Nature-inspired design of motif-specific antibody scaffolds


Aberrant changes in post-translational modifications (PTMs) such as phosphate groups underlie a majority of human diseases. However, detection and quantification of PTMs for diagnostic or biomarker applications often require PTM-specific monoclonal antibodies (mAbs), which are challenging to generate using traditional antibody-selection methods. Here we outline a general strategy for producing synthetic, PTM-specific mAbs by engineering a motif-specific 'hot spot' into an antibody scaffold. Inspired by a natural phosphate-binding motif, we designed and selected mAb scaffolds with hot spots specific for phosphoserine, phosphothreonine or phosphotyrosine. Crystal structures of the phospho-specific mAbs revealed two distinct modes of phosphoresidue recognition. Our data suggest that each hot spot functions independently of the surrounding scaffold, as phage display antibody libraries using these scaffolds yielded >50 phospho- and target-specific mAbs against 70% of target peptides. Our motif-specific scaffold strategy may provide a general solution for rapid, robust development of anti-PTM mAbs for signaling, diagnostic and therapeutic applications.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Design of phospho-specific mAb scaffold.
Figure 2: Selection and characterization of pSer-, pSer/pThr- and pTyr-specific scaffolds.
Figure 3: X-ray crystal structures of phosphoresidue-binding pockets.
Figure 4: Generation of recombinant phospho-specific (PS) mAbs using the pSAb and pSTAb scaffolds.

Accession codes

Primary accessions

Protein Data Bank


  1. 1

    Cohen, P. The regulation of protein function by multisite phosphorylation—a 25 year update. Trends Biochem. Sci. 25, 596–601 (2000).

    CAS  Article  Google Scholar 

  2. 2

    Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646–674 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Blagoev, B., Ong, S.E., Kratchmarova, I. & Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 22, 1139–1145 (2004).

    CAS  Article  Google Scholar 

  4. 4

    Zhou, H., Watts, J.D. & Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat. Biotechnol. 19, 375–378 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Hornbeck, P.V., Chabra, I., Kornhauser, J.M., Skrzypek, E. & Zhang, B. PhosphoSite: A bioinformatics resource dedicated to physiological protein phosphorylation. Proteomics 4, 1551–1561 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Beausoleil, S.A. et al. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc. Natl. Acad. Sci. USA 101, 12130–12135 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Bendall, S.C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Sachs, K., Perez, O., Pe'er, D., Lauffenburger, D.A. & Nolan, G.P. Causal protein-signaling networks derived from multiparameter single-cell data. Science 308, 523–529 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Brumbaugh, K. et al. Overview of the generation, validation, and application of phosphosite-specific antibodies. Methods Mol. Biol. 717, 3–43 (2011).

    CAS  Article  Google Scholar 

  10. 10

    Dopfer, E.P. et al. Analysis of novel phospho-ITAM specific antibodies in a S2 reconstitution system for TCR-CD3 signalling. Immunol. Lett. 130, 43–50 (2010).

    CAS  Article  Google Scholar 

  11. 11

    DiGiovanna, M.P. & Stern, D.F. Activation state-specific monoclonal antibody detects tyrosine phosphorylated p185neu/erbB-2 in a subset of human breast tumors overexpressing this receptor. Cancer Res. 55, 1946–1955 (1995).

    CAS  PubMed  Google Scholar 

  12. 12

    Nita-Lazar, A., Saito-Benz, H. & White, F.M. Quantitative phosphoproteomics by mass spectrometry: past, present, and future. Proteomics 8, 4433–4443 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Marks, J.D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).

    CAS  Article  Google Scholar 

  14. 14

    McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

    CAS  Article  Google Scholar 

  15. 15

    Kang, A.S., Barbas, C.F., Janda, K.D., Benkovic, S.J. & Lerner, R.A. Linkage of recognition and replication functions by assembling combinatorial antibody Fab libraries along phage surfaces. Proc. Natl. Acad. Sci. USA 88, 4363–4366 (1991).

    CAS  Article  Google Scholar 

  16. 16

    Mersmann, M. et al. Towards proteome scale antibody selections using phage display. New Biotechnol. 27, 118–128 (2010).

    CAS  Article  Google Scholar 

  17. 17

    Sidhu, S.S. et al. Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338, 299–310 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Feldhaus, M.J. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat. Biotechnol. 21, 163–170 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Hanes, J., Schaffitzel, C., Knappik, A. & Pluckthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

    CAS  Article  Google Scholar 

  20. 20

    Cobaugh, C.W., Almagro, J.C., Pogson, M., Iverson, B. & Georgiou, G. Synthetic antibody libraries focused towards peptide ligands. J. Mol. Biol. 378, 622–633 (2008).

    CAS  Article  Google Scholar 

  21. 21

    Shih, H.H. et al. An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition. J. Biol. Chem. 287, 44425–44434 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Vielemeyer, O. et al. Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping. J. Biol. Chem. 284, 20791–20795 (2009).

    CAS  Article  Google Scholar 

  23. 23

    Kaneko, T. et al. Superbinder SH2 domains act as antagonists of cell signaling. Sci. Signal. 5, ra68 (2012).

    Article  Google Scholar 

  24. 24

    Pershad, K., Wypisniak, K. & Kay, B.K. Directed evolution of the forkhead-associated domain to generate anti-phosphospecific reagents by phage display. J. Mol. Biol. 424, 88–103 (2012).

    CAS  Article  Google Scholar 

  25. 25

    Malabarba, M.G. et al. A repertoire library that allows the selection of synthetic SH2s with altered binding specificities. Oncogene 20, 5186–5194 (2001).

