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Research Articles
D3S-001, a KRAS G12C Inhibitor with Rapid Target Engagement Kinetics, Overcomes Nucleotide Cycling, and Demonstrates Robust Preclinical and Clinical Activities
D3S-001 Shows Robust Preclinical and Clinical Activity
https://orcid.org/0000-0002-4037-9970
Zhang Jing 1 #
https://orcid.org/0000-0001-7694-1593
Lim Sun Min 2 #
https://orcid.org/0000-0002-2141-0012
Yu Mi Ra 3
https://orcid.org/0009-0008-1657-5016
Chen Cheng 1
https://orcid.org/0009-0009-6561-068X
Wang Jia 1
https://orcid.org/0009-0004-0208-1673
Wang Wenqian 1
https://orcid.org/0000-0003-2787-0834
Rui Haopeng 1
https://orcid.org/0009-0005-4624-0098
Lu Jingtao 1
https://orcid.org/0000-0001-8833-7262
Lu Shun 4
https://orcid.org/0000-0002-8251-0551
Mok Tony 5
https://orcid.org/0009-0001-3164-4920
Chen Zhi Jian 1 ‡ *
https://orcid.org/0000-0002-5562-270X
Cho Byoung Chul 2 ‡ *
1 D3 Bio, Inc., Shanghai, China.
2 Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.
3 Yonsei New II Han Institute for Integrative Lung Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, Seoul, Korea.
4 Department of Medical Oncology, Shanghai Chest Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China.
5 State Key Laboratory of Translational Oncology, Department of Clinical Oncology, Chinese University of Hong Kong, China.
* Corresponding Authors: Zhi Jian Chen, D3 Bio, Inc., 1101 Tower 1, 38 Yuanshen Road, Shanghai 200120, China. Email: George.chen@d3bio.com; and Byoung Chul Cho, Division of Medical Oncology, Yonsei Cancer Center, Yonsei University College of Medicine, 522, ABMRC, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. Email: CBC1971@yuhs.ac
Cancer Discov 2024;14:1675–98

# J. Zhang and S.M. Lim contributed equally to this article.

‡ Z.J. Chen and B.C. Cho are equal senior author contributions and cocorresponding authors.

04 9 2024
07 5 2024
14 9 16751698
06 1 2024
27 3 2024
06 5 2024
©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
https://creativecommons.org/licenses/by-nc-nd/4.0/ This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.

The new-generation GDP-bound KRAS G12C inhibitor D3S-001 overcomes nucleotide cycling with high potency and rapid target engagement kinetics, which is a meaningful and clinically relevant improvement over first-generation GDP-bound KRAS G12C inhibitors.

Abstract

First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic “cycling” of KRAS between its guanosine diphosphate (GDP)– and guanosine triphosphate (GTP)–bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145).

Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs.

National Research Fund, Kenya (NRF) http://dx.doi.org/10.13039/100016400 2022R1A2C3005817 Cho B.C.
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pmcIntroduction

KRAS (Kirsten rat sarcoma viral oncogene homolog) gene mutations represent a distinct molecular subtype and are oncogenic drivers in multiple solid tumors (1). KRAS mutations are dominated by single-base missense mutations, including alterations at codon 12 (G12), codon 13 (G13), or codon 61 (Q61; ref. 2). The glycine (G) to cysteine (C) mutation at codon 12, or KRAS G12C, is predominantly found in NSCLC, manifesting in approximately 14% of adenocarcinomas and 0.5% to 4% of squamous cell carcinomas (3, 4). This mutation is also seen in 3% to 4% of colorectal cancers (CRC) and 1% to 2% of biliary and pancreatic cancers (5). In NSCLC, KRAS G12C mutation is commonly exclusive of other driver oncogenic mutations (6) but can occur concomitantly with tumor suppressor genes such as STK11, KEAP1, TP53, and CDKN2A/CDKN2B (7).

The discovery and development of targeted therapeutic agents against KRAS have been challenging in past decades (8). Unlike protein kinases that utilize ATP (adenosine triphosphate) as substrate, KRAS is a GTPase that hydrolyzes guanosine triphosphate (GTP) to guanosine diphosphate (GDP), and cycles between an active GTP-bound conformation and an inactive GDP-bound conformation during its function as a molecular switch (9). The GTP to GDP hydrolysis process is assisted by GTPase-activating proteins (GAPs), and the exchange of GDP to GTP is facilitated by multiple guanine nucleotide exchange factors (GEF) that are regulated by upstream activation of RTK families (10). The picomolar affinity to GTP/GDP and the lack of an obvious site for drug binding have made it difficult to target KRAS by small-molecule inhibitors. In recent years, medicinal chemistry efforts have inventively identified compounds that form covalent adducts to the mutated cysteine 12 residue (G12C) and induce a cryptic allosteric pocket under the Switch II region of the KRAS protein in its GDP-bound conformation (11–13). Importantly, the KRAS G12C mutant protein still undergoes intrinsic hydrolysis (14). Furthermore, although it is resistant to GAP-induced hydrolysis, it remains sensitive to noncanonical GAPs expressed in cells (15). Early inhibitors such as ARS-853 and ARS-1620 proved that this mode of action can “trap” KRAS G12C protein in its GDP-bound inactive state, resulting in inhibition of the oncogenic signaling and tumor growth (1, 13, 16). The advancement of these covalent G12C inhibitors ultimately led to the clinical evaluation of several orally bioavailable small-molecule agents with improved potency and drug-like properties. Sotorasib (previously called AMG510) is the first KRAS G12C inhibitor approved by the FDA, and the results from CodeBreaK200, a phase 3 clinical trial of sotorasib in KRAS G12C-mutant NSCLC, revealed that sotorasib met the primary endpoint of prolonging PFS as compared with docetaxel (5.6 vs. 4.5 months; hazard ratio 0.66; P = 0.0017; ref. 17). Adagrasib (previously called MRTX849) is another KRAS G12C inhibitor that showed clinical activity in the KRYSTAL-1 phase 1/2 study, with an objective response rate of 43% [95% confidence interval (CI) 34%–53%] and median duration of response of 8.5 months (95% CI, 6.2–13.8; ref. 18). Additionally, adagrasib demonstrated encouraging preclinical and clinical intracranial activity, suggesting therapeutic utility for KRAS G12C mutant NSCLC patients who have developed brain metastases (19). These data have provided the first clinical validation of the allele-specific covalent approach in targeting the GDP-bound form of KRAS G12C and marked a major advancement in targeting this previously considered “undruggable” oncogene.

However, the clinical outcomes of the first wave of KRAS G12C inhibitors seem to be suboptimal, with limited magnitude (ORR ranging from 30%–40% in NSCLC and 10%–20% in CRC) and duration of response (PFS and duration of response of approximately 6 and 8.5 months in NSCLC, respectively; refs. 17, 20). Especially when compared with agents targeting other oncogenic driver mutations in second-line NSCLC, such as osimertinib (21), alectinib (22), and selpercatinib (23), the clinical benefits of these KRAS G12C inhibitors are relatively less compelling. It remains unclear whether this is due to KRAS target/disease biology, the potency/exposure of the compounds, or both. These are critical questions to be addressed for the development of the next-generation KRAS inhibitors as well as for the design of combination strategies aiming to improve the clinical benefits for patients with KRAS G12C-mutated tumors.

Although KRAS target biology in different disease settings is multifaceted, one of the key aspects that makes it difficult to fully block KRAS G12C signaling is the dynamic “cycling” of the oncogenic protein between its GDP-bound and GTP-bound confirmations (24, 25). The drug pocket of current KRAS G12C covalent inhibitors is crypted and only exists when the protein is in its GDP-bound state (11). Therefore, to what extent can an inhibitor “trap” cellular KRAS G12C proteins in their GDP-bound inactive state at clinically achievable pharmacokinetic (PK) exposure is an essential factor that determines the proportion of unoccupied KRAS G12C remaining in the GTP-bound active state that drives downstream oncogenic signaling. The existence of the MAPK pathway feedback mechanisms in tumor cells and the presence of growth factors in human tissues exacerbate this problem (26–29). These stimuli activate RTKs located upstream of KRAS, pushing the KRAS equilibrium in favor of its GTP-bound form, and compromising the activity of the GDP-bound KRAS G12C inhibitors. Hence, it is imperative to understand the target engagement (TE) efficiency of these inhibitors and their effectiveness in depleting cellular active GTP-bound G12C proteins. Ideally, an optimal inhibitor that can “trap” all the KRAS G12C proteins in their inactive GDP-bound form even in the presence of growth factor stimulation is highly desired.

Herein, we investigated the kinetic mechanism of action of earlier KRAS G12C inhibitors ARS-853 and ARS-1620, as well as clinically approved compounds sotorasib and adagrasib. Intending to further improve covalent potency and TE efficiency, we discovered D3S-001, a highly potent, CNS–penetrable covalent small-molecule inhibitor of the GDP-bound KRAS G12C. In preclinical studies, D3S-001 demonstrated substantially improved covalent potency, rapid TE kinetics, and robust antitumor activities. In an ongoing phase 1 trial (NCT05410145) investigating the safety and PK for D3S-001 in patients with advanced or metastatic solid tumors with a KRAS G12C mutation, the clinically meaningful response was observed across all dose cohorts from 50-mg QD to 900-mg QD. Here, we present early promising systemic and intracranial activity of D3S-001 in patients with KRAS G12C-mutant tumors from 50-mg QD dose level, the first cohort of this dose escalation study.

Results

D3S-001 Demonstrated Substantially Improved Covalent Potency and TE Kinetics

To comprehensively understand the molecular activity of D3S-001 relative to other GDP-bound KRAS G12C inhibitors, we profiled the potency of this class of molecules in inhibiting cellular active KRAS, their biochemical/biophysical covalent potency, and cellular TE kinetics. Early KRAS G12C inhibitors ARS-853 and ARS-1620, clinically approved inhibitors sotorasib and adagrasib, and D3S-001 were analyzed in parallel (Fig. 1). First, to evaluate the potency of the inhibitors in reducing cellular active GTP-bound KRAS, the NCI-H358 NSCLC cancer cell line harboring a KRAS G12C mutation was treated with each inhibitor at a series of concentrations for 2 hours. The cellular GTP-bound KRAS G12C levels were detected by an active RAS-binding domain pull-down method followed by KRAS immunoblotting as described previously (12). The levels of active KRAS were then quantified by chemiluminescence intensity and IC50 was calculated for each compound. As shown in Fig. 1A, treatment with KRAS G12C inhibitors decreased cellular active KRAS proteins in a dose-dependent manner, with an IC50 of 5899, 692, 35, 78, 0.6 nmol/L for ARS-853, ARS-1620, sotorasib, adagrasib, and D3S-001, respectively. Consistent with previous reports, sotorasib and adagrasib demonstrated substantially improved potency compared with ARS-853 and ARS-1620 (30, 31). Notably, D3S-001 showed a further improvement in potency with an IC50 of 0.6 nmol/L and 58- and 130-fold more potent than sotorasib and adagrasib, respectively.

