Both chains of the Fv domain are affected during affinity maturation process in anti-protein antibodies. A complete thermodynamic, kinetic and stability dissection

ACIERNO, JP; VILLORDO, S; GOLDBAUM, FA; CAUERFF, A

Boston, Massachusetts, EEUU

Workshop; Fourth Annual PEGS, The Protein Engineering Summit 2008.; 2008

Protein Society

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:6.0pt; font-family:Arial; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Mutations produced during affinity maturation process of anti-protein antibodies usually are found in both CDR and framework regions. This implies conformational and non-covalent bonding changes at the paratope, as well as possible quaternary structure changes and rearrangements at the VH–VL interface. The consequences of the affinity maturation on the stability of the Fv domain were studied in a system composed of two closely related antibodies, F10.6.6 and D44.1, which recognize the same hen egg-white lysozyme (HEL) epitope. The mAb F10.6.6 has an affinity constant 700 times higher than D44.1, due to a higher surface complementarity to HEL. By means of structural comparison, kinetics and thermodynamics of binding and stability studies on Fab and Fv fragments of both antibodies, we have determined that the affinity maturation process of anti-protein antibodies affects the shape of the combining site and the secondary structure content of the variable domain, stabilizes the VH–VL interaction, and consequently produces an increase of the Fv domain stability, improving the binding to antigen. Performing a complete kinetic, thermodynamic and stability dissection of the contribution of affinity maturation process of each chain in the Fv domain by means chain shuffling experiments, we have demonstrated that both chains have evolved to improve the affinity towards antigen. VH maturation improved binding kass and Fv stability, while VL maturation improved binding kdiss and enthalpy but increasing the entropy compensation. Performing point mutation in residues which form both antigen-antibody and VH–VL interface, hot spots were not found to explain the affinity and stability improvement demonstrating that affinity maturation was achieved by a synergic contribution of all the mutations presented in the Fv domain. Also, ANS binding experiments demonstrated that the VH–VL interaction is crucial for the correct folding and stability of Fv domain.