Classifications

• • 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
  • A61K39/001169—Tumor associated carbohydrates
  • A61K39/00117—Mucins, e.g. MUC-1
• • 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
  • A61K39/00118—Cancer antigens from embryonic or fetal origin
  • A61K39/001182—Carcinoembryonic antigen [CEA]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
  • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
• • 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
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • 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
  • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• BACKGROUND OF THE INVENTION Cancer is the second leading cause of death in the United States (behind heart disease), with colorectal cancer (CRC) ranking among the top three carcinogenic causes of morbidity and mortality for 2019 in both men and women of the United States (Siegel et al., CA Cancer J Clin 2019; 69(1): 7-34; Miller et al., CA Cancer J Clin 2019; 69(5): 363- 85).
• CRC colorectal cancer
• Conventional treatment therapies for colon, rectal, and anal cancers typically include surgical resection, radiation, and/or chemotherapy involving an ever-expanding array of constantly improving classes of compounds (Libutti et al., Philadelphia: Wolters Kluwer Health; 2016; Libutti et al., Philadelphia: Wolters Kluwer Health; 2016; Czito et al., Philadelphia: Wolters Kluwer Health; 2016).
• Libutti et al. Philadelphia: Wolters Kluwer Health; 2016
• Libutti et al., Philadelphia: Wolters Kluwer Health; 2016 Libutti et al., Philadelphia: Wolters Kluwer Health; 2016
• Czito et al. Philadelphia: Wolters Kluwer Health; 2016
• Some attenuated vaccine strains have elicited a broad array of immune responses in clinical trials including intestinal secretory IgA antibodies, serum IgG antibodies, and T cell mediated immunity (Tacket et al., Infect Immun 1997; 65(2): 452- 6.; Tacket et al., Infect Immun 2000; 68: 1196-201). The ability of live orally administered S.
• tumor-associated macrophages are anergic (M2 polarization), with reduced phagocytic activity and secretion of inflammatory cytokines to promote tumor clearance otherwise carried out by classically active M1 polarized macrophages (Kather et al., Br J Cancer, (2019), 120:871-82; ME et al., Mol Aspects Med, (2019), 69:123-9; Schmitt et al., Nat Rev Immunol, (2021).
• Typhi-bacterial live vector exhibits enhanced delivery of the fusion protein to the immune system through increased formation of recombinant outer membrane vesicles (rOMVs).
• the S. Typhi-bacterial live vector expresses a ClyA protein that is exported from the live vaccine via rOMVs.
• the invention provides a composition comprising isolated recombinant outer membrane vesicles from Salmonella Typhi comprising one or more cancer antigens, wherein the Salmonella Typhi has been engineered to express the antigen.
• FIG. 11 IFN- ⁇ responses against A3B3-MUC1 in BALB/c mice immunized intramuscularly with 2 ⁇ g of rOMVs on days 0 and 21. Splenocytes were harvested on day 35, pooled for each group, and immediately used for ELISpot analysis without frozen storage.
• FIG. 12 IFN- ⁇ responses against A3B3-MUC1 in C57BL/6 mice immunized as described in Table 4. Splenocytes were harvested on day 21, pooled for each group, and immediately used for ELISpot analysis without frozen storage. See text for further experimental details.
• FIG. 13 IFN- ⁇ responses against A3B3-MUC1 in C57BL/6 mice immunized as described in Table 4. Splenocytes were harvested on day 21, pooled for each group, and immediately used for ELISpot analysis without frozen storage. See text for further experimental details.
• FIG. 13 IFN- ⁇ responses against A3B3-MUC1 in C57BL/6 mice immunized as described in Table 4. Splenocytes were harvested on day 21, pooled for each group, and immediately used for ELISpot analysis without frozen storage. See text for further experimental details.
• FIG. 14 Immunogenicity and therapeutic efficacy of re-engineered rOMVs expressing individual domains of the CRC fusion protein, compared to the un-modified rOMV expressing full-length A3B3-MUC1 fusion protein.
• A graphic describing the re- engineering strategy for construction of rOMVs expressing single CRC domains.
• B anti- A3B3-MUC1 serum IgG titers.
• C ELISPOT assay measuring IFN- ⁇ secretion of isolated splenocytes stimulated with A3B3-MUC1 fusion protein.
• D Tumor volumes measured in C57BL/6 mice challenged on day 0 with MC38-CEAv2 and treated intravenously on days 3, 5, 7, and 9 with 0.75 mg of rA3B3 ⁇ fliC , rMUC1 ⁇ fliC , rOMV ⁇ fliC-CRC , or empty rOMV ⁇ fliC- pagL vesicles.
