Automated Good Manufacturing Practice–compliant generation of human monocyte-derived dendritic cells from a complete apheresis product using a hollow-fiber bioreactor system overcomes a major hurdle in the manufacture of dendritic cells for cancer vaccines

ARTICLE IN PRESS Cytotherapy, 2019; 00: 1 13

Automated Good Manufacturing Practice compliant generation of human monocyte-derived dendritic cells from a complete apheresis product using a hollow-fiber bioreactor system overcomes a major hurdle in the manufacture of dendritic cells for cancer vaccines

UGUR USLU*, MICHAEL ERDMANN*, MANUEL WIESINGER, GEROLD SCHULER & BEATRICE SCHULER-THURNER Friedrich-Alexander-Universit€ at Erlangen-N€ urnberg (FAU), Universit€ atsklinikum Erlangen, Department of Dermatology, Erlangen, Germany Abstract Background: Although dendritic cell (DC) based cancer vaccines represent a promising treatment strategy, its exploration in the clinic is hampered due to the need for Good Manufacturing Practice (GMP) facilities and associated trained staff for the generation of large numbers of DCs. The Quantum bioreactor system offered by Terumo BCT represents a hollow-fiber platform integrating GMP-compliant manufacturing steps in a closed system for automated cultivation of cellular products. In the respective established protocols, the hollow fibers are coated with fibronectin and trypsin is used to harvest the final cell product, which in the case of DCs allows processing of only one tenth of an apheresis product. Materials and Results: We successfully developed a new protocol that circumvents the need for fibronectin coating and trypsin digestion, and makes the Quantum bioreactor system now suitable for generating large numbers of mature human monocyte-derived DCs (Mo-DCs) by processing a complete apheresis product at once. To achieve that, it needed a step-by-step optimization of DC-differentiation, e.g., the varying of media exchange rates and cytokine concentration until the total yield (% of input CD14+ monocytes), as well as the phenotype and functionality of mature Mo-DCs, became equivalent to those generated by our established standard production of Mo-DCs in cell culture bags. Conclusions: By using this new protocol for the Food and Drug Administration approved Quantum system, it is now possible for the first time to process one complete apheresis to automatically generate large numbers of human Mo-DCs, making it much more feasible to exploit the potential of individualized DC-based immunotherapy.

Key Words: adoptive cell transfer, cell therapy, cellular therapy, dendritic cells, dendritic cell vaccine, immunotherapy, monocytederived dendritic cells, vaccines

Background Following the recent success of checkpoint-blockade antibodies in, e.g., patients with metastatic melanoma, and chimeric antigen receptor (CAR)-transfected T cells in hematologic malignancies [1], interest in cancer vaccines is also emerging again [2 4]. Novel vaccine strategies provide the opportunity to induce antigen-specific T cells specific for personalized mutated [5 10], non-mutated neo-antigens [6,11], or defined shared tumor and viral antigens [12,13]. The adoptive transfer of antigen-loaded dendritic cells (DCs) is so far the only vaccine strategy with a proven survival benefit. Sipuleucel-T got

approved by the U.S. Food and Drug Administration (FDA) for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer following two phase 3 trials that demonstrated prolongation of survival. This vaccine consists of apheresis fractions enriched in antigen-presenting cells, including primary DCs that are exposed to a Prostatic Acid Phosphatase (PAP)-granlocyte-macrophage colony-stimulating factor (GM-CSF) fusion protein for antigen loading and activation [14,15]. Most groups now use highly enriched DCs as vaccines, including rare primary DCs isolated from blood, and more frequently DCs generated ex vivo

Correspondence: Gerold Schuler, MD, Universit€atsklinikum Erlangen, Department of Dermatology, Ulmenweg 18, D-91054 Erlangen, Germany. E-mail: [email protected] * These authors share the first authorship. (Received 26 February 2019; accepted 16 September 2019) ISSN 1465-3249 Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jcyt.2019.09.001

