Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Production of human monoclonal antibody in eggs of chimeric chickens


The tubular gland of the chicken oviduct is an attractive system for protein expression as large quantities of proteins are deposited in the egg, the production of eggs is easily scalable and good manufacturing practices for therapeutics from eggs have been established. Here we examined the ability of upstream and downstream DNA sequences of ovalbumin, a protein produced exclusively in very high quantities in chicken egg white, to drive tissue-specific expression of human mAb in chicken eggs. To accommodate these large regulatory regions, we established and transfected lines of chicken embryonic stem (cES) cells and formed chimeras that express mAb from cES cell–derived tubular gland cells. Eggs from high-grade chimeras contained up to 3 mg of mAb that possesses enhanced antibody-dependent cellular cytotoxicity (ADCC), nonantigenic glycosylation, acceptable half-life, excellent antigen recognition and good rates of internalization.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Tissue-restricted expression of mAb from Ov7.5mAbdns and Ov15mAbF1 vectors.
Figure 2: Deposition of mAbF1 into eggs from Ov15mAbF1 chimeras.
Figure 3: Glycosylation of mAbF1 protein produced in chicken tubular gland cells.
Figure 4: Biological activities of mAbF1 produced by chicken tubular gland cells in comparison to that produced by CHO cells.

Accession codes




  1. Ma, J.K., Drake, P.M. & Christou, P. The production of recombinant pharmaceutical proteins in plants. Nat. Rev. Genet. 4, 794–805 (2003).

    Article  CAS  Google Scholar 

  2. Ward, M. et al. Characterization of humanized antibodies secreted by Aspergillus niger. Appl. Environ. Microbiol. 70, 2567–2576 (2004).

    Article  CAS  Google Scholar 

  3. Echelard, Y. Recombinant protein production in transgenic animals. Curr. Opin. Biotechnol. 7, 536–540 (1996).

    Article  CAS  Google Scholar 

  4. Edmunds, T. et al. Transgenically produced human antithrombin: structural and functional comparison to human plasma-derived antithrombin. Blood 91, 4561–4571 (1998).

    CAS  Google Scholar 

  5. Pollock, D.P. et al. Transgenic milk as a method for the production of recombinant antibodies. J. Immunol. Methods 231, 147–157 (1999).

    Article  CAS  Google Scholar 

  6. Kaye, J.S., Bellard, M., Dretzen, G., Bellard, F. & Chambon, P. A close association between sites of DNase I hypersensitivity and sites of enhanced cleavage by micrococcal nuclease in the 5′-flanking region of the actively transcribed ovalbumin gene. EMBO J. 3, 1137–1144 (1984).

    Article  CAS  Google Scholar 

  7. Kaye, J.S. et al. Steroid hormone dependence of four DNase I-hypersensitive regions located within the 7000-bp 5′-flanking segment of the ovalbumin gene. EMBO J. 5, 277–285 (1986).

    Article  CAS  Google Scholar 

  8. Kato, S. et al. A far upstream estrogen response element of the ovalbumin gene contains several half-palindromic 5′-TGACC-3′ motifs acting synergistically. Cell 68, 731–742 (1992).

    Article  CAS  Google Scholar 

  9. Ghirlando, R., Lund, J., Goodall, M. & Jefferis, R. Glycosylation of human IgG-Fc: influences on structure revealed by differential scanning micro-calorimetry. Immunol. Lett. 68, 47–52 (1999).

    Article  CAS  Google Scholar 

  10. Liu, H. et al. Constitutive and antibody-induced internalization of prostate-specific membrane antigen. Cancer Res. 58, 4055–4060 (1998).

    CAS  PubMed  Google Scholar 

  11. Bosselman, R.A. et al. Germline transmission of exogenous genes in the chicken. Science 243, 533–535 (1989).

    Article  CAS  Google Scholar 

  12. Cook, R.F. et al. Liver-specific expression of a phosphoenolpyruvate carboxykinase-neo gene in genetically modified chickens. Poult. Sci. 72, 554–567 (1993).

    Article  CAS  Google Scholar 

  13. Hippenmeyer, P.J., Krivi, G.G. & Highkin, M.K. Transfer and expression of the bacterial NPT-II gene in chick embryos using a Schmidt-Ruppin retrovirus vector. Nucleic Acids Res. 16, 7619–7632 (1988).

    Article  CAS  Google Scholar 

  14. Harvey, A.J., Speksnijder, G., Baugh, L.R., Morris, J.A. & Ivariet, R. Consistent production of transgenic chickens using replication-deficient retroviral vectors and high-throughput screening procedures. Poult. Sci. 81, 202–212 (2002).

