Skip to main content
SpringerLink
Log in

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

Abstract

One of existing strategies to engineer active antibody is to link V H and V L domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or single-chain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned V H and V L domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and ~24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
FIGURE 5
FIGURE 6
FIGURE 7
FIGURE 8
FIGURE 9

Similar content being viewed by others

References

  1. Alfthan, K., K. Takkinen, D. Sizmann, H. Soderlund, and T. T. Teeri. Properties of a single-chain antibody containing different linker peptides. Protein Eng. 8:725–731, 1995.

    Article  CAS  PubMed  Google Scholar 

  2. Alzari, P. M., M. B. Lascombe, and R. J. Poljak. Three-dimensional structure of antibodies. Ann. Rev. Immunol. 6:555–580, 1988.

    Article  CAS  Google Scholar 

  3. Atwell, J. L., L. A. Pearce, M. Lah, L. C. Gruen, A. A. Kortt, and P. J. Hudson. Design and expression of a stable bispecific scFv dimer with affinity for both glycophorin and N9 neuraminidase. Mol. Immunol. 33:1301–1312, 1996.

    Article  CAS  PubMed  Google Scholar 

  4. Bird, R. E., J. W. Jacobson, S. Johnson, B. M. Kaufman, S. M. Lee, T. Lee, S. H. Pope, G. S. Riordan, and M. Whitlow. Single-chain antigen-binding proteins. Science 242:423–426, 1988.

    Article  CAS  PubMed  Google Scholar 

  5. Chesla, S. E., P. Selvaraj, and C. Zhu. Measuring two-dimensional receptor-ligand binding kinetics by micropipette. Biophys. J. 75:1553–1572, 1998.

    Article  CAS  PubMed  Google Scholar 

  6. Davies, G. M., S. Bosze, F. Hudecz, M. R. Price, and S. J. Tendler. Characterisation of a recombinant Fv fragment of anti-MUC1 antibody HMFG1. Cancer Lett 82:179–184, 1994.

    Article  CAS  PubMed  Google Scholar 

  7. Du, J., H. Wang, C. Zhong, B. Peng, M. Zhang, B. Li, S. Huo, Y. Guo, and J. Ding. Structural basis for recognition of CD20 by therapeutic antibody rituximab. J. Biol. Chem. 282:15073–15080, 2007.

    Article  CAS  PubMed  Google Scholar 

  8. Dumaswala, U. J., M. J. Wilson, T. Jose, and D. L. Daleke. Glutamine- and phosphate-containing hypotonic storage media better maintain erythrocyte membrane physical properties. Blood 88:697–704, 1996.

    CAS  PubMed  Google Scholar 

  9. Feng, J. N., Z. G. Xie, N. Guo, and B. F. Shen. Design and assembly of anti-CD16 ScFv antibody with two different linker peptides. J. Immunol. Meth. 282:33–43, 2003.

    Article  CAS  Google Scholar 

  10. Geuijen, C. A. W., M. Clijsters-van der Horst, F. Cox, P. M. L. Rood, M. Throsby, H. H. J. Backus, E. van Deventer, A. M. Kruisbeek, J. Goudsmit, and J. de Kruif. Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery. J. Immunol. Meth. 302:68–77, 2005.

    Article  CAS  Google Scholar 

  11. Glockshuber, R., M. Malia, I. Pfitzinger, and A. Pluckthun. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29:1362–1367, 1990.

    Article  CAS  PubMed  Google Scholar 

  12. Golay, J. T., E. A. Clark, and P. C. Beverley. The CD20 (Bp35) antigen is involved in activation of B cells from the G0 to the G1 phase of the cell cycle. J. Immunol. 135:3795–3801, 1985.

    CAS  PubMed  Google Scholar 

  13. Green, C. E., D. N. Pearson, N. B. Christensen, and S. I. Simon. Topographic requirements and dynamics of signaling via L-selectin on neutrophils. Am. J. Physiol. Cell Physiol. 284:C705–C717, 2003.

    CAS  PubMed  Google Scholar 

  14. Hoedemaeker, F. J., T. Signorelli, K. Johns, D. A. Kuntz, and D. R. Rose. A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution. J. Biol. Chem. 272:29784–29789, 1997.

    Article  CAS  PubMed  Google Scholar 

  15. Horton, R. M., H. D. Hunt, S. N. Ho, J. K. Pullen, and L. R. Pease. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68, 1989.

    Article  CAS  PubMed  Google Scholar 

  16. Huang, J., J. Chen, S. E. Chesla, T. Yago, P. Mehta, R. P. McEver, C. Zhu, and M. Long. Quantifying the effects of molecular orientation and length on two-dimensional receptor–ligand binding kinetics. J. Biol. Chem. 279:44915–44923, 2004.

