Engineered immunostimulatory bacterial strains and uses thereof
Provided are immunostimulatory bacteria and oncolytic viruses, and pharmaceutical compositions containing the bacteria and/or viruses, that act as three prime repair exonuclease 1 (TREX1) antagonists. The bacteria and viruses are for treating tumors that are human papillomavirus (HPV) positive or that have a high tumor mutational burden (TMB). The immunostimulatory bacteria and oncolytic viruses encode therapeutic products such RNAi, such as shRNA and microRNA, that mediate gene disruption and/or inhibit expression of TREX1, or that inhibit TREX1. The bacteria contain additional modifications to enhance their anti-tumor activity. The bacteria and viruses are used for treatment of tumors in which TREX1 expression correlates with the presence of the tumor or properties of the tumor, such that inhibition of TREX1 advantageously treats the tumor.
1. A method for treating a subject with cancer with a three prime repair exonuclease 1 (TREX1) antagonist, comprising:
a) obtaining a tumor sample from the subject;
b) identifying the subject as a subject for whom treatment will be effective, by determining the tumor mutational burden (TMB) or testing for human papillomavirus (HPV) or prior HPV infection in the sample to identify HPV + tumors, wherein:
TMB in a tumor is the number of somatic mutations per megabase (Mb) of the genome of the tumor; and
high TMB is at least 10 mutations per Mb of the genome of the tumor; and
c) administering the TREX1 antagonist only to an identified subject, wherein:
the identified subject is one whose tumor has a high TMB or whose tumor is positive for HPV; and
the TREX1 antagonist inhibits the expression of TREX1 or inhibits TREX1.
2. The method of claim 1 , wherein the TREX1 antagonist is encoded in an immunostimulatory bacterium or oncolytic virus.
3. The method of claim 2 , wherein:
the TREX1 antagonist is encoded on a plasmid in an immunostimulatory bacterium;
the immunostimulatory bacterium contains the plasmid that encodes the TREX1 antagonist under control of a eukaryotic promoter;
the genome of the immunostimulatory bacterium is modified whereby the bacterium lacks flagella, wherein the wild-type bacterium comprises flagella; and
the bacterium is auxotrophic for adenosine.
4. The method of claim 3 , wherein the genome of the bacterium is modified whereby the bacterium is pagP − and msbB − .
5. The method of claim 3 , wherein the immunostimulatory bacterium is a Salmonella species.
6. The method of claim 5 , wherein the Salmonella species is a Salmonella typhimurium strain.
7. The method of claim 3 , wherein the immunostimulatory bacterium is aspartate-semialdehyde dehydrogenase − (asd − ).
8. The method of claim 1 , wherein the TREX1 antagonist is encoded in an immunostimulatory bacterium.
9. The method of claim 2 , wherein the cancer comprises a tumor that has a high tumor mutational burden (TMB).
10. The method of claim 2 , wherein the TREX1 antagonist encoded by the immunostimulatory bacterium or oncolytic virus comprises a sequence of nucleotides encoding RNAi that suppresses, inhibits, disrupts or otherwise silences or reduces expression of TREX1, or comprises an antibody or antigen-binding fragment thereof that inhibits TREX1.
11. The method of claim 1 , wherein the cancer comprises a hematological malignancy, solid tumor, or metastases thereof.
12. The method of claim 1 , wherein the cancer comprises a tumor that has a high TMB or is HPV positive, and is selected from among lung cancer, head and neck cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, ovarian cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, and chronic lymphoblastic leukemia.
13. The method of claim 12 , wherein the cancer is an ovarian cancer or cervical cancer.
14. The method of claim 1 , wherein the cancer comprises a tumor that is HPV positive.
15. The method of claim 1 , wherein the cancer comprises a tumor with a high tumor mutational burden.
16. The method of claim 1 , wherein:
the TREX1 antagonist is an antibody or antigen-binding fragment thereof;
the antibody or antigen-binding fragment thereof binds to TREX1 to inhibit its activity; and
the antibody or antigen-binding fragment thereof is encoded on a plasmid in an immunostimulatory bacterium.
17. The method of claim 1 , wherein:
the TREX1 antagonist is encoded in an immunostimulatory bacterium; and
the genome of the immunostimulatory bacterium is modified so that it preferentially infects tumor-resident immune cells and/or so that it induces less cell death in tumor-resident immune cells.
