Review Article: DNA Vaccines: Developing New Strategies Against Cancer

Hindawi Publishing Corporation
Journal of Biomedicine and Biotechnology

Volume 2010, Article ID 174378, 16 pages
doi:10.1155/2010/174378
Review Article
DNA Vaccines: Developing New Strategies against Cancer
Daniela Fioretti,1 Sandra Iurescia,1 Vito Michele Fazio,2, 3 and Monica Rinaldi1
1 Instituteof Neurobiology and Molecular Medicine, Department of Medicine, National Research Council (CNR),
Via Fosso del Cavaliere 100, 00133 Rome, Italy
2 Section of Molecular Medicine and Biotechnology, Interdisciplinary Center for Biomedical Research,

University Campus Bio-Medico, Via Alvaro del Portillo 21, 00128 Rome, Italy
3 Unit`a Operativa Oncologia, Istituto di Ricovero e Cura a Carattere Scientifico Casa Sollievo della Soerenza,
71013 San Giovanni Rotondo, Foggia, Italy
Correspondence should be addressed to Monica Rinaldi, [email protected]
Received 20 November 2009; Accepted 5 February 2010
Academic Editor: Soldano Ferrone
Copyright 2010 Daniela Fioretti et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines
against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved
to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the
results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods
to make them suciently eective in the clinic. Similarly, it is clear that additional strategies are required to activate eective
immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and
processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several
approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes
of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by
malignant cells to prevent immune cell function. Combined or single strategies to enhance the ecacy and immunogenicity
of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are
substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated.
Some representative DNA cancer vaccine studies are also discussed.
1. Introduction help bodys natural defences to recognise and target cancer

