Development of Automated Separation, Expansion, and Quality Control Protocols for Clinical-Scale Manufacturing of Primary Human NK Cells and Alpharetroviral Chimeric Antigen Receptor Engineering.

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 109 leukocytes (including 1.1-2.3 × 109 NK cells) collected by small-scale lymphapheresis (n = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS® basal medium containing NK MACS® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

Influence of Irradiated Peripheral Blood Mononuclear Cells on Both Ex Vivo Proliferation of Human Natural Killer Cells and Change in Cellular Property.

Clinical studies with adoptive immunotherapy using allogeneic natural killer (NK) cells showed feasibility, but also limitation regarding the transfused absolute cell numbers. First promising results with peripheral blood mononuclear cells (PBMCs) as feeder cells to improve the final cell number need further optimization and investigation of the unknown controlling mechanism in the cross-talk to NK cells. We investigated the influence of irradiated autologous PBMCs to boost NK cell proliferation in the presence of OKT3 and IL-2. Our findings demonstrate a requirement for receptor-ligand interactions between feeders and NK cells to produce soluble factors that can sustain NK cell proliferation. Thus, both physical contact between feeder and NK cells, and soluble factors produced in consequence, are required to fully enhance NK cell ex vivo proliferation. This occurred with an indispensable role of the cross-talk between T cells, monocytes, and NK cells, while B cells had no further influence in supporting NK cell proliferation under these co-culture conditions. Moreover, gene expression analysis of highly proliferating and non-proliferating NK cells revealed important phenotypic changes on 5-day cultured NK cells. Actively proliferating NK cells have reduced Siglec-7 and -9 expression compared with non-proliferating and resting NK cells (day 0), independently of the presence of feeder cells. Interestingly, proliferating NK cells cultured with feeder cells contained increased frequencies of cells expressing RANKL, B7-H3, and HLA class II molecules, particularly HLA-DR, compared with resting NK cells or expanded with IL-2 only. A subset of HLA-DR expressing NK cells, co-expressing RANKL, and B7-H3 corresponded to the most proliferative population under the established co-culture conditions. Our results highlight the importance of the crosstalk between T cells, monocytes, and NK cells in autologous feeder cell-based ex vivo NK cell expansion protocols, and reveal the appearance of a highly proliferative subpopulation of NK cells (HLA-DR+RANKL+B7-H3+) with promising characteristics to extend the therapeutic potential of NK cells.

Shaping of Natural Killer Cell Antitumor Activity by Ex Vivo Cultivation.

Natural killer (NK) cells are a promising tool for the use in adoptive immunotherapy, since they efficiently recognize and kill tumor cells. In this context, ex vivo cultivation is an attractive option to increase NK cells in numbers and to improve their antitumor potential prior to clinical applications. Consequently, various strategies to generate NK cells for adoptive immunotherapy have been developed. Here, we give an overview of different NK cell cultivation approaches and their impact on shaping the NK cell antitumor activity. So far, the cytokines interleukin (IL)-2, IL-12, IL-15, IL-18, and IL-21 are used to culture and expand NK cells. The selection of the respective cytokine combination is an important factor that directly affects NK cell maturation, proliferation, survival, distribution of NK cell subpopulations, activation, and function in terms of cytokine production and cytotoxic potential. Importantly, cytokines can upregulate the expression of certain activating receptors on NK cells, thereby increasing their responsiveness against tumor cells that express the corresponding ligands. Apart from using cytokines, cocultivation with autologous accessory non-NK cells or addition of growth-inactivated feeder cells are approaches for NK cell cultivation with pronounced effects on NK cell activation and expansion. Furthermore, ex vivo cultivation was reported to prime NK cells for the killing of tumor cells that were previously resistant to NK cell attack. In general, NK cells become frequently dysfunctional in cancer patients, for instance, by downregulation of NK cell activating receptors, disabling them in their antitumor response. In such scenario, ex vivo cultivation can be helpful to arm NK cells with enhanced antitumor properties to overcome immunosuppression. In this review, we summarize the current knowledge on NK cell modulation by different ex vivo cultivation strategies focused on increasing NK cytotoxicity for clinical application in malignant diseases. Moreover, we critically discuss the technical and regulatory aspects and challenges underlying NK cell based therapeutic approaches in the clinics.

