Functional diversification of hybridoma-produced antibodies by CRISPR/HDR genomic engineering

Hybridoma technologyis instrumental for the development of novel antibodytherapeutics and diagnostics. Recent preclinical and clinical studies highlight the importance of antibody isotypefor therapeutic efficacy. However, since the sequence encoding the constant domains is fixed, tuning antibodyfunction in hybridomas has been restricted. Here, we demonstrate a versatile CRISPR/HDR platform to rapidly engineer the constant immunoglobulin domainsto obtain recombinant hybridomas, which secrete antibodiesin the preferred format, species, and isotype. Using this platform, we obtained recombinant hybridomas secreting Fab′ fragments, isotype-switched chimeric antibodies, and Fc-silent mutants. These antibodyproducts are stable, retain their antigen specificity, and display their intrinsic Fc-effector functions in vitro and in vivo. Furthermore, we can site-specifically attach cargo to these antibodyproducts via chemoenzymatic modification. We believe that this versatile platform facilitates antibodyengineering for the entire scientific community, empowering preclinical antibodyresearch.