Expressão de anticorpos monoclonais anti-SARS-CoV2 em células CHO

Abstract

Monoclonal antibodies (mAbs) currently represent the most relevant group of biological products in the biopharmaceutical market. Because these molecules are composed of two different polypeptide units — two light chains (LC) and two heavy chains (HC) — several strategies can be implemented to enable their proper expression in heterologous systems. Among these strategies, the use of monocistronic vectors (each containing the coding sequence for either LC or HC) and polycistronic vectors (in which two or more genes or cistrons are expressed from a single mRNA molecule) are well described, being the lader commonly based on internal ribosome entry site (IRES) elements. In this context, two mAbs with neutralizing activity against SARS-CoV-2, previously isolated by other research groups, were selected as model molecules to evaluate the application of a modular expression vector system based on native and adenuated versions of the IRES element from encephalomyocarditis virus (EMCV). The vector design enabled the generation of different types of constructs — mono-, bi-, or tricistronic —, alternating the positions of the LC- and HC-encoding genes relative to the EMCV IRES element. In the tricistronic version, an adenuated version of the EMCV IRES element was introduced upstream of a gene that assists in the detection (encoding green fluorescent protein) or in the amplification of gene expression (encoding the dihydrofolate reductase enzyme). For both mAbs, transient transfection experiments performed in Chinese hamster ovary (CHO) cells of the ExpiCHO™ playorm, showed higher production levels associated with the use of vectors containing the LC gene in the first cistron (for both bicistronic and tricistronic constructs). mAbs obtained by transient transfections using bicistronic vectors with the LC gene in the first cistron were purified by Protein A affinity chromatography and subsequently subjected to physicochemical, structural, and functional characterization. The study allowed the confirmation of the amino acid sequences (by nanoRPLC/MS-MS); the evaluation of secondary, tertiary, and quaternary protein structures (by circular dichroism, intrinsic fluorescence, and SEC-HPLC and TEM, respectively); the determination of electrophoretic migration profiles (by SDS-PAGE and western blot), size distribution (by SEC-HPLC), and N-glycosylation profiles (by HILIC-HPLC and LC/MS). In addition, the ability of the mAbs to bind to different variants of the SARS-CoV-2 spike glycoprotein (by spot blot and LSPR) and to neutralize different viral strains (by in vitro neutralization assays) was also evaluated. Furthermore, one of the mAbs was selected as a candidate molecule for the development, through stable transfection, of constitutively producing cell lines based on the ExpiCHO™ playorm. Two different lipofection protocols were compared, using the cationic lipids Lipofectamine™ 3000 and ExpiFectamine™. Taken together, the results demonstrate that bicistronic vectors based on the EMCV IRES element and containing the LC gene in the first cistron represent an efficient strategy for mAb production in CHO cells, enabling the generation of antibodies that are properly characterized from both structural and functional perspectives, and that the different stable transfection protocols evaluated in ExpiCHO™ cells led to similar volumetric yields (4,1 and 6,4 mg/L).