Avaliação funcional de versões engenheiradas do anticorpo comercial rituximabe

Abstract

Cancer is one of the main challenges facing global public health. Hematological malignancies, such as lymphomas and leukemias, are characterized by high rates of recurrence and resistance to conventional therapies, factors that make the clinical management of thesediseases particularly complex. In this context, monoclonal antibodies (mAbs) have shown high therapeutic potential, and advances in protein engineering have enabled the development of variants with improved structural and functional properties. In this study, we sought to express and purify mutant variants of the commercial monoclonal antibody rituximab in IgG and scFvFc formats. The mutation of interest, called MUT1 (previously selected by phage display), was proposed by directed evolution via PCR and inserted into the native sequence of rituximab. The DNA of the mutated constructs was transformed into Escherichia coli TOP10 cells for plasmid propagation, which were subsequently isolated and subjected to Sanger sequencing to confirm the genetic modifications. After validation, ExpiCHO cells were transfected with the pcDNA™3.4 plasmid containing the constructs of both formats, allowing recombinant expression of the IgG and scFvFc variants. Purification of recombinant antibodies was performed by affinity chromatography on protein A resin, followed by molecular exclusion chromatography, and the structural integrity and purity of the proteins were confirmed by SDS-PAGE and western blotting. Functional assays by flow cytometry demonstrated that both the native antibody and the MUT1 variant specifically recognized Raji cells, exhibiting a dose-dependent binding profile. Functional assays by flow cytometry demonstrated that both the native antibody and the MUT1 variant specifically recognized Raji cells, exhibiting a dose-dependent binding profile. A consistent trend of increased mean fluorescence intensity was observed for the MUT1 variant at higher concentrations, suggesting a possible functional gain associated with the introduced mutation. Thus, the obtaining and characterization of this variant represent an important step toward the standardization of functional assays and the investigation of the structural and biofunctional effects resulting from the mutation, contributing to the rational development of antibodies with improved therapeutic potential.