METHODS published: 20 March 2020 doi: 10.3389/fimmu.2020.00482 Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed Semi-automatic Bioreactor: Experience From an Academic Phase Edited by: I Clinical Trial Rayne Rouce, Baylor College of Medicine, Maria Castella 1,2,3*, Miguel Caballero-Baños 4,5† , Valentín Ortiz-Maldonado 1† , United States Europa Azucena González-Navarro 4 , Guillermo Suñé 1,2 , Asier Antoñana-Vidósola 1,2 , Reviewed by: Anna Boronat 2,4 , Berta Marzal 4 , Lucía Millán 4 , Beatriz Martín-Antonio 1,2 , Joan Cid 2,6 , Pappanaicken R. Kumaresan, Miquel Lozano 2,6 , Enric García 3,7 , Jaime Tabera 3,8 , Esteve Trias 8 , Unai Perpiña 9 , University of Texas MD Anderson Josep Ma Canals 2,9,10 , Tycho Baumann 1 , Daniel Benítez-Ribas 2,4 , Elías Campo 2,10,11,12,13 , Cancer Center, United States Sanjivan Gautam, Jordi Yagüe 2,4,10 , Álvaro Urbano-Ispizua 1,2,10,14,15 , Susana Rives 16,17‡ , Julio Delgado 1,2,10,12‡ National Cancer Institute, National and Manel Juan 2,3,4,5,10,15*‡ Institutes of Health (NIH), 1 Department of Hematology, Institut Clínic de Malalties Hematològiques i Oncològiques, Hospital Clínic de Barcelona, United States Barcelona, Spain, 2 Institut D’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain, 3 Blood and Tissue *Correspondence: Bank (BST), Barcelona, Spain, 4 Department of Immunology, Centro de Diagnóstico Biomédico, Hospital Clínic de Barcelona, Manel Juan Barcelona, Spain, 5 Hospital Sant Joan de Déu, Barcelona, Spain, 6 Department of Hemotherapy and Hemostasis, Institut mjuan@clinic.cat Clínic de Malalties Hematològiques i Oncològiques, Hospital Clínic de Barcelona, Barcelona, Spain, 7 Apheresis Unit, Hospital Maria Castella Sant Joan de Déu de Barcelona, Barcelona, Spain, 8 Unit of Advanced Therapies, Hospital Clínic de Barcelona, Barcelona, mcastella@clinic.cat Spain, 9 Stem Cells and Regenerative Medicine Laboratory, Department of Biomedical Sciences, Production and Validation Center of Advanced Therapies (Creatio), Universitat de Barcelona, Barcelona, Spain, 10 Universitat de Barcelona, Barcelona, † These authors have contributed Spain, 11 Department of Pathology, Hospital Clínic de Barcelona, IDIBAPS, Barcelona, Spain, 12 Centro de Investigación equally to this work Biomedical en Red de Cancer, Barcelona, Spain, 13 Institució Catalana de Recerca i Estudis Avancats, Barcelona, Spain, ‡ Theseauthors share 14 Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute, Universitat de Barcelona, senior authorship Barcelona, Spain, 15 Immunotherapy Unit Blood and Tissue Bank-Hospital Clínic de Barcelona, Barcelona, Spain, 16 Department of Pediatric Hematology and Oncology, Hospital Sant Joan de Déu, Barcelona, Spain, 17 Institut de Recerca Specialty section: Sant Joan de Déu, Barcelona, Spain This article was submitted to Cancer Immunity and Immunotherapy, Development of semi-automated devices that can reduce the hands-on time and a section of the journal Frontiers in Immunology standardize the production of clinical-grade CAR T-cells, such as CliniMACS Prodigy Received: 27 September 2019 from Miltenyi, is key to facilitate the development of CAR T-cell therapies, especially Accepted: 02 March 2020 in academic institutions. However, the feasibility of manufacturing CAR T-cell products Published: 20 March 2020 from heavily pre-treated patients with this system has not been demonstrated yet. Citation: Castella M, Caballero-Baños M, Here we report and characterize the production of 28 CAR T-cell products in the Ortiz-Maldonado V, context of a phase I clinical trial for CD19+ B-cell malignancies (NCT03144583). The González-Navarro EA, Suñé G, system includes CD4-CD8 cell selection, lentiviral transduction and T-cell expansion Antoñana-Vidósola A, Boronat A, Marzal B, Millán L, Martín-Antonio B, using IL-7/IL-15. Twenty-seven out of 28 CAR T-cell products manufactured met the full Cid J, Lozano M, García E, Tabera J, list of specifications and were considered valid products. Ex vivo cell expansion lasted an Trias E, Perpiña U, Canals JM, Baumann T, Benítez-Ribas D, average of 8.5 days and had a mean transduction rate of 30.6 ± 13.44%. All products Campo E, Yagüe J, Urbano-Ispizua Á, obtained presented cytotoxic activity against CD19+ cells and were proficient in the Rives S, Delgado J and Juan M (2020) secretion of pro-inflammatory cytokines. Expansion kinetics was slower in patient’s cells Point-Of-Care CAR T-Cell Production (ARI-0001) Using a Closed compared to healthy donor’s cells. However, product potency was comparable. CAR Semi-automatic Bioreactor: T-cell subset phenotype was highly variable among patients and largely determined by Experience From an Academic Phase I Clinical Trial. Front. Immunol. 11:482. the initial product. TCM and TEM were the predominant T-cell phenotypes obtained. 38.7% doi: 10.3389/fimmu.2020.00482 of CAR T-cells obtained presented a TN or TCM phenotype, in average, which are the Frontiers in Immunology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing subsets capable of establishing a long-lasting T-cell memory in patients. An in-depth analysis to identify individual factors contributing to the optimal T-cell phenotype revealed that ex vivo cell expansion leads to reduced numbers of TN , TSCM , and TEFF cells, while TCM cells increase, both due to cell expansion and CAR-expression. Overall, our results show for the first time that clinical-grade production of CAR T-cells for heavily pre-treated patients using CliniMACS Prodigy system is feasible, and that the obtained products meet the current quality standards of the field. Reduced ex vivo expansion may yield CAR T-cell products with increased persistence in vivo. Keywords: chimeric antigen receptor, CD19, leukemia, lymphoma, immunotherapy, CAR T-cell production, CliniMACS Prodigy INTRODUCTION therapies have proven to be more challenging, so far. Initial clinical trials in solid tumors have shown limited efficacy and high Adoptive T-cell transfer (ACT) immunotherapy is a field in toxicity (21–30). This fact can be attributed to several factors: continuous expansion especially during the last three decades. difficulty in finding tumor-specific antigens to target, poor T ACT involves ex vivo expansion of tumor-specific cells and cell infiltration in the tumor and immunosuppressive tumor reinfusion into the patient. Among these therapies, the use of microenvironment, among others. The scientific community is Chimeric Antigen Receptor (CAR) T-cells