Alloreactive Vs IL15 Ex Vivo Stimulated Natural Killer Cells As Adoptive Cell Therapy in Haploidentical Transplantation for Pediatric Acute Leukemia: A Phase I/II Clinical Trial

Background

Natural Killer (NK) cells play a crucial role in the graft-versus-leukemia effect during haploidentical hematopoietic stem cell transplantation (Haplo-HSCT). Alloreactive NK cells are associated with lower relapse rates, improved engraftment, reduced graft-versus-host disease (GvHD), and decreased viral infection risk. However, not all Haplo-HSCT cases exhibit KIR-HLA incompatibility between donor and recipient. Ex vivo stimulation of NK cells with IL-15 could overcome inhibitory signals. This study aims to evaluate the safety and effectiveness of a single dose of alloreactive or ex vivo NK cells stimulated with IL-15 after Haplo-HSCT.

Methods

Eighteen pediatric patients with high-risk hematological malignancies, transplanted with a haploidentical donor, were enrolled in a prospective, multicenter phase I/II clinical trial. On day +7 after Haplo-HSCT, alloreactive NK cells (n=9) or non-alloreactive but ex vivo overnight IL-15-activated NK cells (n=9) were infused. The NK cell immunophenotype, K562 cytotoxicity, CD107a degranulation in the infused products, and the clinical outcomes, immune reconstitution, and cytokine profile in the patients were analyzed at different time points over the course of one year following Haplo-HSCT.

Results

Ex vivo stimulated L15-NK cells in the infusion product showed higher expression of activating receptors (NKp46, NKp44, NKG2D, CD69) and higher functional capacity than Allo-NK cells. No differences between arms were found in the NK cell phenotype and the functional capacity over time following Haplo-HSCT changes in pediatric patients. Seventeen patients successfully engrafted. The 1-year cumulative incidence (CI) of grade I-II acute GvHD was significantly higher in patients treated with IL-15 NK cells compared to Allo-NK cells (56% vs. 11%, p=0.04). However, similar CI of grade III-IV acute GvHD was observed between the two NK cell infusion groups. The 1-year CI of chronic GvHD was 35% (95% CI: 13%-57%). The 1-year overall survival (OS) and diseased‐free‐survival (DFS) were 77% (95% CI: 60%-99%), and GVHD-free relapse-free survival (GRFS) was 61% (95% CI: 42%-88%), without differences between both groups. The 1-year CI of relapse and transplant related mortality (TRM) for all patients were 5.6% (95% CI: 0.3%-23%) and 17% (95% CI: 3.9%-38%), respectively.

Regardless the type of NK infusion, patients presented rapid immune reconstitution. In the IL-15 NK arm, median CD3⁺ T-cell counts showed a higher trend (671 cells/μL) than in the Allo-NK arm (406 cells/μL) in the first month after Haplo-HSCT and persisted at subsequent time points. However, NK cells showed a trend towards higher expression levels in the Allo-NK arm at 1-month (median: 307 cells/μL) and 6-months post-transplant (median: 242 cells/μL) compared to the IL-15 NK arm (median: 181 cells/μL and median: 196 cells/μL, respectively).

A statistically significant increase in IL-2, IL-17A, and IL-13 at 1 month, as well as in IFN-γ, IL-6, IL-4, IL-9, IL-2, IL-10, IL-17A and IL-13 at 12 months after transplantation, was observed in plasma levels into IL-15 NK arm compared to the Allo-NK arm . In addition, we observed a correlation between higher CD69 expression in total NK cells and an increase in TRM and GvHD. Patients who expressed CD69 on NK cells below the established threshold of 21% at 6 months post-Haplo-HSCT exhibited statistically significant improvements in OS and GRFS, along with a trend towards improved DFS.

Conclusion

Infusion of ex vivo IL-15-stimulated NK cells after Haplo-HSCT was as safe and effective as alloreactive NK cells. T cell immune recovery was faster, and cytokine inflammatory responses were higher compared to alloreactive NK cells. In addition, a higher expression of CD69 on NK cells could be used as a biomarker for TRM.