Figure 1. D3S-001 is a KRAS G12C inhibitor with enhanced covalent potency and TE kinetics. A, Cellular activity of KRAS G12C inhibitors in NCI-H358 NSCLC cells was evaluated through active RAS-GTP pull-down followed by immunoblotting using a KRAS-specific antibody after a 2-hour treatment with different inhibitors at indicated concentrations. The levels of active KRAS were quantified by chemiluminescence intensity, and IC50 values were calculated using GraphPad Prism for each compound. B, Maximal covalent inactivation rate (kinact), the concentrations that achieve a half-maximal rate (KI) and other kinetic parameters of different KRAS G12C inhibitors determined by SPR. The top doses tested were 20 μmol/L for ARS-853 and ARS-1620, 200 nmol/L for sotorasib and adagrasib, and 20 nmol/L for D3S-001, respectively. Three independent experiments were performed, and data are presented as mean ± SD. C, Free–cysteine proteome analysis of D3S-001. NCI-H358 whole-cell lysates were extracted and subjected to proteomics analysis after a 4-hour treatment with 10-nmol/L D3S-001 or DMSO. Criteria for covalent targets (highlighted in gray) were set as < −2.0 log2 fold change with a P value of less than 0.001 across biological replicates (n = 3 replicates). D, Kinetics of cellular active RAS depletion in NCI-H358 cells after treatment with a time course and dose titration of KRAS G12C inhibitors were determined by RAS-GTP ELISA assay. The observed inhibition rate kobs and concentrations to achieve a half-maximal rate [I]50 were determined using GraphPad Prism. Cellular TE efficiency Max kobs/[I]50 values were calculated. E, Cellular kinetic parameters of different KRAS G12C inhibitors determined from time course and dose response studies as illustrated in D. Inhibition rate t1/2 was calculated by ln2/Max kobs.

To dissect the biochemical/biophysical basis underlying the substantially improved cellular activity of D3S-001, a SPR assay was adopted. For covalent inhibitors, the drug–target interaction kinetics are predominantly dependent on two factors: (i) the reversible affinity of the compound to the target protein that can be quantified by the rate constant of association (kon) and rate constant of dissociation (koff), which determine the reversible rate constant of inhibition Ki (=koff/kon), and (ii) the reactivity of the compound to form a covalent adduct to the protein that can be quantified by the rate constant of covalent inactivation (kinact). Another parameter KI [=(koff + kinact)/kon] depicts the affinity of a covalent inhibitor, in which the dissociation rate is dependent on koff and kinact. Combined, the covalent potency parameter kinact/KI reflects both kinact and KI and is commonly used to rank the biochemical/biophysical activity of covalent inhibitors during drug discovery. Although biochemical methods detecting a covalently modified end product are often used to measure kinact/KI, SPR assays provide an additional level of granularity by direct observation of kon, koff, and metrics for covalent reaction efficiency (such as commitment to covalency or Cc [=kinact/(kinact + koff)] that describes the balance between the rate of reversible dissociation and rate of covalent reactivity), resolving inherent ambiguities of biochemical results (32). The binding kinetics of KRAS G12C inhibitors to the purified GDP-bound KRAS G12C protein were therefore evaluated by SPR followed by single-cycle kinetics analysis. D3S-001 exhibited irreversible binding to GDP-bound KRAS G12C protein and limited or no binding to GTP-bound KRAS G12C protein (as depicted in Supplementary Fig. S1A and S1B). The rate constant of reactivity kinact, covalent affinity KI, and overall potency kinact/KI for the KRAS G12C inhibitors are summarized in Fig. 1B. The reversible rate of association kon, rate of dissociation koff, as well as the commitment to covalency parameter Cc are also summarized. As shown in Fig. 1B, early KRAS G12C inhibitors ARS-853 and ARS-1620 demonstrated relatively low covalent potency with kinact/KI of 6.33 × 102 and 8.39 × 102 mol/L−1 second−1, respectively. The kinact/KI of sotorasib and adagrasib are 2.04 × 104 and 7.14 × 104 mol/L−1 second−1, respectively, one to two orders of magnitude improvement from those of ARS-853 and ARS-1620, consistent with previous reports (30, 31). The kinact/KI value of D3S-001 is 1.43 × 106 mol/L−1 second−1, representing another one to two orders of magnitude improvement from adagrasib and sotorasib. Because the increase in covalent potency can be driven either by an increased rate of reactivity, i.e., higher kinact or by affinity, i.e., increased kon or decreased koff, it is essential to understand the individual contributing factors. As illustrated in Fig. 1B, the kinact/KI improvement of ARS-1620 over ARS-853 seems to be mainly driven by prolonged residence time, i.e., lower koff, leading to a more balanced covalent efficiency (Cc from 0.1 to 0.7); the kinact/KI improvement of sotorasib over ARS-1620 is driven by an increased affinity with higher kon and lower koff. The covalent efficiency factor Cc of sotorasib also further improved from 0.7 to 1.0, indicating koff is now negligible compared with kinact; adagrasib demonstrated about 3.5-fold kinact/KI improvement relative to sotorasib with a slight increase in kon and kinact. Notably, the substantial kinact/KI enhancement of D3S-001 over sotorasib and adagrasib is predominantly driven by kon (kon = 2.04 × 104 mol/L−1 second−1 for sotorasib, 7.21 × 104 mol/L−1 second−1 for adagrasib and 1.45 × 106 mol/L−1 second−1 for D3S-001), whereas the kinact value of D3S-001 is comparable to that of sotorasib or adagrasib. All three inhibitors (sotorasib, adagrasib, and D3S-001) have reached covalent reaction efficiency Cc of 1, in which reactivity (kinact) is substantially faster than dissociate rate (koff).

The kon-driven rather than kinact-driven potency enhancement of D3S-001 is important as this indicates its improved biochemical properties are a result of increased affinity to KRAS G12C protein rather than accelerated promiscuous reactivity to nucleophilic cysteine residues, which can compromise selectivity and safety. In a free cysteine–proteome profiling assay using a mass spectrometry method previously described (16), D3S-001 consistently demonstrated a high selectivity to the cysteine 12 of KRAS G12C (Fig. 1C). In this assay, NCI-H358 cells were treated with D3S-001 at 10 nmol/L for 4 hours, cell lysates were collected, and free–cysteine containing peptides were analyzed. The D3S-001 treatment group demonstrated a treated-to-control ratio of 0.05 (5% of DMSO control). Of the 11,522 unique cysteine-containing peptides identified, the peptide containing the cysteine 12 of KRAS G12C was the only one that met the statistical criteria for covalent conjugation, suggesting a significantly low risk for covalent modification of off-target proteins by D3S-001.

Next, cellular TE kinetics of the KRAS G12C inhibitors were investigated. As the mechanism of action of this class of inhibitors is to “lock” the KRAS G12C in its GDP-bound conformation, quantification of either the covalently conjugated GDP-bound protein or the remaining GTP-bound protein can provide an equivalent functional readout of TE. To confirm the consistency of different readouts using different detection methods, the cellular TE IC50 measured by Western blotting, active RAS ELISA, and liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were compared (Supplementary Fig. S2A–D). After NCI-H358 cells were treated with D3S-001 at a series of concentrations for 1 hour, GTP-bound (K)RAS levels in cell lysates were quantified by Western blotting or ELISA. Additionally, covalently modified GDP-bound KRAS G12C was detected by LC-MS/MS. As summarized in Supplementary Fig. S2D, the direct measurement of KRAS G12C protein conjugation IC50 by LC-MS/MS was 2.3 nmol/L. The indirect TE IC50s measured by Western blotting and ELISA were 3.4 and 2.5 nmol/L, respectively, demonstrating highly consistent results from these different readouts and methods. Considering assay throughput, ELISA was utilized in examining cellular G12C engagement kinetics, reflecting the time and concentration dependency of the KRAS G12C inhibitors in depleting cellular GTP-RAS. In these studies, NCI-H358 cells were treated with each inhibitor at various concentrations over a time course from 0 to 480 minutes (8 hours). As shown in Fig. 1D, to reach a TE plateau, ARS-853 and ARS-1620 required concentrations over 10 and 2.5 μmol/L, respectively, consistent with previously reported data for these compounds (13, 16). Near-complete (>95%) TE was achieved at 100 nmol/L for sotorasib and adagrasib. D3S-001, in the same assay, demonstrated a substantial improvement over sotorasib and adagrasib, with near-complete TE achieved at 5 nmol/L within 2 hours and 1 nmol/L at 8 hours post-treatment. This time- and concentration-dependent quantification allows the calculation of the observed rate constant kobs and concentration at a half-maximal rate ([I]50). As summarized in Fig. 1E, the cellular G12C engagement efficiency kobs/[I]50 of D3S-001 was determined to be 5.51 × 104 mol/L−1 second−1, approximately 5- and 12-fold more effective than sotorasib and adagrasib, respectively. Importantly, the rate of target inhibition (Max kobs) of D3S-001 was substantially faster than sotorasib and adagrasib with a rate constant of 2.0 × 10−3 second−1 or a t1/2 of 5.8 minutes. By comparison, the rate of inhibition t1/2 of sotorasib and adagrasib was 44 and 34 minutes, respectively. Notably, the hydrolysis t1/2 of GTP-KRAS G12C to GDP-KRAS G12C was reported to be 23 to 27.4 minutes (16), and the dissociation rate of GDP from KRAS was proposed to be 1.17 × 10−3 second−1 or a t1/2 of 9.9 minutes (16). Together, these data indicate that with a faster inactivation rate, D3S-001 can potentially “lock” KRAS G12C-GDP in its GDP-bound state instantly after GTP hydrolysis and also before GDP is dissociated from the protein for nucleotide exchange, though cellular/disease context needs to be considered.

Unlike Sotorasib and Adagrasib, the TE Kinetics of D3S-001 Are Insusceptible to Epithelial Growth Factor (EGF) Stimulation

Multiple lines of evidence have shown that the nucleotide exchange of mutant KRAS can be further stimulated by upstream RTK activation (16, 26, 27). Upon activation by corresponding growth factors, the intracellular domain of these RTKs recruits GEFs, such as SOS1 (Son of Sevenless Homologue 1), to facilitate the transition of GDP-bound KRAS to its active GTP-bound conformation. Therefore, the activity of the GDP-bound conformation-selective KRAS G12C inhibitors can be compromised. This has been proposed as a disadvantage of this class of inhibitors and hypothesized to contribute to the limited magnitude and duration of tumor response observed in the clinic (33, 34). As D3S-001 demonstrated substantially faster cellular TE kinetics than sotorasib and adagrasib, we next challenged its TE efficiency by using EGF stimulation (Fig. 2A). As expected and consistent with previous reports, the extent and speed of TE of ARS-853 and ARS-1620 were markedly reduced with EGF stimulation (13, 16). At the concentration of 1 μmol/L, the maximal TE for ARS-853 was reduced from 55% to 19%; the maximal TE for ARS-1620 was reduced from 83% to 60%. The cellular TE kinetics of sotorasib and adagrasib were likewise affected in that it now required 8 hours for both compounds at 100 nmol/L to achieve >95% TE in the presence of EGF as opposed to 4 hours without EGF stimulation.

Figure 2. TE of D3S-001 is insusceptible to EGF stimulation. A, Kinetics of cellular active RAS depletion in NCI-H358 cells after treatment with ARS-853 or ARS-1620 at 1 μmol/L, sotorasib, adagrasib, or D3S-001 at 100 nmol/L, in the absence (solid line) or presence (dotted line) of 40 ng/mL EGF was determined by RAS-GTP ELISA assay. B, EGF treatment stimulates the transition of RAS to GTP-bound form. NCI-H358 cells were treated with 40 ng/mL EGF at different times as indicated. Cell extracts were prepared and subjected to (left) pull-down assay and (right) pERK HTRF assay at the indicated time post-treatment. For the pull-down assay, the levels of active KRAS were quantified by chemiluminescence intensity and normalized to vehicle. For pERK HTRF, the levels of pERK were quantified by HTRF and normalized to vehicle. C, NCI-H358 cells were treated with D3S-001, sotorasib, or adagrasib with or without concurrent EGF stimulation (40 ng/mL; top left), HGF stimulation (40 ng/mL; top middle), at 100 nmol/L for 2 hours, or with or without concurrent EGF stimulation (40 ng/mL) and KRAS G12C inhibitors at their corresponding [I]50 concentrations for 1 hour (top right). Extracted cell lysates were subjected to pull-down assay to determine the effect on active KRAS. The levels of active KRAS were quantified by chemiluminescence intensity and normalized to DMSO (bottom). D, NCI-H358 cells were treated with EGF (40 ng/mL) with or without concurrent D3S-001, sotorasib, or adagrasib at their corresponding 5 × [I]50 concentrations for a time course as indicated. Cell extracts were subjected to pERK HTRF assays, and the levels of pERK were quantified by HTRF and normalized to vehicle.

