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

Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance.

The Mediterranean is home to a rich history of medical traditions that have developed under the influence of diverse cultures over millennia. Today, many such traditions are still alive in the folk medical practices of local people. Investigation of botanical folk medicines used in the treatment of skin and soft tissue infections led us to study Castanea sativa (European Chestnut) for its potential antibacterial activity. Here, we report the quorum sensing inhibitory activity of refined and chemically characterized European Chestnut leaf extracts, rich in oleanene and ursene derivatives (pentacyclic triterpenes), against all Staphylococcus aureus accessory gene regulator (agr) alleles. We present layers of evidence of agr blocking activity (IC50 1.56-25 μg mL-1), as measured in toxin outputs, reporter assays hemolytic activity, cytotoxicity studies, and an in vivo abscess model. We demonstrate the extract's lack of cytotoxicity to human keratinocytes and murine skin, as well as lack of growth inhibitory activity against S. aureus and a panel of skin commensals. Lastly, we demonstrate that serial passaging of the extract does not result in acquisition of resistance to the quorum quenching composition. In conclusion, through disruption of quorum sensing in the absence of growth inhibition, this study provides insight into the role that non-biocide inhibitors of virulence may play in future antibiotic therapies

224C-F2 attenuates MRSA-induced dermatopathology in a murine model of skin and soft tissue infection.

<p>C5Bl/6 mice were intradermally injected with 1x10⁸ CFUs of LAC (USA 300 isolate, AH1263) or its <i>agr</i> deletion mutant (AH1292). Mice received a single dose of 224C-F2 (at 5 or 50 μg) or the vehicle control (DMSO) at the time of infection. Significant differences between treatment and vehicle are represented as: *: p<0.05; ‡: p<0.01. <b>(A)</b> Images of abscesses and ulcers on days 2 and 6 post-infection (scale in cm). <b>(B)</b> 224C-F2 attenuates dermatopathology with a single dose of either 5 or 50 μg. <b>(C)</b> 224C-F2 reduces morbidity and mice do not lose weight.</p

Putative structures of compounds other than pentacyclic triterpenes found in the most active region of 224C-F2 (retention time of 21–49 min).

<p>Compounds are listed by Peak number, corresponding to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136486#pone.0136486.t005" target="_blank">Table 5</a>. Peak <b>32</b> was determined to be C₃₅H₅₉O₆ with a relative abundance of 0.30%. Putative structural matches include: (<b>32a</b>) stigmastane and (<b>32b</b>) (3β, 4β, 16α, 21β, 22α) -16, 21, 22, 23, 28- pentamethoxy (9CI) olean- 12- en- 3- ol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136486#pone.0136486.g010" target="_blank">Fig 10</a>). Peak <b>33</b> was determined to be C₂₇H₂₃O₁₈ with a relative abundance of 0.16%. Putative structural matches include: (<b>33a</b>) 1,3,6-tri-O-galloylglucose; (<b>33b</b>) 1,2,6-tri-galloyl-β-D-glucose; (<b>33c</b>) 1,2,3-tri-O-galloylglucose; (<b>33d</b>) 1,2,3-tri-O-galloyl-β-D-glucopyranose; (<b>33e</b>) 2',3,5-tri-O-galloyl-D-hamamelose; (<b>33f</b>) 2- <i>C</i>- [[(3, 4, 5- trihydroxybenzoyl) oxy] methyl]- 1, 5- bis(3, 4, 5- trihydroxybenzoate) D- Ribofuranose; (<b>33g</b>) kurigalin; (<b>33h</b>) 3,4,6-tri-O-galloyl-D-glucose. Peak <b>34</b> was determined to be C₃₉H₃₁O₁₅ with a relative abundance of 0.65%. Putative structural matches include: (<b>34</b>) castanoside B. Peak <b>39</b> was determined to be C₁₇H₁₁O₈ or C₂₀H₁₁O₄N₂ with a relative abundance of 0.72%. Putative structural matches include: (<b>39a</b>) 3,4,3'-tri-O-methylellagic acid and (<b>39b</b>) 3,3',4'-tri-O-methylellagic acid. Peak <b>44</b> was determined to be C₃₄H₂₉O₁₅ with a relative abundance of 0.26%. Putative structural matches included (<b>44</b>) norbadione A.</p

224C-F2 blocks MRSA exotoxin production.