• one or more codons have been optimized to enhance expression.
• the putative ribosome binding sites have been optimized to enhance expression.
• the nucleotide sequence of PagL comprises SEQ ID NOS:1, 3 or 5.
• the amino acid sequence of PagL comprises SEQ ID NOS:2 or 4.
• the S. Typhi-bacterial live vector over-expresses one or more further vesicle-catalyzing proteins such as ClyA responsible for naturally inducing OMV formation in S. Typhi.
• ClyA encompasses full length ClyA as well as biologically active fragments and variants of ClyA.
• ClyA protein can cause hemolysis in target cells.
• the present invention encompasses use of both hemolytically active and hemolytically inactive forms of ClyA, with hemolytically inactive mutant forms being more preferred where preservation of antigen export and immunogenicity of the resulting proteins can be maintained.
• the nucleotide and amino acid sequence of ClyA corresponds to SEQ ID NOS: 15 and 16, respectively.
• the ClyA is mutated to reduce the hemolytic activity of ClyA while still retaining the export function of ClyA.
• the ClyA mutant is ClyA I198N.
• the ClyA mutant is ClyA C285W.
• the cancer is selected from colon, colorectal, leukemia (e.g., chronic lymphocytic leukemia or acute myeloid leukemia) lymphoma (e.g., Non-Hodgkin Lymphoma), breast, prostate, liver, pancreatic, brain, lung (e.g., small cell or non-small cell lung cancer) and skin cancer (e.g., melanoma), uterine, gallbladder, adenocarcinoma, cholangiocarcinoma, esophageal, gastric, glioblastoma, ovarian, urinary bladder cancer, and head and neck cancer.
• the cancer is colon cancer.
• the cancer antigen comprises one or more of the following antigens (or antigenic fragments or derivatives) selected from neo-antigen, carcinoembryonic antigen (CEA), human epithelial mucin MUC-1, the cancer-testis antigen NY-ESO-1, HER2/neu, SART-1, SART-2, KIAA0156, ART-1, ART-4, cyclophilin B, mutated elongation factor 2, malic enzyme, and alpha-actinin-4, eIF4G, aldolase, annexin XI, Rip-1, and NY-LU-12, fibromodulin, RHAMM/CD168, MDM2, hTERT, the oncofetal antigen-immature laminin receptor protein (OFAiLRP), adipophilin, survivin, KW1 to KW14 and the tumor-derived IgVHCDR3 region, RHAMM-derived R3 peptide, B7.1, ICAM-1, LFA-3
• the cancer antigen comprises one or more antigenic fragments of CEA and/or MUC1.
• the CEA antigen and MUC1 antigen are part of a fusion protein.
• the CEA antigen and MUC1 antigen are part of the same fusion protein.
• the CEA antigen comprises domain A3B3.
• the amino acid sequence of domain A3B3 comprises SEQ ID NO:23.
• the nucleotide sequence of domain A3B3 comprises SEQ ID NO:30.
• the MUC1 antigen is a fragment comprising multiple repeat domains.
• the amino acid sequence of the MUC1 fragment comprises SEQ ID NO:24.
• the expression cassette encodes a fusion protein comprising a surface presentation protein and one or more antigens.
• the surface expression protein is selected from Lpp-OmpA, Lpp-OmpT and ClyA.
• the cancer antigen is a fusion protein comprising CEA domain A3B3 and a MUC1 fragment fused to a surface presentation protein such as Lpp- OmpA.
• the cancer antigen comprises a Lpp-OmpA:A3B3:MUC1 fusion protein comprising the amino acid sequence of SEQ ID NO:12.
• the cancer antigen comprises a clyA I198N :a3b3:muc1 fusion protein encoded by SEQ ID NO:19.
• the ClyA I198N amino acid sequence comprises SEQ ID NO:25.
• the polypeptide components of the fusion protein are separated by one or more linker amino acid sequences.
• the linker amino acid sequence is SEQ ID NO:22.
• the cancer antigen exhibits no glycosylation.
• the invention provides a nucleic acid comprising any of SEQ ID NOS:11, 13, 17, or 19.
• the S. Typhi strain can carry one or more additional chromosomal mutations in an essential gene that is expressed on a plasmid.
• the plasmid also encodes a heterologous protein in accordance with the invention, enabling selection and genetic stabilization of the plasmid and preventing loss in S. Typhi.
• the S. Typhi strain carries a mutation in the ssb gene which is encoded on a selection expression plasmid. If heterologous antigens or other proteins are overexpressed using plasmids, plasmid stability can be a key factor in the development of high quality attenuated S. Typhi vaccines.
