Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells.
PDF (676K)
+7 authors
Journal: 2006/July - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
Abstract:
Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAbP) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAbM, the plant mAbP bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAbP and mAbM showed the similar binding to FcgammaRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAbP originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.
Open in
PUBMED | DOI | PMC | Google Scholar | Wikipedia
Relations:
Content
Citations
(13)
References
(33)
Diseases
(1)
Drugs
(1)
Chemicals
(5)
Organisms
(7)
Processes
(3)
Anatomy
(2)
Affiliates
(1)
Similar articles
Articles by the same authors
Discussion board
Proc Natl Acad Sci U S A 103(23): 8804-8809
PMID: 16720700

Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells

+3 authors
*Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, PA 19107; and
Immunotherapy Laboratory, Department of Immunology, University Medical Center Utrecht, 3508 GA, Utrecht, The Netherlands
To whom correspondence should be addressed. E-mail: ude.nosreffej@iksworpok_h

Contributed by Hilary Koprowski, April 16, 2006

.

Author contributions: R.B., J.H.W.L., N.P., M. Golovkin, Z.S., and H.K. designed research; R.B., M. Glogowska, K.B., M.O., D.D., K.K., J.v.d.L., and J.H.W.L. performed research; R.B., Z.S., and H.K. contributed new reagents/analytic tools; R.B., J.H.W.L., N.P., M. Golovkin, Z.S., and H.K. analyzed data; and R.B., K.K., Z.S., and H.K. wrote the paper.

Copyright © 2006 by The National Academy of Sciences of the USA

Abstract

Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Several tumor-associated antigens in tumor cells have been identified as targets for therapeutic mAbs. Here we describe the production of mAb BR55-2 (IgG2a) in transgenic plants that recognizes the nonprotein tumor-associated antigen Lewis Y oligosaccharide overexpressed in human carcinomas, particularly breast and colorectal cancers. Heavy and light chains of mAb BR55-2 were expressed separately and assembled in plant cells of low-alkaloid tobacco transgenic plants (Nicotiana tabacum cv. LAMD609). Expression levels of plant-derived mAb (mAb) were high (30 mg/kg of fresh leaves) in T1 generation plants. Like the mammalian-derived mAb, the plant mAb bound specifically to both SK-BR3 breast cancer cells and SW948 colorectal cancer cells. The Fc domain of both mAb and mAb showed the similar binding to FcγRI receptor (CD64). Comparable levels of cytotoxicity against SK-BR3 cells were also shown for both mAbs in antibody-dependent cell-mediated cytotoxicity assay. Furthermore, plant-derived BR55-2 efficiently inhibited SW948 tumor growth xenografted in nude mice. Altogether, these findings suggest that mAb originating from low-alkaloid tobacco exhibit biological activities suitable for efficient immunotherapy.

Keywords: breast and colorectal cancer, plant biotechnology, transgenic low-alkaloid tobacco, tumor growth inhibition
Abstract

Although current demands for therapeutic mAbs are growing quickly, production methods to date, including in vitro mammalian tissue culture and transgenic animals, provide only limited quantities at high cost. Other available systems, such as bacterial and yeast, do not provide specific machinery for protein posttranslational modifications required for an active or partially active mAb.

The use of mAbs in diagnosis and treatment of various carcinomas has increased in recent years. mAbs against tumor-associated antigens have proven effective in cancer treatment, especially in conjunction with classical chemotherapy and radiotherapy (1, 2). By binding to antigen expressed on the surface of cancer cells, mAbs trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity, which kills abnormal cells (3–5). ADCC requires the presence of tumor cells overexpressing the tumor-associated antigen, efficient binding of the mAb to this antigen, and effector cells, e.g., macrophages that recognize mAbs through their Fc receptors. mAb BR55-2 recognizes the Lewis Y oligosaccharide antigen (LeY), which is overexpressed predominantly on breast, lung, ovary, and colon cancers (6–8). Murine mAb BR55-2 (IgG2a) inhibits tumor growth and kills human cancer cells xenotransplanted in nude mice (9). Under physiological conditions, LeY is expressed predominantly during embryogenesis but is restricted to granulocytes and epithelial surfaces in adult tissue (10).

