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Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display

Abstract

Here we applied ribosome display to in vitro selection and evolution of single-chain antibody fragments (scFvs) from a large synthetic library (Human Combinatorial Antibody Library; HuCAL) against bovine insulin. In three independent ribosome display experiments different clusters of closely related scFvs were selected, all of which bound the antigen with high affinity and specificity. All selected scFvs had affinity-matured up to 40-fold compared to their HuCAL progenitors, by accumulating point mutations during the ribosome display cycles. The dissociation constants of the isolated scFvs were as low as 82 pM, which validates the design of the naïve library and the power of this evolutionary method. We have thus mimicked the process of antibody generation and affinity maturation with a synthetic library in a cell-free system in just a few days, obtaining molecules with higher affinities than most natural antibodies.

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Figure 1: Principle of ribosome display.
Figure 2: Radioimmunoassays of the pools after the fifth and sixth rounds of ribosome display.
Figure 3: Alignment of the amino acid sequences of VH and VL of the scFvs.
Figure 4: Framework usage of the insulin-binding HuCAL scFvs.

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References

  1. Hanes, J. & Plückthun, A. In vitro selection methods for screening of peptide and protein libraries. Curr. Top. Microbiol. Immunol. 243, 107– 122 (1999).

    CAS  PubMed  Google Scholar 

  2. Schaffitzel, C., Hanes, J., Jermutus, L. & Plückthun, A. Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J. Immunol. Methods 231, 119–135 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 –1317 (1985).

    Article  CAS  PubMed  Google Scholar 

  4. Smith, G.P. & Scott, J.K. Libraries of peptides and proteins displayed on filamentous phage. Methods Enzymol. 217, 228–257 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455 ( 1994).

    Article  CAS  PubMed  Google Scholar 

  6. Mattheakis, L.C., Bhatt, R.R. & Dower, W.J. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Hanes, J. & Plückthun, A. In vitro selection and evolution of functional proteins by using ribosome display . Proc. Natl. Acad. Sci. USA 94, 4937– 4942 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H.R. & Plückthun, A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA 95, 14130–14135 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Roberts, R.W. & Szostak, J.W. RNA–peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94, 12297– 12302 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nemoto, N., Miyamoto-Sato, E., Husimi, Y. & Yanagawa, H. In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro . FEBS Lett. 414, 405– 408 (1997).

    Article  CAS  PubMed  Google Scholar 

  11. He, M. et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J. Immunol. Methods 231, 105–117 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Low, N.M., Holliger, P.H. & Winter, G. Mimicking somatic hypermutation: affinity maturation of antibodies displayed on bacteriophage using a bacterial mutator strain . J. Mol. Biol. 260, 359– 368 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Yang, W.P. et al. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254 , 392–403 (1995).

    Article  CAS  PubMed  Google Scholar 

  14. Schier, R. & Marks, J.D. Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections . Hum. Antibodies Hybridomas 7, 97– 105 (1996).

    Article  CAS  PubMed  Google Scholar 

  15. Moore, J.C., Jin, H.M., Kuchner, O. & Arnold, F.H. Strategies for the in vitro evolution of protein function: enzyme evolution by random recombination of improved sequences. J. Mol. Biol. 272, 336–347 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000).

    Article  CAS  PubMed  Google Scholar 

  17. Johnson, G., Kabat, E.A. & Wu, T.T. Kabat database of sequences of proteins of immunological interest. Handbook of experimental immunology, Vol. 1, Edn. 5 (eds Weir, D.M.M., Blackwell, L.A. & Herzenberg, C.) 6.1–6.21 (Blackwell Science Inc., Cambridge, MA; 1996).

    Google Scholar 

  18. Virnekäs, B. et al. Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600–5607 ( 1994).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Malkin, L.I. & Rich, A. Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding . J. Mol. Biol. 26, 329– 346 (1967).

    Article  CAS  PubMed  Google Scholar 

  20. Smith, W.P., Tai, P.C. & Davis, B.D. Interaction of secreted nascent chains with surrounding membrane in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 75, 5922–5925 ( 1978).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Gold, L., Polisky, B., Uhlenbeck, O. & Yarus, M. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64, 763–797 (1995).

