bevacizumab

Bevacizumab, a humanized monoclonal antibody, specifically binds to all VEGF-A isoforms with high affinity, and inhibits its interaction with VEGFR-1 and VEGFR-2. Experimental analysis shows that the EC50 of Bevacizumab to bind VEGF analyzed by ELISA is 0.18 ug/mL. Binding kinetics assays show similar results that Bevacizumab inhibits the VEGF-induced proliferation of HUVEC with an IC50 value of 0.047+/-0.0081 ug/mL.

Kinase Assay: The binding kinetics of Bevacizumab or FD006 to VEGF is measured using Bio-Layer Inter-Ferometry on Octet RED. The assay is conducted at 30C in PBS buffer. Sensor tips are pre-wet for 15 mins in buffer immediately prior to use, and the microplates are filled with 200 uL per well of diluted samples (VEGF) or buffer and agitated at 1000 rpm. The anti-human IgG biosensor are pre-saturated with Bevacizumab or FD006 (10 ug/mL) and washed in buffer for 120 seconds, and then transferred to VEGF at concentrations of 10 ug/mL, 3 ug/mL and 1 ug/mL. The VEGF association and dissociation rates are measured for 5mins and 10mins, respectively. The Kinetics parameters (Kon and Koff) and affinities (KD) are calculated from a non-linear global fit using the Octet analysis software. Multiple independent measurements are performed.

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Cell Assay: Human umbilical vein endothelial cells (HUVECs) (1x104 cells/100 uL/well) are seeded in 96-well plates and cultured at 37 for 14 h with Endothelial Cell Medium supplemented with 5% heat-inactivated FCS, 100 U/mL Penicillin, 100 U/mL Streptomycin, and endothelial cell growth supplement. After low-serum starvation overnight, cells are treated with different concentrations of FD006 or Bevacizumab which are pre-incubated with 10 ng/mL VEGF for 30 minutes and incubated at 37, 5% CO2 for 72 hours. Then, 10 uL CCK8 is added to each well and incubated for another 4 hours. The absorbance is measured by spectrophotometer at 450 nm to determine the cell viability.

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Animal Studies: After modeling, ninety rats are randomly divided into five groups (eighteen rats per group) and receive a subconjunctival injection with 0.05 mL per rat of (1) 0.9% NaCl, (2) solvent, (3) 0.1% Dexamethasone (Dexamethasone sodium phosphate), (4) 25 mg/mL Bevacizumab and (5) 25 mg/mL FD006 in the superior temporal conjunctiva on the day after modeling. All chemical burns and treatments are performed by one investigator. The operator is blinded to the treatment group from which each cornea is derived. At postoperative days 3, 7, 14, 21 and 28, the eyes are harvested for further studies after the rats are sacrificed with an overdose of 10% chloral hydrate.