Chimeric antigen receptor (CAR) therapies are emerging as promising strategies, particularly for metastatic castration-resistant prostate cancer (PCa), as they can act independently of the androgen receptor axis. Adapter CAR-T cell platforms, such as the RevCAR system, offer precise therapeutic control and tumor targeting via small, rapidly eliminated tumor-specific adapters. To enable more convenient late-stage RevCAR-T therapy in PCa patients, allowing for discontinuous reverse target module (RevTM) infusion, we developed novel, larger IgG4-based RevTMs targeting prostate stem cell antigen (PSCA) and benchmarked them against previously described smaller adapter formats. Within the RevCAR system, PSCA-IgG4 RevTMs effectively mediated PCa killing at low effector-to-target ratios and low RevTM concentrations in a strictly antigen-dependent manner. Oncolytic activity was accompanied by a rapid and pronounced release of proinflammatory cytokines across a broad RevTM concentration range, which is particularly advantageous for immunologically cold PCa. Finally, anti-tumor activity was confirmed in a short-term mouse model. Preliminary PET studies further indicate slow blood elimination and tumor-specific accumulation of novel IgG4-RevTMs. Together, these data position PSCA-IgG4 RevTMs as promising candidates for stepwise RevCAR-T treatment in PCa, in which short-lived scFv-RevTMs are initially used to ensure a rapid safety switch, followed by larger IgG4-RevTMs once the risk profile is known.

The Prostate Stem Cell Antigen (PSCA) is not only overexpressed in prostate cancer (PC) but also in many other tumors, including cervical cancer (CC). The adaptor chimeric antigen receptor (CAR) platform RevCAR can be used for immunotargeting of PSCA-expressing tumor cells. We found that the CC cell line SiHa natively expresses PSCA. The expression was sufficient for redirection of RevCAR T-cells using recently described RevCAR target modules (RevTMs) directed against PSCA. Like almost all PC cell lines, however, SiHa cells also downregulate their PSCA expression in cell culture. To overcome this limitation, we established artificially PSCA-overexpressing SiHa cells. Retargeting of RevCAR T-cells strictly depends on PSCA-directed RevTMs and PSCA-expressing target cells. It leads to (i) upregulation of CD69, Perforin and Granzyme B, (ii) lysis of target cells with EC₅₀ values in the picomolar range, and (iii) cytokine secretion of IFN-γ, TNF-α, GM-CSF and IL-2. Phenotypic analysis shows a prominent increase in memory RevCAR T-cells but only a slight increase in exhausted cells, indicating a low level of tonic signaling of RevCAR T-cells. Together, our data supports an efficient and safe immunotargeting of PSCA-expressing CC cells with the RevCAR technology.