Target Occupancy Research Articles

P1247: GB5121 IS A NOVEL, IRREVERSIBLE, COVALENT BTK INHIBITOR WITH HIGH SELECTIVITY AND CNS-PENETRANCE FOR TREATMENT OF CNS MALIGNANCIES

Background: Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) penetration. GB5121 is an orally available, selective, irreversible, covalent small molecule BTK inhibitor that was designed to address these limitations. Aims: To profile GB5121 for selectivity, potency, inactivation kinetics and CNS penetrance in comparison to ibrutinib. Methods: Selectivity of GB5121 and ibrutinib were assessed in kinome scans against 349 kinases at 1 µM compound concentration with 1mM ATP. Irreversible and covalent nature of GB5121 was evaluated using enzymatic assays compared to analogs that lacked an active covalent warhead. Both biochemical and cell-based assays were employed for evaluation of potency and inactivation kinetics of GB5121. For assessment of BTK target occupancy within the CNS, naïve mice with an intact blood brain barrier were dosed with either GB5121 or ibrutinib followed by perfusion, brain dissection and processing into lysates that were analyzed for unoccupied BTK in a probe-based ELISA assay. The pharmacokinetic profile of GB5121 was assessed in multiple species for both intravenous (IV) and oral routes of dosing. Results: In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating >50% inhibition at 1 µM; additionally, GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. Importantly, GB5121 demonstrated rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissues, a critical parameter for covalent irreversible inhibitors. When compared with ibrutinib, GB5121 showed superior CNS target occupancy in the brain of mice receiving three daily oral doses and demonstrated significantly higher brain to plasma ratio. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses. Summary/Conclusion: The unique profile of GB5121 provides differentiation from both FDA-approved BTK inhibitors and those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including in CNS lymphoma. This research has been previously presented at the AACR Annual Meeting 2022.

Abstract 5645: BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity

Abstract Background: BAL0891 is a dual inhibitor of threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1). These kinases collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore (KT) to regulate chromosome alignment and segregation prior to mitotic exit. In vitro, BAL0891 has a combined prolonged effect on TTK and a transient effect on PLK1, leading to rapid disruption of the SAC that potentiates aberrant mitotic progression of tumor cells. In this work, efficacy of BAL0891 was investigated in mouse models of human triple negative breast cancer (TNBC) including evaluation of dose-dependency, drug exposure, target occupancy and a screen of activity across a panel of PDX models. Methods: The MDA-MB-231 cell line was grown sc in nude mice and treated with BAL0891, administered IV weekly (QW) or twice-weekly (2QW). Thirteen sc TNBC PDX models were screened for BAL0891 response using 2QW administration. Efficacy was quantified as deltaT/C (treated/control tumors). Plasma and tumor were analyzed for drug levels or TTK target occupancy by LC-MS/MS. The latter used a biotinylated TTK-specific probe and streptavidin-mediated isolation of unoccupied TTK, trypsin digestion and quantification of TTK-representative peptides. Results: BAL0891 efficacy was tested in the TNBC xenograft model MDA-MB-231 with QW or 2QW IV dosing schedules. All treatments were well tolerated, with no drug-related animal deaths. With MTD dosing, tumor regressions were observed, while different MTD fractions for both QW and 2QW schedules showed dose-dependent anti-tumor activity. The weekly MTD group was followed for an additional 20 days after treatment cessation on day 100. Strikingly, 3 of 8 tumors continued to shrink resulting in 2 (25%) pathologically confirmed cures. Consistent with the potent efficacy of intermittent MTD dosing, and prolonged tumor drug exposure, tumor TTK was fully drug-occupied for ≥ 6 days after the last administration; target occupancy was also dose-and drug exposure-dependent. To further evaluate BAL0891 anti-cancer activity in TNBC, a screen in 13 TNBC PDX models was conducted. Seven models exhibited deltaT/C < 50%, with regressions observed in 3. Of these, 2 models showed persistent regressions ≥ 70% vs. baseline. Interestingly, evaluation of TTK target occupancy in selected models showed high target occupancy independent of tumor response, indicating target dependency rather than drug availability is important for anti-cancer activity. Conclusion: BAL0891 is a novel dual TTK/PLK1 mitotic checkpoint inhibitor with potent anti-cancer activity in TNBC models. Intermittent IV administration is well tolerated and associated with prolonged tumor drug exposure, prolonged TTK inhibition and notable anti-tumor efficacy. These data support further investigation of BAL0891 for the treatment of cancer patients (incl. TNBC). Citation Format: Heidi A. Lane, Felix Bachmann, Elisa Zanini, Paul McSheehy, Karine Litherland, Nicole Forster-Gross, Luc Bury, Diep Vu-Pham, Jos de Man, Wilhelmina E. van Riel, Guido JR Zaman, Rogier C. Buijsman, Laurenz Kellenberger. BAL0891: A novel dual TTK/PLK1 mitotic checkpoint inhibitor (MCI) that drives aberrant tumor cell division resulting in potent anti-cancer activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5645.

Abstract 2921: Probing PRMT5 inhibitors with distinct binding modes using surface plasmon resonance

Abstract Epigenetic modifications are dynamic and reversible processes that regulate gene expression without altering the underlying sequence of the DNA. Gene expression is instead controlled by modification to the chromatin including DNA methylation, histone modifications, and chromatin remodeling. The proteins that perform these epigenetic modifications can be divided into three classes: writers, which perform the modifications; readers, which recognize the modifications; and erasers, which remove the modifications. While essential for normal cellular function, abnormal expression or alteration of the proteins involved in epigenetic regulation can lead to disease, such as cancer. As a result, epigenetic modifiers present attractive targets for the discovery and development of novel therapeutics. At Reaction Biology we offer a suite of services to enable epigenetic drug including the largest panel for epigenetic screening and profiling in the industry and biochemical and biophysical technologies to evaluate inhibition, potency, target occupancy, and even binding modes. Here we highlight our biophysical platform surface plasmon resonance (SPR) using the epigenetic target PRMT5 as an example. SPR was used to measure the binding kinetics of several small molecule inhibitors of PRMT5 and explore their distinct binding modes. Citation Format: Rebecca Eells, Lynn K. Baker, Christopher Bray, Haiching Ma. Probing PRMT5 inhibitors with distinct binding modes using surface plasmon resonance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2921.

