title: Targeting non-small cell lung cancer with chimeric antigen receptor-engineered NK cells
Creators: name: Ben-Horin, Idan
Publisher: name: UNSPECIFIED
Year: 2025

description: Immunotherapy has emerged as a transformative approach in the treatment of cancer, recruiting the body's immune system to target and eliminate malignant cells. However, the heterogeneity of solid tumors presents a significant challenge to the efficacy of these therapies. Tumor heterogeneity refers to the genetic, phenotypic, and microenvironmental variations within and between tumors, which can affect responses to immunotherapeutic agents. This complexity necessitates a personalized approach, as the immune landscape of each tumor influences the effectiveness of immune checkpoint inhibitors, chimeric antigen receptor (CAR)-based cell therapies, and other immunomodulatory strategies. Addressing tumor heterogeneity is crucial for optimizing immunotherapy outcomes, requiring profiling of tumor characteristics. Tumors can also evolve under selective pressures imposed by the immune system and therapeutic interventions, leading to the emergence of resistant clones. 
In previous work by our group, an ErbB2 (HER2)-specific CAR was generated and transduced into clinically usable NK-92 cells (NK-92/5.28.z). The NK-92 cells carrying this second-generation CAR with CD28 and CD3ζ signaling domains demonstrated increased anti-tumor activity against various malignancies in preclinical studies. In addition, it was shown in immunocompetent mouse models that ErbB2-specific NK-92 cells can induce an endogenous immune response against tumor cells. These results led to the initiation of a clinical trial (CAR2Brain) in which the safety and efficacy of this cellular immunotherapy in patients with recurrent ErbB2-positive glioblastoma are currently being investigated. Since ErbB2 also plays a significant role in other tumor entities, including non-small cell lung cancer (NSCLC), an important goal of this work was to investigate the antitumor activity of CAR-NK cells against ErbB2-positive lung cancer cells. These investigations are a prerequisite for the potential future clinical use of NK-92/5.28.z cells for the treatment of patients with lung cancer. In flow cytometry-based and luciferase-based assays, ErbB2-specific NK-92/5.28.z cells indeed displayed high and specific cytotoxicity against both, the human lung cancer cell lines A549 and Calu-3 that naturally express elevated ErbB2 levels, and the ErbB2-transduced murine lung cancer cell line LC6/ErbB2. 
As tumor heterogeneity poses a barrier to antigen-based therapy, another important goal of this work was to examine the use of a dual-CAR approach for the treatment of solid malignancies. This was investigated using two different strategies. Firstly, PD-L1-specific CARs were designed and transduced into NKL, NK-92 and NK-92/5.28.z cells. PD-L1 was chosen as an appropriate target as it serves as an important mechanism in tumor immune evasion. Moreover, PD-L1 directed therapies, notably immune checkpoint inhibitors, have been shown to have a significant effect on the clinical outcome of numerous malignancies, including non-small cell lung cancer. In a second approach, an NKG2D-based CAR (NKAR), previously developed in our laboratory, was transduced as a second receptor into ErbB2-specific NK-92/5.28.z CAR-NK cells. NKG2D-ligands (NKG2D-L), encompassing the MIC and ULBP protein families, are upregulated on transformed cells and play an important role in tumor immune surveillance. Due to their almost complete absence from healthy cells, they are regarded as a potential universal target for antigen-based therapy. Dual-CAR NK cells co-expressing the ErbB2-specific CAR together with a PD-L1-specific (NK-92/5.28.z/PD-L1) or the NKG2D-L-specific NKAR molecule (NKAR-NK-92/5.28.z) had a similar viability and proliferation rate as NK-92/5.28.z cells. Likewise, the natural cytotoxicity of dual-CAR cells, as measured against K562 cells, was not different from that of single-CAR NK cells. This is important, since intrinsic natural cytotoxicity can contribute to the overall response of CAR-NK cells against malignant cells. 
Detailed analysis of PD-L1 as a target antigen in this study showed that unlike the constitutive expression of ErbB2, the expression of PD-L1 is induced by NK-92 cells, and to a larger extent by activated NK-92 cells. As results of transwell assays show, this induction does not require physical contact between effector and target cells, but is mediated by cytokines such as IFN-γ. This underscores the importance of PD-L1 as a target for CAR therapy. As neighboring NK-92 cells induce its expression, cancer cells that initially do not express PD-L1 or are low expressors upregulate it on their cell surface, thus exposing themselves as targets of PD-L1-CAR expressing NK cells.
Cytotoxicity assays with lentivirally transduced NK cells demonstrated the activity of PD-L1-CARs when expressed as a single-CAR, and their contribution to the lysis of dual-target tumor cells when expressed concurrently with the ErbB2-specific CAR. Thereby, NKL, parental NK-92 and NK-92/5.28 cells expressing either a first-generation (PD-L1.z) or a second-generation (PD-L1.28.z) CAR specifically and efficiently lysed PD-L1-positive target cells. However, expression of a PD-L1.TM CAR, which lacks intracellular signaling domains, only activated NK-92 and NK-92/5.28.z cells against PD-L1-positive targets, but not NKL cells. Though the reason for this difference was not investigated further, it appears likely that the CD28 transmembrane domain still present in the PD-L1.TM CAR could mediate dimerization with endogenous CD28 that is only expressed in NK-92 cells but not in NKL cells, thereby acting as a target-specific costimulatory receptor. When dual-CAR NK-92 cells co-expressing ErbB2- and PD-L1-specific CARs were tested, each CAR was shown to work independently, thus serving as a possible solution to heterogeneous antigen presentation and to tumor immune evasion through antigen downregulation. Moreover, in the case of lung cancer cells, simultaneous targeting of PD-L1 and ErbB2 led to a higher specific lysis than achieved with NK-92/5.28.z cells that only recognize ErbB2. Accordingly, in degranulation assays dual-CAR NK-92 cells displayed a higher level of CD107a expression after incubation with ErbB2 and PD-L1 double-positive A549 cells compared to parental A549 cell expressing only ErbB2. When a mixed population of target cells, expressing one or both target antigens, was co-cultured with either dual-CAR NK-92 cells or a combination of two single-CAR (ErbB2- or PD-L1-specific) NK-92 variants, no difference in specific lysis was found, indicating that in this case both approaches for dual-targeting were effective. 