ARTICLE IN PRESS 2 U. Uslu et al. from either CD34+ precursors or CD14+ monocytes (for a recent review see [16]). The monocyte-derived DCs (Mo-DCs) are most often used because they can be readily produced in large numbers. Furthermore, they have already been shown by various groups to induce T cells recognizing mutated and shared cancer as well as microbial antigens if used as vaccines [7,13,17 23] (for reviews see [16,24,25]). They appear to modulate immunity to promote long-term survival [20,21,26], and their combination with checkpoint inhibitors proved promising as expected [13,27]. Mo-DCs offer the additional advantage that the entire antigenic repertoire of a tumor, which appears to include many non-mutated neo-antigens [6,11] can probably be introduced rather simply, either via loading with dying tumor cells [17,18,28 30] or by transfection of total tumor RNA [19,31]. However, despite the apparent potential of this Mo-DC based vaccination strategy, a practical problem is hampering its further exploitation, namely, the lack of a method that allows automatic processing of a complete apheresis product to yield a large number of well-standardized mature DCs without a need for costly class B and A Good Manufacturing Practice (GMP) room facilities and trained staff. The finding that frequent monocytes can be differentiated into DC progeny ex vivo [32 34] was a significant step forward, allowing for pre-clinical as well as initial clinical studies [35 38]. Following this discovery [32 34], the protocol for Mo-DC generation was continuously optimized. Initially, we learned to avoid fetal calf serum and to use monocyte-conditioned medium to induce DC maturation, which is accomplished today by a defined inflammatory cocktail [39]. Next, the production of cryopreserved aliquots of antigen-preloaded, mature DCs ready for clinical use was established [40]. Furthermore, counterflow elutriation using the Elutra cell separation system by Terumo BCT was used for high-level, clean-room independent enrichment of monocytes from apheresis [41, 42]. Despite all the improvements, to generate Mo-DCs several manual steps are still required, e.g., transfering of monocytes into culture bags in appropriate cell concentrations, culturing of monocytes, as well as differentiating and maturing of DCs, harvesting of DCs and antigen loading. All these procedures bear risks of, e.g., contamination and errors, and require costly high-level class A and B GMP room facilities and associated trained staff. To further optimize GMP-compliant Mo-DC generation of elutriated monocytes, particularly by reducing the need for high-level cleanroom resources, we recently extensively tested the CliniMACS Prodigy designed by Miltenyi Biotec for its suitability to generate Mo-DCs, which also

represents a closed and largely automated system integrating a magnetic cell separation system and a cell cultivation chamber [43]. Although the phenotype and functionality of mature Mo-DCs generated by the CliniMACS Prodigy system were equivalent to those generated by our in-house established standard protocol, only one fourth of an apheresis could be processed at once due to the limited size of the cultivation chamber, which in our view represents a significant practical drawback of the CliniMACS Prodigy approach [43]. The Quantum cell expansion system by Terumo BCT is a European Conformity (CE) marked and FDA-listed device that is also a closed, automated and integrated system, allowing a controlled cell culture. At its core is a cylindrical three-dimensional, functionally closed cartridge containing hollow fibers, with flexible protocol management allowing the process optimization for individual cell types [44 48]). The bioreactor hollow fibers of the Quantum system have a total surface area of 2.1 m2 with a total volume of 200 mL inside the hollow fibers (intra-capillary site [IC-site]) and 300 mL outside the fibers (extra-capillary site [EC-site]). The IC-site of the hollow fibers is separated from the EC-site with a membrane impervious for cells, permeable for glucose and lactate, and semipermeable for different cytokines, allowing continuous control over gases, temperature, cell media and waste removal [46,49,50]. It has been already successfully applied by many groups in cell expansion of adherent cells including human embryonic stem cells, human bone marrow derived mesenchymal stromal cells and neural stem cells [44,45,49,51]. Recently, pre-clinical experiments found that the Quantum system is also promising for CAR T-cell expansion [52]. Initial attempts reported in 2014 (available as an abstract and online from Terumo CBT [53]) to generate DCs in the Quantum hollow-fiber system from CD14+ monocytes were successful, but have not been pursued any further. We assume that the fibronectin coating of the hollow fibers to adhere monocytes and the final trypsin incubation step to harvest Mo-DCs appeared too complicated given the fact that only about one tenth of an apheresis product could be processed. Building on our experience in generating DCs from monocytes, we reasoned that it might be possible to circumvent the fibronectinmediated adherence step as well as the use of trypsin to harvest DCs . The aim of the study we describe here was, therefore, to develop a new protocol for the Quantum system that would allow without any fibronectin or trypsin use the automated closed-system manufacturing of human Mo-DCs for the potential clinical use in antigen-specific immunotherapy of

ARTICLE IN PRESS Generation of MoDCs using a hollow-fiber bioreactor system 3 cancer or infections. DC production for a clinical application requires the processing of a complete leukapheresis product to generate a large number of DCs of a single batch that can be aliquoted and cryopreserved as needed for later use. To become acquainted with the new Quantum system, we first performed a series of tests for process development (leukapheresis runs 1 5) followed by experiments for process validation (leukapheresis runs 6 8). Phenotype, antigen presentation and functionality of generated Mo-DCs were analyzed and compared with Mo-DCs generated in parallel in cell culture bags using our established standard protocol [41].

