Avoiding the toll of lung injury while driving CAR T cells to the target tumor

Chimeric antigen receptors (CARs) are genetically engineered synthetic receptors that, when transduced into T cells, recognize and bind tumor-associated antigens, eliciting potent, antigen-specific immune responses (1,2). Although the use of CD19-targeted CAR T cells is standard of care for hematologic malignancies, this approach has had limited clinical success in solid tumors, owing to solid tumor-specific hindrances (3). The first step in translating CAR T-cell therapy to solid tumors is to identify a tumor-specific antigen. Many candidate target antigens are shared with normal tissues, such as the lung or liver, resulting in a high risk of on-target, off-tumor toxicity. Current investigations are focused on targeting differentially expressed antigens by designing CARs with a specific affinity to recognize overexpression on tumor cells while sparing normal cells with low endogenous expression (4). However, both the affinity of CARs and the expression of antigen on normal and tumor cells are dynamic and context dependent. Damage to normal tissue from CAR T cells can result in inflammatory complications related to cytokine release, which, in some patients, can lead to organ damage. Of note, two cases of fatal lung toxicity after CAR T-cell therapy have been reported (5,6). It is critical to investigate the mechanisms that underlie these adverse effects to prevent their occurrence.

In a study published recently in Molecular Therapy, Hou et al. investigated mechanisms of on-target, off-tumor toxicity from CAR T-cell therapy (7). In particular, the authors assessed acute lung injury induced by HER2 antigen-targeted CAR T-cell therapy in a HER2-transgenic mouse model expressing human HER2 under a tissue-wide promoter, with expression of HER2 in normal tissue. Upon intravenous administration of high-dose HER2 CAR T cells, mice developed T-cell accumulation in the lungs, increased extravasation of fluid (assessed by the use of Evans blue), disruption of alveolar architecture (confirmed by histologic assessment), and apoptosis of alveolar epithelial cells (measured by caspase-3 analysis). Interferon-gamma (IFNγ) secreted by CAR T cells induced degradation of the 5' untranslated region of caspase-7 mRNA in alveolar epithelial cells, resulting in pulmonary toxicity. Knockdown of caspase-7 was observed 6 hours after the administration of high-dose CAR T cells (indicated by apoptosis of lung epithelial cells on Annexin V staining). The generation and assessment of CAR T cells with granzyme B knockout ruled out other potential mechanisms of cell death; although levels of apoptosis were not consistent between immune factors, neutralization of IFNγ countered the cytotoxicity of CAR T cells, whereas neutralization of granzyme B did not.

To evaluate the therapeutic effect of IFNγ blockade on the efficacy of CAR T-cell therapy, the authors administered IFNγ-neutralizing antibodies after CAR T-cell infusion in a flank model of colorectal carcinoma. They observed that IFNγ blockade did not impair tumor control within the short period of observation. On the basis of these findings, the authors proposed that transient IFNγ antagonism may provide a therapeutic window in which to mitigate acute lung injury without compromising the antitumor efficacy of CAR T cells.

This study addresses a clinically relevant and challenging aspect of solid tumor immunotherapy by the use of a translationally relevant HER2-targeted CAR to achieve robust antitumor efficacy while minimizing on-target, off-tumor toxicity in the lungs. A strength of this study is the development of a HER2-transgenic immunocompetent mouse model. Unlike traditional xenograft models, which rely on immunodeficient hosts and a xenograft to recapitulate overexpression of tumor-cell antigen, this model allows the simulation of CAR T cell-induced toxicity in a physiologically and immunologically relevant setting. In addition to introducing a relevant preclinical model, this study provides mechanistic insights into IFNγ-mediated tissue toxicity. The authors identified a link between IFNγ signaling and apoptosis of alveolar epithelial cells, highlighting the role of caspase-7 in epithelial cell death (8). The study findings suggest that IFNγ-induced degradation of caspase-7 mRNA sensitizes epithelial cells to premature death, thereby contributing to lung injury. This represents a mechanism of immune-mediated toxicity and enriches our understanding of how proinflammatory cytokine signaling intersects with regulated cell death pathways in the context of CAR T-cell therapy.

Despite its mechanistic depth, this study has several limitations with regard to translational relevance. A central concern lies in the use of a transgenic mouse model that drives artificially high HER2 expression across multiple organs; this likely does not reflect the density or distribution of physiologic antigen observed in patients, and, indeed, no such correlation was demonstrated. This overexpression may exaggerate the severity of toxicities, particularly in the lungs, where CAR T cells are transiently sequestered in the capillaries owing to nonspecific activation and increased granularity and size after manufacturing (9-11). Even modest expression of antigen may provoke immune responses in dense, vascularized tissue. In parallel, the CAR construct used in this study was designed to target a nonnative transduced antigen, rendering recognition and destruction of HER2-expressing tissues essentially inevitable. This design makes it difficult to distinguish specific antigen-driven effects from nonspecific tissue damage, potentially amplifying the clinical risk of on-target, off-tumor toxicity. Without proper controls, such as antigen-negative models or irrelevant CAR constructs, it is difficult to attribute tissue damage to the intended antigen-targeting mechanism or to determine whether it is an artifact of the model, such as the cytokine-induced upregulation of antigen described by other investigators (5). As such, future models should aim to incorporate endogenous antigen expression, immune competence, and spatial tissue complexity to improve clinical relevance.

