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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 pre-clinical models to understand the activity profile of mobocertinib against HER2 exon 20 insertions. In HER2 exon 20 insertion mutant cell lines, the IC(50) of mobocertinib was higher than poziotinib and comparable or slightly lower than afatinib, neratinib, and pyrotinib. Mobocertinib had the lowest HER2 exon 20 insertion IC(50) / WT EGFR IC(50) ratio, indicating that mobocertinib displayed the best selectivity profile in these models. Also, mobocertinib showed strong inhibitory activity in HER2 exon 20(YVMA) allograft and patient-derived xenograft models. In genetically engineered mouse models, HER2 exon 20(G776>VC) lung tumors exhibited a sustained complete response to mobocertinib, while HER2 exon 20(YVMA) 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 20(YVMA) 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 20(YVMA) insertion-mutant lung adenocarcinoma patients.

Characterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/μg gDNA and copy/μL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies. Supplementary Information The online version contains supplementary material available at 10.1208/s12248-021-00560-6.

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