Heath A. Smith

The SSX Family of Cancer-Testis Antigens as Target Proteins for Tumor Therapy

Cancer-testis antigens (CTAs) represent an expanding class of tumor-associated proteins defined on the basis of their tissue-restricted expression to testis or ovary germline cells and frequent ectopic expression in tumor tissue. The expression of CTA in MHC class I-deficient germline cells makes these proteins particularly attractive as immunotherapeutic targets because they serve as essentially tumor-specific antigens for MHC class I-restricted CD8+ T cells. Moreover, because CTAs are expressed in many types of cancer, any therapeutic developed to target these antigens might have efficacy for multiple cancer types. Of particular interest among CTAs is the synovial sarcoma X chromosome breakpoint (SSX) family of proteins, which includes ten highly homologous family members. Expression of SSX proteins in tumor tissues has been associated with advanced stages of disease and worse patient prognosis. Additionally, both humoral and cell-mediated immune responses to SSX proteins have been demonstrated in patients with tumors of varying histological origin, which indicates that natural immune responses can be spontaneously generated to these antigens in cancer patients. The current review will describe the history and identification of this family of proteins, as well as what is known of their function, expression in normal and malignant tissues, and immunogenicity.

PD-1 or PD-L1 Blockade Restores Antitumor Efficacy Following SSX2 Epitope–Modified DNA Vaccine Immunization

Rekoske and colleagues show that an SSX2 DNA vaccine, optimized for MHC class I binding epitopes, led to increased PD-1 expression on antigen-specific CD8+ T cells and inferior antitumor response in mice bearing tumors expressing SSX2, which was reversed when combined with PD-1/PD-L1 blockade. DNA vaccines have demonstrated antitumor efficacy in multiple preclinical models, but low immunogenicity has been observed in several human clinical trials. This has led to many approaches seeking to improve the immunogenicity of DNA vaccines. We previously reported that a DNA vaccine encoding the cancer–testis antigen SSX2, modified to encode altered epitopes with increased MHC class I affinity, elicited a greater frequency of cytolytic, multifunctional CD8+ T cells in non–tumor-bearing mice. We sought to test whether this optimized vaccine resulted in increased antitumor activity in mice bearing an HLA-A2–expressing tumor engineered to express SSX2. We found that immunization of tumor-bearing mice with the optimized vaccine elicited a surprisingly inferior antitumor effect relative to the native vaccine. Both native and optimized vaccines led to increased expression of PD-L1 on tumor cells, but antigen-specific CD8+ T cells from mice immunized with the optimized construct expressed higher PD-1. Splenocytes from immunized animals induced PD-L1 expression on tumor cells in vitro. Antitumor activity of the optimized vaccine could be increased when combined with antibodies blocking PD-1 or PD-L1, or by targeting a tumor line not expressing PD-L1. These findings suggest that vaccines aimed at eliciting effector CD8+ T cells, and DNA vaccines in particular, might best be combined with PD-1 pathway inhibitors in clinical trials. This strategy may be particularly advantageous for vaccines targeting prostate cancer, a disease for which antitumor vaccines have demonstrated clinical benefit and yet PD-1 pathway inhibitors alone have shown little efficacy to date. Cancer Immunol Res; 3(8); 946–55. ©2015 AACR.

Vaccines targeting the cancer-testis antigen SSX-2 elicit HLA-A2 epitope-specific cytolytic T cells.

