High‐level rapid production of full‐size monoclonal antibodies in plants by a single‐vector DNA replicon system

Abstract

Plant viral vectors have great potential in rapid production of important pharmaceutical proteins. However, high-yield production of heterooligomeric proteins that require the expression and assembly of two or more protein subunits often suffers problems due to the “competing” nature of viral vectors derived from the same virus. Previously we reported that a bean yellow dwarf virus (BeYDV)-derived, three-component DNA replicon system allows rapid production of single recombinant proteins in plants (Huang et al. 2009). In this article, we report further development of this expression system for its application in high-yield production of oligomeric protein complexes including monoclonal antibodies (mAbs) in plants. We showed that the BeYDV replicon system permits simultaneous efficient replication of two DNA replicons and thus, high-level accumulation of two recombinant proteins in the same plant cell. We also demonstrated that a single vector that contains multiple replicon cassettes was as efficient as the three-component system in driving the expression of two distinct proteins. Using either the non-competing, three-vector system or the multi-replicon single vector, we produced both the heavy and light chain subunits of a protective IgG mAb 6D8 against Ebola virus GP1 (Wilson et al. 2000) at 0.5 mg of mAb per gram leaf fresh weight within 4 days post infiltration of Nicotiana benthamiana leaves. We further demonstrated that full-size tetrameric IgG complex containing two heavy and two light chains was efficiently assembled and readily purified, and retained its functionality in specific binding to inactivated Ebola virus. Thus, our single-vector replicon system provides high-yield production capacity for heterooligomeric proteins, yet eliminates the difficult task of identifying non-competing virus and the need for co-infection of multiple expression modules. The multi-replicon vector represents a significant advance in transient expression technology for antibody production in plants.

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

“…The amino acid sequence for murine monoclonal antibody 6D8 (5) was used to design plant codon-optimized genes encoding the corresponding HC and LC variable regions; each gene was synthesized commercially, and fused to mouse γ-2a and κ-constant regions, respectively. When HC and LC were coexpressed in tobacco, we observed assembled murine 6D8 (m-6D8) (7). In addition, the sequences for H and L of the murine mAb were used by Biovation (Edinburgh, Scotland) to generate the variable regions of a "humanized" 6D8 (h-6D8) via proprietary peptide threading software on a fee-for-service basis.…”

“…These deimmunized sequences (6) were joined with human IgG1 and λ-chain constant regions, and codon-optimized genes for h-6D8 H and Lchain expression in plants were synthesized commercially. When coexpressed in tobacco, they assembled h-6D8 (7). With the availability of expression vectors for both m-6D8 and h-6D8 in hand, we designed a plant-optimized DNA sequence encoding GP1, by using codons that are preferred in tobacco and removing spurious mRNA-processing signals (14).…”

“…These humanized 13F6 and 6D8 (h-13F6 and h-6D8) heavy-chain (HC) and light-chain (LC) variable regions were joined with human IgG1 and κ-chain constant regions that had been codon-optimized for expression in N. benthamiana. The h-6D8 mAb was found to express at levels as high as 0.5 mg/g of leaf tissue (7). The h-13F6 was also expressed in N. benthamiana to produce various glycoforms of the mAb; these were evaluated for efficacy in a lethal mouse EBOV challenge model (8).…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…In contrast with the stably transformed plants, transient expression systems lead to high-yield of functional recombinant proteins 200-500 mg/kg of fresh tissue in only several days (<2 weeks). [130][131][132] However, the presence of non-human immunogenic glycans, such as β1,2 xylose and core α1,3 fucose constituted the major limitation to their use for the production of therapeutics. 133 Knocked-out lines for xylose transferase (Xylt1) and fucose transferases (Fuct1) of Arabidosis thaliana were also generated, allowing the biosynthesis of recombinant antibodies with homogeneous mammalian-like complex glycans.…”

Agroinfection of tobacco by croton yellow vein mosaic virus and designing of a replicon vector for expression of foreign gene in plant

“…Initial attempts with few bipartite begomovirus [tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV)] as a gene expression vector were encountered with the structural instability of vector, which undermined the potentiality of geminivirus as an efficient vector for expression of foreign gene in plant. However, the change in the design of vector, where the viral genome was used as replicon by deleting coat protein or/and movement protein encoding sequences and by using the 35S promoter to drive the foreign gene expression brought back geminivirus as a potential candidate for the high level expression of foreign protein in plant [4,12]. However, geminivirus based efficient gene expression vector is under the developmental stage.…”