fixed variable.
  • Antibiotic exposure after TO was considered a time-dependent variable.
  • a multivariate model was carried out to isolate the effect of antibiotics targeting anaerobe by comparing patients that received only these antibiotics to those that didn’t receive any antibiotics as outpatients.
  • CT26(FL3) cells were kindly provided by Dr. Maria Marjorette O. Pena at the University of South Carolina.
  • CT26(FL3) cells stably expressing red fluorescent protein (RFP) and luciferase (Luc) were established by transfection with lentivirus vectors carrying RFP and Luc gene and a puromycin resistance gene.
  • RFP red fluorescent protein
  • Luc luciferase
  • CT26(FL3)-RFP/Luc cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose (Gibco) and 10% bovine calf serum (BCS, HyClone) supplemented with 1 pg/mL puromycin (ThermoFisher) and 1% antibiotic- antimycotic (Gibco) at 37°C and 5% CO2 in a humidified atmosphere.
  • MC38 cells was purchased from Kerafast, Inc., which was cultured in DMEM with 10% BCS, 0.1 mM nonessential amino acids and 1% antibiotic- antimycotic.
  • F. nucleatum Bacteria Culture, Inoculation and CFU Quantification Fusobacterium nucleatum subsp. Nucleatum (ATCC®25586TM) (F. nucleatum) was purchased from the American Type Culture Collection (ATCC). F. nucleatum was grown in chopped meat medium with carbohydrates (Anaerobe Systems) overnight in 37 °C in anaerobic Hungate tubes. Bacteria were pelleted anaerobically by centrifugation at 4,000 rpm for 10 min at room temperature and were washed once in sterile phosphate buffered saline (PBS) before use.
  • PBS sterile phosphate buffered saline
  • CFUs colony forming units
  • tissues and tumors samples were homogenized twice (MP Biomedical FastPrep-24) in 40% (vol/vol) glycerol solution at 12 M/s for 20s using 1.4 mm ceramic bead-filled tubes (Fisher).
  • Viable CFUs were enumerated on Fusobacterium selective agar plates (Anaerobe Systems) and data was normalized to the mass of tissue plated.
  • E. coli Nissle 1917 was provided by Dr. Aaron Anselmo lab with integrated a plasmid conferring kanamycin resistance was used for tumor colonization study in mouse. E. coli Nissle 1917 was grown overnight to saturated conditions in kanamycin (50 pg/mL) supplemented LB broth, washed to remove media. Serial dilutions of the E. coli Nissle were plated on selective kanamycin (50 pg/mL) plates. Colony forming units were enumerated after 24 h of growth at 37 °C.
  • the orthotopic and subcutaneous tumor model was established based on the previously work. Briefly, female BALB/cJ or C57BL/6J mice were anesthetized with 2.5% isoflurane in oxygen at supine position. A midline incision was made to exteriorize the cecum. CT26(FL3)- RFP/Luc and MC38 cells at density of 2.0xl0 6 in 50 pL mixture of PBS and Matrigel (1:1) were injected into the cecum wall. The cecum was returned to the peritoneal cavity before the incision was sutured. The tumor burden was monitored by bioluminescent analysis using an IVIS imager (Perkin Elmer) with i.p.
  • the subcutaneous tumor model was established by injection of LOxlO 6 CT26(FL3)- RFP/Luc cells in 100 pL PBS into the right flank of the BALB/cJ mice.
  • CT26(FL3)-RFP/Luc was infected with F. nucleatum prior to inoculation.
  • Tumor volume (V t ) was calculated as following:
  • V t 0.5 X a X b 2 (1)
  • Tn Eq. (1 ), a and b are defined as the major and minor diameter of the tumor.
  • CT26(FL3)-RFP/Luc was infected with F. nucleatum prior to inoculation.
  • Female BALB/cJ mice were anesthetized with 2.5 % isoflurane in oxygen at supine position. An incision was made to exteriorize the spleen below the left rib cage. The spleen was tied and cut into two parts that contain intact vascular pedicle for each half. The distal section of the spleen was inoculated with 1.0 x 10 6 CT26(FL3) cells in 150 pL PBS. The hemi-spleen containing inoculated cells was resected 5 min after inoculation allowing the cancer cells to enter the liver through the portal vein.
  • the other half of the spleen was returned to the peritoneal cavity and the incision was sutured.
  • the tumor burden was monitored by bioluminescent analysis using an IVIS imager (Perkin Elmer) with i.p. injection of 100 pL of D-luciferin (Perkin Elmer, 10 mg/mL).
  • IVIS imager Perkin Elmer
  • D-luciferin Perkin Elmer, 10 mg/mL
  • the dissected liver with tumor was quickly rinsed in PBS and placed in diluted luciferin solution (1 mg/mL) for 1 min. bioluminescence imaging was applied immediately.
  • the cells were then incubated with the stained or unstained bacteria at a multiplicity of infection (MOI) of 20 in a hypoxic incubator for 4 h, followed by three washes in PBS.
  • MOI multiplicity of infection
  • the cells were harvested for inoculation.
  • the cells were staining with phalloidin, and antibodies as described below. Antibodies were incubated at 4°C overnight.
  • Immunofluorescence staining was performed by rinsing with PBS, permeabilizing with 0.1% Triton X-100 (Sigma- Aldrich) in PBS and blocking in 10% goat serum (Life Technologies) at room temperature for 1 h. Antibodies were incubated at 4°C overnight. Finally, the slides were stained with DAPI and mounted with Prolong® Diamond Antifade Mountant (ThermoFisher Scientific). Images were taken using a confocal laser scanning microscopy (CLSM, Zeiss 700 or CLSM, ZeisS610).
  • Tumor Cell Spheroids The CT26(FL3) suspensions were mixed with 0.48% (w/v) methylcellulose. Then drops of cell suspensions were placed onto the lids of 96-well plates, and the lids were inverted over the plates containing sterile deionized water to prevent the droplets from evaporating overnight. The resultant cellular aggregates on the lids were harvested using a pipet and transferred to round-bottom low attachment 96-well plates (Coming, costar® 7007) by centrifugation (1,000g, 3 min). Then the medium was replaced every 48-h using a standard culture medium up to 96 h. After that, cell spheroids were incubated with CFSE-labelled F. nucleatum in hypoxic incubator for 12 h before fixation and DAPI staining.
  • Liposome Preparation Distearoyl phosphatidylcholine (16.0 mg, Avanti), cholesterol (10.0 mg, Avanti) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (5.0 mg, Avanti) were dissolved in chloroform. The solvent was evaporated to form a lipid thin film. Three mL of 300 mM silver nitrate solution (adjusted to pH 3.0 with nitric acid) was added to hydrate the film at 65 °C for 15 min, and then the suspension was sequentially extruded through 400 nm, 200 nm, 100 nm membrane for 10 times, respectively.
  • Liposomal encapsulation efficiency was calculated as the weight of tinidazole entrapped in liposomes versus weight of tinidazole totally added.
  • Liposomal loading efficiency was calculated as the weight of tinidazole entrapped in liposomes versus weight of gross materials added.
  • Cryo-Electron Microscopy Cryo-EM grids were prepared with a Vitrobot Mark (Thermo Fisher Scientific) set to room temperature and 95% humidity. Quantifoil Rl.2/1.3 copper 300 mesh grids were rendered hydrophilic by glow-discharging for 30 seconds at 15 mA with a Pelco easiGlow. Five p L of sample were applied directly to the carbon side of the TEM grid then blotted with Whatman 595 filter paper for 3 sec with the blot force set to -10. Cryo-EM grids were imaged with a Thermo Fisher Scientific 200 kV TEM Tales Arctica G3 equipped with a Gatan K3 direct electron detector. The microscope was aligned, and beam was set parallel at spot size 3 with the 70 pm condenser and 100 pm objective apertures.
  • Drug Release Analysis Drug-loaded liposomes (500 pL) were enclosed into 8-10 kD dialysis devices (Float-A-Lyzer®G2 G235031, Spectra/Por®) floating in 30 mL 5% glucose with or without pH adjusted to 4.5 by nitric acid in 50 mL centrifuge tubes. The tubes were place in a shaker at 100 rpm, 37 °C. Fifty pL of outer medium at 5, 13, 30, 60, 120, 240, 480, 960, 1920 min was sampled and analyzed by LC-MS and ICP-MS.
  • Liposome Characterization The particle zeta potential and size were determined by NTA using NanoSight 500 Version 2.2 (Wiltshire, UK). For the size measurements, liposomes were dispersed in 5% glucose.
  • Antimicrobial Assay Brucella broth (Anaerobe Systems) was used to dilute F. nucleatum culture to 10 5 CFU/mL for antimicrobial assay.
  • One hundred pL of antimicrobial agent (0.1, 0.5, 1, 2, 4 and 8 pM dissolved in Brucella broth) was two-fold diluted by 100 pL F. nucleatum culture in 96-well microtitration plates. After mixing, the plate was incubated under normoxia (20% oxygen) or hypoxia (1% oxygen) condition for 12 h. For quantification of F.
  • samples were serially diluted, drop-plated (10 pL) on Brucella blood agar plates, incubated in an anaerobic chamber (Coy Laboratory Products) at 37 °C for 24-48 h and enumerated for colony forming units (CFUs).
  • CFUs colony forming units
  • the culture medium of CT26(FL3) cells was changed to DMEM supplemented without serum and antibiotics before F. nucleatum infection.
  • the cells were then incubated with F. nucleatum at a multiplicity of infection (MOI) of 20 in a hypoxic incubator for 4 h.
  • MOI multiplicity of infection
  • a serial dilution of liposomes was added and incubated for 12 h in DMEM with 10% BCS.
  • the extracellular bacteria were removed by gentamicin (50 pg/mL) in DMEM with 10% BCS for 2 h. After three washes in PBS, cells were scraped and lysed in sterile water before CFU plating.
  • LC-MS Liquid Chromatography - Mass Spectrometry
  • a Leapsil C18 column (100 mm x 2.1 mm, 2.7 pm) was used, while mobile phase was composed of acetonitrile and water premixed with 0.1% formic acid.
  • the injection volume was 20 pL.
  • ICP-MS Inductively Coupled Plasma Mass Spectrometry
  • mice were anesthetized and the whole blood was collected from the ocular vein using anticoagulant tube (MiniCollect®, Greiner Bio-One) and directly assayed for complete blood count. Serum was obtained after centrifuge at 10,000 g at 4 °C for 10 min. Creatinine (CRE), blood urea nitrogen (BUN), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were assayed as biomarkers of renal and liver functions. Blood tests were performed at the LCCC Animal Histopathology Core at UNC.
  • CRE Creatinine
  • BUN blood urea nitrogen
  • AST serum aspartate aminotransferase
  • ALT alanine aminotransferase
  • haematoxylin and eosin staining was performed at the Animal Histopathology Core at UNC. Organs including heart, liver, spleen, lung, kidney, and tumor were collected and fixed in 4% PFA for haematoxylin and eosin staining.
  • Reverse Transcription PCR Total RNA was extracted from the tissues using the RNeasy Mini Kit (Qiagen). cDNA was reverse-transcribed for each sample using the iScriptTM cDNA Synthesis Kit (BIO-RAD). 500 ng of cDNA was amplified with the TaqManTM Gene Expression Master Mix with addition of primers. GAPDH was used as the endogenous control. Reactions were conducted using the 7500 Real-Time PCR System.
  • FISH Fluorescence In Situ Hybridization
  • Stellaris® RNA FISH buffers (Stellaris® RNA FISH Wash Buffer A, Stellaris® RNA FISH Wash Buffer B and Stellaris® RNA FISH Hybridization Buffer) were used according to Stellaris® RNA FISH protocol for frozen tissue. Briefly, the fresh dissected tumors were quickly embedded in Tissue-Plus O.C.T. Compound and stored in -80 °C.
  • the froze tumors were sliced at thickness of 10 pm, fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. The slices were subsequentially immersed in Wash Buffer A for 5 min, dispensed with 200 pL hybridization buffer containing probe solution of 125 nM followed by incubation in dark at 37 °C for 16 h. The slides were then incubated with Wash Buffer A in dark for 30 min, stained by 5 pg/mL DAPI in Wash Buffer A and immersed with Wash Buffer B for 5 min, and mounted with Prolong® Diamond Antifade Mountant (ThermoFisher Scientific). The uninfected tumors were stained with the probes as negative controls to verify that FISH assay was specifically detecting F. nucleatum.
  • FISH Quantification The distance map indicating minimum distance between FISH signals and DAPI staining was calculated by Euclidean distance function, where fl F and fl D are the image domain of FISH signals and DAPI staining, respectively, x/y denotes the position of each pixel in the two-dimensional coordinate system. For each x of FISH signals, its minimum distance wase computed from position of DAPI staining by Eq. (2). x and d(x) were then fitted with polynomial fourth order curve to better reflect a correlation of distance between FISH signals and DAPI staining, by Graphpad 9.0 prime software.
  • Fecal DNA Isolation and Library Preparation Mouse stool samples were collected on Day 60 following LipoAgTNZ treatment and immediately stored in -80 °C upon collection. Fecal DNA was isolated using QIAamp Fast DNA Stool Mini Kit (Qiagen) following manufacture protocol. 12.5 ng of total DNA were amplified by PCR using primer set (515F-806R) (81, 82) targeting V4 region on 16s rRNA genes, and PCR amplicons were sequenced at V4 region on an Illumina Miseq (Illumina).
  • each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dual-index barcodes (index l(i7) and index 2(i5)) (Illumina) to the amplicon target.
  • the final libraries were purified using the AMPure XP reagent (Beckman Coulter), quantified and normalized prior to pooling.
  • the DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge (Illumina) and on the MiSeq instrument (Illumina). Automated cluster generation and paired-end sequencing with dual reads were performed according to the manufacturer’s instructions.
  • Bioinformatic Analysis Sequencing output from the Illumina MiSeq platform were converted to fastq format and demultiplexed using Illumina Bcl2Fastq 2.20.0 (Illumina, Inc. USA). The resulting paired-end reads were processed with the Quantitative Insights Into Microbial Ecology (QIIME) 2 2021-2 wrapper for DADA2 including merging paired ends, quality filtering, error correction, and chimera detection. Amplicon sequencing units from DADA2 were assigned taxonomic identifiers with respect to the Greengenes and Silva databases, their sequences were aligned using maFFT in QIIME 2, and a phylogenetic tree was built with FastTree in QIIME 2.
  • QIIME Microbial Ecology
  • Rarefaction curve was generated at the depth of 5,000 sequences per subsample. 0-diversity estimates were calculated within QIIME 2 using Bray Curtis distance between samples at a subsampling depth of 5,000. Results were summarized and visualized through principal coordinate analysis as implemented in QIIME 2. Microbiota taxonomy was also applied to classify the organism as a representative OTU. The linear discriminant analysis effect size (LEfSe) Galaxy module was used for analysis examining biologic consistency and effect relevance (huttenhower.sph.harvard.edu/galaxy), which was conducted by coupling standard tests for statistical significance.
  • LfSe linear discriminant analysis effect size
  • the cells were resuspended with 10 mL RPMI 1640 medium (Gibco) with 120 Kunitz unit/mL DNase for 15 min at room temperature and filtered through a cell strainer.
  • the cells were washed with 50 mL PBS with 0.1% BSA and 2 mM EDTA, resuspended by 4 mL PBS with 0.1% BSA and 2 mM EDTA and purified by 3 mL FicolLPaque PLUS at 400 x g for 30-40 min at room temperature.
  • the undisturbed lymphocytes at the interface were collected and used for in vivo (CFSE labelled cells) and in vitro T cell proliferation assay.
  • Pan T cells were further isolated by negative magnetic labeling by using a pan T cell isolation kit (Miltenyi Biotec). T cells were treated with biotin- antibody cocktail and subsequent anti-biotin cocktail and separated by LS column (Miltenyi Biotec) in elution buffer (PBS with 0.5% BSA and 2 mM EDTA) in the magnetic field of the MACS separator (Miltenyi Biotec). The cells were adjusted to IxlO 6 in 200 pL PBS and i.v. injected to the recipient mice on Day 10 and Day 15 after tumor inoculation.
  • lymphocytes were purified as described above.
  • lymphocytes (5xl0 7 ) from the donor mice were labeled with 5.0 pM CFSE (Invitrogen) and washed with PBS containing 5% bovine calf serum.
  • the recipient mice were inoculated with orthotopic CRC tumors with or without F. nucleatum inoculation by oral gavage.
  • Adoptively transferred CFSE-labeled lymphocytes (5xl0 7 per mice in 200 pL PBS) were i.v. injected into the recipient mice. At 18 h after injection, tumors from the recipient mice were harvested and dissociated as described in section of flow cytometry.
  • the cell suspensions were stained with antibodies specific for T cells and analyzed by flow cytometry as described above. Tumor from mice without lymphocytes transfer were used as negative control and splenocytes from mice without CRC tumor and F. nucleatum inoculation were used as positive control for CFSE analysis. The level of CFSE were gated to calculate the percentage of in vivo T cell proliferation.
  • CFSE CFSE
  • the membranes were then blocked with 1% bovine serum albumin in TBST at room temperature for Ih and then incubated with primary antibodies diluted in 1 % bovine albumin in TBST overnight at 4 °C in a shaker. The membranes were washed by TBST and further incubated with a secondary antibody diluted in 1% bovine albumin in TBST at room temperature for Ih, and then detected using the Pierce ECL Western Blotting Substrate (Thermo Fisher). Beta- actin was used as the control. Image Lab 6.0.1 was used to acquire images.
  • EHSpot assay is used to test memory immune response and MHC restricted peptides on splenic CD8 + T cells.
  • Interferon- y (IFN-y) ELISPOT assay R&D SYSTEMS, EL485.
  • IFN-y Interferon- y
  • ELISPOT assay R&D SYSTEMS, EL485.
  • Bone marrow derived dendritic cells (10 5 /well) were pulsed with peptides (20 pg/mL) and were added to CD8 + T cells (2 x 10 4 /well) for 24 h at 37°C and plates area were developed with a biotinylated detection antibody specific for IFN-y for 1 h, followed by streptavidin- alkaline phosphatase for 1 h. Finally, the substrate of alkaline phosphatase (BCIP/NBT buffer) is added for 5-20 min. Spots are imaged by using stereomicroscopy (Olympus BX61).
  • Mass spectrometry Samples were analyzed with a Thermo LTQ Ion Trap (ThermoFisher, Bremen, Germany) mass spectrometer. Samples were introduced via a heated electrospray source (HESI) at a flow rate of 0.3 mL/min. HESI source conditions were set as: source heater temperature 250 °C, sheath gas (nitrogen) 50 arb, auxiliary gas (nitrogen) 15 arb, sweep gas (nitrogen) 0 arb, capillary temperature 200 °C, tube lens voltage 110 V, capillary voltage 10 V, and source voltage 3.50 kV. The mass range was set to 400-1800 m/z and data were collected in the positive polarity. Solutions were analyzed at 0.1 umM or less based on responsiveness to the ESI mechanism. Xcalibur (ThermoFisher, Breman, Germany) was used to analyze the data.
  • HESI heated electrospray source
  • LipoCuTNZ preparation Distearoyl phosphatidylcholine (16.0 mg, Avanti), cholesterol (13.0 mg, Avanti) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (5.0 mg, Avanti) were dissolved in chloroform. The solvent was evaporated to form a lipid thin film. Three mL of 300 mM silver nitrate solution (adjusted to pH 4.0 with copper chloride) was added to hydrate the film at 65 °C for 30 min, and then the liposomes were sequentially extruded through 400 nm, 200 nm, 100 nm membrane for 10 times respectively.
  • T lymphocytes were prepared by standard Ficoll-Paque density gradient centrifugation. CD8+ T lymphocytes were isolated by negative bead selection (Miltenyi Biotec, 130-104-075). The T cell epitopes are evaluated for the percentage of specific CD8+ T lymphocytes by staining with a H2-Db- or H2-Kb-peptide tetramer (5001-1-20, EAGLE bioscience) and anti-CD8 mAb.
  • H2-Db- or H2-Kb-peptide tetramer 5001-1-20, EAGLE bioscience
  • Vaxign2 violinet.org/vaxign2
  • NetMHC 4.0 Server cbs.dtu.dk/services/NetMHC/
  • Example 1 Bacteria Invaded into Tumor Cells in Response to Low Oxygen Level
  • F. nucleatum Gram-negative and anaerobic bacteria, F. nucleatum, is prevalent in human CRC as well as metastasis.
  • CT26(FL3) cells were acclimated to either 1% (hypoxia) or 20% oxygen (normoxia) for 24 h (FIG. 1A).
  • F. nucleatum was able to invade hypoxic CT26(FL3) tumor cells (FIG. IB) with 15-fold higher signals in comparison to cells in normoxia (FIG. 1C).
  • the intracellular F. nucleatum was confirmed by stacking images highlighting the co-localization of F. nucleatum and F-actin labelled by phalloidin, which showed F.
  • nucleatum was able to invade inside the cytoskeleton in response to hypoxia (FIG. ID).
  • F. nucleatum infected CT26(FL3) spheroids bacteria not only adhered on the surface but translocated into the organoids (FIGS. IE and 7A).
  • Spontaneous invasion to hypoxic tumor cell was also found in facultative anaerobic probiotic strain E. coll. Nissle, which suggested that bacteria preferentially invade hypoxic tumor (FIGS. IF and 8A).
  • CRC was established in Balb/C mice infected with F. nucleatum (FIG. 6A).
  • F. nucleatum infection led to over 30-fold higher tumor growth ratio as compared to uninfected controls (FIGS. 6B-6D) without inducing loss of body weight of the BALB/c mice (FIG. 6E).
  • F. nucleatum infection considerably promoted tumor metastasis in proximal lymph nodes and distal metastasis (FIG. 6F). The primary tumors, distal metastases and feces were confirmed to contain F. nucleatum (FIG. 6G).
  • TEM images of F. nucleatum infected CRC tumor demonstrated that F. nucleatum was intracellular in vivo in the tumor region (FIG. 1G).
  • Fluorescence in situ hybridization (FISH) using an RNA probe specific for the 16s ribosomal RNA of F. nucleatum was performed to visualize the bacterium in the CRC tumor sections.
  • the red pixels indicating FISH signals were mapped to the nearest blue pixels indicating DAPI staining, which was calculated by Euclidean distance map.
  • the minimum distance distribution was further fitted by computing the percentage of FISH pixels versus distances to DAPI, which was within the range of 3 /rm, suggesting F. nucleatum appeared near the cell nuclei in the CRC tumor (FIGS.
  • Escherichia coli Nissle 1917 (E. coli Nissle) has been widely studied as a probiotic and engineered bacteria that delivers therapeutics to hypoxic tumor.
  • An orthotopic E. coli Nissle colonized CRC tumor was established in BALB/c mice (FIGS. 8B and 8C).
  • E. coli Nissle colonized in CRC tumor (FIG. 8D), fostered immunosuppressive tumor microenvironment (FIG. 8E).
  • Nitroimidazole is part of a class of antimicrobial prodrugs that is inactive until reduced by the ferredoxin oxidoreductase system in obligate anaerobes.
  • the reductive activation of the nitro group is proposed to form the cytotoxic nitro and other free radical, leading to its structure fragmentation and cytotoxicity to DNA.
  • the presence of oxygen tension inhibits the formation of the cytotoxic daughter drug.
  • Reductive inactivation of the nitro group to the amino group occurs via oxygen-insensitive nitro reductases, rendering nitroimidazole non-toxic.
  • the nitroimidazole coordinates via the ring N3 donor atom with a variety of metal ions including cobalt (II), copper (II), zinc (II) and silver (I) (FIG. 9 A).
  • metal ions including cobalt (II), copper (II), zinc (II) and silver (I) (FIG. 9 A).
  • Ag + ions are potent antibacterial agents used in various forms as antibiotics for centuries.
  • the silver nanoparticles may have the ability to adhere and penetrate into the bacterial cell wall.
