BIP - Expression of the antibody 14D9 in Nicotiana tabacum hairy roots

Financial support: This research was supported by CONICET and the University of Buenos Aires.

Keywords: catalytic antibodies, in vitro cultures, molecular farming, recombinant antibodies, tobacco.

Present address: ^#Fundación Pablo Cassará, Saladillo 2452, Ciudad de Buenos Aires, (C1144OFFX), Tel: 54 11 4886 5242, Fax: 54 11 4065.

Transgenic plants, those that integrate a foreign fragment of DNA in its own genome, are an economical alternative for large-scale production of recombinant proteins for industrial and pharmaceutical uses. Research in the past decades has revolutionized the use of therapeutically valuable proteins in a variety of clinical treatments and diagnosis. Most of the production has been performed in whole plant; however, not many proteins can be produced in plants at high levels. Anyway a 0.1-1.0% total soluble protein (TSP) yield, the typical level obtained for the production of pharmaceutical proteins, is competitive enough in order to make plants economically viable. Besides, protein production in plant cells is safer than traditional techniques (hybridomes and bacterial cell cultures) because of the lack of contamination with strange viral or bacterial materials, mammalian pathogens and other animal cell-culture contaminants. In vitro plant cell cultures, appeared as an interesting alternative when a foreign protein has to be synthesized under controlled environmental conditions or when the product is required in a quicker way than that obtained by agriculture procedure (Hellwig et al. 2004). In addition, another important advantage of in vitro culture is the possibility to recover the foreign protein from the culture medium which reduces the cost of down stream processing. Taking into account all the aspects cited before, protein production costs could be reduced by as much as 1000-fold compared with traditional production practices (Peterson and Arntzen, 2004). On the other hand, as the economic feasibility of plant-based systems depends on the concentration of foreign protein accumulated, preventing the degradation or loss of product after its assembly and/or secretion therefore it is of vital importance. It is also of importance the expression of the foreign protein in a cellular compartment where the degradation were diminished. The fusion of a protein to the C-terminal KDEL sequence for retention in and/or retrieve to the endoplasmic reticulum (ER) has been found to increase protein stability and to avoid addition of complex glycans in plant cells (Ko et al. 2003; Gomord et al. 2004). Also, the use of protein-stabilizing agents such as polyvinylpyrrolidone (PVP) and gelatin is one of the most promising methods for retaining foreign proteins secreted into plant culture medium. If the protein remains attached to biomass, permeabilising agents such as dimethylsulfoxide (DMSO) could be used (Wongsamuth and Doran, 1997; Magnuson, 1996). At present, in vitro cultures and particularly hairy roots have been recognized to be an alternative for foreign protein production. Hairy roots are a stable reproducible system which grows faster than suspension cell cultures and also they are able to grow in plant growth regulator-free culture medium. Considering the proteins that have been produced in plants, perhaps the most promising ones are monoclonal antibodies and antibody fragments. However, there have been few reports on antibody production by hairy root cultures (Wongsamuth and Doran, 1997; Sharp and Doran, 2001). The aim of this work is to study the expression of the 14D9 antibody in Nicotiana tabacum hairy root cultures. 14D9 is a murine antibody IgG1 type which catalyzes the stereoselective transformation of achiralenol ethers having a practical appeal for organic synthesis (Reymond et al. 1993; Zheng et al. 2003).

N. tabacum hairy roots were established at a frequency of above 85%. Among the different clones obtained which present differences regarding growth as well as in antibody expression levels. Clones Ac₂ and AcK₆were selected for their characteristics of growth (high rate) and their capacity to express 14D9 antibody in a stable way. Hairy roots from line AcK₆ grew faster and stronger than the wild type and line Ac₂. That phenomenon is also observed in the whole plants and suspension cell cultures derived from AcK₆ line. It could be attributed to the site of T-DNA integration in that transgenic line.

On the other hand the antibody yield (expressed as µg g^-1FW) in line AcK₆ was 3.21-fold higher than in line Ac_2.The values obtained after 20 days of culture were 0.386 % TSP for clone Ac₂ and 9.34 %TSP for clone AcK₆. These results allow sustaining that preventing the secretion of protein by using the KDEL signal peptide for retention in the ER minimizes antibody fragmentation and improves antibody accumulation in plant cells (Tsoi and Doran, 2002). Also proteases in the secretory pathway and in the apoplast are avoided (Sharp and Doran, 2001). In the culture medium the highest values of antibodies were about 1% of the amounts reached in the biomass for both clones.

Gelatin at 5.0 g l^-1 had a positive effect on antibody level in the culture medium. The maximum antibody concentration in the medium corresponds to 1.44-fold the corresponding level in the control culture. PVP is a versatile polymer which is water soluble, exhibiting excellent complexing, stabilizing and colloidal properties while at the same time being metabolically and physiologically inert. The addition of 1.5 g PVP l^-1 significantly enhanced antibody concentration by 2.38-fold. These results suggest that PVP possibly functions preventing denaturation and adherence to surfaces in the culture vessel of the secreted protein. Finally, 14D9 antibody level in clone AcK₆ increased in about three times at the 20 day of culture using DMSO at a concentration of 5% v/v. The increase of the antibody level into the biomass could be attributed to DMSO producing small channels which do not allow the antibody release. These channels would favor the entrance of small molecules of nutrients which may increase the synthesis of the antibody in the biomass. DMSO could also stabilize proteins against denaturation by forming hydrogen bonds with proton-donor groups on protein molecules (Henderson et al. 1975). However, DMSO was not effective for increasing the release of the antibody in the culture medium; the levels remained approximately the same than those of the control. The size of 14D9, about 150kDa, may be too big to pass through the pores created by the permeabilizing action of DMSO. It has been shown that molecules with a molecular weight greater than 5,000 have difficulty passing through the cell wall even after permeabilization with DMSO concentrations up to 7.5%. Western blots did not show antibody fragment neither in the biomass nor in the culture medium. The influence of additives such as permeabilizant and stabilizers must be studied and optimized according to the plant cell tissue culture for each expressed antibody in order to establish a competitive production system.

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