Expressão heterológica e purificação do fragmento variável de cadeia única do anticorpo monoclonal anti-CD19 FMC63 para estudos de aplicação em imunoterapias do câncer

Abstract

INCA's Population Base data indicates that cancer has become a huge public health problem in Brazil. Besides the high incidence rate, its treatment is complex and costly, since a representative portion of the drugs used in therapy are imported. In this context, immunotherapy has gained increasing prominence, proving to be a promising treatment strategy. A new class of immunotherapics, known as Chimeric Antigen Receptors (CARs), consists of genetically modifying patient-derived T cells to express a receptor with the ability to recognize specific antigens present on target malignant cells. One of the main antigens used as a target is the CD19 membrane receptor, a protein overexpressed in B-cell neoplasms. The domain responsible for antigen recognition in a CAR is usually composed of a single-chain variable fragment (scFv). In FDA-approved CARs targeting CD19, it is used a scFv derived from the murine antibody FMC63, in which protein markers (tags), such as the c-Myc tag, can be inserted to facilitate laboratory manipulation and the execution of detection and purification experiments. Thus, this work aimed to express the scFv derived from the FMC63 antibody in a prokaryotic system, with and without the c-Myc tag, purify them and verify the stability of the expressed proteins. Heterologous expression was performed overnight at 15 °C using pET-SUMO vector, E. coli SHuffle strain and IPTG as inducer. The purification process was performed by immobilized metal affinity chromatography and the purifications were evaluated by SDS-PAGE, and immunodetection (Western blot) and thermal stability assays were also performed. From protein electrophoresis and Western blot, the expression of the anti-CD19 scFv was confirmed. A yield of 0.80 mg/L culture was achieved by expression and purification of the soluble portion for the anti-CD19 scFv and a yield of 0.34 mg/L for anti-CD19 scFv with the c-Myc tag. The thermal stability profile, obtained by intrinsic tryptophan fluorescence, was similar among the expressed scFv. It is concluded that the anti-CD19 scFv with and without the c-Myc tag were able to be expressed and purified with the proposed methodology and that the c-Myc tag does not interfere in the stability of the scFv structure. The prospect is to standardize the process of expression and purification of the scFv, in order to enable the performance of binding capacity and affinity assays, to verify if the c-Myc tag interferes in the antibody-antigen interaction.