cells.
Spontaneous tumour regression has followed bacterial, fun- In the last few years, it is estimated that in Europe there
gal, viral, and protozoal infections. Intratumoral infections were almost 3 000 000 cancer cases diagnosed (excluding non
may reactivate defensive functions, causing tumour regres- melanoma skin cancers) and more than 1 500 000 deaths
sion. from cancer each year [2]. Standard therapeutic procedures
These phenomena inspired the development of numer- currently in practice, including surgery, radiation, and
ous rudimentary cancer immunotherapies, starting with chemotherapy have not greatly impacted the spread and
nonspecific immunostimulatory approaches first used by recurrence of progressive malignancies [3], reducing the
William Coley [1] and leading to the concept of therapeutic ability of the immune system to provoke spontaneous
vaccination against cancer. The recent identification and regressions. Newer strategies are needed to improve upon the
characterization of genes coding for tumour antigens (Ag) current treatment success rate.
has enabled the design of antigen-specific cancer vaccines Historically, Wol and colleagues [4] first demonstrated
based on plasmid DNA and recombinant viral vectors. Gene that long-term gene expression in mouse skeletal muscle
therapy can be used to manipulate the immune system to could be achieved with direct intramuscular injection of
2 Journal of Biomedicine and Biotechnology
plasmid DNA. This and other early studies, demonstrating shared antigens are the cancer-testis antigens [14], human
the feasibility of direct intramuscular gene transfer for DNA epidermal growth factor receptor 2 (HER2)/neu protein
vaccination purpose, propelled the first vaccination studies (reviewed in [16]), and carcinoembryonic antigen (CEA)
utilizing plasmid DNA in protection scenarios involving [17]. Unique tumor antigens result from mutations induced
influenza [5] and HIV-1 [6]. Cellular and humoral immune through physical or chemical carcinogens; they are therefore
responses have been demonstrated after the injection of expressed only by individual tumors. Tumor-specific unique
naked plasmid DNA vaccines into the dermis or muscle antigens encompass melanocyte/melanoma dierentiation
tissue of mice [5, 7]. Such responses have induced protection antigens, such as tyrosinase [18], MART1 [19] and gp100
in preclinical models of infectious disease and malignancies [20], prostate-specific antigen (PSA) (reviewed in [21]) and
(for review, see Donnelly et al. [8]). Idiotype (Id) antibodies [11]. Optimally designed cancer
The DNA vaccine is a prime example of a modern vaccines should combine the best tumor antigens with the
genetic vaccine. The use of naked plasmid DNA as vaccine to most eective immunotherapy agents and delivery strategies
elicit the immune system against disease provides a variety to achieve positive clinical results. An important dilemma for
of practical benefits for large-scale vaccine production that vaccination against overexpressed tumor-associated antigens
are not as easily manageable with other forms of vaccines is how to induce eective immunity against the chosen
including recombinant protein or whole tumor cells [9, 10]. target without leading to damaging autoimmunity. The
The eectiveness to screen for antigens rapidly and to precision oered by DNA vaccines will induce focused
design specific types of expression constructs has made the immunity against selected antigens, and, as they become
study of DNA vaccines a valuable field for immunotherapy more powerful, targets will have to be selected carefully to
of cancer. avoid autoimmunity. Recently, an NCI pilot prioritization
New technologies including gene-expression profiling study produced a well-vetted, priority-ranked list of cancer
has increased the list of candidate tumor antigens. Inves- antigens [22]. Antigen prioritization involved developing
tigators have focused on targets which are either tumour- a list of ideal cancer antigen criteria/characteristics and
specific, including idiotypic antigens of B-cell tumours [11] assigning relative weights to those criteria using pairwise
or tumour-associated antigens [12] that are also expressed by comparisons. The result of criteria weighting was as follows:
the normal cell of origin [13] and that include the so-called (a) therapeutic function, (b) immunogenicity, (c) role of the
cancer-testis antigens [14]. antigen in oncogenicity, (d) specificity, (e) expression level
Examples under intensive investigation are the antigens and percent of antigen-positive cells, (f) stem cell expression,
of melanoma (http://www.cancer.gov/cancertopics/types/ (g) number of patients with antigen-positive cancers, (h)
melanoma), prostate cancer (http://www.cancer.gov/cancer number of antigenic epitopes, and (i) cellular location of
topics/types/prostate), and other epithelial cancers (http:// antigen expression [22]. Such an eort to prioritize cancer
www.cancer.gov/cancertopics/types/skin). antigens represents the logical next step in attempting to
DNA vaccines oer the opportunity to incorporate addi- focus translational eorts on cancer vaccine regimens with
tional genes encoding molecules aimed at overcoming the the highest potential for success.
weak immunogenicity of tumor antigens and the patients A biological issue limiting the ecacy of cancer vaccines
tolerized immune repertoire. is the low immunogenicity of cancer antigens. Strategies to
This paper briefly summarizes findings and key tech- enhance antigen immunogenicity are discussed in a later
nologies that have contributed to the rapid progress of DNA section.
vaccines (mode of action, design, and optimization of DNA
vaccines) as well as the state of the art of some of the 3. Priming the Immune System
more encouraging clinical studies using or against tumor
antigens. DNA vaccines are simple vehicles for in vivo transfection
and antigen production. A DNA vaccine is composed of a
2. Cancer Antigens plasmid DNA that encodes the antigen of interest under
the control of a mammalian promoter (i.e., CMV-intA,
Scientists have identified a large number of cancer-associated CMV immediate/early promoter, and its adjacent intron A
antigens, many of which are now being used to perform sequence) and can be easily produced in the bacteria [23].
cancer treatment vaccines both in basic research and in The optimized gene sequence of interest is delivered to
clinical trials. The list of candidate tumor antigens grows the skin (intradermally), subcutaneum or to the muscle by
daily, largely because of expanding genetic technology one of several delivery methods. Using the host cellular
including human genome sequencing and gene-expression machinery, the plasmid enters the nucleus of transfected
profiling. Tumor antigens have been classified into two local cells (such as myocytes or keratinocytes), including res-
broad categories: tumor-specific shared antigens and tumor- ident antigen presenting cells (APCs). Here, gene expression
specific unique antigens [15]. Shared antigens or tumor- from plasmid is followed by generation of foreign antigens.
associated antigens (TAAs) are expressed by more than one Although the elucidation of all immunological components
type of tumor cells. A number of TAA are also expressed involved following DNA immunization has not been entirely
on normal tissues, albeit in dierent amounts. As reported achieved, the mode of action of plasmid DNA vaccines
in the ocial National Cancer Institute website (NCI, appears twofold. DNA plasmids, which are derived from
http://www.cancer.gov/), representative examples of such bacteria, stimulate the innate immune system by interacting
Journal of Biomedicine and Biotechnology 3
with Toll-like receptor 9 (TLR9) [24]), a receptor found on such as the need to break immunological tolerance, gradual
APCs, although the dierential expression of TLR9 in mice loss of MHC and antigen in tumour cells, regulatory T cells
and primate immune cells makes more complex their role that could negatively influence the induction of antitumour
as adjuvants in primates. This nonspecific immune response responses, systemic defects in dendritic cells, secretion of
augments the antigen-specific immune response, where the immunosuppressive cytokines, resistance to apoptosis [33,
direct and indirect presentations of antigen to APCs are 34] as is discussed elsewhere.
involved. Two overarching models have been proposed. The
antigen encoded by the plasmid is produced in host cells, 4. Advantages and Disadvantages of
either in professional APCs leading to direct priming of DNA Vaccines
immune responses or in nonprofessional cells from where
the antigen can be transferred to APCs leading to cross- The use of DNA vectors represents an important platform for
priming. clinical applications, in which large-scale vaccine production
A series of studies intended to determine how such vac- is not easily manageable with other forms of vaccine
cines could work investigated the source of Ag presentation, including recombinant protein, whole tumor cells, or viral
the immunological properties of the DNA itself, and the role vectors [35].
of cytokines in eliciting the immune responses. Although viral mediated gene transfer by geneti-
Early studies showed that DNA delivery method aected cally modified lentiviruses, adenoviruses, adeno-associated
the cell types that were transfected. Gene Gun (bombard- viruses, and retroviruses is advantageous because of its high
ment of the epidermis with plasmid coated onto gold transfection eciency and stability [36], the largest hurdles
microbeads) tended to directly transfect epidermal ker- using viral vectors are to overcome the immunogenicity of
atinocytes and also Langerhans cells, which were shown to the viral packaging proteins. Furthermore, viral methods are
migrate rapidly to regional lymph nodes [25]. In this case, disadvantageous because of their high expense, toxic side
professional APCs were transfected directly and behave as the eects, limits on transgene size, and potential for insertional
source of Ag presentation. mutagenesis [37].
Alternatively, intramuscular injection of plasmid pre- On the one hand, nonviral vectors are highly flexi-
dominantly led to transfection of myocytes. Myocytes lack ble, are capable of encoding a number of immunological
expression of major histocompatibility complex class (MHC) components, are associated with a lower cytotoxicity, are
II and costimulatory molecules and thus would not be relatively more stable, and are potentially more cost-eective
expected to prime T lymphocytes directly. Instead, immune for manufacture and storage (Table 1).
priming likely occurs by dendritic cells (DCs) [26, 27] Their safety in terms of adverse reactions after injection
that presumably migrate to the site of DNA inoculation in has been demonstrated in animal models [38, 39] as well as
response to inflammatory or chemotactic signals following in human clinical trials.
vaccination [28, 29]. These DCs are thought to present The first clinical trail, initiated to monitor the safety
antigen by cross-presentation of extracellular antigen or and ecacy of a DNA vaccine against HIV-1 infection [40],
following direct transfection of plasmid DNA [26, 30]. demonstrated that DNA plasmid vaccines were safe and were
Thus, in terms of induction of immunity, there is an capable of inducing detectable immune cellular and antibody
influence of the site and procedure used for injection, with responses [4042].
muscle and skin cells clearly able to act as antigen depots The simple plasmid backbone coupled with the tech-
but unable to prime the immune response. It is likely that nology of gene manipulation allows incorporation of genes,
cross-presentation from these sites to APCs is the major which are then expressed by cells transfected in vivo.
route to priming [26], but there is also evidence for direct Although the transfection process is inecient and varies
transfection of APCs, especially when delivery is to skin sites with the target tissue and means of delivery, sucient DNA
through a gene gun [25]. The host-synthesized antigen is is generally taken up to prime the immune response [32].
then processed and presented by APCs in the context of both DNA vaccines are free of the problems associated with
MHC I and MHC II. producing recombinant protein vaccines, and they are also
Antigen-loaded APCs travel to the draining lymph nodes, safer than live attenuated which can cause pathogenic
where they present peptide antigens to nave T cells, thereby infection in vivo. Additionally, studies with DNA vaccines
eliciting both humoral and cellular immune responses. have shown that even after multiple immunizations, anti-
Although plasmid DNA vaccines vectors can induce antibody DNA antibodies are not produced [43].
and CD4+ T cell helper responses, they are particularly The ability to introduce antigen to the host immune
suited to induce CD8+ T cell responses because they express system, thus enabling it to elicit strong Th1 type CD4+ T
antigens intracellularly, introducing them directly into the cells and CD8+ cytotoxic T cells, is a unique feature of
MHC I antigen processing and presentation pathway [31]. DNA vaccines which makes them distinct from conventional
Whatever, the process that conveys antigens to the APC protein or peptide vaccines. Because of this feature, they can
seems highly ecient since DNA vaccines, that produce only readily induce humoral as well cellular immune responses
very low levels of antigen, can induce all arms of the immune [44].
response [32]. Plasmid-based gene transfer can also deliver oligonu-
One lesson learned in the last years is that the devel- cleotides that can alter gene splicing or gene expression, for
opment of plasmid DNA as cancer vaccine raises key issues example, siRNA [35, 45].
Table 1: DNA vaccines main features.

