Cancer is still one of major non-communicable cause of death in the adult population. According to the World Health Organization, cancer occupies the leading position based on mortality rate among people aged under 70 in 112 countries of the world [1]. The most common malignant neoplasms (MNs) by detection rate include breast cancer (BRCA), non-small cell and small cell lung cancer (NSCLC and SCLC, respectively), colorectal cancer (CRC), gastric cancer (GC), liver cancer (LC), prostate cancer (PC), cervical cancer (CC), thyroid cancer (TC), and bladder cancer (BLCA). Melanoma, various types of primary central nervous system cancers (neuroblastoma and glioblastoma) and oncohematological diseases can be considered as the most aggressive MNs. MNs with the highest mortality rate include lung cancer, CRC, LC, GC, BRCA, PC, CC, as well as esophageal cancer, pancreatic cancer, and leukemia [1]. MNs are found in people of various age and gender, different nationalities and professions. The important role in carcinogenesis is played by the genetic predisposition factors, harmful habits (such as tobacco smoking), and environmental factors (such as harsh industrial environment) that significantly increases the risk of MNs [2]. That is why early detection of MNs in the groups with occupational risks, adequate choice and implementation of timely anticancer therapy is important.

The main treatment for solid MNs (stages I–III) is surgical resection of the tumor with adjuvant and/or neoadjuvant therapy [3]. The combination therapy is often used: surgical treatment combined with radiation and chemoradiation therapy [3], as well as the combination with immunotherapy, for example, therapy with immune checkpoint inhibitors (ICIs) [3, 4]. In particular, in 2022 the U.S. Food and Drug Administration (FDA) has approved seven ICIs for the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1(PD-L1) pathway: pembrolizumab, nivolumab, durvalumab, atezolizumab, avelumab, cemiplimab, dostarlimab [4].

The other cancer immunotherapy option is represented by the use of the so-called dendritic cell vaccines (DC-vaccines) [5, 6]. It is believed that clinical efficacy of DC-vaccines is associated with targeting the populations of immunosuppressive cells in the tumor microenvironment and subsequent immunogenic tumor cell death induction [7].

DCs are involved in antigen presentation, immune response regulation, inhibition of immunosuppressive T cells. DCs also can sensitize other effector cells of the innate antitumor immunity [5, 6]. Several DCs subpopulations are distinguished based on the origin and antigen receptors: myeloid DCs, lymphoid DCs, plasmacytoid DCs, Langerhans DCs, and monocyte-derived DCs [5, 6]. As a link of antitumor immune response, DCs are involved in recognition and presentation of the neoantigens, emerging de novo in the tumor cells, to the immunocompetent cells [5, 6]. It is rational to use this ability of the DCs loaded with tumor antigens ex vivo for further activation of the CD4⁺ helper and CD8⁺ cytotoxic T cells in order to determine the directions of the immune responses [8]. Today, only PROVENGE, the autologous cellular product, consisting of the antigenpresenting cells activated by the PA2024 recombinant chimeric protein, has been approved by FDA for treatment of PC based on the phase III clinical trial results () (NCT00779402).

Since the tumor neoantigens (NAs) stimulate specific antitumor immune response in the patient’s body, the new personalized therapeutic approaches in the field of neoantigen vaccines (NA-vaccines) creation have been developed in recent years [9]. Neoantigens are highly specific for tumor cells. They can be divided into common ones, which are produced by the mutations in oncogenes and personalized ones (unique for the tumor found in a certain patient [10]. At least, two NA-based immunotherapy approaches are under activey development: peptide and RNA vaccines. Thus, peptide vaccines may contain the mixtures of synthetic peptides with adjuvants or the DCs loaded with peptides [11, 12].

