title: 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.
authors.affiliation: Program in Molecular Medicine; National Yang-Ming University and Academia Sinica; Taipei, Taiwan; Institute of Biomedical Sciences; Academia Sinica; Taipei, Taiwan.
authors.forename: Yu-Cheng
authors.initials: YC
authors.lastname: Su
authors.affiliation: Institute of Biomedical Sciences; Academia Sinica; Taipei, Taiwan; Taiwan International Graduate Program; National Yang-Ming University; Taipei, Taiwan.
authors.forename: Talal S
authors.initials: TS
authors.lastname: Al-Qaisi
authors.affiliation: Institute of Biomedical Sciences; Academia Sinica; Taipei, Taiwan; Taiwan International Graduate Program; National Yang-Ming University; Taipei, Taiwan.
authors.forename: Hsin-Yi
authors.initials: HY
authors.lastname: Tung
authors.affiliation: Faculty of Biomedical Science and Environmental Biology; MedicoGenomic Research Center; Kaohsiung Medical University; Kaohsiung, Taiwan.
authors.forename: Tian-Lu
authors.initials: TL
authors.lastname: Cheng
authors.affiliation: Graduate Institute of Pharmacognosy; Taipei Medical University; Taipei, Taiwan.
authors.forename: Kuo-Hsiang
authors.initials: KH
authors.lastname: Chuang
authors.affiliation: Institute of Biomedical Sciences; Academia Sinica; Taipei, Taiwan.
authors.forename: Bing-Mae
authors.initials: BM
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Sciences; Academia Sinica; Taipei, Taiwan.
authors.forename: Steve R
authors.initials: SR
authors.lastname: Roffler
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000494:pharmacology*; D041702:BALB 3T3 Cells; D003564:Cytidine Deaminase / Q000235:genetics / Q000276:immunology; D018858:Germinal Center / Q000166:cytology / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000166:cytology / Q000276:immunology*; D017578:Immunoglobulin Class Switching; D016086:Lentivirus; D051379:Mice; D011092:Polyethylene Glycols; D027041:Somatic Hypermutation, Immunoglobulin; D014161:Transduction, Genetic
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D011092:Polyethylene Glycols; D003564:Cytidine Deaminase
keywords: AID; PEG; activation-induced cytidine deaminase; affinity maturation; anti-PEG; class switch recombination; germinal center reaction; hybridoma; polyethylene glycol; somatic hypermutation; temperature-dependent binding; temperature-selective antibody