Heparinized venous blood and tumor samples obtained between June, 2021 and March, 2022 from 9 patients with CRC, 13 patients with NSCLC and 18 patients with BC receiving treatment at the City Clinical Hospital No. 1 (Novosibirsk) and Novosibirsk Regional Oncology Center (Novosibirsk, Russia) were used in the present study (Table I). The inclusion criterion was the lack of history concerning surgery, chemotherapy and/or radiation therapy. Adenocarcinomas of the colon, lung or breast were histologically verified in all patients in accordance with the pathology. The presence of the HLA-A^*02:01 allele was confirmed by genotyping DNA isolated from peripheral blood cells, using an ALLSET™ GOLD HLA A LOW RES SSP kit (54310D; Invitrogen; Thermo Fisher Scientific, Inc.). Voluntary written informed consent was obtained from all patients. All subjects gave their informed consent prior to their participation in the study. The study was conducted in accordance with the Declaration of Helsinki, and the study was approved by the local Research Institute of Fundamental and Clinical Immunology (RIFCI) Ethics Committee (No. 132 from June 4, 2021).

Original DNA vaccine constructs were developed using the corresponding artificial genes based on the pmax plasmid (Addgene) (40). In particular, the following designs were applied: The pmax-CTL_1 construct containing epitopes from MAGE-A10, NY-ESO-1 and MUC-1; the pmax-CTL2 construct containing epitopes from MAGE-A3, PRAME, EpCAM and MUC-1; the pmax-CTL3 construct containing epitopes from EpCAM, CEA, GuanylylCyclase C and 5T4; the pmax-CTL4 construct containing epitopes from Legumain, VEGFR-1, VEGFR-2, FAP and Fos-related antigen-1; the pmax-CTL5 construct containing epitopes from Brachyury, SOX2, Snail1 and Snail2; the pmax-PolyTh construct containing epitopes from HER2, hTERT, p53, WT1, NY-ESO-1, VEGFR-2, survivin and MAGE-A3 (Table II). For DC transfection, a mixture of an equimolar amount of all DNA constructs was used, thereby providing a wide range of antigenic information.

Effective antigenic determinants recognized by cytotoxic T-lymphocytes were predicted using using NetMHC (41) and TEpredict software (42). Fragments containing epitopes capable of binding to the largest number of allomorphs of human MHC class II molecules (HLA-DR) were selected when designing the DNA constructs. The fact that amino acid residues flanking the epitope in the protein (the target antigen) can be important for interaction with the corresponding T-cell receptor was taken into account during fragment selection. The prediction of cytotoxic T-cell epitopes was carried out for the allelic variant of HLA-A^*0201 class I molecules. Peptides for which the predicted pIC50 value (a characteristic of the affinity of the interaction of a peptide with an MHC molecule) was >6.8 were selected for further analysis.

Mononuclear cells (MNCs) were isolated from the peripheral blood of patients with CRC, BC and NSCLC using the standard method of Ficoll-Urografin density gradient centrifugation (ρ=1.077 g/ml) (43). The obtained MNCs were then incubated for 30 min in 5% CO₂ at 37˚C in order to isolate cells with increased adhesion ability. Non-adherent MNCs were isolated with the medium, precipitated by centrifugation at 415 x g for 10 min at room temperature, and cultured at 37˚C and 5% CO₂ in a 75 cm² culture flask (TPP Techno Plastic Products AG) to be further used at a concentration of 2x10⁶ cells/ml, in complete RPMI-1640 medium containing 10% fetal calf serum (FCS; HyClone; Cytiva), 2 mM L-glutamine (BioloT Ltd.), 10 mM HEPES buffer (MilliporeSigma), 5x10^-4 M 2-mercaptoethanol (MilliporeSigma), 40 µg/ml gentamicin (Krka, d. d.) and 200 U/ml benzylpenicillin (OJSC Biosintez) prior to seeding. The adherent fraction was cultured in a 48-well plate (Greiner Bio One; Merck KGaA) at a concentration of 1x10⁶ cells/ml in 0.5 ml of complete RPMI-1640 medium, supplemented with 50 ng/ml recombinant human (rh) granulocyte-macrophage colony-stimulating factor and 100 ng/ml rhIL-4 (PeproTech, Inc.) for 96 h to obtain immature DCs. In order to obtain mature DCs, rhTNF-α (25 ng/ml) (PeproTech, Inc.) was added to the culture of immature DCs, and the cells were incubated for 2 days. In the resulting DCs, the expression of maturation and differentiation markers was assessed using flow cytometry (flow cytometer BD FACS Aria, BD FACS Diva v.6.0 software, BD Biosciences), using primary antibodies labeled with various fluorochromes [CD11c-PE-Cy7 (cat. no. 337216), CD83-APC (cat. no. 305312) and HLA-DR-FITC (cat. no. 327006)] (BioLegend, Inc.).

