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. 2026 Mar 3;26:125. doi: 10.1186/s12935-026-04246-x

Combining an anti-HER2 antibody-drug conjugate with an anti-nectin-4 antibody-drug conjugate enhances efficacy in breast cancer and gastric cancer models

Narjes Yazdi 1,2, Negar Pourjamal 1,2, Hao Li 1,2, Pirjo Laakkonen 3,4,5, Heikki Joensuu 1,2,6,#, Mark Barok 1,2,✉,#
PMCID: PMC12980906  PMID: 41776522

Abstract

Background

Most human epidermal growth factor-2 (HER2)-positive breast and gastric cancers eventually become resistant to HER2-targeting antibody-drug conjugates (ADCs), such as trastuzumab deruxtecan (T-DXd) and trastuzumab emtansine (T-DM1), which are widely used for their treatment. We hypothesized that combination therapy with an HER2-targeting ADC and enfortumab vedotin (EV), an anti-nectin-4 ADC approved for the treatment of advanced urothelial cancer, could be more effective than an HER2-targeting ADC alone, as HER2-positive breast and gastric cancers frequently express nectin-4.

Methods

HER2 and nectin-4 protein expression levels were assessed with flow cytometry. The efficacy of T-DM1 and EV, both as single agents and in combination, was first assessed in breast and gastric cancer cell lines using the AlamarBlue cell proliferation assay. The antitumor activity of T-DM1, EV, and their combination was next evaluated in breast cancer and gastric cancer SCID mouse xenograft models, including a model resistant to T-DM1. Xenograft tumor samples were analyzed by immunohistochemistry. Comparisons between groups were performed using one-way analysis of variance (ANOVA), and two-way repeated measures ANOVA. Survival differences between groups were assessed using the log-rank test.

Results

All studied HER2-positive breast cancer cell lines (SKBR-3, UACC-812, MDA-453, and EFM-192A), the gastric cancer cell line (N87), and their corresponding xenograft tumors expressed nectin-4 protein. The combination of T-DM1 and EV demonstrated greater efficacy than either agent alone in SKBR-3, UACC-812, EFM-192A, and N87 cell lines. Similarly, the combination was more effective at reducing tumor size in xenograft models derived from MDA-453, N87, and RN87 cells, and it significantly prolonged survival in treated mice. Histologically, the combination treatment induced widespread apoptosis and tumor necrosis.

Conclusions

These findings indicate that co-administration of an anti-HER2 ADC and EV may substantially enhance anticancer efficacy compared to either agent alone in HER2-positive breast and gastric cancer cell lines and xenograft models. The results support further evaluation of the T-DM1 and EV combination in clinical trials.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12935-026-04246-x.

Keywords: ADC combination, Antibody-drug conjugate, Breast cancer, Enfortumab vedotin, Gastric cancer, HER2, Nectin-4, Trastuzumab emtansine

Background

The human epidermal growth factor receptor-2 (HER2) is overexpressed in 15–20% of breast and gastric cancers [1, 2]. Trastuzumab deruxtecan (T-DXd, Enhertu®) and trastuzumab emtansine (T-DM1, Kadcyla®) are trastuzumab-based antibody-drug conjugates (ADCs), which selectively deliver a cytotoxic payload to HER2-expressing cancer cells, an exatecan derivative deruxtecan and a maytansine derivative DM1, respectively [3–5]. Both T-DXd and T-DM-1 are widely used in the treatment of HER2-positive breast cancer. The U.S. Food and Drug Administration (FDA) has approved T-DM1 for the treatment of HER2-positive early-stage and advanced breast cancer [6–8] and T-DXd for the treatment of HER2-positive and HER2-low advanced breast cancer, as well as HER2-positive advanced gastric cancer [9–11]. Unfortunately, resistance to each agent eventually develops during treatment [4, 8, 11–13].

The aim of this study was to explore a novel strategy to enhance the efficacy of anti-HER2-targeted ADCs. We found that HER2-positive breast and gastric cancer cells, as well as xenograft tumors, express the cell adhesion molecule nectin-4, the target of enfortumab vedotin (EV, Padcev®). EV is an ADC approved for the treatment of advanced urothelial cancer [14]. We evaluated the effectiveness of a combination therapy using T-DM1 as the HER2-targeting ADC and EV in breast and gastric cancer cell lines and tumor xenografts, including a T-DM1-resistant xenograft tumor model. The rationale behind this approach is that combining two ADCs allows for the delivery of two distinct cytotoxic payloads to cancer cells. We found that the combination of T-DM1 and EV had a stronger anticancer effect than either treatment alone. Moreover, the combination was effective in gastric cancer xenograft tumors that did not respond to either single ADC. To the best of our knowledge, this is the first study to investigate the combination of anti-HER2 and anti-nectin-4 ADCs.