Advantage Drawback
Allowing the introduction of several immunological
Design components; synthetic and PCR methods for simple Low transfection eciency
modifications
Rapid production and formulation; easily engineered,
Manufacture
reproducible, large-scale production and isolation
No pathogenic infection in vivo; no significant adverse
Safety events in any clinical trial; neutralizing immune
responses rarely observed; boost strategy is possible
Long shelf life; relative temperature insensitivity; lack
Stability
necessity of a cold chain stored on a large scale
Lower immunogeni-city in larger animals and
Immunogenicity
human compared to mice
5. Enhancing Efficacy and Immunogenicity of As already discussed, the backbone of bacterial DNA
DNA Vaccines includes cytosine-phosphate-guanine (CpG) unmethylated
regions as sequence motifs that stimulate innate immunity,
Despite immunogenicity of DNA vaccines has been well creating an inflammatory milieu for triggering the adaptive
established in animal models, low immunogenicity has immune response [74]. The role of CpG motifs as adjuvants
been the major deterrent towards the development of DNA of immune response to DNA vaccines is well documented
vaccines in large animal models and human. In order to over- in mice [75]. Preclinical studies showed that the addition
come this hurdle, several approaches have been investigated of CpG motifs in the plasmid can result in the induction
including plasmid design, immunomodulatory molecules, of proinflammatory cytokines, for example, IL-12 or IFN-
delivery techniques, and prime-boost strategy (Table 2). I [75]. CpGs are recognized by TLR9, a receptor found on
APCs, helping cytotoxic T-lymphocyte (CTL) dierentiation
5.1. Plasmid Design. Early in the development of DNA vac- and priming. The coadministration of genes encoding
cines, it became clear that maximizing the expression of the ligands for Toll-like receptors (TLRs) or their signaling
encoded Ag was critical to the induction of potent immune molecules has been shown to improve the immunogenicity
responses. Strong viral promoters, such as CMV-intA, are of DNA vaccines [66, 76].
generally favoured over regulated or endogenous eukaryotic Engineering DNA vaccine design for maximizing
promoters [70]. Furthermore nuclear targeting sequence epitope-specific immunity has allowed epitope enhancement
(NTS) could be introduced to increase the eciency of by sequence modification. The recent molecular under-
nuclear plasmid uptake from cytoplasm after intramuscular standing of the immune response is leading to new strategies
injection [48, 71]. to induce more eective immune responses. Self-tolerance
The utilization of codon-optimized sequences instead might lead to deletion of T cells specific for the most eective
of the wild-type coding sequences is a general and potent epitopes, leaving only low-avidity T cells [77, 78]. Therefore,
method to improve vaccination. An optimal coding sequence not all sequences are optimal antigenic epitopes. A process
is determined back from the amino acid sequence of the termed epitope enhancement is expected to make the
antigen by algorithms that take into account the abun- sequences of many epitopes of cancer more immunogenic
dance of specific tRNAs in the cytosol of human cells [79]. Epitope sequences can be modified to increase the
and the predicted structure of the mRNA. Thereafter the anity of the epitope peptide for the MHC molecule. The
selected gene sequence is constructed in vitro using synthetic knowledge of sequence motifs for peptide binding is the key
oligonucleotides. Adverse rare codons are avoided and to improve the primary and/or secondary anchor residues
secondary structures in the mRNA are minimized. Thereby, that provide much of the specificity of binding to the MHC
the synthetic gene is optimal for expression and consequently molecule [80, 81]. This strategy can greatly increase the
for the induction of an immune response [72]. potency of a vaccine and can convert a subdominant epitope
The flexibility of plasmid design coupled with the into a dominant one by making it more competitive for
technology of gene manipulation allows also gene opti- available MHC molecules, thereby increasing the level of
mization. Indeed, the variable regions of the heavy (VH ) specific peptideMHC complexes on the antigen presenting
and light chain (VL ) of the tumor immunoglobulin, specific cell surface [82]. Epitope enhancement has been used to
for the B-cell malignancies, can be readily cloned and increase the anity for both MHC class I and class II
combined into single-chain variable fragment (scFv) format, molecules (reviewed in [83]).
encoding a single polypeptide consisting of VH and VL genes To enhance the immunogenicity of DNA, vaccines en-
linked together in frame by a short 15-amino acid linker coding immunostimulatory RNA, such as double-stranded
[73]. RNA or replicon RNA, were also generated [84].
Table 2: DNA Vaccine Enhancing Strategies.
Strategy Approach Indication References

