SGN-B6A is a novel investigational antibody-drug conjugate (ADC) directed to integrin beta-6 and uses the clinically validated vedotin drug-linker platform that delivers the microtubule disrupting agent, monomethyl auristatin E (MMAE). SGN-B6A is designed to bind and internalize the integrin beta-6/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. We have previously demonstrated the antitumor activity of SGN-B6A in cell line-derived xenograft models originating from multiple carcinomas as well as patient-derived xenograft models of non-small cell lung cancer (NSCLC). Other clinically validated vedotin ADCs that deliver MMAE have been shown to induce immunogenic cell death (ICD) in preclinical models and have demonstrated promising clinical activity in combination with immunotherapy. Since the induction of ICD appeared to be a consequence of the activity of MMAE, and is independent of the antibody that delivers it, we hypothesized that this mechanism of action may also apply to SGN-B6A. Consistent with this hypothesis, we observed that tumor cells treated with SGN-B6A in vitro showed key hallmarks of immunogenic cell death, including markers of endoplasmic reticulum (ER) stress, exposure of calreticulin, and release of ATP and high mobility group protein B1 (HMGB1). Further, in vivo studies demonstrated that treatment with SGN-B6A led to immune activation and recruitment of immune cells to the tumor environment. In an integrin beta-6-expressing syngeneic model, a vedotin ADC directed to integrin beta-6 has shown combinatorial activity with immunotherapy. Preclinical models suggest that, like other vedotin ADCs, SGN-B6A induces immunogenic cell death which then promotes activation and recruitment of immune cells to the tumor. We have recently reported promising single-agent activity of SGN-B6A in non-small cell lung, head and neck squamous cell, and esophageal cancer observed in interim results of a phase I study (NCT04389632). The combination of SGN-B6A with immunotherapy may be utilized as a potential treatment for integrin-beta-6-expressing tumors including NSCLC, head and neck squamous cell carcinoma, and esophageal carcinoma. Altogether, our preclinical and initial clinical results support the ongoing evaluation of SGN-B6A as a single agent and in combination with immune checkpoint inhibitors. Citation Format: Vivian H. Trang, Rebecca C. Mazahreh, John J. Gosink, Michelle Ulrich, Devra Olson, Allana Ubben, Sean Allred, Li-Ya Huang, Kelly Hensley, Piper M. Treuting, Kerry Klussman, Shaylin Higgins, Patrick Younan, Roma Yumul, Natalya Nazarenko, Robert P. Lyon. SGN-B6A induces immunogenic cell death as an additional mechanism of action [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 1522.

HER2 in tucatinib-treated cells is targeted to the lysosome after a period of delay

Antibody humanization, binding assays, conjugation, mass spec, additional in vivo details

Isobologram analysis of tucatinib with non-targeting IgG1-DM1 shows reduced synergy compared to T-DM1.

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 (A) and BT-474 CDX model (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). C, Summary table of tumor growth inhibition and response rates of subcutaneous-implant xenograft models. D, Mean tumor volume over time in intracranially implanted BT-474-RedLuc xenograft model. E, Kaplan–Meier survival plots of intracranial BT-474-RedLuc xenograft model. F, Summary table of tumor growth inhibition and median survival days of intracranial BT-474-RedLuc xenograft model. All P values were determined by comparing the tucatinib/T-DM1 combination with the closest single agent. Data shown as mean ± SEM.

Tucatinib mediates increased internalization and catabolism of T-DM1. A, Schematic of constant exposure internalization assays with Fabfluor-labeled trastuzumab. B, Fluorescence intensity of SK-BR-3 cells in internalization assays imaged over time. C, 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. D, Schematic of T-DM1 catabolism analysis. E, 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. F, 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.

Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate directed to Nectin-4, which is highly expressed in bladder cancers. Preclinically, EV has demonstrated tumor cell killing by direct cytotoxicity and bystander effect and can induce the hallmarks of immunogenic cell death. EV improves survival in adults with previously treated locally advanced or metastatic urothelial carcinoma (la/mUC) and is approved in the US, Europe, Japan, and others. Most newly diagnosed bladder cancer cases are non-muscle invasive (NMIBC). Standard treatment of high-risk NMIBC involves transurethral resection followed by intravesical Bacillus Calmette-Guerin (BCG) or chemotherapy. Although response to BCG is high, recurrence is common, and treatment options for patients with BCG-unresponsive tumors are limited, underscoring the significant unmet need. Previously, we demonstrated compelling preclinical antitumor activity of EV in NMIBC models with a favorable safety profile and minimal systemic exposure. EV-mediated antitumor activity was confirmed in a mouse model of NMIBC by both bioluminescence imaging and IHC for hNectin-4-expressing cancer cells. Following intravesical administration of EV, tumor growth inhibition ranged 46-96% across the dose range tested. Colocalization of EV to Nectin-4-positive tumor tissues was confirmed by IHC in the engrafted tumor cells. Systemic EV exposure in tumor-bearing mice was low, consistent with previous nonclinical studies, supporting that the antitumor activity is driven by local exposure within the bladder. In a repeat-dose GLP toxicology study in rats, no systemic toxicities were observed at intravesical doses up to 6-fold higher than the maximum tolerated IV dose. This lack of systemic toxicities that can occur with IV administration in rats was likely due to minimal systemic exposure of both EV and unconjugated MMAE. Currently, the safety, tolerability, and antitumor activity of intravesical EV are being evaluated in a Phase 1 study in adults with high-risk, BCG-unresponsive NMIBC (EV-104, NCT05014139). The initial dose level for EV-104 was selected to be active and predicted to have minimal systemic absorption based on preclinical and known clinical IV data. Here, we present confirmatory clinical data demonstrating that EV and unconjugated MMAE are undetectable in the bloodstream at the starting dose. These findings confirm the translatability of our nonclinical models and provide evidence that intravesical administration of EV in NMIBC is a promising approach that limits systemic exposure. These data support the potential for a favorable safety and activity profile and warrant continued investigation of intravesical EV in patients with NMIBC. Citation Format: Devra Olson, Yen Lin Chia, Enaharo Iboi, Lauren Farr, Maddy Burcher, Anthony Lee, Kelly Hensley, Sean Allred, Abbie Wong, Masashi Shimazaki, Masamichi Mori, Sharsti Sandall, Christopher Carosino. Enfortumab vedotin, a nectin-4-directed antibody-drug conjugate, demonstrates compelling antitumor activity in non-muscle invasive bladder cancer models which predicts minimal systemic exposure when administered by intravesical instillation in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB246.

Tucatinib sensitizes HER2-positive cancer cells to T-DM1. A, Summary table of IC50 and Emax values for T-DM1 alone and in combination with tucatinib in CTG Luminescent Cell Viability cytotoxicity assays. 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. C, Heat maps of matrixed CTG drug combination experiments testing the synergistic activity of tucatinib and T-DM1.

The combined suppression of HER2 signaling by tucatinib and T-DM1 is associated with reduced tumor growth. A, ELISAs quantifying phosphorylation of signaling components downstream of HER2 in BT-474 cells (as percentage phosphorylated protein vs. untreated cells with SEMs). B, IHC images of pHER2, pHER3, pAKT, and pMEK staining of the BT-474 xenograft tumor model. Inlay images represent HER2-stained tumor boundaries. C, HALO-based quantification of histologic analysis of HER2 and phospho-epitopes downstream of HER2 in the BT-474 tumor model. IHC images (D) and HALO-based quantification (E) 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. F, HALO-based quantification of histologic analysis of caspase-3 in the BT-474 tumor model. Results are representative of at least 2 independent experiments.

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 in vivo, 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.SIGNIFICANCE: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.

Effect of tucatinib, T-DM1, or combination treatment on mouse body weight in HER2+ xenograft models

Pulsed internalization assay with trastuzumab labeled with quenched fluor demonstrate increased internalization and lysosomal targeting with tucatinib

Tucatinib alters the internalization dynamics of HER2-targeted antibodies. A, Schematic of pulsed internalization assay with AF488 fluorescently-labeled trastuzumab. 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. C, Magnified images of tucatinib-treated cells in internalization assays, counterstained with lysosomal marker LAMP1. Chloroquine, which inhibits lysosomal function (49), was added to show more intense colocalization when lysosomal degradation is impaired. These results are representative of at least 2 independent experiments.

Tucatinib mediates an increase in HER2 through reduced ubiquitination of the receptor. QFACS of total (A) and cell surface (B) HER2 after treatment with tucatinib or lapatinib. Data shown as mean ± SEM. C, Schematic of IP assays/whole-exome sequencing analysis of stable HA-ubiquitin–expressing cell lines treated with TKIs. D, Analysis of HA-ubiquitin incorporated into HER2 (normalized to total HER2 protein levels). Results are representative of at least 2 independent experiments. IP, immunoprecipitation.

