Potential Mechanisms of Interstitial Lung Disease Induced by Antibody–Drug Conjugates Based on Quantitative Analysis of Drug Distribution

Abstract

Antibody–drug conjugates (ADC) are a rapidly advancing category of therapeutic agents with notable anticancer efficacy. However, the emergence of interstitial lung disease as a severe ADC-associated adverse event highlights the need to better understand the underlying mechanisms. In this study, xenograft model mice with tumors expressing different levels of the trophoblast antigen 2 (TROP2) were generated by subcutaneously transplanting the various TROP2-expressing cancer lines. The mice received different doses of TROP2–eribulin, a novel TROP2-targeting ADC, composed of an anti-TROP2 antibody and the eribulin payload, joined by a cleavable linker. The concentration and distribution of TROP2–eribulin, as well as the pharmacokinetics of eribulin release, were assessed in tumor and lung tissues. Analysis of tumor tissue showed that the concentration of released eribulin was approximately 10-fold higher in NCI-H2110 (high TROP2 expression) than in A549 (low TROP2 expression), whereas analysis of lung tissue showed that TROP2–eribulin was distributed in lung tissue in a dose-dependent manner, regardless of TROP2 expression, with significantly more eribulin released in the high-dose group than in the other dose groups (P < 0.05). Immunofluorescence assay analysis showed that TROP2–eribulin localized to alveolar macrophages. In the analysis using human leukemia monocytic cell, the concentration of eribulin released from TROP2–eribulin was significantly reduced by the use of an Fc receptor inhibitor (P < 0.05). These results revealed that Fcγ receptor–mediated uptake by alveolar macrophages releases the cytotoxic payload into lung tissue, helping to clarify the pathogenesis of ADC-induced interstitial lung disease.

Introduction

Cancer is a leading cause of death, accounting for approximately 10 million deaths worldwide in 2020 alone (1). Despite recent advances in cytotoxic chemotherapy, molecular targeted therapy, immunotherapy, and radioimmunotherapy, the long-term prognosis for patients with recurrent or inoperable cancer remains poor; therefore, new therapeutic agents are urgently required.

Antibody–drug conjugates (ADC) are a class of therapeutic drugs, which are composed of an mAb and a cytotoxic agent (the payload), joined via a synthetic linker. The mechanistic principle of ADC is as follows: (i) the mAb component binds to its target antigen; (ii) the ADC–antigen complex is internalized through receptor-mediated endocytosis; and (iii) the cytotoxic agent is released into the cell following the intracellular protease–mediated cleavage of the linker (2–4). The therapeutic efficacy of ADCs does not necessarily depend on the blocking of downstream signaling events. Instead, it is influenced by the levels of the target antigen on the cell surface and the efficiency of ADC internalization. These characteristics allow ADCs to target molecules that have not been traditionally considered targets for antibody therapy (5). In recent years, ADCs targeting HER2 (6–11), trophoblast antigen 2 (TROP2; refs. 12–14), nectin-4 (15, 16), and folate receptor α (17–19) have demonstrated considerable success in the treatment of advanced solid tumors. Consequently, ADCs have become one of the fastest growing classes of drugs used in cancer therapy.

To date, 15 ADCs have been approved by the FDA, and various ADC targets have been investigated in early clinical trials (20). Although ADCs have shown promising therapeutic effects, several ADC-associated toxicities have been reported in clinical trials. For instance, Zhu and colleagues (21) analyzed data from 22,492 patients with cancer, involved in 169 clinical trials of ADCs, and reported that 91.2% of the patients experienced all-grade adverse events, and 46.1% had grade 3 or higher adverse events. Treatment-related deaths occurred in 1.3% of the cases, with respiratory disease being the most common cause. Among the adverse events associated with ADCs, interstitial lung disease (ILD) is of particular concern. For example, a 15.4% incidence of ILD was reported in a study of 1,150 patients treated with trastuzumab deruxtecan (T-DXd), an antibody that targets HER2, coupled with a topoisomerase I inhibitor as the payload (22). In early development trials, a 9.0% incidence of ILD was reported in patients treated with datopotamab deruxtecan, which targets TROP2 and has the same payload as T-DXd (23). Meanwhile, 51.1% of patients treated with farletuzumab ecteribulin, which targets folate receptor α and comprises the microtubule inhibitor eribulin, developed ILD (24). Considering these reports, ILD during treatment with ADCs occurs regardless of the target antigen, suggesting an association between ILD and the cytotoxic payload, as well as other previously reported ADC-related toxicities.

