Genetically-engineered Bacteria Overcome Therapy Resistance

Project Summary: For this proposal in response to PAR-22-085: Microbial-based Cancer Imaging and Therapy - Bugs as Drugs, we will focus on metastatic pancreatic ductal adenocarcinoma (PDAC), for several reasons: PDAC is particularly difficult to target and generally considered recalcitrant, but the pancreatic tumor microenvironment

(TME) features an abundance of tumor-associated macrophages (TAMs) and few T cells. Therefore, methods to reprogram TAMs and enhance T-cell activation and proliferation will likely synergize with radiotherapy and checkpoint immunotherapy to control local and metastatic disease. Bifidobacteria is a commensal that preferentially targets tumors, and oral Bifidobacteria has been found to

translocate from the gastrointestinal tract with subsequent homing to and replication specifically in tumors. Building on these findings, we recently reported that systemically administered Bifidobacterium (i.v.) specifically colonize and replicate in hypoxic tumor regions. Notably, we also found that i.v. administration of

Bifidobacterium also converted non-responding mice into responders, in the context of local immunotherapy and/or ionizing radiation (IR). This data serves as proof-of-principle that systemic administration of Bifidobacterium can enhance tumor control in combination with immunotherapy and/or IR. We have now successfully developed genetic tools to regulate gene expression in Bifidobacterium longum.

We propose to investigate the systemic administration of genetically-engineered Bifidobacterium delivering a novel human IL-2 (SumIL2) to the TME in combination with radiotherapy and/or immunotherapy. This strategy centers on harnessing both innate and adaptive pro-immune responses and is aimed at capitalizing on several

known mechanisms of tumor evasion. In addition, our plan to include a precise gene circuit for precise protein secretion and bacteria self-destruction is a systemic therapeutic delivery innovation. Most importantly, we anticipate that our approach using Bifidobacterium will elicit significant anti- tumor effects for several reasons:

1. Bifidobacterium is selectively taken up by tumors, and SumIL2 secretion is precisely controlled after bacteria colonization, which will obviate the treatment-limiting toxicity commonly associated with IL-2 administration. 2. Our preliminary data presented here indicate that systemic (IV) administration of Bifidobacterium converts

non-responder mice into responders to anti-CD47 immunotherapy and radiotherapy. 3. In contrast to E. coli, Bifidobacterium is a human commensal anaerobic bacterium, giving our approach a less toxic profile and more translational relevance. Our approach takes advantage of the natural features and genetic engineering of Bifidobacterium to

reinvigorate the immune suppressive TME via systemic injection, and thus could have broad applicability in other hard-to-treat cancers.