Computational insights into the pH-dependent behavior of Ipilimumab-CTLA-4

Therapeutic antibodies face on-target/off-tumor toxicity, causing adverse effects such as cardiotoxicity, skin rashes, and organ inflammation. To mitigate these challenges, pH-dependent antibodies have been engineered to preferentially bind in the acidic tumor microenvironment while reducing interactions under physiological pH conditions. Building on experimental work that generated Ipilimumab variants (Ipi95, Ipi105, Ipi106) through charged amino acid substitutions in complementarity-determining regions, we employed molecular dynamics simulations to examine their interactions with CTLA-4 in physiological and acidic conditions. All variants exhibited enhanced binding affinity at acidic pH, with a reasonable agreement between computational and experimental binding free energies (R² = 0.7736; Pearson's r = 0.8795, p = 0.0039; Spearman's ρ = 0.8333, p = 0.0102). Statistical analysis revealed notable differences across conditions, most notably for Ipi95, which demonstrated the highest degree of pH sensitivity. Although no major global structural changes were observed between conditions, our simulations revealed distinct local energetic rearrangements and residue-level interaction changes at the binding interface. Decomposition analysis on binding energy further indicated that the overall antigen-binding mode was maintained, whereas the introduced charged residues modulated local interaction strengths. These results provide mechanistic insights into how targeted mutations modulate pH-dependent recognition, offering a framework for the rational design of safer therapeutic antibodies.