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

Where Is the Drug? Quantitative 3D Distribution Analyses of Confined Drug-Loaded Polymer Matrices

To enhance oral bioavailability of poorly soluble drugs, microfabricated devices can be utilized. One example of such devices is microcontainers. These are cylindrical in shape with an inner cavity for drug loading and with only the top side open for release. Supercritical CO2 (scCO2) impregnation is an interesting technique for loading drugs into polymeric matrices in, for example, microcontainers since it avoids the use of organic solvents and is cheap. One of the main drawbacks of this technique is the unknown three-dimensional drug distribution in the polymer matrix. The aim of this study was to investigate the loading of two poorly soluble drugs, naproxen and ketoprofen, by scCO2 impregnation into confined polymer matrices of different sizes. Three different sizes of microcontainers (small, medium, and large) and, thereby, different surface areas accessible for impregnation were compared. From in vitro studies, the amount of naproxen and ketoprofen loaded into the different microcontainers and their corresponding release profiles were seen to be similar. A custom-made Raman microscope facilitated volumetric Raman maps of an entire microcontainer filled with polyvinylpyrrolidone (PVP) and scCO2 impregnated with either naproxen or ketoprofen. In all microcontainer sizes, the drugs were only detected in the top layer of the polymer matrix, explaining the observed similar release profiles. Using X-ray powder diffraction and Raman spectroscopy, the solid state form of the drugs was evaluated, showing that ketoprofen was amorphous in all microcontainer sizes. Naproxen was found not to be crystalline nor amorphous but in a less ordered configuration than the crystalline state. In conclusion, volumetric Raman mapping is a powerful technology for imaging drug distribution and drug crystallinity in polymers and allowed us to conclude that (i) scCO2 impregnation depth does not depend on surface area and (ii) impregnated drugs are noncrystalline