Dendritic cells transfected with a polyepitope DNA construct stimulate an antitumor cytotoxic response in various tumor diseases

Dendritic cells (DCs) loaded with tumor-associated antigens (TAA) are known to be the important agents in antitumor response realization and still figure in lots of treatment schemes in cancer immunotherapy research. Here, we evaluated a cell-based protocol involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into ex-vivo-generated DCs of cancer patients (breast cancer, colorectal cancer, and non-small cell lung cancer). Direct cytotoxicity assay of effector cells, activated with the transfected DCs, showed a significant increase in the cytotoxicity against autologous tumor cells. The use of DNA-constructs encoding a large number of TAA’s for the in vitro DC loading to activate the T cell response could be a reliable and unified approach for immunotherapy and relapse prevention in patients with epithelial cancers. autologous DCs transfected with a DNA construct encoding the epitopes of tumor-associated antigens to autologous tumor cells (n=18). MNCs – mononuclear cells; MNCs+DCs(0) – mononuclear cells cultured in the presence of dendritic cells not transfected with a DNA construct; MNCs+DCs(C) – mononuclear cells cultured in the presence of dendritic cells transfected with the control plasmid; MNCs+DCs(T) – mononuclear cells cultured in the presence of dendritic cells transfected with a target plasmid (DNA construct). Data are presented as the median ± standard deviation. Arrows indicate statistically significant intergroup differences ( p ≤ 0.05).


Introduction
New technologies in cancer immunotherapy have made significant strides with the emergence of approaches such as blocking checkpoint molecules [16,17] and genetically modifying effector cells [18,19]. Nevertheless, overcoming the limitations appearingand reduction of the cost of such technologies is still to be done, and the study of all possible approaches to the induction of an immune response does not lose its relevance.One of the important aspects of efficient immune response formation isproperantigen presentation to enableendogenous antigen-specific cytotoxic T cell response [1,2]. DCs play a key role in activation of the antitumor immunity [3]. The functional activity of DCs was shown to be significantly reduced in patients with cancers [14,15]. In cancer, the ability of DCs to capture tumor antigens and present them to T cells, as well as to mount an effective cellular response, is impaired [4]. The main reason is considered to be the impairment of DC maturation process [3] as well as the T cell activation mechanisms [4].
Transferring the whole process of the endogenous immune response activation ex vivo, outside the immunosuppressive tumor influence, is shown to be feasible in obtaining T-cells efficient for tumor elimination when administered to patients as part of combination therapy [20,13]. Herewith, a number of studies have confirmed the effectiveness of DC antigen loading using 4 DNA constructs [5][6][7][8]. In this regard, we are still interested in finding the right combination of these approaches to produce the optimized and unified protocol available for wide clinical use for immunotherapy in cancer patients.
Tumor-associated antigens are usually presented in a wide range of epithelial tumors, and each tumor can express a wide range of known antigens. Previously we showed the efficiency of in vitro generation antitumor immune response with the use of DCs transfected with DNA constructs encoding epitopes of particular tumor-associated antigen determinants [12,16]. In the current study, we optimized the approach and tested the hypothesis about the efficiency of using genetic constructs encoding a wide range of TAA's for the activation of T cell cytotoxicity against autologous tumor cells from tumors of different localization

Materials and methods
The study object Heparinized venous blood and tumor samples obtained from 9 patients with colorectal cancer (CRC), 13 patients with non-small cell lung cancer (NSCLC), and 18 breast cancer (BC) patients receiving treatment at the City Clinical Hospital No. 1 (Novosibirsk) and Novosibirsk Regional Oncology Center were used in the study. The inclusion criterion was no history of surgery, chemotherapy and/or radiation therapy. Adenocarcinomas of the large intestine, lung or breast were histologically verified in all patients in accordance with the pathology. Voluntary informed consent was obtained from all patients. This study was approved by the local Research Institute of Fundamental and Clinical Immunology (RIFCI) ethics committee.
All subjects gave their informed consent for inclusion before they participated 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.
Effective antigenic determinants recognized by cytotoxic T lymphocytes were analyzed using specialized software (more than 500 epitopes were used) [7]. 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. Prediction of cytotoxic T cell epitopes was carried out for the allelic variant of HLA-A*0201 class I molecules.