The limitations of DC-vaccines are associated with time- and resource-consuming process of vaccine preparation. Sometimes this is the reason why the disease progression occurs, which reduces the clinical benefit of therapy. Furthermore, some patients might not survive to the end of the therapy course [5, 13]. The high cost of biological stimulators that are critical for correct DC differentiation and loading of DCs with antigens also prevents the timely production of vaccines and their introduction into clinical practice [5, 9, 13]. It is also pertinent to note that, despite the facts of achieving pathomorphological responses of tumors and stabilization of disease while administrating DC-vaccines, together with favourable pharmacological safety data, there is an objective problem of increasing the vaccine efficacy. This can be solved through various modifications of the existing vaccine compositions and combinations with other anticancer drugs [5, 6].

The aim of the review was to systematize the literature data in the approaches to the development of the DC- and NA-vaccines as candidate anticancer drugs in terms of optimizing methodological and some technological aspects of the drug development in order to overcome the abovementioned problem. The review also reports the features of interaction between the DC vaccines and human immune cells and the most advanced developments based on the data of preclinical and clinical trials (PCTs and CTs, respectively).

Clinical trials of the DC- and NA-vaccines for treatment of MNs

As of  December 2022, a total of 410 and 96 records of the clinical trials (CTs) of the DC- and NA-vaccines, respectively, were found in the ClinicalTrials database [14]. Among all CTs focused on DC-vaccines, 191 CTs (46.58%) were completed, 45 CTs (10.97%) were terminated, 24 CTs (5.85%) were withdrawn (suspended). Among a hundred of active CTs, 32 CTs (7.80%) were assigned the status “active, not recruiting”, 57 CTs (13.90%) had the “recruiting” status, and 11 CTs (2.68%) had the “not recruiting” status. The status of another 50 CTs (12.20%) was “unknown”.

Among the successfully completed CTs of anticancer DC-vaccines, a total of 29 CTs (86% — phase II, 14% — phase III). were analyzed tab. 1 provides basic information about the CTs conducted (title, phase, status, disorder, group of patients, DC-vaccine dosing regimen, drug in combinations, etc.). The CTs focused on clinical assessment of safety, tolerability efficacy of the DC-vaccines used in treatment of various cancer types have been distributed as follows. The group of malignant neoplasms (stage III) includes two CTs of DC-vaccines only for treatment of PC. The other two CTs are focused on DC-vaccines in combination with dasatinib for treatment of metastatic melanoma (stage III) or glioma in individuals receiving temozolomide (TMZ). The group of MNs (stage II) includes ten CTs of DC-vaccines used alone and 15 CTs of DC-vaccines used in combination with other pharmacotherapeutics, most often combinations with interleukin 2 (IL2), TMZ or interferon-α (IFNα). Other MNs are  distributed as follows: glioma (five CTs), melanoma (three CTs), sarcoma (three CTs), prostate cancer (three CTs), ovarian cancer (two CTs) and breast cancer (two CTs). It must be acknowledged that the vast majority of clinical trials are focused on assessing the combination of DC-vaccines and ICIs. Information about the active CTs phases II and III is provided in tab. 2 and tab. 3, respectively.

The number of CTs registered in the ClinicalTrials database and devoted to and NA-vaccines was about four times lower than that of the DC-vaccines. Among 96 CTs, 11 CTs were completed, eight were terminated, three were suspended; there were 60 active CTs and 14 CTs with unknown status. By analogy with DC-vaccines, clinical assessment of NA-vaccines involved mostly individuals receiving ICIs, and the spectrum of MNs targeted by CTs was almost the same. The safety and anticancer efficacy of the NA-vaccine in individuals receiving pembrolizumab and nivolumab were confirmed in NCT03633110 (phase II) only. Among eight terminated CTs, three were terminated due to long development time, and the other five were terminated due to underinvestment.

