Introduction:The Chimeric Antigen Receptor (CAR) is recombinant protein expressed in T cells. The Single Chain Variable Fragment (scFv) formed by the VH and VL portions of a monoclonal antibody and the linker, which connects the two domains, is the CAR structure capable of detecting tumor antigens. Two anti-CD22 CARs that are already in clinical trials use the M971 derived scFv, one using a short linker scFv (GGGGS) and the other using a long linker scFv (GGGGS)4, with the short linker scFv having the highest affinity for the CD22, since the short linker in this scFv favors the formation of nanoclusters. Knowing this, it becomes necessary, to begin with assessing whether the linker size in the M971 scFvs affects the structural equilibrium of the scFv, using Molecular Dynamics (DM) simulation.Objectives: Model the structure of two M971 scFvs and submit them to DM simulation, in order to assess whether linker size change in M971 scFvs affects structural equilibrium in aqueous solution.Methodology: 3D scFv structures have been constructed with VH and VL derived from the M971 antibody (code PDB 7O52) connected with a short and long linker in Modeller 10.1 software using the addition of missing residues protocol. Then, each scFv model have been simulated at 500 ns in the GROMACS 2018.3 package, using the CHARMM36m force field, with TIP3P water model and 0.15 M concentration of Na+ and Cl-ions. The structural equilibrium was determined by the Root Mean Square Deviation (RMSD). Results:The RMSD profiles show that in the short linker scFv the VH+VL, VH and VL portions reach structural equilibrium after 50 ns of simulation (RMSDs 0.19 ± 0.02, 0.15 ± 0.01 and 0, 12 ± 0.01 nm, respectively) and in the long linker scFv the VH+VL, VH and VL portions reach structural equilibrium after 100 ns of simulation (RMSDs 0.21 ± 0.02, 0.13 ± 0.02 and 0.17 ± 0.02 nm, respectively). The two types of linker were not considered in the RMSD analyzes because of their structural flexibility. Conclusion:The analysis of the RMSD profiles suggests that the scFv with short linker presented greater apparent structural equilibrium, since the VH+VL and VL portions had less structural modifications. However, the VH domain presented more conformational changes compared to the VH portion of the scFv of long linker. These results indicate that the lower structural movement of the VH+VL domain of the short linker scFv compared to the long linker scFv can justify the formation of nanoclusters. Lastly, both scFvs can be used to study the interaction with CD22, in order to analyze whether the same structural behavior of the scFvs will persist and how the size of the linker in the scFvs will influence the interaction of the scFv/CD22 complexes.

Conclusion:The movements leading to sequential conformational changes in CAR-CD19 are in agreement with the CAR function already described. The proposed mechanism ratifies conformational changes in the intracellular domain that are essential for exposing phosphorylation sites and initiate the function of CD3-ζ, which plays a role in signaling transduction.

Introduction: Chimeric antigen receptors (CARs) are recombinant proteins engineered to be expressed on the surface of cytotoxic lymphocytes to redirect their action in recognizing a specific epitope express on malignant cells, which is usually the CD19 protein. The CAR structure has a domain in the extracellular portion, which is commonly composed of a single-chain variable fragment (scFv), that is responsible for the antigen recognition. The scFv is formed by the light variable (VL) and heavy variable (VH) chains of a monoclonal antibody, which are connected by a linker peptide. The CAR detection can be made by adding a tag, as c-Myc, on its structure, like on the scFv. However, is necessary to evaluate if the c-Myc addition affects the scFv structural stability and if, consequently, it would affect that of the CAR as well, what can be done by employing technics such as molecular dynamics (MD) simulation.Objective: Model the scFv structure to enable the construction of two systems, one only with the scFv and another with the c-Myc tag addition (c-Myc-scFv) to evaluate if the c-Myc incorporation will affect the scFv structural stability. Methodology:The scFv tertiary (3D) structure was constructed with VL and VH derivated of the FMC63 antibody, which were connected with the linker (G4S)3, in the software Modeller 9.20 using the multiple templates protocol. The c-Myc (code PDB 2or9) was added to the scFv (c-Myc-scFv) employing the software PyMol. Each model was subjected to 700 ns of MD simulation in the GROMACS package, at the CHARMM36 force field, with the TIP3P water model, and a 0,15 M concentration of Na+ and Cl-ions. The scFv and c-Myc-scFv structural equilibrium was determined by Root Mean Square Deviation (RMSD). Results:The RMSD shows that in the scFv system, VL-VH and VH achieves structural equilibrium after 250 ns and VL after 100 ns (RMSDs 0,16 ± 0,01, 0,17 ± 0,01 and 0,10 ± 0,01 nm, respectively) and in the c-Myc-scFv system, VL-VH and VH after 150 ns, and VL after 100 ns (RMSDs 0,16 ± 0,01, 0,15 ± 0,01 and 0,09 ± 0,01 nm, respectively). The linker was not considered in the RMSD analyzes due to its structural flexibility. Conclusion:From the RMSD profiles, it was possible to observe that the c-Myc presence did not make the scFv structure unstable, but it has shown apparent stability and less structural modification compared to the beginning. According to this result, both scFvs can be used to study their interaction with the CD19 antigen, to analyze if c-Myc will interfere in the interaction between the structures. Lastly, the complete CAR structure will be constructed to analyze if will exist any difference arising from c-Myc presence.

O presente trabalho tem como objetivo divulgar experiências adquiridas durante a disciplina Estágio Supervisionado I do Curso de Licenciatura em Química do Instituto Federal de Educação, Ciência e Tec

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