for the treatment currently working in finding ways to overcome these challenges of several hematologic malignancies has shown unprecedented (31, 32). As a result, dozens of clinical trials using 4th efficacy rates. Consequently, the development of these therapies generation CARs (Armored CARs, CAR T-cells containing from bench to bedside has been done in an impressively short suicide genes and antibody-producing CARTs) for solid tumors amount of time (1–3). are ongoing (33–36). CAR T-cells are genetically modified cells whose cytotoxic The different clinical applications and the number of patients activity has been redirected toward cancer cells with the aim of waiting to receive a treatment are exponentially growing. eliminating the tumor. CARs are chimeric proteins composed Accordingly, the number of institutions and centers that are of an extracellular region responsible for binding to a particular in need of being capable of performing CAR T-cell therapies antigen and an intracellular region that promotes T-cell cytotoxic is also growing. In this context, developing systems that can activity and proliferation. CAR binding to the selected antigen is facilitate CAR T-cell production is crucial to help CAR T-cell field usually mediated by a single chain variable fragment (scFv) of a move faster, thereby finding effective therapies for all kinds of monoclonal antibody. The scFv-derived region results in a MHC- malignances and other diseases. independent interaction of the CAR with its ligand. This scFv is To this end, we have previously reported the development combined with one or more intracellular co-stimulatory domains and pre-clinical evaluation of a new anti-CD19 CAR, based on (usually CD28 or 4-1BB) and a pro-activator cytotoxic domain the A3B1 antibody (37). Concomitantly, we have also established (CD3ζ) (4–6). a CAR T-cell (ARI-0001) production system at our institution First great successes in the CAR T-cell field came from (Hospital Clinic de Barcelona). Our system is based on the use studies using a 2nd generation CAR (including a co-stimulatory of a closed semi-automatic bioreactor (CliniMACS Prodigy R ) domain) targeting the pan-B-cell lineage marker CD19. In for ex vivo selection, transduction and expansion of CAR T- this studies by National Cancer Institute and University of cells. We are currently conducting a phase I clinical trial using Pennsylvania, one patient with follicular lymphoma experienced our novel anti-CD19 CAR for CD19+ B-cell malignancies partial remission (7) and two out of three patients with (NCT03144583). We have successfully prepared 28 ARI-0001 cell advanced refractory chronic lymphocytic leukemia (CLL) products in the context of this clinical trial. Here, we present obtained complete responses (8). Afterwards, these and several the results and characteristics of the products obtained, thereby other institutions including Memorial Sloan Kettering Cancer demonstrating, for the first time, the feasibility of CAR T-cell Center and the Fred Hutchinson Cancer Research Center production in a relatively wide series of heavily pre-treated pioneered several studies using slightly different CAR19 patients using CliniMACS Prodigy system. constructs, and confirmed response rates between 50 and 80% in CLL, non-Hodgkin’s lymphoma (NHL) and acute lymphoblastic MATERIALS AND METHODS leukemia (ALL) (9–14). Currently, a lot of effort is being put in developing new CARs Patients and Samples to target other types of tumors. Anti-BCMA CAR has also shown At the time of submitting this manuscript, 28 products from impressive response rates (around 90%) for multiple myeloma 27 patients enrolled in phase I clinical trial for CD19+ B-cell (MM) (15–17). Also, other antigens such as CD30 and CD22 malignancies (NCT03144583) have been produced. Among the are currently being explored for the treatment of hematologic 27 patients, 22 had ALL (14 adult and eight pediatric patients), malignancies (18–20). In the field of solid tumors, CAR T-cell four had NHL and 1 CLL. All patients included in the clinical trial Frontiers in Immunology | www.frontiersin.org 2 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing had relapsed of their disease. Patients’ pretreatment regimens are The remaining cells were cryopreserved in bags and vials to summarized in Table 1. be used as control cells for product quality assays and as a Adult patients were subjected to leukocytapheresis at the backup in case of production failure. Cells were cultured using Apheresis Unit, Hospital Clínic, and pediatric patients at the TexMACS R media supplemented with 3% human AB serum Apheresis Unit of Hospital Sant Joan de Déu/BST, after signing an (obtained from the blood bank. BST) and with 155 IU/mL informed consent. Apheresis procedures were performed using IL-7 and 290 IU/mL IL-15 (Miltenyi Biotec #170-076-111 and Amicus device (Fresenius Kabi, Lake Zurich, IL). A minimum #170-076-114, respectively). Cells were immediately activated of 1 × 108 total T-cells diluted in 50 ml of plasma were using TransACT GMP Grade (Miltenyi Biotec, Cat. N. 170- required. This study has been approved by the Research Ethics 076-156) and transduced 24h later using CAR19-containing Comitee (CeIm) of Hospital Clinic. HCB/2017/0001. Clinical lentivirus at MOI = 10. A cell culture wash was programmed trial: CART19-BE-01. Eudra: 2016-002972-29. 48 h after transduction. The cells were then maintained in culture with increasing shaking until the desired cell number was ARI-0001 Cell Production reached (typically 7–10 days after cell culture initiation). Cells Apheresis products were connected to CliniMACS Prodigy R were finally eluted in 100 ml 0.9% NaCl + 1%HSA, aliquoted system (Miltenyi Biotec) tubing set. Erythrocytes and platelets according to the desired ARI-0001 cell dose and cryopreserved were removed by density gradient centrifugation in the until infusion. Centricult unit. The remaining cells were selected using CD4 The aim was to achieve two doses of ARI-0001 cells/patient. and CD8 coated magnetic beads. Selected cells were eluted The planned target cell dose varied depending on the patient’s in the “Reapplication Bag.” After selection, 1 × 108 T-cells disease. Typically, 1 × 106 ARI-0001 cells/kg for patients with (from reapplication bag) were used to initiate cell culture. ALL and CLL, and 5 × 106 ARI-0001 cells/kg for NHL patients. TABLE 1 | Patients’ pretreatment regimens. Pat ID Age Sex Disease Lines of