The cancer-testis antigen synovial sarcoma X breakpoint-2 (SSX-2) is a potentially attractive target for tumor immunotherapy based upon its tissue-restricted expression to germline cells and its frequent expression in malignancies. The goal of this study was to evaluate genetic vaccine encoding SSX-2 to prioritize human leukocyte antigen (HLA)-A2-specific epitopes and determine if a DNA vaccine can elicit SSX-2-specific cytotoxic T lymphocytes (CTLs) capable of lysing prostate cancer cells. HLA-A2-restricted epitopes were identified based on their in vitro binding affinity for HLA-A2 and by the ability of a genetic vaccine to elicit peptide-specific CTL in A2/DR1 (HLA-A2.1+/HLA-DR1+/H-2 class I−/class II-knockout) transgenic mice. We found that SSX-2 peptides p41-49 (KASEKIFYV) and p103-111 (RLQGISPKI) had high affinity for HLA-A2 and were immunogenic in vivo; however, peptide p103-111 was immunodominant with robust peptide-specific immune responses elicited in mice vaccinated with a plasmid DNA vaccine encoding SSX-2. Furthermore, p103-111-specific CTLs were able to lyse an HLA-A2+ prostate cancer cell line. The immunodominance of this epitope was found not to be due to a putative HLA-DR1 epitope (p98-112) flanking p103-111. Finally, we demonstrated that SSX-2 epitope-specific CTLs could be detected and cultured from the peripheral blood of HLA-A2+ prostate cancer patients, notably patients with advanced prostate cancer. Overall, we conclude that SSX-2 peptide p103-111 is an immunodominant HLA-A2-restricted epitope, and epitope-specific CD8+ T cells can be detected in patients with prostate cancer, suggesting that tolerance to SSX-2 can be circumvented in vivo. Together, these findings suggest that SSX-2 may be a relevant target antigen for prostate cancer vaccine approaches.

IgG Responses to Tissue-Associated Antigens as Biomarkers of Immunological Treatment Efficacy

We previously demonstrated that IgG responses to a panel of 126 prostate tissue-associated antigens are common in patients with prostate cancer. In the current report we questioned whether changes in IgG responses to this panel might be used as a measure of immune response, and potentially antigen spread, following prostate cancer-directed immune-active therapies. Sera were obtained from prostate cancer patients prior to and three months following treatment with androgen deprivation therapy (n = 34), a poxviral vaccine (n = 31), and a DNA vaccine (n = 21). Changes in IgG responses to individual antigens were identified by phage immunoblot. Patterns of IgG recognition following three months of treatment were evaluated using a machine-learned Bayesian Belief Network (ML-BBN). We found that different antigens were recognized following androgen deprivation compared with vaccine therapies. While the number of clinical responders was low in the vaccine-treated populations, we demonstrate that ML-BBN can be used to develop potentially predictive models.

Lenalidomide Modulates IL-8 and Anti-Prostate Antibody Levels in Men with Biochemically Recurrent Prostate Cancer

We retrospectively explored changes in immunological parameters in men with biochemically recurrent prostate cancer treated with either 5 or 25 mg of lenalidomide in a randomized phase 2 trial, and determined whether those changes correlated with disease progression.

DNA vaccines encoding altered peptide ligands for SSX2 enhance epitope-specific CD8+ T-cell immune responses

Plasmid DNA serves as a simple and easily modifiable form of antigen delivery for vaccines. The USDA approval of DNA vaccines for several non-human diseases underscores the potential of this type of antigen delivery method as a cost-effective approach for the treatment or prevention of human diseases, including cancer. However, while DNA vaccines have demonstrated safety and immunological effect in early phase clinical trials, they have not consistently elicited robust anti-tumor responses. Hence many recent efforts have sought to increase the immunological efficacy of DNA vaccines, and we have specifically evaluated several target antigens encoded by DNA vaccine as treatments for human prostate cancer. In particular, we have focused on SSX2 as one potential target antigen, given its frequent expression in metastatic prostate cancer. We have previously identified two peptides, p41-49 and p103-111, as HLA-A2-restricted SSX2-specific epitopes. In the present study we sought to determine whether the efficacy of a DNA vaccine could be enhanced by an altered peptide ligand (APL) strategy wherein modifications were made to anchor residues of these epitopes to enhance or ablate their binding to HLA-A2. A DNA vaccine encoding APL modified to increase epitope binding elicited robust peptide-specific CD8+ T cells producing Th1 cytokines specific for each epitope. Ablation of one epitope in a DNA vaccine did not enhance immune responses to the other epitope. These results demonstrate that APL encoded by a DNA vaccine can be used to elicit increased numbers of antigen-specific T cells specific for multiple epitopes simultaneously, and suggest this could be a general approach to improve the immunogenicity of DNA vaccines encoding tumor antigens.

