Generation of affinity matured binder by site directed mutagenesis

Abstract

Antibodies are the macro-molecules produced by vertebrates in response to antigen. Antibodies are present in the lymphoid tissues and blood. Nowadays antibody or its fragments are used in both therapeutics and diagnostics because of their high specificity. Moreover, antibody drug conjugates are also used for target specific drug delivery. High throughput molecular biology and cloning techniques have made production of high affinity binders in vitro. Phage diplay platform, displays the protein of interest on the surface of the phage encoding the antibody. This revolutionized technology has helped in the efficient selection of high affinity engineered antibody. In this study, we are utilizing one of the site directed mutagenesis technique, Kunkel mutagenesis for secondary library preparation. Randomization of CDR L1, L2, L3, H1 and H2 using pre-designed random primers were performed. Different CDR randomized libraries were pooled and high affinity binders were selected by biopannings. Output phages from the biopanning were produced in expression vector and screening of individual clones was done for identification and isolation of high affinity binders