# Datopotamab deruxtecan induces hallmarks of immunogenic cell death

Sabrina Forveille, Marion Leduc, Allan Sauvat, Guido Kroemer, Oliver Kepp

<figure><img src="image_1.png" /></figure>

To cite this version:

Sabrina Forveille, Marion Leduc, Allan Sauvat, Guido Kroemer, Oliver Kepp. Datopotamab deruxtecan induces hallmarks of immunogenic cell death. Cell Stress, 2025, 9, pp.194 - 200. 10.15698/cst2025.08.311. hal-05237869

HAL Id: hal-05237869

https://hal.science/hal-05237869v1

Submitted on 3 Sep 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Datopotamab deruxtecan induces hallmarks of immunogenic cell death

Sabrina Forveille<fnref n="1" />,², Marion Leduc<fnref n="1" />,², Allan Sauvat<fnref n="1" />,², Guido Kroemer<fnref n="1" />,²,³,* and Oliver Kepp<fnref n="1" />,²,*

**ABSTRACT** Antibody-drug conjugates (ADCs) offer a strategy for targeted delivery of cytotoxic agents to cancer cells. In this study, we investigated the mechanism of action of datopotamab deruxtecan, an ADC composed of a monoclonal antibody targeting tumor-associated calcium signal transducer 2 (TACSTD2, also known as trophoblast cell-surface antigen-2 (TROP2)) conjugated to the topoisomerase I inhibitor DXd. Datopotamab deruxtecan reduced the viability of human osteosarcoma U2OS cells engineered to express TROP2, but had no effect on their parental counterparts, which only expressed the CALR-GFP biosensor. In TROP2-expressing cells, it triggered the translocation of CALR-GFP from the ER to the cell periphery. Both datopotamab deruxtecan and its DXd payload elicited several features characteristic of immunogenic cell death (ICD), including detectable calreticulin exposure on the cell surface, release of high-mobility group box 1 (HMGB1), and ATP secretion into the culture medium. Importantly, the TROP2-targeted ADC also exerted a bystander antitumor effect on parental U2OS cells (lacking TROP2 expression) co-cultured with TROP2-expressing U2OS cells. These findings demonstrate that datopotamab deruxtecan delivers a cytotoxic payload capable of inducing hallmark features of ICD *in vitro*.

doi: 10.15698/cst2025.08.311

Received originally: 03.07.2025
in revised form: 28.07.2025,

Accepted 29.07.2025,

Published 11.08.2025.

**Keywords:** anticancer immunotherapy; antibody drug conjugate, bystander effect.

**Abbreviations:**

ADCs – antibody-drug conjugates,

DAMPs – danger-associated molecular patterns,

DCs – dendritic cells,

HR – hormone receptor,

ICD – immunogenic cell death,

ICIs – immune checkpoint inhibitors,

ISO – isotype control,

NSCLC – non-small cell lung cancer.

## INTRODUCTION

Anticancer therapies, including chemotherapies, targeted agents, and antibody-drug conjugates (ADCs), achieve durable efficacy *in vivo* only when they combine direct tumor cell killing with stimulation of the immune system [1, 2]. This immune-mediated component often involves the induction of immunogenic cell death (ICD), a regulated form of cell death that promotes T cell-mediated responses against residual cancer cells [3]. ICD is defined by a coordinated series of stress responses that lead to the emission of danger-associated molecular patterns (DAMPs), which serve as adjuvants to activate dendritic cells (DCs) and support antigen presentation [4-6]. These DAMPs include autophagy-dependent ATP secretion, ER stress-induced calreticulin (CALR) exposure, and HMGB1 release. ATP acts as a chemoattractant for cDCs via P2RY2, CALR engages LRP1 to facilitate phagocytic uptake, and HMGB1 promotes DC maturation through TLR4 [7, 8]. Moreover,

ICD inducers often sensitize tumors to sequential immune checkpoint blockade [2, 9, 10].

ADCs provide a means to selectively deliver cytotoxic payloads to tumor cells while sparing healthy tissues [11]. Notably, a number of ADC payloads, including DXd, maytansinoids, auristatins, and pyrrolobenzodiazepines, have been shown to induce ICD [12-19]. Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancers, with EGFR mutations present in up to 38% of patients [20]. Many patients present with advanced disease, and although EGFR TKIs and platinum-based chemotherapy offer initial benefits, resistance frequently emerges [21]. Here, we evaluated the immunogenic potential of datopotamab deruxtecan, an ADC composed of a human anti-TROP2 IgG1 monoclonal antibody conjugated to multiple molecules of the topoisomerase I inhibitor DXd via a cleavable tetrapeptide linker. In clinical trials, datopotamab deruxtecan has shown durable responses in patients with

<footnote n="1">¹ Metabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Université Paris Saclay, Villejuif, France. ² Centre de Recherche des Cordeliers, Equipe labellisée par la Ligue contre le cancer, Université Paris Cité, Sorbonne Université, Inserm U1138, Institut Universitaire de France, Paris, France. ³ Institut du Cancer Paris CARPEM, Department of Biology, APHP, Hôpital Européen Georges Pompidou, Paris, France.</footnote>

