Anticancer DNA vaccines

Frontiers in Bioscience 11, 1189-1198, January 1, 2006
DNA VACCINES FOR CANCER
Denise R. Shaw, Theresa V. StrongFaculty of Medicine, Department of Hematology and Oncology and Comprehensive Cancer Center of the University of Alabama (Birmingham).

Translation: Evgenia Ryabtseva, portal "Eternal Youth" www.vechnayamolodost.ru

Table of contents 1. Summary

 2. Introduction
 3. Gene transfer for immunization
 4. The mechanism of development of the immune response during DNA vaccination
 5. Factors affecting the induction of an immune response
 6. Strategies to enhance the immune response
 7. Clinical experience of using DNA vaccines
 8. Prospects
 9. Gratitude
10. Literature

1. SummaryDNA vaccines, also called genetic, plasmid or polynucleotide vaccines, are a relatively simple and cost-effective method of applying gene transfer for immunization against tumor-specific antigens.

This review discusses the potential advantages of DNA vaccines for cancer immunotherapy compared to traditional protein vaccines and viral vectors. In addition, the article describes the alleged mechanisms of induction of the immune response that develops in response to DNA immunization. The currently available results of preclinical development of DNA vaccines and clinical experience of DNA immunization in cancer treatment are also analyzed. The low level of toxicity inherent in DNA vaccines indicates in favor of continuing work in this direction, however, for the successful implementation of this approach into clinical practice, it is necessary to develop additional strategies to increase the effectiveness of existing drugs.

2. IntroductionIn the production of the first antitumor vaccines, proteins or cells were used as agents that cause an immune response to tumor-associated antigens.

Gene transfer techniques have provided new opportunities to stimulate the immune response. Among the range of techniques being developed for clinical use, DNA vaccines (also called nucleic, polynucleotide, plasmid or genetic vaccines) have emerged as an effective innovative method for triggering an antigen-specific antitumor immune response.

DNA vaccination is based on the use of plasmid (ring) DNA molecules encoding the target antigen. This delivery method has a number of advantages, the most important of which is perhaps that it triggers both a humoral and cellular immune response. Synthesis of the protein encoded in the plasmid in vivo provides its further processing for presentation in combination with antigens of the major histocompatibility complex (MHC) of class I, which contributes to the formation of cytotoxic T-lymphocytes (CTL) of class I. Cytotoxic T-lymphocytes are considered important mediators of antitumor immune response, and their activation in response to tumor antigens plays a crucial role in the effectiveness of antitumor vaccines. In addition, unlike protein vaccines produced with the help of bacteria, antigen synthesis in vivo ensures proper folding (folding into a three-dimensional structure) and post-translational modification of the protein molecule. DNA-based vaccines also cause long-term antigen expression and, accordingly, a persistent immune response.

Additional factors favoring the development of plasmid DNA immunization strategies include the relative simplicity and low cost of production of such vaccines, as well as their stability. As described in more detail below, DNA vaccines produced with the help of bacteria are immunostimulants by nature, due to the presence of unmethylated cytosine-guanine dinucleotides (cytosine guanine (CpG) dinucleotides) in their structure. This sequence stimulates a non-specific immune response that does not prevent repeated injections of the vaccine. This feature favorably distinguishes DNA vaccines from virus-based vaccines, the effectiveness of which can be greatly reduced due to a pre-existing or vector-induced immune response (1,2). The safety parameters also indicate in favor of polynucleotide vaccines compared to viral ones, since when working with them there is no risk of recombination with wild-type viruses and the risk of insertion mutagenesis is very low. Finally, DNA vaccines can be used for the rapid delivery of several epitopes and even several antigens in one injection, which, given the tendency of tumors to evade immune recognition by losing antigenic variants, is a very important factor (3).

Despite these potential benefits and promising results of preclinical studies, anticancer DNA vaccines have so far shown minimal activity in clinical settings. Many tumor antigens do not contain mutations, so the development of an immune response directed against them implies the ability of the immune system to recognize and launch an effective response against the body's "own" antigen. The results of preliminary studies indicate that this is quite difficult to achieve in relation to human tumors. Therefore, increasing the activity and, accordingly, the clinical effectiveness of polynucleotide vaccines has become the main goal of work in this field of research. The review presented to your attention gives an idea of a number of approaches developed to overcome these limitations and currently being tested on preclinical models.