Analysis of DC-vaccines CTs (phase I and II) details has helped reveal a number of issues in this field. First, a small number of individuals (usually not exceeding 20) enrolled is the main factor of the CTs’ termination. Second, complications with interpretation data obtained on different anticancer treatment regimens in the same CT. Third, specific design of the CT that includes a single cohort of patients or the CT without randomization. Despite the fact of achieving the endpoints of safety and tolerability of the anticancer vaccine, a common trend of moderate efficacy of the DC-and NA-vaccines administrated alone should be noted. It defines the relevance of their combination with other pharmacotherapeutics. However, there are exceptions. For example, the DC-vaccine for intratumoral administration obtained in the presence of IFNα and granulocyte macrophage colony-stimulating factor (GM-CSF) showed high immune responses even in the absence of tumor-associated antigen. It ensured complete regression of follicular lymphoma in some individuals who received low doses of rituximab [15]. It is important to note that the combinations of DC-vaccines with targeted or immunotherapy drugs showed higher efficacy than the DC-vaccines administrated alone. The objective response rate (ORR) reached 50%, and the difference in progression-free survival (PFS) and/or overall survival (OS) was up to 100% depending on the treatment regimen. Thus, DC-vaccines in combinations with other therapy may have a more prominent anticancer effect ensuring higher OS.

The other trend found is — DC- and NA- vaccines are considered as a “last choice therapy” option. It may be the cause of their low efficacy in the CTs in a group of individuals with late-stage cancers. Alternatively, stimulation of the tumorinfiltrating immune cells and local immune responses has all the chance to demonstrate much better efficacy for treatment of early-stage cancers, when it is necessary to prevent metastasis.

Optimization of some manufacture and application steps of biotherapeutic anticancer vaccines

Options of accelerating, simplifying and cost-reducing of the DC-vaccines manufacturing

1. Options for accelerating the DC-vaccines manufacture process

The use of nucleic acids to load the dendritic cells is the first approach to accelerating the DC-vaccine manufacture [9]. Synthesis of nucleic acids is a less time-consuming process than the synthesis of target peptides. Similarly, the nucleic acid purification procedure is less time-consuming than purification of the peptides or polypeptides. Nucleic acids, that are more stable than peptides, are adjuvants that can activate pro-inflammatory molecular pathways involving the Toll-like receptors (TLR) associated with activation of innate immunity [16].

The second approach involves modification of cultivating conditions of manufacturing cell strains. For example, the transfer of murine bone marrow progenitor cells into monolayers of murine OP9 stromal cells expressing the deltalike Notch 1 ligand (OP9-DL1) after three days of incubation with the FMS-like tyrosine kinase 3 ligand (FLT3L) led to the fact that the cells expressed the murine markers (CD103, CD24, DEC205 and CD8α) of myeloid DCs, the population that did not arise after incubation with FLT3L only. The transcriptional gene expression profile of such DCs was most similar to that of autologous DCs of the spleen. Meanwhile, the survival rate of laboratory animals increased, which could be due to enhanced lymphocyte migration to the tumor lesions [6]. The co-culture of human hematopoietic stem and progenitor cells and OP9-DL1 enabled a 20-fold increase in the yield of DCs of all types relative to conventional cell culture methods [17].

The third approach involves stimulation of the cell culture with various cytokines, such as GM-CSF [17, 18]. The transcriptional profiles of the DCs obtained were almost identical to that of primary DCs, while the cells themselves demonstrated normal cytokine responses to TLR agonists, including secretion of IL12, TNFα and IFNγ, and effectively induced the CD4⁺ and CD8⁺ T cell proliferation [17, 18].

The fourth approach was implemented by using the genetic editing technologies. Thus, viral transduction [19] and RNA interference methods [20] together with the CRISPR/CRISPRCas9 genome editing system [21] were used to generate the DC-vaccines. Pre-clinical trials showed that all methods were highly effective and could presumably be scaled to the DC-vaccines manufacture.

