New Cancer Immunotherapy Agents in Development: a report from an associated program of the 31

This report is a summary of \u27New Cancer Immunotherapy Agents in Development\u27 program, which took place in association with the 31st Annual Meeting of the Society for Immunotherapy of Cancer (SITC), on November 9, 2016 in National Harbor, Maryland. Presenters gave brief overviews of emerging clinical and pre-clinical immune-based agents and combinations, before participating in an extended panel discussion with multidisciplinary leaders, including members of the FDA, leading academic institutions and industrial drug developers, to consider topics relevant to the future of cancer immunotherapy

In Vivo Depletion of Lymphotoxin-Alpha Expressing Lymphocytes Inhibits Xenogeneic Graft-versus-Host-Disease

Graft-versus-host disease (GVHD) is a major barrier to successful allogeneic hematopoietic cell transplantation and is largely mediated by activated donor lymphocytes. Lymphotoxin (LT)-α is expressed by subsets of activated T and B cells, and studies in preclinical models demonstrated that targeted depletion of these cells with a mouse anti-LT-α monoclonal antibody (mAb) was efficacious in inhibiting inflammation and autoimmune disease. Here we demonstrate that LT-α is also upregulated on activated human donor lymphocytes in a xenogeneic model of GVHD and targeted depletion of these donor cells ameliorated GVHD. A depleting humanized anti-LT-α mAb, designated MLTA3698A, was generated that specifically binds to LT-α in both the soluble and membrane-bound forms, and elicits antibody-dependent cellular cytotoxicity (ADCC) activity in vitro. Using a human peripheral blood mononuclear cell transplanted SCID (Hu-SCID) mouse model of GVHD, the anti-human LT-α mAb specifically depleted activated LT-expressing human donor T and B cells, resulting in prolonged survival of the mice. A mutation in the Fc region, rendering the mAb incapable of mediating ADCC, abolished all in vitro and in vivo effects. These data support a role for using a depleting anti-LT-α antibody in treating immune diseases such as GVHD and autoimmune diseases

Processed pseudogenes acquired somatically during cancer development

Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5′ truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context

The PREDICTS database: a global database of how local terrestrial biodiversity responds to human impacts

Biodiversity continues to decline in the face of increasing anthropogenic pressures such as habitat destruction, exploitation, pollution and introduction of alien species. Existing global databases of species’ threat status or population time series are dominated by charismatic species. The collation of datasets with broad taxonomic and biogeographic extents, and that support computation of a range of biodiversity indicators, is necessary to enable better understanding of historical declines and to project – and avert – future declines. We describe and assess a new database of more than 1.6 million samples from 78 countries representing over 28,000 species, collated from existing spatial comparisons of local-scale biodiversity exposed to different intensities and types of anthropogenic pressures, from terrestrial sites around the world. The database contains measurements taken in 208 (of 814) ecoregions, 13 (of 14) biomes, 25 (of 35) biodiversity hotspots and 16 (of 17) megadiverse countries. The database contains more than 1% of the total number of all species described, and more than 1% of the described species within many taxonomic groups – including flowering plants, gymnosperms, birds, mammals, reptiles, amphibians, beetles, lepidopterans and hymenopterans. The dataset, which is still being added to, is therefore already considerably larger and more representative than those used by previous quantitative models of biodiversity trends and responses. The database is being assembled as part of the PREDICTS project (Projecting Responses of Ecological Diversity In Changing Terrestrial Systems – www.predicts.org.uk). We make site-level summary data available alongside this article. The full database will be publicly available in 2015

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Immune cell expression of EBI2.

<p><b>A</b>. Quantitative PCR analysis of <i>Ebi2</i> transcript abundance was performed in purified T cells, B cells and indicated myeloid cell populations. <b>B</b>. Expression of <i>Ebi2</i> in TLR-stimulated pDCs and CD11b⁺ myeloid cells. <i>Ebi2</i> expression in purified pDCs and CD11b⁺ myeloid cells that were stimulated for various time points over a 40 hr period with indicated TLR agonist. <i>Ebi2</i> expression in each cell type was normalized to <i>RPL19</i> expression, and data are shown as <i>Ebi2</i> expression relative to unstimulated cells at the corresponding time point. Data are shown as mean ± s.d. of two independent experiments.</p

Immune cell distribution in <i>Ebi2</i>^{−<b>/</b>−} mice. Immune cell distribution in spleen (A) or peritoneal lavage fluid (B) of naïve WT and <i>Ebi2</i>^−/− mice.