  • the silver (I) ions interact with sulfur and phosphorus, thereby causing altered activity of enzymes and DNA.
  • the formation of the silver- tinidazole complex was confirmed by mass spectrometry (FIG. 9B).
  • Tumor hypoxia directly correlates with tumor acidosis due to the Warburg effect favored by cancer cells that metabolized pyruvate into lactate and ethanol.
  • the low pH of hypoxic tumors in result of elevated levels of lactic acid may allow drug release in anerobic bacteria residing region of the tumor.
  • Metal ions can be a trapping agent for loading nitroimidazole into the liposomes (FIG. 2A).
  • a silver-tinidazole complex disassociated in acid, showing a rapid release of both Ag and tinidazole in acidic medium at pH 4.5 (FIG. 2B).
  • the liposomes were spherical with a diameter of -150 nm and zeta potential around -14 mV determined by nanoparticle tracking system (FIGS. 9C and 9D).
  • the liposomes were uniform in morphology imaged by CryoEM (FIG. 2C).
  • the loading of tinidazole (TNZ) into silver containing liposomes was efficient with >80% encapsulation efficiency and >5% loading content (FIG. 2D).
  • TNZ showed quick loading kinetics into the liposomes, over 80% of which was entrapped within 5 min (FIG. 2E).
  • the liposomal formulation has the potential to facilitate intracellular F. nucleatum clearance.
  • Liposomes were labelled with DiD, a fluorescent dye that labels the lipid membrane of liposomes. Distribution of liposomes were determined by quantifying DiD fluorescence in the organs. The liposomes mainly localized to the tumor and the liver, as shown by ex vivo images (FIG. 10D). Liposomes extended the TNZ accumulation in the tumor by more than 10-fold (FIG. 10E). Mice were sacrificed at 24 h post i.v. injection of free drug and liposomes. All major organs including tumor were harvested, homogenized, and measured for both drug contents for biodistribution. Tinidazole encapsulated in liposomes mainly accumulated in the tumor at 24 h after i.v. injection, which suggest the success of tumor targeting (FIGS. 10F and 10G).
  • MTT assays were performed to analyze the in vitro cytotoxicity. Free Ag, TNZ and AgTNZ did not affect the viability of CT26(FL3) cells within 48 h of incubation at concentrations as high as 40 pM (FIGS. 11A and 1 IB). An in vivo toxicity study of liposomal metal-imidazole complexes was also performed. Healthy (non-tumor bearing) mice were i.v. injected (4 mg/kg with respect to TNZ) with either LipoCuTNZ or LipoAgTNZ three times every third day. Treated mice showed no body weight change (FIG. 11C).
  • the monocyte and lymphocyte count were not significantly increased in the LipoAgTNZ treated group (FIG. HE). Toxicity biomarkers were assessed: ALT and AST for liver function, BUN, and creatinine for kidney function (FIG. 1 IF). No obvious alteration in serum biomarkers were observed compared with the untreated mice.
  • the infected CRC mouse model was used to test the therapeutic efficacy of the antibiotic liposomes.
  • Balb/C mice that orthotopically inoculated with tumor and additionally infected with F. nucleatum or E. coli Nissle received antibiotic liposome treatments i.v. (FIG. 3 A).
  • F. nucleatum infected tumor was significantly inhibited by both LipoAgTNZ and LipoCuTNZ at Day 23 (FIGS. 3B, 12A, and 12B), which eradicated the tumor colonizing bacteria (FIG. 3C), LipoAgTNZ induced long-term survival in 6 out of 7 mice (FIG. 3D).
  • Liposomal AgTNZ was also effective in reducing the probiotic E.
  • polymyxin B (pmB) was used to eradicate the Gram-negative bacteria in the gut.
  • Polymyxin B is a cyclic peptide with five positive charges and a logP of -5.6, rendering it low transmembrane permeability and poor oral bioavailability.
  • the orally administered pmB affects the bacteria only in the gut but not in the tumor due to low transmembrane partition and distribution (FIG. 12C).
  • the mice treated with pmB did not show significant reduction in tumor growth (FIG. 12D).
  • the tumor colonizing F. nucleatum promoted tumor progression, which is independent of F. nucleatum burden in the gut.
  • the efficacy of LipoAgTNZ was relative to colonization of F. nucleatum in the tumor, as the uninfected mice did not respond as well as the infected mice (FIG. 12E).
  • CD8 + cytotoxic T cell and CD44 + CD62L + memory T cell were significantly increased in the LipoAgTNZ treated F. nucleatum infected tumors (FIGS. 3H, 14A, and 14B).
  • CD206 + M2 macrophage polarization was reduced following treatment of LipoAgTNZ (FIGS. 3H, 14C, and 14D).
  • LipoAgTNZ treatment induced a substantial normalization of blood vessels and lymphatic vessels, suggesting that the normalization of angiogenesis may contribute to facilitating immune surveillance (FIG. 14E).
  • F. nucleatum interacts with E-cadherin, leading to activation of the P-catenin pathway.
  • F. nucleatum induces DNA damage, which promotes the release of cellular tumor antigen Trp53.
  • Vimentin is also important in epithelial to mesenchymal transition, which was upregulated in F. nucleatum infected tumors.
  • LipoAgTNZ treatment reversed the induction of these oncogenic mediators (Trp53, E-cadherin, Vimentin, P-catenin) and the key inflammatory NF- KB pathway in the F. nuclealum-m' fected tumors (FIG. 14G).
  • rRNA 16S ribosomal RNA
  • liver metastasis the most common distant metastasis in colorectal cancer, afflicts up to 70% of patients.
  • liposomes predominantly accumulated in the tumor and the liver.
  • FIGS. 10D-10F liposomes predominantly accumulated in the tumor and the liver.
  • FIGS. 10D-10F liposomes predominantly accumulated in the tumor and the liver.
  • FIGS. 10D-10F liposomes predominantly accumulated in the tumor and the liver.
  • FIGS. 10D-10F liposomes predominantly accumulated in the tumor and the liver.
  • a F. nucle atum-mf &cted liver metastasis model was established (FIG. 15A).
  • the infected tumor cells were inoculated via the portal vein by hemi-splenic injection, which established uniform metastasis in the liver.
  • the development of liver metastasis was significantly inhibited by treatment of LipoAgTNZ quantified by luciferase imaging in vivo (FIGS. 15B-15D
  • FISH imaging for F. nucleatum 16s RNA revealed a high abundance of bacteria in the untreated liver metastasis, which after the antibiotic treatment was significantly reduced and associated with enhanced infiltration of CD8 + T cells.
  • LipoAgTNZ treatment also reduced anti-inflammatory M2 macrophages, as CD206 expression was much less than the control group (FIG. 15G).
  • the strategy targeted the tumor- associated bacteria in the primary tumor and the distal metastasis.
  • the process of bacterial killing promoted an anticancer response, which restored immune surveillance and inhibited both primary tumor growth and metastatic progression.
  • T cells isolated from survivor mice were compared with age-matched uninfected naive mice.
  • Splenic pan T cells were labelled with the proliferation dye CFSE and injected into the recipient mice with or without F. nucleatum infection.
  • Significant T cell proliferation was found in both infected and uninfected recipient mice that received T cells from survivors, but not from the naive donors (FIGS. 4A and 4B).
  • Incubating survivor-derived donor T cells with uninfected tumor cells in vitro enhanced CD8 + IFN-Y + cell populations (FIGS. 4C and 16A).
  • Splenic pan T cells of long-term survivor mice after liposomal AgTNZ treatment were adoptively transferred to test the efficacy in recipient mice bearing orthotopic CRC tumors with or without F. nucleatum infection (FIG. 4D).
  • Donor T cells from the long-term survivor mice effectively inhibited the growth of tumors infected with F. nucleatum-, Unexpectedly, the same donor T cells also inhibited uninfected tumors (FIG. 4H).
  • Significantly increased numbers of CD3 + and CD3 + CD8 + T cells were found in tumors of both infected or uninfected mice (FIGS. 4E-4G, 16B, and 16C). The data again suggested that survivor T cell recognized both infected and uninfected tumor cells in the recipient mice.
  • F. nucleatum infection decreased the colocalization of calreticulin with cell membrane, downregulating chaperones (e.g., calreticulin) (FIG. 41). It is likely this downregulation is a part of the immune suppression mechanism of the infected tumor cells. It is interesting to note that F. nucleatum infection significantly downregulated both HSP70 and calreticulin, which are stress proteins upregulated by cells in the hypoxia condition. The treatment of liposomal antibiotics restored the chaperone expression by killing of the intracellular bacteria (FIG. 4J). The result suggested that immunogenic cell death may contribute to immune activation of the tumor cells under the treatment of antibiotics.
  • chaperones e.g., calreticulin
  • FIGS. 3 and 4 Data in FIGS. 3 and 4 suggested that LipoAgTNZ induced T cell immunity in the CRC bearing mice. According to identification of transmembrane helices, 1171 out of 2067 proteins are cytoplasmic at F. nucleatum subcellular localization in proteome-wide prediction of Vaxign2 (FIG. 5A). To identify the most abundant protein in cytoplasm, the label-free proteomics of F. nucleatum are mapped to subcellular localization (FIG. 5A). The proteomics data were generated from prior data (See, Ali Mohammed MM, et al., Anaerobe.
  • the MHC class I H-2K d , H-2D d and H2L d binding peptides were predicted from the proteins ranking top in the quantitative proteomics of F. nucleatum, followed by selection of the epitopes with high total score and MHC binding affinity (FIGS. 5B and 5D).
  • the top-ranked cytoplasmic proteins in abundance were selected for epitope prediction.
  • Five top-ranked peptides predicted from each protein were selected using IEDB database applying NetMHCpan algorithm for mouse MHC-I presentation (sequences shown in the upper right comer of FIG. 5B).
  • peptides No. 1-5 are bacteria-derived neoantigens sharing no sequence similarity with mice.
  • Peptides No. 6-8 are homologous epitopes that are shared between bacteria and mice.
  • splenic CD8 + T cell were recovered from Balb/c mice that had survived from CRC tumor infected by F. nucleatum after LipoAgTNZ treatment and restimulated in vitro with the candidate peptides presented by bone marrow derived dentritic cells to measure IFNy production in an Elispot assay. Both homologous antigens and neoantigens showed enhanced IFNy- secreting T cells after LipoAgTNZ treatment (FIGS. 5E and 5F). Splenic T cells from mice infected with F. nucleatum but not treated with LipoAgTNZ did not show significant INFy response to the bacterial cytoplasmic peptides. A peptide from chicken ovalbumin (OVA) was used as a negative control.
  • OVA ovalbumin
  • mice established long-term anti-tumor efficacy, compared with T cells from naive mice.
  • liposomal antibiotics treated mice developed IFNy + CD8 + T cells in response to bacteria-derived neoantigens.
  • IFNy + CD8 T cells in response to homologous epitopes were also developed after antibiotic treatment, suggesting the cellular immunity against the self-epitopes. This result was consistent with the T cell transfer study, in which T cell from survivor mice suppressed the uninfected tumor growth.
  • the bacteria induced CD8 + T cells in response to class I MHC antigens which is the cytotoxic T cell phenotype for antitumor efficacy, were primarily analyzed whereas CD4 + T cells in response to Class II MHC antigens were reported to generate either cytotoxicity or tolerance.
  • Epitope(s) released from dead or dying tumor associated bacteria may prime the immune system to recognize the bacteria infected cells.
  • Bacterial epitopes shared by the tumor cells also elicit anti-tumor effect by priming cytotoxic T cells.
  • Bioinformatics screens and predictions to identify the antigenic T cell epitopes are followed by confirmation of endogenous major histocompatibility complex (MHC) /T cell receptor (TCR) of the tumor-infiltrating T-cells.
  • MHC major histocompatibility complex
  • TCR T cell receptor
  • mRNA encoding the epitopes of both MHC-I and II can be delivered using lipid nanoparticle technology to dendritic cells for immunization and therapy, or prophylactically as a preventive vaccine.
  • Bacteria residing in tumor potentially provide tumor-associated neoepitopes.
  • the most abundant proteins locate in cytoplasm of bacteria and do not share similar sequences if aligned with mammalian proteome.
  • These bacterial epitopes potentially induce CD8+ T cell cross-reactivity with tumor epitopes. Both bacteria-derived neoepitopes and shared epitopes are analyzed and tested for vaccine induced immunogenicity (FIG. 17).
  • Peptide Nos. 5 and 6 in FIG. 18A are two such peptides.
  • two other peptides (Nos. 1 and 2) without sequence similarity to mouse proteome that are predicted as bacteria-derived neoantigens from the top-2 abundant proteins of F. nucleatum analyzed from quantitative proteomics were selected.
  • peptide No. 6 comes from HSP70 chaperone protein (DnaK in gene name), which has 65% similarity with mouse HSP70, two other HSP70 peptides without sequence similarity to mouse proteome (Nos. 3 and 4) predicted to be antigenic for comparison were also added.
  • Listed in FIG. 18A is the scores of these peptides within selection criteria.
  • mice used in the experiment included those infected or uninfected with F. nucleatum and with or without LipoAgTNZ treatment.
  • FIGS. 18B and 18C indicate that peptide No. 5 (LADDNFSTLSEQ ID NO: 105) and No. 6 (RGVPQIEVTF-SEQ ID NO: 106) stood out among the 6 tested by showing high positive ratio in CD8+ T-cells phenotypes suggesting T cell receptor specificities. Both peptides are shared epitopes between bacteria and tumor. Peptide No. 3 also induced tetramer/T cell receptor binding affinity in uninfected tumor, albeit with much lower ratio than the infected tumors. Peptide Nos.
  • VITAFSNI -SEQ ID NO: 111 VITAFSNI -SEQ ID NO: 111
  • 2 SVFVFYPADF-SEQ ID NO: 108
  • 3 ISLPFITM-SEQ ID NO: 109
  • 4 AAIQGGVLM-SEQ ID NO: 110
  • the major tumor infiltrating T-cell specificities are against the bacteria-host shared epitopes, which induced significant response against host epitopes.
  • Bioinformatics prediction of F. nucleatum epitopes that can be presented by the host MHC-I molecules is an effective approach to identifying novel peptide antigens for cancer vaccination.
  • splenic CD8+ T cell were recovered from C57BL/6 mice that had been survived from CRC tumor infected by F. nucleatum after LipoAgTNZ treatment, they were restimulated in vitro with the candidate peptides presented by bone marrow derived dentritic cells to measure IFNy production in an Elispot assay. Both homologous antigens and neoantigens showed enhanced IFNy- secreting T cells after LipoAgTNZ treatment (FIG. 18D). Splenic T cells from mice infected with F. nucleatum but not treated with LipoAgTNZ did not show significant INFy response to the bacterial cytoplasmic peptides. A peptide from chicken ovalbumin (OVA) was used as a negative control.
  • OVA ovalbumin
  • Peptide Nos. 2 and 6 were formulated with DOTAP liposomes as an adjuvant and were given to mice twice by i.m. injection.
  • the presentation of the bacterial epitopes including the epitopes shared with the host epitopes elicited efficient anticancer immunity and resulted in long- term survivals (FIG. 19).
  • An epitope-based vaccine was designed that included a) MHC class I restricted bacterial epitopes from proteins showing top abundancy in F. nucleatum proteomics, b) MHC class II restricted, distinctive bacterial epitopes from proteins showing top abundancy in F. nucleatum proteomics, and c) MHC class I restricted, immunodominant bacterial epitopes with sequence similar to the host proteome.
  • Plasmid templates for in vitro transcription of antigen-encoding RNAs are based on pSTl-MITD vectors. RNA is generated by in vitro transcription and will be synthesized using 1-methyl-pseudouridine instead of uridine (FIG. 20).
  • pSTl-MITD features a signal sequence for routing to the endoplasmic reticulum and the major histocompatibility complex (MHC) class transmembrane and cytoplasmic domains for improved presentation of MHC class I and II epitopes.
  • MHC major histocompatibility complex
  • the peptides are connected by cleavable linkers or 8-12 mer non- immunogenic glycine/serine linkers in comparison with polyepitopes without linker connection.
  • mRNAs are transcribed in vitro by T7 RNA polymerase-mediated transcription according to the manufacturer’s instructions (MEGAscriptTM T7 Transcription Kit, Thermo Fisher).
  • LNP formulations are prepared using a previously described method.
  • LNPs containing mRNA are prepared by rapid mixing through microfluidics as described. Briefly, ionizable lipid, DSPC, cholesterol and DSPE-PEG are dissolved in ethanol at appropriate ratios to a final concentration of 10 mM total lipid. Nucleic acids are dissolved in 25 mM sodium acetate buffer at pH 4.0 to obtain a final mixture with a defined nucleic acid to lipid ratio. The organic and aqueous solutions are mixed at a flow ratio of 1:3 (v:v) and a total flow rate of 28 mL/min using a microfluidic mixing device available to us.
  • Preventive activity of the vaccine will also be tested by immunization before the tumor inoculation.
  • Different CRC patients may be infected with different microbes.
  • the microbiota will be identified from the fecal samples using a database of next generation sequencing (NGS).
  • NGS next generation sequencing
  • microbiome diversity and antigen prediction algorithms are used to generate tailored epitopes based on human HLA haplotypes.
  • mRNA encoding microbiota derived polyepitopes will be developed for each individual patients.
  • R-DOTAP a cationic lipid and an adjuvant, complexes with peptides to form a simple vaccine (See, Vasievich EA, et al., Cancer Immunol Immunother. 201 l;60(5):629-38).
  • the PathSeq algorithm was used to perform computational subtraction of human reads, followed by alignments of residual reads to human reference genomes / transcriptomes and microbial reference genomes (which include bacterial, viral, archaeal, and fungal sequences downloaded from NCBI in October 2015). These alignments result in taxonomical classification of reads into bacterial, viral, archaeal, and fungal sequences in whole genome sequencing (WGS) and RNA sequencing (RNASeq) data.
  • the human reference genome/transcriptome sequences are downloaded from the Ensembl database, female human reference genome, human genome plus transcriptome database and the human reference genome from NCBI.
  • Bacterial, fungal, and archaeal reference sequences are downloaded from NCBI reference sequence database. Viral and phage sequences are downloaded from NCBI nucleotide database using query strings
  • PathSeq is used to remove low quality reads followed by subtraction of human reads by mapping reads to a database of human genomes (downloaded from NCBI in November 2011) using BWA (Release 0.6.1, default settings), MegaBLAST (Release 2.2.25, cut-off E-value 10-7 , word size 16) and BLASTN (Release 2.2.25, cut-off E-value 10-7 , word size 7, nucleotide match reward 1, nucleotide mismatch score -3, gap open cost 5, gap extension cost 2). Sequences with perfect or near perfect matches to the human genome are removed in the subtraction process. In addition, low complexity and highly repetitive reads are removed using Repeat Masker5 (version open-3.3.0, libraries dated 2011-04-19).
  • Taxonomic classification is performed by residual reads alignment using MegaBLAST (Release 2.2.25, cut-off E-value 10-7, word size 16) to a database of bacterial sequences and followed by BLASTN (Release 2.2.25, cut-off E-value 10-7, word size 7) to the human reference genome and microbial reference genomes.
  • BLASTN Release 2.2.25, cut-off E-value 10-7, word size 7
  • Cancer infected by other microbes may also be treated with appropriate antibiotics encapsulated in liposomes or other carriers, depending on microbe identity. Killing of the tumor- associated bacteria may reveal the microbe-associated antigen for anti-cancer immune response similar to what is demonstrated herein for F. nucleatum and E. coli Nissle. Peptide or mRNA vaccines containing epitope(s) from these tumor-associated bacteria may be used to treat the cancer, as illustrated herein for F. nucleatum. Table 1: . Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and Mus mus cuius.
  • Table 2 Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and humans.
  • Table 1 Similar protein sequences (homologous amino acid >7) shared by F, nucleatum and Mus musculus.
  • Table 2 Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and humans.

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Abstract

The present disclosure provides methods, compositions, and systems or kits for treating cancer, promoting an anti-cancer response, and/or increasing anti-cancer immunity. Particularly, the disclosure relates to liposomal antibiotic formulations and compositions comprising one or more peptides comprising at least one tumor-associated bacterial epitope. Establishing immune responses against cancer-derived epitopes has become the mainstay of cancer immunotherapy. Unleashing T cell immunity to elicit antitumor immune responses has led to important clinical advances against cancer, including checkpoint inhibitors, cancer vaccines, and CAR-T therapies.

Description

COMPOSITIONS AND METHODS FOR INDUCING ANTICANCER IMMUNITY
FIELD
[0001] The present disclosure relates to methods, compositions, and systems or kits for treating cancer, promoting an anti-cancer response, and/or increasing anti-cancer immunity. Particularly, the disclosure relates to liposomal antibiotic formulations and compositions comprising one or more peptides comprising at least one tumor-associated bacterial epitope.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] This application claims the benefit of U.S. Provisional Application No. 63/351,641, filed June 13, 2022, the content of which is herein incorporated by reference in its entirety.
SEQUENCE LISTING STATEMENT
[0003] The contents of the electronic sequence listing titled UNC2_40943_601.xml (Size: 106,136 bytes; and Date of Creation: June 12, 2023) is herein incorporated by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0004] This invention was made with government support under Grant No. CAI 98999 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0005] Establishing immune responses against cancer-derived epitopes has become the mainstay of cancer immunotherapy. Unleashing T cell immunity to elicit antitumor immune responses has led to important clinical advances against cancer, including checkpoint inhibitors, cancer vaccines, and CAR-T therapies. Failures to achieve immunotherapy efficacy in colorectal cancer (CRC) have been attributed to both low mutation load, resulting in lack of mutation-derived neoantigens and remarkable immunosuppression of the tumor. Personalized neoantigen vaccines are limited by the rare occurrence of immunogenic mutations in tumors with low mutational burden, especially for patients with microsatellite stable (MSS) CRC tumors. MSS CRC is typically resistant to immune checkpoint blockade and innovative immunomodulating strategies are urgently needed.
SUMMARY
[0006] Disclosed herein are methods of treating cancer, promoting an anti-cancer response, or increasing anti-cancer immunity in a subject in need thereof. The methods comprise administering to the subject an effective amount of one or both of: a liposomal antibiotic formulation; and a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof. [0007] Further disclosed herein are medicaments comprising a liposomal antibiotic formulation. Additionally disclosed are vaccine or medicaments comprising a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof. Also disclosed are methods of using the vaccines and medicaments for the treatment or prevention of cancer.
[0008] In some embodiments, the antibiotic comprises a nitroimidazole antibiotic or a lincosamide antibiotic. In some embodiments, the antibiotic comprises a metal-tinidazole complex. In some embodiments, the antibiotic comprises a silver-tinidazole complex or a copper-tinidazole complex.
[0009] In some embodiments, the at least one tumor-associated bacterial epitope comprises an amino acid sequence have at least 60% similarity with sequences in subject proteome. In some embodiments, the at least one tumor-associated bacterial epitope comprises: a major histocompatibility complex (MHC) class 1 epitope without sequence similarity with subject proteome, an MHC class II epitope without at least 60% sequence similarity with subject proteome, an MHC class I epitope with at least 60% sequence similarity with subject proteome, or a combination thereof.
[0010] In some embodiments, the at least one tumor-associated bacterial epitope is determined by a method comprising: identifying tumor- associated bacteria in a sample from the subject; and selecting the epitopes from tumor associated bacteria proteome based on MHC or human leukocyte antigen (HLA) presentation. In some embodiments, the at least one tumor-associated bacterial epitope is determined by a method further comprising: aligning sequences from bacterial proteome and subject proteome; and determining bacterial epitopes with sequence similarity to subject proteome.
[0011] In some embodiments, the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-112. In some embodiments, the at least one tumor- associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-100. In some embodiments, the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 105-112.
[0012] In some embodiments, the one or more peptides further comprise a signal sequence, major histocompatibility complex (MHC) transmembrane domain, an MHC cytoplasmic domain, or any combination thereof.