Another reported vector-free approach for acceleration of the DC-vaccine preparation is based on the Cell Squeeze® technology which involves forcing the target molecules through the membrane pores emerging due to temporary membrane integrity disruption [22]. It has been shown that this DC loading technique can be used ex vivo and it is suitable for transfer of various antigens to cytosol [23].

2. Options for reducing the cost of the DC-vaccines manufacture process

Among all available options for reducing the cost of DC-vaccines there are exosome preparations obtained from DCs (DEXs). DEXs are considered as more technologically feasible and less expensive compared to conventional DC-vaccine preparation. Both in vitro and in vivo studies have shown that DEXs can activate the CD4⁺ and CD8⁺ Т cells and stimulate the effective antigen-specific responses of cytotoxic lymphocytes. However, the desired anticancer efficacy has not been achieved in several CTs, putting into question the prospects of DEXs application [24]. The DCs pretreatment with interferon — (IFNγ) resulting in the increased expression of CD40, CD80, CD86 and CD54 is an option to increase the DEX efficacy. However, this approach, well proven in PCTs [25], was less effective in the CT (phase II) [26].

3. Options for simplifying the DC-vaccines manufacture process

Preparation of the DC-vaccines based on primary DCs extracted from the patient’s peripheral blood is much simpler than ex vivo DC preparation, with such limitation as the low DC content (less than 1%) in the monocyte fraction [27]. Low circulating DCs counts have been revealed in blood samples of patients with melanoma [28] and breast cancer [29], while abnormal DC differentiation is reported in the breast cancer and pancreatic cancer models [30]. Therefore, the effectiveness of DCs isolation from the peripheral blood of patients with these tumor types was minimal. Since the successful implementation of this approach has yet been demonstrated only in vivo in the murine model with xenotransplantation of B16/F10 and B16-Flt3L cells (melanoma) as well asMC38 cells (CRC) [31], the prospects of preparation the DC-vaccines (DCs type I) against the majority of tumors seem to be hardly feasible.

Options of the anticancer vaccines application in combination therapy

1. Growth factors

The combinations of DC-vaccines and growth factors are designed to enhance the antigen-specific response. GMCSF is most often used in combinations with DC-vaccines because it functions as a hematopoietic growth factor and immunomodulator. GM-CSF was also used as a low-toxic adjuvant during treatment with the DC- or NA-vaccines containing peptides [32]. Another approach based on the use of DC-vaccines and FLT3L has been reported. Thus, a significant increase in the generation of autologous DCs, including plasmacytoid DCs, has been revealed in the murine models in the presence of FLT3L. It is assumed that the increase in the mature DCs functional activity in the presence of FLT3L is mediated through the signaling pathways involving phosphoinositide 3-kinase (PI3K) and mTOR kinase [33].

2. ICIs

The combinations of ICIs and DC-vaccines lead to activation of T cells and NK cells, reduced immunosuppressive activity of regulatory T cells [5, 34], and therefore to the increase in the DC- vaccine efficacy. In turn, the DC-mediated activation of NK cells and DC γδ Т cells [35, 36] can increase the efficacy of ICIs. Synergistic antitumor effect of the combination of nivolumab and DC-vaccine was revealed in individuals with BRCA, myeloma, melanoma, lung cancer, lymphoma and glioblastoma [37]. In addition, the DC-vaccine was proven to be safe for patients; low number of side effects related to the use of nivolumab was reported [37].

3. NK cells

One more promising approach involves the combination of anti-cancer DC-vaccines and NK cell-based vaccines. NK cells present in the tumor microenvironment can produce a number of chemokines that positively affect the DC activity along with the FLT3L that enhances the autologous DC generation [38]. Furthermore, the activated NK cells can kill immature DCs and induce the adaptive immune response in the secondary lymphoid organs. The mature DCs produce cytokines (mainly IL2, IL12, IL18) that stimulate production of IFNγ, TNFα or GMCSF by the NK cells, thereby accelerating the DC maturation process [39].

