Affinity maturation of anti-protein antibodies is modulated through increase in stability of the VH-VL interface.

ACIERNO, JUAN PABLO; GOLDBAUM, FERNANDO ALBERTO; CAUERFF, ANA

Chihuahua, México

Simposio; XVII Congreso Nacional de Inmunología de México; 2006

Sociedad Mexicana de Inmunología

Affinity maturation of antibodies against protein antigens is carried out after the antigen-specific T-cell activation of B-cells, accompanying the switch process from IgM to IgG isotype. Point mutations accumulating in the variable domains of antibodies improve the complementarity of the antibody antigen combining site. The study of the molecular basis of affinity maturation mechanism it is not only important to understand this aspect of the humoral immune response but also for biotechnological and biomedical applications since optimizing the affinity of antibodies to subnanomolar levels could be of great importance in those disciplines. Objective: To study the structural basis of the affinity maturation of two anti-lysozyme antibodies, elicited during secondary immune response, in relationship with the stability of their variable domains. Materials and methods: Two monoclonal antibodies (mAbs) were elicited against lysozyme (HEL), the first, D44.1 was obtained after a short immunization plan, and the second, the mAb F10.6.6 was obtained after a long term immunization plan. Variable domain fragments (Fvs) and chimeras derived from chain shuffling of both antibodies were cloned and expressed in E. Coli. Kinetics and thermodynamics studies of Fvs and chimeras were performed by biosensor and isothermal titration calorimetry respectively, Chemical and thermal stability of all the molecular species was studied by circular dichroism and intrinsic fluorescence measurements. Results F10.6.6 and D44.are derived from the same germlines genes recognizing the same epitope on HEL surface, but with F10.6.6 having ~103 increase in affinity for antigen, compared to D44.1. F10.6.6 showed a 1.7 times larger enthalpy component because of the larger amount of contacts to HEL. However, F10.6.6-HEL reaction has an entropic component around 10 times higher than D44.1-HEL that could be explained by the loss of 40 contacts between VL and VH chains in comparison with D44.1-HEL reaction. Besides, we found that Fv F10.6.6 has an increased thermal and chemical stability than D44.1 and that contacts made by VH domain contribute to the association rates while the VL domain contacts module the dissociation rates. Conclusion: An improvement of the variable domain stability that increases the plasticity of the VH-VL interaction results in the improvement of the binding properties of the antibody towards the antigen.