Generation of nanobodies in E coli using Surface Display Phage Assisted Continuous Evolution (SurPhACE)

Antibodies have a broad range of applications in the industry, medicine and research. However, the development of antibodies is expensive and time consuming. Furthermore, these biological drugs are often proprietary material which makes low-cost distribution difficult in developing countries. In order to address these challenges, we developed Surface Display Phage Assisted Continuous Evolution (SurPhACE), our open-source method that employs the PhiX174 bacteriophage (phage) as a platform to evolve small peptides aided by natural selection. This technique comprises 2 main interacting components: the nanobody, a monomeric version of an antibody, that acts as the spike protein of the engineered phage (EP) and a target protein on the bacterium external membrane which serves as a binding site for viral entry. To complete the system, SurPhACE is paired with an easy-to-build open-source bioreactor to automate the process. This system allows for the evolution of numerous high specificity nanobodies against any target at a low cost. Currently we are developing nanobodies against the human FcR2A receptor due to its importance in the infection mechanism of viruses of the family Flaviviridae. In the process, we have developed two new cloning strategies to produce large quantities of EP DNA in a thermocycler and the construction of an MP6-plasmid-based EP library large enough to carry out natural selection in small scale semicontinuous cultures. Our current goals are to further improve the EP cloning technique, improve the specificity and simplicity of the mutational plasmid, miniaturize the bioreactor and move forward to characterize the binding strength and specificity of the nanobodies produced so far.