Poster X4 - T-cell Biofactory for degrading tumor-associated extracellular matrix

Introduction: The current standard of care for solid tumors is chemotherapy. The present emphasis is focused on immunotherapies, which offer the potential for complete remission with reduced side effects. The tumor microenvironment (TME), harbored within the dense fibrous extracellular matrix (ECM) that is common to many solid tumors, has been implicated as a significant factor in drug resistance and immunosuppression thereby diminishing the performance of these approaches. Increasing the therapeutic dosage in the systemic circulation may not improve efficacy and usually causes cachexia and morbidity. Hence, the challenge is to develop a technology to selectively break down the ECM—in which the TME resides—without affecting normal tissues.

To address this challenge, we have engineered T cells to autonomously synthesize calibrated amounts of ECM-degrading enzymes directly within the TME after engaging the antigen-presenting tumor cells. This will mitigate the undesired systemic effect of directly infusing such ECM-degrading enzymes that, as described earlier, impair the treatment of solid tumors.

Materials and

Methods: To address this challenge, we have engineered T cells to autonomously synthesize calibrated amounts of ECM-degrading enzymes directly within the TME after engaging the antigen-presenting tumor cells, i.e., T-cell Biofactory. This will mitigate the undesired systemic effect of directly infusing such ECM-degrading enzymes that, as described earlier, impair the treatment of solid tumors. The rationale for this technology in treating solid tumors is that it limits the effect of ECM-degrading enzymes with spatiotemporal resolution to within the TME. Thus, degraded ECM will be more easily infiltrated by the innate immune system and many antitumor agents that are currently in the market. It will also allow efflux of the tumor metabolites potentially reducing the immunosuppressive nature of the TME.

Results, Conclusions, and Discussions: In this work, T-cell Biofactory with specificity towards Folate Receptor-alpha (FRα), as previously reported (Repellin et al. (2018) Adv. Biosys. 2(12)), were re-engineered by exchanging the Effector protein with an ECM-degrading enzyme. The resulting ECM-degrading T-cell Biofactory was confirmed for FRα specificity. The enzyme, produced by the T-cell Biofactory upon stimulation by the FRα-presenting target cells, was detected via ELISA and was proportional to the duration of stimulation and the number target cells and the number of T-cell Biofactory. A synthetic fluorogenic substrate, that fluoresces upon degradation, was utilized to validate the activity of the enzyme produced from the T cells after 48 hrs of stimulation. The enzymes secreted by T cell Biofactory was able to degrade ECM coating on cell culture surfaces which was shown to be inhibited by an enzyme-specific inhibitor. A high concentration of degraded ECM components was observed when ECM enriched tumor explants derived from subcutaneous and peritoneal mouse tumors were treated with enzyme enriched cell culture supernatant from T cell Biofactory compared to control CAR T cells. In our pursuit towards clinical translation, we have identified a new combination of the modular components that enables more efficient manufacturing of the T-cell Biofactory. Compared to the previous components, the new composition yields 100-fold expansion of the T-cell Biofactory in 14 days; and as confirmed with the reporter protein, it demonstrated 9-fold higher expression of the desired protein.

The rationale for this technology in treating solid tumors is that it limits the effect of ECM-degrading enzymes with spatiotemporal resolution to within the TME. Thus, degraded ECM will be more easily infiltrated by the innate immune system and many antitumor agents that are currently in the market. It will also allow efflux of the tumor metabolites potentially reducing the immunosuppressive nature of the TME.

As the next step toward clinical translation, we will exchange the reporter protein sequence with the one for ECM-degrading enzyme. Upon completion of the in vitro investigations, we are validating the technology in intraperitoneal mice tumor model of epithelial ovarian cancer.

Acknowledgements (Optional): Research reported in this publication was supported in part by the National Institute of Biomedical Imaging and Bioengineering (DP2EB024245: NIH Director's New Innovator Award Program (https://commonfund.nih.gov/newinnovator); the National Cancer Institute (R21CA236640, R33CA247739) of the National Institutes of Health (NIH); and the Defense Advanced Research Projects Agency (DARPA) (D19AP00024: DARPA Young Faculty Award (https://www.darpa.mil/work-with-us/for-universities/young-faculty-award)