Estudo da interação do scFv do anticorpo Rituximab com a alça do receptor CD20: avaliação da energia livre de ligação pelo método ABF para proposição de biobetters

Given the relevance of cancer in the world, the study of alternatives to its treatment is extremely important, and one of them is to obtain monoclonal antibodies (mAbs) that interact more efficiently with their antigens. Single chain fragment variable (scFv), formed by VH and VL domains of an antibody bound by a flexible peptide (linker), retain the antibodies affinity from which they were originated and have a lower computational cost to Molecular Dynamics (MD) simulation. Then, it is a strategy to seek the optimization of the antibody-antigen interaction. The CD20 receptor, inserted into B lymphocyte membrane, is the antigen recognized by Rituximab mAb. The coordinates of the Rituximab Fab fragment-CD20 loop complex are deposited in PDB, code 2OSL. This study proposes, through MD simulation coupled to Adaptive Biasing Force (ABF) method, to investigate the interaction of the CD20 loop with Rituximab scFv by free energy calculation, in order to compare the results with those obtained experimentally by Li et al. (2009). The scFv model was obtained and simulated for 200 ns in aqueous medium in triplicate (S1, S2 and S3), starting at different initial velocities From stable scFv structures, the scFv-loop complexes of S1, S2 and S3 were constructed and simulated for 100 ns in aqueous medium. Two other systems were constructed: Crystal, structure of the complex not submitted to the DM production stage, only to thermalization; and double mutant D57E/Y102K, obtained from the stable structure of S1 complex. The systems submitted to ABF method showed that the values of DG obtained for S1 and S2 were closer to the experimental ones than those for Crystal, suggesting the importance of previously obtaining a stable structure in aqueous medium by DM and then subjecting it to ABF. Differences were found between ABF results of the interaction of scFv-loop complex and the experimental results of Fab fragment-Daudi cells complex, which contain the complete CD20, but followed the same interaction trend. This suggests that the structure of the entire receptor in cell determines the positioning of the epitope, directly influencing the interaction with the antibody. Thus, the Rituximab scFv gene was cloned and expressed in E. coli, and the CD20 loop will be synthesized to obtain the DG values by radioimmunoassays in order to validate the results obtained by ABF. Further studies of the scFv-loop interaction will enable the development of scFv variants with enhanced interaction affinity, called biobetters.