Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Abstract

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (Crimab^P) had similar binding activity to P-selectin compared to mammalian-derived Crimab (Crimab^M). In surface plasmon resonance, the K_D (dissociation binding constant) and response unit values were lower and higher than Crimab^P, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.