{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T16:59:32Z","timestamp":1777654772070,"version":"3.51.4"},"reference-count":27,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2019,2,19]],"date-time":"2019-02-19T00:00:00Z","timestamp":1550534400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["R44CA187852"],"award-info":[{"award-number":["R44CA187852"]}],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibodies"],"abstract":"<jats:p>To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism.<\/jats:p>","DOI":"10.3390\/antib8010017","type":"journal-article","created":{"date-parts":[[2019,2,20]],"date-time":"2019-02-20T03:05:52Z","timestamp":1550631952000},"page":"17","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries"],"prefix":"10.3390","volume":"8","author":[{"given":"Ang\u00e9lica V.","family":"Medina-Cucurella","sequence":"first","affiliation":[{"name":"Department of Chemical Engineering and Material Science, Michigan State University, East Lansing, MI 48824, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8581-2240","authenticated-orcid":false,"given":"Rena A.","family":"Mizrahi","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9087-0568","authenticated-orcid":false,"given":"Michael A.","family":"Asensio","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert C.","family":"Edgar","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jackson","family":"Leong","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Renee","family":"Leong","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8568-4901","authenticated-orcid":false,"given":"Yoong Wearn","family":"Lim","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ayla","family":"Nelson","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ariel R.","family":"Niedecken","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan Fredrik","family":"Simons","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew J.","family":"Spindler","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kacy","family":"Stadtmiller","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7782-0814","authenticated-orcid":false,"given":"Nicholas","family":"Wayham","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9301-0037","authenticated-orcid":false,"given":"Adam S.","family":"Adler","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David S.","family":"Johnson","sequence":"additional","affiliation":[{"name":"GigaGen Inc., One Tower Place, Suite 750, South San Francisco, CA 94080, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2019,2,19]]},"reference":[{"key":"ref_1","first-page":"495","article-title":"Continuous cultures of fused cells secreting antibody of predefined specificity","volume":"256","author":"Milstein","year":"2011","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1038\/348552a0","article-title":"Phage antibodies: Filamentous phage 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