ABSTRACT
Deruxtecan‐based antibody‐drug conjugates (DXd ADCs) provide substantial clinical benefit across multiple malignancies but are associated with clinically significant interstitial lung disease (ILD), for which the underlying mechanisms remain incompletely understood. To enable mechanistic and translational studies, we established a transgenic rat model expressing human trophoblast cell‐surface antigen 2 (hTROP2) under the control of the surfactant protein C promoter, driving selective expression in alveolar type II (AT2) cells. Repeated administration of a TROP2‐directed DXd ADC with an average drug‐to‐antibody ratio of 7.9 (TROP2‐DXd) reproducibly induced lung injury characterized by alveolar epithelial damage, inflammatory cell infiltration, and fibroblast/myofibroblast proliferation. These pathological changes were accompanied by inflammatory and tissue‐remodeling transcriptomic responses, including increased expression of fibrosis‐associated genes and decreased expression of AT2‐associated genes. These molecular alterations showed partial concordance with molecular programs observed in a cynomolgus monkey ILD model. In hTROP2 transgenic rats, prednisolone markedly attenuated TROP2‐DXd‐related lung injury and inflammatory and tissue‐remodeling transcriptomic responses without altering systemic exposure. Although hTROP2 expression is transgene‐driven and does not fully recapitulate physiological expression patterns and distribution in normal human lungs, this model provides a controlled experimental system for evaluating epithelial injury‐associated mechanisms. Notably, parallel studies in BALB/c and C57BL/6 mice showed no pulmonary findings despite prolonged high‐exposure dosing, suggesting species‐dependent susceptibility. Collectively, this transgenic rat model represents a unique rodent ILD model that recapitulates key pathological and molecular features and provides a scalable, experimentally tractable platform for mechanistic and translational evaluation across DXd ADCs.
Keywords: antibody‐drug conjugate, deruxtecan, interstitial lung disease, rat model, transgenic
hTROP2 Tg rats exhibit key pathological and transcriptomic features of DXd ADC‐related ILD, supporting their utility as a translational model. Rat, alveolus, antibody illustrations were adapted from NIAID NIH BioArt Source.
Abbreviations
- ADC
antibody‐drug conjugate
- AT2
alveolar type II
- BAC
bacterial artificial chromosome
- DAR
antibody‐drug ratio
- DEG
differentially expressed gene
- DXd ADC
deruxtecan‐based antibody‐drug conjugate
- ECM
extracellular matrix
- IgG‐DXd
human IgG1 DXd ADC with an average DAR of 7.9
- IHC
immunohistochemistry
- ILD
interstitial lung disease
- PSL
prednisolone
- RNA‐seq
RNA sequencing
- SP‐C (sftpc)
surfactant protein C
- T‐DXd
trastuzumab deruxtecan
- Tg
transgenic
- TK
toxicokinetics
- TROP2‐DXd
TROP2‐directed DXd ADC with an average DAR of 7.9
- TROP2 (TACSTD2)
trophoblast cell‐surface antigen 2
1. Introduction
Deruxtecan‐based antibody‐drug conjugates (DXd ADCs) have demonstrated substantial clinical benefit across multiple malignancies [1, 2, 3, 4]. Interstitial lung disease (ILD) and pneumonitis have emerged as clinically significant adverse events associated with these agents, posing substantial challenges for patient management [5]. Despite increasing recognition of DXd ADC‐related ILD, its underlying mechanisms remain incompletely understood [6]. A deeper understanding of these processes is essential for improving risk assessment and developing mitigation strategies.
Animal models are indispensable for elucidating the mechanisms of drug‐induced ILD. Although various experimental models have been developed to reproduce pulmonary inflammation and fibrosis, relatively few directly recapitulate drug‐induced lung toxicity [7]. Nonclinical studies in cynomolgus monkeys have provided important insights into the pathophysiological features of DXd ADC‐related interstitial pneumonitis and represent a valuable platform for mechanistic investigation [8, 9]. However, their limited experimental flexibility restricts systematic evaluation of exposure, target dependency, and potential interventions.
Rodent models for drug‐induced ILD could complement these approaches by enabling controlled and scalable investigations. However, in our nonclinical studies, DXd ADCs such as trastuzumab deruxtecan (T‐DXd) and datopotamab deruxtecan did not induce interstitial pneumonitis in rats even at the maximum feasible dose (197 mg/kg for T‐DXd and 200 mg/kg for datopotamab deruxtecan), despite robust systemic exposure, whereas lung toxicity has been observed in cynomolgus monkeys [10, 11, 12]. The establishment of a reproducible rodent model of DXd ADC‐related ILD may therefore facilitate mechanistic investigations and enable systematic evaluation of strategies to modulate this clinically relevant toxicity.
To address these challenges, we established and characterized a rat model of DXd ADC‐related ILD using a bacterial artificial chromosome (BAC) transgenic system. This approach enables genomic integration of large DNA fragments, allowing stable and physiologically relevant expression of human target genes [13, 14]. To facilitate targeted delivery of DXd ADC to alveolar epithelial cells and to model epithelial injury‐driven lung pathology, human trophoblast cell‐surface antigen 2 (TROP2; gene symbol TACSTD2) was introduced under the control of the rat surfactant protein C (SP‐C; gene symbol Sftpc) promoter, which drives expression selectively in alveolar Type II (AT2) cells.
Repeated administration of a TROP2‐directed DXd ADC with an average drug‐to‐antibody ratio (DAR) of 7.9 (hereafter referred to as TROP2‐DXd) reproducibly induced pulmonary lesions that recapitulate key pathological features observed in non‐human primate models of DXd ADC‐related ILD and in established animal models of drug‐induced ILD. Furthermore, the effects of prednisolone (PSL) were evaluated to explore the utility of this model for investigating potential mitigation strategies. This model provides a versatile platform for mechanistic and translational studies of DXd ADC‐related ILD.
