First-in-human evaluation of memory-like NK cells with an IL-15 super-agonist and CTLA-4 blockade in advanced head and neck cancer

Abstract

Background: Cytokine induced memory-like natural killer (CIML NK) cells combined with an IL-15 super-agonist (N-803) are a novel modality to treat relapsed/refractory head and neck cancer.

Methods: We report data from a phase I trial of haploidentical CIML NK cells combined with N-803 with or without ipilimumab (IPI) in relapsed/refractory head and neck cancer patients after a median of 6 prior lines of therapy. The trial adhered to a 3 + 3 dose de-escalation design, with primary endpoint being safety. High-resolution immunophenotypic and transcriptional profiling characterized the NK cells and their interacting partners in vivo.

Results: The primary safety endpoint was established, with dose-limiting toxicity in 1/10 patients. A transient disease control rate correlated with donor NK cell expansion, the latter occurring irrespective of IPI. The combination of CIML NK cells with N-803 and IPI was associated with increased early NK cell proliferation, contraction of Treg: Tcon, rapid recovery of recipient CD8⁺ T cells, and subsequent accelerated rejection of donor NK cells.

Conclusions: CIML NK cells combined with N-803 and ipilimumab to treat head and neck cancer is safe, and associated with a more proliferative NK cell phenotype. However, the combination leads to reduced HLA mismatched NK cell persistence, resulting in an important limitation affecting NK cell combination therapies in clinical trials. These results inform evaluation of CIML NK therapy for advanced malignancies, with considerations for combination with IPI.

Trial registration: NCT04290546.

Keywords: Clinical trial; Head and neck cancer; Interleukin-15; Ipilimumab; NK cells.

Fig. 1

Clinical outcomes of patients with relapsed/refractory head and neck cancer treated with haploidentical donor-derived CIML NK cell therapy. A Clinical trial schema describing CIML NK cell infusion product generation and trial therapy. For each patient, a haploidentical donor underwent non-mobilized apheresis of peripheral blood followed by CD3 depletion and CD56 positive selection. The product was then incubated in a cocktail of IL-12, IL-15, and IL-18 for 12–16 h to induce CIML differentiation and infused on day 0. The patients received lymphodepletion with fludarabine and cyclophosphamide, and IL-15 superagonist (IL-15sa) N-803 on day + 1, with subsequent doses given every 3 weeks (up to 4 doses). After the first 6 treated patients were evaluated for safety, the subsequent patients also received a single dose of ipilimumab (IPI) 1 mg/kg on day − 7 prior to starting lymphodepletion (created using Biorender). B Flow of the clinical trial. Cohort 1 in orange: (no IPI) DL0: 5–10 × 10⁶ cells/kg. One patient was replaced (empty circle) due to NK cells not being received. Cohort 2 in yellow: (with IPI, 3 mg/kg x 1) DL0: 5–10 × 10⁶ cells/kg. *The patient with grade 5 adverse event of F&N (febrile neutropenia) and dose-limiting toxicity (DLT) in Cohort 1 is shown in red C Waterfall plot showing tumor response by RECIST v1.1 criteria on day + 30 following NK cell infusion. The bars in blue represent the first 6 evaluable patients on the trial who did not receive IPI while the green bars represent IPI treated patients. The number above each bar represents the patient ID on the trial. The dashed lines indicate size thresholds per RECIST v1.1 criteria. D Clinical course of all evaluable treated patients. Numbers on the vertical axis represent patient ID on the trial. The horizontal axis refers to days on study from the time of first therapy. D0: the day of CIML NK cell infusion, D30: day + 30 response evaluation time point, CRS: cytokine release syndrome, EOT: end of treatment, PD: progressive disease

