Engineering and characterization of a chimeric anti-platelet glycoprotein Ibalpha monoclonal antibody and preparation of its Fab fragment.
Glycoprotein Ibalpha (GPIbalpha) is a platelet-specific membrane protein. It mediates platelet adhesion to collagen exposed at the vascular injury site by binding to von Willebrand factor (VWF) in plasma. This process is crucial for arterial thrombus formation. Blocking interaction between GPIbalpha and VWF may prevent platelet adhesion and thrombus formation. We previously generated a high affinity monoclonal antibody against human platelet GPIbalpha, SZ2, which inhibits both ristocetin- and botrocetin-induced platelet aggregation in vitro. To convert SZ2 into mouse/human chimeric antibody for anti-platelet therapy in humans, in this study, we constructed a mouse/human chimeric antibody derived from the hybridoma cells producing murine antibody against platelet glycoprotein Ibalpha, conducted its expression in dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cells, and prepared its chimeric Fab fragment. Results from ELISA and Western blot analysis showed that the chimeric antibody was secreted from the cells and that the heavy and light chains were assembled correctly. Flow cytometry analysis confirmed specific binding of the chimeric antibody to the GPIb-expressing CHO cells. In vitro functional studies revealed that the chimeric antibody and its Fab fragment prevented platelet adhesion to VWF under high shear stress and inhibited ristocetin-induced platelet aggregation in a dose-dependent manner. These results demonstrated that the chimeric antibody was successfully engineered and suggested that the Fab fragment of chimeric antibody against GPIbalpha is a promising therapeutic antibody more suitable for prevention and treatment of human arterial thrombosis.
Yang
Jianfeng
J
Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Key Laboratory of Thrombosis and Hemostasis, Ministry of Heath, Suzhou, China.
Ji
Shundong
S
Dong
Ningzheng
N
Zhao
Yiming
Y
Ruan
Changgeng
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000918:Antibody Specificity; D001792:Blood Platelets / Q000276:immunology*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007167:Immunotherapy / Q000379:methods*; D051379:Mice; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000276:immunology*; D013927:Thrombosis / Q000517:prevention & control*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D019038:Platelet Glycoprotein GPIb-IX Complex