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Antibody framework residues affecting the conformation of the hypervariable loops.
Journal of Molecular Biology
|March 20, 1992
Summary
Humanizing antibodies for therapy involves loop transplantation. Framework mutations significantly improved antibody affinity by altering dissociation rates, crucial for successful antibody engineering.
Area of Science:
- Immunology and Biotechnology
- Protein Engineering
- Antibody Therapeutics
Background:
- Antibody humanization or reshaping is critical for therapeutic applications, involving the transplantation of antigen-binding loops from rodent antibodies to human frameworks.
- Achieving high-affinity antigen binding often necessitates additional mutations within the human framework regions beyond the transplanted loops.
- The anti-lysozyme antibody D1.3 serves as a model for studying these framework modifications.
Purpose of the Study:
- To investigate the impact of point mutations in framework regions on the affinity of reshaped antibodies.
- To elucidate the kinetic mechanisms underlying affinity changes induced by framework mutations.
- To assess the contribution of specific framework residue substitutions to antigen binding energy.
Main Methods:
- Generation of reshaped antibody variants with point mutations in framework positions.
- Determination of antibody-antigen affinities using fluorescence quenching upon complex formation and relaxation kinetics.
- Kinetic analysis of antibody-lysozyme complex formation and dissociation using stopped-flow spectrophotometry.
Main Results:
- Reshaped antibodies exhibited a range of dissociation constants for lysozyme, from 3.7 nM to 260 nM.
- Equilibrium constant differences were primarily attributed to variations in the rates of lysozyme dissociation from immune complexes.
- Mutations in heavy chain residues (27, 29, 71) and light chain residue 71 (Phe to Tyr) significantly enhanced antigen binding free energy.
Conclusions:
- Framework region mutations, even without direct antigen interaction, are essential for optimizing reshaped antibody affinity.
- Targeted selection of framework residues is crucial for successful antibody loop transplantation and therapeutic development.
- These framework sites may also play a role in the natural generation of antibody diversity and affinity maturation.