Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia

Abstract

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34⁺ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

Keywords: Adoptive transfer; Cell manufacturing; Good manufacturing practice (GMP); Immunotherapy; Natural killer (NK) cells.

Fig. 1

Translation of HSPC-NK cells with IL15/IL12/SR1-protocol from 6-well plate to a closed system cell culture bag using NK MACS medium. A Representation of the cytokine-based ex vivo culture protocol used for the generation of HSPC-NK cells. B Comparison of fold expansion of CD45⁺ expressing cells of the same donor measured at day 34–36, cultured in 6-well culture plates or cell culture bags using CellGro DC medium (n = 3). C Comparison of % CD45⁺CD56⁺ expressing cells of the same donor measured at day 34–36, cultured in 6-well plates or cell culture bags using CellGro DC medium (p = 0.007, n = 3). D Comparison fold expansion of CD45⁺ expressing cells during cell culture in CellGro DC medium or NK MACS medium, in 6-well culture plates (n = 3). E Comparison of % CD45⁺CD56⁺ expressing cells during cell culture in CellGro DC medium or NK MACS medium, in 6-well culture plates (n = 3). F Comparison of fold expansion of CD45⁺ expressing cells measured at day 35–36, cultured in 6-well culture plates or cell culture bags using NK MACS medium (n = 3–4). G Comparison of % CD45⁺CD56⁺ expressing cells of the same donor measured at day 35–36, cultured in 6-well plates or cell culture bags using NK MACS medium (p = 0.04, n = 3–4). H Mean expression of maturation and activation markers after 35 days, cultured in 6-well plates (n = 3, mean + SD). I Percentage of CD107a and IFNγ expressing CD56⁺Perforin⁺ NK cells, cultured in 6-well plates, with or without 4 h stimulation with K562 E:T ratio of 1.5:1 (n = 3, measured in singlet (unstimulated) or duplo (stimulated), mean + SEM). J Percentage proliferating NK cells, cultured in 6-well plates, after 5 days incubation with 1000 U/ml IL2 or 12,8 ng/ml IL15 (n = 2, measured in triplo, mean + SEM). Glycolytic (K) en mitochondrial (L) stress test of two HSPC-NK donors, each measured in triplo and two PB-NK donors, measured in duplo (PB-NK1) or triplo (PB-NK2). Values normalized to 1 at timepoint 0. Statistical analyses in B, C, F and G performed with two-tailed unpaired T-test

Fig. 2

GMP grade production was successfully validated over seven production runs. Due to technical difficulties in harvesting the cells, no expansion data of the first donor is available. Expansion and differentiation was followed throughout the 35/36 day culture process and phenotype and functionality were determined at the end of culture. A Total number of nucleated cells (NC) during culture (n = 6). B Fold expansion of total nucleated cells (CD45⁺) at day 35/36 of the culture. Line at median (n = 6). C Development of CD45⁺CD56⁺ cells during the culture, indicating differentiation towards NK cells. All donors reached CD56 expression of > 70% (n = 7). D Absolute number of NK cells after harvesting and washing. Line at median (n = 6). E Expression of several activating and inhibitory receptors on CD45⁺CD56⁺ cells at day 35/36 of the culture (n = 4–7). F Percentage of CD107a and IFN-γ expressing CD56⁺perforin⁺ NK cells with and without 4 h stimulation with K562 E:T ratio of 1.5:1 (n = 6), measured in singlet (unstimulated) or duplo (stimulated). All bars represent the mean + SD. Glycolytic (G) and mitochondrial (H) stress test of three HSPC-NK donors, each measured in sixplo and two PB-NK donors, measured in duplo and triplo (mean ± SEM). Values normalized to 1 at timepoint 0

Fig. 3

Mass cytometry of NK SR1/15.12 and NK Hep/15.2 showed that the improved SR1/15.12 resulted in a more mature and activated phenotype compared to the Hep/15.2 product. A Gating strategy used. B NKp46 expression within the CD56⁺CD3⁻ population was similar between culture methods. Both culture methods resulted in distinct phenotypes as visualized in by cluster analysis (C, E, F). D Positive population or Median expression of different markers, measured on CD45⁺CD14⁻CD19⁻ cells. Statistical analyses in B performed with two-tailed Mann–Whitney test, in D for CD56, NKp46, CD94, NKG2A, CD69 and CD25 a two-tailed unpaired T test and for KIR2DL2/2DL3, Ki67, granzyme B and Perforin a two-tailed Mann–Whitney test, in F a two-way ANOVA was used

Fig. 4

Clinical trial study design with RNK001 in AML/MDS-EB2 patients. A This study focusses on patients with either AML or MDS-EB2 de novo or relapsed or refractory disease. Every patient receives a non-myeloablative immunosuppressive conditioning regimen consisting of Cy/Flu, followed by a single infusion of allogeneic NK cells, hypothetically inducing a graft-versus-leukemia (GvL) effect. B The study is divided in two phases. Phase I is designed as a standard 3 + 3 dose escalating study, with increasing dose of IL2. Phase 2a is designed as a Simon’s optimal two stage single arm study. C Schematic overview of the study design. Patients with AML or MDS-EB2 will be screened on eligibility criteria. Five weeks before planned infusion generation of the HSPC-NK cells starts. Close to admission bone marrow investigation will be performed to establish disease status. Five days before infusion patients will receive IV NMA immunosuppression consisting of cyclophosphamide (500 mg/m²/day) and fludarabine (30 mg/m²/day) on days -5, -4 and -3. This Cy/Flu regimen will be administered in an inpatient hospitalized setting. On day 0 patients receive a fixed dose of 1.0–3.0 × 10⁶ NK cells. IL2 will be administered for six doses in total, given every other day, starting 4 h after NK cell infusion. Patients will be admitted to the hospital at least until the last IL2 administration. At day 7 bone marrow examination will be performed to evaluate NK cell homing to the bone marrow. At day 28 bone marrow examination will be performed to evaluate effect on disease status. Follow-up lasts 6 months