Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies

Immunotherapies such as checkpoint inhibitors have revolutionized cancer therapy yet lead to a multitude of immune-related adverse events, suggesting the need for more targeted delivery systems. Due to their preferential colonization of tumors and advances in engineering capabilities from synthetic biology, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we present an engineered probiotic bacteria system for the controlled production and release of novel immune checkpoint targeting nanobodies from within tumors. Specifically, we engineered genetic lysis circuit variants to effectively release nanobodies and safely control bacteria populations. To maximize therapeutic efficacy of the system, we used computational modeling coupled with experimental validation of circuit dynamics and found that lower copy number variants provide optimal nanobody release. Thus, we subsequently integrated the lysis circuit operon into the genome of a probioticE. coliNissle 1917, and confirmed lysis dynamics in a syngeneic mouse model usingin vivobioluminescent imaging. Expressing a nanobody against PD-L1 in this strain demonstrated enhanced efficacy compared to a plasmid-based lysing variant, and similar efficacy to a clinically relevant monoclonal antibody against PD-L1. Expanding upon this therapeutic platform, we produced a nanobody against cytotoxic T-lymphocyte associated protein -4 (CTLA-4), which reduced growth rate or completely cleared tumors when combined with a probiotically-expressed PD-L1 nanobody in multiple syngeneic mouse models. Together, these results demonstrate that our engineered probiotic system combines innovations in synthetic biology and immunotherapy to improve upon the delivery of checkpoint inhibitors.