The magnetic transfection method was used as a method for delivering the DNA construct; unlike the electroporation method, it permits the acquisition of a cell culture with high viability (85.4±6.2%). The essence of magnetic transfection is the preliminary sorption of the DNA construct on the surface of magnetic beads, which penetrate into the cells in the culture of dendritic cells under the influence of a magnetic field. The magnetic transfection of mature DCs was performed in a total volume of 0.5 ml in a 48-well plate. Plasmid pmax (the control plasmid) and pmax-CTL1-8 (the target plasmid) were used for transfection. Magnetic transfection was performed using PromoKine reagents; the procedure was performed according to the manufacturer's protocol. Plasmids were dissolved in DMEM (State Research Center of Virology and Biotechnology ‘Vector’) in separate tubes; component was added in the ratio of 0.3 µg of DNA plasmid per 0.3 µl of MATra-A reagent, and the mixture was incubated at room temperature for 20 min. In parallel, DCs were precipitated by centrifugation at 266 x g for 5 min at room temperature, and RPMI-1640 medium was replaced with 250 µl of DMEM medium. Subsequently, the plasmid-MATra-A complex was added to the cells (25 µl per well); the plate was placed on a magnetic stand for 15 min. The medium was replaced after transfection: the DMEM was removed, and 300 µl complete RPMI-1640 medium were added. The transfected cells were incubated overnight in 5% CO₂ at 37˚C. The obtained dendritic cells were then plated with MNCs. The transfection efficiency was assessed using the Promo-Fluor-500 Nick Translation Labeling kit (PromoKine), with further analysis on a flow cytometer BD FACS Aria, BD FACS Diva v.6.0 software (BD Biosciences) using the Flow-Fish method (44,45).

The obtained DCs transfected with pmax and pmax-CTL1-8 plasmids were co-cultured with the fraction of non-adherent MNCs (at a concentration of 1x10⁶ cells/ml) for 120 h at 37˚C and 5% CO₂ to prime specific antigens (at a 1:10 DC to MNC ratio). Non-adherent MNCs cultured under the same conditions, as well as cells cultured in the presence of DCs not transfected with plasmids [MNCs+DC(0) group], were used as the controls.

Tumor cells were obtained by cold trypsinization of the tumor samples obtained from the patients during the planned surgical intervention. A tumor sample was divided into small fragments in RPMI-1640 medium containing 80 µg/ml gentamicin, 400 U/ml benzylpenicillin and 5 µg/ml amphotericin B (PanEco Ltd.), placed in trypsin (Biolot Ltd.), minced with scissors, and left overnight at >4˚C. To inhibit trypsin, RPMI-1640 medium containing 10% FBS was added and mixed thoroughly; the homogeneous suspension was precipitated (185 x g, 10 min, room temperature), and the cell count was calculated in a Goryaev chamber (Minimed). The cells were frozen in FCS with 10% DMSO and stored at -70˚C. The cells were thawed and cultured in complete RPMI-1640 medium 24 h prior to the cytotoxicity assay. Additionally, the viability of the tumor cell culture was assessed by staining with erythrosin (0.5 mg/ml) (MilliporeSigma) at room temperature within 1 min, prior to the cytotoxicity assay, once the cell suspension has been mixed with the dye, which averaged 67.6±15.4%, and the settlement was calculated based on the number of living cells.

