Lung cancer is one of the most common and deadly cancer types globally (1). Lung cancer is a highly complex, heterogeneous disease with a poor prognosis. The poor survival rate of patients with lung cancer (5-year survival rate: 10%–20%) is a consequence of advanced stage at presentation (2, 3). Histologically, lung cancer is classified as non-small cell lung carcinoma (NSCLC, approximately 85% of cases) or small cell lung carcinoma (approximately 15% of cases). NSCLC, causing a major proportion of lung cancer-related deaths, is classified as adenocarcinoma, squamous cell carcinoma, or large cell carcinoma (4). Furthermore, genomic profiling studies have uncovered driver mutations in lung cancer that support tumor growth and proliferation. The most frequently found driver mutations in lung cancer are Kirsten rat sarcoma viral (KRAS) oncogene homolog and epidermal growth factor receptor (EGFR) mutations (5).

The present main treatment strategies for lung cancer include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy (6, 7). Although lung cancer is curable when diagnosed at an early stage, it even then remains a challenge due to relapse, and poor survival in >70% of patients (8). Over the past two decades, cytotoxic chemotherapies used to treat lung cancer have evolved to platinum-based chemotherapy, cisplatin-based combination therapies, neoadjuvant therapy, and adjuvant therapy (9). In addition, targeted therapies have also been developed to treat patients with lung cancer harboring EGFR or anaplastic lymphoma kinase mutations (10). Recently, immunotherapy has complemented this arsenal with the discovery and targeting of immune checkpoint inhibitors such as anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and anti-programmed cell death-1 (PD-1) therapies (7). Despite the development of various therapeutic regimens for lung cancer, such therapies only provide durable responses and efficacy in a subset of patients. Variable responses observed under treatment in different tumors might be attributable to disease heterogeneity or tumor heterogeneity across patients. Therefore, it is necessary to explore novel therapies to improve clinical outcomes for more patients. In this setting, next-generation immunotherapeutics, such as immunomodulators and adoptive T-cell therapies including classical T-cell receptor (TCR) and chimeric antigen receptor (CAR)-T-cell therapies, bear promise for treating cancers including lung cancer (11–13).

CAR-T cell therapy has emerged as an innovative cancer immunotherapy for lung cancer treatment (13–16). Although CAR-T cell therapy produced remarkable clinical responses in hematological malignancies (17), this therapy has displayed limited anti-tumor activity in solid tumors including lung cancer. Despite targeting a variety of antigens and tumor types, clinical data for CAR-T cell therapy in solid tumors are disappointing (18). While CAR-T cell therapy has shown clinical success in hematological malignancies, severe toxicities such as cytokine release syndrome (CRS), neurotoxicity, on-target/off-tumor toxicity, tumor lysis syndrome (TLS), and anaphylaxis have also been reported in CAR-T therapy (19). Also, some concerns must be addressed including limited efficacy of CAR-T cell therapies in solid tumors, limited persistence, antigen escape, CAR-T cell trafficking, tumor infiltration, and the immunosuppressive microenvironment (20, 21). Recently, several studies proposed strategies to ameliorate efficacy of CAR-T cell therapy and limit its toxicities (22–24). In this review, we focus on CAR-T cell design, present existing preclinical and clinical studies in lung cancer treatment; highlight existing challenges and toxicities; and also discussed strategies to improve the therapeutic efficacy of CAR-T cells in solid tumors.

T cells genetically engineered to carry synthetic CAR bind specifically targeted tumor antigens and kill these targeted tumor cells. CAR are synthetic receptors composed of an antigen-binding domain/hinge motif, transmembrane domain, and intracellular signaling domain. The extracellular antigen-binding domain, composed of a single-chain variable fragment (scFv), recognizes targeted tumor-associated antigens (TAAs) and triggers downstream signaling. The hinge/spacer region provides flexibility to allow the antigen-binding domain to access the targeted antigen. The hinge/spacer region can be adjusted to its optimal length to provide a sufficient distance between CAR-T cells and targeted tumor cells. The transmembrane domain facilitates the distribution of CARs to the T cell membrane, influencing CAR expression, function, and stability. The intracellular domain or endodomain is composed of combinations of signaling domains such as the T-cell activation complex transducer CD3ζ and several costimulatory molecules (25) ( Figures 1A, B ). The design and structure of CAR have been extensively reviewed elsewhere (26, 27).

