Cobra venom factor

Induction immunosuppression for all recipients consisted of a single dose of anti-thymocyte globulin (ATG) (10 mg/kg) (Genzyme, Cambridge, MA, USA) on Day −2, as well as Cobra Venom Factor (CVF) (100 ug/kg) (Complement Technology, Tyler, TX, USA) for complement factor (CH50) depletion on Day −1. Maintenance immunosuppression consisted of a continuous tacrolimus (FK-506) infusion (0.20 mg/kg/day; with target serum levels of 10–20 ng/ml) starting on Day −2 and a methylprednisone taper starting on Day 0. Additional maintenance immunosuppression was provided with belatacept for co-stimulation blockade (n = 3) (20 mg/kg on Days −1, 0, 4, 7, 14 and 21, Bristol-Myers Squibb, New York, NY, USA) or anti-CD40 monoclonal antibody (mAb) (n = 1) (20 mg/kg on Days 0 and 5 and 10 mg/kg on Day 7 and weekly thereafter, NHP Reagent Resource, Boston, MA, USA).

Immunosuppression consisted of Thymoglobulin induction (10 mg/kg) (generously provided by Genzyme, Cambridge, MA, USA) as well as Cobra Venom Factor (100 ug/kg) (Complement Technology, Tyler, TX, USA) for complement factor (CH50) depletion on Day −1. Maintenance immunosuppression consisted of a continuous FK-506 infusion (0.20 mg/kg/day) (target serum levels 10-20 ng/ml) starting on Day −2 and a methylprednisone taper starting on Day 0. Additionally, the historical control (B274) also received LoCd2b (rat anti-primate CD2 IgG2b) and anti-CD154 (25 mg/kg).

Studies of NP mediated complement activation were performed as described with modifications (36 (link)). Briefly, samples containing 10 µl human plasma (non-heat inactivated) (Sigma-Aldrich, lot SLBH6826V), 10 µl veronal buffer with Ca²⁺ and Mg²⁺ (Complement Technology Inc.), 10 µl HP₄A or HP₄A-PEG NP in PBS (final concentration 10ug/ml) were incubated for 2 hr or 8 hr at 37°C. Plasma treated with PBS served as negative control and 50 units of cobra venom factor (Complement Technology Inc.) served as positive control for complement activation. Following treatment, samples were boiled for 5 minutes in 1×SDS sample buffer containing DTT, diluted 1:400 and 12 µl was fractionated on a 4%–10% Bis/Tris gel (Invitrogen) followed by transfer to PVDF membrane (Millipore). Blots were probed with polyclonal goat anti-human C3 (Complement Technology Inc.) using a 1:2000 dilution followed by probing with donkey anti-goat IgG- IRDye® 800CW (Li-Core Biosciences) using a 1:20,000 dilution. Membranes were scanned using Li-Core Odyssey Infrared Imaging system (Li-Core Biosciences).