Production of unique immunotoxin cancer therapeutics in algal chloroplasts

Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved November 19, 2012 (received for review August 24, 2012)
December 10, 2012
110 (1) E15-E22

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.

Author Summary

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.
Here, we demonstrate that a eukaryotic cell is capable of accumulating small monomeric and larger dimeric immunotoxins. These immunotoxins are enzymatically active, bind specifically to cells displaying CD22, and are capable of causing those cells to undergo apoptosis. Furthermore, both immunotoxins produced by algae are capable of inhibiting tumor growth significantly in mice with s.c. tumor-cell xenografts.
To determine if algae are capable of producing functional immunotoxins, we created a recombinant gene encoding a single-chain antibody (scFv) that recognizes CD22, a mammalian B-cell surface molecule, genetically fused to domains II and III of exotoxin A (PE40) from Pseudomonas aeruginosa (αCD22PE40) (represented by the red square in Fig. P1). PE40 inhibits protein synthesis in eukaryotic cells, leading to apoptosis of the targeted cell. A significant problem with immunotoxins similar to αCD22PE40 is their short half-life in the body, resulting from their small size. We therefore engineered a more complex chimeric immunotoxin gene containing the hinge and the CH2 and CH3 domains of a human IgG1 placed between the αCD22 scFv antibody and PE40. This molecule (represented by the green triangle in Fig. P1) should form a dimer through disulfide bonds in the hinge region, making the assembled proteins significantly larger than αCD22PE40 and doubling the number of CD22-binding domains and PE40 toxin molecules to two.
C. reinhardtii contains a single chloroplast that constitutes up to 70% of the cell. Chloroplasts contain ribosomes and translation factors that resemble those of photosynthetic prokaryotes. However, unlike bacteria, the protein-production components of chloroplasts allow them to fold and assemble complex proteins. This machinery also allows these components to fold complex recombinant proteins, such as full-length human antibodies, that accumulate as soluble and functional molecules within the chloroplast (3).
Delivering drug or protein molecules to specific cells is a compelling approach to treating a disease. For example, immunotoxins consist of an antibody domain for binding target cells and a toxin that inhibits their proliferation. Implementation of an immunotoxin therapy suffers from the lack of a suitable production platform that allows their cost-effective production. Recently, algae have gained attention as a potential source of renewable fuel (1) and also have been shown to be capable of producing a wide range of protein therapeutics (2, 3). The chloroplast of the green algae Chlamydomonas reinhardtii contains chaperones, peptidyl-prolyl isomerases and protein disulfide isomerases, that allow them to fold and assemble complex eukaryotic proteins. Additionally, 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 the expression and accumulation of monomeric and dimeric immunotoxin proteins in algal chloroplasts.
This article is a PNAS Direct Submission.
See full research article on page E15 of www.pnas.org.
Cite this Author Summary as: PNAS 10.1073/pnas.1214638110.

References

1
DR Georgianna, SP Mayfield, Exploiting diversity and synthetic biology for the production of algal biofuels. Nature 488, 329–335 (2012).
2
JA Gregory, et al., Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PLoS ONE 7, e37179 (2012).
3
M Tran, B Zhou, PL Pettersson, MJ Gonzalez, SP Mayfield, Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng 104, 663–673 (2009).

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Acknowledgments

This work was supported by Grant CBET-1160184 from the National Science Foundation (to S.P.M.). M.T. was supported by a Skaggs Family Foundation predoctoral fellowship. C.V. was supported by a California Department of Labor Edge Internship.