treatment prior to leukoapheresis Allogeneic HCT T01 27 M ALL PETHEMA 2011, blinatumomab, radiotherapy Yes (+ DLI) T02 25 M PMLBCL R-CHOP, R-ESHAP, autologous HCT, radiotherapy, brentuximab, GSK525762, nivolumab No T03 7 F ALL SEHOP 2013, SEHOP 2016, inotuzumab, methotrexate + cyclophosphamide + anthracyclins + prednisone Yes (+ DLI) T04 19 F PMLBCL R-CHOP, R-ESHAP, autologous HCT, radiotherapy, BURKIMAB, Gemcitabine + vinorelbine + procarbacine No T05 51 M DLBCL BURKIMAB, autologous HCT, cyclophosphamide + prednisone, GSK525762, methotrexate, cyclophosphamide No T06 20 F ALL PETHEMA 2011, PETHEMA 2008 Yes T07 19 M ALL PETHEMA 2011, FLAG-Ida Yes T08 53 F CLL FCR, BR, ibrutinib, venetoclax, obinutuzumab, idelalisib No T09 8 M ALL SEHOP 2008, SEHOP 2013 No T13 20 M ALL GRAAL 2003, FLAG-Ida, blinatumomab Yes (x2) T11 34 F ALL PETHEMA 2011, Hyper-CVAD, inotuzumab Yes T12 3 F ALL SEHOP 2013, SEHOP 2016, vincristine + prednisone Yes T14 27 M ALL PETHEMA 2008, FLAG-Ida, PETHEMA 2011 Yes T15 30 M ALL PETHEMA 2011, FLAG-Ida, FLAG-Ida + blinatumomab, FLAG-Ida, inotuzumab Yes (x2 + DLI) T16 10 M ALL SEHOP 2013 Yes T17 23 M ALL PETHEMA 2011, FLAG-Ida, PETHEMA 2008, BFM-90, inotuzumab Yes T19 9 F ALL PETHEMA, SEHOP 2015, radiotherapy No T20 35 M ALL PETHEMA 2011, FLAG-Ida, PETHEMA 2011, methotrexate + vincristine + dexamethasone Yes T21 13 F ALL SEHOP 2013, SEHOP 2016, prednisone Yes T22 29 M ALL PETHEMA 2008, dasatinib, FLAG-Ida + ponatinib, FCR Yes T24 19 M ALL PETHEMA 2011, FLAG-Ida No T25 47 F ALL PETHEMA 2011, FLAG-Ida, PETHEMA 2011 Yes T26 8 F ALL SEHOP Yes T27 22 M ALL PETHEMA 2008, FLAG-Ida, PETHEMA 2011 No T30 31 M ALL PETHEMA 2008, blinatumomab Yes (+DLI) T32 23 M ALL PETHEMA 2008, PETHEMA 2011, vindesine + prednisone, inotuzumab Yes (x2) T34 45 F DLBCL R-CHOP, R-ESHAP, BURKIMAB, radiotherapy No ALL, acute lymphoblastic leukemia; DLBCL, diffuse large B-cell lymphoma; PMLBCL, primary mediastinal large B-cell lymphoma; CLL, chronic lymphocytic leukemia; DLI, donor lymphocyte infusion; HCT, hematopoietic cell transplantation; FCR, fludarabina + cyclophosphamide + rituximab; BR, bendamustina + rituximab; FLAG-Ida, fludarabina + cytarabine + idarubicin + G-CSF; PETHEMA, Spanish Program of Treatments in Hematology; SEHOP, Spanish Society of Pediatric Hematology & Oncology; GRAAL, Group for Research on Adult Acute Lymphoblastic Leukemia. Frontiers in Immunology | www.frontiersin.org 3 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing Monoclonal Antibodies Alternatively, intracellular cytokine production (IFNγ and CAR19 expression was detected with an APC-conjugated TNFα) was measured by flow cytometry. Briefly, cells were AffiniPureF(ab’)2 -fragment goat-anti-mouse IgG (Jackson first labeled for extracellular markers CD4, CD8, and CD3 and ImmunoResearch Laboratories, 115-136-072). ARI-0001 cell incubated 15 min. Cells were then fixed using 1X BD lysing product composition was determined by flow cytometry using solution (Cat. N. 349202) and incubated for an additional 15 min. staining with the following antibodies (all from BD): CD45-APC, After 2 washes, cells were permeabilized using FACS buffer CD3-BV421, CD4-FITC, CD8-PerCPCy5.5, CD19-PECy7, + 0.1% saponin, and incubated for 15 min. Cells were then CD16-PE, CD56-PE. incubated with anti-IFNγ and anti-TNFα, for 30 min at 4◦ C. For the T cell subset characterization experiments, CAR+ After that, cells were washed in PBS and analyzed. cells were detected using a CD19-Fc recombinant protein chimera (R&D, Cat. N. 9269-CD-050) and a secondary Small-Scale T Cell Expansions antibody FITC-Goat F(ab)2 anti-human IgG (Life Technologies, 0.5 × 106 T-cells were cultured with X-Vivo 15 Cell Medium Cat. N. H10101C). This staining was combined with the (Cultek, Cat. N. BE02-060Q), 5% AB human serum (Sigma, following monoclonal antibodies (all from BD): CD3-BV421, Cat. N. H4522), penicillin-streptomycin (100 µg/ml) and the CD8-APC.Cy7, CD45RA-PECy7, CD45RO-APC, CCR7- indicated cytokine: 50 IU/ml IL-2 (Miltenyi Biotec) or 155 PerCPCy5.5, CD28-BV510, and CD95-PE (or CD27-PE). T IU/mL IL-7 and 290 IU/mL IL-15 (Miltenyi Biotec). Cytokines cell subpopulations were defined as follows: TN : CD45RA+, were added to the media every 48 h. Twenty-seven hours CCR7+; TSCM : CD45RA+, CCR7+,CD95+; TCM : CD45RA–, after thawing cells were activated with Dynabeads Human T- CCR7+; TEM : CD45RA-, CCR7- and TEFF : CD45RA+, CCR7−. Activator CD3/CD28 (Gibco, Cat. N. 11131D) according to For intracellular cytokine measurement, the following the manufacturer’s instructions. Cells were transduced after an antibodies were used, all from BD: CD3-BV450, CD8-APC.H7, additional 24 h with an MOI of 10 and then expanded for 11 days CD4-BV500, IFNγ-PerCP.Cy5.5, TNFα-PE. at a concentration of 0.5 × 106 to 1.5 × 106 T-cells/ml. For repeated challenges experiment, the antibodies used were the following, all from BD: CD3-APC, CD4-BV510, CD8- T-Cell Expansion After Repeated APC.Cy7, CD19-PE. Challenges With Target Cells For flow cytometry analyses, cells were acquired using a FACS To analyze T-cell proliferation capacity after antigen encounter, Canto II, BD and subsequently analyzed using FlowJo Software. we seeded a co-culture of CAR-T cells and NALM6 cells at 1:1 ratio (250,000 cells each). After 4 days of incubation, an aliquot of Product Quality Controls the culture was taken and analyzed to determine T-cell number. Cells were labeled with CD3, CD4, CD8, and CD19, and then 20 Product potency assay was performed by flow cytometry as µl of beads (CountBright, Cat. N. C36950, Invitrogen) was added described in Castella et al. (37). Real-time PCR was used to to the sample to determine absolute cell number. This process measure number of copies/cell and to assess the presence of was repeated 3 times. replication-competent lentivirus (RCL) in the final product. Primer sequences and PCR protocol has been described previously (37). Product sterility, absence of mycoplasma, Statistics Statistical significance was assessed using SPSS software. endotoxin and adventitious virus was determined by a Unpaired T-test was used unless otherwise specified. U-Mann certified laboratory using the technique specified in Table S1. Whitney was used for comparison of variables with non-normal Adventitious virus included the determination of HIV virus distributions. Statistical significance was considered when p- presence among others. Since conventional HIV detection value ≤ 0.05. methods detect also the