<div><p>The oncogenic receptor HER2 is overexpressed in many cancers, including up to 20% of breast cancers. Despite the availability of HER2-targeted treatments, patients’ disease often progresses during therapy, underscoring the need for novel treatment strategies. The addition of tucatinib, a reversible, highly selective HER2 tyrosine kinase inhibitor (TKI), to treatment with trastuzumab and capecitabine significantly improved survival outcomes of patients with HER2-positive metastatic breast cancer, including those with active brain metastases. We rationalized that combining tucatinib with other HER2-targeting agents with complementary mechanisms of action would further increase efficacy against tumors. We characterized the activity of tucatinib with the antibody–drug conjugate T-DM1 in preclinical models of breast cancer, including HER2-positive breast cancer cells and patient-derived xenograft (PDX) models. Mechanistic details on tucatinib activity were obtained in internalization and catabolism studies. In combination, tucatinib and T-DM1 showed an enhanced, often synergistic, cytotoxic response and demonstrated improved antitumor activity <i>in vivo</i>, including in PDX models refractory to T-DM1 single-agent activity. Mechanistically, tucatinib mediated an increase in inactive HER2 molecules at the cell surface through inhibition of HER2 ubiquitination, resulting in increased internalization and catabolism of T-DM1. The combination was correlated with enhanced HER2 pathway inhibition, decreased proliferation, and increased apoptosis. In a xenograft model of brain metastasis, tucatinib penetrated intracranial tumor tissues, inhibiting tumor growth and improving survival. These results suggest that tucatinib may be the optimal TKI partner for HER2-targeted therapies and support clinical studies of its combination with T-DM1, including in patients with brain metastases.</p>Significance:<p>The preclinical findings in breast cancer models presented here demonstrate that combining tucatinib with T-DM1 enhances the antitumor activity of either agent alone, supporting clinical studies of the combination in HER2-positive breast cancer, including in patients with brain metastases, which remains an important unmet medical need.</p></div>
Abstract 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.
<p>Tucatinib alters the internalization dynamics of HER2-targeted antibodies. <b>A,</b> Schematic of pulsed internalization assay with AF488 fluorescently-labeled trastuzumab. <b>B,</b> Images of SK-BR-3 cells in pulsed internalization assays incubated with AF488 fluorescently-labeled trastuzumab and/or treated with tucatinib or neratinib. Inlay images show counterstaining (Hoechst) to signify distribution of cells. <b>C,</b> Magnified images of tucatinib-treated cells in internalization assays, counterstained with lysosomal marker LAMP1. Chloroquine, which inhibits lysosomal function (<a href="#bib49" target="_blank">49</a>), was added to show more intense colocalization when lysosomal degradation is impaired. These results are representative of at least 2 independent experiments.</p>
Abstract The antibody-drug conjugate enfortumab vedotin (EV; AGS22C3E) targets Nectin-4 expressing tumor cells by delivering MMAE, a potent microtubule disrupting agent, to induce cell death. EV has demonstrated single agent activity and encouraging activity (71% ORR) when combined with pembrolizumab (anti-PD-1) in the 1L setting of cis-ineligible metastatic urothelial carcinoma (mUC) (EV-103, NCT03288545). Here we demonstrated that EV may promote multiple mechanisms of action including bystander cell killing and hallmarks of immunogenic cell death (ICD) including ER stress, immune cell recruitment and activation. Two urothelial carcinoma models, T-24 and UM-UC-3, were engineered to express Nectin-4, and both were sensitive to EV in vitro and in vivo. In these Nectin-4 expressing cell lines, EV internalized with Nectin-4, trafficked to lysosomal vesicles, and released intracellular MMAE as shown by intracellular MMAE accumulation. In addition, EV demonstrated a bystander effect by release of the cell permeable MMAE from Nectin-4 positive cells to kill Nectin-4 negative cancer cells in an admixed cellular assay. Furthermore, EV treated cells exhibited early markers of ICD such as induction of the ER stress response through phosphorylation of JNK in Nectin-4 expressing cells. The ER protein calreticulin and protein chaperones like HSP70 are translocated from the intracellular compartments and exposed on the cell surface to signal for immune cell recruitment upon EV treatment. Additional hallmarks of ICD were observed with EV included extracellular release of ATP and HMGB1 in both the T-24 and UM-UC-3 Nectin-4 expressing cell lines. Immune cell recruitment and activation in the T-24 Nectin-4 xenograft model that demonstrated potent anti-tumor activity were measured in vivo using IHC, RNA-seq, flow cytometry, and immune cytokine paneling. Immune profiling assessment showed an enhancement of the immune cell markers in 6 of 7 tumors compared to untreated animals or non-binding ADC control treated animals. Analysis of gene signatures using RNA-seq support our qualitative IHC analysis of immune cell recruitment. Additional gene signature analyses identified altered gene transcripts associated with microtubule disruption, mitotic arrest, and ER stress. In addition, Luminex assessment of mouse cytokines showed coincident changes in RNA expression of genes associated with macrophage activation such MIP1α and MIPβ, suggesting EV treatment can promote APC activation. This mechanistic data and ongoing research by which EV induces cell death to promote immune cell recruitment and activation provides a potential mechanism underpinning the clinical benefit observed with EV as a monotherapy or in combination with pembrolizumab in mUC. Citation Format: Bernard A. Liu, Devra Olson, Katie Snead, John Gosink, Elena-Marie Tenn, Margo Zaval, Anthony Cao, Disha Sahetya, Albina Nesterova, Kelly Hensley, Julia Cochran, Shyra Gardai, Timothy S. Lewis. Enfortumab vedotin, an anti-Nectin-4 ADC demonstrates bystander cell killing and immunogenic cell death anti-tumor activity mechanisms of action in urothelial cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5581.