    Article  CAS  PubMed  Google Scholar 

  17. Huston, J. S., D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotny, M. N. Margolies, R. J. Ridge, R. E. Bruccoleri, E. Haber, and R. Crea. 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, 1988.

    Article  CAS  PubMed  Google Scholar 

  18. Huston, J. S., J. McCartney, M. S. Tai, C. Mottola-Hartshorn, D. Jin, F. Warren, P. Keck, and H. Oppermann. Medical applications of single-chain antibodies. Int. Rev. Immunol. 10:195–217, 1993.

    Article  CAS  PubMed  Google Scholar 

  19. Kofler, R., and G. Wick. Some methodologic aspects of the chromium chloride method for coupling antigen to erythrocytes. J. Immunol. Meth. 16:201–209, 1977.

    Article  CAS  Google Scholar 

  20. Kortt, A. A., O. Dolezal, B. E. Power, and P. J. Hudson. Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting. Biomol. Eng. 18:95–108, 2001.

    Article  CAS  PubMed  Google Scholar 

  21. Kortt, A. A., R. L. Malby, J. B. Caldwell, L. C. Gruen, N. Ivancic, M. C. Lawrence, G. J. Howlett, R. G. Webster, P. J. Hudson, and P. M. Colman. Recombinant anti-sialidase single-chain variable fragment antibody. Characterization, formation of dimer and higher-molecular-mass multimers and the solution of the crystal structure of the single-chain variable fragment/sialidase complex. Eur. J. Biochem. 221:151–157, 1994.

    Article  CAS  PubMed  Google Scholar 

  22. Li, B., J. Chen, and M. Long. Measuring binding kinetics of surface-bound molecules using surface plasmon resonance technique. Anal. Biochem. 377:195–201, 2008.

    Article  CAS  PubMed  Google Scholar 

  23. Li, A. L., Y. Li, Y. X. Sun, M. Yu, J. S. Guo, and B. F. Shen. Studies on apoptosis of Daudi cells induced by monoclonal antibodies against human CD20. Immunol. J. 18:292–295, 2002.

    Google Scholar 

  24. Lilley, G. G., O. Dolezal, C. J. Hillyard, C. Bernard, and P. J. Hudson. Recombinant single-chain antibody peptide conjugates expressed in Escherichia coli for the rapid diagnosis of HIV. J. Immunol. Meth. 171:211–226, 1994.

    Article  CAS  Google Scholar 

  25. Lofblöm, J., J. Sandberg, H. Wernérus, and S. Ståhl. Evaluation of staphylococcal cell surface display and flow cytometry for postselectional characterization of affinity proteins in combinatorial protein engineering applications. Appl. Environ. Microbiol. 73:6714–6721, 2007.

    Article  PubMed  Google Scholar 

  26. Loken, M. R., V. O. Shah, K. L. Datillio, and C. I. Civin. Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development. Blood 70:1316–1324, 1987.

    CAS  PubMed  Google Scholar 

  27. Long, M., H. Zhao, K. S. Huang, and C. Zhu. Kinetic measurements of cell surface E-selectin/carbohydrate ligand interactions. Ann. Biomed. Eng. 29:935–946, 2001.

    Article  CAS  PubMed  Google Scholar 

  28. Petit, S., F. Brard, G. Coquerel, G. Perez, and F. Tron. Structural models of antibody variable fragments: a method for investigating binding mechanisms. J. Comput. Aid Mol. Des. 12:147–163, 1998.

    Article  CAS  Google Scholar 

  29. Press, O. W., F. Appelbaum, J. A. Ledbetter, P. J. Martin, J. Zarling, P. Kidd, and E. D. Thomas. Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas. Blood 69:584–591, 1987.

    CAS  PubMed  Google Scholar 

  30. Schmiedl, A., J. Zimmermann, J. E. Scherberich, P. Fischer, and S. Dubel. Recombinant variants of antibody 138H11 against human gamma-glutamyltransferase for targeting renal cell carcinoma. Hum. Antibodies 15:81–94, 2006.

    CAS  PubMed  Google Scholar 

  31. Selvaraj, P., M. L. Plunkett, M. Dustin, M. E. Sanders, S. Shaw, and T. A. Springer. The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3. Nature 326:400–403, 1987.

    Article  CAS  PubMed  Google Scholar 

  32. Shan, D. M., O. W. Press, T. T. Tsu, M. S. Hayden, and J. A. Ledbetter. Characterization of scFv-Ig constructs generated from the anti-CD20 mAb 1F5 using linker peptides of varying lengths. J. Immunol. 162:6589–6595, 1999.

