Pembrolizumab is a humanized monoclonal antibody targeting programmed cell death protein 1 (PD-1) found on T and pro-B cells. Pembrolizumab prevents PD-1 ligation by both PD-L1 and PD-L2, preventing the immune dysregulation that otherwise occurs when T cells encounter cells expressing these ligands. Clinically, PD-1 blockade elicits potent anti-tumor immune responses and antibodies blocking PD-1 ligation, including pembrolizumab, have recently received federal drug administration approval for the treatment of advanced melanoma, renal cell cancer, and non-small cell lung cancer. Methods In this study, we evaluated the pharmacokinetics, biodistribution, and dosimetry of pembrolizumab in vivo, accomplished through radiolabeling with positron emitter zirconium-89 (89Zr). Positron emission tomography (PET) imaging was utilized to evaluate the whole-body distribution of 89Zr-Df-Pembrolizumab in two rodent models (mice and rats). Data obtained from PET scans and biodistribution studies were extrapolated to humans to estimate the dosimetry of the tracer. As a proof-of-concept, the biodistribution of 89Zr-Df-Pembrolizumab is further investigated in a humanized murine model. Results The tracer remained stable in blood circulation throughout the study and accumulated the greatest in liver and spleen tissues. Both mice and rats showed similar biodistribution and pharmacokinetics of 89Zr-Df-Pembrolizumab. In the humanized mouse model, T-cell infiltration into the salivary and lacrimal glands could be successfully visualized. Conclusion These data will augment our understanding of the pharmacokinetics and biodistribution of radiolabeled pembrolizumab in vivo, while providing detailed dosimetry data that may lead to better dosing strategies in the future. These findings further demonstrate the utility of noninvasive in vivo PET imaging to dynamically track T-cell checkpoint receptor expression and localization in a humanized mouse model.

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. In this report we sought to test if 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 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.

bIn Escherichia coli, FadR and FabR are transcriptional regulators that control the expression of fatty acid degradation and unsaturated fatty acid synthesis genes, depending on the availability of fatty acids. In this report, we focus on the dual transcriptional regulator FadR. In the absence of fatty acids, FadR represses the transcription of fad genes required for fatty acid degradation. However, FadR is also an activator, stimulating transcription of the products of the fabA and fabB genes responsible for unsaturated fatty acid synthesis. In this study, we show that FadR directly activates another fatty acid synthesis promoter, PfabH, which transcribes the fabHDG operon, indicating that FadR is a global regulator of both fatty acid degradation and fatty acid synthesis. We also demonstrate that ppGpp and its cofactor DksA, known primarily for their role in regulation of the synthesis of the translational machinery, directly inhibit transcription from the fabH promoter. ppGpp also inhibits the fadR promoter, thereby reducing transcription activation of fabH by FadR indirectly. Our study shows that both ppGpp and FadR have direct roles in the control of fatty acid promoters, linking expression in response to both translation activity and fatty acid availability.

These data support our claim that PD-1-targeted agents allow for tumor imaging in vivo, which may assist in the design and development of new immunotherapies. In the future, noninvasive imaging of immunotherapy biomarkers may assist in disease diagnostics, disease monitoring, and patient stratification.