• Plasmidless bacterial cells tend to accumulate more rapidly than plasmid-bearing cells.
• One reason for this increased rate of accumulation is that the transcription and translation of plasmid genes imposes a metabolic burden which slows cell growth and gives plasmidless cells a competitive advantage.
• foreign plasmid gene products are sometimes toxic to the host cell.
• the plasmid it is advantageous for the plasmid to be under some form of selective pressure, in order to ensure that the encoded antigens are properly and efficiently expressed, so that a robust and effective immune response can be achieved.
• the plasmid is selected within S. Typhi using a non- antibiotic selection system.
• the fragment ranges from 1-75 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1-20 amino acids, 1- 15 amino acids, 1-10 amino acids and 1-5 amino acids.
• the fragment corresponds to carboxyl-terminal truncation mutant.
• the number of carboxyl terminal amino acids missing from the fragment ranges from 1-100 amino acids.
• it ranges from 1-75 amino acids, 1-50 amino acids, 1-40 amino acids, 1-30 amino acids, 1-25 amino acids, 1- 20 amino acids, 1-15 amino acids, 1-10 amino acids and 1-5 amino acids.
• the fragment corresponds to an internal fragment that lacks both the amino and carboxyl terminal amino acids.
• the fragment is 7-200 amino acid residues in length. In some embodiments, the fragment is 10-100 amino acid residues, 15-85 amino acid residues, 25-65 amino acid residues or 30-50 amino acid residues in length. In some embodiments, the fragment is 7 amino acids, 10 amino acids, 12 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, 50 amino acids 55 amino acids, 60 amino acids, 80 amino acids or 100 amino acids in length. In some embodiments, the fragment is at least 50 amino acids, 100 amino acids, 150 amino acids, 200 amino acids or at least 250 amino acids in length.
• the invention provides a polypeptide comprising any of SEQ ID NOS:12, 14, 18, or 20.
• the polypeptides have an amino acid sequence at least 80, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptides described herein or antigenic or biologically active fragments thereof.
• the variants are those that vary from the reference by conservative amino acid substitutions, i.e., those that substitute a residue with another of like characteristics.
• compositions may be manufactured without undue experimentation in a manner that is itself known, e.g., by means of conventional mixing, dissolving, dragee-making, levitating, emulsifying, encapsulating, entrapping, spray-drying, or lyophilizing processes, or any combination thereof.
• the attenuated S. Typhi-bacterial live vector expressing one or more antigens or isolated recombinant outer membrane vesicles are administered mucosally.
• Suitable routes of administration may include, for example, oral, lingual, sublingual, rectal, transmucosal, nasal, buccal, intrabuccal, intravaginal, or intestinal administration; intravesicular; intraurethral; administration by inhalation; intranasal, or intraocular injections, and optionally in a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted drug delivery system. Combinations of administrative routes are possible.
• the dose rate and suitable dosage forms for the bacterial live vector vaccine compositions or recombinant isolated outer membrane vesicles of the present invention may be readily determined by those of ordinary skill in the art without undue experimentation, by use of conventional antibody titer determination techniques and conventional bioefficacy/biocompatibility protocols.
• the invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject an immunologically-effective amount of isolated recombinant outer membrane vesicles from Salmonella Typhi comprising one or more cancer antigens, wherein the Salmonella Typhi has been engineered to express the antigen, wherein the outer membrane vesicle is delivered to a mucosal tissue of the subject.
• the method comprises administering a combination of live Salmonella Typhi vectors of the invention to a subject.
• the combination of vectors is present in the same composition.
• the vectors are present in separate compositions.
• the method comprises administering a combination of isolated recombinant outer membrane vesicles to a subject.
• the invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject an immunologically-effective amount of isolated recombinant outer membrane vesicles comprising one or more cancer antigens, wherein the recombinant outer membrane vesicles are administered to the subject by an intravenous route, an intramuscular route, or both, wherein the recombinant outer membrane vesicles are administered one or more times.
• the recombinant outer membrane vesicles are from a live Salmonella Typhi vector that has been engineered to express: a.
• the live Salmonella Typhi vectors or isolated recombinant outer membrane vesicles are administered to the subject with one or more additional therapies to treat the cancer.
• the one or more additional therapies are selected from chemotherapy, radiation, surgery, and immunotherapy.
• the live Salmonella Typhi vectors or isolated recombinant outer membrane vesicles are co-administered to the subject with one or more additional therapies as soon as possible after early detection of the target cancer. It is now well appreciated that early detection will enhance the odds of successful treatment of solid tumors prior to progression to large solid masses that can dramatically reduce access of target cancer antigens to therapeutic treatment of any kind.
