Plant-Based Production and Purification of Functional Anti-CD3 Antibody Using Agrobacterium-Mediated Expression

The murine monoclonal antibody OKT3, which targets the CD3 complex on T cells, was the first FDA-approved therapeutic antibody and remains clinically relevant for reversing acute transplant rejection. However, conventional mammalian production systems for monoclonal antibodies are expensive, time-consuming, and prone to contamination. In this study, we investigated the feasibility of using Nicotiana benthamiana as a cost-effective and scalable plant-based platform for transient expression and purification of functional OKT3 antibody. Codon-optimized sequences for the heavy and light chains of OKT3 were designed with signal peptides for ER targeting and a C-terminal SEKDEL sequence to enhance protein folding and retention. These sequences were cloned into a geminiviral vector and transformed into Agrobacterium tumefaciens, followed by co-infiltration into N. benthamiana leaves. Western blot analysis confirmed successful expression and assembly of the antibody. Purification was performed using Protein A affinity chromatography. Functional analysis by flow cytometry demonstrated that the FITC-conjugated plant-produced OKT3 antibody exhibited slightly higher CD3+ T cell binding efficiency and stronger fluorescence intensity than the commercial APC-conjugated counterpart. These findings support the use of N. benthamiana as a viable platform for functional anti-CD3 antibody production, with potential applications in diagnostics and therapeutics.