3. Gene transfer for immunizationA prerequisite for the development of vaccines based on nucleic acids was the observation of Wolff et al., who found that intramuscular injection of deproteinized DNA leads to the expression of the gene encoded in it by muscle fiber cells (4). Subsequent work demonstrated the applicability of this approach to the expression of foreign genes in cells of various species, ranging from fish (5) and ending with primates (except humans) (6). Despite the low efficiency of the process, intramuscularly injected DNA penetrates into muscle fibers through pits on the surface of myocytes and T-tubules (7,8). DNA is stored in the nuclei of receptor cells in extrachromosomal form, but its expression is recorded for a long period of time (9), the duration of which it depends on the immunogenicity of the protein encoded in it.

Using a plasmid encoding a protein of the influenza virus – nucleoprotein A – Ulmer et al. demonstrated for the first time the ability of intramuscular injection of DNA encoding a viral protein presented by antigen-presenting cells (APC) in combination with MHC class I antigens to induce a CD8+ T-lymphocyte-mediated immune response protecting against infection [10]. The results of this study served as proof of the feasibility of developing DNA vaccines for the treatment of various diseases, including cancer.

The induction of cellular and humoral immune response with the introduction of DNA vaccines is not limited to intramuscular administration. The skin contains many APC, such as immature Langerhans cells in the epidermis and mature dendritic cells (DC) in the dermis. Tang et al. demonstrated the ability of DNA penetrating through the skin to trigger an immune response to a protein encoded in it [11]. As part of this work, DNA was deposited on gold microparticles, which were applied to the skin under pressure using a ballistic device [12]. This process, commonly referred to as gene gun delivery, does not injure the skin and requires much less DNA to form an immune response than intramuscular injection (13,14). The induction of effector CTLs mediating tumor rejection was subsequently demonstrated on a mouse model of transplanted tumors [15]. Intradermal immunization can also be carried out by injection of deproteinized DNA or the introduction of DNA-coated nanoparticles using needle-free injection systems (16). The option of applying DNA vaccines to the mucous membranes is also being considered, which is primarily relevant for immunization against infectious diseases (17), but may also be applicable for the treatment of cancer (18). And finally, despite the relatively short half-life in the bloodstream, work on intra-splenic administration of DNA vaccines (19) demonstrated that strategies promoting the uptake of intravenously injected DNA by splenocytes can lead to the formation of a humoral and cellular immune response. The fact that all these delivery methods lead to the synthesis of antigens and the induction of an antigen-specific immune response indicates the universality of DNA vaccination. An important point is also that different methods of vaccine administration can lead to the development of qualitatively different immune reactions (20,21), and their relative effectiveness for humans has yet to be determined.

4. The mechanism of development of the immune response during DNA vaccinationThe study of the ability of DNA vaccines to cause the development of a cellular immune response paved the way for their development as a means of antitumor therapy.

The mechanisms of induction of the immune response during DNA immunization are still not completely clear, however, apparently, they include antigen processing using endo- and exogenous mechanisms that provide presentation of the antigen in combination with class I and II MHC proteins. DNA can transfect both target cells (for example, myocytes with intramuscular injection) and those that are part of the APC tissue. Despite the fact that myocytes synthesize a protein encoded in DNA, only APC provides the costimulatory signal necessary for the effective activation of CTL. The results of a number of studies indicate that the central role in the induction of the immune response to DNA immunization belongs to the APC of bone marrow origin (22-25). These data suggest "cross-presentation" of the antigen by antigen-presenting cells. The antigen is synthesized by myocytes and transferred to the APC in such a way that the processed peptides are presented on their surface in complex with class I MHC proteins, giving the APC the ability to directly activate CTL. This contradicts the usual situation when proteins entering the APC exogenously move into the endolysosomal system for degradation and presentation in complex with MHC class II molecules. Alternatively, or additionally, transfection of the APC itself with nucleic acid can occur (26,27). In vivo, the synthesis of antigens in the cytoplasm of the APC provides the presentation of peptides in complex with Class I MHC molecules. The presentation of the antigen together with class I and II MHC molecules in the presence of the corresponding costimulatory molecules expressed by APC leads to the activation of both CD4+ and CD8+ T cells, providing simultaneous development of cellular and humoral immune response.