[0013] In some embodiments, the one or more nucleic acids comprise mRNA. In some embodiments, the one or more nucleic acids further comprise a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof. In some embodiments, the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
[0014] In some embodiments, the one or more nucleic acids comprise DNA. In some embodiments, the one or more nucleic acids comprise circular RNA.
[0015] In some embodiments, the one or more nucleic acids further comprise at least one chemically modified nucleotide. In some embodiments, the at least one chemically modified nucleotide comprises a modified uracil, a modified cytosine, or a combination thereof.
[0016] In some embodiments, the one or more peptides are encoded on a single nucleic acid. In some embodiments, the single nucleic acid encodes two or more peptides. In some embodiments, the single nucleic acid is an mRNA.
[0017] In some embodiments, the two or more peptides are each separated by a linker. In some embodiments, the linker comprises a cleavable linker, a flexible linker, or a combination thereof. [0018] In some embodiments, the composition further comprises an adjuvant, a delivery vehicle, or a combination thereof. In some embodiments, the adjuvant, the delivery vehicle or the combination thereof comprises a lipid. In some embodiments, the lipid comprises a cationic lipid, an ionizable lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof. In some embodiments, the non-cationic lipid comprises a phospholipid. In some embodiments, the sterol comprises cholesterol or a modification or ester thereof. In some embodiments, the delivery vehicle comprises a lipid particle encapsulating the composition. In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
[0019] In some embodiments, the cancer is associated with tumor-associated bacteria. In some embodiments, the cancer comprises gastric cancer, colorectal cancer, or a combination thereof.
[0020] In some embodiments, the subject was not administered a cytotoxic treatment. In some embodiments, the liposomal antibiotic formulation and the composition are administered simultaneously, separately, or consecutively, in any order separated by a time interval. In some embodiments, the liposomal antibiotic composition is administered prior to a resection.
[0021] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description and accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIGS. 1A-1I show F. nucleatum invaded into CT26(FL3) tumor cell in response to hypoxia. FIG. 1 A is an exemplar)' illustration of F. nucleatum infection of CT26(FL3) cells in vitro. FIG. IB shows CFSE labelled F. nucleatum invaded hypoxic CT26(FL3) cells within 4 h co- incubation. Scale bar = 10 pm. FIG. 1C is quantification of F. nucleatum positive area by CFSE fluorescence, n = 5 experiments. Data are mean ± SD. ****, p<0.0001. FIG. ID is the three- dimensional re-construction of Z projection of stacked images of F. nucleatum infected CT26(FL3) cells by Imaris software. The three arrows in assorted colors indicate intracellular F. nucleatum. Scale bar = 5 pm. FIG. IE is a Z-stack image of CT26(FL3) spheroid co-incubated with F. nucleatum. F. nucleatum covalently labelled by CFSE mostly appeared in intra-spheroid. The right panel is the bright field optical image of the spheroid. Scale bar = 100 pm. FIG. IF shows CFSE labelled E. Coll Nissle invaded hypoxic CT26(FL3) cells within 4 h co-incubation. Scale bar = 10 pm. FIG. 1G is TEM Images of CRC tumor sections. Yellow arrows indicate intracellular F. nucleatum. Scale bar = 1 pm. FIG. 1H is fluorescence in situ hybridization (FISH) visualizing F. nucleatum 16s RNA in mouse CRC tumor sections. Scale bar = 20 pm. FIG. II is fitting distribution of minimum distance of F. nucleatum to cell nucleus for each pixel. F. nucleatum invaded into three-dimensional spheroids in hypoxia.
[0023] FIGS. 2A-2H show the characterization of the pH-sensitive antibiotic liposomes. FIG. 2A is an illustration of remotely loading by silver nitrate gradient and drug release in response to low pH. FIG. 2B is the H NMR characterization of AgTNZ in neutral pH and weak acid. FIG. 2C is images of LipoAg and Lipo AgTNZ by Cryo-EM. FIG. 2D is a summary of loading properties, n = 3 experiments. Data are mean ± SD. FIG. 2E shows the loading kinetics of LipoAgTNZ. n=3 experiments. Data are mean ± SD. FIG. 2F shows the pH-sensitive drug release of LipoAgTNZ in pH4.5 and pH7. Ag and TNZ were determined by ICP-MS and LC-MS, respectively. FIG. 2G shows intracellular F. nucleatum killing of free AgTNZ and LipoAgTNZ in normoxia and hypoxia, n = 3 experiments. Data are mean ± SD. ****, p<0.0001. FIG. 2H shows the in vitro cell uptake of liposome and free drug at Ih, 2h and 4h. n=3 experiments. Data are mean ± SD. *, p<0.05; ****, p<0.0001.
[0024] FIGS. 3A-3K show removing F. nucleatum in the tumor by liposomal antibiotics AgTNZ eradicated colorectal cancer. FIG. 3A is a schematic timeline of F. nucleatum infected CT26(FL3) tumor study followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i. v.). The rechallenge study was performed at Day 60 by inoculating 106 CT26(FL3) cells subcutaneously. FIG. 3B shows tumor growth monitoring by IVIS imaging at six pre-determined timepoints, n = 5 per group. Data are mean ± SD. *, p<0.05; **, p<0.01. FIG. 3C shows colony forming units of F. nucleatum in the tumors at Day 24 by plating tissues in F. nucleatum selective plates, n = 4 per group. Data are mean ± SD. **, p<0.01. FIG. 3D is animal survival curve following the dosing of timeline from day 0 to day 60. n = 7 per group. Data are mean ± SD. **, p<0.01. FIG. 3E is the growth curve of E. coll Nissle infected tumor was monitored by IVIS imaging at six pre-determined timepoints, n = 5 per group. Data are mean ± SD. ****, pcO.OOOL FIG. 3F shows colony forming units of E. coli Nissle in the tumors at Day 24 by plating tissues in F. nucleatum selective plates, n = 4 per group. Data are mean ± SD. *, p<0.1. FIG. 3G shows FISH assays visualizing F. nucleatum 16s RNA, TUNEL staining showing apoptosis. Scale bar = 20 pm. FIG. 3H is the analyses of immune cell population in tumor microenvironment after treatment, n = 5 per group. Data are mean + SD. *, p<0.05. **, p<0.01. FIG. 31 is a tumor growth curve of the rechallenge study. The survivor and control mice were rechallenged by the F. nucleatum infected or uninfected IxlO6 CT26(FL3) cells s.c. after liposomes induced long-term survival, n = 5 per group. Data are mean ± SD. *, p<0.05. FIG. 31 shows anti-CD8 and CD4 antibodies (200 pg/mouse) were given i.p. after the inoculation of rechallenged tumors as black arrows indicated, n = 5 per group. Data are mean ± SD. *, p<0.05. FIG. 3K shows alpha diversity measuring microbiome diversity of each sample, n = 4 per group.
[0025] FIGS. 4A-4J shows T cells from survivors suppressed the growth of both infected and uninfected tumors. FIG. 4A shows an in vivo T cell proliferation assay. T cells were isolated from donor splenocytes and labelled with proliferation dye CFSE before injection into recipient mice. The labelled donor T cells were collected from the primary tumor of the recipient mice and analyzed by flow cytometry. FIG. 4B is quantification of flow cytometry data in FIG. 4A of the percentage of proliferated T cells by gating the fluorescence of CFSE level, n = 4 per group. Data are mean ± SD. *, p<0.05; ***, p<0.001; ****, p<0.0001. FIG. 4C shows the quantification of data of CD8+ IFNy+ T cells after lymphocytes were isolated from survivor or naive mice and incubated with CT26(FL3) cells for 24 h in vitro, n = 4 per group. Data are mean ± SD. **, p<0.01. FIG. 4D is an illustration of the study. Mice with orthotopic CRC tumors with or without F. nucleatum infection received pan CD3+ T cells from either naive mice or long-term survivor mice from LipoAgTNZ treatment described in FIG. 2A. The donor survival mice were pulsed s.c. with 2xl06 CT26(FL3) tumor cells 2 days before their T cells were collected from the spleen. FIG. 4E is the quantification of CD3+ CD8+ T cells, data shown in FIG. 4G. n = 4 per group. Data are mean + SD. **, p<0.01. FIG. 4F is the quantification of CD3+ T cells, data also shown in FIG. 4G. n = 4 per group. Data are mean ± SD. *, p<0.05. FIG. 4G is representative flow cytometry analysis of CD3+ CD8+ T cells in tumors of the recipient mice after adoptive T cell transfer. FIG. 4H shows donor T cells inhibited tumor growth in the recipient mice. At day 10 and day 15 post inoculation, the recipients with or without F. nucleatum infection received i.v. IxlO6 T cells from naive or long- term survival mouse donors, n = 4 per group. Data are mean ± SD. *, p<0.05. FIG. 41 is confocal image of immunofluorescence of Calreticulin (red) in F. nucleatum uninfected and infected CT 26(FL3) tumor cells. The cell membrane is stained with DiO (green). Scale bar = 5 pm. FIG. 4J shows chaperone expression induced by killing intracellular bacteria treatment. CT 26(FL3) cells were infected by F. nucleatum under hypoxia condition followed by treatment with 5 pM drug.
[0026] FIGS. 5A-5F show the killing of tumor-associated bacteria promoted immune recognition of bacterial neoepitopes. FIG. 5 A is topology analysis of the proteome and proteomics of F. nucleatum based on prediction of subcellular localization. The right panel is label-free quantitative proteomics mapped with predicted subcellular localization. The selected proteins are marked with enlarged black circles (KYLSNLGIL (SEQ ID NO: 113), LPMRHFHAF (SEQ ID NO: 114), SYFEWVQNI (SEQ ID NO: 115), RPMIGMHFF (SEQ ID NO: 117), and MPIYQTSTF (SEQ ID NO: 116)). FIG. 5B is a scatter plot showing the rank for total scores and peptide-MHC complex affinity. FIG. 5C is the filters set for alignment of F. nucleatum and mouse genome to generate the homologous epitopes. FIG. 5D shows information of the selected peptides (KYLSNLGIL (SEQ ID NO: 113), LPMRHFHAF (SEQ ID NO: 114), SYFEWVQNI (SEQ ID NO: 115), MPIYQTSTF (SEQ ID NO: 116), RPMIGMHFF (SEQ ID NO: 117), GVPQIEVTF (SEQ ID NO: 53), RQATKDAGTI (SEQ ID NO: 118), and LADDNFSIV (SEQ ID NO: 119) from IEDB database. FIG. 5E is representative images of IFNy Elispot. The bacteria epitopes in FIG. 5D were incubated with bone marrow derived dendritic cells, peptides and T cells isolated from CT26(FL3) tumor bearing Balb/C mice for 24 h. FIG. 5F shows quantification of IFNy spots, n = 4 per group. *, p<0.05; **, pcO.Ol; ***, p<0.001; ns, not significant.
[0027] FIGS. 6A-6I show F. nucleatum promoted colorectal cancer (CRC) growth and fostered immune-suppressive tumor microenvironments. FIG. 6A is a schematic timeline of F. nucleatum infected CT26(FL3) tumor study, F. nucleatum colonized tumor following three oral gavages after tumor inoculation. FIG. 6B shows tumor growth monitored by IVIS imaging at five pre-determined timepoints, n = 5 per group. Data are mean ± SD. ****, p<0.0001. FIG. 6C is IVIS bioluminescence signal of luciferase assay for each mouse at Day 5, 12 and 19. FIG. 6D is the bioluminescence images of FIG. 6B. FIG. 6E shows the body weight change of F. nucleatum infected mice. Mice were inoculated with 108 CFU of F. nucleatum three times by oral gavage. n=5 per group. Data are mean ± SD. FIG. 6F shows the representative primary tumor (green dotted cycles) and metastasis (yellow dotted cycles) under photograph in situ. FIG. 6G shows the F. nucleatum distribution in the organs, tumor, and metastasis at Day 20. The homogenized tissues were plated in F. nucleatum selective agar plates, n = 4 experiments. FIG. 6H shows the analyses of immune cell population in tumor microenvironment, n = 5 per group. Data are mean ± SD. *, p<0.05; ***, p<0.001; ****, p<0.0001 by two-sided t test. FIG. 61 is representative flow cytometry analysis of CD3+ T cells, CD4+ CD44+ CD62L+ T cells, CD3+ CD8+ T cells, CDllb+ F4/80+ CD206+ macrophages, CDllc+ MHCII+ dendritic cells and CDllb+ Grl+ MDSC.
[0028] FIGS. 7A-7C show F. nucleatum colonized in the CRC tumor. FIG. 7A shows Z-stack scanning of CT26(FL3) spheroid co-incubated with F. nucleatum. F. nucleatum labelled by CSFE mostly appears in intra- spheroid layers. FIG. 7B shows Four different tumor samples stained for fluorescence in situ hybridization (FISH) using an RNA probe visualizing F. nucleatum in frozen sections. The bars indicate 20 pm. FIG. 7C is the distance map of the upright panel displaying the minimum distance between the red pixels indicating FISH signals to the blue pixels indicating DAPI staining, where the color from dark to green represents distance range from 0 to 4 pm. [0029] FIGS. 8A-8E show E. coli Nissle infected colorectal cancer and tumor microenvironments. FIG. 8A is stacking images of E. coli Nissle infected CT26(FL3) cells in hypoxia. FIG. 8B is a schematic timeline of E. coli Nissle infected CT26(FL3) tumor study, F. nucleatum colonized tumor following three oral gavages after tumor inoculation. FIG. 8C shows tumor growth monitored by IVIS imaging at five pre-determined timepoints, n = 3 per group. Data are mean ± SD. FIG. 8D is E. coli Nissle biodistribution in the organs, tumor, and metastasis at Day 19. The homogenized tissues were plated in selective agar plates, n = 3 experiments. FIG. 8E is analyses of immune cell population in tumor microenvironment, n = 3 per group. Data are mean ± SD. *, p<0.05; **, pcO.Ol; ***, pcO.OOl; n.s., not significant by two-sided t test.
[0030] FIGS. 9A-9H show the characterization of the pH-sensitive antibiotic liposomes. FIG. 9A is the chemical structure of metal-tinidazole prodrug chelate. FIG. 9B is the mass spectrum of AgTNZ. FIG. 9C is the characterization of size by nanoparticle tracking system. FIG. 9D is the characterization of zeta potential by nanoparticle tracking system. FIG. 9E is the cryo-EM images of LipoCu and LipoCuTNZ. The bar stands for 100 pm. FIG. 9F is the mass spectrum of CuTNZ. FIG. 9G is the characterization of size of LipoCuTNZ by nanoparticle tracking system. FIG. 9H is the characterization of zeta potential of LipoCuTNZ by nanoparticle tracking system. FIG. 91 is an antimicrobial assay of prodrug and parent drug, n = 3 experiments. Data are mean ± SD.
[0031] FIGS. 10A-10G show LipoAgTNZ predominantly accumulated in the tumor. FIG. 10A is the pharmacokinetics profile of LipoAgTNZ (4.0 mg/kg TNZ and 1.1 mg/kg Ag) in mice, n = 4 per group. Data are mean ± SD. FIG. 10B is the pharmacokinetics profile of free TNZ (4.0 mg/kg) in mice. n=4 per group. Data are mean + SD. FIG. 10C is the pharmacokinetic parameters of TNZ and Ag delivered as LipoAgTNZ or free TNZ. FIG. 10D is IVIS images of DiD fluorescence in major organs and tumors at 24 h after i.v. injection. FIG. 10E is fluorescence quantification of the data in FIG. 10D. n = 3 per group. Data are mean ± SD. FIG. 10F is the distribution of LipoAgTNZ in organs, tumors, and colon tissue at 24 h after i.v. injection. TNZ concentration was determined by LC-MS and Ag concentration was determined by ICP-MS. n = 4 per group. Data are mean ± SD. FIG. 10G is the distribution of TNZ (4.0 mg/kg) in organs, tumors, and colon tissue at 24 h after i.v. injection. n=4 per group. Data are mean ± SD.
[0032] FIGS. 11A-11F show LipoAgTNZ showed low toxicity in vitro and in vivo. FIG. 11A is an MTT assay of prodrug and parent drugs, n = 5 experiments. Data are mean ± SD. FIG. 1 IB is an MTT assay in which CT26(FL3) cells were co-incubated with F. nucleatum in hypoxic incubator for 4 h, followed by incubation with LipoAgTNZ at different concentrations for 24 h. n = 5. FIG.
11C is a graph showing changes in body weight. Three doses of liposomes (4.0 mg/kg TNZ and 1.1 mg/kg Ag) were given to mice on Day 12, 16 and 20. n = 5 per group. Data are mean + SD. FIG. 11D is hematoxylin and eosin staining of morphology of major organs after the therapeutic dosing in paraffin sections. Yellow arrows indicate metastatic lesions. Scale bar = 20 pm. FIG. 1 IE is analysis of complete blood count in peripheral blood of mice. n=4 per group. Data are mean ± SD. *, p<0.05; **, p<0.01; ns, not significant. FIG. 11F shows biomarkers indicating liver and kidney functions after liposome treatment in naive mice. There was no statistically significant difference among the biomarkers in the three groups, n = 3 per group. Data are mean ± SD.
[0033] FIGS. 12A-12E show Liposomal AgTNZ eradicated F. nucleatum infected CRC tumor. The orthotopic F. nucleatum infected CRC model is described in FIG. 3 A. FIG. 12A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 18, 33 and 42. FIG. 12B shows CT26(FL3) orthotopic tumor growth with F. nucleatum infection. FIG. 12C shows tumor growth curve. Polymyxin B were given to mice (3.0 mg per mouse, p.o.) at day 15, 18 and 21 following tumor inoculations, n = 5 per group. Data are mean ± SD. *, p<0.05; **, p<0.01, ns, not significant. FIG. 12D shows oral polymyxin B decreased viable F. nucleatum in the feces. FIG. 12E is a luciferase assay indicating tumor growth. The mice were inoculated with tumor without F. nucleatum infection, following three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.) at Day 12, 16 and 20. n = 4 per group. Data are mean ± SD. ns, not significant. The orthotopic CRC model is described in FIG. 3A but without F. nucleatum infection.
[0034] FIGS. 13A and 13B show LipoAgTNZ inhibited tumor growth and induced long-term survival in F. nucleatum infected MC38 tumors orthotopically inoculated in C57BL/6 mice. FIG. 13A is a schematic timeline of F. nucleatum infected MC38 tumor study followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.). The bottom panel shows the images of orthotopic CRC tumor. One mouse was euthanized every 7 days for tumor growth monitoring. FIG. 13B is an animal survival curve following the dosing of timeline (FIG. 13 A) from day 0 to day 90. n = 7 per group. Data are mean ± SD. *, p<0.05; **, p<0.01.
[0035] FIGS. 14A-14G show removing F. nucleatum in the tumor by LipoAgTNZ improved immune surveillance of the tumor. FIG. 14A is immunofluorescence staining of CD8+ T cells in the CRC tumor frozen sections. Scale bar = 20 pm. FIG. 14B is representative flow cytometry analysis of CD3+ T cells, CD4+ CD44+ CD62L+ T cells and CD3+ CD8+ T cells. FIG. 14C is immunofluorescence staining of monocytes and macrophages (F4/80) in the CRC tumor frozen sections. Scale bar = 20 pm. FIG. 14D is representative flow cytometry analysis of CDllb+ F4/80+ CD206+ macrophages in the tumor. FIG. 14E is immunofluorescence staining of blood vessels (CD31) and lymphatic vessels (Lyve-1) in the F. nucleatum infected tumors with or without LipoAgTNZ treatment. Scale bar = 20 pm. FIG. 14F is representative images of the rechallenged and primary tumors at the endpoint of anti-CD8 and CD4 study. The upper panel shows the rechallenged subcutaneous tumors of the survivor mice (blue dotted cycles) grew when CD8+ or CD4+ T cells were compromised. The bottom panel shows the primary tumor inoculated on the cecum (yellow dotted rectangles) were already cured at Day 75 after inoculation. FIG. 14G is relative RNA expression of the CRC tumors by RT-qPCR. n = 3 per group. Data are mean ± SD. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not significant.
[0036] FIGS. 15A-15G show LipoAgTNZ inhibited the tumor growth of F. nucleatum. infected liver metastasis. FIG. 15 A is a scheme of F. nucleatum infected liver metastasis model. The CT26(FL3) cells were pre-infected by F. nucleatum and inoculated to the liver by hemi-splenic injection, followed by three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.). FIG. 15B is a tumor growth curve of CT26(FL3) liver metastasis. n=4 per group. Data are mean ± SD. *, p<0.05. FIG. 15C is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 4, 10 and 15. FIG. 15D is the tumor growth in liver metastasis in each mouse with F. nucleatum infection. FIG. 15E is bioluminescence images of luciferase signals of the representative dissected tumors at day 16. n=4 per group. Data are mean ± SD. *, p<0.05; ****, p<0.0001. FIG. 15F is quantification of the ex vivo luciferase imaging. Scale bar = 20 pm. FIG. 15G is FISH imaging visualizing F. nucleatum 16s rRNA, immunofluorescent staining of CD206+ macrophages and CD8+ T cells in the frozen sections of liver containing metastasis.
[0037] FIGS. 16A-16C show T cell from survivor suppress both infected and uninfected tumor. Lymphocytes were isolated from survivor or naive mice and incubated with CT26(FL3) cells for 24 h in vitro. FIG. 16A is representative flow cytometry data of CD8+IFNy+ T cells after incubation. Tumor growth after adoptive T cell transfer. The adoptive T cell transfer is illustrated in FIG. 5H. FIG. 16B is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 3, 9, 15 and 23. FIG. 16C is CT26(FL3) orthotopic colorectal tumor growth in each mouse with or without F. nucleatum infection with T cells adoptively transferred from naive or survivor mice. [0038] FIG. 17 is a schematic of an exemplary overall approach for epitope discovery and vaccine development.
[0039] FIGS. 18A-18D show bacterial epitope prediction and in vivo confirmation of MHC-I restricted T cells. FIG. 18A shows epitope sequences selected from F. nucleatum for antigen presentation by MHC-I of C57BL6 mice. FIG. 18B is tetramer staining carried out by using flow cytometry. FIG. 18C is quantification of tetramer positive, CD8+ T cells in tumor infiltrating T cells. *p=0.05, **p=0.0I, ***p=0.001, ****p=0.0001, n=3. FIG. 18D is representative images of IFNy Elispot assay. The bacteria epitopes in FIG. 18A were incubated with bone marrow derived dendritic cells, peptides and T cells isolated from MC38 tumor bearing C57BL/6J mice for 24 h. For all of FIGS. 18A-18D, SVFVFYPADF (SEQ ID NO: 108), ISLPFITM (SEQ ID NO: 109), AAIQGGVLM (SEQ ID NO: 110), LADDNFSTI (SEQ ID NO: 105), RGVPQIEVTF (SEQ ID NO: 106), VITAFSNI (SEQ ID NO: 111), and SVFVFYPADFTF (SEQ ID NO: 112).
[0040] FIGS. 19A and 19B show bacteria-derived peptides inhibited tumor growth and induced long-term survival in F. nucleatum infected MC38 tumors orthotopically inoculated in C57BL/6 mice. FIG. 19A is a schematic timeline of F. nucleatum infected MC38 tumor study followed by two doses of DOTAP-peptide treatment. FIG. 19B is animal survival curves following the dosing of timeline from day 0 to day 75 (SVFVFYPADFTF (SEQ ID NO: 112) and RGVPQIEVTF (SEQ ID NO: 106)). n = 5 per group. Data are mean ± SD. *, p<0.05; **, p<0.01.
[0041] FIG. 20 is a schematic of a poly -bacterial-epitope RNA for cancer vaccine. The MHC class I and MHC class II restricted bacterial epitopes without sequence similarity to host proteome are designed to induce cytotoxic T cell responses and helper T cell responses respectively towards neoantigens from cancer microbiota. The MHC class I epitopes with sequence overlapped with host proteome are designed to induce cross reactive anticancer effect.
