Donor memory-like NK cells persist and induce remissions in pediatric patients with relapsed AML after transplant

Abstract

Pediatric and young adult (YA) patients with acute myeloid leukemia (AML) who relapse after allogeneic hematopoietic cell transplantation (HCT) have an extremely poor prognosis. Standard salvage chemotherapy and donor lymphocyte infusions (DLIs) have little curative potential. Previous studies showed that natural killer (NK) cells can be stimulated ex vivo with interleukin-12 (IL-12), -15, and -18 to generate memory-like (ML) NK cells with enhanced antileukemia responses. We treated 9 pediatric/YA patients with post-HCT relapsed AML with donor ML NK cells in a phase 1 trial. Patients received fludarabine, cytarabine, and filgrastim followed 2 weeks later by infusion of donor lymphocytes and ML NK cells from the original HCT donor. ML NK cells were successfully generated from haploidentical and matched-related and -unrelated donors. After infusion, donor-derived ML NK cells expanded and maintained an ML multidimensional mass cytometry phenotype for >3 months. Furthermore, ML NK cells exhibited persistent functional responses as evidenced by leukemia-triggered interferon-γ production. After DLI and ML NK cell adoptive transfer, 4 of 8 evaluable patients achieved complete remission at day 28. Two patients maintained a durable remission for >3 months, with 1 patient in remission for >2 years. No significant toxicity was experienced. This study demonstrates that, in a compatible post-HCT immune environment, donor ML NK cells robustly expand and persist with potent antileukemic activity in the absence of exogenous cytokines. ML NK cells in combination with DLI present a novel immunotherapy platform for AML that has relapsed after allogeneic HCT. This trial was registered at https://ichgcp.net/clinical-trials-registry/NCT03068819" title="See in ClinicalTrials.gov">#NCT03068819.

© 2022 by The American Society of Hematology.

Figures

Graphical abstract

Figure 1.

Treatment schema for clinical trial combining salvage chemotherapy, DLI, and ML NK cell infusion. Patients provided consent and were enrolled in clinical trial NCT03068819. Salvage chemotherapy with fludarabine, cytarabine, and granulocyte colony stimulating factor was administered 2 to 4 weeks before ML NK cell infusion. On day −1, a nonmobilized leukapheresis product was collected from the same donor as HCT. T-cell DLI (1 × 106 T cells per kg) was immediately infused into the patient. The remainder of the apheresis product was enriched for NK cells, which were then stimulated overnight with IL-12, L-15, and -18 to generate ML NK cells, which were infused into the patient the next day (day 0; max dose, 10 × 106 cells per kg). Disease assessment (clinical response) and adverse events were subsequently monitored, and samples for were collected for correlative experiments. SOC, standard of care.

Figure 2.

NK cells expand in patients treated with DLI and ML NK cell infusion. Peripheral blood and bone marrow samples from patients were analyzed by CyTOF. (A) Representative viSNE demonstrating FlowSOM-gated lymphocyte populations (CD34−CD45+CD14−). (B) Heat map of each marker used to annotate FlowSOM-gated lymphocytes in panel A (C) Density map of lymphocytes at the indicated time points from a representative patient’s peripheral blood mononuclear cells. Inset numbers depict the percentage of each population within the indicated cluster. (D) Total number of cells of each lymphocyte population quantitated by CyTOF across the indicated time points in the peripheral blood of patients. (E-F) Percentage of each lymphocyte population quantitated by CyTOF in the peripheral blood (E) and bone marrow (F). CyTOF data were available for patients P-ML002, P-ML003, P-ML005, P-ML007, P-ML008, P-ML009, and P-ML011​. (D-F) Data are expressed as the mean ± standard error. CyTOF, cytometry by time of flight; viSNE, visual stochastic neighbor embedding.

Figure 3.

