Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries

Yang, T.; Veldhoven-Zweistra, J.; Ligtenberg, M.; Erkens, S.; Vredenbregt-van den Berg, M.; Jansen, R.; Chames, P.; Bindels, E. M. J.; Ahmadi, K.; Bangma, C. H.; Kalsbeek, A. M. F.; Leivo, J.; Lumen, N.; van de Werken, H. J. G.; van Weerden, W. M.; Kavousipour, S.; Tooyserkani, R.; Jenster, G.

doi:10.1101/2025.11.17.688677

bioRxiv · 10.1101/2025.11.17.688677

Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries

Yang, T.·Veldhoven-Zweistra, J.·Ligtenberg, M.·Erkens, S.·Vredenbregt-van den Berg, M.·Jansen, R.·Chames, P.·Bindels, E. M. J.·Ahmadi, K.·Bangma, C. H.·Kalsbeek, A. M. F.·Leivo, J.·Lumen, N.·van de Werken, H. J. G.·van Weerden, W. M.·Kavousipour, S.·Tooyserkani, R.·Jenster, G.

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Abstract

There is a strong need for nanobodies targeting 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 on 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), corroborated by epitope competition assays. These findings support the feasibility of non-targeted immunization and panning strategies for isolating clinically relevant cancer nanobodies.

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Yang, T., Veldhoven-Zweistra, J., Ligtenberg, M., Erkens, S., Vredenbregt-van den Berg, M., Jansen, R., Chames, P., Bindels, E. M. J., Ahmadi, K., Bangma, C. H., Kalsbeek, A. M. F., Leivo, J., Lumen, N., van de Werken, H. J. G., van Weerden, W. M., Kavousipour, S., Tooyserkani, R., Jenster, G.. 2025-11-17. Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries. https://doi.org/10.1101/2025.11.17.688677

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