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No targeted treatments are currently approved for HER2 exon 20 insertion–mutant lung adenocarcinoma patients. Mobocertinib (TAK-788) is a potent irreversible tyrosine kinase inhibitor (TKI) designed to target human epidermal growth factor receptor 2 (HER2/ERBB2) exon 20 insertion mutations. However, the function of mobocertinib on HER2 exon 20 insertion–mutant lung cancer is still unclear. Here we conducted systematic characterization of preclinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion–mutant cell lines, the IC50 of mobocertinib was higher than poziotinib and comparable with or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC50/wild-type (WT) EGFR IC50 ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20YVMA allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20G776>VC lung tumors exhibited a sustained complete response to mobocertinib, whereas HER2 exon 20YVMA tumors showed only partial and transient response. Combined treatment with a second antibody–drug conjugate (ADC) against HER2, ado-trastuzumab emtansine (T-DM1), synergized with mobocertinib in HER2 exon 20YVMA tumors. In addition to the tumor cell autonomous effect, sustained tumor growth control derived from M1 macrophage infiltration and CD4+ T-cell activation. These findings support the ongoing clinical development of mobocertinib (NCT02716116) and provide a rationale for future clinical evaluation of T-DM1 combinational therapy in HER2 exon 20YVMA insertion–mutant lung adenocarcinoma patients. Significance: This study elucidates the potent inhibitory activity of mobocertinib against HER2 exon 20 insertion–mutant lung cancer and the synergic effect of combined mobocertinib and T-DM1, providing a strong rationale for clinical investigation.

The design of an optimal combination therapy can be thought of as a set of tradeoffs designed to maximize the efficacy of the combination for a given toxicity budget. As anticancer agents (even targeted ones) often have overlapping toxicities, an efficient approach to making efficacy-toxicity tradeoffs is critical. In this context, a major impediment is that the search space grows exponentially with each additional drug while in vivo studies have a fixed upper size limit. Here, we model the efficacy and toxicity landscapes of multidrug cocktails, and describe an approach to visualize and design efficient studies for three-drug combinations. We further demonstrate the application of our approach in a practical drug development context by showing its impact on actual study design, and validation with in vivo datasets. As our approach relies on equations from first principles, it is in theory extensible to an arbitrary number of drugs, subject to the practical constraints of drug dosing. First, we demonstrate an approach to visually determine the optimal dose combination for a three-drug combination with fully overlapping toxicity. Efficacy and toxicity isoboles visualized as surfaces in three-drug space demonstrate the optimal dose combination, which corresponds to the point of tangency between the MTD and efficacy isobole surfaces. Next, we derive an analytical method to efficiently estimate this point with a minimum of data, and without using graphical methods (important for extending the work beyond three dimensions). The combined efficacy and toxicity of a three-drug combination were modeled from first principles using isobolograms as a sum of the single-agent PK/Efficacy (PK/E) relationships and four combination terms (three binary and one ternary) to account for potential interactions between the drugs. Borrowing from Microeconomic Utility Theory, we found the point of greatest efficacy along the Maximum Tolerated Dose (MTD) toxicity contour, which corresponds to the efficacy isobole tangentially intersecting the MTD contour. Through both analytical and numerical approaches, we determined the the single- and double-agent efficacy parameters uniquely determine the dose escalation path which provides the best estimate of combination efficacy. Simulated experiments demonstrate the optimal observation path up to MTD performs acceptably compared to a uniformly-gridded exposure space. Finally, we demonstrate the application of this approach in a proposed experimental design for three-drug combinations, and validate it with experimental data. While developing multi-drug cocktails poses many scientific and operational challenges, the approach presented here provides a straightforward route to preclinical testing and validation through the design of parsimonious studies that can explicitly define the contribution of each individual drug (and each two drug combination) to the overall efficacy and toxicity landscape of the cocktail. Citation Format: Christopher J. Zopf, Andrew Chen, Santhosh Palani, Rachael Brake, Mark Manfredi, Jeffrey Ecsedy, Wen Chyi Shyu, Arijit Chakravarty. Rational dose optimization for multi-drug cocktails. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 791. doi:10.1158/1538-7445.AM2014-791

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1795. doi:1538-7445.AM2012-1795

Anaplastic lymphoma kinase (ALK) is predominantly expressed in the brain and spinal cord during neonatal development and is subsequently down-regulated in the adult. In cancer, ALK is ectopically expressed as a consequence of one of several different cytogenetic translocations or inversions that lead to the expression of an oligomerized, constitutively active, intracellular tyrosine kinase. The resulting fusion protein has been identified in anaplastic large cell lymphomas, non small cell lung carcinomas and more recently breast and colorectal carcinomas. Preclinical data convincingly demonstrates that fusion transcripts can drive tumorigenesis and inhibition of ALK in vitro and in vivo leads to inhibition of cellular growth. Full length (fl) ALK is also expressed in a variety of other tumor types, and recent data suggest that at least in neuroblastoma, the flALK transcript contains activating point mutations in the tyrosine kinase domain. These data suggest that flALK may also be a therapeutic target in cancer. Here we demonstrate that a significant subset of ovarian carcinoma cell lines and biopsy specimens aberrantly express ALK. Gene expression analysis in 126 ovarian carcinoma samples demonstrated a prevalence of ALK expression of 19%. Analysis of the histological subtypes within this study, showed specific enrichment of ALK transcript in papillary ovarian carcinoma (15 of 42 samples). This subtype showed a range of ALK expression between 2 - 4 fold increase above normal ovarian tissue. We have also confirmed that in a subset of ovarian tumor cell lines and primary tumor samples that ALK transcript is converted into ALK protein. DNA sequence analysis has revealed that in these patient samples and cell lines, ALK is predominantly wild type. Single nucleotide polymorphism (SNP) analysis and fluorescent in situ hybridization (FISH) suggest that over expression may occur as a consequence of low copy number amplification. These preliminary findings suggest that ALK expression in cancer has now been observed in ovarian carcinomas by a mechanism distinct from cytogenetic translocations or inversions. Currently, we are attempting to correlate the aberrant expression of ALK with ovarian cancer prognosis as well as understand the value of inhibiting ALK expression in this cell type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3142.