2. Materials & Methods
2.1. Test Article
TROP2‐DXd as well as an isotype control IgG conjugated with DXd with an average DAR of 7.9 (IgG‐DXd), were manufactured at Daiichi Sankyo. These antibody‐drug conjugates were formulated in 10 mM acetate buffer containing 5% sorbitol (pH 5.5). PSL powder (1%) was purchased from T's Pharma.
2.2. Generation of rSP‐C/hTROP2 BAC Tg Rats
rSP‐C/hTROP2 BAC Tg rats (hTROP2 Tg rats) were generated using a BAC recombineering strategy at Institute of Immunology Co. Ltd. A rat BAC clone spanning the Sftpc locus (CH230‐106P4) and a human BAC clone containing the TACSTD2 locus (CH17‐365A18) were obtained from the BACPAC Resources Center (BACPAC Genomics). Using Red/ET recombination, the genomic region of the rat Sftpc gene from the translation start site through the 3′ untranslated region was replaced with the corresponding TACSTD2 genomic sequence, resulting in the BAC construct (Figure S1A). Correct recombination was confirmed by sequence analysis of junction regions. High‐molecular‐weight BAC DNA was purified, linearized with PI‐SceI, and isolated by pulsed‐field gel electrophoresis to separate and purify the large DNA fragment. Fertilized one‐cell embryos were generated by in vitro fertilization using Crl:CD(SD) rats. Purified BAC DNA (1 ng/mL) was microinjected into the male pronucleus of fertilized embryos, which were subsequently transferred into the oviducts of pseudo‐pregnant recipient females. Founder animals were screened by PCR using transgene‐specific primers. A transgene copy number was estimated by Southern blot analysis. Founder animals with confirmed full‐length BAC integration were used for subsequent characterization and model development.
2.3. Characterization of hTROP2 Tg Lines
Male and female hTROP2 Tg founder rats were bred with wild‐type Crl:CD(SD) rats by natural mating to establish F1 transgenic lines. To assess transgene expression patterns, major organs (lung, heart, liver, kidney, spleen, stomach, and small and large intestines) were collected from F1 transgene‐positive animals after euthanasia by CO2 inhalation in accordance with institutional guidelines. hTROP2 expression was evaluated in formalin‐fixed and paraffin‐embedded sections by immunohistochemistry (IHC) using a rabbit monoclonal anti‐TROP2 antibody (clone SP295, Abcam), which does not cross‐react with rTROP2 protein.
F1 animals from selected transgenic lines were administered a single intravenous dose of TROP2‐DXd or IgG‐DXd at 20 or 60 mg/kg (2 animals/dose/line; dose volume: 10 mL/kg). Major organs were collected 48 h after dosing and processed for IHC using a rabbit monoclonal anti‐DXd antibody (clone 1A3, Immuno‐Biological Laboratories) to assess cellular localization of the TROP2‐DXd and IgG‐DXd. All histopathological examinations in this study were conducted by a board‐certified veterinary/toxicologic pathologist and were performed in an unblinded manner.
2.4. Evaluation of TROP2‐DXd for Establishment of an ILD Model by Repeated Dosing
To establish a DXd ADC‐related ILD model, hTROP2 Tg rats were administered TROP2‐DXd intravenously at 0, 60, or 120 mg/kg every 3 weeks (6–8 animals/sex/dose, 7 weeks of age at the initiation of dosing). Animals were allocated to treatment groups such that mean body weights and variability were balanced across groups at the time of group assignment. Due to treatment‐related findings, repeat dosing was terminated after the third administration, and all remaining animals were euthanized and subjected to necropsy 3 weeks after the 3rd dose.
2.5. Repeated Combination Treatment of TROP2‐DXd and PSL
Following characterization of the rat ILD model by repeated administration of TROP2‐DXd, a combination study with PSL was conducted to further evaluate the utility and characteristics of the model under repeated dosing conditions. Because antifibrotic effects of repeated oral administration of PSL at 3 mg/kg have been reported in a bleomycin‐induced rat pulmonary fibrosis model [15], PSL dose levels of 1, 3, and 10 mg/kg were selected for the present study.
TROP2‐DXd was administered IV at 60 mg/kg every 3 weeks for a total of 3 doses (6‐week treatment period). PSL was administered orally 3 days before the 1st TROP2‐DXd administration for a total duration of 6 weeks, up to the day prior to necropsy. In addition to the combination groups, a TROP2‐DXd monotherapy group was included. Seven male animals were assigned to each treatment group, and all animals were necropsied the day after the 3rd TROP2‐DXd administration.
To evaluate systemic effects of PSL, hematology was performed at necropsy. Under isoflurane anesthesia, blood samples were collected from the abdominal aorta into tubes containing EDTA‐2 K as an anticoagulant. Hematological parameters were analyzed using an automated analyzer (ADVIA 2120i; Siemens Healthcare Diagnostics).
2.6. Toxicokinetics (TK)
Blood samples (approximately 0.4 mL) were collected via the jugular vein from 2 animals per time point at 15 min and 7, 24, 72, 168, and 504 h after dosing. In the repeated dose study, samples were collected after the 1st and 3rd doses of TROP2‐DXd, whereas in the PSL combination study, samples were collected after the 1st and 2nd doses. Plasma concentrations of TROP2‐DXd were quantified using a ligand‐binding assay on a Gyrolab xP Workstation (Gyros Protein Technologies). Plasma concentrations of DXd were quantified by high‐performance liquid chromatography coupled with tandem mass spectrometry using a Nexera X2 system (Shimadzu). TK parameters were calculated with Phoenix WinNonlin version 8.5.2 (Certara).
2.7. RNA‐Sequencing (RNA‐Seq)
RNA‐seq was performed using lung tissues collected in the PSL combination study. Samples were obtained from the right middle lung lobe of the control, TROP2‐DXd monotherapy, and PSL combination (10 mg/kg) groups (n = 7/group). Total RNA was extracted using the miRNeasy Mini Kit (QIAGEN). RNA‐seq libraries were prepared from 100 ng of total RNA and sequenced on a NextSeq 1000 Sequencing System (Illumina) using NextSeq 1000/2000 P1 Reagents (300 cycles). Differential gene expression (DGE) analysis was performed to identify genes with altered expressions among treatment groups. Differentially expressed genes (DEGs) were defined using thresholds of |log2 fold change| > 1 and a false discovery rate of < 0.05. Gene Ontology analysis was performed using the Illumina Correlation Engine platform (Illumina).