Natural Killer Group 2D Ligand Depletion Reconstitutes Natural Killer Cell Immunosurveillance of Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor originating from the epithelial lining of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. The natural killer group 2D (NKG2D) receptors on natural killer (NK) cells and several T cell subsets play an important role for immunosurveillance of HNSCC and are thus targeted by tumor immune evasion strategies in particular by shedding of various NKG2D ligands (NKG2DLs). Based on plasma and tumor samples of 44 HNSCC patients, we found that despite compositional heterogeneity the total plasma level of NKG2DLs correlates with NK cell inhibition and disease progression. Strikingly, based on tumor spheroids and primary tumors of HNSCC patients, we found that NK cells failed to infiltrate HNSCC tumors in the presence of high levels of NKG2DLs, demonstrating a novel mechanism of NKG2DL-dependent tumor immune escape. Therefore, the diagnostic acquisition of the plasma level of all NKG2DLs might be instrumental for prognosis and to decipher a patient cohort, which could benefit from restoration of NKG2D-dependent tumor immunosurveillance. Along these lines, we could show that removal of shed NKG2DLs (sNKG2DLs) from HNSCC patients' plasma restored NK cell function in vitro and in individual patients following surgical removal of the primary tumor. In order to translate these findings into a therapeutic setting, we performed a proof-of-concept study to test the efficacy of adsorption apheresis of sNKG2DLs from plasma after infusion of human MICA in rhesus monkeys. Complete removal of MICA was achieved after three plasma volume exchanges. Therefore, we propose adsorption apheresis of sNKG2DLs as a future preconditioning strategy to improve the efficacy of autologous and adoptively transferred immune cells in cellular cancer immunotherapy.

Standardized and flexible eight colour flow cytometry panels harmonized between different laboratories to study human NK cell phenotype and function.

Advancements in multi-colour fluorescence activated cell sorting (FACS) panel warrant harmonized procedures to obtain comparable data between various laboratories. The intensifying clinical exploration of Natural Killer (NK) cell-based immunotherapy demands standardized and harmonized NK cell FACS panels and acquisition protocols. Eight colour FACS panels were designed to study human NK cell phenotype and function within peripheral blood mononuclear cells (PBMC). The panels were designed around fixed backbone markers and channels, covering antigens for non-NK lineage exclusion (CD3, TCRγδ, CD19, CD14, SYTOX® Blue) and NK cell selection (CD45, CD56, CD16), complemented with variable drop-in markers/channels to study NK cell phenotype (NKG2A, NKG2C, NKG2D and KIR2D) or NK cell function and activation (CD25, NKp44 and CD107a). Harmonized FACS set-up and data analysis for three different flow cytometers has been established, leading to highly comparable and reproducible data sets using the same PBMC reference samples (n = 6). Further studies of NK cells in fresh or cryopreserved PBMC samples (n = 12) confirmed that freezing and thawing of PBMC samples did not significantly affect NK phenotype or function. In conclusion, our data demonstrate that cryopreserved PBMC samples analysed by standardized FACS panels and harmonized analysis protocols will generate highly reliable data sets for multi-center clinical trials under validated conditions.

Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma.

Natural killer (NK) cells are promising antitumor effector cells, but the generation of sufficient NK cell numbers for adoptive immunotherapy remains challenging. Therefore, we developed a method for highly efficient ex vivo expansion of human NK cells. Ex vivo expansion of NK cells in medium containing IL-2 and irradiated clinical-grade feeder cells (EBV-LCL) induced a 22-fold NK cell expansion after one week that was significantly increased to 53-fold by IL-21. Repeated stimulation with irradiated EBV-LCL and IL-2 and addition of IL-21 at the initiation of the culture allowed sustained NK cell proliferation with 1011-fold NK cell expansion after 6 weeks. Compared to naive NK cells, expanded NK cells upregulated TRAIL, NKG2D, and DNAM-1, had superior cytotoxicity against tumor cell lines in vitro and produced more IFNγ and TNF-α upon PMA/Iono stimulation. Most importantly, adoptive transfer of NK cells expanded using feeder cells, IL-2 and IL-21 led to significant inhibition of tumor growth in a melanoma xenograft mouse model, which was greater than with NK cells activated with IL-2 alone. Intriguingly, adoptively transferred NK cells maintained their enhanced production of IFNγ and TNF-α upon ex vivo restimulation, although they rapidly lost their capacity to degranulate and mediate tumor cytotoxicity after the in vivo transfer. In conclusion, we developed a protocol for ex vivo NK cell expansion that results in outstanding cell yields. The expanded NK cells possess potent antitumor activity in vitro and in vivo and could be utilized at high numbers for adoptive immunotherapy in the clinic.

Automation of cellular therapy product manufacturing: results of a split validation comparing CD34 selection of peripheral blood stem cell apheresis product with a semi-manual vs. an automatic procedure.