presence of the lentiviral transgene used to transduce the cells, an alternative PCR assay based on the detection of Env gene was used to discriminate between RESULTS HIV infection and lentiviral transduction. The primers used to amplify Env gene are: Env_F: 5′ CAATGTACACATGGA CAR T-Cell Expansion Twenty-eight apheresis products were obtained from 27 patients ATTAGGCCA 3′ and Env_R: 5′ TTCTGGGTCCCCTCCTGA included in the clinical trial. For one patient, the apheresis GGA 3′ . product was obtained twice due to ARI-0001 cell production failure (T10 and T13 products belong to the same patient). Cytokine Measurement Description of apheresis products is presented in Table 2. Cytokine level was measured using Milliplex MAP Human Patients’ apheresis products were subjected to CD4+ and CD8+ Cytokine/Chemokine Magnetic Bead panels (Millipore). A 10- magnetic selection using the CliniMACS Prodigy system. In all plex kit for IFNγ, IL-10, IL-1β, IL-6, TNFα, IL-12(P40), IL-17, cases except for one (Patient T27), the minimum number of T- IL-2, IL-4, and IP-10, a 3-plex kit for IL-8, IL-15 and MIP1A (Cat cells (100 × 106 ) was obtained (Table 2). In Patient T27, cell N. HCYTOMAG-60K) and a 1-plex kit for GranzymeB (Cat. N. culture was initiated with 50 × 106 cells. HCD8MAG-15K) were used. The assay was performed following Results of cell expansion in CliniMACS Prodigy for the 27 manufacturer’s instructions. Samples were run in a Luminex products are presented in Figures 1A,B and Table S2. Cells were 200 system. expanded for an average of 8.5 days, range 7–10. Average total Frontiers in Immunology | www.frontiersin.org 4 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing TABLE 2 | Apheresis products composition and CD4-CD8 cell selection. Pat. ID Disease Apheresis products CD4-CD8 selection WBC (×109 ) Lymph (×109 ) CD3+ (%)* CD4+ (%)* CD8+ (%)* Total cell number (×106 ) T01 Adult ALL 7.15 6.40 24.80 12.60 10.70 960 T02 NHL 3.31 1.56 32.50 11.40 19.80 1,200 T03 Pediatric ALL 4.59 3.50 49.38 22.67 26.71 1,580 T04 NHL 4.04 0.85 13.50 6.60 6.20 488 T05 NHL 7.71 4.23 30.00 12.10 15.50 1,800 T06 Adult ALL 3.64 3.19 28.80 10.60 16.20 720 T07 Adult ALL 4.91 3.84 29.00 4.40 19.80 1,900 T08 CLL 2.88 2.30 19.70 8.80 10.10 1,560 T09 Pediatric ALL 4.27 3.24 21.50 11.30 8.80 1,442 T10 Adult ALL 1.00 0.72 15.30 7.90 6.10 350 T11 Adult ALL 2.08 0.98 13.90 7.90 4.40 360 T12 Pediatric ALL 8.40 6.21 2.20 1.70 0.50 150 T13 Adult ALL 1.29 0.64 26.90 8.40 15.60 250 T14 Adult ALL 3.22 2.31 38.90 17.80 18.00 2,160 T15 Adult ALL 2.88 1.28 26.10 8.70 14.50 1,050 T16 Pediatric ALL 1.98 1.22 22.60 4.80 15.90 400 T17 Adult ALL 8.55 2.82 27.90 7.40 19.20 1,400 T19 Pediatric ALL 1.09 0.20 22.50 12.80 7.50 250 T20 Adult ALL 3.73 2.23 28.60 7.20 20.70 760 T21 Pediatric ALL 4.50 2.20 36.20 18.60 14.20 400 T22 Adult ALL 8.37 3.16 42.00 21.40 19.50 750 T24 Adult ALL 25.85 22.00 4.10 1.20 0.70 200 T25 Adult ALL 0.83 0.46 32.50 5.50 25.70 1,520 T26 Pediatric ALL 0.84 0.51 6.50 1.40 4.60 200 T27 Adult ALL 0.33 0.10 10.00 3.30 6.10 50 T30 Adult ALL 2.06 1.48 46.50 13.20 28.80 871 T32 Adult ALL 2.23 0.69 52.00 19.30 27.40 2,000 T34 NHL 6.71 3.54 23.50 12.60 9.70 993 *% of cells over WBC. cell number obtained in the final product was 2,548 × 106 , To analyze the percentage of CAR+ cells, we first validated our range 600 × 106 to 5,200 × 106 . In one patient where cell detection method based on the use of an APC-conjugated F(ab’)2 culture was started with 50 × 106 cells, the final product also anti-mouse antibody. To this end, we engineered a vector where met acceptance criteria. In this particular case, cell culture was CAR19 and GFP were co-expressed. As shown in Figure S2, maintained for 13 days, finally obtaining 3,300 × 106 cells. the correlation between GFP+APC+ or GFP–APC– cells was of When compared to healthy donors (used in three previous 93.5%, thereby indicating that the detection method had a good validation runs), patient cells seem to expand more slowly, sensitivity and specificity. even if the number of runs performed with healthy donors is Using this detection system, we assessed the percentage of limited (Figure 1C). CAR+ (ARI-0001) cells in the patients’ products. All products Products were analyzed in terms of appearance, quantity, except one met the specification of >20% ARI-0001 cells. In identity, purity, safety, and potency. A complete list of product one product (T10) only 14.5% ARI-0001 cells were detected. specifications is provided in Table S1. Consequently, this product was considered a production failure. CAR T-cell production was repeated for this patient from Product Purity and Transduction Efficiency a 2nd apheresis (T13). This time, a valid product could be The final product was characterized in terms of cell viability, obtained. Mean (±SD) of percentage of CAR+ cells in this percentage of CD3+ cells and percentage of CAR+ cells. This series was 30.6 ± 13.44 (Figures 1B–D), slightly lower than data is summarized in Table 3. All products met acceptance transduction efficiencies achieved in small-scale expansions criteria for cell viability and percentage of CD3+ cells (>70% for (45.3%) (Figure S1A). No significant differences in efficiency both parameters). The lowest value detected for cell viability was of transduction were observed between healthy donors and 91 and 85.7% for CD3+ cells (Figure 1D). patients (35.8 vs. 30.6%), or among the different diseases Frontiers in Immunology | www.frontiersin.org 5 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing FIGURE 1 | ARI-0001-cell expansion in CliniMACS Prodigy. (A) Expansion kinetics of ARI-0001-cell products (Total cell number). Gray points indicate individual products. Black triangles indicate Mean ± SD and adjusting curve. (B) Expansion kinetics of CAR19+ cells (red) and total cell number (black). Mean ± SD is represented. (C) Expansion kinetics of ARI-0001 cells (Total cell number) comparing healthy controls and different types of disease. Mean ± SEM is represented. (D) Percentage of CD3 and CAR19 positive cells as determined by flow cytometry. Mean ± SD is also indicated. Panels on the right show flow cytometry representative image corresponding to CAR19 and CD3 staining in ARI-0001-cell final products and Control T cells (Untransduced). (Figure S1B). Percentage of CAR+ cells over time during cell and DNA copies/cell was obtained, further validating both expansion was also investigated. A high degree of variability techniques (Figure