Intriguingly, the cellular TE kinetics of D3S-001 were only minimally affected by EGF stimulation (Fig. 2A) despite its also being a GDP-bound KRAS G12C inhibitor with limited or no binding to the GTP-bound form (Supplementary Fig. S1B). We hypothesize that a GDP-bound inhibitor can overcome EGF stimulation only if the compound is efficient enough to compete away the G12C-GDP supply from EGF-induced nucleotide exchange. To understand this, we first evaluated the time course of EGF-stimulated GDP to GTP transition and signal pathway activation. As shown in Fig. 2B, in KRAS G12C-mutant NCI-H358 cells, EGF induced an increase of GTP-bound KRAS in a time-sensitive manner with GTP-KRAS level peaking within 30 minutes, followed by a return to steady-state levels after 2 hours. A time-course experiment detecting downstream activation of ERK (extracellular signal–regulated kinase) also demonstrated a similar pattern of transient activation followed by recovery to steady state (Fig. 2B), which is consistent with a previous report on growth factor-induced RAS-MAPK activation (35). To investigate the efficiency of G12C inhibitors in the presence of EGF, cellular GTP-bound KRAS levels were determined after 2 hours of treatment with the inhibitors and EGF added to cell culture media simultaneously. As shown in Fig. 2C, D3S-001 at 100 nmol/L depleted nearly all detectable GTP-bound KRAS (2% of control). Importantly, the same level of reduction (1% of control) was observed in the presence of 40 ng/mL EGF. By contrast, 100 nmol/L of sotorasib and adagrasib reduced active KRAS to 12% and 43% of control in the absence of EGF. However, in the presence of EGF the active KRAS levels rebounded to 60% and 87% of control, respectively. In addition to EGF, other growth factors, such as HGF, are also known to induce the KRAS GDP to GTP transition and downstream ERK activation (35). The ability of D3S-001 to inhibit KRAS G12C in the presence of HGF was evaluated. Similar to the observations in EGF experiments, D3S-001 was able to overcome HGF-mediated KRAS GDP to GTP transition, whereas target inhibition by sotorasib and adagrasib was largely compromised [Fig. 2C (middle)].

Next, we investigated the time and concentration dependency of D3S-001 in overcoming growth factor-induced nucleotide cycling. In the analysis of cellular TE kinetics (Fig. 1E), D3S-001 depleted cellular GTP-KRAS with a maximal kobs of 2.0 × 10−3 second−1, or a rate of inhibition t1/2 of 5.8 minutes, which means D3S-001 is able to deplete cellular GTP-KRAS by 50% in 5.8 minutes and by >95% within 30 minutes (5 × t1/2), whereas sotorasib and adagrasib require 3.7 and 2.8 hours, respectively, to achieve >95% TE. This kinetics analysis also determined the [I]50 of D3S-001 was 36.3 nmol/L, at which concentration the reaction constant kobs was 1.0 × 10−3 second−1 (half of the Max kobs). Based on these quantitative data, at its [I]50 of 36.3 nmol/L, D3S-001 could deplete GTP-KRAS in 1 hour. To validate this, cellular KRAS-GTP level was detected after treatment with D3S-001 at 40 nmol/L (∼36.3 nmol/L) for 1 hour with or without growth factors. Sotorasib and adagrasib were also included in parallel at corresponding [I]50 concentrations for each compound. As shown in Fig. 2C (right), TE of D3S-001 was marginally affected by EGF, whereas sotorasib and adagrasib demonstrated a suboptimal TE efficiency without growth factor stimulation and their TE was further weakened in the presence of EGF. In addition, neither EGF nor HGF was able to induce downstream activation of ERK in the presence of D3S-001 at 180 nmol/L (5 × [I]50, which was simulated to reach its maximal kobs), whereas ERK activation was not fully blocked by either sotorasib or adagrasib at corresponding 5 × [I]50 concentrations (Fig. 2D; Supplementary Fig. S3A and S3B). Collectively, these data indicate that cellular TE efficiency for GDP-bound KRAS G12C-selective covalent inhibitors is a critical feature that determines the time and concentration required to fully block KRAS activation and signaling in tumor cells. A highly efficient KRAS G12C inhibitor can provide profound advantages, particularly in a physiologic/pathologic environment in which growth factors are present (36), in locking KRAS in its GDP form and interfering with the process of nucleotide exchange.

D3S-001 Demonstrated In Vitro and In Vivo Antitumor Activity at Subnanomolar to Low Nanomolar Concentrations

We next determined the effects of D3S-001 on KRAS downstream signal transduction and cell proliferation in a panel of human cancer cell lines. Inhibitory effects on phosphorylation of ERK were quantified after 2 hours of compound treatment. As shown in Fig. 3A, D3S-001 demonstrated IC50 values of 0.5 and 0.3 nmol/L in NCI-H358 and MIA PaCa2 cells, respectively, which is a 38 to 86-fold increase in potency when compared with sotorasib or adagrasib. To evaluate the antiproliferative activity of D3S-001, cell viability assays were performed in 2D and 3D formats (Fig. 3B). In 2D assays, D3S-001 inhibited cell growth with a median IC50 of 4.35 nmol/L in KRAS G12C-mutant cell lines, and a median IC50 > 10 μmol/L in cell lines without KRAS G12C mutation (Supplementary Table S1-1). In 3D assays, a median IC50 of 0.17 nmol/L was observed in the KRAS G12C-mutant lines and a median IC50 of >1 μmol/L in cell lines without the specific mutation (Supplemental Table S1-2). These data indicate that D3S-001 exhibits high KRAS G12C allele-selective activity with markedly higher potency and selectivity compared with sotorasib and adagrasib (Fig. 3C). The anti-proliferation curves for each cell line were also plotted and shown in Supplementary Fig. S4A and S4B. Notably, several cell lines demonstrated only partial response to the KRAS inhibitors. The partial response does not seem to correlate with KRAS zygosity, as the IC50 difference between heterozygous and homozygous cell lines was not statistically significant (Supplementary Fig. S4C). Co-mutation status may account for the limited response in these cell lines. For example, the nonresponder SW1573 cell line from the 2D assay is an NSCLC cell line that harbors CDKN2A deletion and PIK3CA K111E gain-of-function mutations, both of which are clinically relevant mutations in conferring primary resistance to G12C inhibitors (37).

Figure 3. D3S-001 demonstrated potent antitumor activity in vitro. A, Effect of sotorasib, adagrasib, and D3S-001 on phospho-ERK measured by HTRF assay in NCI-H358 cells (top) and MIA PaCa-2 cells (bottom). Cells were treated with sotorasib, adagrasib, and D3S-001 at indicated concentrations for 2 hours, and then cell lysates were extracted and subjected to pERK HTRF assay (n = 3 replicates). B, Effect of D3S-001 on cell proliferation in a panel of human cancer cell lines by 2D (top) and 3D (bottom) CellTiter-Glo assay (n = 3 replicates). C, IC50 values of each compound tested in 2D (top) and 3D (bottom) assays. Each point represents an individual cell line. Folds of selectivity between the median IC50 of KRAS G12C-mutant cell lines and non-KRAS G12C mutant cell lines were calculated.

The in vivo activity of D3S-001 was then investigated in NCI-H358 xenograft models to assess its PK and pharmacodynamic (PD) properties, efficacy–PK relationships, and predicted efficacious PK exposures. For PD studies, NCI-H358 tumor-bearing mice were administered orally with D3S-001 at serial dose levels of 3, 10, 30, and 100 mg/kg. The tumors were collected 6 hours after treatment, a time point in which maximal phospho-ERK1/2 inhibitions was observed in a pilot time course study. The tumor samples were then analyzed for phospho-ERK1/2 and downstream phospho-ribosomal S6 kinase (RSK), active GTP-RAS, and the expression of 10 MAPK signature genes (38). D3S-001 dose-dependently inhibited the phosphorylation of ERK1/2 and RSK (Fig. 4A), suppressed the expression of all 10 MAPK signature genes (Fig. 4B), and reduced active GTP-bound RAS (Fig. 4C) in the xenograft tumors. Notably, the MAPK pathway inhibition seemed to reach a plateau at 30 mg/kg, correlating with the near-complete active RAS depletion (>95%) at this dose level.

Figure 4. D3S-001 inhibited KRAS signaling and tumor growth in vivo. A, NCI-H358 xenograft model was treated with a single oral dose of D3S-001 at 3, 10, 30, and 100 mg/kg, and tumor samples were collected at 6 hours post-treatment (n = 4 mice). In vivo PD analysis was performed by immunoblotting. The total and phosphorylated levels of ERK1/2 and RSK were analyzed, and GAPDH was used as a loading control. Levels of active ERK1/2 and RSK were quantified by chemiluminescence intensity and normalized to total ERK1/2 and RSK, respectively. B, The effect of D3S-001 on 10 MAPK signature genes was assessed by RNAseq. Tumor tissue samples were collected at 6 hours after a single dose of D3S-001 at different dose levels from the same batch of experiments and subjected to RNA extraction followed by RNAseq analysis. Data are shown as mean ± SEM. C,In vivo depletion of active RAS (bar graph) was determined by RAS-GTP ELISA. Plasma concentrations (red triangles) or tumor concentrations (black open circles) were determined by LC-MS/MS. Plasma and tumor tissue samples were collected at 6 hours after a single dose of D3S-001 at different dose levels from the same batch of experiments of the dose-PD study. Data are presented as mean ± SEM, with n = 4 mice per dose group. D,In vivo tumor growth of the NCI-H358 xenograft model after treatment with D3S-001 at 3, 10, 30, and 100 mg/kg once daily. D3S-001 was administered via oral gavage daily until day 31. Data are shown as mean tumor volume ± SEM. E, Left, Summary of TGI and plasma PK from the NCI-H358 xenograft model in D. Mouse plasma samples were collected at the end of efficacy studies (0.25, 0.5, 1, 2, 4, 8, 24 hours post final dose). Right, Correlation of D3S-001 plasma PK exposure with TGI values was analyzed using a nonlinear regression model in GraphPad Prism.

In an in vivo efficacy study, D3S-001 was administrated once daily by oral gavage in NCI-H358 tumor-bearing mice. Tumor volumes were measured over time and plasma samples were collected after the last dose followed by PK analysis. As shown in Fig. 4D and E, D3S-001 demonstrated a potent and exposure-dependent antitumor effect and led to near-complete tumor regression at a 30-mg/kg daily dose. Based on a free-drug fraction in mouse plasma of 1.5% (Supplementary Table S2), free-drug AUC0-inf was calculated for each dose level. D3S-001’s PK–efficacy relationship was analyzed using the percentage of tumor growth inhibition (TGI) over free-drug AUC0-inf in a nonlinear regression model (Fig. 4E). Our model predicted that free-drug AUC0-inf of 3.8 and 7.7 ng × hours/mL would achieve 60% TGI and 100% TGI, respectively. A free-drug AUC0-inf of 33.6 ng × hours/mL is predicted to result in 100% tumor regression (equivalent to 129% TGI in this experiment). Considering the free-drug fraction of D3S-001 in human plasma is 6.5% (Supplementary Table S2), a total drug AUC0-inf of 58.5 ng × hours/mL (corresponding to a free-drug Cave of 0.23 nmol/L) is the predicted minimal efficacy exposure level, and a total AUC0-inf of 517 ng × hours/mL (corresponding to a free-drug Cave of 2.1 nmol/L) is predicted to be the exposure required to achieve complete tumor regression. Together these data demonstrated robust in vivo antitumor activity of D3S-001 at exceptionally low PK exposures.

