# Patritumab deruxtecan induces immunogenic cell death

Sabrina Forveille, Liwei Zhao, Allan Sauvat, Giulia Cerrato, Marion Leduc, Flora Doffe, Yuhong Pan, Peng Liu, Guido Kroemer, Oliver Kepp

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## To cite this version:

Sabrina Forveille, Liwei Zhao, Allan Sauvat, Giulia Cerrato, Marion Leduc, et al., Patritumab deruxtecan induces immunogenic cell death. OncoImmunology, 2025, 14, 10.1080/2162402x.2025.2514050. [https://hal-05095548](https://hal-05095548)

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# Patritumab deruxtecan induces immunogenic cell death

Sabrina Forveille, Liwei Zhao, Allan Sauvat, Giulia Cerrato, Marion Leduc, Flora Doffe, Yuhong Pan, Peng Liu, Guido Kroemer & Oliver Kepp

**To cite this article:** Sabrina Forveille, Liwei Zhao, Allan Sauvat, Giulia Cerrato, Marion Leduc, Flora Doffe, Yuhong Pan, Peng Liu, Guido Kroemer & Oliver Kepp (2025) Patritumab deruxtecan induces immunogenic cell death, *Oncolmmunology*, 14:1, 2514050, DOI: 10.1080/2162402X.2025.2514050

**To link to this article:** https://doi.org/10.1080/2162402X.2025.2514050

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© 2025 The Author(s). Published with license by Taylor & Francis Group, LLC.

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# Patritumab deruxtecan induces immunogenic cell death

Sabrina Forveille ID a,b, Liwei Zhao ID a,b, Allan Sauvat ID a,b, Giulia Cerrato ID a,b, Marion Leduc ID a,b, Flora Doffe ID a,b, Yuhong Pan a,b,c,d, Peng Liu ID a,b, Guido Kroemer ID a,b,e, and Oliver Kepp ID a,b

ªMetabolomics and Cell Biology Platforms, Gustave Roussy Cancer Center, Université Paris Saclay, Villejuif, France; ᵇCentre 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; ᶜFaculté de Médecine, Université Paris-Saclay, Paris, France; ᵈResearch Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China; ᵉInstitut du Cancer Paris CARPEM, Department of Biology, APHP, Hôpital Européen Georges Pompidou, Paris, France

## ABSTRACT

Antibody-drug conjugates (ADCs) enable targeted delivery of cytotoxic payload to cancer cells. Here, we characterized the mode of action of the ADC patritumab deruxtecan, which is a monoclonal antibody specific for Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3, best known as HER3) coupled to the topoisomerase-I inhibitor DXd. Patritumab deruxtecan decreased viability and induced the relocation of calreticulin fused to green fluorescent protein (CALR-GFP) to the periphery of human osteosarcoma U2OS cells engineered to express HER3 but not in their parental counterparts only expressing the CALR-GFP biosensor. Patritumab deruxtecan as well as its payload DXd induced various traits of immunogenic cell death (ICD) including antibody detectable calreticulin membrane exposure, exodus of high mobility group protein B1 (HMGB1), as well as the release of ATP into cell culture supernatants. Moreover, DXd causes rapid inhibition of DNA-to-RNA transcription, which is a key predictor for ICD. Mouse cancer cells treated with DXd were able to vaccinate syngeneic immunocompetent mice against tumor challenge. Tumor-free mice developed immune memory that led to the rejection of syngeneic tumors after rechallenge. In conclusion, patritumab deruxtecan is equipped with a cytotoxic payload that induces hallmarks of ICD *in vitro* and elicits antitumor immunity *in vivo*.

## ARTICLE HISTORY

Received 17 March 2025
Revised 14 May 2025
Accepted 27 May 2025

## KEYWORDS

Antibody drug conjugate;
anticancer immunity; cancer;
immunotherapy

## Introduction

Many anticancer agents including chemotherapies, targeted therapies, as well as antibody-drug conjugates (ADCs), mediate durable therapeutic effects *in vivo* only when they exhibit a dualistic mode of action that combines direct tumor targeted cytotoxicity with indirect immune system-mediated responses.¹,² Hence, the immunological consequences of the cytotoxic treatment are decisive for the long-term effect of the therapy.³ In the context of an intact immune system, specific anticancer agents induce “immunogenic cell death” (ICD) of malignant cells, thereby stimulating T cell-mediated immune responses against tumor-associated antigens that control residual cancer cells.¹,² ICD is defined as a specific combination of pre-mortem stress responses consisting in the sequential emission of danger associated molecular patterns (DAMPs) that together provide adjuvant signals to stimulate dendritic cell (DC)-mediated T cell priming.³⁻⁶ ICD is characterized by the early onset of the integrated stress response that orchestrates both the autophagy-dependent release of adenosine triphosphate (ATP) as well as the ER stress-dependent exposure of the ER chaperone calreticulin (CALR) on the cell surface. Extracellular ATP acts as a chemoattractant on tumor-infiltrating conventional type I dendritic cells (cDC1) expressing P2RY2 purinergic receptors, and ecto-CALR serves as an

