Optimization of transient anti-CD3 (OKT3) monoclonal antibody expression in Nicotiana benthamiana

Abstract

The research aimed to develop a production platform for the OKT3 (Muromonab-CD3) monoclonal antibody, which specifically targets the CD3 receptor on T-lymphocytes, using Nicotiana benthamiana and the geminiviral transient expression system (pBYR2e). The heavy and light chain genes of OKT3 were codon-optimized for the host plant and engineered with an nSP3 signal peptide and a C-terminal SEKDEL ER-retention motif to facilitate proper protein folding and assembly within the endoplasmic reticulum (ER). Optimization of agroinfiltration parameters indicated that host plant physiology and bacterial density significantly influenced recombinant protein yields. The highest OKT3 accumulation was achieved using 4-week-old plants, an Agrobacterium inoculum density of OD600 0.2, and a harvest time of 5 days post-infiltration (dpi). Under these optimized conditions, the plant-produced antibody was purified via Protein A affinity chromatography, yielding approximately 19.89 microgram per gram of leaf fresh weight. Western blot analysis confirmed the successful assembly of the intact 150 kDa IgG molecule, which resolved into 50 kDa heavy chain and 25 kDa light chain subunits under reducing conditions. Functional characterization via flow cytometry demonstrated that the plant-produced OKT3 exhibited a binding efficiency of 99.2% toward the CD3ε receptor on human peripheral blood mononuclear cells (PBMCs), performing comparably to the commercial standard (98.9%). These findings verify that the plant-based expression system is capable of producing structurally intact and biologically functional antibodies. This research serves as a significant model for the development of cost-effective and scalable diagnostic reagents and biomedical research tools.