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Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

ABSTRACT: Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that were found to be noncompeting. Unlike vectors derived from the same virus, noncompeting vectors effectively coexpress the heavy and light chains in the same cell throughout the plant body, resulting in yields of up to 0.5 g of assembled mAbs per kg of fresh-leaf biomass. This technology allows production of gram quantities of mAbs for research purposes in just several days, and the same protocol can be used on an industrial scale in situations requiring rapid response, such as pandemic or terrorism events.

SUBMITTER: Giritch A

PROVIDER: S-EPMC1566189 | biostudies-literature | 2006 Oct

REPOSITORIES: biostudies-literature

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Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

Giritch Anatoli A Marillonnet Sylvestre S Engler Carola C van Eldik Gerben G Botterman Johan J Klimyuk Victor V Gleba Yuri Y

Proceedings of the National Academy of Sciences of the United States of America 20060914 40

Plant viral vectors allow expression of heterologous proteins at high yields, but so far, they have been unable to express heterooligomeric proteins efficiently. We describe here a rapid and indefinitely scalable process for high-level expression of functional full-size mAbs of the IgG class in plants. The process relies on synchronous coinfection and coreplication of two viral vectors, each expressing a separate antibody chain. The two vectors are derived from two different plant viruses that w ...[more]

PMID: 16973752

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Project description:Agriculture is facing new challenges, with global warming modifying the survival chances for crops, and new pests on the horizon. To keep up with these challenges, gene delivery provides tools to increase crop yields. On the other hand, gene delivery also opens the door for molecular farming of pharmaceuticals in plants. However, towards increased food production and scalable molecular farming, there remain technical difficulties and regulatory hurdles to overcome. The industry-standard is transformation of plants via Agrobacterium tumefaciens, but this method is limited to certain plants, requires set up of plant growth facilities and fermentation of bacteria, and introduces lipopolysaccharides contaminants into the system. Therefore, alternate methods are needed. Mechanical inoculation and spray methods have already been discussed in the literature - and here, we compare these methods with a newly introduced petiole injection technique. Because our interest lies in the development of plant viruses as immunotherapies targeting human health as well as gene delivery vectors for agriculture applications, we turned toward tobacco mosaic virus as a model system. We studied the effectiveness of three inoculation techniques: mechanical inoculation, Silwet-77 foliar spray and petiole injections. The foliar spray method was optimized, and we used 0.03% Silwet L-77 to induce infection using either TMV or a lysine-added mutant TMV-Lys. We developed a method using a needle-laden syringe to target and inject the plant virus directly into the vasculature of the plant - we tested injection into the stem and petiole. Stem inoculation resulted in toxicity, but the petiole injection technique was established as a viable strategy. TMV and TMV-Lys were purified from single plants and pooled leaf samples - overall there was little variation between the techniques, as measured by TMV or TMV-Lys yields, highlighting the feasibility of the syringe injection technique to produce virus nanoparticles. There was variation between yields from preparation to preparation with mechanical, spray and syringe inoculation yielding 40-141 mg, 36-56 mg, 18-56 mg TMV per 100 grams of leaves. Similar yields were obtained using TMV-Lys, with 24-38 mg, 17-28, 7-36 mg TMV-Lys per 100 grams of leaves for mechanical, spray and syringe inoculation, respectively. Each method has its advantages: spray inoculation is highly scalable and therefore may find application for farming, the syringe inoculation could provide a clean, aseptic, and controlled approach for molecular farming of pharmaceuticals under good manufacturing protocols (GMP) and would even be applicable for gene delivery to plants in space.

Project description:IntroductionAnimal models play a vital role in pharmaceutical research and development by supporting the planning and design of later clinical studies. To improve confidence and reliability of first in human dose estimates it is essential to assess the comparability of animal studies with the human situation. In the context of large molecules, it is particularly important to evaluate the cross-species-translatability of parameters related to neonatal fragment crystallizable receptor (FcRn) binding and target mediated drug disposition (TMDD), as they greatly influence distribution and disposition of proteins in the body of an organism.MethodsPlasma pharmacokinetic data of the therapeutic protein efalizumab were obtained from literature. Physiologically based pharmacokinetic (PBPK) models were built for three different species (rabbit, non-human primate (NHP), human). Target binding was included in the NHP and human models. The assumption of similar target turnover and target-binding in NHP and human was explored, to gain insights into how these parameters might be translated between species.ResultsEfalizumab PBPK models were successfully developed for three species and concentration-time-profiles could be described appropriately across different intravenously administered doses. The final NHP and human models feature a common set of parameters for target turnover and drug-target-complex internalization, as well as comparable target-binding parameters. Our analyses show that different parameter values for FcRn affinity are crucial to accurately describe the concentration-time profiles.DiscussionBased on the available data in rabbits, NHP and humans, parameters for FcRn affinity cannot be translated between species, but parameters related to target mediated drug disposition can be translated from NHP to human. The inclusion of additional pharmacokinetic (PK) data including different efalizumab doses would further support and confirm our findings on identifying TMDD and, thus, binding kinetics of efalizumab in NHPs. Furthermore, we suggest that information on target expression and internalization rates could make it possible to develop comprehensive human PBPK models with minimal animal testing. In this project, we compared the pharmacokinetics of a therapeutic protein in rabbit, NHP and human using an open PBPK modeling platform (Open Systems Pharmacology Suite, http://www.open-systems-pharmacology.org). Our findings could support similar translatory studies for first in human dose predictions in the future.

Dataset Information

Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

Publications

Rapid high-yield expression of full-size IgG antibodies in plants coinfected with noncompeting viral vectors.

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