uptake signal for DC expressing the CALR receptor low density lipoprotein receptor-related protein 1 (LRP1).⁷ Furthermore, in the course of ICD, the nuclear high mobility group box 1 protein (HMGB1) is released into the extracellular space and then binds to TLR4 expressed on DC to induce DC maturation and facilitate tumor antigen processing and presentation.⁸ We recently described the inhibition of DNA-to-RNA transcription, morphologically characterized by the condensation of nucleoli (CON) phenotype, as one of the first biochemical alterations that is induced by potent ICD-inducing cytotoxics and hence predicts the propensity of compounds to induce anticancer immunity.⁹,¹⁰ Altogether, the aforementioned cellular alterations constitute the main hallmarks of ICD and can be assessed *in vitro* for the discovery of agents with immunostimulatory properties.¹¹ *In vivo*, subcutaneous vaccination of immunocompetent mice with cells that are undergoing bona fide ICD elicits a protective immune response that precludes the growth of tumors upon rechallenge of the vaccinated mice with cancer cells of the same kind.¹²,¹³ Moreover, it is important to note that ICD-inducing anticancer drugs generally sensitize to subsequent immune checkpoint blockade.²,⁶,¹⁴,¹⁵

To deliver cytotoxicants specifically to cancer cells (and hence to spare normal cells and reduce side effects), it is

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possible to conjugate them to monoclonal antibodies (mAb) targeting tumor-specific surface antigens.¹⁶ Indeed, certain ICD-inducing ADC payloads can ignite anticancer immune responses, as this has been documented for maytansinoid, auristatin, and pyrrolobenzodiazepine.¹⁷⁻¹⁹ In addition, a mAb specific for B-cell maturation antigen (BCMA) conjugated with monomethyl auristatin F (generic name: belantamab mafodotin), as well as a mAb specific for fibroblast growth factor-inducible 14 (Fn14, best known as TWEAKR) coupled to a small molecule inhibitor of kinesin family member 11, effectively induce ICD and stimulate anticancer immunity.¹⁸⁻²¹,²²

Non-small cell lung cancer (NSCLC) accounts for a large majority (85%) of all lung cancers with EGFR-activating driver mutations being detectable in 14% to 38% of all cases worldwide.²³ More than half of the patients present with distant spread at diagnosis. Currently available therapies include EGFR tyrosine kinase inhibitors (TKI) and platinum-based chemotherapy. The frequent emergence of resistance mechanisms limits the efficacy of TKI-based therapy, highlighting the urgent need for new therapeutic approaches.²⁴ Here, we determined potential ICD induction by the ADC combining the mAb patritumab specific for Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3, best known as HER3) with DXd. Patritumab deruxtecan consists of a human anti-HER3 IgG1 monoclonal antibody attached to several molecules of the topoisomerase I inhibitor exatecan derivative DXd via tetrapeptide-based cleavable linkers. In clinical studies, patritumab deruxtecan has demonstrated durable responses in patients with locally advanced or metastatic NSCLC following disease progression on treatment with EGFR TKI and platinum-based chemotherapy.²⁵ Accordingly, patritumab deruxtecan was granted U.S. Food and Drug Administration (FDA) breakthrough therapy designation for the treatment of advanced progressive NSCLC on or after treatment with platinum-based therapies.²⁶ We characterized the induction of immunogenic cell death by patritumab deruxtecan in vitro and then assessed the immunological effects of its payload DXd in suitable mouse models.

# Material and methods

## Cell lines and cell culture

Murine B16-F10 melanoma cells and human U2OS osteosarcoma cells were obtained from the ATCC. Murine MCA205 fibrosarcoma cells came from Merck (Darmstadt Germany). Cell culture plastic came from Corning (New York, NY, USA). B16-F10 and U2OS were kept in Dulbecco's modified Eagle's medium (DMEM, cat. no. 41966-02, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with non-essential amino acids (NEAA, cat. no. 11140-035, Thermo Fisher Scientific) HEPES (cat. no. 15630080, Thermo Fisher Scientific) and 10% fetal bovine serum (FBS, cat. no. F7524, Merck). MCA205 were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium (cat. no. 61870044, Thermo Fisher Scientific) having 10% fetal bovine serum, HEPES and non-essential amino acids. U2OS cells stably expressing calreticulin (CALR) coupled to green fluorescent protein (GFP) were cultured in the presence

of suitable selection antibiotics (5 µg/ml blasticidin, 100 µg/ml zeocin), and HER3 expressing derivatives were generated.²⁷ For cocultures U2OS cells either expressing or not expressing HER3 were mixed at a 1:1 ratio. All cells were kept in a humidified cell culture incubator maintained at 37 °C and 5% CO₂ and the absence of mycoplasma was routinely tested.

## Chemicals and antibodies

Mitoxantrone and dactinomycin came from Merck and oxaliplatin from Accord Healthcare (Ahmedabad, India). DXd, non-targeting IgG-DXd ADC control, anti-HER3 monoclonal antibody (U3-1287) and patritumab deruxtecan were furnished by Daiichi Sankyo. Rabbit polyclonal anti-calreticulin (ab2907), rabbit polyclonal anti-fibrillarin (cat. no. ab5821), mouse monoclonal anti-nucleolin (cat. no. ab13541, clone 4E2) antibodies were all from Abcam (Cambridge, UK). Anti-HER3-antibody (324702) was purchased from BioXcell (West Lebanon, NH, USA) and anti-mouse and anti-rabbit AlexaFluor™ 488- or 647-conjugated secondary antibodies (cat. no. A11029, A11034, A21235) came from Thermo Fisher Scientific.

