Avaliação da interação de fragmentos de anticorpos Fv-Fc derivados do Rituximabe com a proteína de membrana CD20

Abstract

Rituximab was the first monoclonal antibody approved for oncological treatment, targeting the CD20 protein, which is expressed on B lymphocytes but absent in plasma cells and hematopoietic stem cells. It is widely used in the treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphomas (NHL), diseases that together account for a significant number of cancer cases in Brazil. According to the National Cancer Institute (INCA), between 2023 and 2025, approximately 11,450 new cases of leukemia and 12,040 cases of NHL are expected annually, within a total of approximately 704,000 new cancer diagnoses per year. These data highlight the importance of monoclonal antibody (mAb)-based therapies. Despite the established use of Rituximab, its efficacy can be enhanced through antibody engineering, including the development of single-chain variable fragments (scFvs). These fragments are smaller, easier to manipulate in the laboratory, and more cost-effective to produce. Additionally, they can be optimized through directed evolution and selected via phage display, subsequently being fused to crystallizable fragments (Fc). The resulting fragment, termed a variable fragment fused to the crystallizable fragment (Fv-Fc), enables the assessment of both antigen recognition and binding capacity as well as the activation of immune effector functions. In this context, this study aimed to comparatively evaluate the antigen-specific binding capacity of two Fv-Fc fragments derived from Rituximab, expressed on the cell surface: one with the original sequence and another containing a mutation designed to enhance its affinity for CD20. The fragments were expressed in CHO cells and purified by chromatography. Their interactions with CD20 were assessed via flow cytometry using different concentrations of both the native and mutant Fv-Fc fragments. The Fv-Fc fragments were successfully obtained, and the results indicated that the mutant Fv-Fc fragment exhibited significantly higher interaction capacity with CD20. Moreover, no relevant nonspecific interactions were observed, as demonstrated by the low staining of CD20- cells, even at the highest tested Fv-Fc concentrations. These findings suggest that the introduced mutation in the scFvs may contribute both to antigen recognition specificity and to a potential increase in the mutant molecule’s affinity for its target.