Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies

Abstract

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 probiotic E. coli Nissle 1917, and confirmed lysis dynamics in a syngeneic mouse model using in vivo bioluminescent 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. bacteria to grow within the hypoxic and necrotic tumor core (24)(25)(26)(27). At the same time, microbiome research efforts have revealed the widespread prevalence of microbes within malignant tissue that do not cause infections or other long-term detrimental health effects (28, 29). Since bacteria are both inherently present and selectively grow in tumors, they provide a natural platform for the development of programmable therapeutic delivery vehicles.Harnessing the converging advancements in both immunotherapy and synthetic biology, we engineered probiotic bacteria to locally and controllably release PD-L1 and CTLA-4 antagonists in the form of blocking nanobodies. Specifically, we coupled immunotherapeutic expression with an optimized lysing circuit mechanism, such that probiotic bacteria carrying the anti-PD-L1 nanobody homes to the necrotic tumor core, grow to a critical density, and lyse effectively releasing the anti-PD-L1 nanobody to block the PD-1/PD-L1 interaction between tumor and T cells (Fig. 1a). RESULTS Construction and characterization of a probiotically-expressed PD-L1 NbA single-domain antibody, or nanobody (Nb), blocking PD-L1 was chosen from the RCSB Protein Data Bank as therapeutic cargo. Unlike antibodies with a molecular size of approximately 150...

Novel Delivery Systems for Checkpoint Inhibitors

“…While bacteria have been explored as a delivery vehicle for various anti-cancer agents [119,120,121,122,123,124,125], their use as delivery vehicles for checkpoint inhibitors has been limited [126,127]. In a study by Gurbatri et al, the probiotic strain of E. coli ( E. coli Nissle 1917) containing plasmid for single domain antibodies (nanobodies) against either PD-L1 or CTLA-4 was evaluated for efficacy in a murine model of colorectal cancer (CT26) [126]. Gurbatri et al utilized one plasmid system such that a quorum sensing promoter drove transcription of both the quorum sensing genes and phage-derived lysis gene, creating a synchronized lysis circuit (SLC) [126].…”

“…In a study by Gurbatri et al, the probiotic strain of E. coli ( E. coli Nissle 1917) containing plasmid for single domain antibodies (nanobodies) against either PD-L1 or CTLA-4 was evaluated for efficacy in a murine model of colorectal cancer (CT26) [126]. Gurbatri et al utilized one plasmid system such that a quorum sensing promoter drove transcription of both the quorum sensing genes and phage-derived lysis gene, creating a synchronized lysis circuit (SLC) [126]. This allowed for the release of anti-PD-L1 or anti-CTLA-4 nanobodies through bacterial lysis when the bacterial mass reached a critical density, resulting in delivery of a high dose of nanobodies.…”

“…This allowed for the release of anti-PD-L1 or anti-CTLA-4 nanobodies through bacterial lysis when the bacterial mass reached a critical density, resulting in delivery of a high dose of nanobodies. Although bacterial delivery of anti-PD-L1 nanobodies (SLC-PDL1) had similar efficacy as systemic PD-L1 blockade, there were more necrotic areas and neutrophil infiltration into the tumors with SLC-PDL1 treatment [126]. Similarly, the bacterial delivery of anti-CTLA-4 nanobodies (SLC-CTLA4) had efficacy comparable to systemic CTLA-4 blockade.…”

“…Similarly, the bacterial delivery of anti-CTLA-4 nanobodies (SLC-CTLA4) had efficacy comparable to systemic CTLA-4 blockade. The combination of SLC-PDL1 and SLC-CTLA4 led to significantly decreased tumor volumes, compared to each monotherapy, in both the CT26 and A20 tumor models, although comparison with the combination of systemic PD-L1 and CTLA-4 blockade was not performed [126]. The toxicities associated with systemic checkpoint blockade therapies, however, seemed to be significantly eliminated with this delivery method.…”

“…The toxicities associated with systemic checkpoint blockade therapies, however, seemed to be significantly eliminated with this delivery method. In the 4T1 model of low immunogenic breast cancer, Gurbatri et al showed that while systemic anti-PD-L1 antibody treatment led to severe toxicities and deaths, SLC-PDL1 treatment led to no significant toxicities [126]. Similarly, in the colon cancer model (CT26), toxicities observed in the SLC-CTLA4 group were less compared to the group with systemic CTLA-4 blockade (increase in body weight by ~30% compared to ~10%) [126].…”