Xingru Ma

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) targeting HER2, exhibiting significant clinical efficacy in breast cancer (BC) with varying HER2 expression, including HER2-low and HER2-ultralow. However, the precise mechanism underlying its efficacy and the contribution of immune activation in these settings remain unclear. Here, we demonstrate that T-DXd efficacy in HER2-low and HER2-negative BC is independent of HER2 engagement and ADC internalization. Instead, its activity relies on extracellular proteases, such as cathepsin L (CTSL), within the tumor microenvironment. Irrespective of their HER2 status, tumor and stromal compartments of invasive BC abundantly express CTSL, which efficiently cleaves the specialized linker of T-DXd, facilitating payload release and inducing cytotoxicity against HER2-low/negative tumors. In HER2-positive BC, the antibody backbone of T-DXd engages Fcγ-receptors and drives antibody-dependent cellular phagocytosis (ADCP). Concurrently, its cytotoxic payload (DXd) induces immunogenic cell death, further activating myeloid cells via TLR4 and STING pathways to enhance tumor antigen presentation to CD8+ T cells. Notably, T-DXd cytotoxicity also upregulates tumor CD47 expression, dampening immune activation. Combining T-DXd with CD47 checkpoint blockade significantly enhances anti-tumor immune responses in a HER2-transgenic BC mouse model, while also inducing durable CD8+ T cell memory to prevent tumor recurrence after therapy cessation. Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate targeting HER2 but paradoxically efficient even in breast cancers expressing HER2 at very low levels. Here authors show that invasive breast cancers, even if their HER2 expression is negligible, secrete extracellular proteases, such as cathepsin L, which cleave the specialized linker of T-DXd, releasing the drug in the tumour microenvironment, while in HER2 positive breast cancers, T-DXd engages Fcγ receptors to promote phagocytosis of HER2-expressing cells and triggers payload-induced immunogenic cell death.

Abstract Trastuzumab deruxtecan (T-DXd), or fam-trastuzumab-deruxtecan-nxki, is a novel antibody-drug conjugate (ADC) targeting HER2 that has demonstrated profound clinical efficacy across HER2-positive breast cancers (BC) and other cancers. However, the precise mechanisms underlying its superiority over traditional ADCs, such as trastuzumab emtansine (T-DM1), remain poorly understood. In this study, we investigated the immune activation mechanisms mediated by T-DXd cytotoxicity in HER2-positive BC. We found that the T-DXd payload, Deruxtecan (DXd), induces a stronger tumor immunogenic cell death (ICD) phenotype compared to T-DM1. This is evidenced by the elevated release of multiple Damage-Associated Molecular Patterns (DAMPs) and the activation of nearby myeloid immune cells through TLR4 and STING pathways. Additionally, T-DXd’s antibody backbone engages with Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP), similar to trastuzumab. Coupled with DXd-induced ICD, T-DXd enhances tumor antigen uptake by macrophages and stimulates antigen-specific CD8+ T cells better than T-DM1. Importantly, DXd cytotoxicity also upregulates tumor CD47 expression, which engages with SIRPα to suppress ADCP. To assess the importance of this regulatory axis in T- DXd efficacy, we explored the use of CD47/SIRPα checkpoint blockade antibodies in combination with T-DXd. Our in vivo studies revealed that this combination synergistically enhanced anti-tumor efficacy in a HER2-transgenic BC mouse model, while inducing CD8+ T cell immune memory and preventing tumor recurrence after therapy cessation. In sum, these studies highlight the important role of T-DXd in stimulating innate and adaptive immune responses against HER2-positive BC and demonstrate the enhancement of this anti-tumor mechanism through combination with CD47/SIRPα checkpoint blockade. Citation Format: Li-Chung Tsao, John S. Wang, Xingru Ma, Sirajbir Sodhi, Herbert Kim Lyerly, Zachary C. Hartman. Enhancing Antitumor Immune Responses: Trastuzumab Deruxtecan (T- DXd) Induces Immunogenic Cell Death and Phagocytosis, Synergizing with CD47/SIRPα Checkpoint Blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B009.

Tumor antigen vaccination represents an appealing approach for cancer but has failed to materialize as oncologic standard of care. To understand long-term vaccine efficacy, we conducted a retrospective analysis of patients with human epidermal growth receptor 2 + (HER2 + ) breast cancer who received HER2-targeting vaccines and survived for >18 years. PBMC analysis revealed HER2-specific CD27 + memory CD4 and CD8 T cells, suggesting that CD27 signaling supports long-term immune memory. In human CD27 transgenic mice, combining HER2 vaccination with anti-CD27 agonism enhanced HER2-specific responses, particularly long-lived CD4 memory T cells. Murine models demonstrated ~40% tumor regression with combined therapy compared with vaccine alone (~6%). Additional scRNA-seq analysis identified CD4 T cells with a distinct gene expression profile, and depletion/adoptive transfer studies validated that CD4 T cells were essential for this effect. These findings suggest that CD27 agonism enhances vaccine-induced antigen-specific CD4 T cell responses, enabling durable antitumor immunity not entirely dependent on CD8 T cells.

