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Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization

Nature Biotechnology volume 28, pages 1195–1202 (2010)Cite this article

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Abstract

Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ∼10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.

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Figure 1: A spatially addressed antibody library for discovery and optimization.
Figure 2: Screening and identifying hits using ECL detection.
Figure 3: Affinity maturation of F0002 binding to DLL4.
Figure 4: Epitope mapping the anti-DLL4 Fabs binding sites on the DLL4 extracellular domain.
Figure 5: Assays of anti-DLL4 antibodies in cell-based assays.

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Acknowledgements

We thank D. Myszka from Biosensor Tools for measuring binding affinities of our Fabs using surface plasmon resonance, R. Lerner for comments on the manuscript, P. Schultz and W. Huse for useful discussions and M. Sandburg and Y. Jiang from Wintherix for their help with the FACS instrument.

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  1. Byeong Doo Song

    Present address: Present address: Scripps Korea Antibody Institute, Chuncheon-si, Korea.,

Authors and Affiliations

  1. Fabrus LLC, La Jolla, California, USA

    Hongyuan Mao, James J Graziano, Tyson M A Chase, Cornelia A Bentley, Omar A Bazirgan, Neil P Reddy, Byeong Doo Song & Vaughn V Smider

  2. The Scripps Research Institute, La Jolla, California, USA

    Vaughn V Smider

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Contributions

H.M. designed and constructed the plasmid vectors for heavy and light chain library; performed Fab library generation; designed and executed affinity maturations, including alanine-scanning mutagenesis, NNK mutagenesis and cassette mutagenesis, of F0001 and F0002; performed a small subset of library screening using ECL; conducted ECL based epitope mapping competition assay. H.M. also designed the cloning strategy for constructing the VH3-23 library with all possible germline D-J combinations, together with T.M.A.C., J.J.G., C.A.B., O.A.B. and N.P.R. made the VH3-23 library. J.J.G. performed the automated expression and purification on Piccolo. J.J.G. and V.V.S. designed the software for generating the V(D)J recombinant sequences and selecting the representative sequences for gene synthesis. T.M.A.C. performed the majority of the library screening using ECL and performed ELISA on the affinity matured Fabs with DLL4. C.A.B. performed the Fab binding assays on CHO-DLL4 cells using FACS and executed inhibition assays of NOTCH1-DLL4 interaction using ELISA and FACS. C.A.B. and O.A.B. designed and performed the Luciferase reporter assays on inhibition of NOTCH1-DLL4 interaction. N.P.R. made all DLL4 extracellular domain constructs and executed the epitope mapping with western blots, generated CHO-DLL4 cell line and helped H.M. for Fab library transformation. B.D.S. also supervised the construction of DLL4 extracellular domains. O.A.B. made the NOTCH1 reporter plasmid (p6xCBF), which was modified from an earlier reporter plasmid (p4XCBF) made by B.D.S. O.A.B. also constructed the full length IgG eukaryotic expression vectors and expressed and purified the IgGs. V.V.S. conceptualized the spatially addressed antibody library and oversaw the concept development at Fabrus. V.V.S. and H.M. wrote the manuscript. All authors discussed and commented on the manuscript.

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Correspondence to Vaughn V Smider.

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The authors own equity interests in Fabrus LLC.

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Mao, H., Graziano, J., Chase, T. et al. Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization. Nat Biotechnol 28, 1195–1202 (2010). https://doi.org/10.1038/nbt.1694

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