    CAS  Article  Google Scholar 

  26. 26

    Clackson, T. & Wells, J.A. A hot spot of binding energy in a hormone-receptor interface. Science 267, 383–386 (1995).

    CAS  Article  Google Scholar 

  27. 27

    Bogan, A.A. & Thorn, K.S. Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1–9 (1998).

    CAS  Article  Google Scholar 

  28. 28

    Watson, J.D. & Milner-White, E.J. A novel main-chain anion-binding site in proteins: the nest. A particular combination of phi,psi values in successive residues gives rise to anion-binding sites that occur commonly and are found often at functionally important regions. J. Mol. Biol. 315, 171–182 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Landry, R.C. et al. Antibody recognition of a conformational epitope in a peptide antigen: Fv-peptide complex of an antibody fragment specific for the mutant EGF receptor, EGFRvIII. J. Mol. Biol. 308, 883–893 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Hollingsworth, S.A. & Karplus, P.A. A fresh look at the Ramachandran plot and the occurrence of standard structures in proteins. Biomol Concepts 1, 271–283 (2010).

    CAS  Article  Google Scholar 

  31. 31

    North, B., Lehmann, A. & Dunbrack, R.L. Jr. A new clustering of antibody CDR loop conformations. J. Mol. Biol. 406, 228–256 (2011).

    CAS  Article  Google Scholar 

  32. 32

    Alving, C.R. Antibodies to liposomes, phospholipids and phosphate esters. Chem. Phys. Lipids 40, 303–314 (1986).

    CAS  Article  Google Scholar 

  33. 33

    Levine, J.S., Branch, D.W. & Rauch, J. The antiphospholipid syndrome. N. Engl. J. Med. 346, 752–763 (2002).

    CAS  Article  Google Scholar 

  34. 34

    Yaffe, M.B. & Smerdon, S.J. PhosphoSerine/threonine binding domains: you can't pSERious? Structure 9, R33–R38 (2001).

    CAS  Article  Google Scholar 

  35. 35

    Kaneko, T., Joshi, R., Feller, S.M. & Li, S.S. Phosphotyrosine recognition domains: the typical, the atypical and the versatile. Cell Commun. Signal. 10, 32 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Seet, B.T., Dikic, I., Zhou, M.M. & Pawson, T. Reading protein modifications with interaction domains. Nat. Rev. Mol. Cell Biol. 7, 473–483 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985).

    CAS  Article  Google Scholar 

  38. 38

    Bostrom, J. et al. Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610–1614 (2009).

    CAS  Article  Google Scholar 

  39. 39

    Rondot, S., Koch, J., Breitling, F. & Dubel, S. A helper phage to improve single-chain antibody presentation in phage display. Nat. Biotechnol. 19, 75–78 (2001).

    CAS  Article  Google Scholar 

  40. 40

    Thomsen, N.D., Koerber, J.T. & Wells, J.A. Structural snapshots reveal distinct mechanisms of procaspase-3 and -7 activation. Proc. Natl. Acad. Sci. USA 110, 8477–8482 (2013).

    CAS  Article  Google Scholar 

  41. 41

    Luft, J.R. & DeTitta, G.T. A method to produce microseed stock for use in the crystallization of biological macromolecules. Acta Crystallogr. D Biol. Crystallogr. 55, 988–993 (1999).

    CAS  Article  Google Scholar 

  42. 42

    Holton, J. & Alber, T. Automated protein crystal structure determination using ELVES. Proc. Natl. Acad. Sci. USA 101, 1537–1542 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  Article  Google Scholar 

  44. 44

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  45. 45

    Kaufmann, B. et al. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. Proc. Natl. Acad. Sci. USA 107, 18950–18955 (2010).

    CAS  Article  Google Scholar 

  46. 46

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  47. 47

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  Article  Google Scholar 

  48. 48

    Baker, N.A., Sept, D., Joseph, S., Holst, M.J. & McCammon, J.A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

    CAS  Article  Google Scholar 

  49. 49

    Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  Article  Google Scholar 

Download references


We thank members of the Wells laboratory for helpful discussions regarding this manuscript and S. Pfaff for assistance with Biacore experiments. We thank C. Waddling at the UCSF X-ray facility for assistance with generating protein crystals and J. Holton, G. Meigs and J. Tanamachi at the Advanced Light Source beam line 8.3.1 at the Lawrence Berkeley National Laboratory for help with collection of diffraction data. We thank the Court laboratory at the National Institutes of Health for generously providing the recombineering vectors. J.T.K. is a Fellow of the Life Sciences Research Foundation and N.D.T. is the Suzanne and Bob Wright Fellow of the Damon Runyon Cancer Research Foundation. This work was supported by grants from the US National Institutes of Heath (R01 CA154802 to J.A.W. and GM54616 to W.F.D.). J.T.K., J.A.W. and W.F.D. have filed a provisional patent on the technology described in this manuscript.

Author information




J.T.K. designed and executed experiments; N.D.T. assisted with crystallography experiments; B.T.H. and W.F.D. assisted with modeling experiments; J.A.W. designed and supervised experiments. J.T.K. and J.A.W. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to James A Wells.

Ethics declarations

Competing interests

J.T.K., W.F.D. and J.A.W. have filed a provisional patent regarding the technology described in this manuscript.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Tables 1–7 (PDF 5190 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Koerber, J., Thomsen, N., Hannigan, B. et al. Nature-inspired design of motif-specific antibody scaffolds. Nat Biotechnol 31, 916–921 (2013).

Download citation

Further reading