by Rong Deng, and Joseph P. Balthasar Comparison of the effects of antibody-coated liposomes, IVIG, and anti-RBC immunotherapy in a murine model of passive chronic immune thrombocytopenia by Rong Deng, and Joseph P. Balthasar Blood Volume 109(6):2470-2476 March 15, 2007 ©2007 by American Society of Hematology

IVIG effects on the time course of MWReg30-mediated thrombocytopenia in mice after continuous intraperitoneal infusion of antiplatelet monoclonal antibody MWReg30. IVIG effects on the time course of MWReg30-mediated thrombocytopenia in mice after continuous intraperitoneal infusion of antiplatelet monoclonal antibody MWReg30. MWReg30 was administered by intraperitoneal infusion (0.99 μg/d × 7 days). IVIG was given by intraperitoneal bolus injection at 96 hours. Platelet counts were obtained using a Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group and IVIG treatment groups (n = 5 mice/group): sham control group (▾), saline (•), 0.4 g/kg (▪), 1 g/kg (♦), 2 g/kg (▴). Error bars represent the standard deviation. IVIG attenuated MWReg30-mediated thrombocytopenia in a dose-dependent manner. Treatment differences were statistically significant (P < .001). Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

Effects of anti-RBC immunotherapy, antibody-coated liposomes, and IVIG on RBC counts in mice. Effects of anti-RBC immunotherapy, antibody-coated liposomes, and IVIG on RBC counts in mice. MWReg30 was administered by intraperitoneal infusion (0.99 μg/d × 7 days), and treatments were given by bolus injection at 96 hours. RBC counts were obtained using a Cell-Dyn 1700 multiparameter hematology analyzer. (A) Effects of anti-RBC immunotherapy. Symbols represent the sham control group (▾) and animals treated with intravenous injections of saline (•) or TER119 at doses of 5 μg/mouse (▪), 15 μg/mouse (♦), 25 μg/mouse (▴), or 50 μg/mouse (+). Error bars represent the standard deviation associated with the RBC counts. TER119 decreased RBC counts in a dose-dependent manner. Treatment differences were statistically significant (P < .001). (B) Effects of IVIG immunotherapy. Symbols represent the sham control group (▾) and animals treated with intraperitoneal saline (•) or IVIG, administered intraperitoneally, at doses of 0.4 g/kg (▪), 1 g/kg (♦), or 2 g/kg (▴). Error bars represent the standard deviation associated with the RBC counts. RBC counts were not significantly altered (P > .05). (C) Effects of AMI-coated liposomes. Symbols represent the sham control group (▾) and animals treated with intravenous injections of saline (•) or AMI-coated liposomes at doses of 15 μmol/kg (▪), 30 μmol/kg (♦), or 60 μmol/kg (▴). Error bars represent the standard deviation associated with the RBC counts. RBC counts were not significantly altered (P > .05). (D) Effects of IVIG-coated liposomes. Symbols represent the sham control group (▾) and animals treated with intravenous injections of saline (•) or AMI-coated liposomes at doses of 15 μmol/kg (▪), 30 μmol/kg (♦), or 60 μmol/kg (▴). Error bars represent the standard deviation associated with the RBC counts. RBC counts were not significantly altered (P > .05). Please note that all panels include the same sham control group (▾ in each panel) and panels A, C, and D include the same intravenous saline control group (•). Saline was administered intraperitoneally in the saline control group shown in panel B (also symbolized with •). Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

IVIG pharmacokinetics. IVIG pharmacokinetics. IVIG was administered by intraperitoneal injection at doses of 0.4 g/kg (▪), 1 g/kg (♦), and 2 g/kg (▴). IVIG was injected 96 hours after initiation of the MWReg30 infusion. IVIG concentrations in plasma were assessed via ELISA and were found to increase with dose. Systemic clearance, assessed by the ratio of IVIG dose to the area under the mean IVIG plasma concentration versus time curve, was found to increase with increasing doses of IVIG. This finding is consistent with the hypothesis that high-dose IVIG therapy leads to inhibition of the IgG transporter FcRn. Error bars indicate the standard deviation of assayed IVIG concentrations. Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

Effects of AMI-coated liposomes on the time course of MWReg30-mediated thrombocytopenia. Effects of AMI-coated liposomes on the time course of MWReg30-mediated thrombocytopenia. MWReg30 was administered by intraperitoneal infusion (0.99 μg/d × 7 days). AMI-coated liposomes were given by intravenous bolus injection at 96 hours, and platelet counts were obtained using a Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group (▾) and animals treated with saline (•), 15 μmol lipid/kg (▪), 30 μmol lipid/kg (♦), or 60 μmol lipid/kg (▴) (n = 5 mice/group). Error bars represent the standard deviation of platelet count values. AMI-coated liposomes attenuated MWReg30-mediated thrombocytopenia in a dose-dependent manner. Treatment differences were statistically significant (P < .001). Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

Effects of IVIG-coated liposomes on the time course of MWReg30-mediated thrombocytopenia. Effects of IVIG-coated liposomes on the time course of MWReg30-mediated thrombocytopenia. MWReg30 was administered by intraperitoneal infusion (0.99 μg/d × 7 days), and IVIG-coated liposomes were given by intravenous bolus injection at 96 hours. Platelet counts were obtained using a Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group (▾) and animals treated with saline (•), 15 μmol lipid/kg (▪), 30 μmol lipid/kg (♦), or 60 μmol lipid/kg (▴) (n = 5 mice/group). Error bars represent the standard deviation. IVIG-coated liposomes attenuated MWReg30-mediated thrombocytopenia in a dose-dependent manner. Treatment differences were statistically significant (P < .001). Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

Effects of anti-RBC monoclonal antibody therapy on the time course of MWReg30-mediated thrombocytopenia. Effects of anti-RBC monoclonal antibody therapy on the time course of MWReg30-mediated thrombocytopenia. MWReg30 was administered to mice via intraperitoneal infusion (0.99 μg/d × 7 days), and TER119 was given by intravenous bolus injection at 96 hours. Platelet counts were obtained using a Cell-Dyn 1700 multiparameter hematology analyzer. Symbols represent the sham control group (•) and animals treated with saline (▾), 5 μg/mouse (▪), 15 μg/mouse (♦), 25 μg/mouse (▴), or 50 μg/mouse (+). Error bars represent the standard deviation associated with the platelet counts. TER119 attenuated MWReg30-mediated thrombocytopenia in a dose-dependent manner. Treatment differences were statistically significant (P < .001). Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology

Plasma MWReg30 pharmacokinetics following IVIG treatment. Plasma MWReg30 pharmacokinetics following IVIG treatment. MWReg30 was administered by intraperitoneal infusion (0.99 μg/d × 7 days), and IVIG was given by intraperitoneal bolus injection at 96 hours. MWReg30 concentrations were determined via ELISA. Symbols represent treatment groups (n = 5 mice/group) receiving saline (•) or IVIG at doses of 0.4 g/kg (▪), 1 g/kg (♦), or 2 g/kg (▴). IVIG treatment altered the pharmacokinetics of MWReg30, decreasing MWReg30 exposure. This finding is consistent with the hypothesis that high-dose IVIG therapy leads to inhibition of the IgG transporter FcRn. Rong Deng, and Joseph P. Balthasar Blood 2007;109:2470-2476 ©2007 by American Society of Hematology