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US-20260124255-A1 - Salmonella vectored therapies for treatment of cancer

US20260124255A1US 20260124255 A1US20260124255 A1US 20260124255A1US-20260124255-A1

Abstract

A genetically modified Salmonella cell (GMSC) engineered to display pattern-associated and danger-associated molecular patterns to recruit and enhance innate immunity and exhibit specific targeting to cells and regulated delayed lysis in vivo, the GMSC comprising a first heterologous nucleic acid that encodes a first gene product that causes the GMSC to be selectively localized to and/or internalized by a target cell in vivo and a second heterologous nucleic acid that encodes a second gene product that facilitates killing of the target cells following internalization.

Inventors

  • Roy Curtiss, III
  • Shifeng Wang

Assignees

  • UNIVERSITY OF FLORIDA RESEARCH FOUNDATION, INCORPORATED

Dates

Publication Date
20260507
Application Date
20250317

Claims (20)

  1. 1 . A genetically modified Salmonella cell (GMSC) engineered to display pattern-associated and danger-associated molecular patterns to recruit and enhance innate immunity and exhibit regulated delayed lysis in vivo, the GMSC comprising a first heterologous nucleic acid that encodes a first gene product that causes the GMSC to be selectively localized to and/or internalized by target cells in vivo and a second heterologous nucleic acid that encodes a second gene product that facilitates killing of the target cells following internalization.
  2. 2 .- 29 . (canceled)
  3. 30 . A plasmid comprising a first heterologous nucleic acid that encodes a first gene product whose expression in a GMSC causes the GMSC to be localized to and/or internalized by a target cell in vivo, wherein the first heterologous nucleic acid is operably linked to a first promoter that controls expression of the first heterologous nucleic acid in a Salmonella cell; and a second heterologous nucleic acid that encodes a second gene product whose expression facilitates killing of target cells following internalization by the target cell, wherein the second heterologous nucleic acid is operably linked to (i) a second promoter that controls expression of the second heterologous nucleic acid in the target cell and (ii) to a nucleus targeting sequence that directs the plasmid to a nucleus of target cell.
  4. 31 . The plasmid of claim 30 , wherein the first heterologous nucleic acid comprises a nucleic acid sequence encoding OmpA operably linked to a nucleic acid sequence encoding PLZ4.
  5. 32 . The plasmid of claim 31 , wherein the first heterologous nucleic acid comprises a nucleic acid sequence encoding SEQ ID NO: 2, or an amino sequence comprising at least 90% or 95% sequence identity therewith.
  6. 33 . The plasmid of claim 30 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding CXCL11 or KillerRed, or both.
  7. 34 . The plasmid of claim 33 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding KillerRed.
  8. 35 . The plasmid of claim 33 , wherein CXCL11 is fused to KillerRed.
  9. 36 . The plasmid of claim 35 , further comprising a P2A peptide is situated between CXCL11 and KillerRed.
  10. 37 . The plasmid of claim 30 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding KillerRed fused to neuromodulin N-terminal sequence, or KillerRed fused to a mitochondrial targeting sequence.
  11. 38 . The plasmid of claim 37 , wherein the second heterologous nucleic acid comprises a sequence encoding SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO: 52 and SEQ ID NO: 53; or an amino sequence comprising at least 90% or 95% sequence identity therewith.
  12. 39 . The plasmid of claim 30 , wherein the first heterologous nucleic acid comprises a nucleic acid sequence encoding a LHRH peptide or HER2ScFv, or both.
  13. 40 . The plasmid of claim 39 , wherein the first heterologous nucleic acid comprises a nucleic acid sequence encoding at least one selected from the group consisting of SEQ ID NO: 24, SEQ ID NO:25, and SEQ ID NO: 47; or an amino sequence comprising at least 90% or 95% sequence identity therewith.
  14. 41 . The plasmid of claim 30 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding a HAC-PD1 or HaPD1-IgG, or both.
  15. 42 . The plasmid of claim 41 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding at least one selected from the group consisting of SEQ ID NO: 49, and SEQ ID NO: 51; or an amino sequence comprising at least 90% or 95% sequence identity therewith.
  16. 43 . The plasmid of claim 30 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding an HLA peptide.
  17. 44 . The plasmid of claim 43 , wherein the second heterologous nucleic acid comprises a nucleic acid sequence encoding SEQ ID NO: 21; or an amino sequence comprising at least 90% or 95% sequence identity therewith.
  18. 45 . The plasmid of claim 30 , wherein the first promoter comprises a bacterial promoter, optionally further comprising a sequence such as bla SSopt for secretion of the first gene product, and the second promoter a eukaryotic promoter for delivery to the nucleus of the target cells wherein the bacterial promoter optionally comprises P trc , P tac , P lac , or P lpp , wherein the sequence for secretion optionally comprises bla SSopt and wherein the eukaryotic promoter optionally comprises P CMV or P EF1α .
  19. 46 . (canceled)
  20. 47 . (canceled)