Methods Leukapheresis and enrichment of monocytes by using the Elutra cell seperation system Leukapheresis was performed after informed consent and approval by the institutional review board of the Friedrich-Alexander-Universit€at (FAU) ErlangenN€ urnberg (reference number 4602) from healthy blood donors as previously described [54]. We enriched peripheral blood monocytes from leukapheresis as previously described using the Elutra cell separation system [41]. In summary, we loaded white blood cells (WBCs) into the elutriation chambers, and using a constant centrifugation speed of 2400 rpm and increasing the cell medium flow rate step-by-step (37 mL/min, 97.5 mL/min, 103.4 mL/min and 103.9 mL/min), five specific cell fractions were generated [41]. While fraction #3, for instance, mainly contained CD3+ T cells, the final fraction #5 mainly contained monocytes, which we used for generating Mo-DCs [41]. The total elutriation time was 1 h [41]. The percentage of monocytes was determined using flow cytometry (FACSCalibur 4CA and Cellquest software, BD Biosciences, Heidelberg, Germany) and labeling with anti-CD14 antibody (BD Biosciences).

Cultivation of monocytes and differentiation into mature DCs using cell culture bags Enriched monocytes were cultivated at a final concentration of 1 £ 106 cells per mL GMP-grade complete medium (RPMI 1640 [Lonza, Basel, Switzerland] containing 1% autologous human plasma) with up to 1200 £ 106 cells in cell culture bags (Cell-Max GmbH, Munich, Germany) as previously described [43], or by using the Quantum system with about the same cell concentration for appropriate comparison of both cultivation methods. For complete medium preparation, human autologous plasma was heat-inactivated at 56˚C for 30 min and sterile filtered. GMP-compliant cultivation of

enriched monocytes in cell culture bags has been previously described by us [43]. Cultivation of monocytes and differentiation into mature DCs using the Quantum hollow-fiber bioreactor system This section describes the optimized and final Quantum method, which was used for the process validation (leukapheresis run 6 8). The first five runs were performed for process development to optimize the Quantum approach for Mo-DC differentiation, e.g., optimization of the media exchange rate, cytokine concentration and cytokine addition into the EC-site as well as the IC-site. The detailed step-by-step optimization process is described in the Results section. For cultivation of Mo-DCs, the GMP cell expansion set (Terumo BCT, Lakewood, CO, USA) was loaded onto the Quantum system and primed according to the manufacturer’s recommendation with priming medium RPMI 1640 (Lonza) containing 1% human serum albumin. After 18 h overnight incubation, culture medium for DC generation [34] - RPMI containing 1% human plasma, GM-CSF 800 IU/mL (Leukine, Genzyme, Seattle, USA) and interleukin (IL)-4 250 IU/mL (CellGenix, Freiburg, Germany) was connected to the expansion set and the priming medium was exchanged. The incubator was set at 37˚ C and supplied with a premixed gas consisting of 5% CO2, 20% O2 and 85% N2 at approximately 45 psi. DC medium was equilibrated for at least 60 min before cells were seeded. We premixed the Elutra-isolated monocytes with cytokines calculated based on the total cell number (GM-CSF 800 IU/106 cells and IL-4 250 IU/106 cells) per total volume of 150 mL DC medium. We next connected the resulting cell suspension to the expansion set and loaded it into the IC-site of the fiber bioreactor, rinsed with 50 mL of EC medium followed by a 4-h stationary incubation. After that, the EC medium exchange rate was set to 0.5 mL/min, and the bioreactor rocker was set for a slow 180˚ turnover motion every 10 min. EC medium was protected from light, and the EC media flow direction was changed every 12 h for 5 min to minimize the drop-out of cells. After 2 days of incubation, fresh DC medium with different cytokine concentrations was connected to the IC-site (GM-CSF 800 IU/106 cells, IL-4 250 IU/106 cells) and the EC-site (GMCSF 800 IU/mL, IL-4 250 IU/mL). A total of 150 mL IC feeding media was transferred to the ICsite of the bioreactor. The EC media exchange rate was set at 0.5 mL/min, and the bioreactor rocker was set in motion as described before. We repeated the feeding step after 3 days by adding maturation medium to the IC-site (200 mL with GM-CSF 800 IU/106 cells, IL-4 250 IU/106 cells,

