Several recent advances in tumor immunology and molecular biology may provide an opportunity to alter the course of human cancer, namely through the development of tumor vaccines. Therapeutic and prophylactic vaccine strategies that exploit the immune system on a molecular level are being developed. Vaccines are considered active immunotherapy since they elicit an immune response in the patient. Therapeutic cancer vaccines are intended to induce cellular components of the immune system to recognize and attack malignant tissue. Although both the humoral and cellular components of immunity are important, it is now recognized that T-cell responses are the primary causes of tumor rejection.
Prophylactic vaccines elicit humoral immune responses because they induce the production of antibodies capable of neutralizing a viral antigen before it infects the host cell. In this way, virally-induced malignancies may be prevented by inoculation with a vaccine prior to any exposure to the tumor virus. Certain prophylactic vaccines have been enormously effective in preventing infections, such as hepatitis B, measles, mumps, and polio. Recombinant technology has permitted the biosynthesis of the hepatitis B vaccine, and studies have confirmed the production of protective antibodies against the surface antigen of the hepatitis B virus following immunization. These studies have demonstrate the vaccines efficacy in preventing the subsequent transmission of this virus.
Therapeutic vaccines, on the other hand, are administered to reduce or eradicate existing disease or infection. Thus, a therapeutic tumor vaccine attempts to target and destroy cells expressing tumor-associated or tumor-specific antigens on their surface. In the case of cervical neoplasia, the viral peptides derived from high-risk human papillomavirus (HPV) E6 and E7 oncoproteins are the tumor-specific antigens.
T-lymphocytes express receptors specific for small peptides that are presented on the surface of tumor cells in association with major histocompatibility complex (MHC) molecules. This type of immune monitoring is important in defending against many viral infections and virally induced tumors.[4,5] In general, viral proteins or other endogenous cellular proteins are degraded into peptides presented in association with class I MHC molecules on the cell surface of antigen-presenting cells. In the class I pathway, peptides consisting of 8 to 11 amino acids are intracellularly transported via transporter associated with antigen proteins ( TAP1 and TAP2) to the endoplasmic reticulum. After the peptides attach to the class I heavy chain and beta-2-microglobulin, this trimeric structure is translocated onto the cell surface for recognition by CD8+ cytotoxic T-lymphocytes (Figure 1).
In contrast to endogenous proteins, the intracellular processing and presentation of exogenous, endocytosed proteins occurs in the context of MHC class II molecules. The class II molecule and invariant chain complex is formed in the endoplasmic reticulum and transported to a lysosomal compartment. Here, the invariant chain is degraded, allowing for peptide loading of the class II molecule. At the cell surface, the class II molecule and associated peptide are recognized by CD4+ T-cells (Figure 2).
For both MHC class I and II molecules, peptide binding assures structural stability. Without correct configuration, intracellular transport to the cell surface may be compromised. This would allow the peptide to go unrecognized by the cellular immune system. Inhibition of immune responses may also occur with deficiencies in MHC expression. Tumor cells often downregulate MHC class I expression or develop mutations of the beta-2-microglobulin gene, which partially accounts for the ability of tumors to evade immune recognition.[7,8]
A variety of immunologic approaches are candidates for cancer vaccines, including the adoptive transfer of antigen-presenting cells or inactivated whole cancer cells. Other adoptive immunotherapies may involve the ex vivo expansion of specific cytotoxic T-lymphocytes which are then reinfused into the patient. These approaches may include the use of tumor or viral peptides, gangliosides, and heat-shock proteins. Viral expression vectors that encode the gene for a specific tumor antigen can also be used to elicit cellular immune responses. Some candidate immunotherapies are summarized in Table 1.
Antigen-presenting cells are a heterogeneous population of leukocytes found primarily in the skin, lymph nodes, spleen, and thymus. They efficiently present antigens along with MHC, adhesion, and costimulatory molecules on their cell surfaces. Antigen-presenting cells, including Langerhans cells, macrophages, B-cells, and dendritic cells, can engulf exogenous proteins and present the degraded peptide antigens to T cells in an MHC-restricted manner. Dendritic cells can be generated from peripheral blood specimens and can be primed to stimulate antitumor T-lymphocytes by co-incubation with immunologically relevant tumor or viral peptide antigens.
