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Background CAR T-cell therapy for prostate cancer presents several challenges, including poor persistence and proliferation. Prostate-specific membrane antigen (PSMA) is a promising target in advanced prostate cancer, overexpressed in cancer cells, and associated with worse clinical outcomes. Here we have developed PSMA-targeting CAR T-cells, armored to secrete human interleukin-7 and human IgG 1Fc (IL7Fc) fusion protein, and functionality tested in prostate cancer models.Methods Two PSMA CAR T-cells, PSMA2 and PSMA14, were tested and compared with IL7Fc-armor (PSMA2IL7Fc and PSMA14IL7Fc). Immunophenotypic characterization was carried out using flow cytometry, and cytokine measurements were done using ELISA. In vitro cytotoxicity was assessed against LnCap prostate cancer cells (ATCC) in short-term (24 h), medium-term (5 d), and long-term re-challenge assays (12-16 d). In vivo safety and efficacy studies were carried out in tumor-naïve NSG mice, and NSG mice bearing LnCap xenografts, respectively. CAR T-cells were injected intravenously into mice and monitored for body weight change, life span, and tumor progression.Results The IL7Fc-armored PSMA-targeting CAR T-cells showed increased expansion, higher T scm phenotype, and lower exhaustion (LAG3+ and PD-1+) compared to non-armored CAR T-cells. Cytotoxicity analysis in short-, and medium-term co-cultures with LnCap cells showed improved efficacy for the armored CAR T-cells compared to non-armored CAR T-cells. Long-term cytotoxicity also showed increased LnCap killing at the end of 4 rounds of re-challenge by armored CAR T-cells. In vivo, tumor naïve NSG mice injected with PSMA CAR T-cells, with or without IL7Fc armor, did not show safety concerns for the duration of the study. CAR T-cells were detectable in peripheral blood, and mice injected with IL7Fc-armored CAR showed human IL7 expression. Furthermore, PSMA CAR T-cells were highly effective in LnCap xenograft mouse model, causing complete tumor clearance, and showed CAR T-cell expansion in peripheral blood, and improved body weight over the study period. However, mice treated with IL7Fc-armored PSMA CAR T-cells showed a drastic drop in body weight, high level of human IL7 in peripheral blood, and met euthanasia criteria on day 13 after treatment. Cytokine measurements showed significantly higher induction of IFNg and GM-CSF in peripheral blood of IL7Fc-armored PSMA CAR T-cells, compared to non-armored CAR T-cells, suggesting strong activation and contribution to observed lethal toxicity.Conclusions This study identified two PSMA CARs that can effectively target prostate cancer, both in vitro and in vivo. IL7Fc-armored CAR T-cells showed improved in vitro expansion and persistence, however, further studies are needed to design safety switches for inducible expression of IL7Fc.Ethics Approval Safety study in mice was approved by Institutional IACUC review committee at University of Maryland, Baltimore (IACUC protocol no. 1021001: amendment #81). Efficacy study in mice was approved by Labcorp Ann Arbor IACUC, VUF 05.