Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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The Development of Dimerized Chicken Recombinant Single-chain Fv (ScFv) Antibody Using Leucine Zipper Motif

Leucine Zipper Motif를 이용한 닭의 재조합 이량체 Single-chain Fv (ScFv) 항체의 개발

  • Park, Dong-Woon (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Kim, Eon-Dong (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Kim, Sung-Heon (Department of Microbiology, College of Natural Sciences, Changwon National University) ;
  • Han, Jae-Yong (WCU Biomodulation Major, Department of Agricultural Biotechnology, and Research Institute for Agriculture and Life Sciences, Seoul National University) ;
  • Kim, Jin-Kyoo (Department of Microbiology, College of Natural Sciences, Changwon National University)
  • 박동운 (창원대학교 자연과학대학 미생물학과) ;
  • 김언동 (창원대학교 자연과학대학 미생물학과) ;
  • 김성헌 (창원대학교 자연과학대학 미생물학과) ;
  • 한재용 (서울대학교 농업생명과학대학 농생명공학부 WCU 바이오모듈레이션 전공) ;
  • 김진규 (창원대학교 자연과학대학 미생물학과)
  • Received : 2011.09.27
  • Accepted : 2011.11.25
  • Published : 2011.12.31
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Abstract

Leucine zipper motif consists of multiple periodic leucine residues, which forms amphipathic alpha helix. The hydrophobic nature of leucine zipper motif can dimerize proteins which contain this motif. Leucine zipper motif addition at C-terminus of single-chain Fv (ScFv) antibody induces its dimerization. Since the dimeric ScFv antibody contains two antigen binding sites (bivalency) like Y-shaped complete antibody, it could increase avidity. As a result, it could show higher antigen binding activity than monomeric ScFv antibodies. Based on this concept, monomeric chicken 8C3 ScFv antibody previously developed from chicken hybridoma was dimerized by the addition of leucine zipper motif at C-terminus of ScFv antibody. The dimeric 8C3 ScFv antibody specifically reacted with Eimerian sporozoite which causes Avian Coccidiosis. As expected, dimeric 8C3 ScFv antibody showed 3-folds higher antigen binding activity than monomer due to increased avidity. In addition, protien yields of dimer expression were 2-folds higher than monomer.

Leucine zipper motif는 여러 개의 주기적인 leucine 잔기로 구성되어 amphipathic alpha helix형태의 구조를 나타내며 소수성 결합에 의해 이량체를 형성한다. 이 leucine zipper motif를 single chain Fv 항체의 C-terminus에 도입하면 leucine zipper motif의 소수성 결합에 의해 amphipathic alpha helix의 이량체가 형성되면서 융합된 single chain Fv 항체의 이량체 (Dimer) 형성 또한 유도할 수 있다. 이량체 형태의 single chain Fv 항체는 2개의 항원 결합부위를 갖게 되므로 단량체 형태의(monomer) single chain Fv 항체에 비해 항원 결합력(Avidity)이 증가 될 것이다. 이 개념에 기초하여 이전 연구에서 제조된 단량체 형태인 닭 single chain Fv 항체인 8C3 ScFv 항체의 C-terminus에 leucine zipper motif를 도입하여 이량체 형태의 8C3 ScFv 항체를 개발하였다. 이량체 8C3 ScFv 항체는 가금류의 대표적인 기생충 질병인 coccidiosis를 유발하는 Eimerian sporozoite에 특이적으로 결합하는 기능을 나타내었다. 또한 이량체 8C3 ScFv 항체는 avidity 증가로 인하여 단량체에 비해 항원 결합력이 약 3배 증가됨을 확인할 수 있었으며 단백질 회수율 또한 2배 증가되는 부수적인 효과를 얻을 수 있었다.

