Svetlana A. Shestopal

Single-chain variable fragments (scFv) are antigen-recognizing variable fragments of antibodies (FV) where both subunits (V_L and V_H) are connected via an artificial linker. One particular scFv, iKM33, directed against blood coagulation factor VIII (FVIII) was shown to inhibit major FVIII functions and is useful in FVIII research. We aimed to investigate the properties of iKM33 enabled with protease-dependent disintegration. Three variants of iKM33 bearing thrombin cleavage sites within the linker were expressed using a baculovirus system and purified by two-step chromatography. All proteins retained strong binding to FVIII by surface plasmon resonance, and upon thrombin cleavage, dissociated into V_L and V_H as shown by size-exclusion chromatography. However, in FVIII activity and low-density lipoprotein receptor-related protein 1 binding assays, the thrombin-cleaved iKM33 variants were still inhibitory. In a pull-down assay using an FVIII-affinity sorbent, the isolated V_H, a mixture of V_L and V_H, and intact iKM33 were carried over via FVIII analyzed by electrophoresis. We concluded that the isolated V_L and V_H assembled into scFv-like heterodimer on FVIII, and the isolated V_H alone also bound FVIII. We discuss the potential use of both protease-cleavable scFvs and isolated Fv subunits retaining high affinity to the antigens in various practical applications such as therapeutics, diagnostics, and research.