    Article  CAS  Google Scholar 

  15. Harvey, A.J., Speksnijder, G., Baugh, L.R., Morris, J.A. & Ivarie, R. Expression of exogenous protein in the egg white of transgenic chickens. Nat. Biotechnol. 20, 396–399 (2002).

    Article  CAS  Google Scholar 

  16. Rapp, J.C., Harvey, A.J., Speksnijder, G.L., Hu, W. & Ivarie, R. Biologically active human interferon alpha-2b produced in the egg white of transgenic hens. Transgenic Res. 12, 569–575 (2003).

    Article  CAS  Google Scholar 

  17. McGrew, M.J. et al. Efficient production of germline transgenic chickens using lentiviral vectors. EMBO Rep. 5, 728–733 (2004).

    Article  CAS  Google Scholar 

  18. Pain, B. et al. Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities. Development 122, 2339–2348 (1996).

    CAS  Google Scholar 

  19. Petitte, J.N., Liu, G. & Yang, Z. Avian pluripotent stem cells. Mech. Dev. 121, 1159–1168 (2004).

    Article  CAS  Google Scholar 

  20. Acloque, H. et al. Identification of a new gene family specifically expressed in chicken embryonic stem cells and early embryo. Mech. Dev. 103, 79–91 (2001).

    Article  CAS  Google Scholar 

  21. Shields, R.L. et al. Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733–26740 (2002).

    Article  CAS  Google Scholar 

  22. Shinkawa, T. et al. The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278, 3466–3473 (2003).

    Article  CAS  Google Scholar 

  23. Okazaki, A. et al. Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa. J. Mol. Biol. 336, 1239–1249 (2004).

    Article  CAS  Google Scholar 

  24. Niwa, R. et al. Defucosylated chimeric anti-CC chemokine receptor 4 IgG1 with enhanced antibody-dependent cellular cytotoxicity shows potent therapeutic activity to T-cell leukemia and lymphoma. Cancer Res. 64, 2127–2133 (2004).

    Article  CAS  Google Scholar 

  25. Raju, T.S., Briggs, J.B., Borge, S.M. & Jones, A.J.S. Species-specific variation in glycosylation of IgG: evidence for the species-specific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 10, 477–486 (2000).

    Article  CAS  Google Scholar 

  26. Suzuki, N., Khoo, K.H., Chen, C.M., Chen, H.C. & Lee, Y.C. N-glycan structures of pigeon IgG: a major serum glycoprotein containing Galalpha1–4 Gal termini. J. Biol. Chem. 278, 46293–46306 (2003).

    Article  CAS  Google Scholar 

  27. Iwase, H., Kato, Y., Li, S.C., Li, Y.T. & Hotta, K. Comparative study of avian ovalbumins by means of glycosidase treatment and following HPLC analysis of their dansyl glycopeptides. Comp. Biochem. Physiol. B 79, 321–324 (1984).

    Article  CAS  Google Scholar 

  28. Hase, S., Sugimoto, T., Takemoto, H., Ikenaka, T. & Schmid, K. The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts. J. Biochem. 99, 1725–1733 (1986).

    Article  CAS  Google Scholar 

  29. Takahashi, N. et al. A structural study of the asparagine-linked oligosaccharide moiety of duck ovomucoid. Glycoconj. J. 10, 425–434 (1993).

    Article  CAS  Google Scholar 

  30. Kuster, B., Naven, T.J. & Harvey, D.J. Rapid approach for sequencing neutral oligosaccharides by exoglycosidase digestion and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. J. Mass Spectrom. 31, 1131–1140 (1996).

    Article  CAS  Google Scholar 

  31. Harvey, D.J., Wing, D.R., Kuster, B. & Wilson, I.B. Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins. J. Am. Soc. Mass Spectrom. 11, 564–571 (2000).

    Article  CAS  Google Scholar 

  32. Takahashi, N., Khoo, K.H., Suzuki, N., Johnson, J.R. & Lee, Y.C. N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(1)Gal. J. Biol. Chem. 276, 23230–23239 (2001).

    Article  CAS  Google Scholar 

  33. Saba, J.A., Shen, X., Jamieson, J.C. & Perreault, H. Investigation of different combinations of derivatization, separation methods and electrospray ionization mass spectrometry for standard oligosaccharides and glycans from ovalbumin. J. Mass Spectrom. 36, 563–574 (2001).

    Article  CAS  Google Scholar 

  34. Clynes, R.A., Towers, T.L., Presta, L.G. & Ravetch, J.V. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat. Med. 6, 443–446 (2000).

