Nonpathogenic E. coli engineered to surface display cytokines as a new platform for immunotherapy.

Given the safety, tumor tropism, and ease of genetic manipulation in non-pathogenic Escherichia coli ( E. coli ), we designed a novel approach to deliver biologics to overcome poor trafficking and exhaustion of immune cells in the tumor microenvironment, via the surface display of key immune-activating cytokines on the outer membrane of E. coli K-12 DH5α. Bacteria expressing murine decoy-resistant IL18 mutein (DR18) induced robust CD8 ⁺ T and NK cell-dependent immune responses leading to dramatic tumor control, extending survival, and curing a significant proportion of immune-competent mice with colorectal carcinoma and melanoma. The engineered bacteria demonstrated tumor tropism, while the abscopal and recall responses suggested epitope spreading and induction of immunologic memory. E. coli K-12 DH5α engineered to display human DR18 potently activated mesothelin-targeting CAR NK cells and safely enhanced their trafficking into the tumors, leading to improved control and survival in xenograft mice bearing mesothelioma tumor cells, otherwise resistant to NK cells. Gene expression analysis of the bacteria-primed CAR NK cells showed enhanced TNFα signaling via NFkB and upregulation of multiple activation markers. Our novel live bacteria-based immunotherapeutic platform safely and effectively induces potent anti-tumor responses in otherwise hard-to-treat solid tumors, motivating further evaluation of this approach in the clinic.
Competing Interests: Compete interests R.R., J.L., S.Y., and M.S. are named inventors on a patent application that describes the surface display of engineered bacteria. R.R. has a sponsored research agreement with Crispr Therapeutics, Skyline Therapeutics and serves on the scientific advisory board of Glycostem Therapeutics. R.R and J.C are co-founders of the InnDura Therapeutics. J.R. received research funding from Kite/Gilead, Novartis and Oncternal Therapeutics and serves on advisory boards for Akron Biotech, Clade Therapeutics, Garuda Therapeutics, LifeVault Bio, Novartis and Smart Immune. Additional Declarations: Yes there is potential Competing Interest. R.R., J.L., S.Y., and M.S. are named inventors on a patent application that describes the surface display of engineered bacteria. R.R. has a sponsored research agreement with Crispr Therapeutics, Skyline Therapeutics and serves on the scientific advisory board of Glycostem Therapeutics. R.R and J.C are co-founders of the InnDura Therapeutics. J.R. received research funding from Kite/Gilead, Novartis and Oncternal Therapeutics and serves on advisory boards for Akron Biotech, Clade Therapeutics, Garuda Therapeutics, LifeVault Bio, Novartis and Smart Immune.