Tucatinib suppresses growth of CNS tumors

Enfortumab vedotin (EV) is a monomethyl auristatin E (MMAE) containing antibody-drug conjugate directed to Nectin-4, which is highly expressed in bladder cancers. Preclinically, EV has demonstrated tumor cell-killing by direct cytotoxicity, bystander toxicity, and induction of the hallmarks of immunogenic cell death. In EV-301, a phase 3 clinical study, EV monotherapy showed an overall survival (OS) benefit vs chemotherapy in patients with locally advanced or metastatic urothelial carcinoma (la/mUC) who had previously received platinum-based therapy and a PD-1 or PD-L1 inhibitor (Powles 2021). EV also has encouraging activity in combination with pembrolizumab in previously untreated la/mUC (73% ORR)(Friedlander 2021). Most newly diagnosed bladder cancer cases are non-muscle invasive (Chang 2016; Woldu 2017; Kates 2020; Li 2020). Standard treatment of high risk NMIBC involves transurethral resection of the bladder tumor followed by intravesical (IVES) Bacillus Calmette-Guerin (BCG) or chemotherapy. Although response to BCG is high, many patients recur within 1-5 years (Matulay 2021). For patients unresponsive to BCG, treatment options are limited; radical cystectomy remains the standard of care. Due to the morbidity associated with radical cystectomy there remains a significant unmet need for a safe and effective therapy. We demonstrate that NMIBC have a high Nectin-4 expression pattern by RNA analyses and immunohistochemistry (IHC) similar to la/mUC, using an anti-Nectin-4 antibody (clone M22-321b41.1). The anti-tumor activity of EV was demonstrated in vitro and in vivo in human bladder cancer cells overexpressing Nectin-4. The superiority of EV vs MMAE alone was shown via in vitro pulsed (2hr) exposure cytotoxicity assays mimicking IVES administration. In a luciferase expressing orthotopic xenograft mouse model of NMIBC, EV-mediated anti-tumor activity was confirmed by both bioluminescence imaging of tumor burden and IHC for h-Nectin-4 expressing cancer cells. Tumor uptake of EV was further confirmed by IHC detection of EV in the engrafted tumor cells. Repeat-dose IVES administration of EV was well tolerated in a GLP study with minimal local and no systemic toxicities at doses up to 6-fold the intravenous maximum tolerated dose. Consistent with the lack of systemic toxicities, IVES administration was associated with low systemic absorption (<1% of the dose-normalized ADC Cmax observed in intravenous toxicity studies) and undetectable systemic MMAE. In this preclinical model, increased levels of MMAE in the targeted bladder tissue were associated with increasing the total dose and concentration of EV more than changing either the volume instilled or dwell time. These findings provide evidence to support further investigation of intravesical EV in NMIBC patients. Citation Format: Christopher Carosino, Devra Olson, Katie Snead, Anthony Lee, Lauren Farr, Amit Garg, Christine O'Day, Esther Trueblood, Jennifer Wright, Mark Bieda, Charles Caldwell, Kelly Hensley, Sean Allred, Bernard Liu, Masashi Shimazaki, Sharsti Sandall. Enfortumab vedotin, a Nectin-4 directed ADC, demonstrates compelling tolerability and anti-tumor activity with intravesical instillation in preclinical models of non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1140.