NKAR-NK-92/5.28.z cells, generated in this work by lentiviral transduction of ErbB2-specific NK-92/5.28.z cells with a vector encoding an NKG2D-L-specific NKG2D.z CAR (NKAR), were tested in similar experiments. Unlike parental NK-92/5.28.z cells, the dual-CAR NK cells mediated effective lysis of MDA-MB468 cells, a human breast cancer cell line that is ErbB2-negative but NKG2D-L-positive. NKAR-expressing cells were further tested in combination with bispecific antibodies (NKAB molecules) that recognize NKG2D via a single-chain fragment variable (scFv) domain derived from NKG2D-specific antibody Kyk2.0. For tumor targeting, these NKAB molecules contain a second scFv domain either derived from the clinically used antibody cetuximab which recognizes the tumor-associated antigen EGFR (NKAB-EGFR), or atezolizumab which binds to PD-L1 (NKAB-PDL1). As a model, murine glioblastoma cells (GL261) were previously modified by our group to ectopically express either EGFR (GL261/EGFR), ErbB2 and EGFR (GL261/ErbB2/EGFR), or PD-L1 (GL261/PD-L1). As GL261 is a murine cell line, the human NKAR does not recognize murine NKG2D-Ls it might express, ensuring that any response is NKAB-induced. NKAB-EGFR successfully mediated the lysis of EGFR-positive GL261 cells by NKAR-expressing NK-92 cells, while not contributing significantly to the lysis of double-positive GL261/ErbB2/EGFR cells by NKAR-NK-92/5.28.z cells. However, when ErbB2-mediated activity was inhibited with a blocking antibody, imitating downregulation of ErbB2 on the target cells, NKAB-EGFR-mediated activation of the NKAR compensated in dual-CAR NK cells for the loss of ErbB2-CAR activity. Results from these assays also showed that as in the case of PD-L1-CARs, NKAR and ErbB2-CAR activities are independent of each other. Similar experiments conducted with A549 and Calu-3 lung cancer cells revealed that for these targets co-incubation with NKAR-NK-92/5.28.z cells and NKAB-EGFR did not lead to improved cytotoxicity. This may be attributed to the relatively low expression of EGFR by these cell lines, since NKAB-EGFR did also not restore cytotoxicity when ErbB2-mediated killing was blocked. 
The ability of NKAB-PDL1 to activate NK-92 cell via the NKAR receptor was tested in a similar fashion, demonstrating its ability to mediate cytotoxicity against PD-L1-positive target cells. When co-cultured with MDA-MB468/PD-L1 cells, it further enhanced the cytotoxicity mediated by the NKAR alone through interaction with NKG2D-Ls. Importantly, the presence of NKAB-PDL1 did not inhibit lysis of NKG2D-L-positive but PD-L1-negative target cells by NKAR-expressing NK cells. As opposed to the results obtained with NKAB-EGFR, the addition of NKAB-PDL1 fully restored specific lysis of ErbB2 and PD-L1 double-positive A549 and Calu-3 cells when ErbB2-mediated killing was inhibited, which can be attributed to the much higher level of PD-L1 in the target cells when compared to EGFR. 
Subsequently, both NKAB molecules were tested in combination using GL261/EGFR and GL261/PD-L1 cells as targets. Firstly, each target cell line was co-incubated with NKAR-NK-92/5.28.z cells with either NKAB-EGFR or NKAB-PDL1, or both molecules. Thereby, only the relevant NKAB molecule mediated specific lysis. When these assays were repeated with a mixed population of GL261/EGFR and GL261/PD-L1 cells, the use of both NKAB molecules significantly increased cell killing compared to the addition of either NKAB alone, but with each NKAB molecule only mediating lysis of the respective target cell population in the mixture. 
The data obtained in this work contribute to our general understanding of the function of single-CAR and dual-CAR NK cells, and may support the development of novel cellular immunotherapies for lung cancer. As a next step, the specific activity of ErbB2-CAR NK cells against ErbB2-positive lung cancer cells shown in this study in in vitro models should be further investigated in a suitable mouse tumor model in vivo. Furthermore, as demonstrated in this work, dual-CAR NK cells either targeting ErbB2 and PD-L1, or ErbB2 and a second antigen through the NKAB-NKAR system, mediate enhanced cell lysis against double-positive target cells, but with either CAR also working independently against target cells that express only one antigen. This demonstrates a therapeutic benefit for dual-CAR use, which could be employed to address the clinical problem of tumor cell heterogeneity with rationally designed bi-functional CAR effector cells.