TCR-based therapy directed against kallikrein-related peptidase 4 is safe and effective against prostate cancer

Abstract The efficacy of most immunotherapies for prostate cancer is limited by poor tumor immunogenicity, evidenced by minimal T-cell infiltration. Treatment with T cells engineered to express T-cell receptors (TCR) targeting prostate-specific antigens offers a potential solution by bypassing endogenous T-cell repertoire limitations. Through differential gene expression analysis, we have identified kallikrein-related peptidases 2, 3 and 4 (KLK2, KLK3, KLK4) and homeobox B13 (HOXB13) as strictly prostate lineage–specific genes with high expression in prostate cancer and no expression in healthy tissues of risk. Naturally processed peptides derived from these antigens were identified, enabling T-cell enrichment using peptide–MHC multimers. High-avidity T cells targeting these antigens were isolated from allogeneic HLA-mismatched donors. After screening for on-target tumor specificity and absence of off-target reactivity, TCRs recognizing KLK4 in HLA-A*02:01 and KLK3 in HLA-B*35:01 were sequenced and further tested. TCRs were expressed in T cells through TCR gene transfer and TCRs with best performance were selected. Using combinatorial peptide library scanning, the cross-reactive potential of the KLK4-A2 and KLK3-B35 TCRs was analyzed. The KLK3-B35 TCR exhibited cross-reactivity against two additional peptides derived from LOXHD1 and CDH23, with broad tissue-expression, and was therefore excluded. The KLK4-A2 TCR was highly specific for the KLK4 peptide. Further testing confirmed effective cytotoxic killing potential of KLK4-A2 TCR in vitro and in vivo, underscoring its therapeutic potential. These findings highlight the promise of the KLK4-A2 TCR for prostate cancer immunotherapy and demonstrate that prostate-specific antigens can be effectively targeted using TCR-gene transfer strategies.