18. The method of claim 1 , wherein:
the TREX1 antagonist is encoded in an immunostimulatory bacterium; and
the genome of the immunostimulatory bacterium is modified, whereby the bacterium is flagellin deficient and pagP − , wherein the wild-type bacterium comprises flagella.
19. The method of claim 1 , wherein:
the TREX1 antagonist is a therapeutic product that inhibits, suppresses or disrupts expression of TREX1, or that inhibits TREX1;
the therapeutic product is encoded in a plasmid in an immunostimulatory bacterium; and
the plasmid is present in the immunostimulatory bacterium in a copy number of less than 150.
20. The method of claim 19 , wherein the immunostimulatory bacterium is auxotrophic for adenosine.
21. The method of claim 19 , wherein:
the immunostimulatory bacterium has a deletion or disruption or both in a gene(s) encoding the flagella, whereby the bacterium is flagellin deficient; and
the wild-type bacterium comprises flagella.
22. The method of claim 16 , wherein:
the immunostimulatory bacterium that encodes the TREX1 antagonist is a Salmonella species; and
the immunostimulatory bacterium comprises deletions or disruptions or both in the genes encoding both flagellin subunits fliC and fljB, whereby the bacterium is flagellin deficient.
23. The method of claim 12 , wherein the cancer comprises a tumor that has a high TMB.
24. The method of claim 1 , wherein:
the TREX1 antagonist is RNAi that is encoded on a plasmid in an immunostimulatory bacterium; and
the RNAi is short hairpin RNA (shRNA) or micro-RNA (miRNA).
25. The method of claim 1 , wherein:
the TREX1 antagonist is a therapeutic product that inhibits expression of TREX1 or that inhibits TREX1; and
the TREX1 antagonist is encoded in an oncolytic virus.
26. The method of claim 2 , wherein:
the TREX1 antagonist is encoded in an immunostimulatory bacterium; and
the immunostimulatory bacterium is one or more of purI − (purM − ), msbB − , purD − , flagellin − (fliC − /fljB − ), pagP − , adrA − , csgD − , and hilA − .
27. The method of claim 1 , wherein:
the TREX1 antagonist is encoded in an immunostimulatory bacterium; and
the immunostimulatory bacterium is a strain of Salmonella, Shigella, E. coli, Bifidobacteriae, Rickettsia, Vibrio, Listeria, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Cholera, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Bacillus , or Erysipelothrix , or an attenuated strain thereof or a modified strain thereof of any of the preceding list of bacterial strains.
28. The method of claim 27 , wherein the immunostimulatory bacterium is a strain of Salmonella.
29. The method of claim 28 , wherein the immunostimulatory bacterium is a Salmonella typhimurium strain.
30. The method of claim 29 , wherein the Salmonella typhimurium strain is derived from strain AST-100 (VNP20009 or YS1646), or a wild-type strain having all of the identifying characteristics of the strain deposited under ATCC accession number 14028, or is the strain deposited under ATCC accession number 14028.
31. The method of claim 1 , wherein:
the subject has a cancer that comprises a tumor that is human papillomavirus (HPV) positive or that has a high tumor mutational burden (TMB); and
the TREX1 antagonist is encoded in an immunostimulatory bacterium, wherein:
the immunostimulatory bacterium auxotrophic for adenosine;
the immunostimulatory bacterium comprises a modification in its nucleic acid encoding the flagella, whereby the bacterium is flagellin deficient; and
the immunostimulatory bacterium optionally is pagP − .
32. The method of claim 1 , wherein the subject with the cancer is human.
33. The method of claim 1 , wherein administration of the TREX1 antagonist is parenteral.
34. The method of claim 1 , wherein:
the TREX1 antagonist is encoded in an immunostimulatory bacterium or in an oncolytic virus; and
the immunostimulatory bacterium or oncolytic virus is formulated in a pharmaceutical composition that is administered by oral administration, or by rectal administration, or by aerosol into the lung, or by intratumoral, intravenous, intramuscular, or subcutaneous administration.
35. The method of claim 1 , wherein:
the subject has a cancer that comprises a solid tumor or hematological malignancy; and
the TREX1 antagonist is encoded in an oncolytic virus.