## Fluorescence microscopy

U2OS cells wild-type or stably expressing CALR-GFP as well as derivatives thereof expressing HER3, were seeded at a density of 5 × 10² cells per well in 384-well µclear imaging plates (Greiner Bio-one) and let adapt. The following day, cells were treated and were then incubated for 5 days. Next, cells were fixed with 3.7% formaldehyde containing 1 µg/ml Hoechst 33,342 for 20 min. Following images were acquired by automated fluorescence microscopy by means of an IXMc confocal BioImager (Molecular Devices, San Jose, CA, USA) equipped with a Lumencor light source (Lumencor, Beaverton, OR, USA), excitation and emission filters from Semrock (Semrock, Rochester, NY, USA), a 16-bit sCMOS camera (Andor, Belfast, Northern Ireland) and a 20× PlanAPO objective (Nikon, Tokyo, Japan). Next, image analysis was performed using the EBImage package with R.

## Viability assessment

U2OS and MCA205 cells were seeded at a density of 5 × 10² cells per well in 384-well µclear imaging plates (Greiner Bio-one; Kremsmünster, Austria) and let adapt for 24 h. Then cells were treated with patritumab deruxtecan, the non-targeting IgG-DXd ADC and human anti-HER3 IgG1 (patritumab). Next cells were fixed for 20 min in 3.7% formaldehyde (cat. no. F8775, Merck) diluted in PBS supplemented with 1 µg/ml Hoechst 33,342 (cat. no. H3570, Thermo Fisher Scientific). Following images were acquired by automated fluorescence microscopy as described above.

## Calreticulin surface exposure detection

U2OS wild-type cells were seeded at a density of 2 × 10³ cells per well in 96-well µclear imaging plates (Greiner Bio-one). The following day, cells were treated and then incubated for 5
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days. Next, cells were detached and transferred into V-shaped 96-well plates before centrifugation at 200 g for 5 min. The supernatant was discarded, cells were resuspended in blocking buffer (1% BSA in PBS) containing rabbit polyclonal anti-calreticulin antibody (1:100) and incubated for 30 min at 4 °C. Then, cells were washed with PBS, spun at 200 g for 5 min and supernatant was removed. The cells were resuspended in blocking buffer containing secondary AlexaFluor™ 488 goat anti-rabbit IgGs (1:1,000) and incubated again for 30 min at 4 °C. Thereafter, cells were washed with PBS and spun at 200 g for 5 min. The supernatant was removed and cells were resuspended in cold PBS containing 1 µM diamidino-2-Phenylindole (DAPI, cat. no. 62248, Thermo Fisher Scientific). Samples were analyzed using a BD LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, New Jersey) and the percentage of CALR⁺ cells among DAPI⁻ live cells was determined.

## ATP assessment

U2OS wild-type cells were seeded at a density of $2 \times 10^3$ cells per well in 96-well plates (Greiner Bio-one). The following day, cells were treated for 5 days. Next the supernatants were collected, centrifuged at 200 g for 5 min and then transferred into white bottom 96-well plate (Greiner Bio-one). The number of cells per well was assessed by microscopy and ATP content in cell culture supernatants was detected using the ENLITEN ATP Bioluminescence Detection Kit (cat. no. FF2000; Promega, Madison, Michigan, USA) according to the manufacturer's instructions. Luminescence was assessed at 560 nm with a Spectramax i3x. ATP concentration was calculated based on standards and was further normalized with the number of cells.

## Intracellular ATP assessment

Cells were seeded at a density of $2 \times 10^3$ cells per well in a 96-well µclear imaging plates (Greiner Bio-one). The following day, cells were treated for 5 days. Next, cells were incubated with Krebs-Ringer solution (5 mm KCl, 125 mm NaCl, 0.7 mm KH₂PO₄, 1 mm MgSO₄, 2 mm CaCl₂, 6 mm glucose and 25 mm HEPES) at pH 7.4 containing 5 µM quinacrine²⁸ and 4 µM Hoechst 33,342 for 30 min at 37 °C. Then, cells were washed with Krebs-Ringer solution and images were acquired by automated confocal live cell fluorescence microscopy as detailed before. The atmospheric environment used for live cellular imaging was controlled by means of an Ibidi gas mixer (Gräfelfing, Germany). As before analyses were performed with R using the *EBImage* package.

## HMGB1 release ELISA

U2OS wild-type cells were seeded at a density of $2 \times 10^3$ cells per well in a 96-well plate. The following day cells were treated and then incubated for 5 days. Next, cell culture supernatants were collected and the number of cells per well was assessed by microscopy. Supernatants were centrifuged at 200 g for 5 min

and used to assess HMGB1 content by ELISA (cat. no. ST51011; IBL International GmbH, Hamburg, Germany) following the manufacturer's instructions. Absorbance was measured at 450 nm using a Spectramax i3x plate reader (Molecular Devices), the concentration of HMGB1 was calculated based on standards and was further normalized with the number of cells.