Abstract CT202: A first-in-human phase 1 study of LY3537982, a novel, highly selective and potent KRAS G12C inhibitor in patients with KRAS G12C mutant advanced solid tumors (trial in progress)

Abstract Background: KRAS G12C mutations are recurrent oncogenic events in patients (pts) with NSCLC, CRC, and other tumors, and are found in ~14% of NSCLC and 5% of CRC. While KRAS G12C inhibitors have demonstrated activity in pts, overall efficacy may be limited by incomplete target occupancy. LY3537982 is a novel, orally bioavailable, highly selective, and potent inhibitor of the KRAS G12C protein with the potential to deliver >90% target occupancy in pts. In preclinical studies, LY3537982 demonstrated dose-dependent tumor growth inhibition in vivo as a single agent in a KRAS G12C NSCLC PDX model, and robust tumor regression in combination with other agents in vivo in a KRAS G12C sensitive NSCLC model.1 Methods: LOXO-RAS-20001 is an open-label, multi-center, first-in-human Phase 1 study of oral LY3537982 in pts with KRAS G12C-mutant advanced solid tumors. This study consists of 2 parts: Phase 1a dose escalation to evaluate LY3537982 monotherapy and Phase 1b dose expansion to evaluate both LY3537982 monotherapy and in combination. Dose escalation will follow a modified toxicity probability interval-2 (mTPI-2) method, allowing for backfill to previously cleared dose levels that demonstrate therapeutically relevant exposures or direct evidence of clinical activity. DLT evaluation period will be 21 days. The primary objective of Phase 1a is to determine the LY3537982 recommended phase 2 dose (RP2D). Part 1b will enroll pts into dose expansion cohorts based on tumor type (NSCLC, CRC, other solid tumors) and prior treatment including KRAS G12C inhibitor refractory NSCLC. Key objectives of Phase 1b are to determine the safety and tolerability of LY3537982 monotherapy, and in combination with various agents: abemaciclib, erlotinib, cetuximab, an investigational ERK inhibitor (LY3214996), an investigational Aurora A kinase inhibitor (LY3295668), and an anti-PD1 antibody. Key secondary objectives include determining the pharmacokinetic properties, and antitumor activity of LY3537982 based on overall response rate (ORR), progression-free survival (PFS), time to response (TTR), and duration of response (DOR) per RECIST v1.1. Eligible pts must have measurable disease per RECIST v1.1, have a KRAS G12C mutation in tumor tissue or ctDNA, have locally advanced, unresectable and/or metastatic cancer per disease specific criteria, and be appropriate candidates for experimental therapy or refused available therapy. Additionally, pts must be ≥18 years old, have ECOG PS of 0-1, and have adequate organ function. Key exclusion criteria include presence of serious cardiac conditions, interstitial lung disease, symptomatic CNS malignancy, symptomatic CNS metastasis, or carcinomatous meningitis. The trial is currently enrolling patients (NCT04956640).

Abstract 5437: Preclinical characterization of EB160, a novel, highly potent and brain-penetrant KRASG12C inhibitor

Abstract The KRASG12C mutation is present in nearly 13% of lung adenocarcinoma, 3% of colorectal cancer and 2% of other solid tumors. AMG510 developed by Amgen and MRTX849 developed by Mirati Therapeutics both target the KRASG12C mutation through covalent modification of the Cys 12 of the mutated protein. According to the disclosed clinical data, AMG510 and MRTX849 have an ORR of 36-45% in NSCLC and of 7-22% in CRC patients harboring a KRASG12C mutation. Incomplete target occupancy is considered to be a potential reason for only less than 50% of patients with KRASG12C mutation benefiting from those KRASG12C inhibitors. Brain metastases developed from lung cancer, accounting for 40-50% of all brain metastases, were described as the primary or contributing cause of death in a large proportion of patients with brain metastases from NSCLC. About 55% of NSCLC patients with KRAS mutations develop brain metastases. The KRAS mutant patients with untreated brain metastasis from NSCLC have a median OS of only 5 months. Hence, in order to achieve maximal clinical benefit in patients with KRASG12C mutation, a highly potent, brain-penetrant KRASG12C inhibitor with sufficient continuous exposure for the complete dose interval to maximize depth and duration of antitumor activity is required. Here, we report the discovery of EB160, a novel, highly potent and brain-penetrant KRASG12C covalent inhibitor. In a panel of KRASG12C mutant cancer cell lines, EB160 inhibited the proliferation of these cells with higher potency compared to AMG510 and MRTX849. EB160 also inhibited phosphor-ERK in H358 cells with an IC50 value of 17.54 nM at 1 h, while the IC50 values of AMG510 and MRTX849 were 58.98 nM and 41.89 nM, respectively. In vitro ADME, EB160 showed moderate or high stability in liver microsomes across the species tested, and the whole blood t1/2 was greater than 240 min in mouse, rat, dog and human, which was comparable to MRTX849. Following single PO dose (5 mg/kg) in mice, rats and dogs, the mean AUC(0-∞) were 2679 ng*h/mL, 2855 ng*h/mL and 1511 ng*h/mL, respectively, and the absolute bioavailabilities were 86%, 78% and 32%, respectively. A H358 cell line-derived lung cancer xenograft model was selected for in vivo efficacy assay. At 10 mg/kg QD of EB160 for 28 days, the tumor growth inhibition (TGI) was 108.2%, with 5 out of 6 mice showing tumor regression > 80% from initial baseline. And in the 30 mg/kg treatment group, whole 6 mice showed near complete tumor regression. In the H1373Luc KRASG12C brain metastases model, 30 mg/kg QD EB160 oral dose in mice for 28 days results in tumor regression 92%, while in the same dosage of MRTX849 treatment group, the tumor regression was 50%. Citation Format: Xuxing Chen, Jing Li, Yanhong Chen. Preclinical characterization of EB160, a novel, highly potent and brain-penetrant KRASG12C inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5437.

