Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface


Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain–light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.

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Figure 1: Design of an orthogonal CH1-CL interface.
Figure 2: Schematic diagrams, models and X-ray structures of designs VRD1 and VRD2.
Figure 3: Demonstration of the specificity afforded by the heavy chain–light chain interface designed mutants and the dual-binding activity of the resulting BsAb molecules.
Figure 4: Function of IgG BsAbs.

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This work was supported by the Lilly Research Laboratories and the Lilly Research Award Program (LRAP). We thank J. Hannah and B. Gutierrez for their help with transient transfection, R. Yuan and D. He for assistance with protein purification, M. Batt and J. Fitchett for their training and up keep of the mass spectrometry facility at Lilly. B. Stranges provided suggestions for the computational docking protocol. Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Argonne National Laboratory, was supported by the US DOE under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility. We thank S. Wasserman, S. Sojitra, J. Koss for data collection and operation of the beamline.

Author information

All authors contributed to the concepts of the study. S.M.L. and B.K. performed the computational design, with advice from A.L.-F. B.K., A.K.C., S.M.T., S.M.L., S.J.D. and X.W. contributed to the rational designs. X.W., A.S., H.L.R., E.M.C., E.M.S., G.G., C.H., F.H., C.H.-E. and S.J.D. performed the experimental work. A.P. and S.A. performed the crystallization and structure determinations. B.K. and S.J.D. conceived the project. Writing of the paper was done in close collaboration by B.K., S.A., S.M.L. and S.J.D.

Correspondence to Brian Kuhlman or Stephen J Demarest.

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X.W., A.P., A.S., F.H., E.M.S., C.H., A.K.C., S.T., S.A. and S.J.D. are employees of Eli Lilly

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Supplementary Figures 1–10, Supplementary Tables 1–5 and Supplementary Protocols 1–3 (PDF 10445 kb)

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Lewis, S., Wu, X., Pustilnik, A. et al. Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nat Biotechnol 32, 191–198 (2014).

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