Background Enfortumab vedotin (EV) is a first-in-class Nectin-4-directed antibody-drug conjugate (ADC) with demonstrated improved overall survival in patients with previously treated advanced-stage urothelial carcinoma.1 EV is comprised of a fully human Nectin-4-directed monoclonal antibody conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) by a protease cleavable maleimidocaproyl-valine-citrulline linker. EV has a multifaceted mechanism of action. Previously, we demonstrated that EV induces antitumor activity in vitro via direct cytotoxicity on Nectin-4-expressing malignant cells and indirect bystander activity on neighboring Nectin-4­ negative cells, both of which are mediated by MMAE release within target cells. Here, we expand upon the mechanism of action and show EV induces tumor cell killing in a manner leading to immunogenic cell death (ICD) and improves antitumor responses when combined with checkpoint inhibitors. Methods The ability of EV to induce hallmarks of ICD was evaluated in vitro in Nectin-4-expressing human urothelial carcinoma cell lines. Immune activation associated with ICD was assessed in vitro in monocytes co-cultured with EV-treated tumor cells and in vivo by immunohistochemistry, RNA-seq, flow cytometry, and immune cytokine profiling. The effects of EV plus anti-PD-1 on tumor growth inhibition, the tumor microenvironment, and immune memory were evaluated in syngeneic mouse models engineered to express human Nectin-4. Antitumor immune memory was also assessed in mice vaccinated with EV-treated cells. Results In vitro, EV induced ICD via MMAE-mediated microtubule disruption and concomitant endoplasmic reticulum (ER) stress, as evidenced by increased phosphorylation of JNK, extracellular release of inflammatory mediators ATP and HMGB1, and cell surface exposure of calreticulin. Xenograft tumors treated with EV demonstrated upregulation of MHC genes as well as genes involved in ER stress, autophagy, and type I interferon response. Additionally, there were noted increases in both macrophages and dendritic cells along with cytokines involved in chemoattraction and T-cell stimulation. Consistent with ICD induction, vaccination with EV-treated Nectin-4-expressing tumor cells promoted antitumor immunity and provided protection against tumor rechallenge. Lastly, the combination of EV with PD-1 inhibition improved antitumor activity and durable immunity in vivo, consistent with complementary modes of action of these two anticancer agents. Conclusions These data provide insight into the clinical activity observed with EV and bolster the scientific rationale to combine EV with checkpoint inhibitors, which is currently an area of active clinical investigation across multiple studies.2-6 References Powles T, Rosenberg JE, Sonpavde GP, Loriot Y, Duran I, Lee JL, et al. Enfortumab Vedotin in Previously Treated Advanced Urothelial Carcinoma. N Engl J Med. 2021;384(12):1125–35. Epub 2021/02/13. doi:10.1056/NEJMoa2035807. PubMed PMID: 33577729; PubMed Central PMCID: PMCPMC8450892. Friedlander TW, Milowsky MI, Bilen MA, Srinivas S, McKay RR, Flaig TW, et al. Study EV-103: Update on durability results and long term outcome of enfortumab vedotin + pembrolizumab in first line locally advanced or metastatic urothelial carcinoma (la/mUC). Journal of Clinical Oncology 2021;39(15_suppl):4528. doi: 10.1200/JCO.2021.39.15_suppl.4528. Galsky MD, Necchi A, Shore ND, Plimack ER, Jia C, Sbar E, et al. KEYNOTE-905/EV-303: Perioperative pembrolizumab or pembrolizumab plus enfortumab vedotin (EV) and cystectomy compared to cystectomy alone in cisplatin-ineligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol. 2021;39(6_suppl):TPS507. doi: 10.1200/JCO.2021.39.6_suppl.TPS507. Heijden MSVD, Gupta S, Galsky MD, Derleth CL, Lee S, Kataria RS, et al. Study EV-302: A two-arm, open-label, randomized controlled phase 3 study of enfortumab vedotin in combination with pembrolizumab versus chemotherapy in previously untreated advanced urothelial carcinoma (aUC) (trial in progress). J Clin Oncol 2022;40(6_suppl):TPS589. doi: 10.1200/JCO.2022.40.6_suppl.TPS589. Hoimes CJ, Bedke J, Loriot Y, Nishiyama H, Fang X, Kataria RS, et al. KEYNOTE-B15/EV-304: Randomized phase 3 study of perioperative enfortumab vedotin plus pembrolizumab versus chemotherapy in cisplatin-eligible patients with muscle-invasive bladder cancer (MIBC). J Clin Oncol 2021;39(15_suppl):TPS4587. doi: 10.1200/JCO.2021.39.15_suppl.TPS4587. ClinicalTrials.gov [Internet] Bethesda (MD): U.S. National Library of Medicine. 2000 – . ClinicalTrials.gov Identifier: NCT04960709. Treatment Combination of Durvalumab, Tremelimumab and Enfortumab Vedotin or Durvalumab and Enfortumab Vedotin in Patients With Muscle Invasive Bladder Cancer Ineligible to Cisplatin or Who Refuse Cisplatin (VOLGA). 2021 Jul 14 [cited 2022 Jul 22]. Available from: https://clinicaltrials.gov/ct2/show/NCT04960709. Ethics Approval All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee at Seagen, Astellas, or the external testing facilities that conducted the studies.