Abstract A66: Expression and immunotherapeutic targeting of the SSX family of cancer-testis antigens in prostate cancer

Members of the SSX family of proteins were among the first cancer-testis antigens (CTA) identified. Characterized by tissue-restricted expression to MHC class l-deficient germline cells and frequent ectopic expression in tumors of various histological origin, CTA are highly tumor-specific antigens. SSX proteins comprise a family of ten members with mRNA expression often found in advanced-stage disease and associated with worse patient prognosis. We have previously shown that humoral and cell-mediated immune responses to SSX2 can arise spontaneously in patients with prostate cancer, which combined with the expression of these proteins in MHC class l-negative tissue, makes SSX proteins potentially attractive targets for tumor immunotherapy. In the current study, we evaluated SSX family member expression in prostate cancer cell lines and tumor biopsies to identify therapeutic targets for vaccine therapy in prostate cancer. We found by RT-PCR and qRT-PCR that SSX1, SSX2, and SSX5 were frequently expressed in prostate cancer cell lines and could be induced by treatment with epigenetic modifying agents (EMA) such as 5-aza-2′-deoxycytidine. Furthermore, we observed differential SSX protein expression in paraffin-embedded prostate cancer biopsies on tissue microarray by immunohistochemical staining. Interestingly, SSX expression in patient samples was restricted to metastatic prostate cancer lesions (5/22; 23%), while expression in primary prostate tumors was not observed (0/73). Finally, we determined that it is possible to elicit cross-reactive immune responses to a dominant SSX epitope p103–111 shared by the different SSX family members by immunization of A2/DR1 transgenic mice with SSX peptides or a plasmid DNA vaccine encoding SSX2. Overall, these results suggest that multiple SSX family members are expressed in prostate cancer and can be simultaneously targeted using immunotherapeutic vaccines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A66.

Abstract 2397: SSX-2 vaccines with enhanced MHC class I binding epitopes elicit anti-prostate tumor CTL

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The tumor-associated antigen synovial sarcoma X breakpoint 2 (SSX-2) is classified as a cancer-testis antigen (CTA) based upon its tissue-restricted expression to germline cells and its frequent expression in a variety of malignancies. We have previously identified SSX-2 as a potential prostate cancer antigen that can be inducibly expressed in several prostate cancer cell lines. We have also previously identified two nonamer peptides from the amino acid sequence of SSX-2 that have significant affinity for HLA-A2 as assessed by in vitro T2 binding assay. These HLA-2-binding peptides were found to represent HLA-A2-restricted epitopes, able to induce peptide-specific immune responses in peptide- or DNA-immunized HHD-II (HLA-A2.1+/HLA-DR1+ transgenic) mice. Given these observations, the current study was conducted to determine if CTL generated in vaccinated HHD-II mice are capable of lysing HLA-A2+ prostate cancer cells and whether SSX-2 vaccine efficacy could be enhanced using an altered peptide ligand binding strategy. We found that peptides that were designed in silico to have increased or decreased HLA-A2+ affinity had enhanced and reduced HLA-A2 binding by in vitro T2 binding assay, respectively. Additionally, HHD-II mice immunized with modified SSX-2 vaccines developed increased or decreased frequencies of peptide-specific T cells in vivo, which could be detected by IFNγ ELISPOT assay and cytotoxicity assay. Animals immunized with enhancing modified SSX-2 vaccines generated CTL with cross-reactivity to the native SSX-2 epitopes. These CTL were capable of lysing target cells pulsed with both modified and native peptides and could also lyse an HLA-A2-expressing prostate cancer cell line. These results provide support for the further investigation of SSX-2 as an immunotherapeutic target for prostate cancer using vaccines modified to increase MHC class I binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2397.