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Thus, efficient screening methods for agonistic antibodies are urgently needed. OX40 is a promising target for cancer immunotherapy due to its role in enhancing T-cell activation and survival. However, effective anti-OX40 agonistic antibodies have not yet been developed.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>We developed a novel screening strategy that involves the selection of nanobody clone pools enriched by biopanning against gp34-engaged and non-engaged OX40-expressing cells, next-generation sequencing, and computational clustering and subtraction analysis to identify clones recognizing the ligand-receptor interface. Representative nanobody clones underwent in vitro validation, including epitope mapping, binding affinity measurements, and functional assessments. Furthermore, we engineered the selected nanobody to enhance its in vivo efficacy. We also performed structural analysis of the nanobody-OX40 complex.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>Our epitope-directed approach efficiently identified nanobody clones recognizing functionally relevant epitopes distinct from dominant immunogenic regions. Notably, clone Nb479 demonstrated robust agonistic activity, closely mimicking the natural ligand gp34 with extensive OX40-binding interactions. Trimerization of Nb479 facilitated potent OX40 activation without the need for a cross-linking scaffold. Conjugation of the Nb479 trimer with an anti-serum albumin nanobody exhibited significantly improved pharmacokinetics in vivo and enhanced antitumor activity in a mouse model treated with CD19 chimeric antigen receptor T cells.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>This study presents an innovative epitope-directed approach that greatly accelerates the discovery of functionally potent agonistic nanobodies by effectively circumventing enrichment-driven epitope bias. Our approach and engineered multivalent anti-OX40 nanobody offer a powerful platform to advance immunotherapeutic strategies for cancer treatment.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1136\/jitc-2025-014419","type":"journal-article","created":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T12:20:17Z","timestamp":1785759617000},"page":"e014419","update-policy":"https:\/\/doi.org\/10.1136\/crossmarkpolicy","source":"Crossref","is-referenced-by-count":0,"title":["Efficient discovery of an agonistic anti-OX40 nanobody by epitope-directed approach to address enrichment-driven epitope bias"],"prefix":"10.1136","volume":"14","author":[{"given":"Tomoki","family":"Iemura","sequence":"first","affiliation":[{"name":"Department of Hematology, Graduate School of Medicine, Kyoto University, Kyoto, 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