Development and Evaluation of a Bispecific Single-Chain Antibody Targeting PSMA and CD3 to Enhance T-Cell-Mediated Lysis

Prostate cancer is among the most prevalent malignancies in men worldwide and is associated with poor outcomes in advanced stages. Conventional therapies, including androgen deprivation and chemotherapy, provide limited benefit and are often accompanied by significant toxicity. This study aimed to evaluate the efficacy and immunological impact of a novel bispecific single-chain antibody targeting prostate-specific membrane antigen (PSMA) and CD3 in preclinical models. A bispecific single-chain variable fragment (scFv) antibody was engineered to simultaneously bind PSMA on tumor cells and CD3 on T cells. The construct was expressed in mammalian cell lines, and its purity, specificity, and binding affinity were characterized. In vivo efficacy was assessed in murine xenograft models of prostate cancer, with treatment groups receiving human T cells with or without the bispecific antibody. Tumor growth, survival, and immunological responses were evaluated using flow cytometry, immunohistochemistry, and histopathological analysis. The bispecific antibody demonstrated high purity (mean 97.7%), strong binding affinity to PSMA (Kd 0.23 nM) and CD3 (Kd 0.30 nM), and specificity >99%. In vivo, treatment significantly reduced tumor growth (75.45 ± 3.52 mm³ vs. 543.39 ± 44.35 mm³ in controls at Day 28; Student’s t -test, t(28) = 40.52, p < .001) and prolonged survival (59.00 ± 0.84 vs. 31.00 ± 0.84 days; Log-rank test, p < .001). Enhanced T-cell activation, infiltration, and IFN-γ release were observed, accompanied by increased tumor necrosis and apoptosis. The PSMA-CD3 bispecific antibody effectively redirected T-cell cytotoxicity against prostate cancer cells, resulting in robust antitumor activity and survival benefit. These findings support further translational development of bispecific immunotherapies for advanced prostate cancer.