D3S-001 Treatment Resulted in Tumor Regressions in Multiple Cell Line- and Patient-Derived Xenograft Models of Different Cancer Type

To evaluate the antitumor efficacy of D3S-001 across different tumor types and various genetic backgrounds, cell line–derived (CDX) and patient-derived xenograft (PDX) models were utilized. First, KRAS G12C-mutant MIA PaCa-2 pancreatic cancer and SW837 CRC CDX models were treated with D3S-001, sotorasib, or adagrasib. Tumor volumes were monitored over time and plasma samples were collected for PK analysis at the end of the experiments. TGI values and PK parameters were summarized in Fig. 5A and B (top and middle) and waterfall plots representing the percentage of tumor volume change from the baseline of individual mice at the end of the experiment were shown in Fig. 5A and B (bottom). Consistent with its substantially improved covalent potency and TE efficiency, D3S-001 treatment led to deeper antitumor responses at much lower PK exposures than sotorasib and adagrasib.

Figure 5. D3S-001 treatment resulted in tumor regressions in multiple cell line-derived and PDX models of different cancer types. A–C, Top, Tumor growth of MIA PaCa-2 pancreatic cancer xenograft model (A), SW837 colorectal model (B), and YUO142 NSCLC PDO model (C) after oral administration with sotorasib (30 mg/kg, QD), adagrasib (30 mg/kg, QD), and D3S-001 (10, 30, or 100 mg/kg, QD). Middle, TGI and plasma PK summary for each model. 6% and 1% free-drug fractions of sotorasib and adagrasib in mouse plasma were used for free-drug AUC calculation, respectively. Bottom, Individual mouse tumor volume changes at day 52 for the MIA PaCa-2 model, day 35 for the SW837 model, and day 21 for the YUO142 model, respectively. D, D3S-001 was administered via oral gavage at 30 mg/kg every day to mice bearing 10 different KRAS G12C-mutant CRC PDX models as indicated. Tumor volume changes from baseline were calculated after 21 days of treatment. E, D3S-001 (30 mg/kg, QD, p.o.) was co-administered with cetuximab (30 mg/kg, BIW. i.p.) to mice bearing the same panel of CRC PDX models. Tumor volume changes from baseline were calculated after 21 days of treatment.

Next, we investigated the in vivo efficacy of D3S-001 in NSCLC patient-derived organoid (PDO) models (YUO056 and YUO142). Both models were established from patients with KRAS G12C mutant NSCLC who were naïve to any KRAS G12C-targeted therapy. WES analysis of the YUO056 patient’s tumor revealed KRAS G12C mutation (variant allele frequency or VAF 0.979%), and concomitant TP53 mutation (VAF 0.987%). WES analysis of the YUO142 tumor showed KRAS G12C mutation (VAF 0.778%), and concomitant TP53 mutation (VAF 0.931%). The YUO056 and YUO142 PDO models were treated with D3S-001, sotorasib, or adagrasib once daily, and tumor growth was monitored (Fig. 5C; Supplementary Fig. S5A). D3S-001 at 30 and 100 mg/kg resulted in profound tumor shrinkage. Waterfall plots representing the percentage of tumor volume change from the baseline of individual mice were analyzed at the time of tumor volume in vehicle control exceeding 1,500 mm3. Drug treatment was stopped at 40 days, and tumor regrowth in the absence of the inhibitor was monitored. Plasma PK analyses were performed separately in the same strain of mice after 7 days of treatment and the AUC0–last was used to correlate with TGI observed in YUO056 and YUO142 models. D3S-001 treatment resulted in a deeper antitumor response at lower PK exposures than sotorasib and adagrasib at the same dose level (30 mg/kg QD; Fig. 5C; Supplementary Fig. S5A). D3S-001 demonstrated sustained tumor regression with 40% and 80% of the mice remaining tumor-free in the 30 and 100 mg/kg groups, respectively, after treatment was stopped. By contrast, all mice in sotorasib- or adagrasib-treated groups exhibited rapid tumor rebound.

Next, we sought to explore the response to D3S-001 in a panel of 10 genetically defined CRC PDX models. The 10 CRC PDX models were treated with D3S-001 at 30 or 100 mg/kg daily and tumor volumes were measured over time (Fig. 5D; Supplementary Fig. S5B; Supplementary Table S3). The percentage of volume change from baseline was quantified 3 weeks (21 days) post-treatment. As shown in Fig. 5D, D3S-001 demonstrated robust antitumor efficacy with a 40% response rate in this panel of PDX models. A higher dose of D3S-001 at 100 mg/kg only increased the response rate to 50% with one additional model achieving the >30% tumor regression threshold (Supplementary Fig. S5B; Supplementary Table S3). These data are consistent with previous PD analyses in which the 30-mg/kg dose of D3S-001 is correlated with a near-complete target blockade. The results also suggest the existence of bypass or compensatory pathways in CRC that are KRAS G12C-independent. Indeed, there is clinical and preclinical evidence that EGFR activation is a major route conveying primary resistance to KRAS G12C inhibitors in CRC (10, 27). Combination regimens of several KRAS G12C inhibitors with EGFR monoclonal antibodies, such as cetuximab, have resulted in substantially improved ORR in clinical trials (39). Consistently, a combination of cetuximab (30 mg/kg BIW) with D3S-001 (30 mg/kg QD) resulted in an 80% response rate in this PDX panel (Fig. 5E). The high response rate demonstrated a synergy between D3S-001 and EGFR-directed therapy. The preliminary efficacy of D3S-001 in CRC as monotherapy is currently being investigated in the clinic (NCT05410145), and trials evaluating the combination of D3S-001 with an EGFR-directed therapy are planned.

In addition, bioinformatic analysis revealed that the 10 CRC PDX models were representative of the molecular landscape of CRC patients with a KRAS G12C mutation (40). TTN, APC, TP53, and PIK3CA were the most frequently mutated genes (Supplementary Fig. S6A and S6B) in this panel. The consensus molecular subtypes (CMS) were analyzed according to the CMS definition described previously (41). KRAS G12C mutation was found in all CMS subtypes. In the only model, CR6927, that did not exhibit tumor regression in the combination group, bioinformatics analysis indicated low EGFR expression and FGFR1 amplification (Supplementary Fig. S7A), suggesting a potential role of FGFR1-mediated signaling bypass in this model. Indeed, erdafitinib, an FGFR inhibitor, demonstrated synergistic effects with D3S-001 in this PDX model (Supplementary Fig. S7B).

D3S-001 Exhibited Brain Penetration Properties in Preclinical Species and Treatment with D3S-001 Resulted in Durable Intracranial Tumor Regression in Brain Metastasis Mouse Models

To evaluate the CNS penetration properties of D3S-001, two animal species, a beagle dog and a Sprague-Dawley (SD) rat, were utilized in preclinical PK studies. First, plasma and cerebrospinal fluid concentrations of D3S-001 were measured after oral dosing of D3S-001 at 30 mg/kg in beagle dogs. After a washout period, the same beagle dogs were administrated orally with adagrasib at 30 mg/kg as well. PK analysis was performed in these dogs and the Kp,uu,CSF of D3S-001 was determined to be 0.68, indicating good CNS penetrating properties (Fig. 6A). More importantly, at a total plasma AUC0–24 of 7,976 hours × nmol/L (5,416 ng × hours/mL), the average concentration of D3S-001 in CSF is 7.4 nmol/L, exceeding the concentration of 1 nmol/L required to achieve near-complete target inhibition. The Kp,uu,CSF of adagrasib determined in this experiment was 0.42 (Supplementary Fig. S8A), comparable to the values previously reported (19). At a total plasma AUC0–24 of 33,545 hours × nmol/L (20,261 ng × hours/mL), the average concentration of adagrasib in CSF is 6 nmol/L, more than 15-fold below the 100-nmol/L concentration required for this compound to achieve near-complete target inhibition. To determine the Kp,uu in brain tissue, the concentrations of D3S-001 and adagrasib in brain tissue were measured in SD rats after i.v. bolus dosing at 4 and 12 mg/kg, respectively. The dose levels were selected to mimic clinically relevant exposures for each compound. The free-drug fractions in plasma and brain tissue were determined. As shown in Fig. 6B, the Kp,uu of D3S-001 is determined to be 5.3%. At a total plasma AUC0–inf of 1,703 hours × nmol/L (1,156 ng × hours/mL), the maximal free-drug concentration of D3S-001 in brain tissues is 1.2 nmol/kg, close to the concentration of 1 nmol/L required for near-complete target inhibition. The Kp,uu value of adagrasib was determined to be 9.8% (Supplementary Fig. S8B). At a total plasma AUC0–inf of 8,371 hours × nmol/L (5,056 ng × hours/mL), the maximal free-drug concentration of adagrasib in brain tissues is 3.4 nmol/kg, approximately 30-fold below its 100-nmol/L concentration required to achieve near-complete target inhibition. These data indicate that D3S-001 may achieve substantially improved KRAS G12C target coverage in metastatic brain tumors compared with adagrasib at clinically achievable- PK exposures.

Figure 6. Evaluation of the effect of D3S-001 monotherapy or in combination with D3S-002, a clinical-stage ERK1/2 inhibitor, in brain metastatic model and sotorasib-resistant models. A, Plasma and cerebrospinal fluid (CSF) PK analysis of D3S-001 in male beagle dogs after a single oral dose at 30 mg/kg. Plasma and CSF samples were collected at 1, 4, 8, and 24 hours postdose. Drug levels in the plasma (nmol/L) and CSF (nmol/L) are shown from n = 4 beagle dogs as mean ± SD. B, Plasma and brain tissue free-drug analysis of D3S-001 in SD rats after a single i.v. bolus at 4 mg/kg. Plasma samples were collected at 0.25, 0.5, 1, 2, 4, and 6 hours postdose, and brain samples were collected at 0.5, 1, 2, and 6 hours postdose. Drug levels of D3S-001 in the plasma (nmol/L) and brain homogenate (nmol/kg) are shown from n = 3/sex SD rats as mean ± SD. C, Intracranial tumor growth of NC-H1373-Luc model after oral administration with sotorasib (30 and 100 mg/kg, QD), adagrasib (30 and 100 mg/kg, QD), or D3S-001 (30 and 100 mg/kg, QD). Tumor growth was imaged twice per week after grouping by bioluminescent imaging using a living image program (PerkinElmer, IVIS Lumina Series III). Data are shown as mean total flux ± SEM. D, Representative tumor image of NC-H1373-Luc intracranial tumor model presented in C. E, Effect of sotorasib (30 mg/kg, QD, p.o.), adagrasib (30 mg/kg, QD, p.o.), D3S-001 (30 mg/kg, QD, p.o.), D3S-002 (ERK1/2 inhibitor, 25 or 50 mg/kg, QD, p.o.), and the combination of D3S-001 (30 mg/kg, QD, p.o.) + D3S-002 (25 or 50 mg/kg, QD, p.o.) on in vivo tumor growth of sotorasib-resistant MIA PaCa-2 cells with KRAS G12C gene amplification. Data are shown as mean tumor volume ± SEM. F, Event-free survival of sotorasib-resistant MIA PaCa2 CDX model in E using tumor volume >1,000 mm3 as a surrogate endpoint. Survival data were analyzed by Kaplan–Meier statistical analysis. G, Effect of sotorasib (30 mg/kg, QD, p.o.), adagrasib (30 mg/kg, QD, p.o.), D3S-001 (30 or 100 mg/kg, QD, p.o.) on in vivo tumor growth of sotorasib-resistant PDX model. Data are shown as mean tumor volume ± SEM. H, Event-free survival of sotorasib-resistant PDX model in G using tumor volume >500 mm3 as a surrogate endpoint. Survival data were analyzed by Kaplan–Meier statistical analysis.