Abstract 3601: Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein

Abstract KRAS is one of the most frequently mutated genes in cancer with alterations occurring in > 14% of all tumors. Recent advances have led to the discovery and development of inhibitors that bind the inactive (GDP-bound) form of KRASG12C. The most advanced of these first-generation molecules demonstrated clinical response rates of 30-45% and approximately 6-month progression-free survival in lung cancer patients. While significant, a majority of patients failed to achieve a clinical response and acquired resistance can be rapid. One hypothesis to explain tumor resistance is the failure of existing inhibitors to recognize the activated (GTP-bound) form of KRASG12C that can be upregulated in response to these first-generation inhibitors. Here we report the discovery of a series of novel inhibitors that effectively inhibit both the GTP- and GDP-bound forms of KRASG12C. These “dual-acting” inhibitors bind in the switch II pocket of both GTP-bound and GDP-bound KRASG12C and rapidly form a covalent bond with cysteine 12. This results in significantly increased inhibition of RAF1 and PI3Kα effector interactions (IC50 < 5 nM at 2 hrs.) in comparison to inactive state inhibitors. Dual targeting of both GTP- and GDP-bound KRASG12C results in potent cellular activity in models that are both sensitive (NCI-H358 and MIA PaCa-2) and resistant (NCI-H2122) to adagrasib and sotorasib. In contrast to adagrasib and sotorasib which are less effective in the NCI-H2122 cell line model, dual-acting inhibitors of GTP- and GDP-bound KRASG12C elicit rapid inhibition of pERK in < 1 hour with sustained inhibition of MAPK signaling through 48 hours. To model resistance to first generation inhibitors, an A59G mutation was introduced into KRASG12C, abrogating GTPase activity. This decreases the activity of both adagrasib and sotorasib in tumor cell viability assays by more than an order of magnitude whereas dual-acting inhibitors of GTP- and GDP-bound KRASG12C are equally effective in the G12C/A59G and parental G12C cell lines. Evaluation of dual-acting inhibitors of KRASG12C in vivo demonstrated rapid and > 90% KRASG12C target occupancy, resulting in regression of MIA PaCa-2 tumors. Dual-acting inhibitors of both the active and inactive states of KRASG12C may provide the potential for broader and more durable responses in the clinic. Citation Format: Philamer Calses, Sam Clark, Jacob Corpuz, Susan Fong, Phil Gerkin, Mohammad Hekmatnejad, Evan McMahon, Megan Murray, Truc Nguyen, Tony Phan, Allison Roberts, Phillip Schwartz, Mikayla Shanafelt, Hiroko Tanaka, Jennifer Tomczyk, John Widen, Monika Williams, John Eksterowicz, Daniel Erlanson, Marie Evangelista, Johannes Hermann, Richard M. Neve, Snahel Patel, Kevin R. Webster. Discovery of novel dual-acting KRASG12C inhibitors that target both the active and inactive forms of the protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3601.

Abstract 4107: Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor

Abstract KRASG12C is an important oncogenic driver mutation and occurs in about 13% of NSCLC and 1-3% of colorectal and other solid tumors. Lumakras࣪ (sotorasib), a KRASG12C inhibitor was recently approved for treatment of NSCLC, and others, such as, Adagrasib have shown relatively modest activity for KRASG12C.This modest activity may be partly due to incomplete target occupancy or due to unavailability of the drug at the target site, to elicit maximum clinical response. Clearly, this gives path for development of inhibitors which are potent, but also have desired drug like properties to achieve maximal clinical benefit in patients harboring KRASG12C mutation. Here, we report the identification of VRTX126, a novel, highly selective and potent inhibitor of the KRASG12C, discovered using fragment-based drug design. VRTX126 exhibited an IC50 of 10 nM in H358. VRTX126 also inhibited phospho-ERK with an IC50 of <20 nM and <10 nM in H358 and Mia-Paca-2 cell lines respectively, while demonstrating multi fold selectivity over KRAS wild-type or nonG12C mutant cells lines. In xenograft model harboring KRASG12C mutation, VRTX126 exhibited tumor growth inhibition at therapeutically relevant doses. Combination strategies with other targeted therapies such as, our proprietary SOS1 inhibitor, and other marketed or available MEK1/2, SHP2 inhibitors targeting Ras/Raf/MEK pathway is being explored. VRTX126 showed favourable oral properties with dose proportional pharmacokinetics. VRTX126 was found to have no adverse effect on hERG (10 uM). Additionally, early evaluation in acute (>1 g/kg (p.o.) and >0.2 g/kg (i.v.) was well tolerated, with no elevation in liver enzymes and repeat does studies indicated good safety margins, thus supporting further evaluation of VRTX126 in IND enabling studies. A first-in-human Phase 1 clinical study in KRASG12C mutated patients of NSCLC and mCRC will be planned upon completion of through safety evaluations. Citation Format: Prashant Kashinath Bhavar, Uday Kumar Surampudi, Partha Pratim Sarma, Mahesh Mohan Shidore, Anuj Ramesh Kshirsagar. Discovery and preclinical evaluation of VRTX126, a novel, highly selective and potent KRASG12Cinhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4107.

