Supplementary Figure S3. Raw pixel intensities for western blots in Figure 2

Supplementary Table S1. List of antibodies

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that targets cancers expressing HER2. DV consists of an anti-HER2 monoclonal antibody, disitamab, conjugated with the microtubule-disrupting agent monomethyl auristatin E (MMAE) via a cleavable vedotin 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. Released MMAE can also induce immunogenic cell death, which promotes immune cell recruitment to the tumor. In addition, DV stimulates Fc-gamma receptor mediated antibody-dependent cellular cytotoxicity, which can lead to target cell death. DV also inhibits HER2-activated downstream signaling pathways, further blocking cell growth, survival, and proliferation. Previously, we showed the cytotoxic activity of DV against a panel of breast cancer cell lines with varying levels of HER2 expression, including the HER2-low range. Here, we sought to further characterize the preclinical antitumor activity of DV in patient-derived 3D models and patient-derived xenographs (PDX). In cultured breast cancer cells, DV was more potent and internalized to a greater magnitude than the HER2-directed ADCs trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd). In patient-derived 3D models, DV had superior activity compared to T-DXd. We further explored whether dual HER2 targeting with DV in combination with the HER2-selective oral TKI tucatinib improved the antitumor outcomes. In PDX models with varying HER2 IHC levels and models refractory to T-DXd, DV as a monotherapy and in combination with tucatinib showed significant tumor growth inhibition. This result is consistent with previous in vitro results that showed increased cytotoxicity of the combination of DV and tucatinib in cell lines with a wide range of HER2 expression, which may be mechanistically attributed to elevated HER2 cell surface levels upon treatment with tucatinib. Overall, these findings provide scientific rationale to explore DV in HER2-positive and HER2-low breast cancer patients as a monotherapy or in combination with tucatinib. Citation Format: Kelsi Willis, Renee Hein, Katie Snead, Robert Thurman, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy and in combination with tucatinib in preclinical breast cancer models [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-04-07.

Abstract Brentuximab vedotin, a CD30-directed antibody–drug conjugate (ADC), is approved for clinical use in multiple CD30-expressing lymphomas. The cytotoxic payload component of brentuximab vedotin is monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. Preclinical results provided here demonstrate that treatment of cancer cells with brentuximab vedotin or free MMAE leads to a catastrophic disruption of the microtubule network eliciting a robust endoplasmic reticulum (ER) stress response that culminates in the induction of the classic hallmarks of immunogenic cell death (ICD). In accordance with the induction of ICD, brentuximab vedotin–killed lymphoma cells drove innate immune cell activation in vitro and in vivo. In the “gold-standard” test of ICD, vaccination of mice with brentuximab vedotin or free MMAE-killed tumor cells protected animals from tumor rechallenge; in addition, T cells transferred from previously vaccinated animals slowed tumor growth in immunodeficient mice. Immunity acquired from killed tumor cell vaccination was further amplified by the addition of PD-1 blockade. In a humanized model of CD30+ B-cell tumors, treatment with brentuximab vedotin drove the expansion and recruitment of autologous Epstein-Barr virus–reactive CD8+ T cells potentiating the activity of anti–PD-1 therapy. Together, these data support the ability of brentuximab vedotin and MMAE to drive ICD in tumor cells resulting in the activation of antigen-presenting cells and augmented T-cell immunity. These data provide a strong rationale for the clinical combination of brentuximab vedotin and other MMAE-based ADCs with checkpoint inhibitors.

Supplementary figures and table

Supplementary Figure S1. Overview of ICD

Abstract Disitamab vedotin (DV, RC48-ADC) is an antibody-drug conjugate (ADC) that selectively delivers cytotoxic drug to HER2-expressing cancer cells. DV is composed of disitamab, a HER2-directed monoclonal antibody, and a vedotin linker-payload system, which enables conjugation of the microtubule-disrupting agent monomethyl auristatin E (MMAE) to the antibody via a protease-cleavable linker. The proposed mechanism of action (MOA) of DV occurs through direct cytotoxicity of HER2-expressing tumor cells following internalization of DV and intracellular release of MMAE. DV may also induce antitumor activity through bystander-mediated cytotoxicity of neighboring cells and via the inhibition of HER2 signaling pathways. Moreover, cellular response to MMAE includes the induction of immunogenic cell death and recruitment of immune cells to the tumor site. DV is currently being evaluated globally in subjects with HER2-expressing locally advanced or metastatic urothelial carcinoma (la/mUC) and has gained conditional approval in China following an overall response rate of 50.5% (pooled analysis from studies NCT03507166 and NCT03809013). RNA sequencing data from a large cohort of urothelial cancer patient tumors revealed expression of HER2 in many urothelial tumors; however, the relationship between HER2 expression and efficacy of DV is not well understood. Here, we sought to evaluate the antitumor activity of DV monotherapy in preclinical urothelial cancer models with varying levels of HER2 expression. Multiple patient-derived xenografts (PDX) and patient-derived 3D in vitro urothelial cancer models showed potent antitumor response to DV monotherapy in tumors with varying HER2 expression levels including HER2-low tumors. Overall, these findings support ongoing clinical trials in subjects with HER2-expressing la/mUC and provide scientific rationale to further explore DV monotherapy in HER2-positive and HER2-low urothelial cancer clinical settings. Citation Format: Renee Hein, Kelsi Willis, Gina LoMastro, Suhas Vasaikar, Vinita Gupta, Katie Snead, Sharsti Sandall, Anita Kulukian. Disitamab vedotin, a clinical stage HER2-directed antibody-drug conjugate, shows potent antitumor activity as a monotherapy in preclinical urothelial cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1901.