Although ILD is a serious adverse event during ADC therapy, the association between ILD development and ADC characteristics or modes of action has not been adequately studied, in part because of the difficulty in creating preclinical models of ILD development. Thus, the aim of the present study was to explore the mechanisms of ADC-induced ILD. Specifically, we performed a preclinical investigation of the dose-dependent pharmacokinetic profiles of a novel TROP2-targeting ADC and assessed the functions of alveolar macrophages in lung tissues using xenograft mouse models treated with this ADC. In addition, we used THP-1 cells as a model to mimic the function of alveolar macrophages during ADC-induced ILD occurrence.

Materials and Methods

Cell culture

Four human cancer cell lines (A549, HeLa, NCI-H2110, and SKOV3) and one human leukemia monocytic cell line (THP-1) were used in this study. The A549 (RRID: CVCL_0023) cell line was purchased from ATCC in 2019. The HeLa (RRID: CVCL_0030), NCI-H2110 (CVCL_1530), and SKOV3 (CVCL_0532) cell lines were purchased from the ATCC in 2020. The THP-1 (RRID: CVCL_0006) cell line was purchased from the JCRB Cell Bank in 2020. These cell lines were authenticated by short tandem repeat DNA profiling in 2023 by the JCRB Cell Bank. All cells were maintained under a humidified atmosphere with 5% CO₂ at 37°C, tested for Mycoplasma using MycoAlert Mycoplasma Detection Kit (LT07-418), and used within 10 passages. The A549 cells were kept in F-12K medium supplemented with 10% FBS. HeLa cells were kept in Eagle minimum essential medium supplemented with 10% FBS. NCI-H2110 cells were kept in RPMI1640 supplemented with 10% FBS. SKOV3 cells were kept in McCoy’s 5A medium supplemented with 10% FBS. THP-1 cells were kept in RPMI1640 supplemented with 10% FBS, GlutaMAX (Thermo Fisher Scientific), and 55 µmol/L 2-mercaptoethanol (Thermo Fisher Scientific). All media were supplemented with 1% penicillin/streptomycin (Wako).

Compounds and antibodies

The Fc receptor (FcR) blocking reagent (RRID: AB_2892112) was purchased from Miltenyi Biotec. Eribulin and ER-076349 (internal standard) were provided by Eisai Inc.. Anti-TROP2 antibody (HB-187, clone 162-46.2 mAb, RRID: AB_2287132) was purchased from ATCC, and its variable sequence amplified by PCR was fused in human IgG1 antibody. The mouse-derived sequence was then partially humanized. The chimeric anti-TROP2 mAb did not cross-react with murine TROP2. A stable cell line for mAb, named TROP2-xi-162-46.2-IgG1-H4L2, was developed for large production of the antibody. The antibody was then partially reduced using tris(2‐carboxyethyl) phosphine, followed by mixing with Mal‐PEG2‐VCP‐eribulin for conjugation. Unreacted Mal‐PEG2‐VCP‐eribulin was removed by G‐25 chromatography. Conjugated molecules were pooled, formulated, concentrated, enriched, and filtrated, resulting in TROP2-xi-162-46.2-IgG1-H4L2-mal-VCP-eribulin (aka anti-TROP2–eribulin) as the final product. The drug–antibody ratio was analyzed by hydrophobic interaction chromatography–high-performance liquid chromatography and LC/MS, and the percentage of the aggregation of anti-TROP2–eribulin was analyzed using size-exclusion chromatography. The average drug-to-antibody ratio was 3.72. A detailed process relevant to producing this antibody and the conjugation method has also been previously reported (25, 26).

Flow cytometry

TROP2, CD16, CD32, and CD64 expression in cell lines was analyzed using flow cytometry (FCM). To assess TROP2 expression, 1 × 10⁵ cells were incubated with an anti-TROP2 antibody (1/100, ab79976, Abcam, RRID: AB_1603604) for 30 minutes at 4°C. After washing with Dulbecco’s PBS (D-PBS, Thermo Fisher Scientific) containing 0.5% BSA and 2 mmol/L EDTA, the cells were incubated with goat anti–mouse IgG conjugated to Alexa Fluor 647 (1/1,000, Thermo Fisher Scientific, RRID: AB_2535804) for 30 minutes at 4°C. To evaluate CD16, CD32, and CD64 expression, 1 × 10⁶ cells were incubated with Alexa Fluor 488–conjugated anti–human CD16 antibody (RRID: AB_492974), PE-conjugated anti–human CD32 antibody (RRID: AB_314338), and Alexa Fluor 647–conjugated anti–human CD64 antibody (1/20, BioLegend, RRID: AB_528867) for 1 hour on ice. After washing with D-PBS containing 0.5% BSA and 2 mmol/L EDTA, the cells were analyzed by FCM using a Guava easyCyte instrument (Millipore, RRID: SCR_025377). Data were analyzed using FlowJo version 7.6.5 (BD Biosciences, RRID: SCR_008520).

