Since the 1990s, tumor immunology has developed into a distinct discipline with a metamorphosis from clinical observations in oncology to understanding its scientific underpinnings.
This has been particularly relevant to the development of active immunotherapies (vaccines) for cancer.
Traditionally, vaccines have been effective in the induction of protective immunity to bacteria and viruses based on recognition of foreign, or non-self, antigens on these pathogens.
However, cancer cells arise from one's own tissue (self) and this poses a challenge in the development of effective active immunotherapies for cancer.
It also presents a conundrum: can the immune system mount an effective response to reject tumors?
Perhaps the answer to the above question lies in the paradigm that the immune system can distinguish self from 'altered self' rather than the traditional non-self.
While some mutated gene products (altered self) have been identified, surprisingly, the vast majority of antigens on cancers characterized to date are unaltered self-antigens.
These are antigens encoded by genes expressed by both tumor cells as well as their normal cell counterparts.
That cancer immunity exists is observed clinically in the form of spontaneous regressions in melanoma, gastrointestinal tumors, lung and breast cancers.
In addition, histopathology of tumor sections has revealed infiltrating lymphocytes around the tumor bed and recent studies indicate that ovarian cancer patients with such infiltrates around tumors have an improved prognosis, compared with similarly staged patients without lymphocytic infiltrates.
The immune repertoire therefore contains auto-reactive immune cells that may reject tumors, when activated appropriately.
These auto-reactive cells, upon recognizing target molecules on normal cells have the potential to induce tissue destruction leading to toxic autoimmunity.
The molecular characterization of several tumor antigens identified by both by T cells and serology, has provided several candidates for the development of immunotherapy of various malignancies.
Tumor antigens can be broadly categorized into two types – those that are undefined and others that are well defined.
Undefined and unidentified antigens are found in both allogeneic and autologous vaccine settings.
Prominent examples of this type of vaccine based on undefined antigen are intact cells, cell lysate, total (amplified) RNA vaccines and heat-shock proteins.
The underlying principle is that relevant tumor rejection antigens would be present among the thousands of other molecules that would be injected at the same time.
The presence of unique as well as universal (or shared) tumor antigens in the mixture would prevent the expected emergence of antigen loss or escape variants.
Tumors are known to commonly down-regulate or lose key molecules to escape immune surveillance.
Therefore, use of vaccines with numerous targets that induce multiple components of the immune response is advantageous.
An advantage of using defined antigens for immunotherapy is the ability to correlate specific immune responses with the antigen used, thus providing a means to study and improve immunogenicity of the vaccine, though the vaccine will have to be targeted to patients of selected HLA types.
The approach of using defined antigens has been most widely explored in trials of individual antigens though combinations have also been tested.
Using a "cocktail" of defined antigens addresses some of the concerns about the emergence of antigen escape variants.
Table 1: Advantages and disadvantages of vaccines with defined and undefined antigens
||1. Availability of several potential tumor rejection antigens.
||1. Difficulty in correlating clinical response and overall immune response based on select known antigens.
|2. Unrestricted HLA patient population.
||2. Largely dependent on clinical endpoint.
||1. Temporal monitoring of specific immune response.
||1. Limited number of known tumor antigens for use (single or cocktail).
|2. Possibility of correlation of immune response with antigen expression on tumors.
||2. Relatively limited targeting of patient population due to HLA restriction.
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.
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. (Fig. 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 naive T cells.
Alternately, they can be cross-primed when they uptake antigen from dying keratinocytes or myocytes.
They can activate both CD8+ and CD4+ cells in the lymph node via Class I or Class II peptide-MHC complexes.
Abbreviations: DCs - dendritic cells, APC - Antigen presenting cells, MHC - Major Histocompatibility Complex, TcR - T Cell Receptor Complex
The first possibility is the direct transfection of APCs by plasmid DNA.
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.
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 naive T cells in draining lymph nodes.
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.
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'.
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.
An added benefit is the relative ease to design and produce altered forms of the wild type antigen with higher biological potency.
Attempting to generate immune responses to self-proteins raises reciprocal problems of immunological tolerance and potential autoimmune sequelae.
In murine studies using melanosomal differentiation antigens, autoimmune depigmentation was commonly seen (Fig 2).
However, in trials of several immunologic therapies for cancer, autoimmune manifestations remain rare, despite induction of immune responses.
A clinical study in melanoma using adoptive transfer of selected tumor reactive T cells showed that regression of metastatic melanoma was accompanied by autoimmune depigmentation.
However, effector mechanisms for both tumor immunity and autoimmunity could be different.
Murine studies have shown that active immunization with human gp75 induces an antibody response to reject tumor, while depigmentation continued in the absence of this receptor.
Likewise, studies using knockout mice indicated that when TRP2 was used as the immunogen, autoimmunity was dependent on perforin, whereas tumor immunity proceeded in the absence of perforin.
It is now accepted that tolerance to self-antigens on cancer cells can be overcome using active immunization strategies, such as with xenogenetic DNA vaccines.
The hallmarks of a successful vaccine are judged by multiple endpoints, with the most important one being control of dissemination of tumor.
There are several steps involved in the generation of anti-tumor immune responses.
First, there must be efficient uptake of antigen by professional APCs, such as Langerhans cells and DCs, followed by antigen processing and migration to draining lymph nodes.
Precise antigen presentation, leading to induction and expansion of appropriate helper and cytotoxic cells bearing the cognate receptor is necessary.
These effector cells must then traffic to distant tumor sites, recognize and lyse tumor.
Autoimmune depigmention as a result of immunization with human TRP2/DCT.
Abbreviations: hTRP2 - human tyrosinase related protein-2; mTRP2 - mouse tyrosinase related protein-2.
There should be a persistent memory pool of effectors to challenge tumors bearing the same antigen that might grow out over time.
Ultimately, an adaptive response should be generated to control antigen escape variants.
The potency of the response, once induced, must be increased to the magnitude of that as found in infectious disease settings.
A break anywhere in this sequence can give rise to disease progression.
Unfortunately, this frustration is frequently encountered.
Specific immune responses to tumor antigens in vitro can be detected in patients undergoing various immunotherapies that do not translate to a desired clinical response.
A major step forward in understanding and improving vaccine efficacy in cancer immunotherapy is the concordance of clinical outcomes with appropriate, well-timed and accurate immunologic monitoring.
The search for an active immunotherapy for cancer is clearly not easy.
The xenogenetic DNA vaccine approach is only one among the several that has potential in treating cancer.
Research in animal models (inbred mice and outbred companion animals) has shown great promise for this in the treatment of solid tumors.
Based on these results, this immunization strategy is being tested in patients with melanoma and prostate cancers at MSKCC, NY, with further clinical trials proposed for breast cancer and Non-Hodgkin's Lymphoma.