The immunogenicity of antigens delivered via plasmid DNA was first seen in viral studies, where cDNA encoding an influenza viral protein generated specific cytotoxic T cells that could protect against a live influenza viral challenge [66
]. In a plasmid DNA vaccine, the gene of interest is cloned into a bacterial expression vector having a constitutively active promoter for expression of the gene product. The plasmid can be introduced into the dermis or muscle where it is taken up by professional antigen presenting cells (APCs) such as dendritic cells (DCs) as well as by neighboring non-APCs and can be expressed for up to two months [67
]. One of two methods of uptake is possible (Fig. ). The first possibility is the direct transfection of APCs by plasmid DNA [68
]. Even though a relatively small number of cells present at the vaccination site are DCs, their enhanced potential to present and prime T cells can make this feasible. The second mechanism underlying the efficacy of DNA immunization is cross priming [69
]. The DNA transfects neighboring keratinocytes or myocytes that transcribe and translate the antigen. Mature antigen is made available to DCs as secreted protein or through apoptotic transfected cells. The antigen is then processed and presented to naïve T cells in draining lymph nodes.
Figure 1 Methods of antigen presentation that could generate an immune response after DNA immunization. DNA can directly transfect dendritic cells (DCs) which can migrate to the draining lymph node to activate naïve T cells. Alternately, they can be cross (more ...)
DNA vaccines have some properties that help to overcome obstacles encountered with the use of other types of cancer vaccines. Dendritic cells as APCs for peptides, proteins or RNA are known to be effective in generating antigen specific responses [72
]. However, in a clinical setting, autologous cellular vaccines must be custom manufactured for each patient, making them cost prohibitive and labor intensive in a large vaccine trial. Peptide vaccines, while being simpler to manufacture, can be effective only in association with certain HLA molecules. Consequently, only a limited pool of patients bearing the appropriate HLA type is eligible to receive the vaccine. Though immune monitoring to these vaccines is more straightforward, the potential for antigen escape variants is greater, as tumors theoretically only need to alter a single amino acid to abolish presentation of a given epitope. Protein vaccines, on the other hand, are not HLA restricted and can present a variety of epitopes to activate both cell mediated and humoral arms of the immune system. However, large scale manufacturing, which includes purification, can be a challenge.
DNA vaccines encoding full length protein can circumvent some of these problems while having the advantages of purified recombinant protein. First, full length cDNA of the gene of interest provides several potential epitopes to stimulate both cytolytic T cells as well as an antibody response, the latter indicating the presence of strong helper epitopes in the gene sequence. Second, insertion of the antigen coding sequence in a bacterial expression vector provides the vaccine with a 'built-in adjuvant' offered by unmethylated CpG motifs [73
]. Third, transcribing and translating the full length protein also eliminates the need to limit patients of a defined HLA type to be eligible to receive the vaccine. The simplicity and relative economy of producing large quantities of DNA (versus purified recombinant protein) also makes this approach attractive. More importantly, DNA vaccines in human trials for malaria and HIV treatment have shown that they are well tolerated and safe [75
]. An added benefit is the relative ease to design and produce altered forms of the wild type antigen with higher biological potency.
Murine studies to support the use of xenogeneic immunization
The importance of using an 'altered self' form of antigen to induce tumor protection came from studies using lysates of SK-MEL19, a gp75+
human melanoma cell line [46
]. When mice were immunized with human melanoma lysate, autoantibodies that recognized mouse gp75 were produced. Immunization with murine B16 melanoma produced no antibody response, even when potent adjuvants were included. These studies support the idea that ignorance or tolerance to a self protein can be overcome by presenting sources of altered antigen (e.g., homologous xenogeneic protein). A similar study in a rat Her2/neu model, showed that immunization with human intracellular domain segment of the protein generated T cell and antibody responses specific for both rat and human Her2/neu [80
]. This indicates that despite extensive homology between the mouse and human protein, small differences in epitopes between the two are sufficient to overcome immune ignorance or tolerance.
This idea was further tested with a variety of melanosomal differentiation antigens, starting with human TRP1/gp75 [47
]. Human gp75 cDNA, expressed in a plasmid expression vector and introduced into the epidermis via gene gun, protected mice from a syngeneic B16 tumor challenge primary through autoantibodies, while syngeneic (murine) gp75 induced no tumor immunity. Tumor protection required Fcγ receptors (FcγR), CD4+ cells and NK1.1+ cells, but interestingly was independent of CD8+ T cells [47
]. In addition to protection from tumor challenge, many of the mice immunized with human gp75 DNA also developed hypopigmentation of coat, presumably through cross-recognition of endogenous gp75 on melanocytes in the mouse hair follicle. An example of the expression plasmid containing murine tyrosinase DNA vaccine is shown in Fig . This vaccine is currently being used in a clinical trial at Memorial Sloan-Kettering Cancer Center, New York.
Figure 2 Plasmid DNA expressing mouse tyrosinase used in clinical trials at MSKCC. The full length murine tyrosinase cDNA was cloned into a bacterial expression vector having a kanamycin resistance cassette and operating under the host's constitutive CMV promoter (more ...)
