Structural and In Vitro Functional Analyses of Novel Plant-Produced Anti-Human PD1 Antibody

Abstract

Immunotherapy has emerged as a promising and effective treatment for cancer. The frequently used immunotherapy agents are immune checkpoint inhibitors, such as antibodies specific to PD1, PD-L1, or CTLA-4. However, these drugs are highly expensive, and most people in the world cannot access the treatment. The development of recombinant protein production platforms that are cost-effective, scalable, and safe is needed. Plant platforms are attractive because of their low production cost, speed, scalability, lack of human and animal pathogens, and post-translational modifications that enable them to produce effective monoclonal antibodies. In this study, an anti-PD1 IgG4 monoclonal antibody (mAb) was transiently produced in Nicotiana benthamiana leaves. The plant-produced anti-PD1 mAb was compared to the commercial nivolumab produced in CHO cells. Our results showed that both antibodies have similar protein structures, and the N-glycans on the plant-produced antibody lacks plant-specific structures. The PD1 binding affinity of the plant-produced and commercial nivolumab, determined by two different techniques, that is, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), are also comparable. Plant-produced nivolumab binds to human PD1 protein with high affinity and specificity, blocks the PD-1/PD-L1 interaction, and enhances T cell function, comparable to commercial nivolumab. These results confirmed that plant-produced anti-PD1 antibody has the potential to be effective agent for cancer immunotherapy.

Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

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Figure 6 Binding of plant-produced mAb CR3022 to RBD protein. The different concentration of plant-produced mAb CR3022, standard human IgG1, and a plant-produced anti-PD1 antibody (as negative control) 28 were incubated on plates coated either with the plant-produced SARS-CoV-2 RBD ( a ) or a commercial CHO-produced RBD ( c ) and detected with an HRP-conjugated anti-human kappa antibody. In parallel, different dilutions of positive convalescent serum collected from a COVID-19 patient and negative serum were also tested for plant-produced RBD ( b ) and CHO-produced RBD binding ( d ).

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“…An immunofluorescence assay was performed to determine whether mAb CR3022 recognize SARS-CoV-2. SARS-CoV-2 was inoculated onto Vero cells and infected cells were incubated with a known positive serum, CR3022, and a negative control antibody 28 before detection with an anti-human IgG conjugated with FITC. The results showed that the plant produced mAb CR3022 could bind to SARS-CoV-2 in infected cells, similar to the positive control serum (Fig.…”

“…For the purification of recombinant mAb CR3022, the agroinfiltrated leaves were harvested after 3 days and proteins extracted in extraction buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 ) at pH 7.4 using a previously developed method with some modifications 28 . The crude leaf extract was obtained by homogenization and clarified by centrifugation at 15,000 g for 30 min at 4 °C.…”

“…Positive convalescent serum collected from a COVID-19 patient was used as positive control. The commercially available human IgG1 (ab206198; Abcam, UK), a plant produced anti-PD1 antibody 28 and negative serum were used as negative controls.…”

Rapid transient expression of functional human vascular endothelial growth factor in Nicotiana benthamiana and characterization of its biological activity

“…Plant expression systems have many advantages such as rapid scalability, flexibility, improved safety and high-yield of recombinant proteins at short time [ 29 , 30 ]. Growth factors are extensively studied for their skin renewal activities, wound healing properties, cell growth and collagen biosynthesis.…”