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CN-121985958-A - Genetically engineered bacteria for multimodal secretion of neoantigens

CN121985958ACN 121985958 ACN121985958 ACN 121985958ACN-121985958-A

Abstract

A vaccine and method of treatment thereof, wherein the vaccine comprises a recombinant gram-negative bacterium genetically modified to express a first antigen fusion peptide comprising a neoantigen or series thereof associated with a first secretion signal from a trans-bi-membrane secretion system and a second antigen fusion peptide comprising a cognate neoantigen or series thereof associated with a second secretion signal from a trans-outer membrane secretion system. The gram negative bacteria can be further modified for four-mode transport. Specifically, fusion peptides comprising the signal peptide are each associated with a type III secretion system (T3 SS) and a type V secretion system (T5 SS).

Inventors

  • R. Strausman
  • O. Sandler
  • D. G. Rosenberg
  • J. WEISS
  • N. Ellerbaz

Assignees

  • 百赛恩有限公司
  • 耶达研究与开发有限公司

Dates

Publication Date
20260505
Application Date
20240819
Priority Date
20230817

Claims (20)

  1. 1. A vaccine comprising a recombinant gram-negative bacterium genetically modified to express two or more different fusion peptides, the different fusion peptides comprising: a. a first antigen fusion peptide comprising a neoantigen or series thereof associated with a first secretion signal from a transmembrane secretion system, and B. a second antigen fusion peptide comprising a cognate neoantigen or series thereof associated with a second secretion signal from a trans-adventitial secretion system.
  2. 2. The vaccine of claim 1, wherein the transmembrane secretion system is selected from the list consisting of a type I secretion system (T1 SS), a type III secretion system (T3 SS), a type IV secretion system (T4 SS), a type VI secretion system (T6 SS), and a multidrug efflux pump of the drug resistant-nodulation-division (RND) family.
  3. 3. The vaccine of claim 1, wherein the transmembrane secretion system is a type III secretion system.
  4. 4. The vaccine of any one of claims 1 to 3, wherein the trans-adventitial secretion system is selected from the list consisting of a V-type secretion self-transporter system (T5 SS), curli secretion system and a chaperone-guide pathway for pilus assembly.
  5. 5. The vaccine of claim 4, wherein the trans-adventitial secretion system is a V-type secretion system.
  6. 6. The vaccine of claim 5, wherein the trans-bi-membrane secretion system is a type III secretion system and the trans-outer membrane secretion system is a type V secretion system.
  7. 7. The vaccine according to any one of claims 1 to 6, wherein the recombinant gram-negative bacterium is a pathogenic bacterium and/or is recognized by a host pattern recognition receptor.
  8. 8. The vaccine of any one of claims 1 to 7, wherein the recombinant gram-negative bacteria is selected from the list consisting of a plurality of species of Salmonella (Salmonella spp.), a plurality of species of Yersinia (Yersinia spp.), a plurality of species of bordetella (Bordetella spp.), escherichia coli (ESCHERICHIA COLI), a plurality of species of Shigella (Shigella spp.), burkholderia melitensis (Burkholderia mallei), burkholderia melitensis (Burkholderia pseudomallei), and pseudomonas aeruginosa (Pseudomonas aeruginosa).
  9. 9. The vaccine of any one of claims 1 to 8, wherein the recombinant gram-negative bacterium is genetically modified to further express a third antigen fusion peptide comprising a cognate neoantigen or series thereof associated with a third transport signal from a different transport system, wherein the different transport system is selected from the list consisting of a type I secretion system, a type IV secretion system, and an outer cell membrane display system.
  10. 10. The vaccine of claim 9, wherein the recombinant gram-negative bacterium is genetically modified to further express a fourth antigen fusion peptide comprising a cognate neoantigen or series thereof and a fourth transport signal from a different transport system, wherein the different transport system is selected from the list consisting of a Sec or Tat pathway associated with a type II secretion system, a type I secretion signal, a type IV secretion signal, and an outer cell membrane display system.
  11. 11. The vaccine of claim 10, wherein the secretion system or transport system is each an external cell membrane display system, a Sec pathway associated with a type II secretion system, a type III secretion system, and a type V secretion system.
  12. 12. The vaccine of any one of claims 1 to 11, wherein one or more prokaryotic expression cassettes encoding the fusion peptide are operably linked to a constitutive prokaryotic promoter.
  13. 13. The vaccine of any one of claims 1 to 12, wherein the neoantigen forms part of a series, the series further comprising a non-specific tumor antigen.
  14. 14. The vaccine of any one of claims 1 to 13, wherein the neoantigen comprises a cancer-driven mutation.
  15. 15. The vaccine of any one of claims 1 to 14, wherein the neoantigen is tumor specific for a host subject.
  16. 16. The vaccine of any one of claims 1 to 15, wherein a single prokaryotic expression cassette encodes the first and second fusion peptides.
  17. 17. The vaccine according to any one of claims 1 to 16, wherein the recombinant gram-negative bacterium is genetically modified to comprise three nonsense mutations in stm3120, aadA, adI and aac6.
  18. 18. The vaccine of any one of claims 1 to 17, wherein a single bacterial population co-expresses the first and second fusion peptides, and optionally the third and fourth fusion peptides.
  19. 19. Use of the vaccine of any one of claims 1 to 18 in the treatment or prevention of cancer, wherein the method comprises systemic administration of the vaccine to a subject in need thereof, and optionally providing a second therapy against a tumor.
  20. 20. The use of claim 19, wherein the systemic administration is by parenteral administration.