To improve the efficacy and safety of CAR-T cell therapy, CAR-T cells have undergone several progressive changes by modifying the CAR structure based on its intracellular signaling domains ( Figure 1C ). The first generation of CAR, containing the antigen recognition extracellular scFv and CD3ζ signaling endodomain, displayed less efficient T cell activation and a short survival time in vivo (28–30). To improve the persistence and efficacy of CAR-T cells, second-generation CARs contain an additional costimulatory molecule (e.g., CD28, 41BB, ICOS) that enhances T cell proliferation, prolongs T cell survival time, and improves clinical outcomes (31–33). The design of third generation of CAR included CD3ζ and two costimulatory molecules that further enhance CAR-T cell function. The most commonly used third generation costimulatory molecules are CD27, CD28, 41BB, ICOS, and OX-40 (34, 35). The design of fourth-generation CAR-T cells introduced T cells redirected for universal cytokine-mediated killing containing nuclear factor of activated T cells. These fourth-generation CAR-T cells can produce pro-inflammatory cytokines (interleukin [IL]-12, IL-13, and GM-CSF) upon activation and enhance the penetration ability of T cells to overcome the immunosuppressive effect of the hostile tumor microenvironment (TME) (36). The fifth generation of CAR includes an IL-2Rβ fragment that induces JAK production and activates signal transducer and activator of transcription 3/5 (37).

CAR-T cell therapy is an individualized cell-based therapy that involves the modification of a patient’s own T cells to express CAR. The generation of CAR-T cells involves a complex engineering process featuring several steps starting with the collection of T cells from the patients, engineering cells to express tumor-specific antigen-targeted CAR on their surface, CAR-T cell expansion, and purification, and the infusion of CAR-T cells back into the patient with therapeutic intention ( Figure 2 ).

CAR-T cell adaptive cancer immunotherapy has emerged as a promising strategy for the treatment of solid tumors including lung cancer. Synthetic CAR-T cells are independent of major histocompatibility (MHC) complex targeted for TAAs on cancer cells to establish tumor immunity. Similarly, for successful CAR-T therapy in solid tumors, it is important to identify specific TAAs that are highly and selectively expressed in solid tumors but weakly expressed or absent in normal tissue. Several TAAs have been proposed in CAR-T cell research in solid tumors including lung cancer. TAAs currently being investigated in clinical trials of CAR-T cells include carcinoembryonic antigen (CEA), EGFR, human epidermal growth factor receptor 2 (HER2), mesothelin (MSLN), prostate stem cell antigen (PSCA), mucin 1 (MUC1), tyrosine kinase-like orphan receptor 1 (ROR1), programmed death ligand 1 (PD-L1), and CD80/CD86 ( Table 1 ).

CEA is a fetal antigen that is expressed during fetal development but is minimally expressed or absent in adult tissues. CEA is overexpressed in various cancers, including 70% of NSCLC (38). Therefore, CEA has proven useful as a tumor marker and for monitoring the response to CEA-targeted CAR-T therapy. Furthermore, preclinical studies also showed the relevance of serum CEA concentrations as an indicator of brain metastases in patients with advanced NSCLC (39). This led to the establishment of CEA-targeted CAR-T cells in phase I clinical trials to evaluate the efficacy, safety, and maximum tolerated dose of this therapy in various solid tumors including lung cancer (NCT02349724, NCT04348643). In addition, in vivo established human lung cancer model in immune-compromised mice showed treatment with inducible IL8 (iIL8) and CEA-targeted CAR-T cells completely eliminated advanced stage of lung cancer (40).

EGFR, expressed in both epithelial cells and epithelium-derived malignancies, is a transmembrane glycoprotein belonging to the tyrosine kinase receptor family. In addition to EGFR overexpression in solid tumors including NSCLC, it has also been reported that more than 60% of EGFR mutations are associated in NSCLC patients (41). Therefore, EGFR has become a possible therapeutic target in CAR-T cell therapy for NSCLC. In vitro studies revealed that EGFR-targeted CAR-T cells exhibit specific cytolytic activity and produce high levels of cytokine (IL-2, IL-4, IL-10, TNF-α, and interferon-γ [IFN-γ]) against EGFR-positive tumor cells (42). There are two ongoing phase I clinical trials in lung cancer of C-X-C chemokine receptor type 5 modified EGFR-targeted CAR-T cells (NCT04153799, NCT05060796). Furthermore, phase I/II clinical studies in patients with advanced NSCLC revealed no severe toxicity after 3–5 days of EGFR-targeted CAR-T cell perfusion (NCT01869166). These studies indicate the promise of EGFR-targeted CAR-T cells in treating NSCLC.

