Plant production of mAbs employs two main approaches: plant virus-based transient expression and stable transgenic plants. For the latter strategy, the long time frame (months to years) required to create stable transgenic plants is impractical for rapid production of protein samples for pre-clinical studies. In addition, the lack of strong regulatory elements to drive high-level protein accumulation and the position effect associated with the randomness of transgene integration in the plant genome still presents challenges for stable transgenic technology (Chen 2008
). In contrast, transient systems that are focused on production speed have greatly reduced the time needed to obtain material for testing the function of recombinant antibodies in preclinical trials (Chen et al. 2009
; Lico et al. 2008
). In addition, highly efficient viral replicon vectors have enabled high-level antibody expression in plants. For example, it has been recently reported that full-size mAbs can be rapidly produced at levels as high as 0.5 mg of mAb per gram leaf fresh weight (Giritch et al. 2006
). However, the process requires two sets of noncompeting viral vectors derived from TMV and PVX (Giritch et al. 2006
). For the production and assembly of more complex mAbs such as secretory IgA and IgM, it remains a difficult task to find additional viruses compatible with the existing TMV/PVX expression system to allow efficient co-expression of three or more distinct subunit proteins in same cells.
Unlike traditional or “deconstructed” viral expression systems (Giritch et al. 2006
) which rely on in planta
assembly and replication of near-full-length viral genome, the BeYDV-derived DNA replicon system requires only two viral elements, the LIR region and the Rep/RepA, for the initiation and amplification of episomal replicons (Mor et al. 2003
). We hypothesized that this system could be used for non-competing co-expression of multiple protein subunits. We found that co-infiltration of replicons encoding two fluorescence proteins (GFP and DsRed) resulted in the exhibition of both fluorescences, with >80% of cells expressing both proteins in the same cell (), indicating our BeYDV replicon system is indeed noncompeting. Our subsequent effort focused on simplifying the system so that it can be feasible for practical production of multiple-subunit protein pharmaceutical candidates. Our group has previously showed that single replicon vectors containing a gene of interest and the Rep/RepA expression cassettes (such as pBYGFP.R in ) were as efficient as their three-component counterparts in directing the expression of target proteins (Huang et al. 2009
). In this study, we created single vectors containing two tandemly linked replicons expressing distinct genes of interest, with the hypothesis that the two replicons can efficiently replicate without interference. Transient leaf infiltration with one such vector, pBYGFPDsRed.R (), resulted in efficient formation of both GFP and DsRed replicons as evidenced by Southern blotting () as well as co-expression of GFP and DsRed in ~95% cells (), thus proving that our hypothesis is correct. Collectively, our results with model proteins GFP and DsRed indicate the feasibility of using our replicon system for high-level co-expression of two subunit proteins.
To evaluate the utility and capacity of our replicon systems for antibody production, we co-expressed both the heavy chain and light chain molecules of a mAb protective against Ebola virus GP1 using either the three-component noncompeting vectors or the single vector pBY-HL (6D8).R. Our results showed that both systems could rapidly (4 dpi) produce 6D8 mAb at ~0.5 mg of mAb per gram leaf fresh weight (), which is comparable to the highest level ever achieved with a plant-based expression system (Giritch et al. 2006
). Furthermore, the mAb can correctly assemble into its tetrameric structure (). We did not observe any non-assembled or partial assembled 6D8 mAb, suggesting that the assembly of the mAb using the DNA replicon system is highly efficient. We further demonstrated that 6D8 mAb produced with this single vector system not only has the correct assembled tetrameric structure, but also can be readily purified, and is functionally active in specific binding to inactivated Ebola virus. In addition to 6D8, we have also successfully expressed several other mAbs and mAb-fusion proteins twice as large as 6D8 with full assembly and functionality (Chen, unpublished data). Our success in producing the fully-assembled tetrameric functional IgG (two heterooligomeric subunits) with a two-replicon single vector strongly suggest that simultaneous expression of as many as four hetero-subunits can be easily achieved using two of such vectors, or by creating single vectors with three or more tandem linked replicons. To our best knowledge, this technology is the first and the only one that has such a potential, among all plant transient replicon expression systems.
While the BeYDV multi-replicon vector provided high expression levels, there may be additional ways to improve its performance. As suggested by others, molecular chaperones could be included in the expression cassette to accommodate the increasing protein folding and assembly demand by the high-level accumulation of oligomeric mAbs (Nuttall et al. 2002
). Furthermore, the anti-silencing element P19 could be incorporated into the single vector system. We have not fully explored the effect of P19 with the multi-replicon single-vector. Finally, incorporation of translation enhancer elements from cowpea mosaic virus RNA-2 (Sainsbury and Lomonossoff 2008
) have the potential to substantially increase expression of mRNA produced by the DNA replicon system.
In summary, the results presented in this paper collectively demonstrate that the BeYDV-derived DNA replicon expression system is capable of rapidly producing high levels of multi-subunit antibodies in plants. The ability of a simplified multi-replicon single vector to deliver multiple expression cassettes/replicons for production of mAbs and other hetero-oligomeric pharmaceuticals provides some key advantages. Once optimized, this expression system has the potential to provide a technology platform to produce a wide spectrum of pharmaceutical proteins with speed, efficiency, cost-effectiveness and safety.