Description

Genetically engineered bacteria for multimodal secretion of neoantigens RELATED APPLICATIONS The present application claims priority from U.S. provisional application No. 63/533,275 filed on 8 month 17 of 2023 and U.S. provisional application No. 63/556,139 filed on 21 month 2 of 2024, the entire contents of each of which are incorporated herein by reference. Technical Field The present invention relates to recombinant bacteria genetically engineered to express and export cancer-associated antigens, related vaccines and methods of treatment thereof. Background Some tumors are difficult to treat based on conventional methods. Tumors are known to evade host immune responses using advanced mechanisms, such as down-regulating antigen presentation on MHC class I molecules or expressing inhibitory molecules to combat recognition and attack by cytotoxic T lymphocytes. One of the most promising therapeutic strategies is immunotherapy, which aims at enhancing, improving and restoring immune system function to combat cancer. Although immunotherapy is somewhat successful, there are significant challenges that need to be overcome. One of the major challenges is that a significant portion of individuals remain unresponsive to immunotherapy, apparently due to the ability of cancer cells to "hide" from the immune system, and the ability of cancer cells to suppress immune system responses. Thus, there is an urgent need for methods that can improve the efficacy of immunotherapy in individuals who would otherwise be unresponsive to such therapy. In vivo therapeutic cancer vaccine strategies based on bacterial vectors that deliver antigens directly have been developed in academic laboratories. Bacteria can be genetically engineered to express and secrete disease antigens. U.S. patent No. 8,357,373 relates to a method of stimulating an immune response using an antigen delivery system based on avirulent salmonella typhimurium that uses a SopE III type secretion signal to deliver full length cancer testis antigen NY-ESO-1 following oral administration of bacteria. U.S. patent No. 7,842,289 discloses that gram-positive listeria treat cancer by cell wall presentation or alternatively by secreting a cancer-associated antigen. PCT application No. PCT/US2021/065011 discloses commensal bacteria delivered by the local, enteral, parenteral and inhalation routes wherein the bacteria are genetically engineered to have an outer cell wall display signal (e.g., a sortase-derived signal sequence peptide) and/or a single step secretion signal (e.g., a Sec signal sequence or Tat signal sequence). U.S. patent No. 8,669,091 discloses bacteria modified to include fusion proteins having an antigen component, a toxin moiety, and either an external surface display signal or a secretion signal. Systemic administration of recombinant bacteria can be used as a vehicle for targeting and local delivery of tumors-colonization of tumor sites and delivery of therapeutic substances, as well as an adjuvant to stimulate the immune system. Adjuvants are known to non-specifically initiate the immune system. However, little is known about the optimal balance between nonspecific priming of the immune system and systemic clearance of bacteria. Particularly in the case of administration of pathogenic bacteria to cancer subjects, many of the developing recombinant bacteria are significantly attenuated in terms of pathogen-associated molecular patterns (PAMPs) such as LPS, flagellin and peptidoglycans to minimize the risk of systemic toxicity. Thus, although cancer vaccines have been developed for decades, new methods for optimizing the effect of cancer vaccines on tumors are still needed. Summary of The Invention The present invention is based, at least in part, on the discovery that the various output pathways and presentation pathways employed by gram negative bacteria in outputting unnatural antigens in the context of the host circulatory system differentially upregulate specific pathways of the host immune system, increase the clearance rate of bacteria from the host circulatory system, enhance the extent of tumor colonization, and enhance the anti-tumor effect in a cancer mouse model. The inventors have found that repeated secretion of homologous neoantigens or concatamers thereof by different bacterial secretion systems yields a preferred immune system-related profile and circulating bacterial load clearance profile, which is expected to produce more limited systemic side effects. The inventors have found that when presenting tumor-specific antigens to the immune system within the host circulatory system, the tumor-specific antigens are displayed on the surface of bacterial cells and/or secreted from bacteria by a type II or type III secretory system, and are not as effective as employing a transmembrane secretory system and a transmembrane secretory system, particularly a type III secretory pathway and a type V secretory pathway. Enhanced systemic clearance is