[0042] FIG. 21 is a schematic of the filters set for alignment of F. nucleatum and human genome to generate the homologous epitopes.
[0043] FIGS. 22A-22C show that liposomal AgTNZ eradicated F. nucleatum infected CT26 CRC tumor. FIG. 22A is images of F. nucleatum infected CT26 tumors and treatments as shown in FIG. 3A. Images of the primary tumor (green dotted cycles) and metastasis (yellow dotted cycles) at Day 25. n = 5 per group. FIG. 22B is a graph of the number of metastases of FIG. 22A. FIG. 22C is a graph of the tumor weight including primary site and metastases of (E). *, p<0.05; **, p<0.01. [0044] FIGS. 23A-23C show that targeting F. nucleatum in the tumor by liposomal antibiotics AgTNZ eradicated colorectal cancer. FIG. 23A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 14, 23 and 33. F. nucleatum infected CT26FL3(Luc/RFP) tumors were established following the schedule shown in FIG. 3A. Three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.) in combination with anti-CD8 and CD4 antibodies (200 pg/mouse, i.p.). The antibiotics cocktails consisting of Ampicillin (1 g/L), clindamycin (0.5 g/L), and metronidazole (1 g/L) were added in the drinking water from Day 12 to Day 21. FIG. 23B is a graph of tumor growth from FIG. 23A at seven pre-determined timepoints, n = 5 per group. Data are mean ± SD. *, p<0.05. FIG. 23C is a schematic timeline of the F. nucleatum infected CT26FL3(Luc/RFP) tumor study followed by three doses of LipoAgTNZ treatment or antibiotic cocktails in the drinking water from Day 12 to Day 21.
[0045] FIGS. 24A and 24B show the characterization of the pH-sensitive antibiotic liposomes. FIG. 24A is a graph showing the release of LipoAgTNZ in PBS buffer at pH 7. FIG. 24B is graphs of the size, zeta, and PDI of LipoAgTNZ dispersed in 5% glucose solution and PBS buffer at pH 7. [0046] FIGS. 25A-25C show that removing F. nucleatum in the tumor by liposomal antibiotics AgTNZ eradicated colorectal cancer. FIG. 25A is IVIS images of bioluminescence signal of luciferase assay for each mouse at Day 6, 14, 23 and 33. F. nucleatum infected CT26FL3(Luc/RFP) tumors were established following Fig 4A. Three doses of LipoAgTNZ treatment (4.0 mg/kg TNZ and 1.1 mg/kg Ag, i.v.) in combination with anti-CD8 and CD4 antibodies (200 pg/mouse, i.p.). The antibiotics cocktails consisting of Ampicillin (1 g/L), clindamycin (0.5 g/L), and metronidazole (1 g/L) were dissolved in drinking water from Day 12 to Day 20 consecutively. FIGS. 25B is a graph of tumor growth of FIG. 25A at seven pre-determined timepoints, n = 5 per group. Data are mean ± SD. *, p<0.05. FIG. 25C is a schematic timeline of the F. nucleatum infected CT26FL3(Luc/RFP) tumor study followed by three doses of LipoAgTNZ treatment in combination with anti-CD8 or anti-CD4 antibody.
DETAILED DESCRIPTION
[0047] Disclosed herein are methods, compositions, and systems or kits for treating cancer, promoting an anti-cancer response, and/or increasing anti-cancer immunity. Pre-resection liposomal antibiotic formulations targeting anaerobic bacteria significantly improved the disease- free survival rate of colorectal cancer (CRC) patients by 25%. Silver-tinidazole complexes encapsulated in liposomes (LipoAgTNZ) eliminated tumor-associated bacteria in the primary tumor and liver metastasis without causing gut microbiome dysbiosis. A murine CRC model colonized by tumor-promoting bacteria (Fusobacierium nucleatum spp), or probiotics (Escherichia coli Nissle spp) responded to LipoAgTNZ, enabling > 70% long-term survival in two F. nucleatum infected CRC models. Heterologous and homologous bacterial epitopes contributed to immunogenicity, by which T cells were primed to recognize both the infected and uninfected tumors. Provided herein are compositions, including vaccine and medicaments, comprising heterologous and homologous bacterial epitopes and methods of use thereof.
[0048] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.
1. Definitions
[0049] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. [0050] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
[0051] A “chemotherapeutic” or “chemotherapeutic agent” as used herein, refers to a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy. Examples of chemotherapeutic agents include, but are not limited to: cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, carboplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, everolimus, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, erlotinib, toremifene, fulvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, trastuzumab, temozolomide, rapamycin, and tucatinib.
[0052] As used herein, “nucleic acid” or “nucleic acid sequence” refers to a polymer or oligomer of pyrimidine and/or purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively (See Albert L. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)). The present technology contemplates any deoxyribonucleotide, ribonucleotide, or peptide nucleic acid component, and any chemical variants thereof, such as methylated, hydroxymethylated, or glycosylated forms of these bases, and the like. The polymers or oligomers may be heterogenous or homogenous in composition and may be isolated from naturally occurring sources or may be artificially or synthetically produced. In addition, the nucleic acids may be DNA or RNA, or a mixture thereof, and may exist permanently or transitionally in single-stranded or double- stranded form, including homoduplex, heteroduplex, and hybrid states. In some embodiments, a nucleic acid or nucleic acid sequence comprises other kinds of nucleic acid structures such as, for instance, a DNA/RNA helix, peptide nucleic acid (PNA), morpholino nucleic acid (see, e.g., Braasch and Corey, Biochemistry, 41(14): 4503-4510 (2002)) and U.S. Pat. No. 5,034,506), locked nucleic acid (LNA; see Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 97: 5633-5638 (2000)), cyclohexenyl nucleic acids (see Wang, J. Am. Chem. Soc., 122: 8595-8602 (2000)), and/or a ribozyme. Hence, the term “nucleic acid” or “nucleic acid sequence” may also encompass a chain comprising non-natural nucleotides, modified nucleotides, and/or non- nucleotide building blocks that can exhibit the same function as natural nucleotides (e.g., “nucleotide analogs”); further, the term “nucleic acid sequence” as used herein refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single or double-stranded, and represent the sense or antisense strand. The terms “nucleic acid,” “polynucleotide,” “nucleotide sequence,” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
[0053] A “peptide” or “polypeptide” is a linked sequence of two or more amino acids linked by peptide bonds. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms “polypeptide” and “protein” are used interchangeably herein.
[0054] The recitations “sequence identity,” “percent identity,” “percent homology,” “percent similarity,” or, for example, comprising a “sequence 50% identical to” or “sequence with at least 50% similarity to,” as used herein, refer to the extent that sequences are identical on a nucleotide- by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (e.g., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
[0055] Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In certain embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
[0056] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. [0057] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some embodiments, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, (1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package, using an NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Another exemplary set of parameters includes a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller (1989, Cabios, 4: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
[0058] The peptide sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
[0059] As used herein, the terms “providing,” “administering,” and “introducing” are used interchangeably herein and refer to the placement of the nanocomplexes and/or compositions of the present disclosure into a subject by a method or route which results in at least partial localization to a desired site.
[0060] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse model as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non- human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non- human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non- mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.
[0061] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.
[0062] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terns shall include pluralities and plural terms shall include the singular.
[0063] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
2. Methods
[0064] The present disclosure provides methods for treating cancer, promoting an anti-cancer response, or increasing anti-cancer immunity in a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
[0065] The methods disclosed herein are suitable for any cancer type or subtype. In some embodiments, the cancer may be a carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In certain embodiments, the cancer is associated with tumor- associated bacteria e.g., gastric (Helicobacter pylori) and colorectal (Fusobacterium nucleatum and Bacteroides fragilis) cancer. In some embodiments, the cancer comprises gastric cancer, colorectal cancer, or a combination thereof. [0066] In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the methods result in decreased tumor growth. In some embodiments, the methods result in tumor regression. In some embodiments, the methods result in decreased numbers of tumor. In some embodiments, the methods prevent tumor recurrence. In some embodiments, the methods result in increases in overall subject survival.
[0067] The methods include administering to the subject an effective amount of one or both of: a liposomal antibiotic formulation; and a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
[0068] An “effective amount” is an amount that is delivered to a subject, either in a single dose or as part of a series, which is effective for inducing a response in the subject. This amount varies depending upon the health and physical condition of the subject to be treated, the respective formulations of the compositions and antibiotics, the relative stage of the cancer, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined by one of skill in the art through routine trials.
[0069] The compositions, polypeptides, polynucleotides (e.g., mRNA), vaccines, or medicaments disclosed herein can be administered in a wide variety of therapeutic dosage forms. The route and regimen of administration will vary depending upon the population and is to be determined by the skilled practitioner. For example, the formulations, compositions, vaccines, or medicaments disclosed herein may be administered parentally, e.g., intravenous (either by bolus or infusion methods), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form.
[0070] The administration may comprise an initial immunization or dose and at least one subsequent immunization or booster dose, following known standard immunization protocols. The boosting doses will be adequately spaced at such times where the levels of circulating antibody fall below a desired level. Boosting doses may consist of the compositions or vaccines disclosed herein and may comprise alternative carriers and/or adjuvants. The booster dosage levels may be the same or different that those of the initial immunization dosage.
[0071] In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof. In some embodiments, administration includes parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections). In some embodiments, the administration is intratumoral or adjacent to the site of cancer.
[0072] The specific dose level may depend upon a variety of factors including the activity of the polynucleotides (e.g., mRNA), polypeptides, compositions, formulations, vaccine, or medicament, the age, body weight, general health, and diet of the subject, time of administration, and route of administration. For prophylaxis purposes, the amount in each dose is an amount which induces an immunoprotective response without significant adverse side effects.
[0073] A wide range of second therapies may be used in conjunction with the polynucleotides, peptides, formulations, compositions, vaccines, medicaments, and methods of the present disclosure. The second therapy may be administration of a therapeutic agent or may be a second therapy not connected to administration of another agent. Such second therapies include, but are not limited to, surgery, immunotherapy, radiotherapy, or a chemotherapeutic or anti-cancer agent. [0074] The second therapy (e.g., an immunotherapy) may be administered at the same time as the initial therapy. The second therapy may be administered in the same composition or in a separate composition administered at substantially the same time as the initial therapy. In some embodiments, the second therapy may precede or follow administration of the disclosed polynucleotides, peptides, formulations, compositions, vaccines, medicaments, and methods by time intervals ranging from hours to months. In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, cytokine therapy, immunomodulators, or administration of antibodies (e.g., monoclonal antibodies). In some embodiments, the subject was not administered a cytotoxic treatment (e.g., a chemotherapeutic or radiation) prior to the disclosed methods.
[0075] In select embodiments, the methods further comprise full or partial resection of a tumor or cancerous tissue or organ. In some embodiments, the liposomal antibiotic composition and/or the composition (e.g., one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof) is administered prior to the resection. In some embodiments, the liposomal antibiotic composition and/or the composition (e.g., one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof) is administered prior to and following the resection.
[0076] The liposomal antibiotic formulation and the composition may be administered simultaneously, separately, or consecutively, in any order separated by a time interval (e.g., minutes, hours, days, or weeks). The liposomal antibiotic formulation and the composition may be administered at the same time using different methods of administration or the same method of administration. 3. Liposomal Antibiotic Formulation
[0077] Disclosed herein are liposomal antibiotic formulations, e.g., liposomal antibiotic formulations suitable for use in the present methods.
[0078] In some embodiments, the antibiotic targets anaerobic bacteria. In some embodiments, the antibiotic comprises a nitroimidazole antibiotic, a lincosamide antibiotic, or a combination thereof.
[0079] In some embodiments, the antibiotic comprises a nitroimidazole antibiotic. Nitroimidazole antibiotics include those that have a nitro group appended to an imidazole ring. Nitroimidazole antibiotics include, but are not limited to, metronidazole, tinidazole, nimorazole, dimetridazole, pretomanid, omidazole, megazol, azanidazole, and benznidazole.
[0080] In some embodiments, the antibiotic comprises a metal-tinidazole complex. Metal- tinidazole complexes include, but are not limited to, cobalt-tinidazole, copper-tinidazole, zinc- tinidazole, and silver-tinidazole. In select embodiments, the antibiotic comprises a copper- tinidazole complex. In select embodiments, the antibiotic comprises a silver-tinidazole complex. [0081] In some embodiments, the antibiotic comprises a lincosamide antibiotic. Lincosamides consist of a pyrrolidine ring linked to a pyranose moiety (methylthio-lincos amide) via an amide bond. Lincosamide antibiotics include, but are not limited to, lincomycin and clindamycin.
[0082] In some embodiments, the disclosed compounds are incorporated into liposomal compositions comprising one or more vesicle forming lipids. Methods of making liposomal compositions include, for example, lipid film hydration, optionally coupled with sonication or extrusion, solvent evaporation (e.g., ethanol injection, ether injection, or reverse phase evaporation) or detergent removal methods. The disclosed compounds can be combined with the lipid(s) before formation of the vesicles (passive loading) or after vesicle formation (active loading). The liposome compositions may prolong circulation time in vivo, increase stability of the compound, and prevent degradation in the bloodstream. The liposomal composition may increase the distribution of the compounds within the lung, breast, pancreas, and spleen.
[0083] Any naturally occurring or synthetic vesicle forming lipid or combinations thereof can be used, examples of which are provided elsewhere herein. The one or more vesicle forming lipids may be selected from di-aliphatic chain lipids, such as phospholipids; diglycerides; di-aliphatic glycolipids; single lipids such as sphingomyelin or glycosphingolipid; steroidal lipids; hydrophilic polymer derivatized lipids; or mixtures thereof.
[0084] The liposomes may contain other non-vesicle forming lipids or other moieties, including but not limited to amphiphilic polymers, polyanions, sterols, and surfactants. The liposomes contained in the liposome composition can also be targeting liposomes, e.g., liposomes containing one or more targeting moieties or biodistribution modifiers on the surface of the liposomes. A targeting moiety can be any agent that is capable of specifically binding or interacting with a desired target and are generally known in the art, for example ligands such as folic acid, proteins, antibody or antibody fragments, and the like).
[0085] The liposomes can have any liposome structure, e.g., structures having an inner space sequestered from the outer medium by one or more lipid bilayers, or any microcapsule that has a semi-permeable membrane with a lipophilic central part where the membrane sequesters an interior. In some embodiments, the liposome may be a unilamellar liposome, having a single lipid layer. The disclosed compounds may be completely or partially located in the interior space of the liposome or completely or partially within the bilayer membrane of the liposome. In some embodiments, the lipids for a micelle.
[0086] In some embodiments, the liposomal antibiotic formulations are pH-sensitive. pH- sensitive or pH-responsive liposomes can be employed to effect site-specific release of their cargo (e.g., antibiotics or antibiotic formulations). For example, pH changes can trigger changes in permeability of the liposomal membrane by protonation/deprotonation of functional groups that induce morphological changes of the lipid bilayers. In general, pH-responsive liposomes are formulated using a natural phospholipid component that is sensitive to pH changes that may be encountered as they traffic to or are internalized by target cells. Additionally, inclusion of negatively charged moieties into pH-sensitive liposome compositions is a common strategy for destabilizing the liposome structure through the phase conversion, to enable rapid release of the cargo. The liposomal antibiotic formulations may comprise pH sensitive lipids including, but are not limited to phosphatidylethanolamine, palmitoylhomocysteine or analogs or derivatives thereof, such as dioleoylphosphatidylethanolamine, and/or pH sensitive polymers including a pH sensitive moiety, such as, but not limited to alklyacrylic acid, ethylacrylic acid, propylacrylic acid, butylacrylic acid, and methylacrylic acid.
4. Tumor Associated Bacterial Epitopes
[0087] Also disclosed herein are composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof. Thus, the composition may comprise a single peptide comprising one or more tumor-associated bacterial epitopes, multiple peptides (e.g., two or more) each comprising a tumor-associated bacterial epitope, multiple peptides (e.g., two or more) each comprising multiple (e.g., two or more) tumor- associated bacterial epitopes, or a combination thereof, or one or more nucleic acids encoding any of those listed. [0088] In some embodiments, multiple epitopes may be arranged with a linker between the epitopes or directly to one another without a linker between the epitopes in a single peptide. In some embodiments, the two or more epitopes are each separated by a linker. The linker is preferably a polypeptide linker of 1-20 amino acids. The linker may comprise any amino acid sequence. The linker may be flexible such that it does not constrain the epitopes. The linkers may essentially act as a spacer. In some embodiments, the linker is glycine-rich. The linker may comprise glycine-serine polymers, glycine-alanine polymers, alanine- serine polymers and/or other flexible linkers known in the art.
[0089] The linker may be a cleavable linker. Thus, in some embodiments, the linker may comprise an amino acid sequence configured to be cleaved enzymatically or chemically (e.g., at certain pHs, reduction/oxidation conditions or presence of reducing/oxidizing agents, temperatures, etc.). In some embodiments, the cleavable linker comprises a self-cleaving peptide-based cleavage site.
[0090] In some embodiments, the at least one bacterial epitope comprises: a major histocompatibility complex (MHC) class I epitope without sequence similarly with subject proteome, an MHC class II epitope without sequence similarly with subject proteome, an MHC class I epitope with sequence similarly with subject proteome, or a combination thereof. In some embodiments, the at least one bacterial epitope comprises an amino acid sequence have at least 60% similarity (e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or more similarity) with sequences in subject proteome (e.g., the cancer cell genome).
[0091] In some embodiments, the epitopes are neoepitopes. A neo-epitope is a new (or neo) epitope (e.g., antigenic determinant) that has not been previously recognized by the immune system. Neo-epitopes can encompass epitopes on a neoantigen, which is a new antigen.
[0092] In some embodiments, the at least one bacterial epitope is determined by a method comprising identifying tumor- associated bacteria in a sample from the subject and selecting epitopes from the tumor-associated bacteria based on MHC or human leukocyte antigen (HLA) presentation. In some embodiments, the epitopes do not have sequence similarity with a peptide from the subject.
[0093] The sample may be obtained from any suitable source, such as, a physiological fluid including, but not limited to, whole blood, serum, plasma, interstitial fluid, saliva, ocular lens fluid, cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, synovial fluid, peritoneal fluid, vaginal fluid, menses, amniotic fluid, semen, feces, and the like. In some embodiments, the sample is blood or blood products. Blood products are any therapeutic substance prepared from human blood. This includes whole blood; blood components (e.g., red blood cell concentrates or suspensions; platelets produced from whole blood or via apheresis; plasma; serum and cryoprecipitate); and plasma derivatives (e.g., coagulation factor concentrates). In some embodiments, the sample from the subject may comprise a tumor sample, a tissue sample, a blood sample, or any sample that comprises tumor-associated bacteria or tumor-associated bacterial nucleic acids. In select embodiments, the sample is a fecal sample.
[0094] In some embodiments, the methods further comprise aligning sequences from bacterial proteome and subject proteome and determining bacterial epitopes with sequence similarity to subject proteome. In some embodiments, the epitopes have sequence similarity with a peptide from the subject.
[0095] Accordingly, the present invention relates to methods for identifying and/or detecting epitopes. Specifically, the invention provides methods of identifying and/or detecting tumor specific epitopes that are useful for increasing an anti-cancer immune response in a subject. Some of these epitopes are capable of activating anti-tumor CD8 T-cells, e.g., bind to MHC class I polypeptides. Others bind to MHC class II polypeptides and activate CD4+ T helper cells.
[0096] Using computer algorithms, it is possible to predict potential epitopes or peptide sequences, which are bound by the MHC molecules of class I or class II in the form of a peptide- presenting complex and then, in this form, recognized by the T-cell receptors of T-lymphocytes. Examples of programs useful for identifying peptides which will bind to MHC include for instance: Lonza Epibase, SYFPEITHI (Rammensee et al, Immunogenetics, 50 (1999), 213-219) HLA_BIND (Parker et al., J. Immunol., 152 (1994), 163-175), IEDB database (tools.iedb.org/mhci/). Once putative epitopes are selected, they can be further tested using in vitro and/or in vivo assays.
[0097] In some embodiments, the epitope comprises a sequence selected from those shown in Tables 1-4, or a sequence comprising one, two, or three substitutions in reference to a sequence from Tables 1-4. In some embodiments, the epitope comprises a sequence selected from any of SEQ ID NOs: 1-112. In some embodiments, the epitope comprises a sequence comprising one, two, or three substitutions in reference to any of SEQ ID NOs: 1-112.
[0098] In some embodiments, the epitope comprises a sequence selected from any of SEQ ID NOs: 43, 46, and 51-100. In some embodiments, the epitope comprises a sequence comprising one, two, or three substitutions in reference to any of SEQ ID NOs: 43, 46, and 51-100.
[0099] In select embodiments, the epitope comprises a sequence selected from: IINEPTAA (SEQ ID NO: 101), PQIEVTF (SEQ ID NO: 102), RQRVWGV (SEQ ID NO: 103), DIDANGI (SEQ ID NO: 104), GAAIQGGVL (SEQ ID NO: 40), QDPLAELVKI (SEQ ID NO: 45), YTHFTSPIR (SEQ ID NO: 50), IDANGIVHV (SEQ ID NO: 54), NTYGTGCFL (SEQ ID NO: 55), KQRVAIARA (SEQ ID NO: 57), DAFGTAHRA (SEQ ID NO: 63), SQRQATKDA (SEQ ID NO: 65), NAGGVTVSY (SEQ ID NO: 67), GLDPAVYEL (SEQ ID NO: 74), YLAEGFYKA (SEQ ID NO: 75), KLGGKVVAV (SEQ ID NO: 76), KLVEIIAGL (SEQ ID NO: 77), ILFEIVRKI (SEQ ID NO: 78), ILITKTHNL (SEQ ID NO: 79), AISSTLWTV (SEQ ID NO: 80), GIYAEGIASV (SEQ ID NO: 80), RnEITIPV (SEQ ID NO: 82), ILIPMINEA (SEQ ID NO: 83), SQLKAIAAV (SEQ ID NO: 84), ALYEDRESI (SEQ ID NO: 85), KMKGEFPLV (SEQ ID NO: 86), AQTEGFTVV (SEQ ID NO: 87), YVKRNIPEV (SEQ ID NO: 88), FTFEELEAA (SEQ ID NO: 89), GLNDGASFL (SEQ ID NO: 91), FLMPQGRAA (SEQ ID NO: 92), TIPEVVTSI (SEQ ID NO: 93), VLYKKGIVV (SEQ ID NO: 94), GLKDMTGAV (SEQ ID NO: 95), HLDYVIAAV (SEQ ID NO: 96), KLQQLIDAV (SEQ ID NO: 97), YLQERVEQC (SEQ ID NO: 98) and YSQEHLDYV (SEQ ID NO: 99).
[00100] In some embodiments, the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 105-112.
[00101] An amino acid “substitution” or “replacement” refers to the replacement of one amino acid at a given position or residue by another amino acid at the same position or residue within a polypeptide sequence. Amino acids are broadly grouped as “aromatic” or “aliphatic.” An aromatic amino acid includes an aromatic ring. Examples of “aromatic” amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non- aromatic amino acids are broadly grouped as “aliphatic.” Examples of “aliphatic” amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or He), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).
[00102] The amino acid replacement or substitution can be conservative, semi-conservative, or non-conservative. The phrase “conservative amino acid substitution” or “conservative mutation” refers to the replacement of one amino acid by another amino acid with a common property. A functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz and Schirmer, supra).
[00103] Examples of conservative amino acid substitutions include substitutions of amino acids within the sub-groups described above, for example, lysine for arginine and vice versa such that a positive charge may be maintained, glutamic acid for aspartic acid and vice versa such that a negative charge may be maintained, serine for threonine such that a free -OH can be maintained, and glutamine for asparagine such that a free -NH2 can be maintained. “Semi-conservative mutations” include amino acid substitutions of amino acids within the same groups listed above, but not within the same sub-group. For example, the substitution of aspartic acid for asparagine, or asparagine for lysine, involves amino acids within the same group, but different sub-groups. “Non- conservative mutations” involve amino acid substitutions between different groups, for example, lysine for tryptophan, or phenylalanine for serine, etc.