Materials and methods

Cell lines

We studied four HER2-positive human breast cancer cell lines (EFM-192A, MDA-453, SKBR-3, and UACC812), one HER2-negative breast cancer cell line (Hs-578T) as a control, and two HER2-positive human gastric cancer cell lines (N87 and RN87) (Table 1). The T-DM1-resistant HER2-positive gastric cancer cell line, RN87, was developed in our laboratory by exposing N87 cells to gradually increasing concentrations of T-DM1 (Roche Ltd., Basel, Switzerland) [15, 16]. The cells were maintained following the recommended specifications and were mycoplasma-free. The authenticity of all cell lines was confirmed using short tandem repeat analysis.

Table 1.

The cell lines studied

Cell line Origin HER2 status Source Reported sensitivity to T-DM1
EFM-192A Human BC Positive DSMZ Sensitive
MDA-453 Human BC Positive ATCC Sensitive
SKBR-3 Human BC Positive ATCC Sensitive
UACC-812 Human BC Positive ATCC Sensitive
Hs-578T Human BC Negative ATCC Resistant
N87 Human GC Positive ATCC Sensitive
RN87 Human GC, derived from N87 Positive Established in our laboratory Resistant
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ATCC, American Type Tissue Culture Collection (Manassas, VA, USA); BC, breast cancer; DSMZ, German Resource Center for Biological Material, Leibniz Institute (Braunschweig, Germany); GC, gastric cancer; HER2, human epidermal growth factor receptor 2

Cell viability assay

The effects of trastuzumab emtansine (T-DM1, Roche Ltd., Basel, Switzerland) and enfortumab vedotin (EV, Astellas Pharma Europe B.V., Leiden, The Netherlands) on cell growth were studied using the AlamarBlue method (Thermo Fisher Scientific, Waltham, MA, USA) [17, 18]. In brief, cells were trypsinized and seeded into 96-well, flat-bottomed tissue culture plates. The ADCs were initially tested as single agents at concentrations of 0.0001, 0.0006, 0.003, 0.016, 0.08, 0.4, 1, 2, and 10 µg/mL. Next, two ADC combinations were investigated, (1) increasing concentrations of T-DM1 with a fixed concentration of EV, (2) increasing concentrations of EV with a fixed concentration of T-DM1. In these experiments, the increasing concentrations of T-DM1 or EV were 0.0001, 0.0006, 0.003, 0.016, 0.08, 0.4, 1, 2 µg/mL and 10 µg/mL. The fixed ADC concentration was selected to ensure that, when used alone, the ADC had no or only minimal inhibition on cell growth. To investigate the interaction between free monomethyl auristatin E (MMAE) and DM1 (a derivative of maytansine; MedChemExpress, New Jersey, USA), cells were trypsinized and seeded into 384-well black-walled, clear-bottom microplates. Cell seeding and compound dispensing were performed using a high-throughput BioStack 3WR microplate stacking and automated dispensing system (Agilent, Santa Clara, USA). The cells were treated with either MMAE alone (1, 1.5, 3, 6.25, 12.5, 25, 50, 100, 200, 400, 600, 800, and 1,000 pM) or DM1 alone (50, 125, 250, 500, 600, 700, 800, 900, 1,000, and 1,200 pM), or combinations of both (the same increasing concentrations of DM1 with a fixed concentration of MMAE at either 25 pM or 50 pM). After five days of incubation with the ADCs and three days of incubation with either MMAE or DM1, AlamarBlue reagent (Thermo Fisher Scientific) was added to the culture medium. Viable cells were quantified by measuring fluorescence (excitation 540 nm, emission 580 nm) using a VICTOR Nivo Multimode Microplate Reader (PerkinElmer, Shelton, USA). Fluorescence values were normalized to the fluorescence of cell-free culture medium. The proportion of viable cells was calculated by dividing the fluorescence of each test sample by that of the PBS-treated control sample. Drug interactions were evaluated using the DECREASE (http://decrease.fimm.fi) and SynergyFinder (https://synergyfinder.fimm.fi) web applications [19, 20]. Drug interaction analysis maps and the most synergistic area (MSA) scores were calculated using the zero interaction potency (ZIP) model [19, 20]. In this model, MSA scores greater than 10 may be considered to indicate synergy, and scores between 0 and 10 moderate an additive effect.