Given their exquisite immunostimulatory capacity, antigen-presenting cells are essential for effective immunotherapies. Peptide-pulsed dendritic cells have already been used successfully to generate cytotoxic T-lymphocyte immune responses in animal and human models.[12,13] Antigens not presented by antigen-presenting cells can avoid T cell recognition. This is particularly true for noncytopathic viruses, such as HPV, which has a persistent nonlytic infectious life cycle. These viruses evade cytotoxic T lymphocyte-mediated immune responses in part because they generally do not infect antigen-presenting cells; therefore, the relevant peptide antigens are not available for presentation by antigen-presenting cells and specific cytotoxic T-lymphocytes are not induced.
Role of Peptides
Peptides are attractive vaccine candidates because they can be inexpensively synthesized in large quantities and are relatively nontoxic. However, immunologically relevant target peptides must be identified for this strategy to be effective. Viruses, such as the hepatitis B virus, Epstein-Barr virus, and papillomavirus, encode oncoproteins that can interact with normal cellular proteins to induce cell transformation. Several peptides derived from these viral oncoproteins have been characterized as rational immunologic targets.
Other peptides may derive from proto-oncogenes or mutated tumor-sup-pressor genes. Oncogenes and proto-oncogenes commonly associated with human cancers include ras and Her-2/neu (c-erbB-2).[16,17] Mutated tumor-suppressor genes can result in production of excessive or defective protein products that are functionally impaired. Two of the most frequently mutated tumor-suppressor genes in human cancers are p53 and pRb.
However, peptides in general, and specifically most cancer antigens, are often poor immunogens. To overcome this limitation, they can be altered to increase their immunogenicity by modifying the peptide amino acid sequence, conjugating them with immunostimulatory molecules, and administrating adjuvants. Vitiello and coworkers demonstrated that by covalently attaching a tetanus toxoid T-helper peptide epitope and two lipid molecules to an HBV core antigen peptide, primary HBV-specific cytotoxic T-lymphocyte responses could be elicited in human subjects.
Several other T-helper peptides have recently been identified, and the covalent linking of one of these T-helper epitopes (PADRE) and two palmitic acid residues to an HPV-16 E7 peptide epitope is currently being investigated in a phase I clinical trial. Incomplete Freunds adjuvant (IFA) has been used clinically for several years, but it remains a rather nonspecific adjuvant and often induces local inflammatory responses.
Other immunostimulants under investigation include immune-stimulating complexes (ISCOMs), cytokines, and costimulatory molecules. An ISCOM containing a cholesterol matrix into which protein antigens is incorporated has elicited both humoral and cell-mediated responses in immunized animals.[22,23]
Small, synthetic peptides can be polymerized and incorporated into ISCOMs for use as a vaccine. Fernando et al immunized mice with glutaraldehyde polymerized HPV-16 E7 peptides incorporated into an ISCOM vaccine. The mice developed an antigen-specific immune response. In contrast, free peptide was not immunogenic, indicating that ISCOM increased the immunogenicity of the peptides.
Other ISCOM adjuvants consist of cage-like microspheres, prepared by mixing Quillaia, cholesterol, and a phospholipid. Protein or peptide antigens can be incorporated into the microspheres or mixed with the adjuvant. By incorporating the Quillaia into the immune-stimulating complex particles, the local reactivity of Quillaia decreases and the immunostimulating properties increase. The particles are taken up rapidly by the immune system and are transported to the regional lymph nodes. It is believed that the immune-stimulating complex particles deliver antigens into the MHC class I pathway.
Viral Expression Vectors
Viral expression vectors offer another strategy for immunotherapy. Genes encoding relevant antigens can be spliced into recombinant expression vectors allowing for increased cellular production of antigen and induction of cellular and humoral immune responses. Vaccinia virus is perhaps the most widely studied expression vector for immunotherapeutic strategies. The vaccinia virus can accept large gene insertions and is efficient at inducing immunity. Other vectors that may be less pathogenic in immunocompromised individuals are currently under investigation and include attenuated adenovirus, fowlpox virus, and avipoxvirus.
Many of these molecular approaches are now being evaluated in vaccine trials for patients with advanced gynecologic cancers unresponsive to standard treatment (Table 2 and Table 3). Although much of the preliminary data were generated for cancers such as melanoma, more information is emerging regarding the role of immunotherapy in gynecologic cancers. The future holds much promise for the prevention and treatment of certain gynecologic malignancies for which there is currently no effective therapy.
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