<p><b>(A)</b> 224C-F2 demonstrates a dose-dependent effect in inhibition of de-formylated and formylated delta toxin, as illustrated in this HPLC chromatogram. <b>(B)</b> Quantification of delta-toxin confirmed the dose-dependent inhibitory activity of extracts, and the increased activity of the refined fraction 224C-F2 over 224 and 224C. <b>(C)</b> Extracts quench the hemolytic activity of both the <i>S</i>. <i>aureus</i> wild type and Δ<i>hla</i> mutant, demonstrating that in addition to preventing production of α-hemolysin (responsible for the major share of hemolytic activity), that extracts also inhibit PSM production, responsible for the observable hemolytic activity in <i>hla</i> mutant strains. All treated groups are significant in comparison to the vehicle control (<i>p</i><0.001). <b>(D)</b> USA300 (Δ<i>spa</i>) was exposed to increasing doses of 224, 224C, 224C-F2, and vehicle control for 8 hrs. Western blot for α-hemolysin on supernatants demonstrated a dose-dependent decline in protein levels. Significant differences between treatment and vehicle are represented as: *: <i>p</i><0.05; ‡: <i>p</i><0.01; †: <i>p</i><0.001.</p

Schematic of the <i>Staphylococcus aureus</i> accessory gene regulator system.

<p>The <i>agr</i> locus has been investigated in detail and is known to contain two divergent transcripts named RNAII and RNAIII [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136486#pone.0136486.ref009" target="_blank">9</a>]. The RNAII transcript is an operon of four genes, <i>agrBDCA</i>, that encode factors required to synthesize AIP and activate the regulatory cascade. Briefly, AgrD is the precursor peptide of AIP, AgrB is a membrane protease involved in generating AIP, AgrC is a histidine kinase that is activated by binding AIP, and AgrA is a response regulator that induces transcription of both RNAII and RNAIII. The RNAIII transcript yields a regulatory RNA molecule that acts as the primary effector of the <i>agr</i> system by up-regulating extracellular virulence factors and down-regulating cell surface proteins [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136486#pone.0136486.ref065" target="_blank">65</a>]. The <i>agr</i> pathway is illustrated here with potential target sites for 224C-F2.</p

Spent supernatants of <i>S</i>. <i>aureus</i> treated with 224C-F2 exhibit diminished cytotoxic effects against human keratinocytes.

<p><b>(A)</b> Supernatants were applied to HaCaT cells (20% v/v for 24 hrs) to measure the lytic capacity (determined by LDH assay) of a full suite of <i>S</i>. <i>aureus</i> exotoxins. Supernatants from 224C-F2-treated cultures were non-toxic to the mammalian cells, confirming inhibition of exotoxin production. <b>(B)</b> Following exposure to supernatants (14% v/v for 3 hrs) or staurosporine (7.1 μM for 3 hrs), HaCaT cells were imaged by fluorescent microscopy to examine cell integrity. Green cells are live, red are dead. Black regions are indicative of dead cells that have detached from the slide. Significant differences between treatment and vehicle are represented as: *: <i>p</i><0.05; ‡: <i>p</i><0.01; †: <i>p</i><0.001.</p

European Chestnut leaf extracts inhibit all four <i>S</i>. <i>aureus agr</i> alleles a non-biocide manner.

<p><i>S</i>. <i>aureus agr</i> reporter strains were treated with extracts 224, 224C, and 224C-F2 at a dose range of 0.05–100 μg mL^-1. Bioactivity guided sequential fractionation resulted in increased quenching of all 4 <i>agr</i> alleles in a manner independent of growth inhibition. Optical density of the culture is represented by solid black symbols; fluorescence in the <i>agr</i> reporters is indicated by the open symbols. The IC₅₀ and IC₉₀ for quorum quenching impact of each extract are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136486#pone.0136486.t003" target="_blank">Table 3</a>. <b>(A)</b> agr I, AH1677; <b>(B)</b> agr II, AH430; <b>(C)</b> agr III, AH1747; <b>(D)</b> agr IV, AH1872.</p

224C-F2 attenuates virulence without any detectable resistance after 15 days of drug passaging.

<p>Cultures of USA500 isolate NRS385 (<i>agr</i> group I) were passaged for 15 consecutive days in the presence of 16 μg mL^-1 of 224C-F2. <b>(A)</b> The sum total peak area of de-formylated and formylated delta toxin was quantified for the mock vehicle control (DMSO) and treated group. A significant difference (p<0.05) was evident for all treatment days. <b>(B)</b> 224C-F2 inhibited delta-toxin production over the length of the passaging experiment in the absence of growth inhibition. Significant differences between treatment and vehicle are represented as: *: <i>p</i><0.05; ‡: <i>p</i><0.01; †: <i>p</i><0.001.</p

Description of bacterial strains and plasmids used in this study.

<p><b>*Other Characteristics:</b> agr: accessory gene regulator; CA-MRSA: community-associated methicillin resistant <i>Staphylococcus aureus</i>; HA-MRSA: healthcare-associated MRSA; HMP: Human Microbiome Project isolate; PFT: pulsed field type; MLST: multilocus sequence type; MSSA: methicillin sensitive <i>Staphylococcus</i> aureus; SCC: staphylococcal chromosomal cassette; sea: staphylococcal enterotoxin A; seb: staphylococcal enterotoxin B; ST: sequence type; yfp: yellow fluorescent protein.</p><p>Description of bacterial strains and plasmids used in this study.</p