prostate cancer; B-cell
intramuscular/EP [4648]
lymphoma
prostate cancer; colon
intradermal/EP [49, 50]
Routes of cancer
administration gene gun cervical cancer [51, 52]
tattoo perforating needle melanoma [53]
melanoma; renal
intratumor [54, 55]
carcinoma
B-cell lymphoma;
high-pressure liquid delivery [50, 56]
colon cancer
liver cancer; prostate
cytokine cancer; melanoma; B-cell [5662]
Genetic lymphoma
immunomodulators chemokine B-cell lymphoma [63]
as Adjuvants prostate cancer; follicular
T cell helper epitopes lymphoma; colon [46, 47, 64, 65]
carcinoma
Toll-receptor ligands lung carcinoma [66]
heat shock proteins cervical cancer [67]
prostate cancer; colon
plasmid DNA/plasmid DNA+EP [46, 47, 50]
cancer
Prime-boost strategy plasmid DNA/recombinant prostate carcinoma; breast
[68] NCT00363012
protein cancer
liver cancer; melanoma;
plasmid DNA/viral vector [58, 60, 69]
prostate carcinoma
viral vector/plasmid DNA prostate cancer [59]
Engineering vaccine design for manipulating antigen the tumor antigen sequence, it was possible to activate
presentation and processing pathways is one of the most Th cells and to dramatically amplify immunity against
important aspect that can be easily handled in the DNA tumor cells [87]. As discussed in the following section,
vaccine technology. If an antibody response is the goal, it DNA vaccines oer the opportunity to activate Th cells
is clearly desirable to direct antigen expression to the endo- and transform weak and ineective immunity to a powerful
plasmic reticulum (ER), in which folding and secretion can antitumor attack [88].
occur. An appropriate leader (signal) sequence can achieve
this. (reviewed in [47]). For induction of CTLs, addition 5.2. Immunomodulatory and Immunoenhancing Molecules.
of genes encoding molecules such as ubiquitin, aimed to Even though specific antibody and CTL responses could be
enhance degradation and peptide production in the protea- induced in clinical trials with naked DNA vaccines, by the
some, can be eective (reviewed in [85]). Similarly, targeting intramuscular or intradermal route, high doses of DNA were
expression to dierent subcellular pathways such as the necessary to elicit detectable immune responses [89, 90].
endosome or lysosome can amplify CD4+ T cell responses Large quantities, that is, 510 mg, are required to induce only
[85]. Thus, DNA vaccines can be designed to induce an modest immunogenicity [91].
appropriate eector pathway, including antibody against Modifying the microenvironment of the vaccinated site
cell-surface antigens, or CTL response against intracellular by coadministration of genetic, that is, DNA plasmids coding
antigens expressed only as MHC class I-associated peptides. for immunostimulatory molecules, protein, or chemical
Since tumor antigens are often weakly immunogenic and the adjuvants, improves the low immunogenicity of DNA vac-
immune repertoire in patients may have been tolerized, the cines [31].
central question is whether DNA vaccines can activate and Progress has been made in developing improved tech-
maintain the high level of immunity required to suppress niques for encapsulating plasmid DNA (liposomes, poly-
cancer cell growth. mers, and microparticles) although few of these formulations
The pivotal position of CD4 T helper (Th) cells in helping have been shown to elicit immune responses that are superior
B cells to produce antibody and control induction and to those elicited by simple intramuscular plasmid DNA, still
maintenance of CD8 T cells [86] has led some investigators to disappointing in human clinical trials [92].
focus on their importance in responses to DNA vaccination. Considering the ease in design and construction of
By selecting genes encoding microbial proteins fused to plasmid DNA used to target a particular neoplasm, biological
Table 3: Adjuvant molecules employed in cancer clinical trials to to break tolerance is likely due to the presence of some
enhance the immune response. foreign sequences in the xenogeneic antigen that are able
to activate Th cells [100]. Focusing on the antimicrobial
Adjuvant Phase References
repertoire, the principle has been applied to realize the DNA
cytokines NCT00019448 fusion gene vaccines encoding the tumor antigen linked
I/II-II
(GM-CSF, IL-12, IL-2) [56, 62] to an antigen derived from tetanus toxin. Fusion of the
Genetic bacterial toxins Fragment C (FrC) of tetanus toxin amplified the immune
I/II [46, 47, 64, 65]
(pDOM/tetanus toxin FrC) response against a range of tumor antigens, leading to
immunomodulatory suppression of tumor growth [87]. Clinical trials by using
I/II [67] these approaches to breaking self-tolerance for therapeutic
molecules (HSP70)
Protein cytokines (GM-CSF, IL-2) I/II-II [58, 59, 61]; purposes in patients with lymphoma and prostate carcinoma
are discussed elsewhere.
adjuvants can be tailored and encoded within the same DNA 5.3. Route of Administration. It is increasingly apparent that
vector as well [35]. A vast array of molecules able to modulate the immunogenicity of DNA vaccines greatly depends upon
immune responses can be delivered (Table 3). the delivery methods used for immunization [101].
They include chemokines to attract APC [93], activating In a melanoma mouse model, DNA vaccination was
cytokines [94, 95], costimulatory molecules, APC-targeting administered together with intratumoral delivery of antian-
antibodies, and molecules to manipulate antigen presenta- giogenic plasmids, encoding angiostatin, and endostatin.
tion and/or processing [96]. Combined melanoma vaccination resulted in 57% tumor-
One of the common cytokines employed in plasmid DNA free survival over 90 days after challenge [54]. In a modest
vaccine is granulocytemacrophage colonystimulating fac- proportion of patients with malignant disease, intratumoral
tor (GM-CSF), a molecule able to enhance immune injection of DNA led to regression of tumor at distant sites
responses by inducing proliferation, maturation, and migra- [102].
tion of DCs as well as expansion and dierentiation of B and The recent studies have confirmed that physical methods
T lymphocytes [62]. are superior over other delivery methods that administer
In addition to codelivery, DNA vaccines allow fusion of DNA in various chemical solutions [103, 104].
genes encoding activating molecules to the antigen-encoding Biolistic gene gun delivery involves adhering naked DNA
sequence. This is an advantage, and fusion genes can create to gold beads and shooting the particles through a high-
single vaccines capable of multiple functions. pressured instrument. This system delivers DNA directly
Biragyn and colleagues showed that the eciency of into skin and Langerhans cells in a highly ecient process.
DNA vaccination in vivo could be greatly increased by Gene gun immunization has been shown to induce a greater
encoding a fusion protein consisting of scFv fused to a CD8+ T cell response as well as to require less vaccine to
proinflammatory chemokine moiety that facilitates targeting achieve tumor immunity [51].
of APCs for chemokine receptor-mediated binding, uptake, A promising strategy is electroporation (EP), which in
and processing of scFv antigen for subsequent presentation primates increases not only the level but also the breadth of
to CD4+ or CD8+ T cells, or both [63]. In two independent response [105], overcoming the diculty in translating the
models, vaccination with DNA constructs encoding a fusion eectiveness of DNA vaccination from preclinical rodents to
protein consisting of scFv fused to the monocyte chemotactic large animals, including human subjects [106].
protein 3 (MCP-3) or the interferon inducible protein 10 Electroporation-based DNA delivery technology dramat-
(IP-10a) generated superior protection against a large tumor ically enhances cellular uptake of DNA vaccines. EP itself
challenge (20 times the minimum lethal dose), as compared works as an adjuvant to enhance the necessary danger
with the best available protein vaccines [63]. signals that become detectable by the immune system.
Additional strategies to activate eective immunity The tissue damage caused by the application of EP causes
against poorly immunogenic tumor antigens employ the inflammation and recruits DCs, macrophages, and lympho-
DNA fusion genes vaccines to activate T cell help for cytes to the injection site [107, 108] inducing significant
antitumor responses. The CD4+ Th cell, as pivotal cell of the immune responses, including antibody and T-cell responses.
immune response able to induce high levels of immunity and Moreover, it is tolerable without anesthetic and does not
the maintenance of the response, has been extensively studied induce unwanted immune responses against the delivery
by Stevenson and coworkers [97]. The requirement for mechanism, therefore it can be used for repeat administra-
foreign sequences to induce Th for the B-cell response and to tions.
help the CTL response has been known for many years [98, A newly developed intradermal DNA delivery is the
99]. Since Th cells control responses to vaccination, it is quite tattoo technology. The tattoo device has a cartridge of nine
obvious that self-antigens, which do not contain epitopes fine metal perforating needle that oscillate at a constant high
likely to be recognized by available Th cells, are incapable frequency and puncture the skin, leading to DNA transfer