Recently plants have become a prospective replacement bioreactor for currently available production systems to manufacture biopharmaceuticals (11, 12). Moreover, plants offer several advantages as a mAb production system, such as the lack of human pathogens, relatively low-cost manufacturing, and ease of production scale-up. Our group has recently shown that recombinant mAb can be safely purified from tobacco plants (13).

Previously we successfully expressed the human rabies virus-neutralizing mAb SO57 (14) and the murine anticancer mAb C017-1A (15) in planta. Both plant-derived mAbs (mAb) showed excellent in vivo activity similar to that of the parental mAb produced in the mammalian system. However, whereas efficacy of the virus-neutralizing mAb SO57 depends mainly on its activity in binding to virus antigens (16), mAbs for use in cancer immunotherapy require both tumor-associated antigen binding activity and interaction with Fc receptors to exert ADCC effector functions.

Here we report the successful expression and assembly of functional LeY oligosaccharide-specific mAb BR55-2 in transgenic tobacco plants with low alkaloid content (LAMD609). The mAb fusion to the KDEL signal sequence helped to retain the protein inside the endoplasmic reticulum (ER), thus enhancing mAb assembly in plant cells (17). Consequently, it helped to increase the final mAb yields from the plant production system. No significant differences in biological activities suitable for efficient immunotherapy were observed between the mAb and the mAb BR55-2 obtained from the mammalian system (mAb). Our results clearly indicate that plants can be used as an excellent source of fully active mAbs.

Acknowledgments

We thank Dr. W. Crosby (Plant Biotechnology Institute, Saskatoon, SK, Canada) for pBI525 and J. Carrington (Washington State University, Pullman) for pRTL2. We also thank Thomas Jefferson University/Kimmel Cancer Center research and animal facilities for their support. This work was supported by a grant from U.S. Department of Agriculture to Biotechnology Foundation Laboratories (to H.K.).

Acknowledgments

Abbreviations

mAbPplant-derived mAb
mAbMmammalian-derived mAb
HCheavy chain
LClight chain
ADCCantibody-dependent cell-mediated cytotoxicity
LeYLewis Y oligosaccharide antigen
ERendoplasmic reticulum.
Abbreviations

Footnotes

Conflict of interest statement: No conflicts declared.