Mouse xenograft models

To establish xenograft tumors, 5- to 6-week-old female SCID mice (C.B-17/IcrHan Hsd-Prkdcscid, Envigo RMS B.V., Horst, and The Netherlands) were injected subcutaneously with 9 × 106 N87 cells, or with 1 × 10⁷ RN87 cells in 100 µL of the cell culture medium, or with 5 × 106 MDA-453 cells in 50 µL of cell culture medium mixed with 50 µL Matrigel (Merck KGaA, Darmstadt, Germany). To investigate the effects of the single ADCs, T-DM1 (1 mg/kg, 2 mg/kg, 5 mg/kg), or EV (1.25 mg/kg, 5 mg/kg) was administered intravenously at 7-day intervals. T-DM1 plus EV ADC combinations were also investigated: T-DM1 plus EV (1 mg/kg, 1.25 mg/kg), T-DM1 (1 mg/kg) plus EV (5 mg/kg), T-DM1 plus EV (2 mg/kg, 5 mg/kg), and T-DM1 plus EV (5 mg/kg of each). Tumor diameters were measured with a digital caliper, and tumor volume was calculated using the formula Tvol = π/6 × larger diameter × (smaller diameter)2. Complete response was defined as tumor shrinkage to non-palpable sizes. The general condition of the mice monitored through visual inspection, weight measurement, and body condition scoring [21]. Mice were euthanized if they experienced a weight loss 20% or more, showed a decline in body condition score, developed any tumor dimension exceeded 15 mm, or if tumor ulceration was observed. Mice were sacrificed using CO2 inhalation followed by cervical dislocation.

Flow cytometry

To assess cell surface HER2 and nectin-4 expression, the cells were trypsinized and washed with 1% bovine serum albumin in phosphate-buffered saline (PBS). HER2 and nectin-4 receptors were labeled using AlexaFluor647-anti-human HER2 (324412, BioLegend, San Diego, CA, USA) and APC-anti-human nectin-4 (REA967, Miltenyi Biotec, Bergisch Gladbach, Germany) primary antibodies, respectively, for 40 min at 4 °C. Then, the cells were washed twice with PBS and fixed in 2% formaldehyde-PBS. Analysis was performed using using a BD Accuri C6 Plus Flow Cytometer (Becton Dickinson, NJ, USA).

Histology

Xenograft tumor samples were fixed in 4% buffered formaldehyde for 24 h, then processed into paraffin and cut into 4 μm thick sections. These sections were deparaffinized and subjected to antigen retrieval in a sodium citrate buffer (10 mM, pH 6.0) using a 2100 Antigen Retriever (Aptum Biologics Ltd., Southampton, UK) following the manufacturer’s instructions. After blocking for non-specific binding, optimized concentrations of the primary rabbit anti-human monoclonal antibodies, anti-HER2 (SP3, Thermo Fisher Scientific, Waltham, MA, USA), anti-nectin-4 (EPR15613-68, Abcam, Cambridge, UK), and cleaved caspase-3 antibody (IHC Detection Kit #12692, Cell Signaling Technology, Danvers, USA), were applied and incubated overnight at 4 °C. Primary antibody binding was detected using a BrightVision Poly-HRP anti-mouse kit (VWR, Radnor, USA) and 3,3′-diaminobenzidine (ImmPACT DAB, Vector Laboratories, Burlingame, CA, USA), according to the manufacturer’s recommendations. The tissue sections were then counterstained with hematoxylin. Stained slides were imaged with a 20x objective on a Zeiss Axio Scan.Z1 Slide Scanner (Carl Zeiss, Göttingen, Germany).

Apoptotic cells were identified by cleaved caspase-3 antibody staining and counted in at least 20 randomly selected fields containing representative sections of tumor, viewed with a 40x objective on an Olympus BX43 microscope (Olympus, Tokyo, Japan). Necrosis was assessed in hematoxylin and eosin (H&E)-stained sections. Tumor necrosis was defined as homogeneous clusters of degenerating and dead cells or as merging cell groups forming a coagulum in H&E-stained Sects [22, 23]. The H&E-stained slides were imaged with a 20x objective on a Zeiss Axio Scan.Z1 Slide Scanner (Carl Zeiss, Göttingen, Germany). Necrotic areas were measured on the H&E-stained sections using the ZEN 3.1 software (Carl Zeiss, Göttingen, Germany). At least three sections from different xenograft tumors were analyzed per treatment group.

Statistical analysis

In the xenograft tumor models, each treatment group consisted of five to six mice unless otherwise stated. Descriptive data are expressed as the mean ± standard deviation. Groups were compared using one-way analysis of variance (ANOVA), and two-way repeated measures ANOVA. All treated animals were included in the survival analyses. Overall survival was calculated from the date of tumor inoculation to the date of death. Survival differences between groups were compared using the log-rank test. All P values are two-sided. Statistical calculations were carried out using the IBM SPSS version 24 (IBM, Armonk, NY, USA).

