CLAMPs allow single cell tracking of KRASG12C inhibition and endow druggability to KRAS mutants

: 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.

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 5 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 10 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 15 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.
Despite these successes, the consequence of covalent inactivation of KRAS G12C has not been investigated due to the lack of reagents capable of visualizing endogenous KRAS G12C covalent modification in tumor cells. Such reagents could be powerful tools to investigate the dynamics and homogeneity of KRAS G12C covalent modification 20 upon inhibitor treatment in individual cell populations, which may shed light on how tumors cells acquire resistance to KRAS G12C inhibitors.
Synthetic monoclonal antibodies (mAbs) represent an emerging tool to stabilize unique protein conformations and the ability to combine in vitro selections with conformationally-locked targets has enabled the discovery of novel conformational sensors against RAS, caspases, and GPCRs 25-28 . These antibodies have 25 tremendous potential to elucidate the biological role of protein conformations in cells. They may also have utility in drug discovery efforts by serving as structural chaperones to improve structure-based drug design and/or by stabilizing rare protein conformations to increase the success of lead finding efforts.
Here we describe an antibody platform we refer to as Conformation Locking Antibodies for Molecular Probe discovery (CLAMP)s. We applied this platform using covalently modified KRAS G12C and discovered two 30 classes of CLAMPs. Class I CLAMPs specifically recognize a conformational state in KRAS G12C associated with covalent ligand bound to the SWII pocket. One such CLAMP (1A5) enables detection of covalently modified KRAS G12C within individual cancer cells and tumors, providing a direct biomarker for KRAS G12C -inhibitor mediated target engagement, and can be coupled with RAS pathway markers to assess pathway inhibition and subsequent rebound at the cellular level. Class II CLAMPs also exhibit affinity for KRAS G12C -GDP alone and stabilize an open 35 conformation of the SWII pocket in the absence of ligand. This feature significantly improves the affinity of multiple non-covalent inhibitors for KRAS G12C and KRAS WT . Intriguingly, one class II CLAMP (2H11), bound multiple KRAS mutants including KRAS G12V -GDP, KRAS G12R -GDP, and KRAS Q61H -GDP. We propose that KRAS CLAMPs are an important biology and drug discovery tool for investigating KRAS G12C covalent modification in vivo and will provide a unique platform for identifying novel chemical matter targeting the KRAS SWII pocket. We envisage that general application of CLAMPs may enable identification of ligands for otherwise intractable drug targets.

Discovery and characterization of KRAS SWII pocket CLAMPs
We developed a method to identify KRAS CLAMPs using an in vitro selection strategy with synthetic antibody libraries. We applied this method to KRAS, which exhibits conformational heterogeneity in its switch I (SWI) and SWII regions, to identify antibodies that would stabilize an "open" SWII pocket conformation 10 compatible with that induced by KRAS G12C covalent inhibitors (Fig. 1A). To identify KRAS CLAMPs, we the open SWII pocket conformation in the absence of inhibitor, we also performed three rounds of selection with KRAS G12C -GDP+GNE-1952 followed by a fourth round with KRAS G12C -GDP. After phage enzyme-linked immunosorbent assay (ELISA) screens to confirm specificity, we generated IgGs for eleven unique clones and 20 characterized their binding specificity by ELISA (Fig. 1C) and surface plasmon resonance (SPR) (Fig. 1D, Fig. S1B, and Table S1).
The eleven antibodies were assayed for their ability to recognize covalently modified KRAS G12C with additional KRAS G12C covalent molecules ARS-853 and ARS-1620 17,19 (Fig. S1A) to filter out CLAMPs that only recognize GNE-1952 bound KRAS G12C -GDP. All antibodies showed affinity to KRAS G12C -GDP covalently 25 modified by GNE-1952, ranging from ~1-139 nM (Table S1). However, one group of antibodies (five clones) was specific for the GNE-1952-bound KRAS G12C -GDP conformation while a second group of antibodies (six clones) broadly recognized modified KRAS G12C -GDP (GNE-1952, ARS-853, and ARS-1620 bound KRAS G12C -GDP conformations) and were referred to as KRAS CLAMPs since they appeared to be conformation rather than chemotype specific. Epitope mapping analysis revealed that these two groups bound distinct but partially 30 overlapping epitopes on GNE-1952-bound KRAS G12C -GDP (Fig. S1C).
The six KRAS CLAMPs could be further divided into two classes: the Class I CLAMP, 1A5, had high specificity for covalently bound KRAS G12C -GDP with >100-fold improved affinity for this target compared to KRAS G12C -GDP, whereas the Class II CLAMPs, (1E5, 2H11, 2A3, 3A12, and 4G12) recognized KRAS G12C -GDP independent of inhibitor presence, as measured by ELISA and SPR (Fig. 1C, Fig. 1D and Table S1). To further 35 establish the antibody specificity in cells, we performed an immunoprecipitation experiment with 1A5 and 2H11 on lysates from HCC1171 KRAS G12C mutant cells treated with ARS-1620 or DMSO control. We found that both Class I and II CLAMPs specifically immunoprecipitated covalently bound KRAS G12C -GDP but not KRAS G12C -GDP alone ( Fig. 1E) suggesting that the affinity of the CLAMPs was insufficient to bind to KRAS G12C -GDP or KRAS G12C -GTP in cells. To further explore the potential of these antibodies, we focused on representative class I (1A5) and class II (2H11) antibodies.