<footnote>* Corresponding Authors:</footnote>

<footnote>Dr. Oliver Kepp, E-mail: captain.olsen@gmail.com</footnote>

<footnote>Prof. Dr. Guido Kroemer, E-mail: kroemer@orange.fr</footnote>
advanced NSCLC who progressed on EGFR TKIs and chemotherapy, leading to its recent accelerated FDA approval [22]. Moreover, datopotamab deruxtecan also received FDA approval for the treatment of unresectable or metastatic, hormone receptor (HR)-positive, HER2-negative breast cancer [23]. In this study, we demonstrate that datopotamab deruxtecan induces hallmark features of ICD *in vitro*, supporting its dual role as a direct cytotoxic agent and potential stimulator of anticancer immunity.

## RESULTS

### Datopotamab deruxtecan reduces viability and induces ICD traits in TROP2⁺ cells

Datopotamab deruxtecan, a TROP2-targeting ADC incorporating the exatecan-derived pyranoindolizinoquinoline DXd, was evaluated for its cytotoxic and immunogenic effects. Prior studies have shown that compounds in this family can trigger ICD and promote anticancer immunity *in vivo* [24-26]. To assess its mode of action,

we used U2OS osteosarcoma cells, a validated *in vitro* model for ICD characterization [12]. These cells were engineered to express TROP2 via lentiviral transduction. Viability was determined by epifluorescence microscopy, excluding pyknotic nuclei identified by Hoechst 33342. Datopotamab deruxtecan reduced viability in a TROP2-dependent, dose-responsive manner, while parental U2OS cells remained unaffected at equivalent concentrations. Neither the unconjugated anti-TROP2 antibody (datopotamab) nor a non-targeting IgG-DXd isotype control (ISO) induced cytotoxicity in either cell type, underscoring the importance of the DXd payload (Fig. 1A-C). At 5 days post-treatment, viability loss was pronounced in TROP2⁺ CALR-GFP biosensor cells treated with datopotamab deruxtecan but not in TROP2⁻ controls. By contrast, oxaliplatin decreased viability in both cell populations (Fig. 1A-C). Using CALR-GFP-expressing cells, we also evaluated the subcellular localization of CALR. Datopotamab deruxtecan caused a peripheral redistribution of CALR-GFP in TROP2⁺ cells, resembling the phe-

<figure><img src="image_3.png" /></figure>

**FIGURE 1** ● Datopotamab deruxtecan reduces viability and promotes CALR relocalization in TROP2-expressing cells. (A) Schematic representation of the treatment and analysis workflow. (B-D) U2OS osteosarcoma cells stably expressing CALR-GFP and their TROP2-overexpressing counterparts were treated for 5 days with increasing concentrations of a non-targeting IgG-DXd control ADC (ISO), anti-TROP2 monoclonal antibody (datopotamab), or the TROP2-targeted ADC datopotamab deruxtecan (ADC) across a dose range of 0.04-10 µg/mL. Oxaliplatin (OXA; 50 µM) served as a positive control for ICD induction. (B, C) Cell viability was determined by fluorescence microscopy, expressed relative to untreated controls. (D, E) Quantification of GFP signal heterogeneity was used to assess CALR redistribution. Data are shown as mean ± SD of quadruplicates, normalized to untreated controls. Individual data points represent single imaging fields. Statistical significance was calculated using Welch's t-test.
notype induced by oxaliplatin, while no such effect was observed in TROP2+ cells (Fig. 1D-E), suggesting that the ADC induces immunogenic CALR exposure.

## Datopotamab deruxtecan triggers hallmarks of ICD

We next assessed additional ICD markers in TROP2+ and parental

U2OS cells treated with the ADC, datopotamab alone, or ISO. Flow cytometric analysis of viable (DAPI+) cells revealed that datopotamab deruxtecan significantly increased surface CALR exposure exclusively in TROP2+ cells (Fig. 2A-C). It also induced ATP release, as indicated by luciferase-based detection in supernatants (Fig. 2D, E) and confirmed by the loss of quinacrine-positive intracellular

<figure><img src="image_4.png" /><figcaption>**FIGURE 2** ● Datopotamab deruxtecan induces ICD hallmarks in TROP2+ but not parental U2OS cells. TROP2+ and parental U2OS cells were treated for 5 days with 0.16 µg/mL of ISO, datopotamab, or datopotamab deruxtecan (ADC). OXA (5 µM) and mitoxantrone (MTX, 10 nM) served as positive ICD controls. Parental cells were also treated with 0.16 µg/mL of free DXd. (A-C) Surface CALR exposure was assessed via immunostaining and flow cytometry; data show the percentage of DAPI+/CALR+ viable cells. (D, E) Extracellular ATP levels were quantified from supernatants using a luciferase-based bioluminescence assay. (F, G) Intracellular ATP-containing vesicles were assessed by quinacrine staining. (H, I) HMGB1 release into culture supernatants was measured by ELISA. Data represent mean ± SD of triplicate samples, normalized to cell number per microscopy. Statistical significance determined by Welch's t-test.</figcaption></figure>
vesicles (Fig. 2F, G). Moreover, datopotamab deruxtecan stimulated the release of HMGB1 from TROP2⁺ cells, as detected by ELISA (Fig. 2G, H).

