The identification of peptide-specific CD8+ T cells that can recognize and lyse tumor cells is an important component in the development and prioritization of cancer vaccine target antigens. In this report, we have identified pre-existing CD8+ T-cell responses that recognize HLA-A2-restricted peptides derived from the AR LBD using both in vitro and in vivo models. These peptides were found to have varying affinity for HLA-A2, and T cells specific for all of these peptides could be detected in at least one of the HLA-A2+ prostate cancer patients sampled. In particular, two peptide-specific CD8+ T-cell populations (specific for either AR805 or AR811) were found in over half (8/15) of the patients sampled. However, while polyclonal T-cell cultures specific for three peptides (AR761, AR805, and AR811) showed some ability to lyse prostate cancer cells, only AR805 and AR811 peptide-specific CD8+ T-cell clones could recognize and lyse these tumor cells. In addition to the work characterizing these peptide-specific immune responses in vitro, we also utilized an HLA-A2 transgenic mouse to study these responses in vivo. Using the A2/DR1 mouse strain, we found that AR811 HLA-A2-restricted peptide-specific T-cells could be identified in vivo and could lyse HLA-A2-expressing cells expressing the AR, thus further confirming that AR811 is an HLA-A2-restricted epitope, whereas AR805 appears non-immunogenic in this model. In addition, these murine studies demonstrated that AR LBD-specific CTL could be augmented through the use of an AR LBD-based vaccine. These data suggest that the AR LBD is a relevant tumor antigen for prostate cancer, and is unique among commonly targeted prostate antigens in that it is not a secreted or membrane-bound protein, and has a critical function to the survival and proliferation of tumor cells. The frequency of pre-existing immune responses to the AR LBD shows that it is also an immunogenic antigen, and that AR LBD-specific T cells are not deleted during the development of immunological tolerance. The frequency of these responses, as well as the results from the peptide-immunization studies in A2/DR1 mice (a model in which the AR LBD is a self-antigen), also suggests that it may be possible to augment these peptide-specific CTL in vivo through the use of AR LBD-based therapeutics.
The identification of the AR LBD as an immunological target for the treatment of prostate cancer represents a new way of prioritizing potential target antigens for this disease. As the AR LBD has an important molecular function to the survival and proliferation of prostate tumor cells (in both androgen-dependent and castrate-resistant disease settings), it is unique when compared to commonly-targeted antigens such as PAP, PSA, and PSMA, which, though tissue-specific, do not have molecular functions that likely contribute significantly to the oncogenicity of the tumor. This may lead to the outgrowth of tumor escape variants that lack expression of these antigens, whereas tumors that attempt to evade AR LBD-directed therapies by downregulating AR expression could potentially be at a growth disadvantage [21
]. In addition to its crucial oncogenic activity, the AR also benefits from being an intracellular antigen rather than a secreted protein such as PAP or PSA, allowing epitopes derived from the AR LBD to be directed processed and presented via MHC class I. These qualities, in addition to the AR's well-documented expression in all androgen-dependent and most castrate-resistant tumors, sets the AR LBD apart from other prostate cancer antigens under development, and suggests that the AR LBD may be an attractive target for active immunization strategies.
In addition to these qualities, the results from the current study have confirmed previous reports showing that the AR LBD is a highly immunogenic antigen. We have previously reported that at least 17% of patients with prostate cancer have AR LBD-specific antibody responses, a higher frequency of responses than those found against other prostate cancer antigens under active development, such as PSA (5.5% of patients) and PAP (11% of patients), even though these are secreted antigens and as such might be predicted to elicit a predominantly humoral immune response [37
]. Similarly, we found that a high percentage of prostate cancer patients have HLA-A2-specific CTL specific for AR LBD-derived epitopes: in fact, 11/15 (73%) patients sampled had AR805 or AR811 epitope-specific CTL that could be cultured in vitro
, slightly higher than the frequency of PAP-derived HLA-A2-restricted CD8+ T cells we previously identified (9/15, 60%) [32
]. An AR811-bound pentamer (Proimmune, LTD) was synthesized in an attempt to characterize the frequency of AR811 peptide-specific CTL directly ex vivo
; however, a lack of HLA-A2-specificity of this pentamer precluded this analysis. However, the high frequency of peptide-specific CTL detected in the in vitro
culture system suggests that AR LBD-derived T cells are not deleted by the immune system, both in HLA-A2+ prostate cancer patients as well as the A2/DR1 mouse, and are in fact relatively frequent in these patient populations.
