DLL3-targeted CAR T-cell therapy in pre-clinical models for small cell lung cancer: safety, efficacy, and challenges

Chimeric antigen receptor (CAR) T-cell therapy has achieved complete responses in patients with hematological malignancies (1). However, it has shown only limited success in patients with solid tumors, owing to the lack of an ideal antigen to target with no on-target, off-tumor toxicity and to poor infiltration, among other hurdles (2). In solid tumors, such as small cell lung cancer (SCLC), with metastases in multiple organs at the time of presentation, it has been difficult to achieve efficacy without generating on-target, off-tumor toxicity in the normal tissue of the organs that harbor the metastases (3).

Immunotherapies have been investigated to treat patients with metastatic SCLC. Many of these immunotherapies target delta-like ligand 3 (DLL3), which is a notch ligand that is overexpressed on SCLC cells but is expressed in limited amounts on normal cells (4,5). Some of these immunotherapies use bispecific T-cell engagers (BiTEs)—antibodies that can simultaneously bind to an antigen on tumor cells and a surface molecule on T cells to induce tumor lysis (6), thus harnessing the host’s T-cell immune response to enhance antitumor activity. One such BiTE that targets DLL3 is tarlatamab (previously known as AMG 757; Table 1) (7). In preclinical models, tarlatamab showed significant antitumor efficacy against several SCLC cell lines and in patient-derived xenograft models and had manageable adverse events (8). To potentiate the clinical benefit of this agent, combination therapy that includes tarlatamab plus anti-programmed cell death protein 1 (PD1) or anti-programmed death-ligand 1 (PD-L1) checkpoint inhibitors, or both, is being investigated (NCT03319940, NCT04885998, NCT05740566, and NCT05361395). Another T-cell engager that is being investigated in SCLC is HPN328, which is a trispecific T-cell engager designed to engage albumin to increase the half-life of the molecule DLL3 on tumor cells and CD3 on T cells, with the goal of promoting activation and proliferation of T cells (NCT04471727). In an ongoing clinical trial, no grade ≥3 adverse events were observed in patients with SCLC who were treated with HPN328 who were experiencing a >30% decrease in tumor diameter, or in two patients with had stable disease (NCT04471727). Some of the disadvantages of BiTE therapies are a short half-life, the potential to induce cytokine release syndrome, and the inability to promote functional persistence or prevent or rescue T cells from exhaustion (9).

Alternatively, immune-effector cell therapies that target DLL3 are also being investigated. Among the advantages of CAR T-cell therapies are that they can expand in vivo, can functionally persist, and can be engineered to resist exhaustion, in contrast to BiTE therapies (2).

In preclinical studies, DLL3-CAR-natural killer (NK) cells showed significant antitumor efficacy and good tumor infiltration in SCLC subcutaneous xenograft models (10). Nevertheless, flank tumors do not capture the complexity of the tumor microenvironment in the lung; therefore, orthotopic models would be more suitable for these studies. CAR-NK cells that target DLL3 were recently translated into a phase I clinical trial for patients with relapsed or refractory extensive-stage SCLC (NCT05507593). Another DLL3-targeted CAR T-cell therapy of note is AMG 119, which is being investigated in combination with tarlatamab in a phase I clinical trial (NCT03392064) (11). The group, Jaspers et al. (12), investigated DLL3-targeted CAR T cells and found that CAR T-cell efficacy was enhanced with the secretion of interleukin (IL)-18 in both orthotopic and metastatic models of SCLC. This study showed that IL-18-mediated pro-inflammatory response improves the median survival of tumor-bearing mice, compared with treatment with DLL3-targeted CAR T cells alone. Additionally, Chen et al. described in vivo studies evaluating the efficacy of DLL3 targeting CAR T cells and a DLL3 bispecific antibody in xenograft models (13). Interestingly, the group showed that in combination with anti-PD1 checkpoint blockade, their DLL3 bispecific antibody showed increased tumor suppression activity and prolonged survival while DLL3 targeting CAR T cells did not show a significant benefit from the addition of anti-PD1 checkpoint blockade (13) (Table 2).

A recently published study by Zhang et al. (14) demonstrated the safety and efficacy of DLL3-targeted CAR T cells with gene editing in the T-cell receptor α constant locus to avoid graft versus host disease in preclinical models. The authors evaluated more than 50 clones of anti-DLL3 in CAR T cells to later incorporate a rituximab-base, off-switch approach to control the activation of CAR T cells in the case of adverse activity.