## Assessment of RNA transcription via 5-ethynyl uridine (EU) incorporation and Click-iT detection

Alterations in global RNA transcription were assessed by quantifying the incorporation of the Click-iT detectable synthetic thymidine analog 5-ethynyl uridine (EU) (cat. no. C10327, Thermo Fisher Scientific), adapting the manufacturer's advice.²⁹,³⁰ Briefly, osteosarcoma U2OS cells were seeded in 384-well µclear imaging plates (Greiner Bio-one). The next day cells were treated with DXd for 2.5 h. Then 1 mm EU was added for another 1 h before fixation with 3.7% formaldehyde containing 1 µg/ml Hoechst 33,342 for an additional 1 h. Then cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 min and incubated with AlexaFluor™-488-coupled azide for another 2 h at room temperature in the dark. After repeated PBS washes, fluorescence intensity corresponding to EU incorporation in the nucleus was quantified by microscopy as described above.

## Assessment of transcription via fibrillarin and nucleolin co-staining

One day before treatment, U2OS were seeded in 384-well µclear imaging plates and let adapt for 24 h. The next day, cells were treated and incubated for 4 h. Then cells were fixed with 3.7% formaldehyde containing 1 µg/mL Hoechst 33,342 for 30 min at room temperature, washed with PBS and permeabilized with 0.1% Triton X-100 for 10 min. Unspecific antibody binding was blocked by incubation with 2% BSA for 1 h at room temperature. Co-staining with anti-fibrillarin and anti-nucleolin antibodies was performed at 4 °C on a plate rocker overnight, then cells were repeatedly washed with PBS, stained with AlexaFluor™ 647-coupled mouse and 488-coupled rabbit secondary antibodies for 2 h at room temperature. Following cells were washed and images were acquired by microscopy as described above. Nuclear colocalization was assessed by determining the Pearson correlation coefficient (PCC) for fluorescent signals.¹⁰,²⁹

## Assessment of condensation of nucleoli (CON)

U2OS cells were seeded in 384-well µclear imaging plate and let adapt for 24 h. The next day cells were treated and subjected to label-free live cell time-lapse imaging using transmitted-light. Images were acquired at an interval of 30 min over 2 h using an IXMc confocal bioimager. The condensation of nucleoli (CON) phenotype was predicted using a pre-trained convolutional neural network (CNN).¹⁰ Briefly, nuclei were detected and filtered. After centroid extraction, single cell patches were generated and fed into the CON classifier which evaluated the probability of CON phenotype. The CON⁺ mean probability was calculated over time.
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## Targeted mass spectrometric assessment of DXd abundance in cell culture supernatants

U2OS cells wild-type, derivatives thereof stably expressing HER3, or a mix of both at 1:1 ratio were seeded in 6 well plates at let adapt for 24 hours. Cells were then treated with 0.16 µg/ mL patritumab deruxtecan for 5 days. Next the cell culture supernatants were collected and spun at 200 g for 5 min. For each sample, 50 µL of supernatant was mixed with 500 µL of ice-cold extraction buffer (methanol/water, 9/1 spiked with a cocktail of internal standards) and centrifuged for 15 min at 10,000 g and 4 °C. Four hundred and fifty microliters of supernatant were collected and evaporated. Then the dry extracts were spiked with 200 µL of MilliQ water before LC-MS analysis. Pseudo-targeted analysis by ultra-high performance liquid chromatography – high resolution and accurate mass (UHPLC-HRAM) was performed on a U3000 (Dionex)/ Orbitrap q-Exactive (Thermo) coupling, as previously described.³¹,³² All targeted treated data were merged and cleaned with the R package GRMeta (V4.0) (https://github.com/kroemerlab/GRMeta).

## In vivo experiments

Five- to six-week-old female C57Bl/6J mice were obtained from Envigo (Huntington, UK) and kept in a pathogen-free and environment-controlled animal facility with water and food *ad libitum*. Tumor size was measured with a caliper and the ellipsoid area was determined with the formula length x width x π/4. Animals were sacrificed when ethical endpoints were reached (tumor area >200 mm², ulcerating tumors, weight loss > 10% or other signs of discomfort). Tumor growth and survival were assessed with TumGrowth software freely accessible at https://kroemerlab.shinyapps.io/TumGrowth. All in vivo experiments were conducted according to EU Directive 63/2010 and the protocol #40509-2023012516452850 was approved by the local ethical committee (CEEA IRCIV/IGR no.26, registered at the French Ministry of Research).

## In vivo vaccination and rechallenge

Immunocompetent five- to six-week-old female C57Bl/6J mice were inoculated subcutaneously (s.c.) with $1 \times 10^6$ MCA205 cells prior treated with DXd, CDDP or DXd plus CDDP. Mitoxantrone (MTX)-treated cells were used as a positive control and PBS as negative control. One-week later, mice were challenged with $1 \times 10^5$ MCA205 cells injected subcutaneously (s.c.). into the contralateral flank. Then tumor growth was monitored for one month. Tumor-free mice were rechallenged with homologous MCA205 cells injected s.c into one flank and $1 \times 10^5$ heterologous murine melanoma B16-F10 cells into the contralateral flank. Tumor growth was monitored routinely.