Monitoring eribulin release in vitro

A549, NCI-H2110, and THP-1 cells were plated at 2 × 10⁶ cells/well in separate wells of a six-well plate (Corning). The FcR blocking reagent (1/50) was added to block Fcγ receptors on THP-1 cells. A measure of 10 or 25 µg/mL TROP2–eribulin was then added to each well, followed by incubation at 37°C for 6 hours. The cells were then washed with 20 mmol/L ammonium formate, and eribulin was extracted using 1% formic acid in ethanol. The extracts were subjected to LC/MS-MS.

Monitoring TNF-α release in vitro

THP-1 cells were plated at 1 × 10⁴ cells/well in separate wells of a 96-well plate (Corning). A measure of 25 µg/mL TROP2–eribulin was then added to each well, followed by incubation at 37°C for 24 hours. The samples were then centrifuged at 3,000 × g, and the supernatants were collected. After collection, TNF-α levels in the supernatants were measured using AuthentiKine Human TNF-alpha ELISA Kit (Proteintech, RRID: AB_2924421).

IHC and immunofluorescence assays

For IHC, tumor and tissue samples were fixed in 10% neutral-buffered formalin and embedded in paraffin. Formalin-fixed, paraffin-embedded blocks were then sectioned into 4-µm sections using a microtome (REM-710, Yamato Kohki Industrial). During TROP2 staining, peroxidase blocking was performed with 3% hydrogen peroxide in methanol, and antigens were retrieved using Target Retrieval Solution (10×), pH 9.0 (Agilent) over 20 minutes at 95°C. The sections were blocked with 4% Block Ace at room temperature for 1 hour and incubated with an anti-TROP2 antibody (1/20, AF650, R&D systems, RRID: AB_2205667) at 4°C overnight. The sections were then incubated with a horseradish peroxidase (HRP)–conjugated donkey anti–goat IgG (1/500, Jackson ImmunoResearch, RRID: AB_2313587) at room temperature for 90 minutes HRP was detected using the 3,3′-diaminobenzidine substrate solution, and the nuclei were counterstained with hematoxylin. Tissue images were obtained using a VS120 virtual slide system (Olympus, RRID: SCR_018411).

For CD68 and chimeric anti-TROP2 mAb immunofluorescence staining, antigen retrieval was performed using Target Retrieval Solution (10×), pH 6.1 (Agilent) for 20 minutes at 96°C. The sections were then blocked with 4% Block Ace at room temperature for 1 hour and incubated with the anti-CD68 antibody (1/500, Abcam, RRID: AB_10975465) at 4°C overnight. The sections were then incubated with a goat anti–human IgG (1/500, Thermo Fisher Scientific, RRID: AB_228263) at room temperature for 1 hour. The samples were then incubated with the secondary antibodies donkey anti–rabbit IgG conjugated to Alexa Fluor Plus 488 (1/200, Thermo Fisher Scientific, RRID: AB_2762833) and donkey anti–goat IgG conjugated to Alexa Fluor Plus 647 (1/200, Thermo Fisher Scientific, RRID: AB_2762840) at room temperature for 1 hour. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1 µg/mL, Thermo Fisher Scientific), and the sections were mounted with the ProLong Glass Antifade Mountant (Thermo Fisher Scientific). Blue fluorescence (DAPI) was detected using the BZ-X filter (OP-87762, KEYENCE); red fluorescence (Alexa Fluor Plus 647) was detected using the Cy5 filter (OP-87766, KEYENCE); and green fluorescence (Alexa Fluor Plus 488) was detected using the YFP filter (OP-87767, KEYENCE) on the fluorescence microscope (BZ-710X, KEYENCE, RRID: SCR_017202).

