| Autor: |
Angélica V. Medina-Cucurella, Rena A. Mizrahi, Michael A. Asensio, Robert C. Edgar, Jackson Leong, Renee Leong, Yoong Wearn Lim, Ayla Nelson, Ariel R. Niedecken, Jan Fredrik Simons, Matthew J. Spindler, Kacy Stadtmiller, Nicholas Wayham, Adam S. Adler, David S. Johnson |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
|
| Zdroj: |
Antibodies, Vol 8, Iss 1, p 17 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
2073-4468 |
| DOI: |
10.3390/antib8010017 |
| Popis: |
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. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
|
|
Nepřihlášeným uživatelům se plný text nezobrazuje |
K zobrazení výsledku je třeba se přihlásit.
|
