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

A High-Throughput Small Molecule Screen for <i>C</i>. <i>elegans</i> Linker Cell Death Inhibitors

<div><p>Programmed cell death is a ubiquitous process in metazoan development. Apoptosis, one cell death form, has been studied extensively. However, mutations inactivating key mammalian apoptosis regulators do not block most developmental cell culling, suggesting that other cell death pathways are likely important. Recent work in the nematode <i>Caenorhabditis elegans</i> identified a non-apoptotic cell death form mediating the demise of the male-specific linker cell. This cell death process (LCD, <u>l</u>inker <u>c</u>ell-type <u>d</u>eath) is morphologically conserved, and its molecular effectors also mediate axon degeneration in mammals and <i>Drosophila</i>. To develop reagents to manipulate LCD, we established a simple high-throughput screening protocol for interrogating the effects of small molecules on <i>C</i>. <i>elegans</i> linker cell death <i>in vivo</i>. From 23,797 compounds assayed, 11 reproducibly block linker cell death onset. Of these, five induce animal lethality, and six promote a reversible developmental delay. These results provide proof-of principle validation of our screening protocol, demonstrate that developmental progression is required for linker cell death, and suggest that larger scale screens may identify LCD-specific small-molecule regulators that target the LCD execution machinery.</p></div

Compound Structures.

<p>Chemical structures of compounds identified from pilot screen (A, B) and primary screen (B-M). (A) Tyrphostin A9, (B) Tyrphostin AG 879, (C) CB8776, (D) CB0146, (E) EN9834, (F) EN5065, (G) EN1918, (H) EN7212, (I) Leflunomide, (J) EN2416, (K) CB0736, (L) EN1123.</p

Primary screen results and attrition in secondary analyses.

<p>(A) Compounds screened ranked by percent inhibition and normalized to negative and positive control values for each plate screened. All screened compounds are depicted except those with normalized inhibition >160% (23/24298 events). Solid line indicates cutoff for additional testing. Dashed lines indicate percent inhibition of negative controls (0%) and positive controls (100%). (B) Secondary screening resulted in 11 compounds that were further examined.</p

Compounds reproducibly resulting in persistent linker cells.

<p>Compounds reproducibly resulting in persistent linker cells.</p

Screening pipeline.

<p>(A) Assay workflow: male <i>tra-2;xol-1 mig-24p</i>::<i>Venus</i> animals grown on agar plates with OP50 at 25°C for 32 hours are resuspended in S-Basal medium and transferred to plates with screening compounds in most wells (grey squares), positive (blue) and negative (red) controls. Plates are incubated for 12 hours and scanned using a fluorescence cytometer. (B) Image of Tyrphostin A9-treated well. (C) Same as (B), except counted objects marked (yellow). Large debris (red arrow) is not counted by the software, though some smaller fluorescent shapes that are not cells are erroneously labeled as cells (white arrow). “Count” indicates object count output from the MetaXpress software.</p

Assay positives from pilot screen.

<p>Assay positives from pilot screen.</p

Effects of compound treatment on viability and development.

<p>(A) Toxicity following 12-hour incubation with Leflunomide (black), EN5065 (blue), or EN1918 (red) on agar plates with OP50 <i>E</i>. <i>coli</i>, starting 32 hours after L1 arrest. Animals classified as dead did not move and exhibited no pharyngeal pumping. Error bars, SD. (B) Toxicity of CB0146 (squares) and Tyrphostin AG 879 (circles) as in (A). LD50 values calculated at 2.8 μM for CB0146 and 260 nM for Tyrphostin AG 879. (C) Surviving LCs scored in animals after 12-hour treatment with Leflunomide (black), EN5065 (blue), or EN1918 (red) across a range of compound concentrations. Error bars, SD. (D) Surviving LCs scored in animals after 12-hour treatment with CB0146 (squares) and Tyrphostin AG 879 (circles) across a range of compound concentrations. Error bars, SD. (E) Effect of compounds on male tail development in liquid assay. Error bars, SD. *, p<0.01, student’s t-test.</p

Commercial sources of compounds screened.

<p>Commercial sources of compounds screened.</p

Tyrphostin A9 causes linker cell persistence.

<p>(A) Screen of the LOPAC library using Tyrphostin A9 as a positive control. Compounds are plotted with an arbitrary index (X-axis). Percent inhibition (Y-axis) is normalized to negative control and positive control (Tyrphostin A9 treated) counts for each plate screened. The Tyrphostin A9 data point is indicated (arrow). (B) Effects of Tyrphostin A9 on animal viability, development, and linker cell presence are compared to DMSO exposure alone. LC, linker cell. (C) Image of a <i>tra-2(ar221);xol-1(y9) mig-24</i>p::Venus animal, treated with Tyrphostin A9 for 12 hours, showing persistent fluorescent linker cell (arrowhead) and undeveloped tail (arrow). (D) Effect on linker cell persistence after 12 hours of treatment with varying concentrations of Tyrphostin A9. Negative control mean is defined as 0% inhibition and maximum linker cell count in Tyrphostin A9 treated animals is defined as 100% inhibition. The EC₅₀ for Tyrphostin A9 is calculated to be 45.6 nM.</p