Production of unique immunotoxin cancer therapeutics in algal chloroplasts

Abstract

The idea of targeted therapy, whereby drug or protein molecules are delivered to specific cells, is a compelling approach to treating disease. Immunotoxins are one such targeted therapeutic, consisting of an antibody domain for binding target cells and molecules of a toxin that inhibits the proliferation of the targeted cell. One major hurdle preventing these therapies from reaching the market has been the lack of a suitable production platform that allows the cost-effective production of these highly complex molecules. The chloroplast of the green alga Chlamydomonas reinhardtii has been shown to contain the machinery necessary to fold and assemble complex eukaryotic proteins. However, the translational apparatus of chloroplasts resembles that of a prokaryote, allowing them to accumulate eukaryotic toxins that otherwise would kill a eukaryotic host. Here we show expression and accumulation of monomeric and dimeric immunotoxin proteins in algal chloroplasts. These fusion proteins contain an antibody domain targeting CD22, a B-cell surface epitope, and the enzymatic domain of exotoxin A from Pseudomonas aeruginosa. We demonstrated that algal-produced immunotoxins accumulate as soluble and enzymatically active proteins that bind target B cells and efficiently kill them in vitro. We also show that treatment with either the mono-or dimeric immunotoxins significantly prolongs the survival of mice with implanted human B-cell tumors.

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

“…Production of such cytotoxic proteins in eukaryotic hosts such as CHO cells or yeast is not feasible because of the lethal effect of the toxin on the cytosolic translation apparatus, whereas production in prokaryotic systems is challenging because of the difficulty of folding and assembling such complex molecules. In two impressive papers, the group achieved the synthesis of immunotoxins comprising a single-chain antibody recognizing the CD22 surface receptor from B-cells fused either to domain II and III of exotoxin A from Pseudomonas aeruginosa [43] or to the ribosome-inactivating protein, gelonin, from Gelonium multiflorm [44]. Both immunotoxins were capable of specifically binding B-cells in vitro, and in the case of the immunotoxin exotoxin A the life span of mice implanted with a human B-cell tumor was extended.…”

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

“…Moreover, although genetic modification of Chlorella vulgaris (Chow and Tung, 1997) and Chlorella ellipsoidea (Chen et al, 2001) were achieved, none of the Chlorella spp. strains shown to be amenable to transformation are of commercial interest (Tran et al, 2013). Thus transformation of industrial production Chlorella spp.…”

Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines

“…In the near term, proteins that are recalcitrant to production in conventional systems or otherwise biologically suited for production in algae will help drive development of the algae platform. Indeed, algae were recently used to make immunotoxins in a single step (37). Immunotoxins are currently made by chemically conjugating a human antibody, made and purified from CHO cells, to a toxin molecule or protein, followed by a second purification of the chimeric molecule.…”

“…The impending industrial utility of algae is also accelerating the development of genetic tools for metabolic engineering and recombinant protein production as potential coproducts (31). Thus far, C. reinhardtii chloroplasts have been used to make vaccine antigens (32,33), industrial enzymes (34), antibodies (35), human therapeutics (36), and immunotoxins (37). The versatility of the chloroplast is owed to the unique combination of eukaryotic chaperones (38), protein disulfide isomerases (39), and peptidylprolyl isomerases (40) coupled with prokaryotic-like ribosomal machinery (41).…”