LC/MS-MS analysis

For in vivo studies, frozen tissues were sliced into 6-µm sections using a CM1860 cryostat (Leica, RRID: SCR_025772). Eribulin was extracted with 1% formic acid in ethanol during a 30-minute sonication (ASU-6 sonicator, AS ONE) step at room temperature. Proteins were then precipitated using acetone at −20°C overnight. After the collection of supernatants, the samples were dried under reduced pressure in a CC-105 centrifugal concentrator (TOMY) and resuspended in the initial mobile phase. Liquid chromatography was performed on a Nexera-i LC-2040C Plus instrument (Shimadzu) using a Polaris 3 C18-A column (30 × 2 mm; Agilent) and the following conditions: 13% acetonitrile in water with 0.1% formic acid as mobile phase A and 30% tetrahydrofuran in acetonitrile with 0.1% formic acid as mobile phase B. The column temperature was set at 30°C for the entire procedure. The gradient conditions for the separation were as follows: 0.0 to 2.0 minutes, isocratic 0% B; 2.0 to 6.5 minutes, 0% to 35% B linear; 6.5 to 7.0 minutes, 35% to 100% B linear; 7.0 to 8.0 minutes, isocratic 100% B; 8.0 to 8.5 minutes, 100% to 0% B linear; and 8.5 to 10.0 minutes, isocratic 0% B. The flow rate was 0.5 mL/minute but was changed to 0.75 mL/minute from 1.5 to 6.0 minutes. The temperature of the autosampler was set at 15°C. The injection volume was 10 µL. Mass spectrometry detection was performed using an LCMS-8060 triple quadrupole mass spectrometer (Shimadzu, RRID: SCR_020515) by positive Electrospray ionization in Multiple Reaction Monitoring mode. The nebulizer gas, drying gas, and heating gas were set at 3, 10, and 10 L/minute, respectively. Interface temperature, heat block temperature, and Desolvation Line temperature were set at 380°C, 300°C, and 250°C, respectively. The collision-induced dissociation gas was set at 270 kPa. The linear dynamic range of the eribulin analysis was 0.1 to 25 nmol/L for both plasma and tissue bioanalyses. The carryover from ADCs was examined by 125 µg/mL of TROP2–eribulin without sample processing other than acetone precipitation and solvent exchange, and carryover was not observed. The release of the payload from the ADCs during sample processing was examined by pretreating 125 µg/mL of TROP2–eribulin with/without tumor, lung, or cultured medium, and no release of eribulin was observed under measured conditions (Supplementary Table S1). Data acquisition was performed using LabSolutions version 5.9.7 (Shimadzu, RRID: SCR_018241). Concentrations below the lower limit of quantification (0.1 nmol/L) were adjusted to lower limit of quantification/2 for the purpose of the calculation.

ELISA

ELISA plates were prepared using Affinity purified Goat anti-Human IgG-Fc Coating Antibody (Bethyl, RRID: AB_67061) and blocked with D-PBS containing 1% BSA. Plasma samples were diluted with tris-buffered saline with 0.1% Tween 20 containing 1% BSA at 1/50 for the 2.5 mg/kg group, 1/100 for the 5 mg/kg group, and 1/500 for the 12.5 mg/kg group. Samples were then dispensed to coated plates and incubated for 1 hour. Human IgGs were detected with HRP Conjugated Goat anti-Human IgG-Fc Detection Antibody (Bethyl, RRID: AB_67064) and colored by 3,3′,5,5′-Tetramethylbenzidine (DOJINDO). Absorbance was read using SpectraMax Paradigm (Molecular devices, RRID: SCR_025865) at 450 nm for detection and 650 nm for reference.

Experimental mouse model

In this study, 4-week-old female BALB/c nude mice were obtained from The Jackson Laboratory Japan, and 5- or 6-week-old mice were used in this study. They were maintained in cages under specific pathogen-free conditions, provided with standard food, and given free access to sterilized water. Each mouse was monitored daily. The tumor volumes and body weights were measured twice a week. All animal procedures were performed with approval from the National Cancer Center Japan and conducted in compliance with the Guidelines for the Care and Use of Experimental Animals established by the Committee for Animal Experimentation, National Cancer Center, Japan. These guidelines meet the ethical standards required by law and also comply with the guidelines for the use of experimental animals in Japan.

In vivo pharmacokinetic study

To evaluate the pharmacokinetics (PK) of eribulin release from TROP2–eribulin in the tissue, 1.0  ×  10⁷ viable A549 or NCI-H2110 cells, suspended in 0.1 mL of Hank’s Balanced Salt Solution, were subcutaneously injected into the right-side trunk of 5-week-old female BALB/c nude mice. The tumor volume was calculated as (L × W²)/2, in which L is the length and W is the width of the subcutaneous tumor. The mice were randomly divided into several treatment groups (n = 3 per group) when tumor volume reached 200 to 250 mm³. TROP2–eribulin (at 2, 5, or 12.5 mg/kg) was administrated intravenously on day 0. Mice were killed after excision of the tumor and lung under anesthesia; 1% to 5% isoflurane was applied during this procedure. The tumor and lung tissues were not perfused in this study.