References

1
AS Raghavendra Photosynthesis: A Comprehensive Treatise (Cambridge Univ Press, Cambridge, UK, 1998).
2
M Hannon, J Gimpel, M Tran, B Rasala, S Mayfield, Biofuels from algae: Challenges and potential. Biofuels 1, 763–784 (2010).
3
BA Rasala, SP Mayfield, The microalga Chlamydomonas reinhardtii as a platform for the production of human protein therapeutics. Bioeng Bugs 2, 50–54 (2011).
4
JA Gregory, et al., Algae-produced Pfs25 elicits antibodies that inhibit malaria transmission. PLoS ONE 7, e37179 (2012).
5
M Tran, B Zhou, PL Pettersson, MJ Gonzalez, SP Mayfield, Synthesis and assembly of a full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng 104, 663–673 (2009).
6
BA Rasala, et al., Production of therapeutic proteins in algae, analysis of expression of seven human proteins in the chloroplast of Chlamydomonas reinhardtii. Plant Biotechnol J 8, 719–733 (2010).
7
E Specht, S Miyake-Stoner, S Mayfield, Micro-algae come of age as a platform for recombinant protein production. Biotechnol Lett 32, 1373–1383 (2010).
8
BA Rasala, et al., Robust expression and secretion of Xylanase1 in Chlamydomonas reinhardtii by fusion to a selection gene and processing with the FMDV 2A peptide. PLoS ONE 7, e43349 (2012).
9
SE Franklin, SP Mayfield, Prospects for molecular farming in the green alga Chlamydomonas. Curr Opin Plant Biol 7, 159–165 (2004).
10
GL Shen, et al., Evaluation of four CD22 antibodies as ricin A chain-containing immunotoxins for the in vivo therapy of human B-cell leukemias and lymphomas. Int J Cancer 42, 792–797 (1988).
11
E Mansfield, P Amlot, I Pastan, DJ FitzGerald, Recombinant RFB4 immunotoxins exhibit potent cytotoxic activity for CD22-bearing cells and tumors. Blood 90, 2020–2026 (1997).
12
C Bogner, et al., Immunotoxin BL22 induces apoptosis in mantle cell lymphoma (MCL) cells dependent on Bcl-2 expression. Br J Haematol 148, 99–109 (2010).
13
J Yin, G Li, X Ren, G Herrler, Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. J Biotechnol 127, 335–347 (2007).
14
VK Chaudhary, et al., A recombinant immunotoxin consisting of two antibody variable domains fused to Pseudomonas exotoxin. Nature 339, 394–397 (1989).
15
U Brinkmann, Y Reiter, SH Jung, B Lee, I Pastan, A recombinant immunotoxin containing a disulfide-stabilized Fv fragment. Proc Natl Acad Sci USA 90, 7538–7542 (1993).
16
A Selyukh Seattle genetics cancer drug may top $100,000 (Reuters, Washington, DC, Available at www.reuters.com/article/2011/08/22/us-seattlegenetics-idUSTRE77L5EB20110822. (2011).
17
Y Cao, et al., Single-chain antibody-based immunotoxins targeting Her2/neu: Design optimization and impact of affinity on antitumor efficacy and off-target toxicity. Mol Cancer Ther 11, 143–153 (2012).
18
EH Harris Chlamydomonas Sourcebook Introduction to Chlamydomonas and its Laboratory Uses (Academic, New York, 2009).
19
MV Beligni, K Yamaguchi, SP Mayfield, Chloroplast elongation factor ts pro-protein is an evolutionarily conserved fusion with the s1 domain-containing plastid-specific ribosomal protein-7. Plant Cell 16, 3357–3369 (2004).
20
AL Manuell, J Quispe, SP Mayfield, Structure of the chloroplast ribosome: Novel domains for translation regulation. PLoS Biol 5, e209 (2007).
21
M Schroda, The Chlamydomonas genome reveals its secrets: Chaperone genes and the potential roles of their gene products in the chloroplast. Photosynth Res 82, 221–240 (2004).
22
A Danon, SP Mayfield, Light-regulated translation of chloroplast messenger RNAs through redox potential. Science 266, 1717–1719 (1994).
23
A Breiman, TW Fawcett, ML Ghirardi, AK Mattoo, Plant organelles contain distinct peptidylprolyl cis,trans-isomerases. J Biol Chem 267, 21293–21296 (1992).
24
E Mansfield, I Pastan, DJ FitzGerald, Characterization of RFB4-Pseudomonas exotoxin A immunotoxins targeted to CD22 on B-cell malignancies. Bioconjug Chem 7, 557–563 (1996).
25
E Mansfield, MF Chiron, P Amlot, I Pastan, DJ FitzGerald, Recombinant RFB4 single-chain immunotoxin that is cytotoxic towards CD22-positive cells. Biochem Soc Trans 25, 709–714 (1997).
26
T Kondo, D FitzGerald, VK Chaudhary, S Adhya, I Pastan, Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Biol Chem 263, 9470–9475 (1988).