3. Results
3.1. Founder Animal Development
Genotyping by screening PCR and Southern blot analysis of 117 pups obtained from 640 in vitro fertilized embryos identified 7 transgene‐positive animals (two males and five females), all of which were viable. Based on Southern blot analysis using copy‐number reference standards, the integrated BAC copy number in these animals was estimated to range approximately from 3 to 10 copies (Figure S1B). Based on confirmation of full‐length BAC integration, these animals were designated as BAC Tg founder rats and were used for subsequent characterization.
3.2. Selection of the Transgene Line for Subsequent DXd ADC‐Related ILD Studies
Histological examination of lungs from F1 transgene‐positive animals revealed no remarkable findings, except for a slightly increased number of alveolar macrophages compared with the background strain, wild‐type Crl:CD (SD) rats (Figure 1A).
FIGURE 1.
Lung histology, hTROP2 and DXd IHC in hTROP2 Tg rats. (A) Representative H&E‐stained lung section from an F1 transgene‐positive rat. (B) hTROP2 IHC in the lung, demonstrating predominant localization of hTROP2 in cuboidal alveolar epithelial cells, consistent with AT2 cells (arrows). (C) DXd IHC in the lung 48 h after single IV dosing of IgG‐DXd at 60 mg/kg, showing immunoreactivity restricted to stromal compartments, consistent with plasma. (D) DXd IHC in the lung 48 h after single IV dosing of TROP2‐DXd at 60 mg/kg, suggesting preferential alveolar epithelial localization of TROP2‐DXd (arrows) in addition to stromal regions. Bar = 50 μm.
TROP2 IHC demonstrated that hTROP2 was predominantly localized to alveolar epithelial cells, with prominent cytoplasm of cuboidal epithelial cells consistent with AT2 cells in Tg rats (Figure 1B). No detectable hTROP2 immunoreactivity was observed in other organs examined, including the heart, liver, kidney, spleen, or gastrointestinal tract (Figure S2). The strong cytoplasmic staining in AT2 cells is consistent with the replacement of the rat Sftpc locus with human TACSTD2 and reflects transgene‐driven overexpression. In contrast, IHC of normal human lungs demonstrated membranous hTROP2 expression in airway epithelial cells, whereas staining in alveolar regions was relatively weak (Figure S3A,B), consistent with previously reported observations [16, 17]. Absolute RT‐qPCR demonstrated that hTROP2 mRNA levels were substantially higher in hTROP2 Tg rat lungs than in normal human lung tissue, with an approximately 30‐fold difference in transcript abundance (Figure S3C).
At 48 h following single IV administration of IgG‐DXd, DXd immunoreactivity was detected only in stromal compartments, supposedly plasma (Figure 1C). In contrast, after single IV administration of TROP2‐DXd, DXd immunoreactivity was detected in alveolar epithelial cells in addition to stromal regions (Figure 1D). This distribution pattern was consistently observed at both 20 and 60 mg/kg.
As discussed previously, DXd IHC is likely to detect intact DXd ADC because released DXd would be dissolved during fixation and tissue processing due to its physicochemical properties [8, 18]. No parenchymal localization of TROP2‐DXd was observed in other organs examined (Figure S4). Accordingly, preferential parenchymal localization of TROP2‐DXd to alveolar epithelial cells in Tg rats is supported by these findings.
Based on lung‐restricted hTROP2 expression, preferential alveolar localization of TROP2‐DXd, and stable transgene copy number in subsequent generations, the most suitable transgenic line was selected as the parental line for further DXd ADC‐related ILD studies.
3.3. Repeated Administration of TROP2‐DXd Induced Interstitial Pneumonitis
Following repeated administration of TROP2‐DXd, body weight decrease was initially observed in 1 female at each dose level, occurring on Day 8 at 120 mg/kg and on Day 15 at 60 mg/kg after the 1st dose. Subsequently, body weight decreases were also observed in additional animals after the 2nd and 3rd doses. These findings prompted careful clinical assessment, and the affected animals were subjected to unscheduled necropsy. As a result, all animals in the control group (0 mg/kg; n = 12), 6 of 14 animals in the 60 mg/kg group, and 9 of 14 animals in the 120 mg/kg group were euthanized and necropsied 3 weeks after the third dose.
At necropsy, dark red or grayish‐white foci were observed in multiple lung lobes in all animals treated at 60 and 120 mg/kg (Figure 2). Pleural effusion was also noted in a subset of animals. Histopathological examination revealed that all animals at 60 and 120 mg/kg exhibited pulmonary edema characterized by alveolar spaces filled with eosinophilic proteinaceous material, hyperplasia of alveolar epithelial cells (accompanied by airway epithelial regeneration in some cases), and inflammatory cell infiltration composed of neutrophils, eosinophils, lymphocytes, and macrophages within alveolar and interstitial regions. In a subset of animals, fibrotic changes, characterized by proliferation of fibroblasts and myofibroblasts, as well as hyaline membrane formation, were also observed (Figure 2). The incidence and severity of these histopathological findings and TK parameters are summarized in Tables S1 and S2. No apparent differences in the incidence or severity of these pulmonary findings were observed between the 60 and 120 mg/kg groups, despite increased systemic exposure at the higher dose level.
FIGURE 2.
Pulmonary findings in hTROP2 Tg rats following repeated dosing with TROP2‐DXd. (A) Control animal (0 mg/kg), necropsied on Day 65, showing no remarkable changes. (B) 60 mg/kg, necropsied 6 days after the 2nd dose (Day 28). All lung lobes were affected, with subacute alveolar injury characterized by hyaline membrane formation (stars) and alveolar epithelial regeneration (hyperplasia; arrows). (C) 60 mg/kg, necropsied 21 days after the 3rd dose (Day 64). All lung lobes were affected, with prominent alveolar epithelial regeneration (hyperplasia; arrows), alveolar edema (eosinophilic proteinaceous exudates), and intra‐alveolar fibroblast proliferation (stars). Left, gross findings; middle, low‐magnification (bar = 5 mm); right, high‐magnification (bar = 100 μm).