Tracking KRAS G12C covalent modification in cells
Since the 1A5 CLAMP showed the highest specificity for covalently modified KRAS G12C -GDP compared to KRAS G12C -GDP, we tested whether the 1A5 CLAMP could specifically detect inhibitor-bound KRAS G12C in cells.
Immunofluorescence (IF) staining with the 1A5 CLAMP resulted in detection of covalently modified KRAS G12C in HCC1171 KRAS G12C -mutant cells treated with a variety of KRAS G12C covalent inhibitors, including GNE-1952, 10 ARS-853, ARS-1620, and AMG 510 ( Fig. 2A, 2B), thus confirming the ability of 1A5 to recognize a common conformation induced by various KRAS G12C covalent inhibitors. In contrast, there was no detectable 1A5 CLAMP staining in HCC1171 KRAS G12C -mutant cells treated with DMSO or in HCT116 KRAS G13D -mutant cells treated with GNE-1952 (Fig. S2A). The intensity of 1A5 CLAMP staining in ARS-1620-treated HCC1171 cells appeared to be dose-and time-dependent (Fig. 2B, 2C) and was consistent with results from immunoblotting for covalently 15 inhibited KRAS G12C (as measured both by an upward electrophoretic mobility shift of the KRAS G12C protein band migration) and by inhibition of RAS pathway markers pERK and pMEK in a bulk population of cells (Fig. S2B, lane 1, 2). Studies by IF revealed that ARS-1620-induced covalent modification of KRAS G12C in cells occurred in a fairly synchronous fashion occurring at both the plasma membrane as well as in punctate compartments of cells ( Fig. 2A-C). Since antibodies specific for RAS-GDP do not exist, staining with the 1A5 CLAMP likely provides 20 information on the localization of KRAS G12C -GDP in cells upon or shortly after covalent modification. Moreover, the uniformity and kinetics of 1A5 staining indicates that covalent modification of KRAS G12C may occur independent of cell cycle stage. Since covalent modification of KRAS G12C -GDP is dependent on intrinsic GTP hydrolysis (16-17), these findings additionally suggest that intrinsic KRAS G12C -GTP hydrolysis is also not cell cycle dependent (Fig. 2B, 2C). The 1A5 CLAMP was also used to visualize covalent modification of KRAS G12C -GDP by 25 ARS-1620 in a number of KRAS G12C lines expressing very low levels of KRAS G12C protein ( Fig. 2D) highlighting the sensitivity of 1A5 in detecting covalently modified KRAS G12C -GDP.
We next evaluated the utility of combining the 1A5 CLAMP and pS6 antibody to simultaneously measure KRAS G12C covalent modification and RAS signaling in individual cells using flow cytometry. Similar to the IF studies, the 1A5 CLAMP specifically detected covalent modification of KRAS G12C -GDP in a dose-dependent 30 manner (Fig. 2E). This staining correlated with an observed dose-dependent decrease in pS6 levels in most of the population (Fig. 2E). Interestingly, a small population (~1%) of 1A5 + cells remained high for pS6 suggesting this population of cells maintain RAS signaling despite covalent modification of KRAS G12C .
We also surveyed a set of commercially available antibodies to KRAS to determine their conformational specificity. We identified two antibodies (Abcam and Ras10) that had comparable affinity to KRAS G12C , with or 35 without covalent modification, by immunoprecipitation and ELISA ( Fig. 3A and 3B), indicating that these antibodies are not specific to the conformation induced by covalent modification. In contrast, the iDab antibody, 6 which was reported to be highly specific for HRAS-GTP, showed little to no binding to covalently modified KRAS G12C -GDP, but bound to both KRAS G12C -GDP and KRAS G12C -GMPPCP by ELISA ( Fig. 3B and data not shown), and in immunoprecipitation studies (Fig. 3A) 27 . Since the iDab antibody binds an epitope that spans both the SWI and SWII regions, the SWII conformation induced by covalent modification of KRAS G12C -GDP likely prevents iDab binding.