[00104] The one or more nucleic acids may be DNA, RNA, or a combination thereof. In some embodiments, the one or more nucleic acids are DNA. In some embodiments, the one or more nucleic acids are RNA. In some embodiments, the one or more nucleic acids are circular RNA. In some embodiments, the one or more nucleic acids are mRNA.
[00105] In some embodiments, the one or more nucleic acids are optimized for enhanced expression, productive co-translational folding, increased stability, or a combination thereof. [00106] In some embodiments, the one or more peptides are encoded on a single nucleic acid. In some embodiments a single nucleic acid encodes two or more peptides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, etc.). Thus, in some embodiments the composition can comprise one or more polycistronic mRNAs. “Polycistronic” refers to an mRNA encoding more than peptide.
[00107] In some embodiments, the one or more nucleic acids or mRNA are codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide.
[00108] The one or more nucleic acids may further comprise or encode other structures and sequences necessary for proper functionality or stability, including but not limited to a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, a signal sequence, major histocompatibility complex (MHC) class transmembrane and cytoplasmic domain(s), or any combination thereof. Thus, in some embodiments, the disclosure provides a mRNA comprising: a 5’ UTR, an open reading frame encoding the disclosed peptides, and a 3’ UTR.
[00109] The one or more nucleic acids may comprise a 5’ and 3’ untranslated region (UTR). Sequence elements within the UTRs affect translational efficiency and RNA stability but do not encode a polypeptide. The 3 ’ UTR relates to the region located at the 3 ’ end of the mRNA, downstream of the termination codon of a protein-encoding region, which is transcribed but not translated into an amino acid sequence. The 5’UTR refers to the region directly upstream from the initiation codon. Eukaryotic 5’ UTRs contain the Kozak consensus sequence (ACCAUGG), which contains the initiation codon. The eukaryotic 5’ UTR may also contain As- acting regulatory elements called upstream open reading frames (uORFs) and upstream AUGs (uAUGs) and termination codons, which can impact translation regulation. In some embodiments, the 5’ UTR, 3’ UTR, or both is heterologous to the sequence or open read frame encoding the disclosed peptide. [00110] 5’ -cap refers to the structure found on the 5 ’ -end of an mRNA which ordinarily consists of a guanosine nucleotide connected to the mRNA via a 5 ’ to 5 ’ triphosphate linkage. The guanosine may be methylated at the 7-position creating a 7-methylguanosine cap (m7G). In some embodiments, the 5’ cap may be a naturally occurring 5’ cap. In some embodiments, the 5’ cap is a 5’ cap analog or a modified 5’ cap structure which is non-naturally occurring (e.g., phosphorothioate-cap-analogs).
[00111] The one or more nucleic acids may comprise a 3’ tailing sequence. The 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
[00112] The one or more nucleic acids may encode a signal sequence. Signal sequences are generally about 16-30 amino acids (aa) in length, at the N-terminus of a recently synthesized protein that directs the sequence to the secretory pathway (e.g., for routing to the endoplasmic reticulum). The signal sequence may be N- or C-terminal to the one or more peptides.
[00113] The one or more nucleic acids may further encode transmembrane and cytoplasmic domains of an MHC polypeptide. The presence of these domains in the peptide facilitates improved presentation of the epitopes, for example by improving intracellular signaling and trafficking to the plasma membrane. In some embodiments, the transmembrane and cytoplasmic domains are from an MHC class II polypeptide. In some embodiments, the transmembrane and cytoplasmic domains are from an MHC class I polypeptide.
[00114] In some embodiments, the one or more nucleic acids further comprises a triple helix forming sequence. A tiple helix is formed after the binding of a third strand to the major groove of a duplex nucleic acid through Hoogsteen base pairing (e.g., hydrogen bonds) while maintaining the duplex structure of two strands making the major groove. Pyrimidine-rich and purine-rich sequences (e.g., two pyrimidine tracts and one purine tract or vice versa) can form stable triplex structures as a consequence of the formation of triplets (e.g., A-U-A and C-G-C). In some embodiments, the triple helix sequence is derived from the 3’ terminal triple helix sequences of triple helix terminators from a long non-coding RNAs (IncRNAs), e.g., metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). [00115] In some embodiments, the one or more nucleic acids further comprises a tRNA-like sequence. The tRNA-like sequences are those sequences which form similar overall secondary and tertiary structure to tRNA. In some embodiments, the tRNA-like sequence is derived from a long non-coding RNAs (IncRNAs), e.g., MALAT1. The tRNA-like sequence derived from IncRNAs may be truncated or modified as long as they retain the clover secondary structure.
[00116] As the MALAT1 sequences are highly conserved evolutionarily, the MALAT1 sequences for the triple helix or the tRNA-like sequence can be from any species. In one embodiment, the MALAT1 sequences are from a human. In another embodiment, the M AL ATI sequences are from a mouse. In another embodiment, the MALAT1 sequences are from a non- human primate.
[00117] The 3’ poly(A) sequence of mRNA is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA). The poly (A) tail is a segment of RNA at the 3’ end of the molecule that has only adenine bases. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. The poly(A) tail may contain two segments of only adenine bases separated by a linker.
[00118] In some embodiments, the one or more nucleic acids comprise at least one chemical modification or chemically modified base, nucleoside, or nucleotide. The chemical modifications may comprise any modification which is not naturally present in said RNA or any naturally- occurring modification of adenosine (A), guanosine (G), uridine (U), or cytidine (C) ribonucleosides. For example, a single nucleic acid or mRNA may include both naturally-occurring and non-naturally-occurring modifications.
[00119] Chemical modifications may be located in any portion of the nucleic acid or mRNA molecule and the nucleic acid or mRNA molecule may contain any percentage of modified nucleosides (1-100%). In some embodiments, every particular base or nucleoside may be modified (e.g., every uridine is a modified uridine). In some embodiments, at least 80% of any single nucleotide (e.g., uracil) in the of the nucleic acid or mRNA is chemically modified. In some embodiments, a particular modification is used for every particular type of nucleoside or base (e.g., every uridine is modified to a 1-methyl-pseudouridine). Exemplary RNA modifications can be found in the RNA modification database (See mods.ma.albany.edu/home).
[00120] In some embodiments, the at least one chemical modification comprises a modified uridine residue. Exemplary modified uridine residues include, but are not limited to, pseudouridine, 1 -methylpseudouridine, 1 -ethylpseudouridine, 2-thiouridine, 4’- thiouridine, 5-methyluridine, 2- thio-1 -methyl- 1-deaza-pseudouridine, 2- thio-l-methyl-pseudouridine, 2-thio-5 -aza-uridine, 2-thio- dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5- aza- uridine, dihydropseudouridine, 5 -methoxy uridine and 2’-0-methyl uridine.
[00121] In some embodiments, the at least one chemical modification comprises a modified cytosine residue. Exemplary nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-cytidine, 5-formyl-cytidine, N4-methyl- cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5-hydroxymethyl-cytidine, 1-methyl- pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl- cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5 -aza-2-thio- zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5 -methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine, a-thio- cytidine, 2’-O-methyl-cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’ -O-methyl-cytidine, N4,2’- O-dimethyl-cytidine, 5-formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1-thio- cytidine, 2’-F-aracytidine, 2’-F-cytidine, and 2’-OH-aracytidine.
[00122] In some embodiments, the at least one chemical modification comprises a modified adenine residue. Exemplary nucleosides having a modified adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, 2-amino-6-methyl-purine, 8-azido- adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2- amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl- adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl- adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl-adenosine, N6- threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6- threonylcarbamoyl-adenosine, N6,N6-dimethyl-adenosine, N6-hydroxynoryalylcarbamoyl- adenosine, 2-methylthio-N6-hydroxynoryalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl- adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl-adenosine, N6,2’-O-dimethyl-adenosine, N6,N6,2’-O-trimethyl-adenosine, l,2’-O-dimethyl-adenosine, 2’-O- ribosyladenosine (phosphate), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’- F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)- adenosine.
[00123] In some embodiments, the at least one chemical modification comprises a modified guanine residue. Exemplary nucleosides having a modified guanine include inosine, 1-methyl- inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxy wybutosine, hydroxy wybutosine, undermodified hydroxy wybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-cyano-7-deaza- guanosine, 7-aminomethyl-7-deaza- guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio- 7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine, N2- methyl - guanosine, N2,N2-dimethyl-guanosine, N2,7-dimethyl-guanosine, N2,N2,7-dimethyl-guanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2’-O-methyl-guanosine, N2- methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O-methyl-guanosine, l-methyl-2’-O-methyl- guanosine, N2,7-dimethyl-2’-O-methyl-guanosine, 2’-O-methyl-inosine, l,2’-O-dimethyl-inosine, and 2’-O-ribosylguanosine (phosphate).
[00124] The disclosure also provides polynucleotide segments encoding the one or more nucleic acids, vectors containing these segments, and cells containing the vectors. The vectors may be used to propagate the segment in an appropriate cell and/or to allow expression from the segment (e.g., an expression vector). The person of ordinary skill in the art would be aware of the various vectors available for propagation and expression of a nucleic acid sequence.
[00125] In some embodiments, the composition further comprises an adjuvant or a polynucleotide encoding thereof, a delivery vehicle, or a combination thereof.
[00126] Adjuvants are compounds or compositions that either directly or indirectly stimulate the immune system’s response to a co- administered antigen. Suitable adjuvants are commercially available as, for example, Glucopyranosyl Lipid Adjuvant (GLA); Pam3CSK4; Freund’s Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham); mineral salts (for example, aluminum, silica, kaolin, and carbon); aluminum salts such as aluminum hydroxide gel (alum), A1K(SO4)2, AlNa(SO4)2, A1NH4(SO4), and A1(OH)3; salts of calcium (e.g., Ca3(PO4)2), iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polynucleotides (for example, poly IC, poly AU acids, and CpG oligodeoxynucleotides (e.g., Class A or B)); polyphosphazenes; cyanoacrylates; polymerase- (DL-lactide-co- glycoside); bovine serum albumin; diphtheria toxoid; tetanus toxoid; edestin; keyhole-limpet hemocyanin; Pseudomonal Toxin A; choleragenoid; cholera toxin; pertussis toxin; viral proteins; Quil A; aminoalkyl glucosamine phosphate compounds. In addition, adjuvants such as cytokines (e.g., GM-CSF or interleukin-2, -7, or -12), interferons, or tumor necrosis factor, may also be used as adjuvants. Protein and polypeptide adjuvants may be obtained from natural or recombinant sources according to methods well known to those skilled in the art. When obtained from recombinant sources, the adjuvant may comprise a protein fragment comprising at least the immunostimulatory portion of the molecule. [00127] Other known immunostimulatory macromolecules which can be used include, but are not limited to, polysaccharides, tRNA, non-metabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid, polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4’, 4-diaminodiphenylmethane-3, 3’ -dicarboxylic acid and 4-nitro-2- aminobenzoic acid (See, Sela, M., Science 166: 1365-1374 (1969)) or glycolipids, lipids, or carbohydrates.
[00128] In some embodiments, the adjuvantive polypeptide comprises immune activator proteins, such as CD70, CD40 ligand, and constitutively active TLR4, or polycationic peptides (e.g., protamine). In some embodiments, the adjuvantive polypeptide is a flagellin polypeptide. Commercially available mRNA encoding adjuvantive polypeptides are available, for example, as TriMix (See Bonehill, A. et al. Mol. Ther. 16, 1170-1180 (2008), incorporated herein by reference).
[00129] In some embodiments, wherein the adjuvant, the delivery vehicle or the combination thereof comprises a lipid. The lipid may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, polyethylene glycol (PEG)-lipid conjugates, and/or sterols. In some embodiments, the lipid comprises a cationic lipid and/or ionizable lipid, a neutral or non-cationic lipid, and cholesterol.
[00130] Cationic and/or ionizable lipids include, for example, amine-containing lipids that can be readily protonated and may have a positive or partial positive charge at physiological pH due to a pKa value between pH 5 and 8. This class includes lipid molecules which remain neutral at physiological pH, but are protonated at low pH, making them positively charged. The polar headgroup of the cationic lipids preferably comprises amine derivatives such as primary, secondary, and/or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and/or imidazole groups as well as pyridinium, piperazine and amino acid headgroups such as lysine, arginine, ornithine and/or tryptophan. Cationic lipids include, but are not limited to, l,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O- octadecenyl-3 -trimethylammonium propane (DOTMA) and/or l,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl (dimethyl)ammonium bromide (DDAB), l,2-dioleyloxypropyl-3-dimethyl -hydroxyethyl ammonium bromide (DORIE), 3[T[N-(N\N’-dimethylamino-ethane)carbamoyl]cholesterol (DC- Chol) or dioleyl ether phosphatidylcholine (DOEPC). Ionizable lipids include, but are not limited to, l,2-dioleyloxy-3 -dimethylamino-propane (DODMA). In some embodiments, the cationic lipid is DOTAP or R-DOTAP. [00131] The sterol may comprise cholesterol, fecosterol, ergosterol, campesterol, sitosterol, stigmasterol, brassicasterol or a sterol ester, such as cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol.
[00132] A neutral or non-cationic lipid may include one or more phospholipids. Phospholipids include a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety may include, but is not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. A fatty acid moiety may include, but is not limited to, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.
[00133] Phospholipids suitable for use in the compositions may include, but are not limited to, phosphatidylglycerol (PG) including dimyristoyl phosphatidylglycerol (DMPG) and 1,2-dioleoyl- sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG); phosphatidylcholine (PC), including egg yolk phosphatidylcholine, dimyristoyl phosphatidylcholine (DMPC), 1 ,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC), 1 ,2-dilinoleoyl-sn-glycero-3 -phosphocholine (DLPC), 1,2-dioleoyl-sn- glycero-3 -phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1- oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl- sn- glycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3- phosphocholine; phosphatidylethanolamine (PE) including l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-diphytanoyl-sn-glycero-3 -phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn- glycero-3 -phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine; phosphatidic acid (PA); phosphatidylinositol (PI); phosphatidylserine (PS); and sphingomyelin (SM). In some embodiments, the delivery vehicle comprises a lipid particle encapsulation the composition. Lipid particle compositions of the disclosure may include one or more cationic and/or ionizable lipids, phospholipids, neutral or non-cationic lipids, and/or sterols, as described. In some embodiments, the lipid particle comprises a cationic lipid and/or ionizable lipid, a neutral or non- cationic lipid, and cholesterol.
[00134] In some embodiments, the lipid particle further comprises a polyethylene glycol (PEG)- lipid conjugate. A PEG-lipid conjugate may include, but is not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG- modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-DMG (l,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol), PEG-c-DOMG (R-3-[(cu-methoxy poly(ethylene glycol)2000)carbamoyl)]-l ,2-dimyristyloxlpropyl-3 -amine), PEG-DMA (PEG-dimethacrylate), PEG-DLPE (l,2-didodecanoyl-sn-glycero-3-phosphoethanolamine-PEG), PEG-DMPE (PEG- 1,2- dimyristoyl-sn-glycero-3 -phosphoethanolamine), PEG-DPPC (PEG-dipalmitoyl phosphatidylcholine), PEG-N,N-di(tetradecyl)acetamide, or a PEG-DSPE (1, 2-distearoyl-sn- glycero-3 -phosphoethanolamine-poly (ethylene glycol)) lipid. In some embodiments, the lipid nanoparticle comprises PEG-DMG and/or PEG-N,N-di(tetradecyl)acetamide.
[00135] The lipid particles may also include other components typically used in the formation of vesicles (e.g., for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and/or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly.
[00136] The compositions may further comprise excipients or pharmaceutically acceptable carriers. The choice of excipients or pharmaceutically acceptable carriers will depend on factors including, but not limited to, the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. The compositions of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Techniques and formulations may be found, for example, in Remington’s Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
[00137] The compositions may be formulated for any particular mode of administration including for example, systemic administration (e.g., oral, rectal, nasal, sublingual, buccal, implants, or parenteral).
5. Vaccines or Medicaments
[00138] The compositions and formulations described herein may be used to prepare vaccines or another medicament.
[00139] The vaccines or medicaments of the present disclosure may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic and antigenic portions of polypeptides or polynucleotides encoding immunogenic polypeptides, or nucleic acid(s) encoding thereof, may be present within the vaccines or medicaments. The vaccines or medicaments may generally be used for prophylactic and therapeutic purposes. [00140] The vaccines or medicaments may be formulated for any appropriate manner of administration, and thus administered, including for example, oral, nasal, intravenous, intravaginal, epicutaneous, sublingual, intracranial, intradermal, intraperitoneal, intratumoral, subcutaneous, intramuscular administration, or via inhalation.
[00141] The vaccines or medicaments may also comprise buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives. Alternatively, vaccines or medicaments may be formulated as a lyophilizate.
[00142] The compositions, vaccines, or medicaments may be prepared, packaged, or sold in a form suitable for bolus administration or sold in unit dosage forms, such as in ampules or multi- dose containers. In some embodiments, the compositions and vaccines contain a preservative.
6. Systems or Kits
[00143] Also disclosed herein is a system or kit comprising the compositions, formulations, nucleic acids, vaccines, or medicaments disclosed herein.
[00144] The kits can also comprise other agents and/or products co-packaged, co-formulated, and/or co-delivered with other components. The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kit. The materials may include any combination of the following: background information, list of components, brief or detailed protocols for using the compositions, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
[00145] It is understood that the disclosed kits can be employed in connection with the disclosed methods. The kit may further contain containers or devices for use with the methods or compositions disclosed herein.
[00146] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Individual member components of the kits may be physically packaged together or separately.
7. Examples
Materials and Methods [00147] Clinical Data Sources The clinical study was performed by analyzing the French Cancer Cohort from the cancer institute data platform of the French National Cancer Institute (INCa). This prospective cohort is an extraction from the National Health Data System (SNDS) and includes all people diagnosed, treated, or followed up for a cancer in France since 2010. Briefly, health insurance funds were requested by French legislature to develop a national health insurance information system, which encompasses the whole population, in order to more precisely determine and evaluate health care utilization and the health care expenditure of beneficiaries. This dataset includes all drug consumption related to hospitalization care or outpatient care. The French cancer cohort protocol was approved by a national committee (Comite Consultatif sur le Traitement de T Information en Matiere de Recherche dans le Domaine de la Sante) and authorized by the French Data Protection Agency (Commission nationale de 1’informatique et des libertes — Cnil, number 2019-082). Confidentiality in the cohort is guaranteed for all participants with regards to any personal information, as all data are pseudonymized.
[00148] Selection Criteria of the Clinical Cohort The specific methodology regarding the colorectal cohort and antibiotic consumption has been previously reported. In brief, this study included all people aged 18 or older with incident non-metastatic colorectal cancer resected between January 2012 and December 2014 in France. To select incident cases, we excluded people with a previous history of cancer (2010-2011) or long-term disease for cancer (diagnosed before 2012). To obtain a homogeneous population with the same study duration, each patient was followed up for 3 years after surgical resection. The 3-DFS is a validated criterion for adjuvant studies and an accepted surrogate for overall survival in colorectal cancer.
[00149] Measurement of Antibiotic Therapy in the Clinical Cohort All reimbursements for antibiotics were extracted during the perioperative period of 6 months before the surgical resection and until 1 year after. It was assumed that each pill delivered was taken by the patient, the daily delivery dose of each antibiotic was used to convert the quantity reimbursed in number of days of treatment. Because drugs dispensed during a hospitalization were not directly traceable in the Cancer Cohort, consumption of antibiotics in hospital has been evaluated by the presence of a diagnostic code (ICD-10) for infectious diseases. Hospital infections occurring after surgery were split into those occurring within 30 days and those occurring more than 30 days after surgery to isolate post-operative infections.
[00150] For outpatient consumption of antibiotics targeting anaerobe, a list of antibiotics approved for the treatment of Fusobacterium sp. related infection using Anatomical Therapeutic Chemical codes was predefined as following: 1) nitroimidazoles and 2) lincosamides.
[00151] Covariates Used in the Multivariate Model Several potentially predictive factors for survival were identified or reconstructed from the available data: sex, age (18-49, 50-69, 70-79, > 80 years), laterality of cancer Charlson comorbidity index (none, 1-2, >3), nutritional status during the surgical resection (malnutrition vs. no malnutrition), admission to intensive care unit during the surgical resection length of stay (no admission or <2 days, 2-7 days, >7 days), time when antibiotics were used along the time axis to account for a potential time-dependent bias.
[00152] Identification of Recurrences in the Clinical Cohort Recurrences were identified by an algorithm looking for either: the occurrence of an ICD-10 diagnostic code for metastases, a palliative care code, a new cancer-related surgical resection after the first one, treatment with chemotherapy or radiotherapy starting more than 3 months after the surgical resection.
[00153] Statistical Analysis for Clinical Data 3-DFS was defined as the time elapsed between the date of surgery (time origin) and the date of recurrence, death, or the end of 3-year follow-up, whichever occurred first. The 3-DFS has been modelled using Cox models to study the link with antibiotics intake adjusted for all covariates.
[00154] The survival curves were represented using the Kaplan-Meier method and compared with the log-rank test. Hazard ratios (HR) were estimated by Cox proportional regression models. As this study was conducted in the overall population (not a sample) statistical tests for descriptive comparisons were not considered relevant. Antibiotic exposure before TO was considered a fixed variable. Antibiotic exposure after TO was considered a time-dependent variable. A multivariate model was carried out to isolate the effect of antibiotics targeting anaerobe by comparing patients that received only these antibiotics to those that didn’t receive any antibiotics as outpatients.
[00155] Cell Lines The metastatic CT26(FL3) cells were kindly provided by Dr. Maria Marjorette O. Pena at the University of South Carolina. CT26(FL3) cells stably expressing red fluorescent protein (RFP) and luciferase (Luc) were established by transfection with lentivirus vectors carrying RFP and Luc gene and a puromycin resistance gene. CT26(FL3)-RFP/Luc cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L glucose (Gibco) and 10% bovine calf serum (BCS, HyClone) supplemented with 1 pg/mL puromycin (ThermoFisher) and 1% antibiotic- antimycotic (Gibco) at 37°C and 5% CO2 in a humidified atmosphere. MC38 cells was purchased from Kerafast, Inc., which was cultured in DMEM with 10% BCS, 0.1 mM nonessential amino acids and 1% antibiotic- antimycotic.
[00156] Bacteria Culture, Inoculation and CFU Quantification Fusobacterium nucleatum subsp. Nucleatum (ATCC®25586™) (F. nucleatum) was purchased from the American Type Culture Collection (ATCC). F. nucleatum was grown in chopped meat medium with carbohydrates (Anaerobe Systems) overnight in 37 °C in anaerobic Hungate tubes. Bacteria were pelleted anaerobically by centrifugation at 4,000 rpm for 10 min at room temperature and were washed once in sterile phosphate buffered saline (PBS) before use. [00157] For in vitro quantification of F. nucleatum, samples were serially diluted, drop-plated (10 pL) on Brucella blood agar plates (Anaerobe Systems), incubated in anaerobic chamber (Coy Laboratory Products) at 37 °C for 24-48 h and enumerated for colony forming units (CFUs). For in vivo studies, tissues and tumors samples were homogenized twice (MP Biomedical FastPrep-24) in 40% (vol/vol) glycerol solution at 12 M/s for 20s using 1.4 mm ceramic bead-filled tubes (Fisher). Viable CFUs were enumerated on Fusobacterium selective agar plates (Anaerobe Systems) and data was normalized to the mass of tissue plated.
[00158] E. coli Nissle 1917 was provided by Dr. Aaron Anselmo lab with integrated a plasmid conferring kanamycin resistance was used for tumor colonization study in mouse. E. coli Nissle 1917 was grown overnight to saturated conditions in kanamycin (50 pg/mL) supplemented LB broth, washed to remove media. Serial dilutions of the E. coli Nissle were plated on selective kanamycin (50 pg/mL) plates. Colony forming units were enumerated after 24 h of growth at 37 °C.