ARTICLE IN PRESS 4 U. Uslu et al. IL-1b 200 IU/106 cells, IL-6 1000 IU/106 cells, tumor necrosis factor [TNF]-alpha 10 ng/106 cells and Prostaglandin E2 (PGE-2) 1 mg/106 cells) as well as the EC-site (GM-CSF 800 IU/mL, IL-4 250 IU/mL, IL-1b 200 IU/mL, IL-6 1000 IU/mL, TNF-alpha 10 ng/mL and PGE-2 1 mg/mL). The cytokines IL-1b and IL-6 were purchased from CellGenix, TNF-alpha Beromun from B€ ohringer Ingelheim (Ingelheim, Germany) and PGE-2 from Pfizer (Vienna, Austria). The EC media exchange rate was set to 0.5 mL/min with the bioreactor in motion and incubation of approximately 20 h. Mature DCs were harvested by rinsing the system with 2˚C 4˚C phosphate-buffered saline (PBS) ethylenediaminetetraacetic acid 1 mmol/L solution (Lonza) and a 30-min incubation followed by another rinsing step. Morphology and phenotyping of mature DCs For phenotyping, DCs were stained in PBS (Lonza) containing 1% fetal bovine serum (Sigma Aldrich). Cell-surface staining was performed for the expression of CD80 (clone L307.4), CD83 (clone HB15e), CD86 (clone 2331) and HLA-DR (clone G46-6, all from BD Biosciences). For isotype control, immunoglobulin (Ig)G1 kappa (clone MOPC-21) was used for CD80, CD83 and CD86, whereas IgG2a (clone G155-178, both from BD Biosciences) served as a control for HLA-DR. We performed staining with 7-AAD (BD Biosciences) to exclude dead cells. Cell surface staining was performed as described [41]. Mean fluorescence intensity and the number of positive cells was determined by using FACSCalibur 4CA with CellQuest Pro Software (BD Biosciences). An example of the applied gating strategy for flow cytometry data analysis is displayed in Supplementary Figure 1. After setting a gate to exclude debris and to identify Mo-DCs, which due to its size appears at higher side-scattered light/forward-scattered light levels (Supplementary Figure 1; Gate 1), non-viable cells were excluded in the next step by setting a gate into 7-AAD cells (Supplementary Figure 1; Gate 2). Gates for marker positive cells were set according to isotype controls. Images of mature Mo-DCs were taken to illustrate the DC-morphology using the optical microscope Leica DM IL LED (original magnification £ 400). Evaluation of DC stability and survival (“washout test”) For evaluation of DC stability and survival (“washout test”), 1 £ 106 mature DCs were incubated in medium without cytokines for 24 h, as previously established [40]. The yield of viable non-adherent, i.e., mature DCs, was determined by manual cell counting in a Neubauer chamber and staining with

trypan blue. The percentage yield was determined using the following formula: % yield viable = (count of viable trypan blue negative cells after 24 h of incubation in medium without cytokines: count of viable cells before incubation) £ 100. RNA transfection and green fluorescent protein expression of Mo-DCs Mature DCs were electroporated using Gene Pulser Xcell (Bio-Rad) and 4-mm electroporation cuvettes (Peqlab, Erlangen, Germany). DCs were electroporated at 4 £ 107 cells/mL for 1 ms, and 500 V (square-wave pulse) in OptiMem (Life Technologies, Carlsbad, CA, USA) with RNA encoding for green fluorescent protein (GFP) as previously described [55]. Immediately after electroporation, cells were cultured for 4 h in RPMI 1640 medium (Lonza) supplemented with 1% autologous plasma and 2 mmol/L L-glutamine (Life Technologies, Paisley, Scotland). Staining with 7-AAD was performed to exclude dead cells. Electroporated DCs were then analyzed for GFP autofluorescence. For control, we used non-transfected Mo-DCs. Cells were analyzed using flow cytometry using FACS Calibur 4CA and CellQuest Pro Software (BD Biosciences). Primary allogeneic mixed lymphocyte reaction For mixed lymphocyte reaction (MLR) assays, allogeneic CD3+ T cells, obtained from fraction 3 of elutriated leukapheresis products (purity of %CD3+ cells was confirmed beforehand), were labeled with carboxyfluorescein succinimidyl ester (CFSE; 5 mmol/L, CellTrace, Molecular Probes, Eugene, Oregon, USA) according to manufacturer’s recommendations. T cells were then co-incubated in MLR medium (consisting of RPMI 1640 [Lonza] supplemented with 5% heat-inactivated human serum type AB [SigmaAldrich], 1% Penicillin/Streptomycin/l-glutamine [Sigma-Aldrich] and 10 mmol/L HEPES [Lonza]) with mature DCs at a DC:T-cell ratio of 1:20 for 3 days in triplicates. After 1, 2 and 3 days of culture, cells were harvested and stained with anti-CD3 (BD Biosciences) and anti-CD8 (BD Biosciences) antibodies. Staining with 7AAD (BD Biosciences) was performed to exclude dead cells. Cells were analyzed by performing flow cytometry using FACS Calibur 4CA (BD Biosciences) and FCS Express Software (De Novo Software, Glendale, California, USA). 7AAD CD3+CD8+ T cells were selected through gating and analyzed for the intensity of CFSE fluorescence to quantify CD8+ T-lymphocyte proliferation [56]. Unstimulated T cells served as a control. In addition to MLR analysis via CFSE labeling, MLR assays were also performed and evaluated by

ARTICLE IN PRESS Generation of MoDCs using a hollow-fiber bioreactor system 5 pulsing with [3H]-thymidine. To this end, triplicates of 2 £ 105 allogeneic CD3+ T cells were co-cultured in 96-well flat cell culture plates with Mo-DCs at DC:T-cell ratios of 1:6, 1:20, 1:60 and 1:200 for 72 h in MLR medium, and finally pulsed with [3H]thymidine (1 mC/well) for 20 h. Again, unstimulated T cells served as control. We harvested cultures onto glass fiber filtermates using an ICH-110 harvester (Inotech), which were then evaluated in a 1450 microplate counter (Wallac).

