Transient and Stable Expression of Antibodies in Nicotiana Species

Abstract

Expression and purification of recombinant proteins produced in plants is emerging as an affordable alternative to using more costly mammalian bioreactors since plants are capable of producing mammalian proteins at high concentrations. There are two general methods of expressing foreign proteins in plants, namely, transient expression and stable transgenic expression. Both methods have advantages which serve different purposes. Nicotiana benthamiana is primarily used as plant host for transient expression of foreign proteins. This system is capable of producing high yields of antibody in a relatively short period of time (days); however, intensive upstream processing is required as each plant must be infected with Agrobacterium tumefaciens cells by vacuum infiltration. N. tabacum is often used for production of stable transgenic plants through a procedure that requires longer development time (months). Although antibody yields are smaller compared with the transient method, the advantage of using stable transgenic expression is that very little upstream process management is required once homozygous seed lines are developed. In this chapter, we describe the basic methodologies for expressing antibodies in plants using the transient and transgenic systems.

Chimeric Plantibody Passively Protects Mice against Aerosolized Ricin Challenge

“…The technology entails mass infiltration of mature Nicotiana plants with an Agrobacterium tumefaciens suspension carrying T-DNA encoding viral replicons and results in high MAb recovery from original DNA constructs within days (22). The Nicotiana-based RAMP is being used for manufacturing recombinant MAbs or "plantibodies" for clinical studies under good manufacturing practices (GMP) (20,23).In this study, we sought to exploit the Nicotiana-based RAMP for the purpose of identifying and advancing potential immunotherapeutic agents for ricin toxin. In a proof-of-concept study, we recently characterized chimeric GD12 (c-GD12), a partially humanized murine monoclonal antibody (MAb) directed against an immunodominant, linear B cell epitope on RTA (24).…”

“…The technology entails mass infiltration of mature Nicotiana plants with an Agrobacterium tumefaciens suspension carrying T-DNA encoding viral replicons and results in high MAb recovery from original DNA constructs within days (22). The Nicotiana-based RAMP is being used for manufacturing recombinant MAbs or "plantibodies" for clinical studies under good manufacturing practices (GMP) (20,23).…”

Utility of the P19 suppressor of gene‐silencing protein for production of therapeutic antibodies in Nicotiana expression hosts

“…() present data on a modified P19 that does not cause adverse physiological side effects, but displays a mediocre boosting capability because of the overall lower level of expression that is associated with transgenic systems. As transgenes generally express higher in transient as opposed to transgenic settings (Garabagi et al ., ), we showed that the amount of P19 protein produced by transient infiltration at concentrations of OD 600 = 0.02 and lower did not significantly enhance trastuzumab levels. On the other hand, higher P19 concentrations (OD 600 = 0.2) caused a 15‐fold increase in antibody levels (Figure a).…”

When plant virology met Agrobacterium: the rise of the deconstructed clones

“…() then described a variant of the technique known as ‘syringe infiltration’, in which a needle‐less syringe is used to agroinfiltrate the nonexcised leaves of whole plants, without the need for a vacuum chamber. The first description of vacuum infiltration of whole plants, and not just excised leaves, for transient expression purposes, was not published until almost a decade later (Marillonnet et al ., ), with detailed methods described after that (Garabagi et al ., ; Gleba et al ., ).…”