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.

Fig. 1.

Depiction of algal-expressed immunotoxin proteins. (A) Single-chain antibody (scFv) directed against the CD22 cell-surface antigen made by linking the variable domains of the heavy- and light-chain antibodies with a glycine-serine linker. (B) The CD22-scFv is genetically linked to P. aeruginosa exotoxin A domains 2 and 3. Removal and replacement of domain Ia from exotoxin A with an antibody allows cancer cells to be targeted specifically. (C) The CD22-scFv genetically fused to the hinge and constant domains of an IgG1 and to exotoxin A domains 2 and 3 to create a construct that forms a homodimer through disulfide bonds formed between hinge regions. This fusion allows the molecule to have two binding domains as well as two toxin molecules.

Fig. 2.

Integration of genes into the chloroplast genome by homologous recombination. (A) Immunotoxin genes are ligated downstream of the psbA promoter and 5′ UTR and upstream of the psbA 3′ UTR. This construct is placed upstream of an aphA6 gene that confers kanamycin resistance to transformed cells of algae. Regions of chloroplast genome are placed at either end of the transformation vector to allow homologous integration of the entire transformation cassette into the chloroplast genome. (B) Transformation plasmids are precipitated onto gold particles and delivered by particle bombardment into algal chloroplasts, where they recombine into the plastid genome. (C) PCR analysis using primers specific to the αCD22 scFv gene and the psbA 5′ UTR demonstrate that coding sequences for immunotoxins have been integrated into the psbA locus. Lane 1 contains PCR from WT algal cells. Lane 2 contains strains transformed with αCD22. Lane 3 contains strains transformed with αCD22-PE40. Lane 4 contains strains transformed with αCD22-CH23-PE40. (D) PCR analysis is used to confirm homoplasmicity of transformed strains of algae. Primers are used to amplify a control region of the algal chloroplast genome as well the endogenous psbA gene. Loss of the psbA gene (upper band in lane 1) demonstrates homoplasmicity of the transgenic lines.

Fig. 3.

Western blots demonstrating the accumulation of immunotoxin proteins. (A) Samples (each 20 µg o.d) were separated on a SDS/PAGE gel under reducing conditions, transferred to a nitrocellulose membrane, and probed with an anti-Flag antibody that was conjugated with alkaline phosphatase. The lanes contain the following samples: lane 1, WT total protein; lane 2, αCD22; lane 3, αCD22PE40; lane 4, αCD22CH23PE40. (B) The identical samples were separated on a SDS/PAGE gel under nonreducing conditions to keep disulfide bonds intact. Once separated and transferred to a nitrocellulose membrane, samples were probed with an anti-Flag antibody conjugated with alkaline phosphatase and visualized on the nitrocellulose membrane. The black arrow indicates monomeric αCD22CH23PE40; the red arrow indicates αCD22CH23PE40 that has formed a homodimer which is indicative of an assembled antibody; the blue arrow indicates the formation of an assembled product between αCD22CH23PE40 and a degradation product lacking an scFv binding domain. This result demonstrates that algae produce αCD22CH23PE40 as a dimer, making it a divalent protein containing two exotoxin A molecules.

Fig. 4.

The ADP ribosyltransferase assay demonstrates that algal chloroplasts accumulate enzymatically active immunotoxin proteins. Biotinylated NAD⁺ was mixed with eEF2 and purified αCD22, αCD22PE40, or αCD22CH23PE40. Biotinylated ADP was transferred to eEF2 by enzymatically active exotoxin A. After reaction completion, samples were separated on SDS/PAGE and blotted onto nitrocellulose membranes. An anti-biotin antibody conjugated with alkaline phosphatase was used to detect eEF2 that was ribosylated with ADP-biotin. Western blot demonstrates that αCD22 does not ribosylate eEF2 (lane 1) but that αCD22PE40 (lane 2) and αCD22CH23PE40 (lane 3) have enzymatically active PE40 and do ribosylate eEF2.

Fig. 5.

Flow cytometry demonstrates specific binding of algal-produced immunotoxins. αCD22PE40 and αCD22CH23PE40 were incubated with CA-46 B cells, Ramos B cells, or Jurkat T cells. After primary incubation, cells were incubated with anti-exotoxin A produced in rabbit and finally with anti-rabbit DyLight 488. After incubation cells were analyzed by flow cytometry (blue curves). Cells that were not incubated with immunotoxins were used as a baseline of fluorescent intensity (red curves). (A) A shift in the fluorescence spectra demonstrates that αCD22PE40 and αCD22CH23PE40 bind to CA-46 B cells. (B) Fluorescence analysis also demonstrates that αCD22PE40 and αCD22CH23PE40 bind to Ramos B cells. (C) A lack of fluorescence shift demonstrates that algal-produced immunotoxins do not bind nonspecifically to Jurkat T cells.

Fig. 6.

In vitro and in vivo analysis of the effectiveness of algal-expressed immunotoxin against cancer cells. αCD22 (blue traces), αCD22PE40 (red traces), and αCD22CH23PE40 (green traces) were incubated with CA-46 B cells, Ramos B cells, and Jurkat T cells for 72 h in vitro to determine their cytotoxic activity. (A) αCD22PE40 and αCD22CH23PE40 were effective at killing CA-46 B cells, but αCD22 alone was incapable of killing CA-46 cells. (B) Additionally, αCD22PE40 and αCD22CH23PE40 were able to kill Ramos cells, but αCD22 was unable to inhibit Ramos cell proliferation. (C) αCD22, αCD22PE40, and αCD22CH23PE40 were unable to kill Jurkat T cells. (D) The IC₅₀ for each immunotoxin against each cell line was calculated to determine how effective each was at inhibiting cancer-cell proliferation. Both immunotoxins were capable of killing B cells, but dimeric αCD22CH23PE40 was more effective than αCD22PE40 at killing targeted cells in vitro. (E) Ramos cells (3 × 10⁷) were transplanted s.c. into Rag^−/− × gc^−/− mice until they established tumors with a mean diameter of 4 mm. Mice then were treated each day for 3 d with 240 µg/kg of αCD22, αCD22PE40, or αCD22CH23PE40. Both αCD22PE40 and αCD22CH23PE40 inhibited tumor proliferation more effectively than αCD22 alone.

Fig. P1.

Genes encoding monovalent and divalent immunotoxins containing an antibody-binding domain and a eukaryotic toxin were used to transform chloroplasts. (A) Two separate immunotoxins were produced from a single chain fused to exotoxin A (red square) and a divalent immunotoxin with an Fc domain from a human IgG1 between the single-chain antibody and exotoxin A (green triangle). As a control, a single-chain antibody was produced (blue diamond). (B) Both monovalent and divalent immunotoxins that accumulated in the chloroplast as soluble molecules were shown to inhibit cancer cell proliferation, and divalent immunotoxins were more potent than monovalent immunotoxins at inhibiting cancer cell growth.