## Statistical analyses

*In vitro* data are displayed as means ± SD of at least triplicate or quadruplicate assessments. Data were statistically analyzed using the freely available R software (https://www.r-project.org). Data were evaluated by means of Welch's or Student's

t-test using the *t.test* function. *In vivo* data was statistically assessed means of the TumGrowth application freely accessible at https://kroemerlab.shinyapps.io/TumGrowth.³³ Type II analysis of variance (ANOVA) and pairwise Wilcoxon test with multiple comparison and Holm's correction were utilized for tumor growth curves and a log-rank test was performed on survival data.

## Results

### Patritumab deruxtecan decreased viability and induced traits of ICD in cells expressing HER3

Previous studies demonstrated that certain agents of the pyranoindolizinoquinoline family have the capacity to trigger ICD in malignant cells and to induce anticancer immune responses *in vivo*.³⁴⁻³⁶ Here, we characterized the mode of action of the patritumab deruxtecan and its cytotoxic payload DXd, which is a derivative of the pyranoindolizinoquinoline exatecan.

To analyze the cytotoxic activity of patritumab deruxtecan, we took advantage of well human osteosarcoma U2OS cells, a well established model for the assessment of ICD hallmarks in vitro that exhibit a low level of endogenous HER3 expression and engineered cells expressing surface HER3 by lentiviral transduction of a puromycin-selectable expression construct (Figure S1). Viability was quantified by epifluorescence microscopy assessing the number of healthy cells based on size and eccentricity as well as on nuclear morphology (meaning that we excluded pyknotic nuclei accumulating the DNA-intercalating agent Hoechst 33342). Treatment with patritumab deruxtecan (ADC) reduced the viability of HER3-expressing cells in a dose and time-dependent fashion, but did not affect cellular health in parental U2OS controls at low and moderate concentrations, similar to the non-targeting IgG-DXd ADC isotype control (ISO) that failed to kill both HER3-positive and HER3-negative U2OS cells. Human anti-HER3 IgG1 monoclonal antibody (patritumab) alone, without the DXd payload, failed to reduce the viability of HER3-expressing U2OS cells underscoring the essential role of DXd for the cytotoxic effect of patritumab deruxtecan (Figure 1a-c; Figure S2). At 5 days post treatment, patritumab deruxtecan significantly decreased the viability of U2OS calreticulin (CALR) biosensor cells overexpressing HER3, yet did not affect parental cells, whereas the chemotherapeutic agent oxaliplatin similarly diminished the viability of parental and HER3 expressing cells (Figure 1a-c).

Next, we investigated the effect of patritumab deruxtecan on the subcellular distribution of a fluorescent biosensor consisting in the fusion of CALR to green fluorescent protein (GFP), which had been introduced into U2OS cells that were either expressing HER3 or were parental (and hence HER3-negative). In HER3-expressing biosensor cells (but not in HER3-negative controls), patritumab deruxtecan induced the redistribution of GFP-CALR toward granular structures located in the cellular periphery, a phenotype that resembles the one induced by the prototypic ICD inducer oxaliplatin (Figure 1d-g). This finding suggests that patritumab deruxtecan can indeed induce the immunogenic CALR relocation associated with ICD.
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**Figure 1.** Patritumab deruxtecan decreases viability and induces CALR relocation in cells expressing HER3. (a) Scheme depicting the treatment and analysis workflow. (b-g) human osteosarcoma U2OS biosensor cells stably expressing calreticulin (CALR)-green fluorescent protein (GFP) and their derivatives overexpressing HER3 were treated for 5 days with increasing concentrations of non-targeting IgG-deruxtecan ADC control, anti-HER3 monoclonal antibody (U3-1287, patritumab) and patritumab deruxtecan (ADC) in a dose range covering 0.04; 0.16; 0.63; 2.5 to 10 µg/mL. Oxaliplatin (OXA; 50 µM) was used as a prototype inducer of immunogenic cell death. (b,c) Cellular viability was assessed by microscopy and determined by determining the proportion of viable cells relative to untreated controls (d-g) calreticulin relocation was assessed by microscopy measuring GFP-signal heterogeneity as a surrogate marker for vesicular aggregation. Representative images (d,e) are depicted together with CALR relocation (f,g). Results are depicted as mean ± SD of quadruplicate assessments and were normalized to untreated controls. Data points depict single view fields. Significant increase was calculated using Welch's *t*-test. Scale bars equal 10 µm.

