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 Reliable von Willebrand factor: ristocetin cofactor enzyme-linked immunosorbent assay to differentiate between type 1 and type 2 von Willebrand disease

von Willebrand disease (vWD) is the most common hereditary bleeding disorder due to either a qualitative or a quantitative defect in von Willebrand factor (vWF). vWF is a multimeric plasma protein that plays an important role in (1) primary hemostasis, by sustaining indirect platelet adhesion especially at high shear rates, and in (2) secondary hemostasis, by protecting factor VIIIc (FVIIIc) from degradation. A correct diagnosis of vWD is based on the accurate identification of one of the six different subtypes (type 1, 2A, 2B, 2M, 2N, 3). To do this, different laboratory tests are available. One aspect of the identification is the discrimination between type 1 and type 2 (2A, 2B, and 2M) vWD. In type 1 vWD, both vWF levels (vWF:Ag) and vWF activity (vWF:RCof) are decreased; in type 2, the vWF:Ag level is normal or decreased and vWF:RCof is decreased. Thus, ratios of vWF:Ag to vWF:RiCof above 1 allow identifation of type 2 vWD patients. The currently used vWF:RCof test is an agglutination test in which patients' plasma is added to washed fixed control platelets in the presence of ristocetin and the extent of agglutination is measured. This test suffers from high interlaboratory and intralaboratory variability. We have recently shown that the same vWF:RCof can also be measured in an enzyme-linked immunosorbent assay (ELISA) with a low interassay and intraassay variability and can be used to identify patients suffering from vWD. We here show that our test allows the discrimination between type 1 and type 2 vWD patients.status: publishe

Inhibition of platelet glycoprotein Ib, glycoprotein IIb/IIIa, or both by monoclonal antibodies prevents arterial thrombosis in baboons

The antithrombotic efficacy of the monoclonal antibodies 6B4-Fab and MA-16N7C2 against platelet glycoprotein (GP) Ib and GP IIb/IIIa. respectively, on acute platelet-mediated thrombosis was evaluated in a baboon model of femoral artery stenosis, which is a modification of the original Folts model: platelet thrombi form on the injured stenosed artery, producing cyclic flow reductions (CFRs). A dose of 0.6 mg/kg 6B4-Fab significantly reduced the CFRs by 59 +/- 15%, whereas 2 mg/kg 6B4-Fab completely abolished the CFRs without prolongation of the bleeding time. MA-16N7C2 inhibited CFRs by 43 +/- 8% at a dose of 0.1 mg/kg and abolished the CFRs at a dose of 0.3 mg/kg but with a significant prolongation of the bleeding time. Finally, the combination of 0.6 mg/kg, 6B4-Fab and 0.1 mg/kg MA-16N7C2 fully prevented the CFRs without prolongation of the bleeding time. The present study demonstrates that the inhibition of platelet GP Ib function by 6B4-Fab is a powerful intervention to prevent platelet thrombus formation in injured arteries without prolongation of the bleeding time; the latter is in contrast to the result after the inhibition of GP IIb/IIIa. Moreover, we demonstrate that combining a GP Ib blocker with a GP IIb/IIIa blocker can achieve a strong antithrombotic effect without increasing the bleeding time. This provides new information that will be beneficial in designing clinical therapeutic approaches.status: publishe

Inhibition of the von Willebrand (VWF)-collagen interaction by an antihuman VWF monoclonal antibody results in abolition of in vivo arterial platelet thrombus formation in baboons

The interaction between collagen, von Willebrand factor (VWF), and glycoprotein Ib is the first step in hemostasis and thrombosis especially under high shear conditions. We studied the inhibition of the VWF-collagen interaction by using an antihuman VWF monoclonal antibody 82D6A3 to prevent arterial thrombosis in baboons to develop a new kind of antithrombotic strategy and determine for the first time experimental in vivo data concerning the importance of the collagen-VWF interaction. We used a modified Folts model to study the antithrombotic efficacy of 82D6A3, where cyclic flow reductions (CFRs) were measured in the femoral artery. Administering a dose of 100, 300, and 600 microg/kg resulted in a 58.3%, 100%, and 100% reduction in the CFRs, respectively. When 100 microg/kg 82D6A3 was infused into the baboons, 80% of VWF-A3 domain was occupied, corresponding to 30% to 36% ex vivo inhibition of VWF binding to collagen, with no prolongation of the bleeding time. The bleeding time was also not significantly prolonged when the CFRs were abolished at doses of 300 microg/kg and 600 microg/kg. At these doses 100% of VWF was occupied by the antibody and 100% ex vivo inhibition of the VWF-collagen binding was observed. 82D6A3 has a high affinity for VWF; after 48 hours still 68% VWF (300 microg/kg) was occupied with a pharmacologic effect up to 5 hours after administration (80%-100% occupancy). In conclusion, these results clearly indicate that the VWF-collagen interaction is important in vivo in thrombosis under high shear conditions and thus might be a new target for preventing arterial thrombosis.status: publishe