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.

Tumors use multiple mechanisms to actively exclude immune cells involved in antitumor immunity. Strategies to overcome these exclusion signals remain limited due to an inability to target therapeutics specifically to the tumor. Synthetic biology enables engineering of cells and microbes for tumor-localized delivery of therapeutic candidates previously unavailable using conventional systemic administration techniques. Here, we engineer bacteria to intratumorally release chemokines to attract adaptive immune cells into the tumor environment. Bacteria expressing an activating mutant of the human chemokine CXCL16 (hCXCL16 K42A ) offer therapeutic benefit in multiple mouse tumor models, an effect mediated via recruitment of CD8 + T cells. Furthermore, we target the presentation of tumor-derived antigens by dendritic cells, using a second engineered bacterial strain expressing CCL20. This led to type 1 conventional dendritic cell recruitment and synergized with hCXCL16 K42A -induced T cell recruitment to provide additional therapeutic benefit. In summary, we engineer bacteria to recruit and activate innate and adaptive antitumor immune responses, offering a new cancer immunotherapy strategy.

Synthetic biology enables the engineering of interactions between living medicines to overcome the specific limitations of any singular therapy. One major challenge of tumor-antigen targeting therapies like chimeric antigen receptor (CAR)-T cells is the identification of targetable antigens that are specifically and uniformly expressed on heterogenous solid tumors. In contrast, certain species of bacteria selectively colonize immune-privileged tumor cores and can be readily engineered as antigen-independent platforms for therapeutic delivery. Bridging these approaches, we develop a platform of probiotic-guided CAR-T cells (ProCARs), in which T cells are engineered to sense synthetic antigens (SA) that are produced and released by tumor-colonizing probiotic bacteria. We demonstrate increased CAR-T cell activation and tumor-cell lysis when SAs anchor to components of the extracellular matrix. Moreover, we show that ProCARs are intratumorally activated by probiotically-delivered SAs, receive further stimulation from bacterial TLR agonists, and are safe and effective in multiple xenograft models. This approach repurposes tumor-colonizing bacteria as beacons that guide the activity of engineered T cells, and in turn builds the foundation for communities of living medicines.

The specific properties have been examined of the 1,4-beta-glucanase component of Trichoderma koningii that participates in an early and effective stage of random breakdown of native cellulose to short fibres. The enzyme was purified and freed from associated components of the cellulase complex (particularly beta-glucosidase) that interfere with, and complicate interpretation of, the action of such enzymes. Purification increased the specific activity 25-fold over culture filtrates; the enzyme hydrolysed CM-cellulose faster than the purified beta-glucosidase from the same organism hydrolysed any of its substrates (cellobiose or cellodextrins). The specificity of the glucanase was directed towards soluble derivatives of cellulose, CM-cellulose and cellodextrins, and not to insoluble cellulose or alpha-linked polymers. The approximate Km was 2.5 mg of CM-cellulose . ml-1 at 37 degrees C at the optimum pH, 5.5, where enzymic activity was maximal with 6--7 mg of CM-cellulose . ml-1 and inhibited by higher concentrations. The temperature optimum was 60 degrees C. The glucanase attacked larger cellodextrins (cellohexaose to cellotetraose, in that order) much more readily than smaller dextrins (cellobiose and cellotriose) and released a mixture of products, glucose up to cellopentaose, which was quantitatively determined after chromatography on charcoal. Similar examination of hydrolysates of the reduced cellodextrins showed clearly the high specificity of the enzyme for the central bond of its natural substrates (the cellodextrins), whatever their chain length, and indicated the nature of the enzyme as an endoglucanase. Outer bonds shared a weaker, but similar, susceptibility to enzymic cleavage. Transferase activity was absent and no larger dextrins than the initial substrate were formed.

out and the enzyme shows properties similar to that already obtained from several other plant tissues (McCrae, 1971), suggesting that the two pathways for malate oxidation may be a general property of higher-plant mitochondria. The operation of these two pathways may be of importance in controlling the redox equilibrium of NAD in the mitochondrial matrix and in the cytosol. Over the years, biological attack on cellulose has been variously attributed to a single enzyme, CMcellulase (Whitaker, 1953), or to a prime component called Cl thought to produce shorter chains for a more conventional hydrolysis by CM-cellulase (Reese et al., 1950). Other workers support a modified version of the last scheme in which the combined action of both CM-cellulase and component C1 is believed to be essential, since no specific function has so far been assigned to component C1 (Selby, 1968).Recent evidence from this laboratory showed that component C1 hydrolysed native cellulose to cellobiose in a solubilizing reaction promoted more effectively by cellobiase than by CM-cellulase (Halliwell & Riaz, 1971). Later preparations of component C1 indicate that the stimulation of cellulolysis so achieved can be increased even further by up to 80%. Prolonging the incubation beyond the usual period of 7 days had little effect on cellulolysis by component C1 alone. The additional presence of cellobiase, however, proved beneficial in enabling up to 70% solubilization of cotton to be attained in 21 days. The only products recognized chromatographically in the two systems were cellobiose and glucose respectively. The ability of cellobiase, which itself had no action on cellulose, to promote saccharification of the substrate by component C1 suggested that this component was inhibited by its product cellobiose. Inhibition was competitive, being dependent on the relative concentrations of cellulose and cellobiose and overcome completely by increasing the substrate concentration. CM-cellulose behaved similarly. Component C1 exhibits maximum activity and stability at pH5 and 37°C with Km approx. 0.05mg/ml. With cellulose and its derivatives component C1 apparently acts as a cellobiohydrolase with a preference for the ends of chains and in the vicinity of terminal non-reducing groups.