Biochemical limitations to high-level expression of humanized monoclonal antibodies in transgenic maize seed endosperm.

Transgenic plants are potentially valuable systems for the large scale manufacture of therapeutic proteins. To improve this technology, determining the importance of transgene transcript levels on protein accumulation in sink tissues during their development is crucial. In transgenic maize (Zea mays L.) plants expressing humanized monoclonal antibodies (mAbs) in their seed endosperm, steady-state kappa light chain (LC) and gamma heavy chain (HC) mRNA levels were quantified during development and compared to the levels of fully-assembled mAb protein present at seed maturity. RNA blots and non-reducing SDS-PAGE western immunoblots revealed that steady-state LC and HC mRNA and protein levels were undetectable at 10 days after pollination (DAP) but increased quickly thereafter in three transgenic events expressing different mAb molecules. Similar to gamma-zein mRNA, LC and HC messages were highly abundant between 15 and 25 DAP. Quantitative RNA blots and western immunoblots showed that steady-state LC transcript levels during development correlated extremely closely with protein levels in mature seed (r(2)=0.99). For HC, this correlation was not as strong (r(2)=0.85). Consistent with this finding, concomitantly increasing the zygosity levels of the LC and HC transgenes enhanced mAb concentration in mature seed, in contrast to increasing the copy number of the transgene insert, which did not correlate with high seed mAb levels. The results indicate that high-level expression of fully-assembled mAb protein in maize endosperm was favored by high LC and HC mRNA levels and was largely limited by HC protein concentration.

Conservation of receptor antagonist anti-tumor activity by epidermal growth factor receptor antibody expressed in transgenic corn seed.

Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.

Host limits to accurate human growth hormone production in multiple plant systems.

Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.