Stimulation of MelanA- specific cytotoxic T cells Mo-DCs were pulsed with the HLA-A0201-restricted Melan-A.A2 peptide in GMP-quality (ELAGIGILTV, Clinalfa, Laufelfingen, Switzerland) at 2 £ 106 cells/mL and 10 mmol/L peptide in medium supplemented with GM-CSF (800 IU/mL) and IL-4 (250 IU/mL) for 2 h and then co-cultured with autologous CD3+ T cells at a DC:T-cell ratio of 1:20 for 10 days. CD3+ T cells were obtained from fraction 3 of elutriated leukapheresis products. T cells stimulated with Mo-DCs that were not loaded with MelanA.A2 peptide served as a negative control. Cells were harvested at day 4, 7 and 10 and were stained with anti-CD3 (BD Biosciences), anti-CD8 (BD Biosciences) and MelanA.A2 pentamer (ELAGIGILTV, Proimmune, Oxford, United Kingdom). Staining with 7-AAD (BD Biosciences) was performed to exclude dead cells. 7AAD CD3+CD8+ T cells were

selected through gating and analyzed for Melan-A.A2 pentamer binding to quantify antigen-specific CD8+ T-lymphocyte proliferation using FACS Calibur 4CA and CellQuest Pro Software (BD Biosciences). Figure preparation and statistical analysis Graphs were created using Microsoft Excel. We performed statistical analysis using GraphPad Prism, version 7 (GraphPad Software). The Student t test was used to determine P values. Results Schematic representation of Mo-DC generation by the Quantum bioreactor versus the standard cell culture bag system Leukapheresis was performed as previously described [54], and monocytes were enriched by using the Elutra cell separation system. We evaluated the total volume and WBC count of each leukapheresis run and after its elutriation the total cell count and percentage of CD14+ cells in fraction five, which contained the most monocytes (data are shown in Supplementary Table 1). After elutriation, monocytes were then differentiated to Mo-DCs either in the closed Quantum culture system and for direct comparison according to our in-house established standard protocol in cell culture bags (Figure 1A). For each run, we used about the same amount of

Figure 1. Schematic representation of the experimental sequence. (A) After leukapheresis, monocytes were enriched by using the Elutra cell separation system. Enriched monocytes were then differentiated to Mo-DCs in a Quantum hollow-fiber bioreactor or by using an in-house established protocol in cell culture bags. In the next step, phenotypic analysis and functional assays were performed to compare Mo-DCs generated by the Quantum system with Mo-DCs generated by using the standard protocol. (B) The bioreactor hollow fibers of the Quantum bioreactor system have a total surface area of 2.1 m2 with a total volume of 200 mL inside the hollow fibers (IC) and a total volume of 300 mL outside the fibers (EC), separated with a membrane permeable for glucose and lactate and semi-permeable for cytokines. Cytokines were added at regular intervals in the IC as well as the EC and media-exchange was performed as indicated.

ARTICLE IN PRESS 6 U. Uslu et al. monocytes in the Quantum system to ensure proper comparison of DC maturation to the in-house established standard method. Figure 1B shows the final optimized schematic of the Quantum approach, which we used for the leukapheresis runs 6 8 (process validation) described below. Process development (leukapheresis runs 1 5) of the Quantum hollow-fiber bioreactor approach for appropriate Mo-DC generation To get acquainted with the new Quantum system, the first five leukapheresis runs were performed for process development to adapt the Quantum approach for DC differentiation. Initially, we performed a “priming step” with fibronectin as recommended by the manufacturer before loading of the enriched monocyte fraction (Supplementary Table 2). The medium exchange rate was set to 0.1 mL/min and the cytokines GM-

CSF and IL-4 were added to the IC medium at a concentration of 400 IU/106 cells (GM-CSF) and 125 IU/106 cells (IL-4), whereas no cytokines were added to the EC medium (Supplementary Table 2). Mo-DCs were isolated after cultivation using trypsin as used for other cell preparations in the hollow-fiber bioreactor system [44 53] (Supplementary Table 2). First results showed that the lactate concentration, measured with the Lactate Plus Meter by Nova Biomedical, was high in comparison with culture in cell culture bags (Figure 2A, Supplementary Table 3). Also, Mo-DCs generated by Quantum were inferior in the “washout test” with a yield of only 60% as opposed to »90% with our standard protocol (Supplementary Table 3). As a consequence, we performed subsequent runs using RPMI 1640 containing 1% human serum albumin instead of fibronectin as the “priming” medium (Supplementary Table 2). Additionally, the medium exchange rate, as well as cytokine concentrations,