## Patritumab deruxtecan promotes anticancer immune effects in vitro

Intrigued by these results, we next investigated the capacity of patritumab deruxtecan (the ADC) to induce the full spectrum of ICD hallmarks *in vitro*. To this aim, U2OS cells expressing HER3 and parental WT cells were treated with the full ADC, patritumab (αHER3), or the non-targeting IgG-DXd ADC

isotype control mAb (ISO), and CALR exposure was assessed by immunostaining followed by flow cytometric gating on viable cells that exclude the vital dye 4',6-diamidino-2-phenylindole (DAPI). Of note, in this gold standard assay HER3-positive (but not parental) U2OS cells treated with patritumab deruxtecan exhibited a significantly increase in CALR exposure on the cell surface of still viable cells (Figure 2a,b; Figure S3).
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<figure><img src="image_30.png" /><figcaption>**Figure 2.** Patritumab deruxtecan induces hallmarks of immunogenic cell death in HER3 expressing derivatives but not in parental cells. Human osteosarcoma U2OS cells and their derivatives overexpressing HER3 were treated for 5 days with 0.16 µg/mL non-targeting IgG-deruxtecan ADC control, anti-HER3 monoclonal antibody (U3-1287, patritumab) or patritumab deruxtecan (ADC). Oxaliplatin (OXA; 5 µM) and mitoxantrone (MTX, 10 nM) were used as prototype inducers of immunogenic cell death. Parental cells were also treated for 5 days with the cytotoxic payload DXd (0.16 µg/mL). (a,b) calreticulin (CALR) exposure was assessed by surface immunostaining and flow cytometric analysis. The percent of viable DAPI<sup>-</sup>/CALR<sup>+</sup> cells is depicted. (c,d) ATP secretion was measured in cell culture supernatant by detecting bioluminescence of the luciferase conversion assay. (e,f). HMGB1 exodus was assessed by ELISA in cell culture supernatants. Results are depicted as mean ± SD of triplicate assessments. ATP and HMGB1 data were normalized to the number of cells assessed by microscopy. Significant increase was calculated using Welch's *t*-test.</figcaption></figure>
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Furthermore, patritumab deruxtecan caused the release of ATP from HER3-positive (but not HER3-negative) cells, as measured by two complementary methods, namely, (i) a decrease in intracellular ATP-containing vesicles stained with quinacrine, and (ii) the luciferase-based detection of extracellular ATP in cell culture supernatants (Figure 2c,d; Figure S4). Similarly, patritumab deruxtecan induced the ELISA-detectable release of HMGB1 from HER3-positive (but not HER3-negative) cells (Figure 2e,f). Of note, DXd alone was able to induce the hallmarks of ICD in U2OS WT controls cells that do not express HER3 (Figure 2a,c,e). Altogether, these results suggest that patritumab deruxtecan induces several hallmarks of ICD, but only in cells expressing the tumor antigen targeted by patritumab.

## Patritumab deruxtecan exhibits bystander antitumor effects on cells not expressing HER3

The aforementioned comparisons included separate cultures of HER3-positive and HER3-negative cells. We wondered whether patritumab deruxtecan might induce cytotoxic bystander antitumor effects on cells lacking HER3 in mixed cell cultures composed by both HER3-positive and HER3-negative cells. Indeed, in contrast to monoculture assays

(Figure 1a-c; Figure S2), where no cytotoxicity was detectable with patritumab deruxtecan in parental U2OS lacking HER3 expression, in the coculture assays (composed 1:1 by U2OS cell expressing both HER3 and nuclear GFP and U2OS cells that do not), the viability of HER3-negative bystander cells was significantly reduced (Figure 3). Moreover, mass spectrometric analysis of cell culture supernatants confirmed the release of DXd from U2OS cells expressing HER3 alone and in coculture with U2OS that lack HER3 expression treated with patritumab deruxtecan (Figure S5). Altogether, this indicates that patritumab deruxtecan is able to induce nonspecific bystander antitumor effects on cells that are located in the vicinity of their designated immunological target.

## Deruxtecan induces immunogenic cell death in vitro and in vivo

DXd was capable of inducing the condensation of nucleoli (CON), as early as 30 min after its addition to the cells (Figure 4a-d), a phenotype that strongly correlates and reflects the inhibition of DNA-to-RNA transcription.<sup>10,30,31</sup> Indeed, the incorporation of 5-ethynyl uridine (EU) into nuclei, which is a proxy of RNA synthesis resulting from DNA-to-RNA transcription, was significantly inhibited by DXd in

<figure><img src="image_32.png" /><figcaption>**Figure 3.** Patritumab deruxtecan induces bystander antitumor effect in parental cells cocultured with HER expressing derivatives. (a) Scheme depicting the coculture assay and analysis workflow. (b,c) human osteosarcoma U2OS cells and their derivatives overexpressing HER3 differentially tagged by fluorescent markers were cocultured at 1:1 ratio and treated for 5 days with 0.16 µg/mL non-targeting IgG-deruxtecan ADC control, anti-HER3 monoclonal antibody (U3-1287, patritumab); patritumab deruxtecan (ADC) or DXd. Oxaliplatin (OXA; 5 µM) was used as prototype cell death inducer. Cellular viability was assessed by fluorescent microscopy and determined by measuring the proportion of viable cells for each cell line relative to untreated controls. Results are depicted as mean ± SD of quadruplicate assessments. Data points depict single view fields. Significant decrease was calculated using Welch's t-test.</figcaption></figure>
<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /><figcaption>**Figure 4.** The cytotoxic payload DXd induces transcription inhibition. (a-d) human osteosarcoma U2OS cells were treated with 1 µM DXd, the ICD inducer oxaliplatin (OXA; 50 µM), the transcription inhibitor dactinomycin (DACT; 1 µM) or were left untreated (Ctrl) and subsequently subjected to live transmitted light microscopy. The mean probability of nucleolar condensation (CON) phenotype was calculated over time. (e,f) U2OS cells were pre-treated with a dose-range of DXd (0.1, 1, 10 µM) for 2.5 h and treatment pursued for 1.5 additional hours in the presence of 1 mm 5-ethynyl uridine (EU). After staining with an AlexaFluor™-488-coupled azide, the nuclear EU incorporation was quantified as nuclear intensity and normalized to untreated controls. (g, h) U2OS cells were treated with a dose-range of DXd (0.1, 1, 10 µM) for 4 h and co-stained with anti-fibrillin and anti-nucleolin antibodies. Fibrillarin/nucleolin colocalization was quantified by computing the Pearson correlation coefficient (PCC) between fluorescent signals. Results are depicted as mean ± SD of triplicate assessments. ATP and HMGB1 data were normalized to the number of cells assessed by microscopy. Significant increase was calculated using Welch's *t*-test. Scale bars equal 10 µm.</figcaption></figure>

