Autophagosome-based strategy to monitor apparent tumor-specific CD8 T cells in patients with prostate cancer

The immune system plays an essential role in eradicating cancer in concert with various treatment modalities. In the absence of autologous tumor material, no standardized method exists to assess T cell responses against the many antigens that may serve as cancer rejection antigens. Thus, development of methods to screen for therapy-induced anti-tumor responses is a high priority that could help tailor therapy. Here we tested whether a tumor-derived antigen source called DRibbles®, which contain a pool of defective ribosomal products (DRiPs), long-lived and short-lived proteins (SLiPs) and danger-associated molecular patterns (DAMPs), can be used to identify tumor-associated antigen (TAA)-specific responses in patients before or after immunotherapy treatment. Protein content, gene expression and non-synonymous – single nucleotide variants (ns-SNVs) present in UbiLT3 DRibbles were compared with prostate adenocarcinomas and the prostate GVAX vaccine cell lines (PC3/LNCaP). UbiLT3 DRibbles were found to share proteins, as well as match tumor sequences for ns-SNVs with prostate adenocarcinomas and with the cell lines PC3 and LNCaP. UbiLT3 DRibbles were used to monitor anti-tumor responses in patients vaccinated with allogeneic prostate GVAX. UbiLT3-DRibble-reactive CD8+ T-cell responses were detected in post-vaccine PBMC of 6/12 patients (range 0.85–22% of CD8+ cells) after 1 week in vitro stimulation (p = 0.007 vs. pre-vaccine). In conclusion, a cancer-derived autophagosome-enriched preparation, packaging over 100 proteins over-expressed in prostate cancer into microvesicles containing DAMPs, could be used to identify CD8+ T cells in peripheral blood from patients after prostate GVAX vaccination and may represent a general method to monitor anti-cancer T cell responses following immunotherapy

TAF1 Histone Acetyltransferase Activity in Sp1 Activation of the Cyclin D1 Promoter

A missense mutation within the histone acetyltransferase (HAT) domain of the TATA binding protein-associated factor TAF1 induces ts13 cells to undergo a late G(1) arrest and decreases cyclin D1 transcription. We have found that TAF1 mutants (Δ844-850 and Δ848-850, from which amino acids 844 through 850 and 848 through 850 have been deleted, respectively) deficient in HAT activity are unable to complement the ts13 defect in cell proliferation and cyclin D1 transcription. Chromatin immunoprecipitation assays revealed that histone H3 acetylation was reduced at the cyclin D1 promoter but not the c-fos promoter upon inactivation of TAF1 in ts13 cells. The hypoacetylation of H3 at the cyclin D1 promoter was reversed by treatment with trichostatin A (TSA), a histone deacetylase inhibitor, or by expression of TAF1 proteins that retain HAT activity. Transcription of a chimeric promoter containing the Sp1 sites of cyclin D1 and c-fos core remained TAF1 dependent in ts13 cells. Treatment with TSA restored full activity to the cyclin D1-c-fos chimera at 39.5°C. In vivo genomic footprinting experiments indicate that protein-DNA interactions at the Sp1 sites of the cyclin D1 promoter were compromised at 39.5°C in ts13 cells. These data have led us to hypothesize that TAF1-dependent histone acetylation facilitates transcription factor binding to the Sp1 sites, thereby activating cyclin D1 transcription and ultimately G(1)-to-S-phase progression

5 Development of a vaccine to intercept oral cancer

Background Oral cavity cancer is diagnosed in more than 300,000 people each year worldwide and approximately half of these will die within five years despite standard treatment. These cancers are often preceded by the appearance of a premalignant dysplastic lesion, which offers a unique opportunity to identify patients at high risk of developing cancer and offer them a vaccine that may prevent development of this non-viral malignancy. Lesions can be removed but because of a ‘field effect’ their entire oral mucosa is at risk – thus the vaccine. Recent studies have identified genes that are differentially expressed during progression from normal tissue to oral cancer providing a roadmap to developing a preventative vaccine [PMID: 27027432] Methods We have manufactured and performed initial characterization of a DC-targeted microvesicle vaccine, DPV-007, made specifically to intercept the progression of oral dysplasia to oral cancer. DPV-007 is manufactured using platform vaccine technology that is in the clinic. This technology captures short-lived and non-canonical proteins that make up the dominant epitopes presented by HLA and packages them in microvesicles containing DAMPs and molecular chaperones. Characterization of DPV-007 included molecular, proteogenomic, biochemical and functional assessments. Preclinical studies were performed evaluating prevention of tumor development in the 4NQO-induced tumor model. Results The DPV-007 microvesicle vaccine contains more than 200 proteins for genes that are upregulated in oral dysplasia and oral cancer. Preliminary data suggests that the vaccine may contain as many as 30 somatic variants identified as somatic mutations in the COSMIC Database. Additionally, the vaccine contains at least 11 NCI prioritized cancer antigens and has agonist activity for TLR 2, 3, and 9. In the 4NQO preclinical model, vaccination including relevant antigens and agonist activity, provided significant (p\u3c0.02) protection from lesion onset and tumor outgrowth. Conclusions The identification of genes associated with the progression of pre-cancerous lesions to cancer provides targets for active immunotherapy of this disease. In preclinical models we have shown that this vaccine strategy is effective in both protection and therapy studies [PMID: 21810919; PMID: 27874054; PMID: 31747946]. A clinical trial of a similar vaccine, DPV-001, administered as a single agent as adjuvant therapy for NSCLC, documented induction of immunity to a large number of cancer antigens contained in the vaccine and did not identify serious adverse events. Based on data summarized above, we propose to vaccinate patients with dysplastic lesions and investigate whether vaccination reduces lesion recurrence. Acknowledgements Research support was provided by Janssen Research and Development, Johnson & Johnson. Ethics Approval The Institutional Animal Care & Use Committee of the Earle A. Chiles Research Institute approved the above noted studies. Protocol 55