Supplementary Figure S4. pIRE, pJNK, and CHOP upregulation in lymphoma lines treated with brentuximab vedotin or MMAE

Supplementary Figure S2. ICD hallmarks observed from tumor cell lines treated with brentuximab vedotin or MMAE

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

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.

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

Background SGN-B7H4V is a novel investigational vedotin antibody–drug conjugate (ADC) comprising a B7-H4-directed human monoclonal antibody conjugated to the cytotoxic payload monomethyl auristatin E (MMAE) via a protease-cleavable maleimidocaproyl valine citrulline (mc-vc) linker. This vedotin linker-payload system has been clinically validated in multiple Food and Drug Administration approved agents including brentuximab vedotin, enfortumab vedotin, and tisotumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumors, including breast, ovarian, and endometrial tumors, and limited normal tissue expression. SGN-B7H4V is designed to induce direct cytotoxicity against target cells by binding to B7-H4 on the surface of target cells and releasing the cytotoxic payload MMAE upon internalization of the B7-H4/ADC complex.Methods B7-H4 expression was characterized by immunohistochemistry across multiple solid tumor types. The ability of SGN-B7H4V to kill B7-H4-expressing tumor cells in vitro and in vivo in a variety of xenograft tumor models was also evaluated. Finally, the antitumor activity of SGN-B7H4V as monotherapy and in combination with an anti-programmed cell death-1 (PD-1) agent was evaluated using an immunocompetent murine B7-H4-expressing Renca tumor model.Results Immunohistochemistry confirmed B7-H4 expression across multiple solid tumors, with the highest prevalence in breast, endometrial, and ovarian tumors. In vitro, SGN-B7H4V killed B7-H4-expressing tumor cells by MMAE-mediated direct cytotoxicity and antibody-mediated effector functions including antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. In vivo, SGN-B7H4V demonstrated strong antitumor activity in multiple xenograft models of breast and ovarian cancer, including xenograft tumors with heterogeneous B7-H4 expression, consistent with the ability of vedotin ADCs to elicit a bystander effect. In an immunocompetent murine B7-H4-expressing tumor model, SGN-B7H4V drove robust antitumor activity as a monotherapy that was enhanced when combined with an anti-PD-1 agent.Conclusion The immune checkpoint ligand B7-H4 is a promising molecular target expressed by multiple solid tumors. SGN-B7H4V demonstrates robust antitumor activity in preclinical models through multiple potential mechanisms. Altogether, these preclinical data support the evaluation of SGN-B7H4V as a monotherapy in the ongoing phase 1 study of SGN-B7H4V in advanced solid tumors (NCT05194072) and potential future clinical combinations with immunotherapies.

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

TIGIT is an immune checkpoint receptor expressed on activated and memory T cells, immunosuppressive T regulatory cells, and natural killer (NK) cells. TIGIT has emerged as an attractive target for antitumor therapies, due to its proposed immunosuppressive effects on lymphocyte function and T cell activation. We generated an anti-TIGIT monoclonal antibody (mAb) that binds with high affinity to human, non-human primate, and murine TIGIT and through multiple experimental methodologies demonstrated that checkpoint blockade alone is insufficient for antitumor activity. Generating anti-TIGIT mAbs with various Fc backbones we show that muting the Fc-Fcγ receptor (FcγR) interaction failed to drive antitumor activity, while mAbs with Fc functional backbones demonstrate substantial antitumor activity, mediated through activation of antigen-presenting cells (APCs), T cell priming, and NK-mediated depletion of suppressive Tregs and exhausted T cells. Further, nonfucosylation of the Fc backbone resulted in enhanced immune responses and antitumor activity relative to the intact IgG1 backbone. The improved activity correlated with the biased FcγR interaction profile of the nonfucosylated anti-TIGIT mAb, which supports that FcγRIIIa binding with decreased FcγRIIb binding favorably activates APCs and enhances tumor-specific CD8+ T cell responses. The anti-TIGIT mAbs with intact FcγR interacting backbones also demonstrated synergistic enhancement of other standard antitumor treatments, including anti-PD-1 treatment and a model monomethyl auristatin E antibody–drug conjugate. These findings highlight the importance of the anti-TIGIT mAb’s Fc backbone to its antitumor activity and the extent to which this activity can be enhanced through nonfucosylation of the backbone.

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.

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.