27
RJ Kreitman, QC Wang, DJ FitzGerald, I Pastan, Complete regression of human B-cell lymphoma xenografts in mice treated with recombinant anti-CD22 immunotoxin RFB4(dsFv)-PE38 at doses tolerated by cynomolgus monkeys. Int J Cancer 81, 148–155 (1999).
28
I Bertholjotti, [Antibody-drug conjugate—a new age for personalized cancer treatment]. Chimia (Aarau) 65, 746–748 (2011).
29
SS Minich, Brentuximab vedotin: A new age in the treatment of Hodgkin lymphoma and anaplastic large cell lymphoma. Ann Pharmacother 46, 377–383 (2012).
30
Frankel AD, ed (1992) Genetically Engineered Toxins (Mercel Dekker, New York), pp 439–445.
31
VM Gordon, KR Klimpel, N Arora, MA Henderson, SH Leppla, Proteolytic activation of bacterial toxins by eukaryotic cells is performed by furin and by additional cellular proteases. Infect Immun 63, 82–87 (1995).
32
H Xie, C Audette, M Hoffee, JM Lambert, WA Blättler, Pharmacokinetics and biodistribution of the antitumor immunoconjugate, cantuzumab mertansine (huC242-DM1), and its two components in mice. J Pharmacol Exp Ther 308, 1073–1082 (2004).
33
AL Manuell, et al., Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast. Plant Biotechnol J 5, 402–412 (2007).
34
PM Sharp, WH Li, The codon Adaptation Index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res 15, 1281–1295 (1987).
35
P Puigbò, IG Bravo, S Garcia-Vallve, CAIcal: A combined set of tools to assess codon usage adaptation. Biol Direct 3, 38 (2008).
36
RJ Kreitman, I Pastan, Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem J 307, 29–37 (1995).
37
SP Mayfield, J Schultz, Development of a luciferase reporter gene, luxCt, for Chlamydomonas reinhardtii chloroplast. Plant J 37, 449–458 (2004).
38
X Du, R Beers, DJ Fitzgerald, I Pastan, Differential cellular internalization of anti-CD19 and -CD22 immunotoxins results in different cytotoxic activity. Cancer Res 68, 6300–6305 (2008).
39
A Haacke, G Fendrich, P Ramage, M Geiser, Chaperone over-expression in Escherichia coli: Apparent increased yields of soluble recombinant protein kinases are due mainly to soluble aggregates. Protein Expr Purif 64, 185–193 (2009).
40
AR Frand, JW Cuozzo, CA Kaiser, Pathways for protein disulphide bond formation. Trends Cell Biol 10, 203–210 (2000).
41
NS Outchkourov, et al., Correctly folded Pfs48/45 protein of Plasmodium falciparum elicits malaria transmission-blocking immunity in mice. Proc Natl Acad Sci USA 105, 4301–4305 (2008).
42
J Kim, SP Mayfield, Protein disulfide isomerase as a regulator of chloroplast translational activation. Science 278, 1954–1957 (1997).
43
JW Bloom, MS Madanat, D Marriott, T Wong, SY Chan, Intrachain disulfide bond in the core hinge region of human IgG4. Protein Sci 6, 407–415 (1997).
44
TK Bera, J Williams-Gould, R Beers, P Chowdhury, I Pastan, Bivalent disulfide-stabilized fragment variable immunotoxin directed against mesotheliomas and ovarian cancer. Mol Cancer Ther 1, 79–84 (2001).
45
T Ribbert, et al., Recombinant, ETA’-based CD64 immunotoxins: Improved efficacy by increased valency, both in vitro and in vivo in a chronic cutaneous inflammation model in human CD64 transgenic mice. Br J Dermatol 163, 279–286 (2010).
46
P Maliga, Plastid transformation in higher plants. Annu Rev Plant Biol 55, 289–313 (2004).
47
SP Mayfield, et al., Chlamydomonas reinhardtii chloroplasts as protein factories. Curr Opin Biotechnol 18, 126–133 (2007).
48
SC Alley, NM Okeley, PD Senter, Antibody-drug conjugates: Targeted drug delivery for cancer. Curr Opin Chem Biol 14, 529–537 (2010).
49
A Beck, et al., The next generation of antibody-drug conjugates comes of age. Discov Med 10, 329–339 (2010).
50
A Lash, Make the case for antibody-drug conjugates. Pharmaceuticals (Ott) 28, 32–38 (2010).
51
A Younes, U Yasothan, P Kirkpatrick, Brentuximab vedotin. Nat Rev Drug Discov 11, 19–20 (2012).
52
I Pastan, R Hassan, DJ FitzGerald, RJ Kreitman, Immunotoxin treatment of cancer. Annu Rev Med 58, 221–237 (2007).
53
, eds R Kontermann, S Dubel (Springer Lab Manuals, New York) Expression of Full Length Monoclonal Antibodies (mAb) in Algal Chloroplast, pp. 503–516 (2010).
54
J Sambrook, EF Fritsch, T Maniatas Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY, 1989).

