Prostate specific antigen (PSA) is a key marker for early prostate cancer (PCa) detection. Multi-kallikrein immunoassays are commonly utilized to overcome the limitations of PSA in diagnosing aggressive PCa. PSA subforms have been extensively studied, and their usage has been demonstrated to improve PCa detection in the early stages. Early immunoassays for intact free PSA (iPSA) have demonstrated their clinical utility in multiple large PCa screening cohorts. Adding iPSA to a multi-kallikrein panel (total PSA, free PSA, and hK2) to evaluate the risk of clinically significant PCa in apparently healthy men substantially improves diagnostic specificity. From a technical perspective, the iPSA assay uses a unique monoclonal antibody (Mab) 4D4. The performance characteristics of this antibody are however less than ideal when considering the construction of a robust routine assay. The objectives of the thesis were to improve the binding affinity of 4D4 antibody utilizing phage display technology and to develop different assay formats for the sensitive and robust detection of iPSA and nicked PSA (nPSA).

The 4D4 Mab was cloned as recombinant Fab fragment and three mutant Fab libraries were constructed. Phage display technology was used to enrich iPSA-specific antibody Fab fragments from the libraries. Wild-type 4D4 Mab and an affinity-improved mutant 4D4-L3-2 Fab antibody were used as a tracer or capture in iPSA assay. Immunoassays for total PSA, free PSA, iPSA, nPSA, and total and free hK2 were used to assess perioperative plasma samples. The individual PSA and hK2 parameters alone, in different ratios or in the four-kallikrein combinations were specifically analyzed in patients with prostate gland volumes below or equal and above the median.

Several 4D4 mutants showing improved affinity and slower dissociation rate while maintaining the original iPSA specificity were identified from the Fab libraries. Using the mutant 4D4-L3-2 Fab in optimized versions of the iPSA assay yielded several-fold improvements in assay sensitivity compared with wild-type 4D4 Fab. The iPSA assay using 4D4-L3-2 Fab as a capture antibody offered an improvement in separating cancer from non-cancer, benign and low-grade cancers from clinically significant cancers especially in patients with lower prostate gland volumes.