The creation of mouse models with selective disrupted elements of the immune system by knockout of genes made it possible to formulate a clearer definition of factors critical for the induction of an effective immune response [28]. The study of the mechanisms of tumor rejection mediated by a therapeutic DNA vaccine in a transgenic mouse model of breast cancer demonstrated the coordinated functioning of CD4+ and CD8+ cells, antibodies, Fc receptors, perforins, gamma interferon, CD1d-restricted T lymphocytes with the activity of nonspecific killers (NKT) and macrophages, as well as an important role activated neutrophils capable of directly lysing tumor cells and damaging the vascular network feeding tumors (29,30).

5. Factors affecting the induction of an immune responseA number of characteristics of DNA vaccines determine the nature and severity of the immune response caused by them.

DNA composition is the primary parameter characterizing plasmid vaccines. The CpG dinucleotide is relatively poorly represented in the mammalian genome, and CpG-rich DNA regions are often methylated as a result of the functioning of the transcription regulation mechanism. DNA plasmids synthesized by bacteria, on the contrary, contain unmethylated CpG dinucleotides recognized by innate immunity mechanisms as an indicator of the presence of a pathogen [31]. These sequences are recognized by toll-like receptors-9 (toll-like receptor 9, TLR9) and trigger the activation of DC, macrophages, natural killers and NKT cells (32,33). As a result, CpG sequences included in plasmids or purified oligodeoxynucleotides (ODN) are powerful adjuvants and can trigger an immune response mediated by type 1 T-helpers (Th1) [34]. ODN containing CpG also have an anti-apoptotic effect on CD4+ and CD8+ cells (35). The presence of CpG sequences significantly enhances the overall immunogenicity of DNA vaccines.

In addition to the composition of nucleic acids, an important factor determining the immune response is the level of transgene expression. In general, increased expression of the immunogen enhances the immune response. Therefore, a strong promoter is needed to ensure effective transcription of a plasmid-encoded gene, and optimized polyadenylation signals and the presence of untranslated regions can promote transgene expression [36]. The activity of the cytomegalovirus early promoter/enhancer, widely used to trigger the expression of the encoded sequence, can be enhanced by embedding additional sequences, for example, isolated from the DNA of an adeno-associated virus [37].

When creating an optimized vector, the method of its introduction also affects the developing immune response. In the previous section, it was discussed that different methods of DNA immunization lead to the development of a cellular and humoral immune response, but the nature of the immune response that develops when using different approaches to immunization may differ qualitatively (20,21,38,39,40). In general, the introduction of DNA using a gene gun triggers an immune response with a more pronounced participation of T-helper-2 (Th2), characterized by a strong humoral component, not very effective in the treatment of tumors. However, this effect can be modified by simultaneous administration of cytokines that stimulate the activity of Th1 (41). The nature of the immune response can also be influenced by the selection of optimal doses and vaccination regimens (13,42).

The antigenicity of the encoded protein is extremely important for the formation of an effective immune response. The fact that most tumor antigens are "proprietary" is an insurmountable barrier to the development of effective methods of cancer immunotherapy. Changing the antigenicity of the protein or stimulating its absorption by the APC are the key points considered in the aspect of solving this problem. The local cytokine profile also plays an important role in the formation of the final immune response. Optimization of all these factors in order to maximize the effectiveness of the immune response during DNA immunization has become the main goal of research.

6. Strategies to enhance the immune responseDNA vaccines have demonstrated their promise in terms of stimulating an effective CTL response in response to neoantigens, however, the weak antigenic characteristics of many tumors require greater effectiveness of antitumor vaccines to ensure the feasibility of their clinical use.

Therefore, many studies are devoted to strengthening the immune response caused by DNA vaccines. The scientists analyzed every aspect of vaccination, from the introduction of nucleic acid to the modification of the antigen encoded in it and changes in the microenvironment to maximize the immune response and its shift towards the participation of Th1 (Table 1). The versatility of DNA vaccines is an important advantage, since various manipulations can be carried out with both nucleic acid and encoded antigen.