BACKGROUND: Automation of cell therapy manufacturing promises higher productivity of cell factories, more economical use of highly-trained (and costly) manufacturing staff, facilitation of processes requiring manufacturing steps at inconvenient hours, improved consistency of processing steps and other benefits. One of the most broadly disseminated engineered cell therapy products is immunomagnetically selected CD34+ hematopoietic "stem" cells (HSCs). METHODS: As the clinical GMP-compliant automat CliniMACS Prodigy is being programmed to perform ever more complex sequential manufacturing steps, we developed a CD34+ selection module for comparison with the standard semi-automatic CD34 "normal scale" selection process on CliniMACS Plus, applicable for 600 × 10(6) target cells out of 60 × 10(9) total cells. Three split-validation processings with healthy donor G-CSF-mobilized apheresis products were performed; feasibility, time consumption and product quality were assessed. RESULTS: All processes proceeded uneventfully. Prodigy runs took about 1 h longer than CliniMACS Plus runs, albeit with markedly less hands-on operator time and therefore also suitable for less experienced operators. Recovery of target cells was the same for both technologies. Although impurities, specifically T- and B-cells, were 5 ± 1.6-fold and 4 ± 0.4-fold higher in the Prodigy products (p = ns and p = 0.013 for T and B cell depletion, respectively), T cell contents per kg of a virtual recipient receiving 4 × 10(6) CD34+ cells/kg was below 10 × 10(3)/kg even in the worst Prodigy product and thus more than fivefold below the specification of CD34+ selected mismatched-donor stem cell products. The products' theoretical clinical usability is thus confirmed. CONCLUSIONS: This split validation exercise of a relatively short and simple process exemplifies the potential of automatic cell manufacturing. Automation will further gain in attractiveness when applied to more complex processes, requiring frequent interventions or handling at unfavourable working hours, such as re-targeting of T-cells.

Automated CD34+ cell isolation of peripheral blood stem cell apheresis product.

BACKGROUND AIMS: Immunomagnetic enrichment of CD34+ hematopoietic "stem" cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products. METHODS: Granulocyte-colony stimulating factor-mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms. RESULTS: Nine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells. CONCLUSIONS: The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy.

BACKGROUND AIMS: Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. METHODS: Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. RESULTS: Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (± 0.9) × 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. CONCLUSIONS: We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device.

BACKGROUND AIMS: The CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαß(+)) and CD19 positive (CD19(+)) cells from apheresis products. METHODS: Investigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2-10.4) × 10(10) mononuclear cells were used with a median 358 (92-1432) × 10(6) CD34(+) cells. There were 24.8% (6.1-45.7%) median TCRαß(+) cells and 4.4% (1.2-11.7%) median B cells in the apheresis product. RESULTS: After depletion, a median 0.00097% (0.00025-0.0048%) of TCRαß(+) cells could be detected, and B cells, as determined as CD20(+) cells, were reduced to 0.0072% (0.0008-0.072%). TCRαß(+) cells were depleted by log 4.7 (3.8-5.5), and B cells were depleted by log 4.1 (3.0-4.7). Recovery of mononuclear cells was 55% (33-77%), and recovery of CD34(+) cells was 73% (43-98%). Recovery of CD56(+)/3(-) natural killer cells was 80% (35-142%), recovery of TCR gamma/delta positive (TCRγδ(+)) T cells was 83% (39-173%) and recovery of CD14(+) cells was 79% (22-141%). Viability of cells was 98% (93-99%) after separation (all values median). CONCLUSIONS: Profound depletion of TCRαß(+) T cells can be achieved with the CliniMACS system. Recovery of CD34(+) stem cells is in the same range than after CD34(+) enrichment and CD3/CD19 depletion. Transplantation with >4 × 10(6) CD34(+) cells/kg can be performed for every patient with 1-5 × 10(4) TCRαß(+) cells/kg and about 5-10 × 10(6) TCRγδ(+) cells/kg with two rounds of apheresis.

Isolation and enrichment of stem cells.

Stem cells have the potential to revolutionize tissue regeneration and engineering. Both general types of stem cells, those with pluripotent differentiation potential as well as those with multipotent differentiation potential, are of equal interest. They are important tools to further understanding of general cellular processes, to refine industrial applications for drug target discovery and predictive toxicology, and to gain more insights into their potential for tissue regeneration. This chapter provides an overview of existing sorting technologies and protocols, outlines the phenotypic characteristics of a number of different stem cells, and summarizes their potential clinical applications.