Information & Authors

Information

Published in

Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 110 | No. 1
January 2, 2013
PubMed: 23236148

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Submission history

Published online: December 10, 2012
Published in issue: January 2, 2013

Acknowledgments

This work was supported by Grant CBET-1160184 from the National Science Foundation (to S.P.M.). M.T. was supported by a Skaggs Family Foundation predoctoral fellowship. C.V. was supported by a California Department of Labor Edge Internship.

Notes

This article is a PNAS Direct Submission.
See full research article on page E15 of www.pnas.org.
See Author Summary on page 14 (volume 110, number 1).
This article is a PNAS Direct Submission.

Authors

Affiliations

Miller Tran
The San Diego Center for Algae Biotechnology and
Departments of bBiology and
Christina Van
The San Diego Center for Algae Biotechnology and
Departments of bBiology and
Daniel J. Barrera
The San Diego Center for Algae Biotechnology and
Departments of bBiology and
Pär L. Pettersson
The San Diego Center for Algae Biotechnology and
Present address: Tocagen, Inc., San Diego, CA 92109.
Carlos D. Peinado
Pathology, University of California at San Diego, La Jolla, CA
Jack Bui
Pathology, University of California at San Diego, La Jolla, CA
Stephen P. Mayfield2 [email protected]
The San Diego Center for Algae Biotechnology and
Departments of bBiology and

Notes

2
To whom correspondence should be addressed. E-mail: [email protected].
Author contributions: M.T., J.B., and S.P.M. designed research; M.T., C.V., D.J.B., P.L.P., C.D.P., and J.B. performed research; M.T., J.B., and S.P.M. contributed new reagents/analytic tools; M.T., C.V., D.J.B., P.L.P., J.B., and S.P.M. analyzed data; and M.T. wrote the paper.

Competing Interests

Conflict of interest statement: S.P.M. is a founder of Sapphire Energy and has a financial interest in that company. Sapphire Energy has rights to this technology.

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    Production of unique immunotoxin cancer therapeutics in algal chloroplasts
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