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

Trace analysis of cyclophosphamide and its metabolites in urine by liquid chromatography-tandem mass spectrometry

Cyclophosphamide (CP) is an antineoplastic drug used to treat a wide variety of cancers and immune disorders. CP is also a highly toxic alkylating agent, classified as an IARC Group 1 carcinogen. Workers in health-care environments are vulnerable to occupational exposure to CP, primarily via inhalation and dermal absorption. CP is a prodrug; both its therapeutic effectiveness and toxicity are activated through metabolism. To date, however, no study measuring occupational exposure to CP has successfully analyzed its metabolites. The main objective of this work was to develop an analytical method for CP, as well as its metabolites 4-ketocyclophosphamide (KCP) and carboxyphosphamide (CBP), in urine samples collected from health-care workers at risk of CP exposure. A liquid chromatography-tandem mass spectrometry (LC/MS-MS) method was optimized for CP, KCP and CBP on two different instruments. Post-column infusion showed that the matrix effects resulting from synthetic urine could be separated from the analyte peaks by LC. Estimated instrument limits of quantitation for CP, KCP and CBP in neat solvent were respectively 4.2, 8.2 and 57 ng/L. These parameters were sufficient to meet a quantitation target of 50 ng/L CP in urine, but suggested a need to reduce sample volume to reach a 2.5 ng/L target for KCP and CBP. Solid-phase extraction (SPE) was explored as a means to exchange the sample matrix for clean solvent and reduce sample volume. Previously developed SPE methods for CP were not designed to include the more polar metabolites, and thus required modification. The best retention of metabolites was seen on a C¹⁸ sorbent with reduced carbon loading. Retention was improved further under acidic loading conditions, but this had to be controlled carefully since CBP can decompose more rapidly at acidic pH. All three analytes were observed to elute with methanol.Science, Faculty ofChemistry, Department ofGraduat