We have previously reported that tumor antigen-specific DNA vaccination in mice led to an increase in IFNg-secreting T cells and an increase in tumor expression of PD-L1. Further, we demonstrated that increasing the encoded antigen's MHC-binding affinity led to increased PD-1 expression on antigenspecific CD8 C T cells. Together these phenomena provided resistance to antitumor immunization that was abrogated with PD-1/PD-L1 blockade. We consequently sought to determine whether similar regulation occurred in human patients following antitumor immunization. Using clinical samples from prostate cancer patients who were previously immunized with a DNA vaccine, we analyzed changes in checkpoint receptor expression on antigen-specific CD8 C T cells, the effect of PD-1 blockade on elicited immune responses, and for changes in checkpoint ligand expression on patients' circulating tumor cells (CTCs). We observed no significant changes in T-cell expression of PD-1 or other checkpoint receptors, but antigenspecific immune responses were detected and/or augmented with PD-1 blockade as detected by IFNg and granzyme B secretion or trans vivo DTH testing. Moreover, PD-L1 expression was increased on CTCs following vaccination, and this PD-L1 upregulation was associated with the development of sustained Tcell immunity and longer progression-free survival. Finally, similar results were observed with patients treated with sipuleucel-T, another vaccine targeting the same prostate antigen. These findings provide inhuman rationale for combining anticancer vaccines with PD-1 blocking antibodies, particularly for the treatment of prostate cancer, a disease for which vaccines have demonstrated benefit and yet PD-1 inhibitors have shown little clinical benefit to date as monotherapies.KEYWORDS DNA vaccine; PD-1; PD-L1; prostate cancer; prostatic acid phosphatase (PAP)

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.

Prostate cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related death, for men in the United States. Despite the approval of several new agents for advanced disease, each of these has prolonged survival by only a few months. Consequently new therapies are sorely needed. For other cancer types, immunotherapy has demonstrated dramatic and durable treatment responses, causing many to hope that immunotherapies might provide an ideal treatment approach for advanced prostate cancer. However, apart from sipuleucel-T, prostate cancer has been conspicuously absent from the list of malignancies for which immunotherapies have received recent FDA approval. This has left some wondering if immunotherapy will “work” for this disease. In this review we describe current immunotherapy developments, including approaches to engage tumor-targeting T cells, disrupt immune regulation, and alter the immunosuppressive tumor microenvironment. We then describe the recent application of these approaches to the treatment of prostate cancer. Given the FDA approval of one agent, and the fact that several others are in advanced stages of clinical testing, we believe that immunotherapies offer real hope to improve patient outcomes for prostate cancer, especially as investigators begin to explore rational combinations of immunotherapies and combine these therapies with other conventional therapies.

168 Background: We have previously investigated a DNA vaccine encoding prostatic acid phosphatase (PAP, pTVG-HP) in patients with PSA-recurrent prostate cancer, and have demonstrated that this can be safely administered over many months and can elicit PAP-specific T cells. A phase 2 trial is currently underway. In preclinical models, we have found that blockade of regulatory receptors, including PD-1, at the time of T cell activation with vaccination produced anti-tumor responses in vivo. Similarly, we have recently found that patients with prostate cancer previously immunized with a DNA vaccine develop PD-1-regulated T cells. These findings suggested that combined PD-1 blockade with vaccination should elicit superior anti-tumor responses in patients with prostate cancer. Methods: A clinical trial was designed to evaluate the immunological and clinical efficacy of pTVG-HP when delivered in combination or in sequence with pembrolizumab, in patients with mCRPC. Serial biopsies, blood draws, and exploratory FLT PET/CT imaging are being conducted for correlative analyses. Results: While trial accrual continues, 1 of 14 subjects has experienced a grade 3 adverse event. There have been no grade 4 events. Several patients treated with the combination have experienced serum PSA declines, and several have experienced decreases in tumor volume by radiographic imaging at 12 weeks, including one partial response. Expansion of PAP-specific Th1-biased T cells has been detected in peripheral blood samples. Exploratory FLT PET/CT imaging has demonstrated proliferative responses in metastatic lesions and in vaccine-draining lymph nodes. Evaluation of biopsy specimens for recruitment of antigen-specific T cells is currently underway. Conclusions: PD-1 pathway inhibitors have demonstrated little clinical activity to date when used as single agents for treating prostate cancer. Our findings suggest that combining this blockade with tumor-targeted T-cell activation by a DNA vaccine is safe and can augment tumor-specific T cells, detectable within the peripheral blood and by imaging, and result in objective anti-tumor changes. Clinical trial information: NCT02499835.