Evaluation of plant-expressed human PD-1 as an immunogen capable of inducing native PD-1 recognizing antibodies

Abstract Programmed cell death protein-1 (PD-1) is a key immune checkpoint receptor and an important target for therapeutic antibody development. A critical factor in the development of therapeutic monoclonal antibodies is the selection and preparation of the target antigen. Here, we established a plant-based platform for human PD-1 production using Nicotiana benthamiana and evaluated its biochemical properties and immunogenic potential in comparison with bacterial ( E.coli ) and mammalian (HEK293T cells) expression systems. Codon optimization and promoter selection significantly enhanced transient PD-1 expression, with the pFM′M vector producing the highest yield. Although plant-derived PD-1 predominantly accumulated in the insoluble fraction and required denaturing purification, sufficient quantities were obtained for immunological evaluation. Mice immunized with plant-produced PD-1 exhibited strong splenic and lymph-node responses. ELISA and flow cytometry analyses demonstrated that antibodies generated from plant-derived PD-1 recognized native, cell-surface PD-1, although with lower potency than antibodies induced by mammalian cell-derived antigen. While further optimization is required to improve solubility and preserve native protein conformation, this study highlights the potential for generating antigens for therapeutic antibody discovery. Thus, N. benthamiana is a viable platform for producing immunogenic human PD-1 capable of eliciting functional antibodies.