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

Area of Science:

• Biochemistry
• Computational Biology
• Immunology

Background:

• Colorectal cancer (CRC) is a major global health concern, with advanced stages often requiring surgery and chemotherapy.
• Chemoresistance remains a significant challenge, limiting the effectiveness of current CRC treatments.
• Targeting resistance pathways, like CXCR2, with therapeutic antibodies presents a potential strategy to improve patient outcomes.

Purpose of the Study:

• To enhance the binding affinity of the HY29-1 antibody to CXCR2 using structure-guided engineering.
• To assess the conformational stability and binding interactions of the HY29-1 antibody fragment (Fv-HY29-1) with CXCR2 under simulated thermal stress.
• To identify key residues responsible for antibody-antigen binding and rationally engineer improved affinity.

Main Methods:

• Modified heated coarse-grained molecular dynamics (CGMD) simulations were employed to assess antibody stability and binding.
• Deep mutational scanning was utilized over extended simulation trajectories (up to 1000 ns) to identify critical binding residues.
• Rational amino acid substitutions were designed based on simulation and scanning results to improve antibody affinity.

Main Results:

• The Fv-HY29-1 antibody fragment demonstrated remarkable conformational stability and maintained 100% complexation with CXCR2 during 70 ns simulations.
• Deep mutational scanning identified specific residues (A43, L47, F98) in the antibody's light chain as crucial for binding.
• Rational substitutions (A43P, L47V, F98W) significantly increased binding affinity from -36.02 kcal/mol to -94.09 kcal/mol, a twofold improvement.

Conclusions:

• Structure-guided antibody engineering, utilizing CGMD simulations and deep mutational scanning, is an effective method for optimizing antibody binding affinity.
• The identified mutations provide a rational basis for designing high-affinity antibodies targeting CXCR2 and potentially other antigens.
• This cost-effective approach can accelerate the development of novel antibody-based therapeutics for challenging diseases like colorectal cancer.