Modifications of DC- and NA-vaccines

DC-vaccines

The contemporary trend in the development of anti-cancer vaccines is represented by the targeted approach based on the tumor-associated antigens (TAAs). These include overexpressed antigens, normal differentiation antigens and cancer stem cell antigens, as well as NAs. A peptide, chimeric protein, DNA or RNA can be the active ingredient of such vaccines [16].

One approach to modification of DC-vaccines involves the use of nanoparticles that are easily internalized by DCs through endocytosis and can be used as carriers of nucleic acids or peptides [32]. In this context, nanoparticles have some advantages: immunogenicity and the ability to be translocated through lymphatic vessels, if the particle size does not exceed 200 nm. The tumor antigens can be conjugated with nanoparticles by adsorption, encapsulation, chemical conjugation and self-assembly [32].

Another promising approach to modification of DC-vaccines involves genetic reprogramming of somatic cells by inducing the expression of key cell differentiation factors. The moDCs are more appropriate for this approach compared to other DCs. For example, the SmartDC technology enables reprogramming of autologous CD14⁺ monocytes using the lentiviral vector that carries genes encoding GM-CSF, IL4 and TRP2 (dopachrom tautomerase). Transduction with the viral vector triggers differentiation of monocytes into the TRP2⁺ moDCs.The SmartDC technology is simpler and less time-consuming compared to conventional DC-vaccine preparation [19].

NA vaccines

Developments of machine learning algorithms and neural networks allow for rather accurate identification of the patient’s NAs and predicting the protein (peptide) structure [9]. Information about the predicted and tumor NAs is systemized in the specialized databases, such as dbPepNeo [40]. However, not all tumor NAs can be used to develop the NA-vaccines. Such parameters of NAs, as allogeneity, clonal distribution, abundance of the major histocompatibility complexes I and II (MHC-I, MHC-II), affinity of T cells for NAs, and the presence of driver mutation in the gene encoding NAs, have to be taken into account [41]. It is well known that the NA-vaccine efficacy results largely from the tumor mutational burden (TMB), i.e., the number of mutations per DNA fragment with the length of 1 million base pairs, but it can be limited due to low TMB values of some MNs. It should be remembered that TMB is considered to be a predictive biomarker for melanoma and NSCLC only [41, 42]. It was noted that the cultured DCs or DCs isolated from the patient’s blood can be easily loaded with NAs using the routine procedures: electroporation or lentiviral transduction [43]. This contributes to the development of the mixed DC-NAvaccines that have already shown their anticancer efficacy in the PCTs involving the models of PC, BRCA, NSCLC, CRC and Merkel cell carcinoma. Some of these vaccines are being studied in CTs (tab. 2).

CONCLUSION

The DC- and NA-vaccines represent an intensively developed branch of the high-techbiotherapeutic anticancer drugs for the personalized application. Since certain technological aspects of the DC- and NA-vaccine preparation are characterized by considerable duration, high labor and resource intensity, optimization of preclinical developments aimed at accelerating,simplifying and cost reducing the DC-vaccine manufacture processis relevant. These developments will significantly increase the scale of the DC- and NA-vaccines applications in the future.

The approach directed totargeting the vaccines to cancer stem cells (CSCs) and their NAs seems to be ambitious and promising. According to a number of studies, tumors with aggressive phenotypes can contain large populations of CSCs that determine high proliferative potential and the disease progression [44]. However, a more detailed investigation of the CSC molecular genetic profile and the spectrum of the CSC specific biomarkers is needed to improve this approach.

Since the DC- and NA-vaccines have proved to be effective against a number of similarmalignant neoplasms, clinical assessment of the mixed (combined) NA-DC-vaccines should be considered as a promising area, however, the results of such CTs have not yet been published.

According to the analysis of the completed and active CTs, the combinations of DC-vaccines and ICIs currently demonstrate the highest anticancer efficacy along with acceptable safety and tolerability in patients with solid malignant neoplasms.