Fig. 2

Expansion of the NK cells following donor cell product infusion. A Longitudinal evaluation of peripheral blood mononuclear cells (PBMCs) using flow cytometry with a customized panel, single-cell CITE-seq, a multiplexed immunoassay for secreted molecules, and next-generation sequencing of donor chimerism. The number of samples used for each assay at each time point is indicated (created using Biorender). B Size of the NK cell compartment as a proportion of total lymphocytes (left) and as absolute numbers (right) at the indicated time points after donor CIML NK cell infusion. Number of evaluable samples at each time point is shown above each bar. Adjusted p-values are determined using nonparametric Anova (Kruskal-Wallis test) followed by Dunn’s multiple comparisons test. Shown are mean with SD, medians are marked by a red line. C Spearman correlation between NK cell expansion and tumor responses by RECIST v1.1 criteria. NK cell fold change was calculated by taking the ratio of NK cells as a percentage of lymphocytes, in the peripheral blood on day 14 following CIML NK cell infusion to the percentage of NK at the time of screening. The patient ID is indicated at each point. D Donor cells as a proportion of all peripheral blood mononuclear cells at the indicated time points using two different assays. Flow cytometry-based evaluation (Flow) using anti-HLA antibodies targeting recipient-donor disparity in HLA. DNA-based chimerism using next-generation sequencing (NGS) performed by CareDx was also used to evaluate donor chimerism. ≤14 (n = 7) refers to available samples collected between day + 7 and day + 14, ≥28 (n = 4 for Flow, n = 5 for NGS) refers to available samples collected between day + 28 and day + 60. E Percentage expression of interferon gamma (IFNγ) in flow cytometry-based NK cell functional assays using live donor CD3⁻CD56⁺ lymphocytes (gated with donor-specific HLA antibodies) collected at the indicated time points following CIML NK cell infusion. In one recipient there was no donor-specific HLA antibody for flow cytometry gating, but the NGS-based chimerism confirmed that the NK cells were all donor, so the percentage was calculated on total NK cells. p-value for the comparisons in D, E were calculated using Mann-Whitney U. SRN: screening time point for the trial before NK cell infusion

Fig. 3

NK cell populations exhibit markers of proliferation and activation following CIML NK cell infusion. A Distribution of NK cells and T cells in the peripheral blood as a proportion of total lymphocytes assessed with flow cytometry. Data presented is a percentage of total lymphocytes. Gating strategies are indicated in Supplementary Fig. 19. B Distribution of the CD56^dim (Dim) and CD56^bright (Bright) NK cell population assessed with flow cytometry, mean and SD. Adjusted p-values for each time point comparison with SRN are determined using nonparametric Anova (Kruskal-Wallis test) followed by Dunn’s multiple comparisons test. SRN: screening time point C UMAP of NK cell clusters defined using single cell CITE sequencing data, combining all patient samples (n = 4) and all time points following CIML NK cell infusion (D7: day + 7, D28: day + 28). D Pseudotime plots demonstrating the differentiation trajectories of expanding NK cells. The plot on the left shows the same UMAP clustered by time. E Gene set enrichment analysis of pathways in NK cell clusters comparing days + 7 (D7) and + 28 (D28), with the earlier time point showing enrichment of proliferative gene sets while the latter time point showing enrichment of pathways associated with NK cell activation. F Expression of markers of proliferation (MKI67), FCGR3A (gene for CD16) and gene lists of NK activating receptors, inhibitory receptors, activation, and exhaustion. Key genes comprising these lists are described in the supplemental appendix (Supplementary Table 7). The intensity of expression is represented by the color in the legend within each plot

Fig. 4

Ipilimumab is associated with increased early NK cell proliferation but more rapid contraction of the NK cell compartment. A Distribution of NK cells and T cells in the peripheral blood as a proportion of total lymphocytes, stratified by IPI treatment, as evaluated with flow cytometry. The data presented is a percentage of total lymphocytes. B Distribution of peripheral blood mononuclear cells as measured with single cell CITE-seq on day + 7 (D7) and day + 28 (D28), comparing IPI treated (IPI⁺, n = 2) and IPI untreated (IPI⁻, n = 2) patients. C UMAP of NK cell clusters defined with single cell CITE-seq, separated by time points assessed and IPI treatment. The color scheme corresponds to the same populations as shown in B. D Gene set enrichment analysis of pathways in NK cell clusters comparing IPI⁺ (n = 2) and IPI⁻ (n = 2) patients on day + 7 (D7)

Fig. 5

T cell reconstitution following CIML NK cell infusion. A Distribution of the T cell populations assessed by flow cytometry, mean, and SD. Adjusted p-values for each time point comparison with SRN (trial screening time point) are determined using non parametric anova (Kruskal-Wallis test) followed by Dunn’s multiple comparisons test. B Gene set enrichment analysis of pathways in T cell clusters comparing days + 7 (D7) and + 28 (D28), showing enrichment of pathways of activation at the latter time point. C Gene set enrichment analysis of pathways in T cell clusters comparing IPI untreated (IPI⁻) and treated (IPI⁺) conditions. D Expression of exhaustion markers in T cell populations on days + 7 (D7) and + 28 (D28). Boxplots show median and quartiles. Mann-Whitney U test. P-values were calculated per cell type and are detailed in Supplementary Table 15