Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors

Abstract

The discovery of covalent inhibitors binding the switch II (SWII) pocket has enabled therapeutic intervention in KRAS G12C driven tumors and represents a milestone in targeting KRAS-driven cancers. However, the transient nature and high energetic barrier required for binding this pocket has been an obstacle in successfully targeting other KRAS mutant oncoproteins. We report the discovery of KRAS Conformation Locking Antibodies for Molecular Probe discovery (CLAMP)s that specifically recognize the unique conformation of KRAS G12C induced 5 by covalent inhibitors. KRAS CLAMPs enable single cell resolution of covalent inhibitor-bound KRAS G12C in cells and in vivo tumor models, providing a biomarker for direct target engagement of KRAS G12C inhibition. KRAS CLAMPs bind multiple KRAS mutants and stabilize an open conformation of the SWII pocket increasing the affinity of weak non-covalent SWII pocket ligands. This work provides new insights into KRAS G12C upon treatment with covalent inhibitors and offers a path towards targeting the SWII pocket in other RAS mutants. 10 3 Main: RAS proteins are small, membrane-bound guanine nucleotide-binding proteins encoded by three genes (HRAS, NRAS and KRAS). RAS proteins act as molecular switches by cycling between active GTP-bound and inactive GDP-bound conformations 1 . The active GTP-bound conformation allows RAS to signal to a diverse set of downstream effectors including RAF, PI3K, and RAL GDS 2-11 and oncogenic mutations in RAS, frequently at position 12, reduce GTP hydrolysis resulting in constitutively active RAS signaling [12][13][14][15] . The picomolar affinity for GTP or GDP, in addition to the lack of obvious pockets for small molecule binding in RAS have hampered drug discovery efforts against oncogenic mutant RAS for several decades.The landmark discovery of KRAS G12C inhibitors that covalently modify the mutant Cys12 residue has provided a novel and promising opportunity for drugging KRAS G12C mutant tumors 16 . Compound 12, ARS-853, ARS-1620, AIM-4, and clinical molecules AMG 510 and MRTX849 bind and stabilize an "open" conformation in the switch II (SWII) region not previously observed in KRAS-GDP or KRAS-GTP [16][17][18][19][20][21][22][23] . The mechanism of action of such SWII pocket covalent binders is through stabilization of this transient pocket via initial binding to the pocket followed by chemical reaction with Cys12 17 . This modification irreversibly locks KRAS G12C in a GDP-bound inactive state by preventing intrinsic or SOS-mediated exchange, causes tumor growth inhibition in pre-clinical models, and is showing promising clinical activity 17,18 . Discovery of these KRAS G12C inhibitors relied heavily on the covalent reactivity with Cys12 to inhibit KRAS G12C protein 24 . Thus, the viability of strategies targeting this pocket in other KRAS mutants lacking this critical mutant cysteine residue remains to be determined.

Competitive, connective and allosteric Nanobodies that modulate the SOS1•RAS protein-protein interactions and tune the nucleotide exchange rate as a starting point for drug design

“…Remarkably, this RAS-specific Nb further inhibits the intrinsic nucleotide exchange of RAS, locking it in a fully inactive conformation. We hypothesize that the interactions of Nb84 with the switch-1 loop of RAS prevent the conformational changes required to exchange the nucleotide, very similar to the conformation-locking antibodies discovered by the Evangelista lab 15 .…”

“…PPI-stabilizing Nanobodies to facilitate precision drug discovery. Because many Nbs act as structural chaperones that stabilize specific conformations or interactions, they facilitate small molecule/fragment library screenings by enhancing the binding of (weak) ligands that bind the same conformer 15,25,26 . Previously, a series of indolo-4-aminopiperidines was previously identified as compounds that binds the SOS1•RAS interface and elicits the acceleration of SOS1-catalyzed nucleotide exchange 8 .…”

Fragment Optimization of Reversible Binding to the Switch II Pocket on KRAS Leads to a Potent, In Vivo Active KRASG12C Inhibitor

“…Together, these data suggested that it would be feasible to design a covalent KRAS G12C inhibitor from the non-covalent binders discovered in this study. In contrast, a previously described fragment (GNE-2897, 13 ) and also the switch II pocket binding core groups of two reported KRAS G12C inhibitors ( 14 , 15 ) did not display tight binding to KRAS without their covalent warheads. We hypothesized that the use of a KRAS binding core with native affinity to the protein should enable the discovery of a covalent inhibitor with greater selectivity against other cysteines and therefore lower toxicity as well as a higher second-order rate constant and improved KRAS G12C inhibition, resulting in a stronger antiproliferative effect.…”

Assessment of KRAS G12C Target Engagement by a Covalent Inhibitor in Tumor Biopsies Using an Ultra-Sensitive Immunoaffinity 2D-LC–MS/MS Approach

“…After homogenization of the core needle biopsy, an aliquot of the lysate with 20 μg of total protein was incubated with anti-RAS antibody clone RAS10, which was pre-immobilized to Protein A magnetic beads. RAS10 has been shown to possess comparable affinity to KRAS G12C with or without covalent modification by immunoprecipitation . After on-bead tryptic digestion, the concentrations of both free and GDC-6036-bound KRAS G12C were determined with 4 μg of total protein using an ultra-sensitive targeted 2D-LC–MS/MS technology, which enables up to 100-fold increase in sensitivity compared to the conventional LC–MS/MS method .…”

“…RAS10 has been shown to possess comparable affinity to KRAS G12C with or without covalent modification by immunoprecipitation. 13 After on-bead tryptic digestion, the concentrations of both free and GDC-6036bound KRAS G12C were determined with 4 μg of total protein using an ultra-sensitive targeted 2D-LC−MS/MS technology, which enables up to 100-fold increase in sensitivity compared to the conventional LC−MS/MS method. 12 KRAS G12C engagement was calculated as the ratio of GDC-6036-bound KRAS G12C/ (free KRAS G12C + GDC-6036-bound KRAS G12C).…”