High-affinity antibodies are crucial for development of monoclonal antibody (MAb)-based therapeutics for human diseases. Many new detailed methods for affinity maturation have been developed to improve MAb qualities by site-directed mutagenesis, chain shuffling, and error-prone PCR. Site-directed mutagenesis on hotspots in variable heavy (VH) complementary-determining region (CDR) 3 is a commonly used method for improving therapeutic potency and efficacy of targeted MAbs. Strategies for affinity maturation via multi-site-directed mutagenesis in VH-CDR3 described here are for valuable technical tool in the armamentarium of immunologists for development of fast-performance MAbs. Our strategy includes (1) selection of targeted MAb, (2) replacement of certain amino acid residues (e.g., negative or neutral charge to positive amino acids) in VH-CDR3, and (3) determination of binding activity to a target antigen.
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