A versatile genomic transgenesis platform with enhanced λ integrase for human Expi293F cells

Reliable cell-based platforms to test and/or produce biologics in a sustainable manner are important for the biotech industry. Utilizing enhanced λ integrase, a sequence-specific DNA recombinase, we developed a novel transgenesis platform involving a fully characterized single genomic locus as an artificial landing pad for transgene insertion in human Expi293F cells. Importantly, transgene instability and variation in expression were not observed in the absence of selection pressure, thus enabling reliable long-term biotherapeutics testing or production. The artificial landing pad for λ integrase can be targeted with multi-transgene constructs and offers future modularity involving additional genome manipulation tools to generate sequential or nearly seamless insertions. We demonstrated broad utility with expression constructs for anti PD-1 monoclonal antibodies and showed that the orientation of heavy and light chain transcription units profoundly affected antibody expression levels. In addition, we demonstrated encapsulation of our PD-1 platform cells into bio-compatible mini-bioreactors and the continued secretion of antibodies, thus providing a basis for future cell-based 32 applications for more effective and affordable therapies. Agency for Science, Technology and Research (A*STAR) National Research Foundation (NRF) Published version This work was supported through a grant from the National Research Foundation-Competitive Research Programme, Singapore to P.D. (NRF-CRP21-2018-0002), and the Core Fund from the Singapore Immunology Network, Agency for Science, Technology and Research, Singapore to C-I.W.