a dose-dependent fashion (Figure 4e,f). Similarly, DXd induced the separation of nuclear fibrillarin and nucleolin (determined by two-color immunofluorescence staining), which is another surrogate marker of transcription inhibition (Figure 4g,h). Altogether, these data suggest that DXd efficiently induces all stigmata of ICD.

Next, we assessed the capacity of DXd to cause anticancer immune responses *in vivo*. To this aim, we employed murine fibrosarcoma MCA205, a widely accepted model for assessing anticancer immune responses in vivo, treated cells with DXd and observed that their viability decreased in a dose and time-dependent fashion (Figure S6). In the next round of experiments, MCA205 cells were treated with 2 µM DXd for 72 hours and then inoculated subcutaneously into immunocompetent C57BL/6J mice (Figure 5a). One week later, the animals were challenged with untreated MCA205 cells, and the absence of tumor growth was interpreted as the sign of a protective anticancer immune response. DXd-treated cells induced anticancer immunity in 8 out of 10 mice. This effect was comparable to the prototypic ICD inducer mitoxantrone (MTX), which

protected 7 out of 9 mice. In contrast, DXd-treated MCA205 cells were much more efficient in eliciting a protective anticancer immune response than cells killed with the non-ICD-inducing chemotherapeutic cis-diamminedichloroplatinum (II) (CDDP, best known as cisplatin), which could not protect a single mouse against rechallenge with MCA205 cells. The combination of DXd and CDDP did not further modulate anticancer immune responses and was nearly as efficient in inducing ICD (with full protection of 7 out of 10 mice) (Figure 5b-e).

As further proof for the onset of anticancer immune responses, we assessed the establishment of immune memory in mice that remained tumor-free upon challenge with live MCA205 fibrosarcoma cells. Several weeks after the initial vaccination, the mice were rechallenged with living MCA205 cells on one flank while receiving an injection with antigenically distinct B16-F10 melanoma cells into the contralateral side (Figure 6a). MCA205 fibrosarcomas established and expanded aggressively in naïve mice, but always failed to develop in the mice that had been vaccinated with, and protected against, MCA205 cells. In contrast, B16-F10 melanomas
<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

<figure><img src="image_37.png" /></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

<figure><img src="image_40.png" /></figure>

**Figure 5.** Dxd treated cells induce anticancer vaccinating effects in immunocompetent mice. (a) Scheme depicting the experimental design. Murine fibrosarcoma MCA205 cells were treated for 72 hours with DXd (2 µM), 24 hours with cisplatin (CDDP, 150 µM) or the combination of both DXd plus CDDP. Mitoxantrone (MTX, 4 µM for 24 hours) was used as positive control. Cells were inoculated *subcutaneously* (s.c.) into the left flank of C57BL/6J mice and one week later the animals were challenged with untreated MCA205 cells. Absence of tumor growth was interpreted as an onset of anticancer immunity. (b,c) tumor growth was measured and is depicted as means ± SEM and as individual tumor growth curves for each mouse (d,e). Survival and the percent of tumor free mice were assessed and are depicted as Kaplan-Meier plots. Type II analysis of variance (ANOVA) and pairwise Wilcoxon test with multiple comparison and Holm's correction were applied for tumor growth and a log-rank test was performed for survival analysis.

indistinguishably developed in naïve and MCA205-vaccinated mice (Figure 6b-e). Altogether, these results indicate that DXd-killed tumor cells injected into immunocompetent mice elicit a durable specific anticancer immune response.