of inducing immunity. A strategy to activate Th cells for to skin-associated cells. The expression of reporter genes
inducing antitumor immunity is to engage a repertoire results in robust T-cell responses [109]. Recently tattoo-
against nontolerized antigens. The use of xenogeneic antigen immunization was applied in a phase I study to assess
the toxicity and ecacy of inducing tumor-specific T-cell as well as through the production of immune-suppressive
immunity against melanoma [53]. factors by malignant cells themselves.
Many tumor-infiltrating macrophages, referred to
myeloid-derived suppressor cells (MDSCs), have an im-
5.4. Prime-Boost Strategies. Vaccination schedules based on
mune-suppressive phenotype [119]. These macrophages
combined prime-boost regimens using dierent vector sys-
are abundant in many tumors arising in both humans and
tems to deliver the desired antigen (i.e., heterologous prime-
mice and can exert powerful anti-inflammatory eects. In
boost immunization regimen) appear to be a successful
addition to MDSCs, regulatory T cells (Tregs) also heavily
improvement in DNA vaccine platform.
infiltrate many tumors [120]. These cells, characterized by
Actually, prime-boost regimens have shown promise in
the expression of the transcription factor FoxP3 as well as
eliciting greater immune response in humans compared with
CD4 and CD25, play a key role in the regulation of adaptive
DNA vaccination alone [101].
immunity. Tregs can suppress immune responses through
The DNA-prime-viral vector-boost approach focuses on
the secretion of suppressive cytokines like TGF- and IL-
the induction of T-cell immune responses. In this approach,
35 [120, 121]. Tregs are a potential barrier to developing
homologous boost immunization carries the equivalent
productive immune therapies for cancer, and they represent
antigen than the previous immunization. Viral vectors that
an attractive target for enhancing antitumor immunity.
have been tested as booster vaccine include adenovirus,
Cancer immunotherapy is designed to specifically target
vaccinia virus, fowlpox [110, 111] as well as recombinant
cancer types using components of the immune system.
vesicular stomatitis virus [112].
Therefore DNA vaccines are also faced many obstacles that
Likewise, the DNA-prime-protein-boost approach em-
include breaking peripheral T cell tolerance against tumor
ploys recombinant protein antigens that match with the
self-antigens, to elicit appropriate immune reactions, as well
antigens used in DNA prime immunization [68, 113, 114].
as overcoming tumor-derived immunosuppressive networks
This strategy aims to develop balanced humoral and cell-
and evasion tactics. Evasive mechanisms adopted by malig-
mediated immune responses with a focus on eliciting high
nant cells to prevent immune cell function are numerous
quality protective antibody responses.
and lead to the clonal expansion of non-immunogenic
The heterologous prime-boost vaccination regimen
tumor cells, by loss of tumor antigen, and to the apoptosis
exploits the ability of the immune system to generate a
prevention [35].
large number of secondary antigen-specific T cells. Following
Tumour cells can downregulate expression of MHC
a priming immunization, a proportion of the antigen-
and target antigens and often secrete immunosuppres-
specific T cell population transforms into antigen-specific
sive molecules to defend themselves against attack [122].
memory T cells, which have the ability to expand rapidly
Tumours can create a tolerogenic environment which spreads
upon encounter with the same antigen a second-time
to draining lymph nodes and can enhance regulatory T-
round.
cell activity. The hurdles to successful reversal of tolerance
Since the priming and boosting vectors are dierent,
and induction of eective immunity are becoming clear
this strategy allows for greater expansion of the disease
and vaccines must incorporate elements to overcome them
antigen-specific T cell populations [115]. To date, heterol-
[123].
ogous prime-boost regimens are among the most potent
Furthermore cancer cells secrete soluble factors in the
strategies to induce cellular immune responses. Compared
tumor microenviroment, for example, VEGF, IL-10, and
to homologous prime-boost approach with the same DNA
TGF-, that aect the maturation, dierentiation, and
vaccine, boosting a primary response with a heterologous
activity of APCs as DCs [124], interfering with immune cells
vector will result in 410-fold higher T cell responses [116
maturation and eector properties. The tumour microen-
118].
vironment may drive tumour growth and even selectively
On the one end, a combination of DNA vaccines with
support a subset of tumour cells, the cancer stem cells
EP in a homologous prime-boost approach could generate
(CSCs).
antibody responses comparable to those that are induced
The DNA vaccination platform can be capable of
by protein in Complete Freund Adjuvant, and also ampli-
suppressing the progression of already established tumor
fied CTL responses [46]. EP may provide a prime-boost
by targeting those secreted soluble factors in the tumor
combination equivalent to that observed using viral vectors,
microenvironment [125], reversing immunological attenua-
and it is now undergoing testing in the clinic using a DNA
tion mechanisms and improving DNA vaccine potency.
vaccine for patients with prostate cancer. Repeated EP has
The concept of combining cancer vaccination with
been accepted by patients without the need for general or
angiogenesis inhibition is appealing, due to favorable safety
local anaesthesia and with no apparent long-term ill eects
profile of both approaches, as well as possible biological
[47].
synergies [54]. DNA vaccination in mice against the VEGF
receptor, FLK-1, abrogated the tumor vasculature and
5.5. Strategies to Break the Immunosuppressive Networks. protected DNA vaccinated animals from tumor challenge
Immune suppression is a feature of the tumor microen- in prophylactic approach [126]. Expression of the platelet-
vironment and a barrier to tumor immune therapy. The derived growth factor receptor (PDGFR) in stromal cells
microenvironment of tumors is established through the directly correlates with advanced stage disease in human
activity of both myeloid and lymphoid regulatory cells, colorectal cancer. DNA vaccine against PDGFR suppressed
growth and dissemination of human colorectal cancer cells The inclusion of FrC sequence, or other nonself antigens,
injected into mice [127]. activates T-cell help to reverse tolerance and induces high
In vivo coadministration of plasmids encoding the levels of immunity [47, 64, 65]. To further increase immuno-
chemokine macrophage inflammatory protein-1alpha (MIP- genicity of DNA vaccine, the use of molecular adjuvants such
1alpha) and the DC-specific growth factor fms-like tyrosine as cytokines and immunomodulatory molecules has been
kinase 3 ligand (Flt3L) with the plasmid DNA augments the extensively employed in clinical trials [56, 5863, 67].
immunogenicity of the vaccine, mobilizing and activating Clinical trials conducted over the last few years have
large numbers of DCs at the site of inoculation [128]. led promising results, particularly when DNA vaccines
Consistent with the concept that most eective cancer were used in combination with other form of vaccines, as
therapies are multimodal, combining Treg depletion with demonstrated in prostate and liver cancer clinical trials [58
active cancer immunotherapeutic interventions is an attrac- 60, 68]. Delivery of gene-based vaccines by physical methods,
tive prospect, supported by abundant data in mice [129 that is, electroporation and gene gun, has demonstrated
132] and by preliminary human trials [133135]. Lastly, to amplify the immune responses induced by therapeutic
additional strategies aimed to altering regulatory T cell vaccines against cancer [46, 47, 64].
function in cancer immunotherapy, including blocking Treg In the following section, an overview of various types of
tracking, dierentiation, and/or function and reducing clinical trials will be given to highlight the issue for usage of
eector cell susceptibility to suppression, have already proven plasmid DNA in humans. Table 4 provides a brief summary
successful in preliminary studies [136138]. of clinical trials discussed in this review.
6. Human Clinical Trials 6.1. Lymphoma. DNA vaccination is an attractive and eec-
tive approach for active therapeutic vaccination against B-
The goals of the various clinical trials were to demonstrate
cell malignancies given the ease of production compared to
the safety and tolerability of the candidate vaccines, and to
Id protein vaccines.
explore the ecacy of DNA vaccines in humans. Injection
Patient-specific DNA vaccines for therapy of B cell
of the plasmid DNA construct is tolerated well in terms
lymphomas and multiple myelomas based on scFv encoding
of safety in the patient population and rarely involves
a chimeric immunoglobulin molecule consisting of VH and
systemic toxicities. DNA vaccines that are currently being
VL genes derived from each patientss tumor were shown to
tested do not show relevant levels of integration into host
be eective in animal models [47, 73].
cellular DNA [139, 140]. Besides, preclinical studies in