Statistical analyses

All the graph generation, calculations, and statistical analyses were performed using GraphPad Prism (Version 9.1.0., GraphPad Software, RRID: SCR_002798). All the results were expressed as the mean ± SD. Statistical significance was determined using the t test or the ANOVA with the Tukey post hoc multiple comparisons test. Differences for which P < 0.05 were considered significant.

Data availability

The data supporting the findings in this study are available within the article, in the corresponding Supplementary Materials, or upon reasonable request from the corresponding author.

Results

Assessing TROP2 expression in different cell lines

TROP2–eribulin is a novel TROP2-targeting ADC, which is composed of the chimeric anti-TROP2 mAb conjugated to eribulin, a potent cytotoxic microtubule inhibitor, by a cathepsin B–cleavable linker at a drug/antibody ratio of approximately 3.72 (Fig. 1A). The TROP2 expression level on the surface of each cell type was analyzed by FCM and IHC. The A549 cells had low TROP2 expression, the HeLa and SKOV3 cells had intermediate TROP2 expression, and the NCI-H2110 cells had high TROP2 expression (Fig. 1B and C).

Figure 1.

Correlation between the binding of TROP2-eribulin and eribulin concentration in cells with low or high TROP2 expression. A, Molecular structure of TROP2-eribulin. B, TROP2 expression in multiple cancer cell lines was evaluated by flow cytometry. C, Immunohistochemical analysis of TROP2 expression in A549 and NCI-H2110 tumor tissues. D, Intracellular concentrations of eribulin 6 hours after exposure to TROP2-eribulin. Statistical significance of group differences was evaluated using the t test. ****, P < 0.0001.

TROP2–eribulin delivers eribulin in a TROP2-dependent manner to cells in vitro

To assess whether TROP2–eribulin was taken up by cells and eribulin was released in a target-dependent manner, we investigated eribulin release from TROP2–eribulin using cell lines with low (A549) or high (NCI-H2110) expression. Each cell line was treated with TROP2–eribulin at 25 µg/mL. After incubation for 6 hours, eribulin was extracted using 1% formic acid in ethanol and analyzed by LC/MS-MS. The results revealed that the intracellular concentration of eribulin was dependent on the TROP2 expression by cells (P < 0.0001; Fig. 1D).

Eribulin release from TROP2–eribulin increases in a dose-dependent manner in lung tissue

To assess whether the payload concentration in tissues was linked to ADC toxicity, we next examined the PK of eribulin release from TROP2–eribulin in the tumor, lung, and plasma. We established tumor xenograft model mice with low (A549) or high (NCI-H2110) TROP2 expression levels and intravenously injected them with TROP2–eribulin at 2, 5, or 12.5 mg/kg. The tumors and lungs were resected after 6, 24, 48, and 96 hours after TROP2–eribulin administration. Eribulin was extracted from each tumor, lung, and plasma sample using 1% formic acid in ethanol and then analyzed by LC/MS-MS. Analysis of tumor tissue showed that at each dose of TROP2–eribulin, eribulin concentrations were increased over 24 hours and remained at the same concentration until 96 hours after administration. In addition, eribulin concentrations were approximately more than 10-fold higher in NCI-H2110 cell–derived xenograft tumors than in those generated using A549 cells from 6 to 96 hours after administration (Fig. 2A). By contrast, analysis of lung tissue showed that eribulin concentrations decreased gradually after 24 hours of administration and were not associated with target antigen expression (Fig. 2B). In addition, eribulin was not detected in plasma, indicating that TROP2–eribulin remained stable in the blood (Supplementary Fig. S1). These results indicate that eribulin release from TROP2–eribulin is dependent on target antigen expression in the tumor tissue but occurs in a nonspecific manner in the lung tissue.

Figure 2.

PK of eribulin release from TROP2–eribulin in tumor and lung tissues. Concentration of eribulin in the tumors (A) and lungs (B) of xenograft model mice generated by injection of A549 or NCI-H2110 cells. Eribulin concentration in tumor (C) and lung tissues (D) at 24 hours after TROP2–eribulin administration, both which are repeated from A and B, respectively. Statistical significance of group differences was evaluated using the ANOVA with the Tukey post hoc multiple comparisons test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