In tumor protection studies using the other melanosomal antigens, a similar requirement for the xenogeneic antigen was noted; however, there were significant differences in the immunologic mechanisms underlying the tumor immunity. TRP2, another protein in the melanin synthesis pathway led to a potent induction of CD8+ T cells and required both CD4+
effectors for tumor protection [81
]. There was no dependence on antibodies or NK1.1+ cells in this case. Gp100, another melanosomal protein, conferred tumor protection through CD8+
T cells, though without a strict requirement for CD4+
]. While these immunogens were effective in a prophylactic setting, a 'treatment' model to mimic the clinical scenario was also tested. Using TRP2 as the antigen, two models were tested. In the first case, immunization was started 10 days after injecting live tumor intravenously [81
]. In the second case, B16 melanoma was given orthotopically in the foot pad and then surgically excised. Immunization with huTRP2 was then carried out in a 'minimal residual disease' setting that is comparable to adjuvant therapy for micrometastatic cancer. A significant decrease in the development of lung metastases was noted after immunization with human TRP2 [83
Epitope spreading was an interesting phenomenon observed among mice immunized with huTRP2 DNA. In some of the mice, anti-TRP2 antibodies were also specific to gp75, a related protein [84
]. Determinant spreading is a normal feature of protective immune responses to infectious agents, allowing recognition of multiple antigenic targets [85
]. While the immune system depends on diversification to adequately protect against non-self, it is possible that it may also play a role in protection against aberrant processes that are dangerous to 'self', such as cancer. Epitope spreading (both intermolecular and intramolecular) was noted in few cases of clinical responders to peptide vaccines in trials involving patients with melanoma. When immunized with MART-127-35 loaded DCs, one patient developed HLA-A*0201-restricted responses to two additional melanoma antigens (gp100 and tyrosinase) as well as a HLA-DR4-restricted MART-1 epitope [86
]. The patient's tumor was positive for MART-1, gp100 and tyrosinase. In another instance of inter- and intramolecular spreading, the patient (who was a responder), was immunized with DCs loaded with HLA-A*0201 melanoma-derived epitopes MART127–35
, and tyrosinase368–376D
. The patient's T cells showed reactivity to two other HLA-A*0201-binding epitopes (gp100209–217
) and four HLA-DR4 class II epitopes (MART151–73
, and tyrosinase56–70
]. Temporary regression of a melanoma metastasis was also associated with immune reactivity toward a cryptic epitope from the MAGE-12 gene (MAGE-12170–178
), after being immunized with gp100209–2M
]. Spreading of immune reactivity to other melanoma antigens in subjects with clinical response, suggests that determinant spreading may be associated with clinical response to immunotherapy.
In early human clinical trials for infectious diseases, DNA vaccines have not been as potent as might have been expected given pre-clinical mouse studies [76
]. Several studies have shown the benefit of adding cytokines such as GM-CSF (both DNA and soluble protein) to enhance the antigen specific response, perhaps by mobilizing DCs as well as enhancing expression of co-stimulatory molecules [89
]. DNA encoding GM-CSF was shown to improve recruitment of DCs to the local site of injection [89
]) as well as to induce infiltration of inflammatory and Th1 precursors cytokines [90
]). Co-immunization of full length rat neu
cDNA with plasmid DNA coding for co-stimulatory molecules such as CD80, CD86, and CD137 in a rat transgenic mouse model induced both antigen specific T cells and antibodies resulting in an anti-tumor effect [92
]. Local use of GM-CSF DNA can abrogate the inconvenience of multiple injections of soluble GM-CSF protein, while potentially offering the same benefits.
Development of xenogeneic DNA Vaccines for use in canines with spontaneous cancer
As mentioned above, immunotherapies that appear promising in pre-clinical mouse models have often led to clinical trials with disappointing clinical and immunological results. A study was conducted in collaboration with the Animal Medical Center of NY, a tertiary care hospital for pets that has an oncology clinic that sees up to 5000 visits per year. The use of outbred animals with spontaneously arising malignancies may overcome some of the limitations of transplantable tumor systems in syngeneic mice and serve as a translational bridge between standard inbred animal models and human clinical trials.
In dogs, malignant melanoma of the oral mucosa displays a similar natural history to human cutaneous melanoma. This includes early invasion, a predisposition to distant metastasis and relative resistance to standard cytotoxic therapies. Radical surgery followed by radiation is optimal therapy; however, local and distant recurrence is common and difficult to treat. An initial clinical trial using human tyrosinase DNA in 9 dogs with metastatic melanoma was recently completed [93
]. The vaccine was given by the same route and at the same doses that are to be used in the human clinical trial. There has been no toxicity associated with the vaccination. In addition, one dog with numerous lung metastases has had a complete clinical response with disappearance of all detectable disease, lasting over one year. The median actuarial survival for dogs on this trial predicted by Kaplan-Meier analysis is greater than 389 days. Although this is a small single-arm study, this data is encouraging when considered in the context that stage-matched historical controls had a survival of less than 90 days. Similar trials have also been completed using murine tyrosinase and murine gp75 DNA in dogs with melanoma. Follow-up is too short at this time to reach any clinical conclusions for these trials. A trial of GM-CSF DNA alone or in combination with murine tyrosinase DNA is currently underway.
Clinical trials in human using syngeneic cDNA in cancer therapy
In a phase 1 safety study using syngeneic cDNA to CEA, low grade transient toxicity was observed [94
]. While CEA-specific antibodies were not observed, 4 of 17 patients showed lymphoproliferative responses to CEA after vaccination. There was no association with objective tumor regression and sustained declines in circulating CEA, nor a correlation between lymproliferative response with stable disease. In another recent clinical trial, syngeneic cDNA encoding gp100 was used as the vaccine. The results did not demonstrate clinical or immunologic responses to the vaccine [95
]. Several studies have indicated that syngeneic cDNA is immunogenic when used either in prime boost regimens with recombinant viral vectors or with the use of augmentation strategies such as cytokines or costimulatory molecules. The presence of slight differences in epitopes between host 'self' protein and that encoded by xenogeneic DNA plasmid vaccine, along with inherent bacterial unmethylated CpG motifs may be sufficient to boost the immune response to break tolerance to tumors.