Nature-inspired design of motif-specific antibody scaffolds

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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Correspondence to James A Wells.

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

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Koerber, J., Thomsen, N., Hannigan, B. et al. Nature-inspired design of motif-specific antibody scaffolds. Nat Biotechnol 31, 916–921 (2013). https://doi.org/10.1038/nbt.2672

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