Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pah PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.

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Background: The immunosuppressive milieu found within the tumor microenvironment (TME) has long been understood to be a key driver of tumor initiation and progression. More recently it has been appreciated that metabolites derived from biosynthetic pathways are major components in forming this immune-privileged niche. For example, the conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO) or the reduction of adenosine triphosphate to adenosine by the ectoenzymes CD39 and CD73 leads to T cell dysfunction and exhaustion, and a significantly blunted antitumor immune response. At Synlogic we are using synthetic biology in combination with natural probiotics to develop engineered bacteria or “Synthetic Biotic Medicines,” which are programmed with precision to correct disease-causing and -promoting metabolic defects. Here we present results showing the development of two engineered bacterial strains that have been designed to consume either kynurenine or adenosine, two molecules known to play central roles in promoting tumor immune tolerance, with the goal of relieving TME-associated immunosuppression and promoting antitumor immunity. Methods and Results: Synthetic biologic techniques were employed to generate the adenosine-consuming strain (SYN-Ade) or the kynurenine-consuming strain (SYN-Kyn) by introduction of genetic elements that were highly efficient in the metabolism of adenosine or kynurenine, respectively. In in vitro biochemical assays, SYN-Ade and SYN-Kyn were able to deplete test media containing levels of adenosine and kynurenine that are ~100-fold and 20-fold higher than the adenosine or kynurenine levels found in the tumors of cancer patients, (180uM of adenosine or 80uM of kynurenine, respectively) to undetectable levels within 2 hours. For the kynurenine-consuming strain, this in vitro kynurenine consumption translated to robust in vivo pharmacodynamic activity. In mice bearing subcutaneous CT26 tumors, the administration of SYN-Kyn by intratumoral (IT) injection led to significant decreases in tumor kynurenine levels, which was equivalent to small-molecule inhibition of the IDO enzyme. Importantly, the combination of SYN-Kyn with an anti-CTLA4 antibody in the CT26 tumor model or the combination of SYN-Kyn or SYN-Ade with a cocktail of anti-PD1/CTLA4 antibodies in MC38 tumor-bearing mice led to significant antitumor effects over those observed with the antibodies alone. Conclusions: Taken together, these results demonstrate that the application of synthetic biology to engineer nonpathogenic bacteria is a viable approach to deliver profound efficacy in experimental models of cancer, and support the further development of these Synthetic Biotic medicines as potential immuno-oncology therapies. Citation Format: Kip A. West, Adam Fisher, Dan Leventhal, Anna Sokolovska, Ning Li, Chris Plescia, Mary Castillo, Vincent Isabella, Starsha Kolodziej, Paul Miller, Jose M. Lora. Metabolic modulation of the tumor microenvironment using Synthetic Biotic™ Medicines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2920.

Thermobifida fusca is a moderately thermophilic actinobacterium naturally capable of utilizing lignocellulosic biomass. The B6 strain of T. fusca was previously engineered to produce 1-propanol directly on lignocellulosic biomass by expressing a bifunctional butyraldehyde/alcohol dehydrogenase (adhE2). To characterize the intracellular mechanisms related to the accumulation of 1-propanol, the engineered B6 and wild-type (WT) strains were systematically compared by analysis of the transcriptome and intracellular metabolome during exponential growth on glucose, cellobiose, and Avicel. Of the 18 known cellulases in T. fusca, 10 cellulase genes were transcriptionally expressed on all three substrates along with three hemicellulases. Transcriptomic analysis of cellodextrin and cellulose transport revealed that Tfu_0936 (multiple sugar transport system permease) was the key enzyme regulating the uptake of sugars in T. fusca. For both WT and B6 strains, it was found that growth in oxygen-limited conditions resulted in a blocked tricarboxylic acid (TCA) cycle caused by repressed expression of Tfu_1925 (aconitate hydratase). Further, the transcriptome suggested a pathway for synthesizing succinyl-CoA: oxaloacetate to malate (by malate dehydrogenase), malate to fumarate (by fumarate hydratase), and fumarate to succinate (by succinate dehydrogenase/fumarate reductase) which was ultimately converted to succinyl-CoA by succinyl-CoA synthetase. Both the transcriptome and the intracellular metabolome confirmed that 1-propanol was produced through succinyl-CoA, L-methylmalonyl-CoA, D-methylmalonyl-CoA, and propionyl-CoA in the B6 strain.