This study included seven patients diagnosed with acute myeloid leukemia treated at the Hematology and Transfusion Medicine Center of the University of Campinas and two patients with pulmonary adenocarcinoma treated at the Pulmonology Outpatient Clinic of this University Hospital between 2015 and 2019. All patients provided written informed consent. This study was approved by the Ethics Committee of the University and conducted in accordance with the International Conference on Harmonization (ICH) guidelines and applicable regulations governing clinical trials.

The in vitro generation of dendritic cells from peripheral blood mononuclear leukocytes followed a protocol previously developed in our laboratory,17 with modifications. The patients selected for vaccination underwent a leukapheresis procedure to collect mononuclear leukocyte suspension, using a continuous cell separator (Com.Tec, Fresenius Kabi, Bad Homburg, Germany), C4Y kit, program: PBSC-Lymphocyte. The processing of leukapheresis bags and subsequent differentiation and maturation of the DCs were carried out in an ISO 7 clean room (Class-10,000), equipped with Class-100 biological safety cabinets for aseptic handling of the material, located at the Pediatric Research Center of the University (CIPED-UNICAMP).

Cell suspensions collected by apheresis underwent monocyte separation (CD14+ cells) using three different methods:

• i)

  Automated: automatic separation using a tubular system with magnetic beads (commercial system CliniMacs®);

• ii)

  Manual 1: monocyte purification by centrifugation gradients, using the Ficoll® and Percoll® reagents (Sigma Aldrich, USA), achieved by a single passage of the complete amount of leukapheresis material, and;

• iii)

  Manual 2: monocyte purification by centrifugation gradients, using the same procedure used for ‘Manual 1′, however divided into two consecutive passages, each with half of the leukapheresis material, to maintain the cell viability.

The resulting cell suspension, regardless of the separation process, was adjusted to the concentration of 2 × 106 viable cells/ml in the specific culture medium for differentiation of dendritic cells (DendriMACS™ GMP Medium, Miltenyi Biotec, Germany) and subsequently plated in 150 mm treated culture dishes (Corning, NY, USA). For differentiation into immature dendritic cells, adherent cells were cultured at 37 °C in 5% CO2 for five days in DendriMACS Medium containing 100 IU/ml penicillin and streptomycin (10 μg/ml), 50 ng/ml MACS GMP recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 50 ng/ml MACS GMP recombinant human interleukin-4 (IL-4) (Miltenyi Biotec, Germany).

On day 3, the cultures were supplemented with the same cytokines and fresh medium. On day 6, maturation factors were added: 50 ng/ml of MACS GMP recombinant human tumor necrosis factor alpha (TNF-α) (Miltenyi Biotec, Germany) and 2.5 µg/ml prostaglandin E2 (PGE2) (Cayman Chemical, USA), following which the cells were cultured for two more days.

Mature DCs from AML patients were electroporated with the Wilms’ tumor gene 1 (WT1) mRNA on the eighth day of culture, produced through a plasmid kindly provided by Prof. Evelien Smits (Tumor Immunology Laboratory, University of Antwerp, Belgium). A total of 20ηg of WT1 mRNA were added to the cell suspension and then electroporated with a 300 V exponential decay pulse for 7 ms in 0.4 cm cuvettes, using the Electro Square Porator ECM 830 electroporator (BTX Genetronics, USA). After recovery, the DCs were subjected to quality control tests, as described below, and cryopreserved until administration of the vaccine.

Cell lysates from the patients with lung adenocarcinoma were obtained from the patient tumor itself and used as an antigenic source. To produce these cell lysates, a small sample of the tumor removed during surgery was subjected to physical maceration and the cells were then lysed for 5 cycles, alternating freezing (freezer at −80 °C, 30 min.) and washing (room temperature, 10 min.). The total cell disruption was validated microscopically using trypan blue staining. After sonication for 10 min, the lysate was centrifuged at 14,000 rpm (30 min, 4 °C). The supernatant was recovered and stored at −80 °C for later use in cocultures with dendritic cells during the maturation phase (48 h).