To evaluate the antitumor efficacy of D3S-001 in metastatic brain tumors, mice bearing intracranially implanted NCI-H1373-Luc NSCLC tumors were treated with D3S-001 at 30 or 100 mg/kg once daily. Sotorasib and adagrasib were included in this experiment at the same dose levels and dosing schedules. Tumor growth was monitored by measuring the luminescence signal in the brain by an IVIS spectrum imaging system over time. As shown in Fig. 6C and D, all three compounds demonstrated dose-dependent antitumor activity, resulting in TGI or regression in the first 3 weeks. After 3 weeks, however, the tumor luminescent signals start to increase in sotorasib and adagrasib-treated groups. Notably, intracranial tumors in mice treated with D3S-001 exhibited persistent growth inhibition (30 mg/kg group) and regression (100 mg/kg group), even after 93 days. Together, these data demonstrate that D3S-001 is effective in achieving durable tumor regression in a brain metastatic NSCLC model.

D3S-001 Delayed Disease Progression in Xenograft Models that Are Resistant to Sotorasib and Adagrasib

To understand acquired resistant mechanisms and identify new therapeutic agents for patients who have progressed on KRAS G12C inhibitors, an acquired resistance model was established by chronically treating mice bearing MIA PaCa-2 (homozygous KRAS G12C mutant) pancreatic cancer xenografts with sotorasib. NGS analysis identified KRAS gene amplification across all the resistant clones tested (Supplementary Table S4). Because D3S-001 has demonstrated substantially improved KRAS G12C TE efficacy, we hypothesized that D3S-001 may be effective in these cells that express a higher copy number of the oncogene. The antiproliferative effect of D3S-001 in these resistant cell lines was thus evaluated by a 3D cell viability assay. The representative cell growth inhibition curves are shown in Supplementary Fig. S9A and the IC50s from each experiment were summarized. The average IC50 values of sotorasib were 2.1 nmol/L in MIA PaCa-2 parental cells, and 186 and 175 nmol/L in two resistant clones, approximately a 100-fold decrease in potency. A similar shift of IC50s was observed for adagrasib, consistent with the resistant phenotype of these clones. The average IC50 values of D3S-001 were 0.1 nmol/L in parental cells and 4.9 and 4.8 nmol/L for the two resistant clones. Though the potency of D3S-001 also decreased in the KRAS G12C amplified cells, the IC50s of D3S-001 remained in the single-digit nanomolar range, which is a clinically achievable exposure.

The in vivo activity of D3S-001 was further evaluated in one of the resistant clones. Mice bearing sotorasib-R-xMIAPaCa2 clone #2 tumor xenografts were treated with sotorasib, adagrasib, or D3S-001 at 30 mg/kg once daily. Tumor growth was measured over time and TGI was determined (Fig. 6E). For survival analysis, a surrogate endpoint of tumor volume exceeding 1,000 mm3 was used. To investigate if a combination of D3S-001 with downstream MAPK pathway inhibitors could result in enhanced antitumor efficacy and prolonged survival, a clinical-stage selective ERK1/2 kinase inhibitor D3S-002 (NCT05886920) was used. As illustrated in Fig. 6E and F, sotorasib and adagrasib had marginal antitumor effects in this acquired resistant xenograft model, whereas D3S-001 demonstrated a 94% TGI on day 17 and a median survival time of 36.5 days. When D3S-001 is combined with D3S-002, the survival time was further prolonged in a D3S-002 dose-dependent manner to 68 days (D3S-002, 25 mg/kg, QD) and 99.5 days (D3S-002, 50 mg/kg, QD), respectively, and was statistically significantly better than D3S-001 monotherapy (P < 0.01). The combination was well tolerated throughout treatment as indicated by minimal body weight loss in all groups (Supplemental Fig. S9B). Detailed TGI, survival data, and statistical analysis were summarized in Supplementary Table S5. Together, these data demonstrated that D3S-001 as a monotherapy or in combination with ERK1/2 inhibitor D3S-002 may provide clinical benefit to patients who have progressed on KRAS G12C inhibitors, especially if their resistant mechanism involves KRAS G12C gene amplification.

Furthermore, in a xenograft tumor established from a patient whose disease progressed after sotorasib treatment, sotorasib and adagrasib showed no antitumor effect, consistent with the resistance phenotype (Fig. 6G and H). Remarkably, D3S-001 demonstrated robust antitumor activity with 97.4% and 102.9% of TGI and median survival time of 54 days and >54 days at 30 and 100 mg/kg, respectively, without any notable body weight loss (Supplementary Fig. S9C). ctDNA analysis of the patient plasma sample indicated KRAS p.G12C (VAF: 0.359%), KEAP1 p.A474V (VAF: 0.036%), KEAP1 p.P278L (VAF: 0.995%), and STK11 p.D176Y (VAF: 0.995%) mutations and WES analysis of the tumor samples indicated CNV gain for upstream RTK families, including ERBB2 and ERBB3. Though the resistance mechanisms for this patient are yet to be defined, KEAP1 comutation and RTK amplification are likely to have contributed to the disease progression. The robust antitumor effect of D3S-001 in this model highlights that D3S-001, with improved covalent potency and TE efficiency, can be effective in patients with prior treatment with sotorasib or adagrasib.

Clinically Meaningful Response Was Observed in a First-in-Human Clinical Trial of D3S-001

A first-in-human clinical trial of D3S-001 (NCT05410145) to assess the safety, tolerability, PK, and PDs and to identify the recommended phase 2 dose opened in August 2022 in patients with KRAS G12C-mutant solid tumors.

Durable RECIST responses in patients from this trial were observed across all dose cohorts from 50 to 900 mg QD. In addition, durable intracranial activity was seen. Response rate and duration results will be reported upon data maturation at a later date. Here, we report two cases of NSCLC patients enrolled in the first dose cohort of this trial to describe the observed systemic and intracranial antitumor activity of D3S-001 (Fig. 7).• Case 1: A 56-year-old male with recurrent NSCLC with a prior resection of lung cancer harboring KRAS p.G12C (VAF 50.6%) and STK11 frameshift insertion p.Leu282 (VAF 28.1%) via tumor tissue NGS analysis. The patient had received prior anticancer therapies including platinum-based chemotherapy and atezolizumab. The metastasis in the brain was treated with radiotherapy >1.5 years prior to enrolment. He was administered 50 mg D3S-001 once daily. Disease assessment after two cycles of treatment demonstrated a RECIST partial response, with a target lesion decrease of 47.4% of a hepatic lesion [Fig. 7A (top)], and partial response was confirmed on subsequent scans. In addition, intracranial activity was observed with a decreased metastatic lesion in the cerebellum [Fig. 7A (bottom)]. The patient has been on treatment for more than 16 months as of March 2024 with intrapatient dose escalation in cycle 11 to 100 mg daily then to 200 mg daily in cycle 16. The intrapatient dose escalation was based on safety clearance of the higher dose per protocol, not due to disease progression. The patient is currently maintaining PR (target lesion decrease of −86.8%) on 400 mg daily from cycle 19. ctDNA collected at baseline and C1D8 showed a rapid decline in KRAS mutation allele frequency (VAF 8.76% to nondetectable) that remains undetected at C7D1 (Fig. 7B). PK analysis showed an AUC0–24 of 55.2 hours × ng/mL at C1D1 and 47.8 hours × ng/mL at steady state (Fig. 7C) when dosed at 50 mg daily, reaching the predicted efficacious exposure of 58.5 hours × ng/mL derived from preclinical studies.

• Case 2: A 66-year-old male with stage IV NSCLC harboring KRAS G12C mutation. NGS of his tumor showed KRAS p.G12C (VAF 15.4%), STK11 frameshift insertion p.Leu282Afs*3 (VAF 24.2%), and CDKN2A nonsense mutation p.R58* (VAF 9.9%). The patient had received prior treatment with pemetrexed/cisplatin and atezolizumab. He received 50-mg D3S-001 once daily. Disease assessment after two cycles showed a partial response, with a 49% tumor reduction per RECIST (Fig. 7D). Partial response was confirmed on subsequent scans and this patient has been on treatment for a total of 11.1 months with intrapatient dose escalation to 100 mg daily in cycle 10 then to 200 mg daily in cycle 15. The intrapatient dose escalation was based on safety clearance of the higher dose level per protocol, not due to disease progression. The patient experienced disease progression after 8.3 months. ctDNA collected at baseline and C1D8 showed a rapid decline in KRAS mutation allele frequency (VAF 6.49% to nondetectable) that remains undetected at C4D1 (Fig. 7E). PK analysis showed an AUC0–24 of 133 hours × ng/mL at C1D1 and 241 hours × ng/mL at steady state (Fig. 7F) when dosed at 50 mg daily, exceeding the predicted efficacious exposure of 58.5 hours × ng/mL derived from preclinical studies.

Figure 7. Activity of D3S-001 in patients with NSCLC. A, The top shows baseline and C3D1 scans of a pretreated NSCLC patient with a KRASG12C mutation indicating a 47.4% reduction of a target lesion (liver metastasis). Partial response was confirmed on subsequent scans. The patient has been on treatment for more than 16 months as of March 2024 with intrapatient dose escalation in cycle 11 to 100 mg daily then to 200 mg daily in cycle 16. The patient is currently maintaining PR (target lesion decrease of −86.8%) at 400 mg daily from cycle 19. The bottom shows CT scans of the brain at baseline and at C3D1. A decreased metastatic lesion (nontarget lesion) in the cerebellum was observed. B, Baseline, C1D8, and C7D1 ctDNA of the patient. KRAS VAF of 8.76% at baseline decreased to nondetectable at C1D8 and remained undetected at C7D1. C, Plasma PK of the patient on C1D1 (day 1) and C2D1 (steady state). D, The top shows baseline and C3D1 scans of a pretreated NSCLC patient with a target lesion in the left hilar lymph node. The bottom shows baseline and C3D1 scans of another target lesion in the left axilla lymph node, indicating an overall 49% reduction of a target lesion (liver metastasis). Partial response was confirmed on subsequent scans. The patient has been on treatment for a total of 11.1 months with intra-patient dose escalation to 100 mg daily in cycle 10, then to 200 mg daily in cycle 15. The patient experienced disease progression after 8.3 months. E, Baseline, C1D8, and C7D1 ctDNA of the patient. KRAS VAF of 6.49% at baseline decreased to 2.47% at C1D8 and to undetected at C4D1. F, Plasma PK of the patient on C1D1 (day 1) and C2D1 (steady state).

Discussion

The fortuitous discovery of chemical fragments that can covalently conjugate to the cysteine 12 of GDP-bound KRAS G12C marked a turning point in pharmacologically targeting KRAS. Ten years after the publication of this work in 2013 (11), tremendous advances have been made in the field with two KRAS G12C inhibitors, sotorasib and adagrasib, obtained accelerated approval from the FDA for the treatment of advanced KRAS G12C mutant NSCLC in the second-line setting, and more than 10 additional inhibitors in clinical development. With increasing clinical experience from these inhibitors and translation research in understanding KRAS target biology, improving the magnitude and duration of response for patients with KRAS G12C-mutant cancers has become a key goal for the discovery of next-generation G12C inhibitors and the design of new combination strategies.

