Production of monoclonal antibodies (mAb's) in plants has developed greatly over the past two decades. The yields of recombinant proteins produced in plants are generally much lower than those routinely achieved in other systems. High-level production of functional multiplex proteins in plant is not a routine matter, sometimes it is quite challenging. Techniques to optimize heterologous protein overproduction in plants have been explored for host strain selection, plasmid copy numbers, transcription level, promoter selection, mRNA stability, and codon usage, all of which affect the yields of the foreign proteins differently from construct to construct, and from plant species to plant species. When a viral replicon, such as a geminiviral vector, is utilized in order to enhance expression, the performance of the system can be affected by the linear arrangement of DNA elements in the replicon structure, plant developmental stage, or environmental factors, all of which may affect replicon amplification and thereby, protein expression. Oftentimes, the recombinant protein itself plays an important role in determining the yield because some proteins are inherently less stable than others, and some proteins produce unanticipated negative effects on plant growth. In addition, the presence of a signal peptide, the presence of chaperones or protease activity, coexpression of a corresponding antigen, or a stabilizing protein partner, can significantly affect the yield of recombinant protein in plant cells.
Rituximab is a genetically engineered chimeric monoclonal antibody (mAb) anti-CD20 IgG1 licensed in 1997 for treatment of non-Hodgkins lymphoma, under the name Rituxan (Marwick, 1997). Originally isolated as a mouse mAb, the chimeric version replaced most of the mouse-specific regions with human sequences, in order to avoid immune responses in humans. Clinical data from genetic analyses of leukocyte receptor (FcγR) polymorphisms in cancer patients treated with Rituximab showed that antibody-dependent cellular cytotoxicity (ADCC) is a critical mechanism for its clinical efficacy (Weng et al., 2003; Dall'Ozzo et al., 2004). Rituximab-based ADCC is mediated by natural killer (NK) cells that bind the mAb Fc via FcγRIIIa (Dall'Ozzo et al, 2004).
Plants are attractive as pharmaceutical protein factories because they potentially can produce large volumes of products efficiently and sustainably and, under certain conditions, can have significant advantages in manufacturing costs. The unique nicking function of the Rep protein of geminiviruses (Laufs et al, 1995a; Laufs et al, 1995b) allows for replicative release of recombinant viral DNA cloned between a tandem repeat of the LIR from a chromosome-integrated site (Hayes et al, 1988; Grimsley et al, 1987; Kanevski et al, 1992). A previous study has indeed shown that an earlier version, the Bean Yellow Dwarf Virus derived vector and co-expressed with Rep driven by an inducible promoter in stable transgenic potato, produced an 80-fold increase in mRNA and a 10-fold increase in transgenic protein expression (Zhang and Mason, 2006). In order to scale up the productivity of Rituximab in plant, optimization of Rituximab expression in N. benthamiana for plant-produced mAb's with enhanced efficacy is therefore needed. The utilization of advanced and optimized replicon vectors in transient transgenic technologies was explored herein to allow a rapid assessment of pharmaceutical candidates and a scalable platform for commercial production.