The phenotype, viability and expression of DC activation markers were determined by flow cytometry, using antibodies conjugated to fluorochromes: 7AAD, anti-CD14, anti-CD83, anti-CD86, anti-CD80, anti-CD11c and anti-human leucocyte antigen - DR isotype (anti-HLA-DR) (BD-Pharmingem). Samples for microbiological control (aerobic and anaerobic bacteria) were incubated in Bact-alert 120 medium (Organon Teknika, USA) on the final day of cell culture and before freezing. Immunocytochemistry was performed in cytospin preparations of the final product of the vaccine to document antigen expression after electroporation or incubation with tumor lysates: for WT-1 in the case of patients with AML and WT-1, cofilin-1, CEA and TTF1 in the case of lung cancer. Minimum criteria for vaccine release were considered: negative bacteriological control tests, DC phenotype, showing maturation markers with more than 80% viable (7AAD negative) DCs by flow cytometry, and positive immunocytochemistry.

The following data were compared between the 3 methods evaluated: total number of viable cells in the apheresis product, number of monocytes isolated, monocyte separation yield and total number of dendritic cells produced and ready for application, as well as the yield of dendritic cells produced in relation to the number of separated monocytes and the number of initial total leukocytes. Analysis of variance (ANOVA) was performed to compare the 3 groups, using the Vassarstats portal (http://vassarstats.net/), and results with p < 0.05 were considered significant.

Vaccine doses were produced for 7 patients diagnosed with AML and in complete remission after conventional treatment (chemotherapy or chemotherapy + autologous hematopoietic stem cell transplant) and 2 patients diagnosed with stage IIIA-N2 lung adenocarcinoma, treated with surgery, chemotherapy and radiation (clinical and demographic data detailed in Table 1).

For patients with AML, chemotherapy consisted of induction with daunorubicin and cytarabine and consolidation with high-dose cytarabine (HD-ARAC) or HD-ARAC plus autologous hematopoietic stem cell transplant (HSCT). For patients with lung adenocarcinoma, the post-surgery treatment consisted of chemotherapy with carboplatin + paclitaxel, followed by 25 sessions of radiation on the mediastinal region.

Three of the patients had vaccines produced by the automated monocyte separation in the CliniMacs® system and six patients, by the manual separation, using the in-house method with centrifugation gradients. In 3 of these cases of manual separation, the entire product of leukapheresis was subjected to the centrifugation process in gradients at once, whereas, in the other 3, the product of leukapheresis was divided in half, each one processed separately (therefore, in two parallel gradient separation processes).

There were no differences between the 3 groups regarding the total number of viable cells in the apheresis product, number of isolated monocytes, yield of separate monocytes or total number of dendritic cells produced and ready for application, nor in the yield of dendritic cells, in relation to the number of separated monocytes or initial total leukocytes. The comparison between the three methods is presented in Table 2.

Regarding the quality of the final product, the following quality criteria were met for all patients: > 90% cell viability (verified by negativity in 7AAD), > 80% positivity for maturation markers (CD83, CD86, HLA-DR), negative bacterial cultures and positive immunocytochemistry (Figure 1).

Figure 1.

Immunocytochemistry demonstrating DCs with positive cytoplasmic labeling after antigenic pulse: A and B: DCs before and after electroporation with WT-1 mRNA, respectively, and internalization of antigen confirmed by reaction with anti-WT1 antibody; C and D: DCs before and after co-culture with lung cancer lysate positive for cofilin-1; internalization of antigen confirmed by reaction with anti-cofilin-1 antibody (panel D).

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To compare costs between automated and manual methods, we assumed that the indirect costs, such as human resources and infrastructure, were the same for the three methods. Thus, we evaluated the cost differences of reagents and materials used in the processes, from the delivery of the leukapheresis bag to the laboratory until the monocytes were plated in culture, which included the cost of plastic materials, Clinimacs® kits and reagents for centrifugation in gradients. Despite the fact that this is not an accurate evaluation of cost and that even between the two manual methods there may be some variation in relation to the amount of reagents used, we were able to grossly observe a savings of approximately $285 dollars for each dose of vaccine (1 × 107 cells) produced by manual methods.