Abstract 3330: GB5121 is a novel highly potent and selective CNS-penetrant BTK inhibitor for CNS malignancies

Abstract Inhibitors of Bruton’s tyrosine kinase (BTK) are approved treatments for several B cell lymphomas. However, they are characterized by modest selectivity and/or limited central nervous system (CNS) penetrance in instances of CNS disease. GB5121 is an orally available, selective, irreversible small molecule BTK inhibitor. GB5121 was selected based on its potency, specificity, and high brain penetrance in preclinical models. We evaluated its pharmacodynamic properties using both cell-free enzymatic and cell-based functional assays, showing covalent BTK inactivation with a potency in the nM range. GB5121 furthermore demonstrated rapid BTK inactivation kinetics (Kinact/Ki) in both peripheral and CNS tissue, a critical parameter for covalent irreversible inhibitors. In a kinome scan, GB5121 exhibited high kinase selectivity against 349 kinases with only TEC/TXK demonstrating >50% inhibition at 1 µM; GB5121 did not inhibit phosphorylation of EGFR in a cell-based assay. When compared with other BTK inhibitors, GB5121 showed superior CNS target occupancy using a probe-based ELISA measuring free BTK in the brain of mice receiving 3 daily oral doses of 10 mg/kg. GB5121 also demonstrated significantly higher brain to plasma ratio in mice with an intact blood brain barrier. Specifically, GB5121 showed ~2-fold higher brain concentrations with lower plasma levels than ibrutinib. In non-human primates, a 1:1 brain to plasma concentration ratio was demonstrated for GB5121 for up to 8 hours with both oral (30 mg/kg) and IV (2 mg/kg) doses. Together, these features differentiate GB5121 from both FDA-approved BTK inhibitors as well as those currently under clinical investigation. Our data supports the use of GB5121 in clinical trials where BTK is a known driver of malignancies, including in CNS lymphoma. Citation Format: Isharat Yusuf, Sunil Sahdeo, Taylor Ismaili, Zachary Naiman, David Guimond, Theodore Schiff, Bryan Clemons, Krystal Herman, Bryanna Paulson, Kay Hou, Kristen Taylor Meadows, Michael Kennedy, Mark Rose, Laura Carter. GB5121 is a novel highly potent and selective CNS-penetrant BTK inhibitor for CNS malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3330.

Abstract 4026: JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6

Abstract Background: Oncogenic mutations occurring in the KRAS component of the RAS/MAPK pathway slow nucleotide cycling between its active (GTP-bound) and inactive (GDP-bound) states, shifting it towards the active state and increasing oncogenic signaling. Among these mutations, the glycine-to-cysteine mutation of amino acid 12 (KRASG12C), found in ~13% of non-small cell lung cancer (NSCLC) and ~4% of colorectal cancer (CRC), can be specifically targeted and irreversibly locked in the inactive state by covalent modification of Cys12. We have previously reported the discovery and preclinical profile of JDQ443, a selective, oral, covalent inhibitor of KRASG12C that binds under the Switch II loop. Here, we report its antiproliferative and antitumor activity against panels of cancer cell lines as well as cell- (CDX) and patient-derived (PDX) tumor xenografts. Methods: JDQ443 antiproliferative activity was assessed by a high-throughput cell viability assay in a large panel of KRASG12C (n=17) and non-G12C (n=233) cell lines. Single-agent antitumor activity was assessed against a panel of KRASG12C CDX models from NSCLC (LU99, H2122, H2030, and HCC44), pancreatic (Mia PaCa-2), and esophageal (KYSE410) cancer cell lines, plus one non-G12C lung line (H441; KRASG12V). JDQ443 in vivo activity against a panel of KRASG12C NSCLC (n=10) and CRC (n=9) PDX models was assessed either as a single agent or in combination with TNO155 (SHP2 inhibitor [i]), trametinib (MEKi), or ribociclib (CDK4/6i). In vivo combination studies with TNO155 were also performed in CDX models (LU99, H2030, HCC44, and KYSE410). Results: In vitro, JDQ443 demonstrated potent antiproliferative activity selectively towards KRASG12C cell lines. Dose-dependent tumor growth inhibition/regression was observed for all KRASG12C CDX models, but not for the H441 KRASG12V model, and was independent of once-daily (QD) or twice-daily (BID) dosing. Single-agent antitumor activity (best average response, duration of reduction in tumor doubling time) was observed across both PDX panels and was improved by all three combination treatments. The JDQ443/TNO155 combination improved single-agent activity across CDX models, with comparable antitumor benefit maintained at QD or BID TNO155 schedules in two of three models (LU99 and KYSE410). Combination with TNO155 allowed a reduced dose of JDQ443 to achieve similar target occupancy and antitumor activity versus JDQ443 alone. Conclusions: JDQ443 demonstrates significant activity against a broad range of KRASG12C solid tumor models, both in vitro and in vivo, that is increased when combined with agents targeting both upstream and downstream components of the RAS signaling pathway. The combination benefit of JDQ443 + TNO155 over JDQ443 alone is maintained at reduced doses for both agents. Citation Format: Andreas Weiss, Hans Voshol, Diana Graus Porta, Carmine Fedele, Dario Sterker, Ruben De Kanter, Rowan Stringer, Toni Widmer, Alice Loo, Daniel A. Guthy, Kim S. Beyer, Nils Ostermann, Catherine LeBlanc, Marc Gerspacher, Andrea Vaupel, Richard Sedrani, Frederic Zecri, Saveur-Michel Maria, Francesco Hofmann, Peter Hammerman, Jeff Engelman, Edwige Lorthiois, Simona Cotesta, Saskia M. Brachmann. JDQ443, a covalent inhibitor of KRASG12C with a novel binding mode, shows broad antitumor activity in KRASG12C preclinical models as a single agent and in combination with inhibitors of SHP2, MEK or CDK4/6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4026.

