Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2, an oncogenic growth factor receptor that promotes cell proliferation and survival. DV consists of a novel anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable linker. DV has multimodal antitumor mechanisms of action that include direct cytotoxicity of HER2-expressing cancer cells and bystander effect based-cytotoxicity of neighboring cells, both of which are mediated by the intracellular release of MMAE within the targeted cell. Released MMAE can induce immunogenic cell death (ICD), which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity (ADCC), which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth and proliferation. In this preclinical study, we investigated the antitumor activity of DV in breast and gastric cancer models, both as a monotherapy and in combination with tucatinib, a HER2-selective oral tyrosine kinase inhibitor approved in combination with trastuzumab and capecitabine for patients with HER2+ metastatic breast cancer. In vitro, DV demonstrated cytotoxic activity against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range, and was more potent than the HER2-directed ADC trastuzumab emtansine (T-DM1). Internalization assays using time-lapse microscopy of breast cancer cells continuously exposed to labeled naked antibodies showed that disitamab internalized to a greater magnitude than trastuzumab. In a subset of those cell lines, DV showed enhanced internalization compared to other HER2-targeted ADCs, namely T-DM1 and trastuzumab deruxtecan (T-DXd). Similar internalization and cytotoxic activities of DV were observed in gastric cancer cell lines. We explored whether dual HER2 targeting with DV in combination with tucatinib improved the antitumor outcomes. In vitro results demonstrated evidence of enhanced cytotoxicity over single agents when tested in breast and gastric cancer cell lines with a wide range of HER2 expression levels. Investigation of the mechanism of the enhanced cytotoxicity revealed increased DV internalization in the presence of tucatinib, attributable to elevated HER2 levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast and gastric cancer settings as a monotherapy or in combination with tucatinib. Citation Format: Kelsi Willis, Katie Snead, Robert Thurman, Margo Zaval, Anita Kulukian. Disitamab vedotin, an investigational HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 560.
Tubulysins have emerged in recent years as a compelling drug class for delivery to tumor cells via antibodies. The ability of this drug class to exert bystander activity while retaining potency against multidrug-resistant cell lines differentiates them from other microtubule-disrupting agents. Tubulysin M, a synthetic analogue, has proven to be active and well tolerated as an antibody-drug conjugate (ADC) payload, but has the liability of being susceptible to acetate hydrolysis at the C11 position, leading to attenuated potency. In this work, we examine the ability of the drug-linker and conjugation site to preserve acetate stability. Our findings show that, in contrast to a more conventional protease-cleavable dipeptide linker, the β-glucuronidase-cleavable glucuronide linker protects against acetate hydrolysis and improves ADC activity in vivo. In addition, site-specific conjugation can positively impact both acetate stability and in vivo activity. Together, these findings provide the basis for a highly optimized delivery strategy for tubulysin M.
