The IgG1 CH2 domain is involved in Fc-mediated effector functions and is a promising scaffold for development of novel therapeutics. We previously reported that removal of seven unstructured N-terminal residues of an autonomous human IgG1 CH2 domain significantly increased its stability and aggregation resistance. However, the way in which the C-terminal residues affect folding is unclear. Here, we found that the CH2 C-terminus is highly sensitive to truncation although these residues adopt a random coil conformation according to the crystal structure of a CH2 domain. To optimize the C-terminus, we used a phage display platform for high-throughput screening of mutants with improved physicochemical properties. After panning of the CH2 mutant library at high temperature against a CH2-specific antibody recognizing a conformational epitope, we obtained two candidates, B3 and D9, with markedly increased thermal stability. We found that substitution of K338 (EU numbering) by isoleucine is crucial for the increased stability, which might be due to enhanced hydrophobic interactions involving W313. However, the aggregation propensity was also increased. To reduce the aggregation propensity, we further mutated the last two residues A339 and K340 adjacent to residue I338 at the C-terminus by rational design and identified a mutant, CH2-IKS (K338I, A339K, and K340S), with high stability and aggregation resistance. In summary, the C-terminus of CH2 is important for its folding and could be further optimized toward better potential applications for CH2-based therapeutics. Our strategy might be also useful for stabilization of other Ig-like proteins.
Keywords: CH2 domain; CH2-based therapeutics; Ig-like protein; aggregation resistance; phage display; physicochemical property; stability.