Production and characterization of a Pembrolizumab-based anti-PD-1 monoclonal antibody using an Agrobacterium-mediated transient plant expression system

초록

Immune checkpoint inhibitors (ICIs) have demonstrated substantial therapeutic efficacy across multiple carcinomas by restoring T-cell–mediated antitumor immune responses. In particular, monoclonal antibodies targeting the programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) axis effectively overcome tumor immune evasion mechanisms in several cancers. However, conventional mammalian cell-based antibody production platforms are limited in large-scale manufacturing due to high cost and complex processes. Plant-based expression systems have therefore emerged as attractive platforms for therapeutic protein production, owing to lower production cost and the absence of zoonotic infection risks associated with animal cells or bacteria. In this study, a pembrolizumab-based anti-PD-1 monoclonal antibody (mAb) was transiently expressed in Nicotiana benthamiana using an Agrobacterium-mediated protein expression system, and its purification and functional properties were evaluated. Transient expression was achieved by incorporating an endoplasmic reticulum retention signal (KDEL) into pEAQ-HT vector, followed by transformation into Agrobacterium tumefaciens and agroinfiltration of plant leaves. Antibody mRNA expression was confirmed by reverse transcription–polymerase chain reaction (RT-PCR), while protein expression was verified by Western blot analysis. Antibody size and purity were assessed using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and size-exclusion high-performance liquid chromatography (SEC-HPLC) analysis. Furthermore, protein-based enzyme-linked immunosorbent assay (ELISA) demonstrated that the plant-derived anti-PD-1 antibody specifically bound recombinant PD-1 protein. Collectively, these findings demonstrate the feasibility of a plant-based transient expression system for the production and purification of structurally intact, antigen-binding immune checkpoint antibodies.

키워드