Several vaccines designed to elicit antigen-specific immune responses are currently being evaluated in clinical trials as specific, targeted treatments for patients with prostate cancer. Because the prostate is an expendable organ, most immunotherapy studies have targeted tissue-specific antigens. For example, vaccines targeting prostate tumor-associated antigens such as prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been shown to elicit antigen-specific immune responses in patients with prostate cancer [5
]. However, PSA and PAP are not tumor-specific proteins; the identification of tumor-specific targets remains important to the development of immunotherapeutic treatments for prostate cancer, as well as other types of malignancy. Recently, Cheever et. al
. reported that SSX-2 and other sarcoma translocation breakpoint antigens may be higher priority targets for cancer therapy than PSA or PAP based upon certain pre-defined criteria for the prioritization of tumor antigens, such as specificity, oncogenicity, expression in multiple tumor types or advanced disease, and number of identified epitopes [36
]. Since the majority of prostate cancer antigens targeted therapeutically to date have been tissue-specific and not necessarily tumor-specific, and because the evaluation of targets associated with more advanced disease may have relevance to prostate cancer, we sought to further characterize the immunogenicity of SSX-2. In addition, because this cancer-testis antigen is not normally expressed in MHC class I-expressing cells, unlike the tissue-restricted antigens currently being investigated for prostate cancer, we reasoned that it might be possible to evaluate SSX-2 in the future as a prophylactic cancer vaccine antigen, ideally permitting immunization in a setting prior to expression of the antigen in tumor cells and thereby potentially avoiding immune tolerance.
In the present study we used a genetic vaccine to identify and compare SSX-2 HLA-A2-restricted epitopes relevant to prostate cancer. Specifically, we used peptide prediction algorithms and HLA-A2 affinity assays to identify and prioritize potentially immunogenic peptides, followed by in vivo vaccination with SSX-2 peptides or a DNA plasmid encoding SSX-2 to evaluate the immunogenicity of predicted peptides and their ability to elicit anti-tumor CTL. With this approach we identified four SSX-2 peptides that have significant affinity for the HLA-A2 complex in vitro. We subsequently carried out peptide vaccination studies in A2/DR1 mice and found that p41-49 and p103-111 could elicit peptide-specific immune responses in this transgenic model. Using a genetic vaccine encoding SSX-2 we found that significant responses to p41-49 and p103-111 were elicited, demonstrating that both peptides are HLA-A2-restricted epitopes. However, peptide p103-111 appeared to be clearly dominant with robust responses elicited by either direct peptide or DNA vaccination. p103-111 immunization elicited CTL in animals that could lyse both p103-111 peptide-pulsed target cells and LNCaP prostate cancer cells. The dominance of the p103-111 epitope was found to not be due to a putative MHC class II epitope encompassing it. Finally, we also found that HLA-A2+ patients with prostate cancer can have p41-49 and p103-111-specific T cells in their peripheral blood, demonstrating that these cells are within the repertoire of patients with advanced disease in particular.
Several other investigators have also sought to identify MHC class I-restricted epitopes specific for SSX-2. Ayyoub et. al
. previously evaluated SSX-2 peptides by incubating an overlapping library of SSX-2 peptides with standard proteasome complexes in vitro
]. They demonstrated that peptide p41-49 is an HLA-A2-restricted epitope recognized by CTL from melanoma patients [25
]. Peptide p41-49 was also shown to be an epitope presented in hepatocellular carcinoma, and p41-49-specific CTL were found to lyse melanoma cells and the sarcoma cell line SW 872 [25
]. Wagner et. al
. independently demonstrated that SSX-2 peptide p103-111 is an HLA-A2-restricted epitope recognized by CTL from breast cancer patients and presented by melanoma cell lines [21
]. Held et al
. further demonstrated that this epitope is directly presented on melanoma cells using peptide/HLA-A2-specific Fab antibodies [40
]. He et al
. suggested that SSX-2 p57-65 was also an HLA-A2-restricted epitope by demonstrating that peptide-specific CTL cultured from healthy donors are capable of lysing peptide-pulsed T2 cells [41
], however, they did not demonstrate that this peptide is presented as an MHC class I-restricted epitope by tumor cells. Together, these prior studies highlighted these three SSX-2 peptides as possible HLA-A2-restricted epitopes. Prior to our study, a comparison of HLA-A2-restricted epitopes had not been carried out, and it had not been determined whether SSX-2 vaccines can elicit epitope-specific CTL to these peptides. Our studies confirm the work of others that p41-49 and p103-111 are HLA-A2-restricted epitopes. However, while p57-65 can bind HLA-A2, it does not appear to be a presented HLA-A2-restricted epitope, and it does not appear to be immunogenic in our transgenic model.
