451934 Immunogenomic Engineering of a Plug-and-(dis)Play Hybridoma Platform

Monday, November 14, 2016
Grand Ballroom B (Hilton San Francisco Union Square)
Sai T. Reddy, Dept of Biosystems Science & engineering, ETH Zurich, Basel, Switzerland

Hybridomas, a fusion of primary mouse B cells and myelomas, are stable, rapidly proliferating cell lines widely utilized for monoclonal antibody screening and production. The antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell partner. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering. We used CRISPR-Cas9 to generate targeted double-stranded breaks in immunoglobulin loci. Multiplexed-targeting enabled deletion of the native variable light chain and homology directed repair allowed replacement of the endogenous variable heavy chain with a fluorescent reporter protein, mRuby. New antibody genes were then introduced by using Cas9 to target mRuby and promote replacement with a donor construct encoding a light chain and a variable heavy chain, resulting in full-length antibody expression from the immunoglobulin heavy chain locus. Since hybridomas surface express and secrete antibodies, reprogrammed cells were isolated using flow cytometry and cell culture supernatant was used for antibody production. The simplicity of our approach was exemplified in that we generated a homogenous population of antibody-producing hybridoma cells with just a single transfection and screening step. Our hybridoma plug-and-(dis)play platform represents a rapid method for generating antibody-producing stable cell lines.

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See more of this Session: Poster Session: Bioengineering
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division