We have demonstrated, for the first time to our knowledge, that the tumor-stromal antigen FAP can serve as a target for T cell–mediated cancer immunotherapy. This approach has several well-documented advantages over therapies directed against antigens solely expressed by tumor cells. First, stromal fibroblasts are genetically more stable than frequently mutating, heterogeneous tumor cell populations (2
). As such, the expression of the target antigen remains more stable and serves as a more reliable target for immunotherapy. Second, antigen presentation by stromal fibroblasts to the T cell receptor complex is not impaired by downregulated MHC class I antigen expression, as is frequently the case in human tumor cells (27
). Third, tumor cells often become increasingly resistant to T cell–mediated killing because of defects in apoptosis signaling pathways, upregulation of antiapoptotic proteins, or immunosuppressive effects against CTLs (1
). Fourth, targeting FAP, specifically overexpressed in over 90% of colon, breast, and lung carcinomas (18
), allows for a therapeutic approach to treat a number of different malignancies, in contrast to therapies involving antigens that are expressed solely by specific tumor types.
Our experimental data demonstrate that we can break peripheral T cell tolerance against the FAP self-antigen by delivering its cDNA as an oral DNA vaccine with a doubly attenuated strain of Salmonella typhimurium
to APCs in a secondary lymphoid organ, i.e., Peyer’s patches of the small intestine. In prophylactic as well as therapeutic settings, the CD8+
T cell–mediated antitumor immune response induced by pFap vaccination inhibited tumor growth and significantly suppressed the dissemination of established pulmonary metastases in different murine tumor models, i.e., multidrug-resistant CT26 colon and D2F2 breast carcinoma. Additionally, we previously demonstrated that the administration of a DNA vaccine, using attenuated S. typhimurium
as the vaccine carrier, can prolong antitumor effects up to 10 months and also achieve marked upregulation of T cell, NK cell, and dendritic cell activation markers (26
). Furthermore, in vivo depletion of T and NK cells in our current experiments indicates the involvement of CD8+
but not CD4+
T or NK cells. As shown here, the cytotoxic effect mediated by CD8+
T cells in vitro was specifically directed against target cells overexpressing the FAP antigen. We can also rule out nonspecific immune responses, since only CD8+
T cells from pFap-vaccinated mice induced in vitro cytotoxic lysis of tumor cells and fibroblasts overexpressing FAP. Moreover, tumors of pFap-vaccinated mice showed a more pronounced infiltration with CD8+
T cells than tumors of mice vaccinated with the empty-vector control. Even though FAP can also be found transiently overexpressed during wound healing, vaccination against FAP did not impair wound healing, nor did it cause any discernible autoimmune reactions, in the 26 tissues and organs examined histologically. Functional redundancy of proteins with overlapping function may account for this observation (30
We also tested 3 different effector molecules as adjuvants in hopes of improving the efficacy of our vaccine: IL-2, an inducer of T cell proliferation (31
); CCL21, a chemokine that chemoattracts mature dendritic cells (32
) and naive T cells (33
); and CD40LT, a known inducer of dendritic cell maturation (34
) (data not shown). Coadministration of all 3 of these effector molecules failed to improve the efficacy of the vaccine. This indicates that the CTL response induced against tumor-stromal fibroblasts is sufficient to inhibit the growth of different tumors, but not adequate to reject them as a single entity. This may be due to the fact that tumor cells and tumor-associated macrophages can elicit redundancy by synthesizing a variety of tumor-promoting proteins, such as VEGF (35
), and IL-10 (38
), the serum concentrations of which we were unable to lower with our vaccine (data not shown).
The rationale for combining cancer vaccines with chemotherapy has been extensively documented and reviewed elsewhere (40
). In addition to the immunostimulatory potential of various chemotherapeutics, such as doxorubicin, paclitaxel, and cyclophosphamide, which do not cause bone marrow suppression when administered at low doses, the combination of our FAP vaccine with such drugs offers an additional advantage, specifically the downregulation of collagen type I expression, and consequently, increased intratumoral uptake of different chemotherapeutic agents. We demonstrated this with 3 structurally unrelated molecules: the low-molecular-weight dye fluorescein (376 Da), the high-molecular-weight compound Evans blue albumin (68,500 Da), and the chemotherapeutic agent 5-fluorouracil (130 Da). Seemingly, the changes that occur in the pFap-vaccinated animals facilitate both processes responsible for intratumoral transport, i.e., diffusion for small molecules as well as convection for larger molecules (41
). These findings support a novel rationale for improving chemotherapy by combining it with immunotherapy. Indeed, we demonstrated that the combination of a DNA vaccine encoding FAP plus doxorubicin can induce complete rejection of orthotopically grown breast tumors in 50% of mice as well as a more than 3-fold prolongation of the lifespan of mice with experimentally induced breast cancer metastases, a result that was not achieved when either of these treatments was applied alone. Furthermore, we demonstrated that the amount of doxorubicin in the tumor tissue of pFap-vaccinated mice is significantly increased when compared with that in control mice vaccinated with empty vector.
Taken together, our findings demonstrate, for the first time to our knowledge, the efficacy of an oral DNA vaccine in eliciting an immune response mediated by CD8+ T cells against the tumor-stromal antigen FAP. More importantly, our DNA-based vaccine against FAP, when combined with doxorubicin, was able to inhibit tumor growth and lead to complete tumor rejection in 50% of mice. In addition we observed a more than 3-fold prolonged lifespan in a breast cancer model of experimental metastases, which was at least partly due to increased drug uptake at the tumor site. It is anticipated that this and other studies that involve combinations of immuno- and chemotherapies may soon lead to new approaches in clinical practice and improvements in currently available treatments for cancer.