Abstract CT033: KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors

Abstract Background: KRAS G12C oncogenic mutations occur in ~13% of non-small cell lung cancers (NSCLCs) and up to 4% of other solid tumors. JDQ443 is a selective, covalent, orally bioavailable, investigational KRASG12C inhibitor that irreversibly traps KRASG12C in the inactive, GDP-bound state. JDQ443 is structurally unique and forms novel interactions with KRAS in the switch II pocket. Methods: KontRASt-01 (NCT04699188) is a Phase Ib/II, open-label, multicenter, dose-escalation and dose-expansion trial of JDQ443 as monotherapy or in combination with TNO155 (SHP2 inhibitor) and/or tislelizumab (anti-PD-1 monoclonal antibody). Primary objectives of dose escalation are to assess safety and tolerability, and identify the maximum tolerated doses (MTDs) and/or recommended doses (RDs) and regimens for future studies. The primary objective of dose expansion is to assess efficacy. Key inclusion criteria: advanced, KRAS G12C-mutated solid tumors; previous standard-of-care treatment; age ≥18 yrs; ECOG PS 0-1. Key exclusion criteria for the JDQ443 monotherapy arm: active brain metastases, prior KRASG12C inhibitor treatment. Here, we present preliminary results for JDQ443 monotherapy dose escalation. Results: As of Nov 3, 2021, 39 pts were treated with JDQ443 PO continuously across 4 dose levels: 200 mg once daily (QD) (n=10), 400 mg QD (n=11), 200 mg twice daily (BID) (n=11), and 300 mg BID (n=7). Median age was 60 yrs (range 26-76), median prior lines of therapy was 3 (range 1-7), and indications included NSCLC (n=20) and colorectal cancer (CRC) (n=16). Median duration of exposure was 9.1 wks (range 0.9-21), with ongoing treatment in most pts (61.5%) at the time of cut-off. Treatment-related adverse events (TRAEs) occurred in 25 (64.1%) pts. Most TRAEs were Grade (Gr) 1-2. Four Gr 3 TRAEs occurred in 4 (10.3%) separate pts; there were no Gr 4-5 TRAEs. The most common TRAEs (occurring in ≥10% of pts) were fatigue (25.6%), nausea (15.4%), edema (12.8%), pruritus (10.3%), and vomiting (10.3%). There was one DLT (Gr 3 fatigue) and one treatment-related serious AE (Gr 3 photosensitivity reaction), each in separate pts treated at 300 mg BID. TRAEs led to dose reduction in 1 pt and discontinuation in 1 pt. A MTD was not reached. The RD was declared as 200 mg BID. At the RD, PK and PD modeling for JDQ443 predicted average KRASG12C target occupancy of >90% in >82% of pts. Using an efficacy cut-off date of Dec 13, 2021, for the 20 pts with NSCLC among the same 39 pts, the ORR (confirmed complete response or partial response) by RECIST 1.1 was 30.0% (6/20) across dose levels and 43.0% (3/7) at the RD. Additional data will be available at the time of presentation. Conclusions: JDQ443 demonstrates an acceptable safety and tolerability profile, with early signs of clinical activity in pts with NSCLC. Enrollment is ongoing to NSCLC and CRC dose-expansion groups for JDQ443 monotherapy at the RD, and to JDQ443 + TNO155 dose escalation. Citation Format: Daniel S. Tan, Toshio Shimizu, Benjamin Solomon, Rebecca S. Heist, Martin Schuler, Maria J. De Miguel Luken, Anas Gazzah, Martin Wermke, Christophe Dooms, Herbert H. Loong, Neeltje Steeghs, Enriqueta Felip, Conor E. Steuer, Eric van Cutsem, Ross A. Soo, Ashley C. Jaeger, Jaeyeon Kim, Kun Xu, Xueying Chen, Xiaoming Cui, Heather Burks, Anna Farago, Philippe A. Cassier. KontRASt-01: A phase Ib/II, dose-escalation study of JDQ443 in patients (pts) with advanced, KRAS G12C-mutated solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT033.

Abstract 5076: Evaluation of the CNS penetration of a next generation PARP inhibitor, AZD9574, in cynomolgus monkey using positron emission tomography