ML NK cells demonstrate prolonged persistence in vivo in patients with relapsed AML. The NK cell multidimensional phenotype was assessed by CyTOF. NK cell subsets were identified by FlowSOM and annotated based on the expression of known ML NK cell markers. (A) Representative composite viSNE demonstrating FlowSOM-gated CD56dim, CD56bright, and ML NK cells from donors and patient peripheral blood at 7 to 60 days after NK cell administration. (B) Density viSNE plot of donor NK cells (at screening) and NK cells from patient peripheral blood at day 14 after NK cell administration. (C) Overlay viSNE from panel B. (D) Summary of NK cell populations across time points in patient peripheral blood and bone marrow. (E) Representative histograms of NK cell markers from donor and patient peripheral blood NK cells at day 14 after infusion. (F-G) Summary of median (F) and percentage positive (G) of indicated markers on donor vs patient peripheral blood NK cells at day 14 after infusion. Data include peripheral blood for P-ML002, P-ML005, P-ML009, and P-ML011, along with bone marrow from P-ML008 (as peripheral blood was not available for this donor). For perforin, P-ML011 was excluded due to a technical failure. (D,F-G) Data are presented as the mean and standard error of the mean. Data were tested for normal distribution (Shapiro-Wilk) and then compared using paired t test or Wilcoxon matched-pairs signed rank test. P-values are indicated. CyTOF, cytometry by time of flight; n.s., not significant; viSNE, visual stochastic neighbor embedding.

Figure 4.

ML NK cells express a unique transcriptional signature in vivo. Single-cell RNA sequencing was performed on enriched NK cells at baseline (donor) and at time points after infusion of cells from patient samples. NK cell subpopulations were identified by unsupervised cluster analysis. Data are shown for peripheral blood for P-ML008. (A) UMAP visualization of NK cells at the indicated time points. Panels are colored by time point overlaid on composite data from all time points (gray). ML NK cells are outlined with blue dashed lines on the UMAPs. (B) UMAP of indicated NK cell populations within composite data from all time points in panel A. (C) Expression of key NK cell population identifying genes. Cells shown in order on the UMAP by expression level. Black gate denotes ML NK cell population. (D) Percentage of CD56bright, CD56dim, ML, and adaptive NK cells in the donor product (baseline, before infusion) and days 14 and 50 after infusion. UMAP, Uniform Manifold Approximation and Projection.

Figure 5.

ML NK cells are highly functional ex vivo. NK cells from patient peripheral blood (A-B) and bone marrow (C-D) were unstimulated or stimulated with K562 in a standard 6-hour functional assay. (A) Representative data depicting IFN-γ and CD107a in unstimulated and K562-stimulated NK cells from P-ML007 peripheral blood at day 28. Numbers represent percentage of cells in the indicated gate. (B) Summary data from patient peripheral blood NK cells stimulated as in panel A indicated over time. Healthy donor NK cells (collected at screening) stimulated with K562 are included as representative of naive NK cell response. (C) Summary data for CD107a degranulation gated on CD56dim (CD56dim NKG2A+/−) or ML (CD56hi NKG2A+) NK cells in the same patient’s peripheral blood sample stimulated as in panel A. (B-C) Data are expressed as the mean ± standard error of the mean. (D) Representative data depicting IFN-γ and CD107a in unstimulated and K562-stimulated NK cells from bone marrow of P-ML007 at day 14. Numbers represent the percentage of cells in the indicated gate. (E) Summary data from bone marrow NK cells of each patient shown at day 14 stimulated as in panel D. (F) Summary data for CD107a degranulation gated on CD56dim (CD56dim NKG2A+/−) or ML (CD56hi NKG2A+) NK cells in same patient bone marrow sample stimulated as in (D). (B-C,E-F) Data were available for P-ML002, P-ML003, P-ML005, and P-ML007. Unstimulated and stimulated conditions were tested for normal distribution (Shapiro-Wilk) then compared by using 2-way analysis of variance (B-C) or paired t test (E-F). P-values are indicated above the graphs.