<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>
<p>Tucatinib mediates increased internalization and catabolism of T-DM1. <b>A,</b> Schematic of constant exposure internalization assays with Fabfluor-labeled trastuzumab. <b>B,</b> Fluorescence intensity of SK-BR-3 cells in internalization assays imaged over time. <b>C,</b> AUC of fluorescence intensity in internalization assays calculated at 35 hours. Results in panels B and C are representative of at least 2 independent experiments. <b>D,</b> Schematic of T-DM1 catabolism analysis. <b>E,</b> LC/MS-MS analysis of the predominant intracellular catabolite of T-DM1, Lys-MCC-DM1, in BT-474 cells after treatment in the presence or absence of either tucatinib or neratinib. <b>F,</b> LC/MS-MS analysis of the sum of all T-DM1 catabolites, both in intracellular and extracellular fractions. Data in panels E and F shown as mean ± SEM. AUC, area under the curve.</p>
<p>Tucatinib mediates an increase in HER2 through reduced ubiquitination of the receptor. QFACS of total (<b>A</b>) and cell surface (<b>B</b>) HER2 after treatment with tucatinib or lapatinib. Data shown as mean ± SEM. <b>C,</b> Schematic of IP assays/whole-exome sequencing analysis of stable HA-ubiquitin–expressing cell lines treated with TKIs. <b>D,</b> Analysis of HA-ubiquitin incorporated into HER2 (normalized to total HER2 protein levels). Results are representative of at least 2 independent experiments. IP, immunoprecipitation.</p>
<p>Tucatinib mediates increased internalization and catabolism of T-DM1. <b>A,</b> Schematic of constant exposure internalization assays with Fabfluor-labeled trastuzumab. <b>B,</b> Fluorescence intensity of SK-BR-3 cells in internalization assays imaged over time. <b>C,</b> AUC of fluorescence intensity in internalization assays calculated at 35 hours. Results in panels B and C are representative of at least 2 independent experiments. <b>D,</b> Schematic of T-DM1 catabolism analysis. <b>E,</b> LC/MS-MS analysis of the predominant intracellular catabolite of T-DM1, Lys-MCC-DM1, in BT-474 cells after treatment in the presence or absence of either tucatinib or neratinib. <b>F,</b> LC/MS-MS analysis of the sum of all T-DM1 catabolites, both in intracellular and extracellular fractions. Data in panels E and F shown as mean ± SEM. AUC, area under the curve.</p>
<div>Abstract<p>We have developed a highly active and well-tolerated camptothecin (CPT) drug-linker designed for antibody-mediated drug delivery in which the lead molecule consists of a 7-aminomethyl-10,11-methylenedioxy CPT (CPT1) derivative payload attached to a novel hydrophilic protease-cleavable valine–lysine–glycine tripeptide linker. A defined polyethylene glycol stretcher was included to improve the properties of the drug-linker, facilitating high antibody–drug conjugate (ADC) drug loading, while reducing the propensity for aggregation. A CPT1 ADC with 8 drug-linkers/mAb displayed a pharmacokinetic profile coincident with parental unconjugated antibody and had high serum stability. The ADCs were broadly active against cancer cells <i>in vitro</i> and in mouse xenograft models, giving tumor regressions and complete responses at low (≤3 mg/kg, single administration) doses. Pronounced activities were obtained in both solid and hematologic tumor models and in models of bystander killing activity and multidrug resistance. Payload release studies demonstrated that two CPTs, CPT1 and the corresponding glycine analog (CPT2), were released from a cAC10 ADC by tumor cells. An ADC containing this drug-linker was well tolerated in rats at 60 mg/kg, given weekly four times. Thus, ADCs comprised of this valine–lysine–glycine linker with CPT drug payloads have promise in targeted drug delivery.</p></div>
Abstract Auristatins are a class of clinically validated antimitotic agents utilized as payloads in antibody-drug conjugates (ADCs). Auristatins display many of the desirable properties necessary for ADC cytotoxins, such as low nanomolar potency, cell permeability, and activity across multidrug-resistant (MDR+) cell lines. Herein, we report the development of novel auristatins, which have a unique combination of these favorable properties. The hydrophilic monomethyl auristatin F (MMAF) was chosen as the parent structure, and a medicinal chemistry campaign was undertaken to functionalize various sites of the auristatin with hydrophobic moieties in an effort to improve membrane permeability. The structure-activity relationships (SAR) of the new auristatins demonstrated clear trends correlating hydrophobicity, structure, and polarity with permeability and in vitro cytotoxicity. The highest-performing molecules showed a preference for hydrophobic functionalization at the N-terminal dolavaline. The payloads were linked to the C-terminal position of the auristatin with a lysosomally cleavable maleimido-dipeptide linker. Examination of the ADCs revealed low ng/mL activity in CD30+ and CD19+ cell lines in vitro. Anti-CD70 ADCs demonstrated high in vivo efficacy in a 786-O xenograft and complete remissions (CRs) in the efflux-positive renal cell carcinoma model. Anti-CD30 ADCs were dosed in a CD30+ and CD30- admixed Karpas/Karpas-35R xenograft model to demonstrate proof-of-concept in vivo bystander activity. In summary, these novel auristatins showed potential across multiple indications as ADC payloads. Citation Format: Philip N. Moquist, Tim D. Bovee, Andrew B. Waight, Sarah Owen, Jamie A. Mitchell, Margo Zaval, Marsha Quick, Sharsti Sandall, Kim K. Emmerton, Nicole Blesie, Robert P. Lyon, Peter Senter, Svetlana Doronina. Novel auristatins with high activity on efflux-positive models and demonstrable bystander activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2803.