Trastuzumab deruxtecan (T-DXd), also known as fam-trastuzumab-deruxtecan-nxki, is a novel HER2-targeting antibody-drug conjugate (ADC) that has shown remarkable clinical efficacy in breast cancer (BC) and other cancers with varying levels of HER2 expression. However, the mechanisms underlying its activity in HER2-low and HER2-ultralow BC, and the role of immune activation, remain poorly understood. Here, we demonstrate that T-DXd’s efficacy in HER2-low/ultralow BC is predominantly driven by the extracellular tumor-specific protease Cathepsin L (CTSL), but not Ccathepsin B (CTSB). Our in vitro and in vivo studies reveal that extracellular CTSL cleaves T-DXd’s linker, releasing the DXd payload (validated by mass spectrometry) and inducing cytotoxicity within HER2-low/ultralow tumor microenvironments. This mechanism mediates potent anti-tumor effects across multiple HER2-negative cancers. Importantly, analysis of BC biopsies and tissue microarrays from T-DXd-treated patients revealed high CTSL expression in tumor and stromal compartments, independent of HER2 levels, suggesting broad therapeutic potential. In addition to extracellular cleavage, we also found that T-DXd elicits immune activation as an anti-tumor mechanism in HER2-positive BC. The DXd payload induces immunogenic cell death, activating nearby myeloid immune cells via TLR4 and STING pathways, while its antibody backbone engages Fcγ-receptors to stimulate Antibody-Dependent Cellular Phagocytosis (ADCP). This process enhances tumor antigen uptake and drives the expansion of antigen-specific CD8+ T cells. Furthermore, DXd upregulates CD47 on tumor cells, making them more susceptible to immune clearance. Combining CD47 blockade with T-DXd significantly enhanced anti-tumor efficacy in an immune suppressive HER2-transgenic BC mouse model, promoting CD8+ T cell memory and preventing tumor recurrence post-treatment. These studies suggest that strategies targeting the CD47-SIRPa axis may enhance the ability of ADCs to elicit tumor-specific immunity, thus extending the efficacy of these agents and generating long term memory responses against non-immunogenic cancers. Citation Format: Li-Chung Tsao, John S. Wang, Xingru Ma, Sirajbir Sodhi, Joey V. Ragusa, Bushangqing Liu, Jason McBane, Tao Wang, Junping Wei, Cong-Xiao Liu, Xiao-Yi Yang, Gang-jun Lei, Ivan Spasojevic, Ping Fan, Timothy N. Trotter, Michael A. Morse, Herbert K. Lyerly, Zachary C. Hartman. Uncovering the multiple therapeutic mechanisms of trastuzumab deruxtecan (T-DXd): effective extracellular payload release and immunomodulatory stimulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2128.

Abstract Background: T-DXd is an antibody-drug conjugate (ADC) targeting HER2 that has demonstrated superior clinical efficacy over previous HER2-targeting agent Trastuzumab emtansine (T-DM1). Notably, T-Dxd is clinically effective in breast cancers (BC) with a range of HER2 expression. While its purported mechanism of action (MOA) involves its cleavable linker leading to bystander killing, it remains unclear how this potential MOA accounts for efficacy in HER2-low cancers or the role FCGR-mediated signaling and immune induction plays in its clinical efficacy. Method: In vitro studies utilized co-culture, internalization, FCGR activation, ADCP, flow cytometry, and gene expression by QRT-PCR and ELISA assays. Assessments of tumor antigen-specific T cell stimulation involved JEDI T cells. In vivo studies were conducted using in vivo implantation of HER2+ and HER2- cells, PK assessments of T-Dxd/DM1 and therapeutic effects determined using an endogenous HER2 transgenic mouse model that measured HER2-specific adaptive immune responses, in combination with immune checkpoint blockade anti-CD47. Results: T-Dxd elicited a systemic bystander effect that was only partially dependent on HER2 expression, mediated by specific cathepsins in the extracellular TME. Notably, T-Dxd cytotoxicity induced immunogenic cell death (ICD), while also retaining the ability to engage FCGRs to promote ADCP and innate immune activation. ICD was mediated by secretion of specific Dxd driven DAMPs (HMGB1 and eATP), resulting in TLR4/STING activation. ICD and FCGR activation along with antigen ADCP together elicited myeloid cell activation (CD80, CD40) and antigen presentation, resulting in effective expansion of tumor antigen-specific T cells responses, demonstrated by co-culture assays using EGFP/HER2+ BC cells and CD8+ JEDI T cells. T cell activation required TLR4 and STING pathways while in vivo, T-Dxd anti-tumor responses associated with augmented HER2-specific T and B cell responses. However, T-Dxd also induced CD47 to limit phagocytosis. But, the use of CD47/SIRPa blocking antibodies dramatically enhanced CD8+ T cell expansion in vitro and anti-tumor efficacy in vivo upon T-Dxd treatment. Conclusion: Our study demonstrates that T-Dxd is cleavable in HER2 negative BC in vivo, which permits more widespread Dxd activity against HER2 low BC. Critically, Dxd elicits ICD, resulting in innate immune stimulation of myeloid cells, which when combined with FCGR activation and ADCP, leads to a striking induction of tumor-specific adaptive immunity. This combination was unique to T-Dxd, which may underlie its clinical superior efficacy. However, T-Dxd activity also elicits suppressive CD47 expression which could be negated using CD47/SIRPA blockade. We found that this combination dramatically enhanced T-Dxd resulted therapeutic efficacy, thus supporting clinical exploration of these combinations. Citation Format: Li-Chung Tsao, Xingru Ma, John S. Wang, Timothy N. Trotter, Tao Wang, Jun-Ping Wei, Gang-jun Lei, Jason McBane, Ping Fan, Ivan Spasojevic, Zachary C. Hartman. HER2-ADC efficacy is rooted in its induction of immunogenic cell death with FcGR activation and a systemic bystander effect [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 2377.