Collectively, these findings indicate that repeated administration of TROP2‐DXd induces interstitial pneumonitis associated with alveolar epithelial cell injury in Tg rats.
3.4. Amelioration of TROP2‐DXd‐Related Lung Injury by PSL Combination
Compared with the TROP2‐DXd monotherapy group, attenuation of body weight gain was observed in combination groups receiving PSL at 3 mg/kg or higher. In the TROP2‐DXd monotherapy group, 1 animal exhibited decreased body weight and irregular respiration after the second dose and was subjected to unscheduled necropsy 4 days after the 2nd TROP2‐DXd administration. Another animal in the monotherapy group developed irregular respiration from Day 18 after the 2nd dose of TROP2‐DXd, which persisted until the scheduled necropsy on Day 43. Hematological analysis at necropsy showed significant decreases in total white blood cells in PSL combination groups at 1 mg/kg or higher, lymphocyte counts at 3 mg/kg or higher, and eosinophil counts at 10 mg/kg compared with the TROP2‐DXd monotherapy group (Table 1). At necropsy, gross pathology revealed dark red to grayish white lesions in multiple lung lobes in 5 of 7 animals in the TROP2‐DXd monotherapy group. In contrast, similar findings were observed in 1 of 7 animals in the PSL 1 and 3 mg/kg combination groups, whereas no apparent gross abnormalities were observed in the PSL 10 mg/kg group (Figure 3). Histopathological examination demonstrated reduced inflammatory cell infiltration in alveolar and interstitial regions, as well as decreased hyaline membrane formation, in PSL combination groups at doses of 1 mg/kg or higher (Figure 3). In addition, a reduction in fibrotic changes was suggested in the PSL 3 mg/kg group compared with the TROP2‐DXd monotherapy group (Table 2). TK after the 1st and 2nd doses showed comparable systemic exposure to TROP2‐DXd and free DXd between the monotherapy and PSL combination groups (Table S3). Collectively, these findings suggest that PSL mitigates DXd ADC‐related toxicity in Tg rats without altering systemic exposure.
TABLE 1.
White blood cell parameters in blood following combination treatment with TROP2‐DXd and PSL.
| TROP2‐DXd (mg/kg) | 0 | 60 | 60 | 60 | 60 |
|---|---|---|---|---|---|
| PSL (mg/kg) | 0 | 0 | 1 | 3 | 10 |
| Number of animals | 7 males | 6 males | 7 males | 7 males | 7 males |
| WBC (103/μL) | 8.730 ± 3.414 | 7.205 ± 1.392 | 5.226 ± 1.133* | 4.730 ± 0.531* | 3.474 ± 0.780* |
| Lymphocyte (103/μL) | 6.422 ± 2.691 | 4.983 ± 2.086 | 3.966 ± 0.977 | 3.223 ± 0.303* | 1.807 ± 0.411* |
| Neutrophil (103/μL) | 1.923 ± 0.902 | 1.707 ± 1.424 | 0.916 ± 0.168 | 1.170 ± 0.357 | 1.337 ± 0.313 |
| Eosinophil (103/μL) | 0.142 ± 0.030 | 0.197 ± 0.162 | 0.099 ± 0.028 | 0.087 ± 0.029 | 0.066 ± 0.020* |
| Monocyte (103/μL) | 0.127 ± 0.081 | 0.190 ± 0.064 | 0.177 ± 0.047 | 0.194 ± 0.052 | 0.209 ± 0.092 |
p < 0.05 versus the monotherapy group (Dunnett's test).
FIGURE 3.
Pulmonary findings following combination treatment with TROP2‐DXd and PSL in hTROP2 Tg rats. (A) TROP2‐DXd monotherapy. Dark red or grayish‐white foci were observed in all lung lobes. Histopathological findings were characterized by epithelial alterations, including AT2 hyperplasia and bronchiolization (arrows), accompanied by inflammatory cell infiltration and fibroblastic proliferation (stars). (B) TROP2‐DXd plus PSL 1 mg/kg. Dark red foci were observed in several lung lobes. Histopathological lesions were more localized compared with the monotherapy group and were largely limited to relatively early alveolar injury features, including epithelial regeneration (arrows), neutrophilic infiltration, and alveolar hemorrhage. (C) TROP2‐DXd plus PSL 3 mg/kg. Dark red foci were observed only in the left lung lobe. Histopathological lesions were further restricted in distribution, and even within the affected areas, findings were limited to epithelial regeneration (arrow) and alveolar macrophage infiltration. Left, gross findings; middle, low‐magnification (bar = 5 mm for A and B, 2.5 mm for C); right, high‐magnification (bar = 100 μm).
TABLE 2.
Pulmonary histopathological findings following combination treatment with TROP2‐DXd and PSL.
