Characterization of a Phage Display-Selected scFv Against Neuropilin 1 (NRP1) Isolated from a Naïve Human Library

Immune checkpoint inhibitors are established therapeutics for solid tumors. This study isolated a single-chain fragment variable (scFv) targeting neuropilin 1 (NRP1), a promising therapeutic target. A naïve human scFv phage display library underwent five biopanning rounds against NRP1. The lead clone (KZC28) was expressed and characterized. Binding affinity was quantified via ELISA. Specificity was assessed by cross-reactivity testing against structurally similar/unrelated antigens. Sensitivity determined the lower detection limit. Cell-based ELISA and flow cytometry evaluated binding to endogenous NRP1 on U87-MG glioblastoma and PC3 prostate cancer cells. Computational modeling predicted structural stability, solubility, flexibility, and binding energetics, focusing on complementarity-determining region (CDR) interactions. KZC28 exhibited high affinity (K_d = 0.88 nM) and specificity, with no cross-reactivity despite high sequence identity. Sensitivity reached 20 ng/mL for NRP1 detection. Cell-based assays and flow cytometry confirmed robust binding to U87-MG and PC3 cells, with specific binding comparable to a commercial anti-NRP1 mAb and minimal binding to NRP1⁻ cells. In silico analyses predicted favorable solubility, structural flexibility, and energetically stable CDR-mediated binding. KZC28 demonstrates high specificity and affinity for NRP1 with promising biophysical and functional properties. These support its potential as a targeting moiety for solid tumor therapy, particularly in NRP1-overexpressing cancers. Further preclinical evaluation of in vivo efficacy is warranted.