Additionally, key parameters, such as CAR binding affinity, were not reported. Affinity plays a critical role in determining the sensitivity of CAR T cells to varying antigen densities (12). High-affinity CARs are prone to engage and eliminate cells expressing low levels of antigen, contributing to off-tumor toxicity, whereas lower-affinity CARs may provide better selectivity by distinguishing antigen-overexpressing malignant cells from normal tissues. The absence of these data limits the extrapolation of findings across CAR designs and obscures the activation threshold that would be clinically relevant. Moreover, the use of a human single-chain variable fragment within a murine CAR T-cell framework may introduce species-specific differences in CAR signaling or trafficking, further complicating the interpretation of toxicity in a human context.

Although the study mechanistically identifies IFNγ as a key mediator of lung injury and excludes granzyme B-dependent cytotoxicity, it does not explore other major T-cell effector pathways, such as death receptor-mediated apoptosis via Fas/FasL or TRAIL. These established cytolytic mechanisms in T-cell biology may contribute to tissue damage as well (13). Their exclusion leaves an incomplete view of the spectrum of immune effectors potentially responsible for epithelial injury.

The in vivo evaluation of antitumor efficacy was limited to a short-term subcutaneous (flank) model, monitored for only 12 days. This setting fails to capture the architectural and immunosuppressive features of orthotopic tumors or to assess important parameters, including infiltration, persistence, and memory formation of CAR T cells and tumor relapse (9,14). The conclusion that IFNγ blockade does not impair the therapeutic efficacy of CAR T cells should therefore be interpreted with caution, especially given the well-documented role that IFNγ plays in enhancing cytotoxicity and infiltration of T cells, sensitizing tumors via upregulation of the major histocompatibility complex, and limiting residual disease (15,16). Attenuation of IFNγ may reduce acute toxicity but could compromise long-term tumor control, particularly in solid tumors, where immune evasion and recurrence remain major clinical challenges (17).

Finally, the translatability of the described toxicity model to clinical use warrants further investigation. For instance, the body weight of mice, a sensitive indicator of health, remained stable, and no overt signs of clinical distress were reported, even in the setting of elevated IFNγ levels and proposed acute lung injury. Additionally, tumor burden remained similar between IFNγ-blocked and untreated groups, raising further questions about the severity and functional relevance of the postulated toxicity. These inconsistencies underscore the need for more-rigorous physiologic, histopathologic, and longitudinal assessments to substantiate claims of clinically significant toxicity before pulmonary toxicity from CAR T cells can be interpreted in human beings. Nevertheless, this investigation sheds light on a potential mechanism for acute lung injury after CAR T-cell therapy and provides a rationale to investigate such adverse events in clinical trials.

Acknowledgments

We would like to thank Heather Alcorn and David B. Sewell of the Memorial Sloan Kettering Cancer Center Department of Surgery, who provided editorial assistance.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Journal of Thoracic Disease. The article did not undergo external peer review.

Funding: This study was supported, in part, by the National Institutes of Health/National Cancer Institute (Cancer Center Support Grant P30 CA008748 to Memorial Sloan Kettering Cancer Center). P.S.A.’s laboratory work is supported by grants from the National Institutes of Health (UG3CA290241, R01CA292664, R01CA235667, R01CA236615, and T32CA009501), the U.S. Department of Defense (CA200437), the Adolfo F. Sardiña Charitable Foundation, the Batishwa Fellowship, the Baker Street Foundation, the Joanne and John DallePezze Foundation, the Derfner Foundation, the Esophageal Cancer Education Fund, the Memorial Sloan Kettering Technology Development Fund, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, and the Center for Experimental Therapeutics of Memorial Sloan Kettering Cancer Center. P.S.A.’s laboratory receives research support from Novocure.

Conflicts of Interest: The authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-1564/coif). P.S.A. serves as an unpaid editorial board member of Journal of Thoracic Disease from October 2024 to September 2026. P.S.A. declares research funding from Novocure; is a Scientific Advisory Board Member and/or Consultant for Affyimmune Therapeutics, Bio4t2, Carisma Therapeutics, Century Therapeutics, Orion Pharma, Outpace Bio, Pluri-biotech, and Verismo Therapeutics; has patents, royalties, and intellectual property on mesothelin-targeted CAR and other T-cell therapies, an issued patent method for detection of cancer cells using virus, and pending patent applications on PD1 dominant negative receptor, a wireless pulse-oximetry device, and on an ex vivo malignant pleural effusion culture system. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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