5
Given the complementary specificities of the 1A5 CLAMP and iDab, we wondered if the 1A5 CLAMP and iDab antibodies could be used in combination to co-stain and visualize both unmodified and covalently modified KRAS G12C within the same cell. We conducted IF experiments with both 1A5 CLAMP and iDab antibodies on HCC1171 KRAS G12C cells treated with a dose titration of ARS-1620. Similar to previous IF experiments with 1A5, we detected an increase in 1A5 staining that correlated with high concentrations of ARS-1620 treatment.
10 Furthermore, the increased 1A5 staining coincided with decreased staining with the iDab antibody, confirming that these two antibodies stain different KRAS G12C conformations (Fig. 3C). Co-staining with both antibodies allowed us to also monitor the re-synthesis of KRAS G12C . Treatment of KRAS G12C cells with ARS-1620 or ARS-853 for 16 hours resulted in almost complete covalent modification of KRAS G12C based on the appearance of 1A5 and disappearance of iDab staining respectively by IF and was consistent with an electromobility shift of KRAS based 15 on immunoblot analysis ( Fig. 3D and Fig. S2B). Upon drug washout, the appearance of unmodified KRAS G12C was apparent as early as 6 hours and was increased at 24 hours as evident by immunoblot analysis (Fig. S2B). Similar to immunoblot analysis, 24 hour drug washout studies also showed a significant decrease in 1A5 staining and an increase in iDab antibody staining by IF (Fig. 3D).
Tumor cell adaptation to ARS-1620 has been attributed to new synthesis of KRAS G12C protein not bound to 20 drug and subsequent reactivation of pERK, a marker of RAS pathway activity 30 . As such we investigated whether the 1A5 staining signal decreased over time due to new synthesis of KRASG12C that remains unmodified, and whether that decrease correlated with the appearance of a pERK signal. We first treated NCI-H358 KRAS G12C mutant cells with 1 µM ARS-1620 for various time points (4, 24, 48, 72 and 96 hours) to assess both target occupancy, and pERK inhibition and potential rebound. In cells treated with 100 nM and 1 µM ARS-1620, we 25 observed an increase of alkylated KRAS G12C evident by the enhanced intensity of 1A5 signal within the first 48 hours, followed by a drop between 72 to 96 hours, suggesting accumulation of unstained unmodified KRASG12C.
Correspondingly, pERK intensity demonstrated a quick inhibition as early as 4 hours followed by a sharp rebound after 72 hrs suggesting that the rebound of pERK is likely due to insufficient target occupancy by ARS-1620. We then treated cells with 300nM MRTX849 (a sufficiently more potent KRAS G12C covalent inhibitors) at the clinically 30 relevant dose. Unlike ARS-1620, treatment with MRTX849 resulted in a long-lasting increase in 1A5 signal intensity, which correlated with sustained suppression of pERK signaling 96 hours post-treatment. This study not only demonstrates that potent KRAS G12C covalent inhibitors overcome pERK rebound in KRAS G12C mutant tumors but also serves as proof-of-concept for using CLAMPS to study long-term drug dynamics and acquired resistance to therapy.