Table 1. Strategies for enhancing the effectiveness of polynucleotide antitumor therapeutic vaccines.Characteristics of the vaccine

One of the options is the introduction of nucleic acid into liposomes, which can protect it from the action of endogenous nucleases, as well as promote penetration into cells (43). DNA adsorption on cationic microparticles from poly(DL-lactide-co-glycolide), PLG – poly(DLlactide-co-glycolide), PLG – provides the slow release of DNA leads to the formation of a more powerful immune response than the use of deproteinized DNA [44]. Electroporation into the skin(45) or muscle tissue (46), which provides physical facilitation of the transport of nucleic acids into target cells, proved to be a promising approach to increase the efficiency of gene transfer. Another method of increasing transfection efficiency applicable to vaccines is hydrodynamic delivery of plasmid DNA [47]. The application of these approaches in clinical settings requires careful optimization taking into account the characteristics of the human body.

The ease of manipulation of recombinant DNA allows you to change the antigen encoded in it to increase its immunogenicity, while there are a huge number of different types of manipulations. Since the assimilation and adequate presentation of the antigen are mandatory conditions for the induction of an effective immune response, some groups of researchers have modified the encoded antigens in such a way as to increase the efficiency of their absorption by specialized APC [48]. Complexes of antigens with CD40-ligand (49), extracellular domain of Fms-like tyrosine kinase-3 (flt-3) ligand (50) or cytotoxic T-lymphocyte antigen-4 (CTLA4) (51) are examples in which receptors for each of the ligands are present on the surface of DC and provide antigen uptake by cells and the formation of an enhanced immune response. Inside the cell, the encoded antigen can be modified to facilitate degradation using the endosomal-lysosomal mechanism (52,53), which enhances the presentation of the antigen in combination with MHC class II molecules and stimulates CD4+ T-cell reactions. A similar method affecting another mechanism, the proteolytic processing of the encoded antigen, is provided by its fusion with sequences leading to its ubiquitination [54]. The incorporation of heterologous immunogenic sequences, such as the CTL epitope of tetanus toxin, into the tumor antigen sequence led to rapid stimulation of CTL against tumor antigen with simultaneous protection against tumor stimulation [55]. For tumors associated with human papillomavirus, such as cervical cancer, codon optimization of the viral antigen gene has shown itself to be an effective means of increasing protein expression in mammalian cells and enhancing the immune response [56].

Another approach to increasing the immunogenicity of DNA vaccines is the simultaneous introduction of DNA encoding cytokines, justified by the fact that a more powerful immune response is possible if the antigen is presented in a favorable cytokine microenvironment. Therefore, cytokines that stimulate the Th1-mediated immune response, including granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-12 (IL-12) and interlekin-18 (IL-18), are actively They were studied on preclinical models of infectious diseases (57) and cancer (58-60). The research results have demonstrated the ability of this approach to positively influence the nature and severity of the immune response. Based on the assertion that more effective administration of the antigen inside the APC will enhance immunological reactivity, chemokines were used to attract the APC to the antigen synthesis zone. This was achieved either by merging antigen genes and inflammatory chemokines (61), or by simultaneous administration of antigen genes and chemokines (62). Additional strategies include simultaneous administration of anti-apoptotic genes that promote the survival of DNA-transformed DCS (63), and joint administration of antigen-coding DNA and soluble protein encoded by the lymphocyte activator gene-3 (lymphocyte activating gene-3, LAG3), in order to enhance cross-presentation of the antigen (64). An increase in the amount of DC in vivo in order to increase immunological reactivity is stimulated by the introduction of a plasmid encoding the flt-3 ligand [65]. The use of this approach in combination with traditional peptide vaccines enhances the cellular immune response [66].

