Prostatic acid phosphatase has emerged as a target antigen for prostate cancer vaccines, in particular given clinical trials suggesting clinical benefit in patients with prostate cancer treated with vaccines targeting PAP by means of an antigen-presenting cell vaccine [10
]. We report here the identification of three HLA-A2 epitopes derived from the amino acid sequence of prostatic acid phosphatase, p18–26, p112–120, and p299–307. These epitopes were identified by first searching for pre-existing T cells specific for these peptides among HLA-A2-expressing patients with prostate cancer and normal male blood donors, and then characterizing T-cell lines and clones specific for these epitopes. Of these epitopes, p299–307 and p112–120 have been previously identified by others [16
], and p18–26 had not. Direct immunization of HHD-II mice with a DNA vaccine encoding PAP elicited responses to several peptides, suggesting there may not be one immunodominant HLA-A2 epitope. There is currently much interest in the identification of MHC class I-restricted epitopes from tumor antigens. Such epitopes might themselves be useful as peptide antigens for vaccines or adoptive immunotherapies [30
]. Alternatively, the identification of MHC-restricted peptides may be useful to develop immunological monitoring tools to track the development of CD8+ T-cell immune responses resulting from immunization by ELISPOT or multimer-type methodologies. This is particularly important for PAP, given that it is currently being targeted in antigen-presenting cell vaccines [14
], and in earlier clinical trials using DNA vaccines [13
]. The ability to detect effector CD8+ T cells may be relevant to determine whether patients achieved potentially therapeutic immune responses as a biomarker of response to immunization and/or might be relevant to the evaluation of future “booster” immunization schedules. Our results suggest it may be important to monitor immune responses to more than one single immunodominant epitope, at least in the case of PAP-specific HLA-A2-restricted epitopes, since even in an inbred murine strain responses were elicited to separate epitopes in individual animals.
There have been three previous reports of identification of HLA-A2 epitopes derived from PAP [16
]. In the report of Peshwa and colleagues, the authors identified seven potential HLA-A2-restricted epitopes that conformed to the HLA-A2 binding concensus sequence. The seven epitopes were ranked with respect to HLA-A2 binding affinity in T2 assays, and T-cell lines were established for one of these peptides and tested for their ability to lyse HLA-A2-expressing, PAP-expressing cell lines. Curiously, these authors only report seven potential epitopes, whereas we identified ten potential epitopes using similar methods. An eleventh potential epitope arose from use of the on-line algorithms. Three of the peptides not present in their prior analysis had some of the highest binding in vitro
to HLA-A2. From the T-cell culture studies with the seven identified peptides conducted by that group the authors concluded that p299–307 was an HLA-A2 restricted epitope. Our results confirm these findings, and further identified two additional epitopes, p18–26 and p112–120. In a report by Harada and colleagues, the authors screened for IgG responses to a panel of 14 peptides derived from PAP and identified a high frequency of HLA-A2-expressing prostate cancer patients with IgG specific for p112–120, and then demonstrated that they could elicit p112–120 peptide-specific CTL [17
]. Again, our report confirms these findings, but more specifically demonstrates that a clonal line specific for this epitope can lyse PAP-expressing tumor cells in an HLA-A2-restricted fashion. In a report by Machlenkin and colleagues, HHD mice were immunized with peptides derived from PAP, with the identification of p135–143 [18
]. The authors demonstrated that T-cell lines stimulated with this peptide could lyse LNCaP prostate cancer cells [18
]. We cannot explain this discrepancy with our findings -- our results demonstrate that this peptide is not a naturally processed epitope specific for PAP, but rather may potentially be recognized by naïve T cells to which a response was detectable in patient samples. Moreover, this peptide may be particularly immunogenic in the HHD mouse in which we were able to detect responses to this peptide in control immunized mice as well as mice immunized with a DNA vaccine encoding PAP. We believe our data are more definitive, as our conclusions are based on a more controlled animal study and from a clonal T-cell line specific for p135–143, as well as two other peptide-specific clonal lines from other individuals (data not shown), that do not demonstrate lysis of PAP-expressing HLA-A2+
Our data also suggest the importance of evaluating CTL activity to several different antigen-expressing cell lines. The LNCaP cell line, while used by many groups as a model HLA-A2-expressing prostate cancer cell line, expresses very low levels of HLA-A2 [32
]. In our hands, we were unable to detect significant HLA-A2 expression on LNCaP cells even with IFNγ pretreatment (data not shown). For this reason, we used LNCaP cells transfected to express HLA-A2, but with native levels of PAP expression. Conversely, we also demonstrated that cells with native levels of HLA-A2, but transduced to express PAP, were also lysed by PAP-specific CTL. The downregulation of MHC class I in advanced prostate cancer has been previously described in human as well as mouse prostate cancer cells [33
]. These observations suggest that future studies could explore means to increase prostate tumor cell expression of MHC class I as a means to potentially increase the efficacy of CD8+ T cell targeted therapies, particularly in advanced disease where this might be most relevant.
There are many reports identifying peptide epitopes for individual tumor antigens. In the typical application, peptides are first identified based on sequence, and then tested in in vitro culture for the ability to generate peptide-specific and tumor-specific CTL, similar to what we have shown. However, with this approach there is little prioritization, and for a large tumor antigen there could be many potential MHC-restricted epitopes. Ultimately, if the goal of such an approach is to identify epitopes that could elicit an immune response in patients, then there should ideally be some evidence that such T cells specific for these epitopes exist in vivo. That was our rationale for first prioritizing peptides based on whether there were peptide-specific responses detectable in the peripheral blood of patients with prostate cancer. Using ELISPOT, we were able to prioritize three of the eleven peptides identified. While ELISPOT has been used as a robust means of identifying peptide-specific effector cells, our results suggest that it can also identify non-specific immunogenic peptides, as in the case of p135–143. However, the ability to prioritize peptides and culture peptide-specific CTL lines from multiple HLA-A2-expressing patients with prostate cancer permitted the identification of PAP-specific epitopes against which CTL could be expanded from the peripheral blood of patients with prostate cancer. The use of ELISPOT, using peripheral blood mononuclear cells from MHC-restricted individuals, could serve as a general method to prioritize peptides for epitope identification for other tumor antigens.
The finding that individual mice from an inbred transgenic strain had responses to different epitopes suggests to us that there is likely not a single dominant PAP-specific HLA-A2 epitope, and underscores the importance of evaluating responses to multiple epitopes following PAP-specific immunization. The evaluation of PAP-specific CD8+ responses may be particularly relevant given the recent demonstrations that patients with advanced prostate cancer receiving the sipuleucel-T vaccine (Dendreon Corporation, Seattle, WA) have an improved survival compared to placebo-treated patients, and FDA approval is being sought for this vaccine [15
]. Because the sipuleucel-T vaccine targets PAP, and presumably works in part by augmenting PAP-specific CD8+ effector cells, it may be important to develop CD8+ monitoring techniques to identify immunological responders that might have benefited from this treatment and/or to identify optimal schedules of immunization, including whether subsequent immunizations should be considered in individuals with waning immunity. We chose this particular immunization strategy in HHD-II mice with a DNA vaccine to parallel human studies using this same DNA vaccine encoding PAP [13
]. We plan to use these epitopes as a means of evaluating PAP-specific CD8+ T cells following DNA immunization of patients, as well as use this murine model to define more robust immunization schedules, including heterologous immunization strategies, targeting PAP.