Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplantation

Abstract

Relapse has emerged as the most important cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HSCT). To test the hypothesis that natural killer (NK) cells can decrease the risk of leukemia relapse, we initiated a phase 1 dose-escalation study of membrane-bound interleukin 21 (mbIL21) expanded donor NK cells infused before and after haploidentical HSCT for high-risk myeloid malignancies. The goals were to determine the safety, feasibility, and maximum tolerated dose. Patients received a melphalan-based reduced-intensity conditioning regimen and posttransplant cyclophosphamide-based graft-versus-host disease (GVHD) prophylaxis. NK cells were infused on days -2, +7, and +28 posttransplant. All NK expansions achieved the required cell number, and 11 of 13 patients enrolled received all 3 planned NK-cell doses (1 × 10⁵/kg to 1 × 10⁸/kg per dose). No infusional reactions or dose-limiting toxicities occurred. All patients engrafted with donor cells. Seven patients (54%) developed grade 1-2 acute GVHD (aGVHD), none developed grade 3-4 aGVHD or chronic GVHD, and a low incidence of viral complications was observed. One patient died of nonrelapse mortality; 1 patient relapsed. All others were alive and in remission at last follow-up (median, 14.7 months). NK-cell reconstitution was quantitatively, phenotypically, and functionally superior compared with a similar group of patients not receiving NK cells. In conclusion, this trial demonstrated production feasibility and safety of infusing high doses of ex vivo-expanded NK cells after haploidentical HSCT without adverse effects, increased GVHD, or higher mortality, and was associated with significantly improved NK-cell number and function, lower viral infections, and low relapse rate posttransplant.

Figure 1.

NK-cell number, phenotype and function in the first year posttransplant for patients treated with haploidentical stem cell transplantation using posttransplant cyclophosphamide on protocol 2009-0266 (without NK-cell infusions). (A) Absolute lymphocyte count (ALC) was determined from a clinical complete blood count obtained at the indicated time point. (B) Absolute NK-cell counts were determined from PB samples obtained at same time points, from which PBMCs were isolated and cryopreserved for batch testing. CD3⁻CD56⁺ populations were determined from within lymphocyte gates, and absolute NK count derived according to the percent of CD3⁻CD56⁺ cells. (C) NK-cell maturity was determined according to CD16⁺ and CD16⁻ fractions of the NK cells in Figure 3B. (D) NK-cell function at 1 month posttransplant was determined by measuring cytotoxicity against 721.221 targets, wherein PBMCs were applied according to NK-cell content at a 40:1 NK-to-target ratio.

Figure 2.

Treatment schema for the clinical trial 2012-0708 of adding infusions of expanded donor NK cells to haploidentical stem cell transplant with posttransplant cyclophosphamide.

Figure 3.

Comparison of clinical outcomes between patients treated on protocols 2012-0708 (with NK cells [blue line]) and 2009-0266 (no NK cells [red line]). Data from 2009-0266 were censored to include patients with only AML/MDS and CML in morphologic CR at the time of transplant. (A) Time to neutrophil engraftment. (B) Time to platelet engraftment. (C) Cumulative incidence of grade 2-4 acute GVHD. (D) Cumulative incidence of grade 3-4 aGVHD. (E) Cumulative incidence of chronic GVHD limited + extensive. (F) Incidence of BKV cystitis. (G) Incidence of CMV reactivation. (H) Cumulative incidence of relapse. (I) Probability of progression-free survival. Kaplan-Meier survival curves were compared using the log-rank method. BKV, BK polyomavirus.

Figure 4.

Assessment of phenotype and function of mbIL21-expanded NK-cell infusion product (green) or PB NK cells patients. Mononuclear cells were isolated from PBMCs of patients on protocol 2009-0266 (without NK cells [red]) or 2012-0708 (with NK cells [green]) ∼28 days after stem cell transplant. Samples from patients on protocol 2012-0708 were obtained prior to receiving the third dose of NK cells (∼3 weeks after receiving the second dose). (A) Cytotoxicity against 721.221 targets. Cells were applied to the cytotoxicity assay at a 10:1 NK-to-target ratio (according to NK-cell content determined by immune phenotyping). (B) NK-cell responses to stimulation with 721.221 targets at a 2:1 NK-to-target ratio for 3 hours. Degranulation (CD107a), cytokine production, and CD62L cleavage were determined by mass cytometry. (C) Cytotoxicity and IFNγ production from panels A and B stratified according to cell dose received (low, ≤10⁶/kg per dose; high, ≥10⁷/kg per dose). P values shown for unpaired Student t test. (D) Differences in phenotype of NK cells at day +28 posttransplant as determined by mass cytometry. Shown are surface markers on NK cells from supplemental Figure 1 that were significantly different between 2009-0266 (no NK-cell infusions) and 2012-0708 (with NK-cell infusions) using multiple unpaired Student t-test comparisons followed by the 2-stage false discovery approach. Corrected P values are indicated as *P < .01, **P < .001, ***P < .0001, or ****P < .00001. (E) Heatmap with unsupervised clustering analysis of the NK-cell phenotypes from supplemental Figure 1 according to relative expression of each receptor in each sample. Sample origin is indicated along the top row: day +28 PB-NK samples from protocol 2009-0266 (red) or 2012-0708 (blue), or NK-cell product (green). (F) SPADE trees of NK-cell subsets present in the NK-cell product or peripheral blood at day +28 posttransplant. Given the small number of samples in each group, KIR expression was excluded from clustering to avoid bias from individual KIR and HLA genotypes. (G) Heatmap with unsupervised clustering analysis of the NK-cell subsets from each sample according to those identified in panel F (using only nodes constituting at least 1% of any 1 sample). Sample origin color coding is as in panel E. (H) Principal components analysis based on the percentage of NK cells in each node of each sample as in panel G. X-axis and Y-axis show principal component 1 and principal component 2 that explain 31.9% and 24.1% of the total variance, respectively. Prediction ellipses indicate 95% probability that a new observation from the same group will fall inside the ellipse. N = 22 data points. (I) ViSNE clustering analysis of NK cells according to sample origin, showing expression levels of surface markers that were visibly different between 2009-0266 and 2012-0708.