HER2, a member of the tyrosine kinase erythroblastic leukemia viral oncogene homolog (ERBB) family, is also a potential CAR target antigen in lung cancer (43). Studies using an in vivo A549 NSCLC xenograft model and in vitro NSCLC cell lines (A549 and H1650) revealed anti-tumor effects of HER2-targeted CAR-T cells, including decreased tumor growth but not complete tumor elimination (44, 45). In addition, two phase I/II clinical studies of HER2-targeted CAR-T cells in treating NSCLC have been launched (NCT01935843, NCT02713984). However, clinical data have not yet been reported for HER2-targeted CAR-T cell therapy in NSCLC.

MSLN, a cell surfaced glycoprotein, is overexpressed in the majority of cancer types including lung cancer, mesothelioma, pancreatic cancer, and ovarian cancer (46, 47). High expression of MSLN occurs in approximately 69% of lung adenocarcinomas, and it carries an increased risk of recurrence with reduced overall survival in NSCLC (48, 49); therefore, it could be a potential target in CAR-T cell therapy. This prompted the development of MSLN-targeted CAR-T cells, and research using in vivo subcutaneous mouse lung cancer models and ex vivo models revealed a slower tumor growth rate and inhibitory effects on cell proliferation (46, 50). However, phase I/II clinical trials of MSLN-targeted CAR-T cells in MSLN-positive metastatic lung cancer were terminated because of poor accrual (NCT01583686). Furthermore, the intravenous application of mRNA-engineered T-cells expressing MSLN-targeted CARs did not exert effects on metastatic tumors in patients with NSCLC (NCT01355965).

MUC1 is another potential candidate that is aberrantly overexpressed in NSCLC and other cancer types (51). MUC1 is an abnormally glycosylated extracellular transmembrane glycoprotein that is correlated with poor survival and tumor progression (52). Ongoing phase I clinical trial studies are examining Tn glycoform of MUC1-targeted CAR-T cells for the treatment in MUC-1 positive advanced cancers, including NSCLC (NCT04025216). Additionally, phase I/II clinical studies in various solid tumors including lung cancer using MUC-1–targeted CAR-T cells have been launched (NCT03525782, NCT02587689). Meanwhile, an early stage clinical trial is using P-MUC1-ALLO1–targeted CAR-T cells in solid tumors including lung cancer is ongoing (NCT05239143). In contrast, studies using MUC-1–targeted CAR-T cells in patient xenograft model did not reveal significant suppression of NSCLC tumor growth (51).

PSCA is a glycophosphatidylinositol-anchored cell surface protein that is aberrantly overexpressed in NSCLC (51). Using in vivo PDX subcutaneous mouse models and in vitro models, the combination of CAR-T cells targeting MUC-1 and PSCA substantially inhibited tumor growth and PSCA- and MUC-1–expressing NSCLC cell proliferation (51). Meanwhile, an ongoing phase I study is testing safety, efficacy, and tolerance of a combination of CAR-T cells targeting MUC-1 and PSCA in lung cancer (NCT03198052).

ROR1, a tyrosine kinase-like orphan receptor, is highly expressed in NSCLC, breast cancer, and other solid tumors (53, 54). Because of the toxicity of ROR1-targeted CAR-T cells attributable to ROR1 expression in normal tissues, CAR-T cells have been engineered with synthetic Notch receptors EpCAM and B7-H3 to improve selectivity, specificity, and tumor regression in ROR1-expressing tumor cells with less toxicity (55). A phase I clinical study was designed to assess the safety and anti-tumor effects of ROR1-targeted CAR-T cells in ROR-positive NSCLC (NCT02706392). In addition, animal models examining ROR1-targeted CAR-T cells revealed effective elimination of ROR1-positive NSCLC cells (53).

Treatments targeting the PD-1-PD-L1 complex, which blocks the cytotoxic T-cell activity, have made substantial progress in NSCLC and other cancer types (56, 57). In vitro and in vivo studies using PD-L1–targeted CAR-T cells revealed cytotoxic effects and tumor growth inhibition in NSCLC cells (58, 59). However, phase-1 clinical trials of PD-L1 targeted CAR-T cells in advanced lung cancer patients were terminated because of serious adverse effects (NCT03330834). Also, another phase I clinical trial has been ongoing with PD-L1-MSLN targeted CAR-T cells to determine safety and efficacy in PD-L1–positive NSCLC patients (NCT04489862).

The expression of CD80/CD86, costimulatory molecules of the immune system, has been detected in NSCLC (60). CD80 and CD86 bind to CTLA-4 and downregulate T-cell function, making them preferred targets for immune intervention (61). Phase I clinical trial study is ongoing to assess safety and tolerance of PD-L1 and CD80/CD86 targeting CAR-T cells in the treatment of recurrent or refractory NSCLC patients (NCT03060343). In addition, CD80/CD86-targeted CAR-T cell treatment controlled tumors including NSCLC tumors by reversing inhibitory CTLA-4–CD80/CD86 signals (62).