Abstract In efforts to bring forward antibody-drug conjugate (ADC) technologies that complement vedotin and enable new clinical-stage ADCs, we have investigated compounds that interact with topoisomerase 1, an enzyme involved in the unwinding of DNA. In connection with this, we developed a highly active and well-tolerated camptothecin drug-linker technology, in which the lead molecule consists of 7-aminomethyl-10,11-methylenedioxycamptothecin (AMDCPT) attached to a protease-cleavable valine-lysine-glycine (VKG) tripeptide linker unit. A hydrophilic and discrete polyethylene glycol unit was included to improve the properties of the drug-linker, enabling high ADC drug-loading, and reducing the propensity for aggregation. A VKG-AMDCPT ADC, with 8 drug-linkers/mAb (DAR8), displayed a pharmacokinetic profile coincident with parental unconjugated antibody, with a high degree of stability against retro-Michael reaction deconjugation. ADCs based on the VKG-AMDCPT were broadly active against cancer cells in vitro, and in mouse xenograft models, giving tumor regressions and complete responses with a single ≤3 mg/kg dose. These included both solid and hematologic tumor models, and models of bystander killing activity and multidrug resistance. A non-binding DAR8 ADC was well-tolerated in rats at 60 mg/kg, q7dx4. The VKG-AMDCPT drug-linker can be prepared from available materials in eight high-yielding steps, and this drug-linker is being employed in the anti-CD30 ADC, SGN-CD30C; an investigational new drug application is planned for 2020. Citation Format: Ryan Lyski, Lauren Bou, Margo Zaval, Kim Emmerton, Nicole Okeley, Jessica Simmons, Francisco Zapata, David Ortiz, Erica McKinney, David Meyer, Maureen Ryan, Peter Senter, Scott Jeffrey. Discovery of a tripeptide-based camptothecin drug-linker for antibody-drug conjugates with potent antitumor activity and a broad therapeutic window [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2885.
<p>Tucatinib sensitizes HER2-positive cancer cells to T-DM1. <b>A,</b> Summary table of IC<sub>50</sub> and Emax values for T-DM1 alone and in combination with tucatinib in CTG Luminescent Cell Viability cytotoxicity assays. <b>B,</b> CTG assays in which T-DM1 and tucatinib were screened in a panel of HER2-positive breast cancer cell lines. Data shown as mean + SD. <b>C,</b> Heat maps of matrixed CTG drug combination experiments testing the synergistic activity of tucatinib and T-DM1.</p>
<p>Pulsed internalization assay with trastuzumab labeled with quenched fluor demonstrate increased internalization and lysosomal targeting with tucatinib</p>
<div>Abstract<p>Antibody–drug conjugates (ADCs) are a therapeutic modality that enables the targeted delivery of cytotoxic drugs to cancer cells. Identification of active payloads with unique mechanisms of action is a key aim of research efforts in the field. Herein, we report the development of inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) as a novel payload for ADC technology. NAMPT is a component of a salvage biosynthetic pathway for NAD, and inhibition of this enzyme results in disruption of primary cellular metabolism leading to cell death. Through derivatization of the prototypical NAMPT inhibitor FK-866, we identified potent analogues with chemical functionality that enables the synthesis of hydrophilic enzyme-cleavable drug linkers. The resulting ADCs displayed NAD depletion in both cell-based assays and tumor xenografts. Antitumor efficacy is demonstrated in five mouse xenograft models using ADCs directed to indication-specific antigens. In rat toxicology models, a nonbinding control ADC was tolerated at >10-fold the typical efficacious dose used in xenografts. Moderate, reversible hematologic effects were observed with ADCs in rats, but there was no evidence for the retinal and cardiac toxicities reported for small-molecule inhibitors. These findings introduce NAMPT inhibitors as active and well-tolerated payloads for ADCs with promise to improve the therapeutic window of NAMPT inhibition and enable application in clinical settings.</p></div>
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