Modified heated dynamics and deep mutational scanning enhance anti-CXCR2 antibody affinity

Colorectal cancer (CRC) is a leading global malignancy, with many stage IV cases requiring surgery and chemotherapy. However, chemoresistance limits treatment efficacy. Targeting resistance pathways such as CXCR2 with therapeutic antibodies offers a promising solution. This study harnessed deep mutational scanning guided by modified heated coarse-grained molecular dynamics (CGMD) simulations, to enhance the binding affinity of the HY29-1 antibody toward CXCR2. The structural stability and intermolecular binding of the variable heavy (vH) and light (vL) chains of the HY29-1 antibody fragment (Fv-HY29-1) alone and complexed with CXCR2 were assessed using modified heated CGMD simulations under thermal stress conditions. Using a modified heated CGMD protocol, the Fv-HY29-1 antibody fragment, modeled <i>via</i> ClusPro (C7) exhibited remarkable conformational stability, maintaining complete (100%) complexation with CXCR2 over a 70 ns simulation. Meanwhile, a benchmark model of the established Fv-Fab14–canine parvovirus capsid complex retained 100% stability under similar conditions. Extended deep mutational scanning over a 1000 ns trajectory pinpointed critical framework residues in the vL chain (A43, L47, and F98) as key determinants of paratope–epitope binding. Rational substitutions (A43P, L47V, F98W) significantly enhanced binding affinity, improving from −36.02 kcal/mol to −94.09 kcal/mol, approximately two-fold increase in binding strength. This marked affinity gain translated into strengthened CXCR2 engagement, highlighting the promise of structure-guided antibody engineering in optimizing therapeutic interactions. This study demonstrated that a 1 μs modified heated simulation with coarse-grained models and deep mutational scanning enables cost-effective optimization of antibody binding affinity and will guide future design of high-affinity antibodies across diverse antigen targets. The Fv-HY29-1-CXCR2 complex remained 100% stable in modified heated MD simulations.The established Fv-Fab14–canine parvovirus capsid complex model retained 100% stability under similar conditions.Deep mutational scanning through 1000 ns simulation pinpointed crucial residues for stronger antibody-CXCR2 interaction.Mutations in the vL chain (A43P, L47V, F98W) improved binding affinity by approximately two-fold, enhancing CXCR2 engagement. The Fv-HY29-1-CXCR2 complex remained 100% stable in modified heated MD simulations. The established Fv-Fab14–canine parvovirus capsid complex model retained 100% stability under similar conditions. Deep mutational scanning through 1000 ns simulation pinpointed crucial residues for stronger antibody-CXCR2 interaction. Mutations in the vL chain (A43P, L47V, F98W) improved binding affinity by approximately two-fold, enhancing CXCR2 engagement.