<p>Flow cytometric analysis was used to enumerate pDCs, myeloid DCs and B cells in WT (white bars) or <i>Ebi2</i>^−/− (shaded bars) mice (n = 7 per group for spleens; n = 5 per group for peritoneal lavage fluid). Bars represent mean values; white denotes WT, shaded denotes KO; circles represent individual animals. <i>P</i>-values are denoted when considered statistically significant (<i>p</i><0.05).</p

Expression of IRF7 and IDIN genes in TLR-stimulated EBI2-deficient pDCs and monocytes/macrophages.

<p>IDIN gene expression was determined by real-time RT-PCR. Expression in unstimulated or TLR9 agonist CpG-A ODN2216, TLR7 agonist ssPolyU, TLR3 agonist poly(I:C) or TLR4 agonist LPS stimulated pDCs (<b>A</b>) or CD11b⁺ monocytes/macrophages (<b>B</b>) from WT (white bars) or EBI2 KO (shaded bars) mice. Gene expression is presented relative to <i>RPL19</i> expression. Data are shown as mean ± s.d. from a single experiment with duplicate measurements. The experiment was repeated with similar results.</p

EBI2 Is a Negative Regulator of Type I Interferons in Plasmacytoid and Myeloid Dendritic Cells

<div><p>Epstein-Barr virus induced receptor 2 (EBI2), a Gα_i-coupled G protein-coupled receptor, is a chemotactic receptor for B, T and dendritic cells (DC). Genetic studies have also implicated EBI2 as a regulator of an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) associated with autoimmune diseases, although the corollary in primary type I IFN-producing cells has not been reported. Here we demonstrate that EBI2 negatively regulates type I IFN responses in plasmacytoid DC (pDCs) and CD11b⁺ myeloid cells. Activation of EBI2^−/− pDCs and CD11b⁺ cells with various TLR ligands induced elevated type I IFN production compared to wild-type cells. Moreover, <i>in vivo</i> challenge with endosomal TLR agonists or infection with lymphocytic choriomeningitis virus elicited more type I IFNs and proinflammatory cytokines in EBI2^−/− mice compared to normal mice. Elevated systemic cytokines occurred despite impaired ability of EBI2-deficient pDCs and CD11b⁺ cells to migrate from the blood to the spleen and peritoneal cavity under homeostatic conditions. As reported for other immune cells, pDC migration was dependent on the ligand for EBI2, 7α,25-dihydroxycholesterol. Consistent with a cell intrinsic role for EBI2, type I IFN-producing cells from EBI2-deficient mice expressed higher levels of IRF7 and IDIN genes. Together these data suggest a negative regulatory role for EBI2 in balancing TLR-mediated responses to foreign and to self nucleic acids that may precipitate autoimmunity.</p></div

<i>Ebi2</i>^{−<b>/</b>−} mice have stronger pDC-mediαted type I IFN responses to <i>in vivo</i> challenge with TLR agonists or LCMV Cl13.

<p><b>A, B.</b> Type I IFN production in mice challenged i.p. with TLR7 agonist Imiquimod (<b>A</b>) or TLR3 agonist poly(I:C) (<b>B</b>). IFN-α and IFN-β concentrations in peritoneal lavage fluid 4 hr following challenge were determined by ELISA. Bars represent mean values (white denotes WT, shaded denotes KO; n = 5 per group); circles represent individual animals. <b>C</b>. LCMV Cl13 infection in <i>Ebi2</i>^−/− mice. <i>Ebi2</i>^−/− (shaded bars) or WT littermate (white bars) mice were infected with 2×10⁶ PFU LCMV Cl13 i.v. On day 3, serum type I IFN concentrations were determined by ELISA. Bars denote mean values; circles represent individual animals (n = 5 per group). <i>P</i>-values are denoted when considered statistically significant (<i>p</i><0.05). IFN-α concentrations in naïve mice were below assay limit of detection.</p