To improve the outcomes of patients with KRAS G12C-mutant cancers, D3S-001, a GDP-bound conformation-selective small-molecule inhibitor with markedly improved covalent potency over sotorasib and adagrasib, has advanced into clinical trials. We sought to profile D3S-001 from an evolution point of view over earlier KRAS G12C inhibitors, understand its kinetic mechanism of action, and evaluate how covalent potency relates to cellular TE efficiency and antitumor activity of the compound. More importantly, we asked whether improved covalent potency can ultimately translate into augmented clinical benefit manifest by improved objective response characteristics for this class of GDP-bound conformation-selective G12C inhibitors.

Previous studies have reported the biochemical covalent potency, or the kinact/KI values, of several KRAS G12C covalent inhibitors (13, 16, 30, 31, 42). However, most of these data were generated from biochemical assays that detect the end products from the covalent conjugation of KRAS G12C protein by the compound. Although the covalent inactivation constant kinact and the overall affinity of a covalent inhibitor KI were determined from these assays, the reversible affinity parameters, i.e., kon and koff, however, cannot be reflected. Here, we adopted an SPR method that has been used in studying covalent inhibitors (43), in which the individual steps of the reaction can be quantified. By reviewing the reversible affinity (kon and koff) and irreversible covalent reactivity (kinact), the evolution of the kinetics of this class of G12C inhibitors became clearer. For example, the data indicate that sotorasib’s improvement over ARS-1620 is driven by reversible affinity, or lower koff to be more specific, rather than reactivity. The kinact/KI enhancement of D3S-001 over sotorasib and adagrasib is predominantly driven by kon, whereas the kinact value of D3S-001 is comparable to that of sotorasib or adagrasib. The kon-driven mechanism of action is an important feature, suggesting the improvement in covalent potency was not due to a general acceleration of promiscuous reactivity. Results from the free–cysteine proteomics study (Fig. 1C) and GSH (glutathione) stability of the compound with t1/2 > 500 minutes in the presence of liver cytosol (Jing Zhang and Zhi Jian Chen, unpublished data) also support the highly selective profile of D3S-001. The SPR data revealed details of the kinetic mechanism of action with a higher level of granularity than that available in previous reports and provided the first quantitative view of the evolution of this class of KRAS G12C inhibitors. Results from the current study encourage further development of additional kinetic analysis methods to study covalent G12C inhibitors, such as the stopped-flow method used in understanding the covalent kinetics of EGFR inhibitors (44).

From a covalent potency perspective, early KRAS G12C inhibitors ARS-853 and ARS-1620 demonstrated relatively low covalent potency with kinact/KI values in the 102 mol/L−1 second−1 range, whereas the kinact/KI of sotorasib and adagrasib were improved to the 104 mol/L−1 second−1 level. The kinact/KI value of D3S-001 is 1.43 × 106 mol/L−1 second−1, another one to two orders of magnitude improvement from adagrasib and sotorasib, and notably, the highest among all reported KRAS G12C inhibitors to date (30, 31, 42, 45, 46). What we have learned from the evolution of reversible tyrosine kinase inhibitors, such as the development of ALK inhibitors from the first-generation inhibitor crizotinib, to the second-generation alectinib and brigatinib, and then third-generation molecules lorlatinib, is that improvements in biochemical potency and CNS penetration have driven major advances in clinical outcomes (47–49). For covalent tyrosine kinase inhibitors, less preclinical and clinical data are currently available to depict the trend, although the third-generation EGFR inhibitor osimertinib demonstrated higher covalent potency with a kinact/KI of 1.4 × 106 mol/L−1 second−1 in targeting EGFR L858R/T790M when compared with second-generation inhibitors afatinib and dacomitinib. Furthermore, the low covalent efficiency of osimertinib toward wild-type EGFR (kinact/KI of 2.8 × 104 mol/L−1 second−1) resulted in a higher therapeutic index than second-generation inhibitors, which showed less differential in covalent potency between mutant and wild-type EGFRs (44). In the KRAS G12C field, we anticipate a similar pattern of evolution with the progress in drug discovery and clinical research. During the preparation of this manuscript, the phase 1 clinical trial results of GDC-6036 (divarasib) were published, reporting a confirmed response of 53.4% and PFS of 13.1 months in NSCLC patients with a KRAS G12C mutation (50). The clinical benefits were apparently superior to what have been observed for sotorasib and adagrasib. The kinact/KI for GDC-6036 was reported to be 7.1 × 105 (46), or 6.0 × 105 mol/L−1 second−1 in one of our SPR assays, one order of magnitude more potent than sotorasib and adagrasib, though approximately two-fold less potent relative to D3S-001. The recent clinical data of GDC-6036 support the hypothesis that enhanced covalent potency can lead to improvement in the magnitude and duration of clinical responses. As more clinical data from KRAS G12C inhibitors of high covalent potency, such as GDC-6036 and D3S-001, become available, the mechanism(s) by which covalent potency translates into clinical benefit will become unveiled progressively.

In addition to covalent potency, the nature of KRAS biology adds a hurdle for GDP-bound conformation-selective G12C inhibitors to achieve optimal target inhibition. Specifically, the allosteric Switch II pocket of KRAS is crypted and exists only when the protein is in its GDP-bound confirmation. Once KRAS switches to its GTP-bound form, the structure of the pocket is profoundly altered and becomes inaccessible to the GDP-bound confirmation-selective G12C inhibitors. In whole cells, the activity of this class of G12C inhibitors is constrained by the dynamic nucleotide switching of KRAS G12C. Thus, for GDP-bound conformation-selective inhibitors, the rapidity with which it can access the enzyme whereas it is still in its GDP-bound form is critical to its cellular efficacy. Here, we analyzed the cellular TE kinetics of the G12C inhibitors with an ELISA method that quantifies the remaining active GTP-KRAS in NCI-H358 cells. In this study, the speed with which D3S-001 can “lock” GDP-KRAS is faster than the reported rate of KRAS G12C intrinsic hydrolysis and the dissociation rate of GDP from GDP-KRAS (16). This makes it theoretically possible for D3S-001 to occupy all GDP-G12C present in a cell, whereas the slower rate by which sotorasib and adagrasib can accomplish is a deficiency. D3S-001’s high efficiency in achieving target occupancy likely underlies its subnanomolar IC50 in depleting GTP-KRAS 2 hours after treatment. Notably, this subnanomolar IC50 of D3S-001 is comparable or potentially superior to the emerging GTP-bound KRAS(ON) G12C inhibitors of the RMC series, which work as molecular glues in recruiting cellular chaperon protein, i.e., cyclophilin A, to block the interaction of active KRAS with downstream effectors (34). In a recent publication by Schulze and colleagues (34), the reported IC50 values of the KRAS(ON) G12C inhibitor RMC4998 from the same assay condition were in the range of 1 to 10 nmol/L.

Intriguingly, D3S-001 was able to deplete nearly all detectable GTP-KRAS in NCI-H358 cells, a heterozygous KRAS-mutant cell line that expresses one copy of wild-type KRAS. Both the active RAS pull-down and ELISA methods recognize GTP-bound active KRAS regardless of G12C mutation status. These data indicate that even though this cell line is heterozygous for the KRAS G12C mutation, tumor cells preferentially express and activate the mutant form, which was consistent with a previous report from an elegant mass spectrometry-based protein quantification study (51). Nevertheless, additional cell lines with different genetic/disease backgrounds will help expand the observations in NCI-H358 cells from this study. Along these lines, we conducted similar experiments in SW837, a CRC cell line with a heterozygous KRAS G12C mutation. The data suggested that in the SW837 CRC cells, D3S-001 was unable to fully deplete all GTP-bound KRAS, with 15% to 20% remaining compared with <5% remaining in the NCI-H358 NSCLC cell line. This indicates that the activation of wild-type KRAS may be higher in CRC cells. The contributions of KRAS G12C versus KRAS WT and other RAS isoforms are likely to depend on the cellular and disease context and warrant further elucidation.

KRAS nucleotide cycling is tightly regulated by upstream RTKs. Growth factor stimulation of RTKs induces KRAS nucleotide exchange by recruiting GEFs, such as SOS1, to the signaling complex. G12C-mutant KRAS can be further stimulated by growth factors, though to less of an extent from steady state compared with wild-type KRAS (35). Therefore, the cellular activity of the GDP-bound G12C inhibitor can be compromised in the presence of growth factors (16, 26, 52). Given the exceptional cellular TE efficiency of D3S-001, we investigated whether it may overcome or be less susceptible to growth factor-induced nucleotide cycling. Remarkably, the TE kinetics of D3S-001 at 100 nmol/L were minimally impacted with nearly overlying kinetic curves in the presence or absence of EGF. At its [I]50 concentration of approximately 40 nmol/L, D3S-001 was able to deplete nearly all GTP-KRAS in the presence of EGF in 1 hour. At the concentration of 5 × [I]50, which is close to the concentration required to achieve maximal kobs, D3S-001 was able to block growth factor-induced RAS-MAPK pathway activation. More kinetic investigations, such as those evaluating the rate of new G12C protein synthesis and the association rate of the protein to activated GEFs in different cellular contexts, are necessary to fully understand this complex process. Nevertheless, this work provides the first evidence that the rapidity of target occupancy by GDP-KRAS G12C conformation-selective inhibitors is a critical factor that determines the degree to which the activity of the compound is affected by nucleotide exchange, particularly in the presence of growth factors.

Consistent with its high covalent potency and rapid TE kinetics, D3S-001 demonstrated profound antitumor activity in cancer cells and mouse xenograft models. In in vivo PK-PD-efficacy correlation studies, a free-drug AUC0–24 of 33.6 ng × hours/mL, or a free-drug Cave of 2.1 nmol/L, was predicted to achieve tumor remission. The exceptionally low PK exposure required to achieve tumor regression, combined with its high selectivity, may provide D3S-001 with significant pharmacologic advantages in terms of safety when used in combination with other therapeutic agents. Currently, several combination strategies for KRAS G12C inhibitors are undergoing clinical investigation, including combinations with upstream EGFR inhibitors/antibodies, SOS1 and SHP2 inhibitors, downstream MEK and ERK inhibitors, as well as CDK4/6 inhibitors. Considering emerging agents in the NSCLC space, in which KRAS G12C is most prevalent compared with other tumor types, it would be relevant to assess the combination efficacy with these agents. These include amivantamab, an EGFR and c-MET bispecific antibody, and antibody–drug conjugates (ADCs), such as patritumab deruxtecan (HER3 ADC) and datopotamab deruxtecan (TROP2 ADC). Furthermore, combining immune checkpoint inhibitors could leverage antitumor immunity for a prolonged response duration, and has been an important avenue of exploration. D3S-001 may confer substantial safety benefits over other KRAS G12C inhibitors in combination strategies involving either immunotherapy or targeted therapy.