Results

Breast cancer and gastric cancer cells expressed both HER2 and nectin-4

Flow cytometry analysis showed that the HER2-positive breast cancer cell lines SKBR-3, EFM-192A, UACC-812, and MDA-453, as well as the HER2-positive N87 gastric cancer cell line, expressed nectin-4 protein. In contrast, little HER2 or nectin-4 was detected in the Hs-578T breast cancer cells used as a control (Fig. 1).

Fig. 1.

Fig. 1

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HER2 and nectin-4 protein expression in the cell lines studied. Flow cytometric quantification of cell surface HER2 (A) and nectin-4 (B) content (mean fluorescence intensity) in six cancer cell lines. Means ± SD from at least three independent experiments were plotted. (C) Numeric mean fluorescence intensity (MFI) values are shown

Efficacy of single-agent T-DM1 and EV in breast and gastric cancer cell lines

The growth-inhibitory effects of T-DM1 and EV were evaluated in cancer cell lines that were positive for both HER2 and nectin-4, as well as in a cell line negative for both receptors (Hs-578T). Neither T-DM1 nor EV showed activity in the dual-negative Hs-578T cells. In contrast, both ADCs were active in SKBR-3, UACC-812, and EFM-192A cells, while the N87 cells were sensitive to T-DM1 but unresponsive to EV (Supplementary Fig. 1, Supplementary Raw Data S1).

Activity of T-DM1 in combination with EV in breast and gastric cancer cell lines

Next, cells were treated with combinations of T-DM1 and EV, either with increasing concentrations of T-DM1 and a fixed concentration of EV, or with a fixed concentration of T-DM1 and increasing concentrations of EV. The fixed concentrations of T-DM1 and EV used in the combinations were selected to ensure they had only limited single-agent activity at these concentrations. The combination of T-DM1 with a fixed concentration of EV resulted in enhanced efficacy compared with T-DM1 alone in SKBR-3, UACC-812, EFM-192A, and N87 cells, while no added effect was observed in the HER2- and nectin-4-negative Hs-578T cells (Fig. 2). Similarly, EV combined with a fixed concentration of T-DM1 showed slightly improved activity compared to EV alone in SKBR-3 cells, and a more pronounced improvement in UACC-812 cells, but not in Hs-578T cells (Supplementary Fig. 2, Supplementary Raw Data S1).

Fig. 2.

Fig. 2

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Effects of T-DM1, and T-DM1 plus EV combinations in breast and gastric cancer cell lines. (A-E) Cells were treated with increasing concentrations of T-DM1, or with increasing concentrations of T-DM1 plus a fixed concentration of EV. Means ± SD from at least three independent experiments were plotted, with each experiment containing three replicates. Means were compared using a two-way repeated measures ANOVA. *, P ˂ 0.05; **, P ˂ 0.01; ***, P ˂ 0.001. EV, enfortumab vedotin; T-DM1, trastuzumab emtansine

In the T-DM1 + EV combination assays, high MSA scores were observed in the SKBR-3 (13.6), UACC-812 (17.4), N87 (24.9), and EFM-192A (16.2) cancer cell lines indicating a synergistic drug interaction between T-DM1 and EV. A low MSA score was found in the RN87 (2.0) and the control Hs-578T cell lines (2.6) (Supplementary Fig. 3).

Next, we investigated the effect of the combination of the free payloads (DM1 and MMAE) on SKBR-3 breast cancer cells. Both free DM1 and free MMAE were active in SKBR-3 cells, and the combination of DM1 with a fixed concentration of MMAE resulted in enhanced efficacy compared with DM1 alone (Supplementary Fig. 4A-B, Supplementary Raw Data S2). In a combination assay using the free payloads, the MSA score was 7.5, suggesting an additive drug interaction between free DM1 and MMAE (Supplementary Fig. 4C).

The combination of T-DM1 plus EV had a stronger anti-cancer effect compared to the single ADCs in breast and gastric cancer xenograft tumors

The combination of T-DM1 plus EV was then evaluated in N87 gastric cancer xenografts, which were positive for both HER2 and nectin-4 (Supplementary Figure S5). Single-agent T-DM1 has previously been shown to have substantial activity in an N87 gastric xenograft tumor model at a dose of 5 mg/kg [18]. Therefore, a lower dose (1 mg/kg) was used to evaluate the efficacy of the combination treatment. Mice bearing N87 xenograft tumors were treated with 1 mg/kg of T-DM1, or 1.25 mg/kg EV, or the combination of T-DM1 (1 mg/kg) plus EV (1.25 mg/kg). Tumors progressed in all groups, but tumors in the combination group were smaller than those treated with either single-agent T-DM1 or EV (Fig. 3A, Supplementary Raw Data S3). Mice receiving the combination therapy survived longer than those treated with the single ADCs (Fig. 3B, Supplementary Raw Data S4).

