CAR-NK cell(Shanghai Tongji Hospital)

Background: Unlike T cells, natural killer (NK) cells lack a dominant activating receptor analogous to the T cell receptor (TCR) that governs their activation. Whether chimeric antigen receptor (CAR) constructs engineered specifically for T cells can effectively drive NK cell activation remains unresolved. NK cells inherently possess non-specific recognition capacities and exert broad-spectrum cytotoxicity against diverse tumor targets. However, the complexity of receptor-ligand interactions between CAR NK cells and susceptible target cells has impeded efforts to delineate the specific functional contributions of individual CAR constructs. Methods: CAR NK cells were generated via electroporation. The murine B16 melanoma cell line was modified to express various target proteins using lentiviral transduction. In vitro functional assays, including conjugate formation, granule polarization, degranulation, cytotoxicity, and cytokine production, were employed to assess CAR NK cell efficacy. Recombinant protein-coated beads were used to investigate downstream activation signaling pathways. The in vivo antitumor activity of CAR NK cells was evaluated using NPG mouse xenograft models. Results: B16 cell line was first validated to be a suitable model for specifically assessing CAR construct function in CAR NK cells. Among nine distinct CAR molecules generated, the construct incorporating the NKG2DTM-2B4-FCER1G exhibited the most potent capacity to enhance NK cell-mediated functionalities. Consistent with these functional improvements, this CAR construct induced robust phosphorylation of key activation pathways, including AKT, VAV1, ERK, PLCγ1, and NF-κB. Conclusions: The CAR construct incorporating the NKG2DTM-2B4-FCER1G is demonstrated to be the most effective in enhancing NK cell functionality.

Lately, autologous CD19-targeting chimeric antigen receptor (CAR) T cells have shown excellent efficacy in treatment of autoimmune diseases, but with great safety concerns, such as infections. In this study, we aimed to evaluate the safety, tolerability, and efficacy of allogeneic CD19 CAR natural killer (NK)-cell therapy in patients with relapsed or refractory systemic lupus erythematosus (SLE).

In this open-label, single-arm, prospective, first-in-human case series, we evaluated allogeneic CD19 CAR NK-cell therapy in adult patients (aged 18-65 years) with relapsed or refractory SLE at one site in China. Patients who had received at least two previous standard systemic therapies and continued to exhibit moderate-to-severe disease activity were eligible for inclusion. This study consisted of schedule escalation and dose escalation, with schedule escalation from 7 days and dose escalation commencing at 0·75 × 10⁹ CAR NK cells on day 0. All patients received a lymphodepleting conditioning regimen with fludarabine (25 mg/m² per day) and cyclophosphamide (300 mg/m² per day) administered daily from days -5 to -3, followed by three CAR NK-cell infusions within a single treatment cycle at identical dose levels and inter-infusion intervals. Dose-limiting adverse events were monitored in patients for 28 days. The primary endpoints of this study were safety and tolerability, including the incidence of dose-limiting toxicities and adverse events according to National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0. This study was registered with ClinicalTrials.gov (NCT06010472) and follow-up is ongoing.

18 patients with relapsed or refractory SLE with moderate-to-severe disease activity were enrolled between Aug 21, 2023, and June 16, 2024. Of the 18 patients, 17 (94%) were female; the median age was 37·5 years (IQR 32·0-39·8), and the median disease duration was 10·5 years (IQR 4·5-14·8). Patients had received at least two standard systemic therapies, including biological agents (belimumab and telitacicept) in 14 (78%) of 18 patients, and plasmapheresis in one patient. Cytokine release syndrome was reported in one (6%) of 18 patients (grade 1). Neurotoxicity and other CAR NK-cell therapy-related severe adverse events were not observed, and there were no dose-limiting toxicities. Of the nine patients with more than 12 months' follow-up, six (67%) attained DORIS remission and lupus low disease activity state.

This study suggests that allogeneic CAR NK-cell therapy is a potent option for treatment of autoimmune diseases and indicates that such a therapy might address limitations of current autologous CAR T-cell therapy, including manufacturing scale and time, access, safety, and cost.

Shanghai Municipal Health Commission, Changhai Hospital Affiliated to Naval Medical University, and National Natural Science Foundation of China.

For the Chinese translation of the abstract see Supplementary Materials section.