Avaliação in silico dos efeitos de mutação em fragmento do anticorpo M971 na interação com o antígeno CD22: estudo por simulação de dinâmica molecular.

Abstract

Chimeric Antigen Receptors (CAR) associated with T cells (CAR-T) constitute arecent immunotherapy capable of aiding in the treatment of patients with relapsed Acute Lymphoblastic Leukemia (ALL) by redirecting the immune cellular response to neoplastic cells that possess the target antigen. The extracellular domain of the CAR, responsible for recognition, can take the form of a SingleChain Variable Fragment (scFv), which consists of the Heavy Variable (VH) and Light Variable (VL) domains of a monoclonal antibody (mAb) and are joined together by a flexible polypeptide of variable length called a Linker. Human clinical trials already use anti-CD22 CAR designs with scFv derived from the M971 antibody with different Linker lengths: short (5 residues) and long (20 residues). Another study by Ereño-Orbea and collaborators (2021) found a mutation (Y55R) at residue 55 of the VH of the M971 mAb that was capable of increasing affinity with the CD22 antigen by approximately 5-fold. The present work aims to study the interface between the mutant M971 scFv with different Linker lengths and the CD22 membrane glycoprotein in terms of structure and affinity using Molecular Dynamics (MD) simulation techniques and binding free energy (ΔG-binding) calculations. The crystallographic structure of the interface between the Fab of M971 and the d6-d7 domains of CD22 is deposited in the Protein Data Bank under the code 7O52, and from it, the CD22 and mutant scFv models used for the interface in systems with and without a cell membrane were constructed. Changes in the protonation states of the histidine residues of CD22 caused by the mutation were also evaluated using computational prediction methods. The simulations were performed considering systems with and without a membrane, with different scFv Linker lengths (short-scFv and long-scFv) and, only for simulations without a membrane, two different protonation states of histidines 122 and 136 of CD22. The membrane simulations were performed in four replicates considering the state in which the histidines were deprotonated. At the end of the simulations, the ΔG-binding was calculated using the MM/PBSA method. The results of the ΔG-binding curves showed more negative values for systems with the short Linker. In the systems without a membrane, changing the histidines to a deprotonated state resulted in the loss of the interface by the VL domain; however, the average ΔG-binding for the short-scFv system remained morenegative than the protonated state. It was also observed that the systemsconsidering the interface between the mutant scFv and CD22 in a cell membrane obtained average ΔG-binding values closer to those found experimentally with the Fab of the M971 mAb.