Fran?ois Lemonnier (25). 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 Antitumor activity of the optimized vaccine could be increased when combined with Tolnaftate 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 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. Keywords: DNA vaccine, prostate cancer, PD-1, PD-L1, SSX2 Introduction Prostate cancer is the most commonly diagnosed cancer in the United States and the second leading cause of cancer-related death in American men (1). Despite primary therapy with prostatectomy and/or radiation therapy, approximately 1/3 of tumors will recur and can ultimately develop into castration-resistant, metastatic disease, the lethal form of prostate cancer (2,3). In 2010 2010, sipuleucel-T (Provenge?, Dendreon Corp.), Tolnaftate an autologous cellular vaccine targeting prostatic acid phosphatase (PAP), was approved by FDA for the treatment of patients with metastatic prostate cancer based on trials demonstrating an improved overall survival following treatment, underscoring the potential for antigen-specific vaccines to impact the clinical care CD69 of patients with advanced prostate cancer (4). Similarly, encouraging results Tolnaftate observed in randomized phase II trials using PROSTVAC? (rilimogene galvacirepvec/glafolivec, Bavarian Nordic), a viral based vaccine targeting prostate-specific antigen (PSA), has renewed interest in the development of other antigen-specific immunotherapies for the treatment of prostate cancer and other malignancies (5). In fact, due to these and many other recent successes in the cancer immunotherapy field, including clinical results observed from T-cell checkpoint molecule blockade (PD-1, CTLA-4, etc.), the journal named cancer immunotherapy as its Breakthrough of the Year for 2013 (6). We have focused on DNA vaccines as an approach for the treatment of patients with recurrent prostate cancer. We have completed clinical trials evaluating the safety and administration schedule of a DNA vaccine encoding PAP, and a randomized phase II trial is currently ongoing (7,8). However, despite being shown to be safe across many phase I clinical trials, and despite demonstrable efficacy as a treatment for diseases in other animals (including dogs, horses, and fish), no other human DNA vaccines for the treatment of cancer have progressed beyond phase I trials (9C12). As such, much effort has been devoted to better understanding of the mechanisms of action of DNA vaccines and exploring methods to enhance their immunogenicity and possible clinical effectiveness. One such method that has been extensively studied is the Tolnaftate encoding of altered peptide ligands (APL) with point mutations in the presented epitopes to enhance their binding affinity for the major histocompatibility complex (MHC) and/or the T-cell receptor (TCR) (13,14). These types of modifications have been shown to increase the immunogenicity of both peptide and DNA vaccines targeting different viral and tumor antigens that were otherwise weakly immunogenic (15C18). One Tolnaftate vaccine encoding an APL currently in clinical trials is usually PROSTVAC?, the vaccinia- and fowlpox-based vaccine encoding PSA described above (19). In preclinical development of this vaccine it was observed that a native HLA-A2-restricted PSA epitope was weakly immunogenic and that its immunogenicity could be enhanced when encoding an APL with enhanced MHC binding affinity (20,21). We have studied synovial sarcoma X breakpoint 2 (SSX2) as a prostate tumor antigen, and have demonstrated that a DNA vaccine encoding SSX2 was able to elicit HLA-A2-restricted CD8+ T cells with cytolytic activity (22,23). Recently, we identified that point mutations made to these epitopes could be used to immunize HLA-A2-expressing mice to elicit higher frequency of CD8+ T cells that recognized the native epitopes (24). Furthermore, a DNA vaccine encoding these optimized epitopes (pTVG-SSX2opt) was able.