7
We next sought to determine if the 1A5 CLAMP could detect covalently modified KRAS G12C in human tumor xenograft experiments. Detection of covalently inhibited KRAS G12C with the 1A5 CLAMP by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) tissue was not possible, likely due to the harsh formalin treatment that destroys the conformational epitope recognized by 1A5. However, ARS-1620 covalently modified KRAS G12C was readily detectable by IHC in tumor samples prepared as unfixed fresh frozen 5 (FP) tissues (Fig. 4A). In addition, there appeared to be a subtle trend towards stronger staining in samples treated with 200 mg/kg ARS-1620 compared to samples treated with 50 mg/kg ARS-1620 (Fig. 4A). Tumors expressing lower amounts of KRAS G12C covalently modified by ARS-1620 were also detected by the 1A5 CLAMP (Fig. 4B).
Similar to in vitro cell experiments, the 1A5 CLAMP detected KRAS G12C that was covalently modified by ARS-1620 in FACS experiments with ex-vivo tumor samples and could be combined with the pS6 (Fig. 4C). Thus, these 10 results show that the 1A5 CLAMP enables measurement of direct target engagement of KRAS G12C inhibitors in KRAS G12C tumor samples and enables single cell analysis with markers of RAS pathway activation.

KRAS CLAMPs improve the non-covalent affinity of SWII pocket ligands to KRAS G12C and KRAS WT
We hypothesized that the 2H11 class II CLAMP might stabilize the open conformation of the SWII pocket 15 in KRAS G12C -GDP based on its ability to recognize both the unbound and covalently modified conformation of KRAS G12C . Such CLAMPs would then be predicted to improve the weak non-covalent affinity for ligands that bind in the SWII pocket. To test this possibility, we developed an SPR assay to specifically detect binding to the SWII pocket using a SWII-blocked reference (see Materials and Methods). We measured the affinity of various SWII Additionally, we also included KRAS WT -GDP to determine if 2H11 could stabilize the SWII pocket in other KRAS variants. Impressively, we found that inhibitor affinity was greatly enhanced in the presence of the 2H11 CLAMP

Structural analysis of CLAMP:KRAS G12C complex
We determined the crystal structure of KRAS G12C -GDP in complex with 2H11 fragment antigen-binding (Fab) at 2.2Å resolution (Fig. 5B, upper panel) to gain further insights into the mode of action of the 2H11 CLAMP.

30
Rather than binding to the KRAS G12C SWII pocket, the 2H11 Fab contacts the outer surface of the SWII region to stabilize an open conformation of the pocket in an allosteric manner. The 2H11 Fab binding buries ~745 Å 2 of total surface area and contacts residues from SWI, SWII and the center core -sheet (Fig. 5B, lower panel). The 2H11 Fab complementarity-determining regions (CDRs) H1 and H3 contribute to the majority of the direct contacts with KRAS G12C (Fig. 5C). The long 13-residue CDR H3 loop directly engages the SWII region by inserting Trp99 into a 35 small hydrophobic pocket, known as the DCAI pocket 31,32 and is surrounded by KRAS G12C residues Lys5, Leu6, Val7, Ser39, Asp54, Leu56, Tyr71, Thr74, Gly75 (Fig. 5B). Interestingly, 2H11 exploits this site with a chemically similar tryptophan side chain (Fig. S3B). CDR H1 contacts KRAS near the C-terminal end of SWI region and packs against a portion of the RAS-binding domain (RBD) binding site. Unlike iDab (26), 2H11 makes minimal contact with the residues in the N-terminal half of SWI (Fig. S3D), consistent with its observed ability to bind to both KRAS-GDP and GTP states. CDR L2 and H2 also contribute several van der Waals contacts with KRAS. CDR L2 contacts the N-terminal tip of the SWII helix, providing additional stabilization to the SWII loop but without overly 5 restricting the SWII conformation. As shown in Figure 5D, the most flexible part of SWII, Gln60-Ala66, is completely free from direct contact with 2H11, maintaining flexibility in the pocket. Importantly, the SWII pocket lies in a stable open conformation that resembles the shape of previously published ligand bound KRAS structures suggesting easy accessibility for ligand binding 23 .
We then determined the crystal structure of 2H11 in complex with KRAS G12C covalently modified by GNE-

