<p>Combining tucatinib and T-DM1 in HER2-positive breast cancer xenograft models increases antitumor activity. Mean tumor volume over time in T-DM1–resistant breast cancer PDX models (<b>A</b>) and BT-474 CDX model (<b>B</b>). Tucatinib was administered orally at 50 mg/kg twice daily for the duration of the study, while T-DM1 and the IgG1-DM1 nonbinding control ADC were dosed at 10 mg/kg (single dose). <b>C,</b> Summary table of tumor growth inhibition and response rates of subcutaneous-implant xenograft models. <b>D,</b> Mean tumor volume over time in intracranially implanted BT-474-RedLuc xenograft model. <b>E,</b> Kaplan–Meier survival plots of intracranial BT-474-RedLuc xenograft model. <b>F,</b> Summary table of tumor growth inhibition and median survival days of intracranial BT-474-RedLuc xenograft model. All <i>P</i> values were determined by comparing the tucatinib/T-DM1 combination with the closest single agent. Data shown as mean ± SEM.</p>
<p>The combined suppression of HER2 signaling by tucatinib and T-DM1 is associated with reduced tumor growth. <b>A,</b> ELISAs quantifying phosphorylation of signaling components downstream of HER2 in BT-474 cells (as percentage phosphorylated protein vs. untreated cells with SEMs). <b>B,</b> IHC images of pHER2, pHER3, pAKT, and pMEK staining of the BT-474 xenograft tumor model. Inlay images represent HER2-stained tumor boundaries. <b>C,</b> HALO-based quantification of histologic analysis of HER2 and phospho-epitopes downstream of HER2 in the BT-474 tumor model<b>.</b> IHC images (<b>D</b>) and HALO-based quantification (<b>E</b>) of proliferation marker Ki67 in the BT-474 tumor model (7 days after dosing, 1–2 hours after final dose). Inlay images represent HER2-stained tumor boundaries. <b>F,</b> HALO-based quantification of histologic analysis of caspase-3 in the BT-474 tumor model. Results are representative of at least 2 independent experiments.</p>
<p>The combined suppression of HER2 signaling by tucatinib and T-DM1 is associated with reduced tumor growth. <b>A,</b> ELISAs quantifying phosphorylation of signaling components downstream of HER2 in BT-474 cells (as percentage phosphorylated protein vs. untreated cells with SEMs). <b>B,</b> IHC images of pHER2, pHER3, pAKT, and pMEK staining of the BT-474 xenograft tumor model. Inlay images represent HER2-stained tumor boundaries. <b>C,</b> HALO-based quantification of histologic analysis of HER2 and phospho-epitopes downstream of HER2 in the BT-474 tumor model<b>.</b> IHC images (<b>D</b>) and HALO-based quantification (<b>E</b>) of proliferation marker Ki67 in the BT-474 tumor model (7 days after dosing, 1–2 hours after final dose). Inlay images represent HER2-stained tumor boundaries. <b>F,</b> HALO-based quantification of histologic analysis of caspase-3 in the BT-474 tumor model. Results are representative of at least 2 independent experiments.</p>
Abstract Background: Tucatinib is an orally administered, reversible HER2-targeted small molecule tyrosine kinase inhibitor that potently and selectively inhibits HER2 relative to the closely related kinase EGFR. In a phase Ib clinical trial in HER2+ metastatic breast cancer, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in pretreated patients with HER2-positive metastatic breast cancer (Borges VF et al., 2018). Here, we present preclinical data demonstrating tucatinib potentiates the activity of T-DM1 in HER2+ breast cancer models in vitro, and in vivo. In addition, tucatinib also enhanced the activity of a camptothecin-based HER2 ADC comprising trastuzumab conjugated with 8 exatecan moieties (T-Ex). Methods: In vitro assays were conducted to evaluate the potency of tucatinib, T-DM1 and T-Ex as single agents, and in combination, using a panel of breast cancer cell lines expressing various levels of HER2. The combinatorial effects of tucatinib with T-DM1 or T-Ex was assessed by isobologram analysis to determine additivity, synergy or antagonistic properties of the drug combinations. The activity of tucatinib either alone (50 mg/kg BID) or in combination with T-DM1 (10 mg/kg single dose IV) was evaluated in vivo using the HER2+ breast cancer cell line BT-474, and in 3 patient-derived xenograft (PDX) models of HER2+ breast cancer. Results: Tucatinib demonstrated potent anti-tumor activity in HER2 overexpressing cell lines in vitro with a similar selectivity profile as T-DM1 or T-Ex. When co-administered in vitro and subjected to isobologram analysis, tucatinib and T-DM1 or T-Ex combinations produced additive or synergistic effects. Moreover, tucatinib potently inhibited a subset of cell lines that showed reduced sensitivity to either T-DM1 or T-Ex. In BT-474 cells, the co-administration of tucatinib with