S4). was detected among patients, with the percentage of CAR+ cells increasing in some patients while decreasing in others Product Potency (Figure S3). In terms of number of cell doses obtained per Cytotoxic potential was analyzed in vitro for each product before patient, considering a standard weight of 70 kg for adults and infusion. A co-culture of the final product with NALM6 cell line 25 kg for pediatric patients, a minimum of two cell doses were was initiated at different E:T ratios. Percentage of alive-CD19+ quickly obtained (by day 7) for all ALL patients (dose 1 × 106 cells was measured by flow cytometry after 4 h. As a control, the ARI-0001 cells/kg). For NHL patients (dose 5 × 106 ARI-0001 cytotoxic activity of non-transduced CD4+CD8+ cells from the cells/kg), two cell doses were obtained for three out of four same patient was also measured. Products were considered valid patients, by day 9. Indeed, the number of cell doses obtained when the CD19+ cell surviving fraction with ARI-0001 cells, at for ALL far exceeded the need (nine cell doses for adult patients ratio 1:1, was lower than 70%. Results are presented in Table 3 and 25.4 for pediatric patients), indicating that the time of and Figure 2A. All products obtained met the specification of ex vivo cell expansion could be reduced if necessary in these <70% CD19+ surviving fraction at E:T ratio 1:1, indicating that groups of patients. For NHL, the average number of ARI- all products prepared had the intrinsic capacity of eliminating 0001 cell doses obtained was 2.5. Only one CLL patient has CD19+ cells. been produced so far. T-cells from this patient grew slower Cytokine level was also measured in the supernatant of and required 10 days of expansion, finally obtaining 398 × 106 cytotoxicity assays. As expected, increased levels of pro- ARI-0001 cells. inflammatory cytokines such as IFNγ and TNFα was observed CAR19 transduction was also assessed in terms of DNA when ARI-0001 cells were co-cultured with NALM6, compared copies/cell. As shown in Table 3 CAR19 was detected in all to ARI-0001 cells alone. The level of GranzymeB was also products, within a range of 0.4–2.9 copies/cell (all below significantly increased (Figure 2B) consistent with the cytotoxic the limit considered safe of <10 copies/cell). As expected, activity of ARI-0001 cells. The complete set of analyzed cytokines a positive correlation between percentage of CAR+ cells is provided in Figure S5. Frontiers in Immunology | www.frontiersin.org 6 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing TABLE 3 | ARI-0001 product characterization. Pat. ID Cell viability %CD3+ %CAR+ Sterility Mycoplasma Endotoxin Adventitious virus Transgene copies/cell RCL Potency T01 100 94.7 39.3 Sterile Absent <0.5 EU/ml Absent 1.05 Absent 13.9 T02 95 96.5 28 Sterile Absent <0.5 EU/ml Absent 0.92 Absent 6.0 T03 90 96.0 30 Sterile Absent <0.5 EU/ml Absent 0.97 Absent 2.7 T04 98 98.1 22.8 Sterile Absent <0.5 EU/ml Absent 0.66 Absent 17.2 T05 93 98.4 22.9 Sterile Absent <0.5 EU/ml Absent 0.9 Absent 41.4 T06 96 87.1 43.6 Sterile Absent <0.5 EU/ml Absent 1.55 Absent 0.6 T07 96 97.3 20.4 Sterile Absent <0.5 EU/ml Absent 0.73 Absent 18.6 T08 92 98.7 20.7 Sterile Absent <0.5 EU/ml Absent 1.62 Absent 21.4 T09 100 99.1 20.6 Sterile Absent <0.5 EU/ml Absent 0.77 Absent 8.7 T10 98 98.7 14.5* – – – – – – – T11 94 99.7 25 Sterile Absent <0.5 EU/ml Absent 2.31 Absent 0.04 T12 98 99.4 20 Sterile Absent <0.5 EU/ml Absent 0.43 Absent 14.4 T13 95 99.2 20.4 Sterile Absent <0.5 EU/ml Absent 0.57 Absent 3.5 T14 98 99.2 62.2 Sterile Absent <0.5 EU/ml Absent 1.99 Absent 0.01 T15 93 97.5 34.4 Sterile Absent <0.5 EU/ml Absent 1.98 Absent 1.4 T16 91 98.7 26.6 Sterile Absent <0.5 EU/ml Absent 1.46 Absent 0.1 T17 96 94.8 26 Sterile Absent <0.5 EU/ml Absent 1.47 Absent 0.2 T19 93 90.6 22.6 Sterile Absent <0.5 EU/ml Absent 0.99 Absent 0.3 T20 94 98.3 20.8 Sterile Absent <0.5 EU/ml Absent 1.15 Absent 0.02 T21 91 99.4 48.2 Sterile Absent <0.5 EU/ml Absent 1.59 Absent 0.0 T22 82 97.3 44.1 Sterile Absent <0.5 EU/ml Absent 2.1 Absent 0.0 T24 97 99.7 23.8 Sterile Absent <0.5 EU/ml Absent 1.55 Absent 4.18 T25 94 98.2 41.5 Sterile Absent <0.5 EU/ml Absent 2.92 Absent 0.06 T26 97 98.5 23 Sterile Absent <0.5 EU/ml Absent 0.4 Absent 28.0 T27 98 96.5 23.2 Sterile Absent <0.5 EU/ml Absent 0.62 Absent 4.3 T30 95 85.7 26 Sterile Absent <0.5 EU/ml Absent 2.08 Absent 0.2 T32 97 98.2 35.6 Sterile Absent <0.5 EU/ml Absent 1.93 Absent 0.2 T34 94 99.1 71.5 Sterile Absent <0.5 EU/ml Absent 2.93 Absent 0.02 *Value below acceptance criteria. CAR T-cells produced from patients were compared to ratio < 1, the proportion of CD4+ cells tended to increase those obtained from healthy controls in terms of cytotoxic during cell expansion, while in patients where a CD4/CD8 activity and cytokine production. As shown in Figure 2C, ratio > 1 was obtained after cell selection, the proportion patients’ and healthy donors’ CAR T-cells showed similar of CD4+ cells tended to decrease (Figure 3B). Therefore, the cytotoxic potential (even slightly higher for patient’s cells difference in CD4/CD8 ratio (1CD4/CD8) before and after although this was not statistically significant). Production of pro- cell expansion was significantly different depending on the inflammatory cytokines (IFNγ and TNFα) and GranzymeB was initial ratio (Figure S6A). The efficiency of transduction differed also comparable (Figure 2D). between CD4+ and CD8+ subsets, as CD4 showed a significantly higher percentage of CAR+ cells (Figure 3C). T Cell Subset Characterization In terms of TN , TSCM , TCM , TE , and TEM subsets, we observed Product composition was further analyzed in terms of CD4/CD8 a high degree of variability among patients’ final products ratio and TN , TSCM , TCM , TE , and TEM subsets. Consistent with (Figure 3D). This high variability is exemplified by the different previous reports (38), CD4/CD8 ratio was inverted (CD4/CD8 level of CD45RA and CCR7 expression in samples from different ratio < 1) in a large subset of patients that were candidate patients (Figures 3E,F) and cannot be attributed to the different for a CAR T-cell therapy (Figure 3A). Average CD4/CD8 ratio diseases (Figure S1C). Within the CAR+ T-cells of the final was 0.93 ± 0.67 in the apheresis products. This ratio was not product, memory phenotypes (CM and EM) predominated in significantly altered after CD4 and CD8 cell selection in the the vast majority of patients. Average percentage