## Bystander killing in mixed cultures

Finally, we explored potential bystander effects by coculturing TROP2⁺ and TROP2⁻ U2OS cells at a 1:1 ratio. While TROP2⁺ cells were unaffected in monocultures (Fig. 1A-C), their viability was significantly reduced when cocultured with TROP2⁺ cells treated with datopotamab deruxtecan (Fig. 3). These results indicate that the ADC can mediate cytotoxic effects on neighboring TROP2⁺ cells, supporting the existence of a bystander antitumor effect.

## DISCUSSION

This study demonstrates that datopotamab deruxtecan, an ADC targeting TROP2, induces ICD in human cancer cell lines in vitro. In parallel, the topoisomerase I inhibitor payload DXd, derived from a pyranoindolizinoquinoline scaffold and structurally related to exatecan, exhibited potent cytotoxicity against murine tumor cells, leading to long-lasting, antigen-specific immune responses. These findings align with previous studies reporting that pyranoindolizinoquinoline-based compounds, administered either systemically or encapsulated in nanocarriers, can promote ICD and enhance responses to immune checkpoint inhibitors (ICIs) in preclinical models [25, 27, 28].

Datopotamab deruxtecan consists of a fully human IgG1 monoclonal antibody directed against the tumor-associated antigen TROP2 (TACSTD2), conjugated via a tetrapeptide-based cleavable linker to an average of four molecules of DXd. The linker is selectively cleaved by lysosomal proteases after internalization, allowing targeted intracellular delivery of DXd. TROP2 is overexpressed in various epithelial malignancies, including NSCLC, triple-negative breast cancer (TNBC), and urothelial carcinoma, making it an attractive target for ADC therapy [29].

Here, we showed that the unconjugated anti-TROP2 antibody (datopotamab) alone did not reduce the viability of TROP2-overexpressing human U2OS cells, suggesting that these cells are not dependent on TROP2 signaling for survival. Likewise, a non-targeting IgG-DXd isotype control had no cytotoxic effect. In contrast, datopotamab deruxtecan effectively and selectively killed TROP2⁺ cells, while sparing TROP2⁻ cells, indicating a high degree of target specificity and a potentially favorable therapeutic index. In mixed cultures containing both TROP2⁺ and TROP2⁺ cells, datopotamab deruxtecan treatment also reduced the viability of TROP2⁻ cells, an effect consistent with bystander killing. This phenomenon, which is frequently observed with membrane-permeable payloads such as DXd, is mediated by diffusion of the released cytotoxin from targeted cells into the surrounding microenvironment [30, 31]. Importantly, while Dato-DXd requires TROP2-mediated internalization for activation, the local release of a cytotoxic payload capable of crossing cell membranes allows for broader cytotoxicity within the tumor niche. Such activity can be advantageous in tumors with heterogeneous antigen expression or in cases of antigen loss due to immunoselection. The impact on

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**FIGURE 3** ● Datopotamab deruxtecan exerts bystander cytotoxic effects in cocultures of TROP2⁺ and parental U2OS cells. (A) Schematic overview of the coculture assay. (B) Parental and TROP2⁺ U2OS cells labeled with distinct fluorescent markers were mixed 1:1 and treated for 5 days with 0.16 µg/mL of ISO, datopotamab or datopotamab deruxtecan (ADC). Viability was assessed separately for each population using fluorescence microscopy and expressed relative to untreated controls. Results are shown as mean ± SD of quadruplicate samples. Data points represent individual view fields. Significance was assessed using Welch's t-test.</figcaption></figure>

normal tissues appears limited in clinical settings, suggesting that bystander effects are spatially confined and do not broadly harm non-transformed cells, although further studies are warranted to fully understand tissue-specific responses.

Moreover, ADCs like datopotamab deruxtecan may modulate the tumour immune microenvironment through multiple mechanisms, including the depletion of immunosuppressive cell populations like regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which in turn enhances the infiltration, activation, and cytotoxic function of effector CD8⁺ and CD4⁺ T cells; alternatively, the cytotoxic payload, DXd, may directly stimulate DCs, promoting antigen presentation and priming of adaptive immune responses [30, 32, 33].