    CAS  PubMed  Google Scholar 

  33. Sheikholvaezin, A., P. Sandstrom, D. Eriksson, N. Norgren, K. Riklund, and T. Stigbrand. Optimizing the generation of recombinant single-chain antibodies against placental alkaline phosphatase. Hybridoma (Larchmt) 25:181–192, 2006.

    Article  CAS  Google Scholar 

  34. Stashenko, P., L. M. Naler, R. Hardy, and S. F. Schlossman. Characterization of a human B lymphocyte-specific antigen. J. Immunol. 125:1678–1685, 1980.

    CAS  PubMed  Google Scholar 

  35. Tedder, T. F., A. W. Boyd, A. S. Freedman, L. M. Nadler, and S. F. Schlossman. The B cell surface molecule B1 is functionally linked with B cell activation and differentiation. J. Immunol. 135:973–979, 1985.

    CAS  PubMed  Google Scholar 

  36. Tedder, T. F., and P. Engel. CD20: a regulator of cell-cycle progression of B lymphocytes. Immunol. Today 15:450–454, 1994.

    Article  CAS  PubMed  Google Scholar 

  37. Thompson, J. D., D. G. Higgins, and T. Gibson. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. J. Nucleic Acids Res. 22:4673–4680, 1994.

    Article  CAS  Google Scholar 

  38. Turner, D. J., M. A. Ritter, and A. J. George. Importance of the linker in expression of single-chain Fv antibody fragments: optimisation of peptide sequence using phage display technology. J. Immunol. Meth. 205:43–54, 1997.

    Article  CAS  Google Scholar 

  39. van Gunsteren, W. F., and H. J. Berendsen. Computer simulation as a tool for tracing the conformational differences between proteins in solution and in the crystalline state. J. Mol. Biol. 176:559–564, 1984.

    Article  PubMed  Google Scholar 

  40. von Schilling, C. Immunotherapy with anti-CD20 compounds. Semin. Cancer Biol. 13:211–222, 2003.

    Article  Google Scholar 

  41. Wang, Y. G., Y. Huang, X. Gu, M. Yu, J. N. Feng, Y. X. Sun, Y. Li, and B. F. Shen. Construction and expression of chimeric anti-human CD20 monoclonal antibody. J. Cell. Mol. Immunol. 22:363–367, 2006.

    Google Scholar 

  42. Wang, S. H., J. B. Zhang, Z. P. Zhang, Y. F. Zhou, R. F. Yang, J. Chen, Y. C. Guo, F. You, and X. E. Zhang. Construction of single chain variable fragment (ScFv) and BiscFv-alkaline phosphatase fusion protein for detection of Bacillus anthracis. Anal. Chem. 78:997–1004, 2006.

    Article  CAS  PubMed  Google Scholar 

  43. Wu, L., B. T. Xiao, X. L. Jia, Y. Zhang, S. Q. Lü, J. Chen, and M. Long. Impacts of carrier stiffness and microtopology on 2D kinetics of P-selectin-PSGL-1 interactions. J. Biol. Chem. 282:9846–9854, 2007.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Periasamy Selvaraj of Emory University School of Medicine for generous gifts of TS 2/9 mAb. This work was supported by National High Technology Research and Development Program of China Grant 2007AA02Z306, National Key Basic Research Foundation of China Grant 2006CB910303, National Natural Science Foundation of China Grants 30730032, 30771982 and 10332060, and Knowledge Innovation Program of Chinese Academy of Sciences Grant KJCX2-YW-L08 (M.L.), as well as National High Technology Research and Development Program of China Grant 2006AA02A254 and Beijing Grant 5062037 (Y.L.).

Author information

Authors and Affiliations

  1. Institute of Basic Medical Sciences, P.O. Box 130 (3), Taiping Road, Beijing, 100850, P.R. China

    Xin Gu, Jiannan Feng, Beifen Shen, Ying Huang, Shusheng Geng, Yingxun Sun, Yugang Wang & Yan Li

  2. Key Laboratory of Microgravity (National Microgravity Laboratory) and Center of Biomechanics and Bioengineering, Institute of Mechanics, Chinese Academy of Sciences, Beijing, 100190, P.R. China

    Xiaoling Jia & Mian Long

  3. Lab of Cellular and Molecular Immunology, Medical School of Henan University, Kaifeng, 475000, P.R. China

    Jiannan Feng & Yan Li

Authors
  1. Xin Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaoling Jia
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jiannan Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Beifen Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ying Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shusheng Geng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yingxun Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yugang Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Mian Long
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Yan Li or Mian Long.

Additional information

Xin Gu, Xiaoling Jia, and Jiannan Feng contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gu, X., Jia, X., Feng, J. et al. Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity. Ann Biomed Eng 38, 537–549 (2010). https://doi.org/10.1007/s10439-009-9810-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-009-9810-2

Keywords

Search

Navigation