A1 Refereed original research article in a scientific journal
Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries
Authors: Yang, Tong; Veldhoven-Zweistra, Joke; Ligtenberg, Maarten; Erkens, Sigrun; Vredenbregt-van den Berg, Mirella; Jansen, Rick; Chames, Patrick; Bindels, Eric M. J.; Ahmadi, Khadijeh; Bangma, Chris H.; Kalsbeek, Anton M. F.; Leivo, Janne; Lumen, Nicolaas; Werken, Harmen J. G. Van De; Weerden, Wytske M. Van; Kavousipour, Soudabeh; Tooyserkani, Raheleh; Jenster, Guido
Publisher: MDPI AG
Publication year: 2026
Journal: Biomolecules
Article number: 307
Volume: 16
Issue: 2
eISSN: 2218-273X
DOI: https://doi.org/10.3390/biom16020307
Publication's open availability at the time of reporting: Open Access
Publication channel's open availability : Open Access publication channel
Web address : https://doi.org/10.3390/biom16020307
Self-archived copy’s web address: https://research.utu.fi/converis/portal/detail/Publication/515758549
Self-archived copy's licence: CC BY
Self-archived copy's version: Publisher`s PDF
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.
Keywords: folding and docking computational modeling; nanobody; next-generation sequencing (NGS); phage display; prostate cancer; prostate-specific membrane antigen (PSMA); single-domain antibody; target prediction; variable heavy domain of heavy-chain (VHH).
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Funding information in the publication:
This work was made possible through the “IMMPROVE” consortium, sponsored by an Alpe d’HuZes grant of the Dutch Cancer Society (grant #EMCR2015-8022), through the projects CCBC and “Bladder cancer nanobodies” from the Erasmus MC Daniel den Hoed Foundation and through the Chinese Scholarship Council program (grant number 202207650046).