Generation of mature dendritic cells
Mononuclear cells (MNCs) were isolated from peripheral blood of patients with colorectal cancer, breast cancer, and NSCLC using the standard method of Ficoll-Urografin density gradient centrifugation (ρ = 1.077). The obtained MNCs were then incubated for 30 min in 5% CO 2 at 37°C in order to isolate cells with increased adhesion ability. Non-adherent MNCs were isolated with the medium, precipitated by centrifugation, and cultured in a 75 cm 2  with 250 μl of DMEM medium. Next, 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 medium was removed, and 300 μl of complete RPMI-1640 medium was added. Transfected cells were incubated overnight in 5% CO 2 at 37°C.

Co-culturing of dendritic cells and mononuclear cells
The obtained DCs transfected with pmax and pmax-MQ/UB-CTL 1/8 plasmids were cocultured with the fraction of non-adherent MNCs (at a concentration of 1*10 6 cells/mL) for 120 hrs to prime specific antigens (at a 1:10 DCs : MNCs 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 control.

Statistical data analysis
Statistical data analysis was carried out using the GraphPad Prism software. Normality of the sample distribution was assessed using the Kolmogorov-Smirnov test. Non-parametric Friedman test was used for statistical verification in case of non-normal distribution, corrections for multiple comparisons were made. The differences were considered to be significant at error probability p<0,05. Data are presented as median ± standard deviation. The number of 8 individuals per group is indicated by n in figure captions.

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

Evaluation of the cytotoxic activity of mononuclear cells against tumor cells
The LDH-releasing cytotoxicity assay showed that cytotoxicity of MNCs activated by DCs transfected with a polyepitopic DNA construct reached its maximum values (51.2%, 57.2%, and 67.4% for patients with BC, NSCLC, and CRC, respectively) when co-cultured with mature antigen-activated DCs transfected with the original DNA construct ( Fig. 2A, B, C).
A. leads to suppression of the immune response [11]. For this reason, the sensitivity of DC precursors to growth factors is reduced in NSCLC and does not lead to a high expression level of maturation and co-stimulatory molecules.
Direct cytotoxicity assay against autologous tumor cells as target cells is proven to be an objective approach for in vitro assessing the effectiveness of the immune response generated [8].
In our studies, high antitumor cytotoxic activity of effector cells after co-culturing with antigenloaded DCs was assessed on day 5. The tumor cell death rate was more than 51%, significantly exceeding the average values for the control group (MNCs). It was shown that the DNA construct encoding the epitopes of the major tumor-associated antigens of various epithelial cancers can equally enhance the direct cytotoxic immune response against tumor cells of epithelial origin and various localization (in particular BC, CRC, and NSCLC). Previously, we used a similar DC transfection protocol, but using a DNA construct encoding HER2/neu epitopes in order to generate an antigen-specific immune response. The increased population of cytotoxic cells specific for HER2/neu epitopes was shown after co-culturing antigen-specific DCs derived from breast cancer patients with MNCs [12]. In terms of cytotoxicity generated, the results of current research showed comparable efficiency levels comparing with the HER2/neu protocol, thus we can state that the use of DNA constructs encoding the wide range of TAA epitopes can also be efficient in stimulation of antitumor T-cell response, but the wide range of TAA epitopes enables the use of such approach in treatment of more cancer types. We also previously tested a protocol for generation of antigen-primed DCs in breast cancer patients in vivo [13]. There we used autologous tumor cell lysate as an antigen source and showed the increase in the count of CD8+ cells, which exhibit strong antitumor cytotoxic activity [13]. In this regard, we see that autologous tumor lysate can also be a viable option for DC antigen loading, but here we should take into account that lysate requires the preparation of tumor material for every patient separately, and in lots of cases, tumor tissues are not available. At the same time, the use of DNA constructs enables the unification of antigen-loading protocol for different patients and even tumor types, and it could be a reliable solution in situations tumor material is not available.

Data availability
The datasets generated during the current study are available from the corresponding author on request.

Conflict of interests
The authors declare no competing interests.

Funding
The research was carried out at the expense of a grant Russian Science Foundation No. 21-65-00004, https://rscf.ru/project/21-65-00004/.The funding source had no involvement in the study design, data collection, analysis and interpretation of data, writing of the manuscript, or in the decision to submit the article for publication.