Abstract The current clinically approved PARP inhibitors have limited subtype selectivity and are to some degree restricted in their ability to penetrate the central nervous system (CNS) due to efflux transporters, potentially limiting their efficacy in treating metastatic disease or primary tumors in the brain. The current study evaluated the potential of AZD9574, a next generation, PARP1 selective inhibitor/trapper, to penetrate the CNS in cynomolgus monkey, and its occupancy of the PARP1 enzyme, using positron emission tomography (PET). In vitro bidirectional efflux assay data suggested AZD9574 showed minimal substrate potential compared to the clinically approved PARP inhibitors. This was reflected in an increased ratio of unbound brain to unbound plasma concentration (Kpuu) in the rat of ~0.31. Therefore AZD9574 was taken forward into cynomolgus monkey PET studies. Firstly, the Kpuu was determined following dosing of [11C]AZD9574, co-administered with unlabeled drug to minimize the impact of specific binding. The high specific signal observed lead to the development of [11C]AZ3391, a PARP1 selective, CNS penetrant PET tracer, which was subsequently used to directly assess the PARP1 target engagement of AZD9574 in the brain. AZD9574 was found to show a Kpuu in cynomolgus monkeys of 0.79, close to unity with unbound plasma concentrations suggesting minimal CNS restriction. Furthermore, an i.v. infusion dose response study with AZD9574, conducted to examine its ability to block target occupancy by the PET tracer [11C]AZ3391, demonstrated a reduction in [11C]AZ3391 accumulation in whole brain. The resulting calculated occupancy of AZD9574 ranged from 17% for the lowest dose (0.003 mg/kg) to 95% for the highest dose tested (0.05 mg/kg). Comparable reduction in occupancy was seen for peripheral tissue, such as bone marrow, supporting the conclusion that AZD9574 shows minimal CNS restriction. These data show that AZD9574 is the first PARP inhibitor to reach the clinic which combines PARP1 selectivity, trapping and high CNS penetration in a single molecule and supports its development as a potential therapy for the treatment of metastatic disease and primary brain tumors. Citation Format: Andy Pike, Amber Balazs, Zsolt Cselényi, Sébastien L. Degorce, Avipsa Ghosh, Sudhir M. Hande, Jeffrey Johannes, Peter Johnström, Martin J. Packer, Magnus Schou, XiaoLan Zheng. Evaluation of the CNS penetration of a next generation PARP inhibitor, AZD9574, in cynomolgus monkey using positron emission tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5076.

GNUV201, a novel human and mouse cross-reactive PD-1 monoclonal antibody for cancer immunotherapy.

e14509 Background: Several PD-1 antibodies have been recently approved as anti-cancer therapies which work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. However, cross-reactive anti-PD-1 antibodies to both human and pre-clinically relevant animal models are still required to predict more precisely the efficacy and toxicity in humans, especially in various combination settings of anti-PD-1 with other treatments. Methods: Therefore, we have developed an antibody named GNUV201, a highly selective and interspecies cross-reactive humanized monoclonal antibody, with conventional hybridoma technology. Results: GNUV201 equally binds to mouse and human PD-1 (EC50= 32 pM and 28 pM, respectively), while marketed Keytruda and Opdivo bind only to human PD-1. According to the results of co-crystal structure and alanine-scanning mutagenesis to examine the interaction mode between GNUV201 and human PD-1, the epitope recognized by GNUV201 is on the “FG loop” region of human PD-1, which is well conserved in both human and mouse PD-1, supporting GNUV201’s inter-species cross-reactivity, and this position is distinct from those of Keytruda (“C’D loop”) and Opdivo (N-term). Notably, the structural feature in which the protruding epitope loop fits into GNUV201’s binding pocket supports its enhanced binding affinity due to slower dissociation (8.5 times slower than Keytruda in SPR). Furthermore, GNUV201 shows stronger binding affinity at pH 6.0 (KD = 0.9 nM at pH 6.0, 5.8 times smaller KD than at pH 7.4) which mimics more closely the hypoxic and acidic tumor micro-environments (TME), especially due to further improved Koff. This phenomenon is not observed with other anti-PD-1 competitors, implying that GNUV201 achieves better target occupancy in tumors compared to its marketed competitors. GNUV201 was also shown to successfully block the interaction of hPD-1 with hPD-L1 at similar single digit nM levels compared with Keytruda and Opdivo in vitro and strongly suppress tumor growth in syngeneic models. In particular, tumor growth regressed in B16F11 (mouse melanoma) and was inhibited in MC38 (mouse colon cancer) syngeneic models and MC38/hPD-1 knock-in mouse models during treatment, when compared with the original tumor size. Conclusions: In summary, we expect GNUV201 to be an excellent antibody candidate whose efficacy and toxicity in the human system can be predicted from preclinical study results due to its human-mouse cross-reactivity and whose efficacy can be further improved with better target occupancy due to its slower dissociation and preferential binding in low pH tumor microenvironments. We are currently engineering GNUV201 to further improve antigen binding kinetics, which could lead to enhanced efficacy and pharmacokinetics in humans, and to develop the best-in-class PD-1 antibody based on superior comparative data from on-going efficacy studies.

Phase I/IIa study of PM8001, a bifunctional fusion protein targeting PD-L1 and TGF-β, in patients with advanced tumors.