The first phase I/II trial of idiotypic vaccination for fol-
nonhuman primates as well as early studies in humans did
licular B-cell lymphoma using a genetic approach [142] was
not detect increases in antinuclear or anti-DNA antibodies.
conducted by Hawkins and colleagues. Vaccines encoding
Participants in human trials of DNA vaccines are followed
individual DNA idiotypic scFv fused to TTFrC were delivered
for possible signs and symptoms of autoimmunity induced
as naked DNA by i.m. injection in patients with follicular
by DNA vaccination giving no convincing evidence of
lymphoma in clinical remission following chemotherapy,
autoimmunity developing in association with a DNA vaccine
and plasmid DNA were able to develop cellular or/and
[40, 42, 141].
humoral antiidiotype immune responses in 38% of patients
The earliest Phase I clinical trial for a DNA vaccine was
over a period of several months [47].
of an HIV-1 candidate tested in individuals infected by HIV-
In a second study of vaccine therapy for B-cell lym-
1, followed by studies in volunteers who were not infected
phoma, the patients tumor scFv was linked to the IgG2a and
by HIV-1 [40]. Other prophylactic and therapeutic DNA
mouse immunoglobulin heavy- and light-chain constant
vaccine trials followed, including trials that tested DNA vac-
regions chains, respectively. In this phase, I/II trial patients
cines against cancer influenza, malaria, hepatitis B, and other
in remission after chemotherapy received two series of i.m.
HIV-1 candidates [24, 41, 42]. These trials demonstrated
DNA vaccinations and at the end of the second vaccination,
that the DNA vaccine platform is well tolerated and safe,
50% of patients exhibited humoral and/or T-cell anti-Id
as no adverse events were reported and all studies went to
responses; yet, these were cross-reactive with Id proteins
completion.
from other patients tumors. Subsequently, a third series of
The evident safety of DNA vaccines has led to a relaxation
vaccinations was carried out using human GM-CSF DNA
of the requirements for approval by both the United States
mixed with Id DNA: humoral or T-cell responses were
Food and Drug Administration and the national competent
boosted in some cases [56].
regulatory authorities in Europe. This is why many clinical
studies tend to melt the first phase with the second phase.
Then the issue has become the ecacy rather than toxicity. 6.2. Prostate Carcinoma. After the important insights pro-
Since tumour antigens are generally weakly immuno- vided in preclinical studies [49], the department of Oncology
genic, they often induce a low level of spontaneous immunity of the University Hospital of Uppsala is recruiting partici-
or, in other cases, the spontaneous response can lead to pants for a phase I/II trial, where intradermal EP (DERMA
tolerance [47]. The molecular precision oered by gene- VAX) will be used as a delivery system. This study will assess
based vaccines, together with the facility to include addi- the feasibility and safety of vaccination with increasing doses
tional genes to direct and amplify immunity, could lead to an of xenogenic DNA coding for the Rhesus Prostate Specific
ecient methods to use the immune system against cancer. Antigen (rhPSA), a protein that is 89% homologous to
human PSA, administered in patients with relapsed prostate with metastatic melanoma. Rosenberg et al. showed that
cancer. neither intramuscular nor intradermal injection was capa-
A phase I/II, dose escalation, DNA vaccination trial with ble of raising cellular immune reactivity or a significant
plasmid DNA, which carries prostate-specific membrane incidence of antitumor eects [57]. Increasing results were
antigen (PSMA), fused to a domain (DOM1) of Fragment obtained in a phase II study with interleukin-2 cytokines
C of tetanus toxin, delivered either by i.m. or by i.m. as adjuvant used in combination with same vaccination
followed by EP, was performed in patients with recurrent protocols (ClinicalTrials.gov Identifier: NCT00019448) and
prostate cancer. The epitope used in this study, PSMA27 in a phase II trial with human GM-CSF plasmid DNA in
is a short stretch of 9 amino acids tumor-derived epitope conjunction with a multipeptide vaccine encoding gp100 and
belonging to the PSMA. Preliminary analysis of CD8+ T- tyrosinase peptidse [62].
cell reactivity against the prostate-specific membrane antigen
target peptide indicated significant responses in 3 out of 6.4. Cervical Cancer. Current vaccination strategies are based
3 patients and CD4+ T-cell responses against the DOM1. on the induction of neutralizing antibodies against the
These data validated EP as a potent method for stimulating major and minor capsid proteins, L1 and L2, of human
humoral responses induced by DNA vaccination in humans papillomavirus, and Gardasil is only eective against a subset
[46, 47, 64]. of HPV genotypes [146]. Further therapeutic interventions
Results of a phase I/II trial, conducted with DNA vaccine for early-stage and late-stage cervical cancers or HPV-related
encoding human prostatic acid phosphatase (PAP) coad- disease are uneective.
ministered intradermally with GM-CSF, in prostate cancer DNA plasmid platform could represent an ideal vaccine
patients (stage D0) are associated with an increased PSA against HPV infections since it could generate both humoral
doubling time (PSADT), 6.5 months pretreatment versus 9.3 immune response to prevent new infections as well as cell-
months in the 1 year posttreatment [61]. A longer PSADT is mediated immunity to eliminate established infection [146].
associated with an extremely low risk of death from prostate A recent phase I/II clinical trial in patients with
cancer. Besides, 14% of patients developed PAP-specific high-grade squamous intraepithelial lesion associated with
IFN gamma-secreting CD8+ T-cells immediately after the HPV16 provided DNA plasmid expressing a mutated non-
treatment course, and 41% of patients developed PAP- functional E7 incapable of binding retinoblastoma protein,
specific CD4+ and/or CD8+ T-cell proliferation, confirming with no transforming activity, linked to HSP70. A signal
the preclinical studies [143]. sequence was also attached to the hybrid antigen which
Todorova and colleagues [58] enhanced the DNA vaccine results in secretion of the linked E7 antigen [67]. E7 HPV
ecacy by heterologous prime-boost regimen in a Phase antigen as well as E6 antigen are essential for transformation
I/II study. Prostate cancer patients were prime-boosted and are coexpressed in HPV-associated lesions hence they
with alternate injections of recombinant adenoviral vector represent ideal targets for the development of HPV therapeu-
expressing PSMA and plasmid DNA encoding PSMA and tic vaccines.
CD86 alongside receiving GM-CSF proteins as adjuvants.
After 36-month observation period from the first vaccine 6.5. Liver Cancer. In preclinical studies, mice were suc-
injection, 86% of participants developed anti-PSMA anti- cessfully DNA-based immunized [147]. In a prime-boost
body. approach, coadministration of plasmids DNA encoding
murine alpha fetoprotein (AFP) and murine GM-CSF was
6.3. Melanoma. DNA vaccine platform is a promising followed by boosting with an AFP-expressing nonreplicating
therapeutic approach also for the treatment of malignant adenoviral vector [60] leading to tumor protective immunity.
melanoma, as demonstrated by already completed and on- The early studies were applied in a phase I/II clinical
going clinical trials. trial in patients with HLA-A 0201-expressing stage IIIVA
In stage IV melanoma patients, a phase I/II pilot study hepatocellular carcinoma. Vaccine therapy, comprising AFP
of intranodal delivery of Synchrotope MA2M plasmid DNA and sargramostim (GM-CSF) plasmids DNA, followed by
vaccine induced both humoral and CTL responses against AFP adenoviral vector boost determined the dose-limiting
cells expressing tumor two melanoma-associated antigens toxicity and maximum tolerated dose of adjuvant vaccina-
[144]. Synchrotope MA2M plasmid is a bivalent DNA tion (ClinicalTrials.gov Identifier: NCT00093548).
vaccine encoding epitopes for both Melan-A (MART-1) and
tyrosinase with potential antineoplastic activity. 6.6. Breast Cancer. Since HER-2/neu (HER2) oncogenic pro-
The same approach was used in a improved trial con- tein is a tumor antigen in patients with breast and ovarian
ducted with the Synchrovax SEM plasmid DNA vaccine cancer, several vaccine strategies have been developed and
containing a plasmid pSEM that encodes 4 peptide epitope are being evaluated for safety and immunogenicity in phases
sequences, Melan-A (2635), Melan-A (3196), tyrosinase I and II clinical trials (ClinicalTrials.gov). Patients whose
(19), and tyrosinase (369377), resulting in antigen-specific tumors overexpress the antigen have both detectable anti-
immunity even though not induce regression of established body and T-cell immunity directed against HER2. Likewise
disease [145]. preclinical studies suggest that the HER2 protein, particu-
DNA plasmids encoding the gp100 nonmutated melano- larly the intracellular domain (ICD), is a tumor rejection
ma-melanocyte antigen alone were administered in patients antigen [148].
Table 4: Phases I/II-II clinical trials: key summary.
Tumor Study ID Patients no. Objectives Status Response Side eects

