| Autor: |
Chen, Chuan, Li, Nan, Zhao, Yun, Hang, Haiying |
| Zdroj: |
Biotechnology & Bioengineering; Jan2016, Vol. 113 Issue 1, p39-51, 13p |
| Abstrakt: |
ABSTRACT Heterologous expression of activation-induced cytidine deaminase (AID) can induce somatic hypermutation (SHM) for genes of interest in various cells, and several research groups (including ours) have successfully improved antibody affinity in mammalian or chicken cells using AID-induced SHM. These affinity maturation systems are time-consuming and inefficient. In this study, we developed an antibody affinity maturation platform in Chinese hamster ovary (CHO) cells by coupling recombinase-mediated cassette exchange (RMCE) with SHM. Stable CHO cell clones containing a single copy puromycin resistance gene ( PuroR) expression cassette flanked by recombination target sequences (FRT and loxP) being able to highly express a gene of interest placed in the cassette were developed. The PuroR gene was replaced with an antibody gene by RMCE, and the antibody was displayed on the cell surface. Cells displaying antibodies on their membrane were transfected with the AID gene, and mutations of the antibody gene were accumulated by AID-mediated hypermutation during cell proliferation followed by flow cytometric cell sorting for cells bearing antibody mutants with improved affinity. Affinity improvements were detected after only one round of cell sorting and proliferation, mutant clones with 15-fold affinity improvement were isolated within five rounds of maturation (within 2 months). CHO cells are fast growing, stress-resistant and produce antibody with glycosylations suitable for therapy. Our antibody-evolution platform based on CHO cells makes antibody-affinity maturation more efficient and is especially convenient for therapeutic antibody affinity improvement. Biotechnol. Bioeng. 2016;113: 39-51. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
|
Plný text