2512 Background: PM8001 is a bifunctional protein composed of the extracellular domain of the TGF-β RII receptor (a TGF-β “trap”) fused to a humanized anti-PD-L1 IgG1 single-domain antibody. This is the first dose escalation and expansion phase I/IIa study to evaluate the safety and preliminary anti-tumor activity of PM8001 in advanced tumors. Methods: This study is comprised of a standard 3+3 dose escalation (Part A: 1, 3, 10, 20, 30, and 45 mg/kg Q2W) followed by dose expansion (Part B). Primary endpoints include safety for Part A, and ORR per RECIST 1.1 for Part B. Secondary endpoints include pharmacokinetics (PK), immunogenicity, DCR, PFS, and OS. Results: As of 16 December, 2021, a total of 108 subjects had received at least 1 dose of PM8001 with 25 subjects in Part A (0.3 mg/kg [n = 1], 1 mg/kg [n = 3], 3 mg/kg [n = 3], 10 mg/kg [n = 4], 20 mg/kg [n = 7], 30 mg/kg [n = 3], and 45 mg/kg [n = 4]) and 83 subjects in Part B (10 mg/kg Q2W [n = 1], 20 mg/kg Q2W [n = 63], 20 mg/kg Q3W [n = 11]), and 30 mg/kg Q3W [n = 8]). In Part A, only 1 DLT occurred in the 20 mg/kg dose group. MTD was not reached. Finally, 20 mg/kg Q2W was recommended as the RP2D for Part B. Of the 108 subjects, the median duration of PM8001 exposure was 6.9 weeks (range, 1.4-71.9). Any-grade TRAEs occurred in 84 subjects (77.9%), with 18 subjects (16.7%) reported with ≥ Grade 3. The most commonly reported ≥ Grade 3 TRAEs were anemia and rash (3 subjects each). Any-grade irAEs occurred in 66 subjects (61.1%), with 13 subjects (12.0%) reported with ≥ Grade 3. The most commonly reported ≥ Grade 3 irAEs were anemia (3 subjects). PK analysis showed a nearly linear dose-exposure relationship with PM8001 dosing from 1 to 45 mg/kg. Pharmacodynamic analysis demonstrated close to 100% PD-L1 target occupancy on PBMCs from the 20 to 45 mg/kg groups after multiple doses. An adequate inhibition to below the limit of quantitation of TGF-β1 was observed in the 1 mg/kg and higher dose groups. Of the 108 enrolled subjects, 67 subjects completed at least one efficacy evaluation. The ORR per RECIST 1.1 by investigator was 10.4% (7/67; 95% CI, 4.3%-20.4%), with 7 subjects achieving PR; the DCR was 53.7% (36/67; 95% CI, 41.1%-66.0%). Four additional PRs were observed after the data cut-off date. Of the total 11 PRs, 4 PRs occurred in patients who previously progressed after anti-PD-1 treatment. Most responses (10/11) occurred within 6 infusions, which indicates a rapid response towards PM8001 treatment. To date, 8 responders are still on treatment. Conclusions: PM8001 showed an acceptable safety profile and promising anti-tumor activity in advanced solid tumors, especially in patients previously treated with checkpoint inhibitors, which supports further studies to explore the safety and efficacy of PM8001 as a monotherapy and in combination with other anti-tumor agents. Global Phase II monotherapy and combination studies are currently ongoing. Clinical trial information: ChiCTR2000033828.

Shaping health and well-being in a COVID era: the role of design

PurposeIn this study, an interdisciplinary research team at a Midwest US University collaborated with a local county to co-envision interior design strategies for five county buildings: three libraries and two government buildings to reduce in-person contact in response to the COVID-19 pandemic.Design/methodology/approachThe authors' interdisciplinary team used a community-based participatory design process which focused on creating consensus, while seeking out divergent perspectives among stakeholders to serve the needs of diverse users. The design process involved meetings with stakeholders remotely and analyzing survey results from the target occupants collected by the county.FindingsThe county with a population of 550,321 is the second most populous and diverse county in the state. The authors' collaborative efforts resulted in short-and long-term recommendations for the interior space planning to promote health, safety, and well-being for the county's diverse user groups of young children, adults, elderly and vulnerable populations. The short-term recommendations focus on service redesigns that can be implemented as the state shifts out of the stay-at-home order and the community returns to the county's public-facing buildings. The long-term recommendations focus on experiences and design strategies that can be carried forward to future-proof buildings in a post-COVID era and provide models for other counties.Research limitations/implicationsOne limitation is the dynamic nature of how rapidly our team responded to a critical need in the community to develop tangible interior design solutions during the initial stages of the COVID-19 pandemic for the county. The solutions we proposed were based on the scientific evidence available earlier on during that phase of the pandemic. The authors hope to conduct further studies in the future and conduct assessments of our rapid design responses.Practical implicationsThis paper documents a dynamic experience during a once in a lifetime pandemic and thus contributes to further the body of knowledge about the role of interior design in shaping health and well-being during the COVID-19 pandemic.Originality/valueThe article presents a timely interdisciplinary response to the COVID-19 pandemic to promote community safety inside public buildings at the county. The interior design solutions reflected intensive literature reviews, critical space planning, innovative use of lighting, and thoughtful furniture/material selections.

Target occupancy study and whole-body dosimetry with a MAGL PET ligand [11C]PF-06809247 in non-human primates

BackgroundMonoacylglycerol lipase (MAGL) is a key serine hydrolase which terminates endocannabinoid signaling and regulates arachidonic acid driven inflammatory responses within the central nervous system. To develop [11C]PF-06809247 into a clinically usable MAGL positron emission tomography (PET) radioligand, we assessed the occupancy of MAGL by an inhibitor in the non-human primate (NHP) brain. Additionally, we measured the whole-body distribution of [11C]PF-06809247 in NHP and estimated human effective radiation doses.MethodsSeven cynomolgus monkeys were enrolled for brain PET measurements. Two PET measurements along with arterial blood sampling were performed in each NHP: one baseline and one pretreatment condition with intravenous administration of PF-06818883, a pro-drug of a selective MAGL inhibitor (total of seven doses between 0.01 and 1.27 mg/kg). Kinetic parameters K1, k2 and k3 were estimated by a two tissue compartment (2TC) model using metabolite corrected plasma radioactivity as the input function. k4 was set as 0 according to the irreversible binding of [11C]PF-06809247. Ki by 2TC and Patlak analysis were calculated as the influx constant. The target occupancy was calculated using Ki at baseline and pretreatment conditions. Two cynomolgus monkeys were enrolled for whole-body PET measurements. Estimates of the absorbed radiation dose in humans were calculated with OLINDA/EXM 1.1 using the adult male reference model.ResultsRadioactivity retention was decreased in all brain regions following pretreatment with PF-06818883. Occupancy was measured as 25.4–100.5% in a dose dependent manner. Whole-body PET showed high radioactivity uptake values in the liver, small intestine, kidney, and brain. The effective dose of [11C]PF-06809247 was calculated as 4.3 μSv/MBq.Conclusions[11C]PF-06809247 is a promising PET ligand for further studies of MAGL in the human brain.

A guide to enzyme kinetics in early drug discovery.

