Abstract
Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.
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Data availability
Crystal structures have been deposited in Protein Databank (PDB) with the PDB accession codes 7RP2 (https://doi.org/10.2210/pdb7rp2/pdb), 7RP3 (https://doi.org/10.2210/pdb7rp3/pdb), 7RP4 (https://doi.org/10.2210/pdb7rp4/pdb) and 7MDP (https://doi.org/10.2210/pdb7mdp/pdb). Source data are provided with this paper. Antibodies described in this paper are available via material transfer agreement with Genentech. Restrictions on data availability: chemical structures from the fragment library used in Fig. 6b. Structures for all fragment hits cannot be disclosed because they are proprietary.
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Acknowledgements
We thank S. Malek, J. Kiefer, B. St. Onge and A. Shaw for helpful discussions, N. Skeleton for design of the fragment library and the staff at ALS and SSRL for their assistance with data collection.
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Authors and Affiliations
Contributions
Project conceptualization was carried out by M.M.M., J.T.K., W.W. and M.E. Protein production and labeling were performed by A.J.O. and C.L. Antibody selection, SPR and production were performed and overseen by C.W.D. and J.T.K. Cell-based experiments were carried out by R.M., Y.X. and S.F. Advice on, and synthesis of, small-molecule inhibitors were the responsibility of J.R., S.M., S.D., L.G. and H.E.P. Construct generation was performed by Y.F. In vivo tumor studies were conducted by E.C., V.A. and M.M. Immunohistochemical analysis was overseen by S.J. and H.K. SPR with small molecules was performed by M.S., J.M.B., S.C.U., J.D., A.F., M.M.M. and J.G.Q. Crystallography was carried out by A.J.O. and W.W. Structural analysis was performed by W.W. Manuscript writing was undertaken by C.W.D., M.S., M.M.M., J.T.K., W.W. and M.E., M.M.M., J.T.K., W.W. and M.E. are cosenior authors.
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Extended data
Extended Data Fig. 1 Additional characterization of CLAMPs.
(a) Structures of KRASG12C covalent small molecules. (b) Representative SPR traces of 1A5 and 2H11 CLAMPs against KRASG12C-GDP+GNE-1952 and KRASG12C-GDP. (c) Epitope binning results indicate that group 1 and group 2 antibodies bind two overlapping epitopes.
Extended Data Fig. 2 Cell-based assays with 1A5 CLAMP.
(a) No observable KRAS staining by 1A5 CLAMP in HCT116 KRASG13D cells treated with a KRASG12C inhibitor GNE-1952. Scale bar: 200 μM. Results are representative of 2 independent experiments. (b) Immunoblot analysis for covalently modified KRAS and KRAS pathway markers (pERK, pMEK, and pS6) in a bulk population of HCC1171 KRASG12C mutant cancer cells. Washout experiments reveals that KRASG12C covalent modification is maintained for 24 hours after removal of ARS-853. Scale bar: 200 μM. Results are representative of 2 independent experiments. (c) Gating strategy for FACs experiments in Fig. 2e.
Extended Data Fig. 3 Biophysical and structural analysis of 2H11 CLAMP.
(a) Structural alignment of 2H11 CLAMP bound to KRASG12C-GDP and DCAI ligand bound to KRAS (PDB 4DST). KRASG12C is depicted as white ribbons with SWII highlighted in blue. 2H11 CLAMP heavy chain is colored in green, and Trp99 rendered in sticks. DCAI bound KRASG12D is colored in red, with DCAI shown rendered in sticks. DCAI and Trp99 bind to the same site on KRAS. (b) Structural alignment of KRASG12C-GDP/GNE-1952 with and without 2H11 CLAMP bound. KRASG12C/GNE-1952 without 2H11 CLAMP is colored in cyan. KRASG12C/GNE-1952 with 2H11 CLAMP is colored in orange, compound is colored in yellow, and 2H11 CLAMP heavy chain is colored in green. (c) Structural comparison between iDab (orange, PDB 2UZI) and 2H11 CLAMP (green) in SWI (teal) binding suggest less sensitivity to different nucleotide states for 2H11 CLAMP.
Extended Data Fig. 4 2H11 CLAMP enables the discovery of weak affinity SWII ligands.
(a) 2H11 CLAMP enhances the binding of SWII pocket ligands to KRASWT and KRASG12C. KRAS was immobilized on an SPR chip and small molecule was injected at concentrations ranging from 1 to 50 μM in the absence or presence of 2H11 CLAMP. (b) Unbiased omit map, 2Fo-Fc contoured at 1σ of GNE-2897 bound in the SWII region of KRASG12C + 2H11 CLAMP Fab.
Supplementary information
Supplementary Information
Supplementary Tables 1–3 and methods.
Source data
Source Data Fig. 1
Unprocessed immunoblot.
Source Data Fig. 2
Statistical source data.
Source Data Fig. 3
Unprocessed immunoblot.
Source Data Fig. 4
Statistical source data.
Source Data Fig. 5
Statistical source data.
Source Data Fig. 6
Statistical source data.
Source Data Extended Data Fig. 2
Unprocessed immunoblot.
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Davies, C.W., Oh, A.J., Mroue, R. et al. Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors. Nat Biotechnol 40, 769–778 (2022). https://doi.org/10.1038/s41587-021-01126-9
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DOI: https://doi.org/10.1038/s41587-021-01126-9
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