Desenvolvimento de protocolo, in silico, visando avaliação da interface entre um fragmento de anticorpo e o CD22, para aplicação em CAR-T cell

Abstract

CAR-T cell therapy is an immunotherapy aimed at achieving remission of various types of cancer, such as Acute Lymphoblastic Leukemia (ALL). The therapy includes the step of expressing a Chimeric Antigen Receptor (CAR) in the patient's T cell, which has the function of resizing cellular action, making it capable of recognizing specific antigens on the surface of tumor cells, such as CD22. The structure of the CAR is composed of several domains, among them the one responsible for antigen recognition like the single-chain variable fragment (scFv), which is formed by the VL and VH domains of an antibody, linked by a sequence of residues called a linker. Results from clinical trials of two anti-CD22 CARs, named short and long linker scFvs, both using the VH and VL of M971, have been published, identifying that the short scFv has a higher affinity to CD22 than the long scFv. This work aimed to understand the structural differences of scFv and CD22 at the scFv/CD22 interface with CD22 connected to a lipid membrane model, developing a predictive capacity in silico protocol to propose effective mutations for developing new CARs. Molecular Dynamics (MD) simulations were executed, and the trajectory was analyzed to investigate the free binding energy (ΔGbinding) between CD22 and the scFvs. The Fab fragment of M971 and the D6-D7 domains of CD22 are deposited in the PDB, code 7O52, and were used to generate 3D models submitted to simulations in an aqueous solution and lipid bilayer. The scFv/CD22 complexes were constructed, simulated, and evaluated structurally and energetically. The scFv/CD22 systems in the lipid bilayer were simulated in triplicate. As a result, it was observed that short and long linkers scFv/CD22 showed different structural stability from each other in aqueous solutions but were similar when in the cellular membrane environment. The average ΔGbinding between scFv/CD22 is close to the experimental ΔGbinding only when in the lipid membrane model. The results generated a protocol that can be used for the proposition of mutations to be tested in silico to proceed with incredible speed and qualities to the in vitro and in vivo tests, proposing new CARs that are more effective for CAR-T cell therapy.