| TROP2‐DXd (mg/kg) | 0 | 60 | 60 | 60 | 60 | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PSL (mg/kg) | 0 | 0 | 1 | 3 | 10 | ||||||||||||||||||||
| Number of animals | 7 males | 7 males | 7 males | 7 males | 7 males | ||||||||||||||||||||
| Histopathological grade | Total | 1 | 2 | 3 | 4 | Total | 1 | 2 | 3 | 4 | Total | 1 | 2 | 3 | 4 | Total | 1 | 2 | 3 | 4 | Total | 1 | 2 | 3 | 4 |
| Alveolar proteinosis | 0 | 7 | 7 | 7 | 5 | ||||||||||||||||||||
| 0 | 0 | 0 | 0 | 3 | 1 | 2 | 1 | 5 | 2 | 0 | 0 | 6 | 1 | 0 | 0 | 4 | 1 | 0 | 0 | ||||||
| Kruskal‐Wallis | p = 0.080 | ||||||||||||||||||||||||
| Hyperplasia, alveolar epithelium * | 0 | 7 | 7 | 7 | 4 | ||||||||||||||||||||
| 0 | 0 | 0 | 0 | 4 | 0 | 3 | 0 | 7 | 0 | 0 | 0 | 7 | 0 | 0 | 0 | 3 | 1 | 0 | 0 | ||||||
| Kruskal‐Wallis | p = 0.053 | ||||||||||||||||||||||||
| Inflammatory cell infiltration | 0 | 6 | 3 | 2 | 2 | ||||||||||||||||||||
| Mixed, alveolus/interstitium | 0 | 0 | 0 | 0 | 3 | 0 | 3 | 0 | 3 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | |||||
| Kruskal‐Wallis | p = 0.033 | ||||||||||||||||||||||||
| Dunn | p = 0.038 | p = 0.035 | p = 0.035 | ||||||||||||||||||||||
| Fibrosis a | 0 | 4 | 1 | 0 | 1 | ||||||||||||||||||||
| 0 | 0 | 0 | 0 | 1 | 3 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | ||||||
| Kruskal‐Wallis | p = 0.039 | ||||||||||||||||||||||||
| Dunn | p = 0.070 | p = 0.022 | p = 0.070 | ||||||||||||||||||||||
| Hyaline membrane | 0 | 3 | 0 | 0 | 0 | ||||||||||||||||||||
| 0 | 0 | 0 | 0 | 2 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | ||||||
| Kruskal‐Wallis | p = 0.021 | ||||||||||||||||||||||||
| Dunn | p = 0.033 | p = 0.033 | p = 0.033 | ||||||||||||||||||||||
Note: Histopathological grades were analyzed using the Kruskal‐Wallis test followed by Dunn's post hoc test with Holm‐adjusted p values for prespecified comparisons between the monotherapy group and each PSL combination group. p < 0.05 was considered statistically significant. The histopathological grading system is described in Table S1.
Hyperplasia of bronchial epithelial cells was also observed in some areas.
Including fibroblast/myofibroblast proliferation.
3.5. Transcriptomic Alterations in the Lungs
TROP2‐DXd monotherapy resulted in 777 upregulated and 241 downregulated DEGs compared with control. Gene Ontology analysis demonstrated enrichment of pathways associated with inflammatory response and extracellular matrix (ECM) organization in the TROP2‐DXd group (Figure 4A; Table S4). Consistent with these findings, inflammatory genes (Il6, Ccl2, and Cxcl6), fibrosis‐associated macrophage markers (Spp1, Retnla, Trem2, and Gpnmb), and ECM remodeling genes (Mmp12, Mmp7, Serpine1, and Col1a1) were increased, whereas AT2‐associated genes (Abca3, Slc34a2, Sftpc, Etv5, and Tfcp2l1) were decreased (Figure 4B, Table S5).
FIGURE 4.
Transcriptomic alterations in the lungs of hTROP2 Tg rats following TROP2‐DXd treatment. (A) Gene Ontology pathway analysis of top 10 enriched pathways among DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd compared with controls. (B) Representative disease‐associated DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd. (C) Principal component analysis of lung transcriptomes from control, TROP2‐DXd monotherapy, and PSL combination (10 mg/kg) groups. (D) Heatmap of the top 30 annotated PC1‐loading genes. The values in each column represent the fold change relative to those in the vehicle control group, and the color scale represents log2 fold change.
Principal component analysis demonstrated clear separation among the control, TROP2‐DXd monotherapy, and PSL combination (10 mg/kg PSL) groups (Figure 4C). Heatmap analysis of the top 30 annotated PC1‐loading genes revealed increased expression of tissue remodeling‐ and fibrosis‐associated genes (Inhba, Thbs2, Col1a1, and Tnc) and decreased expression of AT2‐associated genes (Sftpc and Slc34a2) (Figure 4D). Several inflammatory and tissue‐remodeling genes, including Il6, Serpine1, and Vcan, were substantially attenuated in the PSL combination group. These findings indicate that PSL partially suppressed TROP2‐DXd‐induced inflammatory and tissue‐remodeling responses.
4. Discussion
The present findings demonstrate that a hTROP2 Tg rat model can reproducibly recapitulate key pathological features of DXd ADC‐related lung injury under controlled experimental conditions. Repeated administration of TROP2‐DXd induced pulmonary lesions characterized by alveolar epithelial injury, hyaline membrane formation, inflammatory cell infiltration, and fibroproliferative changes. These histopathological changes were accompanied by transcriptomic alterations indicative of activation of inflammatory and tissue‐remodeling pathways, and the concordance between morphological and molecular findings supports the biological relevance of this model. Collectively, these findings indicate that the model captures key processes associated with tissue injury, repair, and remodeling following DXd ADC exposure (Figure 5).
FIGURE 5.
Overview of the hTROP2 Tg rat model and key findings. hTROP2 Tg rats exhibit key pathological and transcriptomic features of DXd ADC‐related ILD, supporting their utility as a translational model. Rat, alveolus, antibody illustrations were adapted from NIAID NIH BioArt Source.
No apparent difference in the incidence or severity of pulmonary findings was observed between the 60 and 120 mg/kg groups, despite the dose‐dependent increases in systemic exposure to intact TROP2‐DXd and free DXd. One possible explanation is that target‐mediated delivery and/or intracellular processing of TROP2‐DXd in AT2 cells may have approached saturation at 60 mg/kg in this model, such that increased systemic exposure at the higher dose level did not result in greater pulmonary injury.
Importantly, hTROP2 Tg rats were generated on a Crl:CD(SD) rat background, in which T‐DXd and datopotamab deruxtecan do not cross‐react with endogenous rat targets. In a nonclinical study, repeated intravenous administration of T‐DXd (average DAR approximately 8) at doses up to 197 mg/kg, the maximum feasible dose, every 3 weeks for 3 months did not induce pulmonary findings in Crl:CD(SD) rats [10]. Together with the preferential localization of TROP2‐DXd to alveolar epithelial cells observed in the present study, these findings support the contribution of target‐mediated delivery to the establishment of reproducible lung injury in hTROP2 Tg rats.