The concept of cross-species homologous immunization, also called xenogenic or orthologous immunization, has demonstrated its effectiveness in eliminating tolerance to weakly immunogenic tumor-specific antigens. This strategy involves the isolation of tumor antigen genes from organisms of another species in order to induce a cross-species immune response to the autologous protein of the vaccine recipient. For the numerous proteins studied to date, the orthologous protein of other species has more pronounced immunogenicity than the autologous or its own antigen. This approach leads to the development of immunity, cross-reacting with its own antigen and eliminating tolerance to it. Orthologous immunization has been successfully used in animal models to induce antitumor immune reactions against both endogenous tumor antigens (67-70) and cocancerogenic factors (71). Preliminary clinical studies of a protein-dendritic cell vaccine against prostate cancer have demonstrated stimulation of the immune response to its own antigen, which indicates the possibility of transferring this approach to clinical practice [72]. The simplicity of preparation and the absence of an immune response associated with DNA vaccines directed against viral vectors led to the creation on the basis of this approach of a number of strategies for xenogenic triggering and stimulation of the immune response, which in a number of preclinical models demonstrated higher efficiency compared to DNA immunization in its pure form. These include the use of DNA vaccines in combination with other genetic vectors (73,74) or with proteins, for example, absorbed into microparticles from PLG (75). Despite the fact that such approaches are somewhat more difficult to implement in clinical medicine, increasing the effectiveness of combined vaccines can serve as a decisive factor.

7. Clinical experience of using DNA vaccinesWhile induction of both T- and B-cell immune responses to foreign antigens has been convincingly demonstrated in humans in relation to foreign antigens associated with infectious diseases (76-79), the use of DNA vaccines for cancer treatment has so far been much less successful.

Induction of an effective antitumor immune response is a difficult task, and at the moment attempts at clinical use of DNA vaccines have led to contradictory results. Clinical studies have confirmed the overall safety and low toxicity of the vectors, but the effectiveness of the immune response caused by them was weak, and the antitumor activity was questionable.

To date, several clinical trials have already been completed. Encoding a cloned tumor antigen (carcinoembryonic antigen) DNA was injected intramuscularly into patients with advanced stages of colon cancer (80). Patients were immunized with plasmids simultaneously expressing carcinoembryonic antigen and, as a control, hepatitis B virus surface antigen. As a result, protective levels of antibodies to the hepatitis virus were recorded in some patients, but immunity against carcinoembryonic antigen practically did not develop. Rosenberg et al. published similar data obtained with the introduction of DNA encoding the gp100 melanoma antigen in phase I clinical trials involving patients with metastatic melanoma (81). During these studies, only one out of 22 patients who underwent intramuscular or intradermal immunization had the development of a weak defective specific immune response against the gp100 antigen. The authors concluded that immunization did not cause the development of a significant clinical or immunological response. This fact contradicts the results of earlier clinical studies, in which the gp100 antigen was administered as a transgene in a vaccine based on poultry pox virus or in the form of peptides, and indicates the need to develop strategies to enhance the immune response to DNA vaccines based on plasmids. An alternative method of administration – inside the lymph nodes – was evaluated in 26 patients with progressive melanoma (82). The introduction of plasmid DNA encoding tyrosinase epitopes led to the development of an antigen-specific immune response in 11 patients. At the same time, no clinical changes were observed in patients, except for an unexpectedly high average life expectancy. Plasmid DNA encoding prostate-specific antigen (PSA) was administered to patients with hormone-resistant prostate cancer in combination with cytokines GM-CSF and IL-2 (83). At the same time, two out of three patients of the cohort who received the highest dose had a cellular and humoral immune response to PSA and a decrease in the level of this antigen.

Levy et al. evaluated the immunogenicity of the plasmid DNA vaccine in studies on patients with B-cell lymphoma (84). Earlier clinical studies using proteins representing tumor-specific idiotypes of immunoglobulins for active immunization have demonstrated positive clinical effects for patients (85, 86), however, the preparation of an individual vaccine for each patient is very time-consuming and unacceptable for widespread use. DNA vaccination has the advantage of relatively fast and low-cost manufacturing. The patients were immunized with a DNA vaccine encoding a chimeric molecule consisting of a patient-specific idiotype connected to the a- and k-chains of the constant region of mouse immunoglobulin G2 (IgG2). Cohorts of patients were immunized intramuscularly and intradermally using needle-free devices of the Biojector type with or without the addition of plasmid DNA encoding GM-CSF. In most patients of all cohorts, the development of an immune response to the carrier protein of mouse immunoglobulin was observed, which served as proof of the synthesis of the encoded protein and its ability to elicit an immune response. Induction of an immune response to a tumor-specific identification fragment of the encoded gene was also recorded, although in a smaller number of patients. The clinical efficacy of the vaccine was difficult to assess due to the preliminary and ongoing chemotherapy during the studies and the absence of an unvaccinated control group. Nevertheless, the absence of toxicity and the development of a registered immune response indicate in favor of the expediency of further improvement of this approach to vaccination.

