This article and associated images are based on a poster originally authored by Joel J, Hoffman E, Zeqiri E, Pereira V, and Hutter V and presented at ELRIG Drug Discovery 2026 in affiliation with ImmuONE Ltd. and the University of Hertfordshire.
This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.
Background
Antibody-drug conjugates (ADCs) combine targeted monoclonal antibodies with potent cytotoxic payloads to selectively kill tumor cells while limiting systemic exposure.1 Despite this targeted design, less than 1% of an injected ADC reaches the tumor.2 Most exposure occurs elsewhere in the body, including in immune-rich tissues such as the lung.2 This off-target uptake can cause toxicity, with interstitial lung disease (ILD) emerging as a key concern.
Proposed mechanisms include Fc-γ receptor–mediated internalization by alveolar macrophages and local payload release, while differences in distribution across species complicate toxicity assessment.3
Trastuzumab deruxtecan illustrates these challenges. Preclinical rodent and primate studies have reported pulmonary inflammation, lymphocytic infiltrates, and fibrosis, alongside ADC accumulation in alveolar macrophages.3
As newer ADC formats introduce more complex linker and payload behaviors, human-relevant in vitro systems are increasingly needed to model lung responses and predict off-target toxicity earlier in development.
ImmuPHAGE™ macrophages express Fc receptors, such as CD16, CD32a, CD32b/c, and CD64, which are known to participate in ADC internalization, making these cells a potentially useful in vitro model for assessing ADC off-target toxicity4.
Aims:
- Assess the suitability of a human in vitro system for predicting the pulmonary toxicity observed in in vivo toxicity studies of trastuzumab deruxtecan (ADC), trastuzumab (free antibody), deruxtecan (payload with linker), and Dxd (payload).
- Understand the impact of trastuzumab deruxtecan vs. individual components on human alveolar macrophage responses.
Methods
Image Credit: Image courtesy of Joel J et al., in partnership with ELRIG (UK) Ltd.
Two assays recommended:
- Macrophage health and morphology assessment – morph_ONE assay5
- ADC uptake by macrophages assessment – ADC internalization assay
Results
Individual cell data analysis indicated morphological macrophage changes resulted from ADC or its component treatments
Figure 1. Individual cell analysis displaying phenotype distribution of ImmuPHAGE™ alveolar macrophages after 72 h exposure to trastuzumab-deruxtecan (A), trastuzumab (B), deruxtecan (C) and Dxd (D). Stacked bar charts represent the proportion of cells classified as normal macrophages (purple bars), foamy macrophages (blue bars), or reduced-vacuolation macrophages (green bars) at increasing concentrations. Briefly, cells were fixed and treated with a cocktail of fluorescent dyes staining for cellular features (nucleus, cytoplasm, and mitochondrial activity). Images were captured with a 40x objective in standard 2D imaging mode using the InCell Analyser 6000. Each sample was imaged using 16 fields, representing a total of 50 to 1000 cells per well. Images were analyzed using InCell Developer Toolbox 1.9.2 software and followed by generation of drug-induced phenotypes. Image Credit: Image courtesy of Joel J et al., in partnership with ELRIG (UK) Ltd.
Trastuzumab deruxtecan, trastuzumab, and the untreated group displayed similar phenotype profiles, with 67%-77% of cells showing normal morphology, 20-28% foamy macrophages, and 2%-5% of cells showing reduced vacuolation.
Treatment with deruxtecan (payload with linker) increased the number of cells with reduced vacuolation dose-dependently, from 4% untreated to 34% at 50 µM deruxtecan.
Dxd induced a dose-dependent increase in the number of foamy macrophages, from 20% untreated to 74% at>10 µM Dxd.
Detailed morphological analysis of foamy macrophages allowed identification and quantification of differences in foamy macrophage type
Foamy macrophage is an umbrella term for a macrophage that displays increased vacuolation.
Exposure to Dxd resulted in a 57-fold increase in the subpopulation of cells associated with low mitochondrial activity, smaller area, fewer vacuoles, and a smaller vacuolar area (Figure 2, blue bar).
The subpopulation associated with higher mitochondrial activity, larger cell area, and more vacuoles also increased 16-fold (Figure 2, dark purple bar).
Figure 2. Fold change in phenotype distribution following 72 h exposure to 30 µM Dxd (payload). Each cell was assessed for 4 descriptors (mitochondrial activity, cell size, number of vacuoles, and size of vacuoles), categorized into levels (low, moderate, high) relative to untreated control cells, generating 81 potential phenotypes. Data is expressed as a fold change relative to the untreated cell baseline with fold changes > 5 depicted. Image Credit: Image courtesy of Joel J et al., in partnership with ELRIG (UK) Ltd.
Investigating ADC internalization
Phenotypic changes reflect macrophage responses but do not identify the route of payload uptake
Image Credit: Image courtesy of Joel J et al., in partnership with ELRIG (UK) Ltd.
The internalization assay
Quantifying ADC uptake and intracellular localization in ImmuPHAGE™ could complement phenotypic profiling. FcγR blockade could help establish the contribution of receptor-mediated uptake to macrophage responses.
Implications for ADC development
These data could inform Fc or linker optimization and candidate selection to reduce off-target exposure. Prediction of clinical pulmonary toxicity would require further validation.
Conclusion and impact
High-content image analysis using morph_ONE™ provides a sensitive, human-relevant approach for detecting early macrophage responses to ADCs and their individual components.
Resolving phenotype at the single-cell level identifies altered vacuolation, cell area changes, and metabolic stress that population averages miss, and separates responses elicited by the intact conjugate from those driven by the payload alone.
Work is ongoing to determine whether uptake is FcγR-dependent, which connects this phenotype to an ADC design decision.
References
- Nguyen TD et al.(2023) Cancers (Basel); 15(3):713
- Colombo R et al. (2024) Cancer Discovery; 14(11): 2089 - 2108
- AbuhelwaZ et al. (2022) Drugs; 82(9):979–987
- Aoyama, M., et al. (2022). Fcγ receptor-dependent internalization and off-target cytotoxicity of antibody-drug conjugate aggregates. Pharmaceutical Research, 39(1), pp.89–103. DOI:10.1007/s11095-021-03158-x. https://link.springer.com/article/10.1007/s11095-021-03158-x.
- Hoffman, E., et al. (2023). Profiling alveolar macrophage responses to inhaled compounds using in vitro high content image analysis. Toxicology and Applied Pharmacology, 474, p.116608. DOI:10.1016/j.taap.2023.116608. https://linkinghub.elsevier.com/retrieve/pii/S0041008X23002478.
About ImmuONE Ltd.
ImmuONE specializes in human alveolar macrophage responses, epithelial cell culture platforms and testing services. Our respiratory tissue models closely resemble lung physiology to provide relevant, reliable, and reproducible results.
ImmuONE is partnered with the industry and academia to develop sustainable technologies for the future to support human-relevant, mechanistic driven safety assessments and develop animal product-free cell models. We offer a range of standard and bespoke assessments for inhalation safety assessment needs.
ImmuONE provides innovative solutions for inhalation toxicology, irritation sensitization, alveolar clearance, inflammation and can tailor to your inhalation safety assessment needs.
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Last Updated: Oct 27, 2026