Footnotes

References

  • 1. Stemmler HJ., Kahlert S., Brudler O., Beha M., Muller S., Stauch B., Heinemann V. Clin. Oncol. (R. Coll. Radiol.) 2005;17:630–635.[PubMed][Google Scholar]
  • 2. DeNardo SJ. Semin. Nucl. Med. 2005;35:143–151.[PubMed][Google Scholar]
  • 3. Steplewski Z., Lubeck MD., Koprowski H. Science. 1983;221:865–867.[PubMed][Google Scholar]
  • 4. Plunkett T. A., Miles D. W. Int. J. Clin. Pract. 2002;56:261–266.[PubMed]
  • 5. Adams G. P., Weiner L. M. Nat. Biotechnol. 2005;23:1147–1157.[PubMed]
  • 6. Nichols E. J., Kannagi R., Hakomori S. I., Krantz M. J., Fuks A. J. Immunol. 1985;135:1911.6–1913.6.[PubMed]
  • 7. Fernandes B., Sagman U., Auger M., Demetrio M., Dennis JW. Cancer Res. 1991;51:718–723.[PubMed][Google Scholar]
  • 8. Nemoto-Sasaki Y., Mitsuki M., Morimoto-Tomita M., Maeda A., Tsuiji M., Irimura T. Glycoconjugate J. 2001;18:895–906.[PubMed]
  • 9. Steplewski Z., Lubeck M. D., Scholz D., Loibner H., McDonald S. J., Koprowski H. In Vivo. 1991;5:79–83.[PubMed]
  • 10. Dettke M., Palfi G., Loibner H. J. Leukocyte Biol. 2000;68:511–514.[PubMed]
  • 11. Koprowski H. Vaccine. 2005;23:1757–1763.[PubMed]
  • 12. Ko K., Koprowski H. Virus Res. 2005;111:93–100.[PubMed]
  • 13. Ko K., Tekoah Y., Rudd P. M., Harvey D. J., Dwek R. A., Spitsin S., Hanlon C. A., Rupprecht C., Dietzschold B., Golovkin M., et al. Proc. Natl. Acad. Sci. USA. 2003;100:8013–8018.
  • 14. Ko K., Wei X., Crooks PA., Koprowski H. J. Immunol. Methods. 2004;286:79–85.[PubMed][Google Scholar]
  • 15. Ko K., Steplewski Z., Glogowska M., Koprowski H. Proc. Natl. Acad. Sci. USA. 2005;102:7026–7030.
  • 16. Dietzschold B., Gore M., Casali P., Ueki Y., Rupprecht C. E., Notkins A. L., Koprowski H. J. Virol. 1990;64:3087–3090.
  • 17. Schouten A., Roosien J., van Engelen F. A., de Jong G. A., Borst-Vrenssen A. W., Zilverentant J. F., Bosch D., Stiekema W. J., Gommers F. J., Schots A., et al. Plant Mol. Biol. 1996;30:781–793.[PubMed]
  • 18. Timmermans MC., Maliga P., Vieira J., Messing J. J. Biotechnol. 1990;14:333–344.[PubMed][Google Scholar]
  • 19. Koprowski H., Steplewski Z., Mitchell K., Herlyn M., Herlyn D., Fuhrer P. Somatic Cell Genet. 1979;5:957–971.[PubMed]
  • 20. Torres E., Vaquero C., Nicholson L., Sack M., Stoger E., Drossard J., Christou P., Fischer R., Perrin Y. Transgenic Res. 1999;8:441–449.[PubMed]
  • 21. Ross AH., Herlyn D., Iliopoulos D., Koprowski H. Biochem. Biophys. Res. Commun. 1986;135:297–303.[PubMed][Google Scholar]
  • 22. Balzar M., Winter M. J., de Boer C. J., Litvinov S. V. J. Mol. Med. 1999;77:699–712.[PubMed]
  • 23. Hazenbos W. L., Gessner J. E., Hofhuis F. M., Kuipers H., Meyer D., Heijnen I. A., Schmidt R. E., Sandor M., Capel P. J., Daeron M., et al. Immunity. 1996;5:181–188.[PubMed]
  • 24. Barnes N., Gavin A. L., Tan P. S., Mottram P., Koentgen F., Hogarth P. M. Immunity. 2002;16:379–389.[PubMed]
  • 25. Takai T., Li M., Sylvestre D., Clynes R., Ravetch JV. Cell. 1994;76:519–529.[PubMed][Google Scholar]
  • 26. Bevaart L., Jansen M. J., van Vugt M. J., Verbeek J. S., van de Winkel J. G., Leusen J. H. Cancer Res. 2006;66:1261–1264.[PubMed]
  • 27. Radaev S., Sun PD. J. Biol. Chem. 2001;276:16478–16483.[PubMed][Google Scholar]
  • 28. Shields R. L., Lai J., Keck R., O'Connell L. Y., Hong K., Meng Y. G., Weikert S. H., Presta L. G. J. Biol. Chem. 2002;277:26733–26740.[PubMed]
  • 29. Herlyn D., Powe J., Ross AH., Herlyn M., Koprowski H. J. Immunol. 1985;134:1300–1304.[PubMed][Google Scholar]
  • 30. Sambrook S., Fritsch E. F., Maniatis T. Molecular Cloning: A Laboratory Manual. Plainview, NY: Cold Spring Harbor Lab. Press; 1989. [PubMed]
  • 31. Pang S. Z., Nagpala P., Wang M., Slightom J. L., Gonsalves D. Phytopathology. 1992;82:1223–1229.[PubMed]
  • 32. Carrington J. C., Freed D. D., Leinicke A. J. Plant Cell. 1991;3:953–962.
  • 33. Horsch R. B., Klee H. J., Stachel S., Winans S. C., Nester E. W., Rogers S. G., Fraley R. T. Proc. Natl. Acad. Sci. USA. 1986;83:2571–2575.
  • 34. Daly T., Royal R. E., Kershaw M. H., Treisman J., Wang G., Li W., Herlyn D., Eshhar Z., Hwu P. Cancer Gene Ther. 2000;7:284–291.[PubMed]
  • 35. Herlyn D., Koprowski H. Proc. Natl. Acad. Sci. USA. 1982;79:4761–4765.
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.