RNA‐seq analysis provided additional mechanistic insights into the molecular responses in this model. Among the genes upregulated in the hTROP2 Tg rats, Spp1, Trem2, Gpnmb, and Retnla are notable because related macrophage‐associated signatures have been described in fibrotic lung diseases, including idiopathic pulmonary fibrosis [19, 20]. In parallel, TROP2‐DXd treatment was associated with decreased expression of several genes involved in AT2 cell homeostasis and surfactant biology, including Sftpc, Abca3, Slc34a2, Etv5, and Tfcp2l1. AT2 cells are essential for surfactant production, maintenance of alveolar integrity, and epithelial regeneration following lung injury, and impairment of these functions has been implicated in the pathogenesis of fibrotic ILDs. Experimental and genetic studies have demonstrated that dysfunction of AT2‐associated programs can lead to impaired regenerative capacity, epithelial stress responses, and altered tissue repair [21, 22, 23]. The concurrent induction of macrophage‐associated remodeling genes together with suppression of AT2‐associated genes suggests coordinated epithelial injury and tissue‐remodeling responses in the present model. These molecular alterations are consistent with the histopathological findings and may contribute to the partial transcriptomic concordance observed between the Tg rat and cynomolgus monkey ILD models [9]. In the cynomolgus monkey model for DXd ADCs, three major biological categories associated with disease progression were identified: cell repair and regeneration, inflammatory response, and ECM organization. Figure S5 highlights representative genes commonly associated with inflammatory response and ECM remodeling pathways in both models, supporting shared molecular responses despite species and model differences.
Murine models have also been explored to investigate DXd ADC‐related lung toxicity. Recently, a few murine models using T‐DXd have been reported, in which interstitial inflammation was observed following repeated IV administration in BALB/c mice. In one such model, T‐DXd (5 mg/kg) was administered IV once weekly for up to 3 months, resulting in partial induction of pulmonary lesions [24]. In another study, higher dose regimens (e.g., 200 mg/kg once weekly or 100 mg/kg every 3 days) were employed, and pulmonary changes such as alveolar septal thickening, inflammatory cell infiltration, pneumocyte hyperplasia, and subsequent fibrotic remodeling were reported following repeated dosing [25].
In our experimental evaluations aimed at establishing a DXd ADC‐related murine ILD model, IgG‐DXd was administered to male C57BL/6J and BALB/cJ mice (Jackson Laboratory Japan) starting at 7 weeks of age once weekly at 60 or 200 mg/kg for 3 months (12 doses in total), representing prolonged and high exposure conditions, with intravenous dosing for the first six administrations followed by subcutaneous dosing for the remaining six. No pulmonary findings were observed despite these high exposure conditions (Figure S6, Table S6). Notably, neither T‐DXd nor IgG‐DXd cross‐reacts with murine targets, consistent with previous reports demonstrating a lack of trastuzumab binding to rodent HER2 [26].
These observations indicate that systemic exposure to DXd alone may not be sufficient to induce lung injury and that murine susceptibility may depend on species‐specific biological factors. In this regard, the extent and nature of pulmonary pathology observed in murine models appear variable and may not fully recapitulate the spectrum of ILD, particularly the alveolar epithelial injury‐associated responses observed in other species. This discrepancy across species highlights the importance of host biological context in shaping DXd ADC‐related lung toxicity.
PSL co‐administration attenuated TROP2‐DXd‐induced lung injury in the Tg model at histopathological and transcriptomic levels. Reductions in inflammatory cell infiltration, fibroblast/myofibroblast proliferation, and hyaline membrane formation were observed, together with attenuation of inflammatory and tissue‐remodeling transcriptional responses. Importantly, TK parameters in the PSL co‐administration groups were comparable to those in the TROP2‐DXd‐alone group, indicating that the observed attenuation is unlikely to be explained solely by reduced systemic exposure. Instead, these findings support the interpretation that PSL modulates downstream biological responses associated with lung injury, including inflammatory and tissue‐remodeling responses.
In clinical settings, systemic corticosteroids represent the mainstay of treatment for drug‐induced ILD, including cases associated with T‐DXd, where prompt initiation of steroid therapy is recommended [27, 28, 29]. In this context, the present findings provide experimental support for the steroid responsiveness of DXd ADC‐related lung injury and reinforce the relevance of inflammatory and repair‐associated pathways identified in this model. It should be noted, however, that PSL was administered throughout the dosing period in this study, reflecting a prophylactic experimental design rather than clinical treatment initiated after the onset of lung injury. Therefore, the extent to which these findings directly translate to clinical therapeutic settings remains to be further investigated. Nevertheless, this model may also provide a useful platform for evaluating prophylactic approaches to mitigate DXd ADC‐related lung injury.
In addition, the PSL dose levels used in this study (1–10 mg/kg in the Tg rats), when converted using body surface area‐based scaling, correspond approximately to 0.2–1.6 mg/kg in humans, which broadly overlaps with corticosteroid dose ranges commonly used for the management of mild to moderate drug‐induced ILD [30]. This observation may support the translational relevance of the present findings, although differences in dosing schedule and treatment context should be taken into consideration. Taken together, these observations highlight the utility of the present model not only for mechanistic investigations but also as a platform for evaluating pharmacological modulation of lung injury, while emphasizing the need for careful interpretation in the context of clinical translation.
A potential limitation of the present model is that Tg‐driven expression of hTROP2 may not fully recapitulate the cellular expression patterns and physiological expression levels in normal human lungs. Overexpression of the target antigen, together with its restriction to AT2 cells, could influence ADC distribution or susceptibility to injury and should therefore be considered when interpreting the findings. DXd ADC‐related lung injury is thought to be driven predominantly by target‐independent mechanisms, as suggested by observations across multiple nonclinical and clinical systems [6, 8, 31]. In this context, hTROP2 expression in AT2 cells facilitates target‐mediated delivery of TROP2‐DXd to the alveolar epithelium in Tg rats and serves as an experimental strategy to reproducibly induce alveolar epithelial injury. Therefore, the initiating mechanism of lung injury in this model does not fully recapitulate that proposed for clinical DXd ADC‐related ILD. Nevertheless, this Tg rat model is considered applicable for the investigation of inflammatory and tissue‐remodeling responses following alveolar epithelial injury and for evaluation of potential management strategies relevant to DXd ADC‐related lung injury.