and SD for each vast majority of patients. However, a significant increase in the subpopulation in the CAR+ cells of the final product is as follows: proportion of CD4 cells was detected during cell expansion. TN : 7.71 ± 13.9, TSCM : 5.26 ± 12.0, TCM : 31.01 ± 16.7, TEM : CD4/CD8 ratio increased from 0.64 ± 0.61 after CD4-CD8 cell 35.11 ± 17.7, and TE : 4.2 ± 9.5. Analysis of CD4 and CD8 cells selection, to 1.61 ± 1.04 in the final product. A deeper analysis separately showed that CD8 cells have a more TN , TSCM , and TCM of this data reveled that in patients starting with a CD4/CD8 phenotype than CD4 cells (Figure S6B). We also analyzed how Frontiers in Immunology | www.frontiersin.org 7 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing FIGURE 2 | ARI-0001-cell potency. (A) Cytotoxicity assay after 4 h of ARI-0001 co-culture with NALM6 cells, at the indicated ratios. Mean ± SD of all 27 CAR T cell products is indicated. (#) Dashed line indicates minimum of ARI-0001-cell cytotoxicity level for a product to be considered valid. (B) IFNγ, TNFα, and GranzymeB levels measured in the supernatants of the cytotoxicity assays. E:T ratio 0 indicates no target cells. (*) indicates statistical significance, p < 0.05. (C) Comparison of ARI-0001 cytotoxic potential after 4 h of co-culture with NALM6 cells, at the indicated ratios. Mean ± SD is shown. “n.s.” indicates not statistically significant (Non parametric test). (D) Comparison of IFNγ, TNFα, and GranzymeB levels measured in the supernatants of the cytotoxicity assay at E:T ratio 1:1. “HD” indicates Healthy donors. “n.s.” indicates not statistically significant (Parametric test applied to IFNγ and TNFα and non-parametric test applied to GranzymeB). these subsets varied during ex vivo cell expansion by comparing Small-Scale CAR T Cell Expansions T-cell subsets in the initial (after CD4-CD8 cell selection) and To further evaluate the impact of culture conditions or CAR in the final product, and if CAR expression influenced T-cell expression on the proportion of CD4/CD8 ratio or T-cell subpopulations (CAR– vs. CAR+ cells). As shown in Figure 3D, phenotype, cell expansions from patients’ selected cells were we observed a robust increase in the proportion of TCM during repeated in a small scale experiment, under different conditions. cell expansion while TN and TEFF cells decreased. These changes We selected six of the patients (three adult ALL and three NHL) in T-cell subsets can be attributed to a decrease in CD45RA from which frozen leftover cells after CD4-CD8 cell selection expression which is expected upon cell activation (Figures 3E,F). were available. We expanded patients’ cells in four different No statistically significant changes in the T-cell subsets were conditions: (1a) IL2—Untransduced T-cells, (1b) IL2—CAR T- detected between CAR– and CAR+ cells in the final product, cells, (2a) IL7/IL15—Untransduced T-cells, (2b) IL7/IL15—CAR although a further increase in TCM , and consequent decrease T-cells. As shown in Figure S7A, cells expanded between 17 in TEFF cells, was observed in CAR+ cells compared to CAR– and 100 times over a period of 11 days. CAR19 transduced (Figure 3D). Consistently, a small increase in CCR7 expression T-cells expanded less (or slowly) compared to untransduced was also detected in CAR+ cells vs. CAR- cells. (Figure 3F). counterparts, and IL2 grown cells expanded more than IL7/IL15 The impact of CAR expression on CCR7 was further explored (in both untransduced and CAR19 conditions) (Figure S7B). in independent small-scale expansions (see next section). The Cell transduction or cytokines used did not condition CD4/CD8 changes in expression of CD27, CD28, and CD95 were also ratio in a consistent way. However, as detected previously in assessed by flow cytometry. As shown in Figure S6C, CD95 the products expanded using the Prodigy system, in patients was increased during cell expansion and CD27 decreased. CD28 starting with CD4/CD8 ratio>1 (T04 and T34), the ratio tended did not show significant changes during expansion, although to decrease, while in patients starting with CD4/CD8 ratio presented a higher expression in CAR+ compared to CAR- cells. < 1 (T02, T15, T22, and T34), the ratio tended to increase Frontiers in Immunology | www.frontiersin.org 8 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing FIGURE 3 | ARI-0001 cell subset characterization. (A) CD4/CD8 ratio of apheresis products, after CD4-CD8 cell selection and of the final product. (B) CD4/CD8 ratio variation during cell expansion. Left panel corresponds to products with an initial ratio < 1. Right panel corresponds to products with an initial ratio > 1. (C) CAR19 transduction efficiency in CD4 and CD8 cells. Mean ± SD is shown. (D) Percentage of T-cell subpopulations within initial (CD4-CD8 cell selection) and final products (CAR– and CAR+ cells). (E) Representative flow cytometry plots of three different patients showing T cell populations in initial and final products. (F) Differences in MFI for CD45RA and CCR7 in initial and final products. Lower panel shows paired analysis for CCR7 MFI. (*) indicates statistical significance, p < 0.05. n.s. indicates not statistically significant. Frontiers in Immunology | www.frontiersin.org 9 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing (Figure S7C). Indeed, since the expansions were maintained for functionally comparable, or even slightly more active, than those longer in the small-scale expansions than in the Prodigy system, produced in small-scale expansions. we observed that the ratio CD4/CD8 may fluctuate in a more or Taking all this data together, we conclude that ex vivo cell less pronounced way, but it tends to CD4/CD8 = 1 if the cells are expansion causes a loss of TN and TEFF , which is observed in both cultured for longer periods of time. the Prodigy system and small-scale expansions. On the contrary, Interestingly, significant differences were found in terms of T- TCM cells are largely accumulated, both by ex vivo expansion and cell subsets depending on the culture conditions. The cytokines as a result of CAR expression (in CARs containing 4-1BB as a used in the growth media did not provide significant differences co-stimulatory domain). Cells produced in Prodigy system are in terms of the different subsets in this series of patients. functionally similar to those produced in small-scale expansions. However, a significant and consistent difference was appreciated