Description

REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Jan. 15, 2026, is named “10457-532US2 .xml” and is 338,511 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND Cancer represents a diversity of disease states characterized by unregulated proliferation of cells that are either freely multiplying in blood and/or lymph or organized into tumor masses. After cardiovascular disease, cancer ranks as the second most common cause of death in the US. Bacteria have been used to target cancers since Coley's observation over 100 years ago that tumors regressed in cancer patients infected with Streptococcus pyogenes. Later, he used killed S. pyogenes, known as Coley's toxin, to treat cancer patients. Unfortunately, the trials of using bacteria as cancer therapy agents stopped for almost 70 years. After Malmgren demonstrated that Clostridium tetani could survive and replicate in necrotic tumors in 1955, studies using bacteria as cancer therapy recommenced and now are widespread in preclinical and clinical studies. Many bacteria have been investigated for their anti-cancer ability, including Bifidobacterium infantis, Escherichia coli, C. tetani, Listeria monocytogenes and Salmonella Typhimurium. While obligate anaerobes, such as Bifidobacterium and Clostridium, are highly effective at accumulating and replicating in necrotic tumors, they do not grow in viable tumor tissues, which limits their efficacy as anti-cancer agents. S. typhimurium is a facultative anaerobe, which can survive and grow in anoxic regions as well as in viable oxygenic regions of tumors. Salmonella also has the ability to identify and penetrate tumors by detecting small molecules such as serine and aspartate in tumors, and accumulates in tumors that contain free amino acids, purines and pyrimidines that facilitate Salmonella growth. As Salmonella are easily genetically manipulated, and attenuated Salmonella still retain their tumor-targeting and natural tumor-regressing capabilities, they became safe enough to evaluate in tumor-bearing mice and humans. Therefore, S. typhimurium is widely investigated as an anti-cancer agent. Currently, many researchers use S. typhimurium VNP20009 or its derivatives as the anti-cancer agent or as a vector to investigate the efficacy of anti-cancer activities. While VNP20009 carrying a purine auxotrophic mutation (purI) and lipid A mutation (msbB) and its derivatives demonstrated good anti-tumor efficacy in mice, anti-cancer efficacy in human trials was not achieved in phase I clinical trials in patients with metastatic melanoma and renal carcinoma. In VNP20009-immunized dogs with a variety of malignant tumors, bacterial colonization of tumors was observed but only 4 of 35 dogs tested were completely cured. Even intratumoral injection in humans with cancer only led to colonization in 2 out of 3 patients. The reasons for failure in human clinical trials may be that the parent of VNP20009 is not highly virulent and its genetic construction is not precise. VNP20009 is derived from ATCC 14028, which does not show high virulence and invasiveness compared to other S. typhimurium strains and we demonstrated that an attenuated aroA mutant of 14028 was not as immunogenic and did not induce as high protective immune levels as did an isogenic derivative of the S. typhimurium UK-1 strain and furthermore was not as effective as a UK-1-derived strain in ablating colorectal tumors in mice. In addition, the construction of VPN20009 is based on UV- and Tn 10 transposon-induced mutations, which may result in other mutations and over-attenuation. It has been shown that the design method caused strain VPN20009 to lose chemotactic ability. Also, the msbB mutation in VNP20009 is a bad choice because it leads to production of penta-acylated lipid A, which is a good pro-inflammatory stimulator in mice, but is an antagonist to inhibit stimulating human innate immunity. The second S. typhimurium strain widely used for cancer therapy is A1-R, which is also derived from ATCC 14028 and is a leu-arg auxotroph. Notable, the parent of A1-R, A1 is screened through nitrosoguanidine mutagenesis. A1-R exhibited good tumor-seeking features and has antitumor efficacy against major types of cancer in mice, but no clinical trials in humans or dogs have been performed. The 3rd strain is VXM01, which is based on the S. typhi strain Ty21a vaccine carrying a eukaryotic expression plasmid for VEGFR2, could induce anti-angiogenic activity when delivered by the oral route in pancreatic cancer. But only 1 of 13 patients showed an improved clinical outcome. The 4th strain tested was χ4550 delivering IL-2 to induce responses in dogs and humans, respectively. All these strains lack speci