It should also be noted that TROP2‐DXd used in this study had a higher DAR (7.9) than the clinically administered formulations of datopotamab deruxtecan (approximately DAR 4). Therefore, the present findings should not be interpreted as reproducing the pulmonary toxicity profile of a specific clinical DXd ADC. Rather, TROP2‐DXd was used as a tool compound to reproducibly induce pulmonary injury in this model, enabling investigation of biological processes associated with DXd ADC‐related lung injury.
From a practical perspective, the present hTROP2 Tg rat model provides a versatile platform for controlled and scalable investigations of DXd ADC‐related lung injury. The model enables systematic assessment of dosing regimens, exposure levels, and pharmacological interventions, facilitating mechanistic investigations and evaluation of mitigation strategies. For example, the responsiveness to PSL observed in this study supports its utility for assessing modulation of lung injury. Taken together, this Tg rat model complements non‐human primate models by providing an experimentally tractable platform for translational investigation of DXd ADC‐related ILD.
Author Contributions
Kazuyoshi Kumagai: conceptualization, investigation, writing – original draft, methodology, validation, formal analysis, data curation, project administration, visualization. Yuma Nagai: methodology, investigation, writing – review and editing, formal analysis, data curation, validation, project administration. Yuji Shirai: data curation, investigation, validation, formal analysis, visualization, writing – review and editing. Kazunori Fujimoto: methodology, investigation, validation, visualization, writing – review and editing. Masako Imaoka: data curation, investigation, validation, writing – review and editing. Katsuyoshi Chiba: validation, supervision, writing – review and editing.
Funding
The authors have nothing to report.
Ethics Statement
The generation of Tg rats was conducted at Institute of Immunology Co. Ltd. under containment conditions for recombinant DNA use. Experiments involving genetically modified organisms were approved by the biosafety committees in Daiichi Sankyo and Institute of Immunology Co. Ltd., and performed in compliance with applicable regulations governing the use of recombinant DNA. All animal experiments were approved by the Institutional Animal Care and Use Committee in Daiichi Sankyo.
Conflicts of Interest
All authors are employees of Daiichi Sankyo.
Supporting information
Figure S1: Generation and validation of hTROP2 Tg rats. (A) Construction of the BAC transgene vector using Red/ET recombination and genomic integration. (B) Genotyping of human TACSTD2 by Southern blot analysis in founder candidates. Representative bands corresponding to the transgene are shown, along with a positive control (1.8 kb vector DNA; P.C. in the figure; 1, 3, 10, and 30 copies) and a negative control (wild‐type genomic DNA; N.C. in the figure). The selected founder animal (No. 19), used for subsequent analyses, is indicated by an arrow.
Figure S2: hTROP2 IHC in extrapulmonary organs of hTROP2 Tg rats. IHC showed no apparent hTROP2‐positive staining in the liver (A; bar = 100 μm), kidney (B; bar = 50 μm), heart (C; bar = 50 μm), or ileum (D; bar = 100 μm).
Figure S3: Characterization of hTROP2 expression in normal human and hTROP2 Tg rat lungs. (A) In the normal human lung (65‐year‐old male), hTROP2 showed membranous staining in airway epithelial cells (arrow), whereas staining in alveolar epithelial cells (arrowhead) was relatively weak. IHC was performed using the same antibody and staining protocol as for the Tg rat samples. (B) In hTROP2 Tg rats, strong cytoplasmic staining of hTROP2 was observed in AT2 cells (arrow). No apparent immunoreactivity was detected in airway epithelial cells or alveolar Type I cells. Bar = 50 μm. (C) RT‐qPCR of hTROP2 expression in normal human lungs and hTROP2 Tg rat lungs. hTROP2 mRNA levels were quantified by absolute RT‐qPCR using a standard curve generated from serial dilutions of a human TACSTD2 DNA template (Bio‐Rad Laboratories) and are expressed as copies/ng RNA. Absolute quantification was employed to enable direct cross‐species comparison of hTROP2 transcript abundance, thereby avoiding potential bias introduced by species‐specific differences in housekeeping gene expression. PCR amplification efficiency and assay linearity were confirmed prior to sample quantification. The normal human lung sample consisted of pooled total RNA from the lungs of 3 Asian male donors (Takara Bio). Lung samples from 7 untreated male hTROP2 Tg rats were analyzed individually. The mean hTROP2 mRNA level in hTROP2 Tg rat lungs was approximately 30‐fold higher than that in the pooled normal human lung sample.
Figure S4: DXd IHC in extrapulmonary organs from hTROP2 Tg rats 48 h after single IV dosing of TROP2‐DXd at 60 mg/kg. DXd IHC showed TROP2‐DXd immunoreactivity restricted to the vascular space (blood/plasma), with no detectable staining in parenchymal cells such as hepatocytes in the liver (A; bar = 100 μm), renal tubular epithelial cells in the kidney (B; bar = 100 μm), cardiomyocytes in the heart (C; bar = 50 μm), or intestinal epithelial cells in the ileum (D; bar = 100 μm).
Figure S5: Comparative transcriptomic signatures of DXd ADC‐related ILD models in hTROP2 Tg rats and cynomolgus monkeys. Heatmaps show representative genes associated with inflammatory response and ECM organization that were commonly upregulated in both models. Values represent log2 fold changes relative to control. The transcriptomic profiles demonstrated concordant activation of inflammatory and ECM remodeling pathways across the rat and monkey ILD models.
