SUMMARY PARAGRAPH Synthetic biology is driving a new era of medicine through the genetic programming of living cells 1 , 2 . This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics 3 – 6 . One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo 7 – 11 . Here, we engineered a non-pathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb) 12 , an anti-phagocytic receptor commonly overexpressed in several human cancers 13 , 14 . We show that delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic tumor antigen–specific immune responses that reduce the growth of untreated tumors – providing, to the best of our knowledge, the first demonstration of an abscopal effect induced by an engineered bacterial immunotherapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

Checkpoint inhibitors have revolutionized cancer therapy but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Because of their preferential colonization of tumors, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we engineer a probiotic bacteria system for the controlled production and intratumoral release of nanobodies targeting programmed cell death–ligand 1 (PD-L1) and cytotoxic T lymphocyte–associated protein-4 (CTLA-4) using a stabilized lysing release mechanism. We used computational modeling coupled with experimental validation of lysis circuit dynamics to determine the optimal genetic circuit parameters for maximal therapeutic efficacy. A single injection of this engineered system demonstrated an enhanced therapeutic response compared to analogous clinically relevant antibodies, resulting in tumor regression in syngeneic mouse models. Supporting the potentiation of a systemic immune response, we observed a relative increase in activated T cells, an abscopal effect, and corresponding increases in systemic T cell memory populations in mice treated with probiotically delivered checkpoint inhibitors. Last, we leveraged the modularity of our platform to achieve enhanced therapeutic efficacy in a poorly immunogenic syngeneic mouse model through effective combinations with a probiotically produced cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Together, these results demonstrate that our engineered probiotic system bridges synthetic biology and immunology to improve upon checkpoint blockade delivery.

Co-corresponding authors SUMMARY PARAGRAPHSynthetic biology is driving a new era of medicine through the genetic programming of living cells 1,2 . This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics 3-5 . One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo 6-8 . Here, we engineered a nonpathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb) 9 , an anti-phagocytic receptor commonly overexpressed in several human cancers 10,11 . We show that intratumoral delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic antitumor immune responses that reduce the growth of untreated tumorsproviding, to the best of our knowledge, the first demonstration of an abscopal effect induced by a bacteria cancer therapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

Synthetic biology is transforming therapeutic paradigms by engineering living cells and microbes to intelligently sense and respond to diseases including inflammation, infections, metabolic disorders, and cancer. However, the ability to rapidly engineer new therapies far outpaces the throughput of animal-based testing regimes, creating a major bottleneck for clinical translation. In vitro approaches to address this challenge have been limited in scalability and broad applicability. Here, we present a bacteria-in-spheroid coculture (BSCC) platform that simultaneously tests host species, therapeutic payloads, and synthetic gene circuits of engineered bacteria within multicellular spheroids over a timescale of weeks. Long-term monitoring of bacterial dynamics and disease progression enables quantitative comparison of critical therapeutic parameters such as efficacy and biocontainment. Specifically, we screen Salmonella typhimurium strains expressing and delivering a library of antitumor therapeutic molecules via several synthetic gene circuits. We identify candidates exhibiting significant tumor reduction and demonstrate high similarity in their efficacies, using a syngeneic mouse model. Last, we show that our platform can be expanded to dynamically profile diverse microbial species including Listeria monocytogenes, Proteus mirabilis, and Escherichia coli in various host cell types. This high-throughput framework may serve to accelerate synthetic biology for clinical applications and for understanding the host–microbe interactions in disease sites.

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...