Prostate cancer is the most common cancer in men and continues to have a high mortality rate despite recent advances. Novel therapies are urgently required. To date, immunotherapeutic approaches have had limited efficacy in advanced prostate cancer, hampered by the immunosuppressive microenvironment. T cell-engaging bispecific antibodies may overcome this challenge by initiating a potent T cell response to a specific target on the surface of prostate cancer cells. Target selection is critical to elicit a precise anti-tumour response while protecting healthy tissue from T cell activity. Prostate Specific Antigen (PSA) expression is highly restricted to the prostate, but it is located intracellularly before it is secreted into the bloodstream, making it unsuitable for tumour targeting using antibody-based approaches. However, PSA processed into peptides and presented on the cell surface by Human Leukocyte Antigen (HLA) molecules can be targeted by antibodies. Using the discovery platform, Retained Display (ReD), we have isolated single chain variable fragments (scFv) recognising PSA peptides in the context of two common HLA alleles, HLA-A*02:01 and HLA-A*24:02. These scFv selectively bind HLA complexes loaded with PSA peptide, but not with irrelevant peptides. No binding is observed to potential off-target peptides predicted using in silico analysis that are known to be presented by human cells. In T cell-engaging bispecific format, PSA peptide-specific scFv mediate potent cytotoxicity of human prostate cancer cells via activation of donor T cells in vitro. Furthermore, we have generated affinity matured scFvs and are currently evaluating them for enhanced potency and maintenance of target selectivity. Our data demonstrate the ability to unlock the potential of PSA as a therapeutic target, supporting a powerful new approach to combat prostate cancer.