Fig. 3.

Fig. 3

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T-DM1 plus EV combination is more effective than single ADCs in N87 gastric cancer xenografts. (A) Mice bearing N87 gastric cancer xenografts were treated once (black arrow) with either T-DM1 (1 mg/kg), EV (1.25 mg/kg), or a combination of T-DM1 and EV (1 mg/kg and 1.25 mg/kg, respectively). Tumors treated with the combination were smaller than those in the T-DM1 group (P = 0.010) or the EV (P = 0.032) group (*, two-way repeated measures ANOVA with Tukey’s honestly significant difference test). Three mice in the “no treatment”-group were euthanized on day 48. (B) Overall survival since the day of tumor inoculation. Log-rank test, P = 0.039, comparing combination treatment to single-agent treatments. (C) The combination of T-DM1 and EV was even more effective when EV was administered at a higher dose. Mice were treated once (black arrow) with T-DM1 (1 mg/kg), EV (5 mg/kg), or the combination (1 mg/kg T-DM1 plus 5 mg/kg EV). Tumors treated with EV were smaller than those in the T-DM1 group (P = 0.008), and tumors in the combination group were smaller than those in the EV group (P = 0.001) (**, two-way repeated measures ANOVA). The plots were truncated when ≤ 3 mice remained in the analysis. (D) Overall survival from the day of tumor inoculation. Log-rank test, P < 0.001, comparing combination treatment to single-agent treatments. EV, enfortumab vedotin; T-DM1, trastuzumab emtansine

When a higher dose of EV (5 mg/kg) was used to treat the N87 xenografts, EV alone demonstrated greater efficacy than 1 mg/kg T-DM1. However, combining 1 mg/kg T-DM1 with 5 mg/kg EV further enhanced anti-tumor efficacy, indicating that the combination was more effective than either treatment alone (Fig. 3C, Supplementary Raw Data S5). Mice in the combination group survived longer than those in the single-agent ADC groups (Fig. 3D, Supplementary Raw Data S6). All deaths resulted from euthanasia performed due to the presence of a large or ulcerated tumor.

The combination was next evaluated in MDA-453 breast cancer xenografts, which were positive also for both HER2 and nectin-4 (Supplementary Figure S6). Single-agent T-DM1 showed substantial activity in MDA-453 breast cancer xenograft tumors at a dose of 5 mg/kg (Supplementary Figure S7, Supplementary Raw Data S7). Therefore, a lower T-DM1 dose (2 mg/kg) was used for combination testing. Neither T-DM1 (2 mg/kg) nor EV (5 mg/kg) alone inhibited tumor growth. In contrast, all tumors in the combination group regressed (Fig. 4A). By day 47, the mean tumor volume in the combination group was 7.0 ± 6.3 mm3, and in two mice, tumors became non-palpable by day 55. Tumors of four mice then progressed slowly, while the two mice with non-palpable tumors at day 55 remained tumor-free until the end of the study (day 89) (Fig. 4A, Supplementary Raw Data S8). Mice in the T-DM1 plus EV combination group survived longer than those in the single ADC groups (Fig. 4B, Supplementary Raw Data S9). All deaths resulted from euthanasia. Next, the combination was evaluated in the RN87 gastric cancer xenograft model, which has previously been shown to be resistant to T-DM1 [15]. The RN87 tumors were positive for both HER2 and nectin-4 (Supplementary Figure S8). Tumors treated with either T-DM1 (5 mg/kg) or EV (5 mg/kg) alone continued to grow. In contrast, tumors in the T-DM1 plus EV group were stabilized and remained significantly smaller than those in the single-agent groups (Fig. 4C, Supplementary Raw Data S10).

Fig. 4.

Fig. 4

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T-DM1 plus EV combination is more effective than single ADCs in breast cancer and gastric cancer xenografts. (A) Mice bearing MDA-453 breast cancer xenografts were treated once (black arrow) with T-DM1 (2 mg/kg), EV (5 mg/kg), or a combination of T-DM1 and EV (2 mg/kg and 5 mg/kg, respectively). Tumors treated with the combination were smaller than those in the T-DM1 (P = 0.010) or EV (P = 0.010) groups (*, two-way repeated measures ANOVA with Tukey’s honestly significant difference test). The plots were truncated when ≤ 3 mice remained in the analysis. The transient decrease in mean tumor volume in the “no treatment”-group around d. 42 and in the T-DM1-group around d. 49 is due to euthanasia of the mice with the largest tumor volume. (B) Overall survival from the day of tumor inoculation. Log-rank test, P = 0.002, comparing combination treatment to single-agent treatments. (C) Mice bearing RN87 gastric cancer xenografts were treated (black arrows) with T-DM1 (5 mg/kg), EV (5 mg/kg), or the combination of T-DM1 and EV (5 mg/kg of each). Tumors treated with the combination were smaller than those in the T-DM1 (P = 0.002) or EV (P = 0.007) groups (**, two-way repeated measures ANOVA with Tukey’s honestly significant difference test). EV, enfortumab vedotin; T-DM1, trastuzumab emtansine

The combination therapies were well tolerated, with no changes observed in the physiological condition of the mice. The body weight of the mice treated with the T-DM1 plus EV combination was similar to that of mice treated with a single ADC (Supplementary Figure S9).

