IntroductionThe serine/threonine kinase 17B (STK17B) is involved in setting the threshold for T cell activation and its absence sensitizes T cells to suboptimal stimuli. Consequently, STK17B represents an attractive potential target for cancer immunotherapy.MethodsTo assess the potential of STK17B as an immuno-oncology target, we developed potent and selective tool compounds from starting points in Blueprint Medicines Corporation's proprietary kinase inhibitor library. To characterize these molecules, enzyme and cellular assays for STK17A and STK17B were established to drive chemistry optimization. Mass spectrometry-based phosphoproteomics profiling with tool inhibitors led to the identification of Ser19 on myosin light chain 2 as STK17B substrate, which is then developed into a flow cytometry-based pharmacodynamic readout of STK17B inhibition both in vitro and in vivo.ResultsIn a mouse T cell activation assay, STK17B inhibitors demonstrated the ability to enhance interleukin-2 (IL-2) production. Similarly, treatment with STK17B inhibitors resulted in stronger cytokine secretion in human T cells activated using a T cell bispecific antibody. Subsequent chemistry optimization led to the identification of a highly selective and orally bioavailable tool compound, BLU7482. In vivo, STK17B inhibition led to dose-dependent modulation of myosin light chain 2 phosphorylation and enhanced priming of naïve T cells, as determined by upregulation of CD69, IL-2 and interferon-γ secretion. In line with increased T cell activation, treatment with STK17B inhibitor enhanced antitumor activity of anti–PD-L1 antibody in the MCA205 model.ConclusionsIn summary, we successfully identified and optimized STK17B kinase inhibitors which led to increased T cell responses in vitro and in vivo. This allowed us to evaluate the potential of STK17B inhibition as an approach for cancer immunotherapy.

While epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment landscape for EGFR mutant (L858R and ex19del)-driven non small-cell lung cancer (NSCLC), most patients will eventually develop resistance to TKIs. In the case of first-and second generation TKIs, up to 60% of patients will develop an EGFR T790M mutation, while third-generation irreversible TKIs, like osimertinib, lead to C797S as the primary on-target resistance mutation. The development of reversible inhibitors of these resistance mutants is often hampered by poor selectivity against wild-type EGFR, resulting in potentially dose-limiting toxicities and a sub-optimal profile for use in combinations. BLU-945 (compound 30) is a potent, reversible, wild-type-sparing inhibitor of EGFR+/T790M and EGFR+/T790M/C797S resistance mutants that maintains activity against the sensitizing mutations, especially L858R. Pre-clinical efficacy and safety studies supported progression of BLU-945 into clinical studies, and it is currently in phase 1/2 clinical trials for treatment-resistant EGFR-driven NSCLC.

BACKGROUND: Lung cancer is the leading cause of cancer death globally. EGFR mutations are the most common genomic drivers of non-small cell lung cancer (NSCLC), occurring in ~17% and up to 50% of Caucasian and Asian patients, respectively, with exon 19 deletions (ex19del) and L858R substitutions being the most prevalent activating EGFR mutations (EGFRm). Outcomes have improved in patients with EGFR-driven NSCLC since the introduction of EGFR tyrosine kinase inhibitors (TKIs); however, treatment resistance almost inevitably occurs. It has been reported that patients harboring the EGFR L858R mutation, who represent 3.4% of lung adenocarcinoma cases in North America and 23% in Asia, have poorer outcomes than those with EGFR ex19del, suggesting a significant unmet need in this population. BLU-945 and BLU-701 are investigational, reversible, selective, and orally available TKIs optimized for use as single-agent or combination therapy to effectively suppress activating and on-target resistance EGFR mutants while sparing WT EGFR, and have the potential to treat or prevent central nervous system metastases. In vivo studies previously demonstrated the antitumor activity of BLU-945 against EGFR L858R/T790M and EGFRm/T790M/C797S mutants, and BLU-701 against EGFR ex19del and EGFRm/C797S mutants. Because EGFR L858R-driven NSCLC constitutes a large population of patients who may not be receiving the same benefit from third-generation (3G) TKIs as those with EGFR ex19del, studies were conducted to evaluate the antitumor activity of BLU-945 and BLU-701, as single agents and in combination, in preclinical NSCLC tumor models driven by L858R in the absence of the T790M mutation. METHODS: The in vivo antitumor activities of BLU-945 and BLU-701, as single-agents or in combination, were evaluated in 2 EGFR L858R-driven patient-derived xenograft (PDX) subcutaneous tumor models and in an engineered EGFR L858R/C797S-driven Ba/F3 cell line-derived xenograft (CDX) subcutaneous tumor model. RESULTS: Oral daily administration of single-agent BLU-945 and single-agent BLU-701 resulted in sustained tumor regression in 2 PDX models of EGFR L858R NSCLC. Compared with single agents in an EGFR L858R/C797S Ba/F3 CDX tumor model, combination of BLU-945 with BLU-701 resulted in marked antitumor activity and prolonged tumor regression. CONCLUSION: The in vivo antitumor activities of BLU-945 and BLU-701 in preclinical tumor models suggest that both BLU-945 and BLU-701 have the potential to be used in patients with EGFR L858R-driven NSCLC, including those who are treatment naïve or previously treated with 3G TKI. Combining BLU-945 and BLU-701 may enable coverage of frequent on-target resistance mechanisms, including the EGFR C797S mutation, in addition to the common L858R activating mutation. Citation Format: Luz Tavera, Stefanie Schalm, John Campbell, Jian Guo, Clare Medendorp, Maxine Chen, Faris Albayya, Tom Dineen, Zhuo Zhang, Maria Iliou, Ebby Job, Nisha Perez, Yoav Timsit, Scott Wardwell, Katie McGinn, Richard Woessner, Chiara Conti. Antitumor activity of BLU-945 and BLU-701 as single agents and in combination in EGFR L858R-driven models of NSCLC [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 3328.