Figure 4.

Assessment of phenotype and function of mbIL21-expanded NK-cell infusion product (green) or PB NK cells patients. Mononuclear cells were isolated from PBMCs of patients on protocol 2009-0266 (without NK cells [red]) or 2012-0708 (with NK cells [green]) ∼28 days after stem cell transplant. Samples from patients on protocol 2012-0708 were obtained prior to receiving the third dose of NK cells (∼3 weeks after receiving the second dose). (A) Cytotoxicity against 721.221 targets. Cells were applied to the cytotoxicity assay at a 10:1 NK-to-target ratio (according to NK-cell content determined by immune phenotyping). (B) NK-cell responses to stimulation with 721.221 targets at a 2:1 NK-to-target ratio for 3 hours. Degranulation (CD107a), cytokine production, and CD62L cleavage were determined by mass cytometry. (C) Cytotoxicity and IFNγ production from panels A and B stratified according to cell dose received (low, ≤10⁶/kg per dose; high, ≥10⁷/kg per dose). P values shown for unpaired Student t test. (D) Differences in phenotype of NK cells at day +28 posttransplant as determined by mass cytometry. Shown are surface markers on NK cells from supplemental Figure 1 that were significantly different between 2009-0266 (no NK-cell infusions) and 2012-0708 (with NK-cell infusions) using multiple unpaired Student t-test comparisons followed by the 2-stage false discovery approach. Corrected P values are indicated as *P < .01, **P < .001, ***P < .0001, or ****P < .00001. (E) Heatmap with unsupervised clustering analysis of the NK-cell phenotypes from supplemental Figure 1 according to relative expression of each receptor in each sample. Sample origin is indicated along the top row: day +28 PB-NK samples from protocol 2009-0266 (red) or 2012-0708 (blue), or NK-cell product (green). (F) SPADE trees of NK-cell subsets present in the NK-cell product or peripheral blood at day +28 posttransplant. Given the small number of samples in each group, KIR expression was excluded from clustering to avoid bias from individual KIR and HLA genotypes. (G) Heatmap with unsupervised clustering analysis of the NK-cell subsets from each sample according to those identified in panel F (using only nodes constituting at least 1% of any 1 sample). Sample origin color coding is as in panel E. (H) Principal components analysis based on the percentage of NK cells in each node of each sample as in panel G. X-axis and Y-axis show principal component 1 and principal component 2 that explain 31.9% and 24.1% of the total variance, respectively. Prediction ellipses indicate 95% probability that a new observation from the same group will fall inside the ellipse. N = 22 data points. (I) ViSNE clustering analysis of NK cells according to sample origin, showing expression levels of surface markers that were visibly different between 2009-0266 and 2012-0708.

Figure 5.

Immunologic reconstitution of lymphocyte subsets in the first 6 months posttransplant for patients treated with and without NK cells. Immune subsets were determined by clinical flow cytometry. Absolute cell counts were calculated based on subset percentages and total WBC count obtained at the same time. Red, 2009-0266, previous clinical trial without NK cells; blue, 2012-0708, current phase 1 clinical trial. (A) Total WBC count. (B) Total lymphocyte count (CD3⁺). (C) CD56⁺CD3⁻ NK cells. (D) CD3⁺CD4⁺ T cells. (E) CD3⁺CD8⁺ T cells. (F) CD3⁺CD25⁺ T-regulatory cells. (G) CD3⁺CD45RA⁺ naïve T cells. (H) CD⁺CD45RO⁺ memory T cells. (I) CD19⁺ B cells. D30, day 30 posttransplant; D90, day 90 posttransplant; day 180, day 180 posttransplant; WBC, total WBC count.