    Article  CAS  PubMed  Google Scholar 

  22. Nieba, L., Honegger, A., Krebber, C. & Plückthun, A. Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 10, 435– 444 (1997).

    Article  CAS  PubMed  Google Scholar 

  23. Proba, K., Honegger, A. & Plückthun, A. A natural antibody missing a cysteine in VH: consequences for thermodynamic stability and folding. J. Mol. Biol. 265, 161–172 (1997).

    Article  CAS  PubMed  Google Scholar 

  24. Proba, K., Worn, A., Honegger, A. & Plückthun, A. Antibody scFv fragments without disulfide bonds made by molecular evolution. J. Mol. Biol. 275, 245–253 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Nieba, L., Krebber, A. & Plückthun, A. Competition BIAcore for measuring true affinities: large differences from values determined from binding kinetics. Anal. Biochem. 234, 155–165 ( 1996).

    Article  CAS  PubMed  Google Scholar 

  26. Karlsson, R. Real-time competitive kinetic analysis of interactions between low- molecular-weight ligands in solution and surface-immobilized receptors. Anal. Biochem. 221, 142–151 ( 1994).

    Article  CAS  PubMed  Google Scholar 

  27. Schuck, P. Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules. Annu. Rev. Biophys. Biomol. Struct. 26, 541–566 (1997).

    Article  CAS  PubMed  Google Scholar 

  28. Stemmer, W.P. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 ( 1994).

    Article  CAS  PubMed  Google Scholar 

  29. Cadwell, R.C. & Joyce, G.F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

    Article  CAS  PubMed  Google Scholar 

  30. Zhao, H., Giver, L., Shao, Z., Affholter, J.A. & Arnold, F.H. Molecular evolution by staggered extension process (StEP) in vitro recombination. Nat. Biotechnol. 16, 258–261 ( 1998).

    Article  CAS  PubMed  Google Scholar 

  31. Krebber, C., Spada, S., Desplancq, D. & Plückthun, A. Co-selection of cognate antibody–antigen pairs by selectively infective phages. FEBS Lett. 377, 227– 231 (1995).

    Article  CAS  PubMed  Google Scholar 

  32. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89 (1990).

    Article  CAS  PubMed  Google Scholar 

  33. Reynolds, R., Bermudez-Cruz, R.M. & Chamberlin, M.J. Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho-independent terminators. J. Mol. Biol. 224, 31– 51 (1992).

    Article  CAS  PubMed  Google Scholar 

  34. Pokrovskaya, I.D. & Gurevich, V.V. In vitro transcription: preparative RNA yields in analytical scale reactions. Anal. Biochem. 220, 420–423 (1994).

    Article  CAS  PubMed  Google Scholar 

  35. Krebber, A. et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system . J. Immunol. Methods 201, 35– 55 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Bothmann, H. & Plückthun, A. Selection for a periplasmic factor improving phage display and functional periplasmic expression . Nat. Biotechnol. 16, 376– 380 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Ge, L., Knappik, A., Pack, P., Freund, C. & Plückthun, A. Expressing antibodies in Escherichia coli. In Antibody engineering, Edn. 2 (ed. Borrebaeck, C.A.K) 229–266 (Oxford University Press, New York, NY; 1995).

    Google Scholar 

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Acknowledgements

This work was supported by the Schweizerischer Nationalfonds grant 31-46624.96. C.S. is supported by a predoctoral Kékule fellowship from the Fonds der Chemischen Industrie (Germany). The authors would like to acknowledge Annemarie Honegger, Lutz Jermutus, and Stephen Marino for help, advice, and discussion, and MorphoSys AG for the constructive collaboration on HuCAL.

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Correspondence to Andreas Plückthun.

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Hanes, J., Schaffitzel, C., Knappik, A. et al. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat Biotechnol 18, 1287–1292 (2000). https://doi.org/10.1038/82407

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