Abstract BACKGROUND: Non-small cell lung cancer (NSCLC) represents 80% of lung cancers, the second leading cause of cancer deaths globally. Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) are genomic drivers in a molecular sub-type of NSCLC that is sensitive to tyrosine kinase inhibitors (TKIs). Third-generation, irreversible TKIs, such as osimertinib and lazertinib, have become standard of care and are now widely used as first-line therapy in EGFR-driven metastatic NSCLC. However, resistance invariably arises during treatment. The C797S mutation is the most frequent on-target resistance mechanism in response to third-generation TKIs when used in the first-line setting. There are currently no approved therapies for patients who progress with a C797S mutation. Although first-generation TKIs inhibit EGFR with a C797S mutation, they carry liabilities of 1) insufficient selectivity over wild-type (WT) EGFR, which precludes maximal target coverage and results in dose-limiting adverse events; and 2) limited brain penetration. METHODS: BLU-701 activity was tested in biochemical assays for EGFR mutants and EGFR WT. Cellular activity was evaluated by a phosphorylation specific EGFR AlphaLisa assay in WT cell lines or those expressing EGFR mutations. The in vivo antitumor activity of BLU-701 was assessed in a PC9 ex19del cell line-derived tumor xenograft (CDX) model. RESULTS: We have developed an orally available, single-digit nanomolar, brain-penetrant, WT-sparing, reversible EGFR inhibitor that is active against the C797S resistance mutation. BLU-701 is equally potent against both the sensitizing mutations ex19del and L858R, and the resistance double mutants ex19del/C797S and L858R/C797S. BLU-701 is brain penetrant (Kpu,u >0.9) and demonstrates a selectivity over WT EGFR that compares favorably with osimertinib. Oral administration of BLU-701 to PC9 ex19del tumor-bearing mice resulted in strong and prolonged pathway suppression and tumor regression at tolerated doses. CONCLUSION: Due to its potent pharmacological activity and selectivity for mutant EGFR, BLU-701 has the potential to demonstrate activity in first-line and resistance settings as a single agent and in combination therapy, addressing potential tumor heterogeneity. The pre-clinical data described here supports the clinical development of BLU-701 in EGFR-driven NSCLC. Citation Format: Chiara Conti, John Campbell, Rich Woessner, Jian Guo, Yoav Timsit, Maria Iliou, Scott Wardwell, Alison Davis, Sharon Chicklas, John Hsieh, Meredith Eno, Omar Ahmad, Dilinie Fernando, Kevin Barvian, Joseph Kim, Steven Kazmirski, Emanuele Perola, Tom Dineen, Victoria Brown, Timothy Guzi, Ayşegül Özen, Faith Stevison, Caitlin Utt, Clare Medendorp, Robert Meissner, Marion Dorsch, Klaus Hoeflich. BLU-701 is a highly potent, brain-penetrant and WT-sparing next-generation EGFR TKI for the treatment of sensitizing (ex19del, L858R) and C797S resistance mutations in metastatic NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1262.