Our data show that datopotamab deruxtecan elicits multiple hallmarks of ICD, including surface exposure of CALR, ATP release, and secretion of HMGB1, which together can stimulate dendritic cell activation and T cell priming. This immunostimulatory effect may enhance tumor antigen presentation and support the development of adaptive immunity [34]. ICD inducing chemotherapeutics have synergistic potential when combined with ICIs, as indicated in preclinical experiments [6, 35, 36], as well as in clinical trials [2, 37-39]. Clinical trials are currently evaluating datopotamab deruxtecan in multiple cancer indications, including EGFR-mutant NSCLC (TROPION-Lung01 and TROPION-Lung05), where it has shown promising results even after failure of EGFR TKIs and platinum-based chemotherapy, as well as in breast cancer, where datopotamab deruxtecan shows effect in heavily pretreated ad-
vanced HR-positive/HER2-negative and TNBC (TROPION-PanTumor01) [40, 41]. These observations support further investigation of datopotamab deruxtecan in combination with check-point inhibitors (such as ongoing clinical trials targeting NSCL including Phase 1b TROPION-Lung02 and TROPION-Lung04 and Phase 3 TROPION-Lung07, TROPION-Lung08 and AVANZAR; as well as TNBC such as TROPION-Breast03, TROPION-Breast04, TROPION-Breast05) especially in tumors with high TROP2 expression and immunosuppressive microenvironments.

# MATERIALS AND METHODS

## Cell lines and cell culture

Human U2OS osteosarcoma cells were sourced from the American Type Culture Collection (ATCC). Cell culture vessels were obtained from Corning (New York, NY, USA). U2OS cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Merck, Darmstadt, Germany), non-essential amino acids (NEAA; Thermo Fisher Scientific), and HEPES buffer (Thermo Fisher Scientific). Cells stably expressing a calreticulin-GFP fusion construct were maintained under selective pressure using 5 µg/mL plasticidin and 100 µg/mL zeocin. To generate co-cultures, TROP2-positive and TROP2-negative U2OS cells were combined at a 1:1 ratio. All cultures were grown at 37°C in a humidified atmosphere containing 5% CO₂, and routine testing confirmed cultures were free from mycoplasma contamination.

## Reagents and antibodies

Oxaliplatin was obtained from Accord Healthcare (Ahmedabad, India). Datopotamab deruxtecan (anti-TROP2 ADC), the unconjugated anti-TROP2 monoclonal antibody, and a non-targeting IgG-DXd control ADC were provided by Daiichi Sankyo. The rabbit polyclonal anti-calreticulin antibody (ab2907) was sourced from Abcam (Cambridge, UK), and AlexaFluor™ 488-conjugated goat anti-rabbit IgG secondary antibody was purchased from Thermo Fisher Scientific.

## Fluorescence microscopy

U2OS cells, either wild-type or stably expressing CALR-GFP with or without TROP2, were seeded at 500 cells per well in 384-well µclear imaging plates (Greiner Bio-One, Kremsmünster, Austria). After overnight incubation, cells were treated as indicated and maintained for five days. Cells were fixed with 3.7% formaldehyde containing 1 µg/mL Hoechst 33342 for 20 minutes. Images were acquired using an IXMc automated confocal microscope (Molecular Devices, San Jose, CA, USA) equipped with a Lumencor light source, Semrock filters (Rochester, NY, USA), a 16-bit sCMOS camera (Andor, Belfast, UK), and a 20× Plan APO objective (Nikon, Tokyo, Japan). Image analysis was performed using the EBImage package in R.

## Cell viability assay

U2OS cells were seeded at 500 cells per well in 384-well plates and allowed to adhere for 24 h prior to treatment with datopotamab deruxtecan, the unconjugated anti-TROP2 antibody, or the

non-targeting IgG-DXd ADC. After 5 days of incubation, cells were fixed with 3.7% formaldehyde (Merck) containing 1 µg/mL Hoechst 33342 (Thermo Fisher) for 20 minutes. Fluorescent images were acquired and analyzed as described above.

## Surface calreticulin staining and flow cytometry

Wild-type U2OS cells were seeded at 2,000 cells per well in 96-well µclear plates (Greiner Bio-One). After treatment and 5 days of incubation, cells were detached, transferred to V-bottom 96-well plates, and pelleted by centrifugation at 200 g for 5 minutes. Cells were resuspended in PBS containing 1% BSA and incubated with anti-calreticulin antibody (1:100) for 30 minutes at 4°C. Following two washes, cells were incubated with AlexaFluor™ 488-conjugated secondary antibody (1:1000) for 30 minutes at 4°C, washed again, and stained with 1 µM DAPI (cat. no. 62248, Thermo Fisher Scientific). Flow cytometric analysis was performed using a BD LSR Fortessa (BD Biosciences, Franklin Lakes, NJ, USA). CALR⁺ cells were quantified among DAPI⁺ viable cells.

## Extracellular ATP release

U2OS cells were seeded at 2,000 cells per well in 96-well plates and treated the following day. After 5 days, supernatants were collected, centrifuged (200 g, 5 minutes), and transferred to white-walled 96-well plates. ATP levels were measured using the ENLITEN ATP Bioluminescence Detection Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Luminescence was measured at 560 nm using a SpectraMax i3x plate reader (Molecular Devices) and normalized to cell number.