Determine the safety, Absence of toxicity
Lymphoma UK-007 25 dose, immunogenicity Completed Cellular and/or
humoral responses
Determine the feasibility
and safety
Determine the safety and Open
NCT00859729 18 functionality of DNA recruiting
vaccine delivery system
Determine the Absence of toxicity Brief and
feasibility and safety Open CD8+ T-cell acceptable
UK-112 20 Determine the reactivity against the pain at the
immunological responses target peptide injection site
Prostate cancer PAP-specific IFN
gamma-secreting
NCT00582140 22 Determine the safety Completed CD8+ T-cells
(Aug 2009) PAP-specific
CD4+ and/or CD8+
T-cell proliferation
Bulgarian Characterize the humoral Specific humoral
Drug 52 immune response Completed immune response
Agency Register against PSMA against PSMA
Determine the safety Antigen-specific
and tolerability Immunity
NCT00033228 618 Determine the Completed No regression of Grade I/II toxicity
immunological and (July 2009) established disease
Melanoma clinical responses
Determine the safety Open Immunological
and tolerability Not ecacy
Grade I toxicity
NCT00085137 327 Determine any Recruiting in terms of
antitumor response T-cell response
Determine the feasibility
and toxicity Absence of
Determine the eect toxicity
Determine changes In the highest-dose
in lesion size and Open cohort the number
HPV viral load Not of patients with
Cervical cancer NCT00121173 150 Determine the immune Recruiting complete histologic Transient
responses regression is injection-site
Correlate measures of higher than the discomfort
immune response with unvaccinated cohort,
clinical response but not significant.
Determine the dose-
limiting toxicity and
maximum tolerated dose
Liver cancer NCT00093548 325 Determine the optimal Completed Absence of
biological dose (Feb 2009) toxicity
Determine disease-free
survival of patients treated
Determine and
characterize
Breast cancer NCT00363012 56 the immunologic Open Absence of
memory recruiting toxicity
to the HER2-ICD
Salazar and colleagues are studying the immune response Acknowledgments