Discussion

Here, we present findings that highlight the ability of patritumab deruxtecan to induce ICD *in vitro*, in cultured human cancer cells. We also show that the pyranoindolizinoquinoline payload DXd kills mouse cancer cells in a way that they induce specific anticancer immune responses endowed with long-term

memory. These findings echo prior studies indicating that certain agents of the class of pyranoindolizinoquinolines, which can be administered systemically or as nanovesicle formulations, induce traits of ICD *in vitro* and in preclinical mouse models where they exhibit the potential to enhance the efficacy of immune checkpoint inhibition.<sup>35,37,38</sup>

In this study, the HER3-targeting antibody patritumab did not exhibit any direct cytotoxicity on cultured human osteosarcoma cells engineered to overexpress HER3, meaning that these cells do not rely on trophic HER3-mediated signals. However, at high concentrations, the IgG-DXd ADC isotype control induced some cell killing, and patritumab deruxtecan killed a small
<figure><img src="image_41.png" /></figure>

<figure><img src="image_42.png" /><figcaption>**Figure 6.** Dxd induces immune memory. (a) Scheme depicting the experimental design. (b) Tumor-free mice were rechallenged with homologous MCA205 cells injected subcutaneously (s.c.) into the left flank and heterologous murine melanoma B16-F10 cells into the contralateral flank. (b-e) tumor growth was measured and is depicted as means ± SEM and as individual tumor growth curves for each mouse for homologous MCA205 (b,c) and heterologous B16-F10 tumors (d,e).</figcaption></figure>

fraction of U2OS cells lacking HER3 overexpression. This suggests that, if used at excessive concentrations, DXd exerts target-independent toxicity, which might be explained by HER3-independent nonspecific endocytosis of the ADC.³⁹ However, at lower concentrations, neither patritumab deruxtecan nor IgG-DXd ADC caused detectable off-target effects in conditions in which patritumab deruxtecan efficiently killed HER3-expressing cells only. These findings indicate that the therapeutic window is sufficiently large to enable the safe use of patritumab deruxtecan in HER3-targeted application.

It is interesting to note that, in coculture assays, the targeted deruxtecan delivery to HER3 expressing cells caused the release of free DXd and a loss in viability in neighboring control cells

that did not express HER3. Such bystander antitumor effects have been described for hydrophobic payloads that, once released into the intracellular space of their target cells from ADCs due to the digestion of the cleavable linker, diffuse across cell membranes into the extracellular space and then potentially affect any cell type in the proximity.²⁷,⁴⁰ On one hand, such bystander effect might explain potential systemic toxicity of some ADCs. However, on the other hand, a bystander antitumor effect might offer clinical benefits because the locally released payload can kill malignant cells that have downregulated or suppressed the target antigen due to immunoselection. Moreover, ADCs may deplete regulatory T cells (Tregs) or other immunosuppressive cell types (such as myeloid-derived
<figure><img src="image_43.png" /></figure>

suppressor cells) in the tumor microenvironment, thereby increasing the anticancer effects of CD4⁺ helper and CD8⁺ cytotoxic T lymphocytes infiltrating the tumor.³³,⁴⁰

The findings presented in this paper suggest that the DXd payload induces full blown immunogenic cell death (ICD). Theoretically, DXd-mediated apoptosis may promote the release of tumor-associated antigens, further amplifying adaptive immune responses.⁴¹ Altogether, the immunostimulatory ICD induction by patritumab derutxtecan may lay the groundwork for successful integration with immune checkpoint inhibitor (ICI) therapies, as demonstrated in other instances of ICD induction.⁶,⁴²,⁴³ Hence, future preclinical work involving HER3⁺ cells and clinical trials should establish optimal conditions for combining patritumab derutxtecan with ICIs targeting CTLA-4, PD-1 or PD-L1.

## Acknowledgments

Part of the figures were generated with a licensed version of bioRender (biorender.com). We thank the metabolomics platform of Gustave Roussy, Université Paris-Saclay, INSERM US23/CNRS UAR 3655, for mass spectrometric analyses.

S.F.; L.Z.; G.C.; P.L.; Y.P. and F.D. 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.

## Author contributions

CRediT: **Sabrina Forveille:** Investigation; **Allan Sauvat:** Data curation, Formal analysis; **Giulia Cerrato:** Investigation; **Marion Leduc:** Data curation, Formal analysis; **Flora Doffe:** Investigation; **Yuhong Pan:** Investigation; **Guido Kroemer:** Conceptualization, Supervision, Writing – review & editing; **Oliver Kepp:** Conceptualization, Supervision, Visualization, Writing – original draft, Writing – review & editing.

## Disclosure statement

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 Therastat 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, Merus, Transgene, 9 m, Tusk and Roche, was on the 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.

## Funding

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 labellisée);

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).

## ORCID

Sabrina Forveille ID http://orcid.org/0000-0002-9327-470X
Liwei Zhao ID http://orcid.org/0000-0001-5077-327X
Allan Sauvat ID http://orcid.org/0000-0001-7076-8638
Giulia Cerrato ID http://orcid.org/0000-0003-2755-2860
Marion Leduc ID http://orcid.org/0000-0002-4623-780X
Flora Doffe ID http://orcid.org/0000-0002-0136-424X
Peng Liu ID http://orcid.org/0000-0002-1682-9222
Guido Kroemer ID http://orcid.org/0000-0002-9334-4405
Oliver Kepp ID http://orcid.org/0000-0002-6081-9558

## Ethics statement

All in vivo experiments were conducted according to EU Directive 63/2010 and the protocol #40509-2023012516452850 which was approved by the local ethical committee (CEEA IRCIV/IGR no.26, registered at the French Ministry of Research). The present research adheres to the ARRIVE guidelines.

## Data availability statement

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

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