INTERNATIONAL CONGRESS OF
PharmacoGenetics
Computational Affinity Maturation of Ipilimumab: Site-directed Mutagenesis for Enhanced CTLA-4 Targeting
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zahra sohrabi nasr ,1,*
1. yazd university
- Introduction: Monoclonal antibodies have transformed cancer therapy by targeting specific immune checkpoints, with Ipilimumab representing the first successful antibody against CTLA-4. CTLA-4 is expressed on activated T cells and regulatory T cells, delivering inhibitory signals that suppress T-cell proliferation and cytokine production. By blocking CTLA-4’s interaction with B7 ligands, Ipilimumab restores CD28-mediated costimulation, enhancing T-cell activation and antitumor immunity. In addition, its IgG1 isotype contributes to depletion of intratumoral regulatory T cells via Fc-mediated mechanisms, further promoting effective antitumor responses. Despite its clinical efficacy, limitations remain due to suboptimal binding affinity, which can restrict therapeutic potency and necessitate higher doses.Enhancing antibody-antigen interactions through site-directed mutagenesis offers a rational solution to improve efficacy. Computational approaches, including molecular docking, bioinformatics analysis, and molecular dynamics simulations, provide structural insights into the antibody-antigen interface, allowing identification of key residues critical for binding. By targeting these residues within the complementarity-determining regions for rational mutation, affinity maturation can be achieved in silico, generating optimized antibody variants before experimental validation. Tools that predict the effects of amino acid substitutions on binding free energy, such as coarse-grained statistical potentials, allow rapid screening of numerous mutations and streamline the design of high-affinity antibodies. This integrated computational strategy supports the development of optimized Ipilimumab variants with improved specificity, enhanced binding to CTLA-4, and potential for reduced immunogenicity and lower therapeutic doses. Here, we employ site-directed mutagenesis guided by in silico modeling to refine Ipilimumab’s antigen-binding properties, providing a framework for next-generation immune checkpoint therapies.
- Methods: For the present study, the three-dimensional structure of CTLA-4 bound to Ipilimumab was obtained from the Protein Data Bank (PDB ID: 5XJ3). The complementarity-determining regions (CDRs) of Ipilimumab were mapped using the SAbDab database. Structural visualization and identification of the interacting residues within the heavy chain CDRs and the CTLA-4 antigen were performed with PyMOL. Potential amino acid substitutions to enhance antibody-antigen binding were proposed and their structural plausibility was assessed using the Swiss-Model server. Docking simulations between the mutated antibodies and CTLA-4 were conducted using the HADDOCK server to evaluate changes in binding orientation and affinity. Subsequently, the binding free energy (ΔG) of the complexes was calculated using the PRODIGY server to quantify the effect of the mutations on interaction strength. The stability of the mutated antibody variants was further examined using the I-Mutant server, providing insights into the structural consequences of the introduced modifications. This computational workflow enabled the rational design and in silico evaluation of Ipilimumab variants, highlighting residues critical for affinity improvement and structural integrity.
- Results: PyMOL analysis revealed that mutating the Asparagine (N) residue at position 57 in the CDR2 of Ipilimumab’s heavy chain to Lysine (K) significantly enhances the interaction with the CTLA-4 antigen. This mutation reduces the bond distance between the antibody and antigen from 3.5 Å to 2.1 Å, indicating a stronger and more stable binding interface. The structural integrity and binding affinity of the mutated antibody were further confirmed using Swiss-Model, HADDOCK, PRODIGY, and I-Mutant servers, supporting the improved interaction and stability resulting from this substitution.
- Conclusion: This study demonstrates the in silico optimization of Ipilimumab to enhance its binding to CTLA-4 through site-directed mutagenesis. Mutation of Asn57 to Lys in the CDR2 of the heavy chain decreased the bond distance between the antibody and antigen from 3.5 Å to 2.1 Å, indicating a stronger and more stable interaction. Structural validation using Swiss-Model ensured the feasibility of the mutation, while HADDOCK docking confirmed improved complex formation. The binding free energy assessment via PRODIGY and stability analysis using I-Mutant further supported the enhanced affinity and robustness of the engineered antibody. These results underscore the effectiveness of computational approaches for rational antibody design and provide a valuable framework for developing high-affinity therapeutic antibodies targeting CTLA-4, potentially improving immunotherapeutic outcomes in cancer treatment.
- Keywords: CTLA-4 checkpoint inhibition, Monoclonal antibody engineering , Rational in silico design , Heavy
