Allogeneic induced pluripotent stem cell-derived invariant natural killer T cell therapy: A phase 1 clinical Trial

Abstract Introduction: The invariant Natural Killer T (iNKT) cell is a T cell subset characterized by the expression of invariant T cell receptors (TCRs) composed of Vα24-Jα18 and Vβ11 chains in humans. Unlike other T cell subsets, iNKT cells recognize glycolipids, such as α-galactosylceramide (αGalCer), presented by CD1d, a monomorphic MHC class I-like antigen-presenting molecule that is expressed on dendritic cells (DCs) and other cell types. iNKT cells have been shown to exert anti-tumor activity by activating several anti-tumor effector pathways in mice via αGalCer-pulsed autologous DCs (αGalCer/DCs). Therefore, iNKT cells are expected to play a pivotal role in initiating anti-tumor immunity through their adjuvant activity. They have been recognized as an important target for anti-cancer immunotherapy. Our group and others have performed clinical trials to activate intrinsic iNKT cells by αGalCer/DCs based on proof-of-concept results obtained in mice. These studies highlighted the anti-tumor clinical potential of iNKT cells but also revealed that this potential was dampened by the limited responses of patient-intrinsic iNKT cells to αGalCer/DCs. We therefore regenerated iNKT cells from induced pluripotent stem cells (iPSCs) to overcome this issue. Subsequently, we conducted a phase 1, first-in-human clinical trial to evaluate the safety and efficacy of clinical-grade allogeneic iNKT cells generated from induced pluripotent stem cells (iPSC-iNKT cells) in patients with recurrent head and neck cancer (jRCT2033200116). Methods: This clinical trial was a non-blinded, single-arm Phase I trial targeting patients with recurrent or advanced head and neck squamous cell carcinoma who were unresponsive or intolerant to standard treatments. The trial aimed to investigate the tolerability, safety, pharmacokinetics, pharmacodynamics, anti-tumor effects, and the adjuvant activity of iPSC-iNKT cells. The dosing regimen involved tumor arterial infusion of iPSC-iNKT cells at one of three dose levels: dose level 1 (start dose: 3 × 10⁷ cells/m²), level 2 (1 × 10⁸ cells/m²), or level -1 (1 × 10⁷ cells/m²). These doses were administered at two-week intervals per cycle up to three cycles. Eligible patients were those with recurrent or advanced head and neck squamous cell carcinoma who were refractory or intolerant to standard treatment and had evaluable lesions that could be treated with intra-arterial infusion into the tumor. Patients must not have received any previous therapy for at least 1 month. Any type of prior treatment was acceptable. This clinical trial adhered to the “Declaration of Helsinki”, Pharmaceuticals and Medical Devices Act, and the J-GCP for regenerative medicine, approved by the institutional review board of Chiba University Hospital. To evaluate the iNKT cell adjuvant activity, we performed flow cytometry and single-cell RNA and TCR sequencing analyses with patients' peripheral blood mononuclear cells. Results: iPSC-iNKT cells were administered intra-arterially to 10 patients. At the second dose, one subject developed a grade 3 skin rash, identified as a dose-limiting toxicity. No other severe adverse events, such as cytokine release syndrome, were observed in patients who received at least one injection of iPSC-iNKT cells. Tumor progression was suppressed in two patients. Although no overall changes in the numbers of T cell subsets or NK cells were observed, PD-1 expression significantly increased in both CD4⁺ and CD8⁺ T cells and partially in NK cells according to flow cytometry, peaking at Day 8. In three out of 10 enrolled patients for whom samples were available before administration and 8 and 21 days after administration, peripheral blood T cells and NK cells were submitted for single-cell analysis. Two of them were patients whose tumor growth was suppressed. In all three patients' NK cells and T cells, the IFN-γ pathway was activated after iPSC-iNKT cell administration. In the two effective patients, clonal expansion of CD8 T cells was identified. Conclusions: We have presented safety evidence for iPSC-derived iNKT cells, which could form the foundation for future developments involving various iPSC-derived immune cells. Furthermore, we confirmed the unique immunological function of iPSC-iNKT cells, which activate NK cells and increase anti-cancer T cells. Additionally, we have overcome the manufacturing barrier for iNKT cells, which will accelerate the development of iNKT-cell-based immunotherapy.