The cytotoxic activity was analyzed by assessing the level of lactate dehydrogenase (LDH) in a conditioned medium in the co-culture of the DC and MNC cell population and autologous tumor cells from patients with CRC, BC and NSCLC. The procedure was carried out according to the instructions of the manufacturer of the kit ‘CytoTox96^® Non-Radioactive Cytotoxicity Assayʼ (Promega Corporation). Following the co-culture of the non-adherent fraction of MNCs and DCs transfected with the plasmids, as well as the culture of the cells of the control groups for 120 h, the cell suspension was washed with RPMI-1640 culture medium (Biolot), and the resulting cells were seeded at a cell concentration of 1x10⁶ cells/ml into round-bottom plates (TPP Techno Plastic Products AG; well volume, 50 µl) containing pre-thawed autologous tumor cells at a 10:1 ratio and incubated for 16-18 h at 37˚C and 5% CO₂. Cell seeding, and the experimental protocol were performed in accordance with the instructions for the ‘CytoTox 96 Non-Radioactive Cytotoxicity Assay’ kit (Promega Corporation). The optical density (OD) was measured on an Anthos 2020 spectrophotometer (Biochrom, Ltd.) at a single wavelength (490 nm). The cytotoxic effect was calculated according to the formula proposed by the kit manufacturer and expressed as a percentage: % cytotoxicity={[OD(experimental lysis)-OD(spontaneous lysis of effector cells)-OD(spontaneous lysis of tumor cells)]/[(OD(maximum lysis of tumor cells)-OD(spontaneous lysis of tumor cells)]} x100%. Thus, the formula considered the natural death of the tumor cells and MNCs.

Statistical data analysis was carried out using the GraphPad Prism software (GraphPad Software, Inc.). The normality of the sample distribution was assessed using the Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis test was used for statistical verification in the case of a non-normal distribution. An appropriate post hoc test was used after the Kruskal-Wallis test (e.g., Dunn's test). When comparing only 2 groups, a non-parametric Mann-Whitney U test was used. A value of P<0.05 was considered to indicate a statistically significant difference. Data are presented as the median ± standard deviation. The number of individuals per group is indicated by ‘n’ in the figure legends.

The mature DCs of patients with CRC, BC and NSCLC with a >80% content of CD11c⁺HLA-DR⁺ cells (Fig. 1A) and a >40% content of CD83⁺ cells (Fig. 1B) were obtained in vitro using the protocol described above. The unified protocol for DC generation was used for CRC-, BC- and NSCLC-derived cells, and it was observed to be effective for CRC- and BC-derived dendritic cell maturation. At the same time, in case of NSCLC-derived cells, the absence of a significant increase in the number of CD83⁺ cells was demonstrated.

The described cell-based protocol allows the generation of mature DCs derived from peripheral blood monocytes of patients with CRC, BC and NSCLC. The resulting DCs are characterized by high expression levels of the CD11c⁺HLA-DR⁺ and CD83⁺ markers (Fig. S1).

The following stage involved the magnetic transfection of the obtained DCs with an original polyepitope DNA construct. An average efficiency of the DNA transfection of DCs was ~70%. An example of the evaluation of the efficiency of transfection using flow cytometry is presented in Fig. S2.

Following co-culture of the transfected DCs with the autologous MNCs obtained from cancer patients, final studies were conducted to evaluate the efficacy of the developed cell-based vaccine, which consisted of antigen-activated MNCs (effector cells) and mature DCs transfected with a polyepitope DNA construct. The content of CD4⁺ and CD8⁺ lymphocytes between groups of cancer patients did not exhibit significant differences and amounted to 52-62% (CD4⁺ lymphocytes) and 24-27% (CD8⁺ lymphocytes) (Table III). The efficiency of mounting a specific cytotoxic immune response in the co-culture of MNCs and DCs was evaluated using the cytotoxicity assay against autologous tumor cells.

The LDH-releasing assay revealed that the cytotoxicity of the MNCs activated by DCs transfected with a polyepitopic DNA construct reached its values (45.5±7.1, 51.0±23.7 and 66.2±11.5% for patients with BC, NSCLC and CRC, respectively) when co-cultured with mature antigen-activated DCs transfected with the original DNA construct (Fig. 2). A significant increase in the cytotoxicity of cells in the culture of MNCs co-cultured with antigen-primed DCs, compared with groups of DCs and without transfection with the DNA construct of DCs, indicates the induction of an antigen-mediated cytotoxic immune response in vitro. These results directly demonstrate the effectiveness of the described approach.