in CAR19 expressing cells vs. untransduced T-cells for almost DISCUSSION all subsets. As shown in Figure S7D, CAR19 transduction resulted in a much higher percentage of TN , TSCM and TCM CAR T-cells are complex medical products. Most of the subsets independently of the cytokine used in the culture media. approaches explored so far are based on the use of autologous By contrary, TEM cells were decreased in the CAR19+ cells CAR T-cells, thereby requiring the production of a personalized compared to the untransduced samples. In this case, no difference product for each patient. Moreover, CAR T-cells are gene-therapy in CD45RA MFI between untransduced and CAR19+ cells was products, involving clinical-grade preparation of vectors, and observed that can account for the decrease in TN and TSCM , since subsequent T-cell selection, transduction and expansion. Two in both conditions, cells were activated and proliferated ex vivo. different CAR T-products (Yescarta, Kite-Gilead and Kymriah, However, we observed a significant increase in CCR7 expression Novartis) have already been approved for the treatment of in CAR19+ cells compared to untransduced cells (Figure S7E). pediatric-ALL and NHL. Nonetheless, the development of new This increase explains a higher percentage of TN , TSCM , and CAR T-products, including phase I and II clinical trials, for TCM subsets and lower TEM . Increase in CCR7 expression upon many different targets and diseases is primarily conducted in 4-1BB activation has been previously described in monocytes academic research centers. Manufacturing of clinical-grade CAR (39) and also proposed for CAR T-cells (40). To test if 4-1BB T-cells can pose a challenge for many medium-sized academic activation is responsible for the increase in CCR7 we observe in institutions. We have previously published the development of the CAR+ cells, we modified our CAR construct by changing our own CAR19 product (based on the scFv sequence of A3B1 the co-stimulatory domain to CD28 (Figure S8A). T-cells from antibody) and the establishment of a CAR T-cell production a healthy donor were then left untransduced or transduced protocol and infrastructure, based on the use of CliniMACS with the 4-1BB- or CD28-containing CARs and expanded in Prodigy, a semi-automated closed system by Miltenyi (37). vitro for 10 days. Again, we observed an increase in CCR7 We and others have previously demonstrated the feasibility expression in the CAR-positive fraction of the cells transduced of CAR T-cell manufacturing from healthy donors, using with the 4-1BB-containing construct, compared to untransduced CliniMACS Prodigy (41–45). Now, we report the preparation cells or CD28-containing CAR+ cells (Figure S8B). As expected, and characterization of 27 CAR T-cell products for ALL, CLL, percentage of TCM cells is also higher in 4-1BB-containing CAR+ or NHL patients. cells (Figure S8C). Patients eligible for CAR T-cell therapies have previously Finally, the functionality of CAR T-cells manufactured received multiple lines of therapies. As a result, T-cells from with the Prodigy system and small-scale expansions was these patients have a reduced proliferation capacity and also compared. For this comparison, cells from 3 patients abnormal CD4/CD8 ratio compared to healthy donors (46, expanded with IL-7/IL-15 were used. The production of pro- 47). Nevertheless, we have been able to obtain 27 valid CAR inflammatory cytokines, cytotoxic potential and T-cell expansion T-cell products (according to the specifications indicated in was measured after adjusting for the same percentage of CAR+ Table S1) out of 28 attempts, proving the feasibility of CAR T-cell cells. Production of IFNγ and TNFα was measured after co- manufacturing for heavily pre-treated patients using the Prodigy culture of CAR T-cells with NALM6 at 1:1 ratio, at 4 h system. Cell expansion was maintained for an average of 8.5 days. time-point. Level of these two cytokines was measured both Indeed, patient-derived T-cells expand more slowly compared to by intracellular staining (Figure S9A) and cytokines present the expansions performed from healthy T-cell donors. However, in the media (Figure S9B), yielding consistent results. Cells for ALL patients, where the infused dose is typically 1 × 106 manufactured in the Prodigy system consistently produced CAR T-cells/kg, a minimum of 2 CAR T-cell doses were already slightly more IFNγ and TNFα than cells manufactured in small- achieved by day 7 in a 100% of patients, indicating that it is scale expansions. However, these differences were not statistically possible to reduce the expansion time in this subset of patients significant. In terms of cytotoxic potential, cells produced with (actual expansion time was 8.4 days, due to pre-scheduled both methods showed comparable results (Figure S9C). Finally, work plans). CLL and NHL patients required 9–10 days of T-cell expansion upon repeated challenges with fresh target cells expansion due to reduced intrinsic T-cell proliferation (48, 49) (NALM6) was slightly higher in cells manufactured with the and higher CAR T-cell numbers needed, respectively. Reduced Prodigy system than with small-scale expansions, although it T-cell expansion time is highly desirable, not only to reduce did not reach statistical significance (Figure S9D). Therefore, we the cost of batch production and to shorten the time until the conclude that cells manufactured with the Prodigy system are patient can receive the treatment, but also for the quality of the Frontiers in Immunology | www.frontiersin.org 10 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing product obtained, as discussed below. Despite reduced expansion 4-1BB-containing CAR T-cells have been shown to have an capacity of heavily pretreated patient’s T-cells compared to increased proportion of TCM compared to CD28-containg CARs, healthy donors, we have observed equivalent product potency which have a predominant TEM population (40, 55). We have in terms of cytotoxic potential and pro-inflammatory cytokine shown that the increase in TCM cells is not only the result of production. The method of CAR T-cell production (Prodigy CD45RA loss, but also due to increased CCR7 expression in 4- vs. small-scale) yielded also comparable products in terms of 1BB-containing CAR-expressing cells [our data and Kawalekar product potency, or even slightly more active in cells obtained et al. (40)]. This data correlates with a longer CAR T-cell