We found that in vivo immunization of transgenic mice with peptide p103-111 or a DNA vaccine encoding SSX-2 generated robust p103-111-specific immune responses that could be detected directly ex vivo. Peptide p41-49-specific immune responses generated by peptide or genetic immunization were only rarely detected unless the splenocytes were expanded by in vitro peptide stimulation (). These results were not expected from our in vitro analysis, which showed p41-49 to have the greatest affinity for HLA-A2. It could be that p41-49 is not efficiently processed by the murine proteasome. Interestingly, the Ssxa1 murine peptide corresponding to human peptide p41-49 shares the greatest homology with the human peptide, while the region corresponding to p103-111 is absent in the mouse protein sequence (). Consequently, it is possible that there is some tolerance to the p41-49 epitope in the mouse that contributed to the reduced immunogenicity observed. Peptide p41-49-specific CTL from DNA-immunized mice were able to lyse p41-49-pulsed target cells, suggesting that p41-49 is an HLA-A2-restricted epitope, yet in our studies p41-49-specific CTL failed to lyse SSX-2+ LNCaP cells (). These results were also unexpected but may indicate that LNCaP cells either do not endogenously process and present this peptide, present low levels of this peptide, or perhaps more likely, p41-49-specific CTL generated from vaccination are too low in frequency or avidity to detect this epitope on LNCaP cells. In any case, our results suggest that p103-111 is the dominant HLA-A2-restricted SSX-2 epitope, at least in this transgenic mouse model.
We found that genetic vaccination offered several advantages over direct peptide immunization for epitope identification, including the induction of stronger immune responses and simultaneous recognition of multiple epitopes. Immunization of mice with pTVG-SSX-2 elicited p103-111-specific immune responses in every immunized animal and with higher frequency than p41-49-specific cells as determined by both IFNγ ELISPOT and tetramer staining of splenocytes directly ex vivo. Interestingly, the best in vitro lysis of LNCaP cells and peptide-pulsed T2 cells () was found by splenocytes from an animal that had a robust immune response to both peptides p41-49 and p103-111 by direct ex vivo IFNγ ELISPOT assay (data not shown). This finding may indicate that superior lysis of prostate tumor cells can be achieved by eliciting CTL specific for multiple SSX-2 epitopes. A possible future direction may be to develop the SSX-2 DNA vaccine as an immunotherapeutic tool by optimizing its ability to elicit responses to multiple epitopes simultaneously.
In addition to identifying SSX-2 peptide p103-111 as an immunodominant epitope, we were able to detect T cells specific to this peptide in the peripheral blood of patients with prostate cancer using tetramer staining, with particularly high frequencies of T cells specific to either peptide p41-49 or p103-111 in patients with advanced disease. In concordance with these tetramer results we found that one of seven patients with early-stage prostate cancer had CTL that could specifically lyse p103-111-pulsed target cells, while three of four patients with advanced-stage disease had higher frequencies of tetramer positive CTL that could lyse these target cells or the LNCaP prostate cancer cell line after fewer rounds of in vitro
peptide stimulation. These findings suggest that advanced prostate tumors might have greater SSX-2 expression than earlier stage disease, as has been suggested by findings of increased SSX-2 mRNA expression in metastatic prostate cancer and other tumor types [18
], which could lead to increased cross-presentation of SSX-2 and generation of SSX-2-specific CD8+ T cells. If true, this might permit an opportunity to consider immunization early in the course of disease, more analogous to the prophylactic setting, to ideally prevent the growth of SSX-2-expressing tumors. However, to date the expression and function of SSX-2 in prostate tumors of various stages remains largely unknown. These will be areas of future research.
In conclusion, this work provides direct evidence that SSX-2 is a relevant vaccine target antigen for prostate cancer. In particular these findings highlight the importance of SSX-2 peptide p103-111 as a dominant HLA-A2-restricted immunogenic epitope. To date the majority of the work evaluating SSX-2 has focused on SSX-2 peptide p41-49 as an HLA-A2-restricted epitope in patients with different types of malignancy. Future studies will explore the expression and function of SSX-2, and other SSX family members, in prostate tumors. Other studies will explore modified DNA vaccines as a means to enhance peptide-specific immune responses to this antigen and will assess strategies to augment the anti-tumor potential of these SSX-2-specific vaccines in vivo, such as using these vaccines in combination with epigenetic modifying agents that might increase SSX-2 expression in tumor cells (18).