Background The tumor microenvironment (TME), embedded within the dense fibrous extracellular matrix (ECM) common to many solid tumors, significantly contributes to drug resistance and immunosuppression, thereby reducing the effectiveness of antitumor agents. Increasing the therapeutic dosage in the systemic circulation often fails to improve efficacy and typically results in cachexia and morbidity. Therefore, the challenge lies in developing a technology that can selectively break down the ECM—where the TME resides—without affecting normal tissues.

Methods To address this challenge, we have engineered CD4 T cells to synthesize calibrated amounts of ECM-degrading enzymes after engaging the antigen-presenting tumor cells. This will mitigate the undesired systemic effect of directly infusing such ECM-degrading enzymes and focusing their effect only within the solid TME.

Results Primary T cells were engineered to express ECM-degrading enzymes upon engaging Folate Receptor-alpha (FRα) as the target tumor antigen. Enzyme production by these T cells, triggered by FRα-presenting cells, was quantified using ELISA, showing a correlation with stimulation duration, target cell numbers, and the number of engineered T cells. A synthetic fluorogenic substrate confirmed enzyme activity after 48 hours of stimulation. The enzymes produced by these T cells effectively degraded ECM coatings and were 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 engineered primary T cells that express the ECM-degrading enzymes, compared to those that did not. To streamline clinical translation, we optimized our engineered primary T cells, achieving a 100-fold expansion in 14 days and a 9-fold increase in the expression of the desired protein compared to prior iterations.

Conclusions 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.

Acknowledgements 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); and the National Cancer Institute (R21CA236640, R33CA247739) of the National Institutes of Health (NIH).

Ethics Approval The in vivo validation of our T-cell based delivery system was performed in mice at SRI International in accordance with the guidelines from the Institutional Animal Care and Use Committee (Approval # 22001).