[00159] Animal Models All animal studies were reviewed and approved by the University of North Carolina at Chapel Hill’s Institutional Animal Care and Use Committee. Six-to-eight- week- old female BALB/cJ and C57BL/6J mice were obtained from the Jackson Laboratories. The investigator was blinded to the group allocation during the animal experiments.
[00160] The orthotopic and subcutaneous tumor model was established based on the previously work. Briefly, female BALB/cJ or C57BL/6J mice were anesthetized with 2.5% isoflurane in oxygen at supine position. A midline incision was made to exteriorize the cecum. CT26(FL3)- RFP/Luc and MC38 cells at density of 2.0xl06 in 50 pL mixture of PBS and Matrigel (1:1) were injected into the cecum wall. The cecum was returned to the peritoneal cavity before the incision was sutured. The tumor burden was monitored by bioluminescent analysis using an IVIS imager (Perkin Elmer) with i.p. injection of 100 pL of D-luciferin (Perkin Elmer, 10 mg/ml). Each mouse was inoculated with 108 CFU of F. nucleatum or E. coli Nissle in 100 pL PBS every fifth or forth day by oral gavage. Mice inoculated with tumor and bacteria were randomized blindly into different treatment groups.
[00161] The subcutaneous tumor model was established by injection of LOxlO6 CT26(FL3)- RFP/Luc cells in 100 pL PBS into the right flank of the BALB/cJ mice. CT26(FL3)-RFP/Luc was infected with F. nucleatum prior to inoculation. Tumor volume (Vt) was calculated as following:
Vt = 0.5 X a X b2 (1)
[00162] Tn Eq. (1 ), a and b are defined as the major and minor diameter of the tumor.
[00163] CT26(FL3)-RFP/Luc was infected with F. nucleatum prior to inoculation. Female BALB/cJ mice were anesthetized with 2.5 % isoflurane in oxygen at supine position. An incision was made to exteriorize the spleen below the left rib cage. The spleen was tied and cut into two parts that contain intact vascular pedicle for each half. The distal section of the spleen was inoculated with 1.0 x 106 CT26(FL3) cells in 150 pL PBS. The hemi-spleen containing inoculated cells was resected 5 min after inoculation allowing the cancer cells to enter the liver through the portal vein. The other half of the spleen was returned to the peritoneal cavity and the incision was sutured. The tumor burden was monitored by bioluminescent analysis using an IVIS imager (Perkin Elmer) with i.p. injection of 100 pL of D-luciferin (Perkin Elmer, 10 mg/mL). For ex vivo imaging, the dissected liver with tumor was quickly rinsed in PBS and placed in diluted luciferin solution (1 mg/mL) for 1 min. bioluminescence imaging was applied immediately.
[00164] In Vitro Cell Infection Five mL of overnight F. nucleatum or E. coli Nissle culture was harvested, washed in sterile PBS two times by centrifuge at 4,000 rpm for 10 min in an anaerobic chamber. The optical density was adjusted to 1.3xl09 CFU/mL. For intracellular bacteria imaging, the bacteria were then stained with 5 pM carboxyfluorescein succinimidyl ester (CFSE) (Invitrogen) according to the manufacturer's instructions. The culture medium of CT26(FL3) cells was changed to DMEM supplemented with 10% BCS without antibiotics before bacterial infection. The cells were then incubated with the stained or unstained bacteria at a multiplicity of infection (MOI) of 20 in a hypoxic incubator for 4 h, followed by three washes in PBS. The cells were harvested for inoculation. For intracellular bacteria imaging, the cells were staining with phalloidin, and antibodies as described below. Antibodies were incubated at 4°C overnight.
[00165] Immunofluorescence Staining Dissected tumors were placed in 4% paraformaldehyde (PF A) for 48 h at 4 °C, and then transferred to 30% sucrose solution for dehydration. The tumor was embedded in Tissue-Plus O.C.T. Compound (Fisher Healthcare), and frozen tissues were sectioned on a cryostat microtome (Hacker-Bright, OTF5000). In in vitro experiments, cells were fixed with 4% PFA before immunofluorescence staining. Immunofluorescence staining was performed by rinsing with PBS, permeabilizing with 0.1% Triton X-100 (Sigma- Aldrich) in PBS and blocking in 10% goat serum (Life Technologies) at room temperature for 1 h. Antibodies were incubated at 4°C overnight. Finally, the slides were stained with DAPI and mounted with Prolong® Diamond Antifade Mountant (ThermoFisher Scientific). Images were taken using a confocal laser scanning microscopy (CLSM, Zeiss 700 or CLSM, ZeisS610).
[00166] Tumor Cell Spheroids The CT26(FL3) suspensions were mixed with 0.48% (w/v) methylcellulose. Then drops of cell suspensions were placed onto the lids of 96-well plates, and the lids were inverted over the plates containing sterile deionized water to prevent the droplets from evaporating overnight. The resultant cellular aggregates on the lids were harvested using a pipet and transferred to round-bottom low attachment 96-well plates (Coming, costar® 7007) by centrifugation (1,000g, 3 min). Then the medium was replaced every 48-h using a standard culture medium up to 96 h. After that, cell spheroids were incubated with CFSE-labelled F. nucleatum in hypoxic incubator for 12 h before fixation and DAPI staining.
[00167] Flow Cytometry Freshly harvested tumor tissues were digested into single cell suspensions in PBS with collagenase II (200 U/ml, Invitrogen), DNAase (100 pg/mL, Invitrogen) and 2 mM EDTA (Ultrapure™, Invitrogen) at 37 °C for 60 min. For T cell population analysis, lymphocytes were purified by Ficoll-Paque™ (GE Healthcare) density gradient centrifugation. After that, cells were collected and diluted to IxlO6 cells/mL with FACS buffer (PBS containing 2% Bovine Calf Serum and 2 mM EDTA). One mL of the cell suspension was centrifuged and stained at 4 °C for 30 min by the addition of a cocktail of fluorophore conjugated antibodies. For intracellular staining, cells were permeabilized with Cytofix/Cytoperm buffer (BD Biosciences), and then stained with intracellular antibodies at 4 °C for 20 min. Cells were fixed with 4% PFA and analyzed by flow cytometer (BD LSR II or LSRFortessa).
[00168] Nuclear Magnetic Resonance Nuclear Magnetic Resonance and Mass Spectroscopy. 1 H NMR spectra were recorded on a 400MR Spectrometer instrument equipped with a 400 MHz magnetic field. The proton signals were acquired in DMSO-d6 solution.
[00169] Liposome Preparation Distearoyl phosphatidylcholine (16.0 mg, Avanti), cholesterol (10.0 mg, Avanti) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (5.0 mg, Avanti) were dissolved in chloroform. The solvent was evaporated to form a lipid thin film. Three mL of 300 mM silver nitrate solution (adjusted to pH 3.0 with nitric acid) was added to hydrate the film at 65 °C for 15 min, and then the suspension was sequentially extruded through 400 nm, 200 nm, 100 nm membrane for 10 times, respectively. Gel filtration (Sephadex G-50) was used to remove the silver ions from the outer aqueous phase of the liposomes and to replace it with 5% sucrose with EDTA (Ultrapure™, pH 8.0, 1:25 dilution by sucrose solution) and then with 5% glucose without EDTA. The liposomes were incubated with tinidazole (0.4 mg) at 65 °C for 15 min for remote loading. The gel filtration was repeated to remove the unencapsulated tinidazole. For analysis, the drug-loaded liposomes were dissolved in methanol and measured by LC-MS and ICP-MS for tinidazole and Ag+, respectively. Liposomal encapsulation efficiency was calculated as the weight of tinidazole entrapped in liposomes versus weight of tinidazole totally added. Liposomal loading efficiency was calculated as the weight of tinidazole entrapped in liposomes versus weight of gross materials added.
[00170] Transmission Electron Microscopy The tumors (5x5 mm2) from the infected mice were collected and washed with PBS. The samples were continuously fixed in 2.5% glutaraldehyde plus 2.5% paraformaldehyde in 0.1M PBS buffer and 4% osmium tetroxide solution and transferred into a Beem embedding capsule with micro-embedding resin added to the embedding samples. The obtained blocks were further sectioned with ultramicrotomy to obtain sections with thicknesses of ~70 nm. These sections were retrieved via a copper grid and stained with 1% lead citrate and 4% uranyl acetate separately, and then visualized under a JEOL 1230 TEM electron microscope operated at 100 kV accelerating voltage. Images were taken under 12,000x magnification.
[00171] Cryo-Electron Microscopy Cryo-EM grids were prepared with a Vitrobot Mark (Thermo Fisher Scientific) set to room temperature and 95% humidity. Quantifoil Rl.2/1.3 copper 300 mesh grids were rendered hydrophilic by glow-discharging for 30 seconds at 15 mA with a Pelco easiGlow. Five p L of sample were applied directly to the carbon side of the TEM grid then blotted with Whatman 595 filter paper for 3 sec with the blot force set to -10. Cryo-EM grids were imaged with a Thermo Fisher Scientific 200 kV TEM Tales Arctica G3 equipped with a Gatan K3 direct electron detector. The microscope was aligned, and beam was set parallel at spot size 3 with the 70 pm condenser and 100 pm objective apertures.
[00172] Cellular Uptake Cells were seeded in 6-well plates overnight. Free drug and liposomes were added for the incubation of 1 h, 2h and 4h. At determined timepoints, the cells were washed three times by cold PBS, removed from the bottom of plates by a scraper and dissolved in DI water. TNZ was extracted by liquid-liquid extraction partition described in LC-MS section. The intracellular drug concentration was detected by LC-MS.
[00173] Drug Release Analysis Drug-loaded liposomes (500 pL) were enclosed into 8-10 kD dialysis devices (Float-A-Lyzer®G2 G235031, Spectra/Por®) floating in 30 mL 5% glucose with or without pH adjusted to 4.5 by nitric acid in 50 mL centrifuge tubes. The tubes were place in a shaker at 100 rpm, 37 °C. Fifty pL of outer medium at 5, 13, 30, 60, 120, 240, 480, 960, 1920 min was sampled and analyzed by LC-MS and ICP-MS.
[00174] Liposome Characterization The particle zeta potential and size were determined by NTA using NanoSight 500 Version 2.2 (Wiltshire, UK). For the size measurements, liposomes were dispersed in 5% glucose.
[00175] Loading Kinetics LipoAg, Ag in external water phase of which was removed, was used for loading kinetics study. Four mL of LipoAg was equilibrated at 65 °C. Tinidazole was added with constant stirring and 500 pL of was extracted at 5 min, 10 min, 20 min and 30 min. The samples were centrifuged by Amicon® Ultra-0.5 Insert Vial (MilliporeSigma Filtration). The bottom layers without liposomes were detected for tinidazole concentration by LC-MS as the unentrapped drug.
[00176] Antimicrobial Assay Brucella broth (Anaerobe Systems) was used to dilute F. nucleatum culture to 105 CFU/mL for antimicrobial assay. One hundred pL of antimicrobial agent (0.1, 0.5, 1, 2, 4 and 8 pM dissolved in Brucella broth) was two-fold diluted by 100 pL F. nucleatum culture in 96-well microtitration plates. After mixing, the plate was incubated under normoxia (20% oxygen) or hypoxia (1% oxygen) condition for 12 h. For quantification of F. nucleatum, samples were serially diluted, drop-plated (10 pL) on Brucella blood agar plates, incubated in an anaerobic chamber (Coy Laboratory Products) at 37 °C for 24-48 h and enumerated for colony forming units (CFUs).
[00177] For intracellular CFU quantification, the culture medium of CT26(FL3) cells was changed to DMEM supplemented without serum and antibiotics before F. nucleatum infection. The cells were then incubated with F. nucleatum at a multiplicity of infection (MOI) of 20 in a hypoxic incubator for 4 h. A serial dilution of liposomes was added and incubated for 12 h in DMEM with 10% BCS. The extracellular bacteria were removed by gentamicin (50 pg/mL) in DMEM with 10% BCS for 2 h. After three washes in PBS, cells were scraped and lysed in sterile water before CFU plating.
[00178] Liquid Chromatography - Mass Spectrometry (LC-MS) The whole blood concentration during 12 h after administration was determined by LC-MS (Shimadzu LCMS-2020). TNZ was extracted by liquid-liquid extraction partition. The ethyl acetate (500 pL) was added to the 20 pL blood sample. After vortex for 3 min and centrifugation for 10 min (3,500 rpm), the organic layer was separated and evaporated at 35 °C under vacuum. The residue was dissolved in 20 pL of methanol containing 0.1% formic acid followed by vortex-mixing and centrifugation (13,000 rpm, 10 min). The supernatant was analyzed by LC-MS. A Leapsil C18 column (100 mm x 2.1 mm, 2.7 pm) was used, while mobile phase was composed of acetonitrile and water premixed with 0.1% formic acid. An LC-MS analysis in positive and negative full scan modes combined with SIM channels (m/z 247.85) was applied to monitor the deacylation process. Gradient elution program began at 70% water, and then changed linearly to 50% water (0-2.5 min), then to 5% water (2.5-4.0 min), and at last changed to the initial conditions (4.0-5.0 min) for 1 min. The injection volume was 20 pL.
[00179] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Ag+ concentrations in the dispersions were determined by ICP-MS (NexION 300D, PerkinElmer). Prior to the analysis, each sample aliquots (25 pL) were added to 5 mL 5% nitric acid and digested by microwave to release the silver cations. The sample solution was cooled to room temperature and then centrifuged at 4,000 rpm for 10 min before analysis.
[00180] Image Deconvolution and Reconstruction The noise components of the stacking images are reduced by deconvolution process, which is applied by blind deconvolution algorithms using Autoquant X3.1 software (Media Cybernetics). The subsequent computational images were reconstructed by Imaris 3D reconstruction software (Bitplane Scientific Software) for analyzing the spatial distribution of intracellular bacteria.
[00181] Toxicity Study After treatment, mice were anesthetized and the whole blood was collected from the ocular vein using anticoagulant tube (MiniCollect®, Greiner Bio-One) and directly assayed for complete blood count. Serum was obtained after centrifuge at 10,000 g at 4 °C for 10 min. Creatinine (CRE), blood urea nitrogen (BUN), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were assayed as biomarkers of renal and liver functions. Blood tests were performed at the LCCC Animal Histopathology Core at UNC. To evaluate the organ- specific toxicity and spontaneous metastasis, haematoxylin and eosin staining was performed at the Animal Histopathology Core at UNC. Organs including heart, liver, spleen, lung, kidney, and tumor were collected and fixed in 4% PFA for haematoxylin and eosin staining. [00182] Reverse Transcription PCR Total RNA was extracted from the tissues using the RNeasy Mini Kit (Qiagen). cDNA was reverse-transcribed for each sample using the iScript™ cDNA Synthesis Kit (BIO-RAD). 500 ng of cDNA was amplified with the TaqManTM Gene Expression Master Mix with addition of primers. GAPDH was used as the endogenous control. Reactions were conducted using the 7500 Real-Time PCR System.
[00183] Pharmacokinetics and Biodistribution The concentration of liposomes in the blood were measured by sampling blood at different time (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h). Blood was taken from mice retro-orbi tally at predetermined time points. All the major organs including the tumor were harvested, homogenized, and measured for metal ion and TNZ contents in the tissues for biodistribution of the liposomes. Liposomes were also be labelled with DiD (1%), a lipid soluble fluorescence dye that labels the lipid membrane of the liposomes. At 24 h post injection, the BALB/c mice bearing orthotopic tumor were sacrificed and all major organs including the tumor were harvested and measured for DiD distribution. Pharmacokinetics data were evaluated using DAS 2.0 software.
[00184] Fluorescence In Situ Hybridization (FISH) F. nucleatum in orthotopic colorectal cancer tissues was determined using RNA in situ hybridization. F. nucleatum 16s RNA oligo probe was synthesized by GenScript. The sequence of the F. nucleatum-targeted RNA probe was 5’- CUUGUAGUUCCGCrYUACCUC/3’(rY is a mix of C/U; SEQ ID NO: 120) with CY5 label at 3’ end, which was based on a previously reported F. nucleatum-targeted probe (S-G-Fuso-0664-a-A- 19, probebase.csb.univie.ac.at/pb_report/probe/1346). Stellaris® RNA FISH buffers (Stellaris® RNA FISH Wash Buffer A, Stellaris® RNA FISH Wash Buffer B and Stellaris® RNA FISH Hybridization Buffer) were used according to Stellaris® RNA FISH protocol for frozen tissue. Briefly, the fresh dissected tumors were quickly embedded in Tissue-Plus O.C.T. Compound and stored in -80 °C. The froze tumors were sliced at thickness of 10 pm, fixed with 4% paraformaldehyde and permeabilized with 70% ethanol. The slices were subsequentially immersed in Wash Buffer A for 5 min, dispensed with 200 pL hybridization buffer containing probe solution of 125 nM followed by incubation in dark at 37 °C for 16 h. The slides were then incubated with Wash Buffer A in dark for 30 min, stained by 5 pg/mL DAPI in Wash Buffer A and immersed with Wash Buffer B for 5 min, and mounted with Prolong® Diamond Antifade Mountant (ThermoFisher Scientific). The uninfected tumors were stained with the probes as negative controls to verify that FISH assay was specifically detecting F. nucleatum.
[00185] FISH Quantification The distance map indicating minimum distance between FISH signals and DAPI staining was calculated by Euclidean distance function,
Figure imgf000041_0001
where flF and flD are the image domain of FISH signals and DAPI staining, respectively, x/y denotes the position of each pixel in the two-dimensional coordinate system. For each x of FISH signals, its minimum distance wase computed from position of DAPI staining by Eq. (2). x and d(x) were then fitted with polynomial fourth order curve to better reflect a correlation of distance between FISH signals and DAPI staining, by Graphpad 9.0 prime software.
[00186] Fecal DNA Isolation and Library Preparation Mouse stool samples were collected on Day 60 following LipoAgTNZ treatment and immediately stored in -80 °C upon collection. Fecal DNA was isolated using QIAamp Fast DNA Stool Mini Kit (Qiagen) following manufacture protocol. 12.5 ng of total DNA were amplified by PCR using primer set (515F-806R) (81, 82) targeting V4 region on 16s rRNA genes, and PCR amplicons were sequenced at V4 region on an Illumina Miseq (Illumina). Subsequently, each sample was amplified using a limited cycle PCR program, adding Illumina sequencing adapters and dual-index barcodes (index l(i7) and index 2(i5)) (Illumina) to the amplicon target. The final libraries were purified using the AMPure XP reagent (Beckman Coulter), quantified and normalized prior to pooling. The DNA library pool was then denatured with NaOH, diluted with hybridization buffer and heat denatured before loading on the MiSeq reagent cartridge (Illumina) and on the MiSeq instrument (Illumina). Automated cluster generation and paired-end sequencing with dual reads were performed according to the manufacturer’s instructions.
[00187] Bioinformatic Analysis Sequencing output from the Illumina MiSeq platform were converted to fastq format and demultiplexed using Illumina Bcl2Fastq 2.20.0 (Illumina, Inc. USA). The resulting paired-end reads were processed with the Quantitative Insights Into Microbial Ecology (QIIME) 2 2021-2 wrapper for DADA2 including merging paired ends, quality filtering, error correction, and chimera detection. Amplicon sequencing units from DADA2 were assigned taxonomic identifiers with respect to the Greengenes and Silva databases, their sequences were aligned using maFFT in QIIME 2, and a phylogenetic tree was built with FastTree in QIIME 2. Rarefaction curve was generated at the depth of 5,000 sequences per subsample. 0-diversity estimates were calculated within QIIME 2 using Bray Curtis distance between samples at a subsampling depth of 5,000. Results were summarized and visualized through principal coordinate analysis as implemented in QIIME 2. Microbiota taxonomy was also applied to classify the organism as a representative OTU. The linear discriminant analysis effect size (LEfSe) Galaxy module was used for analysis examining biologic consistency and effect relevance (huttenhower.sph.harvard.edu/galaxy), which was conducted by coupling standard tests for statistical significance.
[00188] T Cell Isolation and Adoptive Transfer The spleens from long-term survivors (pulsed with 2xl06 CT26(FL3) cells subcutaneously in the lower right flank 48 h before T cell isolation) and naive mice were disassociated in PBS with 0.1% BSA and 2 mM EDTA using a syringe plunger and filtered through a 40 pM cell strainer. The splenocytes were centrifuged at 300 x g for 10 min at 4 °C and washed in 50 mL PBS with 0.1% BSA and 2 mM EDTA. The cells were resuspended with 10 mL RPMI 1640 medium (Gibco) with 120 Kunitz unit/mL DNase for 15 min at room temperature and filtered through a cell strainer. The cells were washed with 50 mL PBS with 0.1% BSA and 2 mM EDTA, resuspended by 4 mL PBS with 0.1% BSA and 2 mM EDTA and purified by 3 mL FicolLPaque PLUS at 400 x g for 30-40 min at room temperature. The undisturbed lymphocytes at the interface were collected and used for in vivo (CFSE labelled cells) and in vitro T cell proliferation assay.
[00189] Pan T cells were further isolated by negative magnetic labeling by using a pan T cell isolation kit (Miltenyi Biotec). T cells were treated with biotin- antibody cocktail and subsequent anti-biotin cocktail and separated by LS column (Miltenyi Biotec) in elution buffer (PBS with 0.5% BSA and 2 mM EDTA) in the magnetic field of the MACS separator (Miltenyi Biotec). The cells were adjusted to IxlO6 in 200 pL PBS and i.v. injected to the recipient mice on Day 10 and Day 15 after tumor inoculation.
[00190] T Cell Proliferation The lymphocytes were purified as described above. For in vivo T cell proliferation, lymphocytes (5xl07) from the donor mice were labeled with 5.0 pM CFSE (Invitrogen) and washed with PBS containing 5% bovine calf serum. The recipient mice were inoculated with orthotopic CRC tumors with or without F. nucleatum inoculation by oral gavage. Adoptively transferred CFSE-labeled lymphocytes (5xl07 per mice in 200 pL PBS) were i.v. injected into the recipient mice. At 18 h after injection, tumors from the recipient mice were harvested and dissociated as described in section of flow cytometry. The cell suspensions were stained with antibodies specific for T cells and analyzed by flow cytometry as described above. Tumor from mice without lymphocytes transfer were used as negative control and splenocytes from mice without CRC tumor and F. nucleatum inoculation were used as positive control for CFSE analysis. The level of CFSE were gated to calculate the percentage of in vivo T cell proliferation. [00191] For in vitro T cell proliferation: In each well of 24-well round-bottom plate (Coming® Elplasia® 4441) were seeded with 500 pL of IxlO5 CT26 cells per well. The serial dilutions of the effector cells were prepared at 1:5, 1:50, 1:100 (CT26: lymphocytes) and added to each well of the plates. The coculture was incubated in 5% CO2 at 37 °C for 6 h. The cell suspensions were stained by antibodies specific for T cells and analyzed by flow cytometry.
[00192] Western Blot Cell or tissue samples were lysed by RIPA Lysis buffer (Thermo Scientific™). The Pierce BCA Protein Assay Kit (Thermo Scientific) was used to determine the protein concentration in samples. The protein solution was diluted by NuPAGE LDS Sample Buffer (4X) (Invitrogen) and heated at 95 °C for 5 min. Protein was separated by 4-12% SDS- polyacrylamide gel electrophoresis (Invitrogen), and then transferred to poly vinylidene difluoride membranes (Bio-Rad). The membranes were then blocked with 1% bovine serum albumin in TBST at room temperature for Ih and then incubated with primary antibodies diluted in 1 % bovine albumin in TBST overnight at 4 °C in a shaker. The membranes were washed by TBST and further incubated with a secondary antibody diluted in 1% bovine albumin in TBST at room temperature for Ih, and then detected using the Pierce ECL Western Blotting Substrate (Thermo Fisher). Beta- actin was used as the control. Image Lab 6.0.1 was used to acquire images.