T-DM1 in vitro was synergistic and resulted in an increased intracellular concentration of the TDM-1 catabolite Lys-MCC-DM1. The combination of tucatinib with T-DM1 was also more effective than either single agent alone in BT-474 xenografts in vivo and increased the number of complete tumor regressions. In 2 of 3 PDX models tested, the combination of tucatinib with T-DM1 was significantly more active than T-DM1 or tucatinib alone and produced a higher proportion of partial or complete tumor regressions compared with the single agent treatments. Conclusions: These data demonstrate tucatinib results in selective and potent anti-tumor activity in HER2+ tumor derived cell lines, including cell lines that show reduced sensitivity to T-DM1 or T-Ex in vitro. The results also demonstrate that tucatinib is either additive or synergistic when combined with T-DM1 or T-Ex in vitro. In addition, tucatinib in combination with T-DM1 showed enhanced anti-tumor activity in vivo in models of HER2+ breast cancer when compared to T-DM1 as a single agent. These results, taken together with the early clinical data demonstrating preliminary safety and activity of tucatinib with T-DM1, support continued assessment of tucatinib in combination with T-DM1, as well as other HER2 targeted ADCs, in HER2+ metastatic breast cancer patients. Borges VF, Ferrario C, Aucoin N, Falkson C, Khan Q, Krop I, Welch S, Conlin A, Chaves J, Bedard PL, Chamberlain M, Gray T, Vo A, Hamilton E. JAMA Oncol. 2018;4(9):1214-1220. Citation Format: Anita Kulukian, Janelle Taylor, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Tim S Lewis, Scott R Peterson. Tucatinib, a HER2-selective tyrosine kinase inhibitor, increases the anti-tumor activity of trastuzumab antibody-drug conjugates in preclinical models of HER2+ breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-18-09.
<p>Supplemental tables and figures referenced in main text: Table S1 - Crystallography data collection and refinement statistics; Table S2 - ADC characterization data; Figure S1 - ADC plasma stability data; Figure S2 - Time course for ADC activity in vitro; Table S3 - additional in vitro potency data for ADCs targeting CD30, CD19, and CD123.</p>
Nicotinamide phosphoribosyltransferase (NAMPT) regulates the biosynthesis of NAD from nicotinamide via a salvage biosynthetic pathway. Inhibition of NAMPT depletes cellular NAD levels leading to disruption of energy metabolism and cell death. Non-targeted small molecule NAMPT inhibitors have demonstrated poor tolerability in clinical trials and in preclinical models, including cardiac and retinal toxicities in rats. In an effort to improve the therapeutic window of this drug class, we pursued a targeted-delivery approach using antibody-drug conjugates. Through a medicinal chemistry effort, we identified novel NAMPT inhibitors that incorporate chemical functionality in the solvent-exposed terminus to allow construction of enzyme-cleavable drug linkers. Additionally, we applied a pyridinium-based linker strategy that allows for traceless linker attachment through a conserved nicotinamide-mimetic moiety of NAMPT inhibitors. Candidate molecules were evaluated for NAMPT binding affinity and cellular cytotoxicity as free drugs, and for cellular cytotoxicity as ADCs with the alternate linker strategies. Comparisons across inhibitors and linker strategies provide insight into optimal design of cleavable drug linkers for this class of drugs. In vitro, the ADCs deplete NAD and lead to downstream ATP depletion in a time-dependent manner. In vivo evaluation using human tumor xenografts shows translation of the pharmacodynamic effect resulting in tumor regression in models of Hodgkin lymphoma, non-Hodgkin lymphoma, and acute myeloid leukemia. Toxicology studies in Sprague Dawley rats demonstrate excellent tolerability at active doses, with no observable cardiac or retinal toxicities at the highest tested doses in single- and multi-dose regimens. These findings detail the development of a novel payload class and optimized linker strategy for use with antibody-drug conjugates, and demonstrate a preclinical efficacy and safety profile to support continued efforts toward clinical therapeutics.Citation Format: Chris Neumann, Kathleen C. Olivas, Kung Pern Wang, Andrew B. Waight, David W. Meyer, Luke V. Loftus, Margo C. Zaval, Martha E. Anderson, Steven Jin, Julia H. Cochran, Jessica K. Simmons, Paul G. Pittman, Fu Li, Michelle L. Ulrich, Abbie Wong, Weiping Zeng, Robert P. Lyon, Peter D. Senter. Antibody-drug conjugates of NAMPT inhibitors: Discovery, optimization, and preclinical characterization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 983.
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