Abstract Background: Tucatinib is an orally administered, reversible, highly specific HER2 tyrosine kinase inhibitor recently approved by the FDA in combination with trastuzumab and capecitabine for adult patients with advanced unresectable or metastatic HER2-positive breast cancer (mBC), including patients with brain metastases, that have failed at least one anti-HER2 regimen in the metastatic setting. In a phase IB clinical trial, tucatinib in combination with the HER2-targeted antibody-drug conjugate (ADC) ado-trastuzumab emtansine (T-DM1) was well tolerated and demonstrated activity in heavily pre-pretreated patients with HER2-positive mBC (NCT01983501; Borges VF et al., 2018). We previously presented preclinical data that tucatinib increases the activity of trastuzumab-derived ADCs in HER2-positive breast cancer models. Here, we provide mechanistic insight that tucatinib potentiates the activity of T-DM1 by modulating HER2 protein dynamics and facilitating increased cytotoxic maytansinoid drug delivery. Methods: To assess changes to HER2 protein levels upon treatment with tucatinib, HER2-amplified breast cancer cell lines were analyzed by Western blot and quantitative FACS (qFACS). To probe the dynamics of HER2 at the cell surface upon binding to antibody therapeutics, SK-BR-3 cells were incubated with fluorescently labeled trastuzumab to mark HER2 at the cell surface. Cells were imaged over 72 hours to observe the internalization of surface-bound antibody. Concurrent experiments were conducted with trastuzumab labeled with QF01, a quenched fluor which fluoresces only upon lysosomal processing and can serve as a proxy for antibody catabolism. To directly measure the rates of ADC catabolism, lysates were generated from BT-474 cells treated with T-DM1 in the presence or absence of tucatinib over a 72 hour time course, and were analyzed by mass spectrometry for the T-DM1 adduct, Lys-MCC-DM1. Results: In HER2-amplified breast cancer cell lines, treatment with tucatinib increased overall and cell membrane-localized HER2 levels. As demonstrated by internalization assays, tucatinib had an initial effect that increased the dwell time of HER2 at the cell surface of SK-BR-3 cells. At later timepoints, HER2 bound to trastuzumab was internalized and directed towards lysosomes. These data were supported by parallel intracellular Lys-MCC-DM1 measurements, which demonstrated increased concentration of the adduct when TDM-1 was administered in combination with tucatinib. These data provide a mechanistic rationale as to why the co-administration of tucatinib with T-DM1 in vitro was synergistic by isobologram analysis, and why the combination of tucatinib with T-DM1 was more effective in vivo than either single agent alone in BT-474 xenografts and in PDX models tested, producing a higher proportion of partial or complete tumor regressions. Conclusions: The described preclinical in vitro and in vivo data of simultaneous dual HER2 inhibition with tucatinib and T-DM1, along with the results of the phase IB/2 clinical trial demonstrating preliminary safety and efficacy of the combination, warrant further clinical development of tucatinib in combination with T-DM1. These results also support the evaluation of tucatinib in combination with other HER2-targeted ADCs in patients with HER2-positive mBC. Citation Format: Anita Kulukian, Janelle Taylor, Nishi Jain, Devra Olson, Margo Zaval, Robert Thurman, Shawna Hengel, Lauren Farr, Thomas Pires, Scott R. Peterson. Tucatinib potentiates the activity of the antibody-drug conjugate T-DM1 in preclinical models of HER2-positive breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-08.
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