Preclinical evaluation of homodimeric FAPI target modules for UniCAR-mediated targeting of FAP-expressing tumor and stromal cells.

Due to its overexpression in the tumor microenvironment of most solid malignancies, fibroblast activation protein (FAP) has emerged as an ideal target for (immuno)theranostic applications. The clinically tested UniCAR system represents a promising adapter CAR T-cell approach, in which CAR T-cell activity is regulated through the administration of target modules (TMs). Here, we report the first homodimeric FAP inhibitor (FAPI)-based TMs that enable efficient adapter CAR T-cell immunotherapy of FAP-positive tumors. The novel TMs consist of two UAMC-1110 FAPI moieties, the E5B9 UniCAR epitope, a suitable polyethylene glycol spacer to ensure epitope accessibility, and a functional group that allows for further TM functionalization for diagnostic and radiotherapeutic applications. Following the synthesis of the novel FAPI TMs, we evaluated their functionality using both in vitro and in vivo models. The FAPI TMs successfully redirect UniCAR T-cells and mediate potent lysis of FAP-positive cells in vitro and in an immunodeficient mouse model. In addition, we established a 3D heterospheroid model consisting of tumor cells expressing prostate stem cell antigen (PSCA) alongside FAP-positive stromal fibroblasts. We showed that simultaneous dual-targeting leads to enhanced UniCAR-mediated cytotoxicity. Notably, fibroblast killing is mediated by the release of PSCA from dying tumor cells and its subsequent binding to the surface of neighboring PSCA-negative fibroblasts, thereby making them susceptible to PSCA-directed targeting. Overall, our work not only summarizes the successful development of novel dimeric FAPI adapter molecules for immunotherapeutic applications in solid tumors but also provides novel insights into the targeting of PSCA-positive tumors.