To explore the PK of eribulin released from TROP2–eribulin into tissues in more detail, we next compared eribulin concentrations in tumor and lung tissues resected 24 hours after the administration based on PK analysis of eribulin. Analysis of tumor tissue in the A549 xenograft model (with low TROP2 expression) showed that eribulin concentrations increased significantly in a TROP2–eribulin dose–dependent manner. In the NCI-H2110 xenograft model (with high TROP2 expression), a significant increase in eribulin concentration was observed when comparing the 2 and 5 or 12.5 mg/kg treatment groups but not when comparing the 5 and 12.5 mg/kg treatment groups (Fig. 2C). In the analysis of lung tissue, there was no significant difference in the eribulin concentration between the 2.0 and 5.0 mg/kg treatment groups, but there was a significant difference in the eribulin concentration between the 2.0 and 12.5 mg/kg or the 5.0 and 12.5 mg/kg treatment groups; moreover, these results were unaffected by the TROP2 expression levels (Fig. 2D). These findings indicate that the TROP2-independent eribulin uptake into the lung tissues also increases in a TROP2–eribulin dose–dependent manner. Notably, in the xenograft model with high TROP2 expression, a significant increase in nonspecific eribulin uptake into the lung tissue was observed at the highest concentration of TROP2–eribulin, although no significant increase in tumor tissue uptake was observed.

Target-independent uptake of TROP2–eribulin by alveolar macrophages releases eribulin into the lung tissue

We hypothesize that the release of the payload into the lung tissue was associated with the presence of ADCs in the lung tissue and their subsequent uptake by resident alveolar macrophages. We tested this hypothesis by co-staining lung tissues for ADCs and alveolar macrophages 24 hours after TROP2–eribulin administration. Immunofluorescence analysis revealed that TROP2–eribulin accumulated in the lung tissue in a dose-dependent manner in the xenograft models with low and high TROP2 expression. Notably, the results of anti-CD68 co-staining showed that TROP2–eribulin was localized to CD68⁺ alveolar macrophages (Fig. 3A and B). These findings suggest that ADCs accumulate in alveolar macrophages in a dose-dependent manner irrespective of the target antigen, leading to payload release into the lung tissue.

Figure 3.

Representative photomicrographs of TROP2–eribulin and alveolar macrophage distribution in mouse lung tissue. Xenograft model mice generated using A549 (A) or NCI-H2110 cells (B) were treated with 2, 5, or 12.5 mg/kg TROP2–eribulin. TROP2–eribulin (red), CD68 (green), and nuclei (blue) were detected in mouse lung tissues using immunofluorescence staining.

Fcγ receptor–mediated intracellular uptake of TROP2–eribulin results in the release of eribulin

We next set out to determine whether eribulin was indeed released intracellularly following the nonspecific uptake of TROP2–eribulin by macrophages. We hypothesized that macrophages take up ADCs via the Fcγ receptors and evaluated the intracellular concentration of eribulin released from TROP2–eribulin in vitro using THP-1, a human leukemia monocytic cell line. We first evaluated Fcγ receptors’ expression in THP-1 cell, and FCM analysis revealed that THP-1 cells expressed Fcγ receptors I and II (CD64 and CD32, respectively; Fig. 4A). The THP-1 cell lines were then treated with TROP2–eribulin at 10 or 25 µg/mL in the presence or absence of an FcR blocker. After incubation for 6 hours, eribulin was extracted using 1% formic acid in ethanol and analyzed by LC/MS-MS. The results revealed that the intracellular concentration of eribulin released from TROP2–eribulin was significantly higher in THP-1 cells treated with 25 µg/mL TROP2–eribulin than in those treated with the lower ADC dose (Fig. 4B). Notably, the use of the FcR inhibitor significantly lowered the intracellular concentration of eribulin. Furthermore, compared with THP-1 cells alone, THP-1 cells treated with TROP2–eribulin showed a significant increase in TNF-α in the supernatant (Supplementary Fig. S2). These findings suggest a dose-dependent increase in the Fcγ receptor–mediated uptake of ADCs by monocytic cells, resulting in a higher concentration of payload within the cell and possibly leading to TNF-α–mediated lung injury.

Figure 4.

Target-independent uptake of TROP2-eribulin into THP-1 cell via Fcγ receptors. A, CD64 (Fcγ receptor I), CD32 (Fcγ receptor II), and CD16 (Fcγ receptor III) expression in THP-1 cells was evaluated by flow cytometry. B, Intracellular concentration of eribulin in THP-1 cells 6 hours after their exposure to 10 µg/ml or 25 µg/ml TROP2-eribulin in the presence or absence of Fc receptor (FcR) blocker. Statistical significance of group differences was evaluated using the ANOVA with Tukey’s post-hoc multiple comparisons test. ***, P < 0.001; ****, P < 0.0001.

