Cells that constitutively diversify their immunoglobulin genes can be used for selection of novel antibodies and refining affinities and specificities of existing ones (Cumbers et al., 2002). The chicken derived DT40 B cell line has been adapted for the expression and diversification of humanized antibodies. Human antibodies in the form of scFvs expressed in the surface of these cells have been successfully diversified to produce a scFv library, from which antibodies of desired specificity and improved affinity can be evolved using a combination of flow cytometry (FACS) and magnetic beads (MACS) (Lim et al., 2016). In this thesis I further developed the system by finding an alternative approach to antibody humanization and evolution that overcomes the obstacles encountered during the evolution of human scFvs in DT40. I tested different approaches and, although presenting new challenges, I found that chimeric human/chicken antibodies could be engineered and expressed in this cell line and overcame many of the problems experienced with scFvs evolution. This approach was used to generate a combinatorial library for the screening of novel antibodies and to introduce V genes of antibodies of known specificity that could be evolved for improved affinity. In addition to the engineering of the DT40 cell line, and with the aim to obtain a sustainable antibody diversification rate over time, I generated a cell cycle-regulated, nuclear restricted version of Activation Induced Deaminase AID that yields high rates of mutations in chimeric human/chicken antibodies. To conclude, a new and promising approach for antibody evolution in DT40 was developed. The new system posed new challenges that were identified and either solved or used to outlined future directions for the development and improvement of the technique.
