Brian T. Rekoske

Transcription of the Escherichia coli Fatty Acid Synthesis Operon fabHDG Is Directly Activated by FadR and Inhibited by ppGpp

In 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.

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.

Preclinical Pharmacokinetics and Biodistribution Studies of 89Zr-labeled Pembrolizumab

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 antitumor immune responses, and antibodies blocking PD-1 ligation, including pembrolizumab, have recently received Food and 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 the positron emitter 89Zr. PET imaging was used to evaluate the whole-body distribution of 89Zr-deferoxamine (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 was 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.

Immunotherapy for prostate cancer: False promises or true hope?

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 patients with 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 approval from the US Food and Drug Administration. This has left some wondering whether immunotherapy will “work” for this disease. In this review, the authors describe current developments in immunotherapy, including approaches to engage tumor‐targeting T cells, disrupt immune regulation, and alter the immunosuppressive tumor microenvironment. The authors then describe the recent application of these approaches to the treatment of prostate cancer. Given the Food and Drug Administration approval of 1 agent, and the finding that several others are in advanced stages of clinical testing, the authors believe that immunotherapies offer real hope to improve patient outcomes for men with prostate cancer, especially as investigators begin to explore rational combinations of immunotherapies and combine these therapies with other conventional therapies. Cancer 2016;122:3598‐607.

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.

89 Zr-labeled nivolumab for imaging of T-cell infiltration in a humanized murine model of lung cancer

PurposeNivolumab is a human monoclonal antibody specific for programmed cell death-1 (PD-1), a negative regulator of T-cell activation and response. Acting as an immune checkpoint inhibitor, nivolumab binds to PD-1 expressed on the surface of many immune cells and prevents ligation by its natural ligands. Nivolumab is only effective in a subset of patients, and there is limited evidence supporting its use for diagnostic, monitoring, or stratification purposes.Methods89Zr-Df-nivolumab was synthesized to map the biodistribution of PD-1-expressing tumor infiltrating T-cells in vivo using a humanized murine model of lung cancer. The tracer was developed by radiolabeling the antibody with the positron emitter zirconium-89 (89Zr). Imaging results were validated by ex vivo biodistribution studies, and PD-1 expression was validated by immunohistochemistry. Data obtained from PET imaging were used to determine human dosimetry estimations.ResultsThe tracer showed elevated binding to stimulated PD-1 expressing T-cells in vitro and in vivo. PET imaging of 89Zr-Df-nivolumab allowed for clear delineation of subcutaneous tumors through targeting of localized activated T-cells expressing PD-1 in the tumors and salivary glands of humanized A549 tumor-bearing mice. In addition to tumor uptake, salivary and lacrimal gland infiltration of T-cells was noticeably visible and confirmed via histological analysis.ConclusionsThese 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.

Safety and Immunological Efficacy of a DNA Vaccine Encoding the Androgen Receptor Ligand-Binding Domain (AR-LBD)

The androgen receptor (AR) is a key oncogenic driver of prostate cancer, and has been the primary focus of prostate cancer treatment for several decades. We have previously demonstrated that the AR is also an immunological target antigen, recognized in patients with prostate cancer, and targetable by means of vaccines in rodent models with delays in prostate tumor growth. The current study was performed to determine the safety and immunological efficacy of a GMP‐grade plasmid DNA vaccine encoding the ligand‐binding domain (LBD) of the AR, pTVG‐AR.

Abstract CN04-03: DNA vaccines as treatment for prostate cancer - understanding mechanisms of resistance