Application of CLAMPs to other KRAS mutants
Due to the dynamic nature of the SWII pocket, all previous efforts to discover novel compounds that bind 25 this region have required the use of electrophilic functional groups that can form covalent bonds with Cys12.
However, most compounds in screening libraries typically do not carry chemically reactive groups and thus, such libraries would be of limited use for screening against KRAS G12C. Based on our combined biochemical and structural results, we hypothesized that the 2H11 CLAMP could provide a novel tool for the discovery of new ligands by enabling screens against the KRAS G12C -2H11 complex in which the SWII pocket lies in an open 30 conformation. Furthermore, the lack of convenient reactive residues in other oncogenic KRAS mutants has prevented the targeting of this region more generally. To explore whether 2H11 could also recognize the GDP bound states and potentially stabilize the open conformation of the SWII region in these mutants, we evaluated binding of 2H11 to a panel of KRAS mutants by ELISA (Fig. 5E). Quite strikingly, 2H11 exhibited strong binding to KRAS G12V -GDP, KRAS G12R -GDP, and KRAS Q61H -GDP, and much weaker binding to KRAS G13D -GDP and 35 KRAS WT -GDP (Fig. 5E). The similar binding to G12V, G12R, and Q61H suggests that these mutants may adopt a shared SWI/SWII conformation that is distinct from G13D, G12C, and WT. Given that the 2H11 CLAMP binds multiple KRAS mutants and can increase the affinity of SWII pocket ligands, presumably by stabilizing the open conformation as we observe in our crystal structure, it may enable the identification of novel ligands against other RAS mutants.

5
While RAS proteins are major oncogenic drivers of many cancers, the lack of stable pockets has hindered drug development. The recent discovery of KRAS G12C covalent inhibitors revealed a cryptic pocket within the SWII region and sets a precedent for clinical activity for future ligands of mutant KRAS. Despite this success, whether other KRAS mutants can be targeted via the SWII pocket remains to be determined.
The discovery of KRAS CLAMPs have revealed new insights into KRAS covalent modification and can be 10 used to visualize and track inhibitor-bound KRAS G12C in cells and in vivo tumor models. Although the covalent modification of KRAS G12C appeared to be homogenous in pre-clinical studies, modification of KRAS G12C and subsequent tumor response in patient tumors is likely more heterogeneous. Implementation of a 1A5-based flow cytometry or IHC assay along with markers of KRAS activity pathway activity such as pERK in the clinical trials should enable rapid detection of covalent modification, confirming target engagement, and could also inform 15 potential mechanisms of acquired resistance.
Biophysical and structural studies showed that the 2H11 CLAMP can induce a conformation with an open SWII pocket in KRAS G12C -GDP, can improve the affinity of SWII ligands, and may enable future efforts to target this pocket more broadly across KRAS mutants. While this strategy may be successful for other mutant proteins with sufficient intrinsic GTP-hydrolysis; other KRAS mutants (e.g., KRAS Q61H ) appear to be constitutively bound to 20 GTP making it unclear whether targeting of these mutants is possible 15 . Recent work using a non-natural cysteine suggests that it is possible to identify compounds that engage the SWII pocket in both GDP and GTP bound states 33 .
Future efforts to identify KRAS CLAMPs that also bind and stabilize the GTP state to promote a stable open SWII pocket irrespective of the nucleotide bound state may be possible and provide a foundation for small molecule screens using a CLAMP:KRAS mutant complex to expand the targeting of the SWII pocket to all RAS mutants.

25
Our CLAMP platform has led to the discovery of antibodies that recognize conformations that exist within the dynamic KRAS switch regions. The success of this strategy relied on the presence of a covalent ligand, highlighting the importance of locking conformationally dynamic proteins in different states to enable the discovery of unique conformation-specific antibodies. Additionally, we provide the first experimental evidence that CLAMPs can also induce and lock a conformation in the absence of ligand and thus, enable small molecule discovery efforts 30 against transient pockets within conformationally dynamic proteins. More generally, our work has broad implications for antibody-assisted small molecule drug discovery against highly dynamic biological targets. Wood, K. W., Sarnecki, C., Roberts, T. M. & Blenis, J. ras mediates nerve growth factor receptor modulation of three signal-transducing protein kinases: MAP kinase, Raf-1, and RSK. Cell 68, 1041-1050,       25 Table S1.