Drugs interact with their target of interest to bring about the desired phenotypic outcome that results in disease alleviation. Traditionally, most lead optimization exercises were driven by affinity measures (like IC50 ) to inform structure-activity relationship (SAR)-guided medicinal chemistry. However, an IC50 value is a thermodynamic estimate measured under equilibrium conditions that can vary as a function of substrate concentration and/or time (the latter especially for nonequilibrium modalities). Further, like other thermodynamic estimates, it is a state-function that is indifferent to the path traversed from the initial state to the final state. This can be a cause for concern in drug discovery given the predominance of nonequilibrium interactions and the open thermodynamic nature of the human system. Under such situations, employing rates along with equilibrium constants (or IC50 values) would be far more relevant to capture the time evolution of the small molecule's interaction with the target of interest. These rates are generally typified by the rate of association, rate of dissociation and the residence time of the small molecule on the target (target occupancy). These parameters, when combined with the concept of target vulnerability, therapeutic window, pharmacokinetic profile of the small molecule, estimates of endogenous ligand and target turnover, will shed critical insights into the kinetics and dynamics of a small molecule's interaction with the protein, and allow realistic modelling of the system to enable optimizations and dosing decisions. With that aim, this guide will attempt to introduce the traditional role of mechanistic enzymology within drug discovery and emphasize the importance of kinetics in guiding SAR-based optimizations. It will also present initial ideas on how kinetic investigation should be positioned relative to the temporal span of a drug-discovery pipeline to leverage maximal utility from the investment in time and effort.

Development of High Brain-Penetrant and Reversible Monoacylglycerol Lipase PET Tracers for Neuroimaging.

Monoacylglycerol lipase (MAGL) is one of the key enzymes in the endocannabinoid system. Inhibition of MAGL has been proposed as an attractive approach for the treatment of various diseases. In this study, we designed and successfully synthesized two series of piperazinyl pyrrolidin-2-one derivatives as novel reversible MAGL inhibitors. (R)-[18F]13 was identified through the preliminary evaluation of two carbon-11-labeled racemic structures [11C]11 and [11C]16. In dynamic positron-emission tomography (PET) scans, (R)-[18F]13 showed a heterogeneous distribution and matched the MAGL expression pattern in the mouse brain. High brain uptake and brain-to-blood ratio were achieved by (R)-[18F]13 in comparison with previously reported reversible MAGL PET radiotracers. Target occupancy studies with a therapeutic MAGL inhibitor revealed a dose-dependent reduction of (R)-[18F]13 accumulation in the mouse brain. These findings indicate that (R)-[18F]13 ([18F]YH149) is a highly promising PET probe for visualizing MAGL non-invasively in vivo and holds great potential to support drug development.

Unraveling the origin of higher success probabilities in quantum annealing versus semi-classical annealing

Quantum annealing aims at finding optimal solutions to complex optimization problems using a suitable quantum many body Hamiltonian encoding the solution in its ground state. To find the solution one typically evolves the ground state of a soluble, simple initial Hamiltonian adiabatically to the ground state of the designated final Hamiltonian. Here we explore whether and when a full quantum representation of the dynamics leads to higher probability to end up in the desired ground when compared to a classical mean field approximation. As simple but nontrivial example we target the ground state of interacting bosons trapped in a tight binding lattice with small local defect by turning on long range interactions. Already two atoms in four sites interacting via two cavity modes prove complex enough to exhibit significant differences between the full quantum model and a mean field approximation for the cavity fields mediating the interactions. We find a large parameter region of highly successful quantum annealing, where the semi-classical approach largely fails. Here we see strong evidence for the importance of entanglement to end close to the optimal solution. The quantum model also reduces the minimal time for a high target occupation probability. Surprisingly, in contrast to naive expectations that enlarging the Hilbert space is beneficial, different numerical cut-offs of the Hilbert space reveal an improved performance for lower cut-offs, i.e. an nonphysical reduced Hilbert space, for short simulation times. Hence a less faithful representation of the full quantum dynamics sometimes creates a higher numerical success probability in even shorter time. However, a sufficiently high cut-off proves relevant to obtain near perfect fidelity for long simulations times in a single run. Overall our results exhibit a clear improvement to find the optimal solution based on a quantum model versus simulations based on a classical field approximation.

Optimization of a novel piperazinone series as potent selective peripheral covalent BTK inhibitors

BTK is a tyrosine kinase playing an important role in B cell and myeloid cell functions through B cell receptor (BCR) signaling and Fc receptor (FcR) signaling. Selective inhibition of BTK has the potential to provide therapeutical benefits to patients suffering from autoimmune diseases. Here we report the design, optimization, and characterization of novel potent and highly selective covalent BTK inhibitors. Starting from a piperazinone hit derived from a selective reversible inhibitor, we solved the whole blood cellular potency issue by introducing an electrophilic warhead to reach Cys481. This design led to a covalent irreversible BTK inhibitor series with excellent kinase selectivity as well as good whole blood CD69 cellular potency. Optimization of metabolic stability led to representative compounds like 42, which demonstrated strong cellular target occupancy and inhibition of B-cell proliferation measured by proximal and distal functional activity.

Target identification and occupancy measurement of necrosis avid agent rhein using bioorthogonal chemistry-enabling probes.

Necrosis is an important biomarker, which only occurs in pathological situations. Tracking of necrosis avid agents is of crucial importance toward understanding their mechanisms. Herein, we developed a modular probe strategy based on bioorthogonal copper-free click chemistry. Structural modification of rhein with transcyclooctene (TCO) led to the identification of rhein-TCO2 as the most active probe with specific necrosis affinity. In a systematic evaluation, the colocalization of rhein-TCO2 in the nucleus (exposed DNA and rRNA) of necrotic cells was observed. This work provides a foundation for the development of target-identified of rhein compounds, and binding to exposed DNA and rRNA may be an important target of rhein compounds in necrotic cells.