Expression and functional analysis of anti-human PD-1 monoclonal antibody in transgenic plants

Plant-based biopharmaceutical platforms offer a cost-effective and scalable alternative for therapeutic antibody production. In this study, transgenic Nicotiana tabacum (N. tabacum) plants were generated to express pembrolizumab, an anti-human programmed cell death protein-1 (PD-1) monoclonal antibody (mAb), targeting both classical PD-1 on immune cells and the recently identified intrinsic PD-1 (iPD-1) variant in tumor cells. The plant-derived anti-PD-1 mAb (mAb^P PD-1) was successfully purified and validated through SDS-PAGE and immunoblotting. Functional analyses using ELISA and immunohistochemistry demonstrated that mAb^P PD-1 exhibits strong binding affinity to recombinant human PD-1 and efficiently detects PD-1 expression in human tonsil tissue. Importantly, cell-based assays demonstrated that mAb^P PD-1 binds effectively to iPD-1-expressing bladder urothelial cancer cell lines, resulting in significant inhibition of cell proliferation. Mechanistically, Western blot analysis revealed that mAb^P PD-1 markedly suppresses extracellular signal-regulated kinase (ERK) phosphorylation without altering total ERK levels, indicating direct modulation of the mitogen-activated protein kinase (MAPK) signaling pathway associated with tumor cell proliferation. These findings establish transgenic tobacco plants as a cost-effective and scalable platform for producing functional anti-PD-1 antibodies with potent immunoregulatory and anti-proliferative properties. The dual targeting of immune cell PD-1 and tumor cell iPD-1 underscores the therapeutic potential of plant-derived antibodies in cancer immunotherapy.