Anti-tumor vaccines have demonstrated clinical benefit in patients with advanced prostate cancer, and the first vaccine approved by FDA as a treatment for human cancer was a vaccine targeting the prostate tumor antigen prostatic acid phosphatase (PAP). Plasmid DNA, as another method of antigen delivery, offers significant potential safety and manufacturing advantages over other immunization approaches. DNA vaccines have been approved by USDA as treatments for diseases in fish, horses, dogs, and non-human primates. However, DNA vaccines have generally been perceived as less immunologically potent than other methods of antigen delivery in human clinical trials, leading to efforts to increase their immunogenicity. Most of these efforts have focused on increasing the delivery of vaccine to antigen-presenting cells using different carriers or by electroporation. Our group has evaluated repetitive immunization with plasmid DNA vaccines in rodent models, and has translated this approach to early clinical trials in men with low-volume recurrent prostate cancer with a DNA vaccine encoding PAP. We have demonstrated that persistent Th1-biased PAP-specific T-cell immune responses can be elicited in men with prostate cancer. Moreover, the development of persistent immunity appeared to be associated with favorable changes in tumor growth rate. A randomized phase II trial using this approach is currently ongoing in men with minimal residual prostate cancer to determine whether immunization can delay the time to metastatic disease progression (NCT01341652). In an effort to further improve the immunogenicity of DNA vaccines, we have explored methods to increase antigen presentation by encoding epitopes with increased MHC class I affinity or by increasing the duration of target antigen expression. While such “optimized” vaccines elicited increased numbers of antigen-specific CD8+ T cells, these T cells had less anti-tumor activity in vivo. We determined that immunization with plasmid DNA elicited antigen-specific IFNγ-secreting T cells that led to increased expression of PD-L1 on antigen-expressing tumor cells. However, immunization with the vaccine encoding high affinity epitopes resulted in antigen-specific CD8+ T cells with higher PD-1 expression. In the presence of PD-L1-expressing tumors these CD8+ were less effective. The anti-tumor activity was restored in the presence of PD-1 or PD-L1 blockade. Conversely, immunization with plasmid DNA engineered to increase the duration of antigen expression led to CD8+ T cells with normal PD-1 expression but higher LAG-3 expression. The anti-tumor activity was similarly restored in the presence of LAG-3 blockade. We are currently studying the mechanisms by which different T cell regulatory ligands are expressed following immunization. Notwithstanding, such findings suggest that subtle differences in the method of immunization can affect these different regulatory mechanisms, and hence the choice of optimal combination strategies may depend on identifying the specific mechanisms of resistance to immunization. In the case of patients immunized with a DNA vaccine encoding PAP, we have recently found that PD-L1 expression on circulating tumor (CD45-EpCAM+) cells was increased following immunization, analogous to our murine studies. In addition, PAP-specific secretion of IFNγ and granzyme B by T cells obtained from patients after immunization was increased in the presence of PD-1/PD-L1 blockade. Finally, using a trans vivo delayed type hypersensitivity (tvDTH) assay, we observed that PAP-specific immune responses in T cells obtained from patients after immunization with a DNA vaccine encoding PAP could be “uncovered” with PD-1 blockade but not blockade with anti-LAG-3 or anti-TIM-3. Collectively, these findings suggest that the PD-1/PD-L1 pathway and mechanism of resistance is most relevant to a DNA vaccine encoding PAP, and the efficacy of this vaccine might be augmented with PD-1 blockade. A clinical trial testing this approach in men with advanced prostate cancer, using this DNA vaccine and pembrolizumab, delivered either concurrently or sequentially, is currently underway (NCT02499835). Citation Format: Brian T. Rekoske, Viswa T. Colluru, Douglas G. McNeel. DNA vaccines as treatment for prostate cancer - understanding mechanisms of resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN04-03.

Abstract 243: Expression of T-cell checkpoint ligands on circulating tumor cells is modulated following treatment of patients with an anti-tumor DNA vaccine

Background: We have previously demonstrated in a murine tumor model that treatment with a DNA vaccine could lead to increased PD-1 expression on antigen-specific CD8+ T cells, led to upregulated levels of PD-L1 on tumors, and that the anti-tumor efficacy of the vaccine was enhanced when combined with PD-1/L1 blockade. We therefore examined whether prostate cancer patients previously treated with a DNA vaccine similarly upregulated these immunosuppresive ligands on their tumor cells, or the corresponding receptors on tumor-specific CD8+ T cells, and if augmented Th1-type immune responses were detectable following in vitro stimulation with checkpoint blockade. Methods: Circulating tumor cells (CTCs, CD45-/EpCAM+) from 16 prostate cancer patients with castrate-resistant disease, and antigen-specific (tetramer+) CD8+ T cells from those patients that were HLA-A2+ (6/16), were analyzed for their expression of various checkpoint ligands (PD-L1, galectin-9, HVEM) or receptors (PD-1, TIM-3, LAG-3, BTLA, CD160) using flow cytometry before and after treatment with a DNA vaccine encoding prostatic acid phosphatase (PAP). Immune responses to the PAP target antigen were measured in vitro for cytokine secretion via ELISA, or by a trans-vivo DTH-footpad assay, in the presence or absence of PD-1/L1 blockade. Results: Patients that developed either long-term immune responses or changes in PSA doubling time following vaccination had significantly higher levels of PD-L1 and HVEM, and lower levels of galectin-9, on their post-treatment CTCs than patients who did not develop responses. No significant changes in PD-1 or other regulatory receptors were detectable on tetramer+ CD8+ T cells. However, at least 25% of patients following immunization had augmented PAP-specific cytokine secretion in vitro when combined with PD-L1 blockade. PD-1 regulated PAP-specific responses were also observed in patients using the trans vivo DTH assay. Conclusions: These results demonstrate that the development of an anti-tumor immune response via DNA vaccination can lead to changes in checkpoint ligand expression on tumor cells and checkpoint-regulated immune responses. This suggests that combining DNA vaccines with blockade of one or more of these checkpoint pathways could produce enhanced anti-tumor responses in patients with prostate cancer. Citation Format: Brian T. Rekoske, Brian M. Olson, Douglas G. McNeel. Expression of T-cell checkpoint ligands on circulating tumor cells is modulated following treatment of patients with an anti-tumor DNA vaccine. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2015-243