Discussion

Advances in antibody engineering technology have driven the generation of innovative ADCs, which has ultimately improved the outcomes of patients with solid tumors; however, there is a pressing need to elucidate the mechanisms underlying the side effects of ADC therapy. ILD is the most notable ADC-induced adverse event reported to date. Although several ADC-induced ILD risk factors have been reported by clinical trials, the mechanism of its occurrence has not yet been fully elucidated. In this study, we observed that eribulin was released from an eribulin-conjugated ADC not only in the target tumor tissue but also in lungs. Moreover, the concentration of eribulin increased with ADC dose in both tissue types. We confirmed that in the lung, the ADC was assimilated by CD68⁺ alveolar macrophages in a dose-dependent manner. We then clarified that THP-1 cells, mimicking alveolar macrophages, took up ADCs nonspecifically via their Fcγ receptors and subsequently released eribulin into the surrounding tissue. Collectively, our findings demonstrate that the off-target payload release by ADCs occurs predominantly in alveolar macrophages in an ADC dose–dependent manner (Fig. 5). To the best of our knowledge, this is the first study to analyze ADC distribution and payload PK in the lung tissue and provide a mechanistic explanation for ADC-induced ILD pathogenesis.

Figure 5.

Possible mechanisms of ADC-induced ILD. Alveolar macrophages take up ADCs in a nonspecific manner via their Fcγ receptors. The ADC cytotoxic payload is then released from macrophages into the lung tissue. (1) ADC binds to alveolar macrophages via the Fcγ-receptor (2) Fcγ-receptor-mediated uptake by alveolar macrophages (3) degradation of ADC (4) payload release into the cell and possibly leading to cytokine-mediated lung injury.

Drug-induced lung injury generally occurs via two main mechanisms: (i) drug-induced cytotoxic effects, which occur in a dose-dependent manner; (ii) damage inflicted by the immune response (25–27). Although the detailed mechanism of ADC-induced ILD is not known, Powell and colleagues (22) reported in their analysis of 1,150 patients treated with T-DXd that the risk of ILD was potentially increased at doses of T-DXd ≥6.4 mg/kg, suggesting that lung damage was due to the direct cytotoxicity of the drug. The present study suggests that the concentration of payload released in the lung tissue is significantly dependent on ADC dose, which partially explains the relationship between ADC dose and ILD development in clinical trials. Moreover, in the xenograft model with high TROP2 expression (H2110), a significant increase in nonspecific eribulin uptake into lung tissue was observed at the highest concentration of TROP2–eribulin, in a situation where no significant increase in tumor tissue uptake was observed. Collectively, the relative excess ADCs beyond that needed for full receptor occupancy may lead to higher circulating concentrations in the plasma irrespective of target antigen expression. In antigen-positive models, tumor uptake of ADCs is primarily driven by active targeting and may be sensitive to receptor occupancy compared with antigen-negative models. This finding suggests that the incidence of ILD may be higher when high doses of ADCs are used in targeted antigen-positive patients in early development studies.

The following four theoretical mechanisms of ADC-induced lung injury have been proposed: (i) target antigen–dependent uptake of ADCs by normal cells; (ii) target antigen–independent uptake of ADCs by immune cells; (iii) bystander effects due to free payload released from cancer cells in lung metastases; and (iv) circulation of free payload resulting from the deconjugation of ADC (28). Given that the ADCs administered to the subcutaneous tumor xenograft model mice in this study did not cross-react with murine TROP2 and were stable in plasma, the payload detected in the lung tissue was likely the result of Fcγ receptor–mediated uptake of ADCs by immune cells. Kumagai and colleagues (29) implied that in a study using cynomolgus monkeys, T-DXd, an ADC targeting HER2, was taken up nonspecifically by alveolar macrophages. However, this study and others had not specifically focused on how the relationship between macrophages and the pharmacologic aspects of ADCs may be associated with ILD pathogenesis. In the present study, we confirmed that the ADCs were assimilated nonspecifically by alveolar macrophages. Although we obtained this result with an ADC targeting a different antigen (i.e., TROP2), it aligns with a previous report implying that ADC-induced ILD occurs regardless of the targeted antigen. Furthermore, by assessing the concentration of the payload in addition to the distribution of the ADC, we confirmed that the Fcγ receptor–mediated uptake by alveolar macrophages led to the release of the payload into the lung tissue.

The interaction between the Fc domain of an antibody and an Fcγ receptor expressed on a normal cell has been reported as a mechanism for the nonspecific uptake of mAbs, which may lead to off-target toxicity (30, 31). Similarly, it has been suggested that the binding of the ADC Fc domain to an Fc receptor may induce toxicity through target-independent uptake. For example, preclinical studies of trastuzumab emtansine (T-DM1) have suggested that thrombocytopenia, an adverse event commonly associated with T-DM1 use, may be linked to the Fcγ receptor–mediated, nonspecific uptake of T-DM1 via pinocytosis by megakaryocytes (32). Like megakaryocytes, macrophages express high levels of Fcγ receptors and may, therefore, be responsible for the target-independent toxicity of ADCs (33–35). In the present study, in vitro experiments using the human leukemia monocytic THP-1 cell line showed that blocking Fcγ receptors significantly reduced the release of intracellular payload. This confirms that ADC-induced lung injury may occur via an Fcγ receptor–mediated mechanism.

