(228fa) Production and Purification of Oxidation-Resistant Alpha-1 Antitrypsin By Transient Expression in N. Benthamiana

Alpha-1 antitrypsin (AAT) is an acute phase protease inhibitor. Currently, the only available treatment for patients with COPD caused by AAT deficiency is replacement therapy, requiring weekly infusions of human plasma-derived AAT. While AAT has shown potential when inhaled to treat both emphysema and cystic fibrosis, the oxidation susceptibility of methionine residues at positions 351 and 358 in the reactive site loop have led to difficulties in maintaining AAT activity and stability when aerosolized. Using a novel cucumber mosaic virus-based inducible transient expression system, we have produced a biobetter plant recombinant AAT (prAAT) in Nicotiana benthamiana. This variant replaces the oxidation-susceptible methionine residue at position 358 with a valine residue to increase resistance to oxidation.

N. benthamiana plants were vacuum infiltrated as detached leaves with two strains of Agrobacterium tumefaciens: one carrying the biobetter AAT gene and one carrying a gene for the tomato bushy stunt virus p19 viral RNA silencing suppressor driven by a constitutive cauliflower mosaic virus 35S promoter. Leaves were incubated in humidity chambers at 20 °C in the dark for 6 days before flash freezing in liquid nitrogen, grinding, and protein extraction in a 20 mM Tris, 150 mM NaCl, and 0.01% (v/v) Tween-80 AAT stability buffer. Purification was achieved using Alpha-1 Antitrypsin Select Affinity Chromatography (GE Healthcare) with a 58 ± 8 % yield of active AAT. Activity of purified AAT was confirmed via permanent binding as seen in an enzyme-linked immunosorbent assay, a residual elastase activity inhibitory assay, and a band shift in a Western blot after incubation with excess porcine pancreatic elastase, however, multiple prAAT bands were also observed in all purified extracts. These bands may be due to glycosylation variants or degradation.

Purified prAAT was tested against an analytical standard (Calbiochem) and Prolastin-C (Griffols), a common therapeutic formulation of human AAT, to determine its activity and oxidation resistance. For the purified prAAT, 41 ± 11% of the total AAT was active (able to inhibit porcine pancreatic elastase as determined by a residual elastase activity inhibitory assay) while 72 ± 6% of Prolastin-C AAT was active. Upon exposure to oxidation conditions with 48.9 mM H₂O₂ for 60 minutes at room temperature, prAAT was found to retain 100 ± 20% of its original anti-elastase activity, while Prolastin-C retained 14 ± 5% anti-elastase activity and analytical standard AAT retained 12 ± 8% anti-elastase activity. Future work will focus on increasing the percentage of total prAAT that is active during recovery and purification.