{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,17]],"date-time":"2026-02-17T06:06:39Z","timestamp":1771308399095,"version":"3.50.1"},"reference-count":58,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T00:00:00Z","timestamp":1771027200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004622","name":"Dutch Cancer Society","doi-asserted-by":"crossref","award":["#EMCR2015-8022"],"award-info":[{"award-number":["#EMCR2015-8022"]}],"id":[{"id":"10.13039\/501100004622","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100004543","name":"Chinese Scholarship Council program","doi-asserted-by":"crossref","award":["202207650046"],"award-info":[{"award-number":["202207650046"]}],"id":[{"id":"10.13039\/501100004543","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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\u2013positive 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.<\/jats:p>","DOI":"10.3390\/biom16020307","type":"journal-article","created":{"date-parts":[[2026,2,16]],"date-time":"2026-02-16T12:37:28Z","timestamp":1771245448000},"page":"307","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Cell-Based Immunization Combined with Single-Round Cell Panning Enables Discovery of PSMA-Targeting Nanobodies from Phage Display Libraries"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8561-3441","authenticated-orcid":false,"given":"Tong","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Faculty of Medicine and Human Sciences, Ghent University, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joke","family":"Veldhoven-Zweistra","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-6698-9655","authenticated-orcid":false,"given":"Maarten","family":"Ligtenberg","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sigrun","family":"Erkens","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mirella Vredenbregt-van den","family":"Berg","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rick","family":"Jansen","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Cancer Computational Biology Center, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6104-6286","authenticated-orcid":false,"given":"Patrick","family":"Chames","sequence":"additional","affiliation":[{"name":"Aix Marseille University, CNRS, INSERM, CRCM, Institut Paoli-Calmettes, 13273 Marseille, France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9502-669X","authenticated-orcid":false,"given":"Eric M. J.","family":"Bindels","sequence":"additional","affiliation":[{"name":"Department of Hematology, Erasmus Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Khadijeh","family":"Ahmadi","sequence":"additional","affiliation":[{"name":"Department of Medical Biotechnology, Bushehr University of Medical Sciences, Bushehr 7514633341, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chris H.","family":"Bangma","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anton M. F.","family":"Kalsbeek","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Janne","family":"Leivo","sequence":"additional","affiliation":[{"name":"Department of Life Technologies and InFLAMES Research Flagship Center, University of Turku, FI-20014 Turku, Finland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicolaas","family":"Lumen","sequence":"additional","affiliation":[{"name":"Urology Department, Ghent University Hospital, 9000 Ghent, Belgium"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9794-1477","authenticated-orcid":false,"given":"Harmen J. G. van de","family":"Werken","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Cancer Computational Biology Center, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"},{"name":"Department of Immunology, Erasmus MC Cancer Institute, University Medical Center, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0324-4804","authenticated-orcid":false,"given":"Wytske M. van","family":"Weerden","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Soudabeh","family":"Kavousipour","sequence":"additional","affiliation":[{"name":"Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medical Sciences, Bandar Abbas 7919693116, Iran"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raheleh","family":"Tooyserkani","sequence":"additional","affiliation":[{"name":"Department of Urology, Erasmus MC Cancer Institute, P.O. 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