|Institution:||University of New South Wales|
|Department:||Clinical School - St George Hospital|
|Keywords:||Single-chain variable fragment (scFv); Heparin-Induced Thrombocytopenia; FcgRIIa|
|Full text PDF:||http://handle.unsw.edu.au/1959.4/53031|
Heparin-Induced Thrombocytopenia (HIT) is a life threatening disorder affecting 0.2-5% of patients receiving heparin. HIT is mediated by an autoantibody against platelet factor 4/heparin complex. This antibody/antigen complex binds to platelet FcgRIIa receptors, cross-links the receptors and activates the platelets. Currently, there is no clinically proven treatment that can prevent this platelet activation. IV.3 is a murine monoclonal antibody (MoAb) that binds to the FcgRIIa receptor and inhibits platelet activation induced by the HIT immune complexes. This thesis proposes that a single-chain variable fragment (scFv) derived from the IV.3 MoAb will retain the capacity to inhibit platelet activation caused by HIT antibodies. This will be a novel therapeutic strategy for the treatment of HIT. The scFv was constructed by cloning the variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding domain (Fab). The scFv was formed by joining the VH and VL chains with a flexible linker. This construct was then cloned into a periplasmic expression vector, expressed in bacteria and purified by affinity gel chromatography. The purified scFv was capable of binding human platelets as observed by flow cytometry. Moreover, the protein demonstrated potent inhibitory activity against HIT antibodies in platelet aggregation and 14C-serotonin release assays. The scFv was humanized (mutating as few residues as possible) to avoid potential immune reactions when it is administered to patients, thus improving its therapeutic usefulness. Notably, the activity of the humanized scFv was indistinguishable from that of the parental molecule. To increase its antiplatelet activity and to enhance its therapeutic potential, the molecule was fused to another scFv derived from an anti-GPIbα MoAb. This approach, however, did not progress further due to the observation of spontaneous platelet aggregation induced by the bispecific antibody. In conclusion, this thesis shows that both the murine and the humanized scFv demonstrate binding to the FcgRIIa on human platelets, and exhibit inhibition of platelet activation induced by the HIT antibodies. Therefore, this recombinant protein could potentially improve the treatment of HIT, particularly in preventing the patients serious clinical sequelae.