Keywords

References

  1. Albrecht, H., G.L. Denardo, and S.J. Denardo. 2006. Monospecific bivalent scFv-SH: effects of linker length and location of an engineered cysteine on production, antigen binding activity and free SH accessibility. J. Immunol. Methods 310, 100-116. https://doi.org/10.1016/j.jim.2005.12.012
  2. Bird, R.E., K.D. Hardman, J.W. Jacobson, S. Johnson, B.M. Kaufman, S.M. Lee, T. Lee, S.H. Pope, G.S. Riordan, and M. Whitlow. 1988. Single-chain antigen-binding proteins. Science 242, 423-426. https://doi.org/10.1126/science.3140379
  3. Brinkmann, U., M. Gallo, E. Brinkmann, S. Kunwar, and I. Pastan. 1993. A recombinant immunotoxin that is active on prostate cancer cells and that is composed of the Fv region of monoclonal antibody PR1 and a truncated form of Pseudomonas exotoxin. Proc. Natl. Acad. Sci. USA 90, 547-551. https://doi.org/10.1073/pnas.90.2.547
  4. Brinkmann, U., Y. Reiter, S.H. Jung, B. Lee, and I. Pastan. 1993. A recombinant immunotoxin containing a disulfidestabilized Fv fragment. Proc. Natl. Acad. Sci. USA 90, 7538-7542. https://doi.org/10.1073/pnas.90.16.7538
  5. de Kruif, J. and T. Logtenberg. 1996. Leucine zipper dimerized bivalent and bispecific scFv antibodies from a semi-synthetic antibody phage display library. J. Biol. Chem. 271, 7630-7634. https://doi.org/10.1074/jbc.271.13.7630
  6. Glockshuber, R., M. Malia, I. Pfitzinger, and A. Pluckthun. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29, 1362-1367. https://doi.org/10.1021/bi00458a002
  7. Huston, J.S., D. Levinson, M. Mudgett-Hunter, M.S. Tai, J. Novotny, M.N. Margolies, R.J. Ridge, R.E. Bruccoleri, E. Haber, R. Crea, and et al. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. USA 85, 5879-5883. https://doi.org/10.1073/pnas.85.16.5879
  8. Kim, J.K., M.F. Tsen, V. Ghetie, and E.S. Ward. 1994. Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed mutagenesis. Eur. J. Immunol. 24, 542-548. https://doi.org/10.1002/eji.1830240308
  9. Landschulz, W.H., P.F. Johnson, and S.L. McKnight. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-1764. https://doi.org/10.1126/science.3289117
  10. Lawn, A.M. and M.E. Rose. 1982. Mucosal transport of Eimeria tenella in the cecum of the chicken. J. Parasitol. 68, 1117-23. https://doi.org/10.2307/3281101
  11. Muller, K.M., K.M. Arndt, and A. Pluckthun. 1998. A dimeric bispecific miniantibody combines two specificities with avidity. FEBS Lett. 432, 45-49. https://doi.org/10.1016/S0014-5793(98)00829-1
  12. O'Shea, E.K., R. Rutkowski, and P.S. Kim. 1989. Evidence that the leucine zipper is a coiled coil. Science 243, 538-542. https://doi.org/10.1126/science.2911757
  13. Pack, P., M. Kujau, V. Schroeckh, U. Knupfer, R. Wenderoth, D. Riesenberg, and A. Pluckthun. 1993. Improved bivalent miniantibodies, with identical avidity as whole antibodies, produced by high cell density fermentation of Escherichia coli. Biotechnology (NY) 11, 1271-1277.
  14. Pack, P., K. Muller, R. Zahn, and A. Pluckthun. 1995. Tetravalent miniantibodies with high avidity assembling in Escherichia coli. J. Mol. Biol. 246, 28-34. https://doi.org/10.1006/jmbi.1994.0062
  15. Pack, P. and A. Pluckthun. 1992. Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli. Biochemistry 31, 1579-1584. https://doi.org/10.1021/bi00121a001
  16. Park, K.J., D.W. Park, C.H. Kim, B.K. Han, T.S. Park, J.Y. Han, H.S. Lillehoj, and J.K. Kim. 2005. Development and characterization of a recombinant chicken single-chain Fv antibody detecting Eimeria acervulina sporozoite antigen. Biotechnol. Lett. 27, 289-295. https://doi.org/10.1007/s10529-005-0682-8
  17. Schoonjans, R., A. Willems, S. Schoonooghe, J. Leoen, J. Grooten, and N. Mertens. 2001. A new model for intermediate molecular weight recombinant bispecific and trispecific antibodies by efficient heterodimerization of single chain variable domains through fusion to a Fab-chain. Biomol. Eng. 17, 193-202. https://doi.org/10.1016/S1389-0344(01)00066-1
  18. Shan, D., O.W. Press, T.T. Tsu, M.S. Hayden, and J.A. Ledbetter. 1999. Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. J. Immunol. 162, 6589-6595.
  19. Todorovska, A., R.C. Roovers, O. Dolezal, A.A. Kortt, H.R. Hoogenboom, and P.J. Hudson. 2001. Design and application of diabodies, triabodies and tetrabodies for cancer targeting. J. Immunol. Methods 248, 47-66. https://doi.org/10.1016/S0022-1759(00)00342-2
  20. Verma, R., E. Boleti, and A.J. George. 1998. Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems. J. Immunol. Methods 216, 165-181. https://doi.org/10.1016/S0022-1759(98)00077-5
  21. Winter, G., A.D. Griffiths, R.E. Hawkins, and H.R. Hoogenboom. 1994. Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433-455. https://doi.org/10.1146/annurev.iy.12.040194.002245
  22. Wu, A.M., G.J. Tan, M.A. Sherman, P. Clarke, T. Olafsen, S.J. Forman, and A.A. Raubitschek. 2001. Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange. Protein Eng. 14, 1025-1033. https://doi.org/10.1093/protein/14.12.1025
  23. Yamanaka, H.I., T. Inoue, and O. Ikeda-Tanaka. 1996. Chicken monoclonal antibody isolated by a phage display system. J. Immunol. 157, 1156-1162.
  24. Young, N.M., C.R. MacKenzie, S.A. Narang, R.P. Oomen, and J.E. Baenziger. 1995. Thermal stabilization of a single-chain Fv antibody fragment by introduction of a disulphide bond. FEBS Lett. 377, 135-139. https://doi.org/10.1016/0014-5793(95)01325-3