• the Salmonella Typhi vectors and/or recombinant outer membrane vesicles are administered to a subject following early detection of cancer. This is advantageous because early detection and intervention can improve the probability of curing the cancer in the subject.
• the method comprises a diagnostics screening method to detect the presence of cancer in the subject.
• diagnostic screening methods can include imaging and/or screening tissue or blood samples from the subject for the presence of cancer cells or markers of the cancer. Suitable early diagnostic methodologies are described, for example, in Cohen et al. Science, 359: 926-930 (2016) and Lennon et al., Science 10.1126/science.abb9601 (2020), which are incorporated by reference herein in their entirety.
• cancer can be detected by conducting a liquid biopsy, for example, by taking a blood sample and detecting cancer cells or makers in the sample.
• a positive blood test detecting a cancer can be confirmed by scanning, e.g., PET-CT scanning, to identify a tumor mass.
• protein or nucleic acid markers are detected in the liquid sample to identify the presence of a cancer in the subject.
• the type of cancer that can be identified in blood is not necessarily limiting.
• Such cancers can include lung cancer, ovarian cancer, colorectal cancer, breast cancer, lymphoma, kidney cancer, thyroid cancer, uterine cancer, and cancer of the appendix.
• cancer of the ovary can be detected in blood by detecting the presence of marker TP53, CA19-9, CA125, CA15-3, or a combination thereof.
• cancer of the lung can be detected in blood by detecting the presence of marker KRAS, TP53, CA15-3, HGF, CEA, EGFR, PIK3CA, or a combination thereof.
• cancer of the uterus can be detected in blood by detecting the presence of marker TP53, CA19-9 or a combination thereof.
• cancer of the thyroid can be detected in blood by detecting the presence of marker CEA.
• colorectal cancer can be detected in blood by detecting the presence of marker BRAF, TP53 or a combination thereof.
• breast cancer can be detected in blood by detecting the presence of marker PIK3CA, TP53 or a combination thereof.
• lymphoma can be detected in blood by detecting the presence of marker HGF, NRAS or a combination thereof.
• kidney can be detected in blood by detecting the presence of marker KRAS.
• cancer of the appendix can be detected in blood by detecting the presence of marker CEA.
• Vaccine strategies are well known in the art and therefore the vaccination strategy encompassed by the invention does not limit the invention in any manner. In certain aspects of the invention, the S.
• Typhi live vector vaccines as taught herein can be administered orally or via another mucosal route and subsequently boosted parentally with a vaccine composition comprising isolated recombinant outer membrane vesicles from a S. Typhi vector comprising one or more of the cancer antigens.
• the present invention is directed to methods of inducing an immune response against an antigen in a subject in need thereof, comprising administering to the subject an immunologically-effective amount of a live Salmonella Typhi vector of the invention as a prime, and subsequently administering a boost composition comprising a composition comprising isolated recombinant outer membrane vesicles from a S. Typhi vector comprising one or more of the cancer antigens.
• an "immune response” is the physiological response of the subject's immune system to an immunizing composition.
• An immune response may include an innate immune response, an adaptive immune response, or both.
• the immune response is a protective immune response.
• a protective immune response confers immunological cellular memory upon the subject, with the effect that a secondary exposure to the same or a similar antigen is characterized by one or more of the following characteristics: shorter lag phase than the lag phase resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; production of antibody which continues for a longer period than production of antibody resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a change in the type and quality of antibody produced in comparison to the type and quality of antibody produced upon exposure to the selected antigen in the absence of prior exposure to the immunizing composition; a shift in class response, with IgG antibodies appearing in higher concentrations and with greater persistence than IgM, than occurs in response to exposure to the selected antigen in the absence of prior exposure to the immunizing composition; an increased average affinity (binding constant) of the antibodies for the antigen in comparison with the average affinity of antibodies for the antigen resulting from exposure to the selected antigen in the absence of prior exposure to the immunizing composition; and/
• the term "immunologically-effective amount” means the total amount of a live S. Typhi vector or isolated recombinant outer membrane vesicles that is sufficient to show an enhanced immune response in the subject.
• “immunologically-effective amount” is applied to an individual therapeutic agent administered alone, the term refers to that therapeutic agent alone.
• the term refers to combined amounts of the ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
• the particular dosage depends upon the age, weight, sex and medical condition of the subject to be treated, as well as on the method of administration. Suitable doses can be readily determined by those of skill in the art.
• the dose of the recombinant outer membrane vesicles that is administered is not necessarily limiting.
• the dose of recombinant outer membrane vesicles can be administered one or more times, spaced out over time. In some embodiments, the dose is adjusted up or down over time.
