Advancing chimeric antigen receptor (CAR)-engineered adoptive T cells for the treatment of solid cancers is a major focus in the field of immunotherapy, given impressive recent clinical responses in hematological malignancies. Prostate cancer should be amenable to CAR-based immunotherapy given that several tumor antigens, including prostate stem-cell antigen (PSCA), are widely over-expressed in metastatic disease. The selectivity of CARs for solid cancers is crucial, however, due to the absence of truly restricted tumor antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we show that optimized co-stimulatory signaling and extracellular spacer domains are essential in defining a CAR’s selectivity towards tumor cells that over-express the target antigen. The 4-1BB co-stimulatory domain in PSCA-CARs confers enhanced tumor selectivity with dampened yet dose-responsive cytokine production, reduced T cell exhaustion phenotype, and equivalent tumor killing ability compared to PSCA-CARs containing the CD28 co-stimulatory domain. CARs containing a short extracellular spacer demonstrate the most selective killing of high PSCA-expressing tumors but are unable to produce the inflammatory cytokines important for a complete anti-tumor response. We show that longer extracellular spacers in PSCA-CARs are necessary for both tumor killing and cytokine production. PSCA-CARs exhibit robust in vivo anti-tumor activity in subcutaneous and orthotopic bone-metastatic patient-derived prostate cancer xenograft models, and 4-1BB-containing CARs show superior persistence in controlling metastatic disease compared with CD28-containing CARs. Our study demonstrates the critical impact of CAR components in defining an optimized tumor-selective CAR T cell, and also highlights the importance of clinically relevant animal models in developing effective solid cancer CAR T cell therapies.

Citation Format: Kelly Kennewick, Dileshni Tilakawardane, Ethan Gerdts, John Murad, Anthony Park, Brook Jeang, Yukiko Yamaguchi, Stephen J. Forman, Saul Priceman. Extracellular spacer and co-stimulatory domains define target sensitivity and persistence of CAR T cells for the treatment of PSCA+ bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4981. doi:10.1158/1538-7445.AM2017-4981