with Prodigy. We reasoned that the higher cell densities reached persistence in vivo of 4-1BB- compared to CD28-containing in the Prodigy system may be advantageous for T-cell fitness CARs (11, 12, 49). and potency. In conclusion, we have demonstrated for the first time Average transduction efficiency was 30.6% on average in the feasibility of CAR T-cell manufacturing for heavily pre- our series and does not differ significantly between patients treated ALL, CLL, and NHL patients, using CliniMACS Prodigy. and healthy controls. Similar transduction efficiencies have To our knowledge, this is the first report describing the been reported by other groups using CliniMACS Prodigy for characteristics of the products obtained with this system, production of CAR T-cells from healthy controls (41, 50–52), including a medium-large cohort of patients. Our study shows except one study that reports transduction efficiencies between that CAR T-cell manufacturing can be completed in as 50–60% using very high MOI (MOI = 100) (44). Transduction low as 7 days for ALL patients and that reduced ex vivo efficiency in small-scale CAR-T cell productions is slightly higher expansion time may yield CAR T-cell products with increased (45.3 vs. 30.6%). We can speculate that having the possibility persistence in vivo. The products obtained show potent anti- to put in direct contact cells and lentivirus, i.e., spinoculation, tumor efficacy and are characterized by a predominance of TCM or adding reagents to the media that facilitate the entry of the and TEM phenotypes. lentivirus to the cells (such as polybrene) could help increase transduction efficiency in the Prodigy system. We observed a preference of CD4+ cell expansion over DATA AVAILABILITY STATEMENT CD8+ ex vivo. This is consistent with previous observations from other groups using different expansion systems and CAR All datasets generated for this study are included in the constructs (53, 54). Gomes-Silva and co-workers reported a article/Supplementary Material. preferential expansion of CD4 cells when using a 4-1BB co- stimulatory domain compared to a CD28-containing CAR (55). Therefore, this effect may contribute partially to explain this ETHICS STATEMENT phenomenon. Indeed, we detected a higher fraction of CD4- This study has been approved by the Research Ethics Comitee transduced cells than CD8. Nevertheless, we have clearly shown (CeIm) of Hospital Clinic. HCB/2017/0001. Clinical trial: than CD4/CD8 ratios are highly dependent on the initial ratio CART19-BE-01. Eudra: 2016-002972-29. Written informed and tend to equilibrium in longer ex vivo expansions, indicating consent to participate in this study was provided by the that preferential CD4 expansion may be transitory and fluctuate participants’ legal guardian/next of kin. also after CAR T-cell infusion. TN , TSCM , and TCM phenotypes have been associated to longer persistence in vivo and higher anti-tumor efficacy (38, AUTHOR CONTRIBUTIONS 56–58). We have dissected the factors that determine these T- cell populations with the aim of analyzing if our production MC designed and performed experiments, analyzed data, and system could be optimized. Indeed, there is a very high degree wrote the manuscript. MC-B and VO-M coordinated specific of variability in T-cell phenotypes among patients, which highly parts of the clinical trial and analyzed data. EG-N, GS, AA-V, AB, determines final product composition. In our experimental BM, and LM performed experiments and procedures. BM-A, JCi, setting, both IL-7/IL-15 and IL-2 yielded comparable population ML, EG, JT, ET, UP, JCa, TB, and DB-R supervised procedures. phenotypes. In most patients, ex vivo cell culture led to a decrease EC critically read the manuscript. ÁU-I, JY SR, JD, and MJ in the fraction of CD45RA positive cells in the final product. coordinated and supervised the study. Loss of CD45RA expression is expected upon cell activation and proliferation, and therefore intrinsic to ex vivo cell expansion, resulting in decreased numbers of TN cells (38, 40, 59, 60). In FUNDING this regard, shortening the time of ex vivo cell expansion when possible may help preserve this population. TCM cells have also This work was mainly funded by several public grants been shown to be able to establish persistent T-cell memory PI13/00676, PIE13/00033, PI17/01043, PICIS14/00122 and (58) and have superior anti-tumor effects (38). Therefore, the PI18/00775; included in the Plan Nacional de I + D + I and co- loss of TN cells may not have a major impact in anti-tumor financed by the ISCIII—Subdirección General de Evaluación y efficacy, since most of them are converted to TCM cells. Actually, Fomento de la Investigación Sanitaria—and the Fondo Europeo memory phenotypes are the most represented populations in de Desarrollo Regional (FEDER), and a crowdfunding program, our CAR T final products, consistent with previous reports (53). Projecte ARI. Frontiers in Immunology | www.frontiersin.org 11 March 2020 | Volume 11 | Article 482 Castella et al. Semi-automated CAR T-Cell Manufacturing ACKNOWLEDGMENTS Hospital Sant Joan de Déu. Finally, we also want to thank the support of many people and institutions that encouraged and The authors would like to thank patients and families who helped us develop this proposal in Barcelona. participated in this trial and to those who made it possible. The authors also want to thank Sara Varea responsible for quality SUPPLEMENTARY MATERIAL control of this clinical trial. Carolina España, Juan Ramon Ortega, Carla Guijarro, and Libertad Heredia for their technical support The Supplementary Material for this article can be found in CAR T-cell production. Finally, the authors thank Cristina online at: https://www.frontiersin.org/articles/10.3389/fimmu. Llanos the clinical research nurse of the pediatric patients at 2020.00482/full#supplementary-material REFERENCES 16. Ali SA, Shi V, Maric I, Wang M, Stroncek DF, Rose JJ, et al. T cells expressing an anti-B-cell maturation antigen chimeric antigen 1. Gill S, June CH. Going viral: chimeric antigen receptor T-cell receptor cause remissions of multiple myeloma. Blood. (2016) 128:1688– therapy for hematological malignancies. Immunol Rev. (2015) 700. doi: 10.1182/blood-2016-04-711903 263:68–89. doi: 10.1111/imr.12243 17. Zhao W-H, Liu J, Wang B-Y, Chen Y-X, Cao X-M, Yang Y, et al. A 2. Barrett DM, Singh N, Porter DL, Grupp SA, June CH. 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