Antibody and Fab production
Sequences from lead phage clones were obtained by Sanger sequencing. IgG (human IgG1) expression constructs 35 for the light chain and heavy chain for each clone were obtained by gene synthesis (Genscript, South San Francisco, CA). IgGs were expressed by transient transfection of 293 cells and purified with affinity chromatography followed by SEC using standard methods (MabSelect SuRe; GE Healthcare, Piscataway, NJ, USA).
Fab constructs for bacterial expression were generated by gene synthesis (Genscript, South San Francisco, CA).
Recombinant Fab was generated as previously described 36 .

Antibody ELISA against mutant KRAS-GDP proteins
KRAS-GDP proteins were directly coated in triplicate at 10 µg/mL on Maxisorb plates (Thermo Scientific) in PBS and incubated overnight at 4°C. Plates were blocked for 2 hours at 25 o C using 4% BSA. Serial dilutions of 1A5 and 2H11 antibodies starting at 10 µg/mL were added for 1-2 hours at 25 o C with shaking. Plates were developed and 20 read as described above.

Antibody surface plasmon resonance (SPR)
SPR experiments were carried out on the Mass-1 (Bruker) at 25°C using HBS-P+ (GE Healthcare) running buffer.
1µg/mL of the anti-KRAS antibodies were captured using an anti-HuIgG1 Fc capture kit (GE Healthcare).

30
Sensorgrams were fit to a 1:1 Langmuir model to identify kinetic parameters.

Epitope binning
Epitope binning experiments were performed in HBS-P+ (GE Healthcare) running buffer at 25°C on an array-based imager (IBIS MX96, Netherlands, as described previously 37 . Briefly, 10 µg/mL of anti-KRAS antibody was amine For washout experiments, cells were plated as described above and treated with KRAS G12C inhibitor and incubated with compound for 18-24hrs. One plate was imaged after 24hrs as control and the other plates were washed twice 25 with cold 1X PBS. Plates were then incubated for either 24 or 48 hrs with 150mL of complete compound-free medium and stained and imaged as described above.
For pERK rebound studies, cell were NCI-H358 cells were seeded at cell density of 6000 cells in Poly-D-Lysine

KRAS G12C protein expression and purification
The N-terminal His-tagged KRAS G12C    To prepare for diffraction data collection, 10% glycerol was added to the crystallization buffer as cryobuffer before flash freezing the crystals for above three cases.

Diffraction data collection and structure determination
The diffraction data of KRAS G12C /GNE-1952, KRAS G12C /2H11, and KRAS G12C /GNE-1952/2H11 crystals were collected using monochromatic X-rays at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2 or Advanced Light Source (ALS) beam line 5.0.2 using PILATUS3 6M detector. Rotation method was applied to a single crystal for each of the complete data set. The crystals were kept at cryogenic temperature throughout the data collection process. Data reduction was performed using the program XDS 38 and the CCP4 program suites 39 . Data reduction statistics are shown in Table S2.
The structures were phased by molecular replacement (MR) using program Phaser 39 . A previously 5 published crystal structure of KRAS G12D (PDB code 4DSU) and a Fab structure (PDB code 3R1G) were used as the MR search models. Manual rebuilding was performed with graphics program COOT 40 . The structures were further refined iteratively using program REFMAC5 41 and PHENIX 42 using maximum likelihood target functions, anisotropic individual B-factor refinement and TLS refinement, and to achieve final statistics shown in Supplementary

SPR Experiments with 2H11 CLAMP and small molecules
A series S SA (streptavidin) chip was inserted into a Biacore T200 (GE Health Sciences). The instrument 25 was primed into running buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.2% (w/v) PEG-3350, 0.1% CM-dextran (w/v), 0.1 mM TCEP, 10 mM MgCl2, 100 nM GDP, and 2% (v/v) DMSO). KRAS G12C pre-blocked at the 12 position with a covalent inhibitor was captured to yield 2000-2500 RU on flow channel 1 (FC1) and FC3 to serve as the reference for KRAS WT and KRAS G12C and allow affinity measurement exclusively at the Switch II pocket.
KRAS WT or KRAS G12C was captured on FC2 and FC4 within 100 RU of the reference channel capture level and data 30 was collected in FC 2-1, FC 4-3 mode. All channels were subsequently blocked by injecting 100 μg/mL amine-PEG-biotin (Thermo Fisher). 2H11 was injected 2 times at 200 nM for 120 seconds at the start of the run to saturate FC3 and FC4, and injected every 14 cycles at 100 nM throughout the run to ensure saturation throughout the run.