IL-13Rα2 is overexpressed on certain types of human tumor tissues [15
]. We now provide evidence that IL-13Rα2 is highly expressed in a variety of murine tumor cell lines (Additional file 1
, Figure S1). Although the significance of expression of IL-13Rα2 in cancer is not completely clear, our previous studies indicate that IL-13Rα2 could be linked to oncogenesis and metastasis and may provide a potential target for immunotherapy [23
]. We have extended our prior studies and hypothesized that immunization with a DNA vaccine encoding murine IL-13Rα2, boosted with ECDα2 protein, may work more effectively in syngeneic murine tumor models. We studied three murine tumor models, MCA304 sarcoma, 4T1 breast cancer and D5α2 melanoma. Our results indicate that this strategy can produce significant anti tumor effect in these tumor models using both prophylactic and therapeutic vaccinations.
To our knowledge, this is the first report of using ECDα2 protein in DNA prime-protein boost strategy to enhance the efficacy of DNA vaccine. It has been hypothesized that the use of two versions of the same immunogen may activate different subsets of immune cells. It has been shown that DNA immunization is more effective in inducing CD4+
T-cell responses and priming antigen-specific B cells, whereas protein immunization is more effective in stimulating the proliferation of memory B cells into antibody-secreting plasma cells [34
]. In our study, the IL-13Rα2 DNA prime and ECDα2 protein boost activated CD4+
T cell responses and enhanced antibody response against IL-13Rα2. These T and B cell responses induced by prime-boost strategy correlated with tumor responses causing reduced tumor burden and significantly prolonging mice survival, compared with the IL-13Rα2 DNA vaccine alone.
The involvement of systemic immunity in mediating antitumor effects was confirmed by (a) induction of tumor-specific CTL response, (b) IFN-γ secretion by splenocytes, and (c) infiltration of CD4+ and CD8+ T cells in tumors that secreted tumor reactive chemokines. Splenocytes collected from control mice produced minimal level of IFN-γ when they were restimulated with MCA304 or 4T1 tumor cells. These splenocytes also mediated low level of lysis of each target cells as determined by CTL assays. However, each tumor cell-restimulated splenocytes collected from mice receiving the IL-13Rα2 DNA vaccine boosted with ECDα2 produced substantial levels of IFN-γ in the culture supernatant and was capable of mediating specific lysis of each target cells. In contrast, ovalbumin, an irrelevant protein boost did not further induce CTL response, and thus, we conclude that antitumor effects mediated by this vaccination strategy were murine IL-13Rα2 DNA specific.
It is reported that 4T1 breast tumor is highly metastatic and weakly immunogenic [35
]. Huang et al.
showed that parental 4T1 tumor cells expressing only MHC class I molecules are poorly immunogenic, and immunizations of mice bearing 4T1 breast tumor with the irradiated 4T1 cells alone failed to induce the protective antitumor immune responses [37
]. It has also been reported that 4T1 cell line elaborates a variety of immune suppressive molecules including PGE-2, TGF-β and other factors [38
]. These molecules are the reasons that 4T1 tumor is poorly immunogenic to induce antitumor response. Other examples of poorly immunogenic tumors have been described in the literature. Kjaergaard et al
. explained several reasons for poor immunogenicity of B16/D5 mouse melanoma tumors in response to the therapeutic effects of OX-40R mAb [39
]. These authors proposed that it is possible that B16/D5 tumor cells either lack molecules that can serve sufficiently as tumor antigens recognized by T cells or are deficient in the processing, transportation or presentation of such molecules by APCs. It may also be true for the poorly immunogenic 4T1 tumors of eliciting lower T cell responses. Indeed, in our study, overall CTL activity and IFN-γ production in 4T1 tumor model were lower compared to MCA304 tumor model. However, the IL-13Rα2 DNA and ECDα2 boost vaccination could be effective in reducing tumor burdens and induce or amplify a specific CTL response and IFN-γ release against 4T1 tumors compared with the IL-13Rα2 DNA vaccine alone.
It is noteworthy that the IFN-γ-related chemokines CXCL9 and CXCL10 were expressed in tumors derived from mice receiving the IL-13Rα2 DNA vaccine boosted with ECDα2. CXCL9 is known to function as a potent chemoattractant for tumor infiltrating lymphocytes [31
]. In addition, the CXCL10 displays antitumor properties based on the attraction of monocytes and T lymphocytes [40
]. Our results suggest that chemokines are most likely produced by infiltrating immune cells causing antitumor effect because these chemokines act as potent T cell chemoattracants and angiogenesis inhibitors through their interaction with CXCL3 [31
DNA vaccination and IL-13Rα2 protein boost produced anti-IL-13Rα2 antibody in the serum of mice. This antibody may be directly cytotoxic to tumor cells or mediate growth inhibitory signal to target cells after ligating with IL-13Rα2 antigen. We are currently examining the role of antibody in tumor rejection in the current prime boost model. We have previously reported that vaccination of human IL-13Rα2 cDNA alone in D5α2 model generated antibodies, which were modestly cytotoxic to D5α2 tumor cells in vitro [24
Interestingly, mice vaccinated with therapeutic IL-13Rα2 cDNA vaccine and boosted with ECDα2 protein showed lower percentage of Tregs in the spleen and tumor compared to the PBS control in the MCA304 tumor model. This is an interesting finding as Tregs play a prominent role in the inhibition of anti-tumor immunity. It is possible that the inhibitory effects of IL-13Rα2 DNA boosted with ECDα2 protein vaccination on Tregs expansion will play a potentially important role in clinical efficacy during the treatment of immunocompromised patients, such as those with cancer. The enhanced expansion of Tregs has been reported in a number of solid and hematological cancers [41
]. Our results suggest that IL-13Rα2 cDNA boosted with ECDα2 protein vaccination may enhance anti-tumor-immunity by inhibiting the suppressive effects of Tregs.
We did not observe any visible toxicity in mice vaccinated with IL-13Rα2 DNA alone or in combination with ECDα2 protein. No visual changes in animal behavior, mobility, and body weight were observed after vaccination. Histopathological analyses of vital organs (liver, kidney, lung, spleen, heart, and brain) manifested no abnormalities in vaccinated group compared to no treatment group (data not shown). For future clinical trials, we recommend to carefully observe patients by physical exams, serum chemistry, complete blood count and any sign of autoimmunity.
Many immunotherapy approaches, including therapeutic tumor vaccines targeting specific tumor antigens are being developed [1
]. Our current results may be extrapolated to the clinical setting, and it is possible that both CD4+
T cells will be induced against IL-13Rα2 antigen by the DNA vaccine regimen as observed in this animal study. Although the prime-boost vaccine mediated regression of established tumor, complete responses were not observed in any of three tumor models tested. It is possible that the heterogeneous expression of IL-13Rα2 in tumors is responsible for this effect. Alternatively, a most effective dose of vaccine or schedule of vaccination was not optimized. A more immunogenic vector such as vaccinia virus and/or other virus expressing the IL-13Rα2 and/or an IL-13Rα2 peptide vaccine mixed with adjuvants may be needed to generate robust immune responses. These types of preclinical studies will be needed to translate our observations to the clinic for the treatment of patients with cancer.