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

Nano-Pulse Stimulation is a physical modality that can trigger immunogenic tumor cell death

Abstract Background We have been developing a non-thermal, drug-free tumor therapy called Nano-Pulse Stimulation (NPS) that delivers ultrashort electric pulses to tumor cells which eliminates the tumor and inhibits secondary tumor growth. We hypothesized that the mechanism for inhibiting secondary tumor growth involves stimulating an adaptive immune response via an immunogenic form of apoptosis, commonly known as immunogenic cell death (ICD). ICD is characterized by the emission of danger-associated molecular patterns (DAMPs) that serve to recruit immune cells to the site of the tumor. Here we present evidence that NPS stimulates both caspase 3/7 activation indicative of apoptosis, as well as the emission of three critical DAMPs: ecto-calreticulin (CRT), ATP and HMGB1. Methods After treating three separate cancer cell lines (MCA205, McA-RH7777, Jurkat E6-1) with NPS, cells were incubated at 37 °C. Cell-culture supernatants were collected after three-hours to measure for activated caspases 3/7 and after 24 h to measure CRT, ATP and HMGB1 levels. We measured the changes in caspase-3 activation with Caspase-Glo® by Promega, ecto-CRT with anti-CRT antibody and flow cytometry, ATP by luciferase light generation and HMGB1 by ELISA. Results The initiation of apoptosis in cultured cells is greatest at 15 kV/cm and requires 50 A/cm2. Reducing this current inhibits cell death. Activated caspase-3 increases 8-fold in Jurkat E6-1 cells and 40% in rat hepatocellular carcinoma and mouse fibrosarcoma cells by 3 h post treatment. This increase is non-linear and peaks at 15–20 J/mL for all field strengths. 10 and 30 kV/cm fields exhibited the lowest response and the 12 and 15 kV/cm fields stimulated the largest amount of caspase activation. We measured the three DAMPs 24 h after treatment. The expression of cell surface CRT increased in an energy-dependent manner in the NPS treated samples. Expression levels reached or exceeded the expression levels in the majority of the anthracycline-treated samples at energies between 25 and 50 J/mL. Similar to the caspase response at 3 h, secreted ATP peaked at 15 J/mL and then rapidly declined at 25 J/mL. HMGB1 release increased as treatment energy increased and reached levels comparable to the anthracycline-treated groups between 10 and 25 J/mL. Conclusion Nano-Pulse Stimulation treatment at specific energies was able to trigger the emission of three key DAMPs at levels comparable to Doxorubicin and Mitoxantrone, two known inducers of immunogenic cell death (ICD). Therefore NPS is a physical modality that can trigger immunogenic cell death in tumor cells

Nano-Pulse Stimulation induces immunogenic cell death in human papillomavirus-transformed tumors and initiates an adaptive immune response

<div><p>Nano-Pulse Stimulation (NPS) is a non-thermal pulsed electric field modality that has been shown to have cancer therapeutic effects. Here we applied NPS treatment to the human papillomavirus type 16 (HPV 16)-transformed C3.43 mouse tumor cell model and showed that it is effective at eliminating primary tumors through the induction of immunogenic cell death while subsequently increasing the number of tumor-infiltrating lymphocytes within the tumor microenvironment. <i>In vitro</i> NPS treatment of C3.43 cells resulted in a doubling of activated caspase 3/7 along with the translocation of phosphatidylserine (PS) to the outer leaflet of the plasma membrane, indicating programmed cell death activity. Tumor-bearing mice receiving standard NPS treatment showed an initial decrease in tumor volume followed by clearing of tumors in most mice, and a significant increase in overall survival. Intra-tumor analysis of mice that were unable to clear tumors showed an inverse correlation between the number of tumor infiltrating lymphocytes and the size of the tumor. Approximately half of the mice that cleared established tumors were protected against tumor re-challenge on the opposite flank. Selective depletion of CD8⁺ T cells eliminated this protection, suggesting that NPS treatment induces an adaptive immune response generating CD8⁺ T cells that recognize tumor antigen(s) associated with the C3.43 tumor model. This method may be utilized in the future to not only ablate primary tumors, but also to induce an anti-tumor response driven by effector CD8⁺ T cells capable of protecting individuals from disease recurrence.</p></div

NPS treatment of primary tumors results in significant levels of tumor clearance, enhanced survival, and is effective during multiple applications.

<p>Groups of 10 mice were s.c. challenged with C3.43 tumors. 10-days post tumor challenge mice were given NPS (3 pps, 30kV/cm) treatment at the tumor site. Mice with recurring tumors received a second treatment on day 31 <b>(A)</b> Mean tumor volume (±SEM) of untreated and NPS treated mice (*p<0.05, unpaired students t-test at each time point). Volume measurements of untreated group displayed until there was a loss of 3 or more mice within the group due to euthanasia endpoints met <b>(B)</b> 50-day survival curve of groups with no treatment (naïve, median survival 39 days) or NPS treatment (NPS) (p<0.0012, Mantel-Cox Log Rank test). Data are representative of 2 independent experiments.</p

NPS treatment of tumors results in a CD8-dependent adaptive immune response.

<p>Shown are the individual tumor growth profiles of primary and rechallenge events in mice receiving NPS with or without selective depletion. Growth curves of primary (black) and re-challenge tumors (red) of NPS-treated mice with or without selective depletion of CD4 or CD8 T cells are displayed. <b>(A)</b> Mice received NPS treatment of primary tumor only (3 pps, 30 kV/cm, 70 A). <b>(B and C)</b> Mice received NPS treatment of primary tumor (3 pps, 30 kV/cm, 70A) followed by selective depletion of CD4 cells <b>(B)</b> or CD8 cells <b>(C)</b> with the administration of either an αCD4 mAb (yellow dots) or αCD8 mAb (green dots. The red arrows indicate the day of tumor re-challenge.</p

NPS application.

<p><b>(A)</b> Photo of a typical shaved C3.43 tumor prior to treatment. <b>(B)</b> Pinch electrode used to treat these tumors. <b>(C)</b> Pinch electrode sandwiching a tumor as NPS is applied. <b>(D)</b> Oscilloscope trace of voltage (top) and current (bottom) applied to the tumor in each pulse. <b>(E)</b> Photo of the treated tumor in “A” 11 days later.</p

NPS treatment of C3.43 cells results in significant upregulation of caspase 3/7 activity at lower treatment energies.

<p><b>(A)</b> Measured levels of activated caspase 3/7 in cells at 3h post NPS treatment for a range of NPS energy densities. Data shown as the mean of 4 experiments and the error bars represent the standard error of the mean (**p<0.01 ***p<0.001, One-way ANOVA followed by Dunnett’s multiple comparisons test to untreated cells). <b>(B)</b> Mean distribution of treated C3.43 tumor cells in early and late apoptosis at 3 h post-treatment with indicated NPS energy density <b>(C)</b> Data collected 24 h post NPS treatment.</p