Evaluation of the in vitro biological activity of biosimilar and biobetter versions of rituximab developed in Bio-Manguinhos for lymphoma treatment

Abstract

This work aims to evaluate the biosimilar and biobetters of rituximab developed in Bio-Manguinhos regarding their ability to bind to the CD20 antigen and the in vitro biological activity. Methodology: Through flow cytometry assays it was possible to analyze the binding properties of the constructs to the CD20 antigen on the surface of leukemia cells (K562 CD20+) and the presence of NKG2DL in the constructs. Potential ADCC and CDC were evaluated using the CytoTox96 kit, a non-radioactive cytotoxicity colorimetric assay capable of measuring the lactate dehydrogenase in the medium, and NK cells expanded in vitro as effector cells. Results: Flow cytometry assays have demonstrated that the constructs produced in Bio-Manguinhos are capable of binding to CD20+ cells and that NKG2DLs are present in the biobetters constructs. ADCC and CDC assays demonstrated that the presence of NKG2DLs in the constructs improved the in vitro biological activity of the mAb, evidencing a higher percentage of cell lysis of the biobetter when compared to the biosimilar and rituximab (MabThera) antibodies. Conclusion: The conclusion is that the antibodies developed are capable of binding to CD20 expressing cells and the biobetters presented improved biological activity in vitro when compared to the biosimilar and reference rituximab, suggesting that the addition of the NK-cell ligand to the anti-CD20 mAb may enhance the therapeutic efficacy of rituximab and other therapeutic antibodies. To reinforce this hypothesis, we have established an in vivo model consisting in xenografts of CD20+ the human lymphoma cell line (RAJI) in immunodeficient mice. The immunodeficient animals will be treated with primary human NK cells and/or the mAbs of interest to evaluate the in vivo enhancing function of the new conformations of the anti CD20 mAbs.