Figure 2. Process development of the Quantum bioreactor system for the generation of Mo-DCs. After leukapheresis, monocytes were enriched by using the Elutra cell separation system and then differentiated to Mo-DCs in the Quantum hollow-fiber bioreactor or by using an in-house established protocol in cell culture bags. (A) Lactate concentrations of first and second runs were measured in the cell medium of the Quantum bioreactor system at indicated time points. For comparison, the lactate concentration of cell culture bag medium was analyzed. (B) The concentration of the cytokines GM-CSF and IL-4 was measured in the IC medium, as well as in the EC medium of the Quantum hollow-fiber bioreactor system in the fourth run, 24 h after addition of the cytokines. Arrows indicate the time point of cytokine addition (for run 4: 2400 IU/106 cells [GM-CSF] and 750 IU/106 [IL-4]). For comparison, the cytokine concentration in cell culture bag medium is also shown.

ARTICLE IN PRESS Generation of MoDCs using a hollow-fiber bioreactor system 7 were increased (Supplementary Table 2), and Mo-DCs were harvested by washing them out with cold medium (2˚C 4˚C) instead of using trypsin (Supplementary Table 2). Following the modification, we found that the lactate concentrations (Figure 2A), as well as the stability of the Mo-DCs, became equivalent to the reference Mo-DCs. However, new problems emerged because these Mo-DCs now showed an incomplete phenotypic maturation with much lower CD80, CD83, CD86 and HLADR expression (Supplementary Table 3) when compared with the reference Mo-DCs. By further increasing the cytokine concentrations, as well as the medium exchange rate in the subsequent third and fourth run, no significant additional improvement was observed (Supplementary Tables 2 and 3). Analysis of the cytokine concentrations in both the IC as well as EC compartment of the Quantum system at different time points revealed a lower amount of GM-CSF and IL-4 in the IC and EC medium of the Quantum system when compared with the concentrations in cell culture bag medium (Figure 2B). Also, the cytokine concentrations in the EC medium were higher than in the IC medium (Figure 2B), indicating that the capillary membrane was permeable for the used cytokines and consequently the cytokines got washed out by the medium exchange, resulting in a concentration too low for allowing reliable DC differentiation and maturation. In the fifth run, the cytokines GM-CSF and IL-4 were, therefore, now also added to the EC medium (Supplementary Table 2). Following this rational

step-by-step optimization, all DC tested parameters became equal to our reference Mo-DCs generated by using our in-house established standard protocol. Following these experiments for process development, we then performed further experiments for process validation (leukapheresis runs 6 8) as described below to test the reproducibility of the optimized settings. Mo-DCs generated by the Quantum hollow-fiber bioreactor show a mature morphology and a mature phenotype Cells cultured in the Quantum system resulted in a yield of 27.8% mature DCs (related to input monocytes) with CD83 expression in 92.0% of these cells (Supplementary Table 4). The total yield of mature Mo-DCs was slightly higher when the in-house established standard protocol was used without reaching statistical significance (Supplementary Table 4). The viability of Mo-DCs analyzed in the washout test (developed by us to test the stability of DCs in the absence of added cytokines [40]) was similar in both methods (Supplementary Table 4). The phenotype of DCs generated by our standard protocol indicated a trend toward a more mature phenotype because the cell surface expression of CD80, CD83, CD86 and HLA-DR was in general higher without reaching statistical significance (Figure 3, Supplementary Table 4). Transmission and phase microscopy of Mo-DCs generated by the Quantum hollow-fiber bioreactor or

Figure 3. Maturation marker expression on the surface of Mo-DCs. Monocytes isolated by elutriation were either cultivated in the Quantum hollow-fiber bioreactor system or according to the in-house established standard protocol in cell culture bags. After 7 days of culture we analyzed the mature Mo-DC progeny using cell surface flow cytometry staining: (A) percent positive cells out of living cells and (B) mean intensity cell surface expression of the indicated DC maturation markers. N = 3 § SEM is shown.

ARTICLE IN PRESS 8 U. Uslu et al.

Figure 4. Morphology of generated Mo-DCs. Images show the morphology of mature Mo-DCs by using the Quantum approach and the in-house established protocol using cell culture bags (phase contrast microscopy using a Leica DM IL LED Microscope; original magnification £ 400).

the standard protocol showed a mature morphology of Mo-DCs with both methods, notably non-adherence and typical motile veils (Figure 4). Mo-DCs generated in the Quantum hollow-fiber bioreactor can express proteins encoded by the transfected messenger RNA After isolation and enrichment of monocytes using the Elutra cell separation system and subsequent generation of Mo-DCs via Quantum or the in-house established standard protocol, functionality of Mo-DCs was analyzed. Mo-DCs were electroporated with messenger RNA (mRNA) encoding for GFP and analyzed for GFP expression 4 h after transfection. The results showed that GFP was expressed in Mo-DCs generated by either Quantum or the in-house established standard protocol (Supplementary Figure 2). There was no significant statistical difference in expression efficacy when comparing both methods (Supplementary Table 5). However, electroporated Mo-DCs generated by Quantum showed a trend toward higher GFP expression (Supplementary Figure 2, Supplementary Table 5). There were no significant differences in cell viability after RNA electroporation of Mo-DCs (Supplementary Table 5). Mo-DCs generated in the Quantum hollow-fiber bioreactor can stimulate allogeneic T-cell proliferation In the next step, the capacity of Mo-DCs, generated by the Quantum bioreactor or the in-house