## Intracellular ATP imaging

Cells were seeded at 2,000 cells per well in 96-well µclear imaging plates, treated the next day, and incubated for 5 days. Cells were then incubated in Krebs-Ringer buffer (5 mM KCl, 125 mM NaCl, 0.7 mM KH₂PO₄, 1 mM MgSO₄, 2 mM CaCl₂, 6 mM glucose, 25 mM HEPES, pH 7.4) containing 5 µM quinacrine and 4 µM Hoechst 33342 for 30 minutes at 37°C [42]. After washing, images were captured via automated confocal fluorescence microscopy. Environmental conditions for live imaging were controlled using an Ibidi gas mixer (Gräfelfing, Germany). Image processing was conducted in R using EBImage.

## HMGB1 ELISA

U2OS cells were seeded in 96-well plates (2,000 cells/well), treated the next day, and incubated for 5 days. Cell culture supernatants were collected, centrifuged at 200 g for 5 minutes, and analyzed for HMGB1 content using a commercial ELISA kit (IBL International, Tecan, Männedorf, Switzerland) per the manufacturer's protocol. Absorbance at 450 nm was recorded on a SpectraMax i3x plate reader, and values were normalized to cell number.

## Statistical analysis

All experiments were performed in triplicate or quadruplicate unless otherwise indicated. Data are presented as mean ± standard deviation (SD). Statistical comparisons were made using
Welch's or Student's t-test, implemented via the t.test function in R (https://www.r-project.org).

## Data availability

Data are available from the corresponding authors G.K. and O.K. on reasonable request.

## AUTHOR CONTRIBUTION

S.F. performed investigations. M.L. and A.S. curated the data and performed formal analyses. O.K visualized results and wrote the first draft of the manuscript. G.K. and O.K. conceptualized the study, supervised, reviewed and edited the manuscript. All authors have read and approve the final version of the manuscript.

## ACKNOWLEDGMENTS

OK is supported by Association pour la recherche sur le cancer (ARC), Institut National du Cancer (INCa) and the European Innovation Council (EIC). G.K. is supported by the Ligue contre le Cancer (équipe la-bellisée); Agence National de la Recherche (ANR) - Projets blancs; AMMICa US23/CNRS UMS3655; ARC; Cancéropôle Ile-de-France; Fondation pour la Recherche Médicale (FRM); a donation by Elior; Equipex Onco-Pheno-Screen (ANR 21-ESRE-0028); European Joint Programme on Rare Diseases (EJPRD); European Research Council Advanced Investigator Award (ERC-2021-ADG, ICD-Cancer, Grant No. 101052444), European Union Horizon 2020 Projects Oncobiome, Prevalung (grant No. 101095604) and Crimson (No. 101016923); Fondation Carrefour; INCa; Institut Universitaire de France; LabEx Immuno-Oncology (ANR-18-IDEX-0001); a Cancer Research ASPIRE Award from the Mark Foundation; the RHU Immunolife; Seerave Foundation; SIRIC Stratified Oncology Cell DNA Repair and Tumor Immune Elimination (SOCRATE); and SIRIC Cancer Research and Personalized Medicine (CARPEM).

## REFERENCES

1. Galluzzi L, Guilbaud E, Schmidt D, Kroemer G, Marincola FM (2024). Targeting immunogenic cell stress and death for cancer therapy. *Nat Rev Drug Discov* 23(6): 445-460. doi: 10.1038/s41573-024-00920-9

2. Kroemer G, Chan TA, Eggermont AMM, Galluzzi L (2024). Immunosurveillance in clinical cancer management. *CA Cancer J Clin* 74(2): 187-202. doi: 10.3322/caac.21818

3. Kroemer G, Montegut L, Kepp O, Zitvogel L (2024). The danger theory of immunity revisited. *Nat Rev Immunol* 24(12): 912-928. doi: 10.1038/s41577-024-01102-9

4. Borges F, Laureano RS, Vanmeerbeek I, Sprooten J, Demeulenaere O, Govaerts J, Kinget L, Saraswat S, Beuselinck B, De Vleeschouwer S, Clement P, De Smet F, Sorg RV, Datsi A, Vigneron N, Naulaerts S, Garg AD (2024). Trial watch: anticancer vaccination with dendritic cells. *Oncoimmunology* 13(1): 2412876. doi: 10.1080/2162402X.2024.2412876

5. Janssens S, Rennen S, Agostinis P (2024). Decoding immunogenic cell death from a dendritic cell perspective. *Immunol Rev* 321(1): 350-370. doi: 10.1111/imr.13301

6. Kepp O, Kroemer G (2023). Immunogenic Cell Stress and Death Sensitize Tumors to Immunotherapy. *Cells* 12(24): 2843. doi: 10.3390/cells12242843

7. Guilbaud E, Kroemer G, Galluzzi L (2023). Calreticulin exposure orchestrates innate immunosurveillance. *Cancer Cell* 41(6): 1014-1016. doi: 10.1016/j.ccell.2023.04.015

## CONFLICT OF INTEREST

O.K. is a scientific co-founder of Samsara Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Sutro, Tollys, and Vascage. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is in the scientific advisory boards of Hevolution, Institut Servier, Longevity Vision Funds and Rejuveron Life Sciences. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK's brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim. GK's wife, Laurence Zitvogel, has held research contracts with Glaxo Smyth Kline, Incyte, Lytix, Kaleido, Innovate Pharma, Daiichi Sankyo, Pilege, Mer-us, Transgene, 9 m, Tusk and Roche, was on the Board of Directors of Transgene, is a cofounder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota.