• a dose of from about 0.001 ⁇ g/kg to about 10 ⁇ g/kg of recombinant outer membrane vesicles is administered to the subject, based on the subject’s body weight (in kg). In some embodiments, a dose of from about 0.1 ⁇ g/kg to about 10 ⁇ g/kg of recombinant outer membrane vesicles is administered to the subject.
• a dose of from about 0.25 ⁇ g/kg to about 5.0 ⁇ g/kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, a dose of from about 0.25 ⁇ g/kg to about 2.5 ⁇ g/kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, a dose of from about 0.25 ⁇ g/kg to about 1.0 ⁇ g/kg of recombinant outer membrane vesicles is administered to the subject. In some embodiments, the recombinant outer membrane vesicles are administered via a parenteral route. In some embodiments, the route of administration is intravenous.
• the route of administration is intramuscular.
• the subject is administered multiple doses of the recombinant outer membrane vesicles (or multiple doses of live Salmonella Typhi vector, or a combination of recombinant outer membrane vesicles and live Salmonella Typhi vector) separated by a period of time.
• the subject is administered two doses, three doses, four doses, five doses, six doses or more.
• the doses are spaced from about 2 days-12 weeks apart.
• the doses are spaced from about 2-7 days apart.
• the subject is administered from 2-5 doses spaced 2-7 days apart.
• the subject is administered a priming dose of live Salmonella Typhi vector administered mucosally, e.g., intranasal, followed by a parenterally, e.g., intravenously or intramuscularly, administered boosting dose of the recombinant outer membrane vesicles, e.g., from about 1-4 weeks later.
• the subject is administered a priming dose of recombinant outer membrane vesicles administered parenterally, e.g., intravenously or intramuscularly, followed by a parenterally, e.g., intravenously or intramuscularly, administered boosting dose of the recombinant outer membrane vesicles, e.g., from about 1-4 weeks later.
• the subject is administered a priming dose of live Salmonella Typhi vector administered mucosally, e.g., intranasal, followed by a mucosally administered, e.g., intranasal, boosting dose of the live Salmonella Typhi vector, e.g., from about 1-4 weeks later.
• the S. Typhi-bacterial live vector can be administered intranasally at a dose of about 1 x 10 9 CFU. In some embodiments, the attenuated S. Typhi- bacterial live vector can be administered intranasally at a dose of about 1 x 10 8 CFU to about 1 x 10 10 CFU.
• subject refers to animals, such as mammals. For example, mammals contemplated include humans, primates, dogs, cats, sheep, cattle, goats, pigs, horses, mice, rats, rabbits, guinea pigs, and the like. The terms "subject,” “patient,” and “host” are used interchangeably.
• the subject is administered a multivalent recombinant outer membrane vesicle following either early diagnosis of colorectal cancer, or a subject in the early stages of solid tumor progression with sufficient blood vessel density to potentially allow for passive deposition of recombinant outer membrane vesicles.
• a patient population could be identified through the use of newly developed blood-based diagnostic assays (Lennon et al., Science, (2020), 369(6499).
• a subject that is administered recombinant outer membrane vesicles has an increased risk for colorectal or other cancers in which MUC1 is often overexpressed (see, e.g., Crosby et al., Journal for immunotherapy of cancer, (2020), 8(2); Kimura et al., Cancer Prev Res (Phila) 2013; 6(1): 18-26; Lohmueller et al., Sci Rep 2016; 6: 31740).
• the live Salmonella Typhi vectors or isolated recombinant outer membrane vesicles of the invention may be administered to warm-blooded mammals of any age.
• the live Salmonella Typhi vectors can be administered as a single dose or multiple priming doses, followed by one or more boosters.
• a subject can receive a single dose, then be administered a booster dose up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 or more years later.
• Application of the teachings of the present invention to a specific problem is within the capabilities of one having ordinary skill in the art in light of the teaching contained herein. Examples of the compositions and methods of the invention appear in the following non-limiting Examples.
• EXAMPLES Example 1. Development of a PagL-mediated antigen delivery platform.
• ClyA is a hemolysin with cytopathic characteristics that may reduce the clinical acceptability of candidate vaccine strains in which ClyA is over-expressed
• we sought to develop a non-pathogenic alternative for inducing formation and export of OMVs based on PagL (Ludwig et al., Mol Microbiol 1999; 31(2): 557-67.; Lai et al., Infect Immun 2000; 68(7): 4363-7).
• pagL v1 carries an optimized ribosome binding site (RBS), an ATG start codon, and several optimized codons codon at the beginning of the gene to enhance translation efficiency.
