Antibody Discovery Ex Vivo Accelerated by the LacO/LacI Regulatory Network

Abstract

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.

Harnessing Gene Conversion in Chicken B Cells to Create a Human Antibody Sequence Repertoire

“…Gene conversion generates sequence diversity in the functional light and heavy chain variable regions by using upstream pseudogenes as the sequence donors in a template-driven, unidirectional process to mutate the single rearranged V region in each locus [19] . Wild type DT40 cells have been used to generate antigen-specific antibodies from the endogenous immunoglobulin loci in vitro but the variable regions remained chicken sequence [20] – [22] . The ability of DT40 cells to promote gene conversion has been applied to exogenous genes such as GFP, which was inserted into the immunoglobulin light chain locus [23] , [24] .…”

Directed evolution of human scFvs in DT40 cells

“…Preventing gene conversion, either by inhibiting the early steps of homologous recombination, by removing the pseudogenes or by replacing the rearranged Ig genes, results in diversification solely by SHM ( Sale et al , 2001 ; Arakawa et al , 2004 , 2008 ). The feasibility of coupling continuous Ig diversification with in vitro selection has been demonstrated for both human cell lines, such as the Burkitt lymphoma line Ramos and the chicken cell line DT40 ( Cumbers et al , 2002 ; Seo et al , 2005 ; Yabuki et al , 2012 ). DT40 cells have additional advantages in that they are genetically tractable and can generate higher mutation loads than Ramos, in part due to their very short generation time.…”