As well as detecting AR LBD peptide-specific CTL that can be cultured from patients with prostate cancer, this work has also identified specific antigenic epitopes derived from the AR LBD that are naturally presented on the surface of tumor cells. In particular, we have identified that AR811 appears to be a relatively immunodominant epitope, with AR811 peptide-specific T cells found in a high percentage of prostate cancer patients as well as having the ability to lyse prostate cancer cells in an AR- and HLA-A2-restricted fashion. AR805 was also found to be an HLA-A2-restricted epitope, with AR805 peptide-specific CTL being commonly found in prostate cancer patients and being able to lyse prostate cancer cells (albeit at lower levels than AR811-specific T cells). Intriguingly, these two epitopes, which share three residues of their primary amino acid sequence, are also in the context of several 15-mer peptides (AR802-816, AR805-819, and AR807-821) that are predicted to have strong affinity for HLA-DR1 [38
]. While the importance of CD4+ helper T cells in amplifying peptide-specific CD8+ T-cell responses has long been recognized, the contribution of these hypothesized AR LBD-specific helper T-cell responses in amplifying class I-restricted responses (or even the existence of AR LBD-specific HLA-DR-restricted epitopes) remains a topic of investigation [39
]. Indeed, while our group has previously identified AR LBD-specific CD4+ T cells, we have yet to evaluate the peptide-specificity of these responses and how they may contribute to the generation of CD8+ T-cell responses [30
In the A2/DR1 mouse model, AR811 also appears to be an immunodominant epitope, with all six animals immunized with AR811 developing significant peptide-specific immune responses that could also lyse a human prostate cancer cell line. This is contrasted with the results obtained in the direct immunization studies with AR805, which was found to be non-immunogenic in the A2/DR1 mouse, with only one animal generating a low-level IFNγ ELISPOT response. This suggests that there could be immunological tolerance to this peptide in the A2/DR1 mouse, or that the 9-mer peptide itself is simply not very immunogenic in this model.
We identified that an AR811 CD8+ T cell clone had significant levels of both IFNγ and TNFα cytokine expression. This may contribute to the apparent immunodominance of AR811-specific T cells, as research in a variety of diseases has shown that polyfunctional T cells that express multiple cytokines have higher levels of effector functions and tend to correlate with improved clinical responses [40
]. In fact, we found that two different AR811 CD8+ T-cell clones from separate individuals (data not shown) similarly expressed high levels of both IFNγ and TNFα, whereas AR761 and AR805 T-cell clones expressed predominantly TNFα alone. Additional studies would be beneficial to evaluate the expression of a larger panel of cytokines to determine if these different types of peptide-specific T cells have different overall cytokine profiles and if these profiles correspond with different cytolytic activity. However, the co-expression of two Th1-type cytokines by AR811 CD8+ T-cell clones may be associated with the generation of high avidity AR811 peptide-specific T-cells, and may contribute to the observed higher levels of prostate cancer cell lysis.
While the affinity between peptides and MHC class I has been shown to have an important role in the development of productive CD8+ T-cell responses, the results from our studies have highlighted the importance of actually culturing peptide-specific T cells to conclusively identify naturally processed and presented CD8+ T-cell epitopes. For example, in the process of identifying AR LBD-derived peptides, we found that the AR700 peptide had strong affinity for HLA-A2. However, when we examined prostate cancer patients for the presence of AR700 peptide-specific T cells, we found that only three of the patients sampled had these responses, that AR700 peptide-specific T cells could not lyse prostate cancer cells, and that these responses were not amplified in A2/DR1 mice immunized with AR700 (data not shown). These results agree with previous findings showing that peptide:MHC affinity is necessary, but not sufficient, for a MHC-restricted peptide to be a naturally processed and presented epitope [45
]. In our studies, we found that the three peptides with the weakest HLA-A2 affinity (AR708, AR742, and AR814) were found to be recognized by T cells in the lowest numbers of patients, whereas two of the peptides with the highest affinity for HLA-A2 (AR805 and AR811) were found to be recognized by T cells in more than half of the patients sampled. However, this affinity does not necessarily correlate with whether these peptides are epitopes – of the four peptides with very strong in vitro
affinity for HLA-A2 (AR700, AR761, AR805, and AR811), only AR805 and AR811 were found to be able to lyse AR-expressing cells in an MHC-restricted fashion.
The utilization of A2/DR1 mice to confirm AR811 as a naturally processed and presented prostate cancer epitope also served to establish a potentially ideal model to evaluate AR LBD-based immunotherapies. As mentioned previously, the amino acid sequence of the AR LBD is identical between several species, including mice and humans. When combined with reliance on HLA-A2 for the presentation of MHC class I-derived peptides, the A2/DR1 model allows for the detailed study of the same HLA-A2-restricted immune responses that were characterized in human patients in vitro, and how these responses can be augmented following AR LBD-based immunotherapy. Furthermore, the sequence identity of the AR LBD between these two species could permit the evaluation of the natural generation of AR LBD-specific immune responses, which is not possible for most prostate cancer antigens under active development in which the target antigen is foreign in murine models. This allows the A2/DR1 mouse to serve as a directly relevant model to further study the immunological efficacy of vaccines targeting the AR LBD.