• pagL v2 is similar to v1 but contains a GTG start codon to slightly reduce translation efficiency.
• pagL v3 is essentially identical to the wild type chromosomal sequence of the pagL gene naturally present within Salmonella enterica serovar Typhimurium. Therefore, we expected the highest levels of PagL synthesis from v1, with decreasing levels of synthesis from v2 and the lowest levels of synthesis from v3.
• Each cassette was inserted as a BamHI-NheI fragment into our non-antibiotic low- copy-number expression plasmid pSEC10 digested with BamHI and NheI, replacing the clyA gene to create pPagL; the expected sequence of pPagL v1 is listed in SEQ ID NO:6.
• BamA is an ⁇ 90kDa protein that constitutes an essential component of a 5-protein outer membrane ⁇ -barrel assembly machinery (BAM) complex that catalyzes the insertion of ⁇ -barrel proteins into the outer membrane of Gram negative bacteria (Noinaj et al., Nature reviews Microbiology 2017; 15(4): 197-204.).
• BAM 5-protein outer membrane ⁇ -barrel assembly machinery
• PagL lipid A deacylase
• ClyA cytolysin A
• each plasmid was introduced into the reporter strain CVD 910 ⁇ guaBA::clyA. Strains were then grown under inducing conditions at 37°C into early- log phase growth, and hemolytic activity was measured at OD 540 for approximately 2 x 10 7 CFU of bacteria against sheep red blood cells. As shown in Figure 3, no hemolytic activity was present in the vaccine strain CVD 910 (lane 2). As expected, the hemolytic activity of chromosomally encoded ClyA was not detected in CVD 910 ⁇ guaBA::clyA (lane 3), due to reduced expression levels from the chromosome.
• BAM ⁇ - barrel assembly
• bamA Ab v1 To avoid potentially lethal over-expression of AbBamA, we therefore engineered ribosome binding sites positioned 5 bases (bamA Ab v1) or 4 bases bamA Ab v2) upstream of a GTG start codon to more tightly control translation levels; given that the ideal positioning of an RBS is 7-9 bases away from the start codon, we expected bamA Ab v1 to have slightly higher expression levels than bamA Ab v2 (Ringquist et al., Mol Microbiol 1992; 6(9): 1219-29.).
• bamA alleles As with the pagL alleles, we engineered the bamA alleles under the transcriptional control of a PompC promoter and inserted the resulting cassettes into our low copy expression plasmid to create pAbBamAv1 and pAbBamAv2. These plasmids were then introduced into CVD 910 ⁇ guaBA::clyA. Although ClyA does not possess a ⁇ -barrel structure, we wanted to investigate any potential effect of AbBamA over-expression on OMV formation (Wallace et al., Cell 2000; 100: 265-76.).
• AbBamA can enhance the formation of outer membrane vesicles, phenotypically tagged with ClyA and exported from CVD 910, and may also enhance the export of vesicles carrying the antigen or other foreign antigens relevant to vaccine development.
• This technology is not limited to vaccine development against human pathogens but can also be used in veterinary and other applications, such as the development of immunotherapeutic vaccines against solid tumors as well (Niethammer et al., BMC Cancer 2012; 12: 361; Schmitz-Winnenthal et al., Oncoimmunology 2015; 4(4): e1001217.; Schmitz-Winnenthal et al., Oncoimmunology 2018; 7(4): e1303584).
• Example 4 Development of a candidate bivalent S. Typhi-based colorectal cancer vaccine Using previously attenuated and highly immunogenic S.
• Typhi-based carrier vaccine strains we have recently engineered and functionally tested an osmotically inducible and highly efficient recombinant outer membrane vesicle (rOMV) antigen delivery system driven by over-expression of the lipid A deacylase PagL (Galen et al., J Infect Dis 2009; 199(3): 326-35; Galen et al., Vaccine 2014;32(35): 4376-85; Galen et al., Infect Immun 2015; 83(1): 161-72; Elhenawy et al., mBio 2016; 7(4): pii: e00940-16. doi:10.1128/mBio.-16).
• rOMV outer membrane vesicle
• PagL is encoded by a genetically stabilized low copy number expression plasmid, stabilized through trans-complementation of an otherwise lethal chromosomal deletion of the single stranded binding protein (SSB) ( Figure 5) (Galen et al., Infect Immun 2010; 78(1): 337-47.). We have exported (see FIG.
• the targeted cancer antigens Upon osmotic induction of this vaccine plasmid, the targeted cancer antigens will be expressed on the surface of the S. Typhi-based carrier vaccine, followed by export of these antigens via outer membrane vesicles induced by co-expression of PagL.