## COPYRIGHT

© 2025 Forveille *et al*. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Please cite this article as: Sabrina Forveille, Marion Leduc, Allan Sauvat, Guido Kroemer and Oliver Kepp (2025). Datopotamab deruxtecan induces hallmarks of immunogenic cell death. *Cell Stress* 9: 194-200. doi: 10.15698/cst2025.08.311

8. Bezu L, Kroemer G (2024). High circulating HMGB1 indicates good prognosis in patients with advanced leiomyosarcoma under chemoimmunotherapy. *Oncoimmunology* 13(1): 2432059. doi: 10.1080/2162402X.2024.2432059

9. Pfirschke C, Engblom C, Rickelt S, Cortez-Retamozo V, Garris C, Pucci F, Yamazaki T, Poirier-Colame V, Newton A, Redouane Y, Lin YJ, Wojtkiewicz G, Iwamoto Y, Mino-Kenudson M, Huynh TG, Hynes RO, Freeman GJ, Kroemer G, Zitvogel L, Weissleder R, Pittet MJ (2016). Immunogenic Chemotherapy Sensitizes Tumors to Checkpoint Blockade Therapy. *Immunity* 44(2): 343-354. doi: 10.1016/j.immuni.2015.11.024

10. Yamazaki T, Pitt JM, Vetizou M, Marabelle A, Flores C, Rekdal O, Kroemer G, Zitvogel L (2016). The oncolytic peptide LTX-315 overcomes resistance of cancers to immunotherapy with CTLA4 checkpoint blockade. *Cell Death Differ* 23(6): 1004-1015. doi: 10.1038/cdd.2016.35

11. Dumontet C, Reichert JM, Senter PD, Lambert JM, Beck A (2023). Antibody-drug conjugates come of age in oncology. *Nat Rev Drug Discov* 22(8): 641-661. doi: 10.1038/s41573-023-00709-2

12. Forveille S, Zhao L, Sauvat A, Cerrato G, Leduc M, Doffe F, Pan Y, Liu P, Kroemer G, Kepp O (2025). Patritumab deruxtecan induces immunogenic cell death. *Oncoimmunology* 14(1): 2514050. doi: 10.1080/2162402X.2025.2514050

13. Tsao LC, Wang JS, Ma X, Sodhi S, Ragusa JV, Liu B, McBane J, Wang T, Wei J, Liu CX, Yang X, Lei G, Spasojevic I, Fan P, Trotter TN, Morse M, Lyerly HK, Hartman ZC (2025). Effective extracellular payload release and immunomodulatory interactions govern the therapeutic effect of trastuzumab
deruxtecan (T-DXd). Nat Commun 16(1): 3167. doi: 10.1038/s41467-025-58266-8

14. Bauzon M, Drake PM, Barfield RM, Cornali BM, Rupniewski I, Rabuka D (2019). Maytansine-bearing antibody-drug conjugates induce in vitro hallmarks of immunogenic cell death selectively in antigen-positive target cells. *Oncoimmunology* 8(4): e1565859. doi: 10.1080/2162402X.2019.1565859

15. Rios-Doria J, Harper J, Rothstein R, Wetzel L, Chesebrough J, Marrero A, Chen C, Strout P, Mulgrew K, McGlinchey K, Fleming R, Bezabeh B, Meekin J, Stewart D, Kennedy M, Martin P, Buchanan A, Dimasi N, Michelotti E, Hollingsworth R (2017). Antibody-Drug Conjugates Bearing Pyrrolobenzodiazepine or Tubulysin Payloads Are Immunomodulatory and Synergize with Multiple Immunotherapies. *Cancer Res* 77(10): 2686-2698. doi: 10.1158/0008-5472.CAN-16-2854

16. Sommer A, Berndt S, Lerchen HG, Forveille S, Sauvat A, Mumberg D, Kroemer G, Kepp O (2022). Antibody-drug conjugates harboring a kinesin spindle protein inhibitor with immunostimulatory properties. *Oncoimmunology* 11(1): 2037216. doi: 10.1080/2162402X.2022.2037216

17. Montes de Oca R, Alavi AS, Vitali N, Bhattacharya S, Blackwell C, Patel K, Seestaller-Wehr L, Kaczynski H, Shi H, Dobrzynski E, Obert L, Tsvetkov L, Cooper DC, Jackson H, Bojczuk P, Forveille S, Kepp O, Sauvat A, Kroemer G, ..., Hoos A (2021). Belantamab Mafodotin (GSK2857916) Drives Immunogenic Cell Death and Immune-mediated Antitumor Responses In Vivo. *Mol Cancer Ther* 20(10): 1941-1955. doi: 10.1158/1535-7163.MCT-21-0035