Figure S6: Representative H&E‐stained lung sections in BALB/c and C57BL/6 mice. No noteworthy findings were observed in male BALB/c or C57BL/6 mice administered vehicle (10 mM acetate buffer, 5% sorbitol, pH 5.5) or IgG‐DXd for 12 weeks (once weekly, IV; 12 total doses). (A) Vehicle control, BALB/c mouse. (B) 200 mg/kg IgG‐DXd, BALB/c mouse. (C) Vehicle control, C57BL/6 mouse. (D) 200 mg/kg IgG‐DXd, C57BL/6 mouse. Bar = 100 μm.
Table S1: Pulmonary histopathological findings by the repeated administration of TROP2‐DXd.
Table S2: TK parameters following repeated administration of TROP2‐DXd in hTROP2 Tg rats.
Table S3: TK parameters following combination treatment with TROP2‐DXd and PSL in hTROP2 Tg rats.
Table S4: Gene Ontology enrichment analysis of DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd.
Table S5: DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd versus control.
Table S6: TK parameters following repeated administration of IgG‐DXd in BALB/c and C57BL/6 mice.
Acknowledgments
We thank our colleagues at Daiichi Sankyo: Kyoko Miwa, Satoko Yahagi, and Junya Matsushita for their support in animal experiments; Kumi Honda for clinical pathology; Shinobu Hakamata for preparation of pathological specimens, including IHC in hTROP2 Tg rats; Takanori Maejima and Tomoko Shibutani for hTROP2 IHC in normal human lungs; Noriyo Niino for transcriptomic analysis; and Fuminao Doi for ADC synthesis. We also thank Kyosuke Saito, Yoshikazu Totsuka, and colleagues at the Institute of Immunology Co. Ltd. for their expert support in BAC Tg technologies. We further thank Kotaro Yamashita and colleagues in Mediford Corporation for conducting murine experiments.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: Generation and validation of hTROP2 Tg rats. (A) Construction of the BAC transgene vector using Red/ET recombination and genomic integration. (B) Genotyping of human TACSTD2 by Southern blot analysis in founder candidates. Representative bands corresponding to the transgene are shown, along with a positive control (1.8 kb vector DNA; P.C. in the figure; 1, 3, 10, and 30 copies) and a negative control (wild‐type genomic DNA; N.C. in the figure). The selected founder animal (No. 19), used for subsequent analyses, is indicated by an arrow.
Figure S2: hTROP2 IHC in extrapulmonary organs of hTROP2 Tg rats. IHC showed no apparent hTROP2‐positive staining in the liver (A; bar = 100 μm), kidney (B; bar = 50 μm), heart (C; bar = 50 μm), or ileum (D; bar = 100 μm).
Figure S3: Characterization of hTROP2 expression in normal human and hTROP2 Tg rat lungs. (A) In the normal human lung (65‐year‐old male), hTROP2 showed membranous staining in airway epithelial cells (arrow), whereas staining in alveolar epithelial cells (arrowhead) was relatively weak. IHC was performed using the same antibody and staining protocol as for the Tg rat samples. (B) In hTROP2 Tg rats, strong cytoplasmic staining of hTROP2 was observed in AT2 cells (arrow). No apparent immunoreactivity was detected in airway epithelial cells or alveolar Type I cells. Bar = 50 μm. (C) RT‐qPCR of hTROP2 expression in normal human lungs and hTROP2 Tg rat lungs. hTROP2 mRNA levels were quantified by absolute RT‐qPCR using a standard curve generated from serial dilutions of a human TACSTD2 DNA template (Bio‐Rad Laboratories) and are expressed as copies/ng RNA. Absolute quantification was employed to enable direct cross‐species comparison of hTROP2 transcript abundance, thereby avoiding potential bias introduced by species‐specific differences in housekeeping gene expression. PCR amplification efficiency and assay linearity were confirmed prior to sample quantification. The normal human lung sample consisted of pooled total RNA from the lungs of 3 Asian male donors (Takara Bio). Lung samples from 7 untreated male hTROP2 Tg rats were analyzed individually. The mean hTROP2 mRNA level in hTROP2 Tg rat lungs was approximately 30‐fold higher than that in the pooled normal human lung sample.
Figure S4: DXd IHC in extrapulmonary organs from hTROP2 Tg rats 48 h after single IV dosing of TROP2‐DXd at 60 mg/kg. DXd IHC showed TROP2‐DXd immunoreactivity restricted to the vascular space (blood/plasma), with no detectable staining in parenchymal cells such as hepatocytes in the liver (A; bar = 100 μm), renal tubular epithelial cells in the kidney (B; bar = 100 μm), cardiomyocytes in the heart (C; bar = 50 μm), or intestinal epithelial cells in the ileum (D; bar = 100 μm).
Figure S5: Comparative transcriptomic signatures of DXd ADC‐related ILD models in hTROP2 Tg rats and cynomolgus monkeys. Heatmaps show representative genes associated with inflammatory response and ECM organization that were commonly upregulated in both models. Values represent log2 fold changes relative to control. The transcriptomic profiles demonstrated concordant activation of inflammatory and ECM remodeling pathways across the rat and monkey ILD models.
Figure S6: Representative H&E‐stained lung sections in BALB/c and C57BL/6 mice. No noteworthy findings were observed in male BALB/c or C57BL/6 mice administered vehicle (10 mM acetate buffer, 5% sorbitol, pH 5.5) or IgG‐DXd for 12 weeks (once weekly, IV; 12 total doses). (A) Vehicle control, BALB/c mouse. (B) 200 mg/kg IgG‐DXd, BALB/c mouse. (C) Vehicle control, C57BL/6 mouse. (D) 200 mg/kg IgG‐DXd, C57BL/6 mouse. Bar = 100 μm.
Table S1: Pulmonary histopathological findings by the repeated administration of TROP2‐DXd.
Table S2: TK parameters following repeated administration of TROP2‐DXd in hTROP2 Tg rats.
Table S3: TK parameters following combination treatment with TROP2‐DXd and PSL in hTROP2 Tg rats.
Table S4: Gene Ontology enrichment analysis of DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd.
Table S5: DEGs in lungs of hTROP2 Tg rats treated with TROP2‐DXd versus control.
Table S6: TK parameters following repeated administration of IgG‐DXd in BALB/c and C57BL/6 mice.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.