• the two intended fusion proteins to be co-expressed with PagL are each comprised of a surface expression cassette operationally linked to two additional cancer antigen cassettes, each separated by an engineered linker region [designated as A(EAAAK) 4 A] (Fig. 7); the linker region is designed to fold into a rigid alpha helix which will separate each cancer domain to allow proper folding after translation (Chen et al., Advanced drug delivery reviews 2013; 65(10): 1357-69).
• the surface expression cassette is composed of either a modified and patented non-hemolytic version of the ClyA protein (designated here as clyA I198N ) or a previously published surface expression cassette designated lpp-ompA (designated here as LOA) (Francisco el al., Proc Natl Acad Sci U S A 1992; 89(7): 2713- 7).
• These surface expression cassettes are in turn operationally linked to a cassette encoding a cancer fusion protein comprised of two domains, one from the cancer antigen carcinoembryonic antigen (CEA) and the other from MUC1 (Fig. 7).
• the domain from CEA is designated A3B3 (encoded by a3b3) and encodes a 179 amino acid region from the 6 th and 7 th Ig domains of CEA (Oikawa et al., Biochem Biophys Res Commun 1987; 142(2): 511-8; Hefta et al., Cancer Res 1992; 52(20): 5647-55.; Zaremba et al., Cancer Res 1997; 57(20): 4570-7; Nukaya et al., Int J Cancer 1999; 80(1): 92-7; Gu et al., Gastroenterology 2020; 158(1): 238-52); the MUC1 domain is comprised of 140 amino acids, representing 7 repeat regions from the human MUC1 protein (Engelmann et al., J Biol Chem 2001; 276(30): 27764-9; Soares et al., J Immunol 2001; 166(11): 6555-63; Scheikl-G
• the concentration of rOMVs was rigorously determined using a 3-Deoxy-D-manno-Octulosonic Acid (KDO) assay as prescribed by R.E.W. Hanock (http://cmdr.ubc.ca/bobh/method/kdo-assay/).0.5 ⁇ g of each purified rOMV were 0 then analyzed by Coomassie Brilliant Blue staining and western immunoblot analysis. As shown in Fig.10A, multiple bands are detected in the Coomassie-stained samples, as would be expected with outer membrane vesicles containing the various proteins and lipoproteins found in the outer membrane of S. Typhi.
• KDO 3-Deoxy-D-manno-Octulosonic Acid
• a strong band migrating at approximately 50kDa in lanes 2 and 3 of panel A disappears from lanes 4-7; this is consistent with the 5 known molecular weight of ⁇ 50 kDa for the flagellin FliC, which was deleted from the strains in lanes 4-7.
• the CRC fusion protein is clearly detected in lanes 3, 5, and 7 as expected, but not as a single band.
• the faster running species running below the strong top band are 0 additional species containing one or more of the expected disulfide bridges.
• Typhi carrier strain (and subsequently exported rOMVs) by genetic fusion of the A3B3- MUC1 fusion to an Lpp-OmpA surface localization peptide (Francisco et al., Proc Natl Acad Sci USA, (1992), 89:2713-7); the lpp-ompA-a3b3-muc1 gene encoding this fusion protein was then inserted into pPagL downstream of pagLv1 to create the final expression plasmid pPagL-LOAM.
• This plasmid was electroporated into CVD910 ⁇ guaBA::P ompC - bamA Ab ⁇ fliC::P ompC -lpxE Fn (see Table 1) creating the CRC-targeting carrier strain CVD910 ⁇ guaBA::PompC-bamA Ab ⁇ fliC::PompC-lpxE Fn (pPagL-LOAM), capable of exporting the CRC-targeting vesicles designated rOMV LpxE-CRC .
• mice 50 BALB/c mice (6-8 week old) were randomly assorted into five groups and immunized intramuscularly with either 4.0 ⁇ g of empty unadjuvanted rOMV LpxE empty vesicles as a negative control or unadjuvanted rOMV LpxE-CRC in escalating doses 0.5, 1.0, 2.0, or 4.0 ⁇ g; we also administered PBS to an additional negative control group of 5 mice.
• the concentration of rOMVs was rigorously determined using a 3-Deoxy-D-manno- Octulosonic Acid (KDO) assay. Sera were collected on days 0 and 21, and antigen-specific serum IgG was measured in pooled sera by ELISA.
• KDO 3-Deoxy-D-manno- Octulosonic Acid
• mice el 5 5 5 5 60 BALB/c mice (6-8 week old) were randomly assorted into six groups and immunized intramuscularly with 2.0 ⁇ g of unadjuvanted rOMV vesicles on days 0 and 21; we also administered PBS to an additional negative control group of 10 mice. Sera were collected on days 0, 20 and 35. Antigen-specific serum IgG was measured by ELISA and IFN- ⁇ responses were determined using harvested splenocytes and a MabTech Mouse IFN- ⁇ ELISpotPLUS HRP kit according to manufacturer’s instructions.