Using a subcutaneous tumor model, Zhang et al. showed that three CAR T-cell clones (Clone 4, 7, and 9) successfully controlled tumor growth. Although subcutaneous models are useful for proof-of-principle studies, the limitation of subcutaneous models is that the models do not faithfully mimic clinical disease. In contrast, results from clinically relevant models harboring disease at multiple sites could be more relevant, especially in terms of CAR T-cell efficacy in solid tumor models (15,16). The authors investigated the efficacy of DLL3-targeted CAR T cells in a systemic tumor model established by SCLC cell lines treated via tail vein to mimic a metastatic-stage disease commonly seen in the clinical setting. Even though the same three clones of CAR T cells were able to delay tumor growth, only one clone of CAR T cells was associated with tumor-free survival (Clone 4-RSR).

The authors’ emphasis on evaluating the safety of DLL3-targeted CAR T cells is commendable. Zhang et al. assessed the ability of the soluble binding domains from the three CAR T-cell clones against 36 normal human tissues and found that Clone 7 showed cytoplasmatic binding in pituitary, skin, and ovary tissues, whereas Clone 4 showed binding in the pancreas and placenta. A subsequent study was performed to clarify possible inconsistencies in the tissue cross-reactivity assays. Orthogonal assays revealed that only Clone 7 showed no off-target binding in an array of more than 5,000 membrane proteins. These findings led the authors to investigate the toxicity of DLL3-targeted CAR T cells in an in vivo model, in which tumor-bearing mice treated with CAR T cells showed infiltration into the pituitary gland; however, the hormone-secretion function was not affected. By evaluating the toxicity of human DLL3-targeted CAR T cells in mouse tissue, the authors provided information about the cross-reactivity of scFv-DLL3 antibodies in patients and mice; however, the homology of the epitopes in both species is unknown. These results show that the affinity of scFv-DLL3 antibodies is not the same in all clones, and the in vivo results show that human DLL3-CAR T cells can kill tumor cells with high antigen expression of murine DLL3; but, as the authors mentioned, the pituitary gland does not have the same antigen density. It is plausible that human DLL3-targeted CAR T cells may not be able to kill cells expressing a low density of murine DLL3 as efficiently as they would cells expressing a high density of DLL3.

Along with the translationally relevant strategy to develop effective DLL3-targeted CAR T-cell constructs, Zhang et al. provided important progress in presenting knowledge about safety and efficacy that should be considered before proceeding to a clinical trial.

In summary, CAR therapies that target DLL3 have been shown to be effective in vitro and in vivo in flank tumors and orthotopic and metastatic models of SCLC. These therapies have also been shown to be safe. Taken together, these findings support the use of DLL3-targeted CAR T-cell therapies to treat patients with extensive-stage SCLC in a clinical setting.

Acknowledgments

Funding: P.S.A.’s laboratory work is supported by grants from the National Institutes of Health (grant numbers P30 CA008748, R01 CA236615-01, R01 CA235667, and U01 CA214195); the U.S. Department of Defense (grant numbers CA170630, CA180889, and CA200437); the Baker Street Foundation; the Batishwa Fellowship; the Comedy versus Cancer Foundation, the Cycle for Survival Fund, the DallePezze Foundation; the Derfner Foundation; the Esophageal Cancer Education Fund; the Geoffrey Beene Foundation; the Memorial Sloan Kettering Technology Development Fund; the Miner Fund for Mesothelioma Research; the Mr. William H. Goodwin and Alice Goodwin, the Commonwealth Foundation for Cancer Research; and the Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center. P.S.A.’s laboratory received research support from ATARA Biotherapeutics and Novocure Inc.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Lung Cancer Research. The article has undergone external peer review.

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-820/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-23-820/coif). P.S.A. declares research funding from ATARA Biotherapeutics; scientific advisory board member and consultant for ATARA Biotherapeutics, Bayer, BioArdis, Carisma Therapeutics, Imugene, ImmPactBio, Orion pharma, and Outpace Bio; Patents, royalties, and intellectual property on MSLN-targeted CAR and other T-cell therapies licensed to ATARA Biotherapeutics, issued patent method for detection of cancer cells using virus, and pending patent applications on PD-1 dominant negative receptor, wireless pulse-oximetry device, and an ex vivo malignant pleural effusion culture system. Memorial Sloan Kettering Cancer Center has licensed intellectual property related to MSLN-targeted CARs and T-cell therapies to ATARA Biotherapeutics and has associated financial interests. P.S.A. also serves as an unpaid editorial board member of Translational Lung Cancer Research from September 2023 to August 2025. 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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