Elucidating the structural affinity maturation mechanism of anti-protein antibodies. (Poster)

JUAN P. ACIERNO; SEBASTIÁN KLINKE; BRADFORD C. BRADEN; FERNANDO A.GOLDBAUM; ANA CAUERHFF

Angra dos Reis, estado de Río de Janeiro, Brasil

Congreso; 1st Latin American Protein Society Meeting (LAPSM); 2004

Protein Society

The understanding of the molecular basis of affinity maturation mechanism is a matter of great interest in biomedical and biotechnological applications. Affinity maturation of anti-protein antibodies has been reported to be the result of small structural changes, mostly confined to the periphery of the antigen-combining site. Two monoclonal antibodies (mAbs) elicited against lysozyme (HEL) have been analyzed exhaustively. mAb D44.l is a product of a short term immunization and mAb F10.6.6 was obtained after a long term immunization plan. Both mAbs recognize the same epitope on HEL surface, and their variable domains are probably the product of the same recombination event, with a homology close to 95 % of identity. Kinetic (biosensor) and thermodynamics (ITC) studies were performed for both mAbs, being the KA of F10.6.6 ~ 103 higher than the KA of D44.1. In order to understand the role of water molecules present at the protein-protein interface, several solvent stress conditions were tested affecting the mAb-HEL interaction. D44.1-HEL and F10.6.6-HEL, both reactions are enthalpically driven with different unfavorable entropic contribution. The reaction F10.6.6-HEL has 10 times higher entropic component than D44.1-HEL. Crystal structures of the free and complexed Fab fragments from both antibodies were solved at 2.0 Å, allowing structural comparisons between free and their complexed forms. Antigen¬-antibody interface were analyzed, showing that Fab Fl0.6.6 makes additional and shorter non-covalent interactions with HEL than Fab D44.1, giving a possible explanation about the 1000-fold higher affinity of F10.6.6. The contacts between the VL and VH domains were ca1culated. Upon the complex formation, F10.6.6 loses around 40 interchain contacts, meanwhile D44.1 only loses 10 contacts. These results, the F10.6.6 conformational changes and the water release upon binding would explain the higher entropic component of the F10.6.6-HEL binding reaction. Finally, recombinant F10.6.6 and D44.1 Fv fragments were obtained, and refolded in a combinatory way. The chimeric product of shuffling between the VL and VH chains of D44.1 and F10.6.6 were tested in the biosensor in order to understand the contribution of each chain to the affinity maturation process.