There are several limitations to this study. First, we examined the pathogenesis of ILD using a novel TROP2-targeting ADC, which has not yet been used in clinical practice and may, therefore, not induce ILD. However, given that ADC-induced ILD has been observed in clinical studies using MORAb-202, which is designed using the same linker and payload or datopotamab deruxtecan, which targets the same antigen (TROP2), we believe that our study will help delineate the mechanism of ADC-induced ILD pathogenesis in a clinical setting. Second, the lung tissues of mice were resected and analyzed within a few days after ADC administration. As such, we did not observe evidence of fibrosis suggestive of ILD in the lungs of mice. Further research using a xenograft model better suited for the study of ILD will provide more insights into the pathophysiology of ADC-induced ILD. Finally, the interaction between the Fc domain of TROP2–eribulin and the Fcγ receptor has not been investigated in vivo. Additional evaluation using an Fc-specific IgG, which can abolish Fc binding to the Fcγ receptor, will help validate the mechanism of Fcγ receptor–mediated ADC uptake by alveolar macrophages.

In summary, this study demonstrated that ADC-induced ILD may be mediated by the dose-dependent but target antigen–independent uptake of ADCs in lung tissue via Fcγ receptors expressed on alveolar macrophages. This study offers a new perspective on the mechanism underlying ADC-induced ILD pathogenesis, which will inform approaches geared at developing novel ADCs aimed at minimizing lung injury.

Supplementary Material

Figure S1

Pharmacokinetics of eribulin release from TROP2-eribulin and total TROP2-eribulin concentration in the plasma.

Figure S2

Concentration of TNF-α in the supernatants in THP-1 cells.

Authors’ Disclosures

S. Koganemaru reports grants from Amgen, Bristol Myers Squibb, Daiichi Sankyo, Eisai Inc., AbbVie, MSD, and Incyte outside the submitted work. H. Fuchigami reports grants from Eisai Inc. during the conduct of the study, as well as grants from Eisai Inc. outside the submitted work. Y. Kuboki reports grants and personal fees from Taiho, Kyowa Kirin, and Eli Lilly and Company, grants from Astellas, Daiichi Sankyo, AstraZeneca, Boehringer Ingelheim, Chugai, Genmab, Merck, Hengrui, Novartis, Ono Pharmaceutical, and Bristol Myers Squibb, grants, personal fees, and other support from Takeda, and grants and other support from Incyte, AbbVie, Amgen, and Noile-Immune Biotech Inc. outside the submitted work. K. Furuuchi reports personal fees and nonfinancial support from Eisai Inc. during the conduct of the study, as well as personal fees from Eisai Inc. outside the submitted work. T. Uenaka reports employment with Eisai Co., Ltd., which is the parent company of Eisai Inc., and receiving salary from Eisai. T. Doi reports grants and personal fees from PRA Health Sciences, Daiichi Sankyo, Janssen Pharma, Boehringer Ingelheim, Shionogi, and Chugai Pharmaceutical, grants from MSD, Bayer, Amgen, Taiho, Pfizer, Bristol Myers Squibb, AbbVie, RIN Institute, and Kyowa Kirin, and personal fees from Sumitomo Pharma, Oncolys BioPharma, Takeda, Rakuten Medical, Otsuka Pharma, KAKEN Pharmaceutical, A2 Healthcare, Noile-Immune Biotech, and Mitsubishi Tanabe Pharma outside the submitted work. M. Yasunaga reports grants from Eisai Inc., Daiichi Sankyo, Taiho, Shimadzu, Ajinomoto, Sysmex, Dojin, Johnson & Johnson, and Eli Lilly and Company outside the submitted work. No disclosures were reported by the other authors.

Authors’ Contributions

S. Koganemaru: Conceptualization, formal analysis, writing–original draft, project administration, writing–review and editing. H. Fuchigami: Formal analysis, visualization, writing–original draft, writing–review and editing. C. Morizono: Visualization, writing–review and editing. H. Shinohara: Visualization, writing–review and editing. Y. Kuboki: Supervision, writing–review and editing. K. Furuuchi: Funding acquisition, writing–review and editing. T. Uenaka: Funding acquisition, writing–review and editing. T. Doi: Supervision, writing–original draft. M. Yasunaga: Supervision, project administration, writing–review and editing.