Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts

Abstract

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described. The protocol is based on the CliniMACS Prodigy platform and Natural Killer Cell Transduction (NKCT) (Miltenyi Biotec). NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with IL-2 and IL-15 cytokines. Three CliniMACS Prodigy processes demonstrated the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product. Characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, the NK cells exhibited increased killing efficacy against patient-derived CD33 + AML blasts and NBL cells in vitro, suggesting a potential therapeutic benefit in AML and NBL.

Keywords: AML blast; CliniMACS prodigy; Immunotherapy; Natural killer cells.

Fig. 1

The workflow for the activated NK cell product in the CliniMACS prodigy. Manufacturing steps, sampling points and quality control (QC) analysis scheme are shown. Open phases are performed in the grade A isolator. *In the set-up phase, the sterility analysis was performed on leukapheresis sample directly. **Analyses designed to be performed for the products proceeding to clinical studies but not performed in this study. IPC in-process-control, NTCB-D non-target cell bag depletion, NTCB non-target cell bag, NC nucleocounter, RAB re-application bag, TCB target cell bag.

Fig. 2

The distribution of cell populations at different process stages. The frequencies of different cell populations within CD45 + cells were assessed by flow cytometry at four distinct process stages: starting material, CD3-depleted, CD56-enriched, and end product. Different symbols represent the cell types: T cells (CD3 + CD56-), monocytes (CD14+), B cells (CD19+), NK cells (CD3-CD56+) and two subsets of NK cells characterized by the presence or absence of CD16. The black, purple and blue color represent the three individual Prodigy processes (P_NK1-3). The bars represent means ± SD.

Fig. 3

Phenotype marker expression in activated and non-activated NK cells. The expression of selected phenotype markers was assessed by flow cytometry from the CD56-positive lymphocytes. The black, purple and blue colors represent the three individual Prodigy processes (P_NK1-3) and the circle and star symbols stand for the non-activated and 12-h cytokine-activated NK cells. The bars represent means ± SD.

Fig. 4

CD69 expression in non-activated and activated NK Cells. NK cells were produced in the CliniMACS Prodigy and the expression was studied with flow cytometry from CD56 + NK cells, as part of the quality control panel. The fluorescence histograms for activated NK cells (end product), corresponding non-activated NK cells (after CD56-enrichment), and FMO control are shown.

Fig. 5

Expression of cell surface markers in AML blasts. The expression of cell surface markers was analysed with flow cytometry from cells collected from whole blood (WB) and after CD33 bead isolation from acute myeloid leukemia (AML) patient samples. The orange and blue color represent the patients and the symbol expresses the sample type.

Fig. 6

Functional assessment of NK cells post-production and activation. The luminescence-based assays compare the cytotoxic response of non-activated NK cells (‘non-act’) and 12-h cytokine activated NK cells (‘act’) against (a) K562 and (b) SH-SY5Y NBL targets at different effector to target ratios. Additionally, the P_NK3_act sample underwent post-activation characterization after a freeze/thaw cycle, denoted by an asterisk*. The P_NK1_act* sample was exclusively assessed post-freeze/thaw, with no corresponding non-activated control for comparison. The reported percentage of cell lysis represents the average of five replicates, with standard deviation (SD) indicated by error bars. c) The cytotoxicity of cytokine-activated NK cells targeting AML blast cells from patient samples. d) The degranulation response of NK cells following a 4-hour co-culture with K562 and SH-SY5Y cell lines, compared to NK cells cultured in the media alone (n = 2). P_NK: NK cells were produced in the GMP CliniMACS Prodigy conditions. R_NK and non-activated NK cells were produced in a non-GMP conditions. AML blast_P denotes AML patient.

Fig. 7

Flowchart illustration of the AML blast isolation process. The AML blasts are isolated from the whole blood (WB) of an AML patient for the in vitro experiments. Created with Biorender.com.