18. Lonial S, Lee HC, Badros A, Trudel S, Nooka AK, Chari A, Abdallah AO, Callander N, Lendvai N, Sborov D, Suvannasankha A, Weisel K, Karlin L, Libby E, Arnulf B, Facon T, Hulin C, Kortum KM, Rodriguez-Otero P, ..., Cohen AD (2020). Belantamab mafodotin for relapsed or refractory multiple myeloma (DREAMM-2): a two-arm, randomised, open-label, phase 2 study. *Lancet Oncol* 21(2): 207-221. doi: 10.1016/S1470-2045(19)30788-0

19. Almodovar Diaz AA, Alouch SS, Chawla Y, Gonsalves WI (2024). The Antibody Drug Conjugate, Belantamab-Mafodotin, in the Treatment of Multiple Myeloma: A Comprehensive Review. *Blood Lymphat Cancer* 14: 71-87. doi: 10.2147/BLCTT.S490021

20. Chen R, Manochakian R, James L, Azzouqa AG, Shi H, Zhang Y, Zhao Y, Zhou K, Lou Y (2020). Emerging therapeutic agents for advanced non-small cell lung cancer. *J Hematol Oncol* 13(1): 58. doi: 10.1186/s13045-020-00881-7

21. Janne PA, Baik C, Su WC, Johnson ML, Hayashi H, Nishio M, Kim DW, Koczywas M, Gold KA, Steuer CE, Murakami H, Yang JC, Kim SW, Vigliotti M, Shi R, Qi Z, Qiu Y, Zhao L, Sternberg D, Yu C, Yu HA (2022). Efficacy and Safety of Patritumab Deruxtecan (HER3-DXd) in EGFR Inhibitor-Resistant, EGFR-Mutated Non-Small Cell Lung Cancer. *Cancer Discov* 12(1): 74-89. doi: 10.1158/2159-8290.CD-21-0715

22. Coleman N, Yap TA, Heymach JV, Meric-Bernstam F, Le X (2023). Antibody-drug conjugates in lung cancer: dawn of a new era? *NPJ Precis Oncol* 7(1): 5. doi: 10.1038/s41698-022-00338-9

23. Sibomana O (2025). FDA approves Datroway: a novel therapy for HR-positive, HER2-negative metastatic breast cancer. *Ann Med Surg* 87(4): 2521-2522. doi: 10.1097/MS9.0000000000003122

24. Yao X, Sun C, Xiong F, Zhang W, Yao W, Xu Y, Fan W, Huo F (2024). Polysarcosine as PEG Alternative for Enhanced Camptothecin-Induced Cancer Immunogenic Cell Death. *ACS Appl Mater Interfaces* 16(15): 19472-19479. doi: 10.1021/acsmater.4c00166

25. Wang Z, Li W, Park J, Gonzalez KM, Scott AJ, Lu J (2022). Camptotheso elicits immunogenic cell death to boost colorectal cancer immune checkpoint blockade. *J Control Release* 349: 929-939. doi: 10.1016/j.jconrel.2022.07.042

26. Luo L, Wang X, Liao YP, Chang CH, Nel AE (2022). Nanocarrier Co-formulation for Delivery of a TLR7 Agonist plus an Immunogenic Cell Death Stimulus Triggers Effective Pancreatic Cancer Chemo-immunotherapy. *ACS Nano* 16(8): 13168-13182. doi: 10.1021/acsnano.2c06300

27. Haratani K, Yonesaka K, Takamura S, Maenishi O, Kato R, Takegawa N, Kawakami H, Tanaka K, Hayashi H, Takeda M, Maeda N, Kagari T, Hirotani K, Tsurutani J, Nishio K, Doi K, Miyazawa M, Nakagawa K (2020). U3-1402 sensitizes HER3-expressing tumors to PD-1 blockade by immune activation. *J Clin Invest* 130(1): 374-388. doi: 10.1172/JCI126598

28. Liu X, Jiang J, Liao YP, Tang I, Zheng E, Qiu W, Lin M, Wang X, Ji Y, Mei KC, Liu Q, Chang CH, Wainberg ZA, Nel AE, Meng H (2021). Combination Chemo-

Immunotherapy for Pancreatic Cancer Using the Immunogenic Effects of an Irinotecan Silicasome Nanocarrier Plus Anti-PD-1. Adv Sci 8(6): 2002147. doi: 10.1002/advs.202002147

29. Nelson BE, Meric-Bernstam F (2024). Leveraging TROP2 Antibody-Drug Conjugates in Solid Tumors. *Annu Rev Med* 75: 31-48. doi: 10.1146/annurev-med-071322-065903

30. Salifu I, Singh N, Berraondo M, Remon J, Salifu S, Severson E, Quintana A, Peiro S, Ramkissoon S, Vidal L, Chico I, Saini KS (2023). Antibody-drug conjugates, immune-checkpoint inhibitors, and their combination in advanced non-small cell lung cancer. *Cancer Treat Res Commun* 36: 100713. doi: 10.1016/j.ctarc.2023.100713