Although compound potency and PK exposure drive antitumor efficacy (53), the molecular heterogeneity in KRAS G12C tumors may affect their sensitivity to KRAS G12C inhibitors. KRAS G12C mutations frequently cooccur with TP53, STK11, KEAP1, or CDKN2A mutations in NSCLC. Although no predictive biomarker for primary resistance has been identified to date, clinical data indicates KEAP1 comutation may be correlated with a lower ORR (54). Compared with NSCLC, the correlation between the molecular composition of CRC and response to KRAS G12C inhibitors is less well understood, partly due to a relatively lower mutation frequency (3% in CRC vs. 14% in NSCLC) and limited patient numbers. We here investigated the antitumor efficacy of D3S-001 in 10 CRC PDX models with a genetic background consistent with the genomic landscape of CRC. As highlighted in several preclinical studies, the extent of RTK activation is higher in CRC than in NSCLC (52), and feedback activation of wild-type RAS, i.e., NRAS and HRAS, is involved in CRC (27). In line with these mechanisms, combining a KRAS G12C inhibitor with anti-EGFR antibodies has resulted in improved clinical benefits for multiple KRAS G12C inhibitors (39, 55, 56). We observed a similar trend in which the PDX response rate was 80% when D3S-001 was combined with cetuximab compared with half that from D3S-001 monotherapy. Although D3S-001 is less susceptible to growth factor-induced nucleotide exchange, its activity is clearly restricted to G12C mutant KRAS, thus RTK-mediated activation of wild-type RAS isoforms cannot be overcome by KRAS G12C allele-specific inhibitors. For this reason, a combination of D3S-001 with an anti-EGFR antibody provides a rational combination strategy for CRC.

Aside from primary resistance, patients can develop acquired resistance to KRAS G12C inhibitors. As the median PFS for sotorasib and adagrasib is only 5 to 6 months, resistance may occur not long after treatment with limited subsequent therapeutic choices. The molecular mechanisms underlying acquired resistance and means to delay or overcome resistance are being actively investigated (57, 58). Upstream RTK family gene amplification, KRAS G12C gene amplification, and the reactivation of the downstream MAPK pathway have been identified in ctDNA and/or tissue samples and proposed to be key resistant mechanisms (57, 58). In this study, the utility of D3S-001 in overcoming acquired resistance was explored in two different xenograft models. One was established with chronic treatment of MIA PaCa-2 pancreatic tumor-bearing mice with sotorasib till disease progression, and WES analysis identified KRAS gene amplification, a clinically relevant alteration. The other is a PDX model established from a patient whose disease had progressed on sotorasib. WES analysis indicated gene amplification of the RTK family, including ERBB2 and ERBB3, as well as KEAP1 gene mutation. Impressively, D3S-001 demonstrated robust antitumor effects in both resistant models with substantially prolonged survival time, whereas neither sotorasib nor adagrasib showed antitumor effect. These data highlight that a proportion of the acquired resistance observed in the clinic potentially results from insufficient target blockage. Indeed, based on the clinical PK exposure of sotorasib and adagrasib (FDA review reports), neither of the drugs reaches the 100 nmol/L free-drug concentration required to achieve complete TE at Ctrough. KRAS G12C gene amplification and RTK amplification can further undermine the target coverage ability of the inhibitors. Our data provided the first evidence that a more potent KRAS G12C inhibitor of the same class can still be effective and, when combined with downstream ERK inhibitors, may further delay disease progression in this patient population. Furthermore, brain metastasis in KRAS G12C-mutant NSCLC represents an important area of unmet medical need. D3S-001 demonstrated favorable levels of brain penetration properties and can potentially achieve near-complete TE in the CNS at clinically relevant PK exposures.

In the first-in-human trial of D3S-001, durable RECIST responses were observed across all dose cohorts from 50- to 900-mg QD. In addition, durable intracranial activity was seen. Response rate and duration of response will be reported at a future date as clinical data, including survival assessments, mature. The two patients from the first dose cohort (50-mg QD) of this study highlight the durability of observed clinical responses. One patient has been on treatment for over 1 year, whereas the other patient has had an ongoing partial response for over 8 months. Furthermore, intracranial tumor shrinkage in patients with brain metastasis is encouraging and consistent with preclinical observations of CNS activity. The efficacy of D3S-001 in this patient population will be further explored in expansion cohorts from this study.

In summary, D3S-001 is a next-generation GDP-bound KRAS G12C inhibitor that overcomes growth factor-induced nucleotide cycling. Its high covalent potency and rapid TE kinetics correlate with robust antitumor activity preclinically and promising clinical activity with durable responses observed in an ongoing phase 1 trial. CNS activity is also an important feature of the compound that may offer a clinical advantage over other G12C inhibitors. The kinetic analyses presented in this work provide new insights into critical features that drive efficacy for this class of drugs and reveal for the first time that a GDP-bound conformation-selective KRAS inhibitor can deplete cellular active KRAS as efficiently as recently reported GTP-bound G12C inhibitors. Continued evolution of KRAS G12C inhibitors from both classes is anticipated to further improve clinical benefit for patients with KRAS G12C-mutant cancers.

Methods

Reagents and Cell Lines

D3S-001 was synthesized at WuXi AppTec. ARS-853 (Cat# HY19706), ARS-1620 (Cat# HY-U00418), adagrasib (Cat# HY130149) and sotorasib (Cat# HY114277) were purchased from MedChemExpress. Cetuximab (Lot# G00X3B) was purchased from Merck KGaA. Recombinant Human EGF (Cat# 236-EG200) was purchased from R&D systems. HGF (Cat# 100-39) was purchased from Peprotech.

Human cancer cell lines were obtained from the ATCC, Pricella, CoBioer, and other organizations between 2006 and 2021. NCI-H358 cells used for ELISA and Western blot were obtained from the ATCC (Manassas, VA). For 2D and 3D cell proliferation assay, cell line panels were employed. In 2D cell proliferation assay, SW1463, SW837, TOV21G, NCI-H1373, NCI-H1792, NCI-H2122, NCI-H23, SW1573, UM-UC3, MIA PaCa2, SW756, SW480, NCI-H441, and NCI-H1573 were obtained from ATCC; NCI-H358, A549, and AsPC1 were obtained from Shanghai Institutes for Biological Sciences (Shanghai, China); Calu1 was obtained from CoBioer (Nanjing, China); HCC44 was obtained from Creative Bioarray (Shirley, NY); KYSE410 was from DSMZ (Braunschweig, Germany) and PC9 was from RIKEN (Tsukuba, Japan). In 3D cell proliferation assay, NCI-H1373, NCI-H23, SW837, Calu1, HCC44, NCI-H2030, UM-UC3, SNU1, A427, EBC1, and MIA PaCa2 were obtained from CoBioer (Nanjing, China); KYSE410, AGS, AsPC1, A549, SW480, SW620, HCT116, HCT15, A375, BxPC3, and NCI-H358 were obtained from Pricella (Wuhan, China); LU99, OV56, and LU65 were obtained from Biovector NTCC (Beijing, China). Cell lines were maintained at 37°C in a humidified incubator at 5% CO2, according to the manufacturer’s instructions, and were periodically checked for Mycoplasma. Cell lines used in in vivo study were confirmed pathogen and Mycoplasma-free by MycoAlert Mycoplasma Detection Kit (LONZA, Cat#LT07-318). All cell lines were maintained in cell culture for no more than 15 passages in this work.

Antibodies used for immunoblots were purchased from Sigma-Aldrich, Cell Signaling Technologies, or LI-COR. Detailed information is provided in the table of antibodies in Supplementary Materials and Methods.

In Vitro Studies

Western Blotting and RAS-GTP Pull-down Assay

Cells after different treatments were washed with PBS and lysed on ice for 30 minutes by adding 100 μL of 1× cell lysis buffer (CST, Cat# 9803S) containing PhosSTOP phosphatase inhibitor cocktail (Sigma, Cat# 4906845001) and cOmplete EDTA-free protease inhibitor cocktail (Sigma, Cat# 4693159001). The cell lysate was transferred to a 1.5 mL microcentrifuge tube and centrifuged at 14,000 rpm for 40 minutes at 4°C. Sample supernatants were transferred to clean new microtubes and used for immunoblotting. Cellular active RAS was determined using the active RAS pull-down and detection kit (Thermo Scientific, Cat# 16117) according to the manufacturer’s instructions followed by Western blotting with a KRAS-specific antibody (Sigma-Aldrich Cat#WH0003845M1). Detailed assay procedures are provided in Supplementary Materials and Methods.

SPR

Binding kinetics of test articles to GDP-or GppNHp-KRAS G12C protein were characterized using a single-cycle kinetics program via Biacore 8K+ (Cytiva). The biotinylated protein was immobilized on SA sensor chips and a twofold serial dilution of test articles in running buffer[10-mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid pH 7.4, 150-mmol/L NaCl, 0.05% P20, 1-mmol/L DTT, 1-mmol/L MgCl2, 1-μmol/L GDP, 1% DMSO] was injected at 25°C with a flow rate of 40 μL/minutes for 120 seconds in channel, and dissociation was followed during the 900-second assay time using single-cycle kinetics program. The Biacore sensor grams were analyzed in Biacore Insight Evaluation (v3.0.12.15655, Cytiva) using a “two state reaction model (kinetics analysis).”

Proteomic Studies

NCI-H358 cells were seeded at 2 × 106 cells per 10-cm dish and incubated at 37°C, 5% CO2 for 16 hours. The cells were then treated with 10 nmol/L, 100 nmol/L, and 1 μmol/L D3S-001(n = 3) or DMSO (n = 3) and incubated at 37°C, 5% CO2 for 4 hours. Cells were washed and pelleted before resuspension in lysis buffer [25-mmol/L Tris-HCl (pH 7.5), 150-mmol/L NaCl, 1% SDS, 5% glycerol, 1% protease inhibitors]. Cell lysates were sonicated on an ice bath for 3 minutes followed by centrifugation at 20,000 × g for 10 minutes at 4°C. Clear supernatant was transferred to a new tube and the protein concentration was measured using BCA kit (Beyotime Biotechnology, Cat# P0009) according to the vendor’s instruction. Free cysteine was labeled by mixing 1.5 mg of lysate with biotin polyethyleneoxide iodoacetamide (Sigma-Aldrich, Cat# B2059) at a final concentration of 1 mmol/L and incubating the mixture in the dark for 1 hour at room temperature of 20-22°C. Detailed assay procedures are provided in Supplementary Materials and Methods.

Active RAS Determination Via ELISA

NCI-H358 cells were maintained in RPMI1640 media (ATCC, Cat# 30-2001) supplemented with 10% FBS (Gibco, Cat# 10091148) and 1× Penicillin–Streptomycin (Invitrogen, Cat# 15140122). Cells were plated in 12-well plates (Corning, Cat# 3513) at 500,000 cells in 0.9 mL per well and incubated overnight at 37°C. On the next day, relevant test compounds or growth factors were prepared in a culture medium. Cells were treated with compounds/growth factors or DMSO for indicated time periods and RAS GTPase activity was determined using the RAS GTPase ELISA kit (Abcam, Cat# ab134640) according to the manufacturer’s instructions.

Cellular Phosphor-ERK (pERK) Determination by HTRF

pERK level in cells was determined using the Advanced ERK phosphor-T202/Y204 kit (Cisbio, Cat# 64AERPEG) according to the manufacturer’s instructions.

Cell Proliferation Assay

For 2D cell proliferation assay, cells were seeded in 96-well Flat Clear Bottom Black Polystyrene TC-Treated Microplates (Corning, Cat# 3603). For 3D cell proliferation assay, cells were seeded in 96-well ultralow adhesion plates (Corning, Cat# 7007). After incubation overnight, nine serial dilutions of test articles were prepared using a Biomek FXP laboratory automation workstation (Beckman Coulter, Carlsbad, CA). Cells were treated with compound or DMSO (0.25% v/v) for 120 hours. CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Promega, Cat#G7572) was used to measure cell viability according to the manufacturer’s instructions.