Fibroblast activator protein (FAP), highly expressed in cancer-associated fibroblasts (CAFs), can modulate the tumor microenvironment by ECM remodeling. FAP overexpression on CAFs is associated with poor prognosis in many solid tumors including lung cancer. Targeting FAP is also being evaluated for CAR-T cell therapy in NSCLC. In vitro studies in A549 cells using FAP targeted CAR-T cells showed significant reduction of tumor growth (63, 64). Furthermore, mouse model studies using FAP-targeted CAR-T cells showed 35-50% reduction of tumor growth after treatment (63, 64).

Preclinical CAR-T cell therapy studies in lung cancer by targeting several potential targets, like erythropoietin-producing hepatocellular carcinoma A2 (EphA2), lung-specific X protein (LUNX), variant domain 6 of CD44 gene (CD44V6), melanoma-associated antigen (MAGE)-A1, exhibited significant suppression of tumor growth (65–68). Furthermore, potential targets like MAGE-A1 (NCT03198052 and NCT03356808), AMT-253 (NCT05117138), CD276 [(B7-H3): NCT04864821, NCT05190185), and GPC3-transforming growth factor beta (TGF-β; NCT03198546), are under evaluation for CAR-T cell therapy application in NSCLC in clinical trials.

Although there has been continuous improvement of CAR-T cell therapies and their great promise in the treatment of lung cancer and other solid tumors has been revealed, many challenges and hurdles exist. T cell intrinsic as well as tumor-driven mechanisms and treatment-related toxicities in CAR-T cell limit efficacy and safety in solid tumors including lung cancer.

Despite the success of CAR-T cell therapy against hematologic malignancies, the effects of CAR-T cell therapies on solid tumors such as lung cancer are unsatisfactory because of antigen heterogeneity, an immunosuppressive microenvironment, and insufficient trafficking to tumor tissue. Furthermore, CAR-T therapy in the treatment of solid tumors may result in adverse cytotoxicity in healthy cells because of the presence of targeted TAA on healthy cells. Therefore, it is utmost important to develop strategies to improve safety and efficacy of CAR-T cell therapies in lung cancer and other solid tumors. To overcome these hurdles, several studies adapted genetic engineering approaches to modulate CAR-T cells to enhance their efficacy, functional activity in the immunosuppressive TME, and efficient infiltration into the tumor site.

Over the last decade, CAR-T cell therapy has revolutionized the treatment of hematological malignancies. The clinical application of CAR-T cell therapy and the identification of novel potential target antigens in lung cancer are the subjects of ongoing research. However, the successful use of CAR-T cell therapy against solid tumors including lung cancer is hampered by several hurdles including antigen targeting, tumor heterogeneity, the immunosuppressive TME, CAR-T cell trafficking, associated toxicities, and on-target-off-tumor effects. Several new strategies are being developed to overcome these obstacles and improve the efficacy and scope of CAR-T cell therapies to permit their more widespread use in cancer treatment. In summary, novel strategies of CAR-T cell design with reduced toxicity that efficiently direct CAR-T cells to tumors may provide a path for their safer and more effective use against different cancer types including lung cancer.

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

This work was supported by the Max Planck Society, Cardio-Pulmonary Institute (CPI), the German Center for Lung Research (DZL) and DFG, SFB 1213 (Project A10* to RS). This study also supported by the international doctoral program “i-Target: immunotargeting of cancer” (funded by the Elite Network of Bavaria), Melanoma Research Alliance (grant number 409510 to SK), Marie Sklodowaska-Curie Training Netwrok for Optimizing Adoptive T cell Therapy of Cancer (funded by the Horizon 2020 Program of the European Union: grant 955575), Else Kröner Fresenius-Stiftung, German Cancer Aid, Ernst Jung Stiftung, Institutional Strategy LMU excellent of LMU Munich (within the framework of the German Excellence Intiative), Bunderministerium für Bildung Und Forschung, European Research Council (Starting Grant 756017), Deutsche Forschungsgemeinschaft (DFG), by the SFB-TRR 338/1 2021-452881907, FritzBender Foundation, Jose´Carreras Foundation and Hector Foundation.

SK has received honoraria from TCR2 Inc, Novartis, BMS and GSK. SK is an inventor of several patents in the field of immune-oncology. SK received license fees from TCR2 Inc and Carina Biotech. SK received research support from TCR2 Inc. and Arcus Bioscience for work unrelated to the manuscript.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.