• This MC38-CEA model has been used in preclinical safety studies of a CEA vaccine antigen carrying the immunodominant T cell epitope CEA(6D) (also included in our engineered fusion protein); significant therapeutic effects were reported in an MC38-CEA mouse model which translated well to clinical trials (Crosby et al., Journal for immunotherapy of cancer, (2020), 8(2); Morse et al., J Clin Invest, (2010), 120:3234-41; Osada et al., Cancer Immunol Immunother 2012; 61(11): 1941-51).
• This leaky tumor vasculature allows for passive extravasation of small nanostructures such as OMVs from the blood into tumor tissue (Fang et al., Advanced drug delivery reviews, (2020), 157: 142-60). From within the tumor tissue, these OMVs can then passively drain through the lymphatic system into regional lymph nodes, potentially encountering and activating antigen presenting cells including dendritic cells to elicit tumor-specific immunity (Fang et al., Advanced drug delivery reviews, (2020), 157: 142-60; Kelly et al., Expert review of vaccines, (2019), 18: 269-80).
• splenocytes were plated in mouse IFN- ⁇ ELISpot plates (250,000 cells/well; six replicates per rOMV vaccine) and stimulated with either (1) A3B3- MUC1 (10 ⁇ g/ml) (2) concanavalin A (4 ⁇ g/ml - positive control) or (3) media (negative control).
• the cells were incubated for 40 hours (37oC, 5% CO 2 ), then washed, and an anti- 0 IFN- ⁇ (biotinylated) antibody was added. Following 1h incubation, the plates were washed, streptavidin conjugated to horse radish peroxidase (SA-HRP) was added (1h), and the plates were washed again.
• SA-HRP horse radish peroxidase
• SFC Spot forming cells
• mice (10 mice per group) were implanted subcutaneously with 300,000 tumor cells on day 1 and then treated intravenously with 0.75 ⁇ g of vesicles (based on quantitation of LPS concentration using a KDO assay) on days 3, 5, 7, and 9. Progression of tumors was monitored by calculating tumor volumes through day 28. As shown in Figures 13A and B, all groups treated intravenously with rOMVs experienced reductions in tumor volumes.
• TLRs Toll-like receptors
• ICIs immune checkpoint inhibitors
• TLR agonists have the potential to convert “cold tumors” into “hot tumors” making TLRs in combination with immune checkpoint inhibitors, potential targets for cancer therapies.
• TLR agonists induce cytokine secretion, leading to activation of cytotoxic T lymphocytes (CTLs), resulting in an immune response that mediates inflammation and can reduce tumor burden.
• CTLs cytotoxic T lymphocytes
• TLR Toll-Like Receptor
• rOMV DfliC-CRC were evaluated at four concentrations and compared to control ligands. These steps are performed in triplicate. As shown in Table 6, rOMV DfliC-CRC exhibits a major stimulatory effect on HEK-Blue hTLR2 and HEK-Blue hTLR4 at a dilution of 1/10000 and below. A minor stimulatory effect is also observed on HEK-Blue hTLR3, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8 and HEK-Blue hTLR9 at a 1/10 dilution. Table 6.
• Example 14 Efficacy of rOMVs vaccines administered by intravenous and intramuscular routes.
• TGI Percentage Tumor Growth Inhibition

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Epidemiology (AREA)
• Public Health (AREA)
• Chemical & Material Sciences (AREA)
• Immunology (AREA)
• Medicinal Chemistry (AREA)
• Microbiology (AREA)
• Mycology (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Oncology (AREA)
• Molecular Biology (AREA)
• Developmental Biology & Embryology (AREA)
• Gynecology & Obstetrics (AREA)
• Pregnancy & Childbirth (AREA)
• Reproductive Health (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=90191910

Family Applications (1)

Country Status (5)

Family Cites Families (1)

• 2023
  • 2023-09-05 EP EP23863936.3A patent/EP4583908A2/de active Pending
  • 2023-09-05 CA CA3266756A patent/CA3266756A1/en active Pending
  • 2023-09-05 JP JP2025538207A patent/JP2025529593A/ja active Pending
  • 2023-09-05 AU AU2023338191A patent/AU2023338191A1/en active Pending
  • 2023-09-05 WO PCT/US2023/073489 patent/WO2024054806A2/en not_active Ceased

Also Published As

Similar Documents

Legal Events