Combination of T-DM1 plus EV induces apoptotic and necrotic cell death in breast and gastric xenograft tumors

Formalin-fixed, paraffin-embedded xenograft tumor sections were stained with an anti-cleaved caspase-3 antibody to detect apoptotic cells using immunohistochemistry. More apoptotic cells were observed in N87 gastric xenograft tumors treated once with the T-DM1 plus EV combination than in tumors treated once with either single-agent ADC (Fig. 5).

Fig. 5.

Fig. 5

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The combination of T-DM1 and EV induced apoptosis in N87 gastric cancer xenografts. For histological analysis, N87 xenograft tumors were excised from SCID mice that were either untreated (A) or treated once with EV (1.25 mg/kg) (B), T-DM1 (1 mg/kg) (C), or the combination of T-DM1 and EV (1 mg/kg and 1.25 mg/kg, respectively) seven days after these treatments (D). Arrows indicate the apoptotic cells (brown). Bar = 50 μm. (E) Quantification of apoptotic cells revealed a higher number in tumors treated with the T-DM1 + EV combination compared to other treatment groups (***, P < 0.001; one-way ANOVA with Tukey’s honestly significant difference test). EV, enfortumab vedotin; T-DM1, trastuzumab emtansine

Necrotic areas were assessed in formalin-fixed, paraffin-embedded xenograft tumor sections stained with H&E. The T-DM1 plus EV combination treatment resulted in larger necrotic areas in MDA-453 breast cancer xenograft tumors compared to single-agent treatments (Fig. 6, Supplementary Figure S10).

Fig. 6.

Fig. 6

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The combination T-DM1 and EV induced necrosis in MDA-453 breast cancer xenografts. (A) Quantification of necrotic areas revealed larger necrotic regions in MDA-453 breast cancer xenograft tumors treated once with the combination of T-DM1 and EV (2 mg/kg and 5 mg/kg, respectively) compared to tumors that were untreated or treated once with EV (5 mg/kg) or T-DM1 (2 mg/kg) alone. (***, P < 0.001; one-way ANOVA with Tukey’s honestly significant difference test). (B) An H&E-stained section of an untreated MDA-453 breast cancer xenograft tumor. An MDA-453 xenograft tumor treated once with once either EV (5 mg/kg) (C) or the T-DM1 + EV combination (2 mg/kg and 5 mg/kg, respectively) (D). The tissue samples were excised seven days after the treatments. Necrotic areas are outlined in red. N, necrotic zone; V, non-necrotic, viable zone. EV, enfortumab vedotin; T-DM1, trastuzumab emtansine

Discussion

We found that the anti-cancer effect of trastuzumab emtansine (T-DM1) was enhanced when combined with enfortumab vedotin (EV). The combination of T-DM1 and EV showed stronger anti-cancer effects in most breast cancer and gastric cancer cell lines, as well as in xenograft tumors expressing both HER2 and nectin-4, compared to the single ADCs. Histologically, the cellular response to the T-DM1 and EV combination in the xenograft tumor samples consisted of apoptosis and necrosis. These data indicate that efficacy of T-DM1 can be enhanced by combining it with EV.

T-DM1 has been approved as an adjuvant treatment for patients with HER2-positive early breast cancer who have residual invasive cancer after neoadjuvant taxane and trastuzumab-based therapy, and for advanced HER2-positive breast cancer for patients who previously received trastuzumab and a taxane [6–8]. Unfortunately, most patients experience recurrence despite T-DM1 therapy [8, 12, 13, 24]. T-DM1 is not approved for the treatment of gastric cancer, since it did not demonstrate superiority over taxane chemotherapy in patients with previously treated, HER2-positive advanced gastric cancer [25]. EV is an approved treatment for advanced urothelial cancer [14], but it is not approved for the treatment of either breast cancer or gastric cancer. When EV was evaluated in Phase II trials as monotherapy for the treatment of advanced breast cancer and gastric cancer, the objective response rates achieved ranged from 10% to 19% [26].

