Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Abstract

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.

Keywords: :

• germinal center reaction
• somatic hypermutation
• class switch recombination
• AID
• activation-induced cytidine deaminase
• affinity maturation
• polyethylene glycol
• PEG
• temperature-dependent binding
• hybridoma
• temperature-selective antibody
• anti-PEG

10.4161/mabs.29124

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by Academia Sinica and a research grant from the National Science Council, Taipei, Taiwan (NSC 102–2320-B-001–013-MY3). The authors thank Dr. Shu-Chuan Jao of the Biophysics Core Facility, Scientific Instrument Center at Academia Sinica for assistance in performing Biacore T200 and Nano DSC III experiments. We also thank Ms. Chia-Chen Tai and Ms. Tzu-Wen Tai of the Flow Cytometry Core, Scientific Instrument Center at the Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan for help with the LSR II flow cytometer and FACSAria cell sorter. We appreciate helpful discussions with Dr. Mi-Hua Tao of the Institute of Biomedical Sciences and Dr. Andrew H.-J. Wang and Dr. Meng-Chiao Ho of the Institute of Biological Chemistry at Academia Sinica. We appreciate help with lentivirus production by the National RNAi Core Facility, Institute of Molecular Biology / Genomic Research Center, at Academia Sinica, Taipei, Taiwan.

Author Contributions

Y.-C.S. and S.R.R. conceived the project and designed the experiments and analyses. Y.-C.S., T.S.A.Q., H.-Y.T., and B.-M.C. performed experiments. K.-H.C. cloned recombinant antibody genes. Y.-C.S., T.-L.C., and S.R.R. analyzed data and wrote the manuscript.