established standard protocol, to stimulate T-cell proliferation was tested in MLR assays. Therefore, allogeneic CD3+ T cells were labeled with CFSE and co-incubated with Mo-DCs at a DC:T-cell ratio of 1:20 for 3 days. For readout, viable (i.e., 7AAD negative) CD3+CD8+ T cells were selected through gating and then analyzed for the intensity of CFSE fluorescence to quantify the proliferation of allogeneic CD8+ T cells. The results indicated that MoDCs of three independent donors generated by either method were effective in stimulating allogeneic CD8+ T cells (Figure 5A and 5B and Supplementary Figure 3). Co-incubation with Mo-DCs generated by Quantum resulted in a slightly higher T-cell stimulation and proliferation yet without reaching statistical significance in three independent experiments (Figure 5A and 5B, Supplementary Figure 3, Supplementary Table 6). In parallel, we performed MLRs by co-culturing allogeneic CD3+ T cells with the Mo-DCs of the three donors at a DC:T-cell ratio of 1:6, 1:20, 1:60 and 1:200 for 72 h, and finally pulsed with [3H]-thymidine for 20 h. These results using this readout also revealed that Mo-DCs generated by either method were effective in stimulating allogeneic CD8+ T cells (Figure 5C, Supplementary Table 7). Next, we tested the capacity of Mo-DCs generated in the Quantum bioreactor or in cell culture bags to stimulate cognate antigen-specific T cells. HLA-A0201+ Mo-DCs were pulsed with Melan-A. A2 peptide (ELAGIGILTV) for 2 h and then co-cultured with autologous CD3+ T cells at a DC:T-cell

ARTICLE IN PRESS Generation of MoDCs using a hollow-fiber bioreactor system 9

Figure 5. Allogeneic MLR after incubation of Mo-DCs with T cells. (A+B) Allogeneic CD3+ T cells were labeled with CFSE and co-incubated with Mo-DCs at a DC:T-cell ratio of 1:20 for 3 days. After 1, 2 and 3 days of culture, cells were harvested and stained with anti-CD3, anti-CD8 and 7AAD. 7AAD CD3+CD8+ T cells were selected through gating and analyzed for the intensity of CFSE fluorescence to quantify CD8+ T-lymphocyte proliferation. Unstimulated T cells were used as control. (A) Dot plots of 1 representative of 3 independent experiments and (B) average values of 3 independent experiments § SEM at indicated time points are shown. (C) CD3+ T cells were in parallel co-cultured with allogeneic Mo-DCs at a DC:T-cell ratio of 1:6, 1:20, 1:60 and 1:200 for 72 h and were pulsed with [3H]-thymidine for the final 20 h. Cultures were then harvested in glass fiber filtermates using an ICH-110 harvester, and counted in a 1450 microplate counter. Unstimulated T cells served as control. Average values of 3 independent experiments § SEM are shown.

ratio of 1:20 for 10 days. We then harvested cells at day 4, 7 and 11 to analyze antigen-specific T-cell proliferation. The pentamer staining results demonstrated the expected expansion of Melan A specific CD8+ T cells when either of the two methods was used for Mo-DC generation (Figure 6 and Supplementary Figure 4), without statistically significant differences in three independent experiments (Supplementary Table 8). Mo-DC generation in the Quantum hollow-fiber bioreactor is associated with higher costs for supplies but causes less GMP-related costs A summary of the costs for consumables revealed that Mo-DC generation using the Quantum approach was approximately twice as expensive as Mo-DCs generated using our in-house established standard method in cell culture bags (Supplementary Table 9). Notable are especially a higher amount of cytokine consumption and the purchase of expensive Quantum-related products, which are needed to run the device. It has to be taken in account, however, that the cultivation in culture bags when the standard

protocol is used requires expensive class A and B GMP-compliant clean-room facilities and higher workload for trained personnel, which is not listed in Supplementary Table 9. On the other side, the costs of buying the Quantum device have also to be taken into the equation but constitute a one-time expense. Discussion We describe here a novel protocol for the Quantum hollow-fiber bioreactor system that allows for the first time processing of a complete apheresis product at once to generate a large number of mature, immunogenic human Mo-DCs for potential use as DC vaccines in antigen-specific immunotherapy of cancer or infections. This is a significant advantage because large numbers of DCs must be generated in a clinical setting for repeated vaccinations [20]). To the best of our knowledge, this has not been proven for any other CE-marked and FDA-approved bioreactor. The phenotype of DCs generated by the in-house established standard protocol showed, in general, a trend toward a slightly more mature phenotype compared with those cultivated in the Quantum system

ARTICLE IN PRESS 10 U. Uslu et al.

