Binding Properties of a Human Anti-Ib ScFv Antibody (HIb-1) to Native, Recombinant, and Transgenicially Expressed Human Platelet GP Ibα Antigen.

HIb-1 is a single chain antibody (ScFv) originally obtained from the Griffin.1 library. ScFv from this library are derived from the in vitro recombination of germline human immunoglobulin V_H and V_L chain segments whose diversity has been greatly increased by mutagenesis directed at the CDR3 regions. HIb-1 was selected from the library by sequential biopanning first against CHO cells surface-expressing human GPIbα, and then against human platelets. Previous studies using Western blot analysis have shown that HIb-1 binds to an epitope within the 45 kDa amino-terminal portion of GPIbα (Lapan, Lyle, and Miller, 1999, Thromb. Haemost. 82S:393). HIb-1 displays inhibitory activity against both ristocetin-induced and shear-induced platelet aggregation. Biacore surface plasmon resonance analysis was used to establish dissociation constants between HIb-1and either the extracellular domain of GPIbα (i.e., “glycocalicin” or GC) derived from normal human platelets or the CHO-cell secreted GPIbα_1–483 recombinant extracellular protein. With native GC immobilized on dextran sulfate, the K_D for binding by analyte HIb-1 was ~600nM. With the wild-type recombinant GPIbα_1–483 bound to dextran sulfate, the K_D was only slightly higher at ~900 nM. We additionally studied a double mutant increase-of-function GPIbα_1-483 containing the Gly₂₃₃→Val₂₃₃ and Met₂₃₉→Val₂₃₉ substitituions. The K_D of HIb-1 binding to the double mutant was virtually identical to that of the wild-type, at ~900 nM. Binding of HIb-1 to native GC immobilized in an ELISA assay format was also performed. HIb-1 showed saturable binding, with a half-maximal binding concentration of approximately 170 nM. Flow cytometric analysis was also performed on platelets from murine GPIbα-null (i.e., “Bernard-Soulier”) mice that had been rescued with the human GPIbα transgene (