Nectin-4, which regulates cell adhesion, differentiation, proliferation, migration, and survival [27], is frequently overexpressed in breast and gastric cancers [28]. Although nectin-4 gene amplification is infrequent in breast cancer, most breast cancers and gastric cancers express high or moderate levels of nectin-4 [26]. Nectin-4 is upregulated also in anti-HER2 ADC-induced polyploid multinucleated giant cancer cells (PGCCs), from which drug resistant tumors may arise [29]. High nectin-4 expression has also been detected in HER2-positive breast and gastric cancer cell lines [30, 31]. Additionally, a pan-cancer study investigating the expression of ADC targets found that HER2 and nectin-4 are frequently co-expressed [32], and frequent co-expression of HER2 and nectin-4 has also been observed in breast cancer and urothelial cancer [33, 34]. At the molecular level, nectin-4 co-localizes and interacts with HER2 on the cell membrane, leading to enhanced dimerization and activation of HER2 [27].

We hypothesized that in cancers positive for both HER2 and nectin-4, the efficacy of a HER2-targeting ADC could be enhanced by combining it with a nectin-4 targeting ADC. To test this hypothesis, we treated breast and gastric cancer cells, as well as xenograft tumors positive for both receptors, with a combination of T-DM1 and the anti-nectin-4 ADC, enfortumab vedotin (EV).

Combination regimens are widely used in cancer therapy. However, the combination of ADCs has been investigated in only a few studies. Two recent preclinical studies demonstrated that combining two ADCs targeting different epitopes on the HER2 receptor resulted in a more pronounced anti-cancer effect than using a single ADC [18, 35]. In a phase I clinical trial, the combination of EV and an anti-trop-2 ADC showed activity in patients with advanced urothelial carcinoma; however, the efficacy of the combination was not directly compared to that of the single ADCs [36]. To our knowledge, this is the first study to investigate the combination of an anti-HER2 ADC and an anti-nectin-4 ADC.

Both T-DM1 and EV deliver microtubule assembly inhibitor payloads (DM1 and MMAE, respectively) to cancer cells. However, DM1 and MMAE bind to different sites on tubulin and inhibit microtubule polymerization in distinct ways [37–39]. In the T-DM1 plus EV combination, cancer cells expressing both target receptors can be simultaneously treated with two highly potent cytotoxic payloads. The MSA scores observed in the cancer cell lines in the combination assays indicate that the interaction between free MMAE and free DM1 is likely additive, whereas the interaction between T-DM1 and EV is likely synergistic. These observations suggest that the combined effect of the payloads can be further enhanced when they are delivered as ADC components. Notably, trastuzumab, the monoclonal antibody component of T-DM1, has intrinsic tumor-inhibitory effect that is retained in T-DM1 [4, 40–42] and may therefore contribute to the observed synergy. In contrast, the unconjugated monoclonal antibody used in EV showed little efficacy in a preclinical cancer model [43]. Additionally, while T-DM1 lacks a bystander effect due to the non-cell membrane permeable nature of its cytotoxic metabolite lysine-MCC-DM1 [44, 45], EV exhibits a bystander effect by releasing membrane-permeable MMAE that can diffuse into neighboring cancer cells [46]. Therefore, the bystander effect may contribute to the efficacy of the T-DM1 plus EV combination.

Notably, the T-DM1-resistant RN87 xenograft tumors responded to the T-DM1 plus EV combination. The RN87 cells express the multidrug resistance drug-associated transporters ABCC1, ABCC2, and ABCG2, which may at least partially explain their resistance to T-DM1 [15, 16]. Nevertheless, the RN87 cells are sensitive to anti-HER2 ADCs carrying an MMAE payload [16, 18], suggesting that MMAE may be a less avid substrate for these transporters compared to lysine-MCC-DM1, the active metabolite of T-DM1. However, RN87 tumors did not respond to EV monotherapy, despite EV also delivering MMAE. Similarly, MDA-453 xenograft tumors were nonresponsive to EV monotherapy, while the T-DM1 plus EV combination had a strong effect on both MDA-453 and RN87 xenograft tumors. Nectin-4 has been found to co-localize and interact with HER2 on the cell membrane [27]. The epidermal growth factor receptor (EGFR) also interacts and co-localizes with HER2, and EGFR-directed antibodies promote the internalization and efficacy of anti-HER2 ADCs [47]. Therefore, we hypothesize that simultaneous targeting of cancer cells with EV and T-DM1 might lead to increased internalization of HER2, resulting in higher intracellular concentrations of the payloads. However, the low MSA score observed in RN87 cells treated with the T-DM1 + EV combination does not indicate synergy between the two ADCs in vitro. Nevertheless, the combination resulted in a more pronounced anti-cancer effect than the single treatments alone in RN87 tumor xenografts. Further investigation is warranted to explore the mechanisms underlying the activity of T-DM1 plus EV combination in HER2/nectin-4-positive cancers.