in patients overexpressessing HER2 epitope who have
undergone vaccine therapy in a heterologous prime-boost This work was supported by MUR Grant FIRB 2006
regimen (ClinicalTrials.gov identifier: NCT00363012). After (RBIP0695BB), and by the Italian Banca Marche. D. Fioretti
vaccination with a plasmid encoding HER2 ICD in patients and S. Iurescia. have been supported by MUR Grant FIRB
with advanced stage HER2 overexpressing breast and ovarian 2006 (RBIP0695BB). Both of them contributed equally to
cancers patients receive HER2 ICD protein treatment intra- the preparation of this paper.
dermally at 6 months postvaccination with the pNGVL3-
hICD vaccine. The injection site is biopsied and examined References
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Review Article: DNA Vaccines: Developing New Strategies Against Cancer

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Correspondence should be addressed to Monica Rinaldi, [email protected]

Received 20 November 2009; Accepted 5 February 2010

Academic Editor: Soldano Ferrone

1. Introduction help bodys natural defences to recognise and target cancer

Table 1: DNA vaccines main features.

Table 2: DNA Vaccine Enhancing Strategies.

Strategy Approach Indication References

Table 4: Phases I/II-II clinical trials: key summary.

Tumor Study ID Patients no. Objectives Status Response Side eects

Salazar and colleagues are studying the immune response Acknowledgments

[141] M. L. Bagarazzi, J. D. Boyer, M. A. Javadian, et al., Safety and

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