Figure 6. Expansion of autologous MelanA-specific CD8+ T cells after incubation with peptide-loaded Mo-DCs. Monocytes isolated using the Elutra cell separation system were either cultivated in the Quantum hollow-fiber system or according to the in-house established standard protocol in cell culture bags to obtain mature Mo-DCs. DCs were pulsed with the HLA-A0201-restricted Melan-A.A2 peptide at 2 £ 106 cells/mL for 2 h. Peptide-loaded DCs were then co-cultured with autologous CD3+ T cells at a DC:T-cell ratio of 1:20 for 10 days. After 4, 7 and 10 days of stimulation, T cells were harvested and stained with anti-CD3, anti-CD8 and Melan-A.A2 specific pentamer. 7AAD staining was performed to exclude dead cells. 7AAD CD3+CD8+ T cells were selected through gating and analyzed for Melan-A pentamer binding to quantify antigen-specific CD8+ T-lymphocyte proliferation. Unstimulated T cells served as control. (A) Dot plots of 1 representative of 3 independent experiments with percentage of Melan A positive CD8+ T cells of the whole CD8+ T-cell population are illustrated. (B) Average values of the 3 independent experiments § SEM at indicated time points are shown.

(Figure 3). Of note, we had perceived a marginally lower expression of such markers also for Mo-DCs generated in the CliniMACS Prodigy system [43]. These congruent observations suggest that the absence of an actively supported flow in the standard cell culture bags and/or the distinct biomaterials play a role (the standard bags are made of flexible Ethyl Vinyl Acetate material, whereas the Quantum bioreactor and Prodigy chamber are each made of hard plastic). Both of these explanations would be consistent with our current understanding of DC biology [57] as well as published reports on factors influencing phenotypic maturation of DCs [58 60].

Importantly, functional analysis regarding priming of antigen-specific T cells (Figures 5 and 6) showed that the Mo-DCs generated in the Quantum, as well as those in the Prodigy system [43], are at least equal to those generated by using our in-house established culture bag method. This is consistent with Steinman’s notion that phenotypic maturation is not identical to functional maturation or immunogenicity, and that functional tests are more relevant to assure that the DCs exhibit immunogenicity [57,61]. The latter is the unique feature of functionally mature DCs and required to induce immunity rather than tolerance by vaccines [57,61]. Such functionally

ARTICLE IN PRESS Generation of MoDCs using a hollow-fiber bioreactor system 11 mature DCs typically exhibit a non-adherent phenotype and actively motile veils [57], and indeed the morphology of the Mo-DCs generated in the Quantum bioreactor look similar (Figure 4) despite the slightly lower expression of phenotypic maturation markers. At our department, monocyte enrichment by using the Elutra cell separation system is routinely used in a class C clean room by local GMP requirements. Further cultivation in culture bags and incubators requires, however, class B and A clean-room facilities. Mo-DC generation by using the Quantum bioreactor can be performed in a class C clean room, reducing the need for expensive class B and A cleanroom resources, which is a substantial advantage. Additionally, the Quantum bioreactor, like other closed systems, reduces the risk of contamination and errors, and hands-on time is lower due to the automation. Further aspects of DC generation using the Quantum bioreactor for potential use in cancer immunotherapy not addressed in our current work include antigen-loading and freezing of the final cell product. In our current clinical protocols, we, like others, load DCs with antigens by adding peptides or by electroporation of total tumor mRNA or mRNA coding for defined or mutated antigens. These antigen-loading steps still need to be performed outside of the closed Quantum system at present. Companies are, however, developing and optimizing closed systems for transfecting cells in a GMP-compliant fashion. Miltenyi Biotec Inc. offers the CliniMACS Electroporator designed for fully automated GMPcompliant cell electroporation, but it can only be operated with the CliniMACS Prodigy system described above. Another commercially available and versatile approach represents the MaxCyte GT flow transfection system, which can be used to transfect up to 1 £ 1010 cells within 15 20 min by applying a computer-controlled protocol [62]. The system is pre-configured with protocols optimized for specific cell type and application and uses sterile, singleuse, closed-system processing assemblies [62], and could be used in conjunction with Quantum bioreactor-produced DCs. Conclusions In conclusion, we have worked out a new protocol that allows using the automated Quantum hollowfiber bioreactor system for optimized generation of clinical quality Mo-DCs. It is now possible to produce high-quality Mo-DCs even from one complete apheresis at once. Furthermore, the need for costly class B and A clean-room resources is reduced. The Mo-DCs proved equivalent concerning yield, phenotype and T-cell stimulatory capacity to standard Mo-

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Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j. jcyt.2019.09.001.