Decrease post-transplant relapse using donor-derived expanded NK-cells

Abstract

In this phase I/II clinical trial, we investigated the safety and efficacy of high doses of mb-IL21 ex vivo expanded donor-derived NK cells to decrease relapse in 25 patients with myeloid malignancies receiving haploidentical stem-cell transplantation (HSCT). Three doses of donor NK cells (1 × 105-1 × 108 cells/kg/dose) were administered on days -2, +7, and +28. Results were compared with an independent contemporaneously treated case-matched cohort of 160 patients from the CIBMTR database.After a median follow-up of 24 months, the 2-year relapse rate was 4% vs. 38% (p = 0.014), and disease-free survival (DFS) was 66% vs. 44% (p = 0.1) in the cases and controls, respectively. Only one relapse occurred in the study group, in a patient with the high level of donor-specific anti-HLA antibodies (DSA) presented before transplantation. The 2-year relapse and DFS in patients without DSA was 0% vs. 40% and 72% vs. 44%, respectively with HR for DFS in controls of 2.64 (p = 0.029). NK cells in recipient blood were increased at day +30 in a dose-dependent manner compared with historical controls, and had a proliferating, mature, highly cytotoxic, NKG2C+/KIR+ phenotype.Administration of donor-derived expanded NK cells after haploidentical transplantation was safe, associated with NK cell-dominant immune reconstitution early post-transplant, preserved T-cell reconstitution, and improved relapse and DFS. TRIAL REGISTRATION: NCT01904136 ( https://ichgcp.net/clinical-trials-registry/NCT01904136 ).

Conflict of interest statement

SOC served as advisory board member for Cellularity, CareDx, Kiadis, Spectrum, CytoSen, MolMed, received research funds from Miltenyi Biotech and Kiadis, and has intellectual property and equity in Kiadis. PK served as advisory board member for CareDx. PT has intellectual property licensing to Kiadis Pharma. DAL received consulting fees, equity, and intellectual property in Kiadis Pharma. Consulting fees and equity in Courier Therapeutics, Consulting fees for Caribou Biosciences. All other authors had no potential conflict of interest to declare.

© 2021. The Author(s).

Figures

Fig. 1. Transplant outcomes of NK cell treatment group (cases) and the CIBMTR control group.

A Relapse between cases and controls with the cumulative incidence at 2 years of 4% (95% CI 0–16) vs. 38% (95% CI 30–46), respectively (adjusted P value = 0.014). B DFS of cases vs. controls with the probability at 2 years of 66% (95% CI 51–87) vs. 44% (95% CI 36–53), respectively (adjusted P value = 0.10). C DFS of cases and controls who did not have DSA before transplant. A significantly higher DFS was observed in patients without DSA treated with NK cell infusion compared with controls without DSA with probability at 2 years of 72% vs. 44%, respectively (adjusted P = 0.029). D NRM of cases vs. controls with the probability at 2 years of 30% (95% CI 13–51) vs. 18% (95% CI 12–24), respectively (adjusted P value = 0.37).

Fig. 2. Immunologic reconstitution of patients treated on the clinical trial.

A The median number of absolute lymphocytes (ALC), CD56+, CD3+, CD4+, CD8+, CD19+, CD25+, CD45RO, and CD45RA cells at 30, 90, 180, and 360 days after transplant. Number of all lymphocyte subsets gradually increased and returned to normal by day 90 after transplant. B Number of B and T cell subsets after transplant stratified by NK cell dose level. High number of CD56+ cells was observed in patients who received high NK cell dose (dose level 3: 1 × 108/Kg/dose) compared with low (dose level 1: <1 × 107/Kg/dose) and intermediate dose (dose level 2: 1 × 107–3 × 107/Kg/dose) (P = 0.064). At day 90 and 180, a significantly lower number of CD25+ cells in patients who received higher NK cell dose was observed when compared with low and intermediate dose. The mean number of CD25+ cells at day 90 for patients who received low, intermediate, and high NK dose was 15 cells/mm3 (SD 4.2), 42 cells/mm3 (SD 38) and 6.3 cells/mm3 (SD 5.1), respectively (P = 0.005), and at day 180 were 151 cells/mm3 (SD 108), 66 cells/mm3 (SD 44) and 16 cells/mm3 (SD 14), respectively (P = 0.025). No significant difference in number of CD19+, CD4+, CD8+, and CD3+ cells between patients who received different dose of NK cells. Bars and whiskers represent median ± interquartile range.

