The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in millions of cases and deaths globally. Current treatments, including remdesivir and dexamethasone, have limitations in efficacy. While cellular immunotherapies hold promise, none have yet been approved for COVID-19. Natural killer (NK) cells, innate immune lymphocytes, rapidly lyse infected cells. Chimeric antigen receptors (CARs) enhance NK cell targeting abilities, making them attractive candidates for viral infection treatments. Umbilical cord blood (UCB) is a readily available source of NK cells. A clinical trial using intraoperative IL-15 to augment CAR NK cells targeting CD19 showed promising results in treating lymphoid malignancies. SARS-CoV-2's spike protein binds to the host cell receptor ACE2, mediating viral entry. The S1 subunit mediates receptor binding, and neutralizing antibodies targeting its receptor-binding domain inhibit infection. This study investigates engineering NK cells to express ACE2, enabling them to target SARS-CoV-2 infected cells. The researchers hypothesized that engineering NK cells to express a mutant ACE2 (mACE2) along with soluble IL-15 (sIL15) would create effective CAR NK cells against SARS-CoV-2.

The literature review section details existing treatments for COVID-19, highlighting the limitations of remdesivir and dexamethasone. It also discusses the potential of NK cell-based immunotherapies, citing previous research on CAR NK cells and the use of IL-15 to enhance their efficacy. The review focuses on the mechanism of SARS-CoV-2 entry into host cells via the ACE2 receptor and the spike protein, specifically the S1 subunit. The potential of targeting the spike protein with engineered NK cells is presented as a novel therapeutic strategy.

The researchers isolated NK cells from UCB and engineered them to express both mACE2-CAR and sIL15 using a two-vector retroviral transduction approach. One vector expressed mACE2-CAR and truncated LNGFR (a marker for CAR expression), while the other expressed sIL15 and truncated EGFR (a marker for sIL15 expression). The transduction efficiency was evaluated by measuring LNGFR and EGFR expression via flow cytometry. The phenotype of the mACE2-CAR_sIL15 NK cells was characterized, comparing it to control NK cells transduced with only EGFR or sIL15. The ability of mACE2-CAR_sIL15 NK cells to bind to the SARS-CoV-2 spike protein was assessed using flow cytometry, employing both a recombinant His-tagged spike S1 protein and VSV-SARS-CoV-2 chimeric viral particles. Cytotoxicity assays, using A549 cells expressing the spike protein (A549-spike), were conducted to evaluate the efficacy of mACE2-CAR_sIL15 NK cells in killing infected cells. These assays included real-time cell analysis (RTCA) for long-term cytotoxicity assessment and measurements of NK cell degranulation, TNF-α, and IFN-γ production. IL-15 release was also measured. To assess the clinical potential, the researchers evaluated the stability and efficacy of cryopreserved mACE2-CAR_sIL15 NK cells. In vivo studies were performed using two mouse models: a firefly luciferase (FFLuc)-labeled A549-xenograft model in NSG mice to assess anti-spike activity and a K18-hACE2 transgenic mouse model to evaluate protection against live SARS-CoV-2 infection. In the K18-hACE2 model, endogenous immune cells were depleted before infection with SARS-CoV-2 and treatment with mACE2-CAR_sIL15 NK cells. Viral load was measured via quantitative RT-PCR, and cytokine levels were assessed using a cytokine release Luminex assay. Statistical analysis was performed using appropriate methods for different data types.

The study successfully generated mACE2-CAR_sIL15 NK cells, demonstrating high co-expression of mACE2-CAR and sIL15. These engineered NK cells effectively bound to both recombinant SARS-CoV-2 spike S1 protein and VSV-SARS-CoV-2 chimeric viral particles. In vitro cytotoxicity assays showed significantly enhanced cytotoxicity of mACE2-CAR_sIL15 NK cells against A549-spike cells compared to control NK cells. Increased production of TNF-α and IFN-γ was observed in mACE2-CAR_sIL15 NK cells upon interaction with A549-spike cells. Cryopreserved mACE2-CAR_sIL15 NK cells retained their anti-spike activity. In vivo studies using the FFLuc-labeled A549-xenograft model demonstrated improved tumor control with mACE2-CAR_sIL15 NK cell treatment. The K18-hACE2 transgenic mouse model showed that mACE2-CAR_sIL15 NK cell treatment significantly improved survival, reduced viral load in brain and lung tissues, and did not induce substantial cytokine toxicity.

The findings support the potential of engineered NK cells as an effective therapy against SARS-CoV-2 infection. The inclusion of sIL15 was crucial for NK cell persistence and enhanced antiviral activity. The use of a two-vector approach improved transduction efficiency, though it might be more expensive to manufacture. The efficacy of cryopreserved cells makes it suitable for immediate clinical use. The study's results align with previous work on NK cell clearance of viral infections. The availability of UCB-derived NK cells offers a readily accessible and scalable cell source for this therapy. The data suggest the potential for this approach to target other coronaviruses using the spike protein for cell entry. The absence of significant cytokine storm induction in the mouse model suggests a favorable safety profile.

This study successfully developed and characterized mACE2-CAR_sIL15 NK cells as a promising off-the-shelf therapy for COVID-19. The efficacy and safety data in preclinical models warrant further investigation in clinical trials for critically ill COVID-19 patients, particularly those with limited treatment options. Future research could focus on optimizing the CAR design, exploring different IL-15 delivery strategies, and evaluating the efficacy against emerging SARS-CoV-2 variants.

The study was conducted using mouse models, and the results may not fully translate to humans. The number of mice in some experiments was relatively small. The long-term effects of mACE2-CAR_sIL15 NK cell therapy were not fully assessed. The study did not address the potential for immune escape or the emergence of resistant SARS-CoV-2 variants.