Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3

Abstract

Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with blood-borne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free N-glycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Hydroponic Treatment of Nicotiana benthamiana with Kifunensine Modifies the N-glycans of Recombinant Glycoprotein Antigens to Predominantly Man9 High-Mannose Type upon Transient Overexpression

“…Over the past decade, Nicotiana benthamiana expression systems using viral and non-viral vectors have become viable platforms for the production of recombinant proteins (Matoba et al, 2011 ; Whaley et al, 2011 ; Chen et al, 2013 ; Nandi et al, 2016 ). Taking advantage of the systems' capacity to rapidly express complex proteins within days, a number of novel biopharmaceutical proteins, including monoclonal antibodies and subunit vaccine antigens, have been produced in N. benthamiana and showed protective efficacy in preclinical animal challenge models (Santi et al, 2006 ; Massa et al, 2007 ; Mett et al, 2007 , 2011 ; D'aoust et al, 2008 ; Lai et al, 2010 , 2014 ; Landry et al, 2010 ; Wycoff et al, 2011 ; Karauzum et al, 2012 ; Chichester et al, 2013 ; Petukhova et al, 2013 ; Shoji et al, 2013 ; Garcia et al, 2014 ; Hiatt et al, 2014 ; Qiu et al, 2014 ; Mardanova et al, 2015 ; Pillet et al, 2015 ; Tsekoa et al, 2016 ). Medicago Inc. has recently obtained a U.S. Food and Drug Administration's emergency use authorization for N. benthamiana -expressed hemagglutinin-based virus-like particle vaccine for H5N1 influenza virus, and will soon initiate a multi-center Phase III clinical trial for a quadrivalent seasonal influenza vaccine, highlighting the regulatory and commercial feasibility of the plant expression technology for biopharmaceuticals development ( http://medicago.com ).…”

The study of degradation mechanisms of glyco-engineered plant produced anti-rabies monoclonal antibodies E559 and 62-71-3

“…The mAbs were expressed as described in our previous study [9]. Briefly, tobacco plants ( Nicotiana benthamiana : ΔXT/FT plant line) were used as expression system.…”

“…The high specificity and potency of therapeutic monoclonal antibodies (mAbs) and their clinical and commercial successes have made them an attractive alternative to RIG [4,8]. In previous work [9], we discussed the use of E559 and 62-71-3 in a cocktail as each mAb targets a different site of the rabies virus glycoprotein and as such prevents viral escape [4,6]. These mAbs were expressed in ΔxT/FT plants, a Nicotiana benthamiana mutant that supports production of fructose-free glycans, and were reported to be more efficacious then RIG.…”

“…These mAbs were expressed in ΔxT/FT plants, a Nicotiana benthamiana mutant that supports production of fructose-free glycans, and were reported to be more efficacious then RIG. Expression levels attained in the transient system were higher than transgenic approaches which makes this system a suitable basis for an economically viable manufacturing process [9]. Efficacy studies indicated 62-72-3 to be most efficacious, followed by E559 and HRIG [9].…”

“…Expression levels attained in the transient system were higher than transgenic approaches which makes this system a suitable basis for an economically viable manufacturing process [9]. Efficacy studies indicated 62-72-3 to be most efficacious, followed by E559 and HRIG [9]. This also indicated that a cocktail of 62-71-3 and E559 could be a good replacement for the current commercially available HRIG.…”