It should be noted that the mentioned clinical studies were conducted in conditions of late stages of diseases in which the induction of an immune response is not an optimal approach to treatment. Nevertheless, in general, the experience gained using deproteinized DNA for immunotherapy indicates that the introduction of plasmid DNA vaccines is not enough to form a clinically effective immune response to unmutated own antigens. The transfer of the most promising strategies listed in Table 1 into clinical practice can help overcome the limitations inherent in existing methods.

The results of two phase I clinical trials of a vaccine against malignant diseases associated with human papillomavirus have been published. Immunotherapy of such tumors is facilitated by the fact that their cells express foreign viral antigens. Plasmid DNA encoding peptide epitopes (protein fragments recognized by antibodies) of human papillomavirus type 16 protein E7, presented by APC in combination with MHC class I antigens, was encapsulated in microparticles from a biodegradable PLG polymer and injected intramuscularly [87]. Analysis using ELISPOT test systems showed enhanced T-cell reactions in 10 out of 12 patients with dysplasia. At the same time, some patients from the cohorts who received the highest doses of the vaccine had the development of defective tissue reactions. Intramuscular and subcutaneous administration of the same drug to women with intraepithelial cervical neoplasia resulted in the formation of the majority of patients (73%) the registered immune response to the E7 protein of human papillomavirus type 16 and in 33% of patients – a full-fledged tissue response (88). At the same time, there were no serious side effects associated with vaccination. The results obtained indicate that DNA vaccines against papillomavirus antigens can play an important role in the fight against associated malignant diseases.

8. ProspectsOver the past few years, the rate of identification of tumor antigens has increased significantly (89) and will continue to grow, as the introduction of new techniques, such as expression profiling (90, 91), SEREX technology (serological identification of antigens by recombinant expression cloning) (92) and proteomic analysis (93), provides identification of new potential targets for active immunotherapy.

The use of DNA vaccines in preclinical models is a relatively fast method of evaluating the potential suitability of such candidate antigens for stimulating tumor rejection. In addition to traditional tumor-specific antigens, antigens of the vascular network of tumors can act as targets for DNA vaccines [94, 95]. The work on the creation of DNA vaccines in the field of combating infectious diseases will not lose its value in terms of developing new strategies suitable for use in the creation of antitumor vaccines. The results of a recent clinical study of an infectious disease (malaria) indicate that various immunization strategies aimed at triggering and strengthening the immune response using DNA in combination with other types of vaccination can potentiate the immune response in humans [96]. Despite the fact that definitive clinical evidence of the effectiveness of DNA vaccines in cancer therapy has yet to be obtained, there are reasons to remain optimistic about their potential in the fight against a wide range of malignant neoplasms. As a relatively non-toxic therapy, DNA immunization can find its clinical application as an additional measure in the treatment of minimal residual disease to prevent relapses of the disease. Over time, DNA vaccines can become a means of cancer prevention. The significant advantages of DNA immunization and its proven safety in clinical studies are strong arguments in favor of continuing the development of such vaccines and their introduction into the practice of combating malignant diseases.

9. GratitudeThe study was conducted with the funds of grants NCI 1 P50 CA89019 and NCI 1 P50 CA83591, allocated by the US National Institutes of Health.

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Correspondence address: Dr. Theresa V. Strong, WTI 558, 1530 3rd Avenue South, University of Alabama at Birmingham, Birmingham, AL 35294-3300, Tel: 205-975-9878, Fax: 205-934-9511, E-mail: tvstrong@uab.edu

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