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The Skaggs Institute
for Chemical Biology
Expression of Human Therapeutic Antibodies in Eukaryotic Algae
S.P. Mayfield, P. Lee, P. Pettersson, J. Marìn-Navarro, A. Manuell, M. Muto, M. Tran
We
have developed a system for expressing recombinant proteins, including human therapeutic
proteins, in the chloroplast of the eukaryotic green alga Chlamydomonas reinhardtii.
Expression of therapeutic proteins in eukaryotic algae offers several advantages
over more traditional protein expression systems. Algae are efficient at producing
complex mammalian proteins, stable transgenic lines can be generated in a few weeks,
and algal systems can be scaled to high levels for a fraction of the cost of traditional
fermentation systems.
We
have expressed several recombinant proteins in algae, including human monoclonal
antibodies. Antibodies are complex multiprotein molecules that are difficult to
express in simple expression systems and expensive to produce in mammalian cell
culture. We have had good success in producing these complex proteins in algal chloroplasts.
We have also expressed eukaryotic protein toxins, which we are able to do because
chloroplasts are naturally resistant to these protein toxins. We have now developed
antibody-toxin fusion proteins, a class of recombinant protein molecules that can
target and kill eukaryotic cells, including human cancer and tumor cells. The production
of antibody-toxin fusion proteins is unique to our expression system; bacterial
expression systems cannot produce these complex molecules, and mammalian cell cultures
cannot be used because the toxin would kill the cells during production. These characteristics
make algal chloroplasts potentially the only system that can be used to produce
this type of superior cancer therapeutic agent.
To examine
the potential of antibody-toxin fusions as cancer therapeutics, we fused an antibody
to CD19 with an exotoxin A protein domain to produce an antibody-toxin fusion protein
(CD19-ETA) that targets CD19 on human B-cell lymphomas. This recombinant antibody-toxin
fusion protein was expressed in algae and accumulated as soluble protein. Using
cell-based assays, we showed that isolated CD19-ETA efficiently binds to CD19+
human B-cell lymphoma cells but does not bind to CD19– normal human
cells. Once bound to the tumor cells, CD19-ETA efficiently kills the cells. These
cell-based assays are the first step in demonstrating the potential of these fusion
proteins as human anticancer therapeutic agents.
Eukaryotic
algae offer tremendous potential for the expression of recombinant human therapeutic
proteins, and we have now shown that algal chloroplasts can express classes of proteins
that cannot be produced in other expression systems. The next phase of the project
is to demonstrate the tumor-killing ability of the algal expressed antibody-toxin
fusion proteins in animal models; these studies are under way.
Publications
Fletcher, S.P., Muto, M., Mayfield, S.P. Optimization of recombinant protein expression in the chloroplasts of green algae.
Adv. Exp. Med. Biol. 616:90, 2007.
Manuell, A.L., Beligni, M.V., Elder, J.H., Siefker, D.T., Tran, M., Weber, A., McDonald, T.L., Mayfield, S.P. Robust
expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol. J. 5:402, 2007.
Manuell, A.L., Quispe, J., Mayfield, S.P. Structure of the chloroplast ribosome: novel domains for translation regulation. PLoS Biol.
8:e209, 2007.
Marìn-Navarro, J., Manuell, A.L, Wu, J., Mayfield, S.P. Chloroplast translation regulation. Photosynth. Res. 94:359, 2007.
Mayfield, S.P., Manuell, A.L., Chen, S., Wu, J., Tran, M., Siefker, D., Muto, M., Marìn-Navarro, J. Chlamydomonas
reinhardtii chloroplasts as protein factories. Curr. Opin. Biotechnol. 18:126, 2007.
Merchant, S.S., Prochnik, S.E., Vallon, O., et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions.
Science 318:245, 2007.
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