    Article  CAS  Google Scholar 

  35. Niwa, R. et al. Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 Is independent of FcgammaRIIIa functional polymorphism. Clin. Cancer Res. 10, 6248–6255 (2004).

    Article  CAS  Google Scholar 

  36. Ivarie, R. Avian transgenesis: progress towards the promise. Trends Biotechnol. 21, 14–19 (2003).

    Article  CAS  Google Scholar 

  37. Lillico, S.G., McGrew, M.J., Sherman, A. & Sang, H.M. Transgenic chickens as bioreactors for protein-based drugs. Drug Discov. Today 10, 191–196 (2005).

    Article  CAS  Google Scholar 

  38. Eyal-Giladi, H. & Kochav, S. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick I. General Morphology. Dev. Biol. 49, 321–337 (1976).

    Article  CAS  Google Scholar 

  39. Petitte, J.N. in Handbook of Stem Cells vol 1. (ed. Lanza, R.P.) 471–477 (Elsevier Academic, London, 2004).

    Book  Google Scholar 

  40. Rowlett, K. & Simkiss, K. Explanted embryo culture: in vitro and in ovo techniques for domestic fowl. Br. Poult. Sci. 28, 91–101 (1987).

    Article  Google Scholar 

  41. Carsience, R.S., Clark, M.E., Verrinder Gibbins, A.M. & Etches, R.J. Germline chimeric chickens from dispersed donor blastodermal cells and compromised recipient embryos. Development 117, 669–675 (1993).

    CAS  Google Scholar 

  42. Crooijmans, R.P., Vrebalov, J., Dijkhof, R.J.M., van der Poel, J.J. & Groenen, M.A.M. Two-dimensional screening of the Wageningen chicken BAC library. Mamm. Genome 11, 360–363 (2000).

    Article  CAS  Google Scholar 

  43. Niwa, H., Yamamura, K.-I. & Miyazaki, J.-I. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–200 (1991).

    Article  CAS  Google Scholar 

  44. Dangl, J.L. et al. Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies. EMBO J. 7, 1989–1994 (1988).

    Article  CAS  Google Scholar 

  45. Fishwild, D. et al. High-avidity human IgGk monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat. Biotechnol. 14, 845–851 (1996).

    Article  CAS  Google Scholar 

  46. Holmes, E.H. PMSA specific antibodies and their diagnostic and therapeutic use. Expert Opin. Investig. Drugs 10, 511–519 (2001).

    Article  CAS  Google Scholar 

  47. Mohammed, S.M. et al. Deposition of genetically engineered human antibodies into the egg yolk of hens. Immunotechnology 4, 115–125 (1998).

    Article  CAS  Google Scholar 

  48. Kodama, H. et al. Nucleotide sequences and unusual electrophoretic behavior of the W chromosome-specific repeating DNA units of the domestic fowl, Gallus gallus domesticus. Chromosoma 96, 18–25 (1987).

    Article  CAS  Google Scholar 

  49. Kato, A., Nakamura, R. & Yasushi, S. Changes in the chemical composition of ovomucin during storage. Agric. Biol. Chem. 34, 1009–1013 (1970).

    CAS  Google Scholar 

Download references


The work conducted at Origen Therapeutics and Texas A&M University was supported by the National Institutes of Health Small Business Innovation Research Grant GM064261. Research in the laboratory at UCLA was supported by grants AI29470, AI39187 and AI51415 from the National Institutes of Health.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Robert J Etches.

Ethics declarations

Competing interests

Many of the authors are employees of companies developing these technologies for commerical application.

Supplementary information

Supplementary Fig. 1

Southern blot analysis of cES clones transfected with Ov7.5 and Ov15 expression vectors. (PDF 1575 kb)

Supplementary Fig. 2

The morphology of the female chicken embryonic stem (ES) cell line 440 is shown in the upper panel. (PDF 125 kb)

Supplementary Fig. 3

cIEF data for MAbF1 produced by chicken tubular gland cells and CHO cells. (PDF 377 kb)

Supplementary Fig. 4

Thermal stabilities of chicken and CHO cell derived MAbF1. (PDF 1893 kb)

Supplementary Table 1

The extent of feather chimerism and the number of offspring obtained from male chimeras carrying cES cells derived from Barred Plymouth Rock. (PDF 31 kb)

Supplementary Table 2

Sequences for PCR primers. (PDF 37 kb)

Supplementary Table 3

Sequence analysis of chicken produced and CHO produced MAbF1 by mass spectrometry. (PDF 50 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zhu, L., van de Lavoir, MC., Albanese, J. et al. Production of human monoclonal antibody in eggs of chimeric chickens. Nat Biotechnol 23, 1159–1169 (2005).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing