Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from
Tong Yang1,2, Joke Veldhoven-Zweistra1, Maarten Ligtenberg1
1Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands.
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There is a strong need for nanobodies that target novel cancer-associated antigens to advance radioligand imaging and antibody-based therapeutics. In this study, we investigated whether non-targeted llama immunization using tumor cells, combined with non-targeted phage-display panning of human cell lines, could yield nanobodies specific to Prostate-Specific Membrane Antigen (PSMA). Nanobody selection using both classical three-round PSMA negative-positive panning and single-round panning of cell lines (positive or negative) for PSMA showed clear enrichment for PSMA binders in both strategies. Using shRNA knockdown, flow cytometry, cell-ELISA, immunohistochemistry and structural modeling and docking, we confirmed the PSMA-targeting of selected nanobodies. Two distinct epitopes were predicted to be bound by nanobodies PSMANb9 and A7 (JVZ-007), and this was corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating antigen-targeting cancer nanobodies.