The study has some limitations. The number of cell lines studied was relatively limited and the mouse xenograft tumor models were based on these cell lines, potentially reducing tumor heterogeneity compared to patient-derived xenograft models or human cancers. Additionally, the ADC doses used may not have been optimal. Nevertheless, the T-DM1 plus EV combination was most effective when EV was administered at a dose 5 mg/kg. However, even at a dose of 1.25 mg/kg, the combination showed greater efficacy than either ADC alone. We did not study the type of interaction between T-DM1 and EV – whether synergistic or additive – which remains a topic for further study. Similarly, the relative importance of the three main components of ADCs – the antibody, the linker, and the payload – in determining efficacy remains a topic for further evaluation.

This proof-of-concept study demonstrates that the efficacy of an anti-HER2 ADC can be enhanced by combining it with an anti-nectin-4 ADC. While this study focused on T-DM1 and EV, the principle may extend to other agents. Several anti-nectin-4 ADCs are currently in preclinical or clinical development [48–53], and these could also be evaluated in combination with T-DM1. Moreover, the efficacy of trastuzumab deruxtecan, an ADC approved for the treatment of HER2-positive advanced breast cancer and gastric cancer [11, 13], may likewise be enhanced when combined with an anti-nectin-4 ADC. Several other anti-HER2 ADCs are also under clinical investigation [16, 18, 53].

Conclusion

This study demonstrates that the efficacy of an anti-HER2 ADC can be enhanced by combining it with an anti-nectin-4 ADC. In HER2-positive breast and gastric cancers that also express nectin-4, T-DM1 showed improved efficacy when administered in combination with the anti-nectin-4 ADC EV. The findings support the evaluation of the T-DM1 and EV combination in clinical trials. To our knowledge, no such trial is currently underway.

Supplementary Information

Supplementary Material 1 (6.8MB, docx)

Acknowledgements

We thank Mrs. Marja Ben-Ami, Mrs. Minna Kempas, and Ms Cilla Honkamäki for their skillful help in performing the experiments. We thank the FIMM Digital Microscopy and Molecular Pathology Unit, and the Biomedicum Imaging Unit supported by Helsinki University and Biocenter Finland and the Biomedicum Flow Cytometry Unit. In addition, we acknowledge the support of HiLIFE Laboratory Animal Centre Core Facility, University of Helsinki, Finland.

Abbreviations

ADC

Antibody-drug conjugate

EV

Enfortumab vedotin

HER2

Human epidermal growth factor receptor 2

MSA

Most synergistic area

MMAE

Monomethyl auristatin E

PBS

Phosphate-buffered saline

SCID

Severe combined immunodeficient

T-DM1

Trastuzumab emtansine

Author contributions

N.Y. discussed the hypothesis, designed the experimental approach, performed the experimental work, analyzed data, and drafted the manuscript with H.J and M.B. N.P. performed the experimental work and edited the manuscript. H.L. performed the experimental work and edited the manuscript. P.L. coordinated the project, provided the ethical licenses for animal studies, and edited the manuscript. H.J. discussed the hypothesis, led the project, designed the experimental approach, coordinated the project, drafted the manuscript together with B.M. and N.Y., and provided resources. M.B. conceived the hypothesis, led the project, designed the experimental approach, performed the experimental work, analyzed data, coordinated the project, and drafted the manuscript together with N.Y. and H.J. All authors approved the final manuscript.

Funding

Open Access funding provided by University of Helsinki (including Helsinki University Central Hospital). The study was supported by grants from Sigrid Jusélius Foundation (H.J.), and the Cancer Society of Finland (H.J. and P.L.).

Data availability

All data supporting the findings in this study are included within the manuscript and the supplement figures. The raw data used for analysis is available from the corresponding author on request.

Declarations

Ethics approval

The Committee for animal experiments of the District of Southern Finland approved the animal experiments under the licenses ESAVI/403/2019, ESAVI/11614/2022, and ESAVI/10262/2022. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines (https://arriveguidelines.org/arrive-guidelines).

Consent for publication

Not applicable.

Competing interests

H.J. is the Chair of the Scientific Advisory Board of Orion Pharma, Neutron Therapeutics, and Maud Kuistila Foundation, from which work he has received financial compensation, and owns stock of Orion Pharma and Sartar Therapeutics. M.B. has an advisory relationship at ProBiont Ltd. M.B. and H.J. report grants from Defense Therapeutics Inc. and Merck KGaA outside the submitted work. No potential conflicts of interest were disclosed by the other authors.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Heikki Joensuu and Mark Barok contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (6.8MB, docx)

Data Availability Statement

All data supporting the findings in this study are included within the manuscript and the supplement figures. The raw data used for analysis is available from the corresponding author on request.

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