Fig. 3. Immunophenotyping by mass cytometry with stochastic clustering to compare immune reconstitution in patients compared to healthy controls and FC21-NK cell products.

Blood was obtained at the indicated timepoints, and mononuclear cells (MNC) were isolated, processed, and labeled with a 34-parameter panel of heavy-metal conjugated antibodies (Supplementary Information Table S1), along with healthy subject MNC and expanded FC21-NK cell products as controls. Events were collected on a CyTOF 2 mass cytometer (Fluidigm). Events were filtered by sequential gating on live, singlet (event length vs. 191Ir), non-apoptotic (PARP-negative), and hematopoietic (CD45+) cells, and then clustered by visual interactive stochastic neighbor embedding (ViSNE, CytoBank) on CD3, TIGIT, NKP30, NKP46, CD56, NKG2D, CD94, and CD57, using equal sampling to unbias differences in sample event number. ViSNE clusters corresponding to T-cells, standard NK cells, FC21-NK cells, and any remaining MNC were created using the reference samples. The percentage of cells in clusters 1 through 4 were quantified for each sample. Ki67 staining within the gated populations was determined as a surrogate for proliferation. A shows representative plots from two patients, one healthy subject, and one NK cell product, showing expression of key activating surface markers, perforin, and Ki67 across four broad phenotypic clusters identified. Cluster 1 (bottom left) consisting of CD3+ T cells, cluster 2 (top middle) of CD3−CD56dimNKG2DdimCD57+ “standard” NK cells, cluster 3 (top right) consisting of CD56brNKG2DbrNKp46brCD57− (“superbright”) NK cells corresponding to the phenotype of the infused FC21-NK cell product, and cluster 4 (bottom middle) consisting of all remaining cells. Cluster 3 identifies a unique phenotypic signature associated with the FC21-NK cells that is not present in healthy subjects and persists in patients at day 14 (7 days after adoptive transfer) and later (Supplementary Information Fig. S2). B NK cell immune reconstitution in patients over time maintains FC21-NK “superbright” phenotype with high proliferation, expressed as percent of total cell events, of cluster 3 (superbright FC21-NK cells) in healthy donors, FC21-NK cell products, and in patients receiving FC21-NK cell products (across all timepoints). C Proportion of Cluster 1 (T cells) and total NK cells (Cluster 2 + Cluster 3) in blood of study subjects across time. D The ratio of NK cells and T cells for all patients and timepoints assessed (n = 24). E Ki67 staining in FC21-NK cells (Cluster 3), standard NK cells (Cluster 4), and T cells (Cluster 1) in four representative patient samples obtained at day 14 (7 days after the NK cell infusion at day 7). F, G The percent of Ki67+ and Ki67 mean metal intensity (MMI), respectively, in standard NK cells, FC21-NK cells, and T cells for all patients and timepoints assessed (n = 24). Bars and whiskers represent median ± interquartile range, P < 0.0001. H NK cells in clusters 2 (standard NK) and 3 (FC21-NK) as assessed for expression of NKG2C across all patients at all timepoints, with FC21-NK cell infusion products and healthy subjects shown for reference. I the percent of NKG2C + NK cells from all patients at all timepoints, with early (Days 7 and 14) and late (>day 28) timepoints pooled. J NK cells (CD3−/CD56+) gated and assessed for KIR expression and summed for total percentage of KIR + NK cells, and then K plotted across time, with early and late timepoints pooled.