31. Saini KS, Punie K, Twelves C, Bortini S, de Azambuja E, Anderson S, Criscitiello C, Awada A, Loi S (2021). Antibody-drug conjugates, immune-checkpoint inhibitors, and their combination in breast cancer therapeutics. *Expert Opin Biol Ther* 21(7): 945-962. doi: 10.1080/14712598.2021.1936494

32. Iwata TN, Sugihara K, Wada T, Agatsuma T (2019). [Fam]- trastuzumab deruxtecan (DS-8201a)-induced antitumor immunity is facilitated by the anti-CTLA-4 antibody in a mouse model. *PLoS One* 14(10): e0222280. doi: 10.1371/journal.pone.0222280

33. Iwata TN, Ishii C, Ishida S, Ogitani Y, Wada T, Agatsuma T (2018). A HER2-Targeting Antibody-Drug Conjugate, Trastuzumab Deruxtecan (DS-8201a), Enhances Antitumor Immunity in a Mouse Model. *Mol Cancer Ther* 17(7): 1494-1503. doi: 10.1158/1535-7163.MCT-17-0749

34. Pan H, Liu P, Kroemer G, Kepp O (2024). Preconditioning with immunogenic cell death-inducing treatments for subsequent immunotherapy. *Int Rev Cell Mol Biol* 382: 279-294. doi: 10.1016/bs.ircmb.2023.06.001

35. Heath EI, Rosenberg JE (2021). The biology and rationale of targeting nectin-4 in urothelial carcinoma. *Nat Rev Urol* 18(2): 93-103. doi: 10.1038/s41585-020-00394-5

36. Pan H, Liu P, Zhao L, Pan Y, Mao M, Kroemer G, Kepp O (2024). Immunogenic cell stress and death in the treatment of cancer. *Semin Cell Dev Biol* 156: 11-21. doi: 10.1016/j.semcdb.2023.10.007

37. Liu P, Chen J, Zhao L, Hollebecque A, Kepp O, Zitvogel L, Kroemer G (2022). PD-1 blockade synergizes with oxaliplatin-based, but not cisplatin-based, chemotherapy of gastric cancer. *Oncoimmunology* 11(1): 2093518. doi: 10.1080/2162402X.2022.2093518

38. Martin-Broto J, Diaz-Beveridge R, Moura D, Ramos R, Martinez-Trufero J, Carrasco I, Sebio A, Gonzalez-Billalabeitia E, Gutierrez A, Fernandez-Jara J, Hernandez-Vargas L, Cruz J, Valverde C, Hindi N (2025). Phase Ib Study for the Combination of Doxorubicin, Dacarbazine, and Nivolumab as the Upfront Treatment in Patients With Advanced Leiomyosarcoma: A Study by the Spanish Sarcoma Group (GEIS). *J Clin Oncol* 43(3): 297-307. doi: 10.1200/JCO.24.00358

39. Paz-Ares L, Borghaei H, Liu SV, Peters S, Herbst RS, Stencel K, Majem M, Sendur MAN, Czyzewicz G, Caro RB, Lee KH, Johnson ML, Karadurmus N, Grohe C, Baka S, Csoszi T, Ahn JS, Califano R, Yang TY, ..., investigators IM (2025). Efficacy and safety of first-line maintenance therapy with lurbinectedin plus atezolizumab in extensive-stage small-cell lung cancer (IMforte): a randomised, multicentre, open-label, phase 3 trial. *Lancet* 405(10495): 2129-2143. doi: 10.1016/S0140-6736(25)01011-6

40. Ahn MJ, Lisberg A, Goto Y, Sands J, Hong MH, Paz-Ares L, Pons-Tostivint E, Perol M, Felip E, Sugawara S, Hayashi H, Cho BC, Blumenschein G, Jr., Shum E, Lee JS, Heist RS, Cornelissen R, Chang WC, Kowalski D, Zebger-Gong H, Chargualaf M, Gu W, Lan L, Howarth P, Joseph R, Okamoto I (2025). A pooled analysis of datopotamab deruxtecan in patients with EGFR-mutated NSCLC. *J Thorac Oncol* 12: S1556-0864(25)00761-0. doi: 10.1016/j.jtho.2025.06.002

41. Bardia A, Krop IE, Kogawa T, Juric D, Tolcher AW, Hamilton EP, Mukohara T, Lisberg A, Shimizu T, Spira AI, Tsurutani J, Damodaran S, Papadopoulos KP, Greenberg J, Kobayashi F, Zebger-Gong H, Wong R, Kawasaki Y, Nakamura T, Meric-Bernstam F (2024). Datopotamab Deruxtecan in Advanced or Metastatic HR+/HER2- and Triple-Negative Breast Cancer: Results From the Phase I TROPION-PanTumor01 Study. *J Clin Oncol* 42(19): 2281-2294. doi: 10.1200/JCO.23.01909

42. Forveille S, Humeau J, Sauvat A, Bezu L, Kroemer G, Kepp O (2019). Quinacrine-mediated detection of intracellular ATP. *Methods Enzymol* 629: 103-113. doi: 10.1016/bs.mie.2019.05.050