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Vaccination with Gene-modified Dendritic Cells Protected Transgenic Mice Against Breast Cancer
We hypothesized that the introduction of a gene encoding a TAA into DC using a recombinant viral vector might be beneficial. Potential advantages include the following: (1) There is no requirement for knowledge of the type of MHC molecule expressed, the peptide sequence, or its MHC binding affinity. (2) Gene transfer can provide larger antigenic sequences with more potential targets. (3) Natural antigen processing by DC may improve antigen presentation. (4) Constitutive expression of the antigen may allow for continuous replenishment of low-affinity TAA peptides as they are lost from MHC molecules, and (5) viral proteins expressed by the gene transfer vector may provide signals required for maturation, activation, and increased expression of co-stimulatory molecules on the DC, resulting in a stronger immune response. We used the neu oncogene, the rodent homolog of the human HER-2/neu gene as our targeted TAA. HER-2/neu, an epidermal growth factor receptor family member is overexpressed in cancers of the breast, ovary, uterus, lung, and gastrointestinal tract and is associated with treatment resistance and a poorer clinical outcome. It is a therapeutically important immunological target as evidenced by traztuzumab (Herceptin), a humanized anti-HER-2/neu antibody approved for the treatment of breast cancer. We generated a recombinant adenovirus expressing the extracellular and transmembrane domains of the neu oncogene (Ad.Neu). Studies using mouse bone marrow–derived DC showed that infection and significant neu antigen expression were achieved using our vector (Figure 1). In addition, viral infection increased the surface expression of MHC and co-stimulatory molecules, indicating maturation and activation of the DC. We studied the effectiveness of genetically modified DC vaccination using BALB-neu T transgenic mice. These mice express a neu gene controlled by a mammary-specific promoter. Female mice develop breast tumors at 14 to 15 weeks of age, and progress until all mammary glands are associated with tumors at 24 to 25 weeks. We found that three weekly vaccinations using one million DC modified with Ad.Neu (DCAd.Neu) prevented or delayed the onset of breast tumors compared with mice vaccinated with DC infected with a control vector (DCAd.null), or with unmodified DC alone. DCAd.Neu-vaccinated mice had significantly improved disease-free survival and a reduction in the average number of tumors that appeared. Vaccinated mice free of tumor at 28 weeks were challenged with injections of syngeneic neu+ or neu– tumor cell lines. The mice were protected from growth of the neu+ tumor cells, but not neu– breast cancer cell lines, indicating that immunity was specific for the target antigen. Mice vaccinated with DCAd.Neu had significant increases in anti-neu antibody titers. Surprisingly, induction of tumor-specific CD8+ cytolytic T lymphocytes (CTL) could not be demonstrated, suggesting that the protection was mediated by the development of antibodies and not CTL. On depleting specific immune cell populations, we found that CD4+ T cells, but not CD8+ T cells, played a critical role in the immune response to our vaccine, supporting our hypothesis. Since most adults have antibodies to adenoviruses that might influence vaccine efficacy, we hypothesized that our strategy presented tumor antigens as peptides that would be unaffected by circulating antibodies to adenovirus. Indeed, we found that DC vaccination was equally effective in mice with preexisting immunity to adenovirus. Figure 1. Mouse bone marrow–derived dendritic cells demonstrating high levels of green fluorescence after infection with an adenovirus vector expressing enhanced green fluorescent protein (GFP). Further collaborative work has shown that unlike traztuzumab, whose efficacy depends on the presence of the Fc receptor (FcR), the antibodies induced by the Ad.Neu vaccine protected mice by an FcR-independent mechanism and inhibited the growth of tumor cells in vitro. Therefore, we expect that these antibodies were acting directly through the HER-2/neu receptor on the tumor cells to inhibit growth. Several important questions remain to be answered. DCAd.Neu vaccination was effective in the BALB-neu T model when the mice were less than 7 to 8 weeks of age and prior to the appearance of tumors. Vaccination was less effective in older mice and ineffective once tumors appeared. Increasing the number of DCAd.Neu vaccinations only slightly increased survival once tumors developed. The reason for this is not clear, but could be the result of homeostatic or tumor-specific mechanisms that downregulate the immune response. We plan to examine strategies to overcome the resistance of older mice to anti-neu vaccination, including depletion of T regulatory cell populations and genetically modifying DC with immunostimulatory cytokines. |
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mutations that lead to cancer often result in the expression of novel tumor-associated
antigens (TAA) that can be targeted by the immune system. A successful immune
response to a tumor requires the cooperation of antigen-presenting cells that
process and present antigens to T cells as short peptide fragments bound to
the major histocompatability (MHC) molecules on their surface. These peptides
are recognized by receptors on specific T cells that are then activated to orchestrate
an immunological “attack” on the tumor. Dendritic cells (DC) are
powerful antigen-presenting cells. They express high levels of MHC and other
co-stimulatory molecules, and they secrete the required cytokines that sustain
and direct the immune response after the initial T-cell activation. As a result,
DC are potentially the ideal cells for presenting TAA to vaccinate against cancer.
Tumors, however, attempt to escape the immune response by secreting immunosuppressive
factors that inhibit DC maturation and decrease antigen presentation. These
defects can be overcome by growing DC in culture. DC can be pulse-loaded with
target antigen using peptides, or by incubating them with extracts made from
tumors, and used to vaccinate patients.