[00193] EHSpot Assay Elispot assay is used to test memory immune response and MHC restricted peptides on splenic CD8+ T cells. Interferon- y (IFN-y) ELISPOT assay (R&D SYSTEMS, EL485) are performed in 96-well bottomed sterile plates. The membrane of plates was pre-coated with capture IFN-y antibody and incubated with blocking buffer for 2 h. Bone marrow derived dendritic cells (105/well) were pulsed with peptides (20 pg/mL) and were added to CD8+ T cells (2 x 104/well) for 24 h at 37°C and plates area were developed with a biotinylated detection antibody specific for IFN-y for 1 h, followed by streptavidin- alkaline phosphatase for 1 h. Finally, the substrate of alkaline phosphatase (BCIP/NBT buffer) is added for 5-20 min. Spots are imaged by using stereomicroscopy (Olympus BX61).
[00194] Mass spectrometry Samples were analyzed with a Thermo LTQ Ion Trap (ThermoFisher, Bremen, Germany) mass spectrometer. Samples were introduced via a heated electrospray source (HESI) at a flow rate of 0.3 mL/min. HESI source conditions were set as: source heater temperature 250 °C, sheath gas (nitrogen) 50 arb, auxiliary gas (nitrogen) 15 arb, sweep gas (nitrogen) 0 arb, capillary temperature 200 °C, tube lens voltage 110 V, capillary voltage 10 V, and source voltage 3.50 kV. The mass range was set to 400-1800 m/z and data were collected in the positive polarity. Solutions were analyzed at 0.1 umM or less based on responsiveness to the ESI mechanism. Xcalibur (ThermoFisher, Breman, Germany) was used to analyze the data.
[00195] LipoCuTNZ preparation Distearoyl phosphatidylcholine (16.0 mg, Avanti), cholesterol (13.0 mg, Avanti) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (5.0 mg, Avanti) were dissolved in chloroform. The solvent was evaporated to form a lipid thin film. Three mL of 300 mM silver nitrate solution (adjusted to pH 4.0 with copper chloride) was added to hydrate the film at 65 °C for 30 min, and then the liposomes were sequentially extruded through 400 nm, 200 nm, 100 nm membrane for 10 times respectively. Gel filtration (Sephadex G-50) was used to remove the copper ions from the outer aqueous phase and to replace it with 5% sucrose with EDTA (UltrapureTM, pH 8.0, 1:25 dilution by sucrose solution) and then with 5% glucose without EDTA. The liposomes were incubated with tinidazole (0.2 mg) at 65 °C for 15 min for remote loading. Gel filtration was repeated to remove the unencapsulated tinidazole.
[00196] Statistical Analysis Statistical analysis comparing two groups was performed using unpaired two-tailed t test. Comparisons between three or more groups were done using ordinary two-way ANOVA with multiple comparisons. For survival analyses, log rank test was used for comparison. All statistical analysis was performed using GraphPad Prism 9.0 Software.
Appropriate tests were applied in analyzing these data, meeting assumptions of the statistical methods. P value less than 0.05 was considered significant. Data are presented as means + standard deviation.
[00197] Tetramer Study After 28 days of orthotopic tumor inoculation, surface immunophenotyping of tumor infiltration T lymphocytes was performed as following. Single cell suspension of solid tumor was obtained by standard Ficoll-Paque density gradient centrifugation. CD8+ T lymphocytes were isolated by negative bead selection (Miltenyi Biotec, 130-104-075). The T cell epitopes are evaluated for the percentage of specific CD8+ T lymphocytes by staining with a H2-Db- or H2-Kb-peptide tetramer (5001-1-20, EAGLE bioscience) and anti-CD8 mAb.
[00198] Bacteria Epitope Prediction For bacteria derived neoantigens: Cytoplasmic proteins of F. nucleatum were predicted by Vaxign2 (violinet.org/vaxign2) and then ranked by the abundance in the quantitative label-free proteomics. The most abundent proteins were selected for T cell epitope prediction. T cell epitopes were predicted using open-source tools based on artificial neural networks supported by Immune Epitope Database (IEDB.org) and NetMHC 4.0 Server (cbs.dtu.dk/services/NetMHC/). MHC haplotypes corresponding to strain Balb/C and C57BL/6 were used for this exercise. Predicted epitopes were ranked, and potential epitopes were used for T cell study.
[00199] For bacteria derived homologous antigens: To explore the homologous antigens shared by bacteria and mice, The genome of Fusobacterium nucleatum subsp. nucleatum ATCC 25586 (GCA_000007325.1) was aligned with the mice genome (GRCm39 reference Annotation Release 109) by using Nucleotide Blast. The putative proteins in mouse proteome were selected, among which sequences longer than 7 amino acids were further filtered and aligned in F. nucleatum proteome (UP000002521_190304). The homologous peptide sequences were selected for T cell epitope prediction.
Example 1 Bacteria Invaded into Tumor Cells in Response to Low Oxygen Level
[00200] Gram-negative and anaerobic bacteria, F. nucleatum, is prevalent in human CRC as well as metastasis. To examine whether the infection of F. nucleatum was correlated with tumor hypoxia, CT26(FL3) cells were acclimated to either 1% (hypoxia) or 20% oxygen (normoxia) for 24 h (FIG. 1A). F. nucleatum was able to invade hypoxic CT26(FL3) tumor cells (FIG. IB) with 15-fold higher signals in comparison to cells in normoxia (FIG. 1C). The intracellular F. nucleatum was confirmed by stacking images highlighting the co-localization of F. nucleatum and F-actin labelled by phalloidin, which showed F. nucleatum was able to invade inside the cytoskeleton in response to hypoxia (FIG. ID). In F. nucleatum infected CT26(FL3) spheroids, bacteria not only adhered on the surface but translocated into the organoids (FIGS. IE and 7A). Spontaneous invasion to hypoxic tumor cell was also found in facultative anaerobic probiotic strain E. coll. Nissle, which suggested that bacteria preferentially invade hypoxic tumor (FIGS. IF and 8A).
[00201] CRC was established in Balb/C mice infected with F. nucleatum (FIG. 6A). F. nucleatum infection led to over 30-fold higher tumor growth ratio as compared to uninfected controls (FIGS. 6B-6D) without inducing loss of body weight of the BALB/c mice (FIG. 6E). F. nucleatum infection considerably promoted tumor metastasis in proximal lymph nodes and distal metastasis (FIG. 6F). The primary tumors, distal metastases and feces were confirmed to contain F. nucleatum (FIG. 6G). There was a significant increase in anti-inflammatory M2 macrophages and CDllb+ Grl+ myeloid-derived suppressor cell (MDSC) population compared to uninfected tumors. A significant decrease in CD8+ T cell population, CD3+ CD4+ CD62L+ CD44+ memory T cells and CDllc+MHCII+ dendritic cell was observed compared to the non-F. nucleatum infected control group (FIGS. 6H and 61).
[00202] TEM images of F. nucleatum infected CRC tumor demonstrated that F. nucleatum was intracellular in vivo in the tumor region (FIG. 1G). Fluorescence in situ hybridization (FISH) using an RNA probe specific for the 16s ribosomal RNA of F. nucleatum was performed to visualize the bacterium in the CRC tumor sections. The red pixels indicating FISH signals were mapped to the nearest blue pixels indicating DAPI staining, which was calculated by Euclidean distance map. The minimum distance distribution was further fitted by computing the percentage of FISH pixels versus distances to DAPI, which was within the range of 3 /rm, suggesting F. nucleatum appeared near the cell nuclei in the CRC tumor (FIGS. 1 H, IT, 7B, and 7C). [00203] Escherichia coli Nissle 1917 (E. coli Nissle) has been widely studied as a probiotic and engineered bacteria that delivers therapeutics to hypoxic tumor. An orthotopic E. coli Nissle colonized CRC tumor was established in BALB/c mice (FIGS. 8B and 8C). E. coli Nissle colonized in CRC tumor (FIG. 8D), fostered immunosuppressive tumor microenvironment (FIG. 8E).
Example 2
Liposomal Antibiotics Eliminated Bacteria in the Tumor
[00204] Nitroimidazole is part of a class of antimicrobial prodrugs that is inactive until reduced by the ferredoxin oxidoreductase system in obligate anaerobes. The reductive activation of the nitro group is proposed to form the cytotoxic nitro and other free radical, leading to its structure fragmentation and cytotoxicity to DNA. The presence of oxygen tension inhibits the formation of the cytotoxic daughter drug. Reductive inactivation of the nitro group to the amino group occurs via oxygen-insensitive nitro reductases, rendering nitroimidazole non-toxic. The nitroimidazole coordinates via the ring N3 donor atom with a variety of metal ions including cobalt (II), copper (II), zinc (II) and silver (I) (FIG. 9 A). Among these metal ions, Ag+ ions are potent antibacterial agents used in various forms as antibiotics for centuries. The silver nanoparticles may have the ability to adhere and penetrate into the bacterial cell wall. The silver (I) ions interact with sulfur and phosphorus, thereby causing altered activity of enzymes and DNA. The formation of the silver- tinidazole complex was confirmed by mass spectrometry (FIG. 9B).
[00205] Tumor hypoxia directly correlates with tumor acidosis due to the Warburg effect favored by cancer cells that metabolized pyruvate into lactate and ethanol. The low pH of hypoxic tumors in result of elevated levels of lactic acid may allow drug release in anerobic bacteria residing region of the tumor. Metal ions can be a trapping agent for loading nitroimidazole into the liposomes (FIG. 2A). A silver-tinidazole complex disassociated in acid, showing a rapid release of both Ag and tinidazole in acidic medium at pH 4.5 (FIG. 2B). The liposomes were spherical with a diameter of -150 nm and zeta potential around -14 mV determined by nanoparticle tracking system (FIGS. 9C and 9D). The liposomes were uniform in morphology imaged by CryoEM (FIG. 2C). The loading of tinidazole (TNZ) into silver containing liposomes was efficient with >80% encapsulation efficiency and >5% loading content (FIG. 2D). TNZ showed quick loading kinetics into the liposomes, over 80% of which was entrapped within 5 min (FIG. 2E). The TNZ to Ag ratio in the liposomes was 1.54+0.18 (n=4). This is lower than the theoretical ratio of 2, indicating the liposomes contained some free Ag(I) and/or one-to-one complex. Copper (II) also formed complex with tinidazole and encapsulated TNZ into liposomes (FIGS. 9E-9H). This liposome platform will provide a versatile approach to load the nitroimidazole antibiotics targeting anaerobic bacteria in the infected tumor. The bond between silver and tinidazole readily dissociates in acid, as the ring N3 donor atom (pKa = 4.7) was protonated at pH 4.5 (FIG. 2F). [00206] Antimicrobial assays were performed to determine the minimum inhibitory concentration (MIC) for F. nucleatum by plating and enumerating viable colony forming unit (CFU). The AgTNZ complex inhibited F. nucleatum with MIC95 of 100 nM (FIG. 91). The CT26(FL3) cells were infected with F. nucleatum, incubated with different concentrations of free AgTNZ or LipoAgTNZ for 12 h, and intracellular bacteria were quantified by CFU. Both LipoAgTNZ and free AgTNZ showed higher antimicrobial efficacy in hypoxia. The MIC for effective clearance of intracellular F. nucleatum in hypoxia was of 1.5 pM for LipoAgTNZ versus 7.5 pM for AgTNZ (FIG. 2G) due to the enhanced intracellular accumulation mediated by the liposome delivery (FIG. 2H). Therefore, the liposomal formulation has the potential to facilitate intracellular F. nucleatum clearance.
[00207] To further profile the pharmacokinetic profiles in vivo, i.v. injected LipoAgTNZ were analyzed by sampling blood from mice bearing orthotopic tumors at predetermined time points. The molar ratio of TNZ and Ag remained consistent (approximately 1.5 to 2) within the first 240 min in blood circulation. Compared with free drug, liposomal TNZ showed superior pharmacokinetic profiles, as indicated by increasing area under the curve (AUC) of the drug concentration in circulation (FIGS. 10A and 10B). Tinidazole is metabolized mainly by CYP3A4, which supported the higher level and longer half-life (ti/2) of silver than TNZ in the tissues and blood (FIG. 10C). Liposomes were labelled with DiD, a fluorescent dye that labels the lipid membrane of liposomes. Distribution of liposomes were determined by quantifying DiD fluorescence in the organs. The liposomes mainly localized to the tumor and the liver, as shown by ex vivo images (FIG. 10D). Liposomes extended the TNZ accumulation in the tumor by more than 10-fold (FIG. 10E). Mice were sacrificed at 24 h post i.v. injection of free drug and liposomes. All major organs including tumor were harvested, homogenized, and measured for both drug contents for biodistribution. Tinidazole encapsulated in liposomes mainly accumulated in the tumor at 24 h after i.v. injection, which suggest the success of tumor targeting (FIGS. 10F and 10G).
[00208] MTT assays were performed to analyze the in vitro cytotoxicity. Free Ag, TNZ and AgTNZ did not affect the viability of CT26(FL3) cells within 48 h of incubation at concentrations as high as 40 pM (FIGS. 11A and 1 IB). An in vivo toxicity study of liposomal metal-imidazole complexes was also performed. Healthy (non-tumor bearing) mice were i.v. injected (4 mg/kg with respect to TNZ) with either LipoCuTNZ or LipoAgTNZ three times every third day. Treated mice showed no body weight change (FIG. 11C). Major organs from tumor bearing mice were dissected, fixed, embedded in paraffin, and examined for tissue histology after treatments. Histological changes related to toxicity were not found among the main organs (FIG. 11D). However, metastatic lesions (white arrows in the figure) were found in both control and free TNZ groups. Blood samples were taken and indicated that F. nucleatum and LipoAgTNZ treatment did not induce abnormalities in red blood cell counts or white blood cell counts. F. nucleatum inoculation by oral gavage significantly increased blood platelets, which decreased to the normal level after the LipoAgTNZ treatment. Interestingly, neutrophils, the phagocytes in response to bacterial infection, increased significantly in the blood after treatment. The monocyte and lymphocyte count were not significantly increased in the LipoAgTNZ treated group (FIG. HE). Toxicity biomarkers were assessed: ALT and AST for liver function, BUN, and creatinine for kidney function (FIG. 1 IF). No obvious alteration in serum biomarkers were observed compared with the untreated mice.
[00209] The pH-sensitive liposomes dispersed in PBS at pH 7 were stable for 4 days (FIG. 24A) and less than 10% liposome cargoes were released in 30 hours (FIG. 24B).
Example 3 Killing Intracellular Bacteria Improved Immune Surveillance
[00210] The infected CRC mouse model was used to test the therapeutic efficacy of the antibiotic liposomes. Balb/C mice that orthotopically inoculated with tumor and additionally infected with F. nucleatum or E. coli Nissle, received antibiotic liposome treatments i.v. (FIG. 3 A). Although the growth of F. nucleatum infected tumor was significantly inhibited by both LipoAgTNZ and LipoCuTNZ at Day 23 (FIGS. 3B, 12A, and 12B), which eradicated the tumor colonizing bacteria (FIG. 3C), LipoAgTNZ induced long-term survival in 6 out of 7 mice (FIG. 3D). Liposomal AgTNZ was also effective in reducing the probiotic E. coli Nissle content and inhibiting the growth of infected tumor, compared to the uninfected mice without antibiotic treatment (FIGS. 3E and 3F). The survival study was also performed in F. nucleatum infected MC38 tumor-bearing C57BL/6J mice, in which LipoAgTNZ achieved 71.4% survival rate (FIGS. 13A and 13B) compared to infected mice without treatments.
[00211] To test the hypothesis that the tumor associated bacteria is independent from the gut- colonized F. nucleatum, polymyxin B (pmB) was used to eradicate the Gram-negative bacteria in the gut. Polymyxin B is a cyclic peptide with five positive charges and a logP of -5.6, rendering it low transmembrane permeability and poor oral bioavailability. The orally administered pmB affects the bacteria only in the gut but not in the tumor due to low transmembrane partition and distribution (FIG. 12C). The mice treated with pmB did not show significant reduction in tumor growth (FIG. 12D). The tumor colonizing F. nucleatum promoted tumor progression, which is independent of F. nucleatum burden in the gut. Interestingly, the efficacy of LipoAgTNZ was relative to colonization of F. nucleatum in the tumor, as the uninfected mice did not respond as well as the infected mice (FIG. 12E).
[00212] At Day 24, tumors were excised and sectioned for the FISH assay using a F. nucleatum specific RNA probe. Consistent with quantification by CFU, FISH signals revealed that LipoAgTNZ effectively reduced F. nucleatum burden in CRC tumors. TUNEL assay was performed showing increased apoptotic cells after treatment (FIG. 3G). As AgTNZ did not show cytotoxicity to CT26 (FL3) tumor cells (FIGS. 11A and 1 IB), the treatment induced antitumor immunity in immune competent mice.
[00213] Infiltration of CD3+ T cell, CD8+ cytotoxic T cell and CD44+ CD62L+ memory T cell were significantly increased in the LipoAgTNZ treated F. nucleatum infected tumors (FIGS. 3H, 14A, and 14B). CD206+ M2 macrophage polarization was reduced following treatment of LipoAgTNZ (FIGS. 3H, 14C, and 14D). LipoAgTNZ treatment induced a substantial normalization of blood vessels and lymphatic vessels, suggesting that the normalization of angiogenesis may contribute to facilitating immune surveillance (FIG. 14E).
[00214] To investigate if the treatment was able to trigger an immune memory response, the long- term survivors were subcutaneously rechallenged with tumor cells with or without F. nucleatum infection (FIG. 3A). There was no detectable tumor growth in the survivors, but rapid growth in the naive mice (FIG. 31). The therapeutic efficacy was contingent on the host immune system, as depletion of either CD8 or CD4 cells significantly diminished the anti-tumor activity of LipoAgTNZ (FIG. 3 J). When mice were examined by necropsy after immune compromise by antibody treatment on Day 75, there was no detectable primary tumor left in the long-term survivors (FIG. 14F), indicating that the gut tumors were eradicated by the strong immune response that was dependent on both CD8+ and CD4+ T cells. Therefore, through elimination of the tumor- associated bacteria, immune suppressive microenvironment has been turned into an antitumoral immune activated state.
[00215] The FadA adhesin of F. nucleatum interacts with E-cadherin, leading to activation of the P-catenin pathway. F. nucleatum induces DNA damage, which promotes the release of cellular tumor antigen Trp53. Vimentin is also important in epithelial to mesenchymal transition, which was upregulated in F. nucleatum infected tumors. LipoAgTNZ treatment reversed the induction of these oncogenic mediators (Trp53, E-cadherin, Vimentin, P-catenin) and the key inflammatory NF- KB pathway in the F. nuclealum-m' fected tumors (FIG. 14G).
[00216] To determine the gut microbiota diversity after antibiotics treatment, high-throughput gene-sequencing analysis of 16S ribosomal RNA (rRNA) was performed in fecal bacterial DNA isolated from age-matched control and survivor mice at day 70. Rarefaction analysis comparing bacterial diversity within individual subjects revealed that survivors harbored a similar bacterial community relative to that of controls (FIGS. 3K).
[00217] Liver metastasis, the most common distant metastasis in colorectal cancer, afflicts up to 70% of patients. As shown in FIGS. 10D-10F, liposomes predominantly accumulated in the tumor and the liver. A F. nucle atum-mf &cted liver metastasis model was established (FIG. 15A). The infected tumor cells were inoculated via the portal vein by hemi-splenic injection, which established uniform metastasis in the liver. The development of liver metastasis was significantly inhibited by treatment of LipoAgTNZ quantified by luciferase imaging in vivo (FIGS. 15B-15D). The tumor burdens were reduced by 8-fold at the end point, quantified by ex vivo luciferase signals (FIGS. 15E and 15F). FISH imaging for F. nucleatum 16s RNA revealed a high abundance of bacteria in the untreated liver metastasis, which after the antibiotic treatment was significantly reduced and associated with enhanced infiltration of CD8+ T cells. LipoAgTNZ treatment also reduced anti-inflammatory M2 macrophages, as CD206 expression was much less than the control group (FIG. 15G).
[00218] Unlike chemotherapy or oncogene-specific therapies that induce cytotoxicity to tumor cells, the strategy targeted the tumor- associated bacteria in the primary tumor and the distal metastasis. The process of bacterial killing promoted an anticancer response, which restored immune surveillance and inhibited both primary tumor growth and metastatic progression.
[00219] A similar approach was used for liposomal AgTNZ in the F. nucleatum infected wildtype CT26 tumor model and the same tumor and metastasis inhibition as with the CT26FL3(Luc/RFP) model was observed (FIG. 22).
[00220] An antibiotics cocktail control was included in an experiment with CD8/CD4 depletion treatment. Treatment of the infected CT26FL3(Luc/RFP) tumor with the antibiotics cocktails decreased the tumor load, but cancer relapse occurred after the cocktail treatment stopped (FIGS. 23A-C). Antibiotic cocktails administered orally non- selectively changed the gut microbiota and compromised the immune sensitivity.
[00221] No bacteria colonization was observed in the main organs of mice treated with LipoAgTNZ (FIGS. 25A-C).
Example 4
T Cells from Long-term Survivors Showed Specificity to Both Infected and Uninfected Tumors
[00222] To analyze the T cell specificity to bacterial infection, T cells isolated from survivor mice were compared with age-matched uninfected naive mice. Splenic pan T cells were labelled with the proliferation dye CFSE and injected into the recipient mice with or without F. nucleatum infection. Significant T cell proliferation was found in both infected and uninfected recipient mice that received T cells from survivors, but not from the naive donors (FIGS. 4A and 4B). Incubating survivor-derived donor T cells with uninfected tumor cells in vitro enhanced CD8+ IFN-Y+ cell populations (FIGS. 4C and 16A). These data suggested that the T cells from the long-term survivors recognized tumor cells that were either infected or not.
[00223] Splenic pan T cells of long-term survivor mice after liposomal AgTNZ treatment were adoptively transferred to test the efficacy in recipient mice bearing orthotopic CRC tumors with or without F. nucleatum infection (FIG. 4D). Donor T cells from the long-term survivor mice effectively inhibited the growth of tumors infected with F. nucleatum-, Unexpectedly, the same donor T cells also inhibited uninfected tumors (FIG. 4H). Significantly increased numbers of CD3+ and CD3+ CD8+ T cells were found in tumors of both infected or uninfected mice (FIGS. 4E-4G, 16B, and 16C). The data again suggested that survivor T cell recognized both infected and uninfected tumor cells in the recipient mice.
[00224] F. nucleatum infection decreased the colocalization of calreticulin with cell membrane, downregulating chaperones (e.g., calreticulin) (FIG. 41). It is likely this downregulation is a part of the immune suppression mechanism of the infected tumor cells. It is interesting to note that F. nucleatum infection significantly downregulated both HSP70 and calreticulin, which are stress proteins upregulated by cells in the hypoxia condition. The treatment of liposomal antibiotics restored the chaperone expression by killing of the intracellular bacteria (FIG. 4J). The result suggested that immunogenic cell death may contribute to immune activation of the tumor cells under the treatment of antibiotics.
Example 5
Host T Cells Specifically Targeted Bacteria Epitopes After Antibiotic Treatment [00225] Data in FIGS. 3 and 4 suggested that LipoAgTNZ induced T cell immunity in the CRC bearing mice. According to identification of transmembrane helices, 1171 out of 2067 proteins are cytoplasmic at F. nucleatum subcellular localization in proteome-wide prediction of Vaxign2 (FIG. 5A). To identify the most abundant protein in cytoplasm, the label-free proteomics of F. nucleatum are mapped to subcellular localization (FIG. 5A). The proteomics data were generated from prior data (See, Ali Mohammed MM, et al., Anaerobe. 2021;72:102449 and Mohammed MMA, et al., Anaerobe. 2017;44:133-42). To estimate the potential neoepitopes, the MHC class I H-2Kd, H-2Dd and H2Ld binding peptides were predicted from the proteins ranking top in the quantitative proteomics of F. nucleatum, followed by selection of the epitopes with high total score and MHC binding affinity (FIGS. 5B and 5D). The top-ranked cytoplasmic proteins in abundance were selected for epitope prediction. Five top-ranked peptides predicted from each protein were selected using IEDB database applying NetMHCpan algorithm for mouse MHC-I presentation (sequences shown in the upper right comer of FIG. 5B).