In Vivo Studies

All the procedures related to animal handling, care, and treatment were performed in compliance with all applicable regulations and the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care, and all animal studies were conducted in compliance with applicable regulations and guidelines of the Institutional Animal Care and Use Committee. Animal studies conducted in our research have been approved by an institutional review board.

Mice were housed in specific pathogen-free rooms in individual polysulfone IVC cages (3–5 mice per cage), with constant temperature (21°C–25°C) and humidity (40%–70%). Housing was provided in a 12:12 hours light–dark cycle. The bedding material was autoclaved corncob bedding that would be changed once a week. Mice were provided with autoclaved tap water and food ad libitum. At 6 to 8 weeks old, mice were inoculated subcutaneously with tumor cells in PBS mixed with Matrigel in the right lower flank region for tumor development. For efficacy studies, mice were randomized in groups once tumor volume reached the desired range. Dosing was implemented right after randomization on day 0. Tumor volume was measured twice per week by caliper and expressed in mm3 using the formula: “V = (L × W × W)/2, in which V was tumor volume, L was tumor length (the longest tumor dimension), and W was tumor width (the longest tumor dimension perpendicular to L). The body weight of each mouse was measured twice per week along with the tumor size measurement. Data were presented as mean ± SEM. Detailed information for each model is provided in Supplementary Materials and Methods.

Statistical Analysis

To compare tumor volumes of different groups on a prespecified day, one-way ANOVA was used for analyses implicating multiple comparisons, and a P value of <0.05 was regarded as statistically significant. Survival data were collected for each group and analyzed using the Kaplan–Meier methodology. Survival duration by group was tested for statistical significance using the log-rank test. All statistical analyses were performed by GraphPad Prism 9.

Clinical Trials

D3S-001 clinical trials (NCT05410145) were conducted in accordance with recognized ethical guidelines (Declaration of Helsinki) and per local institutional review board requirements. All patients included in the clinical trial were subjected to written informed consent and consented prior to study enrollment.

Data Availability

The data generated in these analyses are available within the article and its supplementary data files. We will not provide access to patient-level data if there is a reasonable likelihood that individual patients could be reidentified. We will work with qualified requestors to provide summary information where possible with the evaluation of the risk for patient reidentification.

Supplementary Material

Supplemental material and methods addition information for material and methods

Supplemental Table S1 Supplemental Table S1 summarizing the anti-proliferative potency of D3S-001, adagrasib and sotorasib.

Supplemental Table S2 Supplemental Table S2 summarizing plasma protein binding (PPB) characterization of D3S-001 across various species.

Supplemental Table S3 Supplemental Table S3 summarizing the anti-tumor activity of D3S-001 in CDX, PDX or PDO xenograft models

Supplemental Table S4 Supplemental Table S4 summarizing RNAseq and WES analysis of acquired resistant clones from MIA PaCa-2 xenograft model.

Supplemental Table S5 Supplemental Table S5 summarizing tumor growth inhibition analysis of the AMG510-R-xMIA PaCa-2 (clone 2) xenograft model

Supplemental Figure S1 Supplemental Figure S1 shows direct binging of D3S-001 to GDP-bound KRAS G12C but not GTP-bound KRAS G12C.

Supplemental Figure S2 Supplemental Figure S2 shows the percentage of KRAS G12C target engagement determined by different methods.

Supplemental Figure S3 Supplemental Figure S3 shows pERK inhibition by KRAS G12C inhibitors with or without HGF.

Supplemental Figure S4 Supplemental Figure S4 shows dose-dependent curves for each cell line.

Supplemental Figure S5 Supplemental Figure S5 shows D3S-001 additional in vivo efficacy and tolerability data.

Supplemental Figure S6 Supplemental Figure S6 shows baseline mutation landscape of the 10 colorectal cancer PDX models

Supplemental Figure S7 Supplemental Figure S7 shows data from translational exploration of CR6927 PDX model.

Supplemental Figure S8 Supplemental Figure S8 shows brain penetration profile of adagrasib in beagle dogs and SD rats

Supplemental Figure S9 Supplemental Figure S9 shows the in vitro anti-tumor effect and tolerability of D3S-001 as monotherapy or in combination with D3S-002 on sotorasib-resistant models.

Acknowledgments

The authors thank Dr. Peter T.C. Ho for the critical review of the manuscript. The authors thank Crown Biosciences and WuXi AppTec for animal study and bioinformatics support and BioDuro-Sundia and HD Biosciences for molecular and cellular assay support. The authors thank D3 Bio’s clinical development and operation team for clinical trial management. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT; No. 2022R1A2C3005817).

Authors’ Disclosures

C. Chen reports other support from D3 Bio outside the submitted work. H. Rui reports other support from D3 Bio outside the submitted work. S.M. Lim reports grants from AstraZeneca, Hutchison, and Roche and personal fees from AstraZeneca, Boehringer Ingelheim, Hutchison, Roche, Simcere, and Innovent Biologics outside the submitted work. T. Mok reports personal fees from Abbvie Inc., ACEA Pharma, and Adagene; personal fees and other support from Alentis Therapeutics AG; personal fees from Alpha Biopharma Co. Ltd., Amgen, Amoy Diagnostics Co. Ltd., AnHeart Therapeutics Inc., AVEO Pharmaceuticals Inc., Bayer HealthCare, BeiGene, BerGenBio ASA, Berry Oncology, Boehringer Ingelheim, and Blueprint Medicines Corporation; grants and personal fees from Bristol Myers Squibb; personal fees from Bowtie Life Insurance Co Ltd., Bridge Biotherapeutics Inc., C4 Therapeutics Inc., Cirina Ltd., Covidien LP, CStone Pharmaceuticals, and Curio Science; personal fees and other support from D3 Bio Ltd.; personal fees from Da Volterra, Daiichi Sankyo Inc., Daz Group, Eisai, Elevation Oncology, and F. Hoffmann-La Roche Ltd.; personal fees from Fishawack Facilitate Ltd.; grants and personal fees from G1 Therapeutics Inc.; personal fees from geneDecode Co. Ltd, Genentech, Gilead Sciences Inc., GLG’s Healthcare, Gritstone Oncology Inc., Guardant Health, Hengrui Therapeutics Inc., and HiberCell Inc.; personal fees, nonfinancial support, and other support from HutchMed; personal fees from Ignyta Inc., Illumina Inc., Imagene AI Ltd., Gritstone Oncology Inc., Incyte Corporation, Inivata, InMed Medical Communication, IQVIA, Janssen, Janssen Pharmaceutica NV, Jiahui Holdings Co. Limited, and Lakeshore Biotech; personal fees and nonfinancial support from LiangYiHui Healthcare; personal fees from Lilly, Loxo-Oncology, and Lucence Health Inc.; personal fees and other support from Lunit Inc.; personal fees from MD Health Brazil, Medscape LLC, Medtronic, and Merck Pharmaceuticals HK Ltd.; grants and personal fees from Merck Serono; grants, personal fees, and nonfinancial support from Merck Sharp & Dohme; personal fees from Mirati Therapeutics Inc.; personal fees and nonfinancial support from MiRXES; personal fees from MoreHealth; grants, personal fees, and nonfinancial support from Novartis; personal fees from Novocure GmbH, Omega Therapeutics Inc., OrigiMed Co. Ltd., OSE Immunotherapeutics, P. Permanyer SL, and PeerVoice; grants, personal fees, and nonfinancial support from Pfizer; personal fees from Phanes Therapeutics; personal fees and nonfinancial support from Physicians’ Education Resource; personal fees and other support from Prenetics Global Limited; personal fees from PrIME Oncology, Puma Biotechnology Inc., Qiming Development (HK) Ltd., Regeneron Pharmaceuticals Inc., and Research to Practice; grants, personal fees, and nonfinancial support from Roche Pharmaceuticals/Diagnostics/Foundation One; personal fees from Sanofi-Aventis, Schrödinger Inc., and Seagen International GmbH; grants and personal fees from SFJ Pharmaceutical; personal fees from Shanghai BeBirds Translation & Consulting Co. Ltd., Shanghai Promedican Pharmaceuticals Co. Ltd., Simcere of America Inc, Simcere Zaiming Inc., Summit Therapeutics Sub Inc., Synergy Research, and Taiho Pharmaceutical Co. Ltd; grants and personal fees from Takeda; personal fees from Tigermed, Touch Independent Medical Education Ltd, Vertex Pharmaceuticals, Virtus Medical Group, XENCOR Inc., and Yuhan Corporation; personal fees and nonfinancial support from Zai Lab; grants, personal fees, nonfinancial support, and other support from AstraZeneca PLC; personal fees, nonfinancial support, and other support from HutchMed; personal fees and other support from Aurora; personal fees and other support from Insighta; other support from Yinson Capital Pte. Ltd.; personal fees from The Chinese University of Hong Kong; and grants from XCovery outside the submitted work. B.C. Cho reports grants from MOGAM Institute, LG Chem, Oscotec, Interpark Bio Convergence Corp, GIInnovation, GI-Cell, Abion, Abbvie, AstraZeneca, Bayer, Blueprint Medicines, Boehringer Ingelheim, Champions Oncology, CJ bioscience, CJ Blossom Park, Cyrus, Dizal Pharma, Genexine, Janssen, Lilly, MSD, Novartis, Nuvalent, Oncternal, Ono, Regeneron, Dong-A ST, Bridgebio therapeutics, Yuhan, ImmuneOncia, Illumina, KANAPH therapeutics, Therapex, JINTSbio, Hanmi, CHA Bundang Medical Center, Vertical Bio AG, personal fees from Abion, BeiGene, Novartis, AstraZeneca, Boehringer Ingelheim, Roche, BMS, CJ, CureLogen, Cyrus Therapeutics, Ono, Onegene Biotechnology, Yuhan, Pfizer, Eli Lilly, GI-Cell, Guardant, HK Inno-N, Imnewrun Biosciences Inc., Janssen, Takeda, MSD, Janssen, Medpacto, Blueprint medicines, RandBio, and Hanmi; personal fees from KANAPH Therapeutic Inc, Bridgebio therapeutics, Cyrus Therapeutics, Guardant Health, Oscotec Inc, J INTS Bio, Therapex Co., Ltd, Gliead, and Amgen; personal fees from TheraCanVac Inc, Gencurix Inc, Bridgebio therapeutics, KANAPH Therapeutic Inc, Cyrus therapeutics, Interpark Bio Convergence Corp., and J INTS BIO; personal fees from J INTS BIO, Champions Oncology, Crown Bioscience, Imagen, and PearlRiver Bio GmbH; and other support from DAAN Biotherapeutics outside the submitted work. No disclosures were reported by the other authors.

Authors’ Contributions

J. Zhang: Conceptualization, data curation, formal analysis, supervision, validation, investigation, methodology, writing–original draft, project administration. S.M. Lim: Conceptualization, resources, data curation, formal analysis, methodology, writing–review and editing. M.R. Yu: Resources, data curation, validation, investigation, methodology. C. Chen: Data curation, validation, investigation, methodology, writing–review and editing. J. Wang: Data curation, validation, methodology, writing–review and editing. W. Wang: Data curation, formal analysis, validation, investigation, methodology, project administration. H. Rui: Data curation, validation, methodology, writing–review and editing. J. Lu: Data curation, validation, methodology, writing–review and editing. S. Lu: Resources, investigation, writing–review and editing. T. Mok: Resources, investigation, writing–review and editing. Z.J. Chen: Conceptualization, resources, supervision, funding acquisition, validation, investigation, project administration, writing–review and editing. B.C. Cho: Conceptualization, resources, data curation, supervision, validation, investigation, writing–review and editing.

Note Supplementary data for this article are available at Cancer Discovery Online (http://cancerdiscovery.aacrjournals.org/).
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