[00226] To explore the homologous antigens shared by bacteria and mice, the genome of F. nucleatum was aligned with the mice genome by using Nucleotide Blast. Forty-one genes showed similarity including those in the non-open reading frames. Corresponding proteins from the mice proteome were selected; 25 total proteins were selected, of which only 19 contained potential sequence longer than 7 amino acids for the possibility of MHC-I presentation. Ten out of the 19 sequences code for proteins based on F. nucleatum proteome (FIG. 5C). Interestingly, 4 similar sequences out of 10 come from HSP70 chaperone protein (DnaK in gene name), which has 65% similarity with mouse HSP70 (Table 1). Three epitopes outweigh by prediction of the IEDB database according to their likelihood to be presented by H2Kd, H2Dd and H2Ld, the MHC-I haplotype of the Balb/C strain (FIG. 5D). In summary, peptides No. 1-5 are bacteria-derived neoantigens sharing no sequence similarity with mice. Peptides No. 6-8 are homologous epitopes that are shared between bacteria and mice.
[00227] To confirm the activity of the peptides in vivo, splenic CD8+ T cell were recovered from Balb/c mice that had survived from CRC tumor infected by F. nucleatum after LipoAgTNZ treatment and restimulated in vitro with the candidate peptides presented by bone marrow derived dentritic cells to measure IFNy production in an Elispot assay. Both homologous antigens and neoantigens showed enhanced IFNy- secreting T cells after LipoAgTNZ treatment (FIGS. 5E and 5F). Splenic T cells from mice infected with F. nucleatum but not treated with LipoAgTNZ did not show significant INFy response to the bacterial cytoplasmic peptides. A peptide from chicken ovalbumin (OVA) was used as a negative control.
[00228] Overall, these data demonstrated that bacteria epitopes can be presented by the host MHC-I after liposomal antibiotics treatment. The tolerance of self-epitopes that are shared with the tumor associated bacteria is likely reversed by the danger signals induced by the killing of the intracellular bacteria.
[00229] The data suggested that T cells from survivors established long-term anti-tumor efficacy, compared with T cells from naive mice. These liposomal antibiotics treated mice developed IFNy+ CD8+ T cells in response to bacteria-derived neoantigens. Interestingly, IFNy+ CD8 T cells in response to homologous epitopes were also developed after antibiotic treatment, suggesting the cellular immunity against the self-epitopes. This result was consistent with the T cell transfer study, in which T cell from survivor mice suppressed the uninfected tumor growth. The bacteria induced CD8+ T cells in response to class I MHC antigens, which is the cytotoxic T cell phenotype for antitumor efficacy, were primarily analyzed whereas CD4+ T cells in response to Class II MHC antigens were reported to generate either cytotoxicity or tolerance.
Example 6
Microbial Epitopes from Tumor-associated Bacteria as Cancer Vaccine
[00230] Epitope(s) released from dead or dying tumor associated bacteria may prime the immune system to recognize the bacteria infected cells. Bacterial epitopes shared by the tumor cells also elicit anti-tumor effect by priming cytotoxic T cells. Bioinformatics screens and predictions to identify the antigenic T cell epitopes are followed by confirmation of endogenous major histocompatibility complex (MHC) /T cell receptor (TCR) of the tumor-infiltrating T-cells. Several significant epitopes have already been identified. Additional bacterial epitopes are used develop a therapeutic vaccine against F. nucleatum infected colorectal cancer in both mouse model and human. Preliminary data using peptides with the predicted sequence showed promising therapeutic vaccine activity in a mouse model. mRNA encoding the epitopes of both MHC-I and II can be delivered using lipid nanoparticle technology to dendritic cells for immunization and therapy, or prophylactically as a preventive vaccine.
[00231] Bacteria residing in tumor potentially provide tumor-associated neoepitopes. In support with the analysis on the proteomics of F. nucleatum, the most abundant proteins locate in cytoplasm of bacteria and do not share similar sequences if aligned with mammalian proteome. There are few proteins that are conserved from bacteria to eukaryotic cell, e.g., heat shock protein 70 with approximately 60% sequence similarity between F. nucleatum and human. These bacterial epitopes potentially induce CD8+ T cell cross-reactivity with tumor epitopes. Both bacteria-derived neoepitopes and shared epitopes are analyzed and tested for vaccine induced immunogenicity (FIG. 17).
[00232] Peptide Nos. 5 and 6 in FIG. 18A are two such peptides. For comparison, two other peptides (Nos. 1 and 2) without sequence similarity to mouse proteome that are predicted as bacteria-derived neoantigens from the top-2 abundant proteins of F. nucleatum analyzed from quantitative proteomics were selected. Since peptide No. 6 comes from HSP70 chaperone protein (DnaK in gene name), which has 65% similarity with mouse HSP70, two other HSP70 peptides without sequence similarity to mouse proteome (Nos. 3 and 4) predicted to be antigenic for comparison were also added. Listed in FIG. 18A is the scores of these peptides within selection criteria. Peptides Nos. 1-6 were loaded to the tetramers of the corresponding H2 haplotype and tested their binding with tumor infiltrating T-cells isolated from MC38 tumors. Mice used in the experiment included those infected or uninfected with F. nucleatum and with or without LipoAgTNZ treatment.
[00233] Data in FIGS. 18B and 18C indicate that peptide No. 5 (LADDNFSTLSEQ ID NO: 105) and No. 6 (RGVPQIEVTF-SEQ ID NO: 106) stood out among the 6 tested by showing high positive ratio in CD8+ T-cells phenotypes suggesting T cell receptor specificities. Both peptides are shared epitopes between bacteria and tumor. Peptide No. 3 also induced tetramer/T cell receptor binding affinity in uninfected tumor, albeit with much lower ratio than the infected tumors. Peptide Nos. 1 (VITAFSNI -SEQ ID NO: 111), 2 (SVFVFYPADF-SEQ ID NO: 108), 3 (ISLPFITM-SEQ ID NO: 109) and 4 (AAIQGGVLM-SEQ ID NO: 110) are bacteria-derived neoantigens with no sequence similarity with the host, displaying low tetramer binding activity with T-cells isolated from the infected and LipoAgTNZ treated tumors. The major tumor infiltrating T-cell specificities are against the bacteria-host shared epitopes, which induced significant response against host epitopes. Bioinformatics prediction of F. nucleatum epitopes that can be presented by the host MHC-I molecules is an effective approach to identifying novel peptide antigens for cancer vaccination.
[00234] To confirm the activity of the peptides in vivo, splenic CD8+ T cell were recovered from C57BL/6 mice that had been survived from CRC tumor infected by F. nucleatum after LipoAgTNZ treatment, they were restimulated in vitro with the candidate peptides presented by bone marrow derived dentritic cells to measure IFNy production in an Elispot assay. Both homologous antigens and neoantigens showed enhanced IFNy- secreting T cells after LipoAgTNZ treatment (FIG. 18D). Splenic T cells from mice infected with F. nucleatum but not treated with LipoAgTNZ did not show significant INFy response to the bacterial cytoplasmic peptides. A peptide from chicken ovalbumin (OVA) was used as a negative control.
[00235] Peptide Nos. 2 and 6 were formulated with DOTAP liposomes as an adjuvant and were given to mice twice by i.m. injection. The presentation of the bacterial epitopes including the epitopes shared with the host epitopes elicited efficient anticancer immunity and resulted in long- term survivals (FIG. 19).
[00236] An epitope-based vaccine was designed that included a) MHC class I restricted bacterial epitopes from proteins showing top abundancy in F. nucleatum proteomics, b) MHC class II restricted, distinctive bacterial epitopes from proteins showing top abundancy in F. nucleatum proteomics, and c) MHC class I restricted, immunodominant bacterial epitopes with sequence similar to the host proteome. Plasmid templates for in vitro transcription of antigen-encoding RNAs are based on pSTl-MITD vectors. RNA is generated by in vitro transcription and will be synthesized using 1-methyl-pseudouridine instead of uridine (FIG. 20). pSTl-MITD features a signal sequence for routing to the endoplasmic reticulum and the major histocompatibility complex (MHC) class transmembrane and cytoplasmic domains for improved presentation of MHC class I and II epitopes. The peptides are connected by cleavable linkers or 8-12 mer non- immunogenic glycine/serine linkers in comparison with polyepitopes without linker connection. mRNAs are transcribed in vitro by T7 RNA polymerase-mediated transcription according to the manufacturer’s instructions (MEGAscript™ T7 Transcription Kit, Thermo Fisher). CleanCap AG (TriLink) are included in the transcription reaction mixture as a capping reagent that results in a Cap 1 structure at the 5 ’ end of in vitro transcribed mRNA. The resulting mRNAs are purified by using MEGAclear™ Transcription Clean-Up Kit (Thermo Fisher).
[00237] LNP formulations are prepared using a previously described method. LNPs containing mRNA are prepared by rapid mixing through microfluidics as described. Briefly, ionizable lipid, DSPC, cholesterol and DSPE-PEG are dissolved in ethanol at appropriate ratios to a final concentration of 10 mM total lipid. Nucleic acids are dissolved in 25 mM sodium acetate buffer at pH 4.0 to obtain a final mixture with a defined nucleic acid to lipid ratio. The organic and aqueous solutions are mixed at a flow ratio of 1:3 (v:v) and a total flow rate of 28 mL/min using a microfluidic mixing device available to us. The resulting mixtures are dialyzed against a 1000-fold volume of phosphate-buffered saline (PBS) pH7.4 overnight and sterile filtered (0.2 pm). In all experiments a total volume of 200 pL of mRNA vaccine is injected per mouse subcutaneously. Orthotopic tumor is inoculated at day 0. F. nucleatum is orally gavaged at 108 CPU per mouse at days 5 and 10. Mice are vaccinated twice at 15 days apart and re-challenged subcutaneously with fresh tumor cells 4 weeks after the original tumor inoculation to test the memory response.
Preventive activity of the vaccine will also be tested by immunization before the tumor inoculation. [00238] Different CRC patients may be infected with different microbes. The microbiota will be identified from the fecal samples using a database of next generation sequencing (NGS).
Subsequent analysis of microbiome diversity and antigen prediction algorithms are used to generate tailored epitopes based on human HLA haplotypes. mRNA encoding microbiota derived polyepitopes will be developed for each individual patients. R-DOTAP, a cationic lipid and an adjuvant, complexes with peptides to form a simple vaccine (See, Vasievich EA, et al., Cancer Immunol Immunother. 201 l;60(5):629-38).
[00239] The genome of F. nucleatum was aligned with the human genome by using Nucleotide Blast. Corresponding proteins were selected from the bacteria proteome and aligned with those from the human proteome. Because the minimal epitope length for MHC-I presentation is 7 aa, potentially active sequence longer than 7 amino acids were selected. Epitope prediction IEDB database applying prediction algorithm was used to rank these possible peptide sequences according to their likelihood to be presented by HLA molecules (tools.iedb.org/mhci/) (FIG. 21). The homologous epitopes are listed in Table 2. The possible HLA binding peptides predicted from peptides in Table 2. are listed in Table 3. Antigen prediction of heterologous antigens is listed in Table 4.
[00240] The PathSeq algorithm was used to perform computational subtraction of human reads, followed by alignments of residual reads to human reference genomes / transcriptomes and microbial reference genomes (which include bacterial, viral, archaeal, and fungal sequences downloaded from NCBI in October 2015). These alignments result in taxonomical classification of reads into bacterial, viral, archaeal, and fungal sequences in whole genome sequencing (WGS) and RNA sequencing (RNASeq) data. The human reference genome/transcriptome sequences are downloaded from the Ensembl database, female human reference genome, human genome plus transcriptome database and the human reference genome from NCBI. Bacterial, fungal, and archaeal reference sequences are downloaded from NCBI reference sequence database. Viral and phage sequences are downloaded from NCBI nucleotide database using query strings
‘Virusesl Organism | AND srcdb_refseq[PROP] NOT wgsfprop] NOT cellular organisms [ORGN] NOT AC_000001:AC_999999[pacc]” and “Viruses[Organism] NOT srcdb_refseq[PROP] NOT cellular organisms [ORGN] AND nuccore genome samespecies [Filter] NOT nuccore genome [filter] NOT gbdiv syn[prop]”. The curated databases in FASTA format are available at software.broadinstitute.org/pathseq/Downloads.html webpage. Briefly, PathSeq is used to remove low quality reads followed by subtraction of human reads by mapping reads to a database of human genomes (downloaded from NCBI in November 2011) using BWA (Release 0.6.1, default settings), MegaBLAST (Release 2.2.25, cut-off E-value 10-7 , word size 16) and BLASTN (Release 2.2.25, cut-off E-value 10-7 , word size 7, nucleotide match reward 1, nucleotide mismatch score -3, gap open cost 5, gap extension cost 2). Sequences with perfect or near perfect matches to the human genome are removed in the subtraction process. In addition, low complexity and highly repetitive reads are removed using Repeat Masker5 (version open-3.3.0, libraries dated 2011-04-19).
[00241] 16S sequencing analysis. Taxonomic classification is performed by residual reads alignment using MegaBLAST (Release 2.2.25, cut-off E-value 10-7, word size 16) to a database of bacterial sequences and followed by BLASTN (Release 2.2.25, cut-off E-value 10-7, word size 7) to the human reference genome and microbial reference genomes. Following the taxonomic classification of non-human DNA sequencing reads, the relative abundance value for each bacterial organism is calculated by using reads that maps with >=90% sequence identity and >=90% query coverage. Classifications are performed at the domain, then phylum, then genus, then species level, requiring unique alignments (e.g., reads with equivalent Evalues to multiple taxa are removed from the analysis). In the case of WGS data, species level relative abundance (RA) for each organism is calculated as follows: relative abundance of a given organism in a sample= (number of unique alignment positions across the genome x 1,000,000)/ (number of total aligned bacterial reads x bacterial genome size). The RA values are then “per-sample” normalized such that the total relative abundance for each sample sums to one (or 100% for percentage relative abundance).
[00242] Cancer infected by other microbes may also be treated with appropriate antibiotics encapsulated in liposomes or other carriers, depending on microbe identity. Killing of the tumor- associated bacteria may reveal the microbe-associated antigen for anti-cancer immune response similar to what is demonstrated herein for F. nucleatum and E. coli Nissle. Peptide or mRNA vaccines containing epitope(s) from these tumor-associated bacteria may be used to treat the cancer, as illustrated herein for F. nucleatum. Table 1: . Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and Mus mus cuius.
Figure imgf000057_0001
Figure imgf000058_0001
Table 2: . Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and humans.
Figure imgf000058_0002
Figure imgf000059_0001
Table 1: . Similar protein sequences (homologous amino acid >7) shared by F, nucleatum and Mus musculus.
Figure imgf000060_0001
Figure imgf000061_0001
Table 2: . Similar protein sequences (homologous amino acid >7) shared by F. nucleatum and humans.
Figure imgf000061_0002
Figure imgf000062_0001
[00243] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.
[00244] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and may be made without departing from the spirit and scope thereof.

Claims

CLAIMS What is claimed is:
1. A method of treating cancer, promoting an anti-cancer response, or increasing anti-cancer immunity in a subject in need thereof comprising administering to the subject an effective amount of one or both of: a liposomal antibiotic formulation; and a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof.
2. The method of claim 1, wherein the antibiotic comprises a nitroimidazole antibiotic or a lincosamidc antibiotic.
3. The method of claim 1 or claim 2, wherein the antibiotic comprises a metal-tinidazole complex.
4. The method of any of claims 1-3, wherein the antibiotic comprises a silver-tinidazole complex or a copper-tinidazole complex.
5. The method of any of claims 1-4, wherein the liposomal antibiotic formulation is pH-sensitive
6. The method of any of claims 1-5, wherein the at least one tumor-associated bacterial epitope comprises an amino acid sequence have at least 60% similarity with sequences in subject proteome.
7. The method of any of claims 1-6, wherein the at least one tumor-associated bacterial epitope comprises: a major histocompatibility complex (MHC) class 1 epitope without sequence similarity with subject proteome, an MHC class II epitope without at least 60% sequence similarity with subject proteome, an MHC class 1 epitope with at least 60% sequence similarity with subject proteome, or a combination thereof.
8. The method of any of claims 1-7, wherein the at least one tumor-associated bacterial epitope is a ncocpitopc.
9. The method of any of claims 1-8, wherein the at least one tumor-associated bacterial epitope is determined by a method comprising: identifying tumor- associated bacteria in a sample from the subject; and selecting the epitopes from tumor associated bacteria proteome based on MHC or human leukocyte antigen (HLA) presentation.
10. The method of claim 9, wherein the at least one tumor-associated bacterial epitope is determined by a method further comprising: aligning sequences from bacterial proteome and subject proteome; and determining bacterial epitopes with sequence similarity to subject proteome.
11. The method of any of claim 1-10, wherein the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-112.
12. The method of any of claims 1-11, wherein the one or more peptides further comprise a signal sequence, major histocompatibility complex (MHC) transmembrane domain, an MHC cytoplasmic domain, or any combination thereof.
13. The method of any of claims 1-12, wherein the one or more nucleic acids comprise mRNA.
14. The method of any of claims 1-13, wherein the one or more nucleic acids further comprises: a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof.
15. The method of claim 14, wherein the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
16. The method of claim 14 or claim 15, wherein the one or more nucleic acids further comprises at least one chemically modified nucleotide.
17. The method of claim 16, wherein the at least one chemically modified nucleotide comprises a modified uracil, a modified cytosine or a combination thereof.
18. The method of any of claims 1-17, wherein the one or more peptides are encoded on a single nucleic acid.
19. The method of claim 18, wherein the single nucleic acid encodes two or more peptides.
20. The method of claim 19, wherein the two or more peptides are each separated by a linker.
21. The method of claim 20, wherein the linker comprises a cleavable linker, a flexible linker, or a combination thereof.
22. The method of any of claims 18-21, wherein the single nucleic acid is an mRNA, circular RNA, or DNA.
23. The method of cany of claims 1-22, wherein the composition further comprises an adjuvant, a delivery vehicle, or a combination thereof.
24. The method of claim 23, wherein the adjuvant, the delivery vehicle, or the combination thereof comprises a lipid.
25. The method of claim 24, wherein the lipid comprises a cationic lipid and/or an ionizable lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.
26. The method of claim 25, wherein the non-cationic lipid comprises a phospholipid.
27. The method of claim 25 or claim 26, wherein the sterol comprises cholesterol or a modification or ester thereof.
28. The method of any of claims 24-27, wherein the delivery vehicle comprises a lipid particle encapsulating the composition.
29. The method of claim 28, wherein the lipid particle comprises a polyethylene glycol (PEG)-lipid conjugate.
30. The method of any of claims 1-29, wherein the cancer is associated with tumor-associated bacteria.
31. The method of any of claims 1-30, wherein the cancer comprises gastric cancer, colorectal cancer, or a combination thereof.
32. The method of any of claims 1-31, wherein the subject was not administered a cytotoxic treatment.
33. The method of any of claims 1-32, wherein the liposomal antibiotic formulation and the composition are administered simultaneously, separately, or consecutively, in any order separated by a time interval.
34. The method of claim 33, wherein the liposomal antibiotic composition is administered prior to a resection.
35. A vaccine or medicament comprising: a composition comprising one or more peptides comprising at least one tumor-associated bacterial epitope, or one or more nucleic acids encoding thereof; and an adjuvant, a delivery vehicle, or a combination thereof.
36. The vaccine or medicament of claim 35, wherein the at least one tumor-associated bacterial epitope comprises an amino acid sequence have at least 60% similarity with sequences in subject proteome.
37. The vaccine or medicament of claim 35 or claim 36, wherein the at least one tumor-associated bacterial epitope comprises: a major histocompatibility complex (MHC) class I epitope without sequence similarity with subject proteome, an MHC class II epitope without at least 60% sequence similarity with subject proteome, an MHC class I epitope with at least 60% sequence similarity with subject proteome, or a combination thereof.
38. The vaccine or medicament of any of claims 35-37, wherein the at least one tumor-associated bacterial epitope is determined by a method comprising: identifying tumor- associated bacteria in a sample from the subject; and selecting the epitopes from tumor associated bacteria proteome based on MHC or human leukocyte antigen (HLA) presentation.
39. The vaccine or medicament of claim 38, wherein the at least one tumor-associated bacterial epitope is determined by a method further comprising: aligning sequences from bacterial proteome and subject proteome; and determining bacterial epitopes with sequence similarity to subject proteome.
40. The vaccine or medicament of any of claim 35-39, wherein the at least one tumor-associated bacterial epitope comprises a sequence selected from any of SEQ ID NOs: 1-112.
41. The vaccine or medicament of any of claim 35-40, wherein the one or more peptides further comprise a signal sequence, major histocompatibility complex (MHC) transmembrane domain, an MHC cytoplasmic domain, or any combination thereof.
42. The vaccine or medicament of any of claims 35-41, wherein the one or more nucleic acids comprise mRNA.
43. The vaccine or medicament of any of claims 35-42, wherein the one or more nucleic acids further comprises: a 5’ untranslated region (UTR), a 5’ cap, a 3’ UTR, a 3’ tailing sequence, or any combination thereof.
44. The vaccine or medicament of claim 43, wherein the 3’ tailing sequence comprises a polyA tail, a polyG quartet, a stem loop sequence, a triple helix forming sequence, a tRNA-like sequence, or any combination thereof.
45. The vaccine or medicament of claim 43 or claim 44, wherein the one or more nucleic acids further comprises at least one chemically modified nucleotide.
46. The vaccine or medicament of claim 45, wherein the at least one chemically modified nucleotide comprises a modified uracil, a modified cytosine, or a combination thereof.
47. The vaccine or medicament of any of claims 34-46, wherein the one or more peptides are encoded on a single nucleic acid.
48. The vaccine or medicament of claim 47, wherein the single nucleic acid encodes two or more peptides.
49. The vaccine or medicament of claim 48, wherein the two or more peptides are each separated by a linker.
50. The vaccine or medicament of claim 49, wherein the linker comprises a cleavable linker, a flexible linker, or a combination thereof.
51. The vaccine or medicament of any of claims 47-50, wherein the single nucleic acid is an mRNA.
52. The vaccine or medicament of any of claims 47-51, wherein the adjuvant, the delivery vehicle or the combination thereof comprises a lipid.
53. The vaccine or medicament of claim 52, wherein the lipid comprises a cationic lipid, a neutral and/or non-cationic lipid, a sterol, or any combination thereof.
54. The vaccine or medicament of claim 53, wherein the non-cationic lipid comprises a phospholipid.
55. The vaccine or medicament of claim 53 or claim 54, wherein the sterol comprises cholesterol or a modification or ester thereof.
56. The vaccine or medicament of any of claims 52-55, wherein the delivery vehicle comprises a lipid particle encapsulating the composition.
57. The vaccine or medicament of claim 56, wherein the lipid nanoparticle comprises a polyethylene glycol (PEG)-lipid conjugate.
58. A method of treating or preventing cancer in a subject, comprising administering an effective amount of the vaccine or medicament of any of claims 35-57.
59. The method of claim 58, wherein the cancer is associated with tumor- associated bacteria.
60. The method of claim 58 or claim 59, wherein the cancer comprises gastric cancer, colorectal cancer, or a combination thereof.
61. The method of any of claims 58-60, wherein the subject has cancer, has had cancer, is predisposed to cancer, is suspected of having cancer, or has a family history of cancer.
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US20200256877A1 (en) * 2017-10-09 2020-08-13 Enterome S.A. Microbiota Sequence Variants Of Tumor-Related Antigenic Epitopes
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